Advances in Botanical Research, Volume 51: Plant Innate Immunity

Advances in BOTANICAL RESEARCH VOLUME 51 Advances in BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER Laborato...

Author: L.C. van Loon

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Advances in

BOTANICAL RESEARCH

VOLUME 51

Advances in

BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER

Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France

MICHEL DELSENY

Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

PLANT INNATE IMMUNITY Editor L. C. VAN LOON Plant-Microbe Interactions, Institute of Environmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, LondonNW17BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2009 Copyright © 2009, Elsevier Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-374834-8 ISSN: 0065-2296 For information on all Academic Press publications visit our Web site at www.elsevierdirect.com Printed and bound in USA 09 10 10 9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv

CONTENTS OF VOLUMES 35–50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

PAMP-Triggered Basal Immunity in Plants ¨ RNBERGER AND BIRGIT KEMMERLING THORSTEN NU I. II. III. IV. V.

The Concept of Plant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signals Mediating the Activation of Plant Defense Responses . . . . . . . . . . . . . . Receptors Mediating Pattern Recognition in Plant Immunity . . . . . . . . . . . . . . Signal Transduction in PTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppression of PTI—A Major Virulence Strategy of Phytopathogenic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 15 21 25 27 28 28

Plant Pathogens as Suppressors of Host Defense JEAN-PIERRE ME´TRAUX, ROBERT WILSON JACKSON, ESTHER SCHNETTLER AND ROB W. GOLDBACH I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressors Produced by Fungal and Oomycete Pathogens . . . . . . . . . . . . . . . . Suppressors Produced by Bacterial Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA Silencing, the Plant’s Innate Immune System Against Viruses . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40 42 48 65 74 74

From Nonhost Resistance to Lesion-Mimic Mutants: Useful for Studies of Defense Signaling ANDREA LENK AND HANS THORDAL-CHRISTENSEN I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Defense Induction Mediated by PAMPs and EVectors . . . . . . . . . . . . . . . . . . . .

92 93

vi

CONTENTS III. IV. V. VI. VII.

Signaling Downstream of Pathogen Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commonalities in the Defense Response of Host and Nonhost Resistance . . . . What is the Explanation for Nonhost Resistance?. . . . . . . . . . . . . . . . . . . . . . . . . Lesion-Mimic Mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutant Screens Without Pathogens for Finding Genes in Defense Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

96 99 104 107 108 112 112 112

Action at a Distance: Long-Distance Signals in Induced Resistance MARC J. CHAMPIGNY AND ROBIN K. CAMERON I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Time to Flower—Signaling Events in the Vegetative to Flowering Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Mechanisms of Signaling During the Wound Response . . . . . . . . . . . . . . . . . . . . IV. Long-Distance Signaling in SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Systemic Induced Susceptibility (SIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Signaling During ISR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Techniques to Further Elucidate Long-Distance Signaling. . . . . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124 125 132 138 150 151 153 155 156

Systemic Acquired Resistance R. HAMMERSCHMIDT I. II. III. IV. V. VI.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Biological Spectrum of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Induction of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systemic Biochemical Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How SAR Protects Plants Against Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Comments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 177 177 185 188 209 209 209

Rhizobacteria-Induced Systemic Resistance ¨ FTE DAVID DE VLEESSCHAUWER AND MONICA HO I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signalling in Rhizobacteria-Induced Systemic Resistance . . . . . . . . . . . . . . . . . . . Final Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

224 233 258 265 266

CONTENTS

vii

Plant Growth-Promoting Actions of Rhizobacteria STIJN SPAEPEN, JOS VANDERLEYDEN AND YAACOV OKON I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modes of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agricultural Aspects and Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284 285 304 308 309 310

Interactions Between Nonpathogenic Fungi and Plants M. I. TRILLAS AND G. SEGARRA I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Interactions Between Plants and Endophytic Fungi . . . . . . . . . . . . . . . . . . . . . . . III. Interactions Between Plants and Free-Living Opportunistic Symbiotic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Overview of Plant Defense Mechanisms Induced by Nonpathogenic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

322 323 332 347 350

Priming of Induced Plant Defense Responses UWE CONRATH I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priming is a Mechanism of IR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relevance of Priming in Plant Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

362 362 367 379 384 384 384

Transcriptional Regulation of Plant Defense Responses MARCEL C. VAN VERK, CHRISTIANE GATZ AND HUUB J. M. LINTHORST I. II. III. IV.

Plant Immune Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defense Signaling Regulatory Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcription Factors Regulating Plant Defense Gene Expression. . . . . . . . . . . Regulation of Plant Defenses at the Chromosomal Level . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

398 400 407 420 426 426

viii

CONTENTS

Unexpected Turns and Twists in Structure/Function of PR-Proteins that Connect Energy Metabolism and Immunity MEENA L. NARASIMHAN, RAY A. BRESSAN, MATILDE PAINO D’URZO, MATTHEW A. JENKS AND TESFAYE MENGISTE I. Historical Perspective Leading to the Recognition of Innate Immunity in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Roles of PR-proteins Revealed by Studies of PR gene Expression . . . . . . . . . . . III. PR-5 Protein Structure Reveals the Primitive Relationship Between Pathogen Defense and Energy Balance . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Directions in Which Current Classification or Definition of PR-proteins May Change in the Coming Years as Advanced Functional Studies Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

440 444 450 472 474

Role of Iron in Plant–Microbe Interactions P. LEMANCEAU, D. EXPERT, F. GAYMARD, P. A. H. M. BAKKER AND J.-F. BRIAT I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Strategies of Iron Acquisition and Homeostasis by Plants and Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Reciprocal Interactions Between Plants and Microorganisms During Their Saprophytic Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Reciprocal Interactions Between Plants and Microorganisms During Pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

492 494 505 518 530 532

Adaptive Defense Responses to Pathogens and Insects LINDA L. WALLING I. II. III. IV. V. VI. VII. VIII.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co‐evolution of Defense Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Portals of Entry and Activation of Defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perceiving Pathogen and Pest Visitations: The Role of Microbial and Herbivore Elicitors and Molecular Patterns . . . . . . . . . . . . . . . . . Integrating Signals and Activating Defenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptations to Unfriendly Hosts: Effectors and Evasion Tactics . . . . . . . . . . . . Effector-Triggered Immunity: Resistance to Pathogens and Pests . . . . . . . . . . . . Summary and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

552 554 556 564 576 581 589 593 595 595

CONTENTS

ix

Plant Volatiles in Defence MERIJN R. KANT, PETRA M. BLEEKER, MICHIEL VAN WIJK, ROBERT C. SCHUURINK AND MICHEL A. HARING I. II. III. IV. V. VI.

Introduction to Volatile Organic Compounds (VOCs) From Plants . . . . . . . . . Herbivore-Produced Elicitors and Suppressors of Plant VOC Emission . . . . . . Biosynthesis of Plant VOCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile Metabolism in Plant Trichomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile Defence Hormones MeJA, MeSA and Ethylene. . . . . . . . . . . . . . . . . . . VOC Signals Are Influenced by Abiotic Factors and Plant Developmental Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Natural Variation in VOC Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. VOC-Mediated Specificity of Indirect Defences . . . . . . . . . . . . . . . . . . . . . . . . . . IX. VOCs as Alarm Signals for Neighbouring Plants . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

614 616 619 624 627 632 635 639 643 651

Ecological Consequences of Plant Defence Signalling MARTIN HEIL AND DALE R. WALTERS I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signalling at Three Different Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Costs of Induced Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance Induced by Mutualistic Micro-organisms . . . . . . . . . . . . . . . . . . . . . . Defence Signalling at the Level of Plant Individual, Community and Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

668 669 680 687

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

717

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

739

690 698 699 699

CONTRIBUTORS TO VOLUME 51

P. A. H. M. BAKKER Plant–Microbe Interactions, Institute of Environmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands PETRA M. BLEEKER Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands RAY A. BRESSAN Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia, World Class University Program, Gyeonsang National University, Republic of Korea, and Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA J.-F. BRIAT CNRS, Universite´ Montpellier II, SupAgro, INRA, UMR5004, Biochimie et Physiologie Mole´culaire des Plantes, Place Pierre Viala, F-34060 Montpellier cedex I, France ROBIN K. CAMERON Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 MARC J. CHAMPIGNY Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6 UWE CONRATH Plant Biochemistry and Molecular Biology Group, Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany DAVID DE VLEESSCHAUWER Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium D. EXPERT INRA, AgroParisTech, Universite´ Paris 6, UMR217, Interactions Plantes Pathoge`nes, 16 rue Claude Bernard, F-75005 Paris, France CHRISTIANE GATZ Department of General and Developmental Plant Physiology, Albrecht-von-Haller-Institut, Untere Karspu¨le 2, 37073 Go¨ttingen, Germany F. GAYMARD CNRS, Universite´ Montpellier II, SupAgro, INRA, UMR5004, Biochimie et Physiologie Mole´culaire des Plantes, Place Pierre Viala, F-34060 Montpellier cedex I, France ROB W. GOLDBACH Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands R. HAMMERSCHMIDT Department of Plant Pathology, 107 Center for Integrated Plant Systems Building, Michigan State University, East Lansing, MI 48824-1311, USA

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CONTRIBUTORS

MICHEL A. HARING Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands MARTIN HEIL Departamento de Ingenierı´a Gene´tica, CINVESTAV— Irapuato. Km. 9.6 Libramiento Norte, 36821 Irapuato, Guanajuato, Me´xico ¨ FTE Laboratory of Phytopathology, Department of Crop MONICA HO Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium ROBERT WILSON JACKSON School of Biological Sciences, University of Reading, Reading RG6 6AJ, Berks, United Kingdom MATTHEW A. JENKS Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA MERIJN R. KANT Department of Population Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands BIRGIT KEMMERLING Eberhard-Karls-Universita¨t Tu¨bingen, Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany P. LEMANCEAU INRA, Universite´ de Bourgogne, UMR1229, Microbiologie du Sol et de l’Environnement, CMSE, 17 rue Sully, BV 86510, F-21034 Dijon cedex, France ANDREA LENK Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark HUUB J. M. LINTHORST Institute Biology Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands TESFAYE MENGISTE Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA JEAN-PIERRE ME´TRAUX Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland MEENA L. NARASIMHAN Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia ¨ RNBERGER Eberhard-Karls-Universita¨t Tu¨bingen, THORSTEN NU Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany YAACOV OKON Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel MATILDE PAINO D’URZO Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia

CONTRIBUTORS

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ESTHER SCHNETTLER Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands ROBERT C. SCHUURINK Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands G. SEGARRA Departament Biologia Vegetal, Facultat Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, SPAIN STIJN SPAEPEN Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium HANS THORDAL-CHRISTENSEN Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark M. I. TRILLAS Departament Biologia Vegetal, Facultat Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, SPAIN JOS VANDERLEYDEN Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium MARCEL C. VAN VERK Institute Biology Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands LINDA L. WALLING Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA DALE R. WALTERS Crop & Soil Systems Research Group, Scottish Agricultural College, King’s Buildings, Edinburgh EH9 3JG, United Kingdom MICHIEL VAN WIJK Department of Population Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

PREFACE: PLANT INNATE IMMUNITY

Plants flourish in almost all natural environments, even though they are surrounded by potentially harmful organisms. Because of their sessile nature, they have to cope eVectively with these threats. During evolution they have developed eVective mechanisms to counteract microbial invasion and animal attack. Apart from morphological adaptations, they rely on inducible defenses that are activated in response to infection or attack and limit proliferation of, and tissue colonization by the attacker. Most potential pathogens are halted by the expression of an integrated set of defense responses, comprising the reinforcement of plant cell walls, the synthesis of antimicrobial secondary metabolites (phytoalexins, toxins), and the accumulation of so-called pathogenesis-related proteins (peptides and low-molecular-weight proteins with toxic or lytic properties). Plant innate immunity is a collective term to describe this complex of interconnected mechanisms that plants use to withstand potential pathogens and herbivores. The last decade has seen a rapid advance in our understanding of the induction, signal-transduction, and expression of resistance responses to oomycetes, fungi, bacteria, viruses, nematodes, and insects. The present volume is aimed at providing an overview of these processes and mechanisms. It has become clear that plants have evolved sophisticated mechanisms to deal with environmental challenges. To ward oV attack by diVerent types of organisms, they possess a surveillance system that recognizes common microbial components, such as bacterial lipopolysaccharides and flagellin, or fungal constituents comprising ergosterol and chitin. These so-called MAMPS or PAMPS (microbe/pathogen-associated molecular patterns) bind to receptors on or in plant cells, upon which a signaling cascade is activated that results in a basal resistance against potential pathogens. EVective pathogens are able to evade or suppress basal resistance by secreting eVector molecules (see Fig. 1), and may even interfere with plant defense signaling pathways to their advantage. In turn, plants may activate additional defensive mechanisms, triggered either as a result of the presence of specific resistance (R) genes or through boosting of general defense responses. Even harmless organisms that are perceived through conserved MAMPs will activate the innate immune system. This may explain why plants can develop an enhanced defensive capacity upon a primary infection or attack, which may be maintained for long periods. This induced resistance is often expressed systemically and can protect the plant against further infection by the same or unrelated attackers. So-called

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PREFACE

High ETS

PTI

Pathogen effectors

Priming

Amplitude of defense response

Threshold for HR

Level of induced resistance

Level of basal resistance

Low PAMPs

Fig. 1. The basics of plant innate immunity. Healthy plants constitutively express low levels of defenses. Upon contact with a potential pathogen, conserved pathogenassociated molecular patterns (PAMPs) are recognized, leading to inducible defense responses conferring PAMP-triggered immunity (PTI). PTI may be suppressed in turn by the pathogen through the secretion of eVector molecules, resulting in eVectortriggered susceptibility (ETS). Once inducible defense responses have been activated, the tissue is primed to react more eVectively against further infection (induced resistance). Priming leads to an enhanced defensive capacity, in which the amplitude of defense responses typical of basal resistance is shifted to the higher level of induced resistance. Only rarely does induced resistance lead to a hypersensitive reaction (HR) by which the pathogen is completely halted. Adapted by permission from Macmillan Publishers Ltd: Nature 444, 323–329, copyright 2006; courtesy of Christos Zamioudis.

systemic acquired resistance (SAR) results from limited primary infection by a pathogen, whereas induced systemic resistance (ISR) can be triggered by nonpathogenic organisms that colonize root or leaf surfaces. These forms of induced resistance are phenotypically similar in that they both confer an enhanced defensive capacity on plants that is manifested by an earlier and stronger defense response upon pathogen challenge. However, the two forms are mechanistically diVerent in being based on diVerent molecular mechanisms. ISR-eliciting bacteria and fungi can also promote plant growth by mechanisms that may, or may not, be linked to the induced resistance, and in which the element iron can play an important role.

PREFACE: PLANT INNATE IMMUNITY

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Even though all types of resistance to pathogens in plants are genetically determined, the term ‘‘plant innate immunity’’ is used preferentially to denote basal resistance mechanisms. Plants lack the adaptive system of generating specific antibodies against the intruder, with which we as humans are so familiar. It has been pointed out that R gene-mediated resistance is adaptive in the sense that it evolves with time to stabilize host–pathogen relationships in genetically evolving populations. Because the underlying eVector–R protein relationship is highly specific, R gene-mediated resistance appears superficially similar to adaptive immunity in animals. However, in plants, R gene-mediated resistance is expressed through the same defense responses as those that are active in basal resistance, but on a much grander scale, often culminating in a hypersensitive reaction (HR), in which the tissue surrounding the initial point of infection rapidly necroses. The HR is particularly eVective against biotrophic pathogens, which parasitize on living host tissue, and far less so against necrotrophic pathogens that obtain their nutrients from killed tissues. Hence, plants employ diVerent strategies to deal with biotrophic and necrotrophic pathogens, as they also do against insects with diVerent feeding modes. In this book, aspects specific to R gene-mediated resistance are mentioned only briefly, as this type of resistance is essentially superimposed on basal resistance. It has been intensively studied for many years and been the subject of many excellent reviews. In contrast, concepts and mechanisms of basal resistance have emerged only relatively recently. It has become clear that the activation of appropriate defense responses is critically dependent on the integration of diverse signals from the environment: elicitors from pathogenic and nonpathogenic microorganisms, compounds released from plants as a result of damage by insects, and even volatiles from neighboring plants. The various resistance responses rely on the activation, by a combination of signaling compounds, of transcriptional regulators that determine the type of resistance expressed. An important component in the expression of resistance is the synthesis of pathogenesis-related proteins. These are commonly used as markers of resistance, as they possess potential antimicrobial and antiherbivore properties. Pathogens and insects are resisted through partially diVerent signaling pathways that can act either synergistically or antagonistically depending on the plant species and the attacker involved. Activation of these defenses is metabolically costly, and has ecological consequences by influencing plant fitness. These consequences need to be taken into account when aiming at exploiting plant innate immunity to harness the plant’s own natural defense mechanisms for durable and environmental-friendly crop protection. L. C. van Loon

CONTENTS OF VOLUMES 35–50 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN

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Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB

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Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

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Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI

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The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON

Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

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Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER

Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE

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Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER

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Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP

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Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA ZˇALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL

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Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA

Contents of Volume 46 INCORPORATING ADVANCES IN PLANT PATHOLOGY Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development ´ NICA CREUS, MARI´A CELESTE MOLINA-FAVERO, CECILIA MO LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARI´A CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI and LORENZO LAMATTINA How the Environment Regulates Root Architecture in Dicots ´ RIE LEFEBVRE, PHILIPPE MARIANA JOVANOVIC, VALE LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE HARTMANN and MARTIN CRESPI

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Aquaporins in Plants: From Molecular Structure to Integrated Functions OLIVIER POSTAIRE, LIONEL VERDOUCQ and CHRISTOPHE MAUREL Iron Dynamics in Plants JEAN-FRANC ¸ OIS BRIAT Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions VIVIENNE GIANINAZZI-PEARSON, NATHALIE SE´JALON-DELMAS, ANDREA GENRE, SYLVAIN JEANDROZ and PAOLA BONFANTE Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene-Regulated Responses AUDREY COSSE, CATHERINE LEBLANC and PHILIPPE POTIN

Contents of Volume 47 INCORPORATING ADVANCES IN PLANT PATHOLOGY The Plant Nucleolus ´ EZ-VA ´ SQUEZ AND FRANCISCO JAVIER MEDINA JULIO SA Expansins in Plant Development DONGSU CHOI, JEONG HOE KIM AND YI LEE Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIACHI HSU, YA-PING YANG, WEN-HUEI CHEN AND HONG-HWA CHEN

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Molecular Physiology of Development and Quality of Citrus ´ S, JOSE´ M. FRANCISCO R. TADEO, MANUEL CERCO COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A. NARANJO, GABINO RI´OS, ESTHER CARRERA, OMAR RUIZ-RIVERO, IGNACIO LLISO, RAPHAE¨ L MORILLON, PATRICK OLLITRAULT AND MANUEL TALON Bamboo Taxonomy and Diversity in the Era of Molecular Markers MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH, TARCISO S. FILGUEIRAS AND AMITA PAL

Contents of Volume 48 Molecular Mechanisms Underlying Vascular Development JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO AND CHUNG-MO PARK Clock Control Over Plant Gene Expression ANTOINE BAUDRY AND STEVE KAY Plant Lectins ELS J. M. VAN DAMME, NAUSICAA LANNOO AND WILLY J. PEUMANS Late Embryogenesis Abundant Proteins MING-DER SHIH, FOLKERT A. HOEKSTRA AND YUE-IE C. HSING

Contents of Volume 49 Phototropism and Gravitropism in Plants MARIA LIA MOLAS AND JOHN Z. KISS

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Cold Signalling and Cold Acclimation in Plants ERIC RUELLAND, MARIE-NOELLE VAULTIER, ALAIN ZACHOWSKI AND VAUGHAN HURRY Genome Evolution in Plant Pathogenic and Symbiotic Fungi GABRIELA AGUILETA, MICHAEL E. HOOD, GUISLAINE REFRE´GIER AND TATIANA GIRAUD

Contents of Volume 50 Aroma Volatiles: Biosynthesis and Mechanisms of Modulation During Fruit Ripening BRUNO G. DEFILIPPI, DANIEL MANRI´QUEZ, ´ LEZ-AGU ¨ ERO KIETSUDA LUENGWILAI AND MAURICIO GONZA Jatropha curcas: A Review NICOLAS CARELS You are What You Eat: Interactions Between Root Parasitic Plants and Their Hosts LOUIS J. IRVING AND DUNCAN D. CAMERON Low Oxygen Signaling and Tolerance in Plants FRANCESCO LICAUSI AND PIERDOMENICO PERATA Roles of Circadian Clock and Histone Methylation in the Control of Floral Repressors RYM FEKIH, RIM NEFISSI, KANA MIYATA, HIROSHI EZURA AND TSUYOSHI MIZOGUCHI

PAMP-Triggered Basal Immunity in Plants

¨ RNBERGER AND BIRGIT KEMMERLING THORSTEN NU

Eberhard-Karls-Universita¨t Tu¨bingen, Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany

I. The Concept of Plant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Signals Mediating the Activation of Plant Defense Responses . . . . . . . . . . . . . A. Pathogen-Associated Molecular Patterns................................... B. Damage-Associated Molecular Patterns .................................... C. Pathogen-Derived Toxins as Triggers of Plant Immunity................ III. Receptors Mediating Pattern Recognition in Plant Immunity . . . . . . . . . . . . . IV. Signal Transduction in PTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Suppression of PTI—A Major Virulence Strategy of Phytopathogenic Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 4 11 12 15 21 25 27 28 28

ABSTRACT Significant progress has recently been made in our understanding of the molecular mechanisms that underpin a plant’s ability to cope with microbial infection. A new concept has derived thereof that provides evidence for a functional link between different types of microbial resistance in plants and their evolutionary relationship. Research on microbial elicitor-induced plant noncultivar-specific defenses and Corresponding authors: Email: [email protected], [email protected] Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51001-4

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¨ RNBERGER AND B. KEMMERLING T. NU

microbial avirulence factor-induced host plant cultivar-specific defenses had coexisted for a long time, without providing an integrated model for plant disease resistance. Research milestones that have significantly reshaped our view on plant immunity comprise the realization of conceptual and mechanistic similarities in animal and plant immunity (including the adoption of the term ‘‘immunity’’ into the plant literature), the identification of plant pattern-recognition receptors (PRRs) recognizing pathogen- or microbe-derived molecular patterns (PAMP/MAMP), and the finding that PAMP-triggered immunity (PTI) is a biologically important element of plant disease resistance. Moreover, microbial infection strategies that have evolved for the suppression of PTI underline the importance of this element of the plant immune system.

I. THE CONCEPT OF PLANT IMMUNITY The recent literature on plant defense, disease resistance or susceptibility has adopted a terminology that is very different from that of some time ago. This phenomenon is characterized by the use of such terms as innate immunity, pathogen-associated molecular pattern, pattern-recognition receptors (PRRs), effectors etc. This terminology, which has been deliberately chosen because of obvious cross-kingdom parallels in the molecular concept of immunity, has in a large part replaced the phytopathological vocabulary that has dominated the literature for many years. However, is this development a justified one? As there is still some confusion in the field on this issue, we see a need to address the adequacy of an immunity-associated terminology for the description of plant disease resistance. In general, the term immunity describes the ability of an organism to withstand microbial infection, disease or other disadvantageous biological invasion. As this definition is valid for all multicellular eukaryotic systems, it is correct to refer to the ability of plants to cope with microbial infections as an immune response. Notwithstanding early discussions on analogies between plant disease resistance genes and the major histocompatibility complex of animal systems (Dangl, 1992), the true force that initiated this paradigm shift has been the recent reappreciation of innate immunity in insects and jawed vertebrates as a major element for the containment of microbial infection and for sufficient functioning of the adaptive immune system in animals. Since then, countless landmark papers have highlighted striking similarities in the molecular organization of nonself recognition and antimicrobial defense systems in animals and plants and thus, have substantiated and lent justification to this development (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004). Such similarities are found in the nature of the microbial patterns that are recognized by the innate immune systems in both lineages, extend to the molecular architecture of

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pattern recognition complexes, and culminate in the production of antimicrobial products that eventually halt microbial infections. In the light of such information, the adoption of an immunity-based terminology by molecular plant pathologists is a logical consequence—at least in hindsight. Related terminologies in plant and animal immunity should, however, be taken with caution as significant differences between the systems remain. For example, the term ‘‘innate’’ in conjunction with plant immunity appears to be dispensable, as all immunity-associated traits of plants are inheritable. In animal systems, the term is useful as it discriminates between germline-encoded innate immunity and adaptive immunity, the latter being characterized by the large antigen receptor reservoir resulting from genomic recombination and by clonal expansion of particular lymphocyte populations (Medzhitov, 2007). Adaptive immunity, however, does not exist in plants, and the term ‘‘innate’’ in conjunction with plant immunity is misleading because it implies the existence of another type of plant immunity that is of non‐innate nature. Plants provide multiple habitats that can be invaded by microorganisms, including the roots, leaves, flowers or the vascular system. Some of these niches, such as the rhizosphere, are colonized constitutively by a vast microflora. However, the consequence of microbial colonization on host fitness strictly depends on the microbial strategy to adapt to the host environment. Different symbioses represent cases in which the impact of infection is positive and mutually beneficial to both partners. The relationship between two living organisms in which one benefits, and the other is not significantly affected has been described in plant–microbe associations, but the term commensalism (commonly used in animal ecology to refer to this phenomenon) is unusual in the plant literature. In other cases, microbial colonization can be disadvantageous to the host, and microbes that interact in an antagonistic manner with their hosts, are referred to as pathogens. The ability of infectious pathogens to strive in the specific environment of the host is governed by virulence factors that enable host ingress, the establishment of stable infection structures, suppression and evasion of host defenses, microbial nutrition and proliferation. In turn, microbial infections are considered to be the driving force that shapes the plant immune system during evolution. In brief, the plant immune system consists of two evolutionarily linked branches. The evolutionarily more ancient, primary plant immune response is referred to as PAMP-triggered immunity (PTI) and is based upon the recognition of the invariant structures of microbial surfaces termed PAMPs (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004; Zipfel and Felix, 2005). PAMP-induced immune responses are important for immunity to microbial infection of whole plant species (species

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or nonhost immunity) and for basal immunity in susceptible host plant cultivars (Bittel and Robatzek, 2007; Nu¨rnberger and Lipka, 2005). Suppression of PTI by microbial effectors (effector-triggered susceptibility, ETS) is assumed to be the key for successful pathogens to grow and multiply in a potentially hostile plant environment (Alfano and Collmer, 2004; Chisholm et al., 2006; Jones and Dangl, 2006). As a consequence of a co‐evolutionary arms race, co‐evolution between susceptible hosts and virulent pathogens, individual plant cultivars have acquired resistance (R) proteins that guard microbial effector-mediated perturbations of host cell functions and thus trigger plant immune responses. This type of plant defense is referred to as effector-triggered immunity (ETI) and is synonymous with pathogen race/ host plant cultivar-specific plant disease resistance (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006). The following chapters focus on the current knowledge about PTI. Readers interested in the molecular basis of ETS/ETI are referred to the relevant chapters of this monograph, as well as to a wealth of original and review literature that is available on this topic.

II. SIGNALS MEDIATING THE ACTIVATION OF PLANT DEFENSE RESPONSES A. PATHOGEN-ASSOCIATED MOLECULAR PATTERNS

Activation of inducible host defenses strictly depends on the recognition of potential microbial invaders, regardless of their aggressive potential. Microbial sensing is based on the detection of molecular structures (‘‘patterns’’) that are unique to microorganisms and that enable the host to discriminate between microbial nonself and host-derived self (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). In 1997, Medzhitov and Janeway provided a terminology to describe the elements and processes implicated in innate immunity in various animal systems (Medzhitov and Janeway, 1997). The authors referred to PAMPs as triggers of immune responses in organisms as diverse as human, mice, crustaceans, and insects. Immune defenses in jawed vertebrates comprise proinflammatory cytokine production mediating inflammatory responses (referred to as activation of the ‘‘inflammasome’’), as well as the production and secretion of antimicrobial, proteinaceous defensins (Akira et al., 2006; Medzhitov, 2007). Likewise, insects such as Drosophila melanogaster produce a large blend of antimicrobial peptides that constitute the major executive element in insect innate immunity (Ferrandon et al., 2007; Girardin et al., 2002). Lipopolysaccharides (LPS) derived from Gram-negative bacteria, peptidoglycans from both Gram-positive and

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Gram-negative bacteria, eubacterial flagellin, unmethylated bacterial DNA fragments, as well as fungal cell wall-derived glucans, chitins, mannans and proteins are well-characterized patterns that trigger innate immune responses in numerous vertebrate and nonvertebrate organisms (Aderem and Ulevitch, 2000; Ferrandon et al., 2007; Girardin et al., 2002; Medzhitov, 2007). Undoubtedly, the PAMP-terminology had an enormous impact on molecular plant pathology, which was mainly because many of the microbe-associated patterns with immunity-stimulating features were long known as (general) elicitors of cultivar‐nonspecific defenses in many plants (Boller, 1995; Vorwerk et al., 2004; Zipfel and Felix, 2005) (Table I). For example, peptidoglycans and muropeptides derived from Gram-positive and Gramnegative bacteria, different structural elements of LPS from Gram-negative bacteria, or an N-terminal 22-amino acid fragment of eubacterial flagellin (flg22) are potent inducers of defense-associated responses in various plant species (Erbs et al., 2008; Felix et al., 1999; Gust et al., 2007; Newman et al., 2002). A comprehensive summary of such PAMPs with proven immunitystimulating activities in plants is listed in Table I. The intriguing aspect of that insight was that it implies a common evolutionary concept of microbial pattern recognition that generally underlies activation of antimicrobial counter-defense in multicellular eukaryotes. In addition, such insight also suggested a relevant role of elicitor recognition in plant immunity that had been predicted for a long time. However, the mere existence of such recognition events did not provide evidence for a causal link between elicitorinduced plant defenses and plant disease resistance or immunity. This gap was only filled when the contribution of PRR-mediated basal resistance to overall plant immunity was unequivocally documented (Zipfel et al., 2004). It is needless to say that the term PAMP is a misnomer, because such structures are not only found on pathogenic microbes, but are also characteristic of nonpathogenic microorganisms (Ausubel, 2005). There have been attempts in the recent (plant, but not animal) literature to introduce more correct terms, such as MAMP (Ausubel, 2005) or MIMP (microbe-induced molecular patterns) (Mackey and McFall, 2006). While these terms have their merits and justification, we prefer the further use of the ‘‘historical’’ term PAMP simply for the reason of maintaining the understanding among the communities of animal and plant immunologists. PAMPs constitute abundant, conserved structures (patterns) that are typical of whole classes of pathogens (Medzhitov and Janeway, 1997). In addition, such patterns appear to be absent in eukaryotic host organisms, but are indispensable for the microbial lifestyle. Because of such characteristics PAMPs are considered to be favorite determinants for microbe detection by host-encoded nonself recognition systems. General elicitors of plant defenses

TABLE I Known Inducers of PAMP-Triggered Immunity

PAMP Lipopolysaccharide Peptidoglycan Flagellin

Origin Gram-negative bacteria (Xanthomonas, Pseudomonas) Gram-positive and Gram-negative bacteria Gram-negative bacteria

Elongation factor (EF-Tu)

Gram-negative bacteria

Harpin (HrpZ)

Gram-negative bacteria (Pseudomonas, Erwinia)

Cold-shock protein

Gram-negative bacteria Gram-positive bacteria

Necrosis-inducing proteins (NLP)

Bacteria (Bacillus spp.), fungi (Fusarium spp.), oomycetes (Phytophthora spp., Pythium spp.)

Minimal structural motif required for defense activation

Sensitive plants

Lipid A, lipooligosaccharides

Pepper, tobacco

Muropeptides

Arabidopsis, tomato

flg22 (amino-terminal fragment of flagellin) elf18 (N-acetylated amino-terminal fragment of EF-Tu) Undefined

Arabidopsis, tomato

RNP-1 motif (aminoterminal fragment of the cold-shock protein) Undefined

References Meyer et al. (2001); Newman et al. (2002); Zeidler et al. (2004) Erbs et al. (2008); Felix and Boller (2003); Gust et al. (2007) Felix et al. (1999)

Arabidopsis and other Brassicaceae

Kunze et al. (2004)

Arabidopsis, cucumber, tobacco, tomato Solanaceae

He et al. (1993); Lee et al. (2001a); Wei et al. (1992) Felix and Boller (2003)

Dicotyledonous plants

Bailey (1995); Fellbrich et al. (2002); Mattinen et al. (2004); Pemberton and Salmond (2004); Qutob et al. (2002); Veit et al. (2001)

Transglutaminase

Oomycetes (Phytophthora spp.)

Pep-13 motif (surfaceexposed epitope of the transglutaminase)

Cellulose‐binding elicitor lectin (CBEL) Lipid-transfer proteins (elicitins) Xylanase

Oomycetes (Phytophthora spp.) Oomycetes (Phytophthora spp., Pythium spp.) Fungi (Trichoderma spp.)

Conserved Cellulose‐ binding domain Undefined

Invertase

Yeast

-glucans

Sulfated fucans Chitin

Fungi (Pyricularia oryzae) Oomycetes (Phytophthora spp.) Brown algae Brown algae All fungi

Ergosterol Cerebrosides A, C Oligouronides Cellodextrins Cutin monomers

All fungi Fungi (Magnaporthe spp.) Plant cell wall pectins Plant cell wall cellulose Plant cuticle

Siderophores

Pseudomonas fluorescens

TKLGE pentapeptide (surface-exposed epitope of the xylanase) N-mannosylated peptide (fragment of the invertase) Tetraglucosyl glucitol Branched hepta-glucoside Linear oligo--glucosides Fucan oligosaccharide Chitin oligosaccharides (degree of polymerisation > 3) Sphingoid base Oligomers Oligomers Dodecan-1-ol Undefined

Grapevine, Nicotiana benthamiana, parsley, potato, tobacco Tobacco, Arabidopsis Tobacco, turnip

Brunner et al. (2002); Nu¨rnberger et al. (1994)

Gaulin et al. (2006) Osman et al. (2001)

Tobacco, tomato

Enkerli et al. (1999); Ron and Avni (2004)

Tomato

Basse et al. (1993)

Rice, tobacco, Fabaceae

Fliegmann et al. (2004); Klarzynski et al. (2000); Yamaguchi et al. (2000)

Tobacco Arabidopsis, barley, rice, tomato, wheat Tomato Rice Arabidopsis, tobacco Grapevine Apple, cucumber, tomato Tobacco

Klarzynski et al. (2003) Baureithel et al. (1994); Ito et al. (1997) Granado et al. (1995) Koga et al. (1998) Darvill et al. (1994) Aziz et al. (2007) Fauth et al. (1998) Van Loon et al. (2008)

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meet these characteristics (Brunner et al., 2002; Felix and Boller, 2003). Pep-13 (Nu¨rnberger et al., 1994), a surface-exposed peptide motif present in cell wall transglutaminases (TGase) of various Phytophthora species (Brunner et al., 2002) may serve as a recognition determinant for the activation of defense in plants, including parsley, potato, grapevine and Nicotiana benthamiana (Halim et al., 2005; H.-H. Kassemeyer and T. Romeis, personal communication). Pep-13 sequences are conserved among Phytophthora TGases, but are not found in any proteins of higher eukaryotic origin. The Pep-13 motif is essential for elicitor activity and for TGase activity of the protein, and individual TGase isoforms containing the Pep-13 motif are expressed at all stages of the life cycle of Phytophthora infestans, including plant infection, suggesting that these enzymes play pivotal roles in Phytophthora biology (Fabritius and Judelson, 2003). A similar set of investigations was conducted on cold-shock-inducible RNA-binding proteins that are found in various Gram-positive bacteria (RNP-1) and that induce defense responses in tobacco. As shown for Pep-13, this elicitor also met the characteristics of a PAMP (Felix and Boller, 2003). A conserved central peptide (csp22) within RNP-1 was found in all bacterial RNP-1 orthologs tested. This region proved indispensable not only for the RNA-binding activity of the protein, but was also shown to be necessary and sufficient for its defense-inducing potential. It is reasonable to assume that other microbe-specific structures, such as fungal chitin, oomycete glucans, bacterial flagellin and the bacterial elongation factor, EF-Tu (Kunze et al., 2004), are indispensable for the microbial host as well, and are thus supposed to be conceptually equivalent to PAMPs that trigger innate immunity in animal systems (Medzhitov and Janeway, 1997). Indeed, very recently it was shown that the N-terminal fragment of bacterial flagellin, flg22, was not only sufficient to trigger well-known PAMP responses in Arabidopsis thaliana (Felix et al., 1999), but also for the proper function of flagellar stability and bacterial motility (Naito et al., 2008). A mutation in a conserved aspartic acid residue in flagellin of Pseudomonas syringae pv. tabaci was shown to result not only in weaker PAMP activity, but also in reduced bacterial mobility and fitness (Naito et al., 2008). Intimate contact between pathogen and host surfaces during attempted infection inevitably results in near-simultaneous exposure of various microbial patterns to the repertoire of cognate host PRRs. It is envisaged that activation of inducible plant defenses is likely the result of recognition of complex patterns that build the microbial surface (Nu¨rnberger and Lipka, 2005; Zipfel and Felix, 2005) rather than that of individual recognition events. For example, the cell walls of many phytopathogenic fungi harbor chitins, N-mannosylated glycopeptides and ergosterol, all of which have been

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reported to trigger plant defense responses (Basse et al., 1993; Baureithel et al., 1994; Granado et al., 1995). Various phytopathogenic Gram-negative bacteria harbor plant defense-stimulating LPS and flagellin and produce HrpZ (harpins), bacterial effector proteins that may function as pathogenicity factors during bacterial infection of plants and that are translocated into the plant apoplast via the bacterial type III secretion system (TTSS) upon contact with plants (Felix et al., 1999; He et al., 1993; Lee et al., 2001a; Newman et al., 2002; Wei et al., 1992). Moreover, phytopathogenic oomycetes of the genera Phytophthora and Pythium possess defense-eliciting heptaglucan structures, elicitins and other cell wall proteins (Fellbrich et al., 2002; Kamoun, 2001; Mitho¨fer et al., 2000; Qutob et al., 2002; Veit et al., 2001). Although not all plant species may recognize and respond to all of these signals, plant cells have recognition systems for multiple patterns derived from individual microbial species. This is exemplified by tobacco and Arabidopsis cells, which recognize Ps. syringae-derived harpins and flagellin (Desikan et al., 1999; Felix et al., 1999), and tomato cells that have the abilities to perceive fungal chitin fragments, glycopeptides and ergosterol (Boller, 1995). Obviously, complex pattern recognition in plants is another phenomenon that resembles the activation of innate defense responses in animals. For example, innate immune responses in humans are activated by Gram-negative bacteria-derived LPS, flagellin and unmethylated CpG dinucleotides, which are characteristic of bacterial DNA (Aderem and Ulevitch, 2000; Akira et al., 2006; Medzhitov, 2007). It is currently open whether recognition of multiple signals derived from one pathogen may mediate more sensitive perception or, alternatively, if redundant recognition systems may act as independent back-up systems in the same or different tissues. It was shown that peptidoglycans from Gram-positive bacteria act synergistically on inflammatory cytokine production in human mononuclear macrophages when added simultaneously with Gram-negative bacteria-derived LPS (Wolfert et al., 2002). Similarly, simultaneous application of Ps. syringae-derived LPS and HrpZ1 resulted in synergistic activation of antimicrobial phytoalexin production in parsley cells (our unpublished data). In contrast, bacterial flagellin and EF-Tu activate a common set of signaling events and defense responses, but with additive rather than synergistic effects (Zipfel et al., 2006). Further experiments using different PAMP combinations from the same microbial sources, as well as combinations of individual PAMPs at very low concentrations are needed to prove if synergistic or additive effects are triggered by perception of PAMP combinations. In either case, however, may eukaryotic hosts take advantage of concomitant recognition of microbial patterns.

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PAMPs have recently been characterized as triggers of various plant immunity-associated responses that, however, would not include apoptotic-like, programmed cell death (hypersensitive response, HR). This places PAMPtriggered responses in contrast to ETI responses in resistant host plant cultivars that frequently involve HR (Jones and Dangl, 2006). However, like ETI, that does not always rely on host HR, some microbe-derived PAMPs were shown to trigger HR cell death in a plant cultivar-nonspecific manner. For example, a pentapeptide-motif found within fungal cell wall-associated xylanases is sufficient for the HR elicitor activity of the intact protein (Rotblat et al., 2002). Moreover, flagellin preparations from various, but not all Pseudomonas species exhibited cell-death-inducing activities in tobacco and rice (Che et al., 2000; Hann and Rathjen, 2007), but not in Arabidopsis, whereas Pep-13 triggered cell death in potato, but not in parsley (Halim et al., 2005). Very recently, the flagellin peptide flg22 was also reported to trigger cell death responses in Arabidopsis (Naito et al., 2008). This is contrary to previous reports on such activities of flg22, which raises the suspicion that additional, yet elusive environmental conditions may impinge on the occurrence of PAMP-triggered cell death in plants. Clearly, statements regarding cell death activities of PAMPs need to be made with reference to the experimental system, and cannot be made in a grossly generalizing manner. PAMPs are considered to be building blocks of microbial surfaces that are constitutively present. However, phytopathogenic Gram-negative bacterial species of the genera Pseudomonas, Erwinia, and Ralstonia produce HrpZ, HrpN or PopA proteins (‘‘harpins’’), that are massively secreted only upon attempted invasion of plants (Alfano and Collmer, 2004). Although secreted in a TTSS-dependent manner, unlike typical TTSS effectors ‘‘harpins’’ appear not to be translocated into plant host cells, but to be targeted to the apoplastic plant/bacteria interface. Harpins have been shown to contribute to microbial virulence (Bauer et al., 1995) and to form ion-conducting pores in synthetic and plant lipid-bilayer systems (Lee et al., 2001b; Racape´ et al., 2005). This suggested that these proteins are ‘‘helper proteins’’ that facilitate nutrient delivery into the apoplastic space or effector delivery into host cells as part of the TTSS effector translocon. HrpZ has recently been assigned a function in effector delivery in Ps. syringae, thus rendering the latter assumption more likely (Kvitko et al., 2007). Remarkably, all ‘‘harpins’’ have been shown to trigger noncultivar-specific plant immunity, including HR cell death, in various plant species (Alfano and Collmer, 2004; Lee et al., 2001a). A structure–function analysis of the HrpZ protein from Ps. syringae pv. phaseolicola revealed that the full-length protein was required for its virulence-associated pore-forming activity, while a C-terminal portion of the protein was sufficient for the activation of plant immune responses (Engelhardt et al., 2008). Since the C-terminal portion was also sufficient to

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bind to a previously identified HrpZ binding site in tobacco and parsley membranes (Lee et al., 2001a), it is assumed that HrpZ plays dual roles in plant–microbe interactions as a virulence-promoting factor and as a trigger of PTI (Engelhardt et al., 2008). B. DAMAGE-ASSOCIATED MOLECULAR PATTERNS

Breakdown products of the plant cell wall, also called ‘‘endogenous elicitors,’’ have long been known to elicit plant immune responses (Vorwerk et al., 2004). Plant cell wall-derived oligogalacturonide fragments (pectic fragments from primary cell walls), cellulose fragments (cellodextrins) or cutin monomers stimulate plant immune responses that are indistinguishable from those triggered by microbe-derived PAMPs (Aziz et al., 2007; Darvill et al., 1994; Fauth et al., 1998). Such plant-derived elicitors are likely released by glucohydrolytic activities from attacking microbes, and are thought to be conceptually equivalent to animal tissue-derived ‘‘danger’’ or ‘‘alarm’’ signals. Animal host-derived patterns are produced either upon microbial infection, or as a result of mechanical injury or necrotic cell death (called damage-associated molecular patterns, DAMPs), and act as mediators of cell damage or distress, perception of which eventually culminates in the activation of innate immune responses (Gallucci and Matzinger, 2001; Matzinger, 2002; Seong and Matzinger, 2004). There exists a large body of animal hostderived immunostimulators, comprising glucose-starvation proteins, fibronectins, hyaluronan, heat-shock proteins (Hsp), cardiolipin and -defensins, many of which are sensed through PRRs that also recognize ‘‘classical’’ PAMPs. For example, TLR4 (human Toll-like receptor 4) recognizes bacterial LPS, host-derived Hsp70, and breakdown products of host hyaluronan (Matzinger, 2007). Common to all these signals is that they are not released in/to the blood or lymph system in intact and healthy tissues and, therefore, do not normally get into contact with PRRs on specialized immune cells that patrol the body for the presence of microbial patterns as well as for such determinants of damaged host-self. Because these endogenous immunostimulators are difficult to fit into self/nonself discrimination models (referred to as the ‘‘stranger’’ model), it has been proposed that animal innate immune cells may rather recognize ‘‘danger’’ signals that comprise both, nonself representing microbial PAMPs and damaged self-representing DAMPs (‘‘danger’’ model) (Matzinger, 2002). Infection by microbes usually also inflicts tissue damage and, thus, microbial PAMPs together with tissuederived DAMPs might constitute a more powerful trigger of defense responses than either alone. It is quite conceivable that activation of plant immunity by pectin, cellulose or cutin fragments constitutes a phenomenon that is conceptually similar to DAMP-induced animal immunity.

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¨ RNBERGER AND B. KEMMERLING T. NU C. PATHOGEN-DERIVED TOXINS AS TRIGGERS OF PLANT IMMUNITY

Microbial toxin-induced plant immunity is a virtual paradox that has been known for a long time. Phytopathogenic microorganisms produce numerous cytolytic toxins that function as major virulence factors (Glazebrook, 2005; Van’t Slot and Knogge, 2002). Phytopathogenic necrotrophic fungi, for example, synthesize host-selective and host-nonselective compounds that facilitate killing of host plant tissue (Gijzen and Nu¨rnberger, 2006; Qutob et al., 2006; Van’t Slot and Knogge, 2002; Wolpert et al., 2002). An intriguing characteristic of many of these mycotoxins is that they not only cause damage, but also trigger plant immunity-associated cellular responses. Certain Fusarium spp. produce the sphinganine toxin, fumonisin B1 (FB1), that elicits cytolysis of plant and animal cells most probably through competitive inhibition of ceramide synthase, an enzyme involved in sphingolipid biosynthesis (Tolleson et al., 1999; Wang et al., 1996). However, in addition to cell death, FB1 triggers accumulation of reactive oxygen species (ROS), deposition of callose, defense-related gene expression and production of the phytoalexin camalexin in Arabidopsis (Asai et al., 2000; Stone et al., 2000). Likewise, the cell death-inducing toxins fusicoccin from Fusicoccum amygdali, or AAL-toxin from Alternaria alternata trigger expression of pathogenesis-related (PR) genes in tomato or Arabidopsis, respectively (Gechev et al., 2004; Schaller and Oecking, 1999), whilst the host selective cell death-inducing toxin victorin from Cochliobolus victoriae elicits the production of avenanthramide phytoalexins in oat (Tada et al., 2005). It is most important to state though, that it is uncertain in virtually all cases whether toxin-induced plant immune responses are an unavoidable consequence of toxin action or, alternatively, if toxins have a second role as PAMP-like factors that trigger plant defenses in a host-PRRdependent fashion. Necrosis and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs) are proteins that have been scrutinized for their molecular mode of action (Bae et al., 2006; Fellbrich et al., 2002; Mattinen et al., 2004; Pemberton et al., 2005; Qutob et al., 2002; Veit et al., 2001). NLPs trigger a multifaceted plant immune response in various dicotyledonous plants, but not in monocotyledons. NLPs are found in multiple bacterial, fungal and oomycete species, most of which favor a (hemi)biotrophic, necrotrophic or saprophytic lifestyle (Qutob et al., 2006). NLP sequences are not present in higher eukaryotes, including plants, but were shown to be important virulence factors in Erwinia spp. (Mattinen et al., 2004; Pemberton et al., 2005), incapacitation of which resulted in severely reduced bacterial infection rates. Thus, NLPs appear to fulfill the criteria of a ‘‘classical’’ PAMP. However, unlike PAMPs, NLPs are not ‘‘on display’’ on the microbial surface, but are produced strongly at later

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stages of infection. In the hemibiotroph oomycete, Phytophthora sojae, NLP production was strongest during the transition from the biotrophic to the necrotrophic stage of infection (Qutob et al., 2002). It is notable that a Phytophthora-derived NLP restored virulence of NLP-deficient Erwinia carotovora mutants, suggesting that NLPs from both sources share the same molecular mode of action (our unpublished data). Other characteristics of NLPs further distinguish them from ‘‘classical’’ PAMPs. In most PAMPS characterized so far, small ‘‘antigenic’’ epitopes within the intact molecule were identified as sufficient for the immunomodulatory activities of these compounds (Table I). However, no such specific motives could be identified within various NLPs (Fellbrich et al., 2002; Gijzen and Nu¨rnberger, 2006). Moreover, NLPs cause cytolytic cell death in dicotyledonous plants that is genetically different from the programmed cell death that is characteristic of the HR (Qutob et al., 2006), whereas many PAMPs either do not induce HR at all or trigger a HR that involves activation of salicylic acid (SA)-dependent responses similar to those occurring during R gene-mediated resistance (Jones and Dangl, 2006; Kamoun, 2001). NLP-induced plant PR gene expression was also SA-dependent (Fellbrich et al., 2002). Furthermore, NLPs disrupt plasma membrane vesicles prepared from dicot plants, but not those from monocot plants, indicating that an intact cell is not required for NLP-induced cell death (our unpublished data). Altogether, these findings suggest that NLP-induced cell death is a symptom of impending disease, not a plant defense response. Indeed, the cytolytic activity of NLPs and the broad spectrum of NLP-sensitive plants rather suggest that these proteins are microbial toxins with defense-activating potential. The elucidation of the 3D-structure of a Pythium aphanidermatum NLP (C. Oecking and H. U. Seitz, personal communication) and the computational modeling of additional NLP folds revealed a high degree of structural conservation between prokaryotic and eukaryotic NLPs. NLP structures closely resemble those of known proteinaceous cytolytic toxins, called actinoporins. Actinoporins bind to lipid docking sites in animal host target membranes, insert into membranes, form ion-conducting pores and subsequently mediate target cell lysis (Parker and Feil, 2005). Structure–activity relationship analyses performed on amino acid residues that are highly conserved among all NLPs, suggested a close correlation between the ability of mutant proteins to cause cytolysis, to restore virulence in NLP-deficient Erwinia, and to induce plant immune responses (our unpublished data). This is very important as it indicates that a common fold of a cytolytic toxin mediates both, microbial attack and plant immunity. NLPs are distinguished from other known phytotoxins by their wide distribution across taxa and their broad spectrum of activity against

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dicotyledonous plants. In particular, the production of orthologous cytolytic toxins in prokaryotic and eukaryotic phytopathogenic microbes is unprecedented and suggests that NLPs constitute an evolutionarily ancient toxin fold that has been retained preferentially in organisms with a hemibiotrophic or necrotrophic lifestyle. These findings also suggest that toxin-induced interference with cell integrity may culminate in plant immune responses. Importantly, microbial toxin-induced innate immunity is also known from animal systems. Cytolytic bacterial (pneumolysin), fungal (nigericin) or marine (maitotoxin) toxins are triggers of the mammalian inflammasome (Mariathasan et al., 2006; Srivastava et al., 2005). Toxin-mediated activation of mammalian immune responses is based upon the recognition of endogenous, host-derived compounds that are released as a result of toxin-induced host cell damage and subsequent formation of DAMPs (Matzinger, 2002, 2007). Consequently, mammalian inflammasome activation is now considered to be activated not only upon perception of microbial patterns, but also by the action of toxins (Dostert et al., 2008; Mariathasan et al., 2006; Martinon et al., 2004; Srivastava et al., 2005). We suggest that plant cells are also capable of sensing toxin-induced cellular changes. NLP-driven membrane disruption may result in the release of host-derived molecules that serve as endogenous DAMPs. Alternatively, NLP-induced disturbance of the cellular ion homeostasis or membrane potential may signal activation of plant defenses. Remarkably, NLP-induced Ca2þ influx as well as Kþ efflux that mimicked synthetic ionophore-induced ion fluxes in plant cells were reported (Fellbrich et al., 2002), which themselves were shown to trigger plant defense-associated responses in a non-receptor-mediated manner (Jabs et al., 1997). Our structure-based analyses suggest that NLPs are cytolytic toxins that trigger plant immunity-associated defenses through interference with plant tissue integrity. Hence, disturbed host integrity as a common signal for the activation of immune defenses adds to the list of conceptual similarities in the organization of innate immunity in the animal and plant lineages. Peptaibols, the products of nonribosomal peptide synthetases, are linear peptide antibiotics produced by various fungal genera, including Trichoderma. These compounds are assumed to contribute to the protection of fungi against bacterial infections (Engelberth et al., 2000; Viterbo et al., 2007). Several hundred different peptaibols have already been identified, numerous of which were shown to possess plant immunity-stimulating potential (Viterbo et al., 2007). The antibiotic functions of peptaibols have been assigned to their membrane insertion and pore-forming abilities (Engelberth et al., 2000; Viterbo et al., 2007). It is not known whether ion-pore formation is the trigger for immune activation in peptaibol-sensitive plants. Thus, these

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molecules may initiate the release of host-derived DAMPs, may stimulate plant immune responses directly via their ionophore-like activity and disturbance of host cell homeostasis, or, may alternatively be recognized by yet elusive PRRs.

III. RECEPTORS MEDIATING PATTERN RECOGNITION IN PLANT IMMUNITY Vertebrate immune cells sense microbe- or host damage-derived patterns by a family of receptors that resemble the Drosophila Toll protein and are thus referred to as Toll-like receptors (TLRs) (Aderem and Ulevitch, 2000; Akira et al., 2006; Cook et al., 2004; Ferrandon et al., 2007; Girardin et al., 2002; Medzhitov, 2007). TLRs are composed of extracytoplasmic leucine-richrepeat (LRR) domains, a transmembrane domain and a cytoplasmic TIR domain (Drosophila Toll and human interleukin-1 receptor) (Cook et al., 2004; Underhill and Ozinsky, 2002). Human cell types implicated in innate immunity (mucosal epithelia, phagocytes) express a total of 13 different TLRs that are often implicated in the recognition of various structurally different patterns (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). For example, whereas TLR4 recognizes bacterial LPS, host-derived Hsp70 and hyaluronan, TLR2 recognizes approximately 15 microbial and host-encoded agonists (Akira et al., 2006). This is probably brought about by stimulus-specific assembly of multicomponent perception complexes in which individual TLRs represent subunits that supposedly mediate transmembrane signaling. For the sake of correctness, it should be mentioned that several other receptor types have also been implicated in inflammasome activation in animal innate immunity (Ishii et al., 2008) and therefore TLRs represent only one single class of PRRs in animals. Proteinaceous binding sites for microbial patterns have been detected in plasma membrane preparations from various plants, but biochemical purification of these proteins proved notoriously difficult (Montesano et al., 2003; Nu¨rnberger et al., 2004). The first successful purification of a PAMP binding site was reported from soybean membranes that mediated recognition of 1,6--linked, 1,3--branched heptaglucosides (HG) from the cell walls of the phytopathogenic oomycete, Ph. sojae (Mitho¨fer et al., 1999; Umemoto et al., 1997). This 75-kDa HG-binding protein (HGP) conferred glucan binding to transgenic tobacco plants, but lacked a transmembrane or membrane attachment domain (Umemoto et al., 1997). This binding protein was shown to harbor an intrinsic endoglucanase activity that was capable of releasing small oligomeric 1,3--D-oligoglucosides from complex glucans

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(Fliegmann et al., 2004). Thus, during attempted infection plant glucanases may release oligoglucoside fragments from the oomycete cell wall that constitute suitable ligands for a yet unknown transmembrane glucan receptor. The lack of functional domains for transmembrane signaling in HGP and the proposed existence of multimeric glucan recognition systems in different Fabaceae (Mitho¨fer et al., 1999) suggest that plant PAMP perception system architecture may be as multimeric as in animal cells (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). The Arabidopsis FLS2 (FLAGELLIN SENSING 2) gene encodes a plasma membrane LRR-receptor kinase (LRR-RK) that recognizes bacterial flagellin via its extracytoplasmic LRR domain (Go´mez-Go´mez and Boller, 2000). FLS2 is the only flagellin receptor in Arabidopsis because the loss of FLS2 resulted in complete flagellin insensitivity (Go´mez-Go´mez and Boller, 2000), the presence of flagellin binding sites correlated with flagellin sensitivity in all ecotypes tested (Bauer et al., 2001), and the expression of Arabidopsis FLS2 in tomato conferred Arabidopsis flagellin signaling specificity to this plant (flagellin recognition specificities in tomato and Arabidopsis are subtly different) (Robatzek et al., 2007). Flagellin perception is widespread among Solanaceae and Brassicaceae (Go´mez-Go´mez and Boller, 2002), but is lacking, for example, in Umbelliferaceae (our unpublished data). Importantly, flagellin-induced immune responses are necessary for the restriction of the growth of the adapted (virulent) Ps. syringae pv. tomato strain DC3000 (Pst), as fls2 mutants were more susceptible to this pathogen (Zipfel et al., 2004). Thus, bacterial pattern recognition through the PRR FLS2 contributes to basal plant immunity against adapted pathogens (restriction of growth) and likely to species immunity against nonadapted pathogens (halt of growth). This is a most noteworthy finding because this is unequivocal evidence that PTI contributes indeed measurably to plant resistance. It should, however, be stated that not in all cases inactivation of individual PRR may result in statistically significant reduction of overall basal immunity (residual immunity in susceptible hosts) to adapted (virulent) pathogens. This is because the overall aggressiveness of virulent strains may simply override subtle effects brought about by PTI, an immunity that has been rendered insufficient by adapted pathogens. However, the use of partially ‘‘disarmed’’ pathogenic strains (lacking individual effectors, such as Pst AvrPto/AvrPtoB; Shan et al., 2008), nonpathogenic strains (avirulent or TTSS-deficient strains), or nonadapted strains (for which a given plant species is not a host) in infection assays on plants lacking individual PRRs may be a suitable way to demonstrate experimentally the role of these receptors in plant basal immunity. Both plants and animals possess flagellin perception systems that mediate the activation of appropriate innate immune responses (Fig. 1). The human

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Flagellin epitopes

Bacteria

Animals

Plants TLR5 FLS2

BAK1 Plasma membrane MyD88 P

P

IRAK

Innate immune responses

Fig. 1. PAMP perception in plants and animals. Different epitopes of bacterial flagellin are perceived by LRR-type PRRs in plants (FLS2 supported by its co-receptor BAK1) and animals (TLR5). While the plant receptor utilizes cytoplasmic kinase domains for transduction of the signal across the plasma membrane, the animal TLR carries a TIR-(TOLL-Interleukin receptor) domain that interacts via adapter proteins (MyD88, Myeloid differentiation factor 88) with a cytoplasmic kinase (IRAK, Interleukin1-receptor associated kinase). The evolutionary conservation of motives, such as LRR and protein kinase domains, in eukaryotic PPRs probably reflects their biochemical suitability to mediate protein–protein interactions, as well as to initiate intracellular signaling cascades. The fact that both flagellin receptors perceive different epitopes of bacterial flagellin together with the differences in domain structures of FLS2 and TLR5 indicates that flagellin perception in both lineages arose as the result of convergent evolution.

flagellin receptor TLR5 (Hayashi et al., 2001) is the conceptual counterpart of plant FLS2. Beside the striking fact that flagellin perception exists in both lineages, a structural comparison between FLS2 and TLR5 revealed conservation of the modular structure among both receptors. Although amino acid sequences of both receptors differ considerably, both proteins carry extracytoplasmic LRR domains that are linked to a cytoplasmic portion (Fig. 1). Whereas the cytoplasmic portion of FLS2 represents a functional serine/ threonine protein kinase itself, the TIR domain of TLR5 forms a complex with the Interleukin-1-receptor-associated kinase, IRAK, and the adaptor

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protein, MyD88 (Hayashi et al., 2001), and thereby links flagellin perception to cytoplasmic protein kinase activity. Thus, the domain or module structure of flagellin perception appears to be strongly conserved across kingdom borders. However, differences in the cytoplasmic domains of both receptors also suggest that flagellin perception systems arose independently, and are the result of convergent evolution rather than of divergent evolution. This view (Ausubel, 2005) is further substantiated by the absence of LRR receptor-mediated immunity in unicellular eukaryotes (the supposed phylogenetic divergence point of animals and plants), and by the fact that the antigenic epitopes within flagellin required for FLS2 activation (the conserved flg22 motif within the N-terminal region of bacterial flagellin) and TLR5 (two helical structures within the central portion of flagellin) are grossly different (Go´mez-Go´mez and Boller, 2002) (Fig. 1). Plants possess approximately 235 LRR-RKs (Shiu et al., 2004), many of which are expected to serve as PRRs in PAMP perception (Nu¨rnberger and Kemmerling, 2006). This assumption is based on the fact that transcript levels encoding multiple LRR-RK-encoding genes increased upon pathogen infection or PAMP treatment. For example, bacterial infection of Arabidopsis plants resulted in enhanced transcript levels of 49 LRR-RK-encoding genes (Kemmerling et al., 2007), while flagellin treatment led to increased transcript accumulation for 28 LRR-RK-encoding genes (Navarro et al., 2004). Expression of flagellin-responsive LRR-RK genes was also triggered by other PAMPs, including bacterial LPS and fungal chitin (Thilmony et al., 2006; Zhang et al., 2002). The latter was astounding as it suggested that different PAMPs triggered a generic plant response that could potentially facilitate or improve recognition of different microbial species. An N-terminal, acetylated 18-amino acid fragment (elf18) of Escherichia coli elongation factor Tu (EF-Tu) was identified as another PAMP that triggered plant immunity-associated responses in Arabidopsis (Kunze et al., 2004). Based upon identical response patterns observed in Arabidopsis seedlings treated with flg22 or elf18, it was assumed that the EF-Tu receptor might be structurally related to FLS2 (Zipfel et al., 2006). Screening a collection of T-DNA insertion lines impaired in the expression of flg22responsive LRR-RK genes, yielded an elf18-insensitive mutant line (Zipfel et al., 2006). Insensitivity to elf18 was restored by ectopic expression of the EFR (EF RECEPTOR) gene in this mutant. Moreover, expression of the EFR gene in N. benthamiana conferred elf18 sensitivity that was absent from wild-type plants. EFR possesses an extracytoplasmic LRR domain that is linked to a cytoplasmic serine/threonine kinase domain and is, therefore, closely similar to FLS2 (Go´mez-Go´mez and Boller, 2000; Zipfel et al., 2006). As EFR and FLS2 group into the same clade of LRR-RK genes (LRR XII

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clade) (Shiu and Bleecker, 2001; Shiu et al., 2004), it is assumed that more members of this particular clade encode ‘‘orphan’’ PRRs that sense yet unknown microbial patterns. EFR-mediated sensing of bacteria did not contribute measurably to basal immunity of Arabidopsis against Pst infections, but limited Agrobacterium tumefaciens-mediated transformation of this plant (Zipfel et al., 2006). Mutant efr lines consistently showed 50-fold higher expression levels upon transformation with a pBIN1935S::-GLUCURONIDASE (GUS) reporter gene construct. This finding is also important from a biotechnological point of view, as it indicates that suppression of PRR-mediated microbial pattern recognition might be a valuable strategy to develop efficient transformation protocols for crop plants that are difficult to transform by Agrobacterium-mediated technologies. Regardless of the importance of LRR-RKs as PRRs in plant immunity, the existence of other types of plant PRRs is expected (Bittel and Robatzek, 2007; Nu¨rnberger and Kemmerling, 2006). For example, fungal chitin perception in rice occurs by a plasma membrane LysM (lysine motif) receptor protein (LysM-P) carrying an extracytoplasmic LysM domain that is linked to a very small cytoplasmic domain (Kaku et al., 2006). Biochemical evidence suggests that the LysM domain directly mediates binding of oligomeric chitooligosaccharide fragments (Ito et al., 1997; Kaku et al., 2006). It is currently unknown whether the very short cytoplasmic tail of this protein contributes to the initiation of an intracellular signaling cascade. Alternatively, LysM-P may also form complexes with transmembrane proteins carrying cytoplasmic signaling domains. An Arabidopsis LysM-RK (CERK1) has recently been found to be implicated in chitin perception (Miya et al., 2007). Mutants defective in the expression of the CERK1 gene lacked any chitininducible immune responses, such as production of ROS (oxidative burst) or defense-related gene expression. It is unclear whether the LysM domain of CERK1 binds chitin physically. Therefore, it remains to be seen whether or not chitin perception and signaling are brought about by the same or distinct proteins. In any case, chitin recognition in plants appears to engage at least two types of LysM-domain proteins (LysM-P, LysM-RK). The LysM (lysine motif) domain consists of approximately 40 amino acid residues and has originally been found in a variety of bacterial enzymes involved in cell wall biosynthesis and degradation (Bateman and Bycroft, 2000). The finding that plant LysM domain-containing proteins bind fungal chitin (CERK1, plant chitinases) is interesting from an evolutionary point of view, because the bacterial LysM domain is known as a general peptidoglycan-binding module that is present in a number of peptidoglycan-modifying enzymes, such as Es. coli murein transglycosylase D (Bateman and Bycroft, 2000). Peptidoglycans, also known as murein, are polymers consisting of

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sugars and amino acids that form a mesh-like layer in the cell walls of Gram-negative and Gram-positive bacteria. The sugar component consists of alternating residues of -(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid residues (a heteroglycan). Attached to the N-acetylmuramic acid is a peptide chain of three to five amino acids that can be cross-linked to the peptide chain of another strand forming a 3D mesh-like layer. Apparently, during evolution plant LysM domain-containing proteins have acquired the ability to bind fungus-derived homoglycans consisting of -(1,4)-linked N-acetylglucosamine (chitin) that are structurally related to the carbohydrate backbones of bacterial peptidoglycan. Moreover, the LysM motif is present in the Lotus japonicus lipochitooligosaccharide (Nod-factor) receptor kinases LjNFR1 and LjNFR2 that mediate the establishment of symbiosis between leguminous plants and rhizobacteria (Radutoiu et al., 2003). This suggests that plants utilize LysM domain host receptors as a general module for chitin-based self/nonself discrimination in both, symbiotic and antagonistic plant–microbe interactions. A more general assumption would be that carbohydrate ligands are preferentially recognized by LysM domain-containing PRRs, whereas proteinaceous ligands may be recognized preferably by LRR domain-containing proteins. As peptidoglycans are triggers of immune responses in Arabidopsis and tobacco (Erbs et al., 2008; Felix and Boller, 2003; Gust et al., 2007), it will be important to test whether plant LysM domain-containing proteins may (in addition to their ability to sense fungal chitin fragments) also recognize and respond to peptidoglycan. At least six peptidoglycan perception systems in humans [TLR2, NOD1 (Nucleotide binding oligomerization domain), NOD2] and Drosophila immune cells [PGRP (Peptidoglycan recognition protein)-SA, PGRP-LC, PGRP-SC1B] that recognize different fragments of bacterial peptidoglycans (Akira et al., 2006; Ferrandon et al., 2007) are known. However, none of these proteins is a LysM-P or LysM-RK, suggesting that LysMmediated carbohydrate recognition in eukaryotic innate immunity has primarily, and probably independently, evolved in plants. A plasma membrane-anchored extracellular LRR-protein (LRR-P) lacking a cytoplasmic signaling domain has been implicated in the recognition of a fungal xylanase and subsequent activation of noncultivar-specific immunity in tomato (Ron and Avni, 2004). As plant genomes harbor multiple LRR-P-encoding sequences (the Arabidopsis genome contains 57 LRRP-encoding sequences) (Wang et al., 2008), it is conceivable that these proteins constitute another class of PRRs that are mechanistically similar to animal LRR-P-type PAMP receptors (Bittel and Robatzek, 2007; Nu¨rnberger and Kemmerling, 2006). Whether ligand perception and intracellular signal transduction through plant LRR-Ps (similar to LysM-P) requires additional components remains, however, to be shown.

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IV. SIGNAL TRANSDUCTION IN PTI PAMP-mediated activation of PRRs transduces ligand-encoded information across the plasma membrane and initiates a host signaling cascade that culminates in the execution of pathogen-nonspecific immune responses. Previously obtained pharmacological evidence suggested that protein kinase activity is required to trigger very rapid PAMP responses in plants, such as influxes of Hþ and Ca2þ across the plasma membrane (Felix and Boller, 2003; Felix et al., 1991, 1999; Nu¨rnberger et al., 1994). The fls2-17 allele of the flagellin receptor FLS2 carries a point mutation in the protein kinase domain that confers insensitivity to flagellin and loss of autophosphorylation activity of FLS2 in vitro (Go´mez-Go´mez et al., 2001). This mutation also abolished the binding of flg22 to the LRR domain of FLS2, suggesting that the overall stability or conformation of the receptor was altered by this single mutation. A point mutation in a putative phosphorylation site of FLS2 also led to flagellin insensitivity, which further documents the importance of protein kinase activity for the activation of flagellin-inducible plant responses (Robatzek et al., 2006). Moreover, overexpression of KAPP, a kinase-associated protein phosphatase that is supposed to regulate the function of several transmembrane RKs, resulted in flagellin insensitivity and reduced flagellin binding to FLS2 (Go´mez-Go´mez et al., 2001). Thus, the current knowledge suggests that phosphorylation of FLS2 (by its own intrinsic PK activity or by another, yet unknown, PK) is a crucial element of flagellin sensing/signaling. More recently, BAK1 (BRI1-associated receptor kinase 1), an LRR-RK that was previously shown to control plant growth by hormone-dependent heterodimerization with the plant brassinosteroid (BR) hormone receptor, BRI1 (brassinosteroid-insenstive 1; an LRR-RK itself) (Li et al., 2002; Wang et al., 2001), has been implicated in FLS2 and EFR function (Chinchilla et al., 2007). BAK1 mutants were (partially) insensitive to both flg22 and elf18. Flg22-dependent rapid heterodimerization of FLS2 and BAK1 was demonstrated by co-immunoprecipitation experiments (Chinchilla et al., 2007), suggesting that BAK1 function follows the same mode of action in activation of both, FLS2 and BRI1. In addition to its role as a positive regulator of PTI and plant growth, BAK1 appears to fulfill other functions. bak1 mutants were recently shown to have altered disease-resistance phenotypes to biotrophic and necrotrophic pathogens, that are likely to be the consequence of infection-induced deregulated cell death control (Kemmerling et al., 2007). Thus, in addition to its role as a positive regulator of PTI, BAK1 may act as a negative regulator of plant cell death. Both plant immunityassociated functions of BAK1 are independent of the BR activity, because

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several mutants impaired either in BR sensitivity or biosynthesis were not impaired in flagellin sensitivity or infection-induced runaway cell death (Chinchilla et al., 2007; Kemmerling et al., 2007). Thus, BAK1 has BRindependent, immunity-associated functions in addition to its well-established, BR-dependent role in plant development (Morillo and Tax, 2006). BAK1 represents a second example of a plant LRR-RK with dual functions in plant development and immunity, as the LRR-RK ERECTA was previously implicated in both flower development and plant pathogen resistance (Godiard et al., 2003; Llorente et al., 2005). Dual roles for receptor proteins in development and immunity are also known from animal systems. For example, the Drosophila receptor TOLL controls embryonic patterning in larvae and immunity against fungal infections in adult insects (Lemaitre et al., 1996). Attenuation and termination of PRR function in animal immunity is achieved mainly by the activities of negative regulators. In Arabidopsis, KAPP-mediated inactivation of FLS2 has been proposed to be such a mechanism (Go´mez-Go´mez et al., 2001). More recently, proteasomedependent, ligand-induced endocytosis of FLS2 has been demonstrated and proposed to be an additional way to shut down PRR activity in plant immunity (Robatzek et al., 2006). In addition, internalization of FLS2 into endosome-like compartments may contribute to flg22 signaling. PTI signaling pathways further employ altered cytoplasmic Ca2þ levels, ROS, nitric oxide (NO) and several mitogen-activated protein kinase (MAPK) cascades (Jonak et al., 2002; Nu¨rnberger et al., 2004; Zhang and Klessig, 2001). Many of these components are important for PAMP-induced activation of innate immune responses in animal cells (Barton and Medzhitov, 2003) also, lending further support to the view of conceptual and mechanistic conservation in the molecular architecture of eukaryotic innate immunity across kingdom borders. NO production has been observed in both PAMP-treated plants and during ETI in resistant host plants (Clarke et al., 2000; Delledonne et al., 1998; Durner et al., 1998). Although there is no evidence for a plant ortholog of human NO SYNTHASE (hNOS ), pharmacological hNOS inhibitors blocked both infection and elicitor-stimulated NO production, cell death and defense gene activation in plants (Delledonne et al., 1998; Durner et al., 1998). Zeidler et al. (2004) reported that AtNOS1, a plant-specific NOS previously associated with hormone signaling in plants (Guo et al., 2003), mediated LPS-induced NO production and PR gene expression in Arabidopsis. Importantly, inactivation of the AtNOS1 gene did not only abrogate LPS-induced NO production in these plants, but also dramatically enhanced susceptibility of the mutant to

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Pst infection. However, the role of AtNOS1 as a bona fide plant NO synthase was recently questioned, as the 3D-fold of the protein suggested similarities to mitochondrial GTPase involved in mitochondrial biogenesis (Zemojtel et al., 2006). Thus, it appears likely that reduced levels of NO in Atnos1 mutants and the observed immunity-associated phenotypes are due rather to mitochondrial dysfunction affecting NO production (mitochondria are a major source of NO) than to a lack of NO synthase activity (Zemojtel et al., 2006). NO-mediated protein nitrosylation has been found in PAMPtreated plant cells (Lindermayr et al., 2005, 2006). Recently, NO-mediated nitrosylation of NPR1 (Nonexpressor of PR genes 1), a key molecule in the establishment of SA-dependent systemic acquired resistance, and of AtSABP3, a SA-binding protein with intrinsic carbonic anhydrase activity and a proven role in basal immunity to bacterial infection, were shown to be necessary for the biological contribution of both proteins to different types of disease resistance (Tada et al., 2008; Wang et al., 2009). This strongly suggests that infection-induced nitrosylation bursts mediate redox changes in the plant cell that affect biological activities of proteins through posttranslational nitrosylation events (Wang et al., 2009). MAPKs are central points of cross-talk in plant signaling cascades, including those that protect against microbial invasion (Go´mez-Go´mez and Boller, 2002; Jonak et al., 2002; Nakagami et al., 2005; Zhang and Klessig, 2001). Various fungus- or bacteria-derived PAMPs and phytopathogenic microbes activate MAPK enzyme activities in a transient fashion. For example, Arabidopsis AtMPK3 and AtMPK6 are responsive to PAMP treatment or infection (Jonak et al., 2002; Zhang and Klessig, 2001). Silencing of MPK6 resulted in remarkably compromised disease resistance in Arabidopsis (Menke et al., 2004). In PAMP-treated parsley cells, PcMPK3 and PcMPK6 translocate into the nucleus (Lee et al., 2004; Ligterink et al., 1997) and contribute to WRKY transcription factor-dependent PR gene expression (Eulgem et al., 1999; Kroj et al., 2003). Using an Arabidopsis protoplast transient expression system, Asai et al. (2002) identified a flg22induced MAP kinase cascade consisting of the MAPK kinase kinase MEKK1, the MAPK kinases MKK 4/5 and MAPK 3/6, and WRKY transcription factors acting downstream of FLS2, and proposed a role of this cascade in bacterial and fungal resistance (Asai et al., 2002). Importantly, PAMP-triggered MAPK pathways regulate PTI-associated responses both in a positive and a negative manner. The flg22 peptide was recently shown to activate the MEKK1/MKK1/MPK4 pathway that suppresses various pathogen defense responses, including callose deposition and PR gene expression (Ichimura et al., 2006; Suarez-Rodriguez et al., 2007). Thus, fine-tuning of

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PTI-associated responses is brought about by two MAPK pathways that exert positive and negative regulatory activities. Surprisingly, flg22-induced activation of the MPK4 pathway did not require MEKK1 kinase activity, suggesting that the protein may exert biological functions as a scaffold or structural protein (Suarez-Rodriguez et al., 2007). The only known substrates that are directly phosphorylated by AtMPK6 are two isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene (ET) biosynthesis (Liu and Zhang, 2004). Phosphorylation of ACS2 and ACS6 by MPK6 led to the accumulation of the ACS proteins, elevated levels of cellular ACS activity, increased ET production and ET-induced plant phenotypes. Causal links between MAPK activation, expression of PR genes, and the initiation of programmed cell death were suggested by a set of loss- and gain-of-function experiments performed in tobacco or Arabidopsis, respectively (Jonak et al., 2002; Nu¨rnberger et al., 2004; Ren et al., 2002; Zhang and Klessig, 2001). Surprisingly little is known about the role of plant hormones in the activation of PTI-associated immune responses. Classical hormones implicated in the establishment of ETI, such as SA, jasmonic acid (JA) or ET, appear not to be required for flg22-induced defense responses and basal immunity to Pst (Zipfel et al., 2004).Thus, although flg22 triggers ET biosynthesis in Arabidopsis (Felix et al., 1999), this may not be necessary for the activation of PTI in PAMP-treated cells, but might contribute to the activation of defenses in plant cells remote from the site of PAMP application. Flagellin treatment further induces a plant microRNA (miRNA) that negatively regulates transcript levels of the auxin receptors TIR1, AFB (Auxin binding factor) 2, and AFB3 (Navarro et al., 2006). Moreover, repression of auxin signaling restricted growth of Pst, suggesting that the growth-promoting hormone, auxin, is a disease susceptibility factor and that miRNAmediated suppression of auxin signaling is an element of PTI. Suppression of auxin-mediated growth as a consequence of activated immunity might reflect the functionality of an innate, built-in trade-off between plant immunity and growth programs. Expression of genes encoding several components implicated in basal immunity of host plants or species resistance is enhanced by various PAMPs (Bittel and Robatzek, 2007). Gene products, such as PENETRATION 1 (PEN1), PEN2, PEN3, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4), are thus likely to contribute to PAMP-triggered immune responses in infected plants. For more details on these proteins the reader is referred to the relevant chapters of this issue.

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V. SUPPRESSION OF PTI—A MAJOR VIRULENCE STRATEGY OF PHYTOPATHOGENIC BACTERIA Infection of host plants by virulent pathogens requires tolerance to or active evasion of the host immune system. A major strategy of virulent pathogens to facilitate infections in susceptible host plants is effector-mediated suppression of PTI. This phenomenon was first documented by showing that TTSSdeficient mutants of Pst (incapable of effector delivery into host cells) triggered a number of plant defenses (callose deposition, PR gene expression) that were suppressed by virulent Pst (Hauck et al., 2003). Activation of these responses by TTSS-deficient bacteria was proposed to be mediated through PAMP perception/PRR activities, and, later on, such responses were indeed reported to be triggered by various PAMPs (He et al., 2007). Among the first bacterial effectors shown to suppress PTI-associated responses were AvrPto and AvrPtoB (He et al., 2006). In a screen that aimed at identifying Pst effectors that suppress flg22 responses in Arabidopsis protoplasts, both effectors, but not several others, were identified to block PAMP-induced signal transduction cascades upstream of MAPK activities (He et al., 2006). In these experiments, it remained unclear whether these effectors inhibited a regulator of PTI pathways or interfered directly with components of PAMPtriggered signaling pathways. The 3D-structure elucidation-based identification of AvrPto as a Ser/Thr protein kinase inhibitor (Xing et al., 2007) favored the idea that soluble protein kinases and/or cytoplasmic protein kinase domains of transmembrane LRR-RKs could serve as direct targets for bacterial effector activities in planta. Indeed, AvrPto has very recently been shown to bind to BAK1 in vivo, thereby inhibiting flg22-induced heterodimerization of FLS2 and BAK1 (Shan et al., 2008) (Fig. 2A). As BAK1 is implicated in the function of additional PRRs, such as EFR (Chinchilla et al., 2007), interference with BAK1 appears to be a powerful strategy pursued by virulent pathogens to suppress PTI and to infect susceptible host plants (Shan et al., 2008). Very recently, the ubiquitin ligase activity of AvrPtoB was shown to mediate ubiquitinylation and subsequent destabilization (presumably by degradation via the host 26S proteasome) of FLS2 (Go¨hre et al., 2008) (Fig. 2B). Thus, multiple microbial effectors target PRR function, thereby underlining the major importance of plant basal defenses for plant immunity, as well as suppression of PTI as a major step towards the establishment of infection (Fig. 2). Different modes of interference with PTI were proposed for Ps. syringae effectors AvrRpt2 and AvrRpm1 that inhibit PAMP-induced signaling and compromise host basal immunity through manipulation of a regulator of PTI, RPM1-INTERACTING PROTEIN 4 (RIN4) (Kim et al., 2005).

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A

FLS2

flg22

BAK1

AvrPto B P

P

effectors Suppression of PTI P

Ub

P

AvrPtoB

Fig. 2. Suppression of PAMP-triggered immunity at the receptor level. PAMPtriggered defense responses are suppressed by Pseudomonas syringae pv. tomato effectors AvrPto and AvrPtoB that are injected into the plant cell via a TTSS. (A) The bacterial effector protein AvrPto is a kinase inhibitor that suppresses downstream responses by the inhibition of PRR complex formation. Interactions of AvrPto with the protein kinase domains of both FLS2 and its co-receptor BAK1 were shown. However, in planta AvrPto appears to target BAK1 preferentially. This results in suppression of all PRR activities that are regulated by the co-receptor BAK1. (B) Interference with FLS2 activity is also mediated by the bacterial effector AvrPtoB that harbors intrinsic E3-ligase activity. Ubiquitinylation of FLS2 promotes degradation of FLS2 by the 26S proteasome, and thereby interferes with PTI.

Arabidopsis RIN4 is a target for proteolytic degradation by the cysteine protease, AvrRpt2 (Coaker et al., 2005; Mackey et al., 2003), while AvrRpm1 mediates phosphorylation-dependent inactivation of this protein (Mackey et al., 2002). Strikingly, resistant Arabidopsis ecotypes that harbor the resistance (R) proteins RPS2 or RPM1, develop AvrRpt2/AvrRpm1mediated ETI, that is based upon R-protein-mediated sensing (‘‘guarding’’) of the manipulation of PTI by these effectors (Mackey et al., 2002, 2003). This is an impressive case for a mechanistic link between both PTI and ETI, the two major evolutionary forms of plant immunity. Recently, it was demonstrated that abscisic acid-dependent stomatal closure is observed upon bacterial infection and is due to PPR-mediated perception of bacterial patterns (Melotto et al., 2006). Infection of Arabidopsis with TTSS-deficient Pst DC3000 resulted in FLS2-dependent stomatal

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closure. Initial closure of stomata was also observed upon infection by Pst, but was reversed at later times of infection, which suggested that virulent pathogens have evolved effectors to reopen closed stomata. Indeed, stomatal reopening was accounted for by the TTSS-independent activity of the bacterial toxin, coronatine (Melotto et al., 2006). Taken together, the mere fact that several types of effectors (secreted toxins and TTSS effectors delivered into the plant cell) have evolved to act in concert to suppress PTI documents the importance of basal defenses for plant immunity and indicates that evasion of host immunity is an inevitable requirement for microbial proliferation on host plants. Further details on microbial effector-mediated suppression as a general virulence strategy of microbial phytopathogens is provided in the following chapter of this volume (Me´traux et al., 2009).

VI. CONCLUDING REMARKS There is now ample knowledge that plants and animals deploy innate immune (PTI) systems that share a similar logic (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004; Zipfel and Felix, 2005). Self/ nonself discrimination is based on the detection of invariant, microbe-specific patterns through host PRRs. As a result, nonspecific antimicrobial defenses are triggered that are supposed to arrest and terminate microbial ingress. Moreover, both plants and animals appear to possess sensor systems that facilitate recognition of host-derived determinants of host damage (nonintact self). Common principles in the organization of innate immunity in different kingdoms are also documented by similar microbial patterns that are recognized, by similar PRR types and by similar signaling cascades. However, significant differences in the molecular organization of immunity in plants and animals remain. Plant cells respond to microbial infection in a cell-autonomous manner, whereas in animals specialized cell types protect host tissues against microbial invasion (Jones and Dangl, 2006; Medzhitov, 2007). Moreover, adaptive immunity, which evolved in jawed vertebrates most likely as a result of insufficient innate immunity (Medzhitov, 2007), is missing in plants (Ausubel, 2005). In plants, insufficiency of PTI brought about by suppressive activities of microbial effectors might rather have driven the evolution of ETI (Alfano and Collmer, 2004; Chisholm et al., 2006). Although there is currently no experimental evidence that supports the latter assumption, there is evidence that PTI is an important element of basal immunity against adapted pathogens and of species immunity against nonadapted pathogens (Bittel and Robatzek, 2007; Nu¨rnberger and Lipka,

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2005). Moreover, resistance protein-mediated ETI has recently been shown to operate through derepression and potentiation of PAMP-inducible gene expression, thus demonstrating a functional interdependence between the two types of plant immunity (Shen et al., 2007).

ACKNOWLEDGMENTS We thank Jen Sheen and Georg Felix for critical discussions and comments. Research in the lab of T.N. and B.K. is supported by the Deutsche Forschungsgemeinschaft (AFGN, SFB 446, SFB 766), the European Community and the German Ministry of Education and Research (BMBF).

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Plant Pathogens as Suppressors of Host Defense

´ TRAUX,*,1 ROBERT WILSON JACKSON,{ JEAN-PIERRE ME ESTHER SCHNETTLER{ AND ROB W. GOLDBACH{,w

*Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland { School of Biological Sciences, University of Reading, Reading RG6 6AJ, Berks, United Kingdom { Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Suppressors Produced by Fungal and Oomycete Pathogens . . . . . . . . . . . . . . . A. Suppressors Comprise a Wide Group of Metabolites ................... B. Race-Specific Elicitors Turn Out to Suppress Defenses ................. C. Concluding Remarks.......................................................... III. Suppressors Produced by Bacterial Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bacterial Evolution to Overcome Plant Resistance ...................... B. Bacterial Suppression of PTI ................................................ C. Type III Protein Secreted Effectors are Used to Suppress PTI......... D. Multifunctional Effectors .................................................... E. RNA and RNA-Binding Protein Targeting ............................... F. Attack of Negative Regulators of PTI ..................................... G. Targeting Hormone Signaling? .............................................. H. Disruption of Vesicle Trafficking ........................................... I. Targeting MAP Kinase Signaling........................................... J. Other Effectors Involved in PTI Suppression for Which Targets are Unknown.........................................................

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Corresponding author:Email: [email protected] Prof. R. W. Goldbach tragically died in India on 7 April 2009

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Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51002-6

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K. Other Effectors Involved in PTI Suppression, but Lacking Functional Information..................................................................... L. Other Potential Mechanisms—Type VI Secretion........................ M. Complexity and Evolution of PTI Suppression by Bacterial Pathogens IV. RNA Silencing, the Plant’s Innate Immune System Against Viruses. . . . . . . A. The Discovery of RNA Silencing as the Plant’s Innate Immune System Against Viruses ................................................................ B. Current Views of RNA Silencing as Antiviral Mechanism in Plants .. C. Viral Suppressors of RNA Silencing ....................................... D. Possible Interactions Between Plant Viruses and the miRNA Pathway .............................................................. E. Is Antiviral RNAi Restricted to Plants and Insects? ..................... Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT This chapter reviews our current knowledge about mechanisms of suppression developed by pathogens to avoid host defense responses. In general, plants perceive pathogens by diverse pathogen- or microbe- or even damage-associated molecular patterns (PAMPs, MAMPs, DAMPs) and induce a variety of defense mechanisms referred to as horizontal or basal resistance, nowadays designated PAMP-triggered immunity (PTI). In addition, plants can also recognize specific pathogen-derived effectors and have derived a highly specific defense response termed effector-triggered immunity (ETI), classically called R gene-mediated, specific or vertical resistance. Both PTI and ETI are responses to potential dangers and have common components. Fungal, oomycete, and bacterial pathogens have evolved various effector-based mechanisms of suppression that interfere with such components. Plants strongly depend on RNA gene silencing to interfere with viral pathogens. Plant viruses counteract this response by encoding suppressor proteins of RNA silencing.

I. INTRODUCTION The notion that chemical interactions take place between plants and pathogens goes back to the early days of plant pathology when De Bary studied the disease development caused by Sclerotinia sclerotiorum. The first concept that emerged from such studies is that plant pathogens use various factors to invade their hosts. These factors were proposed to comprise cell wall-degrading enzymes, the presence of which was first reported in bacterial soft rot of potato by Jones (1909). At about the same period, Hitchinson (reviewed in Dimond, 1955) proposed that wilt-inducing fungi release toxins, thus opening another area in the field of pathogenicity factors. Research on toxins has followed a vigorous course and numerous chemical structures have been identified (Walton, 1996). The introduction of genetic analyses has greatly helped to determine the biological relevance for several toxins (Scheffer, 1991;

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Yoder, 1980). Besides cell wall-degrading enzymes and toxins, yet another group of substances has made its appearance. The discovery of pathogeninduced antimicrobial compounds in plants, the phytoalexins, by Mu¨ller and Bo¨rger (1940) opened the way to a vigorous research effort in induced resistance. Later, besides pathogens, chemicals from fungal cultures were shown to induce phytoalexins. A first example was provided by monilicolin A, a partly characterized peptide factor first isolated from the mycelia of Monilinia fructicola, which induces phaseollin, a phytoalexin from bean (Cruickshank and Perrin, 1968). In the early 1970s, Noel Keen proposed the term elicitor for metabolites from pathogens that induce phytoalexins (Keen et al., 1972). This concept was later expanded, and elicitors were proposed to induce various resistance mechanisms (De Wit, 1986). A number of reviews relate the progress of what became a very busy and fruitful area of molecular plant pathology (Boller, 1995; Ebel and Mitho¨fer, 1998; Hahn, 1996; Yoshikawa et al., 1993). In fact, elicitors were shown to include common microbial molecules, such as bacterial lipopolysaccharide (LPS), flagellin, elongation factor Tu, cold-shock protein and peptidoglycan (Aslam et al., 2009; Nu¨rnberger and Kemmerling, 2009), as well as products associated with damage of plant components by pathogen virulence factors, for example, oligogalacturonides and cellulose byproducts. These general elicitors are nowadays named using acronyms such as PAMPs (pathogen-associated molecular patterns), MAMPs (microbe-associated molecular patterns) or DAMPs (damage-associated molecular patterns; see Boller and Felix, 2009). The elicitor concept has received strong support with the characterization of the structure and biological relevance of several receptors for elicitors. These receptors are part of a pathogen surveillance system of proteins coupled to a set of antipathogen defense mechanisms via mitogen-activated protein kinase (MAPK) and various hormone signaling pathways. This defense system has the hallmarks of the innate immunity known in animals and serves as the basic frontline defense against potential pathogens, regardless of whether the interacting organism is a pathogen or not. Traditionally, this has been called basal resistance, but it has recently been designated as PAMP-triggered immunity (PTI) (Chisholm et al., 2006; Jones and Dangl, 2006). The second pathway is a more advanced and specific form of resistance that detects pathogen effectors leading to sacrificial programmed cell death, known as the hypersensitive response (HR), at sites of infection—and is known as effector-triggered immunity (ETI). This relates to what was formerly called R gene-based or vertical resistance. Typical defense responses in innate immunity include the closure of stomata (Melotto et al., 2006), the strengthening of the plant cell wall (thickening of the wall occurs by formation of papilla, an apposition composed of phenolics, lignin, and callose among others) (Keshavarzi et al., 2004), and the release of antimicrobial products, for example,

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reactive oxygen species (ROS), phytoalexins, glucosinolates (Clay et al., 2009), and enzymes such as glucanases and chitinases belonging to a wide variety of so-called pathogenesis-related (PR) proteins (Sels et al., 2008; Van Loon et al., 2006). While MAMPs provide a convenient explanation for the induction of resistance in response to avirulent pathogens, the scenario of a compatible interaction remained unanswered. This is especially puzzling as virulent pathogens produce PAMPS similar to the MAMPs of avirulent microbes. How do virulent pathogens avoid the detrimental effects of being recognized by their PAMPs? An older concept already hinted at by Ga¨umann (1946), proposed that virulent pathogens, besides their ability to tolerate or detoxify phytoalexins, also produce suppressors of phytoalexin accumulation (see also Heath, 1981). Suppressors interfere with the induced defenses of the infected plant and promote virulence of the pathogen. Suppressor activities from virulent pathogens had been described earlier on by Japanese colleagues (Doke, 1975; Oku et al., 1977). This initial spurt of activity was soon to be followed up by other laboratories worldwide as reviewed by Shiraishi et al. (1994). This functional definition of suppressors has been the conceptual basis for further research and has gained considerable support in recent years. Nowadays, the notion of suppressor is often used in alternation with effectors of virulence (Da Cunha et al., 2007; Ma and Guttman, 2008). This chapter will review the current state of research on suppressors of plant defenses in interactions of plants with fungi, bacteria, or viruses.

II. SUPPRESSORS PRODUCED BY FUNGAL AND OOMYCETE PATHOGENS A. SUPPRESSORS COMPRISE A WIDE GROUP OF METABOLITES

Preliminary infection of plants with a virulent pathogen can lead to an increased susceptibility to subsequent inoculation with avirulent pathogens. This was observed in the potato/Phytophthora infestans, barley/Blumeria graminis and oat/Bl. graminis, and Puccinia coronata interactions (as reviewed in Staples and Mayer, 2003). These observations provided experimental support for the hypothesis that virulent pathogens release suppressors of plant defenses, and led to an active search for such suppressors (initially coined as impedins (Ouchi and Oku, 1986), then supprescins (Shiraishi et al., 1992)) in fungal culture filtrates (Shiraishi et al., 1997). Supprescins A and B secreted by the phytopathogenic fungus Mycosphaerella pinodes are good examples of such compounds. Application of these small glycoproteins to the

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epicotyls of peas delayed the transcription and activity of elicitor-induced phenylalanine ammonia-lyase (PAL), as well as the accumulation of pisatin, the pea phytoalexin (Yamada et al., 1989). Similarly, the expression of the BvPAL and cinnamic acid 4-hydroxylase (BvC4H) genes in sugar beet was repressed at both the transcript and the enzyme activity levels upon infection by virulent Cercospora beticola. This fungal repression was shown to reside at the core promoter of PAL (Schmidt et al., 2004). The nature of the fungal suppressor responsible for this repression remains to be determined but a possible control by the phytohormone abscisic acid (ABA) was suggested for the infection of sugar beets by Ce. beticola (Schmidt et al., 2008). In fact, several examples illustrate how ABA might be a virulence factor that suppresses defense responses (Asselbergh et al., 2008; De Torres-Zabala et al., 2007). Fungi of the genera Botrytis, Ceratocystis, Fusarium, and Rhizoctonia can produce ABA, making it likely that this hormone is involved in pathogen virulence (Do¨rffling et al., 1984; Siewers et al., 2006, reviewed in Tudzynski and Sharon, 2002). The precise chemical structure of suppressors was mostly unknown, making it difficult to study their interaction with a potential binding site, and their mode of action as well as their biological relevance has remained elusive. A first example of a chemically characterized suppressor came with the study of a yeast elicitor. Cleavage of yeast invertase by -chymotrypsin leads to highly active glycopeptide elicitors that stimulate the biosynthesis of ethylene (ET) and the activity of PAL in tomato cells. Release of the mannosecontaining side chain of one purified elicitor-active glycopeptide by an endo -N-acetylglucosaminidase yielded a mannose-rich oligosaccharide that acts as a specific suppressor of the glycopeptide-induced ET biosynthesis and PAL activity. Based on the structural requirements of the glycopeptides mannose side-chains for elicitor activity and mannose oligomers for suppression, it was concluded that elicitor and suppressor compete for the same binding site (Basse et al., 1993). This opens up the possibility of determining the relevance of this suppressor in the larger context of a plant–fungus interaction. A study on the oomycete pathogen Phytophthora sojae has led to the cloning of a glucanase-inhibiting protein (GIP) that was shown to form a complex in vitro and in vivo with a soybean endoglucanase (PR-2 protein) and to inhibit its activity. This interaction also inhibits the release of a glucan elicitor from Ph. sojae cell walls in vitro. Thus, GIP1 is an example of a suppressor of a plant defense response produced by a virulent pathogen of soybean (Rose et al., 2002). Suppression of disease resistance in the form of a two-step process was reported for the soft-rot fungus Septoria lycopersici, a pathogen of tomato. Tomato accumulates an antifungal glycoalkaloid, the saponin -tomatine that can be hydrolyzed to -tomatine by a detoxifying enzyme, tomatinase,

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secreted by Se. lycopersici. Mutants of Se. lycopersici deficient in tomatinase induced plant defense genes and plant cell death in contrast to wild types. They were not otherwise impaired in their ability to cause disease on tomato leaves. This led to a model, in which -tomatine produced after hydrolysis of -tomatine by tomatinase in wild-type strains, acts as a suppressor of plant defenses (Martin-Hernandez et al., 2000). Support for this hypothesis was obtained by studying the interaction between Se. lycopersici and Nicotiana benthamiana. When defense reactions of the host were suppressed by virusinduced gene silencing (VIGS), the tomatinase-deficient mutant caused disease compared to plants with intact defenses. Infiltration of leaves with purified tomatinase or -tomatine, but not -tomatine, led to susceptibility to tomatinase-deficient mutants of Se. lycopersici. Infiltration of tomatinase or -tomatine reduced the HR induced by transient expression of the bacterial effector gene AvrPto (AVIRULENCE GENE OF PS. SYRINGAE PV. TOMATO) in N. benthamiana expressing the matching resistance (R) gene Pto of tomato. Together these results show a dual effect of tomatinase in saponin detoxification and in suppression of the resistance response (Bouarab et al., 2002). Since it is not known if N. benthamiana accumulates -tomatine, the products of tomatinase activity in N. benthamiana remain to be determined. It will be interesting to learn about the existence of other cases of suppressors formed by the action of fungal detoxifying enzymes. Botrytis cinerea and Sc. sclerotiorum produce oxalic acid during infection (Germeier et al., 1994; Godoy et al., 1990). Oxalic acid is a pathogenicity factor necessary for the infection of various plant species by Sc. sclerotiorum (Godoy et al., 1990) and to a certain extent for Bo. cinerea (Schoonbeek et al., 2007). Oxalate deserves special attention since this molecule was shown to suppress the elicitor-induced oxidative burst in soybean and tobacco cultured cells (Cessna et al., 2000). An oxidative burst was shown to be effective in defense against Bo. cinerea in ABA-deficient tomato lines (Asselbergh et al., 2007). It appears therefore reasonable to ask if oxalate could act in planta as a non-proteinaceous suppressor of the oxidative burst. B. RACE-SPECIFIC ELICITORS TURN OUT TO SUPPRESS DEFENSES

Our knowledge about the structure and biological relevance of fungal suppressors has leaped forward as a result of studies on elicitors and their role in the molecular basis of host specificity. Gene-for-gene interactions as studied in tomato infected with the leaf mold fungus Cladosporium fulvum turned out to be particularly rewarding since they allowed the chemical characterization of so-called race-specific elicitors. Cl. fulvum avoids breaching the cell wall,

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enters through stomata and dwells exclusively in the intercellular spaces of the plant tissue. In this interaction, elicitors released from the fungus are located in the apoplast. Very ingeniously, Pierre De Wit realized the unique opportunity offered by the lifestyle of this fungus, and centrifuged vacuuminfiltrated leaf tissue to extract race-specific elicitors from Cl. fulvum-inoculated tomato leaves. A number of small-sized proteins were isolated that induced a HR race-specifically when injected into leaves of tomato plants containing the cognate R genes (De Wit and Spikman, 1982; De Wit et al., 1985). The elicitors were referred to as products of fungal Avr genes, in agreement with pathogen avirulence (Lauge´ and De Wit, 1998). For quite some time, researchers pondered about the real function of the evolutionarily conserved avirulence gene products. Their sole function is very unlikely to make its bearer recognized by plants, and in the late 1990s a concept emerged whereby pathogen avirulence gene products are in fact effectors that target plant proteins to promote disease. Products of R genes, if present, guard these targets and when interacting with the cognate Avr gene product, initiate a defense reaction often in the form of a HR (Dangl and Jones, 2001; Dixon et al., 2000; Mackey et al., 2003). Detailed studies were mainly carried out on specific interactions between plants and bacterial pathogens notoriously known for their large number of secreted effectors (see Section III). Among the main reasons for the strong breakthroughs in plant–bacteria interactions, is that bacteria were far more amenable to molecular studies with the advent of a plethora of molecular techniques. Eventually, tools such as whole genome sequences and transformation protocols also became available for fungal and oomycete pathogens. The host target systems perturbed by various bacterial effectors include protein ubiquitination, MAPK signaling, vesicle trafficking, hormone signaling and transcriptional regulation (Da Cunha et al., 2007). Several effectors that target plant defenses have now also been identified in pathogenic fungi and oomycetes. A well-studied case is the Cl. fulvum AVR2 protein. AVR2 is a cysteine proteinase inhibitor that binds to, and inhibits RCR3, an extracellular papainlike Cys protease (PLCP) that is induced and secreted by tomato as a defense response against pathogens (Kru¨ger et al., 2002). Such secreted proteases can be involved in various ways in plant defenses: for example, by acting directly on the invading pathogen, or by being part of signaling cascades for the induction of the HR (Van der Hoorn and Jones, 2004). AVR2 is a pathogen effector with a suppressor activity: in the absence of the tomato resistance gene Cf2, AVR2 suppresses the action of a PLCP; but in the presence of Cf2 the AVR2 /RCR3 complex triggers ETI-dependent HR (Rooney et al., 2005). AVR2 expression in tomato causes susceptibility to races of Cl. fulvum defective in AVR2 and also towards Bo. cinerea and Verticillium dahliae. In addition, heterologous

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expression of AVR2 in Arabidopsis thaliana leads to enhanced susceptibility against Bo. cinerea and Ve. dahliae (Van Esse et al., 2008). It turns out that RCR3 is not the major PLCP targeted by AVR2. The tomato apoplast was shown to contain a diversity of PLCPs with up to seven proteins clustering into four different subfamilies. Sequencing of PLCP alleles showed that only two relatives, RCR3 and a PLCP produced by tomato leaves infected with Ph. infestans named Phytophthora-inhibited protease (PIP1), undergo selection leading to diversified residues around the binding site of the substrate at the protein surface. This variance might help the plant to prevent inhibition of its defenses by pathogen-derived proteinase inhibitors. The higher number of variants observed at the surface of AVR2 tends to show that its gene is under stronger selection. Inhibition by AVR2 is mainly targeted at RCR3 and PIP1. As PIP1 is more abundant in the apoplast than RCR3, PIP1 was proposed to be the main target of AVR2 and to contribute to the fitness of the pathogen in the absence of Cf2. The major contribution of the less abundant RCR3 (referred to as a decoy) is to bind AVR2 and lure it into a recognition event with subsequent activation of ETIdependent HR in Cf2-bearing tomato (Shabab et al., 2008). This example is one of the few cases illustrating the so-called decoy model, where the binding of an effector with its target is not the main contributor to pathogen fitness in the absence of a corresponding R gene (Van der Hoorn and Kamoun, 2008). Effectors with protease inhibitor activity are also secreted by the oomycete Ph. infestans, a pathogen of potato and tomato. A family of Kazal-like Ser protease inhibitors (extracellular proteinase inhibitors, EPIs) of at least 35 members was found among five different Phytophthora species (Tian et al., 2004). Two of these, EPI1 and EPI10, inhibit the tomato serine protease P69B (PR-7) (Tian et al., 2004, 2005). Another family includes four EPIs with cystatin-like domains (EPICs), EPIC1-4. Of these, EPIC2B was shown to interact with and inhibit PIP1 (Tian et al., 2007). A recent study shows that EPIC1 and EPIC2B of Ph. infestans can also target the RCR3 protease from tomato, like AVR2 from Cl. fulvum. However, in contrast to AVR2, the interaction of EPIC1 and EPIC2B with RCR3 does not induce a HR in tomato harboring the resistance gene Cf2. These results demonstrate that effectors from phylogenetically unrelated pathogens target the same host defense protein (Song et al., 2009). The importance of EPICs in the virulence of Ph. infestans remains yet to be documented. Another apoplastic effector of Cl. fulvum is AVR4, which is characterized by a chitin-binding motif. AVR4 was shown to bind specifically to fungal chitin but not to plant cell-wall preparations in vitro. AVR4 can protect fungal walls against chitin hydrolysis by plant chitinases (PR-3 type proteins) (Van den Burg et al., 2006). When Avr4 was expressed in Arabidopsis,

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increased virulence of several fungal pathogens containing chitin in their cell wall can be observed. Similarly, expression of Avr4 in tomato increased virulence of Fusarium solani pv. lycopersici. To complete this study, Avr4 was silenced in Cl. fulvum leading to decreased virulence on tomato. These results demonstrate the requirement of AVR4 as an effector for pathogenicity. AVR4 antagonizes the defensive function of plant chitinases and illustrates another form of suppression of the action of the plant against an invading fungus (Van Esse et al., 2007). The smut fungus Ustilago maydis was shown to produce PEP1, a protein secreted into the apoplast that is required for the establishment of the fungal haustorium and for further biotrophic development of the fungus within its host, maize. Deletion of the PEP1 gene in U. maydis prevents fungal penetration and unleashes a combination of defense responses by the plant. A PEP1 orthologue with similar function was also observed in Ustilago hordei, the smut pathogen of barley. PEP1 from barley can substitute for PEP1 in U. maydis, as evidenced by the restoration of full virulence to pep1 mutants. Unlike AVR2 of Cl. fulvum, the central domain essential for the function of PEP1 is highly conserved, making it reasonable to speculate that it is an enzyme inhibitor with little specificity or that it affects the deployment of plant defenses in some other ways (Doehlemann et al., 2009). It will be very interesting to learn more about the action of this newly identified fungal suppressor. Fusarium oxysporum f.sp. lycopersici, a fungus invading the xylem, secretes the small cysteine-rich protein SIX1 during the colonization of tomato (Rep et al., 2005). Tomato harboring the I-3 resistance gene develops an incompatible interaction with Fu. oxysporum containing the SIX1 gene. SIX1 can be equated to Avr3, given its gene-for-gene relation with I-3. Fu. oxysporum also produces the avirulence protein AVR1, which matches the product of the tomato resistance gene I-1. However, Avr1 has the additional virtue of suppressing the protective effect of resistance genes I-2 and I-3. As I-2 is cytoplasmic, and AVR2 is apoplastic, AVR1 has been tentatively proposed to interfere with the uptake of AVR2 or AVR3. It might also possibly interfere with the signaling downstream of I-2 or I-3 (Houterman et al., 2008). In addition to apoplastic effectors, a number of cytoplasmic effectors defined by a conserved RXLR amino acid motif have been identified in oomycetes. These molecules are delivered into the cytoplasm of the host cell where they suppress cell death and contribute to the virulence of the pathogen. A well-studied example is the AVR3aKI protein of Ph. infestans. The interaction between R3a, the product of the cognate R gene of Avr3aKI, and AVR3aKI was studied in N. benthamiana using concurrent agro-infiltration. Interaction of AVR3aKI with R3a triggers cell death, but AVR3aKI also

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suppresses cell death induced by INF1, a major elicitin of Ph. infestans (Bos et al., 2006). Elicitins are small, highly conserved proteins secreted by phytopathogenic oomycetes (Phytophthora and Pythium); they induce necrosis in infected plants and elicit an incompatible HR-like reaction. INF1 is a conserved PAMP in oomycetes (Vleeshouwers et al., 2006). Mutants of Avr3a were identified that activate R3a but do not suppress INF-induced cell death. Distinct amino acids in AVR3a determine recognition by R3a but not suppression of INF-induced cell death (Bos et al., 2009). AVR3a can therefore be considered a suppressor of immunity triggered by PAMPs. The importance of the amino acid residues identified in this study will eventually allow for resolving the three-dimensional structure of AVR3a. C. CONCLUDING REMARKS

The weight of the evidence accumulated in the past years demonstrates the validity of the concept of suppressors of disease resistance. As far as fungi and oomycetes are concerned, molecular insights have been obtained by studies on pathogens like Cl. fulvum or Ph. infestans with partly biotrophic lifestyles. In a majority of cases described so far, the interplay of suppressors with their targets is mainly taking place in the apoplasm, as illustrated in Fig. 1. It will be particularly interesting to follow up these studies in the future as we gain more and more information on the molecular targets of suppressors. Only a limited number of cases have been studied so far and it will be exciting to learn more about suppressors produced by other types of fungal pathogens, for example necrotrophs or basidiomycetes.

III. SUPPRESSORS PRODUCED BY BACTERIAL PATHOGENS A. BACTERIAL EVOLUTION TO OVERCOME PLANT RESISTANCE

Bacteria have evolved to employ a variety of virulence factors including toxins, enzymes, hormones, polysaccharides, and most famously, type three protein secretion effectors (T3SE). Many of them are used for the suppression of plant resistance and the release of nutrients; these physiological conditions are thus conducive to the establishment and replication of bacterial cells. The pathogen we know today, however, would have been a different beast in the distant past. A co-evolutionary arms race has seen the development of increasingly sophisticated strategies by pathogens to infect plants and for plants to prevent infection. By considering the ZigZag model defined by

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Pathogen

Pathogen

Pathogen

Chitinbinding protein

AVR3a, SIX1 Protease Glucanase inhibitor inhibitor

Apoplasm

ABA

Elicitin

Chitin or other PAMPS

Oxalate

?

?

ROS MAPK cascade

HR HR

?

Chitinase

Extracellular protease Glucanase

PR-Proteins PAL,... Activation of gene expression Nucleus

Effector targets Plant cell defense mechanisms Plant cell cytosol

Fig. 1. General overview of the mechanisms of defense suppression by fungal and oomycete pathogens. The suppressors and their targets are represented by gray symbols and connected by dotted lines. The targets include interference with various aspects of PAMP-triggered immunity (PTI) (reactive oxygen species (ROS), expression of defense-related genes, or the action of defense proteins) or effector-triggered immunity (ETI). Plant defense mechanisms, PTI and ETI, are in black (symbols and lines). See text for details.

Jones and Dangl (2006), it can be envisaged that a nonpathogenic bacterium interacts with a plant. Changes in the bacterial genotype, through mutation, recombination and/or gene acquisition that allow the bacterium to manipulate the plant to obtain nutrients then occurs. The detrimental effect on the health of the plant (disease) induced by this emerging pathogen imposes strong selection pressure on the plant host for the selection of varieties that are resistant—the varieties develop sensing systems to detect conserved molecules of the bacterium. Consequently, the resistant plant imposes selection for bacteria that can overcome the resistance system and so on. McCann and Guttman (2008) defined this arms race as analogous to the Red Queen principle in Lewis Caroll’s Through the Looking Glass—both pathogen and host are evolving as fast as possible just to maintain the equilibrium. Over a period of time pathogens have evolved to first overcome PTI and then ETI. As an example of the paradox that bacteria face, the flagellum chokepoint is considered here.

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Plants deploy cell-surface pathogen recognition receptors (PRR) that can detect specific, conserved patterns in proteins expressed by bacteria, that is PAMPs. The function of the bacterial proteins encoding the PAMP is often important for bacterial cellular function or for ecological success (fitness). For example, the flg22 peptide is part of the flagellin protein that is the main unit of the bacterial flagellum filament. The flagellum plays a critical role in bacterial motility, enabling the bacterium to move towards nutrients and away from antagonists; in several cases the flagellum has been implicated as important for bacterial virulence in plants (e.g., Antu´nez-Lamas et al., 2009; Jahn et al., 2008; Naito et al., 2008), probably for entry into the intercellular areas of plant tissue. Therefore, many bacteria, both pathogens and nonpathogens, are under strong selective pressure to maintain flagellum function. If the bacterium maintains flagellum function, then it inevitably runs the risk of being detected by plant PRR sensors and exposure to host resistance. One consequence of this plant-induced stress is selection for strains that do not trigger resistance (Arnold et al., 2007; Pitman et al., 2005). At least three outcomes are envisaged: (1) The bacterium downregulates flagellum expression inside the plant; (2) Mutations in the PAMP occur so that the PAMP is no longer recognized by the PRR; (3) The bacterium obtains a mechanism that acts downstream of PRR-PAMP detection, thereby removing PAMP recognition as an issue. Little is known about the expression status of the flagellum system in planta or the role the flagellum might play. However, it is clear that mutations to the PAMP will be limited to a select number of sites so that the function of the PAMP-encoded protein is not lost (Naito et al., 2008). Over a period of time, it is likely that PRRs will evolve to recognize PAMP variants. PAMP mutations that have a significant detrimental effect on bacterial function will not be maintained within the population. This strong selective pressure imposed on the bacteria has inevitably led to the evolution of new gene systems or the acquisition of other genes by horizontal transfer. These potentially include polysaccharides and host-specific toxins, which appear to have limited effects and are only partially effective. Undoubtedly, the single most important evolutionary leap made by bacteria is the acquisition of the type III protein secretion system (TTSS). B. BACTERIAL SUPPRESSION OF PTI

When bacteria enter the vicinity of plant cells, either on the plant’s external surface or within the plant tissue, they express a wide variety of proteins including various PAMPs that betray the bacterial presence in the plant. Plant cells deploy cell-surface PRRs, such as FLS2 (Flagellin sensitive 2), EFR (Elongation factor-Tu receptor) and CERK1 (Chitin elicitor receptor

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kinase 1, a PRR receptor-like kinase (RLK) that detects the fungal elicitor chitin to trigger PTI), to detect distinct PAMPs. Some studies, described below, have now shown that detection of PAMPs leads to association of PRRs with the RLK BAK1 (BRI1 (Brassinosteroid-insensitive 1)-associated receptor kinase 1), subsequent activation of MAPK signaling and WRKY transcription factors and the expression of a wide range of genes, many of which contribute to the rapid defense response—of note here are NHO1 (NONHOST RESISTANCE TO PS. SYRINGAE PV. PHASEOLICOLA 1) and FRK1 (FLG22-INDUCED RECEPTOR-LIKE KINASE 1), which are commonly used markers for PTI. NHO1 encodes a glycerol kinase that is necessary for Arabidopsis resistance to non-host pathogens, but it is not effective against the pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst); FRK1 is a flg22-induced RLK. The signaling pathways are accompanied by ion channel openings leading to calcium influx from the apoplast to the plant cytosol. It is immediately apparent that a number of plant mechanisms are potential targets for bacterial suppression. These are described in detail below. 1. Calcium signaling suppression by extracellular polysaccharides (EPS) Calcium is a key secondary messenger that binds to calcium-binding proteins such as calmodulin, leading to triggering of signaling mechanisms for activation of PTI and ETI (Ma and Berkowitz, 2007). Thus, calcium signaling naturally poses one potential target for suppression by pathogens. Recent evidence by Aslam et al. (2008) showed that the polyanionic EPS produced by bacterial symbionts and pathogens of plant and animals were able to specifically bind calcium ions. It has long been known that EPS is an important virulence factor, for example in the production of bacterial biofilms and for protection against antimicrobials, but a role in calcium sequestration is relatively new. Aslam et al. (2008) found mutations in Xanthomonas and Pseudomonas xanthan and alginate genes, respectively, which resulted in loss of virulence. Concomitantly, there was an increase of callose deposition, production of ROS and upregulation of the defense-related PR-1 and PDF1.2 (PLANT DEFENSIN 1.2) genes. Critically, there was also an increase in cytosolic calcium levels. These calcium surges were shown to be PAMP-inducible and suppressible by purified polyanionic EPS from various symbiotic and pathogenic bacteria, but not by the neutral EPS, levan. Examination of the ultrastructure of infected sites showed that pathogen cells are embedded within high quantities of EPS, which also appears to be interacting with cell wall fibrils and therefore, is in direct contact with this calcium store. The implication of this discovery is that bacteria have evolved to use EPS for sequestration of apoplastic calcium to interrupt signaling and douse PTI.

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Considering the context of EPS suppression with that of T3SE-mediated suppression of PTI and the more advanced ETI, this may therefore be considered to be one of the pathogens’ most primitive PTI suppression systems. That the expression and function of EPS is maintained may reflect its constant role in PTI suppression or its multifaceted protective and structural roles (e.g., biofilms, UV and desiccation resistance). 2. Coronatine toxin suppression of stomatal closure A recent study by Melotto et al. (2006) showed that in Arabidopsis a variety of plant and animal pathogenic bacteria triggered stomatal closure whereas Pst has evolved a mechanism to suppress the closure. Stomatal closure is clearly an organelle-scale PTI mechanism to stop invasion of the internal plant tissue by bacteria. Further investigation found that the PAMPs flg22 and LPS were triggering stomatal closure and clearly indicated that Pst carried a suppressor mechanism. A detailed analysis revealed that the polyketide toxin coronatine was responsible for the inhibition of closure. Coronatine toxin is composed of coronamic acid and coronafacic acid and is structurally similar to jasmonic acid (JA), a plant hormone that activates plant defenses. Coronatine toxin inhibited ABA-induced stomatal closure, but not in coronatine-insensitive (coi1) mutant plants, indicating that the action of coronatine is downstream of ABA signaling. Thus, coronatine is used to open stomata to allow ingress of the bacteria into the plant tissue. A recent paper has identified that Xanthomonas also produces a diffusible signal factor-dependent molecule to suppress stomatal closure and that the MAPK MPK3 is involved in the closure response (Gudesblat et al., 2009). The gene clusters containing the coronatine biosynthetic genes have only been found in a handful of plant-pathogenic bacteria (Ps. syringae pvs alisaliensis, atropurpurea, glycinea, maculicola, morsprunorum, and tomato; Xanthomonas campestris pv. phormiicola; Pectobacterium atrosepticum) (Bender et al., 1999; Cintas et al., 2002; Mitchell, 1991; Toth and Birch, 2005) including the model pathogen, Pst. Why the toxin cluster is not more widely distributed is unclear—possibly coronatine acts as a PAMP in other plants, its usefulness has been superseded by other virulence factors (e.g., toxins), or there is an element of plant or environment specificity associated with its action. C. TYPE III PROTEIN SECRETED EFFECTORS ARE USED TO SUPPRESS PTI

The TTSS gene system is comprised of 20–30 hrp/hrc (HYPERSENSITIVE REACTION AND PATHOGENICITY/HYPERSENSITIVE RESPONSE CONSERVED) genes clustered in several operons on a pathogenicity island in the chromosome or on plasmids (Jin et al., 2003). The expression of the

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genes is tightly regulated and occurs primarily inside the plant intercellular spaces, that is once a bacterium arrives inside plant tissue and makes contact with the plant cells. The proteins encoded by the TTSS genes are similar to the flagellum proteins, with evolutionary analyses showing that the two systems actually evolved from a common ancestor (Gophna et al., 2003). Like the flagellum, the TTSS forms a pore complex that spans the inner and outer membranes of the bacterium. A pilus is formed from the pore that extends away from the bacterial cell to neighboring plant cells. The function of the TTSS is to deliver type III secreted effector (T3SE) proteins into plant cells. Once inside the plant cells, the effectors target plant mechanisms to suppress plant defenses and implement release of nutrients to the bacterium. Current knowledge indicates that effectors target PTI pathways, but also ETI pathways as a consequence of plants evolving to recognize PTI-suppressing effectors. The following sections will review the various effectors found that suppress PTI; because effectors can often have multiple roles, effectors will be considered individually.

D. MULTIFUNCTIONAL EFFECTORS

1. avrPto avrPto was first identified in Ps. syringae pv. tomato as an avirulence gene detected by varieties of tomato expressing the resistance gene Pto (Ronald et al., 1992); an avirulence gene is an effector that has become recognized by a plant host as a consequence of plant evolution to detect bacterial virulence factors. Although AvrPto is recognized by the resistance protein Pto in tomato, AvrPto suppresses basal defenses in Arabidopsis and N. benthamiana. Several important studies demonstrated this role: firstly, expression of avrPto in transgenic Arabidopsis plants prevented the formation of papillae and deposition of callose (a PTI response) when the plants were challenged with a nonpathogenic (TTSS-minus) Ps. syringae that still expresses PAMPs (Hauck et al., 2003). In N. benthamiana, AvrPto can suppress flagellin-induced cell death and callose deposition (Hann and Rathjen, 2007). Secondly, AvrPto suppresses both expression of Arabidopsis NHO1 and FRK1, genes that are induced by the PAMP, flg22 (He et al., 2006; Li et al., 2005a), and activation of the MAPKs, MPK3 and MPK6. These kinases lie at the bottom of a signaling pathway that follows: FLS2 activation leads to activation of MAPKKK activating MAPKK activating MAPK. Further analysis showed that overexpression of the MAPKKK, MEKK1, could block AvrPto suppression of MPK3 and MPK6—this indicates that AvrPto must act at some stage between the FLS2 activation and MEKK1.

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Two recent studies indicate that AvrPto acts at the level of the receptor kinases (Shan et al., 2008; Xiang et al., 2008). Xiang et al. (2008) showed that AvrPto can bind to FLS2 and EFR PRRs and Shan et al. (2008) showed that AvrPto targets BAK1, a RLK that associates with FLS2 and EFR after PAMP recognition. BAK1 was originally found because of its role in brassinosteroid signaling. Although Xiang et al. (2008) showed that the FLS2–AvrPto interaction still occurred in bak1-1 protoplasts, Shan et al. (2008) provided convincing evidence that AvrPto makes a stronger association with BAK1. Indeed, AvrPto disrupts FLS2-BAK1 association and BAK1 is important for multiple PAMP-triggered FRK1 expression. Taken together, these data show that AvrPto binds to protein kinases, potentially acting as a kinase inhibitor. AvrPto has also recently been found to interfere with microRNA (miRNA) accumulation, which plays a key role in RNA silencing defense responses against Pst (Navarro et al., 2008). Taken together, Ps. syringae uses AvrPto to target the BAK1 RLK and deactivate PAMP signaling at the top tier. One interesting consequence of AvrPto function is increased ET production, as discussed below for avrPtoB. 2. avrPtoB (hopAB2) avrPtoB (hopAB2, hrp outer protein AB2) has a number of overlapping functions with avrPto including the targeting of RLK PRRs and miRNA interference (He et al., 2006). However, one intriguing difference between AvrPto and AvrPtoB defense suppression is that, unlike AvrPto suppression, AvrPtoB suppression is dependent upon RAR1 (Required for Mla resistance 1), SGT1 (Suppressor of G2 allele of Skp1), both chaperone proteins that stabilize NBARC-LRR (Nucleotide binding domain shared by Apaf-1, certain R gene products and CED-4, fused to C-terminal leucine-rich repeats), R-proteins, and EDS1 (Enhanced disease susceptibility 1), indicating AvrPtoB targets different defense pathways (Hann and Rathjen, 2007). The 553-amino acid AvrPtoB was first identified in Pst AvrPto as an interactor with Pto and demonstrated to be the product of an avirulence gene recognized by tomato plants expressing the Pto and Prf kinase genes (Kim et al., 2002); it shares 52% identity to the Ps. syringae pv. phaseolicola effector HopAB1 (VirPphA) (Jackson et al., 1999, 2002). Like AvrPto, AvrPtoB also plays a role in interfering with miRNA levels (Navarro et al., 2008). Several seminal studies by Greg Martin and coworkers have shown that AvrPtoB contains domains that contribute to both virulence and avirulence (e.g., Abramovitch et al., 2003, 2006; Kim et al., 2002; Rosebrock et al., 2007) and that it shares many of the same roles and functions as AvrPto, described above. The C-terminus encodes an E3 ubiquitin ligase that is involved in suppression of ETI (Abramovitch et al., 2006; Janjusevic et al., 2006; Rosebrock et al., 2007)

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by directing host proteins to the proteasome. The N-terminal amino acid residues 1–307 are sufficient for eliciting Pto/Prf-dependent immunity. Upon delivery into the plant cell, AvrPtoB is phosphorylated on a key serine residue in the N-terminus (Xiao et al., 2007a); alanine substitution of this residue abolishes the virulence function of AvrPtoB. Phosphorylation is independent of the Pto and Prf protein kinases, indicating the activity of a novel kinase interaction. The AvrPtoB N-terminus also promotes virulence by two distinct mechanisms: residues 1–307 contribute to virulence in tomato lines lacking Pto/Prf by increasing ET production via upregulation of ET biosynthetic proteins (Cohn and Martin, 2005; Xiao et al., 2007b); residues 308–387 are functionally distinct in being involved in a second virulence function to suppress tomato PTI. AvrPtoB can also suppress PTI in Arabidopsis and N. benthamiana (De Torres et al., 2006; Hann and Rathjen, 2007) including suppression of callose deposition and PTI-induced genes, for example, NHO1 and FRK1. In a manner similar to AvrPto, AvrPtoB targets BAK1 and FLS2 RLKs (Go¨hre et al., 2008; Shan et al., 2008), with the kinase domains being essential for the interaction. Two recent reports have shown that the E3 ubiquitin ligase domain also contributes to PTI by ubiquitinating FLS2 and CERK1, thus directing them for degradation (Gimenez-Ibanez et al., 2009; Go¨hre et al., 2008); interestingly, CERK1 appears to operate independently of BAK1. Two studies have now shown that AvrPtoB manipulates plant hormone biosynthesis pathways to promote virulence. Cohn and Martin (2005) found that AvrPtoB (and also AvrPto) upregulates ET-related genes during the interaction of Pst with its host, tomato. ET production enhanced the severity of necrotic symptoms in infected leaves. Pst also modulates the ABA signaling pathway in Arabidopsis, driving production of ABA and subsequent enhanced growth of the pathogen in the plant tissue (De Torres-Zabala et al., 2007). Since plant hormones, including ET and ABA, are important for plant stress response and disease resistance, the pathogens have evolved a strategy to alter plant hormone levels to their benefit. Clearly, there are a number of interesting mechanisms that need to be elucidated for this remarkable, multifunctional effector. 3. avrRpt2 avrRpt2 was isolated as an avirulence gene recognized by the RPS2 gene in resistant Arabidopsis plants (Dong et al., 1991; Innes et al., 1993; Whalen et al., 1991). Pst expressing avrRpt2 grows to higher cell densities in susceptible Arabidopsis ecotypes lacking a functional RPS2 gene (Chen et al., 2000), indicating that AvrRpt2 enhances virulence probably by suppression of PTI. AvrRpt2 can also suppress hypersensitive resistance caused by avrRpm1/

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RPM1 indicating it can also suppress ETI (Ritter and Dangl, 1996). The AvrRpt2 protein is a cysteine protease that degrades the plant protein RIN4 (RPM1 interacting protein 4) (Axtell and Staskawicz, 2003; Axtell et al., 2003; Kim et al., 2005a,b), which is a negative regulator of PTI (overexpression of RIN4 led to inhibition of flg22-triggered callose deposition). The possibility exists that AvrRpt2 interferes with FLS2 signaling. However, RIN4 is not essential for AvrRpt2 virulence function (Belkhadir et al., 2004). AvrRpt2 also alters Arabidopsis auxin physiology which appears to promote virulence, although the mechanisms are yet to be determined (Chen et al., 2007). AvrRpt2 is also important for virulence of Pst in tomato (Lim and Kunkel, 2005); taken together, these findings indicate AvrRpt2 probably has other host targets. 4. xopD The X. campestris pv. vesicatoria TTSS effector XopD is a 545-amino acid cysteine protease that targets tomato small ubiquitin-related modifier (SUMO) protein precursors (Hotson et al., 2003). The N-terminus of XopD contains a nuclear localization signal that causes it to localize to the plant cell nucleus. A recent study has shown that XopD can suppress PTI by dampening salicylic acid (SA) synthesis, which inhibits X. campestris pv. vesicatoria growth in planta (Kim et al., 2008). XopD was also shown to be able to bind to the promoters and to suppress expression of the defense-related genes PR-1 and PDF1.2 that are upregulated by SA. Considering the multifunctional nature of this effector, it would appear to have the potential to interact with multiple DNA and protein targets. Intriguingly, this study also found that XopD appears to delay the onset of senescence, such that the pathogen is effectively ‘‘farming’’ the plant host to extend the period that nutrients are available. E. RNA AND RNA-BINDING PROTEIN TARGETING

1. hopU1 (hopPtoS2) hopU1, along with hopO1-1 (hopPtoS1) and hopO1-2 (hopPtoS3), encode proteins containing ADP-ribosyltransferase (ADP-RT) active sites (Fu et al., 2007). Compared to the wild type, a Pst hopU1 mutant was more effective at triggering a HR in the non-host N. benthamiana, indicating that HopU1 plays a role in suppressing non-host HR. Transgenic Arabidopsis Col-0 plants expressing HopU1 deposited less callose than wild-type plants when treated with flg22, and also exhibited a delayed HR to Pst expressing AvrRpt2. These observations indicate that HopU1 can interfere with both PTI and ETI pathways of innate immunity. Biochemical analysis demonstrated that HopU1 exhibits ADP-RT activity that ADP-ribosylates plant host proteins at arginine

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residues. In Arabidopsis, HopU1 targets at least two glycine-rich RNA-binding proteins (RBP), including GRP7. GRP7 regulates mRNA levels in plants at the posttranscriptional level, but was previously characterized in circadian rhythm functions. HopU1 specifically ADP-ribosylates GRP7 at one of the two arginine residues, both in vitro and in planta, which is believed to block binding of the protein to RNA. An Arabidopsis grp7-1 mutant line that does not produce GRP7 exhibited enhanced disease susceptibility to wild-type Pst and its hrcC mutant, confirming that GRP7 is important in plant PTI. Taken together, this elegant study demonstrated a novel target of bacterial effectors, namely RBP, for defense suppression. 2. hopT1-1 HopT1-1 has been shown to interfere with miRNA-dependent RNA silencing-based defense (Navarro et al., 2008). Small RNA’s, including miRNAs, guide Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs) to suppress the expression of genes at either transcriptional or posttranscriptional levels, that is AGO-RISC targets the transcripts of genes and slices them. HopT1-1 appears to act in two ways: it suppresses AGO1mediated slicing and also miRNA-mediated translational inhibition. The exact mechanism of HopT1-1 action remains elusive. F. ATTACK OF NEGATIVE REGULATORS OF PTI

1. avrB avrB was the first avirulence gene isolated from a plant pathogen (Staskawicz et al., 1984). It was identified from Ps. syringae pv. glycinea race 6 and recognized by soybean R gene Rpg1-b (Ashfield et al., 1995). In susceptible soybean and Arabidopsis plants, AvrB enhances virulence (Ong and Innes, 2006) indicating that it plays a role in PTI suppression. AvrB, like AvrRpm1, can interact with and phosphorylate RIN4 (Desveaux et al., 2007; Mackey et al., 2002). From an elegant screen to identify Arabidopsis host proteins required for AvrB function, Shang et al. (2006) identified the chaperone RAR1, previously linked to ETI. By using wild-type and rar1 mutant lines encoding dexamethasone-inducible AvrB it was found that AvrB expression reduced callose deposition in wild-type plants treated with flg22, whereas there was no effect in the mutant line. RAR1 was found to be a negative regulator of PTI through the use of rar1 mutant lines expressing AvrB, which showed higher levels of callose deposition than the wild-type line expressing AvrB. Both AvrB and RAR1 proteins were found as a complex in immunoprecipitation experiments, although yeast two-hybrid analysis did not detect

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a direct physical interaction in vitro. Thus, AvrB appears to target RAR1 to enforce the accumulation of negative regulators of PTI. 2. avrRpm1 avrRpm1 was originally found as an avirulence gene in Ps. syringae pv. maculicola recognized by the resistance gene RPM1 in Arabidopsis (Dangl et al., 1992; Debener et al., 1991; Grant et al., 1995). It also has a virulence function (Ritter and Dangl, 1995; Rohmer et al., 2003). Expression in plants of avrRpm1 can suppress PTI and promote the growth of a Pst hrcC mutant (Kim et al., 2005a,b). Like AvrB, AvrRpm1 can interact with and phosphorylate RIN4, the negative regulator of PTI (Mackey et al., 2002). Two studies have shown that AvrRpm1 has a significant effect on the Arabidopsis proteome and can suppress accumulation of proteins associated with PTI (Jones et al., 2006; Kaffarnik et al., 2009). G. TARGETING HORMONE SIGNALING?

1. hopAN (avrE1/wtsE/dspA/dspE) Homologues of the hopAN family are well characterized in Ps. syringae (avrE), Erwinia amylovora (dspA/E) and Pantoea stewartii (wtsE). It was first identified as a Ps. syringae pv. tomato avirulence gene recognized by soybean to trigger a HR, but also as a virulence factor for strain PT23 on tomato (Lorang and Keen, 1995). The gene is found in the conserved effector locus (CEL) of the TTSS pathogenicity island, located close to hopM1 described below, and encodes a very large protein (ca. 1800 amino acids). The hopAN effector was later recognized as an important pathogenicity gene for Erwinia/Pantoea on apple, gypsophila, maize and pear (Bogdanove et al., 1998; DebRoy et al., 2004; Gaudriault et al., 1997; Mor et al., 2001) and acts synergistically with hopM1 in promoting Ps. syringae virulence in Arabidopsis (Badel et al., 2006) and suppressing PTI in tomato and N. benthamiana (DebRoy et al., 2004; Oh and Collmer, 2005). An elegant study by Debroy et al. (2004) showed that an E. amylovora dspA/E and Pst CEL mutant caused limited disease symptoms in apple and Arabidopsis, respectively, but caused increased callose deposition in the cell walls of the host plant indicating the mutants trigger PTI. Moreover, the Pst CEL mutant was able to grow better in Arabidopsis transgenic NahG and mutant eds5 lines compared to wild-type plants; NahG and eds5 plants are unable to accumulate SA, confirming the importance of SA in disease resistance of Arabidopsis against Pst. Further analysis showed that the Pst CEL mutant could not suppress SA-mediated PTI, while the wild type could do

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so—however, the action of the CEL effectors did not affect SA-responsive genes in the plant, implying that they operate by a different mechanism. Complementation of the Pst CEL mutant with avrE partially restored suppression of the SA-mediated PTI defense, observed as reduced callose deposition compared to the CEL mutant itself. More recently, it has been shown that dspA/E and wtsE are important for early growth of E. amylovora and Pa. stewartii in the non-host N. benthamiana (Ham et al., 2008; Oh et al., 2007). Inoculation of Arabidopsis Col-0 with Ps. syringae pv. phaseolicola expressing wtsE was sufficient to downregulate PR-1 gene expression and callose deposition compared to an empty vector control, further implicating the role of wtsE in PTI suppression; the function of this class of effectors is yet to be determined. 2. hopAM1 (avrPpiB) The hopAM1 effector was first identified in Ps. syringae pv. pisi as an avirulence gene recognized by the R3 resistance gene in certain cultivars of pea (Arnold et al., 2001; Cournoyer et al., 1995). During a screen of effectors that could enhance the virulence of the weak Arabidopsis pathogen Ps. syringae pv. maculicola M6CDE, HopAM1 was found to significantly improve bacterial growth (Goel et al., 2008). Furthermore, HopAM1 improved bacterial growth in plants growing in water-stressed conditions compared to an empty vector control and water-sufficient grown plants. Since ABA is well known for its role in plant responses to abiotic stresses, the use of the abi5-1 (aba insensitive 5) regulatory mutant line was used to show that HopAM1 virulence effects are enhanced by ABA. HopAM1 had a wide range of effects on Arabidopsis plants, including inducing chlorosis in newly emergent leaves, enhancing ABA sensitivity, reducing salt stress, stimulating stomatal closure and inhibiting seed germination. Furthermore, transgenic hopAM1 Arabidopsis plants enhanced proliferation of a Pst hrcC-minus mutant and showed suppressed papilla formation and callose deposition compared to wild-type plants. Taken together these findings indicate that HopAM1 can suppress PTI in Arabidopsis. Thus, like AvrPtob, HopAM1 can enhance the defense suppressive effects of ABA and appears to be active at various stages of bacterial infection. H. DISRUPTION OF VESICLE TRAFFICKING

1. hopM1 (hopPtoM) hopM1 is located close to avrE in the CEL of the TTSS pathogenicity island in Pst. Deletion of the CEL from Pst leads to loss of pathogenicity on tomato, which can be restored by complementation with hopM1 and

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its cognate chaperone, shcM (Badel et al., 2003). HopM1 can fully suppress SA-mediated callose deposition triggered by a Pst CEL mutant in Arabidopsis (DebRoy et al., 2004), unlike the partial suppression seen by AvrE (described above); HopM1-mediated PTI suppression was also observed in a vascular staining assay in which leaves detached from the plant 6 h after inoculation were placed with their petiole in Neutral Red solution. Basal resistance leads to less dye accumulation and this PTI-based reduced vascular flow into the leaf veins was suppressed by HopM1 (Oh and Collmer, 2005). In a seminal study by Nomura et al. (2006) it was found that HopM1 targets immunity-associated AtMIN7 (A. thaliana HopM1 interacting protein 7). Transgenic Arabidopsis plants expressing hopM1 allowed the Pst CEL mutant to grow to near wild-type levels—HopM1 localized to the endomembrane compartments. A series of transgenic plants expressing Nand C-terminally truncated versions of HopM1 were used to discover that the N-terminus is important for virulence. The N-terminal protein HopM11–300 was used to isolate 21 plant-interacting AtMIN proteins in a yeast twohybrid screen—none of these were identified from a screen with full-length HopM1 due to HopM1-dependent destabilization; whether this is a direct or indirect effect remains to be tested. AtMIN protein destabilization was observed in both Arabidopsis and N. benthamiana, where ubiquitination of the proteins was enhanced by HopM1. Inoculation of the Pst CEL mutant into T-DNA knockout lines of AtMIN genes showed that only mutation of AtMIN7 allowed the bacterial strain to grow to higher levels. AtMIN7 is one of eight members of an ADP-ribosylation guanine nucleotide exchange factor that are important in vesicle trafficking in eukaryotes. Increased vesicle trafficking is associated with cell-wall defenses—testing the AtMIN7 mutant line for callose deposition showed that it was not able to deposit as much callose as the wild-type plant when challenged with the Pst CEL mutant. Thus, HopM1 targets this vesicle trafficking system as part of PTI suppression. More recently, Ham et al. (2007) used the plant defense gene mutant lines sid2 (SA induction deficient 2), npr1 (nonexpressor of PR genes 1), pad4 (phytoalexin deficient 4), rar1 and pmr4 (powdery mildew resistant 4) in combination with Ps. syringae pv. phaseolicola with and without ectopically expressed hopM1 to unravel the basis of PTI in Arabidopsis. They showed that Ps. syringae pv. phaseolicola carrying HopAM1 suppresses non-host PTI in Arabidopsis, observed as a reduction in small and large callose deposits and also PR-1 expression, and concluded that a multilayered defense is expressed by Arabidopsis to resist Ps. syringae pv. phaseolicola infection.

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I. TARGETING MAP KINASE SIGNALING

1. HopAI1 The role of HopAI1 in suppression of PTI was first found during a screen for suppressors of NHO1 expression (Li et al., 2005a). The protein shares 35% identity to the Salmonella enterica serovar typhimurium effector VirA. Transgenic expression of HopAI1 in Arabidopsis plants enabled a TTSS mutant to grow in planta although knockout of hopAI1 in Pst had no discernible effect on virulence. Further analysis of other Ps. syringae pv. tomato strains indicated that knockout of hopAI1 in strain 0288-9 led to a reduction in virulence (Zhang et al., 2007). It was revealed that HopAI1 directly interacts with MPK3 and MPK6 MAPKs and uses a phosphothreonine lyase domain to dephosphorylate the proteins and prevent further phosphorylation of the protein. The consequence of this was the blocking of the MAPK-signaling pathway, callose deposition and suppression of FRK1 expression. Thus, HopAI1 acts downstream of the PRR action of effectors such as AvrPto and AvrPtoB.

J. OTHER EFFECTORS INVOLVED IN PTI SUPPRESSION FOR WHICH TARGETS ARE UNKNOWN

1. avrRps4 This effector was originally identified from Ps. syringae pv. pisi as an avirulence gene recognized by the RPS4 resistance gene in some Arabidopsis ecotypes (Hinsch and Staskawicz, 1996); AvrRps4 also triggers an HR in turnip cv. Just Right (Sohn et al., 2009). After secretion into plant cells via the TTSS, the 28 kDa full-length AvrRps4 is processed to a smaller 11 kDa form. Only the C-terminal 88 amino acids of the 221-amino acid AvrRps4 are required to trigger a HR in turnip but processing is not necessary to trigger the HR. A KRVY motif was identified that is located just downstream of the in planta processing site within the 88-amino acid processed peptide—the KRVY motif is required for avirulence in Arabidopsis, but was also found to be necessary for AvrRps4-dependent increased virulence of Ps. syringae pv. tabaci in N. benthamiana; expression of AvrRps4 in N. benthamiana does not trigger a HR. Creation of transgenic Arabidopsis ecotype RLD plants (that lack a functional RPS4 gene) expressing AvrRps4 led to increased growth of wild-type and hrcC-minus strains of Pst and suppression of flg22-induced callose deposition and production of ROS, that is suppression of PTI. Interestingly, an AvrRps4 orthologue, XopO, was identified in X. campestris pv. vesicatoria that shows 41% identity and has a KRVY motif—the protein is processed in planta, but does not elicit an HR in turnip. Taken together,

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these observations suggest this to be an important effector, with an important role in PTI suppression; however, the dual activities and processing phenotype may indicate that two distinct pathways are involved in promoting virulence and triggering resistance. 2. hopAO1 (hopPtoD2) HopAO1 is a protein tyrosine phosphatase and contributes to the virulence of Pst in Arabidopsis—deletion mutants lacking the gene exhibit reduced virulence (Bretz et al., 2003; Espinosa et al., 2003). Underwood et al. (2007) showed that the TTSS hrpA mutant of Pst was able to multiply to higher levels in transgenic plants expressing HopAO1 compared to wild-type plants. Concomitant with this phenotype, the HopAO1 transgenic plants suppressed PTI immunity, as observed by reduced callose deposition upon challenge with the Pst hrpA mutant, compared to wild-type plants. This was further confirmed when using flg22 versus water treatment on wild-type and HopAO1 transgenic plants—the HopAO1 plants suppressed flg22-induced immunity compared to controls. In both experiments, the phosphatase activity of HopAO1 was shown to be essential for the suppression phenotypes. The site of action of HopAO1 is likely to be the plant cell cytosol due to the lack of transmembrane regions or myristoylation sites in the protein sequence. Indeed, HopAO1 protein was discovered to be present in the soluble fraction of plant cell extracts. The MAPKs MPK3 and MPK6 were ruled out as targets for dephosphorylation since transgenic plants expressing HopAO1 had no reductive effects on kinase activity. A microarray analysis of genes expressed in HopAO1 transgenic plants compared to wild-type plants, upon challenge with a Pst hrpA mutant, indicated that HopAO1 blocks only a subset of PAMP-induced genes, possibly by altering JA responses or signaling. K. OTHER EFFECTORS INVOLVED IN PTI SUPPRESSION, BUT LACKING FUNCTIONAL INFORMATION

In the screen carried out by Li et al. (2005a) that identified avrPto, hopAI1, hopM1 and hopT1-1 described above, seven other effectors were found that suppressed NHO1 expression: hopAA1-1, hopAF1, hopC1, hopF2, hopG1, hopS1, hopT1-2. Of these, HopF2 and HopG1 were separately shown to suppress PTI using the vascular staining assay described in Section III.H.1, but the precise function of these effectors remains unknown (Oh and Collmer, 2005) Metz et al. (2005) used a genetic screen to identify X. campestris pv. vesicatoria elicitors that trigger the non-host defense response in

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N. benthamiana. X. campestris pv. vesicatoria cosmid clones were expressed in X. campestris pv. campestris (that only causes mild chlorosis) and screened on N. benthamiana to identify clones that caused cell death—the effector gene xopX was identified from the screen. Although expression of the gene on its own in X. campestris pv. campestris was able to trigger cell death, Agrobacterium-mediated transient expression in N. benthamiana did not trigger cell death, indicating a synergistic effect of XopX and another X. campestris pv. campestris effector. Furthermore, transgenic expression of XopX in N. benthamiana allowed increased growth of non-XopX Xanthomonas and Pseudomonas strains; this increased disease susceptibility is likely due to the suppression of PTI although the precise mechanism remains unknown. Further experiments using xopX knockout strains demonstrated that XopX is important for X. campestris pv. vesicatoria virulence on host plants. L. OTHER POTENTIAL MECHANISMS—TYPE VI SECRETION

The VAS/vgrG type VI secretion system (T6SS) was discovered initially in Vibrio cholerae as an anti-amoeba system (Pukatzki et al., 2006). Since then it has also been described in Pseudomonas aeruginosa (Mougous et al., 2006), Ps. syringae (Arnold et al., 2009) and Pe. atrosepticum (Liu et al., 2008). In Pe. atrosepticum, the T6SS is regulated by quorum sensing and T6SS gene knockout mutants display a reduced virulence on potato tubers and in potato stems, indicating a role in plant pathogenicity. The exact function of the T6SS has yet to be defined, but a role in PTI suppression cannot be ruled out. M. COMPLEXITY AND EVOLUTION OF PTI SUPPRESSION BY BACTERIAL PATHOGENS

It is clear that bacteria target a number of components of plant PTI including: calcium stores in the apoplast; PRR-RLKs that detect PAMPs; the signaling pathways that are activated by the PRRs; hormone signaling pathways, including ABA, auxin, SA and JA; and interference with RNA and protein structure, function and activity including regulators of PTI. This vast array of targets illustrates the complexity of the evolution of plant-pathogenic bacteria and why they have such large arsenals of virulence factors. A general overview is depicted in Fig. 2. Without a deeper evolutionary analysis, it is unclear how the bacteria have evolved to overcome plant innate immunity other than the prediction that they first overcame PTI and then suppressed ETI. Based on the nearubiquitous spread of EPS gene clusters among most free-living bacteria, it is possible that EPS production was the first true PTI suppression system.

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Fig. 2. General overview of the mechanisms for suppression of PAMP-triggered immunity (PTI) by bacterial pathogens. The effectors and their targets are represented by filled gray symbols and connected by dotted lines. Mechanisms for PTI suppression include calcium chelation by extracellular polysaccharides (EPS), opening of stomata by coronatine, hormone signaling, blockage of vesicle trafficking, subjugation of surveillance and defense systems by effectors injected into the cell by the Type III secretion system. Plant defense mechanisms, PTI and effector-triggered immunity (ETI), are in black (symbols and lines). See text for details.

As EPS suppression was overcome, possibly due to EPS acting as a PAMP (Aslam et al., 2008), a subset of pathogens may then have acquired the coronatine biosynthesis genes so that the pathogens could interfere with JA-dependent stomatal closure. Further rounds of evolution, either in parallel or after pathogens acquired toxins and EPS, would see the emergence of the TTSS to secrete effectors into the plant to douse resistance (Jones and Dangl, 2006). Pathogens would then have evolved further by the acquisition/ evolution of more effectors to overcome ETI. Clearly, there is still considerable effort required to understand the functions and targets of all the effectors and then how these integrate together to subjugate host defenses. However, understanding of the different mechanisms employed in suppression of PTI is making good progress.

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IV. RNA SILENCING, THE PLANT’S INNATE IMMUNE SYSTEM AGAINST VIRUSES A. THE DISCOVERY OF RNA SILENCING AS THE PLANT’S INNATE IMMUNE SYSTEM AGAINST VIRUSES

The plant’s innate immune system against viruses is very different from that against fungi and bacteria. The basal antiviral defense relies on the recognition and sequence-specific breakdown of (double-stranded, ds) viral RNA rather than targeting the pathogen’s proteins. In this section, this recently disclosed defense system, which is generally referred to as antiviral RNA silencing or RNA interference (RNAi), will be discussed, as well as the strategy how viruses may counteract this innate defense system. Unlike fungal and bacterial pathogens, viruses are exclusively intracellular parasites, multiplying in either the cytoplasm or the nucleus of plant cells. As a consequence, the interplay between host defense systems and the virus is strictly an intracellular event. Only since the mid 1990s, plant molecular biologists and virologists have become aware that the plant possesses a sequence-specific RNA breakdown mechanism, often referred to as posttranscriptional gene silencing (PTGS) or RNA silencing, and that this mechanism acts as the major innate immune system against viruses. The discovery of this defense system occurred accidentally, by encountering unexpected results during attempts to obtain virus-resistant plants through genetic engineering approaches. In the 1980s, several groups were investigating whether transgenic forms of virus resistance could be obtained according to the concept of ‘‘pathogen-derived resistance’’ (PDR). This concept was first described by Grumet et al. (1987), who proposed the possibility to exploit pathogenderived genes as a means to obtain resistance in a variety of host–parasite systems. It was suggested that deliberate expression of such genes in an altered form, level or developmental stage, could interfere with pathogen replication resulting in specific host resistance. Among possible targets for PDR-mediated virus resistance, the most broadly exploited viral genes were those coding for the coat protein (CP), replicase and movement protein (Baulcombe, 1996; Powell et al., 1990; Prins and Goldbach, 1996). Following the demonstration that expression of a viral CP confers a level of resistance to the pathogen (Abel et al., 1986; Powell et al., 1990), it was observed in control experiments that nontranslatable CP transgenes conferred similar levels of resistance as the functional gene. For years, this phenomenon was referred to as RNA-mediated resistance, and only in 1993, William Dougherty and coworkers (Lindbo et al., 1993) linked this phenomenon to ‘‘cosuppression’’ in plants and ‘‘quelling’’ in fungi (Cogoni and Macino, 1999a,b),

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which involve sequence-specific degradation of transcripts from both transgenes and their homologous endogenous counterparts. In turn, cosuppression was discovered when transgenic petunia plants with additional copies of endogenous genes involved in flower pigmentation, became completely white due to a dramatic decrease in expression level of the respective genes (Napoli et al., 1990; Van der Krol et al., 1990). Identification of (induced) RNA silencing as the principle mechanism of transgenic resistance to viruses has—in retrospective—been a major break-through. Rapidly, multiple publications appeared providing evidence that RNA silencing is a naturally occurring, ancient mechanism having a major function in regulating gene expression, transposon behavior, and viral infections (Carthew and Sontheimer, 2009). Moreover, RNA silencing occurs not only in plants and fungi, but has later been found also in invertebrate (Fire et al., 1998) and vertebrate animals, including humans (Carthew and Sontheimer, 2009; Elbashir et al., 2001; Hammond et al., 2000; Zamore et al., 2000) where this phenomenon is usually referred to as RNAi. A crucial discovery was the finding of short, virus-derived dsRNA molecules in infected host plants, explaining the sequence specificity of the RNA breakdown mechanism (Hamilton and Baulcombe, 1999). These short dsRNA species are commonly referred to as short interfering RNAs (siRNAs). Next to the discovery of virus-specific siRNAs, it was demonstrated that plants that are deficient in essential RNA silencing genes, show enhanced viral pathogenicity (Dalmay et al., 2001; Morel et al., 2002; Mourrain et al., 2000). These, and the fact that all tested plant viruses encode proteins that interfere with, and suppress the RNA silencing pathway, supported the idea that RNA silencing acts as innate antiviral defense system in plants. The viral proteins antagonizing RNA silencing, often already known as ‘‘virulence factors,’’ are commonly referred to as RNA silencing suppressor (RSS) proteins (Brigneti et al., 1998; Kasschau and Carrington, 1998). B. CURRENT VIEWS OF RNA SILENCING AS ANTIVIRAL MECHANISM IN PLANTS

With increasing insights, it was found that RNA silencing (RNAi) is not one single RNA breakdown pathway but encompasses two major ones, the siRNA pathway and the miRNAs. The former includes the antiviral defense branch of the system, while the miRNA pathway is primarily involved in regulating (host) gene expression. Figure 3 presents a simplified scheme of the RNA silencing pathways in the plant (most data have been obtained from Arabidopsis). As visualized in the scheme, RNA silencing starts with the recognition of long dsRNA by a type III endonuclease, called Dicer-like protein (DCL) in plants

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(the term Dicer was coined for a similar enzyme in the fruitfly Drosophila melanogaster (Bernstein et al., 2001)). It will be obvious that in particular RNA viruses are excellent targets to provoke (antiviral) RNA silencing: they replicate through (partially) dsRNA intermediates, while also the singlestranded (ss) genome contains extensive secondary structures. For viruses with a DNA genome, like the caulimo- and geminiviruses, the viral transcripts are the targets for RNA silencing, induced by secondary structures (e.g., the 35S RNA transcript of cauliflower mosaic virus, CaMV) and/or by overlapping sense–antisense transcripts (Chellappan et al., 2004; Du et al., 2007; Moissiard and Voinnet, 2006; Molna´r et al., 2005; Sharp and Zamore, 2000). 1. The siRNA pathway The siRNA pathway represents the antiviral branch of RNA silencing and this process takes place entirely in the cytoplasm (Covey et al., 1997; Ratcliff et al., 1997) (Fig. 3). It is known that plants encode different DCLs; in Arabidopsis DCL-4 is the most important one in the antiviral siRNA pathway while DCL-3 is needed for long-distance silencing. When DCL-4 is inactivated, its function is partly replaced by DCL-2 (Gasciolli et al., 2005). DCL-4 cleaves the viral dsRNA target molecules into short viral specific dsRNA molecules (siRNA) of 21–30 nucleotides (nt) in length with 2-nt overhangs at their 30 ends (Dunoyer et al., 2005; Gasciolli et al., 2005; Hamilton et al., 2002). After cleavage by DCL, the 21-nt siRNAs are incorporated into the RNA induced silencing complex (RISC) complex, which harbors a member of the Ago protein family, a key molecule of RISC (Tanaka Hall, 2005). After unwinding and degradation of the passenger siRNA strand (or siRNA*), the guide siRNA strand is used to identify complementary ss viral RNA sequences. After duplex formation between the guide siRNA strand and viral ssRNA, RISC (more specifically the Ago protein) facilitates target cleavage of the viral ssRNA molecule, resulting in sequence-specific RNA degradation of the viral RNA (Tomari and Zamore, 2005b). A special feature of the silencing pathway in plants is the possibility to amplify the silencing signal, in order to extend silencing along the target gene, using a host-encoded RNAdependent RNA polymerase (hRdRp). The hRdRp is able to produce new dsRNA molecules in an either primer-dependent or -independent manner; those can again enter the siRNA pathway, resulting in secondary siRNA molecules (Baulcombe, 2004; Sijen et al., 2001; Vaistij et al., 2002) (Fig. 3). RNA silencing is not only induced within the infected cell; plants are able to preprogram not yet infected cells by spreading the silencing signal beyond the site of initiation. This feature is called systemic silencing and can be divided in short-distance spread (10–15 cells) and phloem-dependent longdistance transport. It is believed that the short-distance silencing is performed

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Fig. 3. Schematic representation of the siRNA and miRNA pathways in plants and the inhibitory action (indicated ‘‘Stop’’) by some selected viral RNA silencing suppressor proteins (tombusviral p19, auriusviral p14, potyviral HC-Pro, cucumoviral 2b and tenuiviral NS3). RISC, RNA-induced silencing complex; DCL, Dicer-like protein; Ago, Argonaute protein; vRdRp, viral RNA-dependent RNA polymerase; hRdRp, host-encoded RNA-dependent RNA-polymerase.

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by 21-nt siRNAs and dependent on the activity of the hRdRp (Dunoyer et al., 2005; Himber et al., 2003). The long-distance silencing is suggested to rely on the activity of DCL-3, producing 24-nt siRNA molecules (Voinnet, 2005b; Yoo et al., 2004). However, the precise mechanism for both shortdistance and long-distance systemic silencing remains to be further resolved. 2. The miRNA pathway The miRNA pathway has no primary function in antiviral defense; it rather represents a gene expression regulation mechanism, shared with animals, to downregulate plant genes. Comparing the siRNA and miRNA pathway (Fig. 3), it is obvious that there is a high degree of parallelism: both start with the processing of longer dsRNA substrates into small dsRNA species, of which the guide strands are incorporated into RISC (often denoted miRISC) and searching for complementary ssRNA molecules. A fundamental difference is that while the siRNA pathway occurs entirely in the cytoplasm, the miRNA pathway starts in the nucleus: the miRNAs are endogenous RNA species, encoded by host genes. Chromosomal miRNA genes are transcribed mostly by RNA polymerase II to deliver the primary miRNA (pri-miRNA) that are folded into a partly double-stranded stem-loop structure, and become a substrate for DCL-1, thus producing precursor miRNA (pre-miRNA). Cleavage of premiRNA, again performed by DCL-1, generates mature, 21–22 nt miRNAs, which, unlike siRNA, are not completely double-stranded (Bartel, 2004; Voinnet, 2009). The miRNAs are then exported from the nucleus, by the nuclear export receptor HASTY (the product of the Arabidopsis orthologue of EXPORTIN5/MSN5). In the cytoplasm, the miRNAs are incorporated into RISC, unwound and used as guide to find perfectly or partly complementary ssRNA sequences, resulting in degradation or translational inhibition, respectively, of target mRNAs. These target mRNAs often encode transcription factors that, in turn, are in charge of regulating multiple genes (Chen, 2005). Most miRNAs are expressed in a tissue-specific manner and some are able to downregulate the expression of key RNA-silencing proteins, like DCL and Ago. The complementary miRNA target sequences in the host mRNAs can be present in the coding sequence or in the 30 untranslated regions (Voinnet, 2009). C. VIRAL SUPPRESSORS OF RNA SILENCING

During a compatible interaction between a virus and its host plant, infected plant tissues contain significant amounts of virus-derived siRNAs, indicating that the invading virus is actively targeted by the antiviral silencing machinery.

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Plant viruses would not exist if they had not generated an efficient strategy to counteract this antiviral RNA silencing. Indeed, they do so by encoding RSS proteins that are able to suppress RNA silencing. Among the first viral RSS proteins identified was HC-Pro of potyviruses, a multifunctional protein involved in aphid-mediated virus transmission, genome amplification, polyprotein processing, viral long-distance movement and RNA silencing suppression (Anandalakshmi et al., 1998; Be´clin et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998). This protein was already known as ‘‘virulence factor,’’ as there was a causal linkage between HC-Pro expression and severity of disease symptoms (Atreya and Pirone, 1993; Atreya et al., 1992). This effect of HC-Pro can now be readily explained in view of its function to suppress the host’s antiviral RNA silencing. Mutational analysis has revealed that the RSS and proteolytic activity are independent, separable properties within HC-Pro, in contrast to genome amplification and long-distance movement functions, which seem to be related to the RSS activity (Kasschau and Carrington, 2001). At least part of these activities can be explained by the affinity of HC-Pro for siRNA and its interference with their methylation, reducing siRNA stability (Ebhardt et al., 2005; Lakatos et al., 2006; Li et al., 2005b). To date, for an increasing number of plant viruses the encoded RSS protein has been identified mostly based on transgenic suppressor assays in Arabidopsis or Nicotiana spp. (Li and Ding, 2006; Roth et al., 2004). These include positive-, negative- and dsRNA viruses as well as the geminiviruses, which have a ssDNA genome. Most of these viruses encode only a single RSS protein, which acts on a single step in the siRNA pathway, resulting in partial suppression (Li and Ding, 2006). The situation for geminiviruses is more complicated though, as among different viral species the RSS activity appears to reside in different proteins (Bisaro, 2006; Voinnet et al., 1999). The closterovirus citrus tristeza virus is also a special case, as this virus encodes three proteins involved in RSS action (Lu et al., 2004; Satyanarayana et al., 2002). Since RNA silencing has been recognized more and more as an ancient cellular mechanism shared by most living organisms (Dı´az-Pendo´n and Ding, 2008; Li and Ding, 2006), it is obvious that viruses and antiviral RNA silencing will have co-evolved over a very long period, and hence viral RSS proteins are expected to form one or more clusters of similar proteins containing conserved sequence motifs. This is, surprisingly, not the case. In genomic position, in molecular size and in amino acid sequence, the reverse is true: viral RSS proteins are extremely variable among viruses, which is most prominently illustrated by the situation within the family Tombusviridae: depending on the species, the RSS function may reside in the viral CP, the viral polymerase, or in a separate viral protein (Fig. 4, see also Me´rai et al., 2005, 2006; Takeda et al., 2005). The general picture, which

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Aureusvirus P84 (replicase)

P27 (MP)

PoLV

P41 (CP) P25 (repl)

P14 (RSS)

siRNA and dsRNA binding

Carmovirus P88 (replicase) TCV

P9 (MP) P8 (MP)

P28 (repl)

siRNA and dsRNA binding P38 (CP) (RSS)

Dianthovirus P88 (replicase) (RSS) RNA-1

RCNMV RNA-2

DCL interaction

P37 (CP)

P27 (repl) (RSS) P35 (MP)

Tombusvirus P92 (replicase)

P22 (MP) siRNA binding

P41 (CP)

TBSV/CymRSV P33 (replicase)

P19 (RSS)

Fig. 4. Schematic representation of the genome organization of four different viral species belonging to the Tombusviridae. Open reading frames (ORFs) in the respective RNA genomes are indicated as open bars. ORFs and names in gray represent the identified RNA silencing suppressor (RSS) proteins and their functional activity. PoLV, Pothos latent virus (genus Aureusvirus); TCV, turnip crinkle virus (genus Carmovirus); RCNMV, red clover necrotic mosaic virus (genus Dianthovirus); CymRSV, cymbidium ringspot virus (genus Tombusvirus). (After Takeda et al., 2005).

emerges when comparing different plant viruses, is that their encoded RSS activity is often part of a multifunctional protein. Viral RSS proteins not only come in very different shapes, but their mode of action may also differ. Some RSS proteins act by sequestering dsRNA molecules, either size-specifically, like tombusviral P19 (exclusively binding siRNAs), or nonspecifically, like aureusviral P14 (also binding longer dsRNAs) (Lakatos et al., 2006; Me´rai et al., 2005, 2006). Others bind protein factors of the RNA silencing pathway, like cucumber mosaic virus (CMV) protein 2b. While most RSS proteins interfere with only a single step in the RNA silencing pathway, some are able to block at different points, including again CMV protein 2b, which is able to both sequester siRNAs and to interact with Ago within the RISC complex, as demonstrated in infected Arabidopsis (Goto et al., 2007; Zhang et al., 2006). RSS proteins encoded by different virus families often share no homology at the amino acid sequence level, even if they have a similar mode of action (Lakatos et al., 2006; Me´rai et al., 2006). So far not a single sequence motif characteristic for (a subclass of) RSS proteins has been identified. This is surprising as RNA silencing is generally regarded to be an ancient mechanism. One explanation for this could be that the long-lasting evolutionary interplay with the plant’s antiviral RNA silencing mechanism has driven

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viruses to continuously change and adapt their suppressor protein sequences (and their coding sequences) to keep ahead of the host defense system. The RNA silencing’s selective pressure would then act as a major evolutionary driving force resulting in extreme speciation. This would also explain the overwhelming excess of RNA virus species versus DNA virus species within the plant kingdom. A similar situation may have occurred in bacteria where the great majority of phages have DNA genomes, targets for the (DNA-based) restriction/modification system in bacteria. Another explanation might be that RSS genes have been introduced into viral genomes through multiple independent evolutionary events. An argument in favor of this alternative is the observation that RSS genes often overlap with another viral gene, including in some cases the polymerase gene. In evolutionary terms, it is believed that overlapping genes are created by overprinting, meaning that an existing coding sequence is translated in a different reading frame (Ding et al., 1995; Keese and Gibbs, 1992). According to this scenario, the lack of sequence homology between different RSS proteins would be explained by multiple independent introductions into viral genomes. Plant viruses also replicating in their insect vectors, such as the rhabdo-, tospo- and tenuiviruses, need to counteract RNA silencing in two very distinct types of organisms. It is to be expected that this may be achieved by specifying an RSS protein which blocks a step in the RNA silencing mechanism that is conserved between plants and insects. Indeed, both tospoviral NSs and tenuiviral NS3 proteins exert their RSS function by sequestering siRNAs (Hemmes et al., 2007, 2009), a key molecule shared by plant and insect. D. POSSIBLE INTERACTIONS BETWEEN PLANT VIRUSES AND THE miRNA PATHWAY

During the infection process the presence of the viral RSS protein may, in addition to blocking the antiviral siRNA pathway, interfere also with the miRNA pathway. Both pathways share similar key molecules, either RNA or proteins, and nearly all tested RSS proteins which act by sequestering siRNA molecules are equally able to bind miRNAs in vitro. Examples are tombusviral p19 and tenuiviral NS3 (Dunoyer et al., 2004; Hemmes et al., 2007; Silhavy et al., 2002). Further research confirmed the ability of RSS proteins to suppress the miRNA pathway in vivo, resulting in virus diseaselike symptoms. Drastic effects in phenotypes, reminiscent of virus disease symptoms, have also been observed in transgenic Arabidopsis plants expressing viral RSS proteins (Chapman et al., 2004; Dunoyer et al., 2004). These observations point to a prominent role of viral RSS proteins in the induction

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of disease symptoms upon infection. Whether this symptom induction is intended or happens accidentally due to the high similarities between the siRNA and miRNA pathways is not known yet. E. IS ANTIVIRAL RNAi RESTRICTED TO PLANTS AND INSECTS?

After the ground-breaking work of Andrew Fire and Craig Mello (Fire et al., 1998), who discovered RNAi in nematodes, increasing evidence indicated that RNAi is an ancient gene regulation mechanism occurring in almost every eukaryotic organism, from algae and plants to insects and humans (Sontheimer and Carthew, 2005; Tomari and Zamore, 2005a; Voinnet, 2005a). The mechanism shared between all these organisms is the miRNA pathway, whereas the siRNA pathway may be shared only among plants and invertebrates and probably not by mammals. For insects, the existence of a separate antiviral siRNA branch within RNAi has been well established, and insect-infecting viruses, in turn, have been shown to encode RSS proteins (e.g., protein B2 of Flock House virus (Li et al., 2002)) to combat this antiviral response. In infected mammalian cells, virus-derived siRNAs have so far not convincingly been detected (Pfeffer et al., 2004), but antiviral RNA can be readily induced upon transfection with dsRNA (hairpin RNA) containing viral sequences (Haasnoot et al., 2007; Lo´pezFraga et al., 2008; Marques and Carthew, 2007). While a separate antiviral siRNA branch may be absent in mammals there is increasing evidence that also mammalian, for example, human viruses encounter antiviral RNAi and this may exclusively occur through the miRNA pathway, resulting in the thought that human viruses encode RSS proteins too (Berkhout and Jeang, 2007; Grassmann and Jeang, 2008; Murakami et al., 2009; Triboulet et al., 2007). In mammalian cells, a Human Immunodeficiency Virus (HIV)-1 mutant lacking its proposed RSS, HIV-1 Tat (Transactivator of transcription) could be complemented in its viral production by transfection with a plant viral RSS. Thus, it was convincingly demonstrated that HIV-1 specifies an RSS protein, HIV-1 Tat, that is capable of suppressing the action of miRNA in vivo and that this suppression is essential for efficient virus production (Schnettler et al., 2009; Qian et al., 2009). PTGS, or shortly RNA silencing, not only represents a major regulatory mechanism in most, if not all, eukaryotes, in plants this sequence-specific RNA breakdown mechanism, through a separate siRNA branch, also represents the basal defense system against viruses. In turn, plant viruses have been able to counteract this host response by encoding antagonizing RSS

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proteins. Some of these RSS proteins also interact with the miRNA pathway, explaining some of the observed disease symptoms in infected plants. Whether this interaction is on purpose or represents a non-specific side effect, remains to be established.

ACKNOWLEDGMENTS The authors thank Jens Boch for his comments on the manuscript.

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From Nonhost Resistance to Lesion-Mimic Mutants: Useful for Studies of Defense Signaling

ANDREA LENK AND HANS THORDAL-CHRISTENSEN1

Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Defense Induction Mediated by PAMPS and Effectors. . . . . . . . . . . . . . . . . . . III. Signaling Downstream of Pathogen Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The SA-Signaling Pathway.................................................. IV. Commonalities in the Defense Response of Host and Nonhost Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Penetration Resistance of Arabidopsis..................................... B. Nonhost Resistance to Bacteria ............................................ V. What is the Explanation for Nonhost Resistance?. . . . . . . . . . . . . . . . . . . . . . . . VI. Lesion-Mimic Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Mutant Screens Without Pathogens for Finding Genes in Defense Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SSD Mutants .................................................................. B. SFD Mutants ................................................................. C. MOS Mutants................................................................. VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51003-8

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ABSTRACT Nonhost and host resistance are very similar phenomena which employ the same defense mechanisms, spanning from passive defense, through PAMP-triggered immunity to responses toward pathogen eVectors. The diVerence between an unsuccessful and a successful pathogen lies in its eVector portfolio. It has become apparent that only if the pathogen’s eVectors are able to suppress defense responses, and do so without being recognized, can the attacked plant serve as a host for the pathogen. Thereby, the sum of eVectors determines the host range of a given pathogen. Another approach for investigating defense mechanisms, using lesion-mimic mutants, is attracting interest. These plants have enhanced resistance to biotrophic pathogens, and due to their constitutively active defense-signaling pathways, lesion-mimic mutants are valuable for identifying genes involved in defense signaling.

I. INTRODUCTION Plants are surprisingly healthy considering the vast number of potential pathogens in their surroundings. To a large extent, this is due to nonhost resistance, where all genotypes of a plant clade are resistant to all genotypes of a pathogen (Mysore and Ryu, 2004; Thordal-Christensen, 2003). Nevertheless, significant application of pesticides is required to minimize crop damages resulting from the few pathogens that are able to cause disease. Pathogenic microbes are an expense factor, causing immense harvest losses and quality reduction in agriculture every year. As such, there is good reason to investigate the nature of nonhost resistance to pathogens in order to understand and exploit this durable form of plant protection. Nonhost and host pathogen resistance appear to be based on a complex interplay involving the same plant and pathogen components. Therefore, we summarize the most essential elements of this interplay, and present what is currently known about how this results in nonhost resistance. In Arabidopsis thaliana, nonhost resistance toward the nonadapted barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) is mediated to a large extent by ‘‘penetration resistance.’’ In this review, we will address how studies of this system have identified three PEN (PENETRATION) genes required for resistance to fungal penetration. The first of these to be identified, PEN1, encodes a syntaxin (Collins et al., 2003) which is required for vesicle fusion at the plasma membrane. Combining mutations in PEN1 and its closest homolog, SYP122 (SYNTAXIN IN PLANTS 122), has demonstrated an overlapping function of these genes as negative regulators of defenses, resulting in a severe lesionmimic phenotype of this syntaxin double mutant. Constitutive production of diVerent defense-signaling compounds, including salicylic acid (SA), makes

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these plants resistant to biotrophic pathogens. Such lesion-mimic mutants (LMMs) have visible hypersensitive response-like symptoms, and therefore can be considered to have genetic diseases with significant similarity to diseases caused by pathogens. In classical defense expression studies, many important mutants have been discovered with altered response to pathogens. This has led to the identification of numerous defense-signaling genes and has provided most of our current insight into defense mechanisms. However, we and others have found that screening for suppressor mutants in LMMs can circumvent pathogen-based mutant screens, which are often time-consuming and problematic. Thereby, it is possible to simplify our continuous eVorts to unravel new components in defense-signaling pathways in plants. Furthermore, combining defense-signaling mutations in LMM backgrounds permits investigation of signaling gene interrelationships and allows signaling networks to be proposed.

II. DEFENSE INDUCTION MEDIATED BY PAMPS AND EFFECTORS Protection against pathogen attack occurs in diVerent layers of defense and can be divided into passive and active defense mechanisms. Passive defense against attacking microorganisms does not require pathogen recognition, but instead consists of permanent physical barriers such as plant hairs, wax layers, and strong cell walls. Moreover, pathogens can be resisted by the constitutive presence of toxic secondary metabolites as well as antimicrobial proteins. All these are preformed mechanisms of defense. Importantly, plants can also actively mount resistance upon recognition of pathogens. Microbes possess highly conserved structures, called ‘‘pathogenassociated molecular patterns’’ (PAMPs) (Chisholm et al., 2006; Ingle et al., 2006). PAMPs are indispensable for microbial life, and their recognition by the plant predominantly occurs preinvasively via plant receptors in the plasma membrane, leading to activation of PAMP-triggered immunity (PTI). PTI is often referred to as ‘‘basal defense.’’ Through MAPK (mitogen-activated protein kinase) cascades, defense mechanisms are turned on, including responses such as callose deposition, ion fluxes, production of reactive oxygen species (ROS), and secretion of antimicrobial compounds (Altenbach and Robatzek, 2007). Receptors for the fungal PAMPs, chitin and xylanase, have been identified (Kaku et al., 2006; Ron and Avni, 2004; Wan et al., 2008). However, the best characterized PAMP receptors are FLS2 and ERF, two leucine-rich repeat receptor-like kinases (LRR-RLK) which

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recognize bacterial flagellin and translation elongation factor TU, respectively (Go´mez-Go´mez and Boller, 2000; Zipfel et al., 2006). It has been shown that FLS2 directly interacts with flagellin and that it accumulates in intracellular endocytotic vesicles upon ligand activation (Robatzek et al., 2006). During co‐evolution with their hosts, pathogens have been able to circumvent this PTI by avoiding recognition or by suppressing PAMP-mediated defense mechanisms using eVector proteins (Chisholm et al., 2006; Da Cunha et al., 2006). EVectors are small molecules that are released by the pathogen to their potential host to promote the process of infection. Pathogenic bacteria use the type III protein secretion system (T3SS) to translocate their eVectors into the plant cell (Schechter et al., 2006), whereas some fungal pathogens deliver eVectors into the apoplast or through their feeding structure, the haustorium, into the host cell (Catanzariti et al., 2006; Kemen et al., 2005). Oomycetes secrete eVectors containing a specific RXLR-dEER-motif necessary for their translocation into the plant cell (Dou et al., 2008; Morgan and Kamoun, 2007; Whisson et al., 2007). In addition to eVectors, some pathogens produce plant hormones or hormone analogs to aVect plant defense. As some signaling pathways in plants act antagonistically, these pathogens aim at producing hormones antagonizing the defense pathway directed against their own propagation. A well-documented example is the SA-signaling pathway antagonized by the jasmonic acid (JA)-signaling pathway. The SA-signaling pathway is eVective against biotrophic pathogens, relying on living host tissue. Therefore, some pseudomonads produce coronatine, a bacterial analog of the JA-derivative, JA-isoleucine, in order to suppress the SA-signaling pathway (Cui et al., 2005; Laurie-Berry et al., 2006; Zhao et al., 2003). In attempts to counteract pathogen eVectors, plants have evolved genotypespecific disease resistance (R) genes. Resistance proteins directly or indirectly recognize microbial eVectors, which were previously referred to as avirulence (Avr) proteins. Avr genes are pathogen-race specific. R gene-mediated resistance is usually accompanied by a localized programmed cell death (PCD) called the hypersensitive response (HR) (Lam, 2004). During the HR, hydrogen peroxide (H2O2) accumulates, acting a dual role such as in preventing pathogen growth, due to its toxicity, and a signal for promotion of the PCD. Although R genes mediate resistance to diVerent types of pathogens, they share conserved characteristics. The most prominent class of R genes, with approximately 125 genes in Arabidopsis, encode NB-LRR proteins with a central nucleotide binding site (NB) and a C-terminal leucine-rich repeat (LRR) domain (Martin et al., 2003). These NB-LRR proteins are localized in the cytoplasm and can be divided into two subclasses based on their N-terminal domain. The region either contains a coiled-coil structure

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(CC-type) or has homology to the Drosophila Toll and mammalian Interleukin-1 receptor (TIR-type). The LRR domain is responsible for specific protein recognition and the CC or TIR domains control downstream signaling (Martin et al., 2003). The NB domain is proposed to act as a molecular switch that hydrolyzes ATP. Thereby, the NB-LRR protein undergoes conformational changes, which activate regulatory elements further downstream and trigger the HR (Takken et al., 2006). Autoactive mutant versions of NBLRR proteins have been identified, in which ATP hydrolysis appears to be impaired. Plants expressing autoactive R-proteins are more resistant to pathogens, but on the other hand, suVer from spontaneous necrosis (Howles et al., 2005; Shirano et al., 2002; Zhang et al., 2003). Other eVectors capable of suppressing eVector-triggered immunity (ETI) and the accompanying cell death have evolved during the arms race between plants and pathogens. There are documented examples of eVectors suppressing both PTI and ETI by utilizing diVerent strategies, ranging from host-protein ubiquitination, phosphorylation, and dephosphorylation to alteration of the RNA metabolism (Block et al., 2008; Da Cunha et al., 2007). Throughout the past decades, diVerent models have been proposed to explain how R-proteins and their corresponding Avr-proteins interact. In 1956, Flor hypothesized the ‘‘gene-for-gene model,’’ with one R gene for every Avr gene (Flor, 1956). In a few cases direct interactions have been documented, thereby supporting a ‘‘receptor-ligand-model’’ (Deslandes et al., 2003; Dodds et al., 2006; Jia et al., 2000; Ueda et al., 2006). However, in most cases, no direct interaction between the R-protein and the Avrprotein occurs. An explanation for this is provided by the ‘‘guard model’’ in which R-proteins are monitoring the action of eVectors/Avr-proteins by surveying whether eVector targets are being perturbed (Dangl and Jones, 2001; Van der Biezen and Jones, 1998). Additionally, the guard model can explain how only a few R genes can monitor various unrelated eVectors at once. This can occur when a single R-protein guards multiple eVector targets, or when a single target is attacked by diVerent eVectors. For example, the Arabidopsis Avr-protein RPM1, which is activated upon RIN4 hyperphosphorylation, detects two unrelated Pseudomonas syringae eVector proteins which both target RIN4 (Bisgrove et al., 1994; Chisholm et al., 2006). In tomato, the R-protein Mi recognizes organisms which are as remotely related as nematodes and aphids (Rossi et al., 1998). A recently proposed ‘‘decoy model’’ suggests that many eVector targets mimic real targets and only serve to recognize the eVector, which then can be detected by the R-protein to activate defense (Nandi et al., 2003; Van der Hoorn and Kamoun, 2008; Zhou and Chai, 2008). Decoys may have evolved from eVector targets through gene duplication (Van der Hoorn and Kamoun, 2008).

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Plants successfully employ both PTI and ETI to repel microbial invaders. These two layers of immunity result in the activation of similar subsets of genes (Lin and Martin, 2007; Lindeberg et al., 2006). In a transcription profiling study, comparing tomato plants treated with a PAMP elicitor (flg22) and an eVector (Avr9) from Cladosporium fulvum, similar genes were induced (Navarro et al., 2004). Another example of how PAMP- and eVector-mediated resistance are linked is the barley MLA resistance protein-mediated response to the powdery mildew fungus (Shen et al., 2007). Through PAMP-mediated signaling, WRKY transcription factors are activated and PTI is triggered. However, at the same time, defense protein production is reduced by the activation of other WRKY factors that suppress transcription. After eVector recognition, the activated MLA protein acts positively on PTI by interfering with the repression mechanism of these additional WRKY factors, thereby amplifying PTI and killing the pathogen. Initially, PTI responses are weak, not to damage the host. As soon as MLA protein activation occurs, responses become much stronger (Shen et al., 2007). This fascinating set of interactions between several components in the plant and the pathogen must have a long co‐evolutionary history. Each time either the plant or the pathogen has managed to develop an eYcient tool in its favor, the other organism has evolved a method to overcome this obstacle. The final result as we see it today is likely to have evolved one step at the time, alternating between the two organisms.

III. SIGNALING DOWNSTREAM OF PATHOGEN DETECTION Most work investigating signaling pathways has been done in mutant studies, notably in Arabidopsis. Proteins such as EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and its interacting partners, PAD4 (PHYTOALEXIN DEFICIENT 4) and SAG101 (SENESCENCE-ASSOCIATED GENE 101), act downstream of TIR-NB-LRR-type R-proteins in ETI (Wiermer et al., 2005). However, EDS1 is also a positive regulator of PTI. The Arabidopsis eds1 mutant has, for example, a reduced penetration resistance against barley and wheat powdery mildew fungi (Lipka et al., 2005; Wiermer et al., 2005; Yun et al., 2003; Zimmerli et al., 2004). While EDS1, PAD4, and SAG101 are important for signal transduction from TIR-NB-LRR-type R-proteins, NDR1 (NONRACE-SPECIFIC DISEASE RESISTANCE 1) acts in concert with the CC-NB-LRR-type R-proteins (Aarts et al., 1998; Century et al., 1997). SGT1b (SUPPRESSOR OF G2 ALLELE OF SKP1), RAR1 (REQUIRED FOR MLA RESISTANCE 1), and HSP90 (HEAT SHOCK PROTEIN 90),

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act as chaperones and stabilize R-protein complexes rather than acting in the downstream signaling per se (Azevedo et al., 2002; Holt et al., 2005). A. THE SA-SIGNALING PATHWAY

SA is a key signaling molecule in R gene-mediated resistance and PTI that is activated in response to biotrophic pathogens (Glazebrook, 2005). Mutants with reduced SA levels, or attenuated SA signaling, show decreased resistance to biotrophic pathogens, such as the oomycete Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica). The phytohormone SA is synthesized in response to pathogen infection. In Arabidopsis, pathogen-induced SA synthesis requires the gene SID2 (SA INDUCTION DEFICIENT 2), encoding isochorismate synthase 1 (ICS1) (Nawrath and Me´traux, 1999; Wildermuth et al., 2001). SID2 is located in the chloroplast and catalyzes the conversion of chorismate to isochorismate. An unknown, probably chloroplast-located enzyme is needed for conversion of isochorismate to SA (Strawn et al., 2007). sid2 mutant plants are more susceptible to pathogens, but still contain a small amount of residual SA after stress stimulation. The only other isochorismate synthase in Arabidopsis, ICS2, is responsible for production of a small part of the stress-induced SA in the sid2 mutant background. Double-mutant analysis revealed the existence of an ICS-independent SA biosynthesis pathway. However, this pathway only plays a minor role (Garcion et al., 2008). Similarly, eds5 (enhanced disease susceptibility 5) mutant plants fail to accumulate SA. EDS5 has homology to members of the MATE (multidrug and toxin extrusion) transporter family, and is believed to be involved in translocation of intermediates for SA biosynthesis across the chloroplast membrane (Nawrath et al., 2002). The functional order of EDS5 and SID2 is not yet established. Altered SA levels are also observed upon mutation of ALD1 (AGD2LIKE DEFENSE RESPONSE PROTEIN 1) and its close homolog AGD2 (ABERRANT GROWTH AND DEATH 2). However, mutations in ALD1 and AGD2 cause opposite phenotypes (Song et al., 2004a). While a specific agd2 mutant produces elevated SA levels and is resistant to P. syringae pv. maculicola, ald1 mutants show reduced SA accumulation and are more susceptible (Song et al., 2004a,b). ALD1 gene expression is highly upregulated in response to infection by P. syringae. ALD1 is likely to be a part of an EDS1/PAD4-controlled SA-independent pathway, because its expression is dependent on PAD4, but not on SA (Song et al., 2004b). Both ALD1 and AGD2 are predicted to be located in the chloroplast and show aminotransferase activity on several amino acid substrates in vitro (Song et al., 2004a).

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AGD2 catalyzes a step in lysine biosynthesis, while recombinant ALD1 does not have this particular activity (Hudson et al., 2006). Another gene, believed to be part of the EDS1/PAD4 regulated SAindependent pathway, is FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE 1). FMO1 transcription is upregulated after pathogen attack and in lesion-mimic mutants such as acd11 (accelerated cell death 11), lsd1 (lesions simulating disease resistance 1), and the pen1 syp122 double mutant. FMO1 was identified independently by several groups as a positive regulator of defense (Bartsch et al., 2006; Koch et al., 2006; Mishina and Zeier, 2006; Olszak et al., 2006; Zhang et al., 2008). FMO proteins catalyze the transfer of hydroxyl groups to nucleophilic heteroatom-containing substrates, and have been speculated to function in changing the cellular redox state by production of ROS. But the FMO1 substrate is still elusive (Schlaich, 2007). The enzymatic activity of FMO1 is required for plant defense (Bartsch et al., 2006). PBS3 (avrPphB SUSCEPTIBLE 3) is a member of the GH3-like family of acyl-adenylate/thioester-forming enzymes which are known as phytohormone-amino acid synthetases (Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007). pbs3 mutants have decreased transcript levels of PATHOGENESIS-RELATED (PR)-1, and are more susceptible to several virulent and avirulent P. syringae strains. After inoculation with bacterial pathogens, pbs3 mutant plants exhibit lower levels of glucoside-conjugated SA (SAG), which is the primary storage form of SA. Total SA levels are decreased, while contradictory results were reported for accumulation of free SA in response to various P. syringae strains carrying diVerent Avr genes. Nevertheless, external SA application can restore PR-1 expression and resistance toward pathogens (Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007). It is speculated that PBS3 has a role in the SA-signaling pathway, acting as a regulator on SA and SAG accumulation, or even upstream of SA synthesis (Nobuta et al., 2007). In Arabidopsis and tobacco, many studies have employed plants expressing the bacterial transgene NahG from Pseudomonas putida. NahG encodes a SAhydroxylase that converts SA to catechol. Phenotypical diVerences between NahG plants and SA-deficient mutant plants raised the question whether NahG plants have a distinct phenotype caused by the production of catechol (Van Wees and Glazebrook, 2003). Furthermore, data suggest that SA is unlikely to be the only substrate of the NahG SA-hydroxylase, and those particular aspects of the NahG phenotype are neither SA dependent nor due to catechol (Heck et al., 2003). In addition, based on LMMs, evidence has been provided that NahG inactivates potential defense‐signaling compounds other than SA (Brodersen et al., 2005; Zhang et al., 2008).

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A central downstream component of the SA-signaling pathway is NPR1 (NONEXPRESSOR OF PR GENES 1) (Dong, 2004). In the cytosol, NPR1 is present in an oligomeric form. Upon increase of SA levels, the redox state of the cell is altered and internal disulfide bridges of the NPR1 protein are reduced. This leads to monomeric NPR1, capable of entering the nucleus (Mou et al., 2003). Here, it interacts with TGA transcription factors, modulating their binding activity to as-1-like promoter elements of PR genes; thereby, PR gene expression is induced (Despre´s et al., 2000). PR-1, the function of which is still unknown, is a widely utilized marker for SA signaling. Another common DNA sequence motif in PR promoter regions is the W-box, which is recognized by WRKY transcription factors (Eulgem and Somssich, 2007; Eulgem et al., 2000). In fact, many PR genes are more likely to be regulated by WRKY transcription factors than by TGA transcription factors (Maleck et al., 2000). WRKY factors act both as negative and positive regulators in pathogen defense, and interference with the negatively functioning WRKY factors has been documented to promote plant defense (Shen et al., 2007). In addition to local defense responses at the site of pathogen attack, a systemic, long-lasting disease resistance can occur. This form of induced resistance, termed ‘‘systemic acquired resistance’’ (SAR), is functional against a broad spectrum of pathogens. In many plant species, SA-dependent SAR can be induced by necrotizing pathogens and various chemical compounds. PR gene expression in uninfected leaves functions as marker for SAR (Durrant and Dong, 2004). Identification of the mobile signal inducing SAR in distal leaves has challenged scientists in the field for many years. It has been suggested to be a lipid-based molecule (Maldonado et al., 2002) or JA (Truman et al., 2007). In tobacco, the mobile signal has been identified as SA-derived methyl salicylate (MeSA), not excluding a lipid-based signal in Arabidopsis (Park et al., 2007). Many mutants that are deficient in the SAsignaling pathway are also unable to activate SAR. This is true for the mutants sid2, eds1, eds5, pad4, ald1, fmo1, pbs3, and npr1, and for plants expressing the transgene NahG.

IV. COMMONALITIES IN THE DEFENSE RESPONSE OF HOST AND NONHOST RESISTANCE In an attempt to describe how nonhost resistance functions, we find it interesting that many examples are available, documenting similarities between how plants respond to host and nonhost pathogens. This has been shown convincingly using transcript expression profiling (Stein et al., 2006;

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Tao et al., 2003; Truman et al., 2006). Moreover, a number of examples described below have utilized functional studies to demonstrate that the mechanisms underlying these two types of resistance overlap. A. PENETRATION RESISTANCE OF ARABIDOPSIS

Plants have a distinct type of PTI against powdery mildew fungi, manifested as ‘‘penetration resistance.’’ The powdery mildew fungus, Golovinomyces cichoracearum (Gc), in general overcomes this defense on its host plant, Arabidopsis (Zhang et al., 2007). Meanwhile, when the barley powdery mildew fungus Bgh attacks Arabidopsis, it only overcomes the penetration resistance at 10–20% of the attack sites (Fig. 1). Powdery mildew fungi are obligate biotrophs. Their life cycle is dependent on living host tissue, and they are often specialized to survive only on a specific host plant clade. Bgh proliferates on plants from the genus Hordeum, and is not able to complete its life cycle on other plants. Nevertheless, Bgh spores germinate and develop normally on

% Bgh penetration

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Fig. 1. (A) Penetration frequency of the nonhost barley powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh), on Arabidopsis Columbia (Col-0) wild type and its pen1 mutant. (B) Growth of Bgh on Arabidopsis plants. Germinating conidiospores (co) stopped by papilla (pap) or by single epidermal cell death (HR) after generating haustorium (h) and secondary hyphae (sh). Stained with Trypan Blue 48 h postinoculation.

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Arabidopsis. When specialized infection hyphae, the appressoria, attempt to penetrate through the cuticle and cell wall of an epidermal cell of this nonhost plant, only a minority succeeds. In about 80–90% of the cases, the appressorium is stopped at this preinvasive stage by a reinforcement of the cell wall, the socalled ‘‘papilla’’. The few spores that manage to penetrate develop a haustorium, an invasive feeding structure, inside the epidermal cell. In some cases, they even develop secondary hyphae, which reflects that the haustorium is functioning and able to take up nutrients from the host cell. However, the growth of Bgh is eventually stopped by an epidermal single cell HR, serving as a second layer of defense (Fig. 1) (Collins et al., 2003). This backup defense layer is predicted to be mediated by a gene-for-gene interaction between fungal eVector/Avr-proteins and R-proteins in this nonhost plant. Interestingly, both Bgh penetration and secondary hyphal growth rates are significantly higher in Arabidopsis eds1 mutants (Zimmerli et al., 2004). The same EDS1-dependence has been observed for penetration resistance and epidermal HR in Arabidopsis after attack by the wheat powdery mildew fungus, B. graminis f.sp. tritici (Yun et al., 2003). The need for the EDS1-signaling step in the single cell HR has been confirmed by its requirement for the EDS1-interacting proteins, PAD4 and SAG101 (Lipka et al., 2005). In summary, it appears that two layers of defense, PTI and ETI, prevent these nonhost fungi from completing their life cycle on Arabidopsis. In a genetic screen for mutants that are impaired in stopping B. graminis at the first layer of defense, an ethyl methane sulfonate (EMS)-mutagenized Arabidopsis population was analyzed. Bgh entry into epidermal cells was analyzed microscopically. The first penetration resistance mutant to be identified was pen1 (Collins et al., 2003). The PEN1 gene codes for the syntaxin SYP121 (PEN1), necessary for exocytosis. Syntaxins are SNARE proteins (Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) involved in vesicle traYcking, where they are located in the target membrane. Together with two other SNARE proteins, SNAP (soluble NSF attachment protein) and VAMP (vesicle-associated membrane protein), they form SNARE complexes that mediate fusion of vesicles to their target membranes. In Arabidopsis, the SNARE complex members corresponding to PEN1 have been identified as SNAP33 and VAMP721/VAMP722 (Kwon et al., 2008). When inoculated with Bgh, the pen1 mutant allows about 80% of the germinating Bgh spores to penetrate the epidermal cells (Fig. 1) (Assaad et al., 2004; Collins et al., 2003; Kwon et al., 2008; Zhang et al., 2007). Subsequently, Bgh is stopped by HR. This loss of penetration resistance can be explained by a delay of approximately 2 h in papilla formation around 11 h after inoculation, which also is the approximate time of penetration (Assaad et al., 2004; Kwon et al., 2008). Silencing of the VAMP721/VAMP722 proteins also results in increased Bgh penetration rates (Kwon et al., 2008).

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The role of SNAP in penetration resistance was documented in barley by silencing the SNAP33 ortholog, HvSNAP34 (Collins et al., 2003; Douchkov et al., 2005). The barley ortholog of PEN1, ROR2 (REQUIRED FOR mloSPECIFIED RESISTANCE 2), has been identified in a mutant screen for loss of mlo (mildew resistance locus O) resistance against Bgh (Collins et al., 2003; Freialdenhoven et al., 1996). In barley, Bgh cannot penetrate the host successfully in the absence of the MLO protein. However, the mlo ror2 double mutant has an intermediate penetration rate, demonstrating the importance of ROR2 in penetration resistance in this host–pathogen interaction. In the same way, a mutation in the PEN1 gene in Arabidopsis attenuates penetration resistance against the host powdery mildew fungus, Gc, in specific longitudinal epidermal cells along the leaf midrib (Zhang et al., 2007). The ROR2 gene is also required for nonhost penetration resistance to the wheat powdery mildew fungus (Trujillo et al., 2004). This illustrates that nonhost and host resistance have shared mechanisms in this type of PTI. The mlo resistance is the most commonly used powdery mildew resistance in barley production. Although employed for several decades, this broad-spectrum resistance against powdery mildew has not been overcome until now. A serious disadvantage linked to mlo resistance is an early leaf senescence and subsequent yield reduction (Jørgensen, 1992; Panstruga, 2005). It has attracted considerable attention that the barley MLO and ROR2 proteins, and the Arabidopsis PEN1 protein, all focally accumulate at the site of attempted penetration by the powdery mildew fungus (Assaad et al., 2004; Bhat et al., 2005; Kwon et al., 2008; Lipka et al., 2008). However, since mutation of PEN1 causes a delay in papilla formation at around 11 h after inoculation, and the earliest documented focal accumulation of PEN1 is 10–14 h after inoculation (Bhat et al., 2005), the accumulation is likely to occur too late to play a role in penetration resistance. We predict that the plasma membranelocated nondynamic PEN1 can be important for this PTI mechanism. More interesting, perhaps, is the physical interaction between MLO, which is a calmodulin-binding seven-transmembrane protein, and ROR2 syntaxin (Bhat et al., 2005). Even though these two proteins play counteracting roles in penetration resistance, their interaction is likely to hide the explanation for cellular functions of MLO and ROR2. It is noteworthy that Arabidopsis also has MLO genes that when mutated confer penetration resistance. However, due to gene redundancy in Arabidopsis, mlo single mutants do not show much diVerence in penetration resistance. Only the triple mutant, mlo2 mlo6 mlo12, has a strong powdery mildew resistance (Consonni et al., 2006). In addition to PEN1, two other genes (PEN2 and PEN3) have been identified to play a role in nonhost penetration resistance (Lipka et al., 2005;

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Stein et al., 2006). pen2 mutants allow a significantly higher Bgh penetration rate than wild type, but the attenuation of this PTI is less severe than in pen1. PEN2 is not only necessary for resistance against Bgh, but also against other biotrophic pathogens, as well as hemibiotrophs and necrotrophs (Lipka et al., 2005). PEN2 encodes a family 1 glycoside hydrolase and may catalyze the hydrolysis of O- or S-glycosidic bonds of metabolites. Penetration resistance is dependent on this PEN2 enzymatic activity (Lipka et al., 2005). GFP-labeled PEN2 has been shown to localize to peroxisomes, which accumulate at fungal entry sides. A peroxisome-derived product of PEN2 is predicted to have antifungal activity (Lipka et al., 2005). Analysis of a pen1 pen2 double mutant revealed higher fungal entry rates into host cells than in the two single mutants. Therefore, PEN1 and PEN2 appear to be involved in diVerent defense pathways, both contributing to penetration resistance (Lipka et al., 2005). The third gene required for penetration resistance, PEN3, encodes an ABCtransporter (ATP-binding cassette), also called PDR8 (PLEIOTROPIC DRUG RESISTANCE 8). The PEN3 protein is located at the plasma membrane, and like PEN1, accumulates at sites of fungal penetration (Stein et al., 2006). It has been suggested that PEN3 mediates the export of a toxic compound to stop fungal growth. This compound could be the product of PEN2, which is also located closely to sites of fungal attack. Surprisingly, pen3 mutants have higher resistance to the adapted powdery mildew fungus, Gc, and become chlorotic upon infection. This resistance can be explained by a higher level of SA in pen3 and is abolished by introduction of mutant alleles of SA-signaling genes (Stein et al., 2006). B. NONHOST RESISTANCE TO BACTERIA

Mutant studies have shown that the glycerol kinase NHO1 (NONHOST RESISTANCE 1) is required for Arabidopsis nonhost resistance to P. syringae pv. phaseolicola (Pph) (Kang et al., 2003). NHO1 expression is flagellininduced and can be suppressed by at least nine Pseudomonas eVectors and partially by the phytotoxin coronatine (Li et al., 2005). During ETI, induction of NHO1 gene expression is reestablished, leading to resistance. NHO1 overexpression diminishes bacterial growth, highlighting the importance for host bacteria to suppress NHO1 expression (Kang et al., 2003). As discussed below, ETI can play a role in nonhost resistance to bacteria. Therefore, not only PAMP-triggered, but also eVector-triggered expression of NHO1 is likely to be essential in nonhost resistance. As has been known for viruses, it is now proven that bacteria counteract host plant defense by suppressing gene silencing through interfering with

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small RNA pathways. This mechanism plays a role in both host and nonhost resistance to bacteria. Very recent data suggest that bacterial eVectors suppress plant gene silencing by interfering with the microRNA (miRNA) pathway. Most miRNAs act as negative regulators and downregulate gene expression by degradation of complementary mRNA molecules or by inhibition of their translation. A single miRNA can target several mRNAs, often coding for transcription factors (Novina and Sharp, 2004). The first evidence that small RNAs are involved in defense against bacteria was provided in a study where P. syringae, carrying the AvrRpt2 eVector, induced a small interfering RNA (siRNA) in plants carrying the cognate host R gene RPS2. This specific siRNA was able to silence a negative regulator of the RPS2-mediated defense response, thereby enhancing ETI (Katiyar-Agarwal et al., 2006). In a similar manner, the flagellin-derived peptide flg22 induces expression of miRNA393, which represses auxin signaling, consequently enhancing PTI. This also illustrates the role of auxin as a negative regulator of defense responses (Navarro et al., 2006). In another study, miRNA-deficient mutants failed to prevent growth of T3SS-defective virulent P. syringae pv. tomato (Pst), nonhost Pph and even nonpathogenic P. fluorescens and Escherichia coli (Navarro et al., 2008). It was demonstrated that the eVector AvrPtoB decreases miRNA precursor accumulation and that several other eVectors suppress transcriptional activation of a number of PAMP-responsive miRNAs. Finally, it has been observed that attack by several pathogens at once has synergistic eVects on infection. This could be demonstrated by co‐inoculation using either T3SS-defective Pst or Pph together with turnip mosaic virus (TuMV). TuMV induced suppression of siRNAs and miRNAs, thereby reducing nonhost resistance and allowing the nonhost Pph to grow (Navarro et al., 2008). The plant RNA-silencing pathways are dependent on specific members of the ARGONAUTE (AGO) protein family. Studies of the DNA methylation mutant ago4 confirm the involvement of RNA silencing in defense since this mutant has increased susceptibility to host bacteria and decreased resistance against nonhost P. syringae pv. tabaci (Agorio and Vera, 2007). In summary, small RNA-mediated gene silencing appears to play a role in both host and nonhost resistance.

V. WHAT IS THE EXPLANATION FOR NONHOST RESISTANCE? Since downstream defenses are similar in host and nonhost resistance, the discrimination between a host and a nonhost pathogen appears to occur at the stage of recognition or during early signaling. Host pathogen eVectors are

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characterized by their eVectiveness in suppressing host defense, while there are indications in the literature suggesting that nonhost pathogens are unable to deliver appropriate eVectors into the plant in order to suppress defense responses eVectively. Defense signaling is induced immediately upon pathogen recognition by the plant. For example, Arabidopsis expression of the essential defense component NHO1 is rapidly induced upon perception of flagellin (Li et al., 2005). In this case, host and nonhost pathogens can be distinguished by their ability to suppress NHO1 induction by eVector molecules. Unlike host Pst bacteria, nonhost Pph bacteria are incapable of suppressing NHO1 expression (Kang et al., 2003; Li et al., 2005). The importance of eVectors for the suppression of plant defense mechanisms was also demonstrated in another study. It was shown that Pph was able to proliferate moderately when co‐inoculated with Pst on Arabidopsis, since Pst suppresses Pph-activated defense responses (Ham et al., 2007). Furthermore, it was shown that eVectors AvrRpm1 and HopM1 from Pst are able to suppress plant defense, and that Arabidopsis expressing AvrRpm1 allows moderate growth of Pph (Ham et al., 2007). This clearly illustrates that an appropriate set of eVectors can turn a nonhost pathogen into a host pathogen. It is known that a given pathogen might use a few dozen eVectors to evade or suppress defense signaling and to promote virulence. This, together with the fact that heterologous expression of a single eVector from Pst did not result in maximum growth of the nonhost bacterium Pph, implies that adaption of one eVector alone is not suYcient to turn a nonhost pathogen into a host pathogen when the evolutionary distance between host and nonhost pathogen is large. On the other hand, defense-signaling-deficient Arabidopsis mutants, such as sid2 and pad4, are still resistant to Pph, as no elevated growth can be observed (Ham et al., 2007; Mishina and Zeier, 2007; Van Wees and Glazebrook, 2003). However, heterologous expression of single Pst eVectors in Pph enables enhanced proliferation of this nonhost bacterium on defense-signaling-deficient mutants of Arabidopsis. Moreover, Arabidopsis becomes susceptible to Pph, with proliferation rates comparable to those observed for Pst on wild-type plants, when several diVerent signaling pathways are disrupted simultaneously (Ham et al., 2007). In theory, the expression of several heterologous eVectors in a nonhost pathogen would lead to the same results as the disruption of multiple defense-signaling pathways. Pph obviously holds potent eVectors against target proteins in its host, bean, but its inability to suppress defense responses in Arabidopsis renders Pph a nonhost bacterium for this plant species (Ham et al., 2007). A similar situation is seen for powdery mildew fungi, where disruption of PTI increases the susceptibility of Arabidopsis to nonhost fungi (Lipka et al., 2005;

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Wiermer et al., 2005; Yun et al., 2003; Zimmerli et al., 2004). The fact that the host powdery mildew fungus, Gc, is able to penetrate at a very high rate, most likely reflects that it expresses eVectors that eYciently suppress PTI in the form of PEN gene-mediated defenses. There is evidence in the literature that some nonhost pathogens fail to infect a plant because their eVectors are recognized by the plant’s R proteins, which results in HR (Thordal-Christensen, 2003). For instance, three diVerent Avr gene products from Pst are recognized in the nonhost soybean (Kobayashi et al., 1989) and an Avr gene product from Pph is recognized in the nonhost pea (Arnold et al., 2001). In each case, the products activate HR only in nonhost plants with the corresponding R genes. A study in tomato documents the importance of the R genes Pto and Prf for resistance against 10 diVerent nonhost pathovars of P. syringae. It showed that pto and prf mutant plants allow moderate bacterial proliferation and display disease symptoms upon bacterial infiltration (Lin and Martin, 2007). There are also indications from plant–powdery mildew fungus interactions that ETI plays a role in nonhost resistance. This may, for instance, explain the formae speciales concept. It has been possible to cross wheat and rye powdery mildew isolates, which showed that the rye isolate has six Avr genes corresponding to known R genes in wheat (Matsumara and Tosa, 1995). Lipka et al. (2005) also showed that attenuation of ETI is important for resistance against nonhost powdery mildew fungi in Arabidopsis. Combining mutant alleles of PAD4 and SAG101 with a pen2 mutation reduces single cell HR and allows proliferation of barley and pea nonhost powdery mildew fungi to an extent not seen with the pen2 mutation alone. This stresses the additional importance of multiple gene-for-gene interactions that simultaneously contribute to nonhost resistance. Activation of R gene-mediated resistance in nonhosts probably occurs due to homology between eVector molecules in related pathogens (Lin and Martin, 2007; Lindeberg et al., 2006). For example, Arabidopsis recognizes an eVector protein homologous to AvrRpt2 from the nonhost pathogen Erwinia amylovora, which normally causes disease on rosaceous plants such as apple and pear (Zhao et al., 2006). Similarly, the soybean R gene product Rpg1-b recognizes AvrB from Pst, which is a nonhost pathogen on this plant (Ashfield et al., 2004). In the Pst host plant, Arabidopsis, AvrB is recognized by the corresponding R protein RPM1. The respective R genes are not related, meaning that AvrB recognition has developed convergently in the host and in the nonhost plant (Ashfield et al., 2004). Since nonhost resistance is durable and diYcult to overcome, most plants remain healthy. The outcome of whether a pathogen will be successful in colonizing a plant relies on the interplay between plant receptors for both

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PAMPs and eVectors, and the eVectors’ ability to suppress or evade defense responses and to avoid recognition. Essentially, nonhost resistance is the consequence of co‐evolutionary specialization of a given pathogen on its host plant. During their co‐evolution with host plants, pathogens have been challenged with one obstacle at the time, which has made it possible for them to develop an eVector to overcome this obstacle. In this way, an array of obstacles has accumulated in the host, all of which have been overcome by their pathogens. The nonhost pathogen, on the other hand, is hopelessly behind in the race with the nonhost plant, in that it has not developed the necessary eVectors in pace with the evolving defense mechanisms in the plant. Meanwhile, having to develop more eVectors simultaneously is an insurmountable task, which stabilizes the nonhost plant-pathogen situation. ETI is apparently a diVerent molecular level that contributes to nonhost resistance. EVectors in nonhost pathogens appear to maintain an ability to trigger ETI. This may also be explained by evolution. Some eVectors are likely to be maintained due to their role in targeting the host plant. However, in host plants, pathogens are forced to modify certain characteristics in the individual eVector in order not to cause R-protein recognition, and at the same time maintain suppression of PTI. Since there is no selection pressure against R-protein recognition in nonhost plants, resistance can remain intact. This could be the way AvrRpt2EA from Erwinia amylovora has maintained the same ability to trigger Arabidopsis ETI, mediated by the R-protein RPS2, as its Pst homolog AvrRpt2 (Zhao et al., 2006). It will be interesting to see in the future whether the ‘‘decoy model’’ plays a role in these aspects of nonhost resistance.

VI. LESION-MIMIC MUTANTS LMMs are plants that develop spontaneous lesions without pathogen infection, stress, or injury. The first LMMs were found in maize and were named ‘‘disease mimic mutants’’ (NeuVer and Calvert, 1975), which illustrates the link between LMMs with a genetic cause, and diseases caused by pathogens. Most LMMs have an elevated resistance to pathogens with a biotrophic lifestyle. They have elevated SA levels, and defense markers are upregulated (Lorrain et al., 2003). This makes LMMs an excellent tool for analysis of pathogen defense mechanisms. The lesion-mimic phenotype of LMMs is the consequence of defects in genes controlling programmed cell death (PCD). PCD is an active form of cell death, orchestrated by an internal cellular program. Introducing mutations from the SA pathway can rescue the lesion-mimic phenotype of

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many LMMs. Therefore, LMMs are used by several research groups to uncover novel aspects in defense signaling. Examples of prominent LMMs include lsd1 (Dietrich et al., 1997), acd2 (Mach et al., 2001), acd6 (Lu et al., 2003), acd11 (Brodersen et al., 2002), cpr22 (constitutive expresser of PR genes 22) (Yoshioka et al., 2006), ssi2 (suppressor of salicylic acid insensitivity 2) (Shah et al., 2001), and pen1 syp122 (Zhang et al., 2007). Also other mutants with constitutively activated defense pathways are studied, which do not necessarily lead to cell death. These include dnd1 (defense, no death 1) (Clough et al., 2000), dnd2 (Balague´ et al., 2003; Jurkowski et al., 2004), mpk4 (map kinase 4) (Petersen et al., 2000), cpr1 (Bowling et al., 1994), and snc1 (suppressor of npr1-1, constitutive 1) (Zhang et al., 2003).

VII. MUTANT SCREENS WITHOUT PATHOGENS FOR FINDING GENES IN DEFENSE SIGNALING Many defense-signaling genes have been found by screening mutated populations of wild-type plants following pathogen inoculation, and searching for less resistant mutants. However, by remutating LMMs and other mutants with constitutively active defense signaling, mutants with deficient signaling can be found simply by searching for individuals with improved performance. This eliminates the need for laborious pathogen inoculations. Below, three such strategies are described. Some of the mutations that rescue LMM plants have occurred in genes previously known to be involved in pathogen defense. Such mutations validate the method of the suppressor screen. Others have not previously been connected to pathogen defense, which demonstrates the value of this alternative approach in enriching our understanding of pathogen defense signaling. A. SSD MUTANTS

The closest homolog of the Arabidopsis PEN1, required for penetration resistance to the nonhost powdery mildew, Bgh, is SYP122. syp122 mutant plants show no phenotype regarding penetration resistance. However, the pen1 syp122 double mutant has an LMM phenotype. It is dwarfed and necrotic, and it has elevated SA and PR-1 transcript levels (Assaad et al., 2004; Zhang et al., 2007). The pen1 syp122 double mutant also has the reduced penetration phenotype against Bgh, like the pen1 single mutant, but it is resistant against the host powdery mildew, Gc. By introducing the known mutations in the SA pathway, sid2, eds5, and npr1, or the bacterial gene NahG, the LMM phenotype was partially rescued (Zhang et al., 2007).

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Mutations in the JA ( jar1 ( jasmonate resistant 1), coi1 (coronatine insensitive 1)) and ethylene (ein2 (ethylene insensitive 2)) pathways could not rescue the dwarfed and necrotic phenotype (Zhang et al., 2008). pen1 syp122 double-mutant plants were remutagenized, and in a screen for rescued phenotype, genes contributing to the lesion-mimic phenotype were identified. These genes were named SUPPRESSORS OF SYNTAXINRELATED DEATH (SSD) (Fig. 2). Complementation analysis of the obtained pen1 syp122 ssd triple mutants identified novel alleles of SID2 and EDS5. Furthermore, map-based cloning has revealed the identity of four other SSD genes. These turned out to be the already known defense-signaling genes FMO1, ALD1, PAD4 (Zhang et al., 2008), and PBS3 (S. M. Mørch, C. Pedersen, A. Lenk, Z. Zhang and H. Thordal-Christensen, unpublished data). In all of these genes, we have identified several novel mutant alleles. In addition to the six defense-signaling genes mentioned, mutant alleles of six others have been demonstrated to rescue pen1 syp122. Of these, EDS1, NDR1, RAR1, and SGT1b are known (Zhang et al., 2008), while two are not hitherto implicated in defense signaling (C. Pedersen, M. X. Andersson, Z. Zhang, A. Lenk and H. Thordal-Christensen, unpublished data). It is presumed that the SSD genes in their active form contribute to lesion formation. By crossing the rescued triple mutants, and combining mutations in diVerent SSD genes, it is possible to study the relationship of these SSD genes and to draw statements relating to the signaling network. In the pen1 syp122 background, quadruple and quintuple mutants were obtained. When a quadruple mutant performs significantly better than the parent triple mutants, it indicates that the mutations in SSD genes contribute additively to the rescuing of pen1 syp122. This, in turn, suggests that these SSD genes are on diVerent signaling pathways leading to the lesion-mimic phenotype.

Col-0

pen1 syp122

pen1 syp122 ssd1

Fig. 2. Suppression of the pen1 syp122 lesion-mimic mutant (LMM) phenotype by ssd1. Morphology of Arabidopsis Columbia (Col-0) wild type, syntaxin double mutant pen1 syp122, and rescued triple mutant pen1 syp122 ssd1 plants after 4 weeks of growth.

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On the other hand, when the quadruple mutant performs as the triple mutants, then the genes are on the same signaling pathway. By this signaling-network analysis, plant size permits statements about the relationship of proteins on defense-signaling pathways to be made. As an example, a cross was made between pen1 syp122 eds5 and pen1 syp122 sid2, and the quadruple mutant pen1 syp122 eds5 sid2 was generated. Remarkably, this quadruple mutant performed significantly better than the parental triple mutants, even though the eds5 and sid2 mutations both largely prevent SA accumulation. This observation demonstrates that EDS5 and SID2 are involved in diVerent SA-independent processes, illustrating that a more complex network exists in association with SA signaling (Zhang et al., 2008). npr1 mutants are hypersensitive to exogenously applied SA and show a bleaching phenotype (Cao et al., 1997). When spraying older plants with SA, stem and leaves turn white and the npr1 genotype can be determined by eye. Because of the high level of SA in the syntaxin double mutant, the triple mutant pen1 syp122 npr1 has a spontaneous bleaching phenotype. Additional knockout of, for example, sid2 causes the bleaching to disappear, while in the quadruple mutant with npr1 and ndr1 bleaching still occurs. This confirms that NDR1-mediated signaling is SA independent. Therefore, the npr1 bleaching phenotype allows statements about the involvement of SA in signaling processes and can be used as a simple and uncomplicated tool (Zhang et al., 2008). B. SFD MUTANTS

The npr1 mutant is SA insensitive and has reduced defense responses. However, remutagenesis of npr1 plants led to the identification of the mutation ssi2, which reestablishes the defense responses. The npr1 ssi2 double mutant and ssi2 single mutant are lesion-mimics due to a constitutive NPR1-independent defense pathway (Shah et al., 2001). SSI2 encodes a stearoyl-acyl carrier protein desaturase that catalyzes the conversion of stearic acid to oleic acid in plastids. ssi2 mutant plants possess altered fatty acid compositions (Kachroo et al., 2001). This shows that fatty acid desaturation is involved in defense responses. In a suppressor screen in the ssi2 npr1 genetic background, a number of sfd (suppressor of fatty acid desaturase deficiency) mutants were found that suppress the ssi2 LMM phenotype (Nandi et al., 2004). SFD1 is involved in the synthesis of plastidial glycerolipids and the sfd1 mutant is impaired in SAR, but its local defense responses are not significantly diVerent from wild type (Nandi et al., 2004). sfd4 is a mutant allele of the FAD6 gene that encodes an !6-desaturase that is involved in the synthesis of plastidial lipids containing polyunsaturated fatty acids (Nandi et al., 2003). The SFD2 gene is not yet cloned, but it confers

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altered fatty acid composition as in the other sfd mutants. All three sfd mutant alleles suppress the LMM phenotype and the increased resistance of ssi2 toward P. syringae pv. maculicola (Nandi et al., 2003). C. MOS MUTANTS

A mutation in the TIR-NB-LRR-type R gene SNC1 renders it constitutively active, and snc1 mutant plants are resistant to virulent pathogens (Zhang et al., 2003). In a suppressor screen of snc1, genes were identified with functions downstream of R genes without the use of pathogens (Zhang and Li, 2005). Mutations in seven MOS (MODIFIER OF snc1) genes have been found to rescue the snc1 phenotype. Three of these genes encode components of the nuclear traYcking machinery. MOS6 is an importin 3, necessary for the import of specific proteins across the nuclear envelope (Palma et al., 2005). MOS3 and MOS7 are nucleoporins and are involved in the transport of RNA, proteins, ribonucleoprotein particles, and other cargo across the nuclear envelope (Wiermer et al., 2007; Zhang and Li, 2005). At the same time, other discoveries connecting nuclear traYcking and plant innate immunity have been made. There are a few examples showing that R-proteins are imported into the nucleus. It was shown that the tobacco TIR-NB-LRRtype R-protein N functions inside the nucleus, and also the barley CC-NBLRR-type R-protein MLA10 shuttles between the cytoplasm and the nucleus, where it turns on defense (Burch-Smith et al., 2007; Shen et al., 2007; Wiermer et al., 2007). Whether the MOS proteins are required for transport of autoactivated snc1 or downstream components into the nucleus, still remains to be clarified. Other MOS genes include MOS2, that encodes a protein showing RNA-binding activity and acting inside the nucleus, and MOS4, which encodes a protein of a DNA-binding complex, probably controlling transcription of defense regulators (Palma et al., 2007; Zhang et al., 2005). This DNA-binding complex consists of two other compounds, AtCDC5 (a transcription factor) and PRL1 (PLEIOTROPIC REGULATORY LOCUS 1). Both are involved in pathogen resistance in Arabidopsis. An Atcdc5 mutation rescues the snc1 phenotype, and MOS4, AtCDC5, and PRL1 all are required for R-protein-mediated resistance of both CC- and TIR-NB-LRR-types (Palma et al., 2007). MOS5 and MOS8 are involved in protein ubiquitination and farnesylation, respectively (Goritschnig et al., 2007, 2008). Nearly all mos mutant alleles suppress the constitutive defense responses in snc1 completely and confer enhanced disease susceptibility toward oomycete and bacterial pathogens. mos6 only partially rescues the snc1 phenotype and confers moderately reduced resistance toward an oomycete pathogen, but no altered resistance to bacterial pathogens.

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VIII. CONCLUSION We have summarized current data relating to nonhost resistance and believe that the diVerence between host and nonhost resistance lies in the ability of eVectors to suppress activated defenses, and in the extent to which eVectors are recognized by R-proteins. Mutations identified in screens for nonhost resistance have turned out to expose downstream-signaling events. Defense needs to be under tight control, not only to protect healthy cells from damage, but also to conserve plant resources. A number of mutant lines exhibit autoactivated and permanent defense responses. Some of these are LMMs which suVer from uncontrolled defense, leading to unintended cell death. However, studying these plant systems can deepen our understanding of plant cell death, responses to pathogens, and defense mechanisms, without the need of pathogens. Some LMM mutant screens identify genes in specific processes, others in more general mechanisms. Depending on the starting material, outputs of screens have varied and resulted in, for example, novel genes involved in lipid saturation or transport across the nuclear envelope, which have now been linked to pathogen defense. Others, such as the SSD genes, are more generally involved in signaling pathways. By using LMM as a tool, additional genes in defense signaling will reveal new insights into the large field of pathogen–plant interactions.

ACKNOWLEDGMENTS We are grateful to Drs. Dale Godfrey and Ingo Lenk for critically reading the manuscript. Andrea Lenk was supported by a grant from the Faculty of Life Sciences, University of Copenhagen.

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Action at a Distance: Long-Distance Signals in Induced Resistance

MARC J. CHAMPIGNY* AND ROBIN K. CAMERON{,1

*Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6 { Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Time to Flower—Signaling Events in the Vegetative to Flowering Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Flowering Time as a Model for Long-Distance Signaling ............. B. Control of Flowering Occurs in Distinct Stages ......................... C. The Long-Distance Flowering Signal is Phloem Mobile and Highly Conserved ....................................................... D. Candidates for the Floral Long-Distance Signal—The Identity of ‘‘Florigen’’ ..................................................................... E. Salicylic Acid and Flowering—Convergence of Signaling Mechanisms?................................................... III. Mechanisms of Signaling During the Wound Response . . . . . . . . . . . . . . . . . . A. Role of Systemin in Systemic Wound Signaling ......................... B. Wound-Response Mutants are Deficient in the Biosynthesis or Perception of JA, or in Systemin Functioning........................ C. Systemin and JA Production in Wounded Leaves and JA Perception in Distant Tissue.............................................................. D. JA Biosynthesis Occurs in the Sieve Element/Companion Cell Complex ..................................................................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

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E. JA-Mediated Wound Response is Modulated by Other Signals ...... F. Mechanism of JA Action on Effector Genes............................. Long-Distance Signaling in SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SAR Develops in Distinct Stages .......................................... B. Role of SA and NPR1 in SAR ............................................. C. SAR Signal Transport ....................................................... D. Candidates for the SAR Long-Distance Signal .......................... E. Other Genes Involved in SAR Long-Distance Signaling............... F. Role of ET in SAR Long-Distance Signaling ............................ G. SAR Long-Distance Signaling Across Species ........................... Systemic Induced Susceptibility (SIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling During ISR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Induction of ISR.............................................................. B. Signal Perception and Priming During the Development of ISR ..... Techniques to Further Elucidate Long-Distance Signaling. . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Plants perceive environmental changes in one tissue and inform distant tissues of these changes using long-distance signals. These signals are implicated in developmental processes such as the transition to flowering as well as in responses to abiotic and biotic stresses, including pathogen infection and herbivory. In this review, we discuss research findings in the regulation of flowering time as well as in induced defense responses from the perspective that long-distance signaling progresses in a series of distinct stages: (1) initiation or induction, (2) synthesis and movement of a signal to distant tissues, (3) perception of the signal, and (4) establishment or manifestation of the appropriate response. We highlight recent studies that implicate DIR1 and lipids, methyl salicylate, and jasmonic acid (JA) as long-distance signals during systemic acquired resistance (SAR). Additionally, it appears that a requirement for JA is common to the SAR, induced systemic resistance, and wound response pathways. Finally, we discuss future avenues of research to further elucidate the mechanisms of long-distance signaling in plants.

I. INTRODUCTION Plants are rooted to the earth and therefore cannot escape from adverse environmental conditions. Instead, they have evolved sophisticated mechanisms that allow them to survive both biotic and abiotic environmental stress. Plants respond locally at the individual cell level and systemically in distant tissues. The environmental condition is perceived in one part of the plant followed by transport of this information to distant tissues to alert the entire plant. Long-distance signaling pathways are often divided into a number of stages. The first stage has been termed induction or initiation in response to the stress in the initially affected tissue and production of (a) long-distance signal(s). Secondly, the signal(s) move(s) from the induced or initial tissue to distant tissues via the vascular system, air, or cell-to-cell movement.

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Thirdly, the distant tissues must perceive or interpret the newly arrived longdistance signal(s) and set in motion the appropriate response. A number of plant long-distance signaling pathways are being studied, including perception of nutrient limitation (reviewed in Forde, 2002) and drought stress in the roots followed by signaling to the shoots to produce the appropriate response (reviewed in Schachtman and Goodger, 2008). In this review, the most thoroughly studied long-distance signaling pathway, the photoperiodic flowering pathway will be discussed in relation to long-distance signaling in four defense response pathways (wound response, induced systemic resistance (ISR), systemic acquired resistance (SAR), systemic induced susceptibility (SIS)).

II. TIME TO FLOWER—SIGNALING EVENTS IN THE VEGETATIVE TO FLOWERING TRANSITION A. FLOWERING TIME AS A MODEL FOR LONG-DISTANCE SIGNALING

The transition between vegetative and reproductive growth is a critical developmental switch that is tightly regulated in plants to ensure that production and dispersal of seeds occur in environmental conditions most favorable for reproductive success. Different plant species utilize a variety of autonomous and environmental cues to trigger this transition to flowering, including the developmental stage of the plant, amount of rainfall, temperature, and photoperiod—the relative duration of light in a single day (Hastings and Follett, 2001). The contribution of photoperiod to flowering time was first reported early in the twentieth century with the observation that flowering in spinach occurred when the leaves were in long-day (LD) conditions and the shoot apex in short-day (SD) conditions, but not vice versa (Knott, 1934). Based on grafting experiments, Chailakhyan (1936) proposed the existence of ‘‘florigen,’’ a photoperiod-dependent, graft-transmissible signal that travels from leaves to the shoot apex to promote flower development. More recent work has enriched the simple florigen concept to include multiple mobile signals that may be involved in inhibiting flowering as well as promoting floral initiation (Bernier et al., 1993; Pe´rilleux and Bernier, 2002). The mechanism of florigen translocation and investigation into its identity have become valuable research models to understand long-distance signaling in plants. B. CONTROL OF FLOWERING OCCURS IN DISTINCT STAGES

Control of flowering time has historically been divided into a series of discrete steps: (i) induction of a floral signal within the leaves, (ii) commitment to flowering at the shoot apical meristem (SAM), and (iii) reprogramming of

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the meristem for floral morphogenesis (Pe´rilleux and Bernier, 2002; Sua´rezLo´pez, 2005). The broad mechanisms involved in the control of flowering time clearly have much in common with the induced defense responses, which progress in a similar series of steps that we and others have highlighted— induction of a signal, translocation of the signal to distant tissues, and perception of the signal, culminating in manifestation of the defense response. The primary, or induction, stage in the transition to flowering occurs in leaves. Multiple experiments have demonstrated that applying the inductive photoperiod only to leaves can induce flowering (Hempel et al., 2000; Zeevaart, 1976). Most tellingly, grafting of a single induced leaf was sufficient to elicit flowering in Perilla (Zeevaart, 1985). In many species, sensing of photoperiod can occur in immature leaves. Defoliation experiments established that the floral signal was produced in cotyledons of Impatiens balsamina (Pouteau et al., 1997) and Chenopodium rubrum (King, 1972). In addition, Arabidopsis thaliana grown in LD conditions flowers before any rosette leaf reaches maturity (Bradley et al., 1997). There exists great species variability in the observed capacity of the SAM to commit to flowering. Lolium and Xanthium were induced to flower after exposure to a single inductive photoperiod (Zeevaart, 1976), but other species such as soybean and some cultivars of Impatiens reverse the flowering process if inductive conditions cease (Pouteau et al., 1997; Washburn and Thomas, 2000). In most cases, it has not yet been determined whether the observed variation in commitment to flowering is due to species differences in the dosage of the flowering signal, or a change in the ability of the SAM to perceive the signal. In one case, I. balsamina becomes irreversibly committed to flowering because its leaves constitutively produce the flowering signal after induction (Tooke et al., 1998). C. THE LONG-DISTANCE FLOWERING SIGNAL IS PHLOEM MOBILE AND HIGHLY CONSERVED

The physiological experiments described above clearly demonstrated that the flowering signal is graft-transmissible, suggesting that it travels through the vasculature. Further studies established that the signal is phloem mobile because the speed of signal movement out of induced leaves, as well as the pattern of signal movement, was similar to the transport of photoassimilates (Zeevaart, 1976). Clever intercultivar and interspecies grafting experiments led to the suggestion that flowering signals must be very similar or identical in higher plants (Zeevaart, 1976). For example, grafting of day-neutral tobacco scions onto rootstocks of SD tobacco or rootstocks of the LD Nicotiana sylvestris caused acceleration of flowering when the rootstocks were exposed

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to the appropriate inductive conditions (Lang et al., 1977). The same signal(s) produced in response to an inductive photoperiod may participate both in the transition to flowering at the shoot apex and in the transition to tuberization (Sua´rez-Lo´pez, 2005), as grafted tomato scions promoted tuberization of potato rootstocks only when the scions were induced to flower (Chailakhyan et al., 1981). D. CANDIDATES FOR THE FLORAL LONG-DISTANCE SIGNAL—THE IDENTITY OF ‘‘FLORIGEN’’

Two contrasting approaches have been employed to uncover the molecular nature of the flowering signal. The first approach involved the physiological study of compounds transmitted to the shoot apex in response to changing photoperiod. The second approach, largely carried out in the genetic model Arabidopsis, was to identify and characterize mutants that showed either accelerated or delayed flowering time in response to various environmental stimuli. 1. Sucrose, cytokinins, and gibberellins Several different plant hormones and small molecules have been proposed as candidates for the floral stimulus, largely on the basis of their ability to promote flowering at the SAM or to complement the deficiencies of various flowering mutants. Among cytokinins, gibberellins, and sucrose, there is widespread agreement that gibberellins promote flowering in several different species (Pe´rilleux and Bernier, 2002). Although cytokinins accumulated at the shoot apex of Arabidopsis plants induced to flower (Jacqmard et al., 2002), exogenous application of cytokinins did not induce flowering in Arabidopsis or in Sinapis alba (Bonhomme et al., 2000). Sucrose was also found to accumulate in Arabidopsis phloem exudates collected from photoperiod-induced leaves (Bernier et al., 1993; Corbesier et al., 1998) and this transient increase in phloem sucrose concentration preceded cell division at the SAM (Corbesier et al., 1998). However, high levels of sucrose delayed flowering in wild-type Arabidopsis and rescued the deficiency of several important late-flowering mutants but not others (Ohto et al., 2001), making the precise role of sucrose in flowering promotion difficult to define. The role of gibberellins as flowering signals has been best studied in the LD plant Lolium temulentum, in which gibberellins are synthesized in the leaves and large increases in gibberellin levels are observed at the shoot apex, suggesting the long-distance movement of gibberellins to promote flowering (King and Evans, 2003; King et al., 2006). Gibberellins are also involved in the transition to flowering that eventually occurs under SD conditions in the

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quantitative LD plant Arabidopsis (Eriksson et al., 2006). Gibberellins are not, however, a universal floral stimulus, as their activity was not required in several species studied such as S. alba (Corbesier et al., 2004), and can inhibit flowering in others (Bernier, 1988; Zeevaart, 1976).

2. Characterization of genes involved in the regulation of flowering time Much of our knowledge concerning the genetic control of flowering has come from analysis of Arabidopsis mutants exhibiting premature or delayed flowering in response to environmental stimuli such as vernalization and photoperiod. To date, the photoperiod-dependent pathway is the best understood. For interesting discussions of genes involved in other pathways and crosstalk between pathways, refer to several recent reviews (Corbesier and Coupland, 2006; Imaizumi and Kay, 2006; Zeevaart, 2008). Photoperiod is perceived in leaves and several genes have been identified that regulate the transition to flowering in terms of their ability to discriminate light quality or intensity. GIGANTEA (GI) encodes a nuclear protein of unknown biochemical function (Fowler et al., 1999; Park et al., 1999), but overexpression of GI promoted early flowering (Mizoguchi et al., 2005). GI regulates flowering time in part by regulating the expression of a zincfinger transcription factor called CONSTANS (CO) (Sua´rez-Lo´pez et al., 2001). Transcription of GI and CO is regulated by the circadian clock: mRNA abundance of each gene is highest at approximately 12 h after dawn when plants are grown in LD conditions (16 h light) (Park et al., 1999; Sua´rez-Lo´pez et al., 2001). In addition, CO is controlled at the posttranslational level, such that the protein is rapidly degraded by proteasome-targeted ubiquitination, but stabilized through a mechanism involving signaling through phytochrome A and cryptochrome photoreceptors (Valverde et al., 2004). These complex regulatory steps result in CO protein accumulation only during long days and its degradation in the absence of light, pointing toward a mechanism through which CO promotes flowering only in long days. CO and an additional important floral regulator, FLOWERING LOCUS T (FT), are expressed in phloem companion cells of leaves (An et al., 2004; Corbesier and Coupland, 2006; and see below). FT encodes a protein similar to animal RAF kinase inhibitors (Kobayashi et al., 1999). FT is thought to be the major target of CO in leaves because expression of only this gene was highly upregulated in wild-type plants compared to co mutants grown in long days (Wigge et al., 2005). Moreover, CO-dependent regulation of flowering time required FT because expression of FT by companion cell-specific promoters rescued the late-flowering co phenotype (An et al., 2004).

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A fascinating spatial difference between the site of CO and FT transcription and the site of FT action was observed in studies expressing these genes under tissue-specific promoters. Expression of CO in companion cells of leaf minor veins using the minor vein-specific GAS1 promoter (Ayre and Turgeon, 2004), or in major veins using the phloem-specific SUC2 promoter (An et al., 2004) complemented the co-1 mutation, but CO expression at the SAM had no effect on flowering. Conversely, FT induced flowering when expressed in epidermal cells, companion cells, or in meristematic tissue (An et al., 2004). These results collectively suggested that the physiological role of CO is to activate transcription of FT, whose activity is required at the shoot apex. FT interacts with the bZIP transcription factor FLOWERING LOCUS D (FD) to activate transcription of the floral meristem-identity gene APETALA1 (AP1) and of SUPPRESSION OF OVEREXPRESSION OF CO1 (SOC1), thereby reprogramming the shoot apex for floral initiation (Abe et al., 2005; Wigge et al., 2005). 3. FT protein is phloem-mobile Having established that phloem-specific expression of CO induced expression of FT (Takada and Goto, 2003) and that FT acts at the shoot apex to promote flowering (Wigge et al., 2005), many studies have been conducted to understand the spatial discrepancy between the site of FT synthesis and the site of FT action. An early report suggested that production of FT mRNA in transgenic Arabidopsis leaves by activation of a heat-shock promoter led to detection of FT mRNA at the shoot apex (Huang et al., 2005), implicating the mRNA as the long-distance signal. This was a timely hypothesis, as several mRNAs, small RNA molecules and viruses have been reported to travel long distances in the phloem (Lough and Lucas, 2006). However, detection of FT mRNA in the phloem could not be reproduced and the original report was retracted (Bo¨hlenius et al., 2007a,b). Additional grafting experiments failed to establish that rootstock-produced FT mRNA molecules crossed into the scion and message encoding a translational fusion of FT with a green fluorescent protein (GFP) tag was not detected either in grafted Arabidopsis ft-7 mutant shoots (Corbesier et al., 2007). Additionally, messages encoding SINGLE FLOWER TRUSS (SFT), the tomato orthologue of FT, did not cross a graft junction (Lifschitz et al., 2006). The presence of FLOWERING LOCUS T-LIKE mRNA was not detected by RT-PCR in phloem sap collected from Cucurbita moschata (Lin et al., 2007). Finally, in an elegant complementary experiment, tissue-specific reduction of FT using artificial microRNAs established that FT mRNA is dispensable at the SAM (Mathieu et al., 2007). These data firmly established that FT mRNA was not the phloem-mobile floral stimulus.

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Additional studies strongly supported a model in which FT protein translocates from its site of synthesis in leaf phloem cells to the SAM. For example, experiments in which a FT:GFP fusion protein was expressed in Arabidopsis companion cells under the control of the SUC2 promoter revealed that the GFP signal could be detected both in the phloem and at the shoot apex (Corbesier et al., 2007). Similar results were obtained using Myc–FT fusions in Arabidopsis (Jaeger and Wigge, 2007) and GFP fusions with a rice FT orthologue (Hd3a-GFP) (Tamaki et al., 2007). One could argue that these results are not physiologically relevant for several reasons. First, FT is a low abundance protein and exogenous expression of FT using phloem-specific promoters may not reflect its natural movement. Second, diffusion of small proteins from companion cells into sieve elements is widespread (Lough and Lucas, 2006) and may explain the observed results. These objections were partially resolved by the observation that the GFP fusions mentioned above were biologically active; that is, phloem-specific expression of FT–GFP fusions in leaves promoted early flowering. Furthermore, native FLOWERING LOCUS T, a cucurbit orthologue of FT, could be identified by mass spectrometry in phloem sap collected from Cu. moschata (Lin et al., 2007). Elegant experiments were performed to demonstrate that the physical movement of FT protein from leaves to the shoot apex is a necessary and sufficient condition for flowering. In these studies, FT or an FT–GFP fusion protein was expressed via the minor vein-specific GAS1 promoter and the ability of these constructs to rescue the late-flowering phenotype in ft mutant grafted scions was assessed. Surprisingly, only FT on its own resulted in early flowering, suggesting that the FT–GFP protein was too large to move into minor veins. However, the fact that FT could rescue the ft late-flowering phenotype in these experiments meant that FT traveled a considerable distance through a graft junction (Corbesier et al., 2007). Similar experiments employed an FT protein fused to tandem yellow fluorescent protein (YFP) molecules under the direction of the phloem-specific SUC2 promoter. In this case, FT:3xYFP did not affect flowering time, presumably because it was too large to translocate into sieve elements, but release of FT from the fusion protein using a viral endopeptidase initiated early flowering (Mathieu et al., 2007). 4. FT, a near universal flowering signal: ‘‘florigen’’ revealed In addition to its ability to move in the phloem from induced leaves to the shoot apex and to reprogram the SAM, FT fulfills another key requirement of the florigen hypothesis, namely that it acts as a near universal floral stimulus. In many species, notably Arabidopsis and monocots, grafting

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experiments are technically challenging or physically impossible, respectively. Therefore, recent studies have focused on the ability of overexpressed FT transgenes to promote flowering in many different species. To date, overexpression of Arabidopsis FT or various orthologues promoted accelerated flowering in poplar (Bo¨hlenius et al., 2007a; Hsu et al., 2006), tobacco (Lifschitz et al., 2006), winter wheat (Yan et al., 2006), Arabidopsis (Abe et al., 2005), and Pharbitis nil (Hayama et al., 2007). In similar experiments, FT orthologues from Cucurbita maxima (Lin et al., 2007), rice (Kojima et al., 2002), grapevine (Carmona et al., 2007; Sreekantan and Thomas, 2006), and tomato (Lifschitz et al., 2006) induced early flowering when overexpressed in Arabidopsis. The role of FT as a universal floral stimulus is further supported by reports demonstrating delayed flowering time in distantly related rice and Arabidopsis plants in which the FT orthologue was disrupted by mutation or knock-down methods (Kojima et al., 2002; Komiya et al., 2008; Yamaguchi et al., 2005). Unlike the so-called autonomous and vernalization pathways regulating flowering time (reviewed in Corbesier and Coupland, 2006), the gibberellin pathway does not seem to converge on FT as a floral stimulus. Recent work in Arabidopsis demonstrated that application of gibberellin to either leaves or the apical region of plants grown in SD conditions caused flowering by upregulating expression of LEAFY and SOC1 (Eriksson et al., 2006; Moon et al., 2003), but expression of FT was unaffected. Although LEAFY is highly conserved in plants, exogenously applied gibberellin seems to induce flowering only in the subset of SD plants (Maizel et al., 2005). Further research is required to explain this phenomenon and to explore the redundancy of gibberellin and FT as leaf-produced, phloem mobile floral signals (Zeevaart, 2008). E. SALICYLIC ACID AND FLOWERING—CONVERGENCE OF SIGNALING MECHANISMS?

Some plant species, such as duckweeds, flower in response to treatment with the pathogen defense hormone salicylic acid (SA) (Zeevaart, 1976). Fortuitously, Arabidopsis is an excellent model to study the role of SA in regulating the control of flowering time because flowering is accelerated upon exogenous application of SA and mutants deficient in accumulation of the hormone exhibit delayed flowering (Martinez et al., 2004). SA was reported to affect flowering time by negatively regulating the floral repressor FLC and by stimulating expression of the floral long-distance signal, FT (Martinez et al., 2004). A recent study has shown a convergence for the requirement of the small ubiquitin-like modifier (SUMO) E3 ligating protein AtSIZ1 in regulating

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plant innate immunity as well as flowering time. Arabidopsis siz1 mutants have a pleiotropic phenotype including low tolerance to drought stress (Catala et al., 2007), heightened innate resistance to bacterial pathogens (Lee et al., 2007a), and accelerated flowering (Jin et al., 2008). The accelerated flowering phenotype in siz1 mutants was shown to be SA-dependent as demonstrated by the absence of early flowering in SA-deficient and siz1 double mutants. SIZ1 was proposed to delay flowering by facilitating the sumoylation of FLD, resulting in increased transcription of the floral repressor FLC (Jin et al., 2008). Further research will identify the specific roles of sumoylation in regulating SA-dependent pathogen defense and flowering pathways. In the meantime, these intriguing findings hint at a relationship between the regulation of flowering time and defense against pathogens. This may reflect conservation in long-distance-signaling mechanisms shared between the two processes, but may also simply be an example of SA-mediated cross-talk during the induction stages of flowering and pathogen defense.

III. MECHANISMS OF SIGNALING DURING THE WOUND RESPONSE Plants combat wounding by herbivorous insects and foraging animals with an array of chemical defense strategies. Included among these are a variety of noninducible defenses such as the synthesis of bitter or unpalatable secondary metabolites, including the tannins and saponins found in potato, as well as more toxic compounds, for instance nicotine in tobacco and pyrethrum produced by members of the genus Chrysanthemum. Wounding of several agriculturally important plant species such as corn and cotton results in the induction and emission of volatile substances that attract insect predators. Volicitin, present in oral secretions of feeding beet armyworm caterpillars was found to induce a variety of complex secondary metabolites, including monoterpenes, sesquiterpenes, homoterpenes, lipoxygenase products, and indole (Alborn et al., 1997). Local and systemic production of these volatiles caused increased predation of caterpillars by the parasitic wasp Hyposoter exiguae (Ro¨se et al., 1996; Turlings and Tumlinson, 1992). Members of the Solanaceae exhibit an additional important inducible defense against herbivory. Wounded potato and tomato plants express proteinase inhibitors (PIs or PINs) that limit the ability of foraging insects to extract nutrients by reducing the activity of digestive enzymes in the insect midgut (Green and Ryan, 1972).

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Inducible responses against wounding share the notable feature of being expressed not only in local tissue but also in tissue distant from the site of attack, implicating mechanisms of long-distance communication that may be shared between various species’ wound responses. The discovery of robust PIN expression by wounded tomato plants (Green and Ryan, 1972) has made the tomato leaf a valuable model for the study of signaling events leading to the wound response. Wounding causes the systemic reprogramming of tomato plant tissue resulting in expression of over 20 defense-related proteins, including several classes of PINs, proteinases, and signaling molecules, as well as several mitogen-activated protein kinases (MAPKs) (Ryan, 2000; Stratmann and Ryan, 1997).

A. ROLE OF SYSTEMIN IN SYSTEMIC WOUND SIGNALING

An 18-amino acid peptide hormone, systemin, was purified from wounded tomato leaves and discovered to be a potent inducer of PIN formation (Pearce et al., 1991). This, and the observation that jasmonic acid (JA) biosynthesis via the octadecanoid pathway is required for PIN expression (Farmer and Ryan, 1992) established JA and systemin as key players in the wound response. What roles do these hormones play in the wound response and how do they interact to establish systemic protection against herbivory? Systemin is produced as a specific response to insect feeding or mechanical wounding by the proteolytic cleavage of a 200-amino acid precursor called prosystemin (McGurl et al., 1992). Additional prosystemin-like proteins have been identified in tomato (Pearce and Ryan, 2003) and tobacco (Pearce et al., 2001) and all are cleaved into small peptides capable of inducing PIN expression. Transformed tomato plants expressing an antisense prosystemin cDNA under the direction of the cauliflower mosaic virus 35S promoter were deficient in systemic induction of PIN proteins (McGurl et al., 1992) and were consumed to a greater extent by Manduca sexta larvae (Orozco-Cardenas et al., 1993). Conversely, plants overexpressing prosystemin in its sense orientation demonstrated constitutive production of defenserelated proteins even in the absence of wounding, presumably from ectopic overproduction of the systemin hormone (McGurl et al., 1994). Purified systemin or bacterially expressed recombinant prosystemin applied to freshly wounded tomato stems rescued the deficiency of prosystemin-antisense plants in that the wound response approached wild-type levels in distant tissues (Dombrowski et al., 1999). This discovery, along with the observation that radioactively labeled systemin applied to wounded tomato leaves translocated from the site of application to the leaf petiole, suggested that

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systemin itself may be a crucial long-distance signal to establish the systemic wound response (Narva´ez-Va´squez et al., 2005; Pearce et al., 1991). Biochemical purification of a transmembrane systemin receptor SR160, a leucine-rich-repeat (LRR) receptor kinase (Scheer and Ryan, 2002), provided an important clue as to the requirement for systemin in the wound response. Interestingly, SR160 plays dual roles in the perception of both brassinosteroid and systemin hormones (Montoya et al., 2002; Scheer et al., 2003). Interaction of systemin with its receptor results in a suite of events including depolarization of the plasma membrane (Moyen and Johannes, 1996) and an increase in intracellular Ca2þ (Moyen et al., 1998), as well as activation of MAPKs (Kandoth et al., 2007; Stratmann and Ryan, 1997; Usami et al., 1995) and a phospholipase A2 (Lee et al., 1997; Narva´ezVa´squez et al., 1999). The culmination of these biological processes is the release of linolenic acid from membranes and its biochemical conversion via the octadecanoid pathway to the oxylipins 12-oxyphytodienoic acid (OPDA) and JA (Farmer and Ryan, 1992; Schilmiller and Howe, 2005). B. WOUND-RESPONSE MUTANTS ARE DEFICIENT IN THE BIOSYNTHESIS OR PERCEPTION OF JA, OR IN SYSTEMIN FUNCTIONING

To identify additional components of the wound signaling pathway, a number of forward genetic screens were undertaken, searching specifically for mutants that failed to express PINs in response to wounding (Lightner et al., 1993) or failed to respond to the constitutive signal provided by a 35S: PROSYSTEMIN transgene (Howe and Ryan, 1999). Phenotypic analysis of these mutants and cloning of some of their deficient alleles revealed that wound response mutants are defective in distinct classes of biological functions, chiefly the biosynthesis of JA (defenseless, def1, Li et al., 2002a; acylCoA oxidase, acx1, Li et al., 2005; allene oxide cyclase, aoc, Stenzel et al., 2003; !3-fatty acid desaturase, spr2, Howe and Ryan, 1999), perception of JA, (JA insensitive, jai1, Li et al., 2004), and systemin-specific functions (suppressed in prosystemin-mediated responses, spr1, Lee and Howe, 2003). JA is a plant hormone that regulates many processes during development and in response to a broad array of mainly necrotizing pathogens and herbivorous insects. The importance of JA in the wound response was clearly illustrated by the observations that JA-biosynthetic mutants were highly impaired in expression of defense-related genes in response to wounding and in their ability to combat herbivory (Howe et al., 1996; Stenzel et al., 2003). For detailed discussions of the diverse roles of jasmonates in plants, the reader is referred to several excellent recent reviews (Balbi and Devoto, 2008; Wasternack, 2007).

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C. SYSTEMIN AND JA PRODUCTION IN WOUNDED LEAVES AND JA PERCEPTION IN DISTANT TISSUE

Elegant grafting experiments were performed to shed light on the mechanism of long-distance signaling during the wound response, in particular how systemin and JA interact and in which tissues each activity is required. Two contrasting models were postulated. The first model assumes that systemin is in fact a crucial component of the long-distance wound signal. In this case, systemin produced at the site of insect attack is translocated through the vasculature and stimulates the expression of jasmonates and defense genes in distant tissues. The contrasting model assumes that systemin signaling is only required in locally wounded tissue, such that locally synthesized JA or another octadecanoid is transported to distant tissues to promote wound resistance. In either scenario, a functional JA-perception mechanism must be in place in systemic tissue and indeed, JA-insensitive jai mutant scions were unable to respond to graft-transmissible signals from rootstocks (Li et al., 2002b). Grafting experiments involving a suite of tomato JA-biosynthetic mutants established that production of JA was required in the wounded rootstock but not in the grafted scion, indicating that de novo synthesis of JA was dispensable in perceiving and responding to a systemic wound signal (Li et al., 2002b). Taken together, these results demonstrated that a member of the octadecanoid pathway is an essential component of the long-distance wound signal. Grafting experiments utilizing the OPDA-forming but JA-deficient mutant acx1 as rootstock were similarly deficient in the generation of the systemic wound signal, strongly implicating JA itself or one of its conjugates as the signal (Li et al., 2005). Similar experiments utilizing spr1, a mutant deficient in systemin function, established that SPR1 is required for generation or transmission of the longdistance signal, but not for its perception (Lee and Howe, 2003). Although systemin was detected in phloem, these results established that it is not a component of the mobile signal because its activity was not required in distant tissues. However, spr1 demonstrated a novel phenotype in that the wound response of undamaged leaves was severely affected but the response of attacked leaves was nearly normal, suggesting that signaling events leading to wound resistance in local and systemic tissue operate differently (Lee and Howe, 2003). In addition, expression of several defense-related genes was maintained in the spr1 mutant, pointing toward the existence of redundant wound signaling pathways. JA is modified in planta into a number of bioactive conjugates including methyl-JA (MeJA) and JA-isoleucine (JA-Ile). An Arabidopsis mutant defective in conjugating JA to isoleucine, jar1 (Staswick and Tiryaki, 2004), was

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compromised in its response both to necrotrophic fungi (Staswick et al., 1998) and to feeding insects (Kang et al., 2006), suggesting that this conjugation is important in manifesting the wound response. There is currently no evidence that JA-Ile is transported in the vasculature. Therefore, it is not a compelling candidate for the long-distance wound signal. However, isotopically labeled MeJA was detected both in xylem and in phloem upon application to tobacco leaves (Thorpe et al., 2007). Tantalizing recent results suggest that exogenously applied MeJA is converted to JA and JA-Ile within plant leaves (Tamogami et al., 2008). Some reports suggest that damaged leaves emit volatiles that alter the resistance of neighboring plants (Heil and Silva Bueno, 2007; Karban et al., 2006; Kessler and Baldwin, 2001). Highly volatile MeJA could conceivably be an interplant signal for heightened wound response. For example, tomato leaves accumulated PIN proteins when incubated in the same growth chamber with sagebrush, Artemisia tridentata, a plant that contains MeJA in leaf surface structures (Farmer and Ryan, 1990). However, contrary evidence has been reported in that exogenously applied MeJA failed to induce PIN expression or induce protection to M. sexta in wild tobacco, Nicotiana attenuata (Preston et al., 2004). The role of atmospheric MeJA in regulating various biotic and abiotic plant stresses is discussed in more detail elsewhere (Cheong and Choi, 2003; Heil and Ton, 2008).

D. JA BIOSYNTHESIS OCCURS IN THE SIEVE ELEMENT/COMPANION CELL COMPLEX

Studies on the tissue-specific localization of prosystemin function and the JAbiosynthetic complex have shed light on the transport mechanism of the longdistance wound signal. Transgenic tomato expressing PROSYSTEMIN: -GLUCURONIDASE (GUS) reporter constructs originally revealed that prosystemin was particularly enriched in vascular bundles within the center vein of a leaf (Jacinto et al., 1997). Further analysis with more sensitive methods showed prosystemin mRNA and protein accumulation solely within phloem parenchyma cells (Narva´ez-Va´squez and Ryan, 2004). Complementary research using specific antibodies directed against enzymes of the JA-biosynthetic pathway established that 13-lipoxygenase, allene oxide synthase, and allene oxide cyclase were all localized to phloem companion cells and sieve elements (Hause et al., 2003). These observations also describe an amplification loop for JA accumulation within the vasculature, on the basis of the observation that prosystemin expression itself is induced by JA accumulation (Stenzel et al., 2003).

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These results suggest a model in which systemin is processed from its prosystemin precursor in phloem parenchyma cells, followed by its binding to the SR160 receptor on companion cell plasma membranes to initiate production of JA via plastidial and peroxisomal enzymes of the octadecanoid pathway (Schilmiller and Howe, 2005). JA itself or a JA-conjugate moves into sieve elements via plasmodesmatal connections for long-distance transport. Although compelling, there are several important gaps in our knowledge that will be filled only after further research: (1) Enzymes involved in the proteolytic processing of prosystemin have not been described, nor do we understand how systemin might travel to companion cells, although the recent discovery that prosystemin is embedded within the plant cell wall (Narva´ez-Va´squez et al., 2005) suggests that it or systemin may reach companion cells through the apoplast. (2) A receptor for JA has only recently been described (see below). Therefore, we are only beginning to understand how the long-distance wound signal is perceived and future data must also explain the observation that PIN proteins are solely expressed in mesophyll cells (Orozco-Ca´rdenas et al., 2001). (3) The importance of JA in long-distance signaling is clear, but it is likely that long-distance translocation of the wound signal follows a more complex route than traveling from source to sink through the phloem. Although translocation of the wound signal to induce PIN expression was highly correlated with the strength of vascular connections within and between tomato leaves (Orians et al., 2000; Rhodes et al., 1999), phloem girdling experiments cannot rule out cell-to-cell movement of the signal (Malone, 1996; Nelson et al., 1983). Furthermore, studies on the systemic response to M. sexta larval regurgitant in wild tobacco showed bidirectional movement of a wounding signal through orthostichies, from sink to source leaves as well as from source to sink leaves (Schittko and Baldwin, 2003). (4) Finally, one report demonstrated that phloem transport could be inhibited in tomato without effect on the systemic accumulation of PIN proteins (Wildon et al., 1992). The authors provided evidence that chilling of cotyledons did not prevent the transmission of an electrical signal required for a systemic wound response. E. JA-MEDIATED WOUND RESPONSE IS MODULATED BY OTHER SIGNALS

Prosystemin orthologues are not found outside of the Solanaceae but several other groups, particularly the model crucifer Arabidopsis, exhibit robust and well-characterized JA-dependent responses, highlighting the importance of signals other than systemin in influencing the core JA wound response pathway. JA-regulated gene expression is induced by several plant-derived compounds such as H2O2 (Jih et al., 2003; Orozco-Ca´rdenas et al., 2001),

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ethylene (ET) (O’Donnell et al., 1996), and abscisic acid (Herde et al., 1996), environmental stimuli including ultraviolet light (Conconi et al., 1996), and substances such as the fatty acid conjugate volicitin present in the oral secretions of feeding insects (Alborn et al., 1997). SA is a potent negative regulator of the wound response, whose activity inhibits the local accumulation of JA during wounding (Doares et al., 1995). Combinations of these compounds are reported to act cooperatively (O’Donnell et al., 1996, 2003) but may also antagonize one another’s action (Orozco-Ca´rdenas and Ryan, 2002) in order to fine-tune the wound response. F. MECHANISM OF JA ACTION ON EFFECTOR GENES

Recent experiments have attempted to identify a receptor for jasmonates and to elucidate the downstream signaling events leading to activation of JAresponsive target genes. As it is outside the aims of this review, only a brief outline of recent progress will be outlined here, but for more comprehensive discussion of these topics please refer to several excellent reviews (Katsir et al., 2008; Kazan and Manners, 2008; Staswick, 2008). Our current understanding of JA-regulated expression of target genes involves a quartet of different molecular players: the intracellular JA signal, transcription factors that specifically upregulate transcription of target genes, JA ZIM-domain (JAZ) transcriptional repressor proteins, and the E3 ubiquitin ligase COI1. JA-responsive genes are repressed in cells with low JA levels and this repression occurs largely through the binding of JAZ family transcriptional regulators. External cues such as wounding or abiotic stress lead to the intracellular accumulation of bioactive jasmonates, particularly JA-Ile. JA-Ile binding to the E3 ubiquitin ligase COI1 recruits JAZ proteins to this complex, resulting in JAZ polyubiquitination and subsequent degradation via the 26S proteasome. Transcription factors such as MYC2/JIN1 are thus freed from JAZ-mediated repression and JA-responsive genes are highly transcribed. These molecular, genetic, and biochemical studies suggest that COI1 is a molecular receptor for jasmonates (reviewed in Katsir et al., 2008).

IV. LONG-DISTANCE SIGNALING IN SAR SAR has been described in the literature for over 100 years. An early review on the SAR phenomenon was published in 1933 by Kenneth Chester. This review described many observations and experiments suggesting that plants often display partial or complete immunity to reinfection after recovery from an initial pathogen attack. During this time plant scientists were trying to

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determine if plants had circulating antibodies and phagocytic cells that could explain the immunity to reinfection that was being observed (Chester, 1933). It was subsequently determined that plants do not possess antibodies or moving cells and that instead each plant cell is capable of responding to pathogen infection. Studies in the second half of the twentieth century carefully dissected the physiological aspects of SAR in tobacco and cucumber (reviewed in Kuc, 1982), providing the definition of SAR still in use today. The term SAR was first used by Ross (1961) to describe systemic resistance induced by necrosis-causing viruses in tobacco. SAR is defined as an initial infection in one part of a plant that leads to resistance in distant tissues to normally virulent pathogens. SAR also provides broad-spectrum resistance in that the initial infection, for example by a bacterial pathogen, will result in subsequent resistance to viruses, bacteria, fungi, and even nematodes (Kuc, 1982). A. SAR DEVELOPS IN DISTINCT STAGES

Like other long-distance-signaling pathways, SAR develops in distinct stages (Table I). Evidence from tobacco, cucumber, and Arabidopsis suggests that there are four stages in the SAR response. 1. Induction A SAR-inducing pathogen infects a leaf typically leading to the formation of localized necrotic lesions and local resistance (Hypersensitive Response, HR) or disease-induced necrosis (reviewed in Kuc, 1982). The SAR-inducing lesion elicits the expression of a set of genes encoding pathogenesis-related (PR) proteins in tobacco, cucumber, and Arabidopsis (Uknes et al., 1992, 1993; Van Loon, 1997; Ward et al., 1991). A study using ET-insensitive tobacco suggests that ET perception is required for production of the SAR long-distance signal(s) (Verberne et al., 2003). Accumulation of SA (10–50fold increase over background levels) is also associated with this stage (Delaney et al., 1994; Lawton et al., 1995; Malamy et al., 1990; Uknes TABLE I Long-Distance-Signaling Stages During Defense 1. Induction of response in local tissue (includes production of long-distance signal) 2. Movement of the long-distance signal 3. Molecular perception of signal in distant tissue immediate response in Wound Response and SIS plant becomes ‘‘primed’’ to withstand subsequent attack in SAR and ISR 4. Manifestation of response upon subsequent attack in SAR and ISR

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et al., 1992; Yalpani et al., 1991). A recent study by Mishina and Zeier (2007) suggests that cell death is not required to induce SAR in Arabidopsis as a number of nonhost Pseudomonas strains and infiltration with compounds acting as microbe-associated molecular patterns (MAMPs) did induce SAR. During the SAR induction stage, microscopic cell death levels were similar to cell death levels in mock-inoculated plants (no SAR induction) as measured by Trypan Blue staining. However, it is still possible that a few cells undergoing pathogen-induced cell death were enough to induce SAR. It is also possible that the MAMPs, bacterial flagellin, and lipopolysaccharide used in this study, are phloem mobile and moved from the infiltrated leaf to distant tissue and induced a basal defense response. This could also be the case for induction of SAR with high doses of nonhost flagellin-bearing Pseudomonas strains. 2. Movement of a long-distance signal(s) A signal or signals are produced and thought to move from the induced leaf through the phloem (Guedes et al., 1980; Jenns and Kuc, 1979; Kiefer and Slusarenko, 2003; Tuzun and Kuc´, 1985) or cell-to-cell via plasmodesmata to the rest of the plant to establish SAR. Shulaev et al. (1997) suggested that airborne methyl-SA (MeSA) may also participate in long-distance signaling during SAR in tobacco. A study using 18O-labeling of tobacco mosaic virus (TMV)-inoculated tobacco leaves is consistent with SA itself being the mobile SAR signal (Shulaev et al., 1995). However, other studies using cucumber (Rasmussen et al., 1991), or grafting experiments with transgenic tobacco with reduced levels of SA, have led to the suggestion that SA is not the mobile signal (Pallas et al., 1996; Vernooij et al., 1994). These results also suggest that SA accumulation may not be required in the induction phase of SAR, but that SA is required in distant tissues during the establishment stage of SAR in tobacco. 3. Establishment of the ‘‘primed’’ plant The establishment stage of SAR involves the arrival and perception of the long-distance signal in the distant leaves, the accumulation of SA in distant Arabidopsis (1- to 2-fold, Cameron et al., 1999; Delaney et al., 1994; Lawton et al., 1995) and tobacco leaves (10-fold, Yalpani et al., 1991) and in cucumber phloem sap (8- to 200-fold, Me´traux et al., 1990), as well as the expression of PR genes. A receptor is postulated to interact with the long-distance signal (s) to ‘‘prime’’ the plant to respond rapidly and effectively to future pathogen attacks. In one Arabidopsis study, establishment of SAR in distant tissues was associated with the occurrence of low frequency microscopic HRs (Alvarez et al., 1998).

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4. Manifestation The final phase is the manifestation stage which occurs when the plant is challenged with a second, normally virulent pathogen such that the plant responds with a rapid and effective resistance response (Kuc, 1982). Evidence for this final stage comes from a number of studies (Kuc, 1982, 1983). For example, PR-proteins accumulate earlier and more rapidly after challenge with Peronospora tabacina in tobacco induced with Pe. tabacina or TMV (Ye et al., 1989), or in Arabidopsis induced with avirulent Pseudomonas syringae pv. tomato (Pst) (Cameron et al., 1999). Additionally, Siegrist et al. (1994) observed increased deposition of phenolic compounds and increased chitinase activity at the site of secondary challenge with Colletotrichum lagenarium in induced cucumber hypocotyls. B. ROLE OF SA AND NPR1 IN SAR

As discussed above, SA was believed to be the long-distance signal in SAR because it is found in phloem sap and exudates collected from SAR-induced cucumber and tobacco leaves, respectively, and its presence is required in distant tobacco tissue for a successful SAR response. In other words, SA is in the right place at the right time to be a long-distance-signaling molecule. Although the elegant cucumber leaf detachment (Rasmussen et al., 1991) and SA-deficient transgenic plant studies (Gaffney et al., 1993; Pallas et al., 1996) strongly suggest that SA is not the signal moving from induced to distant leaves, these studies cannot entirely eliminate SA as the long-distance signal. SA-deficient transgenic tobacco lines still contain uninduced levels of SA (10–40 ng g fw 1) and it is possible that this is sufficient for long-distance signaling. Additionally, undetectable amounts of SA in cucumber phloem could be sufficient for long-distance signaling during SAR. More recent studies have unraveled the role of SA and NONEXPRESSOR OF PR GENES-1 (NPR1) during SAR in Arabidopsis. Studies of npr1 mutants indicate that NPR1 is a key regulator in SAR, basal resistance, and the ISR response in Arabidopsis (reviewed in Dong, 2004; Grant and Lamb, 2006). There is mounting evidence that SA acts as a local signaling molecule during defense by inducing the reduction of key intermolecular disulphide bonds in NPR1, allowing translocation of NPR1 into the nucleus, where NPR1 interacts with TGA transcription factors which subsequently upregulate defense genes like PR-1 (Fobert and Despre´s, 2005). In the Arabidopsis-Pst SAR model, SAR is often induced in the initial leaf by inoculation with Pst (avrRpt2) (Cameron et al., 1994). The AvrRpt2 bacterial effector is recognized by the RPS2 resistance receptor, initiating a signal cascade that results in the HR (Bent et al., 1994; Leister and Katagiri, 2000;

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Mindrinos et al., 1994). This RPS2-AvrRpt2 pathway is SA-dependent suggesting that SA accumulation is required during the HR and the induction stage of SAR in Arabidopsis. Moreover, the RPS2-avrRpt2 HR is NPR1independent in ecotype Columbia-0 suggesting that NPR1 is not required for the HR (Rairdan and Delaney, 2002; Zhang et al., 2004) or the induction stage of SAR in Arabidopsis. However, functional NPR1 is required for the establishment and manifestation of the SAR response in Arabidopsis (Cao et al., 1994) suggesting that NPR1 functions in the distant leaves to upregulate PR genes during SAR. Yet, in the Arabidopsis ecotype Nossen the RPS2-AvrRpt2 pathway is compromised in the npr1-5 mutant (Nandi et al., 2004), suggesting that NPR1 is required for the SAR induction stage in this ecotype. C. SAR SIGNAL TRANSPORT

Grafting experiments with cucumber provided evidence that a long-distance signal does move from induced rootstocks to distant scions (Jenns and Kuc, 1979). Girdling with hot cotton wool in cucumber (Guedes et al., 1980) or removing the stem sheath in tobacco (Tuzun and Kuc´, 1985) prevented signal transport to distant leaves, suggesting that the SAR long-distance signal moves via the phloem. However, these techniques reduce both phloem and cell-to-cell movement, indicating that the SAR long-distance signal could travel using either or both transportation routes. Source–sink relationships (orthostichies) in the Arabidopsis rosette were investigated in relation to SAR-competence (Kiefer and Slusarenko, 2003). Movement of the SAR signal from induced to distant leaves to establish and manifest SAR as measured by PR-1 expression and reduced growth of Pst, indicated that leaves outside the orthostichy of the induced leaf were also SAR-competent. These data suggest that the Arabidopsis long-distance SAR signal(s) moves via the phloem and other means, perhaps cell-to-cell. In the cucumber–Ps. syringae pv. syringae SAR model, leaf detachment experiments indicated that the long-distance signal moves out of the induced leaf by 4 h after inoculation (Rasmussen et al., 1991, 1995) and distant leaves become SAR-competent by 24 h after SAR induction. In contrast, Arabidopsis leaf detachment experiments demonstrated that it takes between 36 and 48 h for the SAR signal to move and render distant leaves SAR-competent (Cameron et al., 1994). In tobacco it takes between three and nine days for the signal to move and render distant leaves SAR‐competent (Ross, 1961; Tuzun and Kuc´, 1985; Vernooij et al., 1994). There is a large range in the time it takes the SAR signal to move and render distant leaves SAR-competent in these SAR model systems suggesting that in cucumber the rapid movement of the

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SAR signal occurs mainly in the phloem, while Arabidopsis and tobacco use a combination of phloem and cell-to-cell movement. D. CANDIDATES FOR THE SAR LONG-DISTANCE SIGNAL

Since the discovery of DIR1 (Defective in Induced Resistance), a putative lipid transfer protein (LTP) involved in long-distance signaling during SAR (Maldonado et al., 2002), a number of candidate SAR long-distance signals have been identified. Our work with the dir1-1 SAR-defective mutant indicated that dir1-1 petiole exudates (enriched for phloem sap) do not contain the SAR mobile signal, leading to the hypothesis that DIR1-LTP could be involved in production of the SAR signal or act as a chaperone for a lipid signal (Maldonado et al., 2002). Plant LTPs contain eight cysteine residues forming four disulphide bonds that participate in forming an internal hydrophobic tunnel that has been shown to bind long-chain fatty acids in vitro. Plant LTPs are associated with numerous plant developmental and defensive processes (reviewed in Yeats and Rose, 2008). Recently the structure of DIR1 has been solved and in vitro lipid binding studies indicate that DIR1 can bind two monoacylated phospholipids side-by-side in its large internal tunnel (Lascombe et al., 2008). These researchers identified two proline-rich regions on the DIR1 surface. These proline-rich regions are involved in protein‐ protein interactions in other signaling pathways leading to the idea that these regions of DIR1 interact with the long-distance signal receptor upon arrival in distant tissues (Lascombe et al., 2008). DIR1, which was identified as a putative LTP, was the first and, to date, only plant LTP that has a genetically defined function. This discovery led to the idea that lipid signals may be important in SAR long-distance signaling. Both EDS1 (Falk et al., 1999) and PAD4 (Jirage et al., 1999), putative lipases involved in both basal and R gene-mediated defense, were postulated to produce lipid-derived signals that might be chaperoned to distant tissues by DIR1 (Maldonado et al., 2002). Subsequent studies demonstrated that EDS1 and PAD4 are required for a successful SAR response (Truman et al., 2007; R. K. Cameron, unpublished data; J. E. Parker, personal communication) and therefore provide further evidence that EDS1 and PAD4 may be supplying an essential lipid for DIR1. Recent experiments in the group of Robin Cameron support the idea that DIR1 may be a SAR long-distance signal. Agrobacterium-mediated transient expression of DIR1 in one leaf is sufficient to rescue the dir1-1 SAR defect, but not the npr1-2 SAR defect, supporting the idea that NPR1 is required in the distant tissue during Pst (avrRpt2)-induced SAR. Moreover, DIR1 protein was found in petiole exudates collected from leaves transiently

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expressing DIR1 only after SAR induction, suggesting that DIR1 moves via the petiole to distant tissues during SAR (our unpublished data). Lipid involvement in SAR is further supported by the identification of the sfd1 (suppressor of fatty acid desaturase deficiency 1) mutant that, like dir1-1, displays normal local defense (basal and R gene-mediated) but is defective in SAR long-distance signaling (Maldonado et al., 2002; Nandi et al., 2004). The SFD1 gene encodes a dihydroxyacetone-phosphate reductase that functions in plastid glycerolipid metabolism, pointing yet again to the importance of lipids in SAR signaling (Nandi et al., 2004). In a recent follow-up article, Jyoti Shah and coworkers have gone on to demonstrate that SFD1 and FAD7 (FATTY ACID DESATURASE-7), genes involved in glycerolipid synthesis, are required to produce the SAR long-distance signal (Chaturvedi et al., 2008) along with DIR1. Petiole exudates collected from sfd1 and fad7 mutants did not contain a SAR-inducing activity. However, these mutants were responsive to exudates collected from wild-type SARinduced leaves, suggesting that they perceive the SAR long-distance signal. SAR-induced exudates from either the sfd1 or fad7 mutant combined with SAR-induced exudates from dir1-1 were infiltrated into lower untreated leaves such that resistance was restored in distant leaves challenged with virulent Pseudomonas (Chaturvedi et al., 2008). It is tempting to speculate that DIR1 may bind a glycerolipid supplied by SFD1 and FAD7 and chaperone this lipid signal to distant tissues to establish SAR. JA, another lipid-derived molecule, has also recently been implicated in SAR long-distance signaling. A study by Truman et al. (2007) demonstrated that the JA biosynthesis mutant opr3 and the JA signaling mutant jin1 are defective in SAR. Induction of SAR in wild-type plants using Pst (avrRpm1) led to an increase in JA levels in petiole exudates and distant leaves. Additionally, spraying JA on leaves led to increased basal resistance to virulent Pst. These data suggest that JA could be a long-distance signal during SAR as JA appears to move to distant tissues to establish SAR. Unfortunately, Arabidopsis SAR marker gene expression (PR-1, PR-2, PR-5) was not monitored to demonstrate that classical SAR was induced in these experiments. An additional problem with these experiments is that Pst produces coronatine that may act as a JA mimic (reviewed in Katsir et al., 2008) and therefore, Pst-produced coronatine could induce JA production that has nothing to do with the SAR response. It is interesting to note that Truman et al. (2007) observed that the JA signaling mutant jin1 was SAR-defective and upon attempted induction supported wild-type levels of virulent Pst growth. JIN1 (MYC2) has been shown to be a negative regulator of SAmediated resistance to Pst such that jin1 mutants are resistant to virulent Pst and accumulate wild-type levels of JA (Laurie-Berry et al., 2006; Lorenzo

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et al., 2004; Nickstadt et al., 2004). Experiments by Cui et al. (2005) also indicated that the JA pathway mutant coi1-1 is SAR-competent, suggesting that JA signaling is not required for SAR. These seemingly conflicting data suggest that the JA-mediated systemic resistance observed by Truman et al. (2007) requires further study before it can be called a classical SAR response. Chaturvedi et al. (2008) concluded that JA is not a long-distance signal during SAR because they observed that petiole exudates accumulating JA did not possess SAR-inducing activity, whereas exudates with SAR-inducing activity accumulated only basal levels of JA. However, it is possible that basal levels of JA are sufficient to participate in SAR long-distance signaling. JA has also been shown to bind to the tobacco LTP1 protein that is involved in disease resistance in tobacco to Phytophthora parasitica (Buhot et al., 2004), leading to the hypothesis that DIR1 may bind JA during SAR longdistance signaling (Buhot et al., 2004; Grant and Lamb, 2006; Truman et al., 2007). Volatile MeSA has been implicated in SAR signaling between induced and distant leaves and in plant-to-plant signaling in tobacco (Shulaev et al., 1997). As discussed by Shulaev et al. (1997), the extent of volatile signaling in field-grown plants is not known and has not been investigated further. However, in a recent study, MeSA accumulated to high levels in Arabidopsis, eliciting PR-1 expression in neighboring plants supporting a role for volatile MeSA in plant-to-plant signaling (Koo et al., 2007). Additionally, an elegant study by Park et al. (2007) provided evidence that SA‐binding protein 2 (SABP2) (Kumar and Klessig, 2003) is involved in the accumulation of MeSA in tobacco leaves and petiole exudates and long-distance signaling during SAR. SABP2 is a methyl esterase that converts MeSA to SA, while SA inhibits the esterase activity of SABP2 (Forouhar et al., 2005). Park et al. (2007) performed a number of grafting experiments in which wild-type and mutant versions of SABP2 were silenced either in tobacco rootstocks or scions, combined with MeSA determination in leaves and petiole exudates. Together these experiments indicated that the methyl esterase activity of SABP2 is inhibited in the induced leaf by the accumulation of SA that occurs during induction of SAR. Therefore, MeSA levels rise in the induced leaf, followed by MeSA increases in phloem exudates and distant leaves, suggesting that MeSA is a SAR long-distance signal. In the distant leaves of plants induced for SAR, SA levels are lower leading to activation of SABP2’s methyl esterase activity, such that MeSA is converted to SA. As discussed above, SA produced locally in the distant leaves activates NPR1 leading to PR gene expression.

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Park et al. (2007) speculate that SABP2 generates MeSA as a SAR longdistance signal, and also perceives MeSA in distant tissues. However, it is not known if SABP2 participates directly in the ‘‘priming’’ mechanism that allows the distant uninfected tissue to respond to the next pathogen challenge in a rapid and effective manner. It is possible that SA produced from MeSA via SABP2 in distant leaves acts as a local signal to activate the ‘‘primed’’ state in distant leaves. The nature of the ‘‘primed’’ state is totally unknown, but it seems reasonable to speculate that a long-distance signal receptor(s) becomes activated in some way and upon subsequent pathogen challenge, the distant uninfected leaves respond in a resistant manner. NPR1 function is required during SAR and NPR1 becomes activated by SA leading to PR gene expression in distant leaves (Cao et al., 1994; Fobert and Despre´s, 2005); therefore, it is possible that NPR1 may act as a long-distance signal receptor in distant leaves during SAR (Fig. 1). Vlot et al. (2008) have determined that a number of SABP2 family members in Arabidopsis also participate in SAR. The quest for SAR long-distance signal(s) has been very fruitful in the past few years. A number of small hydrophobic molecules including MeSA, JA, and lipids are potential candidates. It is tempting to speculate that DIR1 with its large hydrophobic tunnel could possibly form a complex with any or all of these small hydrophobic molecules to act as a chaperone or as part of a longdistance SAR signal complex (Fig. 1). E. OTHER GENES INVOLVED IN SAR LONG-DISTANCE SIGNALING

A number of other genes have been implicated in the SAR pathway in the last few years, including Arabidopsis CDR1 (CONSTITUTIVE IN DISEASE RESISTANCE‐1) (Xia et al., 2004). Intercellular washing fluids collected from SAR-induced leaves contain a CDR1-dependent elicitor that induces PR gene expression when infiltrated into untreated healthy leaves. Additionally, grafting experiments with an inducible CDR1 transgenic line suggest that expression of CDR1 in lower leaves leads to production of a signal that moves to the grafted scion to elicit PR gene expression. However, SAR competence in these leaves was not measured (Xia et al., 2004). Moreover, antisense-CDR1 plants were more susceptible to virulent and avirulent Pst and Ps. syringae pv. maculicola (Psm) compared to wild type. CDR1 encodes an aspartic protease leading Xia et al. (2004) to hypothesize that intercellular CDR1 (Suzuki et al., 2004) may generate a peptide involved in local and systemic defense responses. Moreover, given the evidence that supports phloem and cell-to-cell SAR signal movement in Arabidopsis (Kiefer and Slusarenko, 2003), it will be important to determine how an apoplastic

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4. Manifestation SA, NPR1, PRs

Pathogen

Other putative Stage 3 & 4 genes DTH9, EDR1 NDR1, FMO1 WIN3

3. Perception of signals/priming (SABP2, NPR1, ?) Volatile meSA

DIR1 Lipids CDR1 MeSA JA

SABP2 Me

SA

SA

SFD1, FAD7 Lipids

DIR1 CDR1

Lipids

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MeSA JA

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Fig. 1. SAR long-distance-signaling model. Putative SAR long-distance signals are produced (MeSA, JA, lipids, CDR1) and may bind DIR1, a putative signal chaperone, in the induced leaf (Stage 1). The signals and/or DIR1þsignals move from the induced leaf to distant tissues (Stage 2, via the phloem and/or cell-to-cell), where they are perceived (Stage 3) by SAR long-distance signal receptors which could include SABP2, NPR1 and unknown receptors such that the plant becomes primed. Volatile MeSA could be perceived in distant tissues of induced or neighboring plants. Upon subsequent pathogen challenge the activated/primed SAR long distance receptor(s) allows the distant leaves to respond in a resistant manner (Stage 4). See SAR Signal Transport section for details. SA, salicylic acid; MeSA, methyl-SA; CDR1, Constitutive defense response-1; DIR1, Defective in induced resistance-1; SABP2, SA‐binding protein 2; NPR1, Nonexpressor of PR genes-1; SFD1, FAD7, glycerolipid biosynthesis enzymes; PRs, pathogenesis-related proteins.

CDR1-derived peptide moves to distant tissues as it is currently believed that proteins in the phloem arrive intracellularly via companion cell plasmodesmata (Lough and Lucas, 2006). It is interesting to note that DIR1 also has a signal sequence that directs it into the intercellular space (R. K. Cameron, unpublished data). Moreover, we have evidence that DIR1 with its signal sequence removed can complement the SAR defect in dir1-1. These data suggest that upon SAR induction, DIR1 remains in the cytoplasm and then moves cell-to-cell and/or through the phloem to distant leaves (our unpublished data). Another gene that implicates peptides in SAR

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signaling is ALD1 which encodes an aminotransferase that is required for SAR, as well as R gene-mediated and basal resistance, and may be involved in generating an amino acid-derived signal (Song et al., 2004). FMO1, a flavin-dependent monooxygenase, is required for a successful SAR response in Arabidopsis (Mishina and Zeier, 2006). An fmo1 mutant displayed a wild-type local response to SAR-inducing Psm (avrRpm1) in terms of SA accumulation, HR cell death, and defense gene expression; however, there was little accumulation of SA or expression of defense genes in distant leaves, suggesting that FMO1 function is required in distant leaves for the establishment of SAR. Additionally, the ndr1-1 (no disease resistance-1-1) (Century et al., 1995, 1997) mutant was shown to be SAR-defective and FMO1 expression was abolished in distant leaves of ndr1-1 plants induced with Psm (avrRpm1) (Mishina and Zeier, 2006). NDR1 is a plasma membrane associated protein required for RPS2‐ and RPM1-mediated resistance (Coppinger et al., 2004). Therefore, NDR1 function may be required during the induction stage of SAR elicited by Pst (avrRpt2) or Psm (avrRpm1). Moreover, plants overexpressing NDR1 display enhanced resistance to Pst in the absence of constitutive expression of PR-1 suggesting that NDR1 may be involved in the priming stage of SAR (Coppinger et al., 2004). Taken together, these results suggest that NDR1 function is required for FMO1 expression in distant leaves during SAR. It is interesting to note that in another report, the ndr1-1 mutant did display SAR in response to Pst (avrB) (Zhang and Shapiro, 2002). Additional genes have also been implicated in the priming response of SAR. The ENHANCED DISEASE RESISTANCE-1 (EDR1) gene encodes a putative MAP kinase kinase (Frye et al., 2001) that is hypothesized to negatively regulate the priming response in Arabidopsis (Conrath et al., 2006). The SARdefective dth9 (detachment 9) mutant displays wild-type levels of PR-1 gene expression in SAR-induced and distant leaves, suggesting that DTH9 function is required in distant leaves during priming or for the manifestation stage of SAR. Most recently, WIN3, a protein required downstream of RIN4 in the RPS2-avrRpt2 resistance pathway, was demonstrated to be required for the RPS2-avrRpt2-mediated HR and for SAR in Arabidopsis (Lee et al., 2007b). Lee et al. (2007b) speculate that WIN3 function is important in the induction as well as the establishment stages of SAR, as WIN3 expression was abolished in distant leaves of the win3-T mutant. The effect of the environment, a key component of the disease triangle, (Agrios, 2005) must be kept in mind when comparing data from various labs and among various species, and may explain why some mutants (ndr1-2, eds1-1) appear SAR‐defective in some labs and SAR-competent in others. Additionally, the environment may affect how well petioles exude phloem sap and may explain why Jyoti Shah and co-workers (Chaturvedi et al., 2008)

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observed that infiltration of Arabidopsis petiole exudates collected from SAR-induced leaves, did induce SAR in naı¨ve Arabidopsis leaves, while these types of experiments have not been successful in other labs (R. K. Cameron, unpublished data; L. C. van Loon, personal communication). F. ROLE OF ET IN SAR LONG-DISTANCE SIGNALING

ET is an important plant hormone involved in a number of developmental, as well as biotic and abiotic stress responses (Binder, 2008). Responsiveness to ET is required in the wound response (see above) and ISR (De Vleesschauwer and Ho¨fte, 2009). However, its role in the SAR response has not been studied as thoroughly. ET is synthesized prior to necrotic lesion formation and the development of SAR in TMV-infected tobacco (Knoester et al., 2001). Tobacco plants expressing the Arabidopsis etr1-1 dominant mutant ET receptor are insensitive to ET and when SAR was induced with TMV, PR gene expression in induced leaves was observed. However PR gene expression in distant tissue was abolished (Knoester et al., 1998), leading to the suggestion that ET signaling may be important for the expression of SAR in tobacco. Subsequently Verberne et al. (2003) performed grafting experiments using the same ET-insensitive tobacco plants demonstrating that the SAR induction stage requires a functional ET response to generate the long-distance signal, whereas ET responsiveness is not required in distant leaves to establish and manifest SAR. In contrast, Arabidopsis ET-insensitive mutants etr1-1 and ein2-1 were SAR-competent when induced for SAR using SA or the SA analog, 2,6dichloroisonicotinic acid (INA), followed by inoculation with virulent Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica), suggesting that ET signaling is not required for SAR in Arabidopsis (Lawton et al., 1995). In a subsequent study, SAR was induced with Pst (avrRpt2) and distant leaves were challenged with H. arabidopsidis. Mutant etr1-1 and wild-type plants were similarly competent for SAR. When other SAR-inducing molecules (vitamin B1, harpin, BTH (benzo(1,2,3)thiadiazole7-carbothioic acid S-methyl ester) were applied to Arabidopsis plants, SAR was induced in an ET-independent manner (Ahn et al., 2007b; Dong et al., 1999; Lawton et al., 1996). Taken together, these data strongly suggest that SAR in Arabidopsis does not require a functional ET-signaling pathway. G. SAR LONG-DISTANCE SIGNALING ACROSS SPECIES

SAR has been mechanistically studied in cucumber, tobacco, and Arabidopsis. In the last few years a number of studies in both tobacco and Arabidopsis have contributed to elucidating SAR long-distance signaling and to extend these

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findings to other plant species. Chaturvedi et al. (2008) were able to demonstrate that Arabidopsis petiole exudates collected from SAR-induced leaves, when infiltrated into wheat spikelets or tomato leaves, induced SAR to Fusarium graminearum or Pst, respectively. Additionally, Vlot et al. (2008) determined that like tobacco, Arabidopsis possesses a family of genes encoding SABP2 methyl esterases (AtMES). A number of these genes have methyl esterase activity and silencing of multiple AtMES genes reduced the SAR response. In our lab we have developed a cucumber–Arabidopsis model system to aid in our SAR studies. SAR-induced cucumber exudates can rescue the SAR defect in dir1-1 Arabidopsis and protein gel blot analysis indicates that these cucumber exudates contain a DIR1-like protein (R. K. Cameron, M. J. Champigny, and J. Faubert, unpublished data). These studies indicate that there appear to be shared components between tobacco and Arabidopsis and between cucumber, wheat, tomato, and Arabidopsis, hinting that longdistance-signaling mechanisms are conserved in diverse plant species.

V. SYSTEMIC INDUCED SUSCEPTIBILITY (SIS) Plants employ many defense responses to combat and withstand pathogen infection and in turn pathogens have evolved mechanisms to suppress or evade plant defense and manipulate plant metabolism (reviewed in Melotto et al., 2008; Speth et al., 2007). Recently it was discovered that in Arabidopsis, Psm not only suppresses plant defense in the infected leaf, but also in distant or systemic leaves, rendering the distant leaves more susceptible to subsequent infections (Cui et al., 2005). This SIS is dependent on the Psm phytotoxin coronatine as demonstrated by the inability of coronatinedeficient Psm to elicit SIS. A SIS long-distance signal has been detected in experiments in which petiole exudates enriched in phloem sap collected from Pst(avrRpt2)-induced wild-type leaves were infiltrated into the SAR-defective mutant plants sid2 and npr1-2. These SAR-defective mutants proved not only SAR-defective, but also exhibited SIS (Chaturvedi et al., 2008). Whether coronatine itself acts as the long-distance signal or instead stimulates JA long-distance signaling is not known (Cui et al., 2005). Coronatine is very similar in structure to JA and is known to act as a molecular mimic to activate downstream JA signaling and at the same time suppress SA-mediated resistance in infected leaves (reviewed in Katsir et al., 2008). The fact that JA is known to be a long-distance signal in the wound response leads to the idea that coronatine may also act as a long-distance signal in SIS.

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VI. SIGNALING DURING ISR Several soil-borne microorganisms are beneficial to plants in that they increase nutritional capacity and enhance resistance to biotic and abiotic stresses, including drought and pathogen infection. Colonization of roots by certain strains of nonpathogenic plant growth-promoting rhizobacteria (PGPR) results in enhanced resistance in aerial plant organs to normally virulent pathogens, a phenomenon known as rhizobacteria-induced systemic resistance (Van Loon et al., 1998). ISR has been observed for a variety of plant root/ microbe interactions, such as carnation/Fusarium oxysporum f. sp. dianthi (Van Peer et al., 1991), bean/Bacillus pumilis SE34 (Benhamou et al., 1996), Arabidopsis/Pseudomonas fluorescens WCS417r (Pieterse et al., 1996), as well as in the mycorrhizal symbiosis tomato/Glomus mosseae (Cordier et al., 1998). Signaling events during the development of ISR have features in common with other induced defense responses, particularly in that ISR occurs as a series of discrete steps temporally and spatially separated in plant tissues: First, during an induction step, beneficial microbes colonize and are recognized by plant roots. This stimulates the synthesis of a long-distance signal that is transported systemically and perceived in aerial tissues. Following perception, plants become ‘‘primed’’ to assert a more aggressive response to virulent pathogens and subsequent pathogen attack is met with a more abundant and/or earlier stimulation of defense-related genes (Conrath et al., 2006). The phenomenon of ISR has been reviewed recently (Conrath et al., 2006; De Vleesschauwer and Ho¨fte, 2009; Van Wees et al., 2008); therefore, we will highlight only issues relevant to long-distance signaling during ISR here. A. INDUCTION OF ISR

The initial, or induction stage in ISR involves the recognition of certain microbe-derived molecules by plant roots. Different classes of molecules, including bacterial flagellin and lipopolysaccharides (Bakker et al., 2007), antibiotics (Iavicoli et al., 2003), surfactants (Tran et al., 2007), and ironchelating siderophores (Meziane et al., 2005), have been implicated as inducers of ISR, largely on the basis of the inability of mutant microbes deficient in these compounds to promote systemic disease resistance in the host plant. It should be noted that many of these molecules are also recognized during plant immune responses against virulent pathogens suggesting that beneficial microbes and virulent pathogens are recognized in a similar way. Compared to SAR, little is known about plant-derived molecules required for the induction of ISR. Transcriptome analysis of Arabidopsis colonized by ISRinducing Ps. fluorescens WCS417r revealed significant changes in expression of

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97 genes in roots, but only subtle changes in gene expression within leaves (Verhagen et al., 2004). This observation suggested that, unlike SAR, ISR is not associated with massive plant-wide transcriptional reprogramming. The above-mentioned microarray study identified that the transcription factor MYB72 was upregulated in roots after induction of ISR (Verhagen et al., 2004). Null myb72 mutants, when treated with WCS417r, did not develop resistance against a variety of foliar pathogens, suggesting that MYB72 was required for an early ISR signaling step in plant roots (Van der Ent et al., 2008). Furthermore, MYB72 interacted with the Ethylene-insensitive3-like-3 (EIL3) protein in a yeast two-hybrid assay, associating the function of MYB72 with the ET-response pathway (Van der Ent et al., 2008). Although the importance of this interaction is not understood and the target genes of these transcription factors have not yet been identified, they may be required for production or transmission of an ISR long-distance signal. Currently there is little experimental evidence that implicates any molecule(s) as a long-distance signal(s) required for the development of ISR. In particular, grafting experiments or transgenic technologies have not yet been employed to specifically identify substances whose site of production, presumably in roots, is spatially separated from their site of action in aerial tissues. JA is a priori a compelling candidate because of its phloem mobility and the fact that ISR in Arabidopsis requires elements of the JA signaling pathway (see below). However, as is the case for SA in SAR, JA may be required to mount an effective ISR response in distant tissue but not itself be a long-distance signal. B. SIGNAL PERCEPTION AND PRIMING DURING THE DEVELOPMENT OF ISR

In Arabidopsis, ISR signaling events downstream of the initial induction step share features with both SAR and the wound response. Like the wound response, ISR is abolished in many JA- or ET-responsive mutants (Ahn et al., 2007a; Hossain et al., 2008; Pieterse et al., 1998) and manifestation of enhanced resistance to virulent pathogens during ISR was found to involve a variety of JA- and ET-responsive genes (Ahn et al., 2007a; Van der Ent et al., 2008; Van Oosten et al., 2008). Furthermore, SA is not required for most instances of ISR as demonstrated by the fact that ISR could be established in mutants deficient for SA synthesis or accumulation (Pieterse et al., 1996; Ton et al., 2002; Van Wees et al., 2008). A requirement for SA was observed in the Arabidopsis ISR response to Verticillium dahliae (Tjamos et al., 2005), demonstrating variation in ISR signaling requirements in different plant/microbe associations.

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Like SAR, ISR in Arabidopsis requires the master defense regulator NPR1 (Knoester et al., 1999; Pieterse et al., 1998; Van Wees et al., 2000). However, in ISR, NPR1-directed upregulation of PR genes is not observed (Pieterse et al., 1996; Van Wees et al., 1999). NPR1 can thus be considered an integral part of the molecular machinery involved in perceiving or ‘‘interpreting’’ both the SAR and ISR long-distance signals because NPR1 discriminates between signals originating from the action of SAR-inducing pathogens and soil-borne beneficial microbes in ISR and orchestrates a different suite of SA-dependent or JA/ET dependent defense genes for either case. Although the requirement for NPR1 was not examined, a recent report provided evidence that the transcription factor MYC2 is required for the priming step of ISR because MYC2 was upregulated in WCS417r-treated Arabidopsis plants and mutants deficient in MYC2 were unable to mount a systemic response to bacterial or fungal challenge (Pozo et al., 2008).

VII. TECHNIQUES TO FURTHER ELUCIDATE LONG-DISTANCE SIGNALING To date, a number of molecules appear to be in the right place at the right time to be candidates for the SAR long-distance signal (DIR1, MeSA, JA, lipids, CDR1-derived peptide). The evidence is convincing that JA is a long-distance signal during the wound response, although it is possible that additional signal molecules will be discovered in the future. The ISR and SIS long-distance signals are unknown; however, coronatine or JA appear to be reasonable candidates for a SIS signal and JA pathway genes are required for ISR. Although petiole exudates enriched for phloem sap collected from Arabidopsis are rather dilute, these exudates do contain both SAR-inducing (Chaturvedi et al., 2008; Maldonado et al., 2002) and SIS-inducing properties (Chaturvedi et al., 2008). Therefore, it should be possible to further characterize SAR and SIS long-distance signals by metabolomic and proteomic analysis of petiole exudates. For example, if DIR1 binds a lipid or other hydrophobic molecule in a covalent manner, as has been demonstrated for barley LTP1b (Bakan et al., 2006), then 2-D gel electrophoresis of SARinduced petiole exudates combined with protein-ligand identification via mass spectrometry analysis, may identify the DIR1 ligand. It may also be possible to detect the ISR signal by collecting petiole exudates from leaves that are receiving the ISR signal from the roots. Highly pure phloem exudates can be collected in abundance from species with robust SAR responses such as cucumbers. Mass peptide fingerprinting of uninduced

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and SAR-induced phloem exudates from cucumbers could potentially identify all proteins present in a sample and thereby discover new proteins acting as long-distance signals or chaperones. Another approach to identify the DIR1 ligand includes collection of petiole exudates from SAR-induced leaves and subsequent immunoprecipitation/mass spectrometry analysis of DIR1 plus its ligand. This has been attempted in the Cameron lab, but with little success probably because of the very low levels of DIR1 present in exudates even in DIR1 overexpressing lines and the use of an antiserum raised against unfolded DIR1 produced in transformed Escherichia coli cells. DIR1, like most LTPs is thought to enter the secretory system, obtain its four disulphide bonds in the endoplasmic reticulum and be secreted to the plant cell wall. Therefore, to enrich for DIR1-ligand complexes during immunoprecipitation, antibodies should be generated to correctly folded DIR1 produced in a eukaryotic system such as the methanotrophic yeast Pichia pastoris. DIR1 is expressed in all cells in Arabidopsis leaves (R. K. Cameron, M. J. Champigny, H. Shearer, J. Faubert, K. Haines, A. Mohammad and M. Neumann, unpublished data) making it challenging to design experiments to observe DIR1 move from induced to distant leaves during SAR. To address this question we have developed a transient Agrobacterium-SAR assay using the transient transformation method of Wroblewski et al. (2005). DIR1 fused to the gene encoding YFP is expressed in one dir1-1 leaf by Agrobacterium-mediated transient transformation, followed by SAR induction with avirulent Pst in the same leaf. Expression of DIR1:YFP in one leaf does rescue the SAR defect in dir1-1 and native DIR1 protein can be detected in petiole exudates of SAR-induced leaves using protein gel blot analysis. However, DIR1:YFP cannot be detected, suggesting that YFP is being cleaved from the DIR1:YFP fusion protein, making it impossible to follow DIR1 movement using fluorescence microscopy. In preliminary experiments using this transient Agrobacterium-SAR assay, we detected DIR1 protein in the petiole exudates of distant leaves suggesting that DIR1 does move to distant tissues during SAR (R. K. Cameron, M. J. Champigny and J. Faubert, unpublished data). To shed light on the long-distance signal perception mechanism in the wound response, SAR, ISR, or SIS, it should be possible to take advantage of mutants that are wild-type in the respective induction stages, but are defective in the establishment and manifestation stages. Comparing wildtype and mutant gene expressions (microarray or transcriptome sequencing) in distant leaves may identify important genes whose expression changes in the perception or establishment stage and give some clues as to the identity of the various long-distance signal receptors.

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VIII. CONCLUDING REMARKS The molecular receptor of FT in the SAM is FD, a bZIP transcription factor that interacts with FT, the long-distance signal, to initiate the gene expression cascade that induces expression of the floral meristem-identity genes resulting in the switch from vegetative to flowering meristems (reviewed in Kobayashi and Weigel, 2007). In the wound response pathway, emerging evidence suggests that COI1 is a molecular receptor for JA in distant leaves leading to COI1 interaction with the JAZ repressor proteins, their subsequent polyubiquitination and degradation, thus releasing the JIN1 (MYC2) transcription factor to upregulate the JA responsive genes (reviewed in Katsir et al., 2008). Thus, in both the photoperiodic flowering and wound response pathways, long-distance signals interact with intracellular receptors that are directly linked to upregulation of key response genes. This may also be occurring in SAR as NPR1 appears to be an intracellular receptor of SA leading to activation of TGA transcription factors that upregulate expression of the PR genes in distant leaves during SAR (Fobert and Despre´s, 2005). It is notable that the signaling molecule JA is a long-distance signal in the wound response and has been implicated in both SAR and ISR, while the JA mimic, coronatine, is required in SIS. This suggests that JA may be a common component of long-distance signaling during defense and that long-distance communication has been co‐opted to aid in bacterial pathogenesis (Table II). In the wound response pathway, systemin does accumulate in the phloem, as does SA during SAR; however, neither appears to be a long-distance signal. Are systemin and SA accumulation in the phloem necessary for the wound response and SAR or are they present in the phloem because of a high capacity to accumulate as has been demonstrated for SA (Rocher et al., 2006) and in fact serve no function during SAR or the wound response?

TABLE II Signaling Components in SAR, SIS, WR, and ISR Signaling components

SAR

SIS

Long distance signals

DIR1? JA? MeSA? CDR1 Peptide? SABP2? NPR1? ?

Long distance signal receptors

WR

ISR

JA? COR?

JA

JA?

COI?

COI?

COI?

COR, coronatine; WR, wound response; SA, salicylic acid; MeSA, methyl-SA; CDR1, Constitutive defense response-1; DIR1, Defective in induced resistance-1; SABP2, SA-binding protein 2; NPR1, Nonexpressor of PR genes-1; COI, Coronatine insensitive.

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Tobacco grafting experiments indicate that SA accumulation in the induced leaf is not required for production and movement of the long-distance signal to distant tissues (Pallas et al., 1996; Vernooij et al., 1994), However, SAR in Arabidopsis is most often induced via R gene-mediated resistance and this response does require SA accumulation (Delaney et al., 1994) suggesting that SA accumulation is required during the induction stage of SAR in Arabidopsis. Identification and characterization of mutants that cannot accumulate SA in the induced leaf, but still retain SAR-inducing ability could shed some light on this question. Many questions concerning long-distance signaling in SAR, ISR, SIS, and the wound response remain unanswered. Molecular perception and dissemination of the long-distance signal(s) in distant leaves is unknown. The signal arrives in the distant leaves via the phloem and/or cell-to-cell movement and thus in both cases the signal(s) will be present in the cytoplasm with the ability to move to neighboring cells using plasmodesmata. It is possible to speculate that some cells at the leaf base are exposed to the long-distance signal(s) eliciting production of a secondary local signal that can spread readily via the phloem and cell-to-cell. In the case of long-distance signal perception during the wound response, PIN expression is restricted to mesophyll cells (Orozco-Ca´rdenas et al., 2001), raising the question of how the phloem-mobile signal (presumably JA or a JA-conjugate) gets access to and exerts its effects in mesophyll cells in the leaf. Hydrogen peroxide could act as a secondary local signal in systemic leaves on the basis of the observation that H2O2 travels through the apoplastic space of wounded tissue and is itself a potent inducer of PIN expression (Orozco-Ca´rdenas et al., 2001). Further research is warranted to investigate the role of hydrogen peroxide in systemic as well as locally wounded tissue. During the perception or manifestation stages of SAR, SA is a possible secondary signal as it is phloem mobile (Rocher et al., 2006) and SA levels do increase substantially in distant leaves. Additionally, evidence from Arabidopsis indicates that SA may act in a feedback loop to increase its own accumulation (reviewed in Shah, 2003). Therefore, sufficient SA could accumulate and spread to neighboring cells in a reiterative fashion to disseminate the SA signal to cells in the distant leaf leading to the ‘‘primed’’ state which probably involves NPR1 and other unidentified players.

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Van Loon, L. C., Bakker, P. A. H. M. and Pieterse, C. M. J. (1998). Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36, 453–483. Van Oosten, V. R., Bodenhausen, N., Reymond, P., Van Pelt, J. A., Van Loon, L. C., Dicke, M. and Pieterse, C. M. J. (2008). Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis. Molecular Plant-Microbe Interactions 21, 919–930. Van Peer, R., Niemann, G. J. and Schippers, B. (1991). Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 81, 728–734. Van Wees, S. C. M., Luijendijk, M., Smoorenburg, I., Van Loon, L. C. and Pieterse, C. M. J. (1999). Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct affect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge. Plant Molecular Biology 41, 537–549. Van Wees, S. C. M., De Swart, E. A. M., Van Pelt, J. A., Van Loon, L. C. and Pieterse, C. M. J. (2000). Enhancement of disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 97, 8711–8716. Van Wees, S. C. M., Van der Ent, S. and Pieterse, C. M. J. (2008). Plant immune responses triggered by beneficial microbes. Current Opinion in Plant Biology 11, 443–448. Verberne, M. C., Hoekstra, J., Bol, J. F. and Linthorst, H. J. M. (2003). Signaling of systemic acquired resistance in tobacco depends on ethylene perception. The Plant Journal 35, 27–32. Verhagen, B. W. M., Glazebrook, J., Zhu, T., Chang, H.-S., Van Loon, L. C. and Pieterse, C. M. J. (2004). The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Molecular Plant-Microbe Interactions 17, 895–908. Vernooij, B., Friedrich, L., Morse, A., Reist, R., Kolditz-Jawhar, R., Ward, E., Uknes, S., Kessmann, H. and Ryals, J. (1994). Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. The Plant Cell 6, 959–965. Vlot, A. C., Liu, P.-P., Cameron, R. K., Park, S.-W., Yang, Y., Kumar, D., Zhou, F., Paddukavidana, T., Gustafsson, C., Pichersky, E. and Klessig, D. F. (2008). Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana. The Plant Journal 56, 445–456. Ward, E. R., Uknes, S. J., Williams, S. C., Dincher, S. S., Wiederhold, D. L., Alexander, D. C., Ahl-Goy, P., Me´traux, J.-P. and Ryals, J. A. (1991). Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3, 1085–1094. Washburn, C. F. and Thomas, J. F. (2000). Reversion of flowering in Glycine max (Fabaceae). American Journal of Botany 87, 1425–1438. Wasternack, C. (2007). Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Annals of Botany 100, 681–697. Wigge, P. A., Kim, M. C., Jaeger, K. E., Busch, W., Schmid, M., Lohmann, J. U. and Weigel, D. (2005). Integration of spatial and temporal information during floral induction in Arabidopsis. Science 309, 1056–1059.

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Systemic Acquired Resistance

R. HAMMERSCHMIDT1

Department of Plant Pathology, 107 Center for Integrated Plant Systems Building, Michigan State University, East Lansing, MI 48824-1311, USA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Systemic Acquired Resistance ................................................ B. Other Forms of Induced Resistance ......................................... II. The Biological Spectrum of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The Induction of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Necrotizing Pathogens ......................................................... B. The Hypersensitive Response................................................. C. Is Pathogen-Induced Necrosis Needed for SAR Induction? ............. D. Pathogen-Produced Inducers of SAR ....................................... E. Chemical Induction of SAR .................................................. IV. Systemic Biochemical Changes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pathogenesis-Related Proteins................................................ B. Other Proteins................................................................... C. SA Accumulation............................................................... V. How SAR Protects Plants Against Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Priming ........................................................................... B. Protection Against Fungi and Oomycetes .................................. C. Protection Against Bacteria................................................... D. Protection Against Viruses .................................................... E. Mechanisms of Defense in Summary........................................ B. What Don’t We Know? ....................................................... VI. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51005-1

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ABSTRACT Systemic acquired resistance (SAR) is a form of induced resistance that is activated by pathogens that induce localized necrotic disease lesions or a hypersensitive response. A major characteristic of SAR is the broad spectrum nature of the protection it confers against a wide range of pathogens, although recent studies suggest that the resistance is most effective against biotrophic and hemibiotrophic pathogens and less effective against necrotrophs. SAR is dependent on salicylic acid signaling and is typically associated with systemic expression of pathogenesis-related protein genes and other putative defenses. Once induced, SAR-expressing plants are primed to respond to subsequent pathogen infection by induction of defenses that are localized at the site of attempted pathogen ingress. Finally, SAR typically does not provide full resistance to disease indicating that the practical application of this form of resistance will require the use of other disease management tools. On the basis of these types of observations, it is likely that SAR and other forms of induced resistance are based on the enhanced ability to express basal defenses.

I. INTRODUCTION It is probably safe to assume that all plants have the genes needed to mount an effective defense against pathogens. This is illustrated by the diversity of plant defenses and the fact that plants resist most pathogens (Hammerschmidt, 1999a,b; Hammerschmidt and Nicholson, 1999; Hu¨ckelhoven, 2007; Van Loon et al., 2006). Thus, one difference between a resistant and a susceptible plant may reside in the timely expression of these defenses. This is certainly true for resistance controlled by the interactions between pathogen effectors and host resistance (‘‘R’’) genes. In this case, plant R genes provide the plant with the ability to detect the pathogen and then rapidly express defenses after infection. This is also true for the nonhost resistance expressed in plants that are under attack by pathogens of other plant species. In these cases, the failure of the pathogen to invade and colonize the nonhost is associated with the expression of a number of defenses. The observations that even susceptible plants can mount some degree of defense against pathogens plays into the overall concept that plants come equipped with defense genes. This form of defense, known as basal disease resistance (Jones and Dangl, 2006), is induced in susceptible plants upon infection with compatible pathogens. Although not effective enough to stop the pathogen, basal defenses may help limit the spread of the disease in the infected tissue. These defenses are likely the same as those induced in other forms of resistance, though they may often be expressed too late or at too low a level to be totally effective (phytoalexin accumulation is one good example of this type of defense response) (e.g., Hammerschmidt, 1999b).

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The assumption that plants have all necessary defenses also provides support for the general phenomenon of induced resistance and forms the basis for the reason why induced resistance exists. In induced resistance, certain biotic or abiotic treatments of a susceptible plant alter its defensive abilities in such a way that resistance is enhanced to one or more pathogens (Hammerschmidt, 1999a; Kuc´, 1982). It is important to note that the induced plants may still become diseased, indicating that induced resistance does not provide the level of resistance mediated by major R genes. Depending on the type of inducing agent and the signaling pathways involved, induced resistance can be classified in different ways. The two forms of induced resistance that have been best characterized are systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Van Loon et al., 1998). However, it is likely that other forms of induced resistance exist. There has been an explosion of information published on induced resistance and its many forms in the last few decades (e.g., see reviews by Durrant and Dong, 2004; Sticher et al., 1997; Vallad and Goodman, 2004; Van Loon et al., 1998; chapters in Walters et al., 2007). As such, it is impossible to cover all the aspects of this type of resistance in one chapter; here, the main focus will be on the various mechanistic and resistance characteristics of the SAR form of induced resistance. The reader is directed to other reviews in this volume and elsewhere for a more detailed discussion of the signaling pathways found in SAR and in other forms of induced disease resistance (e.g., Bostock, 2005; Durrant and Dong, 2004; Glazebrook, 2005; Pieterse and Van Loon, 2004, 2007; Van Loon et al., 1998). A. SYSTEMIC ACQUIRED RESISTANCE

SAR can be broadly defined as a form of induced resistance that is activated throughout a plant typically following infection by a pathogen that causes localized necrotic lesions. The necrosis can be the result of disease induced by a pathogen or a hypersensitive response (HR) (e.g., Kuc´, 1982; Kuc´ et al., 1975; Ross, 1961b). Multiple rounds of inducing inoculations (i.e., ‘‘booster’’ inoculations) can also increase the level of SAR (Kuc´, 1982). SAR is dependent on salicylic acid (SA) signaling (Gaffney et al., 1993). Although the role of SA as a mobile signal for SAR is still debatable (Rasmussen et al., 1991; Shulaev et al., 1995; Vernooij et al., 1994), there is little doubt that this simple phenol is essential for the expression of SAR (Delaney et al., 1994). The development of SAR throughout a plant takes several days and is accompanied by the systemic expression of genes encoding pathogenesisrelated (PR) proteins and the accumulation of their protein products (Hammerschmidt, 1999a; Van Loon, 1997; Van Loon and Van Strien, 1999;

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Van Loon et al., 2006). Another characteristic of SAR is the requirement of the NPR1 (Nonexpressor of PR genes 1) protein, functioning downstream of SA, as a major regulatory factor in the expression of SAR, PR-protein accumulation, and priming. SA accumulation in the cytoplasm affects the cellular redox state. This change is involved in the reduction of cytoplasmic NPR1 protein that then enters the nucleus and interacts with TGA transcription factors (Pieterse and Van Loon, 2004, 2007). Plants expressing SAR are ‘‘primed’’ to respond to subsequent infections by expression of additional defenses, such as the oxidative burst, cell wall alterations at the site of attempted infection, and phytoalexin production (Conrath et al., 2000, 2002, 2006). A final characteristic of SAR is that the resistance is effective against a broad range of pathogens that include bacteria, true fungi, oomycetes, and viruses (Deverall, 1995; Hammerschmidt and Kuc´, 1995; Kuc´, 1982). Within this range, recent studies with model systems (e.g., Arabidopsis thaliana) suggest that SAR, and SA-mediated resistance in general, may be most effective against biotrophic and hemibiotrophic pathogens and not against necrotrophs (Glazebrook, 2005; Oliver and Ipcho, 2004). However, as discussed later in this review, SA induces resistance to viruses by an NPR1-independent mechanism (e.g., Singh et al., 2004). B. OTHER FORMS OF INDUCED RESISTANCE

Another form of induced resistance is ISR. Like SAR, ISR is systemic and can be effective against a number of pathogens. Unlike SAR, ISR induction is associated with the interaction of roots with certain plant growth-promoting rhizobacteria that do not elicit a necrotic response or cause any type of visible symptoms. In addition, ISR is dependent on jasmonic acid (JA)- and ethylene (ET)-signaling pathways, and its induction does not result in systemic expression of PR genes (Van Loon et al., 1998). As a contrast to SAR, studies in Arabidopsis have suggested that ISR is most effective against necrotrophic pathogens (Glazebrook, 2005). Like SAR, plants expressing ISR are also primed to express additional defenses upon infection (Conrath et al., 2006) and also require NPR1 (Pieterse and Van Loon, 2004, 2007). More details on the ISR form of induced resistance can be found elsewhere in this volume (De Vleesschauwer and Ho¨fte, 2009). Induced resistance can also be localized to the tissue that is treated with an inducing agent. Some of the earliest studies on induced resistance were conducted by treating entire plants or parts of plants with an inducing agent, such as an avirulent race of a pathogen or a pathogen of another plant species, followed by inoculating the treated tissues one or more days later with a virulent pathogen (e.g., Hammerschmidt et al., 1976; Rahe et al.,

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1969; Ross, 1961a; Skipp and Deverall, 1973). In these cases, it is difficult to determine what types of mechanisms are involved in the resistance. It is possible that defenses induced by the initial inoculation may be sufficient to block infection by a subsequent inoculation with a pathogen. This is probably what occurs in the resistance activated by the herbicide lactofen in soybean to Sclerotinia sclerotiorum (Dann et al., 1999). Lactofen induces the accumulation of the glyceollin phytoalexins, and upon subsequent inoculation leaves that have accumulated glyceollin are more resistant to infection. No resistance is detected several weeks later when the content of glyceollin returns to baseline levels (Dann et al., 1999). However, prior localized infections may protect through the activation of resistance potential in surrounding host cells. For example, the resistance of cucumber to Cladosporium cucumerinum, the cause of scab, is associated with the rapid deposition of lignin at the site of attempted infection (Hammerschmidt et al., 1984). Preinoculation of scab-resistant cucumber seedlings with Cl. cucumerinum induces local resistance to Colletotrichum orbiculare (syn.: Colletotrichum lagenarium). A histochemical analysis of the tissues revealed that an active defense against Co. orbiculare was expressed in cucumber epidermal cells from seedlings preinfected with Cl. cucumerinum (Hammerschmidt and Kuc´, 1982). The host cells that Co. orbiculare appressoria were attempting to invade were several cells away from any lignification induced by Cl. cucumerinum. This suggested that the resistance was not merely the result of physical blocking of infection by the lignin induced by the inducing inoculation.

II. THE BIOLOGICAL SPECTRUM OF SAR Systemic resistance induced by necrotizing pathogens or the HR has been observed in many plant species (e.g., Deverall, 1995; Hammerschmidt and Kuc´, 1995; Kuc´, 1982; Sticher et al., 1997; Walters et al., 2007). Thus, it is likely that SAR is a general phenomenon throughout the plant kingdom. However, detailed studies on SAR have been limited to only a handful of plant species and some species, especially non‐angiosperm plants such as the conifers (Bonello et al., 2006), have received only cursory examination.

III. THE INDUCTION OF SAR A. NECROTIZING PATHOGENS

After inoculation with a resistance-inducing microbe, SAR takes several days to develop and the time of appearance is associated with the development of necrosis produced by the inducing organism (Kuc´, 1982). For example, early

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work on the tobacco-Peronospora tabacina interaction demonstrated that necrotic stem lesions caused by Pe. tabacina would systemically protect the foliage from infection by Pe. tabacina (Cruickshank and Mandryk, 1960; Pont, 1959). Cohen and Kuc´ (1981) later confirmed the observations with tobacco and Pe. tabacina. Using cucumber, Kuc´ and coworkers demonstrated that localized necrotic lesions produced on the first true leaf of cucumber plants following inoculation with Co. orbiculare or Pseudomonas syringae pv. lachrymans resulted in systemic resistance to these and other pathogens of cucumber (Caruso and Kuc´, 1979; Kuc´ et al., 1975). Increasing the number of necrotic lesions on the leaf used for induction also increased the expression of SAR (Caruso and Kuc´, 1979; Kuc´ and Richmond, 1977).

B. THE HYPERSENSITIVE RESPONSE

The necrotic local lesions typical of the HR induced by tobacco mosaic virus (TMV) on N gene tobacco are also effective in inducing resistance to TMV (Ross, 1961a,b) and other tobacco pathogens (e.g., Friedrich et al., 1996). Increasing the number of TMV lesions on the leaf used for induction also increased the level of systemic resistance (Ross, 1961b). Similarly, Jenns and Kuc´ (1977, 1980), reported that inoculation of cucumber plants with tobacco necrosis virus (TNV) induced systemic resistance to Co. orbiculare, Ps. syringae pv. lachrymans and to itself. In addition to viruses, the HR elicited by bacteria can also induce SAR. Cameron et al. (1994) successfully induced a SAR response in Arabidopsis by inoculating one leaf with Ps. syringae pv. tomato carrying avrRpt. This interaction resulted in a HR and, by 2 days, the expression of resistance in other leaves to inoculation with a strain of Ps. syringae pv. tomato lacking avrRpt and of Ps. syringae pv. maculicola. Smith et al. (1991) were able to induce SAR in cucumber with a wheat isolate of Ps. syringae pv. syringae that caused a rapid HR in cucumber leaves. Using Co. orbiculare as the challenge pathogen, the resistance induced by Ps. syringae pv. syringae was reported to be fully expressed within 24 h of the inducing inoculation. These authors also confirmed the observations of Caruso and Kuc´ (1979) that the cucumber pathogen Ps. syringae pv. lachrymans also induced SAR. Ps. syringae pv. lachrymans took several days longer to induce systemic resistance as compared to Ps. syringae pv. syringae. This may be because the necrotizing response to the pathogen was much slower.

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C. IS PATHOGEN-INDUCED NECROSIS NEEDED FOR SAR INDUCTION?

Although the formation of necrotic lesions has been correlated with the induction of SAR by pathogens, is necrosis really necessary for induction by these microbes? Some early indication of the need for pathogen-induced necrosis was the observation of Jenns and Kuc´ (1980) who found that nonnecrotic, yet localized, ‘‘starch lesions’’ induced by TMV in cucumber cotyledons did not induce SAR in this plant against Co. orbiculare (although it was later reported by Roberts (1982) that TMV would induce systemic resistance in cucumber to TNV). Neither heat- or formalin-killed cells of Ps. syringae pv. lachrymans nor cell-free sonicates of this bacterium induced necrosis or SAR in cucumber (Caruso and Kuc´, 1979). Furthermore, necrotic areas induced by dry ice damage were ineffective or SAR in inducing SAR in cucumber (Hammerschmidt et al., 1982). Dean and Kuc´ (1986a) indirectly addressed this question by inoculating the first true leaf of cucumber plants with Co. orbiculare and then excising the inoculated leaf at intervals over the next 6 days. The second (true) leaf of all plants was challenge inoculated with the same pathogen 7 days after the inoculation of the first leaf. They found that the first leaf needed to be on the plant for at least 72 h for any expression of SAR in the second leaf. This time is when necrotic lesions caused by Co. orbiculare are first visible. The longer the first leaf remained on the plant (and thus time for additional necrosis development as lesions expanded), the stronger was the observed SAR response in leaf two. Using the HR induced by Ps. syringae pv. syringae as the inducing treatment, Smith et al. (1991) found that resistance could be induced in cucumber within 24 h. Taking advantage of this rapid response, Jennifer Smith and colleagues essentially repeated the experiments of Dean and Kuc´ (1986a), but removed the Ps. syringae-inoculated leaf at 0, 3, 6, 9 and 24 h after inoculation. The second leaf of all plants was challenged with Co. orbiculare at 24 h after the induction inoculation. Smith et al. (1991) reported that the first leaf only needed to be on the plant for as little as 6 h for a measurable amount of SAR to be expressed. Visual observations of the Ps. syringae-inoculated tissue showed initial signs of tissue collapse by 6 h after inoculation, but no clearly visible necrosis until several hours later (unpublished observation). Inoculation of cucumber plants with hrp (hypersensitive response and pathogenicity)-minus mutants of Ps. syringae pv. syringae did not result in the expression of the HR or SAR. Restoration of HR-inducing ability in the mutants by complementation restored the ability to induce SAR (Smith et al., 1991). Further studies on this system by Rasmussen et al. (1991) demonstrated that systemic induction of SA by Ps. syringae pv. syringae

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required that the inoculated first leaf only be on the plant for a few hours. Thus, confirming that the induction of SAR may occur prior to full expression of host necrosis. Further supporting a role for HR-like necrosis as part of the induction phase was the observation of Strobel et al. (1996) who used Ps. syringae 61 (Pss61) and HrpZPss protein from this bacterium to induce SAR in cucumber. Both the pathogen and the protein induced SAR that was effective against Co. orbiculare, Ps. syringae pv. lachrymans and TNV with the degree of protection comparable to that induced by Co. orbiculare. A hrpH mutant of Pss61 was unable to elicit SAR (possibly because it was unable to secrete the HrpZPss protein). Another Hrp protein, harpin (HrpNEa) was also reported to induce the HR and SAR response when sprayed onto tobacco or Arabidopsis leaves (Peng et al., 2003). Although no visible necrosis was observed, microscopic observations demonstrated that micro‐HRs were induced in both plant species, thus further supporting some association of pathogenesis-induced necrosis with the induction of SAR. However, is pathogen-induced necrosis really necessary to induce SAR? The experiments described thus far show possible correlations but little proof. In fact, the research by Smith et al. (1991) suggests that perhaps events leading up to necrosis (i.e., part of the initial phase of plant–microbe interaction) are what trigger SAR and that host cell death is not needed. Recently, the need for pathogen-induced necrosis for SAR induction has been questioned by Mishina and Zeier (2007). They found that Ps. syringae pv. glycinea and pv. phaseolicola do not elicit a HR in Arabidopsis but do induce SAR at a level similar to the HR-inducing Ps. syringae pv. tomato. Interestingly, hrp mutants of Ps. syringae pv. tomato and pv. phaseolicola also induced SAR in Arabidopsis. The authors showed that lipopolysaccharides (LPS) and flagellin protein, two pathogen-associated molecular patterns (PAMPs) (Nu¨rnberger and Kemmerling, 2009), were able to induce SAR in Arabidopsis. These results differ from other reports in which hrp mutants are unable to induce SAR (e.g., Smith et al., 1991). However, it may be that Arabidopsis may respond differently than other plant species as also reflected by the observation that necrosis induced in Arabidopsis by Botrytis cinerea did not result in SAR expression (Govrin and Levine, 2002). Another line of evidence that suggests that necrosis is not needed for the induction of SAR comes from the effect of systemic mosaic virus infection on disease resistance. Mu¨ller and Munro (1951) reported that infection of potato with potato virus X (PVX) or potato virus Y (PVY) increased resistance of the plants to infection by Phytophthora infestans. Both viruses were systemic and produced mosaic patterns in the potato plants.

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Van Loon (1975) provided further evidence that systemic infection with mosaic-inducing viruses would induce resistance. Inoculation of tobacco with PVYº or cucumber mosaic virus (CMV) did not induce necrosis, yet both viruses induced resistance and the accumulation of PR-proteins. It would be of great interest to determine if infection by these viruses also induces the accumulation of SA. The question still remains: Is necrosis needed for the induction of SAR? The answer is probably ‘‘no.’’ However, the events involved in the early stages of pathogenesis that lead to necrosis might be. It is also possible that what is true in one plant system may not be valid in another. This is shown by the differences in induction observed in the work of Smith et al. (1991) with cucumber and that of Mishina and Zeier (2007) with Arabidopsis. Application of molecular tools derived from studies on Arabidopsis SAR may prove very useful asking the same questions in other plant species. D. PATHOGEN-PRODUCED INDUCERS OF SAR

As described above, there are pathogen-derived molecules, such as LPS (Newman et al., 2001), that can induce systemic resistance and possibly SAR. Other known inducers are the unsaturated fatty acids arachidonic acid and eicosapentaenoic acid from Phy. infestans that were first identified as an elicitor of sesquiterpenoid phytoalexins in potato tuber tissue (Bostock et al., 1981). These fatty acids were later shown to induce systemic resistance to Phy. infestans in potato plants (Cohen et al., 1991). Other SAR-activating compounds that could be released from oomycetes and true fungi include cell wall fragments composed of chitosan, chitin, and -1,3-glucan (Lyon, 2007; Reglinski et al., 2007). These elicitors could be released from invading pathogens by action of host chitinases and -1,3-glucanases and act as activators of local, if not systemic, resistance. Other examples can be found in the reviews by Lyon (2007) and Reglinski et al. (2007). E. CHEMICAL INDUCTION OF SAR

In addition to pathogens, induced resistance can also be activated by chemicals. Many naturally occurring and synthetic chemicals have been shown to increase disease resistance, and the reader is directed to Lyon (2007) and Reignault and Walters (2007) for more comprehensive lists. Kessmann et al. (1994) provide an interesting background perspective on the use of chemicals as SAR activators. Whether or not all chemicals induce a SAR type of response is not known, and this section/chapter will focus on a few chemistries that appear to induce SAR.

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1. Salicylic acid One of the first chemicals shown to induce resistance is SA (2-hydroxybenzoic acid). Well before this simple hydroxybenzoic acid was demonstrated to be an endogenous signal for SAR (Malamy et al., 1990; Me´traux et al., 1990), White (1979) reported that the application of SA or acetyl salicylic acid (ASA) induced resistance to TMV in N gene tobacco. Further studies showed that application to tobacco would also induce the formation of PR-proteins (another characteristic of SAR) (Van Loon and Antoniw, 1982). Mills and Wood (1984) demonstrated that SA induced resistance in cucumber to Co. orbiculare, and Narusaka et al. (1999) reported that SA induced resistance in this species to Cl. cucumerinum. 2. 2,6-Dichloroisonicotinic acid DCINA (2,6-dichloroisonicotinic acid and its methyl ester) was the first synthetic compound produced as a resistance inducer (Me´traux et al., 1991). This compound induced resistance in cucumber, pear, pepper and rice (Me´traux et al., 1991). Ward et al. (1991) reported that DCINA induced resistance in tobacco to TMV and also elicited the same spectrum of PR genes induced by TMV and SA. These results provided very good evidence that DCINA was an inducer of SAR. Treatment of Arabidopsis with DCINA induced resistance to both Ps. syringae pv. maculicola and Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica) (Uknes et al., 1992). Based on these observations, DCINA appears to induce SAR by mimicking the activity of SA. Treatment of the unifoliate leaves of bean (Phaseolus vulgaris) with DCINA or inoculation with Colletotrichum lindemuthianum (the cause of bean anthracnose) induced resistance in the trifoliate leaves to Co. lindemuthianum and the bean rust pathogen, Uromyces appendiculatus (Dann and Deverall, 1995). DCINA also induced resistance to Ps. syringae pv. phaseolicola but not to root rotting Fusarium solani or Rhizoctonia (Dann and Deverall, 1995). Further studies showed that DCINA would also protect field grown beans against rust (Dann and Deverall, 1996). Treatment of unifoliate bean leaves with DCINA or Co. lindemuthianum induced an increase in activity of chitinase and -1,3-glucanase in upper, trifoliate leaves, thus further demonstrating the SAR-inducing ability of DCINA (Dann et al., 1996). Dann et al. (1998) tested the efficacy of DCINA for inducing resistance in soybean to the white mold pathogen, Sc. sclerotiorum in both the greenhouse and in the field. Multiple applications of DCINA were needed to result in a reduction of the lesions’ size in greenhouse studies. Three years of field trials showed some efficacy of DCINA in reducing white mold severity after multiple applications of the compound.

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3. Acibenzolar-S-methyl Acibenzolar-S-methyl (S-methyl benzo [1,2,3]thiadiazole-7-carbothioate, ASM; synonym: benzothiadiazole-7-carbothioic acid S-methyl ester, BTH) is another synthetic compound that induces SAR (Kunz et al., 1997; Tally et al., 1999). This material is commercially available as a plant activator and sold under the names of BionÒ , ActigardÒ , and BoostÒ (Leadbeater and Staub, 2007). Like DCINA, ASM is thought to be a functional analog of SA as it induces resistance to multiple pathogens and elicits the expression of PR genes associated with SAR (Oostendorp et al., 2001). In 1996, several papers demonstrating the SAR-inducing ability of ASM were published. Lawton et al. (1996) showed that treating Arabidopsis plants with ASM induced resistance to turnip crinkle virus, Ps. syringae pv. tomato and Hy. arabidopsis. ASM was also capable of inducing expression of PR-1, PR-2 and PR-5, thus further demonstrating that ASM induced a SAR-like response. Through the use of NahG plants that are unable to accumulate SA (Gaffney et al., 1993), these authors were able to show that ASM action did not require SA and thus likely functioned downstream of, or at the same site as SA. Using tobacco, Friedrich et al. (1996) found that both TMV and ASM induced SAR against TMV, Cercospora nicotianae, Erwinia carotovora, Ps. syringae pv. tabaci and Phytophthora parasitica. They also reported that ASM induced resistance to Pe. tabacina. The ability of ASM to induce SAR was further demonstrated by Friedrich et al. (1996) by the ability of this compound to induce expression of PR genes as well as genes for peroxidase and other defense-associated enzymes. Recent work by LaMondia (2008) noted that ASM, especially when combined with traditional fungicides, was an effective management tool for Pe. tabacina under field conditions. Go¨rlach et al. (1996) further expanded the diversity of plants induced by ASM by testing its effect on wheat. ASM induced resistance to the powdery mildew pathogen Blumeria graminis and induced the expression of a novel set of genes in wheat (WCI genes). Interestingly, two PR-1 genes from wheat, PR1.1 and PR1.2, were induced by both compatible and incompatible isolates of Bl. graminis but not by ASM, DCINA or SA. This suggested that ASM-modulated defense in wheat may differ from dicots. Smith-Becker et al. (2003) induced resistance in melons (Cucumis melo) to both CMV and Co. orbiculare. ASM greatly reduced the spread of CMV from infected leaves to young developing leaves and nearly completely eliminated the lesion development after challenge inoculation with the fungus. Huang et al. (2000) were able to protect the fruit of Hami and rock melons from postharvest fungal diseases. The ability of ASM to induce resistance is not restricted to herbaceous plants. Brisset et al. (2000) and Maxson-Stein et al. (2002) treated

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orchard-grown apple trees with ASM and were able to suppress the fire blight disease caused by Erwinia amylovora. More than one application was needed to reduce fire blight severity, but the control was similar to that observed with streptomycin. Local and systemic accumulation of peroxidase and -1,3-glucanase activity (Brisset et al., 2000) and induction of PR-1, PR-2, and PR-8 gene expression (Maxson-Stein et al., 2002) following ASM treatment accompanied the induction of resistance. ASM also induced resistance in Japanese pear trees against the scab pathogen Venturia nashicola (Ishii et al., 1999). They reported that the ASM-induced resistance to scab was similar to control by polycarbamate fungicides. There are many other examples of ASMinduced resistance in the literature, and reviews by Reglinski et al. (2007) and Reignault and Walters (2007) provide much of this information. 4. Tiadinil A new plant activator, Tiadinil (TDL, N-(3-chloro-4-methylphenyl)-4-methyl1,2,3-thiadiazole-5-carboxamide), was developed for rice blast management (Yasuda et al., 2004, 2006). TDL also induces resistance in tobacco to Ps. syringae pv. tabaci and TMV as well as expression of PR genes. TDL is metabolized in rice to 4-methyl-1,2,3-thiadiazole-5-carboxylic acid which is also capable of inducing resistance and PR gene expression in tobacco. This metabolite is also effective in tobacco expressing NahG suggesting that these compounds, like DCINA and ASM, act downstream of SA. 5. Other chemical inducers There are many other synthetic and natural compounds that have been reported as activators of disease resistance, and there are several reviews that provide information on these materials (Deverall, 1995; Kessmann et al., 1994; Leadbeater and Staub, 2007; Lyon, 2007). In many cases, the mode of action of the compounds or materials that have been reported to induce resistance is not known. Some, like the nonprotein amino acid -aminobutyric acid (BABA) have been reported to induce resistance in a number of plant species (Cohen, 2002). The mode of action of this chemical suggests that it may function through both SA-mediated resistance (Siegrist et al., 2000) as well as abscisic acid signaling (Ton and Mauch-Mani, 2004). Thus, BABA may induce SAR (albeit via SA induction) and certainly other defense signaling pathways. The harpin protein from Erw. amylovora induces resistance in Arabidopsis to Hy. arabidopsidis and Ps. syringae pv. tomato and also induces expression of PR-1 and PR-2 genes (Dong et al., 1999). Because of PR gene expression and the observation that harpin did not induce resistance in NahG plants, harpin is likely an inducer of SAR through the induction of SA biosynthesis.

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The ability of harpin to induce microlesions which trigger the SAR response also suggests that this protein induces SAR (Peng et al., 2003). Another compound that activates resistance through induction of SA production is the fungicide probenazole (Leadbeater and Staub, 2007). Probenazole and its metabolite 1,2-benzoisothiazole-1,1-dioxide (saccharin) were shown to induce SAR by their ability to induce PR gene expression (Yoshioka et al., 2001). Unlike ASM or DCINA, these activators appear to act upstream of SA as both induce the accumulation of SA (Nakashita et al., 2002). Probenazole or saccharin treatment of NahG tobacco plants did not result in the induction of SAR, thus demonstrating the need for SA accumulation as part of the mode of action of these compounds (Yoshioka et al., 2001). Saccharin is also able to induce resistance in barley to powdery mildew (Boyle and Walters, 2006) and in broad bean to rust (Boyle and Walters, 2005). The strobilurin fungicide pyraclostrobin has also been reported to induce resistance. Injection of tobacco leaves with this compound induced resistance to TMV and Ps. syringae pv. tabaci (Herms et al., 2002). Pyraclostrobin by itself did not induce PR-1 protein accumulation. However, the fungicide did prime the tissue to more rapidly express PR-1 and accumulate PR-1 protein after inoculation with TMV. This suggests that pyraclostrobin does not induce SAR in the classical sense. However, the observation that the tissues were primed suggests that pyraclostrobin is an inducer of SAR or a SAR-like response. This study also illustrates the type of work that is needed to determine if and how materials reported to induce resistance actually function in disease suppression.

IV. SYSTEMIC BIOCHEMICAL CHANGES It is logical to assume that if plant tissues at some distance from an induction treatment are more effective in stopping pathogens than noninduced tissues, it follows that there are measurable biochemical changes in the induced tissues. This chapter will describe some of the biochemical changes that have been reported. A. PATHOGENESIS-RELATED PROTEINS

In the early 1970s, the systemic increase in novel proteins in N gene tobacco infected with TMV was reported (Van Loon, 1975; Van Loon and Van Kammen, 1970). These proteins were not found in uninfected tissues and were not peroxidases or other enzymes known to increase systemically after

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TMV infection (e.g., Simons and Ross, 1970). Van Loon and Van Kammen (1970) proposed that these proteins were involved in the SAR response. Following the work of White (1979) who found ASA and SA would induce resistance in tobacco, Van Loon and Antoniw (1982) reported that SA was also capable of inducing these SAR-associated proteins. Over a period of time, 17 families of proteins, now known as PR-proteins have been identified in a number of plant species and putative functions such as chitinase and -1,3-glucanase have been determined for some of these proteins (for reviews see Van Loon, 1997; Van Loon et al., 2006). B. OTHER PROTEINS

In addition to the classical PR-proteins, the accumulation of other putative defense-related proteins or expression of their genes has been associated with SAR. In some of A. Frank Ross’s early work on SAR induction in tobacco, he and colleagues reported on TMV-induced systemic increases in peroxidase, catalase and glucose-6-phosphate dehydrogenase activities (Simons and Ross, 1970, 1971a) but not polyphenoloxidase (Bozarth and Ross, 1964). A change in peroxidase activity was also observed by Van Loon and Geelen (1971). These results indicate an increase in oxidative metabolism as well as a possible increase in respiratory metabolism mediated by the increase in glucose-6-phosphate dehydrogenase. In cucumber and other cucurbits, a set of acidic, apoplastic peroxidases accumulates systemically upon infection of one leaf with necrotizing pathogens (Hammerschmidt et al., 1982; Smith and Hammerschmidt, 1988). These same peroxidases and their transcripts also accumulate in response to SA treatment (Rasmussen et al., 1991, 1994). Systemic increases in a microsomal callose synthase have been reported in cucumber plants expressing systemic resistance (Schmele and Kauss, 1990). As cell wall alteration involving callose deposition may be part of the defense of plants, activation of this enzyme may be part of the defense against cell wall penetrating pathogens. Hydroxyproline-rich glycoproteins (HRGP) are structural components of plant cell walls that have been implicated in host defense responses (e.g., Hammerschmidt et al., 1984; Stermer and Hammerschmidt, 1987). Accumulation of these proteins increases the resistance of cell walls to cell wall-degrading enzymes (Stermer and Hammerschmidt, 1987). Induction of resistance in tobacco by TMV results in systemic accumulation of HRGP in the host cell walls and resistance to Pe. tabacina (Ye et al., 1992) and Erysiphe cichoracearum (Raggi, 1998). Similarly, PVYN, which causes veinal necrosis in tobacco, also induced resistance to Ery. cichoracearum and caused a systemic increase in HRGP (Raggi, 2000). Treatment of tobacco leaves

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with SA or ASM resulted in local increases in HRGP content of cell walls. ASM, but not SA, also caused a systemic increase in HRGP (Raggi, 2007). This difference is likely the result of the different mobility of SA as compared to ASM after topical application. Since treatments with ASM and SA also result in increases in cell wall associated peroxidases, it is possible that one function of the induced peroxidase activity is to facilitate the incorporation of newly synthesized HRGP into cell walls (Sommer-Knudsen et al., 1998). In addition to proteins considered to be part of defense responses, induction of SAR also results in expression of genes encoding types of proteins used for pathogen detection. In 1999, Sakamoto et al. reported that induction of resistance in rice by probenazole was associated with the expression of a gene, RICE PROBENAZOLE-RESPONSIVE 1 (RPR1), which codes for a protein with a nucleotide binding site and leucine-rich repeats (NBS-LRR). SA treatment of rice resulted in induction of the NBS-LRR Pib rice-blastresistance gene family (Wang et al., 2001). Finally, a similar result was reported by Faize et al. (2007) who found that ASM treatment of Japanese pear resulted in expression of a gene for a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Taken together, these reports suggest that one aspect of resistance induction may involve the expression of genes involved in pathogen detection and, therefore, may allow the induced plants the ability to more rapidly respond to attempted infections. C. SA ACCUMULATION

Induction of SAR in tobacco and cucumber results in the systemic accumulation of SA (Malamy et al., 1990; Me´traux et al., 1990). Because of the resistance-inducing properties of SA and the observation that this simple phenol was found in the phloem of cucumber during the induction process, it seemed plausible that SA was the mobile signal required for SAR development. In fact, there is evidence for the systemic transport of SA out of infected leaves of cucumber (Mo¨lders et al., 1996) and tobacco (Shulaev et al., 1995). Although some SA may be transported out of leaves undergoing necrotic lesion formation, work by Rasmussen et al. (1991) with cucumber and Vernooij et al. (1994) with tobacco cast doubt on the role of SA as the primary systemically transported signal, and suggested that it may be synthesized systemically in response to another, yet to be described, mobile signal. This concept was supported by observations that some SA was synthesized at a distance from inducing inoculations. Meuwly et al. (1995) found that both locally inoculated and systemically induced cucumber tissues produced more SA from phenylalanine than did control plants. The conversion of phenylalanine to SA was blocked by the

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phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindan-2-phosphonic acid. Also working with cucumber, Smith-Becker et al. (1998) found an increase in PAL in petioles of leaves inoculated with Ps. syringae pv. syringae and stem tissue above the inoculated leaf at 9 and 12 h after inoculation. Both SA and 4-hydroxybenzoic acid could be detected in petiole and stem phloem exudates at a time corresponding to increased PAL activity. More details on the role of SA and systemic signals can be found in the chapter by Champigny and Cameron (2009).

V. HOW SAR PROTECTS PLANTS AGAINST PATHOGENS How plants actually stop pathogen development and suppress disease symptoms in plants expressing SAR is slowly being revealed, and progress has been made since I asked this question a decade ago (Hammerschmidt, 1999a). The production of PR-proteins as a result of resistance induction is likely part of the defense process (Van Loon, 1997; Van Loon et al., 2006) and possibly serving as a ‘‘preformed’’ defense against infection. However, plants expressing high levels of these proteins may not be protected against disease. For example, the activity of chitinase, a major PR-protein in cucumber (Me´traux et al., 1988, 1989), remains high in leaves even after the acquired resistance levels decline (Dalisay and Kuc´, 1995). This work and other lines of evidence (see Van Loon, 1997, for review) suggest that the PR-proteins play some, but perhaps not a decisive defense role in SAR. A. PRIMING

Plants that are expressing SAR respond to infection by rapidly deploying defenses suggesting that the induced state also sensitizes the plant to detect new infections. This phenomenon, known as priming, has been demonstrated for both the SAR and other forms of induced resistance (Conrath et al., 2002, 2006). As the molecular regulation and other aspects of priming are discussed in detail by Conrath (2009), the remaining part of this section/chapter will examine which defenses are induced in SAR-expressing plants after infection with pathogens. B. PROTECTION AGAINST FUNGI AND OOMYCETES

1. Cucurbits The first report of SAR in cucumber described the ability of Co. orbiculare to induce resistance to itself (Kuc´ et al., 1975). Characteristics of the induced resistance to Co. orbiculare are the formation of both fewer and smaller

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lesions (Kuc´ and Richmond, 1977). These authors also reported that the SAR response was seen in cucumber cultivars that are susceptible as well as resistant to Co. orbiculare. Further work by Joe Kuc´ and others showed the induced resistance in cucumber was effective against other fungal pathogens (reviewed in Hammerschmidt and Yang-Cashman, 1995; Kuc´, 1982). The reason why SAR-expressing plants developed fewer lesions caused by Co. orbiculare was addressed by Richmond et al. (1979) using light microscopy. Prepenetration effects on expression of the resistance were ruled out by noting that the numbers of melanized appressoria produced by the pathogen were the same on both controls and SAR-expressing plants. Thus, the resistance could not be explained by the formation of a preformed antimicrobial compound on the surface of the plants. What Richmond et al. (1979) did find was a significant reduction in the number of successful penetrations from appressoria on the induced ones as compared to the control plants. At 60 h after inoculation, up to 40% of the appressoria on the control plants were associated with successful penetration into the host while only around a 5% success rate was found on the induced plants. This phenomenon was observed in several cucumber cultivars including some carrying genetic resistance to Co. orbiculare. Inoculation of resistant plants not expressing SAR resulted in the penetrated cells undergoing necrosis (possibly a HR). When the resistant plants were induced, there were far fewer penetrations, although those cells that were penetrated did become necrotic. Damage to SARexpressing leaves with Carborundum or needle punctures resulted in a loss of resistance to Co. orbiculare, suggesting that an intact epidermis was required for SAR expression. Removing the epidermis effectively eliminated the SAR-mediated resistance. Similar results were obtained by Jenns and Kuc´ (1980) using TNV as the inducing pathogen, thus demonstrating that the ability to reduce penetration efficiency in induced cucumber, like the reduction in disease severity, was independent of the inducing pathogen. A reduction in penetration is also a very likely explanation for why increasing inoculum concentration increased severity of disease development on both control and SAR-expressing cucumber (Dean and Kuc´, 1986b). Although the results discussed above show there was reduced penetration efficiency into SAR-expressing plants, they do not explain why. Histochemical staining of cucumber epidermal peels from induced and control plants inoculated with Co. orbiculare revealed the deposition of a lignin-like material under appressoria that had not penetrated into the induced plants (Hammerschmidt and Kuc´, 1982). The number of lignified sites was very similar to the numbers of successful penetrations into control tissues (as measured over time). Thus, it can be inferred that the pathogen was attempting to penetrate into the induced epidermal cells but was blocked

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by the formation of a structural barrier. Attempted infection of another cucumber pathogen, Cl. cucumerinum, into induced tissues also resulted in deposition of lignin at and near the site of penetration (Hammerschmidt and Kuc´, 1982). Papillae formation has also been associated with blocked penetration of Co. orbiculare into induced cucumber leaves. Stumm and Gessler (1986) reported that failure to penetrate was associated with fluorescent papillae directly under appressoria. Where penetration occurred, no papillae or very small, fluorescent papillae formed. Kovats et al. (1991a) confirmed the previous observations of Hammerschmidt and Kuc´ (1982) and Stumm and Gessler (1986) by showing the reduced penetration of Co. orbiculare was directly related to lignified papillae. Using Aniline Blue staining, they also found that the papillae contained callose. The latter is not surprising since callose is a common component of papillae (Aist, 1976). The deposition of callose also provides a role for the callose synthase that Schmele and Kauss (1990) reported to be more active in plants expressing SAR. An ultrastructural study of the response of induced cucumber to Co. orbiculare also supported the role of cell wall modifications and papillae in the resistance (Stein et al., 1993). Ultrastructural histochemistry coupled with energy dispersive X-ray analysis revealed that the cell wall alterations contained phenolic compounds. Silicon was also detected in the cell wall directly under appressoria that failed to penetrate into induced tissues (Kauss et al., 2003). Hyphae that successfully penetrated into the induced host were found to be encased in phenolic compounds (Stein et al., 1993). Diaminobenzidine staining provided evidence for peroxidase activity localized in the same regions where phenolic compounds were deposited (B. D. Stein and R. Hammerschmidt, unpublished data). However, it is not known if these peroxidases are the same as those induced systemically in cucumber after induction of SAR (Hammerschmidt et al., 1982; Smith and Hammerschmidt, 1988). SAR can also be induced in cucumber against powdery mildew caused by Sphaerotheca fuliginea using TNV as the inducing inoculum (Bashan and Cohen, 1983; Conti et al., 1990). In the former study, TNV induction reduced the numbers of powdery mildew colonies that formed on inoculated leaves as compared to the control. In addition, lignification of epidermal cells after Sp. fuliginea inoculation was observed much more frequently in the induced as compared to the control plants. In the latter study, histological observations revealed that papillae formed at infection sites on induced plants were very fluorescent and stained positively for phenolic compounds and lignin. In a subsequent study (Conti et al., 1994), Sp. fuliginea conidial germination, germ tube length and numbers of haustoria were reduced on TNV-induced plants as compared to controls. The decrease in germination and germ tube

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length on the surface of induced leaves suggested the presence of an inhibitor on the leaf surface. Increases in epidermal lignification as part of the response to Sp. fuliginea were also confirmed. Finally, treatment of cucumber plants with SA resulted in resistance against Sp. fuliginea that was very similar to the resistance induced by TMV in terms of disease suppression and cell wall modifications (Conti et al., 1996). SAR may not always protect plants against infection. Although it is now widely known that different forms of induced resistance ‘‘target’’ different pathogens types, one of the first ‘‘failures’’ was reported by Bashan and Cohen (1983). In the same paper in which successful resistance to powdery mildew of cucumber was reported, they also reported that TNV did not induce systemic resistance to the oomycete Pseudoperonospora cubensis (the cause of downy mildew). The reason for the failure to induce resistance was not elucidated, but the authors suggest that it may reside in the fact that this pathogen penetrates through stomata. This bypasses the epidermis which is where defense responses to true fungi, as discussed above, have been described. However, exogenous application of SA was later shown to induce resistance to Ps. cubensis (Okuno et al., 1991), thus contradicting the report of Bashan and Cohen (1983). The nature of the differences in these reports needs clarification since both TNV and SA should induce SAR in cucumber. However, Okuno et al. (1991) suggested that the concentration of inoculum used in the challenge may explain the differences between their results and those of Bashan and Cohen (1983). ASM also induces resistance in cucumber to Pythium (Benhamou and Be´langer, 1998a). In this interaction, roots from ASM-treated plants responded to infection by Pythium ultimum with the accumulation of phenolic materials in the cortex and vascular parenchyma cells. The phenolic materials appeared to block the progress of the pathogen toward the stele whereas the pathogen appeared to easily colonize root tissue in the noninduced plants. The observation that induced cucumber plants readily produce phenolic materials such as lignin suggests that these plants respond to infection by induction of enzymes involved in this process. ASM-treated cucumber plants respond to infection by Co. orbiculare by a rapid induction of genes encoding peroxidase and PAL (Cools and Ishii, 2002). As would be predicted by previous work on the cucumber SAR response (e.g., Hammerschmidt et al., 1982; Rasmussen et al., 1994), ASM treatment induced peroxidase gene expression. Cools and Ishii (2002) also reported the induction of PR-1 gene expression in response to ASM. PAL, however, was not induced by ASM, but its expression was enhanced in ASM-treated tissues after inoculation with Co. orbiculare. The effect of ASM as a priming agent for pathogen

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response in cucumber was later illustrated by the induction of a lignin peroxidase and a callose synthase gene after inoculation with Co. orbiculare (Deepak et al., 2006). Expression of these genes would be important for the production of papilla. Interestingly, genes for cinnamyl alcohol dehydrogenase and caffeoyl-CoA 3-o-methyl transferase were not induced after pathogen challenge (Deepak et al., 2006). Since the lignin-like material produced by cucurbits after infection is low in methoxylated components (Hammerschmidt et al., 1985), the lack of induction of the o-methyl transferase is perhaps not surprising. It would be interesting to determine the activity of the other enzymes that catalyze steps in lignin biosynthesis in induced plants before and after challenge. 2. Legumes Induced resistance (local, at a short distance and systemic from the point of induction) has been demonstrated in green bean (Pha. vulgaris) against the anthracnose fungus (Co. lindemuthianum). Early studies by Rahe et al. (1969) demonstrated that prior inoculation of bean hypocotyls with Helminthosporium carbonum, Alternaria sp. or incompatible races of Co. lindemuthianum induced resistance against compatible races of Co. lindemuthianum. Similarly, Skipp and Deverall (1973) reported that incompatible races of Co. lindemuthianum would induce resistance in green tissues of bean, including immature pods. However, as the inoculations in these studies were performed by inoculating entire plants or plant parts with inducer and challenge fungi, it is difficult to determine from these experiments if the induced resistance was due to the observed accumulation of phytoalexins elicited by the inducer inoculations (Rahe et al., 1969) or activation of the potential for resistance in tissues not directly affected by the inducing fungi. The question raised above was answered, in part, through a series of papers by Elliston et al. In 1971 they reported that inoculation of specific sites on bean hypocotyls with incompatible races of Co. lindemuthianum induced resistance in neighboring tissue to subsequent attack by compatible races of the fungus. Induced resistance was expressed if 18 or more hours elapsed between the time of the inducing inoculation and that of the challenge inoculation. Although the type of induced resistance described by Elliston et al. (1971) is not truly systemic in the sense in which the term is currently used (they challenged the tissue only 0.5 cm from the site of the inducer inoculation), this work did demonstrate that cells at a distance from the inducer site had become "conditioned" or ‘‘primed’’ to behave in a resistant manner. In a subsequent study, Elliston et al. (1976a) demonstrated that local and systemic induced resistance could be elicited by a number of different

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Colletotrichum species. Nonpathogenic races of Co. lindemuthianum as well as Co. orbiculare and Colletotrichum trifolii, induced a high level of local resistance to cultivar‐pathogenic races of Co. lindemuthianum. However, only Co. lindemuthianum and Co. orbiculare were able to elicit ‘‘systemic’’ resistance. This may be a reflection of the failure of Co. trifolii to cause a hypersensitive necrosis in bean tissue, whereas the other fungi elicited the hypersensitive response. Histological studies revealed that the development of compatible races of Co. lindemuthianum into control and induced tissues was initially the same (Elliston et al., 1976b). In induced tissue, however, the pathogen developed a limited amount of primary mycelium, but then was unable to develop any further. They found that the induced resistance was very similar to mature tissue resistance found in hypocotyls of all bean cultivars, and was distinctly different from the HR observed in tissue responding to an incompatible race of Co. lindemuthianum. Elliston et al. (1977) also reported that fungi capable of inducing systemic resistance elicited a localized accumulation of phytoalexins. However, there was no systemic change in phytoalexin content of the tissue. Induced tissue did, however, produce phytoalexins upon challenge with compatible races of Co. lindemuthianum. Thus, the induced resistance sensitizes tissues in a way that they will more rapidly produce phytoalexins in response to an attempted infection. Induction of phytoalexins by the inducer fungi may be involved in local induced resistance (Elliston et al., 1977). Local resistance was induced by Co. trifolii, even though it does not elicit phytoalexin accumulation. Although phytoalexin accumulation and an enhanced ability to produce phytoalexins may play a role in local and systemic induced resistance, respectively, it is obvious from the resistance induced by Co. trifolii that phytoalexin production was not the sole answer. True SAR also occurs in legumes. Sutton (1979) and Cloud and Deverall (1987) demonstrated that inoculation of primary leaves of Pha. vulgaris with Co. lindemuthianum induced resistance in upper trifoliate leaves against the same pathogen. In addition, inoculation of the hypocotyl also induced resistance in the leaves. This suggests that the induced resistance observed by Elliston et al. (1971) in hypocotyls may be a similar phenomenon. Fewer successful penetrations from appressoria occurred in leaves from plants with acquired resistance and cell wall appositions appeared to be part of this process (Cloud and Deverall, 1987). More rapid cell death of invaded cells occurred in the induced plants as compared to controls. SAR induced by ASM has been reported for the cowpea-Colletotrichum destructivum interaction (Latunde-Dada and Lucas, 2001). Penetration by Co. destructivum into ASM-induced tissues was greatly reduced as compared

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to controls, and, therefore, was similar to what is observed in both bean and cucumber. Where penetration into induced host tissues occurred, the invaded cells responded with a hypersensitive-like response and there was no transition by the invading pathogen to the more necrotrophic, secondary hyphal phase of pathogenesis. Challenge of ASM-induced tissues with Co. destructivum also resulted in a rapid, albeit transient, increase in activity of PAL and chalcone isomerase, both key enzymes in flavonoid biosynthesis. These two enzymes reached peak activity at 18 and 24 h postchallenge inoculation, respectively. Increases in the accumulation of the phytoalexins kievitone and phaseollidin also occurred more rapidly in the induced tissues. Results presented by Latunde-Dada and Lucas (2001) showed an increase in phaseollidin by 24 h and maximum accumulation at 96 h after inoculation. Phaseollidin was detected in inoculated control tissues at 48 h, and accumulated to a lower amount than observed in the induced tissues. Thus, it is likely that the expression of SAR in this host is associated with the ability to rapidly activate enzymes involved in phenolic compound biosynthesis and the accumulation of isoflavonoid phytoalexins. Other details on induced resistance in legumes can be found in Deverall and Dann (1995). 3. Solanaceous species In the early 1960s, systemic protection of tobacco foliage against Pe. tabacina was found to develop following stem infection by the same fungus (Cruickshank and Mandryk, 1960; Pont, 1959). In later studies, Cohen and Kuc´ (1981) further demonstrated that stem injection or inoculation of the stem–root interface of tobacco with conidia of Pe. tabacina induced resistance to foliar attack by the same pathogen. The induction of resistance was associated with necrosis of the external phloem and cambium of the stem. As in other induced resistance systems, a certain period of time was required between the inducing inoculation and expression of systemic resistance. Cohen and Kuc´ (1981) reported that 3 weeks were required to attain 95% protection. Inducing resistance in tobacco against Pe. tabacina by the methods of Cohen and Kuc´ (1981) and of Cruickshank and Mandryk (1960) also resulted in plant stunting and production of suckers. However, inoculating stem tissue exterior to the cambium resulted in an expression of induced resistance against Pe. tabacina and also induced increased growth of the plants (Tuzun and Kuc´, 1985). Thus the stunting, and therefore yield reduction, is not necessarily associated with the induction of resistance in tobacco to this disease. Although tobacco has been a model for many studies on SAR, most of the research has focused on the induction phase and the systemic regulation and

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accumulation of PR- and other proteins. There are a few studies that do provide some insight into how SAR-expressing tobacco and other solanaceous species respond to infection by fungi. A possible mechanism for the acquired resistance to Pe. tabacina was reported by Stolle et al. (1988) and Ye et al. (1992). In the former report, colonization of induced host tissues ceased after about 5 days and sporangia production was reduced by 70% as compared to noninduced controls. Sesquiterpenoid phytoalexin production was observed in both induced and control tissues, but higher concentrations of these compounds occurred in the Pe. tabacina-challenged controls. Further study is needed to determine if the phytoalexins are a component of resistance in SAR-expressing tissues. Using light microscopy, Ye et al. (1992) found that development of Pe. tabacina was restricted in the induced tissue and some of the hyphae exhibited cellular disorganization. Ultrastructural analysis indicated that host cell wall appositions and increased electron-opaqueness of the cell walls were also characteristic of the induced host response to the pathogen. Ribonuclease activity increased in induced plants more rapidly after infection than was observed in control plants (Lusso and Kuc, 1995). The relationship between an increase in this enzyme and defense was not determined. SAR in potato can be induced by a prior inoculation with Phy. infestans (Stromberg and Brishammar, 1991). Challenge inoculations of the induced plants with Phy. infestans revealed that the resistance was associated with a more rapid deposition of papillae and decreased efficiency of penetration (Stromberg and Brishammar, 1993). The development of hyphae that were able to enter the mesophyll tissues was much more restricted in the induced as compared to control plants (Stromberg and Brishammar, 1993). SAR can also be induced in tomato by prior inoculation with TNV (Anfoka and Buchenauer, 1997). In this case, the acquired resistance appeared to be associated with reduced penetration by Phy. infestans, but this apparently was not associated with callose deposition. In addition, a hypersensitive-like response occurred more frequently in the mesophyll of induced plants as compared to controls (Kovats et al., 1991b). Jeun et al. (2000) likewise induced resistance in tomato to Phy. infestans with TNV and also found that reduced penetration occurred into induced plants. However, these authors reported that the decrease in penetration was associated with callose deposition. Recently, the effect of NahG on basal resistance in potato to Phy. infestans has been reported (Halim et al., 2007). Although there was little difference in susceptibility observed at a macroscopic level, NahG expression in the plants did allow more growth of the pathogen in the tissue. Treatment of NahG plants with DCINA reversed the ability of the plants to support more pathogen growth. Callose deposition, a defense associated with SAR in

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potato (see preceding paragraph), was also decreased in NahG plants infected with Phy. infestans. Taken together, these results suggest that SA-mediated defenses may be needed for both basal and systemic resistance responses. ASM-induced resistance in tomato to Fusarium oxysporum f.sp. radicislycopersici was reported to be associated with rapid deposition of callose and phenolic materials in host cells walls (Benhamou and Be´langer, 1998b). This further demonstrated that SAR can be induced in roots and provided additional evidence for the role of cell wall modifications in the defense responses elicited by pathogens in induced tissue. 4. Arabidopsis Induction of SAR against the oomycete Hy. arabidopsidis was first reported by Uknes et al. (1992) who used DCINA as the inducing agent. Depending on the amount of DCINA applied to the plants, challenge of the plants with Hy. arabidopsidis resulted in single-cell host necrosis (HR) at the site of penetration to ‘‘delayed’’ necrosis of host cells along hyphae growing in the host. Arabidopsis SAR against Hy. arabidopsidis was further demonstrated by Mauch-Mani and Slusarenko (1994) who used F. oxysporum as the inducing agent. If the plants were challenged more than 4 days after the inducing inoculation, Hy. arabidopsidis induced single host cell hypersensitivity at the site of attempted penetration. If the plants were challenged before that time, the pathogen was able to infect, but there was the developing of host cell necrosis (‘‘trailing necrosis’’) along the pathogen hyphae. The deposition of lignin, based on histochemical staining, was reported to be associated with the trailing necrosis observed in Hy. arabidopsidis-infected plants (Mauch-Mani and Slusarenko, 1996). These authors concluded that lignification was part of the defense but not the deciding factor. These studies show that expression of SAR primes Arabidopsis to respond to Hy. arabidopsidis with a hypersensitive-like host response. Inoculation of Arabidopsis nim1 (non‐inducible immunity 1) mutants (allelic to npr1 in tobacco) or NahG plants resulted in increased susceptibility to Hy. arabidopsidis and greatly reduced expression of PR-1 (Donofrio and Delaney, 2001). In NahG plants, there was more pathogen biomass as well as less callose deposition around haustoria of Hy. arabidopsidis. Treatment of NahG plants with DCINA reversed the increased susceptibility and decreased ability to deposit callose. nim1 mutants were not as susceptible nor showed as much callose reduction when compared to NahG plants. These results support the role of SA in basal defenses and further indicate that SAR may be an enhancement of basal resistance that includes cell wall modifications.

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5. Japanese pear ASM also induces resistance in Japanese Pear to the scab fungus, V. nashicola (Ishii et al., 1999). Inoculation of ASM-treated pear leaves with V. nashicola resulted in the rapid, though transient, expression of transcripts encoding polygalacturonase inhibiting protein (PGIP) (Faize et al., 2004). Other defense-associated enzymes such as chitinase and peroxidase as well as certain PR-proteins were also expressed more rapidly after fungal infection in ASM-treated leaves as compared to controls. Ultrastructural analysis of V. nishicola-infected ASM-induced pear leaves (Jiang et al., 2008) revealed a higher incidence of fungal hyphae collapse and less pectin degradation in host cell walls suggesting that the induced chitinase and PGIP reported by Faize et al. (2004) may be components of the ASM-induced resistance expression. 6. Cereals Although there are numerous reports describing induced resistance in cereals, relatively few have documented the induced resistance as SAR and/or have examined the defenses activated after subsequent pathogen inoculations (Kogel and Langen, 2005). Using DCINA as the SAR activator, induced barley was shown to respond to infection with Bl. graminis f.sp. hordei in a way that resembled race-specific resistance conditioned by the Mlg gene (Kogel et al., 1994). In the induced plants, the numbers of fungal attacked host cells that produced papillae with strong autofluorescence increased over the noninduced controls. There was also a significant decrease in haustorium formation in the epidermal cells of induced plants, which was associated with a large increase in the number of attacked host cells responding hypersensitively. The induction treatment increased expression of peroxidase transcripts prior to inoculation, and the expression of this gene was increased even higher after challenge of the induced plants as compared to controls. In a subsequent study, DCINA‐induced barley seedlings were used to determine if the induced response was associated with superoxide radical anions (Kogel and Hu¨ckelhoven, 1999). As in the previous study (Kogel et al., 1994), much of the induced resistance response was associated with failure of Blumeria to invade the epidermal cells, an increase in hypersensitively responding host cells, and a decrease in successful haustorium formation. Although the formation of autofluorescent papillae and expression of hypersensitive cell death suggests that reactive oxygen species (ROS) were produced, no superoxide was detected in induced epidermal cells under attack by the pathogen. This was similar to what was observed in Mlg gene-mediated resistance to Bl. graminis in barley (Hu¨ckelhoven and Kogel, 1998).

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Saccharin-induced resistance of barley to Bl. graminis f.sp. hordei was associated with increases in peroxidase activity after challenge of induced plants (Boyle and Walters, 2006). These authors also reported an increase in cinnamyl-alcohol dehydrogenase after saccharin treatment that increased further upon challenge with Bl. graminis. Not surprisingly, the changes in activity were dependent on the amount of time between saccharin treatment and challenge.

C. PROTECTION AGAINST BACTERIA

1. Examples of induced resistance to bacterial pathogens Induction of resistance against bacterial pathogens is also well known. Although not truly systemic, Lovrekovich et al. (1968) found that inoculating the apical half of tobacco leaves with TMV induced resistance in the basal half of the leaf to Ps. syringae pv. tabaci. In this system, the line of tobacco used responded hypersensitively to TMV. In 1970, Lozano and Sequeira reported that the HR of tobacco leaves elicited by race 2 of Ralstonia solanacearum (formerly: Pseudomonas solanacearum) could be prevented if the area to be inoculated (by infiltration) with race 2 was previously infiltrated with heat killed cells of race 1 or race 2. Heat killed cells of other bacterial pathogens (e.g., Ps. syringae pv. lachrymans) also caused this effect. The induced resistance against the HR could be observed by 18 h after infiltration with the heat killed cells. From this time onward, the induced resistance was found to spread to neighboring cells and eventually (within 48 h) to become systemic. The induced resistance response was also effective against disease caused by a compatible isolate of race 1 of R. solanacearum (Lozano and Sequeira, 1970). Whether or not this resistance is SA-mediated has not been addressed in this system. In cucumber plants, inoculating one leaf with Ps. syringae pv. lachrymans, the angular leaf spot pathogen, induced resistance to Ps. syringae pv. lachrymans and to Co. orbiculare (Caruso and Kuc´, 1979). In this system, resistance was induced by a compatible interaction between the host and inducing pathogen and was thus similar to the induction of resistance in cucumber by Co. orbiculare against Co. orbiculare (Kuc´ et al., 1975). Systemic induction of resistance against Ps. syringae pv. lachrymans by Co. orbiculare was also reported by Doss and Hevisi (1981). SAR that is effective against bacterial pathogens has also been reported for Arabidopsis using both chemical (Uknes et al., 1992) and biological inducers (Cameron et al., 1994; Uknes et al., 1993).

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2. How SAR protects against bacterial pathogens How induced resistance reduces bacterial pathogen symptoms, whether the necrotic lesion associated with disease or with the HR, is not well understood (Lozano and Sequeira, 1970; Summermatter et al., 1995). Lovrekovich et al. (1968) and Caruso and Kuc´ (1979) found decreased populations of bacteria in the inoculated induced leaves as compared to controls. In the localized induced resistance described first by Lozano and Sequeira (1970), there was a much more rapid decrease in bacterial numbers in the induced plants (Sequeira and Hill, 1974). In Arabidopsis, SAR also resulted in a decrease in the growth of bacterial pathogens in induced tissues (Cameron et al., 1994; Uknes et al., 1993). These observations were further supported by Pieterse et al. (1998) who found that SAR induced in Arabidopsis by avirulent Ps. syringae pv. tomato decreased growth of virulent Ps. syringae pv. tomato in induced tissues. Suppression of symptoms, instead of reducing bacterial populations, has also been suggested as a factor in the induced resistance against these pathogens. Doss and Hevisi (1981) reported that systemic resistance in cucumber to Ps. syringae pv. lachrymans was not associated with an effect on bacterial growth in the induced tissues, thus contradicting the work of Caruso and Kuc´ (1979). In Arabidopsis, induction of SAR by the HR-inducing Ps. syringae pv. syringae resulted in a suppression of hypersensitive necrosis after challenge by the same bacterium in induced upper leaves (Summermatter et al., 1995). Similar to the observation of Doss and Hevisi (1981), there was little difference in bacterial growth in induced as compared to control leaves. These results suggest that the SAR phenomenon may be a focus of tolerance to disease rather than resistance. Suppression of disease symptoms was also reported in the systemic induction of resistance in tomato by both virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria against the same pathogen (Block et al., 2005). PR-1a and PR-1b genes were expressed systemically after an inducing inoculation with either strain and both genes showed strong expression in systemically induced leaves at 24 h after challenge with a virulent strain of X. campestris pv. vesicatoria. Systemic resistance could also be induced by Ps. syringae pv. tomato against itself and X. campestris pv. vesicatoria. Xanthomonas also induced systemic resistance to Ps. syringae pv. tomato. These results are very much in line with one of the characteristics of SAR in which multiple pathogens can be used to induce resistance against different pathogens in the same host. What was interesting about these results is that the resistance induced by the bacteria did not result in the restriction of growth of either pathogen in the systemically induced tissues as compared to growth in the controls. Symptom suppression (i.e., reduced tissue damage)

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was observed, and the authors referred to this phenomenon as systemic acquired tolerance rather than SAR. Notably, treatment with ASM induced resistance that was characterized by a suppression of X. campestris pv. vesicatoria and Ps. syringae pv. tomato growth in induced tissues. This suggests that different mechanisms of disease and/or symptom suppression may be functioning depending on the nature of the inducer. An important question to address is how induced plants are able to actually resist or stop bacterial pathogens. In the case of reduced growth of bacteria in induced plants, it is not known which factor or factors may be involved. Rathmell and Sequeira (1975) reported that induced tobacco tissues produced an antimicrobial substance(s). The nature of this antimicrobial was not determined. Since this was also localized induced resistance, the nature of this defense may or may not be relevant to systemic responses. However, since this localized resistance is known to be induced by LPS (Graham et al., 1977), it is possible that the inhibitors reported by Rathmell and Sequeira (1975) are low molecular weight compounds such as feruloyl tyramines (Newman et al., 2001). It would be interesting to see if these types of inhibitors are induced systemically by inducing treatments or induced to a greater extent after challenge of SAR-expressing plants. As described above, an interesting systemic response is the lack of symptom/damage in induced plants after challenge that is not associated with a decrease in bacterial populations (e.g., Block et al., 2005; Doss and Hevisi, 1981; Summermatter et al., 1995). It is not at all clear why this may occur, although a suppression of necrosis as a result of increased antioxidant status of the induced plant has been suggested to reduce necrosis in SAR-expressing plants that have been challenged by necrotic-lesion inducing pathogens (e.g., Barna et al., 2003). Plant cells can respond to bacterial pathogens with cell wall modifications such as callose deposition (Kim et al., 2005), and an enhanced ability to structurally block the delivery of bacterial effector molecules to host cells may also be a cause of suppressed symptoms. As such, it would be interesting to determine if this interference in a pathogenicity mechanism, such as effector delivery, is part of the SAR defense response. The nonhost resistance response of Arabidopsis to Ps. syringae pv. phaseolicola is also associated with PR-1 protein accumulation and callose deposition. Inoculation of Arabidopsis mutants npr1 or sid2 (SA induction deficient 2) resulted in increased growth of Ps. syringae pv. phaseolicola suggesting a connection between SA signaling and basal defenses. There is no reason to believe that this would not also be the case for SAR responses to this bacterium. Another possibility is an ability of the induced host to interfere with the expression of genes controlling the bacterial type III secretion pathway.

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Although not dealing with a SAR type of response, Minardi (1995) reported that the resistance induced by protein-LPS in tobacco to the HR induced by Erw. amylovora was associated with altered expression of the bacterium’s hrp genes. Thus, interference with the basic mechanisms of pathogenicity may also be a part of the expression of resistance after challenge.

D. PROTECTION AGAINST VIRUSES

1. Decrease in lesion size and number SAR in N gene tobacco against TMV is expressed as a reduction in the number and size of local lesions caused by this virus (Ross, 1961b) and is, in essence, an increase in the expression of major gene resistance to this virus. In an attempt to explain the resistance, biochemical changes following challenge inoculations of SAR-expressing tobacco leaves with TMV were reported by Simons and Ross (1970, 1971a) and Van Loon and Geelen (1971). Challenge of the induced tissues resulted in faster increases and higher final peroxidase activity than in control leaves. Simons and Ross (1971b) also demonstrated that extractable PAL activity was greater after challenge of leaves with induced resistance than with control leaves. Challenge of both control and induced tissues resulted in peak PAL activity at about the same time after inoculation followed by a rapid decrease in extractable activity. The content of o-dihydroxyphenolic compounds decreased in the challenged tissue, with the greatest decrease in the induced leaves (Simons and Ross, 1971b). These results suggested that the resistance, as expressed by smaller and fewer necrotic lesions may be the result of viral localization by formation of phenolic structural barriers or perhaps highly reactive quinones formed from the phenolic compounds. This, of course, would be in line with what has been observed in defenses expressed against fungal pathogens in plants expressing SAR in which cell wall alterations appear to be a major factor. Another explanation for the decrease in disease severity after challenge is simply the suppression of necrosis formation by increases in antioxidant activity of SAR-expressing tissues. Activity of glutathione reductase and glutathione-S-transferase as well as glutathione content increased systemically in tobacco after inoculation of lower leaves with TMV or treatment with SA (Fodor et al., 1998). Further investigations on this system revealed that SAR-expressing leaves had increased superoxide dismutase activity. Challenge with TMV resulted in reduced hydrogen peroxide levels at infection sites (Barna et al., 2003). However, it is not clear from these studies if the reduced necrosis is, indeed, the cause or result of SAR. It is apparent from other studies that necrosis does not restrict the spread of virus in N gene

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tobacco (Wright et al., 2000). Thus, host cell death may be more of a result of resistance than an active cause. 2. Inhibition of virus replication One possible explanation for reduced lesion size and number is a decrease in viral replication in SAR-expressing tissues. Van Loon and Antoniw (1982) found that treatment of susceptible ‘‘Samsun’’ (nn genotype) tobacco with SA reduced TMV accumulation following inoculation. Similarly, White et al. (1983) reported that treatment of tobacco with ASA induced resistance to TMV in tobacco lines with and without the N gene for hypersensitive resistance. Associated with the induced resistance was a decrease in the accumulation of virus in both genotypes of tobacco. The results of Van Loon and Antoniw (1982) and White et al. (1983) suggested that SAR type resistance may be expressed by inhibiting the replication of the virus. Following up on this observation, Chivasa et al. (1997) found that SA pretreatment of an nn genotype of tobacco was able to greatly reduce the accumulation of TMV coat protein and RNAs after inoculation with the virus. Inhibition of virus replication has also been proposed as a mechanism of SA-induced resistance in tobacco to PVX (Naylor et al., 1998) and in Arabidopsis to turnip vein clearing virus (TVCV) (Wong et al., 2002). 3. Inhibition of cell-to-cell movement Murphy and Carr (2002) provided further evidence that SA can reduce TMV replication and that it can also reduce cell-to-cell movement of the virus in tobacco. What is interesting about this report is that the SA-induced resistance to TMV in tobacco is also tissue specific with cell-to-cell movement being affected in the epidermis while virus replication was reduced in mesophyll cells. The basis for inhibition of cell-to-cell movement was not elucidated, but it was shown not to be caused by changes in the size of the plasmodesmata (Murphy and Carr, 2002). Inhibition of cell-to-cell movement was also suggested to be the mechanism behind SA-induced resistance of squash to CMV (Mayers et al., 2005). 4. Inhibition of systemic movement Inoculation of lower leaves of cucumber plants with Co. orbiculare, Ps. syringae pv. lachrymans or TNV induced resistance to challenge inoculation with CMV (Bergstrom et al., 1982). Challenge with CMV by mechanical inoculation reduced the number of chlorotic primary CMV lesions on the challenged leaf and delayed appearance of systemic mosaic symptoms. Challenge of induced plants with CMV-infested melon aphids resulted in a delay in systemic spread of the virus but found no differences in symptoms on the

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aphid-infested leaves on control or induced plants. Using ASM as the inducer, Smith-Becker et al. (2003) found a reduction in systemic spread of CMV in cantaloupe following mechanical inoculation with the virus. In tobacco and Arabidopsis, SA treatment also induces resistance to CMV (Mayers et al., 2005; Naylor et al., 1998). These authors found that the induced resistance was based on inhibition of systemic movement of the virus and not due to inhibition of replication. In tobacco, SA did not affect normal translocation processes of photosynthetic assimilates as demonstrated by feeding plants 14CO2 and following the transport of the label in treated and control plants (Naylor et al., 1998). This demonstrated that SA-induced resistance to systemic movement was not due to changes in source–sink relationships or other normal functions of translocation. As noted above, the induction of resistance to CMV in squash was associated with reduced viral replication, and this indicates different mechanisms of resistance against the same virus are present in different plant species (Mayers et al., 2005).

5. Does SA induce a different form of resistance to viruses? SAR is typically associated with SA functioning upstream of NPR1 and the expression of PR genes. However, the role of SA in the SAR response to viruses may be different (Murphy et al., 1999). In the report by Chivasa et al. (1997), SA was found to inhibit TMV replication by a mechanism that is sensitive to salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. This suggested that SA, a known inducer of the alternative oxidase pathway (Raskin, 1992), was inducing resistance via another route. Treatment of TMV susceptible (nn genotype) tobacco with SHAM had no effect on TMV replication as compared to SA which blocked replication (as based on TMV coat protein accumulation). However, co‐incubation of leaf tissue with SHAM and SA resulted in no inhibition of coat protein production. SHAM also delayed the SA-induced delay in systemic symptoms in n gene tobacco as well as resistance in N gene tobacco (Chivasa et al., 1997). In further support of the role of the alternative oxidase pathway in SA-mediated resistance, a respiratory inhibitor, cyanide, was able to restore the normal response of N gene tobacco to TMV in plants expressing NahG (Chivasa and Carr, 1998). Further support for the role of alternative oxidase in resistance to TMV was the ability of antimycin A, another inhibitor of respiration, to also induce resistance (Chivasa and Carr, 1998). These same authors also reported that SA and antimycin A induced transcripts of the alternative oxidase gene, thus further suggesting a role for the alternative oxidase pathway in induced resistance to TMV.

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To test the role of alternative respiration on SA-induced resistance, transgenic tobacco with increased or decreased alternative oxidase expression were generated (Gilliland et al., 2003). Altered alternative oxidase activity had no effect on SA-induced resistance to TMV in susceptible tobacco. Interestingly, antimycin A-induced resistance based on virus accumulation in inoculated leaves was inhibited in transgenic plants with enhanced alternative oxidase activity. Decreasing alternative oxidase, however, allowed short-term induction of resistance following SA or antimycin A treatment. Based on these results, Gilliland et al. (2003) suggested that SA-induced resistance to TMV is controlled by more than one mechanism, of which alternative oxidase plays one role. These authors further hypothesize that ROS in the mitochondria, the concentration of which would be affected by altered alternative oxidase expression as well as by treatment with SA or antimycin A, are likely important signals in the induced resistance response. SA-mediated resistance to viruses also differs from SAR described for bacteria and fungi in the expression of PR genes. Chivasa et al. (1997) found that SA induction of PR-1 protein accumulation was not inhibited by SHAM and was even induced by this inhibitor. The SA-induced resistance of Arabidopsis to TVCV was also shown to be separate from the induction of PR-proteins (Wong et al., 2002). These authors and Kachroo et al. (2000) also demonstrated that SA-induced resistance was also independent of NPR1 function as SA was able to induce resistance in npr1-1 mutants. Thus, SA-induced resistance to viruses appears to follow a signaling pathway that is different from that observed for fungi and bacteria (Murphy et al., 1999). This is of course an interesting revelation in light of the fact that classical SAR often uses the work of Ross (1961b) as a primary example. What these studies do demonstrate is that signal molecules that induce a similar ‘‘resistance phenotype’’ may do so by different pathways. Further details on the role of SA and related signaling molecules for induced resistance to viruses can be found in recent reviews by Singh et al. (2004) and Palukaitis and Carr (2008). E. MECHANISMS OF DEFENSE IN SUMMARY

1. Enhancing basal defense Having described a number of examples of how plants expressing SAR are thought to defend themselves against infection, it might be good to again consider the basic foundations and characteristics of this form of resistance. First of all, it should be remembered that SAR (and other forms of induced resistance) is functioning in a plant that is susceptible to multiple pathogens. Second, because of the diversity of pathogens that are resisted by a plant

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expressing SAR, it is logical that the resistance is associated with the expression of multiple defenses that are induced as a result of pathogen or chemical activator induction and those induced after a challenge. In other words, the nonspecificity of SAR is likely related to a diversity of defenses. Looking back over the defenses described in this review, it is clear that these can be broken into three stages representing SAR induction and expression. The first, or induction stage, requires inoculation of one leaf with a compatible pathogen that causes a necrotic lesion or an incompatible pathogen that elicits a HR. Both of these plant–pathogen interactions generate a systemically transported signal (or signals) that trigger(s) SA accumulation throughout the plant. Based on current information, the accumulated SA induces resistance to biotrophic or hemibiotrophic fungi and bacteria through an NPR1-mediated process that is accompanied by PR-protein accumulation. Although resistance induced by SA against viruses is also accompanied by PR-protein accumulation, the mechanism(s) involved appear to be different. At least in tobacco, SA induces resistance to virus through a SHAM-sensitive pathway. It is interesting to note that in the necrotic lesions that result from compatible interactions and lead to SAR, basal defenses such as lignification and phytoalexin accumulation occur (Hammerschmidt, 1999b; Hammerschmidt and Nicholson, 1999). In NahG plants, compatible interactions with necrotizing pathogens expand more rapidly than in untransformed controls (Delaney et al., 1994). It is important to note that SA-mediated defenses are not effective against necrotrophs which are typically resisted by the JA/ET pathway (Glazebrook, 2005; Thomma et al., 2001). This suggests that basal defenses are involved in limiting lesion spread. However, it seems unlikely that locally induced basal defenses are a major player in induction of the systemic resistance as inoculated NahG rootstocks were still capable of generating a systemic signal that induced SAR in wild-type scions (Vernooij et al., 1994). The second stage of SAR expression is the systemic accumulation of PR- and other proteins. Although the precise role of these proteins in defense is not clear (Van Loon et al., 2006), one possible function is to slow the development of the pathogen so that the SAR-expressing host has the needed time to induce additional defenses. However, there is little evidence to suggest this is happening. This is illustrated by the failure of plants constitutively expressing PR-proteins to be consistently more resistant (Van Loon et al., 2006). However, because it is known that slowing pathogen development by fungicides can result in the expression of defenses at the site of infection (Lazarovits and Ward, 1982), retarding pathogen growth (thus giving the plant more time to react) is still a possibility. Another alternative is that fungicide-sensitive pathogens release elicitors which trigger the

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localized defenses (Cahill and Ward, 1989). As some of the PR-proteins are chitinases or -1,3-glucanases that can act on fungal or oomycete cell walls, it would seem possible that the PR-proteins could also function in SAR by releasing cell wall elicitors that trigger localized defenses (Okinaka et al., 1995). However, there must be more to this than the presence of enzymes that can release elicitors from pathogens as extracellular chitinase activity remains high in cucumber even after SAR is no longer evident (Dalisay and Kuc´, 1995). Other systemic protein changes, such as the accumulation of HRGP in the cell wall, may function as structural barriers that slow the penetration of pathogens through cell walls and may provide an additional explanation for decreased fungal penetration into SAR expressing tissues. The last stage is the expression of defenses at the site of pathogen challenge. These types of responses clearly show that the SAR-expressing tissues are primed to respond to new infections (Conrath et al., 2000, 2002, 2006). As early as 1961, Ross (1961b) manipulated the size of TMV lesions produced in induced and noninduced leaves by varying environmental conditions. He found that the effects were similar in induced and noninduced leaves, and concluded that the mechanisms of lesion restriction were the same in noninduced and induced plants. With fungi, a general response observed in many hosts is modification of cell walls. These modifications (e.g., lignin and callose deposition) are not novel responses, but might, as suggested by Ton et al. (2006), be an enhancement of basal defenses primed by SAR. For example, one response of SAR expressing cucumber to Colletotrichum is the rapid deposition of lignin in outer epidermal cells walls (Hammerschmidt and Kuc´, 1982). Lignification also occurs, although more slowly and after penetration, in the necrotic lesions that result from the compatible interaction between Colletotrichum and cucumber. Other defenses, such as phytoalexin production, are enhanced in SAR-expressing tissue (Hammerschmidt, 1999a,b). Since these compounds are also produced in many compatible interactions (Hammerschmidt, 1999b), these observations further support the concept that SAR is an enhancement of basal defense responses. The idea that SAR and other forms of systemic induced resistance is an enhancement of basal defenses is supported in another, albeit a somewhat indirect, way: the failure of SAR to provide complete resistance. This should not be surprising since a SAR-expressing plant is really a susceptible plant with a layer of added defenses (i.e., the newly induced PR-proteins) and additional defenses elicited after challenge inoculation. Since basal defenses alone are not sufficient to stop compatible pathogens, there is no reason to expect these same defenses (even if enhanced) would be 100% effective. This is illustrated by the observation that SAR-expressing cucumber are only partially effective in blocking penetration by Co. orbiculare and infections

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that are successful result in typical, although smaller, anthracnose lesions (Jenns and Kuc´, 1980; Richmond et al., 1979). Furthermore, increasing the concentration of inoculum essentially eliminates the ability of SARexpressing cucumber to reduce anthracnose lesion development (Dean and Kuc´, 1986b). Thus it is not surprising that SAR is not always highly effective under field conditions (reviewed in Vallad and Goodman, 2004). B. WHAT DON’T WE KNOW?

There are many things that are not known about induced resistance and plant defense, in general. What follows are a few thoughts on this topic based on questions that arose while preparing this review. First, it is not known with absolute certainty which SAR-associated defenses are needed to stop pathogens from invading. There are obviously many good correlations, but it is important to further dissect and evaluate how each putative defense contributes to SAR. Studies using transgenic plants expressing individual PR-proteins have shown that one component is not sufficient to provide resistance, but this may not be surprising as SAR and other inducible defense responses are multicomponent. Obviously, a strategy using multiple defenses suggests there might be interactions between defenses that make the whole much more effective than individual responses. It may also be of use to look at defense expression as a process and not solely at how metabolic ‘‘end products’’ function in resistance. Ride (1978), in discussing the role of lignin in host defense, indicated that the process of lignification may be as important in defense as the static structural barrier of lignified walls. Of course, it is also possible that all of the ‘‘defenses’’ identified thus far are just host responses and have nothing to do with stopping the pathogen. This is illustrated by the work on SA-mediated resistance to viruses which appears to function differently from what was expected based on early studies. The similarities between the expression of defenses in SAR-expressing plants and those of basal resistance in noninduced plants suggest that comparative studies of these types of resistance might be helpful in evaluating which defenses are most important. The use of mutants in genes that are directly involved in the production of specific defenses is another approach that needs to be more thoroughly integrated into this line of research. Second, it is not known for certain why plants expressing SAR respond more quickly to pathogens. It is well known that SAR-expressing plants are primed to respond, but this does not explain how, or even whether the plant recognizes the presence of the pathogen more quickly. Perhaps the answer lies in the expression of genes encoding NBS-LRR proteins such as those

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induced by SAR activators (e.g., Faize et al., 2007; Sakamoto et al., 1999; Wang et al., 2001). Induction of these types of proteins might enable the induced plant to detect PAMPs more efficiently and thus more rapidly induce defenses at the infection site. This would also further support induced resistance as an enhancement of basal disease resistance. An equally likely explanation is that the primed, SAR-expressing plants respond faster to infection because of the enhancement of defense signaling pathways. Induced resistance also is not 100% effective. It is now clear that there are differences in systemic resistance expressed against biotrophs/hemibiotrophs as compared to necrotrophs that can be based on signaling pathways (Glazebrook, 2005), and this explains why SAR might not work against all pathogens. However, this does not explain why, for example, SAR does not fully protect plants such as cucumber against hemibiotrophs like Colletotrichum (e.g., Dean and Kuc´, 1986b). Histological studies have shown that a certain percentage of appressoria on SAR-expressing leaves produce penetrations that result in successful infections (e.g., Richmond et al., 1979). This suggests there is nonuniform expression of priming in the epidermis. Since SAR can be considered to be an enhancement of basal resistance to pathogens and some pathogens can suppress this type of resistance (e.g., Cooper et al., 2008), it is also possible that part of the ‘‘failure’’ of acquired resistance may be the result of suppression of defense responses by the ‘‘challenge’’ pathogen. Another item to consider is whether the apparent differences between SA-mediated (SAR) and JA/ET-mediated defense signaling that differentiates between biotrophs and necrotrophs (Glazebrook, 2005; Oliver and Ipcho, 2004) is a widespread phenomenon in the plant kingdom. This certainly appears to be the case with Arabidopsis, but whether it is universal needs to be determined. For example, the SAR activators ASM and DCINA induce resistance in soybean to Sc. sclerotiorum, a necrotroph (Dann et al., 1998). This is not only of intellectual interest, but this type of information would be of value in implementing induced resistance as part of practical disease management. Finally, it is important to determine how best to take advantage of SAR and other forms of induced resistance in disease management. As might be expected from the resources needed to synthesize PR-proteins, there are metabolic costs associated with SAR expression (Heil, 2007; Walters and Boyle, 2005; Walters and Heil, 2007). Thus, the use of SAR in practical management must take this into account. One way to possibly reduce the metabolic costs would be to combine SAR with other management approaches. It is interesting to note that fungicides are less effective in Arabidopsis plants impaired in SA signaling (Molina et al., 1998). This suggests that basal resistance may work in concert with fungicides in

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protecting plants against disease, and indicates that fungicides might act to enhance disease protection conferred by SAR. As reviewed by Reglinski et al. (2007), there are examples where activators such as ASM have been shown to improve the efficacy of standard fungicides. Understanding the biochemical bases for such interactions could shed further light on how to use induced resistance in the field.

VI. CONCLUDING COMMENTS Over the last 50 years, the study of SAR and other forms of induced resistance has evolved from a rather curious phenomenon studied by only a few researchers to a line of research that is providing new insight into signaling pathways that regulate disease resistance. In addition, the development of synthetic and natural plant resistance activators for use in production agriculture demonstrates that SAR may have a place in practical disease management. Based on a number of lines of evidence, it appears that SAR is an enhancement of the basal defenses that all plants have the capacity to express. Although this helps to explain why plants have the genetic capacity to resist disease, it does not help narrow down which defenses are most important. Thus, it is still important to determine the relative contribution of known defenses to disease resistance and to be open to other forms of defense that may not fit current thinking of what a defense looks like. Finally, one of the shortcomings of SAR research has been the limited number of plant species that have been studied. It is important to survey for the expression of SAR as defined by model systems in other species, to determine if the models are correct. This will also help determine the best strategies for implementing SAR and other induced resistance types in disease management.

ACKNOWLEDGMENT I wish to thank the Michigan Agricultural Experiment Station for its support.

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Rhizobacteria-Induced Systemic Resistance

¨ FTE1 DAVID DE VLEESSCHAUWER AND MONICA HO

Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. PAMP- and Effector-Triggered Immunity ................................. B. Systemic Acquired Resistance or Salicylic Acid-Induced Systemic Resistance ............................................................ C. Rhizobacteria-Induced Systemic Resistance ............................... D. Rhizobacteria Known to Trigger ISR....................................... E. Scope of this Review ........................................................... II. Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Flagella........................................................................... B. Lipopolysaccharides............................................................ C. Biosurfactants ................................................................... D. N-acyl-L-homoserine lactone.................................................. E. N-alkylated benzylamine ...................................................... F. Siderophores .................................................................... G. Antibiotics ....................................................................... H. Volatiles .......................................................................... I. Exopolysaccharides ............................................................ J. Other Bacterial Determinants ................................................ III. Signalling in Rhizobacteria-Induced Systemic Resistance . . . . . . . . . . . . . . . . . . A. The Arabidopsis–Pseudomonas fluorescens WCS417r System: A Paradigm for SA-Independent ISR Signalling .......................... B. SA-Dependent ISR Signalling ................................................ C. SA-Dependent and SA-Independent Signalling ...........................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51006-3

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IV. Final Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

ABSTRACT Rhizobacteria-induced systemic resistance (ISR) is a type of systemically enhanced resistance against a broad spectrum of pathogens that is triggered upon root colonization by selected strains of non-pathogenic bacteria. Over the past decade, a myriad of bacterial traits operative in triggering ISR have been identified, including flagella, cell envelope components such as lipopolysaccharides, and secreted metabolites like siderophores, cyclic lipopeptides, volatiles, antibiotics, phenolic compounds, and quorum sensing molecules. This review provides an in-depth overview of these determinants, thereby focusing specifically on the molecular recognition processes in the plant. The putative mechanisms involved in microbial perception include high- and low-affinity membrane receptors, membrane bilayer perturbation, and siderophoremediated alterations in cellular iron homeostasis. In addition, details about the various defence signalling pathways reported to underpin rhizobacteria-mediated ISR are presented. Evidence is accumulating that there is not one definitive resistance pathway to ISR but that various hormone-dependent signalling conduits may govern the induced resistance phenotype depending on the rhizobacterium and the plant–pathogen system used.

I. INTRODUCTION A. PAMP- AND EFFECTOR-TRIGGERED IMMUNITY

Plants have evolved a powerful immune system to resist their potential colonization by microbial pathogens and parasites. Over the past decade, it has become increasingly clear that this innate immunity is, in essence, composed of two interconnected branches, termed PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Eulgem and Somssich, 2007; Jones and Dangl, 2006). PTI is triggered by recognition of pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), which are conserved molecular signatures decorating many classes of microbes, including non-pathogens. Perception of MAMPs by pattern recognition receptors (PRRs) at the cell surface activates a battery of host defence responses leading to a basal level of resistance (Chisholm et al., 2006). However, during the evolutionary arms-race between plants and their intruders, many microbial pathogens acquired the ability to dodge PTI-based host surveillance via secretion of effector molecules that intercept MAMP-triggered defence signals (Go¨hre and Robatzek, 2008). In turn, plants have adapted to produce cognate R (resistance) proteins by which they recognize, either directly or indirectly, these pathogen-specific effector proteins, resulting in a superimposed layer of defence variably termed ETI, gene-for-gene resistance, or R gene-dependent resistance (Jones and Dangl, 2006).

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In many cases, effector recognition culminates in the programmed suicide of a limited number of challenged host cells, clearly delimited from the surrounding healthy tissue. This hypersensitive response (HR) is thought to benefit the plant by restricting pathogen access to water and nutrients and is correlated with an integrated set of physiological and metabolic alterations that are instrumental in impeding further pathogen ingress, among which exists a burst of oxidative metabolism leading to the massive generation of reactive oxygen species (ROS) (Glazebrook, 2005; Greenberg and Yao, 2004). B. SYSTEMIC ACQUIRED RESISTANCE OR SALICYLIC ACID-INDUCED SYSTEMIC RESISTANCE

Systemic acquired resistance (SAR) or salicylic acid-induced systemic resistance (ISR), is a phenomenon whereby disease resistance to subsequent microbial infection is induced at the whole plant level by a localized pathogen inoculation (Durrant and Dong, 2004). Development of tissue necrosis used to be considered a common and necessary feature for SAR activation. This tissue necrosis can result from ETI-associated HR formation or originate from disease symptom development following infection by virulent pathogens. Mishina and Zeier (2007), however, have demonstrated that in Arabidopsis thaliana, SAR can also be triggered without tissue necrosis or HR by non-host or type III secretion-deficient Pseudomonas syringae strains or by local leaf infiltration of typical PAMPs such as bacterial lipopolysaccharides (LPS) and flagellin (Mishina and Zeier, 2007). These results indicate that both PTI and ETI can lead to SAR. PTI-induced SAR, however, is less pronounced than its ETI-induced counterpart (Mishina and Zeier, 2007). SAR requires endogenous accumulation of the signal molecule salicylic acid (SA) and is marked by the transcriptional reprogramming of a battery of SA-inducible genes encoding pathogenesis-related (PR) proteins. These PR-proteins, of which some possess antimicrobial activity, serve as hallmarks of SAR in several plant species and are thought to contribute to the state of resistance attained. The expression of a PR-1 gene or protein in particular is usually taken as a molecular marker to indicate that SAR was induced. All PR-1 genes in plants appear to be inducible by SA (Van Loon et al., 2006). Transduction of the SA signal requires the function of NPR1 (also known as NIM1), a master regulatory protein that was identified in Arabidopsis through genetic screens for SAR-compromised mutants (Cao et al., 1994; Shah et al., 1997). SAR is abolished in SA-non-accumulating NahG plants that express the bacterial SA-hydroxylase gene NahG, which converts SA to catechol (Lawton et al., 1996). NahG transgenes are available in various plants including Arabidopsis (Delaney et al., 1994), tobacco

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(Gaffney et al., 1993), tomato (Brading et al., 2000), and rice (Yang et al., 2004). Accumulation of SA is required for SAR, but only in the signalperceiving systemic tissue. Recently, it was demonstrated that the volatile molecule methyl salicylate (MeSA) can act as a long-distance mobile signal for SAR. MeSA itself appears to be biologically inactive, but in the systemic tissue MeSA is hydrolysed to SA by the MeSA-esterase activity of SA-binding protein 2 (Park et al., 2007; Vlot et al., 2008a,b). It is likely that MeSA can travel by both air and vascular transport to mediate long-distance induction of resistance in distal leaves that lack a direct vascular connection to the attacked leaf and in neighbouring plants (Heil and Ton, 2008). Besides MeSA, lipid-derived signals also play a role in triggering SAR. Vlot et al. (2008a) state that two lipid-associated signals may work in parallel with each other and with MeSA to regulate SAR. Possible candidates are galactolipids and jasmonic acid (JA). Recent findings show that auxin-related genes are repressed in the systemic tissue of SAR-induced Arabidopsis, and it was concluded that SA enhances resistance by inhibiting auxin signalling via SA-dependent stabilization of auxin repressor proteins (Wang et al., 2007). C. RHIZOBACTERIA-INDUCED SYSTEMIC RESISTANCE

Since the 1980s, numerous reports have ascribed a beneficial effect of root colonization by specific bacteria on plant development, and they were therefore, termed plant growth-promoting rhizobacteria or PGPR (Kloepper et al., 1980). Plant-growth promotion can result from a direct effect on plant growth, but is mostly related to a PGPR-mediated biological control of deleterious soil micro-organisms that can be on the basis of various mechanisms such as competition for nutrients, siderophore-mediated competition for iron, or antibiosis (Schippers et al., 1987). In 1991, three groups independently demonstrated that PGPR could also reduce pathogen infections on above-ground plant parts such as leaves and stems (Alstro¨m, 1991; Van Peer et al., 1991; Wei et al., 1991). Given the spatial separation of PGPR and pathogen, this effect had to be plant-mediated. Later on, similar results were obtained when root tips were treated with the PGPR, and the pathogen was inoculated on the root base (Leeman et al., 1995). Thus, colonization of plant roots by selected PGPR can lead to a type of systemic resistance that has been termed ‘induced systemic resistance’ (ISR) (Kloepper et al., 1992; Van Loon et al., 1998). To avoid a Babel confusion of terminology, we propose ISR to depict induced systemic resistance induced by non-pathogenic rhizobacteria or PGPR irrespective of the signalling pathway involved in this process, while the term SAR will be used to describe SA-dependent induced resistance triggered by a localized pathogen infection as explained above.

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Generally, the onset of ISR, unlike SAR, is not accompanied by the concomitant activation of PR genes. Even though colonization of the roots by ISR-triggering bacteria leads to a heightened level of resistance against a diverse set of intruders, often no defence mechanisms are activated in aboveground plant tissues upon perception of the resistance-inducing signal. Rather, these tissues are sensitized to express basal defence responses faster and/or more strongly in response to pathogen attack, a phenomenon known as priming (Conrath et al., 2002). As demonstrated recently, priming of the plant’s innate immune system confers broad-spectrum resistance with minimal impact on seed set and plant growth (Van Hulten et al., 2006). Hence, priming offers a cost-efficient resistance strategy, enabling the plant to react more effectively to any invader encountered by boosting infection-induced cellular defence responses (Beckers and Conrath, 2007; Conrath et al., 2006). In a series of influential studies using the reference rhizobacterial strain Pseudomonas fluorescens WCS417r, Pieterse et al. (1996, 1998, 2000) demonstrated that, at least in Arabidopsis, WCS417r-mediated ISR functions independently of SA, but requires components of the JA and ethylene (ET) response pathways. Like SAR however, WCS417r-mediated ISR is dependent on NPR1. In recent years, though, it is becoming increasingly clear that not all rhizobacteria trigger ISR by a JA-, ET-, and NPR1-dependent pathway. Table I shows that all kinds of variations can be found depending on the rhizobacterium and the plant–pathogen system used. The role of plant hormones in ISR signalling will be discussed further below. D. RHIZOBACTERIA KNOWN TO TRIGGER ISR

The first comprehensive review about rhizobacteria-mediated ISR was written by Van Loon et al. (1998). In this seminal paper ISR was reported in seven dicotyledonous plants (Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato) and inducing bacteria were limited to 15 Pseudomonas strains and two Serratia strains. Since 1998, ISR has been reported in many different plant–pathogen systems. A comprehensive overview of PGPR able to trigger ISR is given by Bent (2006). A review by Van Loon and Bakker (2006) reports ISR in 19 different plant species including, beside the plant species listed above, banana, pea, chickpea, white clover, pepper, pine, Eucalyptus, potato, rice, tall fescue, and sugarcane. Besides Pseudomonas and Serratia, many Bacillus strains have the capacity to elicit ISR. The literature about ISR by Bacillus spp. up to 2004 has been reviewed by Kloepper et al. (2004). Numerous rhizobacteria able to trigger ISR appear to be endophytes that can be isolated from surface-sterilized plant tissue. Endophyte-mediated ISR has been summarized by Kloepper and Ryu

TABLE I Rhizobacteria that Induce Systemic Resistance in Plant–Pathosystems for Which Information is Available about Bacterial Determinants and/or Defence Pathways Involved Bacterial strain Arthrobacter oxidans BB1 Bacillus amyloliquefaciens IN937a Bacillus pumilus SE34

Plant species Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis

Bacillus pumilus T4

Tobacco Tomato Arabidopsis Arabidopsis Tobacco

Bacillus sp. L81

Arabidopsis

Bacillus sp. N11.37

Arabidopsis

Bacillus subtilis GB03

Arabidopsis

Bacillus subtilis S499 Burkholderia gladioli IN26

Bean Cucumber Tobacco

Pathogen Pseudomonas syringae pv. tomato Erwinia carotovora Cucumber mosaic virus Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Peronospora tabacina Phytophthora infestans Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Pseudomonas syringae pv. tabaci Pseudomonas syringae pv. tomato Xanthomonas campestris CECT95; 4480 Erwinia carotovora subsp. carotovora Botrytis cinerea Colletotrichum orbiculare Pseudomonas syringae pv. tabaci

Determinanta

Pathwayb

Plant lines tested/method usedc

Reference

ND

SA-dependent; JA-independent

NahG, jar1

Barriuso et al. (2008)

2R,3R-butanediol

ET/JA/NPR1/SA-independent

Ryu et al. (2004a)

ND ND

ND

SA-independent ET/JA/NPR1-dependent; SA-independent NPR1/SA-dependent; ET/JAindependent SA-independent ET/JA-dependent; SA-independent ET-dependent; SA/JA/NPR1independent ET-dependent; SA/JA/NPR1independent Induces PR-1a

ein2, coi1, npr1, NahG, cpr1 NahG ein2, fad3-2 fad7-2 fad8, npr1, NahG npr1, NahG, ein2, fad3-2 fad7-2 fad8 NahG Nr/Nr, def1, NahG ein2, NahG, fad3-2 fad7-2 fad8, npr1 ein2, NahG, fad3-2 fad7-2 fad8, npr1 GUS activity

ND

SA-dependent; JA-independent

NahG, jar1

Park and Kloepper (2000) Barriuso et al. (2008)

ND

SA/ET-dependent; JA-independent

NahG, etr1-1, jar1-1

Domenech et al. (2007)

2R,3R-butanediol

ein2, NahG, cpr1, coi1, npr1 ND ND

Ryu et al. (2004a)

Surfactin; fengycin EPS

ET-dependent; SA/JA/NPR1independent ND ND

ND

Induces PR-1a

GUS activity

ND ND ND ND ND

Ryu et al. (2004b) Ryu et al. (2003b) Ryu et al. (2003b) Zhang et al. (2002) Yan et al. (2002) Ryu et al. (2003b) Ryu et al. (2003b)

Ongena et al. (2007) Park et al. (2008a) Park and Kloepper (2000)

LPS

Induces PR-1

Quantitative PCR

Solano et al. (2008)

Arabidopsis

Pseudomonas syringae pv. tomato Verticillium dahliae

ND

Arabidopsis

Erwinia carotovora

ND

Pseudomonas aeruginosa 7NSK2

Arabidopsis

Unknown

Timmusk and Wagner (1999) Ran et al. (2005b)

Tobacco

Pseudomonas syringae pv. tomato Tobacco mosaic virus

sid2, eds5/sid1, npr1, jar1-1, etr1-1, pad3-1, pad4-1, NahG RNA differential display; RT-PCR NahG

Tjamos et al. (2005)

Paenibacillus polymyxa

SA/NPR1-dependent; JA/ET/ phytoalexin/NahGindependent Induces PR-1, HEL, VSP, ERD15, RAB18 SA-independent SA-dependent

NahG

De Meyer et al. (1999a)

Tomato

Botrytis cinerea

Tomato

Chryseobacterium balustinum AUR9 Paenibacillus alvei K165

Pseudomonas aeruginosa KMPCH

Pseudomonas chlororaphis O6

Arabidopsis

SA-dependent

NahG

Audenaert et al. (2002)

Meloidogyne javanica

Salicylic acid (role of pyocyanin not tested) Salicylic acid or pyochelin þ pyocyanin Unknown

SA-independent

NahG

Rice

Magnaporthe oryzae

Pyocyanin

SA-dependent

NahG

Bean

Botrytis cinerea

Salicylic acid þ pyocyanin

ND

ND

Bean

Salicylic acid (role of pyocyanin not tested) Salicylic acid þ pyocyanin

ND

ND

Tomato

Colletotrichum lindemuthianum Botrytis cinerea

SA-dependent

NahG

Cucumber

Corynespora cassiicola

ND

Northern hybridisation

Tobacco

Erwinia carotovora

Tetr18, NahG

Tobacco

Pseudomonas syringae pv. tabaci

2R,3R-butanediol; phenazines, other determinants 4-(aminocarbonyl) phenylacetate

Primed induction of galactinol synthase ET-dependent; SA-independent

Siddiqui and Shaukat (2004) D. De Vleesschauwer et al. (2006), D. De Vleesschauwer (unpublished data) De Meyer and Ho¨fte (1997) Bigirimana and Ho¨fte (2002) K. Audenaert et al. (2002), K. Audenaert et al. (unpublished data) Kim et al. (2008)

ET- and SA-independent

Tetr18, NahG

Spencer et al. (2003), Han et al. (2006b), Kang et al. (2007) Spencer et al. (2003), Park et al. (2008b)

(continues)

TABLE I Bacterial strain Pseudomonas fluorescens 89B-61 (¼ G8-4)

Plant species

Pseudomonas fluorescens Q2-87 Pseudomonas fluorescens SS101 Pseudomonas fluorescens WCS374

Determinanta

Pathwayb

Plant lines tested/method usedc

Reference

Arabidopsis

Pseudomonas syringae pv. maculicola

ND

NPR1-dependent; SA/JA/ETindependent

npr1, NahG, fad3-2 fad7-2 fad8, ein2

Ryu et al. (2003b)

Arabidopsis

Pseudomonas syringae pv. tomato Peronospora tabacina Pseudomonas syringae pv. tabaci Phytophthora infestans Hyaloperonospora arabidopsidis

ND

NPR1-dependent; SA/JA/ETindependent SA-independent Induces PR-1a

npr1, NahG, fad3-2 fad7-2 fad8, ein2 NahG GUS activity

Ryu et al. (2003b)

ND 2,4-diacetylphloroglucinol

ET/JA-dependent; SA-independent JA/NPR1/EIR1-dependent; SA/ET/ phytoalexin-independent

Tobacco

Tobacco necrosis virus

ND

Tomato

Meloidogyne javanica

Pyoverdine; unknown determinant 2,4-diacetylphloroglucinol

Nr/Nr, def1, NahG jar1, npr1, eir1, NahG, sid2-1, ein2-1, etr1-1, pad2-1 ND

SA-independent

NahG

Arabidopsis

Pseudomonas syringae pv. tomato Phytophthora infestans

2,4-diacetylphloroglucinol

ND

ND

Siddiqui and Shaukat (2003) Weller et al. (2004)

Massetolide A

SA-independent

NahG

Tran et al. (2007)

Flagella

ET/JA/NPR1-dependent; SA-independent SA/NPR1-dependent; ET/JA-independent ND

etr1, jar1, npr1, NahG, sid1 NahG, sid1, npr1, etr1, jar1 ND

Ran et al. (2005b), Djavaheri (2007) Djavaheri (2007)

ND

ND

ET/JA-dependent; SA-independent

471, hebiba, NahG

Leeman et al. (1995, 1996) De Vleesschauwer et al. (2008)

Tobacco Tobacco

Pseudomonas fluorescens CHA0

Pathogen

(continued)

Tomato Arabidopsis

Tomato Arabidopsis Arabidopsis

Pseudomonas syringae pv. tomato Turnip crinkle virus

Eucalyptus

Ralstonia solanacearum

Radish

Fusarium oxysporum f. sp. raphani Magnaporthe oryzae

Rice

ND ND

Pseudobactin þ salicylic acid Pseudobactin; unknown determinant Pseudobactin; LPS Pseudobactin

Zhang et al. (2002) Park and Kloepper (2000) Yan et al. (2002) Iavicoli et al. (2003)

Maurhofer et al. (1994)

Ran et al. (2005a)

Pseudomonas fluorescens WCS417

Pseudomonas syringae pv. tomato

LPS; other unknown metabolites

ET/JA/MYC2/NPR1/MYB72dependent; SA-independent

ein2, ein3 to ein7, eir1, etr1, eto1, jar1, myc2, npr1, myb72, NahG

Arabidopsis

Hyaloperonospora arabidopsidis Botrytis cinerea Alternaria brassicicola Fusarium oxysporum f. sp. dianthi Ralstonia solanacearum Fusarium oxysporum f. sp. raphani Botrytis cinerea

ND

MYC2/MYB72-dependent

myc2, myb72

ND ND LPS

MYB72-dependent MYB72-dependent ND

myb72 myb72 ND

LPS LPS; unknown ironregulated determinant N-alkylated benzylamine

ND ND

ND ND

SA-independent

Enzymatic assays

N-alkylated benzylamine ND ND

ND SA-independent ET/JA/NPR1-dependent; SA-independent SA-independent

ND Enzymatic assays etr1, jar1, npr1, NahG

Pseudobactin; LPS Pseudobactin; LPS

ND ND

ND ND

Meziane et al. (2005), Van Wees et al. (1997) Meziane et al. (2005) Meziane et al. (2005)

Pseudobactin Pseudobactin Pseudobactin; LPS LPS

ND ND ND ND

ND ND ND ND

Ran et al. (2005a) Van Loon et al. (2008) Meziane et al. (2005) Reitz et al. (2000, 2002)

Arabidopsis Arabidopsis Carnation Eucalyptus Radish Pseudomonas putida BTP1

Pseudomonas putida LSW17S Pseudomonas putida WCS358

Bean Cucumber Tomato Arabidopsis Arabidopsis

Bean Bean

Rhizobium etli G12

Van Wees et al. (1997), Pieterse et al. (1998), Knoester et al. (1999), Pozo et al. (2008), Van der Ent et al. (2008) Van der Ent et al. (2008)

Arabidopsis

Eucalyptus Tobacco Tomato Potato

Colletotrichum lagenarium Botrytis cinerea Pseudomonas syringae pv. tomato Pseudomonas syringae pv. tomato Botrytis cinerea Colletotrichum lindemuthianum Ralstonia solanacearum Erwinia carotovora Botrytis cinerea Globodera pallida

Pseudobactin; LPS; flagella

NahG

Van der Ent et al. (2008) Van der Ent et al. (2008) Van Peer and Schippers (1992) Ran et al. (2005a) Leeman et al. (1996) Ongena et al. (2004, 2005) Ongena et al. (2008) Akram et al. (2008) Ahn et al. (2007)

(continues)

TABLE I Bacterial strain

Plant species

Pathogen

(continued)

Determinanta

Pathwayb

Serratia liquefaciens MG1

Tomato

Alternaria alternata

N-acyl homoserine lactone

Probably SA- and ET-dependent

Serratia marcescens 90-166

Arabidopsis

Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Cucumber mosaic virus

ND

Catechol-type siderophore ND ND

JA/NPR1-dependent; SA/ETindependent JA/NPR1-dependent; SA/ETindependent JA-dependent; SA/NPR1independent ND SA-independent SA-independent

EPS

Induces PR-1b

ND

SA-dependent; ET/JA-independent

ND

SA-dependent; ET/JA/NPR1independent SA/NPR1-dependent; ET/JAindependent

Arabidopsis Arabidopsis Cucumber Tobacco Tobacco Serratia sp. strain Gsm01 Stenotrophomonas sp. N6.8 Streptomyces sp. EN27

Tobacco Arabidopsis Arabidopsis Arabidopsis

Colletotrichum orbiculare Peronospora tabacina Pseudomonas syringae pv. tabaci Cucumber mosaic virus Xanthomonas campestris CECT95; 4480 Erwinia carotovora subsp. carotovora Fusarium oxysporum

ND ND

ND

Plant lines tested/method usedc Macroarray with pure N-acyl homoserine lactone fad3-2 fad7-2 fad8, npr1, NahG, ein2 fad3-2 fad7-2 fad8, npr1, NahG, ein2 fad3-2 fad7-2 fad8, NahG, npr1 ND NahG NahG

Reference Schuhegger et al. (2006a) Ryu et al. (2003b) Ryu et al. (2003b) Ryu et al. (2004b) Press et al. (2001) Zhang et al. (2002) Press et al. (1997)

Reverse transcriptasePCR NahG, etr1-1, jar1-1

Ipper et al. (2008)

NahG, npr1, etr1-3, jar1

Conn et al. (2008)

NahG, npr1, etr1-3, jar1

Conn et al. (2008)

Domenech et al. (2007)

ND, not determined; LPS, lipopolysaccharides; EPS, exopolysaccharides. ND, not determined; SA, salicylic acid; ET, ethylene; JA, jasmonate; PR-1, Pathogenesis-related protein 1, SA-dependent; VSP, Vegetative storage protein, JA-responsive; HEL, Hevein-like protein, ET-responsive; ERD15, drought stress-responsive; RAB18, ABA- and drought stress-responsive. c See Table II for explanation about plant lines. a b

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(2006), who merely focus on Pseudomonas, Serratia, and Bacillus endophytes. A non-exhaustive list of other rhizobacteria with ISR-triggering capacities includes Lysobacter enzymogenes (Kilic-Ekici and Yuen, 2003), Paenibacillus alvei (Tjamos et al., 2005), Acinetobacter lwoffii (Trotel-Aziz et al., 2008), Chryseobacterium balustinum, Azospirillum brasilense (Ramos Solano et al., 2008), Curtobacterium sp., Arthrobacter oxidans (Barriuso et al., 2008), Stenotrophomonas (Domenech et al., 2007), and endophytic Actinobacteria (Conn et al., 2008). Although outside the scope of this review, it should be mentioned that various root-colonizing plant growth-promoting fungi also have ISR-eliciting capacities (see Bent, 2006, for an overview).

E. SCOPE OF THIS REVIEW

Rather than aiming at an exhaustive overview of different rhizobacteria able to elicit ISR, in this review we specifically focus on mechanisms of rhizobacteria-mediated ISR. We highlight recent progress in the identification of bacterial determinants, produced by rhizobacteria, that can trigger ISR, and pay special attention to how plants may recognize and respond to these resistance-inducing stimuli. In addition, details about the role of phytohormones in the various defence signalling pathways reported to underpin rhizobacteria–mediated ISR are presented. The plant defence responses that are being triggered during the onset and/or maintenance of ISR have been the subject of some excellent recent reviews (Van Loon, 2007; Van Loon and Bakker, 2005) and will therefore not be discussed in detail. A cursory overview of rhizobacteria for which information is available about bacterial determinants and/or signalling pathways involved in ISR, is listed in Table I.

II. RECOGNITION The only bacterial determinants mentioned in the review by Van Loon et al. (1998) are LPS and siderophores, including SA. Since that time a variety of additional resistance-inducing molecules have been described, including flagella, biosurfactants, N-acyl-homoserine lactones (AHL), N-alkylated benzylamines, antibiotics, and exopolysaccharides (EPS) (Table I). In the next paragraphs ISR-eliciting bacterial determinants and possible mechanisms by which plants can recognize or interfere with these compounds are discussed in detail.

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Bacterial motility is based on the flagellum and is important for the virulence of bacterial pathogens (Ramos et al., 2004) and for root colonization by rhizobacteria (De Weger et al., 1987b). The flagellum consists of a long helical filament and a rotary motor which is anchored in the cell surface. Flagellar filaments consist of thousands of flagellin protein subunits. The central part of flagellin, which forms the surface of the flagellum, is highly variable in sequence and length, while the N- and C-termini, which face the inside of the flagellar tube, are well conserved among bacteria. Flagellin is transported to the cell surface through the hollow core of the flagellum and assembled at the distal tip. Flagellin, however, can also accumulate in the environment as a result of leaks and spillover during the construction of flagellae, as well as by active shedding during biofilm formation and during cell death (Go´mez-Go´mez and Boller, 2002). In the case of Pseudomonas putida WCS358, the major secreted protein appears to be flagellin. Apparently, flagella from strain WCS358 are easily sheared from the cells and appear in the extracellular fraction (De Groot et al., 1996). In contrast, only a small amount of flagellin has been detected in the supernatant of Ps. fluorescens WCS374 (De Weger et al., 1987b). The conserved part of flagellin is recognized as a PAMP by the innate imune systems of plants and animals. In animal cells, a specific D1 domain of the conserved part of the flagellin polypeptide is recognized by the Toll-like receptor TLR5 (Ramos et al., 2004; Zipfel and Felix, 2005). Plant cells recognize a stretch of 15–22 amino acids close to the conserved N-terminal domain of flagellin. Flg22, a 22-amino acid peptide spanning the conserved domain is an extremely potent elicitor in cell cultures of different plant species such as Arabidopsis, tomato, potato, and tobacco. In tomato cells, flg22 was active at a threshold of about 1 pM, and the concentration required for half-maximal activity was 30 pM (Felix et al., 1999). In Arabidopsis, flagellin is perceived through its direct interaction with the transmembrane leucine-rich-repeat receptor kinase (LRR-RK) FLS2 (Chinchilla et al., 2006). FLS2 is normally present on the plasma membrane and was found to be internalized upon flg22 stimulation (Robatzek et al., 2006). Orthologues of FLS2 have been identified in Nicotiana benthamiana (Hann and Rathjen, 2007), tomato (Robatzek et al., 2007), Brassica spp. (Dunning et al., 2007), and rice (Takai et al., 2008). Flg22-type sequences can be found in flagellins from widely divergent bacteria, including Ps. putida and Pseudomonas aeruginosa. The flagellins of the plant-associated bacteria Agrobacterium and Rhizobium, however, have a highly divergent flagellin sequence, and their flagellins or the corresponding flg22-type sequences do not stimulate the

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flagellin perception system (Felix et al., 1999). Infiltration of 200 nM flg22 into lower leaves of Arabidopsis accession Col-0 or Ler-0 resulted in induced resistance against Ps. syringae pv. maculicola in distal leaves. Induced resistance could not be triggered by flg22 in the Ws-0 ecotype, a natural fls2 mutant insensitive to flagellin as well as to ET (Ton et al., 2001). Flg22induced systemic resistance was lost in the SAR-deficient Arabidopsis mutants sid2, npr1, ndr1, fmo1, eds1, and pad4 (Mishina and Zeier, 2007; see Table II for further information about these mutants). In contrast, when whole plants were treated with 1 M flg22, mutants npr1, eds1, pad4, and other mutants impaired in ET- or JA-dependent defence signalling, still showed a significant flg22-induced reduction in bacterial growth upon leaf infection with the bacterial speck pathogen Ps. syringae pv. tomato (Zipfel et al., 2004). It was suggested that whole plant treatment with flg22 induces the activation of the SA, JA, and ET pathways in parallel and that knocking out a single pathway alone does not abolish the induction of resistance (Zipfel et al., 2004). Recently, it was demonstrated that the early response to flg22 involves activation of several components of the JA-, ET-, and SAdefence signalling pathways, while the late response involves activation of SA-regulated processes. Activation of the late response may be dependent on signal strength reaching some threshold, as SA-dependent PR-1 expression was observed when flg22 was applied to seedling medium at a final concentration of 1 M, but not when applied at a concentration of 10 nM (Denoux et al., 2008). Up to now, a role of flagellin in rhizobacteria-mediated ISR has only been reported in Arabidopsis. Soil inoculation with Ps. putida WCS358 or a root dip in a solution containing 8.35 g/ml isolated flagella (corresponding to 108 colony-forming units (cfu)/ml) from this strain triggered ISR to Ps. syringae pv. tomato in Arabidopsis. However, a non-motile Tn5 mutant of WCS358 lacking flagella (strain GMB6), was equally effective in triggering ISR as the wild-type strain (Meziane et al., 2005). This can be explained by the fact that WCS358 has additional determinants that are recognized by Arabidopsis and that can trigger ISR, such as LPS and the iron-chelating fluorescent siderophore PSB (or pyoverdine) (see further). It is likely that flagellin from WCS358 is recognized by the FLS2 receptor in Arabidopsis, because the amino acid sequence of the conserved part of the N-terminal domain of Ps. putida flagellin is identical to flg22 and, as stated above, the major extracellular protein of WCS358 corresponds to flagellin. It would be interesting to test the ability of Ps. putida WCS358 or its isolated flagella to trigger ISR on fls2 Arabidopsis mutants. Isolated flagella of Ps. putida WCS358, however, did not induce systemic resistance to Botrytis cinerea and/or Colletotrichum lindemuthianum in bean or tomato (Meziane et al., 2005) or to the rice-blast fungus Magnaporthe oryzae in rice (D. De Vleesschauwer, unpublished data).

TABLE II Description of Plant Mutants or Transgenic Lines Mentioned in Table I and/or in the Text of this Review Mutant/ transgenic line

Phenotypeb

Wild-type gene product/functionb

Reference

Arabidopsis coi1

Coronatine- and JA-insensitive

F-box protein, activation of JA-dependent responses

Feys et al. (1994), Xie et al. (1998)

coi1-16a

Coronatine- and JA-insensitive

Ellis and Turner (2002)

cpr1 eds1

SA overproducer, constitutive expressor of PR genes Impaired in SA signalling

F-box protein, activation of JA-dependent responses Negative regulator of SAR

Parker et al. (1996), Falk et al. (1999)

eds5/sid1

SA-induction deficient

eds8

Reduced sensitivity to MeJA

Lipase-like protein, SA and oxidative stress signalling MATE transporter, SA biosynthesis JA signalling

ein2

ET-insensitive

ein3 ein4 ein5, ein7

ET-insensitive ET-insensitive ET-insensitive

Positive regulator of ET responses Transcription factor ET receptor Exoribonuclease

ein6 eir1

ET-insensitive ET-insensitive in the roots

Involved in ET signalling Auxin transporter

eto1

ET overproducer

etr1

ET-insensitive

Negative regulator of ET biosynthesis ET receptor, ET sensitivity

Bowling et al. (1994)

Nawrath and Me´traux (1999), Nawrath et al. (2002) Glazebrook et al. (1996), Ton et al. (2002a) Guzma´n and Ecker (1990), Alonso et al. (1999) Kieber et al. (1993), Solano et al. (1998) Roman et al. (1995), Hua et al. (1998) Roman et al. (1995), Potuschak et al. (2006) Roman et al. (1995) Roman et al. (1995), Luschnig et al. (1998) Guzma´n and Ecker (1990), Wang et al. (2004) Bleecker et al. (1988), Chang et al. (1993)

jar1

Deficient in linolenic acid, no JA accumulation Flagellin-insensitive Impaired in SAR, no systemic SA accumulation JA-insensitive

jin1-1, jin1-2/myc2

JA-insensitive

myb72-1/myb72-2

Impaired in ISR signalling

NahG

No SA accumulation

ndr1

Impaired in SAR

npr1/nim1

SA-insensitive, non-expressor of PR genes SA induction-deficient

fad3-2 fad7-2 fad8 fls2 fmo1

sid2

pad3-1

Reduced camalexin levels, glutathione-deficient Camalexin-deficient

pad4-1

Impaired in SA signalling

pad2-1

Trienoic fatty acid biosynthesis

McConn and Browse (1996)

Flagellin receptor Flavin-dependent monooxygenase

Go´mez-Go´mez and Boller (2000) Mishina and Zeier (2006)

JA-amino synthetase, JA signalling MYC-type helix-loop-helix transcription factor MYC2 R2R3-MYB-like transcription factor Transformed with SA hydroxylase, breaks down SA to catechol Plasma membrane-localized protein, SA signalling Ankyrin-repeat protein, SA and JA/ET response regulator Isochorismate synthase 1, SA biosynthesis

-glutamyl cysteine synthetase, glutathione synthesis P450 monooxygenase, camalexin biosynthesis Lipase-like protein, SA and oxidative stress signalling

Staswick et al. (1992), Staswick and Tiryaki (2004) Lorenzo et al. (2004) Van der Ent et al. (2008) Delaney et al. (1994) Century et al. (1995, 1997) Cao et al. (1994) Nawrath and Me´traux (1999), Wildermuth et al. (2001) Glazebrook and Ausubel (1994), Parisy et al. (2007) Glazebrook and Ausubel (1994), Schuhegger et al. (2006b) Glazebrook et al. (1996), Jirage et al. (1999) (continues)

TABLE II Mutant/ transgenic line

Phenotypeb

Rice hebiba 417

JA-deficient ET-insensitive

NahG

Reduced SA accumulation

Tobacco NahG

No SA accumulation

Tetr18

ET-insensitive

Tomato def1 NahG

Impaired in JA biosynthesis No SA accumulation

never-ripe (nr)

ET-insensitive

a b

(continued)

Wild-type gene product/functionb

Reference

JA biosynthesis Transformed with OsEIN2 antisense construct Transformed with SA-hydroxylase gene, breaks down SA to catechol

Riemann et al. (2003) Jun et al. (2004)

Transformed with SA hydroxylase, breaks down SA to catechol Transformed with mutant ET-receptor gene etr1-1 from Arabidopsis

Gaffney et al. (1993)

Octadecanoid metabolism Transformed with SA-hydroxylase gene, breaks down SA to catechol ET receptor, ET sensitivity

Carries additional mutation in pen2, a gene involved in non-host resistance (Westphal et al., 2008). ET, ethylene; JA, jasmonate; MeJA, methyl jasmonate; SA, salicylic acid.

Yang et al. (2004)

Knoester et al. (1998)

Howe et al. (1996) Brading et al. (2000) Lanahan et al. (1994)

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Although rice has a flagellin receptor, OsFLS2, flg22 induces only a very weak immune response without cell death in cultured rice cells (Takai et al., 2008), which may explain why flagella of WCS358 are not recognized by rice roots. It is presently unclear why purified flagella of WCS358 fail to induce resistance in tomato, as a high-affinity binding site for flagellin, flg22, and its 15-amino acid peptide flg15 has also been identified in tomato (Robatzek et al., 2007). One explanation is that the resistance pathway induced upon flagellin-receptor recognition in tomato is not effective against Bo. cinerea. In this context, it should be mentioned that purified flagella of Ps. fluorescens WCS374, Ps. fluorescens WCS417, and Ps. aeruginosa 7NSK2 did not trigger resistance to Bo. cinerea in tomato either (Meziane, 2005). It remains to be investigated whether purified flagella of WCS358 or other rhizobacteria can induce resistance to other pathogens in tomato. Van Wees et al. (1997) reported that unlike Ps. putida WCS358, Ps. fluorescens WCS374r did not induce resistance to Ps. syringae pv. tomato in Arabidopsis. But recently it was shown that Ps. fluorescens WCS374r does trigger ISR against Ps. syringae pv. tomato in Arabidopsis when the inoculum was grown at 33 8C rather than at 28 8C (Ran et al., 2005b), or when bacteria were applied at a low inoculum density (Djavaheri, 2007). When applied at an initial density of 103 cfu/g of soil, WCS374r reached a population density on Arabidopsis roots of around 107 cfu/g of root after 3 weeks. Apparently, active multiplication of WCS374r is required for bacterial elicitors of ISR to be produced or perceived by the plant. Two non-motile mutants of WCS374, L30 and L36 (De Weger et al., 1987b), lacking flagellin and defective in flagellin polymerization, respectively, failed to induce resistance to Ps. syringae pv. tomato. Isolated flagella from a culture of WCS374r significantly reduced disease incidence when applied to Arabidopsis roots 7 or 4 days prior to inoculation with the pathogen. Preparations of mutants L30 or L36, however, were not effective, but it should be noted that root colonization by these mutants was significantly impaired, which might be the reason for the lack of ISR-eliciting activity of these mutants. These results suggest that flagella of actively growing Ps. fluorescens WCS374r cells can induce systemic resistance to Ps. syringae pv. tomato in Arabidopsis, but it cannot be excluded that other determinants produced by actively growing cells are also involved in this process. It was hypothesized that WCS374r, when applied at high density, loses its flagella, whereas, if the number of cells is low, they would be incited to propel themselves towards the nutrient-rich zone of the rhizosphere through active flagellar movement. Perception of the flagella by the roots would then result in elicitation of the plant. Loss of flagella at high cell densities has been shown for Ps. syringae cells on moist bean leaves (Djavaheri, 2007). In this

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context, it is noteworthy that the defence pathway against Ps. syringae pv. tomato triggered by WCS374r applied at low densities in Arabidopsis was SA-independent (tested in genotypes NahG and sid1), while dependent on JA, ET, and NPR1 and probably similar to the signalling pathway described by Pieterse et al. (1998) for WCS417-mediated ISR (Djavaheri, 2007). These results may indicate that the flagella dose perceived by the plant is too low to trigger the late SA-mediated defences seen by Denoux et al. (2008) or the SA-dependent SAR described by Mishina and Zeier (2007). It is not known by which signalling pathway(s) isolated flagella or pure flg22 induce systemic resistance when applied to plant roots and this should be tested. As stated before, it cannot be excluded either that other determinants of WCS374r besides flagella are recognized by the plant. Ran et al. (2005b) showed that at cell densities of 5 107 cfu/g of soil Ps. fluorescens WCS374 triggered ISR to Ps. syringae pv. tomato in Arabidopsis when the inoculum was grown at 33 8C, but not when grown at 28 8C. It is not clear whether growth temperature may have an influence on flagella formation or on other bacterial determinants that may be implicated in WCS374-elicited ISR. Interestingly, mutants of Ps. aeruginosa 7NSK2 that failed to induce systemic resistance in tomato, tobacco, and bean, still induced systemic resistance to Ps. syringae pv. tomato in Arabidopsis by an SA-independent signalling pathway (Ran et al., 2005b). It is not unlikely that flagellin from Ps. aeruginosa 7NSK2 is involved in this elicitation, as flg22like sequences are found in the flagellin of Ps. aeruginosa. B. LIPOPOLYSACCHARIDES

LPS constitute the major structural component of the outer membrane of Gram-negative bacteria. LPS are complex molecules possessing both hydrophilic and lipophilic properties. LPS consist of three different components: a lipid, a core oligosaccharide, and an O-linked polysaccharide. The lipophilic part, called lipid A, is embedded in the outer membrane. Its structure is highly conserved among Gram-negative bacteria. The core oligosaccharide consists of a short chain of sugars, which connects the lipid A part to the hydrophilic O-antigen, which is composed of repeating oligosaccharide subunits made up of three to five sugar molecules. The individual chains can vary in length ranging up to 40 repeating units. The O-polysaccharide is much longer than the core oligosaccharide and extends out into the environment. The composition of the O-antigen part is highly variable between species and even between strains of Gram-negative bacteria (see Lerouge and Vanderleyden, 2002, for a comprehensive review). LPS has a structural function in stabilizing the outer membrane of the bacterium but also play a

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number of important roles in the interactions of bacteria with eukaryotic hosts. LPS are important virulence factors in animal pathogenesis. Lipid A is the toxic component of LPS and lipid A molecules are detected at picomolar concentrations by the innate immune system of animals. LPS molecules can dissociate from the surface of Gram-negative bacteria. The hydrophilic O-polysaccharide may allow diffusion or delivery of the toxic lipid in the hydrophilic environment. LPS first binds the serum LPS-binding protein. Then, the LPS, bound to the LPS-binding protein, is perceived by a complex that consists of the Toll-like receptor TLR4, the membrane receptor CD14, and an extracellular accessory protein MD-2 (Underhill and Ozinsky, 2002). LPS play an important role in bacterial interactions with plants also. The most studied effect of LPS on plant cells is their ability to prevent the HR induced in plants by either avirulent or nonhost bacteria. Following adhesion to cell wall components, the LPS bind to yet to be defined plasma membrane receptors. This response can be observed in various plants including tobacco, pepper, turnip, and Arabidopsis and is referred to as ‘localized induced resistance’ or ‘response’ (LIR) (see Dow et al., 2000, for a review). The minimal structure of LPS required for the prevention of the HR appears to be the lipid A attached to a truncated core oligosaccharide. LPS from plantpathogenic bacteria can also directly induce or activate defence-related responses in plants (Newman et al., 2007). In Arabidopsis, LPS from animal and plant pathogens at concentrations between 10 and 200 g/ml induced a rapid burst of nitric oxide (NO), a hallmark of innate immunity in animals. Lipid A was as effective as most LPS preparations. In addition, LPS of Burkholderia cepacia at 100 g/ml activated an array of defence genes in Arabidopsis plants and suspension cells, including glutathione S-transferases, cytochrome P450 and many genes encoding PR-proteins, such as PR-1, -2, -3, -4, and -5. Several of the LPS-induced genes were activated in systemic leaves too (Zeidler et al., 2004). Recently it was shown that the intact lipooligosaccharide (LOS) (LPS without the O-chain) of Xanthomonas campestris pv. campestris and the lipid A moiety, as well as the core oligosaccharide derived from it, could trigger the LIR response and the expression of PR-1 and -2 in Arabidopsis. In response to X. campestris pv. campestris LOS transcript levels showed an early but transient accumulation at 12 h and a later more substantial accumulation after 20 h. The core oligosaccharide only induced the early accumulation, while lipid A only induced a substantial response after 20 h. These data suggest that LOS is recognized through independent mechanisms involving the core oligosaccharide and lipid A moieties, respectively (Silipo et al., 2005). Infiltration of LPS preparations from Ps. aeruginosa or Escherichia coli at a concentration of 100 g/ml into primary leaves triggered resistance to

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Ps. syringae pv. maculicola in systemic leaves. This systemic resistance response was not observed when a de-esterified LPS lacking the lipid A part of the molecule was used. The LPS-elicited ISR response did not develop in the SAR-deficient Arabidopsis mutants sid2, npr1, ndr1, fmo1, eds1, and pad4 (Mishina and Zeier, 2007). To date, no LPS receptor has been identified in plants. However, fluorescently labelled LPS from X. campestris pv. campestris rapidly bound to tobacco cells and became internalized into endocytic vesicles, consistent with a receptor-mediated process and reminiscent of the mammalian system (Gross et al., 2005). In general, it can be stated that putative LPS receptors in plants are probably of a low-affinity type as micrograms-per-millilitre concentrations of LPS are needed to trigger plant defence responses (Zeidler et al., 2004). In the case of PGPR, the O-antigen of the LPS rather than the lipid A or the core oligosaccharide appears to be the moiety that triggers ISR in plants. In radish Ps. fluorescens WCS374 and Ps. fluorescens WCS417 triggered ISR against Fusarium oxysporum f. sp. raphani (Leeman et al., 1995). Under conditions of high iron availability, the O-antigen-minus mutants of these strains no longer reduced disease incidence, while their purified LPS were as effective as the wild-type strains. Ps. putida WCS358 or its isolated LPS were unable to trigger ISR against Fusarium wilt in radish. However, this strain triggered resistance to Bo. cinerea and C. lindemuthianum in bean and to Bo. cinerea in tomato. In these three cases, an O-antigen-minus mutant of WCS358 no longer triggered ISR, while purified LPS applied to plant roots was very active (Meziane et al., 2005). In Arabidopsis, LPS of Ps. fluorescens WCS374 is not effective, but LPS of both Ps. fluorescens WCS417 and Ps. putida WCS358 appear to be involved in ISR against Ps. syringae pv. tomato. Cell wall preparations of WCS417r triggered ISR, while cell walls of a mutant lacking the O-antigenic part of the LPS were less effective (Van Wees et al., 1997). Likewise, crude LPS of WCS358 triggered ISR (Meziane et al., 2005). The O-antigen-minus mutants of these strains, however, were equally effective as the wild-type strains (Meziane et al., 2005, Van Wees et al., 1997). These results indicate that other determinants of WCS417 and WCS358 that are still produced by the O-antigen-minus mutants are recognized in Arabidopsis. It cannot be excluded that the lipid A part or core oligosaccharides of the LPS molecules of WCS358 are recognized in Arabidopsis, as cell walls of its O-antigen-minus mutant were not tested. Alternatively, for Ps. putida WCS358 it is known that its flagella and PSB are also recognized in Arabidopsis. Ps. fluorescens WCS374 and Ps. putida WCS358 differ in their LPS composition; the LPS composition of Ps. fluorescens WCS417 is not known. The LPS of Ps. fluorescens WCS374 contains fucose and rhamnose (De Weger

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et al., 1987a), two sugars that are not found in the LPS of WCS358 but are typically present in O-chain structures from phytopathogenic bacteria (Newman et al., 2007). The O-antigenic side chain of WCS358, however, contains the aminosugar quinovosamine, which is absent in the LPS of WCS374 (De Weger et al., 1987a). In this context, it is interesting to note that quinovosamine is also present in the O-antigen of the LPS from Rhizobium etli. Mutants of R. etli and other Rhizobia that lack the O-antigen portion of the LPS are ineffective in infection and cause aberrant development of nodules on their host legumes. The LPS of R. etli mutant strain CE166 lacks quinovosamine and is as ineffective in symbiosis as mutants that lack the O-antigen completely. It was suggested that the symbiotic role of LPS requires a structural feature conferred by quinovosamine (Noel et al., 2000). Interestingly, the LPS of R. etli strain G12 acts as the inducing agent of systemic resistance against the potato cyst nematode Globodera pallida in potato roots (Reitz et al., 2000). In a follow-up study, however, it was shown that the oligosaccharides of the core region, rather than the O-antigen are the main trigger of systemic resistance in potato roots towards G. pallida infection (Reitz et al., 2002). C. BIOSURFACTANTS

Recently, it was shown that also biosurfactants, and more specifically cyclic lipopeptides, can act as bacterial determinants for ISR in plants. Cyclic lipopeptides are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain (Raaijmakers et al., 2006). Cyclic lipopeptides are produced by several plant-associated bacteria, including pathogenic and antagonistic Pseudomonas bacteria (reviewed by Raaijmakers et al., 2006) and antagonistic Bacillus strains (reviewed by Ongena and Jacques, 2008). Bacillus subtilis produces cyclic lipopeptides from the surfactin, iturin, and fengycin families. Surfactins are heptapeptides interlinked with a -hydroxy fatty acid with a length that may vary from 13 to 16 carbon units. Surfactins display haemolytic, anti-viral, anti-mycoplasma, and anti-bacterial activities but no marked fungitoxicity. Surfactins can readily associate with, and tightly anchor into lipid bilayers and thereby, interfere with biological membrane integrity in a dose-dependent manner. At low concentration, surfactins insert exclusively in the outer leaflet of the membrane, inducing only limited perturbation. At intermediate concentrations, surfactins cause a transient permeabilization, but membranes reanneal. At higher concentrations, irreversible pore formation and complete disruption and solubilization of the lipid bilayer can occur. Fengycins are lipodecapeptides with an internal

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lactone ring in the peptidic moiety and with a -hydroxy fatty acid chain ranging from 14 to 18 carbon units that can be saturated or unsaturated. Fengycins show strong fungitoxic activity, specifically against filamentous fungi. Mechanistically, the action of fengycins is less well known than that of surfactins, but they also readily interact with lipid bilayers and to some extent also possess the potential to alter cell membrane structure and permeability in a dose-dependent manner. As the presence of cyclic lipopeptides at concentrations of about 5 M was not associated with any phytotoxicity or adverse effect on bean or tomato plants (Ongena and Jacques, 2008), it is suggested that these molecules could interact with plant cells by inducing disturbance or transient channelling in the plasma membrane that can in turn activate a biochemical cascade of molecular events leading to defensive responses. Iturins are heptapeptides linked to a -amino fatty acid chain with a length of 14–17 carbons. They display a strong in vitro antifungal action against a wide variety of yeasts and fungi, but only limited antibacterial and no antiviral activities. Fungitoxicity is based on osmotic perturbation owing to the formation of ion-conducting pores in the fungal membranes and not on membrane disruption or solubilization. In addition, the presence of ergosterol in fungal membranes is important for iturin activity. The lack of effect of iturin on plants cells could be because of a different composition in phytosterols (Ongena and Jacques, 2008). Pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants against Bo. cinerea, similar to the one induced by living cells of the strain Ba. subtilis S499 (Ongena et al., 2007). In addition, a significant protective effect against Bo. cinerea was gained by treating bean or tomato plants with surfactin and/or fengycin overproducing derivatives generated from wild-type Ba. subtilis Bs168, which is not able to synthesize cyclic lipopeptides and does not induce systemic resistance in plants. On tomato, surfactin appeared to be more effective than fengycin. However, fengycins, but not surfactins, could induce a defence response in potato tuber cells, while both surfactants elicited major defence-associated changes in tobacco cells. Iturin, however, does not show any ISR-eliciting activity in tomato plants, potato tuber slices, or tobacco cells. None of the surfactants had an effect on cucumber (Ongena and Jacques, 2008; Ongena et al., 2007). Raaijmakers et al. (2006) have classified the cyclic lipopeptides of Pseudomonas spp. into four major groups: the viscosin, amphisin, tolaasin, and syringomycin groups. Massitolide A is a member of the viscosin group which harbours cyclic lipopeptides with 9 amino acids linked with a 10-carbon hydroxy fatty acid chain. The massitolide-producing Ps. fluorescens strain SS101 was effective in preventing infection of tomato leaves by Phytophthora infestans and significantly reduced the expansion of existing

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late blight lesions. Ps. fluorescens SS101 or massitolide A could only prevent late blight infections through direct antagonism. Ps. fluorescens SS101 or massitolide A at a concentration of 50 g/ml (44 M) significantly reduced lesion area but not disease incidence when applied to the lower leaves of tomato plants, 24 h prior to challenge inoculation of the upper leaves with zoospores of Ph. infestans. A massitolide-negative mutant of Ps. fluorescens SS101 completely lost the ability to induce systemic resistance. Ps. fluorescens SS101 significantly reduced lesion area and sporangia formation per unit lesion area, but not disease incidence when applied to tomato seeds 5 weeks prior to inoculation with Ph. infestans zoospores on the tomato leaves. The massitolide-negative mutant showed effects intermediate between the control and SS101 treatments. These results show that massitolide A is a bacterial determinant of ISR in tomato (Tran et al., 2007). How massitolide A induces resistance in plants is not known, but it is likely that it can also be explained by effects on the plant plasma membrane. Cyclic lipopeptides produced by plant-pathogenic Pseudomonas spp. cause the formation of ion channels in the host plasma membrane. At high concentrations, cyclic lipopeptides can solubilize plasma membranes. Cell suspensions of Ps. fluorescens SS101 or massitolide A cause lysis of zoospores of oomycete pathogens within 60 s (De Souza et al., 2003). Cell suspensions of Ps. putida WCS358 also show a clear biosurfactant activity, such as lowering of the surface tension of water and drop collapse, but they do not have an adverse effect on zoospores (De Souza et al., 2003). There are no indications that biosurfactants of Ps. putida WCS358 play a role in ISR. It would be interesting to test whether there is a correlation between the ability of a biosurfactant to cause zoospore lysis and and its capacity to trigger ISR. D. N-ACYL-L-HOMOSERINE LACTONE

AHLs occur in various Gram-negative bacteria and act as signal molecules to control expression of various functions in a cell density-dependent manner. This phenomenon is known as quorum sensing (Miller and Bassler, 2001). Serratia liquefaciens MG1 produces two AHL molecules: N-butanoyl and N-hexanoyl homoserine lactones. S. liquefaciens MG1 can induce systemic resistance to Alternaria alternata when applied at a concentration of 1010 cfu/ ml around the shoots of tomato plants 3 days prior to challenge inoculation with the pathogen. An AHL-negative mutant of S. liquefaciens MG1 slowed down the development of A. alternata-induced cell death, but infected plants showed no significant alterations in response to the fungal pathogen when compared with the non-inoculated control. Inoculation with the AHL-producing Ps. putida strain IsoF also resulted in a marked reduction of leaf

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damage following challenge with A. alternata. This strain produces at least four different 3-oxo-AHL molecules with acyl side chains varying from 6 to 12 carbon units. An AHL-negative mutant, F117, was less effective than the wild-type strain IsoF, but still reduced necrotic cell death by about 50%, suggesting that additional factors which are not regulated by quorum-sensing compounds contribute to the biocontrol activity of Ps. putida IsoF. S. liquefaciens MG1 and pure N-hexanoyl homoserine lactone (10 M) significantly increased free and conjugated SA levels in tomato leaves, while this increase was not observed for the AHL-negative mutant. In addition, a macroarray analysis of defence gene expression in tomato leaves after application of 10 M N-hexanoyl or N-butanoyl homoserine lactone to the roots showed, among others, enhanced expression of PR-1a (P4), PR-1b (P6), and a 26 kDa acidic chitinase (Schuhegger et al., 2006a). In this study, however, the effect of application of pure AHL to plant roots on A. alternata leaf infection is not shown. It cannot be excluded that the bacterial determinant triggering resistance to A. alternata is not the homoserine lactone but a secondary metabolite regulated by quorum sensing. E. N-ALKYLATED BENZYLAMINE

Ps. putida BTP1 induces resistance to Bo. cinerea in bean and tomato and to Pythium aphanidermatum and Colletotrichum lagenarium in cucumber (Ongena et al., 1999, 2002, 2008). At least in bean and cucumber, N, N-dimethyl, N-tetradecyl-N-benzylammonium (NABD) appears to be the bacterial determinant responsible for ISR (Ongena et al., 2005, 2008). Pure benzylamine at a concentration of 1 M was also effective in triggering induced resistance in bean and cucumber, indicating that the aromatic amino part is important for the biological activity of the entire molecule. It was hypothesized that the aromatic phenol group, which is also present in thiamine (according to Ahn et al. (2005) another inducer of systemic resistance in plants), SA, and 2,4-diacetylphloroglucinol (DAPG), could constitute a general motif widely recognized by specific plant cell receptors (Ongena et al., 2008). F. SIDEROPHORES

Siderophores are low-molecular-weight molecules that are secreted by most aerobic and facultative anaerobic microorganisms to trap traces of ferric iron [Fe(III)] in the environment and deliver the iron to the cell. According to current concepts, ferric ion-loaded siderophores are selectively recognized and bound by high-affinity receptor proteins that are present on the outer

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bacterial membrane. Although competition for ferric iron between biocontrol bacteria and plant deleterious microorganisms is considered to be the main mode of action of siderophores, there is accumulating evidence that these compounds also function as important triggers of the plant immune response (Ho¨fte and Bakker, 2007).

1. Pseudobactins So far, research aimed at deciphering the role of siderophores in systemically induced resistance has tended to focus on the involvement of pseudobactin-type elicitors. Pseudobactin (PSB), also termed pyoverdin or fluorescein, designates an extensive group of diffusible green-fluorescent pigments that are historically recognized as the distinctive phenotypic trait of the rRNA homology group I species of the genus Pseudomonas (Visca et al., 2007). Following the seminal work of Meyer (Meyer and Abdallah, 1978; Meyer and Hornsperger, 1978), the structures of more than 50 PSBs from different strains have now been determined. All appear to comprise a conserved fluorescent dihydroxyquinoline chromophore joined to a highly variable peptide and acyl side chain (for review see Visca et al., 2007). Synthesis of PSBs and the corresponding membrane receptors occurs exclusively under conditions of iron starvation, and is repressed under iron-rich conditions (Meyer and Abdallah, 1978). In addition, some PSBs are known to trigger their own synthesis and uptake in a cell-density dependent manner, indicating a complex autoregulatory system that enables maximal expression of the cognate synthesis and receptor genes only when the siderophore is effective in Fe3þ delivery to the cell (Visca, 2004). A role for PSBs as determinants of rhizobacteria-triggered resistance has been reported in several systems. For instance, a clear-cut role for PSB in ISR was reported for Ps. putida WCS358 in the suppression of Ralstonia solanacearum in Eucalyptus urophylla (Ran et al., 2005a), Erwinia carotovora in tobacco (Van Loon et al., 2008), and Bo. cinerea in tomato (Meziane et al., 2005). In all three cases, the purified PSB358 was as effective as the wild-type strain, whereas a PSB-deficient mutant lost the ability to cause ISR. In bean, however, the situation appears to be more complex in that wild-type bacteria, the PSB-minus mutant, and isolated PSB358 all induced a significant level of ISR against Bo. cinerea and C. lindemuthianum, indicating redundancy of ISR-triggering traits in this system (Meziane et al., 2005). A similar phenomenon was observed for the ISR elicited by Ps. fluorescens WCS374r in Eu. urophylla. Although infiltration of the lower leaves with a siderophoreminus mutant of WCS374r 3–7 days before challenge inoculation with Ra. solanacearum, caused a similar reduction of bacterial wilt as the parental strain, purified PSB induced resistance as well (Ran et al., 2005b). Both the

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siderophore and another, yet unidentified, inducing determinant of WCS374r thus seem to be capable of causing ISR in Eucalyptus. In Arabidopsis, Ps. fluorescens WCS374r induced resistance to Turnip crinkle virus (TCV) irrespective of the bacterial density applied to the soil. Mutants impaired in PSB and/or SA production lost the ability to trigger ISR to TCV, suggesting that both compounds are required in this pathosystem (Djavaheri, 2007). In rice, however, PSB seems to be the only determinant responsible for elicitation of WCS374r-mediated ISR. Assessing the effect of several well-defined mutants and testing of the purified compound in the microgram range unambiguously demonstrated that WCS374r-afforded protection against the rice blast-causing ascomycete M. oryzae is based on PSBmediated priming for a multifaceted cellular defence response, comprising among others the concerted expression of a diverse set of physiological and biochemical defences, as well as a hyperinduction of H2O2 generation in the epidermis (De Vleesschauwer et al., 2008). Strikingly, the isolated PSBs of Ps. fluorescens WCS358 and Ps. aeruginosa 7NSK2 failed to induce ISR in rice against M. oryzae, whereas PSB of Ps. fluorescens C7R12 was as effective as that of WCS374 (our unpublished data; Fig. 1). Such variability between a

Ctrl BTH PSB374 PSBC7R12

b b b a

PSB358 PSB7NSK2

a 0 10 20 30 40 50 60 Sporulating blast lesions on leaf 5

Fig. 1. Effectiveness of benzothiadiazole (BTH; 0.05 mM)- and pseudobactin (PSB)-induced resistance in rice against the blast pathogen Magnaporthe oryzae. To trigger resistance, rice seedlings (5-leaf stage) were hydroponically fed with the various compounds by including the desired concentration in the half-strength Hoagland nutrient solution 3 days before challenge. Six days post inoculation, disease was rated by counting the number of susceptible-type lesions on leaf 5. All pseudobactins were isolated from the respective bacterial cultures and applied at a concentration of 100 g per root system, except for the purified PSB of Pseudomonas fluorescens WCS374, which was applied at a concentration of 70 g per plant. Different letters indicate statistically significant differences between treatments (Kruskal–Wallis and Mann–Whitney test, ¼ 0.05).

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PSBs has also been observed in radish, where the PSB siderophore of WCS374, but not those of Ps. fluorescens WCS417 and WCS358, was found to trigger ISR against Fusarium wilt (Leeman et al., 1996). In contrast, Van Loon et al. (2008) recently reported the capacity of all three of these siderophores to enhance defence against Er. carotovora in tobacco. Finally, PSB-conferred disease suppression was also found to be effective against Tobacco necrosis virus in tobacco, on the basis of the observation that CHA400, a PSB-deficient mutant of Ps. fluorescens CHA0, was less effective in reducing numbers and size of viral lesions than the parental strain (Maurhofer et al., 1994). Although the phenomenon of PSB-mediated resistance has received steadily increasing attention over the past decade, it is still unknown how PSB-type siderophores are perceived by the plant and ultimately give rise to ISR. An alternative to direct recognition of PSB elicitors by the plant is the perception of microbially induced alterations in the plant’s immediate environment, i.e. the rhizosphere. Given the scarcity of bioavailable iron [Fe(III)] in the rhizosphere, and the high affinity of PSBs for the ferric ion, PSBproducing rhizobacteria are thought to interfere with the iron acquisition of other soil organisms, including the host plant (Vansuyt et al., 2007). In this context, our recent observation that Ps. fluorescens WCS374r aggravates chlorosis symptoms of young rice plants grown under iron-limiting conditions is of particular interest (our unpublished data; Fig. 2). Strikingly, enhanced iron deficiency-induced chlorosis was not observed in response to root colonization by rhizobacteria producing non-ISR-eliciting PSBs, such as Ps. aeruginosa 7NSK2 or Ps. putida WCS358 (our unpublished data). Furthermore, purified PSB374, but not PSB358, was found to trigger intracellular iron depletion in systemic leaves as evidenced by the down-regulated expression of the iron homeostasis marker gene OsFer1 (our unpublished data). These findings suggest that the ability of a given PSB to increase blast resistance is related to its potential to deprive rice from iron. In a recent microarray study on iron-deficient rice, Kobayashi et al. (2005) found that iron deficiency in roots strongly elicits the transcriptional activation of nearly all genes involved in the methionine cycle, both in root and leaf tissue. Furthermore, several studies point to a role for the methionine cycle and its main intermediate, the universal substrate S-adenosyl-L-methionine (SAM), in rice defence to M. oryzae. Most tellingly in this regard, Seguchi et al. (1992) reported that the activity of the SAM-utilizing enzyme SAM decarboxylase was suppressed by as much as 50% in M. oryzae-inoculated rice plants, whereas such suppression was not observed in plants pretreated with the blast resistance-inducing chemical N-cyanomethyl-2-chloroisonicotinamide. Similarly, SAM-SYNTHETASE, a SAM-biosynthesis gene, was shown to be

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Distribution over classes

100%

80%

60%

Severe chlorosis Slight chlorosis No chlorosis

40%

20%

0% Ctrl

WCS374r

AT12

Fig. 2. Root colonization by the wild-type bacterium Pseudomonas fluorescens WCS374r aggravates iron-deficiency-induced chlorosis of young rice seedlings (5-leaf stage), while strain AT12, a salicylic acid-positive but pseudobactin- and pseudomonine-negative mutant derivative of WCS374r, does not induce chlorosis.

dramatically upregulated upon treatment of rice with probenazole, a blast fungicide and well-characterized inducer of resistance in several plant species (Shimono et al., 2003). The link between the methionine cycle, SAM metabolism, and resistance to M. oryzae is further strengthened by the rapid and specific expression of OsBISAMT1, encoding a putative SAM-methyltransferase, in incompatible rice–M. oryzae interactions (Xu et al., 2006), and the observation that topical application of methionine not only induces production of the rice phytoalexins sakuranetin and momilactone A, but also increases resistance to subsequent blast attack (Nakazato et al., 2000). Taking these facts into account, one may hypothesize that PSB-type siderophores enhance defence against M. oryzae by depriving rice roots from iron, leading to cytosolic iron depletion and resultant activation of the methionine cycle. This hypothesis has received further support recently from work by Liu et al. (2007). In line with disease-related alterations in iron homeostasis in animals (Hissen et al., 2005; Schaible and Kaufmann, 2004), these authors elegantly demonstrated that targeted redistribution of redox-active Fe inflicted by powdery mildew attack, acts as an underlying factor associated with the oxidative burst and regulation of disease resistance in cereals. A model implying PSB-inflicted iron stress on the roots as a primary event in the activation of rhizobacteria-mediated resistance may also

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hold for WCS417r-triggered ISR in Arabidopsis, as MYB72, a transcription factor gene required for the early onset of ISR (Van der Ent et al., 2008), was reported to be activated exclusively in response to low-iron conditions (Colangelo and Guerinot, 2004; Van de Mortel et al., 2006). Nevertheless, it should be noted that experiments conducted in radish indicate that iron deficiency alone is not sufficient to enhance defence against Fusarium wilt, implicating the necessity for at least one additional component to induce systemic resistance in this system (Leeman et al., 1996).

2. SA and SA-containing siderophores Under conditions of iron limitation, several Pseudomonas spp. not only produce PSBs but also the phenolic compound SA, which functions as a signalling hormone in the development of SAR (Durrant and Dong, 2004; Loake and Grant, 2007). Accordingly, the increased effectiveness of the pseudomonads WCS374r and WCS417r to suppress Fusarium wilt in an iron-deficient radish system was originally attributed to bacterial SA production (Leeman et al., 1996). Implicit here was the view that the ISR triggered by these strains would be reliant on SA signalling in the plant. However, Hoffland et al. (1995) failed to observe any activation of the SA-dependent signalling conduit in radish after treatment with WCS374. Moreover, root treatment with 5 107 cfu/g of soil of strain WCS374r did not induce systemic resistance in Arabidopsis against Ps. syringae pv. tomato, whereas exogenously administered SA did (Djavaheri, 2007; Van Wees et al., 1997). Additionally, in some elegant work on Arabidopsis, Ran et al. (2005b) demonstrated that many SA-producing rhizobacteria, among which are Ps. aeruginosa 7NKS2 and Ps. fluorescens WCS417, elicit systemic protection against Ps. syringae pv. tomato in an SA-independent manner (Ran et al., 2005b). In keeping with these results, several other studies describe that rhizobacterial SA production and triggering of ISR are not necessarily interrelated. For instance, elicitation of ISR in tobacco and cucumber by the versatile resistance-inducer Serratia marcescens 90-166 was shown to function independently of bacterially synthesized SA, while requiring production of a catechol-type siderophore (Press et al., 1997, 2001). Together, these reports constitute a large body of evidence suggesting that SA, despite being a potent elicitor of SAR, does not generally contribute to initiation of rhizobacteria-mediated ISR. This apparent discrepancy can be addressed by considering that bacterial SA often serves as a precursor in the biosynthesis of other siderophores

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containing a SA-moiety, such as pseudomonine in Ps. fluorescens WCS374r and pyochelin in Ps. aeruginosa 7NSK2. In this perspective, it is tempting to speculate that in the rhizosphere, where iron-limiting conditions tend to prevail, SA is not excreted by the bacteria but channelled into the production of SA-containing siderophores. Indeed, measuring SA levels on the roots of tomato colonized by either 7NSK2 or its chemical mutant KMPCH, which is unable to convert SA into pyochelin, demonstrated that only KMPCH produces nanogram amounts of SA in the rhizosphere, whereas 7NSK2 does not (Audenaert et al., 2002). In line with the latter observation, Audenaert et al. (2002) postulated that elicitation of ISR by 7NSK2 does not depend on SA per se, but rather is on the basis of a synergistic interaction between SA-derived pyochelin and the phenazine antibiotic pyocyanin. This notion was borne out by the observations that PHZ1, a mutant of 7NSK2 no longer able to produce pyocyanin because of an insertion in the phzM gene encoding an O-methyltransferase, failed to induce resistance in tomato against Bo. cinerea, whereas complementation for pyocyanin production or co-inoculation with the pyocyanin-overproducing but SA-, pyochelin-, and ISR-deficient mutant 7NSK2-562 restored induced resistance. Induction of resistance by mutant KMPCH, on the other hand, does appear to involve SA. Contrary to wild-type bacteria, treatment of bean and tomato roots with KMPCH resulted in elevated activity of phenylalanine ammonia-lyase (PAL), a key enzyme involved in the phenylpropanoid-driven SA biosynthetic pathway, and elicitation of ISR by KMPCH was blocked in SA-deficient NahG tomato plants (Audenaert et al., 2002; De Meyer and Ho¨fte, 1997; De Meyer et al., 1999b). Additional evidence supporting the involvement of bacterially produced SA in ISR comes from a 10-year-old report by Maurhofer et al. (1998), demonstrating that transfer of the SA-biosynthetic cluster from Ps. aeruginosa PAO1 to the non-SA-producing strain Ps. fluorescens P3 rendered this strain capable of SA production in vitro and significantly improved ISR in tobacco against Tobacco necrosis virus. Surprisingly, however, a pyocyanin-negative mutant of KMPCH lost the ability to trigger ISR against Bo. cinerea in bean and tomato (K. Audenaert and M. Ho¨fte, unpublished data), suggesting that also in strain KMPCH, SA and pyocyanin, rather than SA alone, are the determinants for induced resistance. SA has also been implicated in 7NSK2- and/or KMPCHmediated ISR against Tobacco mosaic virus (TMV) in tobacco (De Meyer et al., 1999a) and C. lindemuthianum in bean (Bigirimana and Ho¨fte, 2002). Nevertheless, pyocyanin-minus mutants were not tested and it cannot be excluded that also in these pathosystems a combined production of pyocyanin and SA (or pyochelin) is needed to trigger ISR.

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G. ANTIBIOTICS

1. 2,4-Diacetylphloroglucinol Like siderophores, antibiotics can play a dual role in rhizobacteria-mediated biocontrol by exerting a direct inhibitory effect on pathogens (Raaijmakers and Weller, 1998; Weller et al., 2002) as well as by triggering ISR. One of the most conclusive pieces of evidence for the involvement of antibiotics in rhizobacteria-triggered systemic resistance came from work carried out in the laboratory of Jean-Pierre Me´traux, where it was demonstrated that 2,4-diacetylphloroglucinol (DAPG) functions as a key determinant of ISR in Arabidopsis. In this plant system, DAPG produced by Ps. fluorescens CHA0 was shown to induce resistance against the oomycete Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica), whereas mutations interfering with DAPG production led to a significant decrease in ISR (Iavicoli et al., 2003). In trans complementation of the mutant restored the ability to induce resistance, further supporting the contribution of DAPG to CHA0-mediated ISR. In tomato, Ps. fluorescens CHA0 induces resistance against the root knot nematode Meloidogyne javanica. Also in this case, ISR seems to depend on the production of DAPG, as a DAPG-negative mutant was not effective in reducing the disease in split-root assays, and effectiveness could be restored by complementation of the mutant (Siddiqui and Shaukat, 2003). DAPG has likewise been implicated in ISR in Arabidopsis against Ps. syringae pv. tomato by Pseudomonas chlororaphis Q2-87, suggesting that DAPG is a major determinant of ISR in DAPG producers (Weller et al., 2004). To date, research aimed at elucidating the mode of action of DAPG has been confined to the study of Iavicoli et al. (2003). By screening an extensive set of Arabidopsis mutants and transgenic lines impaired in various structural components of known defence-signalling pathways, it was shown that, unlike ISR triggered by the wild-type bacteria, resistance induced by DAPG follows a signalling route that requires neither the master transcriptional regulator NPR1, nor a functional JAR1 protein. Particularly interesting, however, was the analysis of the eir1 (ethylene-insensitive root-1) mutant, which is ET insensitive in the roots only (Roman et al., 1995). This eir1 mutant was incapable of mounting ISR upon DAPG feeding, leading the authors to suggest that an intact ET signalling pathway is required for the establishment of DAPG-inducible resistance (Iavicoli et al., 2003). What should be noted, however, is that the EIR1 protein is not only obligatory for ET-signalling, but also is implicated in regulating the plant’s response to endogenously synthesized auxin (Luschnig et al., 1998; Sieberer et al., 2000). Especially noteworthy in this regard is a recent report by Brazelton et al. (2008)

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suggesting a connection between auxin signalling and DAPG-induced morphological alterations in tomato and tobacco. In these plant species, DAPG was shown to inhibit primary root growth and to stimulate lateral root formation. Indeed, roots of the auxin-resistant diageotropica mutant of tomato displayed reduced sensitivity to DAPG with regard to inhibition of primary root growth and induction of root branching, which is suggestive of a link between auxin signalling and DAPG sensitivity. Moreover, application of exogenous DAPG repressed the activation of an auxin-inducible GH3 promoter::luciferase reporter gene construct in transgenic tobacco hypocotyls in a manner similar to treatment with DAPG-producing Ps. fluorescens strains, whereas a DAPG-minus mutant displayed less inhibitory effects compared to the parental strain. These results clearly indicate that DAPG can alter root architecture by interacting with an auxin-dependent signal transduction pathway. Considering the well-documented role of auxin as a virulence factor for biotrophic microbes (Chen et al., 2007; Navarro et al., 2006; Spoel and Dong, 2008; Wang et al., 2007), it is not inconceivable that DAPG may induce systemic resistance through interference with pathogeninduced auxin signalling. 2. Pyocyanin Another antibiotic that has been identified as a crucial determinant of rhizobacteria-elicited ISR is the N-containing heterocyclic blue phenazine pigment pyocyanin. A well-characterized virulence factor in clinical isolates of Ps. aeruginosa (K. Britigan et al., 1992, 1997), pyocyanin produced by the rhizobacterium Ps. aeruginosa 7NSK2 is postulated to join forces with the SA-derivative pyochelin in triggering systemic resistance in bean and tomato against Bo. cinerea (Audenaert et al., 2002; unpublished data), as described in detail in the section on siderophores. In rice, however, no evidence could be found for the involvement of pyochelin in ISR against the rice blast pathogen, M. oryzae. This pyochelin-independency of 7NSK2-mediated ISR in rice was borne out by the observation that bacterial inoculum prepared from iron-rich medium was as effective as that prepared from iron-poor medium, and was further confirmed by the resistance-inducing potential of the pyochelin-negative mutant KMPCH (De Vleesschauwer et al., 2006). In contrast, treatment with the pyocyanin-deficient strains 7NSK2-phzM and KMPCH-phzM did not reduce the number of sporulating lesions after infection with M. oryzae. Because the inability of these strains to induce resistance did not result from insufficient rhizosphere populations, these results strongly suggested that pyocyanin production is an integral component of 7NSK2-mediated ISR against the pathogen. Additional support for the involvement of pyocyanin was

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provided by complementation experiments, as well as by the protective effect obtained upon hydroponic feeding of the purified compound at pico- and nanomolar concentrations. Strikingly, in bioassays with Rhizoctonia solani as challenging pathogen, a necrotrophic fungus which causes sheath blight, the situation appeared to be entirely different in that the wildtype bacteria were not effective, whereas the pyocyanin-negative mutants 7NSK2-phzM and KMPCH-phzM did induce resistance. In accordance with these findings, pyocyanin-treated rice seedlings exhibited a heightened level of susceptibility to Rh. solani. When considered together, these data clearly indicate that pyocyanin plays a dual role in 7NSK2-triggered ISR, acting as a positive regulator of resistance to M. oryzae while rendering plants hypersusceptible to attack by Rh. solani. Transient generation of low-level micro-oxidative bursts by redox-active pyocyanin in planta most likely accounts for the differential effectiveness of this phenazine antibiotic in 7NSK2-mediated ISR, as exogenous application of sodium ascorbate, a major anti-oxidant buffer and free radical scavenger (Foyer and Noctor, 2005), alleviated the contrasting effects of pure pyocyanin on M. oryzae and Rh. solani pathogenesis (De Vleesschauwer et al., 2006). In conclusion, it can be stated that while in bean, tomato, and tobacco, pyocyanin and SA/pyochelin act synergistically to induce resistance, in the monocot rice pyocyanin alone is sufficient to activate the host immune response. The outcome of pyocyanin-steered defence responses on disease resistance, however, seems highly dependent on the type of challenging pathogen. Numerous pharmacological studies have revealed that pyocyanin can undergo redox-cycling, with resultant formation of superoxide and H2O2 (Britigan et al., 1997; Hassan and Fridovich, 1979). These active oxygen species (AOS) apparently suffice to induce resistance against blast in rice. Yet, in dicot plants such as bean and tomato, establishment of ISR requires AOS to be converted into highly toxic OHradicals through the Haber–Weiss reaction in the presence of an iron-chelating catalyst such as ferrated pyochelin or Fe-SA (Audenaert et al., 2002). H. VOLATILES

A relatively new addition to the list of rhizobacterially produced compounds with a putative role in eliciting host defence is a class of volatile organic compounds (VOCs). Unlike airborne VOCs, such as C6 green-leaf volatiles that can be easily sampled by headspace collections of the living plant, rhizosphere emissions by root-colonizing bacteria present the complication of de-adsorbing low-molecular-weight compounds from the soil matrix (Pare´ et al., 2005). However, by physically separating plant seedlings

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from VOC-emitting rhizobacteria on divided Petri dishes, Ryu et al. (2004a) were able to unequivocally delineate the role of airborne bacterial metabolites in eliciting ISR. Exposure of Arabidopsis seedlings to volatile blends from Ba. subtilis GB03 and Bacillus amyloliquefaciens IN937a for 4 days was sufficient to activate ISR, as reflected by a marked reduction in the number of symptomatic leaves 24 h after inoculation with the soft-rot pathogen Er. carotovora. Extensive gas-chromatographic analysis of the complex bacterial bouquet emitted by the Bacillus strains revealed the release of a series of low-molecular-weight hydrocarbons, the most abundant component being the growth-promoting volatile 2R,3R-butanediol. The importance of this 2R,3R-butanediol in ISR was surmised in Arabidopsis when pre-exposure of plants to low doses (pg to ng range) of the pure compound activated resistance, while this ability was alleviated in bacterial mutant derivatives emitting reduced levels of 2,3-butanediol and acetoin (Ryu et al., 2004a). Additional support for a role of VOCs in activating ISR came from Han et al. (2006b). In this study, activation of systemically induced resistance against Er. carotovora in tobacco by Ps. chlororaphis O6 was found to be inherently linked to the production of 2R,3R-butanediol, the latter being subject to positive regulation by the global sensor kinase GacS (Han et al., 2006b). It remains to be investigated whether the site of plant VOC perception is above or below ground for soil-grown plants and how plants perceive VOC signals. It is not clear either whether an endogenous signal is involved in VOC-mediated ISR (Pare´ et al., 2005). Heil and Ton (2008) hypothesized that green-leaf volatiles may provoke changes in transmembrane potentials and thereby induce gene activity. Possibly, rhizobacterially produced VOCs may be perceived by a similar mechanism. Although our understanding of the molecular mechanisms underpinning volatile-mediated ISR is still rudimentary, it is evident from the limited data available that, at least in some plant–bacterium interactions, responsiveness to ET is key to optimal manifestation of the VOC-triggered immune response (Ryu et al., 2004a; Spencer et al., 2003). Considering the role of microbial volatiles in regulating an array of cellular processes, including plant growth and development, pathogen defence, and abiotic stress adaptation (Cho et al., 2008; Han et al., 2006b; Ryu et al., 2003a, 2004a), there may be yet other signalling pathways extant. For instance, VOCs might also operate through an auxin-dependent mechanism, as was suggested for the antibiotic DAPG. In support of this assumption, Zhang et al. (2007) recently demonstrated that volatile blends from the Ba. subtilis strain GB03 orchestrate cell expansion in Arabidopsis by modulating the auxin-signalling infrastructure.

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I. EXOPOLYSACCHARIDES

EPS are a group of high-molecular-weight carbohydrates secreted by both pathogenic and beneficial bacteria. In pathogenic bacteria, EPS are necessary to cause disease symptoms such as wilting and water-soaking. EPS also promote survival and colonization within host tissues (Denny, 1995). In beneficial bacteria, EPS can function as signalling molecules that trigger a developmental response in the plant or suppress host defence responses (see, for instance, Jones et al., 2008). In Arabidopsis, leaf infiltration with bacterial EPS from plant, insect, and human pathogens and symbionts (10 mg/ml) suppressed calcium influx and defences induced by MAMPs such as flg22 (Aslam et al., 2008). In contrast, EPS from Pantoea agglomerans activated the oxidative burst response of tobacco and parsley cell cultures directly and primed rice and wheat cells for augmentation of H2O2 accumulation after subsequent induction by a chitin hexamer elicitor (Ortmann et al., 2006). Foliar-applied EPS from two pathovars of X. campestris induced local and systemic protection against coffee rust caused by Hemileia vastatrix. Protection was also observed when coffee plants were treated with different concentrations of commercially available xanthan gum (Guzzo et al., 1993). Recently it was shown that EPS from the PGPR Burkholderia gladioli IN26 can induce systemic resistance to Colletotrichum orbiculare on cucumber when infiltrated in leaves or applied via seed soaking. The most effective concentration appeared to be 200 ppm. In addition, EPS induced the PR-1a promoter in transgenic tobacco plants in a concentration-dependent manner (Park et al., 2008b). EPS from Serratia sp. strain Gsm01 sprayed at a concentration of 200 ppm on tobacco leaves affected Cucumber mosaic virus (CMV) accumulation. EPS-treated plants showed an enhanced accumulation of PAL, peroxidase, and phenols, and PR-1b expression was increased (Ipper et al., 2008). The culture filtrate of Serratia Gsm01 also controlled CMV infection in tobacco. In these experiments, no bacterial mutants impaired in EPS production were used, so it is not clear to what extent EPS production is important in rhizobacteriamediated ISR. From the examples cited above, it is clear that depending on the conditions, EPS can either trigger or suppress resistance responses in plants. Aslam et al. (2008) found that while pure xanthan suppressed defence responses, calcium-saturated xanthan elicited PR-1, PDF1.2, and PAL genes in Arabidopsis and also xanthan oligomers could function as MAMPs. Structural differences in EPS may explain their differential effects on plants.

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Park et al. (2008a) identified 4-(aminocarbonyl) phenylacetate as an ISReliciting metabolite produced by Ps. chlororaphis O6 against the wildfire pathogen Ps. syringae pv. tabaci in tobacco by ISR bioassay-guided fractionation of O6 culture supernatant. Application of 68 mM 4-(aminocarbonyl) phenylacetate on in vitro grown tobacco plants yielded protection against Ps. syringae pv. tabaci. This concentration appears to be very high; the effect of lower concentrations was not reported. It remains to be investigated whether this compound can trigger ISR at physiologically realistic concentrations and what role it plays in Ps. chlororaphis O6-mediated ISR. Han et al. (2006a) isolated a variety of mutants of Ps. chlororaphis O6 impaired in ISR against Er. carotovora in tobacco. Genes disrupted in these mutants were involved in various functions including biosynthesis of purines, phospholipase C, transport of branched-chain amino acids, chemotaxis, and ABC transporters. Most of these mutants, except the ABC-transporter mutants, were seriously impaired in root colonization, indicative of the importance of this trait in ISR.

III. SIGNALLING IN RHIZOBACTERIA-INDUCED SYSTEMIC RESISTANCE A. THE ARABIDOPSIS–PSEUDOMONAS FLUORESCENS WCS417R SYSTEM: A PARADIGM FOR SA-INDEPENDENT ISR SIGNALLING

Following the perception of resistance-inducing stimuli, plants activate an elaborate matrix of signal-transduction pathways in which phytohormones such as SA, JA, ET, and abscisic acid (ABA) act as key signalling molecules. A mechanistic understanding of ISR in terms of signalling has derived largely from studies in Arabidopsis where, as with pathogen-induced SAR, mutant and transgenic lines have identified the key roles of phytohormonal signals, effectors, and potential points of cross-talk among the signals. Driving this research initially was the observed disconnection between resistance induced by the Dutch reference strain Ps. fluorescens WCS417r and the accumulation of transcripts for certain PR genes that are hallmarks of SAR and SA action (Hoffland et al., 1995; Pieterse et al., 1996; Van Wees et al., 1997). Measurements of SA levels in ISR-expressing Arabidopsis plants revealed that ISR, unlike SAR, is not associated with alterations in endogenous SA content, neither before nor after challenge inoculation (Pieterse et al., 2000). Moreover, WCS417r-mediated ISR was maintained in SA non-accumulating Arabidopsis NahG transformants (Pieterse et al., 1996; Van Wees et al., 1997), leading the

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authors to suggest that WCS417r-mediated ISR is a SA-independent resistance mechanism and that WCS417r-mediated ISR and pathogen-induced SAR are regulated by largely distinct signalling conduits. The non-involvement of SA in WCS417r-mediated ISR prompted Corne´ Pieterse and colleagues to investigate the possible requirement for other regulatory factors implicated in plant defence. To this end, the Arabidopsis JA-response mutant jar1 and the ET-response mutant etr1 were tested for their ability to mount ISR. Intriguingly, both mutants were unable to develop ISR against Ps. syringae pv. tomato upon colonization of the roots by WCS417r bacteria (Pieterse et al., 1998), illustrating the dependency of ISR signalling on these phytohormones. Bioassays with other mutants in JA and ET signalling yielded similar results. For instance, Arabidopsis mutant eds8, which was previously shown to display increased susceptibility to Ps. syringae pv. maculicola (Glazebrook et al., 1996), was impaired in both WCS417r-mediated ISR (Ton et al., 2002a) and JA signalling (Glazebrook et al., 2003; Ton et al., 2002a). Furthermore, of a large set of well-characterized ET-signalling mutants, none displayed enhanced resistance against Ps. syringae pv. tomato upon root treatment with WCS417r (Knoester et al., 1999), indicating that an intact ET signalling pathway is a prerequisite for the establishment of ISR. Bioassays with the eir1-1 mutant, which is insensitive to ET in the roots only, shed additional light on the pathway by which ISR is induced. In this mutant, application of WCS417r to the roots did not result in an ISR response, whereas infiltration of the inducing bacteria into the leaves did. The eir1-1 leaves are normally sensitive to ET, in contrast to the etr1-1 and several ein mutants, which display complete ET insensitivity. These results demonstrated that for induction of ISR in Arabidopsis by WCS417r, responsiveness to ET is required at the site of application of inducing bacteria, be it roots or leaves (Knoester et al., 1999). Further evidence for the involvement of the ET-response system came from the identification of the Arabidopsis ISR1 locus (Ton et al., 1999, 2002b). Genetic analysis of the progeny from crosses between WCS417rresponsive and non-responsive accessions demonstrated a single dominant locus, designated ISR1, to be important for ISR signalling against different pathogens (Ton et al., 2002b). Interestingly, accessions carrying the recessive isr1 allele display reduced sensitivity to ET (Ton et al., 2001), as well as enhanced susceptibility to Ps. syringae pv. tomato (Ton et al., 1999). These findings suggest that ISR1 encodes a novel component in the ET signalling pathway that is integral to both basal and rhizobacteria-induced resistance in Arabidopsis. Treatment of wild-type Arabidopsis plants with JA, ET, or the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced systemic

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protection identical to that elicited by WCS417r rhizobacteria, and protected plants against Ps. syringae pv. tomato (Pieterse et al., 1998). However, etr1-1 plants failed to respond to either ACC or JA, whereas the jar1-1 mutation abrogated the response to JA but not to ACC, indicating that JA operates upstream of ET in the ISR signalling cascade (Pieterse et al., 1998). Surprisingly, the ISR response does not appear to depend on changes in the endogenous levels of JA or ET, as significant changes in the levels of these phytohormones did not occur during induction of resistance by WCS417r (Pieterse et al., 2000), and transcripts of classic JA- and ET-regulated genes (e.g., LOX1, LOX2, PAL1, HEL, CHI-B, PDF1.2) were not upregulated, neither before nor after pathogen challenge (Van Wees et al., 1999). However, it is not inconceivable that activation of ISR by WCS417r requires responsiveness to JA and ET, rather than increased levels of these defence regulators. In this scenario, the sensitivity to JA and ET is likely to be boosted as a result of ISR elicitation. Two lines of evidence support this hypothesis. First is the observation that in ISRexpressing plants the capacity to convert ACC to ET is significantly enhanced, providing a greater potential to produce ET upon pathogen attack (Hase et al., 2003; Pieterse et al., 2000). Secondly, in induced plants, after pathogen challenge JA- and ET-responsive gene expression is enhanced relative to non-induced plants (Pozo et al., 2008; Verhagen et al., 2004). Thus, it seems that induced plants are sensitized for perception of pathogen-induced JA and ET, a notion which is consistent with priming or potentiation of the plant to respond more rapidly and with greater intensity to subsequent pathogen attack (Conrath et al., 2006). Recent findings pinpoint the helix-loop-helix transcription factor MYC2 as a potential regulator in this rhizobacteria-induced priming for enhanced defence (Pozo et al., 2008). Promoter analysis of ISR-primed and methyl jasmonate-responsive genes revealed over-representation of a G-box-like motif known to serve as a docking-site for MYC2. In addition, the MYC2impaired mutants jin1-1 and jin1-2 were unable to express ISR against Ps. syringae pv. tomato and the downy mildew pathogen H. arabidopsidis upon elicitation by WCS417r, further highlighting the important regulatory function of this transcription factor in priming for enhanced JA-responsive gene transcription during WCS417r-mediated ISR. Intriguingly, mutant jin11 plants were recently shown to be compromised also in the ability to express pathogen-induced SAR, a finding which is consistent with the view of JA as an important early signal establishing broad-spectrum systemic immunity (Truman et al., 2007). To delineate the possible role of the SAR master regulatory protein NPR1 in ISR response triggered by WCS417r, bioassays were performed with the

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Arabidopsis npr1 mutant. In contrast to other mutations in the SA-signalling pathway, npr1 failed to express ISR upon elicitation by WCS417r (Pieterse et al., 1998). Hence, NPR1 seems to play a pivotal role in reaching the induced state, whether triggered by avirulent pathogens or benign rhizobacteria. Further analysis placed NPR1 downstream of the requirements for both JA and ET in the ISR-signalling cascade. Considering that SAR is associated with NPR1-controlled PR-transcript accumulation, and ISR is not, one would expect the action of NPR1 in ISR to be substantially different from that in SAR. Especially noteworthy in this regard is that these supposedly distinct activities are not mutually exclusive, as simultaneous activation of pathogen-induced SAR and WCS417r-governed ISR was found to act independently and additively to increase resistance of Arabidopsis against Ps. syringae pv. tomato (Van Wees et al., 2000). To date, the function of NPR1 in ISR is still unresolved, although some studies portend a role for NPR1 in the stress-induced augmentation of the protein-secretory pathway (Van der Ent et al., 2009; Wang et al., 2005). Recently, the R2R3-MYB-like transcription factor gene MYB72 was identified as a novel player in WCS417r-mediated ISR (Van der Ent et al., 2008). T-DNA knockout mutants myb72-1 and myb72-2 were incapable of mounting ISR against a fairly broad range of pathogens with different lifestyles (i.e. biotrophs, hemibiotrophs, necrotrophs), indicating that MYB72 is essential to establish broad-spectrum ISR. However, overexpression of MYB72 did not result in enhanced resistance against any of the pathogens tested, leading the authors to suggest that MYB72 acts in concert with another signalling component. A clue for the identity of this accomplice was provided when yeast-two hybrid analysis revealed MYB72 to physically interact in vitro with the EIN3-like transcription factor EIL3. This interaction with EIL3 links MYB72 to the ET response pathway, further establishing this transcription factor as a crucial signalling intermediate required in early signalling steps of WCS417r-mediated ISR (Van der Ent et al., 2008). Emerging from this extensive series of studies on WCS417r-induced Arabidopsis is a model providing fascinating insights into the various aspects of ISR signalling, ranging from the early ET-regulated and MYB72-dependent onset of ISR following perception of resistance-inducing stimuli in the roots, to long-distance signalling and ET-, JA-, and NPR1-dependent priming of effector responses in systemic leaves. Priming by several other rhizobacteria, such as Ps. putida LSW17S, also is reliant on ET, JA, and NPR1, and not on SA. However, augmented expression of PR genes was observed in Arabidopsis treated with LSW17S and challenged with Ps. syringae pv. tomato. Pathogen challenge likewise resulted

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in higher H2O2 accumulation and enhanced callose deposition in bacterized plants compared to control ones (Ahn et al., 2007). These reactions have not been observed in WCS417r-primed plants, suggesting that the molecular and biochemical events associated with ISR elicited by LSW17S overlap only partly with those defined for ISR against Ps. syringae pv. tomato, as induced by WCS417r. Ryu et al. (2003b) found that Bacillus pumilus SE34 triggered ET/ JA- and NPR1-dependent ISR against Ps. syringae pv. maculicola in Arabidopsis. Surprisingly though, ISR against Ps. syringae pv. tomato triggered by the same bacterium appeared to be JA/ET-independent. Ps. fluorescens WCS374r-mediated ISR against Ps. syringae pv. tomato in Arabidopsis is also mediated by a signalling pathway that depends on ET, JA, and NPR1. In rice, WCS374r-elicited ISR against M. oryzae is maintained in SA-non-accumulating NahG transformants, but completely abolished in the ET-insensitive antisense line 471 and the JA-deficient mutant hebiba, suggesting that in the latter plant–pathogen system also, WCS374r-mediated ISR derives primarily from JA/ET-driven effects (De Vleesschauwer et al., 2008). Consistently, root treatment with the purified PSB siderophore of WCS374r, which faithfully mimicks living bacteria in activating ISR, does not lead to direct transcriptional activation or priming of SA-responsive PR-like genes, such as OsPR-1b and OsPBZ1. Plant growth-promoting fungi such as Penicillium sp. GP162 (Hossain et al., 2008) and Trichoderma asperellum T203 (Shoresh et al., 2005) have been shown to induce systemic resistance in an ET- and JAdependent manner as well. Moreover, T. asperellum T34-mediated ISR required, besides NPR1, the MYB72 transcription factor, suggesting that MYB72 functions as a node of convergence in induced defence triggered by soilborne beneficial microorganisms (Segarra et al., 2009; Van Wees et al., 2008). Whereas JA- and ET-dependent signalling mechanisms are co-required for the elicitation of, among others, Ps. fluorescens WCS417r- and Ps. putida LSW17S-mediated ISR in Arabidopsis (Ahn et al., 2007; Pieterse et al., 1998), as well as Ba. pumilus SE34-mediated ISR in tomato (Yan et al., 2002), alternative signalling pathways have been demonstrated in other plant–bacterium interactions. For instance, in Arabidopsis resistance against H. arabidopsidis triggered by DAPG-producing Ps. fluorescens CHA0 follows a pathway that is JA- and NPR1-dependent and needs a functional EIR1 protein in the roots (Iavicoli et al., 2003). Pure DAPGtriggered resistance, however, only depends on a functional EIR1 protein and is NPR1-independent. As discussed above, it is possible that the mutation in EIR1 acts independently of ET signalling and is rather implicated in auxin signalling. ISR induced by strain CHA0 was ineffective against Ps. syringae and Bo. cinerea (Iavicoli et al., 2003), a further

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indication that the induced signal-transduction pathway is different from that underpinning WCS417r-mediated ISR. S. marcescens 90-166 likewise seems to elicit a defence pathway that is mediated by NPR1 and JA, but is SA- and ET-independent (Ryu et al., 2003b; Table I). This strain was not tested on an eir1 mutant of Arabidopsis. However, unlike Ps. fluorescens CHA0, S. marcescens 90-166 is effective against Ps. syringae in Arabidopsis, indicating that these two strains do not trigger the same defence pathway. Still other variations are indicated by the capacity of rhizobacteria such as Ba. pumilus T4, Ba. subtilis GB03, and S. marcescens 90-166 to induce disease resistance signalling that depends only on either ET or JA (Ryu et al., 2003b, 2004a,b).

B. SA-DEPENDENT ISR SIGNALLING

Although rhizobacteria-mediated ISR is perhaps the best-studied example of induced resistance that is not regulated by SA, it is becoming increasingly clear that not all rhizobacteria-triggered ISR is mediated by JA/ET (Table I). The role of SA in ISR was first studied for Ps. aeruginosa 7NSK2 and its SA-producing mutant Ps. aeruginosa KMPCH. Both strains clearly induce resistance in a SA-dependent manner as the resistance response to TMV is no longer expressed in NahG tobacco (De Meyer et al., 1999a), while resistance to Bo. cinerea can no longer be triggered in NahG tomato (Audenaert et al., 2002). Although some strains of Bacillus spp. operate through a JA/ET-dependent mechanism and require NPR1 similar to Ps. fluorescens WCS417r, ISR induced by other Bacillus strains requires SA but not JA and NPR1 (Barriuso et al., 2008; Ryu et al., 2003b). Also ISR against Verticillium dahliae in Arabidopsis in response to root inoculation with the rhizobacterial strain Pa. alvei K165 requires a SA-dependent resistance mechanism, as evidenced by the ISR-minus phenotype of the SA-signalling mutants eds5 and sid2 (Tjamos et al., 2005). However, Pa. alvei K165 was still fully active in NahG Arabidopsis plants. Although difficult to explain, this result indicates that other plant lines besides NahG should be tested in order to unambiguously exclude a role for SA in ISR. There are other examples in Arabidopsis where diseases are diminished by a SA-dependent rhizobacteria-induced resistance. For instance, Bacillus strain N11.37 induces ISR to X. campestris in Arabidopsis by a SA- and ETdependent pathway, while only SA-signalling seems to be operative in the case of resistance induced by the Stenotrophomonas strain N6.8 (Domenech et al., 2007). Soil inoculation with Bacillus sp. strain L81 or Art. oxidans

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strain BB1 protected Arabidopsis plants against Ps. syringae pv. tomato and increased plant tolerance to salt stress. The SA-dependent pathway was involved in the defence response against biotic and abiotic stress induced by both strains. Inoculation with Art. oxidans strain BB1 increased PR-1 gene transcription, while this was not observed for Bacillus sp. strain L81. The authors, however, did not analyze gene expression after stress challenge (Barriuso et al., 2008). Interestingly, also Ps. fluorescens WCS374r-mediated ISR against TCV in Arabidopsis is SA- and NPR1-dependent (Djavaheri, 2007).

C. SA-DEPENDENT AND SA-INDEPENDENT SIGNALLING

From Table I and the examples cited above, it is evident that both SA-dependent and SA-independent signalling conduits can contribute to rhizobacteria-mediated induced resistance, making it difficult to assign a definitive signalling pathway to ISR. Rather, it appears that plants display a remarkable flexibility in the molecular processes governing the perception and subsequent transduction of resistance-inducing stimuli produced by root-colonizing bacteria. Another challenge in finding coherence among models for ISR signalling is added by the fact that even within the same plant–rhizobacterium interaction, different signal transduction pathways can be activated depending on the type of intruder encountered. For instance, Conn et al. (2008) recently demonstrated that inoculation of Arabidopsis with selected endophytic actinobacteria results in a low-level induction of both SA and JA/ET marker genes in systemic healthy leaves. However, whereas resistance to the bacterial pathogen Er. carotovora subsp. carotovora required the JA/ET pathway, resistance to the fungal pathogen F. oxysporum proved to involve primarily the SA-regulated SAR pathway. The endophytic actinobacteria thus appear to be able to prime both the SA and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Induction of resistance in Arabidopsis by Ps. fluorescens WCS374r is another case in point. In the case of TCV, to which defence responses in Arabidopsis are SA-dependent, ISR is abolished in NahG and sid1 plants, as well as in npr1, but not in jar1 and etr1 mutant plants. In contrast, WCS374r-mediated ISR against Ps. syringae pv. tomato is abrogated in npr1, jar1, and etr1 plants, but still functional in NahG and sid1 plants (Djavaheri, 2007). Hence, similar to actinobacteria, root colonization by WCS374r appears to sensitize Arabidopsis for augmented infection-induced activation of multiple defence signalling pathways that all add to establish broad-spectrum WCS374r-triggered ISR.

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IV. FINAL REMARKS A detailed understanding of the complex, yet fascinating mechanisms by which resistance-inducing bacterial stimuli elicit host immune responses may open new doors to design innovative strategies for biologically based, environmentally friendly, and durable disease control. Emerging from the numerous studies dealing with ISR elicitation is a greater awareness that plants are able to perceive a plethora of resistance-inducing bacterial components reminiscent of the large variety of chemical or pathogen-derived elicitors that stimulate the plant’s immune response (Schreiber and Desveaux, 2008). Some bacterial determinants such as flagellin, SA, pyocyanin, DAPG, N-alkylated benzylamine, and VOCs are recognized at concentrations in the pico- to nanomolar range, whereas other compounds only trigger protective effects in the g/ml range, with examples of the latter including LPS, EPS, PSB, and biosurfactants. Moreover, a clear dose-response effect is typically observed for the determinants that need higher concentrations to be effective, whereas for determinants recognized at very low doses, no such relationship seems to hold. A lack of dose-response is characteristic of recognition by high-affinity receptors such as the LRR-RK FLS2 (Jones and Dangl, 2006). This would imply that plants possess high-affinity receptor kinases able to recognize and respond to compounds such as SA, pyocyanin, DAPG, and N-alkylated benzylamine. Considering that all of these metabolites contain an aromatic phenol group, it is tempting to speculate that the latter moiety may constitute a general motif widely recognized by specific plant receptors (Ongena et al., 2008). On the other hand, compounds such as LPS that require higher doses to be effective may possibly be perceived by low-affinity receptors (Gross et al., 2005; Newman et al., 2007). These resistance determinants may, however, also enhance the plant’s defensive capacity by inducing disturbance or transient channelling in the plasma membrane, thereby activating a cascade of molecular events culminating in the primed or direct activation of immune responses, as was suggested for biosurfactants (Ongena and Jacques, 2008). Alterations in intracellular iron homeostasis caused by siderophore-mediated iron deprivation in the rhizosphere can be put forward as an alternative mechanism by which rhizobacteria activate the plant’s immune response. Interestingly, these proposed types of non-self recognition all have equivalents in the mammalian immune system (Nu¨rnberger et al., 2004; Zipfel and Felix, 2005), suggesting that fundamental modes of microbe sensing and subsequent resistance elicitation have been evolutionarily conserved across biological kingdoms. In general, ISR leads to a level of resistance that is less pronounced compared to ETI or ETI-triggered SAR. In this perspective, it can be stated that ISR resembles, to some extent, PTI-triggered SAR (Mishina and Zeier, 2007).

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Considering that PGPRs do not produce virulence factors such as toxins and pectinolytic enzymes that inflict tissue necrosis, and probably do not inject effector proteins into host cells to circumvent, or attenuate MAMP-triggered defences, rhizobacterial ISR is not expected to be associated with a full-blown ETI or SAR response in the absence of a challenging pathogen. However, in order to establish a compatible interaction with their hosts, PGPRs are likely equipped with diverse mechanisms to alleviate the local plant defence response triggered upon recognition of the aforementioned bacterial traits. Plant hormones such as auxin and JA are possibly involved in this process. Auxins are commonly produced by PGPRs (Spaepen et al., 2007) and besides their growth-promoting effects, they are known to suppress SA-dependent defence responses (Robert-Seilaniantz et al., 2007; Wang et al., 2007). In a similar vein, local triggering of the JA response may be a strategy to suppress SA-regulated defences in the root, while leading to the enhancement of the defensive capacity in naive systemic leaves similar to what has been reported in the context of plant–mycorrhiza interactions (Pozo and Azco´n-Aguilar, 2007). Alternatively or in addition, LPS or EPS production may also fulfil such a dual role in the ISR response. In this respect, the observation that a mutant of Ps. fluorescens WCS417r lacking the O-antigenic side chain of its outer membrane LPS, displays a reduced capacity to colonize the interior but not the exterior root tissues of tomato, is of particular interest (Duijff et al., 1997). Moreover, suppression of local defences by bacteria-produced LPS or EPS may also explain why purified compounds are often more effective in triggering ISR and reducing disease severity relative to treatment with living bacteria. It is also apparent that rhizobacteria-mediated ISR is not conditioned by one definitive signalling pathway. Rather, the pathway triggered is depending on the PGPR, the plant, and the challenging pathogen. In many cases, and especially in Arabidopsis, multiple bacterial determinants tend to be involved in the elicitation of the ISR response, resulting in the sensitization of various signalling conduits. The enhanced signalling capacity in these primed plants not only facilitates a faster and stronger activation of basal defences upon microbial infection, but also confers flexibility to fine-tune the plant’s immune response to the type of invading pathogen.

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Plant Growth-Promoting Actions of Rhizobacteria

STIJN SPAEPEN,*,1 JOS VANDERLEYDEN* AND YAACOV OKON{

*Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium { Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Modes of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Growth-Promoting Substances........................................ B. Nitrogen Transformations .................................................... C. Phosphate and Micronutrient Availability ................................. D. Emerging Signals ............................................................... E. Biocontrol in the Rhizosphere................................................ III. Agricultural Aspects and Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. PGPR and Endophytes—Role of Bacterial Numbers .................... B. PGPR and Other Symbiotic Systems such as Rhizobium-Legumes..... C. Vegetative Growth and Grain Filling ....................................... D. Inoculant Technology.......................................................... E. Probiotics in Agriculture ...................................................... IV. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51007-5

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ABSTRACT The rhizosphere as compared to bulk soil is rich in nutrients because of root exudation and deposits. As a consequence, the number of bacteria surrounding plant roots is 10–100 times higher than in bulk soil. These rhizobacteria, based on their effects on plants, can be largely divided into beneficial, deleterious, or neutral bacteria. The beneficial bacteria, also called plant growth-promoting rhizobacteria (PGPR), exert their beneficial effect through either direct or indirect mechanisms or both. In this chapter, the different mechanisms of plant-growth promotion and their impact are discussed. The mechanisms comprise the production of plant growth-promoting substances, nitrogen transformations, increasing bioavailability of phosphate and micronutrients, and biological control, as the best documented cases. In addition, the bacterial production of some molecules with recently described plant growthpromoting effects is discussed. To indicate the impact of PGPR, the applications and relevance of these bacteria in agricultural practices are highlighted. The importance of the plant genotype, inoculum density and technology, and co‐inoculation practices, in terms of plant responsiveness are discussed. It is concluded that basic research should remain a priority in order to be able to develop performing and reliable bacterial inocula as a means to support sustainable agriculture.

I. INTRODUCTION The term ‘‘rhizosphere’’ was for the first time defined by Lorenz Hiltner in 1904 as ‘‘the soil compartment influenced by the root’’ (Hiltner, 1904). Rhizodeposition describes the total carbon transfer from the plant roots to the soil and comprises exudates (small molecules), secretions (macromolecules such as enzymes), lysates from dead cells, and mucilage. Rhizodeposition promotes microbial abundance and activities in the rhizosphere, which can therefore be described as the most active microbial habitat of the soil (Burdman et al., 2000; Dobbelaere and Okon, 2007; Hartmann et al., 2008; Lucy et al., 2004; Smalla et al., 2006; Van Loon, 2007). The rhizosphere is a highly dynamic open system with temporal and spatial changes of biotic (such as resulting from physiological and morphological changes of the growing root system) and abiotic (such as rain, irrigation, drought) factors. Consequently, it is difficult to understand the microbial adaptations to each particular situation. The rhizosphere is often depicted as a soil cylinder of given radius around the root, drawing a boundary between the rhizosphere and the bulk soil. In the last decade, there have been many developments and new theories trying to understand the architectural features and gradients of the rhizosphere (Hinsinger et al., 2005). Novel in situ techniques and modeling will help in providing a holistic view of the rhizosphere function. However, the new knowledge about root architecture, root growth, function, and rhizosphere is not yet ready for application in the management and manipulation of the rhizosphere.

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Through the dynamic rhizodeposition, it is suggested that roots can regulate the soil microbial community in their immediate vicinity. Some bacterial species living in the rhizosphere and inside the roots can affect growth in either a positive or a negative way. Bacteria that favorably affect plant growth and yield of commercially important crops are denominated as plant growthpromoting rhizobacteria (PGPR) (Burdman et al., 2000; Dobbelaere and Okon, 2007; Lucy et al., 2004; Steenhoudt and Vanderleyden, 2000). The well-known PGPR include members of the genera Azospirillum, Bacillus, Paenibacillus, Pseudomonas, Enterobacter, Klebsiella, Burkholderia, Serratia, Gluconacetobacter, Herbaspirillum, Azoarcus, Arthrobacter, among others. Beneficial bacteria that are able to establish a nitrogen-fixing symbiotic relationship with leguminous plants (and collectively called Rhizobia) are usually not considered within this group. However, recently some of these bacteria have been shown to be plant-growth promoting on non‐legumes, through mechanisms different from nitrogen fixation. Nevertheless, these will not be considered further, as the mechanisms invoked are not different from those of the well-known and better documented PGPR. Within the PGPR group, it is also common to discriminate between bacteria that are usually found at the plant root surface and those that are found most often or exclusively within the plant root tissue, the so-called endophytes. For the latter, we refer to recent reviews (Hardoim et al., 2008; Rosenblueth and MartinezRomero, 2006; Ryan et al., 2008). Direct plant-growth promotion can be derived from phosphorus solubilization, production of plant growth regulators such as auxins, gibberellins (GAs), and cytokinins, by eliciting root metabolic activities and/or by supplying biologically fixed nitrogen. Indirect plant growth-promoting mechanisms used by PGPR include induced systemic resistance (ISR), antibiotic protection against pathogens, reduction of iron availability in the rhizosphere by sequestration with siderophores, synthesis of fungal cell wall-lysing or lytic enzymes, and competition for nutrients and colonization sites with pathogens (Burdman et al., 2000; Dobbelaere and Okon, 2007; Lucy et al., 2004). These direct and indirect mechanisms will be discussed in detail in this chapter and in other chapters in this volume.

II. MODES OF ACTION A. PLANT GROWTH-PROMOTING SUBSTANCES

Plants synthesize several hormones, which act as chemical messengers to regulate plant growth and development. Phytohormones or plant growthpromoting substances are chemical compounds that in small amounts

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promote and influence the growth, development, and differentiation of cells and tissues. In view of this, changes in hormone concentrations alter plant growth and development in a drastic way (possibly with a positive or negative outcome, as in the case of auxins for which an optimal dose-response curve exists). Although phytohormones have been intensively studied over many years, the exact mode of action of some molecules in plants is still not very clear. It is generally accepted that there are five major classes of plant hormones: auxins, cytokinins, GAs, abscisic acid (ABA), and ethylene (ET) (Table I). However, more phytohormones are presently known, such as strigolactone, a carotenoid-derived molecule that inhibits shoot branching and acts as signal molecule in symbiotic interactions with arbuscular mycorrhizal fungi; new ones are still be discovered (Gomez-Roldan et al., 2008; Umehara et al., 2008). Other identified plant growth regulators include: brassinolides, salicylic acid (SA), jasmonates (JA), polyamines, plant peptide hormones, and nitric oxide (for a recent review: Santner et al., 2009). The production of plant growth-promoting substances by bacteria has been reported for many bacterial species, and ideas that this production contributes to the growth-promoting effects of some bacteria have been launched more than 50 years ago (Barea et al., 1976). Although the list of soil and plant-associated bacteria, capable of producing phytohormones, is extensive, the direct evidence for the role of phytohormones in the plant growth-promoting capacities of these bacteria is scarce. The lipochitooligosaccharides (LCOs) produced by rhizobia that evoke nodule formation in legumes, however, are by now very well documented in terms of receptor and signal transduction pathway (D’Haeze and Holsters, 2002; Geurts et al., 2005; Oldroyd and Downie, 2008) and will not be discussed here. This part of the chapter aims to discuss the bacterial production of several phytohormones and highlights the evidence for their role in plant growth promotion. For background information on the biosynthesis, signal transduction, and action of plant hormones, we refer to the excellent book edited by Davies (2004). 1. Auxins Auxins are an important class of phytohormones, driving different plant processes, such as embryogenesis, organ differentiation, root and shoot architecture, apical dominance, and tropistic responses (Teale et al., 2006). The best characterized and most abundant member in the auxin family is indole-3-acetic acid (IAA). The biosynthesis of IAA by plants has been an intensive research topic over many years, but till now some pathways are still poorly characterized or under debate. The aromatic amino acid tryptophan is the main precursor for IAA synthesis, but also a tryptophan-independent

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TABLE I Plant Hormones, Produced by Plant Growth-Promoting Rhizobacteria Class

Example

Structure

Effect on plant

N H

Root and shoot architecture Apical dominance Tropistic responses

HO

Auxins

IAA

O

HO

H3C

Cytokinins

NH

Zeatin

Inhibition of root elongation Leaf expansion by cell enlargement Delay of senescence

N

N

N H

N

H

Gibberellins

Ethylene

GA3

O

OH

HO O

H CH3 O

H2C

CH2

H3C CH3 OH

Abscisic acid

O

CH2

OH

CH3

CH3

O

OH

Seed germination Stem and leaf growth Floral induction and fruit growth Stress and ripening hormone Flower and leaf senescence and abscission Adaptation to abiotic and biotic stresses Stomatal closure Bud dormancy Adaptation to abiotic and biotic stresses

Abbreviations: IAA, indole-3-acetic acid; GA3, gibberellic acid.

pathway has been described (Woodward and Bartel, 2005). Based on feeding studies and mutant analyses, multiple pathways have been suggested, although for most pathways identification of single steps is still lacking, as exemplified by the recent identification of the aromatic amino acid

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transferase 1 in Arabidopsis thaliana, involved in the IAA biosynthetic pathway via indole-3-pyruvate (Stepanova et al., 2008; Tao et al., 2008). For diverse bacteria, production of IAA has been reported. Around 80% of the bacteria isolated from the rhizosphere are estimated to be capable of producing IAA (Khalid et al., 2004; Patten and Glick, 1996), indicating a potential role in the interaction with the plant. As in plants, different biosynthetic pathways have been described in bacteria, and these are mostly similar to those in plants (Fig. 1). The best characterized pathways are those via indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) intermediates. The former pathway consists of two distinct steps. In a first step, tryptophan is metabolized to IAM by a tryptophan monooxygenase (encoded by iaaM). IAM is further converted to IAA by an IAM hydrolase (encoded by iaaH). This pathway has been characterized in various plant pathogens and some rhizobial strains (Clark et al., 1993; Glickmann et al., 1998; Manulis et al., 1998; Morris, 1995; Sekine et al., 1989; Theunis et al., 2004). There is no evidence for the presence of this pathway in plants, although IAM could be detected in Arabidopsis (Pollmann et al., 2002). In the IPyA pathway, tryptophan is firstly transaminated to IPyA by an (aromatic) aminotransferase. In the next step, IPyA is decarboxylated by an indole-3-pyruvate or phenylpyruvate decarboxylase to

Nitrilase

Indole-3-acetonitrile

Trp mono-oxygenase

OH O

Indole-3-acetamide

IAM-hydrolase

HO O

NH2

N H

N H

Tryptophan side-chain oxidase

Indole-3-acetic acid

Tryptophan

Amino transferase

Trp decarboxylase

Indole-3-pyruvate

Tryptamine

IPDC/PPDC

Amine-oxidase

IAAId dehydrogenase

Indole-3-acetic acid IAA conjugates

Fig. 1. Indole-3-acetic acid biosynthetic pathways in bacteria. Most biosynthetic pathways for IAA start from the main precursor tryptophan (Trp). The pathways are mostly named by the molecule that is used as an intermediate. It should be noted that not all pathways are characterized to the same extent and that multiple pathways can exist in a single organism. The pathways via indole-3-acetamide and indole-3-pyruvate are well known, as they are the predominant pathways in bacterial pathogens and PGPR. IAA can be conjugated to sugars (ester linked) or amino acids (amide linked). The functions for these conjugates are storage, transport, compartmentalization, and protection against degradation (Cohen and Bandurski, 1982), although the process of conjugation is not well characterized in bacteria. Abbreviations: IAAld, indole-3acetaldehyde; IAM, indole-3-acetamide; IPDC, indole-3-pyruvate decarboxylase; PPDC, phenylpyruvate decarboxylase.

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indole-3-acetaldehyde, which is subsequently oxidized to IAA by non‐ enzymatic oxidation or by an aldehyde oxidase/dehydrogenase (Costacurta and Vanderleyden, 1995; Patten and Glick, 1996). The IPyA pathway has been identified in several bacteria, such as Pantoea agglomerans, Bradyrhizobium, Azospirillum, Rhizobium, Enterobacter cloacae, and Pseudomonas putida, mostly associated with the plant growth-promoting capacities of these bacteria. The key enzyme in the IPyA pathway is the indole-3-pyruvate decarboxylase or phenylpyruvate decarboxylase, and these enzymes and genes have been characterized from Azospirillum brasilense, E. cloacae, Pa. agglomerans, and Ps. putida (Brandl and Lindow, 1996; Costacurta et al., 1994; Koga et al., 1991; Patten and Glick, 2002; Schu¨tz et al., 2003). Based on biochemical studies and a phylogenetic analysis, the group of indole-3-pyruvate decarboxylases and phenylpyruvate decarboxylases can be divided into two subgroups, with distinct activities (Spaepen et al., 2007b). Besides the two major pathways via IAM and IPyA, other pathways have been described (Fig. 1). In the pathway via tryptamine (TAM), tryptophan is decarboxylated to TAM and subsequently converted to indole-3-acetaldehyde by an amine oxidase. By the conversion of exogenous TAM, this pathway could be demonstrated in Azospirillum; however, further biochemical and genetic data are missing (Hartmann et al., 1983). In Pseudomonas fluorescens strain CHA0, the tryptophan side-chain oxidase (TSO) pathway was identified (Oberhansli et al., 1991) and some nitrilases, catalyzing a key step in the pathway via indole-3-acetonitrile (IAN), were characterized (Kobayashi et al., 1993, 1995; Nagasawa et al., 1990). For many years, it has been accepted that the production of auxins by PGPR is the main factor in the growth-promoting capacities of these bacteria. In Az. brasilense, the mechanism of plant-growth promotion by the production of IAA has been intensively studied. In early reports, the importance of bacterial IAA production in plant stimulation could be shown by inoculation studies with mutant (mostly mutants in IAA biosynthetic genes) or overexpressing strains (chemical mutants selected for resistance to the toxic compound 5-fluorotryptophan) (Barbieri and Galli, 1993; Barbieri et al., 1986; Harari et al., 1988). A direct link between IAA production and altered root morphology was demonstrated by Dobbelaere et al. (1999). Inoculation with the wild-type strain results in a shortening of the root length and enhanced root hair formation, and this effect could be mimicked by the addition of pure IAA. A mutant strain, strongly reduced in IAA production by mutation in the ipdC gene, failed to induce these changes. Mutant strains in which the native ipdC promoter was exchanged for a constitutive or plantinducible promoter, showed the same effects as the wild-type strain at lower inoculum concentrations (Spaepen et al., 2008).

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It is of interest to mention the environmental regulation of the ipdC gene of Az. brasilense in relation to plant root colonization. In Az. brasilense IAA production and expression of the key gene ipdC have been shown to be increased under carbon limitation, during reduction in growth rate, and at acidic pH values (Ona et al., 2003, 2005; Vande Broek et al., 2005). As Az. brasilense cells are colonizing plant roots, the bacterial cells are using root exudates for proliferation. When root exudates are becoming limited for bacterial growth, Az. brasilense increases IAA production, thereby triggering lateral root and root hair formation resulting in more root exudation. In this way a regulatory loop connecting plant root proliferation and bacterial growth is created. All mutants in specific genes involved in IAA biosynthesis, still show residual IAA production, even though levels are reduced by 90–99%. Attempts to isolate null mutants failed, indicating the redundancy of IAA biosynthetic pathways in bacteria. The presence of multiple pathways in a single organism suggests a distinct contribution of pathways to the IAA pool, for example, by differential regulation (Barbieri et al., 1986; Carreno-Lopez et al., 2000; Hartmann et al., 1983; Prinsen et al., 1993; Spaepen et al., 2007a). The exact contribution and regulation of different IAA biosynthetic pathways has clearly been demonstrated in the pathogen Pa. agglomerans. Mutants in genes of the IAM pathway cause a large reduction in gall size, while mutants in ipdC genes show no decreased pathogenicity. The latter mutants have reverse effects on the epiphytic fitness of the bacterium. The difference in both pathways is also reflected in the gene regulation: the ipdC gene is enhanced during plant colonization, whereas the iaaM gene is upregulated at later phases of the interaction (e.g., during gall formation) (Manulis et al., 1998). Although it is quite clear that rhizobacterial auxin production drastically affects plant root morphology, information on other plant processes affected by bacterial IAA synthesis is still fragmentary, or even speculative. Nevertheless, recent publications on the role of auxin in defense of Arabidopsis against the leaf pathogenic bacterium Pseudomonas syringae pv. tomato (Chen et al., 2007; Navarro et al., 2006) could open new research perspectives to better understand the role of IAA produced by PGPR. Both studies illustrate very well a prominent role for host auxin signaling in a particular aspect, that is, effector-triggered susceptibility and pathogen-associated molecular pattern (PAMP)-triggered immunity, respectively, of the ZigZag model of the plant immune system proposed by Jones and Dangl (2006). Both studies demonstrate that exogenous application of auxin to the host promotes susceptibility to the pathogen and disease development. The link to the ability of some bacterial pathogens to produce free IAA in culture was

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made in both publications. It is tempting to postulate that auxin production by PGPR could promote the colonization of plant roots by these PGPR as bacterial IAA production might contribute to circumvent the host defense system by derepressing auxin signaling (Remans et al., 2006). However, there is no direct evidence so far, and the dynamic differential distribution of auxin within plant tissues should be taken into account when comparing events in the phyllosphere versus the rhizosphere. Several research groups are currently studying the transcriptome of Arabidopsis roots colonized by PGPR. This together with quantitative imaging of roots colonized by IAA-producing PGPR and IAA-minus mutants, respectively, will allow testing of this hypothesis. Biotrophic pathogens and plant-beneficial bacteria are possibly coming closer to each other when taking an auxin perspective, recently referred to as an ‘‘information-processing system’’ (Vogel, 2006). Clearly, answering questions in one direction (phytopathology) raises new fascinating questions in another direction (phytostimulation). 2. Cytokinins The balance between auxins and cytokinins regulates the outcome on cellular differentiation in plants: if the balance is shifted toward auxin, root development is favored, while in the case of more cytokinin shoot growth is induced. When equimolar amounts of cytokinin and auxin are added in in vitro plant tissue cultures, undifferentiated callus cells proliferate. In contrast to the group of auxins, cytokinins are a broad group, mainly identified in bioassays as inducers of cell division, and are derived from 6N-substituted aminopurines. After biosynthesis in root tips and developing seeds, cytokinins are transported to the shoot via the xylem, where they regulate several processes, such as cell division, leaf expansion, and delay of senescence. The main representatives are zeatin and kinetin (Table I). As for auxins in bacteria, cytokinins were first discovered in pathogens, before cytokinin production was identified in PGPR. Exogenous application of this phytohormone can cause different responses in plants, including abnormal differentiation, promotion of tillering, and activation of seed germination. For several pathogens, the massive production of both auxins and cytokinins is an important virulence factor as they induce gall formation, as exemplified for the Agrobacterium tumefaciens oncogenes that are transferred into the plant DNA, Pa. agglomerans and Ps. syringae (Barash and Manulis-Sasson, 2007; Costacurta and Vanderleyden, 1995; Jameson, 2000). In parallel to auxin production, the capacity to produce cytokinins is widespread among rhizosphere bacteria and the spectrum of cytokinins does not differ from that in plants (Barea et al., 1976; De Salamone et al., 2001; Frankenberger and Arshad, 1995; Tien et al., 1979). Cytokinins are synthesized from isopentenyl

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pyrophosphate and 50 -AMP and several genes involved in this catalysis have been characterized, mainly from pathogenic bacteria such as Pa. agglomerans, Rhodococcus fascians and several Agrobacterium strains (Akiyoshi et al., 1984; Crespi et al., 1992; Lichter et al., 1995a,b). In a next step, isopentenyladenosine-50 -monophosphate is converted to isopentenyladenosine, which can be further modified (Kakimoto, 2003; Prinsen et al., 1997). It was even hypothesized at some time that all cytokinins present in plants are produced by microbial symbionts and are not plant-derived (Holland, 1997). Proof for the mode of action or mechanistic insights into the role of cytokinins in microbe–plant interactions is very scarce due to the lack of mutants that would allow measuring the contribution of bacterial cytokinin production to the plant growth-promoting effect. It is thought that bacterial cytokinin production contributes to the plant cytokinin pool, thereby influencing its growth and development. Recently, it has been shown that bacterial cytokinins are perceived by plant cytokinin receptors. In combination with the inadequate elimination of bacterial cytokinins by the plant, this ultimately leads to developmental changes and tissue proliferation (Pertry et al., 2009). Many PGPR produce both auxins and cytokinins. Consequently, the effect of these PGPR on plant growth and development depends on the balance between these two. Van Laer (2003) has made a quantitative survey of the auxin and cytokinin production by different PGPR.

3. Gibberellins The phytohormone group of the GAs consists of more than 100 members. These compounds are mainly involved in division and elongation of plant cells and influence almost all stages of plant growth, including seed germination, stem and leaf growth, floral induction, and fruit growth. As with auxins and cytokinins, they mainly act in combination with other phytohormones. Due to the use of diverse species and experimental models and the existence of over 120 different gibberellinic compounds, determination of the exact role of GAs is challenging, as it is difficult to discriminate between biologically active and precursor compounds (Yamaguchi, 2008). GAs can be classified as tetracyclid diterpenoic acids, with the typical ent-gibberellane basis synthesized from mevalonic acid. In bacteria, not much is known about GA biosynthesis. Therefore, most suggested pathways are based on those typical for plants and fungi. For Bradyrhizobium japonicum, a biosynthetic pathway different from plants and fungi was demonstrated by biochemical characterization and sequence comparison with known players in early steps of fungal and plant GA biosynthesis, such as diterpene cyclases, cytochromes

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P450 and dioxygenases, which are involved in the formation of the GA precursor ent-kaurenoic acid (Morrone et al., 2009). In the culture medium of some (plant-associated) bacteria, low concentrations of GAs could be measured, and it was demonstrated that GAs are released into the rhizosphere (Frankenberger and Arshad, 1995; Rademacher, 1994). For rhizobial strains, it was observed that root nodules contain higher amounts of GAs in comparison with noninfected roots. Since the GA spectrum in the nodules was identical to that of the bacteria, the nodule-derived GAs appeared to originate from the rhizobia. However, it cannot be ruled out that the rhizobial strains also induce GA biosynthesis in the nodules (Dobert et al., 1992; Ferguson and Mathesius, 2003). Several Azospirillum species are capable of producing different GAs and in addition metabolize exogenously applied GAs. Based on the observation that Azospirillum can hydrolyze GA conjugates, it has been suggested that the plant growth-promoting effect of rhizobacteria is a combination of both bacterial production of GAs and release of GAs from stored plant conjugates (Piccoli et al., 1997). The strongest evidence for the effect of bacterial GAs was demonstrated in an Azospirillum–maize system. The dwarf phenotype of the dwarf-1 line of maize or dwarfism induced by inhibitors of GA biosynthesis could be reversed by inoculation with Az. brasilense or Azospirillum lipoferum. However, whether bacterial GA biosynthesis or bacterial production of enzymes that release GA from conjugates is involved in this process is not clear (Lucangeli and Bottini, 1996, 1997). As GAs can act at several stages of plant growth and development, the bacterial production of GAs can interfere in very different ways. The exact mechanism of plant growth promotion by GAs is not known, although root-colonizing PGPR probably act by promoting root growth, more particularly by increasing root hair density in root zones involved in nutrient and water uptake, as shown for Az. lipoferum inoculation of maize seedlings (Fulchieri et al., 1993). 4. Ethylene The phytohormone ET was first named the ripening hormone, as it induces fruit ripening. Later, it was shown that the molecule also has a role in other processes, such as seed germination, cell expansion, leaf and flower senescence and abscission, and plant–pathogen interactions. In addition, ET is produced under both abiotic and biotic stress conditions and is therefore known as the stress hormone. High ET concentrations have an inhibitory effect on root growth, resulting in reduced plant growth (Abeles et al., 1992; Mattoo and Suttle, 1991). ET is produced by shuttling methionine out of the methionine cycle yielding S-adenosylmethionine (SAM) by SAM synthetase. SAM is converted in the rate-limiting step to 1-aminocyclopropane-1-carboxylate

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(ACC) and 50 -deoxy-50 methylthioadenosine (MTA) by the enzyme ACC synthase. MTA can be recycled to methionine. Finally, ACC is converted to ET, CO2 and cyanide by ACC oxidase (Chae and Kieber, 2005; Glick et al., 2007). ET biosynthesis is highly regulated mostly at the key step catalyzed by the enzyme ACC synthase at different levels, such as transcriptional regulation, enzyme degradation by the 26S proteasome, and phosphorylation. Factors altering ET biosynthesis are abiotic and biotic stresses (e.g., drought, wounding, and pathogen infection), hormones (cytokinins, auxins, and brassinosteroids), and developmental cues. Their action is exerted mainly through regulation of ACC synthase (Argueso et al., 2007; Chae and Kieber, 2005). Some PGPR express the enzyme 1-aminocyclopropane-1-carboxylate deaminase (AcdS), which can degrade ACC to -ketobutyrate and ammonia. The acdS gene is probably more widespread in bacteria than originally thought, and has been found in both pathogenic and plant-beneficial bacteria (Blaha et al., 2006). Inoculation of diverse plant species with bacteria expressing ACC deaminase activity stimulates plant growth, probably by lowering the level of ET inside the plant (Glick, 2005; Glick et al., 2007; Saleem et al., 2007). A model was proposed by Glick et al. (1998) to explain the role of bacterial ACC deaminase in the plant growth-promoting effect of these bacteria. As ACC may be exuded by plant roots (Bayliss et al., 1997), it can be taken up and hydrolyzed by bacteria with AcdS activity to -ketobutyrate and ammonia, which are then metabolized by the bacteria. The ACC concentration outside the roots decreases and more ACC is exuded by the plant. As a result, ACC levels in the plant are lowered and the ET content is reduced as biosynthesis of ET is inhibited by the lack of precursor (Glick et al., 1998). This results in an increased plant root and shoot length, increased biomass, and reduction of inhibitory effects of ET as a consequence of diverse stresses (Contesto et al., 2008; Glick et al., 2007; Saleem et al., 2007). In addition to expressing AcdS activity, most PGPR are capable of producing IAA. IAA excreted by these bacteria can contribute to the plant IAA pool, thereby inducing the effects described in Section II.A.1, as well as synthesis of plant ACC synthase, leading to more root-excreted ACC (Argueso et al., 2007). Bacteria with ACC deaminase activity have been extensively used for alleviating diverse stresses in plants. By reducing the stress hormone ET, these bacteria are able to protect plants from the growth inhibition caused by ET under several stress conditions, such as flooding, toxic compounds (both organic compounds and heavy metals), high salt concentrations, drought, and pathogenic attack. Transgenic expression of bacterial ACC deaminase genes in plants results in tolerance toward those stresses, although to a lesser

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extent compared to bacterial inoculation, as the bacteria possess also other mechanisms for plant-growth promotion (for reviews: Glick et al., 2007; Saleem et al., 2007). Production of ET has also been reported for microorganisms, including bacterial pathogens, as exemplified by Ps. syringae. In different pathovars ET production is mostly contributing to bacterial virulence, probably by creating a hormonal imbalance in the infected plant. Higher levels of ET found in diseased plant tissues could be correlated to ET production by inoculated bacteria. (Weingart and Volksch, 1997; Weingart et al., 2001). Bacterial ET biosynthesis differs from plant biosynthesis. Two distinct pathways have been described. In the first, the transaminated derivative of methionine, 2-keto-4-methyl-thiobutyric acid, is formed, which is further converted to ET by hydroxyl radicals formed by an NADH:Fe(III)EDTA oxidoreductase. In the second pathway, 2-oxoglutarate is the precursor for ET production by the ET-forming enzyme. The latter pathway is mainly used by ET-producing pathogens and some fungi (Tsavkelova et al., 2006; Weingart et al., 2001). ET also activates several defense responses in plants and is a necessary component in the induction of systemic resistance by rhizobacteria. The dual role of ET as a virulence factor of fungal and bacterial pathogens and as a signaling compound in disease resistance makes it difficult to define the exact role of ET produced by different strains of PGPR (Van Loon et al., 2006).

5. Abscisic acid Also ABA is involved in plant responses to biotic and abiotic stresses. As a hormone, it induces stomatal closure, inhibits seed germination and fruit ripening, and is involved in bud dormancy. Moreover, it mediates protective responses against adverse growth conditions such as drought, salt stress, and metal toxicity. ABA is synthesized in all plant parts and can be readily translocated through the plant. In plants, ABA is synthesized from the isoprenoid molecules isopentenyl diphosphate and dimethylallyl diphosphate via terpenoid intermediates (Taylor et al., 2005). Production of ABA in the culture medium of bacteria has been shown for Az. brasilense and some Br. japonicum strains, but a mechanistic proof or a biosynthetic pathway has not yet been provided (Boiero et al., 2007; Cohen et al., 2008). It can be hypothesized that ABA-producing bacteria can increase plant growth by interfering with the plant cytokinin pool, as ABA inhibits the synthesis of cytokinins (Miernyk, 1979). In addition, under stress conditions bacterially produced ABA can alleviate plant stress as suggested by Boiero et al. (2007).

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1. Biological nitrogen fixation Following reports of the isolation of rhizosphere and endophytic diazotrophs and their potential biological nitrogen fixation (BNF) activities (Dobereiner and Campelo, 1971; Dobereiner et al., 1972), there has been a large effort to demonstrate substantial BNF in different associative (nonsymbiotic) systems and their contribution to the nitrogen needs of plants. All the bacterial species studied were found to be vigorous nitrogen fixers in pure culture when an appropriate energy source, optimal temperatures, pH, and O2 concentration in the growth medium were available (Elmerich, 2007). There are probably many more N-fixing, nonculturable species, as revealed by the presence of the nifH and other marker genes in DNA extracted from soil (Schmid and Hartmann, 2007). BNF has been measured variously by the acetylene-reduction assay, 15N dilution technique, 15N fixation, and Kjeldahl N-content measurements (Okon, 1985). In most systems and conditions, the nitrogen fixation values for many grain and forage grass crops, when extrapolated to fixed nitrogen, generally did not amount to more than 10 kg N ha1 year1. Under some growth conditions, in few cultivars of sugar cane and rice (colonized by rhizosphere bacteria and endophytes representing several bacterial species) and in Kallar grass in Pakistan, BNF values indicated potentially significant BNF of up to 50% of the N content of the plant (Elmerich, 2007). From 15N dilution studies it was concluded that in annual crops, the fixed N probably remained in the bacterial cells, and that the bacterial cells could not be readily mineralized, preventing the uptake of combined nitrogen by the plant (Okon, 1985). In most cases, the contribution of biologically fixed N is far too low to contribute significant amounts of N to field crops. For example, a hybrid maize cultivar is commonly fertilized with 250 kg N ha1 per growth season. 2. Nitrate uptake by roots as affected by bacteria Enhanced mineral uptake in plants inoculated with PGPR has been reported repeatedly, both in greenhouse experiments (Lin et al., 1983) and in the field (Dobbelaere and Okon, 2007; Dobbelaere et al., 2003). The major nutrient involved was nitrogen in the form of nitrate (Dobbelaere and Okon, 2007). Using a hydroponic system containing NO3, during plant growth both the surface area of wheat roots and the uptake of NO3 from the mineral nutrient solution were found to increase upon inoculation with Az. brasilense. However, no significant changes were obtained in the NO3 uptake/root surface area ratio, indicating that the increased NO3 ion uptake by wheat was due to a general increase in root surface area, and not because of

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an increase in specific uptake rate (Kapulnik et al., 1985). On the other hand, direct effects on specific uptake mechanisms cannot be excluded, as it was observed that Azospirillum–inoculated corn and sorghum plants take up NO3 from solutions at faster rates than non‐inoculated plants (Lin et al., 1983). Section II.B.3. 3. Denitrification A large number of PGPR species that are used for inoculation in greenhouse and field experiments and commercially are also capable of dissimilatory nitrate reduction (denitrification). Under soil conditions of high NO3 content and low O2 availability all bacterial denitrifiers, and not just PGPR denitrifiers, will be active in soil denitrification (Steenhoudt and Vanderleyden, 2000). Denitrification is the respiratory process whereby NO3 is successively reduced to NO2, N-oxides (N and N2O), and dinitrogen (N2) (Fig. 2). (Steenhoudt and Vanderleyden, 2000). The first step, the reduction of nitrate to nitrite, can be catalyzed by a membrane-bound respiratory, or a periplasmic dissimilatory nitrate reductase. It has been proposed that bacterial nitrate reductase activity plays a significant role in PGPR–plant associations. It was also proposed that nitrite could mimic the effects

NO3 − (1) (2)

(3)

(6)

NO2 − (6) NO, N2O, N2 (4)

NO2 −

(2) (3)

NH4 + (5) Organic N

Fig. 2. The biological nitrogen cycle. Nitrogen can be transformed into different chemical forms by microbes, ranging from oxidation state þ5 (NO3) to 3 (NH4þ). The different reactions, carried out by different microbial groups under different ecological conditions, are indicated by numbers: (1) denitrification, (2) nitrification, (3) assimilatory and dissimilatory nitrate reduction, (4) biological nitrogen fixation, (5) ammonium assimilation, and (6) anaerobic ammonium oxidation (anammox). Processes (1), (3), and (4) require an electron donor, whereas process (2) requires an electron acceptor. NO3 and NH4þ are the main forms of nitrogen that can be readily used by plants.

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of IAA in several plants tested (Zimmer et al., 1988). However, all these observations have not been further substantiated (Steenhoudt and Vanderleyden, 2000). C. PHOSPHATE AND MICRONUTRIENT AVAILABILITY

1. Phosphate Phosphorus is an important nutritional element for growth and development of plants. The concentration of bioavailable phosphorus is rather low in soil (<5% of the total P pool). Under P deficiency, microorganisms can solubilize insoluble mineral phosphorus by releasing phosphatases (to release organic-P) or producing organic acids (to release inorganic-P). Organic acids such as acetate, succinate, citrate, and gluconate, acidify the surrounding soil, thereby releasing phosphate ions. Acidification is not the only mechanism for P solubilization. The organic acids can also chelate Fe and Al associated with P. Mineralization of organic phosphate by phosphatases involves dephosphorylation through the hydrolysis of phosphoester or phosphoanhydride bonds (Gyaneshwar et al., 2002; Rodriguez and Fraga, 1999). The process of P solubilization is very common for soil bacteria: up to 40% of the culturable population of the rhizosphere is capable of solubilizing P in culture. However, a clear demonstration of the role of phosphate solubilization in microorganism–plant associations is still not evident because of a lack of well-defined mutants. 2. Vitamins Under normal conditions, healthy plants produce enough vitamins to support their growth. However, in suboptimal or under stress conditions plants can suffer from vitamin deficiency, and therefore bacterial strains that produce vitamins can promote plant growth under these conditions. Some PGPR produce B-group vitamins, such as pantothenic acid, thiamine, riboflavin, and biotin, which can be absorbed by plant roots. However, further studies are necessary to prove the contribution of vitamin production to the plant growth-promoting effect (Dobbelaere et al., 2003). 3. Iron and other microelements Iron is very abundant in the soil, although it is mostly not available for assimilation, as it occurs mainly as Fe3þ oxides with a low solubility. Bacteria have developed a strategy for efficient uptake of iron by producing and secreting low-molecular-weight iron-chelating molecules, known as siderophores. Upon binding of Fe3þ to these molecules, they are transported back into the cells, readily available for microbial metabolism. Some

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microorganisms are able to take up siderophores from other organisms. In this way, a competition for iron between organisms occurs in the rhizosphere (Raaijmakers et al., 1995). The biosynthesis and mechanism of iron uptake by siderophores has been studied intensively in Pseudomonas species. In other PGPR, such as Azospirillum species and Rhizobia, the production of siderophores has also been reported (Robin et al., 2008; Whipps, 2001). Biosynthesis of siderophores is mostly tightly regulated by several systems, such as iron-sensing Fur proteins, the two-component regulators GacS and GacA, the stationary sigma factor RpoS, and quorum-sensing (QS) systems. Environmental factors, such as pH, iron level and ionic form, and nutrient availability, are important modulators of their production (Duffy and De´fago, 1999; Ravel and Cornelis, 2003). In addition, it has been suggested that bacterial siderophores may be taken up by plants and thus serve as an iron source, mainly in soils with low available iron. However, some reports question these assumptions (Marschner and Ro¨mheld, 1994). D. EMERGING SIGNALS

1. Signals related to QS QS is a mechanism by which small bacterially produced signal molecules accumulate to a certain threshold concentration (‘‘quorum’’) at which changes in gene expression can be triggered in a population density-dependent way. Several QS systems have been described in (Gram-negative) bacteria (Hooshangi and Bentley, 2008). The N-acyl-homoserine lactone (AHL) system, documented for many Gram-negative bacteria, is not generally present in Gram-negative PGPR. For instance, in Azospirillum only a few strains give a positive test result (Vial et al., 2006). QS enables bacteria to coordinate their behavior. Therefore, it is involved in several processes such as virulence, symbiosis, biofilm maturation, motility, and survival (Hooshangi and Bentley, 2008). Eukaryotes respond to QS molecules produced by bacteria. This phenomenon has been well described in mammalian hosts (Bauer et al., 2005; Hughes and Sperandio, 2008), but has also been reported for plants (Bauer and Mathesius, 2004). Via a proteomic analysis, the global response of the model legume Medicago truncatula to bacterially derived AHL was investigated. AHLs, in the concentration range produced by bacteria, can affect a wide set of functions, such as protein processing, hormone signaling, primary and secondary metabolism, and plant defense. The plant response depends on the structure of the AHL; in addition, tissuespecific responses can be observed (Mathesius et al., 2003). Other groups have shown that AHL-producing rhizobacteria can trigger systemic resistance in plants against pathogens by inducing SA- and ET-dependent defense

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genes, indicating a role of AHLs in the biocontrol activity of rhizobacteria (see below) (Schuhegger et al., 2006). Recently, it was shown that AHLs affect root architecture in a similar way as exogenously applied auxins, although the AHL response is independent of an auxin-signaling pathway (Ortı´z-Castro et al., 2008). Plants can also interfere with the bacterial QS communication by secreting QS-mimicking compounds. The exact role of these compounds is not known, but it is speculated that they can benefit the host by prematurely interfering with the bacterium to counteract the virulence of a pathogen or to initiate symbiosis (Pierson and Pierson, 2007; Teplitski et al., 2004). 2. Volatile compounds Some bacteria are able to release volatile organic compounds (VOCs) that promote plant growth. For Bacillus subtilis, acetoin and 2,3-butanediol were shown to be the active compounds inducing plant growth. This was demonstrated by using a knockout mutant in the biosynthetic pathway of these VOCs, be it only in a soil-less system (Ryu et al., 2003). The promotion of plant growth by rhizobacterial volatiles was also shown for Bacillus amyloliquefaciens and Pseudomonas chlororaphis. The production of these compounds is induced under low atmospheric oxygen partial pressure as an alternative electron sink for the regeneration of the NADþ that is needed (Cho et al., 2008; Ryu et al., 2003). Besides growth promotion, VOCs can also induce systemic resistance in plants under in vitro conditions, or tolerance to drought (Cho et al., 2008; Ryu et al., 2004). A microarray study to identify Arabidopsis transcripts regulated by the VOC-producing PGPR strain Ba. subtilis GB03 revealed that the expression of genes involved in auxin synthesis is upregulated by bacterial inoculation. In addition, inoculated plants contained less auxin in leaves and more auxin in roots, and the regulation of genes encoding cell wall-loosening enzymes, was predominantly upregulated, while some genes involved in cell wall rigidification were downregulated. Bacterial VOCs regulated auxin homeostasis and cell expansion, thereby inducing an optimized coordination between root and leaf development, including promotion of leaf expansion and lateral root formation (Zhang et al., 2007). 3. Nitric oxide Nitric oxide (NO) participates in several physiological and developmental processes in organisms. In plants NO is involved in root growth and proliferation through IAA-dependent signaling for adventitious root development. Az. brasilense cells produce NO in aerobic conditions using ammonium as a sole nitrogen source, or via a nitric oxide synthase with arginine as a substrate (Creus et al., 2005; Molina-Favero et al., 2008).

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Earlier reports suggested NO production by denitrification (Hartmann and Zimmer, 1994). The different biosynthetic pathways may indicate adaptation of the metabolism of Az. brasilense to the presence of different nitrogen sources. Az. brasilense is capable of influencing root architecture in tomato and inoculated tomato plants respond in a dose-dependent manner to NO production. An Az. brasilense mutant in the periplasmic nitrate reductase gene produced less NO and was not able to promote root formation. Moreover, treatment with a NO scavenger blocked the Azospirillum-induced root proliferation, indicating the importance of NO production in the plant growth-promoting capacity of Azospirillum (Molina-Favero et al., 2008). E. BIOCONTROL IN THE RHIZOSPHERE

Besides stimulating plant growth by direct mechanisms, such as the production of phytohormones or phosphate solubilization, PGPR can also indirectly induce plant growth by protecting plants against soil-borne pathogens, a process known as biocontrol activity (Bloemberg and Lugtenberg, 2001; Mercado-Blanco and Bakker, 2007; Schippers et al., 1987). Biocontrol by PGPR has mainly been described for Pseudomonas and Bacillus species, although a few reports on diazotrophic bacteria also cover biocontrol activity. Different mechanisms for biocontrol activity have been described. These can be classified as competition for nutrients and binding sites on the root (niche exclusion), production of antimicrobial compounds, and induction of systemic resistance (De Vleesschauwer and Ho¨fte, 2009; Van Loon and Bakker, 2003). 1. Competition Plant exudates are part of the available nutrients for rhizosphere organisms as up to 40% of plant photosynthetic products can be found in root exudates. These nutrient-rich zones along the root attract bacteria, including pathogens. Competition for suitable root niches is a main mechanism for excluding pathogens from the root by PGPR. Bacteria are attracted to the root by chemotaxis toward different compounds in the root exudates (organic acids, sugars, and amino acids) (Miller et al., 2009). PGPR move toward the root by flagellar motility and attach to the roots in a biphasic process (De Weert et al., 2002; Michiels et al., 1991; Vande Broek and Vanderleyden, 1995). Upon colonization, potential binding sites for pathogens are occupied, excluding potentially deleterious microorganisms from the plant root. Most suitable places are the nutrient-rich junctions between root epidermal cells.

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As the nutrient composition and the availability of the rhizosphere are very dependent on exudation by the plant root, microorganisms with a versatile metabolism and high-affinity transporters are highly favored in this environment. Most PGPR can use a wide variety of molecules as carbon and nitrogen sources in an efficient manner. In this way, they can scavenge away nutrients from deleterious microorganisms and reduce the ability of pathogens to proliferate in the rhizosphere, thereby protecting the plants from soil-borne pathogens. This mechanism is also known by the term antagonism (Weller, 1988, 2007; Weller et al., 2002). The competition for the limiting element iron (Section II.C.3) has been intensively studied in the competition potential of biocontrol and PGPR strains. Related to antagonism, the term ‘‘disease-suppressive soils’’ has been used to indicate soils in which pathogens do not establish, nor persist and cause little or no damage. Suppression is gained by the activity of the microbial population in the soil, which inhibits the growth and activity of pathogens (Weller et al., 2002). Several studies have tried to identify the microbial ecology in such soils in order to gain insights into the microbial agents and mechanisms involved in disease suppression (Mazzola, 2004). 2. Antibiosis Bacteria can produce a wide range of secondary metabolites that can inhibit the growth of bacteria and fungi (antimicrobial compounds). The action of antimicrobial compounds has been studied extensively for pseudomonads and bacilli, and their role in the biocontrol activity could be shown by the use of mutant and overexpressing strains. The best characterized compounds are phenazines, 2,4-diacetylphloroglucinol, pyrrole compounds (e.g., pyoluteorin and pyrrolnitrin), cyclic (lipo-) peptides, and hydrogen cyanide (HCN) (Table II). As the biosynthesis genes for the production of these secondary metabolites have been identified, the biosynthetic pathways have been described (Chin-A-Woeng et al., 2003; Cook et al., 1995; De Weert and Bloemberg, 2006; Dubuis et al., 2007; Dwivedi and Johri, 2003; Haas and Keel, 2003; Raaijmakers et al., 2002; Weller, 2007). The best characterized group of antimicrobial compounds produced by biocontrol strains are the phenazines, heterocyclic nitrogen-containing compounds, with a broad antimicrobial spectrum. The production of phenazines has been mainly investigated in Pseudomonas, but has also been reported for Streptomyces, Brevibacterium, Burkholderia, and other bacterial species. The exact mode of action of phenazines is unknown; however, it is assumed they can act as reducing agents and thus uncouple oxidative phosphorylation, and generate toxic oxygen species. Therefore, they are toxic to a wide range of

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TABLE II Examples of Antimicrobial Compounds, Produced by Biocontrol Strains Class

Example

Structure O

HO

Phenazine

Phenazine-1-carboxylic acid

N

N

HO

Pyrroles

Pyoluteorin

CI

NH OH O

CI

NH

Pyrrolnitrin

N+ O−CI

CI O

OH

HO

Phloroglucinols

2,4-Diacetylphloroglucinol

H3C

CH3 O

OH

O O

N

O

N

R

Cyclic oligopeptides

Iturin A

N

O O

O

N O

N N

O

O

N N

O

O

N

O

N

O N

O H

O N

Hydrogen cyanide

HCºN

organisms, including bacteria, fungi, and algae. Phenazines are synthesized by the condensation of derivatives of chorismic acid molecules. Further steps in the pathway modify the phenazine-1-carboxylic acid to other derivatives. The biosynthetic genes phzABCDEFG are clustered in the genome and highly conserved among Pseudomonas species. Their expression is regulated by

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two-component regulatory systems (quorum sensing) and by environmental conditions (Chin-A-Woeng et al., 2003; Dubuis et al., 2007; Mavrodi et al., 2006). Pyoluteorin is a polyketide antibiotic, linked to a bichlorinated pyrrole. The biosynthesis mostly starts from proline by the addition of three acetate molecules, followed by cyclization. The biosynthetic genes pltLABCDEFG, clustered in the genome, were characterized in Ps. fluorescens Pf-5. Pyrrolnitrin is synthesized from the precursor tryptophan by enzymes encoded by the prnABCD genes, and the expression of the prn operon is regulated by the global response regulator GacA (Costa et al., 2009; Dwivedi and Johri, 2003; Van Pee and Ligon, 2000). 2,4-Diacetylphloroglucinol is also a polyketide, produced by the condensation of 3 acetyl-CoA molecules with malonyl CoA. Subsequent acetylation results in 2,4-diacetylphloroglucinol. As for the other antibiotics described, the biosynthetic genes phlACBDE in various Pseudomonas species are clustered in the genome. A negative regulator gene, phlF, is located upstream of this cluster and is part of the complex regulation of phloroglucinol biosynthesis (Dubuis et al., 2007; Dwivedi and Johri, 2003; Weller et al., 2007). Other antimicrobial metabolites that are produced by plant-associated bacteria are HCN and cyclic (lipo-)peptides, such as viscosinamide, putisolvin, and iturin (Haas and Keel, 2003; Ongena and Jacques, 2008).

III. AGRICULTURAL ASPECTS AND RELEVANCE It is clear that for a substantial commercial and agronomical impact of the use of PGPR, the products need to be adapted and formulated based on the accumulated knowledge of plant growth-promoting mechanisms and rhizosphere microbial ecology. In this way, use of PGPR in a more comprehensive scientific rather than in an empirical manner, as it has been the case so far, becomes possible. A. PGPR AND ENDOPHYTES—ROLE OF BACTERIAL NUMBERS

For any mechanism of plant-growth promotion to operate, bacterial population densities on the roots need to be substantial. For BNF, it has been clearly shown that if there are less than 106–107 cells per gram of root tissue, the fixed amount of N will probably be too small to have an impact on plant N-nutrition (Dobbelaere and Okon, 2007). For plant growth substances acting in small concentrations it is conceivable that lower bacterial numbers suffice, but to observe a clear effect the population density needs to reach at

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least 107 cells per plant. It is not known what the optimal number of cells actually colonizing and growing on the root surface or in the root tissue is or should be, but 103–104 cells per gram root tissue seems reasonable to postulate. For the production of antibiotics and competition for colonizing sites, the numbers needed are at least 105–107 colony-forming units (cfu). Therefore, it has been proposed that only very efficient root colonizers should be used as commercial inoculants (Weller, 2007). One important issue is whether the PGPR colonize the root surface or the inside of the plant as endophytes without causing disease symptoms. As in the case of the Rhizobia–legume symbiosis, living inside the plant would be more efficient, but if bacterial numbers are too high, it could affect the development of the plant similar to pathogens reaching 107–108 cfu g1 diseased tissue. In this respect, it is often said that endophytes could be considered as weakened biotrophic pathogens. So far numbers of endophytic PGPR are reported to be relatively low (103–104 cfu g1 tissue) and to be present only in specific plant tissues (small parts of the root system) (Dobbelaere et al., 2003). In contrast, rhizosphere colonization usually occurs at much higher levels, as in the case of some antibiotic-producing Bacillus and Pseudomonas spp., with 106–107 cfu g1 root (Weller, 2007). Endophytes may secrete plant growth substances inside the plants and alter their metabolism in a positive or negative manner, but promotion of growth while living in the rhizosphere has been mainly and clearly observed in the case of auxin-producing Azospirillum (Spaepen et al., 2007a). B. PGPR AND OTHER SYMBIOTIC SYSTEMS SUCH AS RHIZOBIUM-LEGUMES

Evidence has accumulated that co-inoculation with two beneficial organisms having different mechanisms of plant-growth promotion can have additive or synergistic effects on plant growth and crop yield. One of the best studied systems is the co-inoculation with Azospirillum and Rhizobium. It has been observed that in legumes Azospirillum promotes root development and mineral uptake but at the same time enhances secretion of flavonoid compounds by the plant, which induce the expression of nodulation (nod) genes in Rhizobium, resulting in early and faster nodulation, earlier onset of nitrogen fixation in the nodule, and higher crop yield (Burdman et al., 1998; Dardanelli et al., 2008; Remans et al., 2008). Dual inoculation with Rhizobium and Azospirillum and other PGPR was shown to significantly increase both upper (i.e., those nodules formed in the upper 5 cm of the root system) and total nodule number of several legumes. BNF was higher, as evidenced by increased acetylene-reduction activities, faster 15N dilution, and higher contents of total N and mineral

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macro- and micronutrients as compared to inoculation with Rhizobium alone (Burdman et al., 1998; Rodelas et al., 1996, 1999; Sarig et al., 1986). An indole-3-pyruvate decarboxylase-minus (ipdC) mutant of Az. brasilense strain Sp245, producing only 10% of the IAA produced by the wild-type strain caused far less nodulation and nitrogen fixation when co‐inoculated with Rhizobium on common bean (Phaseolus vulgaris) (Remans et al., 2008). Therefore, the positive effect on flavonoid production could likely be attributed to IAA and other plant growth substances produced by Azospirillum in the rhizosphere (Spaepen et al., 2007a). C. VEGETATIVE GROWTH AND GRAIN FILLING

The utilization of PGPR in agronomy should take into account the agronomic conditions and the type of crop to be harvested: forage crops for shoot-leaves-biomass production or for grain production. In the latter case, the accumulated photosynthate in the leaves has to be translocated to the inflorescence for forming the seeds. Sometimes if vegetative growth is promoted and translocation to seeds is inhibited, as in the case of early drought, inoculation with PGPR can result in lower yield as compared to non‐inoculated plots. For example, in non‐irrigated early cultivars of wheat in Israel, there was significant increase in yield upon inoculation with Azospirillum, while in late cultivars that filled their grains after the raining season has ended, the higher shoot weight observed did not translate into higher grain yield and actually resulted in lower yield (shoots dried faster) in inoculated plots compared to non‐inoculated controls (Kapulnik et al., 1987). D. INOCULANT TECHNOLOGY

Bacterial inoculant preparations for agricultural uses are commercial formulations containing PGPR that can be applied to the seed or the soil during planting. During several stages, these inoculants must survive stress conditions such as acidity, desiccation, chemical pesticides, and possibly strongly fluctuating temperatures (Ben Rebah et al., 2007). In this framework, an inoculant that possesses a high capacity to maintain high survival rates of the bacterial cells within the carrier itself is desirable. Thus, an appropriate material for carrying microbes must offer special properties such as chemical and physical uniformity, high water-holding capacity, and lack of toxicity, and it must be environmentally friendly. Commercial inoculants are sold as solid, in powder from peat, or in granular form, or as liquid, in broth formulations (Ben Rebah et al., 2007; Stephens and Rask, 2000). Many of the microbial inoculants are based on solid peat formulations due to the peat protective properties. Most of

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the research done in the field of formulation aims at testing new carriers or improving carrier properties by adding elements that can prolong microbial survival, such as nutrients, or other synthetic products. The vast amount of information gathered on Azospirilla and Rhizobia throughout the years suggests that for an inoculant to be successful, that is, for it to provide efficient root colonization or invasion, the type of carrier material, the metabolic state of the cells, and their capability to use intracellular storage material are important for survival within the carrier itself. This knowledge originated from studies showing that while the carriers may vary, plant growth-promoting effects were more consistent with Az. brasilense inoculants containing high amounts of polyhydroxyalkanoates (PHAs) (Fallik and Okon, 1996). Corroboration was obtained in maize and wheat field experiments carried out in Mexico, where increasing crop yield was obtained using peat inoculants prepared with PHA-rich Azospirillum cells (Dobbelaere et al., 2001). In bacteria, PHAs are intracellular energy and carbon storage compounds that can be mobilized and used when carbon is a limiting resource. Intracellular accumulation of PHA enhances the survival of several bacterial species under environmental stress conditions in water and in soil, such as UV irradiation, salinity, thermal and oxidative stress, desiccation, and osmotic shock (Kadouri et al., 2005). The ability to endure these stresses is linked to a cascade of events concomitant with PHA degradation and the expression of genes involved in protection against damaging agents. The fact that PHA production is a widespread trait supports the assumption that PHA accumulation plays a central role in survival, notably when bacteria are faced with starvation. In PHA-producing bacteria, PHA is a major determinant for overcoming periods of carbon and energy starvation, and may represent a basic feature for so-called ‘‘environmental bacteria’’. Production of PHA is of critical importance for improving the shelf life, efficiency, and reliability of commercial inoculants (Kadouri et al., 2003). There has been a large improvement in the technology of PGPR inoculants, particularly the use of liquid inoculants instead of solid supports and bacterial carriers such as peat. The use of osmoprotectants in the liquid inoculant permits the maintenance of 109 cfu ml1 for storage periods of at least 1 year, such as in the case of Br. japonicum (Ben Rebah et al., 2007; Stephens and Rask, 2000). Also new liquid inoculants were developed for Azospirillum, Bacillus, and Pseudomonas spp. Liquid inoculants are more easily stored, applied, and less expensive than the conventional inoculant products. Liquid inoculants colonizing roots to higher population densities have resulted in more consistent plant-growth promotion in fields in Argentina in the past 5 years.

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Basic investigation of features for bacterial survival such as PHA accumulation, exopolysaccharide production, and the involvement of gene regulatory factors could have a significant impact on prolonging shelf life, thereby improving root colonization at the time of the PGPR application, allowing higher probabilities of successful root colonization by the PGPR.

E. PROBIOTICS IN AGRICULTURE

The concept of using bacteria to promote health of higher organisms is not unique to plants. A striking parallel can be noticed when looking at the first publications reporting the beneficial effects of bacteria on humans and plants. In 1907 the Nobel Prize winner Elie Metchnikoff postulated yogurt for the first time as a health-promoting food (Metchnikoff, 1907). Three years before, Lorenz Hiltner defined for the first time the term rhizosphere as the soil compartment influenced by the root. He concluded that ‘‘the plant attracts on one hand beneficial bacteria through its root exudations, but it attracts also uninvited guests’’ (Hiltner, 1904). The term probiotics refers to live microorganisms, which when administered in adequate amounts, confer a health benefit to the host (FAO/WHO, 2001). As such, the term could also be used to refer to live microorganisms that confer a health benefit to plants. The two areas of research, being gut microbiology and rhizosphere microbiology, have very much in common regarding the scientific questions (structure and dynamics of the microbiological community, identification of the probiotic factors and host responses), and the technological tools that are presently used to address these questions. Consequently, it can be stated that knowledge from one area can be used in the other. This becomes particularly striking when focusing on the identification of probiotic factors (Lebeer et al., 2008). Although the dominant bacterial species present in the gastrointestinal tract of humans are very different from those usually recovered from the rhizosphere, some species are common, such as the lactic acid bacteria. As more and more common themes are disclosed regarding the innate immune systems in humans (animals) and plants (Ausubel, 2005), exciting new developments lie ahead.

IV. PERSPECTIVES The mechanisms and effects of PGPR, as summarized in this chapter, have been studied within the confines of knowledge on root architecture and function, plant hormone metabolism, and many other related processes.

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The accumulating scientific advances in understanding root morphology and physiology, the sequencing and annotation of entire genomes of plants, such as rice, sugarcane, maize, and wheat, and of PGPR will support very soon important advances in the comprehensive use of PGPR in agriculture. Furthermore, the use of modern techniques for high-throughput analysis of transcriptomes, proteomes, etc. will detect much faster and more accurately changes taking place during PGPR–plant associations. Critical functions could then be enhanced by molecular manipulations and genetic engineering of plants and bacteria, and negative or deleterious trends could be diminished. Although the effort in developing microbial inoculants (including rhizobia) for plant growth promotion in agriculture (Dobbelaere and Okon, 2007; Dobbelaere et al., 2001) has been increasing for the past 40 years, the scientific basis is far away from being complete and the research effort must be continued. There is a very good possibility that various developed PGPR systems will be used extensively in agriculture, particularly when practice demands a clear and substantial reduction of both fertilizer and pesticide use (Dobbelaere and Okon, 2007). The potential of PGPR in terms of enhancing plant vigor, increasing plant biomass, and providing plant protection against pathogens has been repeatedly demonstrated. PGPR as biocontrol agents deserve special attention since bacteria in general are a rich and well-known source of complex metabolites for use in (phyto-) pharmaceutical applications. Still, fungal diseases cause significant losses (estimated at 7%, at the least) in plant production worldwide. The search for antifungal compounds produced by bacteria has resulted in some active compounds, such as 2,4-diacetylphloroglucinol produced by Ps. fluorescens (Walsh et al., 2001). A more systematic screening of bacterial antifungal compounds can result in the identification of broad and narrow spectrum compounds. With more such compounds at hand, PGPR can be optimized for better biocontrol. Furthermore, PGPR-mediated ISR offers a huge potential to enhance the defensive capacity of crop plants against both soil-borne and foliar pathogens.

ACKNOWLEDGMENTS Jos Vanderleyden and Stijn Spaepen acknowledge financial support of the FWO-Vlaanderen (G.0632.08). Stijn Spaepen thanks the Research Fund K. U. Leuven for a postdoctoral fellowship grant.

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Interactions Between Nonpathogenic Fungi and Plants

M. I. TRILLAS1 AND G. SEGARRA

Departament Biologia Vegetal, Facultat Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, SPAIN

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Interactions Between Plants and Endophytic Fungi . . . . . . . . . . . . . . . . . . . . . . . . A. Plants and AM Fungi.......................................................... B. Plants and Other Endophytic Fungi......................................... III. Interactions Between Plants and Free-Living Opportunistic Symbiotic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plants and Trichoderma spp. BCAs.......................................... B. Plants and Nonpathogenic F. oxysporum................................... C. Plants and Nonpathogenic Penicillium spp., Phoma spp., and Pythium oligandrum ............................................................ IV. Overview of Plant Defense Mechanisms Induced by Nonpathogenic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT This chapter deals with the interactions established between nonpathogenic fungi/ biocontrol agents and plants and their involvement in the reinforcement of plant defenses. Among the nonpathogenic fungi, one can distinguish between the true endophytic and the free-living opportunistic symbiotic fungi. As for the first group, the majority of the literature available corresponds to mycorrhizal fungi. Historically, these studies were focused on the influence of the mycorrhizal symbiosis on plant nutrition and later evolved to studies on plant immunity. Other endophytic fungi that 1

Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51008-7

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trigger enhanced plant resistance correspond to nonpathogenic binucleate Rhizoctonia spp. and Piriformospora indica. The second group includes fungi that were considered to be free-living microorganisms that reduce plant diseases by direct competition with plant pathogens through different antagonistic mechanisms. More recently, such fungi were proven to opportunistically establish symbiotic relationships with plants and to enhance plant defense by inducing systemic resistance. The most studied genus corresponds to Trichoderma spp., but information is also available on the mode of action of nonpathogenic Fusarium oxysporum, Penicillium spp., Phoma spp., and Pythium oligandrum strains. Information about the mechanisms of action of this broad range of nonpathogenic fungi is summarized, paying special attention to the genes, proteins, and signaling pathways involved, and common patterns of defense mechanisms induced by beneficial fungi in plants are proposed.

I. INTRODUCTION Interactions between nonpathogenic fungi and plants can often enhance plant nutrient uptake, promote growth, and suppress disease. Like pathogens, these nonpathogenic fungi live on/in plants, and despite differences between their lifestyles, they share some common features. The nonpathogenic microorganisms are capable of establishing themselves in a changing environment, compete with the stable resident microbial community, thereby maintaining a constant population, and interact with the plant. The oldest relationship studied between nonpathogenic fungi and plant hosts is mycorrhizal symbiosis. Components of soil communities in many temperate and tropical ecosystems, these fungi colonize the roots of most plant species. These root symbionts can benefit plant nutrition by exchanging host carbohydrates for phosphorus as well as nitrogen, potassium, calcium, iron, copper, zinc, etc. under limiting environmental conditions (Franken et al., 2007; Smith, 1988). Seven categories of mycorrhiza have been distinguished on the basis of their morphological characteristics and the fungal and plant species involved: arbuscular mycorrhizal (AM) fungi of the order Glomales of the Zygomycota; ericoid mycorrhizal fungi of the Ascomycota and Basidiomycota belonging to clade B Sebacinales; ectomycorrhiza, Basidiomycota, and Ascomycota, several distinct fungal species; orchid mycorrhiza, basidiomycetous Rhizoctonia spp.; monotropoid mycorrhiza, arbutoid mycorrhiza, and ectendomycorrhiza (Finlay, 2008). Historically, mycorrhizal research focused on AM fungi and improved plant uptake of dissolved mineral nutrients; however, given that the plant–fungal association is more common than can be explained by facilitation of phosphorus uptake alone, several studies have addressed the mitigating effects of AM fungi on plant diseases and the stimulation of local/systemic resistance in their hosts (Borowicz, 2001; Garcı´a-Garrido and Ocampo, 2002; Pozo and Azco´n-Aguilar, 2007; Volpin et al., 1994).

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More recent research has described specific interactions between other nonpathogenic endophytic fungi and plants, such as those involving Piriformospora indica and binucleate Rhizoctonia. Also, various nonpathogenic, free-living, soil-borne fungi that are beneficial for plants by suppressing diseases have been studied, such as Trichoderma spp., avirulent strains of Fusarium oxysporum, Penicillium spp., Phoma spp., and Pythium spp. In the past, studies of the mechanisms of action of these biological control agents (BCAs) focused on mycoparasitism and other direct effects on target pathogens. However, recent research has demonstrated that these beneficial fungi can also increase root and shoot growth of plants, enhance nutrient availability, colonize roots, and induce local/systemic resistance (De Cal et al., 2000; Harman et al., 2004a; Hase et al., 2008; Hossain et al., 2007; Howell, 2003; Olson and Benson, 2007; Segarra et al., 2007; Shoresh et al., 2005; Waller et al., 2008). In this chapter, we will describe the metabolic changes induced by these nonpathogenic fungi in plants and the enhancement of plant defense against pathogens. Given the involvement of these fungi in plant defense, we will also address plant colonization by these microorganisms and their contribution to improved plant nutrition. As demonstrated later on, these beneficial fungi share some common features in their interaction with plant roots. Most prime the plant to respond more strongly after subsequent pathogen infection. Furthermore, the induction of direct effects on plant metabolism is also a common feature of these fungi.

II. INTERACTIONS BETWEEN PLANTS AND ENDOPHYTIC FUNGI A. PLANTS AND AM FUNGI

AM fungi establish a symbiotic association with plant roots. These microorganisms penetrate the root and form special structures with their host, which serve as the interface for nutrient exchange between the two partners. Mycorrhizal fungi may improve plant health by enhancing nutrition status, promoting growth and development of soil microbes, competing with pathogens (space/nutrients), ameliorating abiotic plant stress (metal toxicity, water stress), and inducing plant defenses (local/systemic). Most studies show that AM fungi alleviate the damage caused by soil-borne pathogens (fungi and nematodes). However, their impact on above-ground plant diseases is less clear. AM fungi have been associated with no effect on plant diseases (Baath and Hayman, 1983, 1984; Chandanie et al., 2006), enhanced susceptibility to

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biotrophic pathogens, and enhanced resistance against Alternaria solani, Xanthomonas campestris, and Pseudomonas syringae (Davis and Menge, 1981; Finlay, 2008; Pozo and Azco´n-Aguilar, 2007; Vaast et al., 1998). Resistance to fungal pathogens might affect the interaction of plants with AM fungi, as the latter must avoid host defense responses. In this regard, corn inbreds that are generally more resistant to a variety of fungal pathogens contain fewer AM arbuscules in their roots (Smith and Goodman, 1999). 1. Plant root colonization AM symbiosis is characterized by the development of highly branched fungal structures (arbuscules) that grow intracellularly, while ericoid mycorrhizae form coiled structures within each cell, without penetrating the host plasmalemma. Ectomycorrhizal fungi and orchid mycorrhizae are ectomycorrhizal symbionts, which do not penetrate the host cells. Their symbiosis is characterized by the presence of a fungal mantle or sheath around each of the short roots, and intercellular hyphae that penetrate between epidermal and cortical cells, forming a Hartig net. Monotropoid mycorrhizae structurally resemble ectomycorrhizae but with a more superficial Hartig net, with single hyphae growing into the epidermal cells, forming peg-like structures. Arbutoid mycorrhizae and ectendomycorrhizae are also ectomycorrhizal (Finlay, 2008). In the interaction between mycorrhizal fungi with their hosts, plant flavonoids and fungal auxins act as signals for the initiation of morphogenetic events, which include appressorium development (Franken et al., 2002). In addition, genes that encode hydrophobins and other structural proteins of the cell wall are specifically upregulated in mycorrhizal fungi. These proteins facilitate the attachment of hyphae to hydrophobic surfaces. A correlation between endoxyloglucanase activity of the external mycelia of several Glomus spp., root colonization, and stimulation of plant growth has been reported. This observation indicates that this enzyme is a key factor influencing fungal colonization and plant growth (Garcı´a-Garrido et al., 2000). The intercellular colonization by AM fungi involves cytological events and intracellular modifications. A new interface compartment comprising membranes from both the plant host and the fungus is created, surrounded by molecules common to the plant primary cell wall (cellulose, pectin, xyloglucan, and hydroxyproline-rich glycoproteins), yet not assembled into a fully structured wall (Bonfante, 2001). 2. Improvement of plant nutrition The plant response induced by AM fungi varies depending on phosphate availability. Variation in phosphorus uptake has been found among plant species and cultivars, and among strains of mycorrhizal fungi (Smith and Goodman, 1999). Plant phosphate concentrations are negatively correlated

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with root colonization by AM fungi and positively influenced by plant carbohydrate status. A significant correlation between the level of colonization by the fungus and an increase in phosphorus uptake/yield has been described for maize and onion, but no such effect was found in wheat and alfalfa (Azco´n and Ocampo, 1981; Lambert et al., 1980; Toth et al., 1984). Carbohydrates from the plant are taken up by the fungus for growth and arbusculated cells are greater sucrose sinks than cortical cells without arbuscules (Vierheilig et al., 1993). A number of authors have postulated (Blee and Anderson, 2000; Herbers et al., 1996) a relationship between sugar regulation, the expression of defense genes, and the activation of systemic resistance, which is linked to the ethylene (ET) signal transduction pathway (Zhou et al., 1998). LeST3 is a putative sugar transporter isolated from mycorrhizal tomato roots. Its expression increases in the leaves of plants colonized by the AM fungi Glomus mosseae or Glomus intraradices, or by the root pathogen Phytophthora parasitica. It is involved in the allocation of carbon compounds to the AM fungi and in increased metabolic activity of the root pathogen. An alternative hypothesis regarding the function of LeST3 is that it is a component of a signal transduction pathway that triggers defense responses in either a successful mycorrhizal association or in a plant– pathogen interaction (Garcı´a-Rodrı´guez et al., 2005). Besides sugar and ET metabolism, cytokinin concentrations are altered: levels of this hormone are increased in AM alfalfa roots (Van Rhijn et al., 1997). Elevated levels of cytokinins in AM roots could suppress the induction of genes encoding PR (pathogenesis-related)-proteins such as chitinase and glucanase. However, it is not clear whether cytokinins are directly involved in the process of mycorrhization (Garcı´a-Garrido and Ocampo, 2002). A number of studies support the hypothesis that enhanced plant tolerance to pathogens is a direct consequence of the improved root nutrition, growth, and function in AM symbioses (Hussey and Roncadori, 1982; Smith, 1988). Moreover, AM fungi can directly compete with soil microorganisms for space and nutrients. These fungi also suppress pathogen growth by nutrient competition with soil-borne nematodes and fungal pathogens and promote the growth of soil microbes that antagonize pathogens (Muchovej et al., 1991; Smith, 1988; Thomas et al., 1994). AM fungi reduce pathogen activity by altering root exudations (quality or quantity). This strategy is used against the pathogen Gaeumannomyces graminis (Graham and Menge, 1982). Although AM fungi, soil pathogens, and nematodes occupy similar root tissues, direct competition for space is not evident since many root pathogens infect at the root tip, where AM fungal structures do not occur. However, the amount of mycorrhizally colonized root tissue is reduced when the pathogen and AM fungi are in direct association (Davis and Menge, 1981; Smith, 1988).

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3. Induction of plant resistance Borowicz (2001) proposed that AM fungi increase host resistance by stimulating a defense response. An overview of studies conducted from 1976 to 1996 (Morandi, 1996) on the interactions between plants and AM fungi and linked to the metabolism of phenolics leads to the conclusion that mycorrhizal fungi cause an accumulation of phenolic compounds in the roots of their hosts, in particular phytoalexins and associated flavonoids and isoflavonoids. The magnitude of the plant reaction to AM fungi is consistently lower than to pathogens. It has been proposed that the accumulation of phenolics makes mycorrhizal roots less suitable for pathogens (Morandi, 1996; Volpin et al., 1994, 1995). Conjugated isoflavonoids that are nontoxic and that accumulate at higher concentrations in mycorrhizal roots may serve as a storage form of phytoalexins, which could be quickly hydrolyzed in response to a pathogen attack (Morandi, 1996). Several steps of mycorrhizal development have been considered in relation to plant defense responses, from mutual recognition during early contact to interactions during later stages of mycorrhizal development in root cells (Garcı´a-Garrido and Ocampo, 2002; Morandi, 1996). A requirement of a well-established AM symbiosis for induction of resistance is generally accepted (Pozo and Azco´n-Aguilar, 2007). The primary contact of the AM fungus with its host during root colonization induces a weak and transient activation of plant defenses. This induction is triggered by elicitors secreted by the mycorrhizal fungi (Garcı´a-Garrido and Ocampo, 2002), or generated as a result of physical/chemical cleavage of plant cell wall components (Somssich and Hahlbrock, 1998). Plant defense responses against mycorrhizal colonization may result from a specific activation of host defense genes, from a nonspecific activation caused by a low capacity of the fungus to trigger the response, and from suppression of the activated plant cell response. Plant defense can be attenuated by the degradation of exogenous elicitor molecules produced by AM fungi and by the prevention of endogenous elicitor release from the plant cell wall (Garcı´a-Garrido and Ocampo, 2002). Another possible mechanism to weaken host defense is by blocking components of the signal transduction pathway that activate the plant response, such as salicylic acid (SA) and reactive oxygen species (ROS). Alterations in the activities of antioxidative enzymes (catalase and peroxidase) may also indicate that oxidative compounds are produced (Blilou et al., 2000a,b). Like obligate biotrophs, AM fungi share similarities with biotrophic pathogens and, thus, may be sensitive to SA-regulated defenses (Pozo and Azco´nAguilar, 2007). In this regard, in NahG tobacco plants, that are unable to accumulate free SA, the extent of AM fungal colonization is greater than in

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wild-type plants, thereby suggesting a link between SA accumulation and fungal infectivity in compatible mycorrhizal associations (Garcı´a-Garrido and Ocampo, 2002). Studies using legume mutants unable to support nodulation and nitrogen fixation by Rhizobia revealed similarities between AM symbiosis and rhizobial colonization/nodule formation. Indeed, a suppression of SA responses is also required for the establishment of the Rhizobium–legume symbiosis (Pozo and Azco´n-Aguilar, 2007). These findings are of interest since microorganisms that differ in host specificity have the capacity to activate similar genes and plant responses in a wide range of plant species. In legume plants carrying mutations in SYM8, SYM9, SYM19, and SYM30 genes, the penetration of AM fungi is blocked at the level of appressoria formation (myc mutants), and the fungi are also unable to form infection threads upon contact with Rhizobium (Marsh and Schultze, 2001). Interestingly, in sym30 mutants, resistance to AM fungi and rhizobial fungi correlates with enhanced concentrations of endogenous SA in the roots (Blilou et al., 1999). In mutants in three other loci (sym2, sym3, and sym4), which show no induction of early nodulin genes, early interaction with AM fungi is also affected. As only a local, weak, and transient defense response is activated during early interactions between AM fungi and plants, AM fungi must repress SA-dependent defense responses in the host in order to achieve a compatible interaction (Pozo and Azco´n-Aguilar, 2007). At later stages of AM fungi–plant symbioses, when arbuscules are fully functional, the activation of plant defense responses becomes stronger in cells that contain fungal structures. These responses include enhanced expression of genes encoding hydroxyproline-rich glycoproteins, enzymes of phenylpropanoid metabolism, enzymes involved in the metabolism of ROS, and plant hydrolases (Blee and Anderson, 1996, 2000; Volpin et al., 1994, 1995). Induced expression of phenylalanine ammonia-lyase (PAL), chalcone synthase, and flavonoid compounds (Bonanomi et al., 2001; Harrison and Dixon, 1993, 1994; Volpin et al., 1994, 1995) has also been described. Lanfranco et al. (2005) reported a functional AM CuZn superoxide dismutase that may provide protection against localized host defense responses involving ROS. The AM fungus colonizes the upper parts of the roots, and the response is significantly greater in this section of the roots than in lower parts (Volpin et al., 1994). On the basis of these data, it can be concluded that mycorrhizae do induce local defense responses. Dehne and Scho¨nbeck (1979) studied the effect of the mycorrhizal fungus G. mosseae on resistance to Fusarium wilt in tomato plants. Simultaneous infection with G. mosseae and F. oxysporum increases PAL, -glucosidase activity, and total phenol content of roots to a greater extent than either the

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mycorrhizal fungus or the pathogen alone. Together with the increased lignification of the endodermis and stele by mycorrhizal infection, these findings support the hypothesis that enhanced resistance caused by mycorrhizal infection to Fusarium wilt is mediated by induced phenolic metabolism in the roots (Dehne and Scho¨nbeck, 1979). In the Medicago truncatula–X. campestris pv. alfalfae interaction, Liu et al. (2007) found 26% of the genes induced in the shoots by AM fungi to be involved in signaling or responses to abiotic or biotic stress. These include six genes similar to those encoding Avr9/ Cf9 proteins, seven genes encoding ET-responsive element-binding proteins, and five similar to syringolide-induced genes. The gene expression response of the roots and shoots differed in magnitude and with respect to the predicted functional categories of genes induced by the AM fungi (Liu et al., 2007). On the basis of these data, it can be concluded that plants show two defense responses to AM fungi: an early and limited response in which defensive mechanisms are activated, and an enhanced response similar to the priming of defense reactions that is associated with induced resistance (Conrath et al., 2006), when the plant is subsequently attacked by a pathogen. In split roots of tomato plants infested with G. mosseae and challenged with the necrotrophic soil fungus Ph. parasitica, plant protection occurred due to a combination of local and systemic mechanisms (Pozo et al., 2002). Local induction of chitinase, chitosanase, and -1,3-glucanase, as well as superoxide dismutase activities were observed. The local induction of certain chitosanase isoforms appears to be a specific response to the AM symbiosis, since these isoforms were not induced by the pathogen Ph. parasitica itself (Pozo et al., 1998, 1999). Yet, the plant response was systemic, because the pathogen was controlled in distant leaves. However, a putative effect of the mycorrhizal fungus on plant nutrition was also documented. Yet, in the same system, a systemic induction of plant resistance associated with cell wall thickening with nonesterified pectins, PR-1a protein production, and formation of callose-rich encasement material around Ph. parasitica hyphae has also been reported (Cordier et al., 1998). The authors hypothesized a link between the jasmonic acid (JA) pathway and primed deposition of callose. JA-responsive genes and genes involved in JA biosynthesis were expressed in arbuscules-containing cells, and mycorrhizal roots had increased levels of JA. The elevated JA production could be related to the increased resistance of mycorrhizal plants to pathogens and insects (Pozo and Azco´n-Aguilar, 2007). The emerging picture is that colonization by AM fungi suppresses SAdependent plant responses, but enhances JA-regulated responses. This mode of action would explain the effectiveness described in mycorrhiza-induced resistance in plants: namely increased susceptibility to biotrophs and increased resistance to necrotrophs and certain hemibiotrophs.

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B. PLANTS AND OTHER ENDOPHYTIC FUNGI

1. Piriformospora indica Pir. indica, recently described to be related to the Sebacinales [ordo nov.] (form genus Rhizoctonia; Hymenomycetes, Basidiomycota) on the basis of an alignment of nuclear DNA sequences from the D1/D2 region of the large ribosomal subunit, is a cultivatable root endophyte that promotes plant growth (Waller et al., 2005). The fungus has growth-promoting effects on a broad range of plants and has the added trait (compared with AM fungi) that it can grow in axenic culture (Deshmukh et al., 2006; Varma et al., 1999). The fungus–plant interaction is detectable by the appearance of strong autofluorescence in the roots, followed by the colonization of root cells by fungal hyphae and the generation of chlamydospores. Even before root colonization is detectable, root growth has been promoted. The promoting effect is observed during the whole life of Arabidopsis thaliana plants (PeskanBergho¨fer et al., 2004). After contacting roots, the fungal hyphae develop appressoria and colonize root cells intercellularly. The fungus colonizes cortex cells intracellularly by forming coiled branches and chlamydospores. However, it never invades the stele or traverses upward into the shoot, and neither does it induce visible cell wall reinforcements, a typical host defense response (Varma et al., 1999; Waller et al., 2008). Deshmukh et al. (2006) showed that plant cell death is required for fungal proliferation. In this study, the root tip meristem showed no colonization and the elongation zone presented mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. Most of the hyphae were present in dead rhizodermal and cortical cells, which were filled with chlamydospores. The authors proposed that the fungus either kills cells or senses cells undergoing endogenous programmed cell death. In this regard, the same authors showed that transgenic barley plants overexpressing GFP-tagged HvBI-1 (a gene capable of inhibiting plant cell death) are significantly less colonized by this endophyte than the wild-type plants. Barley roots infested with Pir. indica show a kind of cell death that differs from that observed when microorganisms induce tissue necrosis, since defense marker genes encoding PR-1b, PR-5, and -1,3-glucanase are weakly and transiently upregulated only at early interaction stages, as opposed to the pronounced induction during a pathogeninduced hypersensitive response. The observation that Pir. indica suppresses the accumulation of H2O2 in colonized tissues may be crucial to achieve and maintain compatibility with their hosts plants (Scha¨fer et al., 2007). Roots of Arabidopsis plants treated with Pir. indica, compared with untreated controls, show differences in the expression of several proteins:

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-subunit of glucosidase II, -glucosidase PYK10, two glutathione-Stransferases, and a number of uncharacterized proteins (Peskan-Bergho¨fer et al., 2004). Barley plants colonized by Pir. indica and subjected to moderate salt stress behaved like nonstressed controls but under high salt stress showed the same biomass reduction as stressed controls. Root and shoot fresh weight were reduced only twofold by the soil pathogen Fusarium culmorum in Pir. indica-infested plants, while plants infected with F. culmorum alone showed a 12-fold decrease (Waller et al., 2005). Pir. indica induced systemic resistance when challenged with the barley powdery mildew pathogen Blumeria graminis f.sp. hordei on the leaves (Waller et al., 2005). In this study, the authors proposed that the reduction in disease severity was related to increased antioxidant metabolism. Pir. indica-infested plants showed enhanced ascorbate concentrations, but the concentrations of dehydroascorbate were reduced and the activity of dehydroascorbate reductase was increased in roots. Similarly, leaf glutathione and glutathione reductase content were increased (Waller et al., 2005). Pir. indica did not induce the expression of a large set of PR, or JA-inducible genes. Gene markers indicative of SA and JA accumulation were not expressed during the interaction between Pir. indica and its host. No difference in the expression of the JA-inducible protein gene JIP23 was observed during the Pir. indica-pathogen–plant interaction compared with the pathogen–plant interaction, whereas PR-5 , indicative of pathogen infections, was strongly expressed 24 h after powdery mildew inoculation in both the pathogen–plant interaction and the Pir. indica—pathogen–plant interaction (Waller et al., 2005). Based on these findings the authors concluded that the mechanism of action triggered by Pir. indica differs from either systemic acquired resistance (SAR) (since no PR gene expression was found) or induced systemic resistance (ISR) (since no priming for JA-responsive genes was found). However, later results changed this perception. PR-17b was upregulated to a higher extent in Pir. indica-infested plants after challenge with B. graminis than when non‐infested plants were challenged with the pathogen alone. This points to priming being involved in Pir. indica-mediated plant responses (Waller et al., 2008). Moreover, HvHSP70 (Hordeum vulgare heat-shock protein 70) was upregulated in the leaves of plants infested with strains of Pir. indica and Sebacina vermifera, indicating that systemic effects of fungal colonization did occur (Waller et al., 2008). Pir. indica did not alter a large set of genes in systemic leaves of barley and thus, its action resembles ISR in Arabidopsis triggered by plant growth-promoting rhizobacteria, which in the absence of a challenging pathogen, is accompanied by systemic up- or downregulation of low numbers of transcripts (Verhagen et al., 2004; Waller et al., 2008).

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2. Binucleate Rhizoctonia The genus Rhizoctonia comprises anamorphic and teleomorphic species that can lead to extensive crop damage, and, consequently, to economic loss. However, species of Thanatephorus and Ceratobasidium also form beneficial mycorrhizal associations in a large number of orchid species. Several authors have reported that uninucleate Rhizoctonia cause damping-off in Scots pine and Norway spruce, while binucleate Rhizoctonia are not responsible for these symptoms (Hietala, 1995; Lilja, 1994). Since the binucleate Rhizoctonia were not pathogenic but potentially endophytic, research by Gro¨nberg et al. (2006) showed stimulation of early (86 days postinoculation) Pinus sylvestris seedling growth in nitrogen-limited nursery soil. Induced adventitious rooting in young seedlings inoculated with binucleate Rhizoctonia was also observed (Kaparakis and Sen, 2006). The binucleate Rhizoctonia grew intercellularly on the outer cortical cells of long roots, while forming intracellular fungal monilioid cellular infection units. The binucleate Rhizoctonia isolates (teleomorph of Ceratobasidium spp.) are potential BCAs and have been demonstrated to control diseases caused by Rhizoctonia solani Ku¨hn, Pythium spp., Phytophthora spp., and Fusarium spp. in multiple host plants (Muslim et al., 2003; Olson and Benson, 2007). Binucleate Rhizoctonia isolates exhibit several characteristics that support the induction of plant resistance as a likely mechanism of biological control. Hwang and Benson (2002a,b) described that binucleate Rhizoctonia strain P9023 induced systemic resistance to Rhizoctonia stem rot in poinsettia plants. Stock plants were treated with strain P9023, and the cuttings were later inoculated with the pathogen; thus, the BCA and the pathogen remained spatially separated. Depending on the plant cultivar, 7, 10, or 14 days were required before resistance was expressed in the cuttings, while an increase in root colonization by the binucleate Rhizoctonia was observed in all cultivars between days 5 and 7. The level of protection obtained with P9023 was not related to the dose applied as increasing the number of propagules did not lead to an increase in host colonization or in the level of protection (Honeycutt and Benson, 2001). Using light microscopy as well as scanning and transmission electron microscopy, Poromarto et al. (1998) demonstrated that binucleate Rhizoctonia AG-K isolate 8-3 grew over hypocotyls, roots, and root hairs of soybean, colonizing only epidermal cells. It entered plant cells, forming penetration pegs without any distinct appressoria, and degraded the cuticle at the point of penetration. Cell necrosis was also observed in adjacent cells where there was no evidence of hyphae. Cell walls were not destroyed. Attempted penetration into cortical cells was observed and papillae formed on the inside of cortical cell walls. Preinoculation of soybean seedlings with Rhizoctonia AG-K 24 or 48 h before inoculation with Rh. solani (1 cm between inocula) impaired the

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growth of the pathogen on plant tissue. Thus, disease incidence and severity were reduced. In vitro and in vivo, there was no evidence of antagonism between hyphae (lysis, mycoparasitism, inhibition of growth), thereby suggesting that the biocontrol mechanism exerted by Rhizoctonia AG-K toward Rh. solani in soybean was induced resistance. Olson and Benson (2007) described induction of resistance in geranium to Botrytis blight when the plants were transplanted in potting mixture amended with formulations of binucleate Rhizoctonia (isolate P9023) or Trichoderma hamatum (strain 382) two weeks before leaf inoculation with Botrytis cinerea. Both BCAs reduced disease to a comparable extent as a foliar fungicide control consisting of weekly alternate applications of fenhexamid and chlorothalonil. Using binucleate Rhizoctonia (isolate 232-CG) to inoculate hypocotyls of bean plants, which were later challenged with the foliar pathogen Colletotrichum lindemuthianum or the root pathogen Rh. solani, Xue et al. (1998) induced disease suppression and biochemical changes in the host. This study found local and systemic increases in peroxidase activity, chitinase, and -1,3-glucanase in all tissues examined when plants had been treated with the isolate 232-CG and challenged with the pathogen, as compared to nontreated diseased and control plants. The interaction between a tomato mutant with reduced mycorrhizal colonization (rmc) and two pathogenic Rh. solani isolates (anastomosis groups AG4 and AG8) or a binucleate Rhizoctonia has been addressed by Gao et al. (2006). The rmc mutant excluded most AM fungi but did not affect interactions with the three Rhizoctonia isolates. The binucleate Rhizoctonia colonized root epidermal cells in a similar way as AM fungi, thereby causing minimal tissue damage. In contrast to Rh. solani, binucleate Rhizoctonia did not induce defense-related gene expression, or induced it to a far lesser extent. The necrotrophic feeding strategy of Rh. solani implies that it is unlikely to induce the same responses in roots as biotrophic root invaders, including AM fungi (Gao et al., 2006).

III. INTERACTIONS BETWEEN PLANTS AND FREE-LIVING OPPORTUNISTIC SYMBIOTIC FUNGI A. PLANTS AND TRICHODERMA SPP. BCAs

Trichoderma (Hypocreales, Ascomycota), a genus of soil fungi found worldwide and abundant on decaying wood, has various relevant applications in industry (enzymes) and is widely used to interfere with plant pathogens.

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Identification of Trichoderma species is complex because only a few morphological characters are available and these show little variation. In the last 35 years, the number of Trichoderma species identified has increased from 9 to 89 (Samuels, 2006), or 100 (Druzhinina et al., 2006) on the basis of molecular phylogenetic analyses. This advance is leading to the reclassification of several strains. It is noteworthy that the biological control properties of Trichoderma strains of a given species do not correlate with phylogenetic lineages. For example, the lineages in Trichoderma asperellum based on variations in the sequences of the translation elongation factor 1- (TEF-1) and RNA polymerase II second large subunit (RBP2) genes and the random amplified polymorphic DNA technique (RAPD) do not correlate with the biological control abilities of each strain (G. J. Samuels, personal communication). Trichoderma spp. are free-living fungi and early studies considered that the effectiveness of the diverse strains results from several modes of action, such as competition with other soil microorganisms for nutrients (Chet et al., 1987) or production of antibiotics and lytic enzymes to parasitize or inhibit other fungi (Harman et al., 1981; Howell, 2003; Lorito et al., 1996; Schirmbo¨ck et al., 1994; Zimand et al., 1996). Recent findings have shown that some strains of Trichoderma spp. colonize plant roots, enhance plant growth and nutrient uptake, and systemically elevate resistance to several types of pathogens in various plant species (De Meyer et al., 1998; Djonovic´ et al., 2006, 2007; Harman et al., 2004a; Korolev et al., 2008; Segarra et al., 2007; Shoresh et al., 2005). 1. Plant root colonization In the case of the BCA T. asperellum strain T-203 (formerly Trichoderma harzianum), the formation of hyphal coiling and appressorium-like structures that attach to a cucumber root surface have been described. This strain penetrates the root epidermis and progresses toward the cortical area mainly by intercellular growth, thereby suggesting active degradation of plant cell walls. Lytic zones are limited to the immediate area surrounding the penetrating hyphae. The host reacts at the sites of potential fungal penetration. Cucumber plants respond by strengthening epidermal and cortical cell walls and also by depositing a newly formed barrier, comprising wall appositions containing large amounts of callose and infiltrations of cellulose (Yedidia et al., 1999, 2000). In a model to study the changes in Trichoderma gene expression during root colonization, cucumber seedlings grown in aseptic hydroponic medium were inoculated with a spore suspension of T. asperellum strain T203. This strain secreted cell wall lytic enzymes (two arabinofuranosidases (ABF1 and ABF2) and an aspartyl protease). The proteases identified were induced in

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T203-treated seedlings in response to plant root attachment and interestingly were also expressed in the plant (Viterbo et al., 2004). In a more recent study, the same group analyzed the proteins secreted in the co‐culture of cucumber seedlings and T203 in hydroponic medium. In addition to the identification of cellulolytic enzymes and glucoamylase, a swollenin was distinguished among the differentially secreted proteins. Trichoderma transformants in which the TasSwo (T. asperellum Swollenin) gene was either overexpressed or silenced, showed involvement of this protein in root colonization by T203. Swollenin peptide (carbohydrate binding module family 1 domain: CBD with cellulose-binding function) stimulated local defense responses in cucumber roots and leaves and afforded local protection against infection by Bo. cinerea and Ps. syringae pv. lachrymans (Brotman et al., 2008). The authors proposed that swollenin disrupts the plant cell wall and thereby facilitates root colonization. Preliminary results have indicated specific upregulation of hydrophobins during tomato root colonization by T. harzianum strain 2413 (Benı´tez et al., 2004). T. asperellum strain T34 applied at a concentration of 107 colony-forming units (cfu)/ml colonized the cucumber root surface by growing in the intercellular spaces between the epidermal cell walls (Fig. 1). Transverse sections of the root hair zone showed that strain T34 was not inside the cortex and its presence inside the plant was limited to the outermost epidermis. Strain

E

C

F

F

C E

5 mm

2 mm

Fig. 1. Transmission electron micrographs of 7-day-old roots of cucumber seedlings 72 h after being transferred to a hydroponic system inoculated with Trichoderma asperellum strain T34 at 107 cfu/ml. Root cortical cells (C) and epidermal cells (E) of cucumber seedlings; fungal hyphae of T34 (F). Note the intercellular presence of T34 only in the epidermal cells and the reinforcement of cortical cells with undisrupted plant cell cytoplasm.

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T203, inoculated at 105 germinated spores/ml, colonized the epidermis of cucumber seedlings and outer cortex more profusely than strain T34, in spite of the similarity of the hydroponic systems used and the higher concentration of T34 (Segarra et al., 2007; Yedidia et al., 1999). It is generally accepted that Trichoderma strains must colonize plant roots before stimulating plant growth and protection against pathogens (Benı´tez et al., 2004; Olson and Benson, 2007). This implies that these strains have the capacity to recognize, adhere to, and penetrate the plant, and to withstand toxic metabolites produced by the plant in response to invasion. The resistance of Trichoderma strains to the products released by the plant (phytoalexins, flavonoids, terpenoids, phenolic derivates, etc.) may be an essential requirement for plant colonization and has been associated with the presence of ABC transporter systems in these strains. However, data supporting the capacity of mycelial extracts or purified molecules from Trichoderma strains to induce effective protection against plant pathogens (swollenin, SM1, peptaibols, etc.) suggest that chemical elicitation plays a key role in Trichoderma spp.-induced resistance. 2. Improvement of plant nutrition Trichoderma strains have frequently been reported to enhance host root growth and development as well as nutrient uptake. T. harzianum strain T22 was shown to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate). Acidification was not the main mechanism of solubilization and organic acids were not detected. T22 also produced diffusible metabolites with the capacity to reduce Fe(III) and Cu(II) (Altomare et al., 1999). Maize plants grown from seeds treated with T22 have been found to give maximum yield with as much as 40% less nitrogen-containing fertilizer than similar plants that were not treated with this BCA (Harman et al., 2004a). Shoots and roots of maize seedlings grown in the presence of T22 had increased root size and area, and the root hair area was also larger than that of control plants (Harman et al., 2004b). T. asperellum strain T203 promoted seedling emergence (day 8 after sowing), and increased root area, root length, dry weight, shoot length, and leaf area (on day 28). Similarly, increased P and Fe contents were found in an experiment in which cucumbers were grown in soil. In a gnotobiotic system strain T203 enhanced the growth and dry weight of roots and shoots (5 days postinoculation). Similarly, increases were found in Cu, P, Fe, Zn, Mn, and Na in the roots, and in the shoots increases were found for Zn, P, and Mn (Yedidia et al., 2001). T. asperellum strain T34 likewise enhanced fresh and dry weight of cucumber and pepper plants grown in peat/perlite (Fig. 2) and improved a number of parameters associated with plant growth (height, fresh/dry weight

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Fig. 2. Plant growth-promoting effect of Trichoderma asperellum, strain T34, on pepper plantlets germinated in a greenhouse, in peat covered with perlite substrate, not fertilized. Back tray: T34-treated seeds; front tray: nontreated. Photograph obtained with permission of Biocontrol Technologies, S.L.

ratio, chlorophyll content) and plant nutrient uptake (Fe, S, Cu, Si, B) of tomato plants grown in peat (Idrissi, Segarra, and Trillas, to be published). There are very few reports on Trichoderma strains producing growth factors that have been detected and identified in the laboratory, such as indole acetic acid, cytokinin-like molecules, gibberellins, or ET (Benı´tez et al., 2004). 3. Induction of plant resistance Bigirimana et al. (1997) showed that treating soil with T. harzianum strain T39 conferred to the leaves of bean plants resistance to diseases caused by the fungal pathogens Bo. cinerea and C. lindemuthianum, even though T39 was present only on the roots and not on the foliage. Since then, several Trichoderma strains have been claimed to induce local and/or systemic resistance to plant pathogens in diverse species, such as bean, cotton, cucumber, maize, and Arabidopsis (Bigirimana et al., 1997; Harman et al., 2004b; Howell et al., 2000; Segarra et al., 2009; Shoresh et al., 2005; Yedidia et al., 2003). Cucumber plants treated with T. asperellum strain T203 are protected against Ps. syringae pv. lachrymans by a mechanism that reduces bacterial population densities in the leaves. This protection is associated with the accumulation of the mRNA of two defense genes: the phenylpropanoid pathway gene encoding PAL and the lipoxygenase pathway gene encoding

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hydroxyperoxide lyase (HPL). The presence of T203 in cucumber roots activated transiently PAL and HPL expression in leaves and roots (up to 24–48 h) without pathogen inoculation. In addition, plants treated with T203 showed a primed HPL expression in leaves 48 h after interaction with the pathogen, compared to plants treated with either T203 or with the pathogen alone. However, T203-treated plants showed no priming for PAL after pathogen inoculation (Yedidia et al., 2003). A different strain, T. harzianum strain T22, increased the activities of -1,3-glucanase, exochitinase, and endochitinase in roots and shoots. Upon infection with Pythium ultimum some of these enzymes showed highest activity in the combination of T22 and the pathogen (Harman et al., 2004b). These results suggest that strain T22, like strain T203, induces a direct effect on plant defense metabolism in the absence of the pathogen and, at the same time, both strains also prime the plant to respond more strongly after pathogen attack. Inoculation of cucumber roots with T203 at doses of 105 spores/ml induced an increase in peroxidase and chitinase activities in roots and leaves within 48 and 72 h, respectively (Yedidia et al., 1999). In a following study, activation of chitinase, -1,3-glucanase, cellulase, and peroxidase activity up to 72 h after the interaction in the roots was demonstrated. Plants prevent the penetration of T203 by depositing dense materials to restrict fungal dispersion and proliferation, and by synthesizing phenolic compounds (Yedidia et al., 2000). After 48 h, a significant increase (35%) in the concentration of total phenolics was found for both free and conjugated phenols in the leaves (Yedidia et al., 2003). Activation of plant chitinase isozymes (proteins of 33 kDa) was observed only in intercellular spaces of epidermal and cortical cells of the roots, although the hyphae of T203 were found over the entire root surface (Yedidia et al., 2000). In another study using the T. asperellum T203–cucumber–Ps. syringae pv. lachrymans system, treatment of plants with JA and ET synthesis inhibitors reduced or eliminated the resistance induced by the BCA. This study showed involvement of both plant hormones in the induction of systemic resistance (Shoresh et al., 2005). However, the authors did not find differences in the concentrations of ET between control plants and plants treated with T. asperellum strain 203. These observations suggest that the involvement of ET in the induction of resistance is more related to the sensitivity of the plant tissue than to an increase in hormone levels. Moreover, no differences were found in the concentrations of SA between control and T203treated plants (Shoresh et al., 2005). The absence of an increase in SA was also confirmed upon treatment with T. asperellum strain T34 at a concentration of 105 cfu/ml. Furthermore, in this system no increase was found in JA (Segarra et al., 2007). T203 enhanced the expression of ETR1 and CTR1 in

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the roots at 24 h postinoculation. ETR1 and CTR1 are gene products that negatively regulate the ET response in the absence of ET. Binding of ET to its receptor downregulates the activity of the ETR1/CTR1 complex. The enhanced expression of these negative ET regulators in T. asperellum-treated plants suggests that the ET response in roots is inhibited (Shoresh et al., 2005). Such a phenomenon is well documented in symbiotic systems where, for example, the AM fungus–plant interaction results in local silencing of the plant defense response in order to allow a symbiotic interaction to develop (Penmetsa et al., 2008). In the leaves, ETR1 expression peaked at 24 h post‐ inoculation and decreased below control levels by 48 h. CTR1 expression in leaves showed a similar pattern. The downregulation of these two genes indicates that the negative regulation of the ET response is reduced in leaves and ET sensitivity is enhanced. Genes coding for chitinase, -1,3-glucanase and peroxidase were significantly upregulated in the T. asperellum strain 203–cucumber–Ps. syringae pv. lachrymans interaction compared with cucumber–Ps. syringae alone. On the basis of findings by Yedidia et al. (1999, 2000, 2003) and Shoresh et al. (2005), it appears that during early interaction (48 h post T203 inoculation) expression of defense genes is elevated and 48–72 h later decreases to control levels. After 48 h of pathogen infection, the expression of defense genes is higher in T203-treated plants than in plants infected with the pathogen alone or in plants treated with T203 alone. These data show that a priming effect is evident 48 h after pathogen infection. In the same plant species and under the same growth conditions, T. asperellum strain T34 did not induce peroxidase activity in the leaves when it was applied at 105 cfu/ml to the roots (Segarra et al., 2007). However, in the same system, a higher concentration of T34 (106 and 107 cfu/ml) applied to the roots caused an increase in peroxidase activity in the leaves after 12 and 6 h, respectively. At the same T34 doses, an increase in SA was detected 24 and 6 h after treatment, respectively. At the highest T34 concentration, a peak of JA was recorded 3 h after T34 inoculation (Segarra et al., 2007). It is notable that the concentration of T34 affected the way the plant perceived the microorganism. It seems that at a higher dose the plant responds as if it were attacked by an avirulent pathogen, while at lower doses its response reflects the interaction with a beneficial microorganism. Strain T34 at 107 cfu/ml induced systemic resistance against Ps. syringae pv. lachrymans in cucumber plants. Under these conditions, the use of twodimensional electrophoretic protein profiling and mass spectrometry analysis allowed the identification of 28 proteins whose expression was affected in cotyledons after root colonization by T34. Systemically induced proteins involved in ROS scavenging, stress response, isoprenoid and ET biosynthesis, and in photosynthesis, photorespiration and carbohydrate metabolism

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were differentially regulated by T34. These data point to a switch from assimilatory metabolism to defensive metabolism (Segarra et al., 2007). T. harzianum strain T22 systemically induced large changes in the proteome of maize seedling shoots (7 days old), even though it was present only in the roots upon application at a concentration of 7 105 cfu/5 g of kernels. Most of the upregulated proteins were involved in carbohydrate metabolism; in addition, numerous proteins induced in response to T22 participated in stress and defense responses, genetic information processing, and amino acid metabolism. The upregulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by T22 inoculation (Shoresh and Harman, 2008). Interestingly, in the proteome studies for T22 (on maize; monocot) and T34 (on cucumber; dicot), both peroxidase and heat-shock protein 70 were upregulated; in the same way PAL was upregulated upon application of T22 and also upon treatment with T203 (Segarra et al., 2007; Shoresh and Harman, 2008; Shoresh et al., 2005). Trichoderma atroviride strain P1 systemically induced changes in the proteome of bean leaves when the fungus was applied to the rhizosphere but separated from the roots by a cellulose membrane that allows the diffusion of macro- and micromolecules but not fungal mycelia. An NBS-LRR type resistance protein and ribonuclease-like PR-10a were upregulated. Strain P1 induced also local changes in the proteome (thaumatin-like protein, PR-5b) in bean leaves when the fungus was applied to the leaves, separated by a cellulose membrane (Marra et al., 2006). In the same study, a comparison between two two-way interactions (Trichoderma P1—bean vs. Botrytis/Rhizoctonia—bean) indicated a distinct proteomic response of the plant to the antagonist and the pathogens. The presence of the beneficial microorganism altered the expression pattern of plant genes responding to pathogen attack. Some spots were more upregulated locally in the combination of Trichoderma P1—pathogen—plant than in the P1—plant or pathogen–plant interactions (resistance candidate RPP8-like protein and SGT1-like protein) (Marra et al., 2006). These data point to the local priming of defense responses by Trichoderma. The BCA T. hamatum strain 382 established at 2 105 cfu/g substrate (peat/perlite), provided protection against bacterial spot of tomato caused by Xanthomonas euvesicatoria, even though this agent colonized only the roots and the pathogen was present in the leaves. High-density oligonucleotide microarrays were used to determine the effect of T382 on the expression pattern of 15925 genes in the leaves just before they were inoculated with the pathogen. The genes induced by T382 have functions related to biotic and abiotic stress, as well as RNA, DNA, and protein metabolism. Four extensin and extensin-like proteins were induced. These results are in agreement with

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the observation that BCAs lead to alterations in plant cell walls before root penetration by pathogens (Yedidia et al., 1999). PR-5 was the only main marker of SA-dependent defenses to be upregulated. The expression of marker genes for the JA/ET defense pathway, such as LOX1, ETR1, and CTR1, was not significantly affected (Alfano et al., 2007). These data are consistent with findings by Segarra et al. (2006, 2007), who reported that the concentrations of SA and JA in cucumber roots and cotyledons were not altered by T34. These findings contrast with those mentioned earlier that T203 induces peaks of ETR1 and CTR1 expression in roots and leaves. The differences due to distinct Trichoderma spp. strains could be explained by temporal variations in the expression of marker genes or the concentration of the BCAs. T382 downregulated the expression of an MYB transcription factor in the leaves. Notably, Arabidopsis myb72 mutants are incapable of developing ISR against diverse foliar pathogens when treated with either the plant growth-promoting, ISR-inducing rhizobacterial strain Pseudomonas fluorescens WCS417 or T. asperellum strain T34 (Segarra et al., 2009; Van der Ent et al., 2008). By testing mutants of Trichoderma virens on cucumber plants growing in axenic hydroponic systems, it was demonstrated that the mitogen-activated protein kinase (MAPK) TmkA of T. virens is required to induce full systemic resistance in the plant against Ps. syringae pv. lachrymans, although this protein is not required for effective root colonization (Viterbo et al., 2005). Similarly, a Trichoderma-induced MAPK (TIPK) was found to be required for systemic resistance to be induced by T. asperellum T203 against the same pathogen. TIPK overexpressors were more resistant to pathogenic bacterial attack than control plants even in the absence of T203. Furthermore, mutant plants with no TIPK expression revealed increased sensitivity to pathogen attack and were not protected by T203 (Shoresh et al., 2006). In the same system but using a PCR differential mRNA display analysis conducted on Trichoderma interacting with plant roots, a hydrophobin-like clone (TasHyd1), was found to be expressed. Overexpresser mutants were not affected in mycoparasitic activity against Rh. solani and retained root colonization capacity. In contrast, deletion mutants showed no significant reduction in mycoparasitic activity, but wettability was altered and root attachment and colonization were impaired (Viterbo and Chet, 2006). In a hydroponic system similar to that described by Yedidia et al. (1999), T. virens Gv29-8 expressed and secreted a protein called SM1 (Small protein 1), which shows significantly higher expression in the presence of cotton plants. SM1 lacks toxic activity against plants and microbes but triggers the production of ROS and induces the expression of the defense-related genes -1,3-glucanase, chitinase, peroxidase, and lipoxygenase both locally and systemically (Djonovic´ et al., 2006). SM1 is a hydrophobin-like elicitor that

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when applied on detached leaves induces protection against the fungal pathogen Colletotrichum spp. Mutants of T. virens lacking SM1 did not protect maize plants; conversely, SM1 overexpressors elicited enhanced protection compared to wild-type plants. SM1 expression by T. virens was required to systemically activate the genes encoding PAL, OPR7 (12-Oxophytodienoic acid reductase 7), AOS (Allene oxide cyclase) and HPL (Djonovic´ et al., 2007). T. virens strain Gv29-8 also produces peptaibols of three lengths (11, 14, and 18 residues long, with several isoforms each). Disruption of the NRPS (Non‐ribosomal peptide synthetase) gene TEX 1 resulted in the loss of production of all forms of 18-residue peptaibols. TEX 1 is expressed at all stages of Trichoderma development (germination and sporulation of conidia and in nonsporulating mycelia). Expression analysis by reverse transcriptase PCR showed that the abundance of the TEX 1 transcript in the wild-type strain Gv29-8 is greater during co‐cultivation with cucumber seedling roots than when grown in the absence of the host plant. T. virens strains disrupted in TEX 1 have a significantly reduced capacity to induce resistance against the leaf pathogen Ps. syringae pv. lachrymans and to induce the production of phenolic compounds, compared with the wild type. A synthetic 18-residue peptaibol identical to the one from Gv29-8 induced systemic protection against the leaf pathogenic bacterium when applied to cucumber seedlings, and upregulated HPL, PAL, and peroxidase gene expression (Viterbo et al., 2007). In tobacco seedlings, an extract from the mycelium of nonpathogenic Trichoderma longibrachiatum induced resistance to the pathogen Ph. parasitica. This induction did not involve a hypersensitive response, but led to expression of PR-1b and PR-5c (osmotin) at higher levels than those induced by mycelial extracts of the pathogenic fungus. ET synthesis inhibitors blocked the induction of resistance by T. longibrachiatum (Chang et al., 1997). Interestingly, strains of T. virens differing in effectiveness as BCAs and in pathogenicity to seedling disease in cotton show few differences in phytotoxin production or lytic enzyme activity. However, effective BCAs induce higher levels of the phytoalexins hemigossypol and desoxyhemigossypol in cotyledons than pathogenic strains (Howell and Puckhaber, 2005). To better understand the role of signaling molecules in T. harzianum strain T39-mediated ISR, Arabidopsis mutants with altered sensitivity to, or production of phytohormones were studied. Ecotype Col-0 was relatively resistant to Bo. cinerea and showed an inducible phenotype (which means that inoculation of the roots with T39 rendered the systemic tissue more resistant against Bo. cinerea). Conversely, ecotypes with low levels of basal resistance against Bo. cinerea were not induced by T39. The inducibility of Col-0 mutants varied depending on the type of mutant. SA- and auxin-impaired mutants were

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inducible, while JA-, ET-, abscisic acid-, and gibberellin-impaired mutants were not. It is noteworthy that defense-regulatory npr1 mutants had an inducible phenotype, suggesting that NPR1 is not required for T39-elicited ISR. T39 enhanced the growth of Arabidopsis plants regardless of genotype or ISR inducibility (Korolev et al., 2008). Colonization of Arabidopsis roots by T. asperellum strain T34 results in the activation of a systemic defense response, which is effective in reducing diseases produced by the necrotrophic fungal pathogen Plectosphaerella cucumerina, the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica), and the bacterial pathogen Ps. syringae pv. tomato. T34 primed the leaf tissue for enhanced expression of the JA-responsive gene LOX2 and increased the formation of callose-containing papillae upon attack by Hy. arabidopsidis. T34-triggered ISR was fully expressed in the SA-impaired mutant sid2, but blocked in the npr1 mutant. The dependence of T34-elicited ISR on NPR1 differs from the results described by Korolev et al. (2008) for ISR triggered by strain T39. However, the results from T34 are similar to those obtained upon elicitation of ISR by Ps. fluorescens strain WCS417r. The root-specific transcription factor MYB72, which is essential in early signaling steps of Ps. fluorescens strains WCS417r (Van der Ent et al., 2008), is also required for T34-elicited ISR. Together, these results indicate that the defense pathways triggered by T34 and WCS417r are similar and based on priming, and that MYB72 functions as an early node of convergence in the signaling pathways induced by different types of beneficial microorganisms (Segarra et al., 2009). B. PLANTS AND NONPATHOGENIC F. OXYSPORUM

Nonpathogenic F. oxysporum strain Fo47 colonized the root surface of tomato plants by direct competition with the plant-pathogenic strain F. oxysporum f.sp. lycopersici Fol8 not only for the same sites at the root surface, but also inside the plant tissues. The use of confocal laser scanning microscopy allowed researchers to observe that both pathogenic and protective strains of F. oxysporum grew in close proximity in the root, with no exclusion of the pathogenic strain by the presence of the nonpathogenic strain when Fo47 was introduced at a higher concentration than Fol8. These data support the hypothesis that competition occurs for nutrients rather than for infection sites and suggest that the production of antifungal metabolites contributes to their interaction (Nahalkova et al., 2008; Olivain et al., 2006). In response to fungal invasion, the defense response of the plant involves cell wall thickening and intracellular plugging in order to prevent the nonpathogen from reaching the vessels (Benhamou and Garand, 2001;

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Mandeel and Baker, 1991; Olivain and Alabouvette, 1999). In inoculated pea roots, Fo47 induced host metabolic changes which led to the development of structural barriers at sites where the fungus attempted to penetrate. Fo47 followed a well-defined scheme of events, including proliferation along the elongating root and penetration of the epidermis. Establishment in the root tissues resulted in a massive elaboration of hemispherical wall appositions containing callose, and in the deposition of electron-opaque material (phenolic-like compounds) frequently encircling the hyphae of the pathogen F. oxysporum f.sp. pisi, and accumulating in the noninfected xylem vessels (Benhamou and Garand, 2001). Given that Fo47 becomes established in the cortical tissues, it could be considered a fungal endophyte that triggers the plant defense system. Biles and Martyn (1989) were the first to attribute the control of Fusarium wilt of watermelon achieved by several strains of nonpathogenic F. oxysporum to ISR. According to Fravel et al. (2003), when competition is the main mode of action of a nonpathogenic Fusarium strain, the population of the biocontrol fungus must be at least as large as that of the pathogen in order to achieve control; in contrast, when ISR is the main mode of action, control can often be achieved when the pathogen population is much greater than that of the biocontrol fungus. Accordingly, these authors concluded that the mode of action of Fo47 is mainly competition, since Fo47 was effective at reducing Fusarium wilt in tomato only at doses 10–100 times higher than the pathogen F. oxysporum f.sp. lycopersici. However, the mode of action of strain CS-20 was ISR, since significantly reduced wilt was obtained when the pathogen was present at levels up to 1000 times greater than CS-20 (Larkin and Fravel, 1998). To study the involvement of induced resistance, several experiments were designed in which Fo47 and F. oxysporum f.sp. lycopersici were separated, either physically or in time. In a spatial separation of the pathogen and the biocontrol strain, Fo47 protected tomato against Fusarium wilt; in addition, inoculation with Fo47 increased chitinase activity in stems and leaves, as well as -1,3-glucanase and -1,4 glucosidase activities in stems. Thus, it was concluded that Fo47 induced resistance in tomato (Fuchs et al., 1997). When five more strains of nonpathogenic F. oxysporum (5a1, N1.5, 11V, T, and Fop2) were tested for efficacy in controlling tomato wilt disease, the most efficient strain (5a1) induced increases in laminarinase, chitinase, N-acetyl-glucosaminidase, and -1,4-glucosidase activities compared with non‐inoculated plants growing in conducive soils (Tamietti et al., 1993). By a split-root system, two nonpathogenic F. oxysporum isolates (Fo47 and Fo52) were shown to induce systemic resistance against F. oxysporum f.sp. ciceri in chick-pea plants. In this system, plants colonized by the

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nonpathogenic strains showed significant improvement in shoot length and fresh weight, as well as decreased wilt incidence compared with control plants (Kaur and Singh, 2007). In studies on the suppression of Fusarium wilt in tomato by a combination of the fungal strain Fo47 and the bacterium Ps. fluorescens WCS417r in a system with and without spatial separation between pathogen and BCAs, the suppression of Fusarium wilt by WCS417r was ascribed to ISR, although induction of PR-1 and chitinases was not detected. In contrast, the suppression achieved by Fo47 was ascribed mainly to microbial antagonism but also, to a lesser extent, to ISR associated with accumulation of PR-1 and chitinases (Duijff et al., 1998). Like WCS417r, T. asperellum strain T34 induces systemic resistance without direct transcriptional activation of SA-regulated genes, such as PR-1 (Segarra et al., 2009). C. PLANTS AND NONPATHOGENIC PENICILLIUM SPP., PHOMA SPP., AND PYTHIUM OLIGANDRUM

De Cal et al. (1995) reported that Penicillium oxalicum reduces Fusariuminduced tomato wilt. Disease control did not correlate with a reduction in the population density of F. oxysporum since the pathogen remained in the rhizosphere but did not induce severe symptoms. By applying the pathogen and the BCA at separate sites on tomato plants or in the soil, these researchers demonstrated that plant-mediated phenomena are responsible for the decrease in disease severity (De Cal et al., 1997a; Sabuquillo et al., 2006). A possible involvement of PR-proteins (chitinases, -1,3-glucanases, and PR-1) was studied, but no evidence was found (Garcı´a-Lepe et al., 1999). Other alterations in the plants were examined in hydroponic cultures. Pe. oxalicum-treated plants took up more nutrient solution than nontreated plants when both were infected with Fusarium. This observation suggests that Pe. oxalicum partially prevents the blocking or collapse of xylem vessels in infected plants (De Cal et al., 1997b). Furthermore, Pe. oxalicum treatment protected the plants infected with Fusarium from losing their cambium, thus favoring the formation of additional secondary xylem while reducing the number of bundles (De Cal et al., 2000). Hossain et al. (2007, 2008a) reported that Penicillium simplicissimum isolate GP17-2 or its culture filtrate (CF) and Penicillium spp. isolate GP16-2 or its CF induced systemic resistance against infection by Ps. syringae pv. tomato in Arabidopsis plants, as evidenced by reduced disease development and pathogen growth. Root colonization by isolate GP16-2 promoted plant growth and was not involved in the direct activation of known defense-related genes, but potentiated the activation of JA/ET-inducible ChitB after infection by

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Ps. syringae pv. tomato. However, the CF of this isolate stimulated systemic expression both before and after pathogen inoculation by directly activating marker genes for both the SA- and the JA/ET-signaling pathways; in addition, priming was evident by enhanced activation of the JA/ET-inducible ChitB and Hel genes upon pathogen challenge (Hossain et al., 2008a). Isolate GP17-2 of Pe. simplicissimum also promoted plant growth. Both isolate GP17-2 and its CF had direct effects on the expression of SA- and JA/ET-regulated defense responses. Subsequent challenge of GP17-2 with Ps. syringae pv. tomato was accompanied by activation of the SA-inducible PR-2 and PR-5 genes and the JA-inducible VSP gene (encoding a vegetative storage protein). Treatment with CF of GP17-2, followed by infection with the pathogen resulted in elevated expression of PR-1, PR-2, PR-5, PDF1.2 (encoding plant defensin), and Hel genes, with an initial elevation of SA responses followed by late induction of JA responses. The activation of SA-, JA-, and ET-inducible genes led the authors to suggest involvement of multiple pathways in the induction of systemic resistance in Arabidopsis by GP17-2 (Hossain et al., 2007). Similar results were obtained by the same authors working with the CF of another fungus, Phoma spp. isolate GS8-1 in the same pathosystem, indicating a partial involvement of the SA- (PR-1, PR-2, and PR-5 genes), JA /ET(ChitB), and ET-inducible (Hel) marker genes (Hossain et al., 2008b). Induction of systemic resistance was also achieved by substrate inoculation with Phoma spp. isolates GS8-2 and GS8-3 in a model system consisting of cucumber and the anthracnose pathogen Colletotrichum orbiculare (Chandanie et al., 2006). In this study, plants were also inoculated with G. mosseae, but a significant reduction in disease development was not observed. However, combined inoculation with Phoma GS8-3 or GS8-2 with G. mosseae induced higher levels of protection, even though root colonization of both Phoma spp. isolates was suppressed by the presence of the AM fungus. In previous work, Meera et al. (1994) had tested sixteen fungal isolates from the rhizosphere for their effectiveness as inducers of systemic resistance in the same cucumber– anthracnose model. Four fungal isolates (Trichoderma GT3-2, Fusarium GF19-2, Penicillium GP17-2, and the sterile fungus GU23-3) or their CFs applied to the roots promoted plant growth and induced systemic resistance. The CFs of these isolates induced lignification in the epidermal tissues at fungal penetration points and elicited superoxide generation (Koike et al., 2001), indicative of cell wall reinforcement. The BCA Py. oligandrum, a soil-borne oomycete, colonizes the rhizosphere of many crop species and reduces diseases produced by a number of soil-borne pathogens. Cytological studies of samples from Py. oligandrum-inoculated tomato roots revealed that the fungus colonizes root tissues without inducing

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extensive cell damage. Py. oligandrum ingress in root tissues is associated with host metabolic changes, which culminate in the development of structural barriers at the sites of potential fungal penetration. In control roots, the pathogen multiplies abundantly throughout many of the tissues, whereas in roots colonized by Py. oligandrum, pathogen growth is restricted to the outermost root tissues, which is accompanied by deposition of newly formed barriers beyond the infection sites. Wall appositions contain a large amount of callose and phenolic compounds (Benhamou et al., 1997; Rey et al., 1998). Disease suppression by Py. oligandrum can result from mycoparasitism, competition for nutrients, and the production of antimicrobial substances. In addition to these direct antagonistic activities emerging evidence suggests that Py. oligandrum also produces elicitors that activate plant defense mechanisms (Picard et al., 2000; Takenaka et al., 2003). Oligandrin, an elicitin-like protein with a molecular mass of 10 kDa, is secreted by Py. oligandrum and triggers the cytological and biochemical modification of tomato cells, thereby inducing resistance to Ph. parasitica (Picard et al., 2000). This elicitor shows similarities with several elicitins from other Phytophthora and Pythium spp. Although oligandrin does not induce hypersensitive necrosis, a significant decrease in the incidence of disease is observed in plants treated with oligandrin against Ph. parasitica. These plants react by restricting pathogen growth to the outermost tissues. This effect is achieved by plugging of the intercellular spaces and by the formation of wall appositions (Picard et al., 2000). Another elicitin from the cell-wall protein fraction (CWPF) of Py. oligandrum contains two major proteins, POD-1 and POD-2. This elicitin induces resistance to Rh. solani in sugarbeet plants by activating PAL and chitinase activities as well as synthesis of phenolic compounds, without inducing hypersensitive-like cell death. Treatment of tomato roots with CWPF or a mycelial homogenate of Py. oligandrum induces an increase in ET and expression of genes encoding PR-proteins and ET-responsive proteins (Hase et al., 2006). Py. oligandrum and also its mycelial homogenate can suppress bacterial wilt disease in tomato caused by Ralstonia solanacearum. Roots treated with Py. oligandrum homogenate did not accumulate SA, nor did they show an upregulated expression of PR-1 compared with control plants. NahG transgenic plants (unable to accumulate SA) were induced by the Py. oligandrum homogenate. Furthermore, the expression of the JA-responsive gene PR-6 was induced by this homogenate. Mutant plants defective in JA signaling were not induced by the Py. oligandrum homogenate, indicating that the JA-signaling pathway is required for Py. oligandrum-induced resistance against Ra. solanacearum (Hase et al., 2008).

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IV. OVERVIEW OF PLANT DEFENSE MECHANISMS INDUCED BY NONPATHOGENIC FUNGI The nonpathogenic fungi/BCAs described in this review show several common features (Table I). They colonize the root inter- and/or intracellularly at the epidermis/cortical cells, thereby triggering plant defense mechanisms that prevent fungi from entering the endodermis and the vessels. This early response to fungal colonization is usually transitory, weak, and complex, and varies depending on how the plant perceives the fungus. In this regard, AM fungi behave as biotrophic microorganisms and thus activate SA-dependent responses. Plants may perceive other fungi as necrotrophs and consequently activate JA-dependent responses. The concentration of the BCA to which the plant is exposed is highly relevant in tuning the plant response. A high concentration of nonpathogenic fungi may be perceived by the plant as an avirulent pathogen, while a low concentration of the same fungus can be interpreted by the same plant as a beneficial fungus. In addition, given the transitory nature of this early response, the time course of the studies performed affects the plant responses observed. It is generally accepted that colonization of the host by the BCA is required to induce systemic resistance before challenge by a pathogen. However, mycelial extracts or purified molecules from certain BCAs and fungal CFs have the capacity to induce effective protection against plant pathogens. These data suggest that the presence of the fungi inside the plant is not always required and chemical elicitation may play a relevant role in the resistance induced by nonpathogenic fungi. From the literature reviewed, three plant response patterns can be distinguished. The first is the standard ISR pattern, where no direct effects of BCA colonization are observed and later challenge with the pathogen is counteracted by enhanced defenses (priming). The second is a hybrid pattern where transitory direct effects of BCA colonization are observed and later challenge with the pathogen is counteracted mainly by priming of defense mechanisms that are not necessarily the same as those previously activated by the BCA. Finally, the third is the standard SAR pattern, where direct effects of the BCA are likely to be the same mechanism that protects the plant from the later pathogen attack. It is unlikely that this latter pattern is prevalent among nonpathogenic fungi with plant-protective properties, as it is metabolically costly and the BCAs usually have beneficial effects on plant growth and yield. However, as studies of plant responses induced by biological control fungi are often incomplete (for example no studies on gene expression before and after pathogen inoculation) and little information is available on plant growth, the occurrence of SAR cannot be excluded. Despite phenotypic

TABLE I Summary of Interactions Between Nonpathogenic Fungi and Plants AM fungi Plant nutrition

Exchange of plant photoassimilates

Piriformospora

Binucleate

indica

Rhizoctonia

Facilitation of plant

Fusarium Trichoderma spp.

oxysporum

Facilitation of plant

nutrition

Pythium Penicillium spp.

Phoma spp.

oligandrum

Facilitation of plant

nutrition

nutrition

Plant growth

Plant growth

for facilitation of mineral uptake

promotion Plant root colonization

Intra- and

Intra- and

Intra- and

Some strains:

promotion Establishment in

Plant growth promotion Intra- and

intercellular

intercellular

intercellular

intercellular

epidermis and

intercellular

without

without cell wall

monilioid cells

(epidermal and

cortical cells.

colonization

plasmalemma

reinforcements

(inflated

cortical cells)

Inter/

without

intracellular

extensive cell

growth. Callose

damage

penetration

subapical

Highly branched

hyphal cells)

arbuscules

Callose, cellulose, and phenolic compound

depositions and

depositions at the

structural

penetration site

barriers at the penetration site

Interface

Coiled, branched

Cell necrosis in

compartment to

hyphae and

separate both

chlamydospores

colonization of

partners

in dead

epidermal cells

rhizodermal and cortical cells

adjacent cells

Other strains: intercellular

Induction of

Local and weak

Local and systemic

SA-independent

plant

transient

and transiently

induction of

responses

resistance

activation and

upregulated

resistance

Defense genes weak

SAR

Systemic induction

SA-independent

of resistance

responses

later suppression of SA-dependent responses No SAR (no PR expression)

Importance of the

Some strains:

concentration of the

development of new

BCA

secondary xylem

JA-dependent responses

vessels Activation of local

No JA-dependent

JA/ET-dependent

SA- and JA/ET-

and systemic

responses

responses

dependent

JA-dependent

responses

responses Direct response and priming

Direct response and priming

Direct response and priming

Direct response

Some strains: priming Other strains: direct response and priming

Direct response

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M. I. TRILLAS AND G. SEGARRA

similarities between plant colonization and the induction of resistance by nonpathogenic fungi, considerable differences even between strains of the same species of nonpathogenic fungi can be found in the literature. Nonspecificity of the BCAs with regard to the pathogens controlled is a common feature among BCAs that induce systemic resistance. This feature is well documented in the strains of the genus Trichoderma that control several soil-borne, as well as foliar plant pathogens, both necrotrophic and biotrophic. However, the percentage of disease reduction that can be explained by induced resistance is not sufficiently defined. In the case of aerial plant pathogens that are spatially separated from the BCA, disease reduction can be attributed mainly to ISR, but also to improved plant nutrition. In the case of soil-borne plant pathogens, the complexity is greater, as there is usually no spatial separation between the BCA and pathogen. By split-root and other techniques, direct antagonistic effects of the BCA toward the pathogen can be prevented. However, the implication of induced resistance in the reduction of disease observed remains difficult to evaluate.

REFERENCES Alfano, G., Lewis Ivey, M. L., Cakir, C., Bos, J. I. B., Miller, S. A., Madden, L. V., Kamoun, S. and Hoitink, H. A. J. (2007). Systemic modulation of gene expression in tomato by Trichoderma hamatum 382. Phytopathology 97, 429–437. Altomare, C., Norvell, W. A., Bjorkman, T. and Harman, G. E. (1999). Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Applied and Environmental Microbiology 65, 2926–2933. Azco´n, R. and Ocampo, J. A. (1981). Factors affecting the vesicular arbuscular infection and mycorrhizal dependency of thirteen wheat cultivars inoculated with Glomus mosseae. New Phytologist 87, 677–685. Baath, E. and Hayman, D. S. (1983). Plant growth responses to vesicular arbuscular mycorrhiza. New Phytologist 95, 419–426. Baath, E. and Hayman, D. S. (1984). No effect of VA mycorrhiza on red core disease of strawberry. Transactions of the British Mycological Society 82, 534–536. Benhamou, N. and Garand, C. (2001). Cytological analysis of defense-related mechanisms induced in pea root tissues in response to colonization by non-pathogenic Fusarium oxysporum Fo47. Phytopathology 91, 730–740. Benhamou, N., Rey, P., Che´rif, M., Hockenhull, J. and Tirilli, Y. (1997). Treatment with the mycoparasite Pythium oligandrum triggers induction of defencerelated reactions in tomato root when challenged with Fusarium oxysporum f.sp. radicis–lycopersici. Phytopathology 87, 108–122. Benı´tez, T., Rinco´n, A. M., Limo´n, M. C. and Codo´n, A. C. (2004). Biocontrol mechanisms of Trichoderma strains. International Microbiology 7, 249–260. Bigirimana, J., De Meyer, G., Poppe, J., Elad, Y. and Ho¨fte, M. (1997). Induction of systemic resistance on bean (Phaseolus vulgaris) by Trichoderma harzianum.

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Van Rhijn, P., Fang, Y., Galili, S., Shaul, O., Atzmon, N., Wininger, S., Eshed, Y., Lum, M., Li, Y., To, V., Fujishige, N., Kapulnik, Y. et al. (1997). Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved. Proceedings of the National Academy of Sciences of the United States of America 94, 5467–5472. Varma, A., Verma, S., Sudha Sahay, N., Bu¨tehorn, B. and Franken, P. (1999). Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Applied and Environmental Microbiology 65, 2741–2744. Verhagen, B. W. M., Glazebrook, J., Zhu, T., Chang, H.-S., Van Loon, L. C. and Pieterse, C. M. J. (2004). The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Molecular Plant-Microbe Interactions 17, 895–908. Vierheilig, H., Alt, M., Neuhaus, J. M., Boller, T. and Wiemken, A. (1993). Colonization of transgenic Nicotiana sylvestris plants, expressing different forms of Nicotiana tabacum chitinase, by root pathogen Rhizoctonia solani and by the mycorrhizal symbiont Glomus mosseae. Molecular Plant-Microbe Interactions 6, 261–264. Viterbo, A. and Chet, I. (2006). TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization. Molecular Plant Pathology 7, 249–258. Viterbo, A., Harel, M. and Chet, I. (2004). Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots. FEMS Microbiology Letters 238, 151–158. Viterbo, A., Harel, M., Horwitz, B. A., Chet, I. and Mukherjee, P. K. (2005). Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance. Applied and Environmental Microbiology 71, 6241–6246. Viterbo, A., Wiest, A., Brotman, Y., Chet, I. and Kenerley, C. (2007). The 18mer peptaibols from Trichoderma virens elicit plant defence responses. Molecular Plant Pathology 8, 737–746. Volpin, H., Elkind, Y., Okon, Y. and Kapulnik, Y. (1994). A vesicular arbuscular mycorrhizal fungus (Glomus intraradix) induces a defense response in alfalfa roots. Plant Physiology 104, 683–689. Volpin, H., Phillips, D. A., Okon, Y. and Kapulnik, Y. (1995). Suppression of an isoflavonoid phytoalexin defense response in mycorrhizal alfalfa roots. Plant Physiology 108, 1449–1454. Waller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Heier, T., Hu¨ckelhoven, R., Neumann, C., Von Wettstein, D., Franken, P. and Kogel, K.-H. (2005). The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proceedings of the National Academy of Sciences of the United States of America 102, 13386–13391. Waller, F., Mukherjee, K., Deshmukh, S. D., Achatz, B., Sharma, M., Scha¨fer, P. and Kogel, K.-H. (2008). Systemic and local modulation of plant responses by Piriformospora indica and related Sebacinales species. Journal of Plant Physiology 165, 60–70. Xue, L., Charest, P. M. and Jabako-Hare, S. H. (1998). Systemic induction of peroxidases, 1,3- -glucanases, chitinases, and resistance in bean plants by binucleate Rhizoctonia species. Phytopathology 88, 359–365. Yedidia, I., Benhamou, N. and Chet, I. (1999). Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Applied and Environmental Microbiology 65, 1061–1070.

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Yedidia, I., Benhamou, N., Kapulnik, Y. and Chet, I. (2000). Induction and accumulation of PR proteins activity during early stages of root colonization by the mycoparasite Trichoderma harzianum strain T-203. Plant Physiology and Biochemistry 38, 863–873. Yedidia, I., Srivastva, A. K., Kapulnik, Y. and Chet, I. (2001). Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants. Plant and Soil 235, 235–242. Yedidia, I., Shoresh, M., Kerem, Z., Benhamou, N., Kapulnik, Y. and Chet, I. (2003). Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Applied and Environmental Microbiology 69, 7343–7353. Zhou, L., Jang, J., Jones, T. L. and Sheen, J. (1998). Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Plant Biology 95, 10294–10299. Zimand, G., Elad, Y. and Chet, I. (1996). Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity. Phytopathology 86, 1255–1260.

Priming of Induced Plant Defense Responses

UWE CONRATH1

Plant Biochemistry & Molecular Biology Group, Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Types of IR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Systemic Acquired Resistance (SAR) ....................................... B. Resistance Induced by Beneficial Microorganisms ........................ C. Resistance Induced by Chemicals ............................................ D. Resistance Induced by Wounding ........................................... E. Resistance Induced by Modifications of Primary Metabolism .......... III. Priming is a Mechanism of IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History ........................................................................... B. Elucidation of Priming in Parsley Cell Cultures ........................... C. Priming in SAR ................................................................. D. Priming Induced by Beneficial Microorganisms ........................... E. Priming in BABA-IR .......................................................... F. Priming in Wound-Induced Resistance ..................................... G. Priming by Modifications in Primary Metabolism ........................ IV. Relevance of Priming in Plant Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Costs and Benefits of Priming ................................................ B. Exploiting Priming in Greenhouse and Field .............................. V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51009-9

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ABSTRACT Upon infection by a pathogen, colonization of the roots by certain beneficial microbes, or after treatment with various chemicals, plants can establish a unique physiological situation which is called the ‘‘primed’’ state of the plant. In the primed condition, plants respond faster and/or more strongly with the activation of defense responses when subsequently challenged by microbial pathogens, herbivorous insects, or abiotic stresses. The potentiated activation of defense responses in primed plants is frequently associated with enhanced resistance to the challenging stress. Although priming has been known as a component of various induced resistance phenomena for decades, most of the progress in the understanding of the phenomenon has been made over the past decade. Here, I summarize the current knowledge of priming, its role in various forms of induced resistance, and its relevance for plant protection in the greenhouse and in the field.

I. INTRODUCTION Most plants are sessile organisms unable to escape unfavorable changes in their environment. To counter harmful conditions in their surroundings, plants therefore have evolved elaborate mechanisms to sense stress and adapt to it by rapid, dynamic, and complex alterations in their physiology. At best, the physiological switch leads to enhanced resistance against a given stressor without causing major fitness costs. A thorough understanding of the molecular and physiological basis of induced resistance (IR) to disease will lead to a better understanding of signal perception and transduction in plants and allow for effective and sustainable pest management in the field.

II. TYPES OF IR A. SYSTEMIC ACQUIRED RESISTANCE (SAR)

When a plant becomes infected by a pathogen, it can develop resistance to a broad and distinctive spectrum of pathogens (Durrant and Dong, 2004; Ryals et al., 1996). The pathogen-induced resistance can be established in the tissue surrounding the site of initial infection and also in the distant, uninfected parts of the plant. The former type of IR has been called ‘‘localized acquired resistance’’ (LAR), while the latter was named ‘‘systemic acquired resistance’’ (SAR) (Hammerschmidt, 2009; Ross, 1961a,b). It has been known for many years that both LAR and SAR are frequently associated with the accumulation of so-called ‘‘pathogenesis-related (PR) proteins’’ (Van Loon et al., 2006). Some of these proteins have antimicrobial activity and, therefore, may contribute to the resistance (Van Loon et al., 2006;

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see also Section III.A). The identity of the long-distance signal(s) that travel(s) from the site of primary infection to the remote parts of the plant to induce PR gene expression and SAR, however, is still unclear (Conrath, 2006; Durrant and Dong, 2004, Grant and Lamb, 2006; Heil and Ton, 2008; Ryals et al., 1996). In the early 1990s, studies with transgenic tobacco and Arabidopsis thaliana plants that constitutively accumulate a salicylic acid (SA) hydroxylase of bacterial origin clearly demonstrated that the plant hormone SA is required in the distal tissue for SAR to be expressed (Delaney et al., 1994; Gaffney et al., 1993; Vernooij et al., 1994). More recent work with Arabidopsis mutants affected in either the biosynthesis of SA or in SA signaling confirmed this conclusion (Dong, 2001). While the important role of SA in the development of SAR was without any controversy, it has remained unclear whether SA is the long-distance signal that travels from the site of primary pathogen infection throughout the plant to induce SAR (Champigny and Cameron, 2009). Some findings argued in favor of SA as the long-distance signal (Shulaev et al., 1995; Yalpani et al., 1991) while others argued against it (Meuwly et al., 1995; Rasmussen et al., 1991; Smith-Becker et al., 1998; Vernooij et al., 1994). Over the past few years, several other signaling molecules have emerged as possible candidates for the endogenous long-distance signal for SAR (reviewed by Vlot et al., 2008). These include methyl salicylate (MeSA), which is the methyl ester of SA (Park et al., 2007), lipid-derived signaling molecules (Maldonado et al., 2002; Nandi et al., 2004), including jasmonic acid (JA) (Truman et al., 2007) and azelaic acid (Jung et al., 2009), peptides (Xia et al., 2004), and reactive oxygen species (ROS) (Alvarez et al., 1998). Together, these findings argue that a complex and possibly variable combination of systemic signals may be required to fully induce the bona fide SAR response. B. RESISTANCE INDUCED BY BENEFICIAL MICROORGANISMS

1. Induced systemic resistance (ISR) Colonization of plant roots by selected strains of nonpathogenic plant growth-promoting rhizobacteria (PGPR), such as various species of the genera Pseudomonas (Ahn et al., 2007a; Van Loon, 2007; Van Loon et al., 1998), Bacillus (Kloepper et al., 2004), or Bradyrhizobium (Cartieaux et al., 2008), can induce a distinct broad-spectrum resistance response in both below- and above-ground parts of the plant. This type of IR was named ‘‘rhizobacteria-mediated ISR’’ (De Vleesschauwer and Ho¨fte, 2009; Van Loon, 2007; Van Loon et al., 1998). In contrast to SAR, ISR is not associated with PR gene expression or SA accumulation (Pieterse et al., 1996). It rather requires responsiveness to the plant hormones JA and

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ethylene (ET) (Pieterse et al., 1998). Thus, although having similar phenotypes, SAR and ISR recruit different, yet partly overlapping sets of plant hormones for their establishment. 2. Resistance induced by symbiotic fungi Interactions of plants with beneficial microorganisms other than those causing ISR can also result in systemic, broad-spectrum resistance. The symbiosis between barley roots and the endophytic basidiomycete Piriformospora indica, for example, confers systemic resistance to various root and leaf pathogens (Waller et al., 2005). These include the necrotrophic root-rot fungus Fusarium culmorum and the biotrophic fungus Blumeria graminis f. sp. hordei (Waller et al., 2005). The signaling mechanism by which Pi. indica induces resistance to these two pathogens in barley is not known, but it seems to be independent of SA and JA while being associated with the activation of the glutathione–ascorbate cycle, indicating an increase in antioxidative capacity in Pi. indica-elicited IR (Waller et al., 2005). Systemic resistance induced by the endophytic fungus Trichoderma asperellum T34 protected Arabidopsis against a wide range of pathogens through engagement of the same signaling components as used in Pseudomonas fluorescens (strain WCS417r)-mediated ISR (Segarra et al., 2009). IR to pathogens was also reported for plants whose roots have been colonized by mycorrhizal symbionts (Pozo and Azco´n-Aguilar, 2007; Pozo et al., 2005). For example, transcript profiling of the shoots of Medicago truncatula plants whose roots had been colonized by the arbuscular mycorrhizal fungus Glomus intraradices revealed both systemic expression of various defense-associated genes and establishment of an IR response to the bacterium Xanthomonas campestris pv. alfalfae (Liu et al., 2007). C. RESISTANCE INDUCED BY CHEMICALS

1. Synthetic SA analogs Besides pathogens and beneficial microorganisms, many different inorganic and organic compounds have been reported to induce resistance in plants (Kuc´, 2001). These include some synthetic SA analogs. 2,6-Dichloroisonicotinic acid and its methyl ester (both are referred to as INA) were the first synthetic compounds reported to activate a phenocopy of bona-fide SAR (Kessmann et al., 1994). Some years later, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; synonym: acibenzolar S-methyl, ASM) was introduced as another synthetic and highly potent activator of SAR (Friedrich et al., 1996; Go¨rlach et al., 1996; Lawton et al., 1996). SA, INA (methyl ester), and BTH are assumed to activate SAR via a same signaling pathway (Ryals et al., 1996).

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2. -Aminobutyric acid Various amino acids have been shown to induce resistance in plants (Kuc´, 2001). Among these, the nonprotein amino acid -aminobutyric acid (BABA) has received a lot of attention. The compound was shown to be a potent inducer of resistance against abiotic stress (Jakab et al., 2005; Zimmerli et al., 2008), nematodes (Oka et al., 1999), insects (Hodge et al., 2005), and microbial pathogens (Cohen, 2002; Jakab et al., 2001). In Arabidopsis, the BABA-IR to the oomycete Hyaloperonospora arabidopsidis does not require SA accumulation (Zimmerli et al., 2000), while BABAinduced protection against the bacterium Pseudomonas syringae pv. tomato strain DC3000 (Pst) (Zimmerli et al., 2000) and the gray-mold fungus Botrytis cinerea (Zimmerli et al., 2001) is SA-dependent. Research on the induction by BABA of resistance to Plectosphaerella cucumerina revealed that, in this case, the induction does not require ET or SA-mediated signaling. It rather depends on the plant hormone abscisic acid (ABA) (Ton and Mauch-Mani, 2004). In sum, the BABA-IR response in Arabidopsis seems to ward off various types of pathogens by recruiting distinct signaling pathways. D. RESISTANCE INDUCED BY WOUNDING

It is generally assumed that physical injury can make living plant tissue prone to pathogen invasion. However, over the past few years it has become increasingly clear that wounding, whether caused by mechanical damage or feeding by herbivorous insects, can also serve as an effective stimulus for the induction of local and systemic resistance to microbial pathogens or herbivorous insects (Chassot et al., 2007, 2008; Francia et al., 2007; Green and Ryan, 1972; Ryan, 1990). This wound-induced resistance (WIR) involves direct activation of many genes, including those encoding protease inhibitors. These proteins can inactivate enzymes with important roles in either disease symptom development or digestion of plant tissue in the insect gut (Green and Ryan, 1972; Ryan, 1990). Genetic studies have shown that several compounds of the octadecanoid pathway (e.g., JA) can act as endogenous mediators for wound-induced defense gene activation and resistance against herbivorous insects (Howe, 2004). However, it is also known that an extensive number of wound-activated genes can be induced via a signaling pathway that is independent of JA (Reymond et al., 2000), indicating a complex nature of the wound response in plants. Systemin, the first plant peptide for which a signaling function has been demonstrated, is involved in the wound-induced activation of protease inhibitor (PIN) genes in tomato (Pearce et al., 1991; Ryan, 1992) and other species of the Solanaceae family, such as potato, bell pepper, and black

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nightshade (Constabel, 1999; Constabel et al., 1998). At wound sites, the 18-amino acid peptide systemin is processed from a 200-amino acid precursor protein, called ‘‘prosystemin,’’ and transported throughout the plant (Narva´ez-Va´squez et al., 1995). Because protease inhibitor gene expression was constitutive in transgenic tomato plants that overexpressed the PROSYSTEMIN gene, it was assumed that systemin is the endogenous signal that would move within the plant to mediate the systemic wound response (McGurl et al., 1994). However, a series of grafting experiments with the suppressed in prosystemin-mediated responses-1 (spr1) mutant of tomato, which is defective in systemin perception, revealed that JA, rather than systemin, is responsible for systemic signaling of the wound response (Lee and Howe, 2003). Systemin is needed locally in the damaged tissue but not in the systemic, undamaged tissue of wounded plants for signal transduction. Thus, it seems now that wound-induced systemin locally contributes to the biosynthesis of JA, which regulates the production of, or acts as, a mobile wound signal that is transported throughout the plant to trigger the systemic wound response (Lee and Howe, 2003). E. RESISTANCE INDUCED BY MODIFICATIONS OF PRIMARY METABOLISM

While it is obvious that exposure of plants to biotic or abiotic stresses can affect photosynthesis, partitioning of assimilates, and source-sink relationships (Schwachtje and Baldwin, 2008), little is known about the impact of primary plant metabolism on IR in plants. One frequently reported resistance phenotype in plants is the so-called ‘‘high-sugar resistance.’’ This type of IR is associated with elevated levels of soluble carbohydrates which result from certain alterations in primary metabolism (Horsfall and Dimond, 1957). The concept of ‘‘high-sugar resistance’’ has been supported by various studies demonstrating that application of sugar to various plant tissues, or provoking the accumulation of sugar in transgenic plants, can lead to activation of various PR genes (Herbers et al., 1996a,b; Johnson and Ryan, 1990). Similarly, tubers of transgenic potato plants with decreased activity of the plastid ATP/ ADP transporter AATP1(St) not only display reduced starch content, but they also have altered levels of primary metabolites, such as glucose and other carbohydrates (Geigenberger et al., 2001; Tjaden et al., 1998). The alterations in primary metabolism coincide with an IR response to the soft-rot causing bacterium Pectobacterium atrosepticum (Linke et al., 2002) and the fungal pathogen Alternaria solani (Conrath et al., 2003) in the tubers, and with IR to the late blight-causing oomycete Phytophthora infestans in the leaves (Conrath et al., 2003). Thus, certain alterations in plant primary metabolism can cause tissue-specific resistance to a variety of biotic challenges.

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III. PRIMING IS A MECHANISM OF IR A. HISTORY

For many years, IR in plants has been suggested to be on the basis of the direct activation of defense responses in systemic tissue of pathogen-infected plants. In case of SAR, these directly induced responses in the systemic tissue include the accumulation of PR-proteins (Durrant and Dong, 2004; Ryals et al., 1996). Many PR-proteins display antimicrobial activity presumably through hydrolytic activities on cell walls of potential microbial pathogens and contact toxicity, and maybe also as compounds involved in plant defense signaling (Van Loon et al., 2006). However, as the expression of cloned genes for PR-proteins in transgenic plants does not generally lead to enhanced resistance against diverse pathogens, the actual contribution of PR-proteins to IR appears to be minor (Van Loon, 2000). As research on IR had focused primarily on the role of PR-proteins and other directly induced defense-related compounds, it has not been widely appreciated that the enhanced defensive capacity characteristic of IR is also associated with a sensitized state in which the plant responds more rapidly and/or more robustly with the activation of defense responses after exposure to a biotic or abiotic stressor (Conrath and Go¨llner, 2008; Conrath et al., 2002, 2006; Kuc´, 1987) (Fig. 1). The state of enhanced capacity to activate stress-induced defense responses has been called the ‘‘primed’’ (or ‘‘sensitized’’) state of the plant. As a matter of fact, as early as the 1980s, Kuc´ (1987) had already argued that priming would be an important component of SAR. Yet, although priming could be a unifying mechanism for the different types of IR in plants, the phenomenon did not attract much attention at the time (Van Loon, 2000). In the 1990s, an important role of priming in SAR was supported by the finding that there is close correlation between the capability of various chemicals to activate resistance against tobacco mosaic virus (TMV) in tobacco (Conrath et al., 1995) and their capacity to prime for enhanced PHENYLALANINE AMMONIA-LYASE (PAL) gene expression induced by microbe-associated molecular pattern (MAMP) elicitor treatment in cultured parsley cells (Katz et al., 1998; Thulke and Conrath, 1998), or upon infection of Arabidopsis plants with Pst (Kohler et al., 2002). B. ELUCIDATION OF PRIMING IN PARSLEY CELL CULTURES

Only in the early 1990s, Uwe Conrath and associates provided the first biochemical analyses of the priming phenomenon in plants (Conrath et al., 2002, 2006). In their initial studies, the authors employed a model system of a

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(no SAR)

B

(SAR)

Fig. 1. Priming for more robust induction of defense gene expression and reduced lesion formation are part of the SAR response of plants. Leaves of tobacco (Nicotiana tabacum cv. Samsun NN) plants transformed with a chimeric gene that is composed of the promoter of a PR-10 gene of Asparagus officinalis fused to the coding sequence of the GUS reporter gene, were infiltrated with either buffer as the control (A), or with Ps. syringae pv. syringae to induce SAR (B). Seven days later, systemic tissue (photo) of each plant was infected with lesion-causing TMV and after another 7 days histochemically assayed for GUS reporter gene activity (blue) and lesion development. Bar ¼ 1 cm. Photograph courtesy of Luis Mur (University of Wales, UK).

parsley cell culture and a MAMP elicitor from Phytophthora sojae to elucidate molecular aspects of priming and the associated amplification of MAMP elicitor-induced defense responses. They showed that pretreatment with low concentrations of compounds that would not directly induce cellular defense responses but did induce SAR in plants, such as SA, INA, or BTH, primed parsley cells in a time-dependent manner for stronger activation of various cellular defense responses by low MAMP elicitor doses that would not per se significantly induce these defenses directly. These reactions were also induced by the low doses of the MAMP elicitor in nonprimed parsley cells, but to a much lesser extent. The analyses encompassed the socalled ‘‘oxidative burst’’ (a term describing the production of various ROS), rapid changes in ion transport across the plasma membrane, the synthesis and secretion of antimicrobial secondary metabolites (phytoalexins), various cell wall phenolics, a lignin-like polymer, as well as the expression of various defense-related genes (Katz et al., 1998; Kauss and Jeblick, 1995; Kauss et al., 1992a; 1993; Thulke and Conrath, 1998). In subsequent studies using the parsley cells, it was demonstrated that the effect of the SAR inducers on defense gene activation depended on the dose of the inducer applied and the gene that was assayed (Katz et al., 1998; Thulke and Conrath, 1998). While some defense genes were found to be directly responsive already to moderate concentrations of SA or BTH, others were not induced at all. This second set of defense genes rather displayed very strong activation after a priming treatment of the cells with moderate concentrations of SA or BTH followed

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by challenge treatment with a very low, suboptimal dose of the MAMP elicitor (Katz et al., 1998; Thulke and Conrath, 1998). These results attested to a dual role for inducers of SAR in the activation of plant defense responses: on the one hand, moderate doses of SA or BTH directly activated a specific set of defense genes, while, on the other hand, they primed cells for enhanced expression of a different set of defense genes when the cells were subjected to challenge treatment with the MAMP elicitor. C. PRIMING IN SAR

1. Tobacco In 1996, the group of John Draper reported the first molecular analysis of the priming phenomenon in intact plants (Mur et al., 1996). The authors demonstrated that a soil-drench pretreatment with SA of transgenic tobacco plants expressing either a chimeric gene composed of the promoter of an Asparagus PR10 gene and the coding sequence of the reporter gene -GLUCURONIDASE (GUS), or the promoter of the defense-related PAL3 gene fused to GUS, did not significantly induce gene activation (Mur et al., 1996). However, after infection with Ps. syringae pv. syringae or after wounding, activation of the reporter gene was much stronger in the SA-pretreated plants than it was in plants that had not been pretreated with SA (Mur et al., 1996). A few years later, the same group demonstrated that the loss of resistance to avirulent bacterial pathogens in SA hydroxylase-expressing transgenic tobacco was associated with an attenuation of the SA-potentiated oxidative burst (Mur et al., 2000). Together, these findings confirmed, at the molecular level, that priming is a part of IR in whole plants. 2. Arabidopsis In 1999, Van Wees et al. demonstrated that induction of SAR by infection of Arabidopsis leaves with avirulent PstavrRpt2 resulted in priming of the systemic tissue, exhibited as elevated expression levels of PR genes. Pretreatment with BTH likewise primed Arabidopsis for more robust induction of the PAL gene by Pst (Kohler et al., 2002). Together, these two reports allowed for in-depth molecular and genetic studies of the priming phenomenon in plants. Thus, the Arabidopsis mutant enhanced disease resistance-1 (edr1) constitutively displays systemically enhanced resistance to Pst as well as to the fungus Erisyphe cichoracearum (Frye and Innes, 1998). It is interesting that edr1 differs from other known Arabidopsis mutants with constitutively enhanced disease resistance in that it does not display constitutive activation of the PR-1 and -2 genes, although their transcripts accumulated to higher levels after pathogen attack (Frye and Innes, 1998). This observation and the finding

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that edr1, when compared to the wild type, displays more robust induction of defense responses upon pathogen infection strongly suggest an engagement of the EDR1 protein in priming. In response to infection with avirulent pathogens the Arabidopsis nonexpresser of PR genes-1 (npr1) mutant (also named noninducible immunity-1 (nim1) or salicylic-acid insensitive-1 (sai1)) accumulates SA levels that are similar to those found in the wild type. However, the npr1 mutant is unable to express biologically or chemically induced SAR (Cao et al., 1994; Delaney et al., 1995, Shah et al., 1997). Upon pretreatment with BTH the enhanced activation of Pst-induced PAL gene expression is absent in the npr1 mutant, while PAL gene expression per se is not abolished in this mutant (Kohler et al., 2002). This result demonstrates that a functional NPR1 gene is required for this priming in Arabidopsis. In a converse manner, the constitutive expresser of PR genes-1 (cpr1) and cpr5 mutants of Arabidopsis both express SAR in the absence of pretreatment with activators of SAR (Bowling et al., 1994, 1997). This is probably because the cpr1 and cpr5 mutants are both in a state of ‘‘enhanced defense readiness’’ that resembles SAR in that both express defense responses in addition to exhibiting the primed state. Not only is a set of defense genes constitutively expressed (Bowling et al., 1994, 1997), but both these mutants are also sensitized for higher PAL gene activation upon infection by Pst (Kohler et al., 2002). Thus, it is likely that because of the enhanced SA levels in cpr1 and cpr5 (Bowling et al., 1994, 1997) these plants are permanently primed. Because of permanent priming, cpr1 and cpr5 might be able to quickly and strongly activate various cellular defense reactions upon attack by pathogens. In this context it is worth mentioning that the constitutively enhanced disease resistance of another Arabidopsis mutant referred to as cpr5-2 has been ascribed to the boosted activation of the PR-1 defense gene in these plants (Boch et al., 1998). Since priming leads to more robust induction of defense responses and ultimately to resistance, it can be anticipated that the phenomenon includes improved perception and/or amplification of the defense response-inducing signal from the pathogen (Conrath et al., 2006). It has been proposed that priming is associated with increased accumulation, and/or posttranslational modification, of inactive cellular signaling proteins that play an important role in signal amplification (Conrath et al., 2006). Subsequent exposure to stress could activate, or modulate, these ‘‘dormant’’ signaling proteins, thereby initiating signal amplification leading to faster and/or stronger activation of defense responses and IR (Conrath et al., 2006). However, the identity of these hypothetical proteins has remained obscure. Recently, two members of the mitogen-activated protein kinase (MAPK) family of

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signaling enzymes, MPK3 and MPK6, were found to accumulate upon priming in Arabidopsis without displaying enzyme activity (Beckers et al., 2009). Possibly because of the enhanced level of inactive MPK3 and MPK6 in primed plants, the capacity for subsequent signaling is increased, and upon stimulation by biotic or abiotic stresses MPK3 and MPK6 activity is augmented, associated with enhanced induction of defense responses and IR (Beckers et al., 2009). These findings argue that pre-stress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR. Future genetic and biochemical analysis will probably yield more candidates for cellular components with an important role in priming for enhanced disease resistance. 3. Other species Observations similar to those made with the parsley cell culture and tobacco and Arabidopsis plants have been reported for many other plant species. For instance, pretreatment with physiological concentrations of SA had negligible effects on soybean cell suspension cultures. However, when the SA-pretreated cells were challenged with an avirulent strain of Ps. syringae pv. glycinea, the activation of defense genes, the oxidative burst, and cell death were markedly enhanced (Shirasu et al., 1997). In a similar manner, BTH primed Agastache rugosa suspension cells for augmented production of rosmarinic acid upon induction by a yeast extract elicitor (Kim et al., 2001). BTH also sensitized sunflower plants for increased production and excretion of phytoalexins and phenolic compounds upon infection with the sunflower rust fungus Puccinia helianthi (Prats et al., 2002), and it also primed cucumber plants for enhanced activation of various defense genes as well as resistance to Colletotrichum orbiculare (Cools and Ishii, 2002). In addition, BTH sensitized cowpea (Vigna unguiculata) plants for rapid, transient increases in PAL and chalcone isomerase activity, potentiated accumulation of the isoflavonoid phytoalexins kievitone and phaseollidin, and resistance to damping-off disease caused by Colletotrichum destructivum (Latunde-Dada and Lucas, 2001). Furthermore, treatment with SA primed intact Asparagus officinalis plants for faster and stronger induction by Fusarium oxysporum f. sp. asparagi of peroxidase or PAL activity, and for quicker and more robust lignin deposition associated with enhanced resistance to F. oxysporum f. sp. asparagi (He and Wolyn, 2005). Moreover, vitamin B1 (thiamine) was shown to prime rice, Arabidopsis, and several vegetable crop plants for faster and stronger activation of defense-related genes and IR to various fungal, bacterial, and viral pathogens (Ahn et al., 2005). In Arabidopsis the vitamin B1-induced priming required hydrogen peroxide and the NPR1 gene (Ahn et al., 2007b). In cucumber plants, infection by the anthracnose-causing

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fungus Colletotrichum lagenarium induced resistance to further infection by the same pathogen. This SAR response was associated with enhanced deposition of a lignin-like polymer in cell wall sites under fungal appressoria (Hammerschmidt and Kuc´, 1982), indicative of priming of cell wall reinforcement in this species. D. PRIMING INDUCED BY BENEFICIAL MICROORGANISMS

1. Priming in ISR Most of the studies that investigated priming by beneficial microorganisms made use of ISR-eliciting PGPR. The first evidence that priming of plant defense responses is involved in ISR came from experiments with carnation (Dianthus caryophyllus). Inoculation with F. oxysporum f. sp. dianthi of carnation plants displaying ISR led to a faster rise in phytoalexin levels than in noninduced control plants (Van Peer et al., 1991). In a similar manner, Bacillus pumilus (strain SE34) induced systemic resistance against the root-rot fungus F. oxysporum f. sp. pisi in bean (Benhamou et al., 1996). Upon challenge infection with the same fungus, the walls of root cells were rapidly strengthened at sites of attempted fungal penetration through apposition of callose and phenolic material (Benhamou et al., 1996). In Arabidopsis, priming associated with the systemic resistance induced by root-colonizing Ps. fluorescens strain WCS417r has been studied at the molecular level. Although WCS417r-elicited ISR is effective against a broad and distinctive sprectrum of pathogens, it is not associated with the activation of genes encoding PR-proteins (Pieterse et al., 1996). Analyses of the Arabidopsis transcriptome have shown that locally in the colonized roots, WCS417r bacteria induce the expression of 94 genes (Le´on-Kloosterziel et al., 2005; Verhagen et al., 2004). However, in the systemic leaves, no significant alteration in gene expression was observed. Thus, WCS417r-elicited ISR is not associated with obvious changes in gene expression in distant leaves (Verhagen et al., 2004). In Arabidopsis expressing WCS417r-mediated ISR, 81 genes showed enhanced expression upon infection of the leaves with Pst, indicating that these plants were primed to respond in a faster and/or more robust manner to pathogen attack (Van Wees et al., 1999; Verhagen et al., 2004). Most of the genes with potentiated induction have been described as being regulated by either JA or ET, or both. The findings confirmed earlier results demonstrating that colonization of the roots by WCS417r primed Arabidopsis for augmented induction of the JA- and/or ET-responsive genes VEGETATIVE STORAGE PROTEIN-2 (VSP2), PLANT DEFENSIN-1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL), and ACC (1-Aminocyclopropane-1-carboxylic acid) OXIDASE (ACO) (Hase et al., 2003; Van Wees et al., 1999). In contrast to

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gene expression, significant alterations in the production of either JA or ET have not been observed in the plants exhibiting ISR (Pieterse et al., 2000). These observations argue that the state of ISR is based on an enhanced sensitivity to these plant hormones rather than just on an increase in their production (Pieterse et al., 2000). Two recent studies by Corne´ Pieterse and associates suggest that the Ps. fluorescens WCS417r-induced priming and the associated ISR of Arabidopsis require the transcription factor MYB72 in the roots (Van der Ent et al., 2008) and the helix-loop-helix transcription factor MYC2 in the leaves (Pozo et al., 2008). MYC2 plays a role not only in the regulation of JA-responsive genes (Berger et al., 1996), but also in ABA and drought signaling (Abe et al., 2003). Studies with other PGPR on different plant species generally confirm that ISR is associated with primed expression of defense genes (reviewed by Van Wees et al., 2008). Ryu et al. (2004) demonstrated that some plant-growth promoting Bacillus spp. can prime plants by the release of volatile organic compounds (VOCs). Bacillus subtilis GB03 produces the green leaf volatiles 3-hydroxy-2-butanone and (2R,3R)-(–)-2,3-butanediol, which can prime Arabidopsis for augmented defense responses against herbivore attack or pathogen infection. In that case, a signaling pathway that is independent of SA, JA, and the NPR1 gene, yet requiring ET, is triggered (Ryu et al., 2004). 2. Priming in beneficial interactions other than ISR In addition to SAR and ISR, the primed state is a common feature also of resistance responses that are induced by beneficial microorganisms other than PGPR. For example, colonization of tomato roots by mycorrhizal fungi protected the plant systemically against Phytophthora parasitica with no detectable accumulation of PR-proteins before pathogen assault. Only after Ph. parasitica attack, mycorrhizal plants accumulated significantly more PR1a and basic -glucanases than non-mycorrhizal plants (Cordier et al., 1998; Pozo et al., 1999, 2002). Ultrastructural studies revealed that plants with established mycorrhizal symbiosis also displayed pectin-rich, callosecontaining cell wall depositions at the sites of attempted pathogen infection, whereas non-mycorrhizal plants did not (Cordier et al., 1998; Pozo et al., 1999, 2002). Certain plant growth-promoting fungi can similarly induce priming in plants. For example, challenge inoculation with the leaf pathogen Ps. syringae pv. lachrymans of cucumber plants that had been preinoculated with the plant growth-promoting fungus Trichoderma asperellum (strain T203) led to augmented PR gene expression (Shoresh et al., 2005). Also, like SA, infection by a nonpathogenic isolate of the fungus F. oxysporum primed As. officinalis plants for faster and stronger induction of defense reactions and enhanced resistance to F. oxysporum f. sp. asparagi (He et al., 2002).

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3. Priming by bacterial lipopolysaccharides Unspecific, so-called ‘‘basal’’ defense responses can be induced in plants and animals by various MAMP elicitors. Among other molecules, these include small peptides, lipooligosaccharides and lipopolysaccharides (LPS) (Newman et al., 2007). The latter are ubiquitous, indispensable components at the cell surface of Gram-negative bacteria. They have diverse effects in plants (Newman et al., 2007). These include prevention of the hypersensitive response, synthesis of nitric oxide, phosphorylation of MAPKs, and priming for more effective induction of various defense responses (Erbs and Newman, 2003; Newman et al., 2001, 2007). In pepper leaves, for example, the genes coding for the PR-protein P6 (an ortholog of PR-1 of tobacco), and acidic and basic -glucanase were not or only weakly induced by LPS from Salmonella enterica serovar minnesota or X. campestris pv. campestris, respectively (Newman et al., 2000, 2002). However, pretreatment of the pepper leaves with the LPS augmented the expression of the genes encoding the P6 protein and acidic and basic -glucanase following infection with X. campestris pv. campestris (avirulent) or X. campestris pv. vesicatoria (virulent) (Newman et al., 2002, 2007). The authors also reported that LPS pretreatment led to faster and more robust induction of coumaroyl tyramine and feruloyl tyramine, which have antimicrobial activity and serve to strengthen the cell wall upon inoculation with X. campestris pv. campestris. Yet, LPS treatment alone did not induce either coumaroyl tyramine or feruloyl tyramine production (Newman et al., 2007). Thus, at least in plants LPS-activated IR seems to be mediated by LPS-induced priming for enhanced activation of defense responses upon challenge infection (Newman et al., 2002, 2007). The molecular mechanism of the LPS-induced priming, however, is still unclear. It is noteworthy that the effects of various beneficial bacteria on plants, such as activation of ISR by PGPR, at least in some cases are believed to be a consequence of LPS perception (Leeman et al., 1995a). Thus, it would be interesting to know whether ISR is mediated by LPS-induced priming for enhanced activation of defense responses and resistance. E. PRIMING IN BABA-IR

1. Biotic stress Research on the mechanism(s) of BABA-IR in Arabidopsis has shown that this type of IR, just like SAR and ISR, is frequently associated with priming for various pathogen-induced defense responses. For example, the induction of the PR-1 gene is faster when BABA-pretreated Arabidopsis plants are challenged with Pst than when non-pretreated plants (Zimmerli et al., 2000). As a matter of fact, the induction kinetics of the PR-1 gene by Pst in

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BABA-primed Arabidopsis plants are very similar to the induction kinetics of PR-1 gene expression in the resistance response to the avirulent strain PstavrRpt2 (Zimmerli et al., 2000). This priming of PR-1 expression by BABA is dependent of NPR1, indicating that a pathway similar to SA signaling in SAR is activated by BABA (Zimmerli et al., 2000). In Arabidopsis BABA-IR to H. arabidopsidis coincided with fast and robust deposition of callose-containing papillae (Zimmerli et al., 2000). This correlation between BABA-IR and augmented papillae formation was intensively studied using the interaction of Arabidopsis with the two necrotrophic fungi Alternaria brassicicola and Pl. cucumerina. The use of various Arabidopsis mutants indicated that neither the phytoalexin camalexin, nor SA-, JA-, or ET-dependent defense responses seem to play a critical role in BABA-IR to these two necrotrophic pathogens (Ton and Mauch-Mani, 2004). Cytological investigations at sites of attempted penetration by A. brassicicola and Pl. cucumerina demonstrated that the formation of callose-rich papillae was increased in attacked epidermal cells of BABA-pretreated plants (Ton and Mauch-Mani, 2004). Pharmacological studies with 2-deoxy-Dglucose, an inhibitor of callose synthesis, indicated that in BABA-pretreated plants the enhanced callose deposition plays a key role in the induction of resistance to A. brassicicola (Ton and Mauch-Mani, 2004). In a consistent manner, the callose-deficient powdery mildew-resistant-4-1 (pmr4-1) mutant of Arabidopsis was completely blocked in BABA-IR to Pl. cucumerina. Priming for enhanced papillae formation after fungal infection was absent in the Arabidopsis mutants abscisic acid-insensitive-4-1 (abi4-1) and abi1-5 (Ton and Mauch-Mani, 2004). In addition, exogenous application of ABA mimicked the effect of BABA with respect to increased formation of callose-rich papillae and resistance to fungal ingress. Priming for enhanced callose deposition by BABA involves the augmented transport of vesicles through the Golgi apparatus to release precursors at the plasma membrane. In this process, different soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins are involved. SNARE proteins have also been linked to disease-resistance mechanisms in the plant cell wall. Mutations in the SNARE genes PENETRATION-1 (PEN1) (encoding syntaxin) and REQUIRED FOR mlo RESISTANCE-2 (ROR2) (Collins et al., 2003; Lipka et al., 2005) caused a partial loss of resistance at the level of the plant cell wall, resulting in a loss of ‘‘basal resistance’’ to the nonadapted parasites B. graminis f. sp. hordei and Erysiphe pisi that in nature colonize barley and pea, respectively. Both, PEN1 and ROR2 are thought to play a role in cellular targeting of vesicles carrying phytoalexins or callose synthase II to sites of attempted fungal penetration. Together, these findings suggest a prominent role of ABA in the accelerated formation of callose-rich

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papillae through enhanced ABA-dependent transcription, or augmented induction of SNARE genes upon pathogen attack (Leyman et al., 1999; Zhu et al., 2002). Indications for interaction-dependent complexity of the BABA-induced priming mechanism(s) have been provided by Hamiduzzaman et al. (2005). Using BABA as the inducer of resistance to downy mildew (Plasmopara viticola) in grapevine (Vitis vinifera) the authors showed that in this interaction the primed deposition of callose and phenylpropanoid-derived phenolics is associated with resistance to the pathogen but is dependent on JA rather than ABA (Hamiduzzaman et al., 2005). 2. Abiotic stress It is known that SA and its derivative acetyl-SA (aspirin) can protect various plants from abiotic stresses, such as chilling, heat, drought, and wounding (Janda et al., 1999; Kohler et al., 2002; Senaratna et al., 2000). However, much more information is available for the BABA-induced protection from abiotic stress. For example, BABA is known to protect Arabidopsis from heat (Zimmerli et al., 2008), drought, and salt stress (Jakab et al., 2005). The BABAinduced tolerance to the latter two abiotic stresses correlated with primed expression of SA- and ABA-responsive genes upon exposure of the BABApretreated plants to drought or salt (Jakab et al., 2005). Arabidopsis mutants with defects in ABA signaling could not be protected by BABA, while BABApretreated SA-signaling mutants showed a resistance response that was similar to wild-type plants. In the wild type, pretreatment with BABA did not lead to ABA accumulation. Rather, the production of this plant hormone was more rapidly induced after exposure of the plants to osmotic stress. The accelerated ABA production resulted in more robust expression of ABA-responsive genes and quicker closure of stomata (Jakab et al., 2005). These findings demonstrated that BABA-induced tolerance to osmotic stress is based on priming for enhanced adaptation responses rather than on direct activation of stress defense responses. The latter is commonly observed during acclimation treatment in which plants are gradually exposed to an increasingly stressful situation. Together, ABA seems to have a crucial role in BABA-induced priming for stronger responses to both biotic and abiotic stresses, at least in Arabidopsis. F. PRIMING IN WOUND-INDUCED RESISTANCE

1. Priming in IR to herbivores In 1972, Green and Ryan were the first to report that insect feeding on potato and tomato plants activates local and systemic accumulation of proteinase inhibitors that disrupt the activity of digestive proteases in the insect gut

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(Green and Ryan, 1972). Wound-induced local and systemic resistance to pathogenic microorganisms or herbivorous insects is illustrated by a recent study of Chassot et al. (2008), who demonstrated that wounding leaves either by squeezing with a pair of forceps or by puncturing holes with a needle induces resistance to the gray-mold fungus B. cinerea in Arabidopsis. This WIR seems to require neither SA, nor JA or ET. It rather needs glutathione and the phytoalexin camalexin (Chassot et al., 2008). The wound stimulus per se did not lead to camalexin production. Wounding rather primed Arabidopsis for enhanced camalexin biosynthesis after B. cinerea inoculation (Chassot et al., 2008). These observations not only revealed that glutathione and camalexin play a likely key role in WIR of Arabidopsis to B. cinerea, but also demonstrated that priming is a likely mechanism of wound-induced pathogen resistance, at least in the Arabidopsis–B. cinerea interaction. Earlier studies with cell cultures and the wound signals MeJA and systemin had already suggested that priming represents the basis of WIR to pathogenic microbes and insects. For example, pretreatment with MeJA primed parsley suspension cells for enhanced secretion of phytoalexins and augmented incorporation of hydroxycinnamic acid esters and ‘‘lignin-like’’ polymers into the cell wall (Kauss et al., 1992b). In a similar manner, pre-exposure of cultured tomato cells to the wound-signaling peptide systemin, but not an inactive systemin analog, led to a time-dependent enhancement of the oxidative burst that was subsequently induced by oligogalacturonides (Stennis et al., 1998). In response to wounding or herbivore attack, plants often release extrafloral nectar or VOCs. While some of these serve to attract parasitic or predatory natural enemies of the herbivores (Pare´ and Tumlinson, 1999), others have a role in the activation of resistance in the same (Heil and Silva Bueno, 2007) or even nearby, unharmed plants (Baldwin and Schultz, 1983; Heil and Kost, 2006). During the past years, there was strong evidence that VOC-mediated IR is mediated by priming (see Section III.D.1). In a pioneering study, Engelberth et al. (2004) showed that maize seedlings when exposed to certain volatiles from neighboring plants and subsequently challenged by a combination of mechanical damage and exposure to regurgitant of caterpillars of the beet armyworm (Spodoptera exigua), show higher production of volatile sesquiterpenes and JA when compared to triggered plants not exposed to the volatiles before. In a follow-up study, it was shown that the VOC-induced priming for augmented induction of defense genes and emission of aromatic and terpenoid volatiles in maize correlates with reduced caterpillar feeding and enhanced attraction of the parasitoid wasp Cotesia marginiventris (Ton et al., 2006).

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Further work revealed that the green leaf volatile cis-3-hexenyl acetate serves as a wound signal that primes leaves of hybrid poplar saplings for higher concentrations of JA and linoleic acid following gypsy moth (Lymantria dispar) feeding, and for improved oxylipin signaling, terpene volatile release, and defense responses (Frost et al., 2008). In field studies, Heil and Kost (2006) demonstrated that Lima bean plants respond to leaf damage with the secretion of extrafloral nectar, and this response was much higher in plants that had been exposed before to a complex artificial volatile blend that mimics the herbivore-induced bouquet of Lima bean plants both quantitatively and qualitatively. In sum, these findings strongly suggest that VOCs play a key role in priming during the wound response of plants. 2. Priming between plant species Over the past few years, it has become increasingly clear that priming can be the result of plant–plant communication in the wild when VOCs serve as priming-inducing signals even between plant individuals of different species. Kessler et al. (2006) reported that VOCs from clipped sagebrush (Artemesia tridentata) prime nearby wild tobacco (Nicotiana attenuata) plants for quicker production of trypsin inhibitors, and this was associated with lower herbivore damage and higher mortality rate of young tobacco hornworm (Manduca sexta) caterpillars (Kessler et al., 2006). Using tobacco as the model plant Shulaev et al. (1997) proposed that MeSA, which is synthesized from SA and acts by being reconverted into SA, may function as an airborne signal that activates disease resistance and the expression of defense genes in healthy tissues of infected plants and also in neighboring plants. However, whether the resistance induced by MeSA is mediated by priming or whether it is due to direct activation of defense responses has not been addressed in these studies. In any case, it seems that plants can use chemical signals in their environment to assess the risk of herbivory and integrate this information to adjust and fine-tune their overall defense strategy. G. PRIMING BY MODIFICATIONS IN PRIMARY METABOLISM

Although application of sugar to various plant tissues, or provoking the accumulation of sugar in transgenic plants, can lead to activation of various PR genes (Herbers et al., 1996a,b; Johnson and Ryan, 1990), the ‘‘high-sugar concept’’ of resistance (Horsfall and Dimond, 1957) has been called into question by recent work which demonstrated that expression of a yeast invertase in the cytoplasm of potato tuber cells leads to decreased levels of starch and enhanced levels of glucose, yet to drastic susceptibility to the softrot pathogen Pe. atrosepticum (Conrath et al., 2003). In addition, the

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enhanced resistance to Ph. infestans in leaves of transgenic potato plants with decreased activity of the plastid ATP/ADP transporter was not associated with obvious changes in carbohydrate accumulation, in contrast to the enhanced disease resistance in the tubers (Conrath et al., 2003). These results suggested that the resistance of plant tissue with elevated levels of carbohydrates is not due to enhanced sugar levels. This suggestion is supported by findings demonstrating that increased glucose levels are not associated with constitutive expression of PR genes in potato tubers with reduced activity of the plastid ATP/ADP transporter (Conrath et al., 2003). Detailed analyses on the timing and extent of defense responses in these same plants provided an alternative explanation for the IR phenotype observed. Upon exposure of leaf or tuber tissue to culture supernatants of Pe. atrosepticum or pep13, a 13-amino acid MAMP elicitor from oomycete cell walls, there was enhanced activation of defense responses, including defense gene activation and the oxidative burst (Linke et al., 2002). Thus, the IR of transgenic plants with reduced ATP/ADP transporter activity seems to be mediated by metabolic priming for enhanced induction of defense responses rather than by the associated elevation in carbohydrate levels in these plants. A correlation between elevated sucrose levels and priming of defense responses was reported recently also for rice overexpressing the PRms gene from maize, which encodes a PR-1 type protein (Casacuberta et al., 1991). In these plants elevated levels of sucrose were associated with quicker and more robust induction of defense responses during pathogen infection and broad-spectrum disease resistance (Go´mezAriza et al., 2007).

IV. RELEVANCE OF PRIMING IN PLANT PRODUCTION A. COSTS AND BENEFITS OF PRIMING

The direct stimulation of defense responses by external application of high doses of SA or JA, or by the action of resistance genes reduces certain fitness characters, such as growth and fruit or seed set, under pathogen-free conditions (Agrawal et al., 1999; Baldwin, 1998; Cipollini, 2002; Heidel et al., 2004, Heil et al., 2000, Korves and Bergelson, 2004; Tian et al., 2003; Van Dam and Baldwin, 2001). Also, plants transformed with genes encoding SA biosynthetic enzymes (Mauch et al., 2001), or gain-of-resistance mutations in Arabidopsis such as cpr1, cpr5 and cpr6, which all contain constitutively elevated levels of SA, permanently express PR genes, have a dwarf

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phenotype, which is associated with reduced fitness (Bowling et al., 1994, 1997). These observations were also made in the field. Heidel et al. (2004) demonstrated that Arabidopsis mutants blocked in SA-inducible defenses, as well as mutants showing constitutive expression of these defense responses, all were affected in growth and seed set. The authors argued that optimal fitness would be reached at a certain level of resistance that balances fitness and defense (Heidel et al., 2004). Similar conclusions were made from research on the costs of JA-inducible defenses, which seem to be affordable only when the plant is actually exposed to herbivore attack (Agrawal et al., 1999; Baldwin, 1998). The dilemma between resistance and costs of defense can probably be overcome by priming. In a recent study by Van Hulten et al. (2006), the costs and benefits of priming in Arabidopsis were determined and compared to those of the direct activation of defense. Application of low doses of BABA induced the primed state, caused minor growth reduction, and had no obvious effect on seed production. In contrast, direct induction of defense responses by high doses of either BABA or BTH strongly affected both these fitness parameters (Van Hulten et al., 2006). The effects in primed plants, although minor, could be due to enhanced expression of genes for signaling compounds with an important role in priming (Maleck et al., 2000; Van Hulten et al., 2006) (see Section III.C.2). Consequently, it was suggested that priming causes less fitness costs than the direct induction of defense (Van Hulten et al., 2006). Intriguingly, when attacked by pathogens, primed plants showed even higher fitness than non-primed ones. Thus, in pathogenfree environments, the low fitness costs of priming seem to be outweighed by its benefits after pathogen attack (Van Hulten et al., 2006). B. EXPLOITING PRIMING IN GREENHOUSE AND FIELD

Many natural and synthetic compounds can prime plants (summarized in Conrath et al., 2006) (Fig. 2). In addition to those mentioned above, these include Brotomax, a commercial product that contains aluminum lignosulphonate (Fuster et al., 1995; Ortun˜o et al., 1997). Brotomax, BABA, and some other priming-inducing compounds were shown to be potent inducers of stress tolerance in the greenhouse and in the field (Cohen, 2002). Foliar application of BABA, for example, protected field-grown grape against Pl. viticola and suppressed disease symptoms induced by Ph. infestans on potato and tomato plants in the field. BABA also protected melon from sudden wilt disease by Monosporascus cannonballus (Cohen, 2002), and peanut from Cercosporidium personatum in both greenhouse and field trials (Cohen, 2002). As the compound also displays synergistic interactions with

Naïve plant with normal defense capacity is subject to a priming-inducing treatment Natural and synthetic compounds

Primed plant with enhanced defense capacity

Wounding Pathogen attack

‘Priming’ Pathogen attack

Beneficial microorganisms

Expression of induced resistance (reduced disease symptoms)

Natural and synthetic compounds

Pathogen attack

Naïve plant with normal defense capacity

Fig. 2.

(continued )

Dramatically diseased plant

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certain fungicides (Cohen, 2002) BABA has a high potential to become a part of sustainable disease management in the field, even though at higher doses the compound induces female sterility (Kocsis and Jakab, 2008). INA was the first synthetic compound shown to induce both priming of defense responses in the lab (Kauss et al., 1992a) and SAR to fungal and bacterial diseases on various crops in greenhouses and in the field (Kessmann et al., 1994). However, INA and SA were insufficiently tolerated by some crops, which prevented their practical use as plant protection compounds (Ryals et al., 1996). The SAR inducer BTH, which primes plant cell cultures and intact plants for defense responses (see Section II.C.1) was shown to protect various crops against many diseases in the field (Ryals et al., 1996). In contrast to INA and SA, BTH was sufficiently tolerated by most crops. Therefore, the compound became attractive for practical agronomic use. In 1996, BTH was introduced as a ‘‘plant activator’’ (Ruess et al., 1996) with the trade names BionÒ , ActigardÒ , or BoostÒ . However, BTH’s economic success was only limited because it has protective rather than curative activity. Thus, to serve as a protectant the compound must be applied some time before a potential pathogen attack, and even then is seldom as effective as commercial fungicides. Because of this restricted activity, BTH was not sufficiently accepted by farmers who were in favor of using curative standard fungicides. Because of the repudiation of BTH, it became an opportune time to identify plant-protecting compounds combining both direct action on the pathogen and priming-inducing activity in the plant. Some strobilurin fungicides seem to team both these activities (for a review on strobilurins, see Sauter, 2007). In the laboratory, for example, the strobilurin fungicide Pyraclostrobin (trade names: HeadlineÒ , CabrioÒ ) primed the tobacco cultivar ‘‘Xanthi-nc’’ for faster accumulation of PR-1 proteins after infection with TMV or the wildfire pathogen Ps. syringae pv. tabaci. The Pyraclostrobin-induced priming for enhanced PR-1 accumulation after pathogen attack was associated with IR to disease (Herms et al., 2002). IR to viral and bacterial pathogens in Pyraclostrobin-treated plants was also observed in various crops and ornamental plants both in the greenhouse and field Fig. 2. Ways to induce ‘‘priming’’ and resistance to biotic and/or abiotic stress in plants. Spray or drench pretreatment of plants with certain natural or synthetic compounds (SA, some of its analogs, BABA, LPS, and others), wounding, or colonization of the roots by beneficial microorganisms (mycorrhizal fungi, growthpromoting fungi and rhizobacteria), causes a primed state. In the primed condition, plants are able to respond with more robust and/or faster induction of defense responses upon exposure to pathogen attack. Ultimately, priming causes a reduction of disease symptoms through enhanced resistance (upper row) which is not seen in non-primed plants (lower row).

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(Koehle et al., 2003, 2006). In the field the Pyraclostrobin-induced priming was associated with induced tolerance also to abiotic stresses, including drought (www.agweb.com/aims/files/HeadlineAdvantage.pdf). Together, the findings with Pyraclostrobin suggest that this compound, in addition to exerting direct antifungal activity, may also protect plants by priming their defense responses to biotic, as well as abiotic stresses. This conclusion is consistent with an earlier report demonstrating that another commercial fungicide, OryzemateÒ , enhanced the resistance to TMV in tobacco (Koganezawa et al., 1998) and to a bacterial and an oomycete pathogen in Arabidopsis (Yoshioka et al., 2001). OryzemateÒ contains probenazole as the active ingredient which is metabolized to saccharin in treated plants. The latter compound seems to be responsible for the induction of priming in OryzemateÒ -treated plants (Siegrist et al., 1998). In Arabidopsis plants with impaired disease resistance signaling, such as SA-deficient NahG plants or the npr1 mutant, the fungicides metalaxyl, fosetyl-Al, and Cu(OH)2 are much less effective than they are in plants with an intact disease resistance signaling pathway (Molina et al., 1998). This finding suggests that these substances possess some resistance-inducing activity besides their fungicidal properties. The fungicide-mediated resistance could be on the basis of priming because fungicide application alone does not lead to any obvious changes in gene induction in Arabidopsis. A role for NPR1 in priming by fungicide or chemical inducer action is also supported by the fact that NPR1 overexpressing Arabidopsis plants display both potentiated disease resistance and enhanced efficacy of fungicides (Friedrich et al., 2001). Similar observations have recently been made in lab and field trials with the insecticide Imidacloprid. One of its major degradation products, 6-chloronicotinic acid, has a structure very similar to INA. It is supposed to cause the so-called ‘‘stress shield effect’’ on crops by priming them for augmented expression of defense genes, enhancing their tolerance to biotic and abiotic stresses, and increasing plant growth and yield (Thielert, 2006). Various associations of plants with beneficial microbes in the soil have also been demonstrated to induce the primed state and/or resistance to aboveground pathogens and abiotic stresses in the greenhouse and field. For example, growth-promoting Ps. fluorescens (strain WCS417r) induces priming and ISR in plants (see Sections II.B.1 and III.D.1) and Ps. fluorescens (strain WCS374) was demonstrated to suppress Fusarium wilt disease and improve yield in greenhouse-grown radish (Leeman et al., 1995b). Furthermore, seed treatment of various crops and ornamental plants with a mixture of endo- and ectomycorrhizal fungi (available as MycoGrowTM Micronized Endo/Ecto Seed Mix) enhanced their growth and induced

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resistance in the greenhouse as well as under field conditions (www.fungi. com/index.html). Similarly, Pi. indica systemically primes barley against biotic and abiotic challenges and increases growth and yield of Spilanthes calva and Withania somnifera in the field (Rai et al., 2001).

V. CONCLUSIONS Over the past decade it has become increasingly clear that priming is a complex mechanism that is part of various types of IR in plants. Priming allows plants to activate defense responses more quickly and/or effectively when exposed to biotic or abiotic stress. Because of its advantageous economic features, priming represents an ecologically important adaptation to withstand environmental challenges. The phenomenon can contribute to new concepts for disease control as priming provides broad-spectrum resistance without significantly affecting growth and fruit or seed set. Priming offers a smart, effective, and realistic option for effective plant protection, especially when combined with conventional pesticides. Taking advantage of the natural, broad-spectrum defense capacity of plants in the field will be facilitated by a better understanding of the molecular, physiological, and ecological aspects of priming, which represents an exciting challenge for future research.

ACKNOWLEDGMENTS Research on priming in my laboratory is supported by the Peter and Traudl Engelhorn Foundation and, in part, by BASF and the German Science Foundation (DFG).

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Transcriptional Regulation of Plant Defense Responses

MARCEL C. VAN VERK,* CHRISTIANE GATZ{ AND HUUB J. M. LINTHORST*,1

*Institute Biology Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands { Department of General and Developmental Plant Physiology, Albrecht-von-Haller-Institut, Untere Karspu¨le 2, 37073 Go¨ttingen, Germany

I. Plant Immune Signaling Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Defense Signaling Regulatory Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Jasmonate Signal Transduction .............................................. B. Ethylene Signal Transduction ................................................ C. SA Signal Transduction ....................................................... III. Transcription Factors Regulating Plant Defense Gene Expression . . . . . . . . A. AP2/ERF Transcription Factors ............................................. B. MYB Transcription Factors .................................................. C. MYC Transcription Factors .................................................. D. bZIP Transcription Factors................................................... E. WRKY Transcription Factors ............................................... IV. Regulation of Plant Defenses at the Chromosomal Level . . . . . . . . . . . . . . . . . . A. Chromatin Modifications and Gene Expression........................... B. Chromatin Modifications in Plants .......................................... C. Chromatin Modifications at Promoters Involved in Innate Immunity............................................................. Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51010-5

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ABSTRACT Plants possess constitutive as well as inducible defense systems to oppose attack by pathogens and herbivores. Signal-transduction pathways mediated by the plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are involved in regulating appropriate defense responses. Extensive cross-talk between these different signal-transduction pathways allows the plant to fine-tune its defenses against different types of pathogens and insect attackers. This review presents brief overviews of the separate JA, ET, and SA signal-transduction pathways, followed by a description of the main classes of transcription factors involved in defense gene activation. The last part is devoted to recent work highlighting the regulation of plant defense responses by transcriptional reprogramming at the chromosomal level.

I. PLANT IMMUNE SIGNALING PATHWAYS As plants are sedentary organisms, they possess elaborate mechanisms to defend themselves against attack by pathogens and pests. Successful defense relies on early recognition of the attackers and activation of appropriate defense responses. Different defense strategies have evolved against biotrophic and necrotrophic pathogens and insect attack. Defense against biotrophic pathogens is typically mediated by a signal-transduction route in which the endogenous plant compound salicylic acid (SA) plays a prominent role (Dong, 1998). In contrast, attack by necrotrophic pathogens and herbivorous insects triggers a signal-transduction pathway that is characterized by the signal molecule jasmonic acid (JA) (Howe, 2004). Both signaling pathways affect each other through extensive cross-talk occurring at different levels, while additional modulation of the defense response is brought about by the effects of a third signal transduction cascade triggered by ethylene (ET) produced upon attack (Koornneef and Pieterse, 2008; Leon-Reyes et al., 2009; Reymond and Farmer, 1998; Spoel and Dong, 2008). Although this system of interacting signaling pathways may underscore the ability of the plant to specifically, efficiently, and effectively cope with the multitude of biotic threats from its environment, it is evident that the immense complexity of this signaling network stands in the way of an easy and clear-cut understanding of how exactly plant defense works. Nevertheless, the end result of the signal transduction is the induced production of defense proteins that directly or indirectly inhibit proliferation of the attacker. Upon infection or attack, various defense responses are induced, requiring these proteins to be newly synthesized. Figure 1 shows that in Arabidopsis thaliana, each of the signal-transduction pathways acts to activate a distinct set of

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Control ET 3 6 12 24 3 6 12 24

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JA SA 3 6 12 24 3 6 12 24 PR-3 / ChiB PR-4 / HEL VSP1 GRX480 PDF1.2 PR-2 / BGL2 PR-1 GST1 Actin3

Fig. 1. Time course showing expression of ethylene-, jasmonic acid-, and salicylic acid-inducible defense-related genes in Arabidopsis thaliana. A total of five plants per time point per treatment were mock treated, or sprayed with 10 mM ethylenereleasing ethephon (ET), 250 m jasmonic acid (JA), or 5 mM salicylic acid (SA). After 3 h the plants were sprayed with tap water. Plants were harvested 3, 6, 12, or 24 h after treatment, after which RNA was extracted and loaded on denaturing agarose gels, subjected to electrophoresis and blotted. The blots were hybridized to cDNA probes corresponding to the various marker genes, as indicated. A cDNA probe for Actin 3 was used to check for equal loading. Abbreviations: BGL, -GLUCANASE; ChiB, CHITINASE B; GRX, GLUTAREDOXIN; GST, GLUTATHION-S-TRANSFERASE; HEL, HEVEIN-LIKE; PDF, PLANT DEFENSIN; PR, PATHOGENESIS-RELATED; VSP, VEGETATIVE STORAGE PROTEIN.

defense genes. Marker genes are specifically expressed via a single pathway, as for example VEGETATIVE STORAGE PROTEIN 1 (VSP1) by JA, GLUTAREDOXIN 480 (GRX480) by SA, and PATHOGENESIS-RELATED 3/CHITINASE B (PR-3/ChiB) and PR-4/HEVEIN-LIKE (HEL) by ET. Others respond to two signals, like PLANT DEFENSIN 1.2 (PDF1.2) to ET and JA, or PR-1 and GLUTATHION-S-TRANSFERASE 1 (GST1) to ET and SA. From the increase in mRNA levels upon signal molecule application, it is evident that gene expression must require specific transcription factors that are activated or produced at the end of the signaling pathways. Without trying to cover all the details that have accumulated in the past decades concerning transcription factors involved in biotic stress responses, this chapter aims at providing a timely overview of the most important classes of transcription factors engaged in the defenses that are mediated through the three signal-transduction pathways described above. Obviously, the model species Arabidopsis plays a central role in this review, although, where appropriate, results from other plant species are also described.

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II. DEFENSE SIGNALING REGULATORY COMPOUNDS A. JASMONATE SIGNAL TRANSDUCTION

Induced defense against necrotrophic pathogens and herbivorous insects involves a signal-transduction pathway in which the plant hormone JA plays a central role. Jasmonates are oxylipins that have an important function in the positive regulation of secondary metabolites like alkaloids, glucosinolates, phenylpropanoids, and terpenoids. Three types of stress activate the JA-signaling pathway. In tobacco, it was shown that perception of a primary wound results in accumulation of JA and its methyl ester, MeJA. An important factor in positive regulation of JA biosynthesis upon wounding is the wound-inducible protein kinase (WIPK), a member of the class of mitogen-activated protein kinases (MAPK). When WIPK is impaired, accumulation of JA or MeJA upon wounding no longer occurs (Seo et al., 1995). Overexpression of WIPK leads to accumulation of JA and proteinase inhibitor 2 (PIN2) (Seo et al., 1999). In Arabidopsis, a similar MAPK, MPK4, which is also rapidly activated upon wounding and is involved in JA signal transduction, was found (Ichimura et al., 2000). Mutant screens for phenotypes showing impaired characteristics of JA signaling or impaired resistance against a variety of biotic stresses have revealed a number of genes involved in JA biosynthesis and signal transduction. In the fatty acid desaturase triple mutant fad3-2 fad7-2 fad8, no production of -linolenic acid occurs. -Linolenic acid is the precursor for jasmonates that are synthesized via the octadecanoid pathway (McConn and Browse, 1996). This pathway consists of a number of steps of which most enzymes have been identified. The -linolenic acid is oxygenated by lipoxygenases (LOXs) to 13-hydroperoxy-linolenic acid (13-HPOT), which is then released from chloroplast membranes by lipases. In the plastids, 13HPOT is converted by allene oxide synthase (AOS) and allene oxide cyclase (AOC) to 12-oxo-phytodienoic acid (OPDA). Next, OPDA is transported to the peroxisomes, where it is reduced by OPDA-reductase 3 (OPR3) and further converted by three cycles of -oxidization by acyl-CoA oxidase (ACX), multifunctional protein (MFP), and 3-ketoacyl-CoA thiolase (KAT) to JA (Wasternack, 2007). JA can be metabolized in the cytoplasm to several derivatives. The best characterized are the volatile MeJA, synthesized by JA carboxyl methyltransferase (JMT) (Seo et al., 2001), and JAamino acid conjugates, synthesized by the AMP-transferase activity of JA conjugate synthase Jasmonate resistant 1 (JAR1) (Staswick and Tiryaki, 2004). Active signaling molecules are the JA precursor OPDA, JA itself

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and its derivatives MeJA, and JA-isoleucine (JA-Ile). JA regulates its own synthesis positively by stimulating the expression of most of the JA biosynthetic genes. Overexpression of ORA47, an APETALA2/Ethylene-response factor (AP2/ERF) type transcription factor, results in an increased amount of the JA precursor OPDA. This accumulation of OPDA is caused by the enhanced expression of various JA biosynthetic genes (LOX2, AOS, AOC2) by ORA47, which is induced by JA, and therefore, having a positive feedback regulatory role for JA biosynthesis (Pre´, 2006). Treatment of plants with JA overcomes mutations in any of the biosynthetic genes. The constitutive expression of VSP1 mutant (cev1) acts at an early step in the JA and ET signal-transduction pathways, overproducing JA and ET and displaying constitutive expression of JA-inducible genes (Ellis et al., 2002). CEV1 encodes a cellulose synthase, indicating the involvement of the cell wall in defense responses. Root growth is inhibited by JA. Therefore, it is a useful selection feature for mutant screens to identify genes involved in JA signaling. In an ethyleneinsensitive (ein3) mutant background, where ET is not able to repress JAregulated responses to stress, five JA-insensitive (jai1-5) mutants were identified (Lorenzo et al., 2004). JAI1, also known as JIN1, encodes the basic helixloop-helix (bHLH) transcription factor AtMYC2, which is rapidly induced by JA. AtMYC2 controls two main branches of JA signaling. The first branch consists of genes that are activated by AtMYC2 in the systemic responses to wounding or chewing herbivores. The second branch results in repression of genes involved in defense responses against pathogens and in this way acts as an integrator of different environmental stress responses (Lorenzo et al., 2004). The JAI2 locus corresponds to JAR1, as described above. In a screen for Arabidopsis mutants insensitive to coronatine and MeJAmediated growth inhibition, the mutant coronatine insensitive 1 (coi1-1) was isolated (Feys et al., 1994). COI1 encodes a JA-receptor protein that is required for responses mediated through JA-Ile. Coronatine is an effector produced by several pathovars of Pseudomonas syringae (Mitchell and Young, 1978). An example of a fungus that directly triggers COI1 without inducing the JA biosynthetic pathway is Fusarium oxysporum. It has been proposed that F. oxysporum is capable of producing an oxylipin-like chemical just as Ps. syringae (Thatcher et al., 2009). By mimicking JA-Ile it induces JA signal transduction resulting in suppression of SA-mediated defense against the biotrophic Ps. syringae. The COI1 gene was mapped to a small region of the genome and located by complementation. COI1 corresponds to the JAI5 locus. The amino acid sequence of the COI1 protein contains an F-box motif and has similarity with Transport inhibitor response 1 (TIR1),

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an F-box protein that is part of an SCF complex and functions as an auxin receptor (Ruegger et al., 1998; Xie et al., 1998). F-box proteins associate with Cullin (Cul1), Rbx1, and Skp1 to form an Skp, Cullin, F-box (SCF) complex, also known as E3 ubiquitin ligase. The F-box protein in this complex functions as a receptor to target interacting proteins to be ubiquitinated and degraded by the 26S proteasome. In vivo interaction of the COI1 F-box protein with Cul1, Skp1, and Rbx1 was shown by co-immunoprecipitation, linking COI1 to the SCF complex. Genetic and molecular analyses show the involvement of AUXIN RESISTANT 1 (AXR1), CONSTITUTIVE PHOTOMORPHOGENIC 9 (COP9) and SUPPRESSOR OF THE G2 ALLELE OF SKP1 VARIANT B (SGT1b) (corresponding to the JAI4 locus) as regulators of the SCF complex. Mutations in these regulators result in a reduced response in JA signaling (Feng et al., 2003; Lorenzo and Solano, 2005; Tiryaki and Staswick, 2002). The coi1-1 mutant fails to express JA-regulated genes and is defective in resistance against necrotrophic pathogens and insects (Turner et al., 2002). This indicates the importance of ubiquitination in the JA-signaling pathway. The function of COI1 is specific to the JA pathway, whereas other parts of the SCFCOI1 complex (SGT1b/ JAI4 and AXR1) are shared by other pathways. A breakthrough in understanding how COI1 mediates JA signaling via the SCFCOI1 complex came with the discovery of JA ZIM-domain (JAZ) repressor proteins. One member of this group, JAZ3 (corresponding to the JAI3 locus), interacts directly with MYC2 and acts as a negative regulator of MYC2-dependent gene expression. JA-Ile produced after biotic stress or coronatine are proposed to bind the leucine-rich repeat (LRR) domain of COI1, thereby generating a high affinity-binding site for JAZ3. Polyubiquitinylation of JAZ3 by the SCFCOI complex results in its degradation through the 26S proteasome. The release of the JAZ3 repressor frees MYC2 to activate transcription of its target genes. As one of MYC2 target genes is JAZ3, this process constitutes a direct negative regulatory loop to dampen MYC2 activity in cells with low levels of JA (Chini et al., 2007; Thines et al., 2007). B. ETHYLENE SIGNAL TRANSDUCTION

The simplest hormone in plants is the gaseous ET. ET is involved in various developmental processes, such as plant growth and fruit ripening. Besides these processes, ET is also involved in environmental stress signaling upon wounding or pathogen attack. The biosynthesis of ET proceeds via a short pathway. First, methionine is activated by ATP through the action of methionine adenosyltransferase,

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resulting in S-adenosylmethionine (SAM). In the next step SAM is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). Finally, production of ET from ACC is catalyzed by ACC oxidase (ACO). The biosynthesis of ET is regulated through a range of positive and negative factors. Formation of ACC is the rate-limiting step in the pathway. Arabidopsis contains nine ACS genes encoding three types of enzymes, which are under strict regulatory control. The first type consisting of ACS2 and ACS6 can be phosphorylated by MAPK 6 (MPK6). This phosphorylation stabilizes the protein, which results in increased ET production. Presumably, phosphorylation of ACS2 and ACS6 protects these proteins from recognition and breakdown by the 26S proteasome pathway (Liu and Zhang, 2004). ACS4, ACS5, and ACS9 are members of the second type of ACSs, while ACS7 comprises the third type. The type II ACSs are under control of ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-LIKE1 and 2 (EOL1/EOL2). ETO1 is a member of the Broad complex/Tramtrack/Bric-a-brac (BTB) proteins, and together with Cul3a/b and Rbx1 forms an E3 ubiquitin protein ligase. Binding of type II ACSs by ETO1 (and EOL1/EOL2) targets them for ubiquitination and degradation by the 26S proteasome pathway, thereby negatively regulating ET production. It is likely that type I ACSs can also be recognized by a BTB protein and, vice versa, that the type II enzymes can be phosphorylated to prevent them from being targeted for degradation (Christians et al., 2009; Guzma´n and Ecker, 1990; Wang et al., 2004). After production, ET is perceived by a group of (hybrid) histidine kinases that are membrane-bound ET receptors: Ethylene response 1 (ETR1)/Ethylene insensitive 1 (EIN1), ETR2, EIN4, Ethylene-response sensor 1 (ERS1) and ERS2 (Bleecker et al., 1988; Chang et al., 1993; Hua et al., 1995, 1998; Roman et al., 1995; Sakai et al., 1998). Pull-down experiments and yeast twohybrid interaction assays show that Constitutive triple response 1 (CTR1), a Ser/Thr kinase, is in the same signaling complex as ETR1 and can interact with ERS1 and ETR2 (Cancel and Larsen, 2002; Clark et al., 1998; Gao et al., 2003; Kieber et al., 1993). CTR1 has a negative regulatory function on ET signaling by actively suppressing the signaling pathway in the absence of ET. Upon binding of ET to the receptors, CTR1 is no longer capable of repressing EIN2. EIN2 is a membrane-bound protein that directly or indirectly prevents the key ET response transcription factors EIN3 and EIN3like 1 (EIL1) to bind to EIN3 binding F-box protein 1 and 2 (EBF1, EBF2) that are part of a SCF E3 ligase complex (SCFEBF1/2), with the result that EIN3 and EIL1 are no longer degraded through the 26S proteasome pathway (Binder et al., 2007; Guo and Ecker, 2003; Potuschak et al., 2003). EIN3 and EIL1 regulate the downstream targets of the ET-signaling pathway among which is the Ethylene-response factor 1 (ERF1) (Solano

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et al., 1998). Besides directly targeting downstream targets, EIN3 and EIL1 also induce transcription of EBF1 and EBF2 (Konishi and Yanagisawa, 2008). This results in a negative feedback loop that targets EIN3 and EIL1 for degradation when ET levels decrease. This feedback loop is under control of EIN5, a 50 !30 exoribonuclease (XRN4) that acts downstream of CTR1. In the presence of ET, EIN5 promotes EBF1 and EBF2 mRNA decay, which allows the accumulation of EIN3 (Olmedo et al., 2006). In addition to being regulated by the proteasome pathway, EIN3 can, just like the ACSs, be stabilized by phosphorylation. This occurs via a MAPK cascade consisting of CTR1 (a MAPK kinase kinase), MKK9, and MPK3/ MPK6. It remains unclear how CTR1, a dominant negative regulator, can positively activate the phosphorylation of EIN3 via MKK9—MPK3/MPK6 (Yoo et al., 2008). C. SA SIGNAL TRANSDUCTION

For the defense response launched after attack by biotrophic pathogens genetic data from Arabidopsis have led to a signal-transduction model in which SA plays a central role. Tissue colonization and pathogen proliferation are caused by pathogen effectors, previously named avirulence (Avr) proteins, which are targeted (in)to the host tissues to promote pathogen virulence (Jones and Dangl, 2006). In incompatible plant–pathogen interactions these effectors are recognized by specific R gene-encoded receptors. Examples are, for instance, the Ps. syringae effector AvrRps4, which is recognized by the Arabidopsis Toll/Interleukin1 receptor—nucleotide binding site—leucine-rich repeat (TIR-NBS-LRR) receptor RPS4 (Aarts et al., 1998). Innate immunity or basal defense has been found to have significant overlap with R gene-mediated resistance responses, including production of SA and expression of SA-regulated defense genes (Tsuda et al., 2008). In this case, pathogen-associated molecular patterns (PAMPs), such as conserved fragments of bacterial flagellin or elongation factor Tu, or microbe-induced molecular patterns (MIMPs), that are released from the host by pathogen activity, function as elicitors that are recognized by LRR receptor kinases, that is, the flagellin receptor Flagellin sensing 2 (FLS2) and the EF-Tu receptor EFR (Kunze et al., 2004; Mackey and Mcfall, 2006; Turner et al., 2002; Zhao et al., 2005). Subsequent signal transduction from the flagellinactivated FLS2 receptor involves MAPK cascades with intricate positive and negative regulation on the establishment of immunity (Asai et al., 2002; Chinchilla et al., 2007). In Arabidopsis, the biosynthesis of pathogen-induced SA depends on isochorismate synthase (ICS), the product of the ICS1 gene that converts part of

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the ubiquitous chorismate into isochorismate. Isochorismate is an intermediate in the synthesis of phylloquinone (vitamin K1), which is an essential component of the plant’s photosynthetic machinery (Verberne et al., 2007; Wildermuth et al., 2001). In noninfected cells SA is present only at very low concentrations, but upon pathogen attack its level increases rapidly. Apparently, after attack isochorismate is channeled away from phylloquinone synthesis toward synthesis of SA. Also bacteria synthesize SA from isochorismate in a single-step reaction involving the enzyme isochorismate pyruvatelyase (IPL) (Gaille et al., 2002). However, no such activity has yet been found in plants. Genetic evidence has indicated that upstream of ICS1, several more genes are necessary to mount the defense response. Genes involved in the earliest steps of the signal-transduction pathway upstream of SA, that is, PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) encode proteins with similarity to lipases. EDS1 is probably activated upon elicitor recognition by R gene-encoded cytoplasmic TIR-NB-LRR receptors (Wirthmueller et al., 2007). How exactly this activation is linked to induction of SA biosynthesis is not known, however, heterodimerization of EDS1 and PAD4 and their nuclear localization may be important for subsequent steps in the signaling pathway (Feys et al., 2001). Recently, it was found that EDS1 expression is repressed by the Ca2þ/ calmodulin-binding transcription factor Serine/threonine protein kinase 1 (AtSR1) binding to a conserved CGCG element in the EDS1 promoter, indicating that SA levels are regulated by Ca2þ (Du et al., 2009). Situated downstream of EDS1, but upstream of SA synthesis is EDS5 (Rogers and Ausubel, 1997). Pathogen infection strongly induces the accumulation of the EDS5 transcript in an EDS1- and PAD4-dependent manner. The increase in EDS5 mRNA precedes SA accumulation, supporting a role for EDS5 in this process. Furthermore, EDS5 gene expression is also induced by treatment with exogenous SA, suggesting a positive feedback loop for enhanced SA production during the defense response (Nawrath et al., 2002). Increased levels of SA induce a state of enhanced defensive capacity, called systemic acquired resistance (SAR), that confers broad-spectrum resistance to subsequent pathogen infection (Ross, 1961). eds5 mutant plants cannot mount the SAR response and are unable to accumulate high levels of SA (Nawrath and Me´traux, 1999). The EDS5 protein has homology to bacterial multidrug and toxin extrusion (MATE) antiporters that export toxic compounds across the plasma membrane in a process energized by Hþ or Naþ electrochemical gradients. EDS5 contains a number of transmembrane domains suggesting that the protein is membrane-localized and might likewise function as a

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transmembrane transporter of small compounds (Nawrath et al., 2002). Chloroplast localization of EDS5 was recently confirmed by transient transformation experiments with GREEN FLUORESCENT PROTEIN (GFP)tagged EDS5 (Ishihara et al., 2008). Based on its homology to MATE transporters and the initial placement of the EDS5 gene before ICS1 in the SA signaling pathway, Nawrath et al. (2002) suggested that EDS5 functions as a transporter of precursors of SA synthesis. Alternatively, EDS5 could be the chloroplast-cytosol translocator of SA. This would equally rightfully explain the inability of the eds5 mutant to mount the SAR response due to lack of sufficient SA in the cytosol. AVRPPHB SUSCEPTIBLE 3 (PBS3), of which the pathogen-induced expression is highly correlated with ICS1, is acting downstream of SA. In the pbs3 mutant accumulation of SA-glucoside and expression of PR-1 are drastically reduced. PBS3 is a member of the auxin-responsive GH3 family of acyl-adenylate/thioester forming enzymes of which some have been shown to catalyze hormone–amino acid conjugation, like JAR1 in the JA pathway. This has led to speculation that SA–amino acid conjugates are involved in SA signal transduction (Jagadeeswaran et al., 2007; Nobuta et al., 2007). Upon a local primary infection with a necrotizing pathogen, SAR primes distal tissues for defense against secondary infections (Conrath et al., 2006). Methyl SA (MeSA) was identified as a mobile signal that is critical for the development of SAR in tobacco. SA produced at the primary infection site is converted by a SA methyl transferase (SAMT) to MeSA and loaded into the vascular system for transport to distant plant tissues. Upon arrival in these systemic tissues, MeSA is converted back to active SA by the esterase SAbinding protein 2 (SABP2), which triggers defense gene expression in these tissues (Park et al., 2007). Recently, it was found that in Arabidopsis MeSA is not the systemic signal for SAR. Instead, azelaic acid was identified as a mobile signal for priming defense, for which also the gene AZELAIC ACID INDUCED 1 (AZI1) is required (Attaran et al., 2009; Jung et al., 2009). One of the effects triggered by SA is the elicitation of an imbalance in the redox state of the cell, which results in reduction of specific disulfide bridges in the ankyrin-repeat protein Nonexpressor of PR genes 1 (NPR1). NPR1 plays a central role in defense responses and is required for the establishment of SAR and the expression of SA-dependent defense genes like PR-1. NPR1 exists in the cytoplasm as a multimeric complex. Reduction results in release of NPR1 monomers and their subsequent translocation into the nucleus, where they interact with TGA transcription factors and activate defense gene expression (Kinkema et al., 2000; Mou et al., 2003). NPR1 contains an ankyrin-repeat domain, which facilitates protein–protein interactions (Cao et al., 1997). Moreover, it harbors a BTB domain, which might be

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ubiquitinylated by an E3 ubiquitin ligase complex and targeted for degradation by the proteasome. Recently, it was found that upon initiation of PR gene transcription by the TGA–NPR1 complex NPR1 is phosphorylated, possibly by a factor of the basal transcription machinery, and becomes inactive. Phosphorylation results in enhanced affinity for CUL3 and consequently rapid degradation by the proteasome. This will clear the promoter to reinitiate transcription, resulting in a pulse-wise activation of gene expression as long as nuclear NPR1 is available (Spoel et al., 2009). Based on these results, NPR1 seems to act as a co-activator that is recruited to the promoter by interaction with TGA transcription factors (Rochon et al., 2006). However, it still has to be considered that NPR1 is only necessary if a functional SUPPRESSOR OF NPR1 (SNI1; Li et al., 1999) allele is present. SNI1 is an armadillo repeat protein that may form a scaffold for interaction with proteins that modulate transcription (Mosher et al., 2006), leading to transcriptional repression. The Whirly protein AtWhy1 is a transcription factor that is also involved in SA-dependent basal resistance. Interestingly, AtWhy1 acts independently of NPR1 (Despre´s et al., 2000). In the npr1-1 mutant not only the SA signaling pathway is disrupted, but also JA/ET signaling is affected, indicating a role of NPR1 in both SA and JA/ET signal transduction (Pieterse et al., 1998).

III. TRANSCRIPTION FACTORS REGULATING PLANT DEFENSE GENE EXPRESSION As indicated in the above sections, the transcription factors involved in the various defense pathways mostly belong to five main groups. Many transcription factors involved in JA and ET signal transduction are members of the AP2/ERF group, while for example, SA signal transduction involves mostly WRKY and bZIP members. Venn diagrams have been created using publicly available microarray datasets of SA-, ET-, and MeJA-treated Arabidopsis plants (Fig. 2). Although the numbers of genes of which the expression was found to change are rather small, it is evident that considerable overlap occurs in the induction characteristics of these transcription factors. This overlap allows integration of different signals and, thereby, the fine-tuning of plant defense responses to attackers activating different signaltransduction pathways. The diagrams show that there is no strict correlation between signaling pathway and transcription factor type and that transcription of the genes encoding these transcription factors can be either up- or downregulated by the treatments. In the next sections these main types of transcription factors are being discussed.

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Fig. 2. Venn diagrams of transcription factor-encoding genes in Arabidopsis thaliana responsive to treatment of the plants with salicylic acid (SA), methyl jasmonate (MeJA), or ethylene (ET). Separate Venn diagrams for AP2/ERF-, bHLH-, bZIP-, MYB-, and WRKY-encoding genes were generated. Transcription factor gene IDs were obtained from the Database of Arabidopsis transcription factors (DATF) (Guo et al., 2005) and loaded in Genevestigator V3, where they were used in a bicluster analysis, sorted on stimulus, and analyzed using the bimax algorithm (Hruz et al., 2008). Expression data from 3 h SA, 3 h ET, or 2 h MeJA treatment were selected and the numbers of upregulated or downregulated genes determined. For each set of genes the left diagrams (dark grey) correspond to genes that are upregulated by the various treatments, the diagrams at the right (light grey) show the genes that were downregulated by the treatments.

A. AP2/ERF TRANSCRIPTION FACTORS

With over 140 predicted members in Arabidopsis the AP2/ERF family of plant transcriptional regulators is one of the largest. AP2/ERF transcription factors are characterized by a 58- to 60-amino acid DNA-binding domain first identified in APETALA2 (AP2) and the Ethylene-response factors (ERF) (Jofuku et al., 1994; Ohme-Takagi and Shinshi, 1995). It has been demonstrated that members of this family have important functions in a broad range of biological processes, from growth and development to the response to environmental stimuli (Nakano et al., 2006, and references therein). Within the AP2/ERF family, members can be divided into AP2like transcription factors containing two AP2 domains, and ERF-like factors with a single AP2 domain. In the last subfamily, the proteins Related to ABI/ VP1 1 (RAV1) and RAV2 are classified as a separate group because in

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addition to the AP2 domain they contain a second DNA-binding domain, B3. Both domains bind autonomously to the DNA motifs CAACA and CACCTG, respectively, and together they achieve a high DNA-binding affinity of the protein (Kagaya et al., 1999). The other ERF-like members are separated into a class that is responsive to drought and/or low temperature. They bind the C-repeat (CRT) or dehydration-responsive element (DRE) in the promoters with the core sequence CCGAC. The other class of AP2/ ERF proteins with a single AP2-domain is responsive to ET and bind ethylene-response elements (ERE), also known as the GCC-box (GCCGCC) (Allen et al., 1998). The GCC-box is found in many promoters of biotic stress genes that are inducible by ET. The GCC-box also occurs in the promoters of SA-inducible PR genes, indicative of possible cross-talk between ET and SA. AP2/ERF proteins are also involved in JA-inducible gene expression. STRICTOSIDINE SYNTHASE (STR) gene expression in Catharanthus roseus depends on the Octadecanoid-responsive C. roseus AP2/ERF transcription factors ORCA2 and ORCA3. These transcription factors bind to a GCC-like box that is a JA- and elicitor-responsive element (JERE) in the promoter of the STR gene (Menke et al., 1999). STR is an important enzyme in the terpenoid indole alkaloid (TIA) biosynthetic pathway. TIAs offer the plant protection against pathogens and UV radiation (Meijer et al., 1993; Van der Fits and Memelink, 2000). In Arabidopsis the subgroup of AP2/ERF transcription factors that are rapidly induced by JA is known as Octadecanoid-responsive Arabidopsis AP2/ERF (ORA). Multiple ORA transcription factors have a key role in disease resistance and signaling. As described above, ORA47 plays a role in the positive feedback regulation of JA biosynthetic genes by JA. ORA37/AtERF4 acts as a negative regulator of various defense genes, including PDF1.2, ChiB and -GLUCOSIDASE 2 (BGL2) upon treatment with JA and/or ET (McGrath et al., 2005; Pre´, 2006; Yang et al., 2005). Contrary to ORA37, ORA59 positively regulates expression of these defense genes, integrating both JA and ET signals. Overexpression of ORA59 results in increased resistance against the necrotrophic fungus Botrytis cinerea. Induction of defense genes PDF1.2 and HEL in ORA59-overexpressing Arabidopsis is also observed in the coi1-1 background, placing ORA59 downstream of COI1. Silencing of ORA59 using RNA interference (RNAi) results in reduced resistance against B. cinerea. Besides ORA59, also ERF1 has been reported to integrate JA and ET signals and to synergistically induce PDF1.2 downstream of COI1 (Lorenzo et al., 2003). Although ORA59 and ERF1 appear to have similar functions, RNAi-silenced lines of ORA59 that still activate ERF1 upon application of JA or ET, nevertheless are impaired in PDF1.2 expression, indicating an essential role of ORA59 in this signaling branch. Another

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difference between ERF1 and ORA59 is that after induction, ERF1 represses JA-related marker genes such as VSP, while ORA59 does not (Pre´ et al., 2008). Apart from factors that activate gene expression by binding to the GCCbox (AtERF1, 2, and 5), the AtERF group of transcription factors also contains GCC-box-binding repressors (AtERF3, 4, and 7–12; cf. ORA37/ AtERF4 described above) (Fujimoto et al., 2000; Yang et al., 2005). AtERF14 induces PDF1.2 relatively late in comparison to ORA59 and ERF1; its expression is induced only by ET. This indicates that Arabidopsis AP2/ERF transcription factors can be divided into a group that integrates JA and ET pathways to activate defense gene expression, a group that selectively represses JA-responsive genes, and a group that induces gene expression through ET only (On˜ate-Sa´nchez et al., 2007). Interactions of AP2/ERF proteins with other transcription factors may also play an important role in defense gene regulation. In a screen for interactors of the bZIP transcription factor TGA4, AP2/ERF transcription factor ERF72 was identified. ERF72 binds a GCC-box in the promoter of PRB-1b, encoding a basic PR-1 type protein from tobacco (Bu¨ttner and Singh, 1997; Sessa et al., 1995). Another screen for interactors of the tomato Ser/Thr kinase Pto—the product of the R gene recognizing the Ps. syringae effector AvrPto—using the yeast two-hybrid system resulted in several AP2/ ERF proteins, which were named Pto‐interacting (Pti) proteins Pti4, Pti5, and Pti6 (Tang et al., 1996; Zhou et al., 1997). ET treatment resulted in rapid induction of Pti4. Pti4 can be phosphorylated by the Pto kinase, enhancing its ability to activate defense gene expression (Chakravarthy et al., 2003; Gu et al., 2000). Overexpression of Pti4 in transgenic Arabidopsis resulted in increased gene expression of GCC-box containing PR genes (Wu et al., 2002). That phosphorylation can be important for AP2/ERF transcription factor activity was also shown for the rice AP2/ERF transcription factor Ethylene-responsive element binding protein (OsEREBP1), which after phosphorylation showed an enhanced binding to GCC-boxes (Cheong et al., 2003). B. MYB TRANSCRIPTION FACTORS

The first MYB factor identified was v-MYB from Avian myeloblastosis virus. It probably originated by capture from a vertebrate gene, which was subsequently converted into an oncogene. Vertebrates contain three Myb genes (c-Myb, A-Myb, and B-Myb) that all have MYB DNA-binding domains. The MYB DNA-binding domain contains up to three repeats that each form a helix-turn-helix structure characterized by a series of regularly spaced

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tryptophan residues. In c-MYB there are three different versions of these repeats, referred to as R1, R2, and R3. Other MYB proteins are characterized based upon their similarity with these repeats. MYB factors that have one repeat are referred to as MYBR1, MYB proteins with two repeats named R2R3-MYB, and proteins with three repeats named MYBR3. Plants have very large MYB families; for example, Arabidopsis contains 125 MYB genes. Most plant MYB factors belong to the R2R3 group, which is divided in two types that can bind different DNA sequences. Type I binds the DNA sequence (T/C)AAC(T/G)G, while type II binds to G(G/T)T(A/T)G(G/T)T (Eulgem, 2005; Stracke et al., 2001). In Arabidopsis, only a few R2R3-MYB proteins are involved in defense-related pathways. Many MYB transcription factors are involved in the regulation of the biosynthesis of both primary and secondary metabolites. As an example of secondary metabolites, the group of the glucosinolates contains important defense compounds against herbivores and microorganisms. There are two main branches in glucosinolate biosynthesis. One leads to the formation of aliphatic glucosinolates derived from methionine, while the other branch leads to indole glucosinolates, which are derived from tryptophan. MYB 28, also known as Production of methionine-derived glucosinolate 1 (PMG1), plays a key role in MeJA-induced biosynthesis of the aliphatic glucosinolates. In addition, MYB29/PMG2 has a modest role in regulation of this pathway (Hirai et al., 2007). MYB34/Arabidopsis P450 reductase (ATR1) has a similar function for the tryptophan-derived glucosinolates as MYB29/PMG2 has for the methionine-derived glucosinolates (Celenza et al., 2005). Pathways for other secondary metabolites are also regulated by MYB transcription factors. For instance, the flavonoid biosynthetic pathway is positively regulated by AtMYB75 (Borevitz et al., 2000). During the hypersensitive response (HR) to the bacterial pathogen Xanthomonas campestris pv. campestris, AtMYB30 is activated early and independently of NPR1. Overexpression of AtMYB30 results in a stronger HR response against avirulent bacterial pathogens and increases the resistance against a variety of bacterial pathogens, while silencing of AtMYB30 in Arabidopsis using antisense lines strongly decreases the HR response against avirulent bacterial pathogens. This indicates a role for AtMYB30 in promoting HR-related cell death and resistance against bacterial pathogens (Daniel et al., 1999; Raffaele et al., 2006; Vailleau et al., 2002). Besides resistance against HR-inducing pathogens, MYB transcription factors also play an important role in resistance against necrotrophic pathogens like B. cinerea and Alternaria brassicicola, for example, BOTRYTIS SUSCEPTIBLE 1 (BOS1)/AtMYB108. This pathogen-induced MYB factor functions to reduce spread of the pathogen through the plant tissue. The expression of

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AtMYB108 is severely impaired in the coi1-1 mutant, indicating an important role for the JA-signaling pathway in this defense response (Mengiste et al., 2003). AtMYB72 has been shown to be an essential component of rhizobacteria-mediated induced systemic resistance (ISR). Induction of this gene in roots by nonpathogenic Pseudomonas fluorescens WCS417r bacteria is necessary for priming of systemic JA/ET-dependent defense responses against various pathogens (Van der Ent et al., 2008). Defense responses regulated by MYB transcription factors seem to cover all signaling pathways and act against many types of pathogens. MYB transcription factors also play roles in the defense response against insects. Caterpillars of the small cabbage white, Pieris rapae, induce local expression of AtMYB102. Overexpression of AtMYB102 results in upregulation of a large number of genes that are involved in cell wall modifications. On T-DNA insertion lines lacking AtMYB102 the development of Pi. rapae proceeds faster than on wild-type plants. Possibly, plants lacking AtMYB102 can no longer support the induction of cell wall modifications that interfere with Pi. rapae feeding (De Vos et al., 2006). C. MYC TRANSCRIPTION FACTORS

The MYC family of transcription factors is part of a large transcription factor family that consists of more than 120 basic helix-loop-helix (bHLH) proteins, which has been intensively studied in mammals. The most characteristic attribute of the family is the presence of a bipartite bHLH domain consisting of about 60 amino acids. This domain contains a region with a large number of basic residues at the N-terminal side, which is involved in DNA binding. The HLH part located at the C-terminal part of the domain consists of two hydrophobic regions that play a role in homo-and/or heterodimerization. Binding to DNA occurs at the core DNA hexamer sequence CANNTG, named the E-Box or G-Box after the most frequently observed variant (CACGTG). Other less frequently occurring variants of these motifs are known as H-Box, N-Box, and Z-Box (Murre et al., 1994; Toledo-Ortiz et al., 2003). Of the current 162 annotated bHLH transcription factor genes in Arabidopsis not many have been studied to an extent that a function is known. The bHLH proteins that have been characterized function mainly in anthocyanin biosynthesis, phytochrome signaling, seed globulin expression, fruit dehiscence, and carpel and epidermal development (Buck and Atchley, 2003). Only a limited number of bHLH transcription factors characterized so far have been found to be involved in defense against pathogens. One important member is conserved in many plant species and in Arabidopsis is named AtMYC2/JIN1, which plays a central role in both JA- and abscisic acid

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(ABA)-regulated signaling. AtMYC2 is induced by wounding and herbivory. The response to these types of stresses is mediated through the JA pathway and results in the induced expression of a subset of JA-responsive genes, including VSP1, LOX, and THIONIN 2.1 (THI2.1). Upon infection with necrotrophic pathogens, genes like PDF1.2, ChiB, and HEL are regulated by both the JA- and ET-signaling pathways. AtMYC2 negatively regulates the induced expression of these genes. This negative regulation is suggested not to be a direct effect of AtMYC2 on the downstream targets, and might be caused by a negative regulation of the expression of transcription factors such as ERF1, that positively regulate these genes (Boter et al., 2004; Dombrecht et al., 2007; Lorenzo et al., 2004; Reymond et al., 2004). AtMYC2 is also important for ISR-associated priming for enhanced JAresponsive gene expression upon pathogen or insect attack (Pozo et al., 2008). D. BZIP TRANSCRIPTION FACTORS

bZIP transcription factors are characterized by their basic leucine zipper (bZIP) domain. This is a bipartite region enriched in basic amino acid residues that are in direct contact with the DNA and involved in DNA binding. In close proximity of this region is a leucine zipper region consisting of regularly spaced leucine residues. This region is important for the homoand heterodimerization of the bZIP proteins (Schindler et al., 1992). Two of the 10 groups of bZIP transcription factors (Jakoby et al., 2002) in Arabidopsis have been implicated to play a role in plant innate immunity. AtbZIP10, a member of group C, is a positive regulator of basal defense responses, R gene-mediated hypersensitivity, and reactive oxygen-induced cell death. AtbZIP10 is controlled by Lesions simulating disease resistance 1 (LSD1), a plant-specific zinc-finger protein that negatively regulates cell death by inhibiting nuclear translocation of AtbZIP10 (Kaminaka et al., 2006). Likewise, the tobacco bZIP transcription factor BZI-1, which is related to Arabidopsis group C transcription factors, regulates cell death. Again, nuclear localization is regulated, in this case through the interacting ankyrin-repeat protein ANK1. In addition, BZI-1 transcription is upregulated in response to pathogen attack and pathogen-induced phosphorylation of BZI-1-related proteins has been described (Kuhlmann et al., 2003). The second group of Arabidopsis bZIP transcription factors involved in innate immunity is group D, which harbors the 10 members of the TGA family of transcription factors. So far, six of them, TGA1, TGA2, TGA3, TGA4, TGA5, and TGA6, have been shown to be involved in defense

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responses against pathogen attack (Kesarwani et al., 2007; Zhang et al., 2003). The first TGA factor to be identified was TGA1a from tobacco, which binds to activation sequence-1 (as-1). This element, which is characterized by two TGACG motifs in a tandem arrangement, was first identified in the 35S promoter of cauliflower mosaic virus (CaMV) (Katagiri et al., 1989). When acting independently of other enhancers, this element confers SA- and auxin-dependent expression in leaves (Qin et al., 1994; Xiang et al., 1996) and constitutive expression in roots (Benfey et al., 1990). With the discovery of TGA factors interacting with NPR1, which has a central role in SA-regulated gene expression (see above), the importance of TGA factors in SA-regulated gene expression and their role in development of SAR was established (Despre´s et al., 2003; Zhang et al., 1999). The Arabidopsis PR-1 and the tobacco PR-1a promoters, which are studied as model systems to understand SA-induced transcriptional regulation, each contain an as-1-like element in a region of the promoter that is important for SA-inducible gene expression (Lebel et al., 1998; Strompen et al., 1998). In Arabidopsis, linker scanning analysis revealed that one of the TGACG motifs is a positive regulatory element (LS7), whereas the other functions as a constitutive negative element (LS5) for induced expression (Lebel et al., 1998). TGA2 and TGA3 were found to bind to the PR-1 promoter in vivo (Johnson et al., 2003; Rochon et al., 2006), with TGA3 acting as a transcriptional activator of PR-1 expression, whereas TGA2 represses expression in the uninduced state. Conflicting data concerning the mechanism of action of the TGA/NPR1 complex have been reported. Based on studies involving chromatin immunoprecipitation analysis (Johnson et al., 2003), electrophoretic mobility shift assays (Despre´s et al., 2000) and transgenic plants expressing the C-terminal domain of TGA2 as a fusion with the DNA-binding domain of the yeast transcriptional activator protein Gal4 (Fan and Dong, 2002), it was first hypothesized that NPR1 serves to facilitate binding of TGA factors at the promoter. Later, it was found that at least TGA2 binds constitutively to the PR-1 promoter and that yet unknown factors already recruit NPR1 to the promoter in the uninduced state. NPR1 interacts with TGA factors only under inducing conditions to form an enhanceosome, a protein complex that binds DNA in the enhancer region of the gene (Rochon et al., 2006). Although it is generally accepted that TGA factors are crucial for the regulation of many SA-dependent processes, the importance of the different members of the TGA family is controversial. First, it was reported that TGA2, TGA5, and TGA6 are redundant and essential activators of PR-1 expression (Zhang et al., 2003). Later, other studies documented that PR-1 expression is only delayed in the tga2 tga5 tga6 triple mutant (Blanco et al., 2009),

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and that additional mutation of TGA3 is necessary to get a more stringent knockout phenotype (Kesarwani et al., 2007). TGA1 and TGA4 are essential for SA-dependent basal resistance (Kesarwani et al., 2007). Disulfide bridges of Arabidopsis TGA1 are reduced after a SA-mediated redox change, which allows interaction with NPR1. However, more information is needed to unravel the in vivo function of TGA1 and TGA4 with respect to the regulation of SA-inducible genes. The so-called class II TGA factors TGA2, TGA5, and TGA6 are not only known to activate gene expression in the presence of enhanced levels of SA, but they are also necessary for the negative cross-talk that is exerted by SA on the JA/ET pathway. A yeast two-hybrid screen of an Arabidopsis library with tobacco TGA2.2 as a bait identified Glutaredoxin 480 (GRX480) as an interactor of TGA factors. Overexpression of GRX480 interfered with the induction of PDF1.2 (Ndamukong et al., 2007), indicating that this interaction is functional with respect to SA/JA/ET signaling. Furthermore, TGA4 was found to interact with ERF72/AtEBP (see above). Recently, we found that tobacco NtWRKY12, a WRKY transcription factor required for high-level expression of PR-1a, specifically interacts in vitro and in vivo with tobacco TGA2.2 (M. C. van Verk and H. J. M. Linthorst, unpublished data). More details are described in the next section. E. WRKY TRANSCRIPTION FACTORS

WRKY proteins are characterized by a stretch of the amino acids tryptophan (W), arginine (R), lysine (K), and tyrosine (Y), followed by a typical zincfinger domain, and constitute a large class of DNA-binding proteins in plants (Zhang and Wang, 2005). In Arabidopsis, more than 70 WRKY genes have been identified. The first WRKY-cDNA clone was characterized in 1994 from sweet potato (Ishiguro and Nakamura, 1994), and their description as a class of transcription factors followed soon afterwards (Eulgem et al., 2000). Many WRKY proteins have specific binding affinity for the consensus W-box motif TTGAC(T/C). In parsley it was shown that clustering of Wboxes is important for a strong transcriptional response (Eulgem et al., 1999; Rushton et al., 1996). Based on their domain structure, WRKY proteins can be divided into three major groups. Proteins with two WRKY domains belong to group I. WRKY proteins containing one WRKY domain belong to groups II or III, depending on the type of zinc-finger motif (Eulgem et al., 2000). The importance of WRKY factors for SA-mediated gene expression was first shown for the Arabidopsis SAR marker gene PR-1, in which a Wbox motif conferred a strong negative effect on gene expression (Lebel et al., 1998). W-box motifs are overrepresented in the promoters of Arabidopsis

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genes that are coregulated with PR-1. Yet, TGA transcription factor-binding as-1 elements occur at statistically expected frequencies in these promoters (Rowland and Jones, 2001). Besides the consensus W-box, WRKY factors have been identified to bind to other motifs. Recently, we identified tobacco NtWRKY12 as a WRKY protein with a variant WRKYGKK amino acid sequence in the WRKY domain instead of the WRKYGQK sequence of the majority of WRKY proteins (Van Verk et al., 2008). NtWRKY12 is involved in transcriptional activation of the PR-1a promoter and binds to WK-boxes, TTTTCCAC, in this promoter, while it is unable to bind to the consensus W-box (Van Verk et al., 2008). A WRKY protein from barley (SUSIBA) was found to bind to SURE, a SUGAR-RESPONSIVE CIS ELEMENT in the promoter of the ISOAMYLASE 1 (ISO1) gene (Sun et al., 2003). The authors did not further delineate the binding site of SUSIBA in SURE, although the presence of the sequence TTTTCCA in this element suggests that it could be the WK-like sequence. WRKY proteins have been found as transcriptional activators at the end of the PAMP signaling cascade involved in the response of Arabidopsis to the flagellin fragment flg22. In this case, signal transduction via the MAPK cascade MEKK1–MKK4/MKK5–MPK3/MPK6 leads to the activation of downstream WRKY22 and WRKY29. These WRKY factors are suggested to amplify their expression levels via multiple WRKY binding sites in their own promoters, thereby creating a positive feedback loop. The induced expression of these WRKY factors would then allow induction of resistance to both bacterial and fungal pathogens (Asai et al., 2002). Activation of the WRKY factors could possibly occur via targeted degradation of bound suppressors, as has been found for the activation of WRKY33. Another MAPK cascade (MEKK1–MEK1/MKK2–MAPK4), induced by challenge inoculation with Ps. syringae or treatment with flg22 leads to phosphorylation of Map kinase substrate 1 (MKS1), through which WRKY33 and possibly WRKY25 are bound to MAPK4. Upon phosphorylation of MKS1, WRKY33 is released in the nucleus to initiate positive regulation of JA-induced defense genes and negative regulation of SA-related defense genes. Also other WRKYs, like WRKY11 and WRKY17, act as negative regulators of basal resistance responses. Moreover, overexpression of the flagellin-inducible WRKY41 abolishes the inducibility of PDF1.2 by MeJA. In all these cases the mechanisms underlying these antagonistic effects are as yet unknown (Andreasson et al., 2005; Brodersen et al., 2006; Higashi et al., 2008; Journot-Catalino et al., 2006; Qiu et al., 2008). Activation of the MAPK pathway by flagellin leads to increased levels of SA, which is strongly dependent on the pathogen-inducible ICS1. Activation

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of ICS1 gene expression is likely to occur via WRKY transcription factors. WRKY28 is rapidly induced to very high levels upon flg22 treatment (Navarro et al., 2004). We have found that transient overexpression of WRKY28 in Arabidopsis protoplasts leads to induction of a -GLUCURONIDASE (GUS) reporter gene under control of the 1 kb ICS1 upstream promoter region as well as elevated levels of endogenous ICS1 mRNA. This may indicate a link between PAMP signaling and the biosynthesis of SA. From public databases it appears that WRKY28 is the only WRKY protein of which the expression is suppressed by both JA and ET. As the 1 kb ICS1 promoter lacks a consensus W-box, WRKY28 probably exerts its effect through binding to a different sequence motif. A WK-like box (TTTTCCA) is present in the 1 kb upstream region and this might function as the WRKY28 binding site (M. C. van Verk and H. J. M. Linthorst, unpublished data). The PBS3 gene is induced by avirulent strains of Ps. syringae and has an important role in SA accumulation (Nobuta et al., 2007). By a similar approach as described above, we found that the 1 kb PBS3 promoter directs reporter gene expression in Arabidopsis protoplasts upon transient expression of WRKY46. WRKY46 is a transcription factor that is rapidly induced downstream of avirulence effectors. These results suggest an involvement of WKRY46 in the signaling cascade of avirulence effector recognition and the subsequent accumulation of SA (He et al., 2006; M. C. van Verk and H. J. M. Linthorst, unpublished data). A more direct link between defense responses and WRKY proteins is supported by the structure of WRKY52/Resistance to Ralstonia solanacearum 1 (RRS1). In addition to a WRKY domain, this protein contains a domain that is characteristic of TIR-NBS-LRR R proteins. In the nucleus, WRKY52 interacts with the R. solanacearum effector PopP2. Plants challenged with strains of R. solanacearum that lack the popP2 gene are highly susceptible to the pathogen, indicating the importance of WRKY52 in resistance against this pathogen (Deslandes et al., 2002, 2003). The barley R protein Mildew A (MLA) appears to interfere with the PAMP-inducible repressors of basal resistance HvWRKY1 and HvWRKY2. In this manner the repressor effect of the PAMP-induced WRKY genes is derepressed, thereby triggering basal defense responses (Shen et al., 2007). The important function of NPR1 in defense pathways is evident by the requirement of this cofactor for the development of SAR, ISR, and defenserelated PR gene expression. Eight WRKY genes (WRKY18, 38, 53, 54, 58, 59, 66, and 70) have been identified as direct targets of NPR1 (Spoel et al., 2009; Wang et al., 2006). Most of the encoded WRKYs are known to have a function in the regulation of PR genes and in SAR. Negative regulators are WRKY58, a direct negative regulator of SAR, and WRKY38, which, similar

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to WRKY62, can activate repressors of PR-1. WRKY62 also acts in the cross-talk between SA and JA signaling by repressing downstream JA targets such as LOX2 and VSP2 (Kim et al., 2008; Mao et al., 2007). Both WRKY18 and WRKY53 are positive regulators of PR-gene expression and SAR. Functional WRKY18 is required for full induction of SAR and is linked to the activation of PR-1 (Wang et al., 2006). Together with WRKY18, WRKY40 and WRKY60 play partly redundant roles in regulating disease resistance. These three WRKY proteins can interact physically and functionally in their responses to different microbial pathogens. While WRKY18 enhances resistance against Ps. syringae, co-expression of WRKY40 or WRKY60 renders plants more susceptible to this pathogen (Xu et al., 2006). WRKY70 and the functional homolog WRKY54 have dual roles in SA-mediated gene expression and resistance. Upon high accumulation of SA, WRKY54/70 act as negative regulators of SA biosynthesis, probably by direct negative regulation of ICS1. Besides this negative role, they activate other SA-regulated genes (Kalde et al., 2003; Wang et al., 2006). WRKY70 also acts as a key regulator between the SA and JA defense pathways by inducing SA-dependent responses and repressing JA-dependent responses, such as expression of VSP, LOX, and PDF1.2. WRKY70 expression is repressed by the JA-signaling regulator COI1 to overcome the negative effect of SA on JA signaling (Li et al., 2004, 2006). Tobacco NtWRKY12 activates PR-1a gene expression via the WK-box in its promoter. Mutation of this box has a far more severe effect on PR-1a gene expression than mutation of the nearby as-1 element, implying that TGAs are not the predominant activators of PR-1a expression (M. C. van Verk et al., 2008). This is supported by our finding that in npr1-1 mutant protoplasts NtWRKY12-induced PR-1a expression is still fully operative (M. C. van Verk and H. J. M. Linthorst, unpublished data). NtWRKY12 gene expression is induced upon PAMP elicitation and tobacco mosaic virus infection. It is arguable that NtWRKY12 expression requires NPR1-dependent activation via TGAs, which would lend support for an indirect rather than a direct role of NPR1 in PR-1a expression. As many WRKY transcription factors can bind similar cis elements, the question arises how the different WRKYs can specifically activate or suppress their respective target genes. Possibly, fine-tuning of specific gene regulation involves interactions between different transcription factors binding to proximal binding sites at the promoter. We found that NtWRKY12 can specifically interact with tobacco TGA2.2 both in vitro and in vivo. This suggests a role of TGA2.2 in PR-1a expression as a recruiter of NtWRKY12 to the promoter or to stabilize its binding.

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A graphical summary of the various pathways from pathogen perception to transcriptional activation of defense gene expression as described in this chapter is presented in Fig. 3.

Flg22 RPP2/4

PAD4

Fad3/7/8

AVR4

MPK6

Methionine

a-linolenic acid

EDS1

P

LOX

FLS2

CEV1

SAM ACS4/5/9

ACS2/6

13-HPOT CPR1/5/6

W46

AOS

MAPKKK/MEKK1

EOL1/2 ETO1

ACC ACO

AOC ET EDS5

MKK4/5

MPK3/6

OPDA OPR3

PBS3

MFP KAT ORA47

W54/W70

W22/W29

ETR1/2 ERS1/2 EIN4

ACX

WIPK

ICS1

W28

ER Membrane

MAPKK

JA

SA

CTR1

MKK9 JMT

JAR1

MAPK

Defense genes

Ja-lle SA defense genes

P

W25/33

MPK3/6

MeJA

W11/17 EIN2

EIN5 MKS1 MPK4 MKS1 W25/33

MEK1/MKK2

SA

Ja-lle

NPR1

ORCA2

EBF 1/2

MEKK1

EIN3/EILs

P

JA defense genes

Myb30

Pseudomonas syringae

Fusarium oxysporum

MeJA

HR Induction

P NPR1 P TF NPR1 target genes

Myb34

Myb28/29

Indole GSL

Aliphatic GSL

NPR1 target genes

Pulse

TIA biosynthesis

NPR1 target genes

W54

+

Pto

W40/60

Pti4

W18

AtMYC2 SNI1

W41

P

GCC-Box

SCFCOl1

AtMYC2 JAZ

W70

TGA1/3

TGA2/5/6 W18/53

Coronatine

Ja-lle

SCFCOl1 Ja-lle JAZ

NPR1 TF

W58

Fo oxylipin ?

ORCA3

EIN3/EILs

ORA37

ERF1

P

ORA59

GRX480 TGA

Other PR Genes

SSN

PR1 Repressor

W54/W70 SAR

W38/62

W62

VSP LOX Thi21

W70

PDF1.2 ChiB HEL

HDA19

Fig. 3. Graphical summary of the pathways from pathogen perception to transcriptional activation of defense gene expression described in this chapter. See text for details. Pointed arrows indicate positive regulation, blunted arrows indicate negative

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IV. REGULATION OF PLANT DEFENSES AT THE CHROMOSOMAL LEVEL A. CHROMATIN MODIFICATIONS AND GENE EXPRESSION

Considering the large-scale transcriptional reprogramming events that occur in plants upon pathogen infection, a relevant question is whether chromatin structure is altered either as a means to regulate transcription or as a consequence of ongoing transcription. Chromatin is the complex combination of DNA, RNA, and protein that makes up chromosomes. One of the functions of chromatin is to compact the DNA, but it also provides mechanisms to control gene expression. In the basic ‘‘beads on a string’’ structure, the DNA is wrapped around the nucleosomes, histone octamers consisting of two copies each of the core histones H2A, H2B, H3, and H4. Linker histones such as H1 and its isoforms are involved in chromatin compaction, as found in heterochromatin, which contains primarily nontranscribed DNA. Noncondensed nucleosomes without the linker histone resemble ‘‘beads on a string of DNA’’ and are typical for euchromatin, which contains regions with actively transcribed genes (Li et al., 2007). Chromatin structure is influenced by a number of different mechanisms including: methylation of cytosine residues residing in CpG sequences of the DNA; acetylation, phosphorylation, methylation, ubiquitination, and ADPribosylation of histones; incorporation of histone variants; histone eviction; and chromatin remodeling, which utilizes ATP hydrolysis to alter histoneDNA contacts (Li et al., 2007). The combination of histone modifications at a promoter is called ‘‘histone code’’ (Strahl and Allis, 2000). The histone code is likely to have at least two roles: to provide heritable epigenetic marks and to facilitate reversible control over events on chromatin in real time. Some of these modifications are known to act as sites for recruitment of regulatory proteins and enzymes (code readers), that can either repress or activate transcription (Seet et al., 2006). The histone code is established mainly by the posttranslational modification pattern of the flexible N-terminal domains of histones H3 and H4 which

regulation. Green arrows: regulation under inducing conditions. Red arrows: Regulation under noninducing conditions. Dashed arrows: hypothetical regulation. Black arrow: pointing away—signal continues elsewhere in the figure, pointing towards— continued signal from elsewhere in the figure. Lightning arrow: elicitor. Blue hexagon: enzyme. Yellow rectangle: MAP kinase. Brown rectangle/star: downstream target. Yellow star with a P: phosphorylation. Blue ellipse: hormone/signaling molecule. Pink ellipse: transcription factor (TF, transcription factor; W, WRKY). Green ellipse: modulator of transcriptional activity.

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protrude from the globular nucleosome. In general, transcriptionally inactive heterochromatin as well as silenced promoter regions are characterized by hypoacetylation of lysine residues in histones H3 and H4 and by methylation of lysines 9 and 27 at histone H3, and lysine 20 at histone H4 (Table I). Active genes are associated with hyperacetylation of lysine residues as well as methylation of lysines 4, 36, and 79 on histone H3. Often, hyperacetylation of histones at promoter regions is necessary but not sufficient for transcriptional activation (Berger, 2002; Cosma, 2002; Narlikar et al., 2002; Ng et al., 2006). Modifications can be detected by chromatin immunoprecipitation experiments using commercially available antibodies directed against specifically modified histones. Enzymes responsible for establishing/maintaining the steady-state balance of histone acetylation are histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Strahl and Allis, 2000). Histone methyl transferases (HMTs) and histone demethylases are responsible for reversible histone methylation (Mellor, 2006). A crucial question is how these enzymes are recruited to the promoters depending on the developmental stage or environmental conditions. Most likely, regulatory trans factors binding to specific cis elements function either directly or indirectly as anchor proteins. The resulting changes in chromatin structure can again be the prerequisite for binding of other transcription factors: For example, the yeast protein General control nonrepressed protein 5 (GCN5), which exhibits histone acetyl transferase activity, is targeted to specific promoter regions by the transcriptional activator GCN4 (Kuo et al., 2000) under conditions of amino acid starvation. Subsequently, the relaxation of the chromatin structure allows recruitment of general transcription factors (Narlikar et al., 2002). B. CHROMATIN MODIFICATIONS IN PLANTS

In plants, histone modifications have been demonstrated to be involved in the control of various developmental processes. Classical and well-studied examples are the chromatin modifications at the FLOWERING LOCUS C (FLC)

TABLE I Simplified Overview of the Histone Code

Acetylation Methylation

Transcriptionally inactive heterochromatin

Transcriptionally active euchromatin

Low Dimethylation of H3-K9, H3-K27, H4-K20

High Di- and trimethylation of H3-K4

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in Arabidopsis and at the -PHASEOLIN (PHAS) promoter in bean (Phaseolus vulgaris). Histone H3 and H4 acetylation as well as trimethylation of K4 of histone H3 at the FLC locus are associated with active FLC expression, whereas histone deacetylation and dimethylation at lysines K9 and K27 are involved in FLC repression after vernalization (Bastow et al., 2004; He and Amasino, 2005; He et al., 2004). Vernalization-induced changes in chromatin structure are ‘‘remembered’’ through subsequent rounds of replication of the meristem allowing FLC repression at higher temperatures following the cold period. Thus, histone modifications at the FLC locus constitute a heritable epigenetic mark. At the embryo-specific PHAS promoter, dimethylation of histone H4 at lysine K20 is suggested to contribute to the establishment of the heterochromatic PHAS chromatin. The transcription factor ABI3-like factor (ALF), which is only expressed in the embryo, may function as an anchor protein that promotes recruitment of histone acetyl transferases that acetylate H3-K9 and H4-K12 residues. However, this modification is not sufficient for transcription. Instead, it renders the PHAS promoter more accessible for the assembly of other factors after activation of the ABA signal transduction cascade leading to the activation of transcription (Ng et al., 2006). In this situation, the histone code serves to ‘‘prime’’ promoters for rapid transcriptional activation. C. CHROMATIN MODIFICATIONS AT PROMOTERS INVOLVED IN INNATE IMMUNITY

1. The SA pathway As described in Section III.D, one of the best studied promoters that is being analyzed as a paradigm for understanding regulatory events of the innate immune response is the SA-inducible PR-1 promoter from Arabidopsis (Lebel et al., 1998). Increased methylation at lysine K4 and acetylation at lysines K9 and K14 of histone H3 are observed in the sni1 mutant, which indicates that SNI1 is required for histone modifications related to transcriptional repression (Mosher et al., 2006). In wild-type plants, K4 methylation and K9K13 acetylations can be induced after 48 h of treatment with the SAanalog benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; synonym: acibenzolar S-methyl, ASM). However, using the same antibody (anti-dimethyl-histone H2 (Lys4), Alvarez-Venegas et al. (2007) were not able to detect significant changes in chromatin marks at the PR-1 promoter after 24 h of SA treatment. Likewise, in our hands SA-induced changes on histone acetylation were not detected after 3 and 12 h of SA treatment when deploying a polyclonal antiserum against hyperacetylated lysines 5, 8, 12, and 16. Thus, chromatin modifications at the PR-1 promoter might depend

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on the growth conditions and might not be essential for appropriate regulation. Still, PR-1 seems to be under the control of at least indirect effects on chromatin structure. For instance, its expression is reduced in atx1 plants, which have a disruption at the ARABIDOPSIS HOMOLOG OF TRITHORAX locus (Alvarez-Venegas et al., 2007). ATX1 carries a highly conserved SET domain. SET domain peptides (named after the three Drosophila proteins SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR) 3-9], ENHANCER OF ZESTE [E(Z)] and TRITHORAX (TRX)) of the Trithorax-family can methylate K4 of histone H3 (Rea et al., 2000) and functional analysis of the atx1 mutant supports the notion that ATX1 is involved in methylation of histones at specific promoters (Alvarez-Venegas et al., 2007). Ps. syringae pv. tomato-induced expression of the WRKY70 gene is reduced in the atx1 mutant, which correlates with a decreased level of trimethylated H3-K4. As increased levels of WRKY70 lead to the constitutive accumulation of an abnormal PR-1 transcript (Li et al., 2004), the authors concluded that decreased WRKY70 expression in the atx1 mutant leads to decreased PR-1 expression. However, as the wrky70 mutant is not affected in SA-induced PR-1 transcription (Ren et al., 2008), unknown effects of ATX1 on proteins regulating PR-1 expression have to be postulated. Ps. syringae pv. tomato-induced PR-1 transcription is also reduced in the hda19 mutant, which is deficient in a HDAC that physically interacts with WRKY38 and WRKY62 (Kim et al., 2008). It was hypothesized that WRKY38 and WRKY62, which presumably activate transcription of negative regulators of PR-1 expression, are inhibited by HDA19. The notion that increased histone acetylation (as mimicked by a mutation in a HDAC gene) leads to decreased PR-1 expression is supported by pharmacological studies: treatment of plants with trochostatin A (TSA), a chemical that blocks histone deacetylation, also leads to reduced basal levels of PR-1 expression (Chang and Pikaard, 2005). The target genes of WRKY38 and WRKY62 are likely candidates to be acetylated in hda19 mutants or in TSA-treated plants. When acetylated, their expression might be increased, which in turn would inhibit PR-1 expression. PR-1 expression has been analyzed in the splayed (syd) mutant, which has a defect in the SWI/SNF class chromatin remodeling ATPase SYD (Walley et al., 2008). Chromatin remodeling complexes use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes and are thus of major importance to facilitate transcription (Clapier and Cairns, 2009). In contrast to wild-type plants, syd mutant plants show a strong induction of PR-1 expression after infection with the necrotrophic fungus B. cinerea.

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As many JA-dependent responses, such as the expression of PDF1.2 and VSP2, are compromised in the syd mutant, it is hypothesized that the negative cross-talk between JA and SA is affected by a SYD-dependent mechanism. In addition to the Arabidopsis PR-1 gene, the tobacco PR-1a gene has been studied by different groups (Buchel et al., 1999; Gru¨ner and Pfitzner, 1994; Gru¨ner et al., 2003; Uknes et al., 1993). Like the Arabidopsis PR-1 promoter, the tobacco PR-1a promoter contains a functionally important TGA binding site (Strompen et al., 1998) and its expression depends on TGA (Thurow et al., 2005) and WRKY transcription factors (Van Verk et al., 2008). Chromatin immunoprecipitation experiments unraveled increased histone H4 hyperacetylation at the PR-1a promoter at 3 h and even more at 12 h after SA treatment, which correlates with the kinetics of transcript accumulation (Butterbrodt et al., 2006). In RNAi plants with reduced levels of TGA2.2, histone acetylation also increased after SA treatment, although the promoter was not activated. This indicates that histone acetylation is not sufficient for transcription. In addition, this histone hyperacetylation does not seem to be triggered by TGA2.2 binding. Indeed, it might even be the prerequisite for binding of TGA2.2 to the promoter, as TGA2.2 is recruited to the PR-1a promoter only after SA treatment. In contrast, constitutive histone acetylation and constitutive binding of TGA2.2 were observed at the truncated CaMV35S promoter that contains the TGA2.2 binding element as-1 as the only regulatory cis element. The truncated CaMV35S promoter, which can be activated by SA independently from NPR1, is activated with faster kinetics (maximum at 3 h after SA treatment) than the PR-1a promoter (maximum after 12 h of SA treatment), which might be due to the fact that the chromatin of the truncated CaMV35S promoter is already in an ‘‘open (potentiated)’’ chromatin configuration, allowing constitutive binding of TGA2.2 and subsequent fast activation by an as yet unknown activation mechanism (Fig. 4).

2. The JA pathway Few data are available with respect to the analysis of the histone code at JA-dependent promoters. No significant changes in the amount of acetyl groups were detected at the PDF1.2 promoter when performing chromatin immunoprecipitation experiments with antibodies directed against diacetylated histone H3 (Koornneef et al., 2008). However, chromatin remodeling seems to play a role in the positive regulation of JA-dependent genes. In the

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P_as-1 T T GG AA

+ SA

T T GG AA

as-1

as-1

“Open” accessible chromatin structure P_PR-1a + SA as-1-like

TT GG AA

as-1-like

“Closed” inaccessible chromatin structure

Fig. 4. Proposed model of the histone acetylation status and the status of TGA binding at the ‘‘immediate early’’ truncated CaMV 35S (P-as-1) and ‘‘late’’ PR-1a promoter in tobacco as suggested by results of ChIP experiments. Histones at promoters that respond with a fast kinetics (within 3 hours) are constitutively acetylated, thus allowing binding of TGA2.2. At the same time, hypoacetylation of promoters which respond with slower kinetics to SA makes the DNA inaccessible to TGA2.2. After induction with SA, the histones at the PR-1a promoter get acetylated, leading to a decondensation of the chromatin, allowing binding of TGA2.2. The cylinders represent histone octamers, flags stand for acetyl groups and TGA binding sites are indicated by grey boxes. As hyperacetylation of histones often leads to a loss of histones (Reinke and Horz, 2003), the decondensed state of the chromatin in the presence of SA is schematically represented by three instead of four nucleosomes.

syd mutant background, PDF1.2 expression is severely compromised after B. cinerea infection (Walley et al., 2008), but recruitment of SYD to this promoter was not detected. Induction of the transcriptional activator AtMYC2/JIN1 and its downstream gene VSP2 was reduced in the syd mutant after wounding. Chromatin immunoprecipitation experiments suggest that these promoters are direct targets of SYD. The atx1 mutant, which is deficient in a putative HMT, reveals constitutive THI1.2 expression, which is most likely due to the reduced expression of WRKY70 and other genes that are direct targets of ATX1 (Alvarez-Venegas et al., 2007). Interestingly, ectopic expression of the HDAC gene AtHDAC19, which is induced by JA, leads to increased expression of ERF1 and its target genes (Zhou et al., 2005). This suggests a similar indirect positive effect of decreased histone acetylation on defense gene expression as already observed for PR-1. Likewise, loss of function analysis of plants deficient in AtHDAC6 leads to reduced expression of JA-responsive genes such as JIN1, ERF1, PDF1.2, and VSP2 (Wu et al., 2008). As speculated for the PR-1 promoter, hyperacetylation of a negative regulator might be responsible for

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the compromised JA response. AtHDAC6 interacts with the F-box protein COI1 which is of critical importance for all JA-mediated responses (Devoto et al., 2002). A plausible model would be that COI1 activates AtHDAC6 by forming a complex which would lead to the silencing of a general negative regulator of the JA response. In conclusion, the increased susceptibility of hda19 (Kim et al., 2008), atx1 (Alvarez-Venegas et al., 2007), and syd (Walley et al., 2008) plants indicates that chromatin structure plays a crucial role in pathogen responses. However, it seems that transcription of key regulators (e.g., WRKY70) is under direct control of chromatin modifications, whereas downstream genes like PR-1 and PDF1.2 are misregulated as a consequence of these primary events. The identification of promoters which show strong and robust changes in epigenetic marks under inducing conditions is a major goal that will help our understanding of the role of chromatin structure for innate immunity.

ACKNOWLEDGMENT We gratefully acknowledge Steven Spoel for providing a copy of his latest manuscript before publication.

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Unexpected Turns and Twists in Structure/Function of PR-Proteins that Connect Energy Metabolism and Immunity

MEENA L. NARASIMHAN,* RAY A. BRESSAN,*,{,{,1 MATILDE PAINO D’URZO,* MATTHEW A. JENKS{ AND TESFAYE MENGISTE}

*Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia { World Class University Program, Gyeonsang National University, Republic of Korea { Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA } Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

I. Historical Perspective Leading to the Recognition of Innate Immunity in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Immunity Involves Pathogenesis-Related (PR) Proteins .......... B. Definition and Classification of PR-proteins............................... II. Roles of PR-proteins Revealed by Studies of PR gene Expression . . . . . . . . . A. Cross-talk Between Overlapping Biotic and Abiotic Stress Response Pathways and Hormone Signaling Precludes Identification of Clear Roles for PR-proteins.......................................................... B. Nutrient Acquisition Strategies of Pathogens Are Associated with Distinct PR gene Sets .......................................................... C. The Connection Between Energy Balance and Immunity................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51011-7

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III. PR-5 Protein Structure Reveals the Primitive Relationship Between Pathogen Defense and Energy Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structural Features of PR-5 Proteins........................................ B. Function of PR-5 Proteins in Plants......................................... C. Information on Plant PR-5 Proteins in Genomic Databases ............ D. Comparison of THN Domain with C1q-TNF Domains ................. E. THN Domain Proteins and C1q-TNF Domains Across Phyla ......... IV. Directions in Which Current Classification or Definition of PR-proteins May Change in the Coming Years as Advanced Functional Studies Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Innate immunity in plants is manifested by a complex array of antimicrobial processes that includes induction of sets of pathogenesis-related (PR) proteins. The availability of genomic data has made clear that each PR-protein family in a species is represented by several genes. Microarray data in public databases show that in most families, including the PR-5 family surveyed here, the expression of only few family members is defense associated. Genetic studies show that depending on their nutrient acquisition strategy, pathogens induce distinct but overlapping sets of PR genes, suggesting a connection to energy or resource allocation. PR-5 proteins have a clearly recognizable structure that is referred to as the thaumatin (THN) domain, which can be overlapped with mammalian Complement 1q-tumor necrosis factor (C1q-TNF) domains such as that of the mammalian hormone adiponectin. The occurrence of THN domain proteins is widespread. Similarities between THN domain proteins and mammalian C1q-TNF family proteins include their ligands and their subcellular locations. Osmotin (tobacco PR-5c) regulates energy balance signaling in mammalian cells by interaction with adiponectin receptors by a pathway that shares components with plant energy and stress signaling pathways. These data suggest additional roles for PR-5 proteins, as scaffolds and/or in signaling, particularly in regulating energy balance.

I. HISTORICAL PERSPECTIVE LEADING TO THE RECOGNITION OF INNATE IMMUNITY IN PLANTS A new era in our understanding of biology began in the 1800s with Louis Pasteur’s research demonstrating that microbes arose by biogenesis, not spontaneous generation, which eventually led to a new perspective on diseases (Debro´, 1998). The conclusive demonstration by Robert Koch that microbes could cause diseases led to the famous Koch’s postulates and marked the beginning of modern pathology theory (Nobel Lectures, 1967). It is interesting to note that the direction of our efforts and eventual bursts of understanding of the etiology of microbial diseases dramatically hinged on the discovery of immunization by Edward Jenner and others (Cartwright, 2005). Jenner’s demonstration that cowpox infection could protect (immunize) against smallpox began our revelation and understanding of the

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adaptive immune response. The fascinating and dramatic function of mammalian adaptive immunity that is able to retain a memory of specific microbes, and its underlying genetic and molecular bases, dominated scientific thinking of microbial pathology for considerable time. Not until the introduction of evidence for a more primitive and general immune system that is present in all cellular organisms, referred to as innate immunity and championed by Charles Janeway in the 1990s, did the true dual and interactive nature of the total immune process become appreciated (Murphy et al., 2005). Indeed, only a generation ago it would have been nearly heretical to claim that plants (and other lower organisms) had any immune system at all. It is now well established that the innate immune system of plants recognizes attempted pathogen infection and that defense responses are activated to limit the extent of damage (Bittel and Robatzek, 2007). A. PLANT IMMUNITY INVOLVES PATHOGENESIS-RELATED (PR) PROTEINS

It is only the very rare combination of microbe–plant or microbe–animal interactions out of the millions of possible combinations that initiates a disease response. Thus, the homeostatic condition of plants, as for animals, is immunity to the vast majority of microbes. This nonhost resistance, long ignored by plant scientists as a form of immunity, is now considered to have arisen from a long history of co-evolution between microbes and plants that probably reflects the need of microbes not to begin colonization in an environment that they cannot exploit nutritionally. Inability of the pathogens to surmount preformed physical and chemical barriers, such as the plant cuticle, cell wall, and antimicrobial compounds, is a relevant component of plant immunity. The first important clue that plants also possessed an immune system that could respond to the presence of a pathogen emerged with the discovery by Leendert C. van Loon and Albert van Kammen (1970) that pathogens were able to induce the expression of host proteins, called PR-proteins, later shown to be defense related (reviewed in Van Loon et al., 2006). As a result of intense research following this discovery, particularly from the analysis of mutants of the model plant Arabidopsis thaliana, it is now well established that the innate immune system of plants recognizes attempted pathogen infection and that defense responses are activated to limit the extent of damage. The mechanisms of recognition and the genetic components of the signaling pathways are complex and depend on the nature of the pathogen encountered, although it is now understood that most signaling pathways converge onto an overlapping/common set of defense responses. These complex response patterns and the different plant defense strategies were recently reviewed (Alfano and Collmer, 2004; Ausubel, 2005;

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Jones and Dangl, 2006). The conclusion from these studies is that variation in host plant susceptibility and resistance is mainly determined by the rate, magnitude, and specific location at which these responses are activated (Tao et al., 2003; Van Loon et al., 2006). There is some evidence that host plant susceptibility may also be determined by combinatorial interactions between defense factors, both induced and constitutive (Veronese et al., 2003). Notably, perturbation of PR gene expression is a commonly observed component of all of these defense responses. B. DEFINITION AND CLASSIFICATION OF PR-PROTEINS

Proteins that were structurally and serologically similar to the originally identified PR-proteins (Van Loon and Van Kammen, 1970) were subsequently found to be widespread in plants. Some were found to be products of genes that are associated with systemic acquired resistance (SAR), a broad-spectrum wholeplant disease resistance following an initial pathogen encounter. Some had demonstrable antimicrobial activity and many conferred some level of protection against diseases when overexpressed in transgenic plants (Broekaert et al., 2000; Datta and Muthukrishnan, 1999; De Lucca et al., 2005; Sels et al., 2008; Van Loon et al., 2006; Veronese et al., 2003). Today, PR-proteins are defined as ‘‘inducible defense-related proteins’’ (Van Loon et al., 2006). There are 17 families of PR-proteins widely recognized based on primary structure, serological relatedness, enzymatic functions, and biological activities, although not every family is represented in every plant species (Van Loon et al., 2006) (Table I). An additional PR-18 family is being considered that consists of proteins with antimicrobial activity, and occurring in Helianthus annuus and Amaranthus caudatus (Van Loon et al., 2006). Many PR-proteins are extracellular and others have been localized in the plant cell vacuole. As can be seen from Table I, many of the PR-proteins have demonstrable antimicrobial activity that would support a role in plant defense. However, some members of each PR-protein family have no demonstrated antimicrobial activity (reviewed in Veronese et al., 2003). The defense function of chitinases (PR-3, PR-4, PR-8, and PR-11) and -1,3-glucanases (PR-2) can be ascribed to their ability to degrade major cell-wall constituents of most filamentous fungi. PR-6 includes proteinase inhibitors that may reduce the ability of the pathogen or pest to obtain nutrition, complete its replication cycle, or generate secondary elicitors (reviewed in Sels et al., 2008). PR-9 represents a peroxidase thought to be involved in plant cell-wall reinforcement through increased lignification (Passardi et al., 2004). PR-10s are ribonuclease-like proteins, some with weak activity that could affect translation and viral replication (Bufe et al., 1996; Park et al., 2004). PR-15, PR-16,

TABLE I Recognized Families of Pathogenesis-Related (PR) proteinsa Family PR-1 PR-2 PR-3 PR-4 PR-5 PR-6 PR-7 PR-8 PR-9 PR-10 PR-11 PR-12 PR-13 PR-14 PR-15 PR-16 PR-17 a

Sources Nicotiana tabacum, and many other species Hordeum vulgare, Ni. tabacum, Oryza sativa Ni. tabacum, Phaseolus vulgaris, Triticum aestivum Ho. vulgare, Ni. tabacum, Solanum tuberosum, Solanum lycopersicum, Zea mays Cucurbita maxima, Ho. vulgare, Ni. tabacum, Zea mays So. lycopersicum So. lycopersicum Cucumis sativus Ni. tabacum Capsicum annuum, Lupinus albus, Oxalis tuberosa, Petroselinum crispum Ni. tabacum Raphanus sativus Arabidopsis thaliana Ho. vulgare Ho. vulgare Ho. vulgare Ho. vulgare, Ni. tabacum, Tr. aestivum

Protein

Recognized targets

Unknown (antifungal)

Phytophthora infestans

-1,3-Glucanase (antifungal)

Colletotrichum lagenarium, Fusarium solani, Rhizoctonia solani Fusarium oxysporum, Physalospora piricola

Chitinase (antifungal) Chitinase (antifungal)

Alternaria radicina, Fusarium moniliforme, Fu. solani, Trichoderma viride

Thaumatin-like (antifungal)

Candida albicans, Fu. oxysporum, Neurospora crassa, Phy. infestans, Trichoderma reesei, Trich. viride

Proteinase inhibitor Endoproteinase Chitinase (antifungal) Lignin-forming peroxidase Ribonuclease-like (antifungal) Chitinase (antifungal) -Defensin (antifungal) Thionin (antifungal) Lipid-transfer protein Oxalate oxidase (antifungal) Oxalate-oxidase-like Unknown (antifungal)

Co. lagenarium Colletotrichum gloeosporioides, Fu. oxysporum, Phytophthora capsici, Rh. solani Al. radicina, Trich. viride Aspergillus flavus, Fu. moniliforme Fu. solani Blumeria graminis Bl. graminis

Compiled from http://www.bio.uu.nl/fytopath/PR-families.htm; Flores et al. (2002). More complete lists of targets can be found in Veronese et al. (2003).

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and PR-18 are enzymes that produce toxic hydrogen peroxide, which can also elicit additional plant defense responses (Bernier and Berna, 2001; Custers et al., 2004; Donaldson et al., 2001; Hu et al., 2003). The defense function of thaumatin (THN)-like proteins (PR-5), defensins (PR-12), thionins (PR-13), and lipid-transfer proteins (LTPs; PR-14) are usually ascribed to their ability to permeabilize membranes, although as we shall see, PR-5 proteins may also have non‐defense functions. In fact, proteinase inhibitors (PR-6) could also have non‐defense roles such as in the control of seed dormancy and reserve protein mobilization in seeds, whereas thionins (PR13) may function as storage proteins and in redox regulation of enzymes, and LTPs (PR-14) in lipid catabolism. There is evidence suggesting that some defensins (PR-12) might function in zinc tolerance and that defensin-like polypeptides function as chemokines (Okuda et al., 2009; reviewed in Sels et al., 2008). Non‐defense functions in normal embryogenesis, pollen development, abscission, and antifreeze activity have also been suggested for PR-1, -2, -3, and -4 proteins, and hormone carrier functions have been suggested for PR-10-type proteins (Hon et al., 1995; reviewed in Van Loon et al., 2006). Genome sequence data from many plant species are revealing that the PRprotein families in plants are large. The PR-1, -6, -12, -13, and -14 families have been reviewed based on genome sequence data (Sels et al., 2008; Van Loon and Van Strien, 1999; Yokoyama and Nishitami, 2004). As a complement to these studies, we present a review of the PR-5 family with special emphasis on its role in maintaining plant energy balance during infection. This body of evidence supports the notion that expression of only a few genes from every PR-protein family is associated with plant defense responses. Many PR genes are expressed in a tissue-specific manner and many PRproteins might have functions outside of defense. Thus, it appears likely that we have only uncovered the tip of the proverbial iceberg when it comes to unraveling the functions of plant PR-proteins.

II. ROLES OF PR-PROTEINS REVEALED BY STUDIES OF PR GENE EXPRESSION A. CROSS-TALK BETWEEN OVERLAPPING BIOTIC AND ABIOTIC STRESS RESPONSE PATHWAYS AND HORMONE SIGNALING PRECLUDES IDENTIFICATION OF CLEAR ROLES FOR PR-PROTEINS

As mentioned earlier, the mechanisms of recognition of attempted pathogen infection by the innate immune system of plants and the genetic components of the signaling pathways that are activated are complex and depend on the nature of the pathogen encountered. Host-defense strategies can include morphological barriers such as thickened cuticles and lignified cell walls,

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as well as the induction of PR-proteins and secondary metabolites. According to the ZigZag model of Jones and Dangl (2006), plants initially recognize pathogens through interactions between pathogen recognition receptors (PRRs) and conserved pathogen-associated molecular patterns (PAMPs) that serve as non‐self recognition entities. Chitin fragments, glucans, glycoprotein peptides, oligosaccharides, bacterial lipopolysaccharides (LPS), and flagellin are examples of PAMPs from various plant pathogens. In the case of necrotrophic pathogens, toxins, damage to plant cell walls by hydrolytic enzymes and the resulting release of oligosaccharides are likely to play important roles in the perception and activation of defense responses. Oligogalacturonide fragments released from plant cell walls during pathogen attack or insect herbivory can induce plant defense responses and may also serve as host-derived defense elicitors (Doares et al., 1995; Ferrari et al., 2003). Plants may then ‘‘sense’’ damage to the cell wall or recognize oligogalacturonide fragments through PRRs. A well-studied example is the interaction between bacterial flagellin (PAMP) and the Arabidopsis FLagellin Sensing 2 (FLS2) protein (PRR) that activates downstream signaling cascades regulating transcription of several genes encoding defense-related proteins, including PR-1 and PR-5 (Asai et al., 2002). The Arabidopsis FLS2 and its co-receptor BRI1Associated receptor Kinase 1 (BAK1) proteins are both leucine-rich repeatreceptor-like kinases (LRR-RLKs) involved in recognition and signaling of flagellin (Chinchilla et al., 2007). Potential receptors for chitin, a component of fungal cell walls recognized as a PAMP by plant cells, have been documented from Arabidopsis and rice (Kaku et al., 2006; Miya et al., 2007; Wan et al., 2008). LysM Receptor-Like Kinase 1 (LysM RLK1) from Arabidopsis is required for responses to chitin. PRR-mediated immunity responses, also called PAMP-triggered immunity (PTI), are basal defense responses that have to be overcome by the pathogen to establish disease. Thus, disruption of LysM RLK1 impairs resistance to Alternaria brassicicola and Erysiphe cichoracearum. PTI responses are triggered by diverse pathogens regardless of their lifestyle. Plants also recognize pathogen race-specific effectors (Avrproteins) directly or indirectly by a corresponding plant resistance (R) protein, resulting in effector-triggered immunity (ETI), which can be amplified by further effector recognition events and culminate in a hypersensitive response (HR) (Flor, 1956; Van der Biezen and Jones, 1998). The first Avr gene was discovered in Pseudomonas syringae by Staskawicz et al. (1984) and the first R gene, conferring resistance to Ps. syringae in tomato, was cloned by Martin et al. (1993). Many bacterial, fungal, and oomycete effectors are known (Jones and Dangl, 2006). The HR is a particularly strong form of resistance to specific races (genotypes) of mostly biotrophic pathogens.

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Interactions involving effectors and PAMPs result in SAR and the expression of associated PR-proteins, although the strength and speed of the activation varies significantly (Mishina and Zeier, 2007; Ryals et al., 1996). SAR is initiated by limited pathogen infection and confers resistance to secondary infection. SAR is effective against a broad spectrum of mostly biotrophic and hemibiotrophic pathogens including viruses, bacteria, fungi, and oomycetes (Dempsey et al., 1997; Sticher et al., 1997). SAR and other systemic and induced responses are thus superimposed on ETI and PTI in a given plant genotype. It is now recognized that most signaling pathways converge onto an overlapping and common set of defense responses. PR gene expression constitutes an extensively studied component of these defense responses that correlates with resistance, and yet the relative contribution of many individual PR-proteins in resistance is not well understood. B. NUTRIENT ACQUISITION STRATEGIES OF PATHOGENS ARE ASSOCIATED WITH DISTINCT PR GENE SETS

Regardless of their nature, the perception of pathogen-derived elicitors initiates a variety of downstream events, including activation of mitogenactivated protein kinase (MAPK) cascades, phosphorylation of downstream targets, and activation of general and specific defense responses (Asai et al., 2002). Downstream of MAPKs are transcription factors and defense molecules. The last decade or so has seen the detailed interrogation of the SAR pathway leading to PR gene expression. The plant hormone salicylic acid (SA) is an important component of SAR and basal defense. Impairment of SA accumulation, biosynthesis, or responsiveness alters PR gene expression, basal defense, and SAR (Durrant and Dong, 2004; Gaffney et al., 1993; Nawrath and Me´traux, 1999). The Arabidopsis Nonexpressor of PR genes 1 protein (NPR1), a central regulator of SAR, and other components of the SAR pathway are among the well-defined elements. In addition to controlling the expression of PR genes, NPR1 also directly controls the expression of protein secretory pathway genes (Dong, 2004). Upregulation of secretory pathway genes is essential for SAR, because mutations in some of these genes diminish the secretion of PR-proteins, including PR-1, resulting in reduced resistance (Wang et al., 2005a). PR-1 expression correlates strongly with the activation of SAR in Arabidopsis. Just as upregulation of PR-1 and SA are hallmarks of SAR and resistance to biotrophs in Arabidopsis, the plant hormones jasmonic acid (JA) and/or ethylene (ET), as well as unique sets of PR-proteins, are associated with interactions involving necrotrophic fungi. Intriguingly, infection of Arabidopsis by Botrytis cinerea, a typical necrotrophic fungus, induces

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PR-1 expression, but still results in susceptibility (Veronese et al., 2004, 2006). Infection by some necrotrophic fungi and exogenous applications of JA and/or ET induce a subset of defense genes, including those encoding plant defensins (PR-12), thionins (PR-13), proteinase inhibitors (PR-6), and basic members of other families of PR-proteins (Penninckx et al., 1996, 1998). The Arabidopsis PLANT DEFENSIN 1.2 (PDF1.2) and the tomato PROTEINASE INHIBITOR 2 (PI-2) genes are induced by necrotrophic fungi, methyl jasmonate (MeJA), and ET (AbuQamar et al., 2008; O’Donnell et al., 1996; Penninckx et al., 1998). In both Arabidopsis and tomato, the JA receptor mutant coronatine insensitive 1 (coi1)/jasmonate insensitive 1 (jai1) fails to induce PDF1.2/PI-2 gene expression (PR-12/PR6 family, respectively) and shows enhanced susceptibility to infection by necrotrophic fungi (AbuQamar et al., 2008; Thomma et al., 1998). In both plant systems, mutations blocking JA or ET signaling and biosynthesis impair PR gene expression and enhance susceptibility to necrotrophic fungi as well as to some insect pests. In tomato, the Tomato Protein Kinase 1b (TPK1b) protein regulates responses to pathogens and herbivorous insects through the regulation of the tomato PI-2 gene in an ET-dependent but JA-independent manner (AbuQamar et al., 2008). Expression of PR genes and disease resistance are modulated by cross-talk between the different signaling pathways involving several plant hormones, like JA, ET, abscisic acid (ABA), and SA. Genetic studies in Arabidopsis have described many mutations that show contrasting disease phenotypes and contrasting PR gene expression. For example, the Botrytis susceptible mutants botrytis susceptible 3 (bos3) and botrytis induced kinase 1 (bik1) show increased susceptibility to the necrotrophic fungus Bo. cinerea, but not to biotrophic pathogens (Veronese et al., 2004). In the bos3 mutant, the PR-1 gene is expressed at elevated levels, both constitutively and in response to pathogen challenge, but the plant defensin gene PDF1.2 is expressed at generally lower levels in bos3 as compared to wild-type plants. BIK1 is a positive regulator of resistance to necrotrophic fungi (Veronese et al., 2006). Inactivation of BIK1 causes severe susceptibility to Bo. cinerea and Al. brassicicola. In contrast, bik1 plants display enhanced resistance to the virulent strains of the bacterial pathogen Ps. syringae. JA- and ET-regulated defense response pathways, generally associated with resistance to necrotrophic fungi, are attenuated in bik1, as measured by the expression of the plant defensin PDF1.2 gene transcript. The Arabidopsis WRKY33 transcription factor regulates antagonistic interactions between defense pathways (Zheng et al., 2006). Mutations in the Arabidopsis WRKY33 gene cause enhanced susceptibility to Bo. cinerea, concomitant with reduced expression of PDF1.2 and other JA-regulated

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PR genes. Overexpression of WRKY33, on the other hand, increases resistance to the two necrotrophic fungal pathogens, but enhances susceptibility to Ps. syringae. The susceptibility of WRKY33 overexpressing plants to Ps. syringae is associated with reduced expression of the SA-regulated PR-1 gene. Thus, WRKY33 is an important transcription factor that regulates the antagonistic relationship between defense pathways mediating responses to Ps. syringae and necrotrophic fungi. Accordingly, in many mutants the reduced expression of PDF1.2 is mirrored by increased expression of PR-1 and contrasting disease resistance phenotypes, indicative of antagonistic relationships between pathways. Although diverse PR genes have been identified and their regulation has been studied in various plant species, including Arabidopsis, maize, soybean, and tomato, the extent of functional conservation of signaling pathways upstream of PR-proteins in various plants is not clear. Some general observations on defense signaling have emerged, however. Defense responses to necrotrophic pathogens tend to be mediated by JA/ET, whereas responses to biotrophs are usually mediated by SA. Herbivory involves wounding and damage similar to that exerted by necrotrophic fungi, and induced resistance to herbivory is JA dependent (Howe and Jander, 2008). Moreover, the activity of both signaling systems overlaps with a shared set of PR genes. Finally, it is possible that certain mechanisms of pathogen resistance act independently of classical PR-proteins. Mutation of Arabidopsis BOS1 and HISTONE MONOUBIQUITINATION 1 (HUB1) genes increases susceptibility to necrotrophic fungi without affecting the expression of PR genes (Dhawan et al., 2009; Mengiste et al., 2003). Also, expression of PR-1 and PR-2 (-1,3-glucanase), which are SAR markers in Arabidopsis, can be uncoupled from Ps. syringae resistance phenotype (Greenberg et al., 2000). Thus we see that while PR gene expression correlates with resistance and constitutes an extensively studied component of these defense responses, the relative contribution of many individual PR-proteins in resistance is still not well understood. The positive correlations between high PR-1 expression with increased susceptibility to necrotrophic fungi and with resistance to biotrophic pathogens suggest that PR-1 may have a crucial role in HR, necrosis, and other forms of cell death, together with many other attenuating or enhancing factors. Elucidation of the function of PR-proteins from genetic studies is confounded by the fact that the path from signal perception to PR gene activation is not well defined. While some PR genes are tightly regulated by specific pathways, others respond to divergent, convergent, or even antagonistic signals that originate from biotic sources (such as PAMPs, effectors, and nutrition acquisition strategies of the pathogen or pest) and abiotic sources (such as wounding, low temperature, drought, salinity, herbicides,

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fungicides, and reactive oxygen species, (ROS), indicating extensive overlap or cross-talk of immune response signaling with other stress responses (Graham, 2005; Romeis, 2001; Yoshioka et al., 2001; Zhang and Klessig, 2001; Zhang et al., 1998). The relative nonspecificity in the regulation of some of the PR-proteins raises the possibility that their induction may be considered a general inflammatory response. In animal systems, general stress perception (wounding, temperature, chemicals, ROS) and pathogen perception (PAMPs, toxins) activate overlapping signaling pathways leading to inflammation (Carlsen et al., 2004; Carmichael et al., 2009; Chung et al., 2001; Elenkov and Chrousos, 2002, Freche et al., 2007; Golden et al., 2009; Li and Stark, 2002; Lotze et al., 2007). The inflammatory response in animals is a component of innate immunity reflecting the effort of the animal to contain infection and repair injury. It is an ‘‘alarm system’’ composed of extracellular hormone-like polypeptides that function to restrain continued insults (McIntire et al., 2009). Inflammation has now been shown to be tightly linked to energy homeostasis, especially fat and sugar metabolism (Hotamisligil, 2006; Roubenoff, 2008). By analogy, could it be that the extensive overlap of stress signaling pathways leading to PR-protein expression in plants is related to the need to coordinate energy balance with immunity? C. THE CONNECTION BETWEEN ENERGY BALANCE AND IMMUNITY

By relying on induced resistance responses such as SAR rather than constitutive resistance, plants remain vulnerable to pathogen attack. Thus it has been postulated that the very existence of SAR must indicate that constitutive resistance has fitness costs (Heil and Bostock, 2002). Indeed, Arabidopsis mutants expressing SAR constitutively show stunted or dwarfed phenotypes and are less fertile, suggesting nutrient or energy limitation (reviewed by Heil and Bostock, 2002). Similarly, stunted and premature senescence phenotypes in Arabidopsis are associated with impaired energy balance signaling (BaenaGonza´lez et al., 2007). In animals, it has been firmly established in the last decade that energy homeostasis and immunity response signaling are extensively and intricately overlapping (Hotamisligil, 2006). The apparent evolutionary basis for this association is the primitive struggle between host and pathogen for control of energy use. The pathogen needs to redirect host energy to itself, and the host needs to redirect energy to combat the pathogen. Remarkably, this struggle may underlie and provide an explanation for a multitude of human diseases, apparently instigated by the chronic hypernutrition that is sweeping global human populations as a result of modernization and industrialization of the food supply (Hotamisligil, 2006).

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In fact, considerable evidence that many PR-proteins also serve as signal molecules has emerged, and that even antimicrobial activities may involve signaling functions (Narasimhan et al., 2005). Thus, we shall explain how the unexpected relationship of PR-5 protein structure, relative to both immune response and energy metabolism signaling, has provided crucial insight into both plant and human pathology.

III. PR-5 PROTEIN STRUCTURE REVEALS THE PRIMITIVE RELATIONSHIP BETWEEN PATHOGEN DEFENSE AND ENERGY BALANCE A. STRUCTURAL FEATURES OF PR-5 PROTEINS

PR-5 proteins are also called THN-like proteins (TLPs) because their amino acid sequence has high homology to THN, a very sweet-tasting protein that was first isolated from the fruit of the West African shrub Thaumatococcus danielli (Cornelissen et al., 1986; Van der Wel and Loeve, 1972). A large number of plant PR-5 proteins have been isolated (Chu and Ng, 2003, Ho et al., 2007; Krebitz et al., 2003; Ng, 2004 and references therein; Veronese et al., 2003 and references therein; Vitali et al., 2006) and several have also been crystallized (Batalia et al., 1996; De Vos et al., 1985; Ghosh and Chakrabarti, 2008; Jami et al., 2007; Koiwa et al., 1999; Leone et al., 2006; Menu-Bouaouiche et al., 2003; Min et al., 2004; Van der Wel et al., 1975). This body of literature establishes that all PR-5 proteins have a similar topology, consisting of a central lectin-like domain of antiparallel -strands surrounded by one or two disulfide-stabilized domains. Sequences bearing features recognizable and conserved in PR-5 proteins are annotated in protein databases as the THN domain (THN; Pfam 23.0 database accession number PF00314; http://pfam.sanger.ac.uk/; Finn et al., 2008).

B. FUNCTION OF PR-5 PROTEINS IN PLANTS

Many lines of evidence support a role in antimicrobial defense for plant PR-5 proteins. The expression of many PR-5 genes is induced by pathogens and hormones (Van Loon et al., 2006 and references therein). There are many examples where significant delays in disease symptom development were observed in greenhouse/controlled-environment grown transgenic plants that overexpress PR-5 genes, particularly when the PR-5 gene was overexpressed in a heterologous plant species (Table II).

TABLE II Stress Tolerance of Transgenic Plants Overexpressing PR-5 Genes Plant species

PR-protein

Donor

Agrostis palustris

PR-5

Oryza sativa

Brassica napus

PR-5

Hordeum vulgare

Citrus sinensis

PR-5

Daucus carota

PR-5

Solanum lycopersicum Or. sativa

Dianthus caryophyllus Fragaria x ananassa

Osmotin

Nicotiana tabacum

Thaumatin

Gossypium hirsutum Hyacinthus orientalis

Tolerance (change)a

Stressor

Reference

Sclerotinia homeocarpa Plasmodiophora brassicae Phytophthora citrophthora Alternaria dauci, Aternaria petroselini, Alternaria radicina, Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii Fusarium oxysporum

þ

Fu et al. (2005)

þ

Reiss et al. (2009)

þ

Fagoaga et al. (2001)

þ

Chen and Punja (2002), Punja (2005)

þ

Zuker et al. (2001)

Bo. cinerea

þ

Osmotin

Thaumatococcus daniellii Ni. tabacum

Salt

þ

Osmotin

Ni. tabacum

Drought

þ (greenhouse)

Schestibratov and Dolgov (2005) Husaini and Abdin (2008) Parkhi et al. (2009)

Thaumatin

Th. danielliii

Fusarium culmorum

þ

Popowich et al. (2007) (continues)

TABLE II Plant species

PR-protein

(continued)

Donor

Tolerance (change)a

Stressor

Reference

Osmotin

Ni. tabacum

Drought, salt

þ

Thaumatin

Th. daniellii

þ

PR-5

Or. sativa

Pythium aphanidermatum, Rhizoctonia solani Alternaria alternata

Olea europaea

Osmotin

Ni. tabacum

Cold

þ

Oryza sativa

PR-5 Chitinase þ TLP þ serinethreonine kinase Osmotin þ chitinase

Or. sativa Or. sativa

Rhizoctonia solani Rh. solani, Sarocladium oryzae, Xanthomonas oryzae Fusarium oxysporum

þ þ

þ

Ouyang et al. (2005)

Phytophthora infestans Phy. infestans

þ (greenhouse)

Korneeva et al. (2008)

þ

Fusarium graminearum Fusarium Salt

(field) þ (greenhouse) þ þ

Li et al. (1999), Liu et al. (1994) Anand et al. (2003) Chen et al. (1999) Mackintosh et al. (2007) Noori and Sokhansanj (2008)

Nicotiana tabacum

Solanum lycopersicum

Solanum tuberosum Triticum aestivum

a

Thaumatin

Ni. tabacum, Phaseolus vulgaris Th. daniellii

Osmotin

Ni. tabacum

PR-5

Or. sativa

Osmotin

Ho. vulgare Ni. tabacum

þ

Barthakur et al. (2001), Sokhansanj et al. (2006) Rajam et al. (2007) Velazhahan and Muthukrishnan (2003) D’Angeli and Altamura (2007) Datta et al. (1999) Kalpana et al. (2006), Maruthasalam et al. (2007)

Unless specifically indicated, tests on overexpressing transgenic plants have been performed under laboratory conditions. Symbols: þ, increase; , decrease.

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Resistance was enhanced by co-expression of PR-5 with other PR or disease-resistance genes but, as with transgenic plants overexpressing other PR genes, a significant disease resistance phenotype has not been demonstrated in field-grown plants (Table II; reviewed in Van Loon et al., 2006). Antisense suppression of PR-5 gene expression did not result in increased disease susceptibility in potato (Zhu et al., 1996), potentially due to compensation by one or more of the multiple isofunctional orthologous PR-5 proteins, compensation by other defense proteins, or other factors not yet recognized (Beffa et al., 1993; Veronese et al., 2003). Many PR-5 proteins have in vitro antifungal activity, supporting a role in plant defense (De A Campos et al., 2008; Ho et al., 2007; Jami et al., 2007; Krebitz et al., 2003; Ng, 2004 and references therein; Veronese et al., 2003 and references therein; Vitali et al., 2006). However, not all PR-5 proteins have demonstrable in vitro antifungal activity (reviewed in Veronese et al., 2003). It has been suggested that this could be due to the fact that PR-5 proteins exhibit target selectivity and these microbial targets have still not been identified. The ability of PR-5 proteins to permeabilize fungal membranes is well documented (Van Loon et al., 2006 and references therein). However, many of these activities were measured for basic proteins in very dilute buffers, under which conditions most polyanions will lyse cell membranes (Narasimhan et al., 2001), and must therefore be interpreted with caution. Another mechanism of action has been reported for osmotin, the tobacco PR-5c protein. Osmotin induces apoptosis in the yeast Saccharomyces cerevisiae, a model fungus (Narasimhan et al., 2001, 2005). Studies with purified PR-5/THN-like proteins show that many of them bind -1,3-glucans, such as those integral to fungal cell walls (Trudel et al., 1998). The -1,3-glucan binding sites have been identified and appear to have conserved recognizable features (Menu-Bouaouiche et al., 2003). Many of the -1,3-glucan binding PR-5 proteins have weak -1,3-glucanase activity. However, there is no evidence that -1,3-glucan binding or -1,3-glucanase activity has any bearing on the antifungal activity of these proteins (Van Loon et al., 2006). Osmotin does not bind -1,3-glucan. It binds yeast cell wall phosphomannoproteins and this interaction facilitates antifungal activity (Ibeas et al., 2000). A wheat TLP has xylanase inhibitor activity and binds non-starch polysaccharides (Fierens et al., 2008, 2009). Zeamatin, an antifungal TLP from maize, was reported to inhibit trypsin and insect -amylase, although these enzyme inhibitory activities may be artifacts (Van Loon et al., 2006). A tobacco PR-5 protein was shown to bind viral coat proteins and viral movement proteins, but there is no evidence that this activity has a role in plant defense (Kim et al., 2005).

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Several lines of evidence suggest roles for plant PR-5 proteins besides defense against microbes. A tobacco PR-5 protein has cytokinin-binding activity and a potato PR-5 protein has actin-binding activity (Igarashi et al., 2001; Kobayashi et al., 2000; Takemoto et al., 1997). In rye, PR-5 proteins have antifreeze activity (Hon et al., 1995), as do other PRs (e.g., PR-3 type chitinases) (Griffith and Yaish, 2004; Hon et al., 1995). As shown in Table II, transgenic plants overexpressing PR-5 genes can exhibit resistance to cold, drought, salt, and osmotic stresses, all of which are abiotic. It has been documented that overexpression of upstream components of plant innate immunity signaling, such as transcription factors and NPR1, a regulatory component of multiple plant defense pathways, affect both biotic and abiotic stress responses (Chen and Guo, 2008; Guo et al., 2004; Luo et al., 2005; Quilis et al., 2008). The data in Table II indicate that overexpression of PR-5, an end product of plant innate immune responses, affects also abiotic stress responses, which cannot be attributed to the antimicrobial activity of the expressed PR-protein. Although some of these phenotypes have only been studied in laboratory conditions and could result from the unique genetic backgrounds of the recipient plants, they support the notion that THN domain/PR-5 proteins may have signaling or protective roles besides antimicrobial activity. C. INFORMATION ON PLANT PR-5 PROTEINS IN GENOMIC DATABASES

As a consequence of genome sequencing projects undertaken in several plant species, it has become apparent that most green plants encode large PR-5 protein families. PR-5 proteins/TLPs are encoded by 21 loci in Arabidopsis and 30 loci in rice (Shatters et al., 2006; www.arabidopsis.org; http://www. gramene.org). Poplar and grape genomes encode PR-5 proteins at 51 and 28 loci, respectively (http://genome.jgi-psf.org/, grape genome browser at http:// www.genoscope.cns.fr/). Genomes of papaya, maize, sorghum, and alfalfa also encode large numbers of PR-5 proteins (17–57 proteins; Superfamily 1.69 database, http://supfam.org/SUPERFAMILY/hmm.html). To better understand the structural features and functions of PR-5 proteins in plants, information available on open reading frames (ORFs) encoding PR-5 proteins in the Arabidopsis genome is summarized in Figs. 1 and 2. Microarray data show that all these PR-5 ORFs are expressed in Arabidopsis, with the expression level of each dependent on the tissue being examined. Thus acidic, basic, and neutral PR-5 proteins can be predicted to occur in Arabidopsis, just as in tobacco (Koiwa et al., 1994). Proteins corresponding to nine of these loci have been identified in various plant parts by mass spectrometry. Of the 21 hypothetical PR-5 proteins, only

Fig. 1.

(continued )

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2 (encoded by At1g18250 and At1g75030) have been expressed as recombinant proteins and were found to be antifungal (Hu and Reddy, 1995, 1997). The protein encoded by At1g18250 (AtTLP1) is basic and that encoded by At1g75030 (AtTLP3) is acidic. This agrees with earlier observations that acidic, basic, and neutral PR-5 members can have antimicrobial activity, mainly antifungal (Chen et al., 2006; Cheng et al., 2004; Kim et al., 2002; Koiwa et al., 1997; Onishi et al., 2006; Stintzi et al., 1993; Tachi et al., 2009; Vitali et al., 2006). The PR-5 transcript that is considered as a marker for SAR is the one encoded by locus At1g75040, but the antifungal activity of the corresponding protein has not been examined (Bowling et al., 1994; Lawton et al., 1996; Uknes et al., 1992). Except for the proteins encoded by At2g24810 and At4g36000, which are truncated near the N-terminus, the PR-5 ‘‘cores’’ of the remaining predicted Fig. 1. Predicted PR-5 proteins of Arabidopsis thaliana: characteristics and tissue-specfic expression. Sequence information and gene expression data for the 21 Arabidopsis PR-5/thaumatin-like proteins were obtained from The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org/). The in vitro activities recorded represent tests performed on recombinant AtTLP-1 and -3 (Hu and Reddy, 1997). The PR-5 transcript associated with systemic acquired resistance (SAR; Uknes et al., 1992) occurs in a cluster of PR-5 genes on chromosome 1. Two other PR-5 gene clusters in the Arabidopsis genome are indicated by different colors. An osmotin-like salinity stress associated gene AtOSM34 has been reported and is indicated (Capelli et al., 1997). Proteomics data represent peptides identified by mass spectrometry from plant organs and subcellular locations as indicated. These data were culled from AtProteome V.0.81 at http://fgcz-atproteome.unizh.ch/, The Plant Proteome Database (PPDB) at http://ppdb.tc.cornell.edu/, and literature (Baerenfaller et al., 2008; Bayer et al., 2006; Bindschedler et al., 2008; Boudart et al., 2005; Carter et al., 2004; Charmont et al., 2005; Feng et al., 2009; Jamet et al., 2006; Jiang et al., 2007; Mitra et al., 2007; Schmidt et al., 2007; Sun et al., 2009; Wienkoop et al., 2004). For ORF-based predictions, the conserved core was first identified by alignment with the conserved core of PR-5 proteins, i.e., from the N-terminal amino acid of mature PR-5 proteins up to the C-terminal amino acid of secreted PR-5 proteins (Melchers et al., 1993). The isoelectric point (pI) for the conserved core domain was then calculated at http://ca.expasy.org/. Subcellular location (Loc) was predicted using TargetP 1.1 server (http://www.cbs.dtu. dk/; Emanuelsson et al., 2000). The predictions are annotated as S for secretory pathway, i.e., the sequence contains a signal peptide, I for any other location, M for mitochondrion and C for chloroplast, with the reliability class of prediction indicated in parentheses from 1 to 5 where 1 indicates the strongest prediction. The lengths of the N- and C-terminal extensions (N-ext and C-ext, respectively) are shown. Any signal sequences (S) or transit peptides (T) predicted by the SignalP 3.0 server (http://www.cbs.dtu.dk/; Bendtsen et al., 2004), and TargetP 1.1 server, respectively, are shown in parentheses. Occurrence of probable transmembrane sequences (TM), GPI attachment (GPI) and membrane association (MA) sites in the extensions is indicated. Organ-specific gene expression data were taken from the Arabidopsis eFP browser at TAIR which is based on Affymetrix ATH1 GeneChip data and indicated numbers represent the eFP outputs in absolute mode (Arabidopsis eFP browser at bar.utoronto.ca; Winter et al., 2007). Abbreviations: apo, apoplast; cw, cell wall; fl, inflorescence or floral organs; mic, microsome; po, pollen; ros, rosette leaves; rt, roots; sd, seed; si, silique; tri, trichome; vac, vacuole.

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Fig. 2. Predicted PR-5 proteins of Arabidopsis thaliana: gene expression in response to stress, senescence, and nutrients. Data were derived from the Arabidopsis Microarray Database and Analysis Toolbox GENEVESTIGATOR Response Viewer and are based on Affymetrix ATH1 GeneChip data. Gene expression was analyzed after inoculation with Phytophthora infestans (non‐host fungus), Erysiphe cichoracearum (biotrophic fungus), Botrytis cinerea (necrotrophic fungus), Myzus persicae (green peach aphid) or Pseudomonas syringae pv. tomato (bacterium), and treatments with salicylic acid (SA, 10 M), methyl jasmonate (MeJA, 10 M), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC, 10 M), mannitol (300 mM; Osmotic), NaCl (150 mM; Salt), 10 % water loss (Drought), or 4o C (Cold). Senescence refers to programmed cell death in cell cultures. Nutrient treatments were Dextrose (3 % glucose versus no sugar), potassium deprivation (Kþ low; deprivation induced by addition of 2 mM CsCl to 1/10 Murashige and Skoog nutrient medium), and low nitrogen (N low, 1/20 of total N).

AtTLPs have similar structure. Unlike the truncations in Arabidopsis TLPs, those reported in some monocot PR-5 proteins occur near the C-terminus (Reiss et al., 2006). The pathogen-inducible acidic PR-5a and PR-5b proteins in tobacco are secreted into the apoplast, whereas the basic PR-5c (osmotin) and neutral PR-5d proteins have been localized to the vacuole. Besides, PR-5 proteins

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have been identified in vascular bundles, phloem exudates, and xylem fluid (Charmont et al., 2005; Dore et al., 1991; Le´on-Kloosterziel et al., 2005; Melchers et al., 1993; Rep et al., 2002; Singh et al., 1987; Uknes et al., 1992). Most Arabidopsis PR-5 proteins are predicted to enter the secretory pathway and proteomic studies have localized two of these to the cell wall/apoplast or the vacuole (Fig. 1; Bayer et al., 2006; Jamet et al., 2006). Both Arabidopsis (At1g20030 and At1g77700) and rice (Os10g05660) encode PR-5 transcripts lacking a signal sequence, and so these proteins could be cytoplasmic (Fig. 1; Shatters et al., 2006). Bioinformatic analyses predict that few of the Arabidopsis PR-5 proteins are anchored, either by transmembrane helices or glycosylphosphatidylinositol (GPI), to membranes (Fig. 1). There is some probability that chloroplast- and mitochondrion-localized PR-5 proteins may also exist in Arabidopsis (Fig. 1). Gene expression data collated in Fig. 2 show that of the 21 Arabidopsis PR-5 genes, only 4 are induced by the indicated biotic stress or defense hormones, whereas 8 are repressed significantly by the same treatments, with the remaining having no significant change in expression levels. A similar distribution is seen in response to abiotic stresses, nutritional signals, and senescence. The ‘‘constitutively expressed’’ gene at locus At5g24620 is overexpressed in the papillar cells of the stigma and is presumed to influence pollen tube growth (Tung et al., 2005). Thus, PR-5 proteins also appear to function in plant growth and development. One piece of evidence supporting a signaling role for PR-5 proteins is the fact that in addition to the PR-5 genes listed in Fig. 1, the genome of Arabidopsis also encodes three RLKs with THN domains (Afzal et al., 2008; Shiu and Bleecker, 2001). The structures of these PR-5 receptor-like kinases (PR-5Ks) are shown in Fig. 3. The kinase domains of the three proteins share about 80% sequence similarity at the nucleotide level and the protein kinase domain encoded by At5g38280 (referred to in the literature as AtPR-5K) has enzymatic activity (Wang et al., 1996). Heterologous expression of AtPR-5K in creeping bentgrass increased resistance to Sclerotinia homeocarpa, indicating a role in defense (Guo et al., 2003). The proteins encoded by At1g70250 and AtPR5K have N-terminal signal sequences of 15 and 22 amino acids, respectively, and are predicted to be secreted. The protein encoded by At4g18250 does not have a signal sequence and could be targeted to the chloroplast. The two THN domains of the protein encoded by At4g18250 have no intervening sequence separating them. The RLK protein encoded by At1g70250 contains a bifunctional trypsin -amylase inhibitor/LTP/seed storage protein domain in addition to the THN domain. Expression data in TAIR (www. arabidopsis.org) show that the three AtPR-5Ks have tissue-specific

At1g70250 (799 aa) At4g18250 (853 aa) At5g38280 PR5K (665 aa) Plant LTP PF00234

THN PF00314

Transmembrane

Locus

Loc

THN pl

THN MW (Da)

At1g70250

S (2)

7.5

23346

Moderate expression in rosette leaves, roots, flowers and pollen

At4g18250

C (5)

6.1, 5.5

23466, 23906

Low expression in above tissues

Organ-specific gene expression

Protein kinase PF00069

Inducer/repressor

Inducer: Bo. cinerea, My. persicae, salicylic acid. Repressor: Ps. syringae.

At5g38280

S (1)

6.71

24645

Moderate expression in roots, floral stem and pollen

Fig. 3. PR-5 kinases of Arabidopsis thaliana. The sequence information and gene expression data for the Arabidopsis PR-5 kinases were obtained from The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org/). The domain structure was obtained by querying the predicted proteins against InterPro at http://www.ebi.ac.uk/ (Hunter et al., 2009; Quevillon et al., 2005). Pfam accession numbers of the domains are shown (Pfam 23.0 database; http://pfam.sanger.ac.uk/) (Finn et al., 2008). Subcellular location (Loc) was predicted using TargetP 1.1 server at http://www.cbs.dtu.dk/services/TargetP/ (Emanuelsson et al., 2000). The predictions are annotated as S for secretory pathway, that is, the sequence contains a signal peptide, and C for chloroplast, with the reliability class of prediction indicated in parentheses from 1 to 5, where 1 indicates the strongest prediction. The isoelectric point (pI) and molecular weight (MW) for the conserved core thaumatin (THN) domain (defined as in Fig. 1) was calculated at http://ca.expasy.org/tools/pi_tool.html. Organ-specific gene expression data were taken from the Arabidopsis eFP browser at TAIR, which is based on Affymetrix ATH1 GeneChip data (Arabidopsis eFP browser at bar.utoronto.ca) (Winter et al., 2007). Treatments eliciting greater than twofold induction or repression are listed and were identified based on data derived from the Arabidopsis Microarray Database and Analysis Toolbox GENEVESTIGATOR Response Viewer that is based on Affymetrix ATH1 GeneChip data.

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expression (Fig. 3). The At4g18250 transcript responds to biotic stresses, suggesting a role in defense. Genomic information indicates that a variety of THN domain fusions occur in plants. THN domain fusions in grapevine, maize, and rice are detailed in Fig. 4. The domains fused with THN domain(s) detected so far in higher plants include protein kinase, anticodon loops involving His-, Gly-, Thr-, and ProtRNA synthases, cytochrome P450, etc. Some of the THN domain fusion proteins have signal sequences and are presumably secreted, whereas others lack signal sequences. Although these data do not shed light on the probable function of THN domains, they strongly suggest that both intra- and extracellular THN domain proteins play an important role in plants. Another piece of evidence suggesting that PR-5 proteins may be signaling molecules comes from a recent study showing that osmotin is a homolog of the mammalian hormone adiponectin that controls energy homoeostasis by regulating the activity of AMP-dependent protein kinase (AMPK) (Narasimhan et al., 2005). This and other studies have shown that osmotin and THN induce signaling that alters the expression of stress-responsive genes, metal ion uptake genes, and cell differentiation in Sa. cerevisiae, and that this signaling is mediated by their interaction with the yeast homologs of adiponectin receptors (Jin et al., 2008; Kupchak et al., 2007, 2008; Lyons et al., 2004; Narasimhan et al., 2005; Smith et al., 2008; Villa et al., 2009). Components of this signaling pathway also regulate the ability of osmotin to induce apoptosis in yeast (Narasimhan et al., 2005). Heterologous overexpression and activation of adiponectin receptors in yeast activates the same pathways (Kupchak et al., 2008; K. Kim, personal communication); conversely, osmotin induces energy balance signaling in mammalian cells through adiponectin receptors (Narasimhan et al., 2005). Adiponectin receptors and their yeast homologs have characteristics of G-protein coupled receptors and belong to the same family as the plasma membrane progesterone receptor. Members of this family of progestin and AdipoQ (adiponectin) receptors (PAQR) are ubiquitous in all phyla, and include five members in Arabidopsis (Hsieh and Goodman, 2005; NCBI conserved domain database at http://www.ncbi.nlm.nih.gov, query pfam03006). Disruption of Arabidopsis HEPTAHELICAL PROTEIN 1 (HHP1), a PAQR-encoding gene, results in higher sensitivity to ABA and osmotic stress (Chen et al., 2009). HHP1 was found to control expression of stress-responsive genes. This parallels the situation in Sa. cerevisiae, where PAQR overexpression and PR-5 signaling through PAQRs, control expression of stress-responsive genes, raising the possibility that also in Arabidopsis some PR-5 proteins might be involved in signaling via these receptors (Kupchak et al., 2008; Narasimhan et al., 2001, 2005). THN

Species

Accession numbers

Domains with Pfam accession numbers (in order from Nterminus)

Protein structure

Domain function

ssp. indica BGIOSIBCE000106.1 ssp. japonica Q656X4, LOC_Os01g02310

PF00314 thaumatin, PF00069 protein kinase

ssp. indica BGIOSIBCE000110.1 Ssp. japonica Q656X3

PF00314 thaumatin, PF00069 protein kinase

ssp. japonica Q0JR93 LOC_Os01g0113500

PF00069 protein kinase, PF00314 thaumatin, PF00069 protein kinase

Oryza sativa

ssp. indica BGIOSIBCE035972.1 LOC_Os12g08280

PF00314 thaumatin, PF00459 inositol monophosphatase, signal sequence

PF00459 : inositol polyphosphate 1-phosphatase dephosphorylates inositol phosphate to inositol as part of the phosphatidylinositol signalling pathway

Oryza sativa

ssp. indica BGIOSIBCE012847.1 LOC_Os03g52010 Q851F0

PF00314 thaumatin, PF02450 lecithin acetyltransferase, signal sequence

PF02450: lecithin acetyltransferase is involved in cholesterol esterification. It interacts with lipoproteins and functions in cholesterol transport by helping to sequester cholesterol esters in lipoprotein particles.

Vitis vinifera

>GIDVvT00015510001_ PF000697 P450, 1 PF00314 thaumatin, signal sequence A5AEX9

Oryza sativa

PF00069: protein kinase : transmembrane

Fig. 4.

PF00067: cytochrome P450 enzymes are heme containing mono-oxygenases that are important in plants for the biosynthesis of hormones, defensive compounds and fatty acids

(continued )

Species

Accession numbers

Domains with Pfam accession numbers (in order from N-terminus)

Protein structure

Domain function

PF01151: ELO is a family of membrane proteins involved in the synthesis of ceramides and sphingolipids. They may have roles in controlling glucose signaling and plasma membrane H+-ATPase.

Vitis vinifera

>GSVIVT00026571001 A7PYT6

PF01151 ELO, PF00314 thaumatin, signal sequence

Zea mays

AC187883.4_FGT009

PF00314 thaumatin, PF00069 protein kinase, signal sequence

PF00069: protein kinase

PF00314 thaumatin, PF03129 HGTP anticodon, signal sequence

PF03129: this is believed to be the anticodon binding domain of His-, Gly-, Thr- and Pro-tRNA synthases

: transmembrane

AC212473.1_FGT011 Zea mays

AC193988.3_FGT028 Q8SA98

Fig. 4. Thaumatin domain fusion proteins in plants. Domain searches were performed with THAUMATIN or PF00314 as query terms at Pfam 23.0 database (http://pfam.sanger.ac.uk/) (Finn et al., 2008), Superfamily 1.69 database (http://supfam.mrc-lmb.cam.ac.uk/) (Gough et al., 2001), and manually at Grape genome browser (http://www.genoscope.cns.fr/). Domains predictions were verified manually by querying the predicted proteins against InterPro at http://www.ebi.ac.uk/ (Hunter et al., 2009; Quevillon et al., 2005). Domain descriptions can be found at the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (Marchler-Bauer et al., 2009). Accession numbers are from UniProtKB (http://www.uniprot.org/uniprot/; The UniProt Consortium, 2009), MaizeSequence.org Release 3b.50 (http://maizesequence.org/), the Grape Genome Browser (http://www.genoscope.cns.fr/), J. Craig Venter database (http://www.tigr. org/), and BGI Rise Rice Genome Database (http://rice.genomics.org.cn/). Some of the indicated signal sequences have transmembrane domains and others do not. Domains and their descriptions are in identical colors. Truncated domains are indicated with jagged edges. These data were verified on March 24, 2009 and may change as annotations in the databases are revised.

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interacts with G-protein-coupled taste receptors to induce the sensation of sweet taste, lending further support to the notion that the THN domain has broad, yet poorly understood, target recognition functions (Temussi, 2006).

D. COMPARISON OF THN DOMAIN WITH C1q-TNF DOMAINS

Several intriguing concepts regarding the role of PR-5 proteins can be established by comparing similarities in the properties, location, and functions of adiponectin and adiponectin-like mammalian proteins with those of PR-5 proteins. Adiponectin is a protein hormone secreted by adipocytes, that is, mammalian fat cells (Diez and Iglesias, 2003; Fang and Sweeney, 2006). It is an abundant plasma protein. Many PR-5 proteins share a similar extracellular location (Bayer et al., 2006; Charmont et al., 2005; Melchers et al., 1993; Rep et al., 2002; Uknes et al., 1992) (Fig. 1), as the mammalian plasma is equivalent to apoplastic fluid, xylem, and phloem sap of plants. Adiponectin is composed of two domains: an N-terminal collagen domain and a C-terminal globular domain that has a clearly recognizable fold, annotated in databases as the C1q domain (Pfam23.0 database, accession PF00386; http://pfam.sanger.dc.uk; Finn et al., 2008). The adiponectin C1q domain is necessary and sufficient for interaction with adiponectin receptors (Tomas et al., 2002; Yamauchi et al., 2003a,b). Although the sequence identity between osmotin and adiponectin is insignificant (<10 %), crystal structures show that the core lectin-like domain of osmotin and the C1q domain of adiponectin are composed of a compact jelly roll of antiparallel strands that can be significantly overlapped. It has been shown that osmotin is not only the functional equivalent, but also the structural equivalent of the C1q domain of adiponectin (Narasimhan et al., 2005). C1q domain containing proteins constitute a large family of mammalian proteins that have several similarities with THN domain proteins (Table III). Extracellular mammalian proteins containing the C1q domain include hormones (adiponectin, hibernation proteins); proteins that promote interaction of cells with the Extracellular matrix and have roles in tissue development and remodeling (the elastin microfibril interface located proteins, known as EMILINs; Collagen VIII and Collagen X; the C1q tumor necrosis factor (TNF)-related protein 5, known as CTRP5); proteins involved in target recognition for immune responses (Complement 1q); and brain proteins implicated in building memory (cerebellins and precerebellins) that regulate synapse development (development of connections between neurons) and synaptic plasticity (usage-associated changes in strength of connection between neurons) (Innamorati et al., 2006 and references therein;

TABLE III Comparison of Plant Thaumatin (THN) Domain Proteins with Mammalian C1q-TNF Superfamily of Proteins THN domain PF00314 PR-5

Property Structure

Location

Multimer formation

Interaction of THN/C1q domain with non-protein molecules Interaction of THN/ C1q/TNF domain with proteins or peptides

a

C1q-TNF superfamily PF00386 and PF00229 Adiponectin

b

Complement 1qb C1q domain fusion with collagen-like domain

Other C1q proteinsb Single C1q domain

TNFb

Single THN domain

C1q domain fusion with collagen-like domain

Multiple THN domains THN domain fusion with kinase/anticodon loop/ cytochrome P450/other domains Extracellular Possibly intracellular Possibly membrane anchored Some proteins crystallize as dimer; osmotin exists in solution mainly as monomer along with small amounts of multimers (two to six) -1,3-Glucans Arabinoxylans Phosphomannan (Ca2þ-dependent) Cytokinin Viral coat protein Immunoglobulin E (IgE)

Multiple C1q domains C1q domain fusions to cysteine/ Gly-rich/coiled-coil/Leu zipper/EGFc-like/collagenlike domains Extracellular Extracellular Extracellular Extracellular Intracellular Possibly membrane anchored The C1q–TNF domain trimerizes. Higher order structures depend on collagen domain (or other) fusion partner

Bacterial lipopolysaccharide (LPS)

LPS Lipid A Phospholipids

Antifreeze proteins

Growth factors AdipoR1/R2 (GPCR-like) receptors T-cadherin

Actin Xylanase

Chemokines Early apoptotic cell debris

Viral proteins Vasopressin receptor (GPCR) Bacterial outer membrane proteins Serum IgM, IgG, C-reactive Other receptors? protein Apoptotic cells Alzheimer -amyloid peptide

C1q domain fusion with collagen-like domain

TNF receptors

Familial dementia peptide

Adiponectin receptors R1/ R2 (GPCRc-like) receptors Taste receptors (GPCR)

Function

Defense

Energy metabolism; induces lipid and carbohydrate catabolism

Antidiabetic, induces insulin sensitivity

Anticancer Anti-atherosclerotic, Anti-inflammatory

C1s and C1r proteases (Ca2þ-dependent) Adiponectin Defense

Clearance of toxic cell debris

Many members are involved in extracellular matrix development and function. Family members are implicated in development and stability of smooth muscle, blood vessels, Purkinje cell synapses, bone, retinal epithelium, inner ear membrane, etc

Defense and energy metabolism

Involved in apoptosis, survival, and induction of insulin resistance Proinflammatory

a See Fig. 1 and Fig. 4; Narasimhan (unpublished work): osmotin solutions of pH 6.5–7.4 were examined for evidence of oligomerization by mass spectrometry and by two-dimensional gel electrophoresis; Batalia et al. (1996), Bublin et al. (2008), Dore et al. (1991), Fierens et al. (2008, 2009), Grenier et al. (1999), Ibeas et al. (2000), Igarashi et al. (2001), Kim et al. (2005), Kobayashi et al. (2000), Krebitz et al. (2003), Min et al. (2004), Narasimhan et al. (2005), Salzman et al. (2004), Soman et al. (2000), Takemoto et al. (1997), Temussi (2006), Villa et al. (2009), Yu and Griffith (1999). b Doliana et al. (1999), Duus et al. (2007), Ghai et al. (2007), Hug et al. (2004), Innamorati et al. (2006), Kishore et al. (2004), Masaie et al. (2007), Peake et al. (2006, 2008), Takemura et al. (2007), Wang et al. (2005b), Yuzaki (2008). c EGF, epidermal growth factor; GPCR, G-protein-coupled receptor.

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Kishore et al., 2004 and references therein; Lafontan and Viguerie, 2006 and references therein; Yuzaki, 2008 and references therein). Intracellular mammalian C1q domain proteins are just being identified and their functions are not well understood. One such protein interacts in vitro with the G-protein-coupled vasopressin receptor that regulates water permeability, and overexpression of another induces apoptosis (Innamorati et al., 2006). The C1q domain is the target recognition module in all of the above proteins. Structure–function studies have shown that another class of mammalian proteins, the TNFs, have a globular domain that shares almost no sequence similarity, but has high three-dimensional similarity with C1q domains (Innamorati et al., 2006 and references therein; Kishore et al., 2004 and references therein; Yuzaki, 2008 and references therein). The conserved globular TNF and C1q domains also have an evolutionary link. C1q-TNF domain proteins are recognized as a superfamily of proteins with target recognition domains. It should be re-emphasized here that the compact jelly roll of -strands that is the hallmark of the C1q-TNF domain superfamily, has three-dimensional overlap with the compact jelly roll of -strands forming the core lectin-like domain of osmotin and THN domain proteins (Narasimhan et al., 2005). Just as the THN domain family is large in Arabidopsis and in several other plant species, the C1q and TNF domain families are large in mammals, and consist of 13 and 32 members, respectively, in humans (Fig. 1, Innamorati et al., 2006 and references therein; Kishore et al., 2004 and references therein; Shatters et al., 2006; Yuzaki, 2008 and references therein). The adiponectin gene is expressed only in adipocytes, whereas the cerebellins are expressed only in brain, exhibiting tissue-specific expression much like THN domain proteins (Fig. 1). Many C1q domain proteins are extracellular, like THN domain proteins. EMILIN and multimerin are C1q domain extracellular matrix proteins with adhesive properties (Jeimy et al., 2008; Kishore et al., 2004). Complement 1q is a blood protein that induces intracellular signaling and protease cascades for host defense. Whereas PR-5 can either possess or be devoid of antimicrobial activity, Complement 1q itself is not antimicrobial (De A Campos et al., 2008; Ho et al., 2007; Jami et al., 2007; Krebitz et al., 2003 and references therein; Ng, 2004 and references therein; Veronese et al., 2003 and references therein; Vitali et al., 2006). Membrane-anchored members of THN domain proteins are predicted to exist (Wang et al., 1996) (Fig. 1) and two intracellular C1q proteins are suspected to be membrane anchored. TNFs occur as membrane-anchored proteins that mediate signaling initiated by cell contact and they also exist as soluble extracellular forms released by proteolytic cleavage of the membrane-bound versions. The soluble forms of TNFs can act as agonists or antagonists of the membrane-bound

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versions. Some TNFs induce apoptosis while some promote survival and others induce proliferation. TNFs play important roles in inflammation, immunity, development of hair follicles and sweat glands, bone homeostasis, and angiogenesis (creation of blood vessels). The crucial roles of the C1q-TNF domain superfamily of proteins in energy homeostasis, immune responses, cell growth, cell survival, and cell death inferred by in vitro studies, have been supported by pathologies observed in knockout mice or in humans with genetic disorders. The C1q domain protein adiponectin and the PR-5 protein osmotin require the same receptors to activate AMPK-dependent signaling in mammalian cells and in yeast (Kupchak et al., 2007; K. Kim, personal communication; Narasimhan et al., 2005). However, a role of PR-5/THN domain proteins in plant energy metabolism remains to be established. By analogy with animal systems and as suggested by the data in Fig. 2, their roles in cell growth survival, differentiation, and death are also possible. There are also instances where a member of the C1q domain family will influence the effect of a member of the TNF family. For example, obesity has opposite effects on the expression of adiponectin and TNF (Whitehead et al., 2006). The similarity of this observation with data (Fig. 2) showing that a given defense or nutritional stimulus can have opposite effects on the expression of different TLPs in Arabidopsis is to be noted. Adiponectin and TNF proteins elicit opposite effects in insulin resistance and arteriosclerosis (Rondinone, 2006), and it remains to be seen whether some Arabidopsis TLPs affect the function of other members of their family. Most C1q-TNF domain proteins occur as fusions of the C1q-TNF domain with other domains. The C1q-TNF domain spontaneously forms trimers that may be homomers (adiponectin) or heteromers (Complement 1q). Formation of higher order oligomers is usually due to the fused domain, which could be a collagen triple-helix repeat (Complement 1q, adiponectin, TNF), the cysteine-rich EMI or epidermal growth factor (EGF)-like motifs (EMILIN, multimerin), coiled-coil domain (EMILIN, multimerin), leucine zipper (EMILIN), another C1q domain, or cysteine residues (Innamorati et al., 2006 and references therein; Kishore et al., 2004 and references therein; Yuzaki, 2008 and references therein). Oligomerization of the C1q domain is critical for ligand recognition, avidity of interaction with receptors, and signaling outcome (Callebaut et al., 2003; Fang and Sweeney, 2006; Innamorati et al., 2006; Kishore et al., 2004). The compact jelly roll of -strands that is the hallmark of the C1q-TNF superfamily is conserved in THN domains and in several viral capsid proteins, suggesting that this architecture promotes protein oligomerization (Kishore et al., 2004). Osmotin can form oligomers, but the relevance of oligomerization for its biological

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activity is unknown. It must also be noted here that basic proteins such as osmotin have a tendency to polymerize at acidic pH, so oligomerization of acidic PR-5 proteins needs to be demonstrated in order to confirm that it is an innate property of the THN domain. THN domain fusions that occur in higher plants seem to be species specific (Fig. 4). For example, RLKs with THN domains are found in Arabidopsis, rice, maize, and sorghum, but not in grape. Conversely, THN domain fusion proteins of the types that occur in grape cannot be found in Arabidopsis. THN domain fusions in higher plants occur mostly with domains found in enzymes (Fig. 4). Bioinformatic analyses show a strong statistical association of the PR-5K kinase domains with innate immunity in animals and plants (Dardick and Ronald, 2006). The kinase domains of AtPR-5Ks are very similar and are paralogous to mammalian Interleukin-1 Receptor Associated Kinases (IRAKs) that play critical roles in mammalian innate immune responses (Gan and Li, 2006). In particular, they are involved in Toll-like receptor-mediated signaling upon PAMP detection, which shares common structural elements with plant defense signaling (Ausubel, 2005). Bioinformatic predictions of a role in immunity are supported by data demonstrating that heterologous expression of AtPR-5K in bentgrass protects against Sclerotinia homeocarpa (Guo et al., 2003). Inositol polyphosphate 1-phosphatase is involved in linking stress signaling to sugar signaling in Arabidopsis (Ananieva et al., 2008; Xiong et al., 2001). The fatty acid ELOngation factor (ELO) domain that occurs in fusion with a THN domain (Fig. 4) is found in proteins involved in the biosynthesis of ceramides and sphingolipids. Yeast ELO3 also functions in glucose signaling and regulation of plasma membrane proton ATPase activity. An Arabidopsis ELO-like gene, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 3 (HOS3) controls abiotic stress responses (Quist et al., 2009). The occurrence of a THN domain fusion with an ELO domain or inositol polyphosphate 1-phosphatase domain therefore strongly suggests a function in energy and/or stress metabolism. Cytochrome P450s are heme mono-oxygenases involved in detoxification and the biosynthesis of hormones, lignin, and secondary metabolites in plants. They are associated with plant growth and antimicrobial defense, suggesting that the cytochrome P450-THN domain fusion protein could have a role in plant defense (Schuler and Werck-Reichhart, 2003). The C1q domain of Complement 1q is a versatile ligand-binding domain (Gasque, 2004). It binds to a large variety of ligands (immune complexes of immunoglobulins IgG and IgM, the complement-reactive protein known as CRP, PAMPs, apoptotic cell molecular patterns) that bear a remarkable similarity to molecules known to bind to PR-5 proteins (Table III). Binding of immune complexes of IgG or IgM to the C1q domain of Complement 1q

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causes a conformational change that exposes the fused collagen domain and allows it to recruit protease activity (Innamorati et al., 2006; Kishore et al., 2004). In other C1q domain proteins, the C1q domain has been suggested to function in promoting activity of the fused domain or in targeting the activity of the fused domain to the proper intracellular location (Innamorati et al., 2006). Thus, by analogy, THN domains in fusion proteins are probably ‘‘ligand’’ recognition domains that affect activity or location of the fused partner domains. E. THN DOMAIN PROTEINS AND C1q-TNF DOMAINS ACROSS PHYLA

Proteins with THN domains occur in plants, insects, many species of ascomycete fungi, some bacteria, and even viruses (Table IV; Shatters et al., 2006; Superfamily 1.69 database, http://supfam.org/SUPERFAMILY/hmm). The genomes of these species encode families of THN domain proteins ranging from 1 to 20 members. Clearly, such highly conserved features must be significant. THN domain fusion partners encoded in fungal genomes include LRR, lectin, THN, ankyrin repeats, and a Zn2Cys6 DNA-binding domain. A PR-5 protein with -1,3-glucanase activity was identified in senescing fruiting bodies of the basidiomycete Lentinula edodes, showing that fungal and plant PR-5/THN domain proteins have common functions (Sakamoto et al., 2006). Simultaneous deletion of Aspergillus nidulans calA and cetA, encoding two secreted PR-5 proteins, severely inhibits conidial germination, showing that these proteins have essential and overlapping roles in development (Belaish et al., 2008). For THN domain proteins, a role in plant growth and development has been suggested (Fig. 1), but not yet demonstrated. cDNAs encoding a PR-5 family protein were induced in the beetle Tribolium castaneum by bacterial LPS (Altincicek et al., 2008). There are four PR-5 proteins encoded in the Tribolium genome. Of these, recombinant Tr. castaneum THN-1 (Genbank number XP_968724) that had antifungal activity against the plant pathogen Fusarium culmorum and the beetle-parasitizing fungus Beauveria bassiana. In the larvae of the beetle Dendroides canadensis, a PR-5 protein, TLP-Dcan1 (Genbank number DQ023319), was identified as a specific enhancer of the activity of two larval antifreeze proteins, DAFP-1 and -2 (Wang and Duman, 2006). TLP-Dcan1 by itself had no antifreeze activity and had no enhancing effect on larval antifreeze proteins besides DAFP-1 and -2. Thus, plant and insect PR-5 family proteins appear to have similar functions. Expression of TLP–Dcan1 was detected in fat body and gut epithelial cells of De. canadensis (Wang and Duman, 2006). A second PR5 gene, TLP-Dcan2, was also expressed in the fat body of De. canadensis larvae, but not studied further. To underline once more the parallelism

TABLE IV Species Distribution of THN and C1q-TNF Domain Proteins Organism Kingdom, phylum

Class, division or group

Plantae

Angiospermae Pinophyta Bryophyta Chlorophyta

Protista Amoebozoa Fungi Ascomycota Basidiomycota

Heterokontophyta Animalia Chordata

Echidermata Mollusca

Oomycetes

C1q-TNF superfamily of proteins (PF00386 and PF00229)

THN domain proteins (PF00314) Flowering plants (30 species) Conifers (13 species) Mosses: Physcomitrella patens, Selaginella moellendorfii Green algae: Chlamydomonas reinhardtii Single celled phytoplankton: Emiliana huxleyi Dictyostelium discoideum, Dictyostelium purpureum

Dictyostelium discoideum

34 species, including Candida, but not Saccharomyces or Schizosaccharomyces Coprinopsis cinerea, Cryptococcus neoformans var. neoformans, Laccaria bicolor, Lentinula edodes, Moniliophthora perniciosa, Sporobolomyces roseus Phytophthora infestans, Phtophthora parasitica, Phytophthora ramorum, Phytophthora sojae

Phytophthora sojae

Mammals Birds Amphibia Bony fishes

None None None None

Lampreys Cefalochordata Urochordata Echinoidea

None None None None None

None

30 species, including Homo sapiens Anas platyrhynchos, Anser anser, Gallus gallus, Taeniopygia guttata Xenopus spp. Carassius auratus, Cyprinus carpio, Danio rerio, Dissostichus mawsoni, Epinephelus coioides, Lepomis macrochirus, Lithognathus mormyrus, Oncorhynchus keta, Oncorhynchus masou formosanus, Oncorhynchus mykiss, Oryzias latipes, Psetta maxima, Salmo salar, Salvelinus fontinalis, Siniperca chuatsi, Tetraodon nigroviridis Lethenteron japonicum Branchiostoma floridae, Branchiostoma belcheri Ciona intestinalis Strongylocentrotus purpuratus Cepaea hortensis, Chlamys farreri, Haliotis discus, Helix pomatia, Mytilus edulis, Pinctada fucata

Anellida Choanozoa Arthropoda

Clitellata Choano-flagellatea Insecta

Branchiopoda Arachnida Nematoda

Secernenta

Bacteria

None None Beetles: Biphyllus lunatus, Dendroides canadensis, Diaprepes abbreviates, Tribolium castaneum, Tribolium confusum Bees and wasps: Lysiphlebus testaceipes Butterflies and moths: none Flies and mosquitos: none Aphids: Acyrthosiphon pisum, Toxoptera citricida Locust: Schistocerca gregaria None Mites: Aleuroglyphus ovatus, Glycyphagus domesticus, Suidasia medanensis, Tyrophagus putrescentiae Caenorhabditis elegans, Caenorhabditis briggsae Geobacter lovleyi, Legionella pneumophila, Methylocella silvestris, Sorangium cellulosum, Syntrophobacter fumaroxidans

Cyanobacteria Viruses

Cyanothece spp. Ectocarpus siliculosus virus 1, Feldmannia species virus

Helobdella robusta, Hirudo medicinalis Monosiga brevicollis Beetles: Tribolium castaneum Bees and wasps: Apis mellifera, Nasonia vitripennis Butterflies and moths: Bombyx mori Flies and mosquitos: Aedes aegypti, Anopheles gambiae, Culex pipiens, Culex quinquefasciatus, Drosophila spp. Aphids: Acyrthosiphon pisum Locust: none Daphnia pulex Mites: none Caenorhabditis elegans Bacillus spp., Enterococcus spp., Lactococcus spp., Nitrobacter hamburgensis, Oligotropha carboxidovorans, Paenibacillus spp., Paracoccus denitrificans, Pediococcus spp., Prochlorococcus marinus, Roseobacter denitrificans, Shewanella woody, Xanthobacter autotrophicus Prochlorococcus marinus Bacillus phage SPO1, Phormidium phage

Data were obtained from the Pfam 23.0 database with thaumatin, C1q, and TNF as query terms (http://pfam.sanger.ac.uk/; Finn et al., 2008), Superfamily 1.69 database with the same query terms (http://supfam.mrc-lmb.cam.ac.uk/; Gough et al., 2001), and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) using C1q and thaumatin as query terms and then checking all hits on the list of linked taxonomic groups. Insect data were also obtained from Shatters et al. (2006). These data were verified on March 24, 2009, and since they are based on nucleotide sequences, some of the data may change as annotations in the databases are revised. Many of the listed species have more than one predicted THN or C1q-TNF domain protein. Species that are predicted to have both THN and C1q-TNF family proteins are highlighted.

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between THN-domain and C1q-TNF superfamily domain proteins, mammalian adiponectin and TNF proteins that affect energy metabolism and defense are expressed in fat cells (adipocytes), just like the PR-5 proteins of De. canadensis (Kishore et al., 2004; Wang and Duman, 2006). An examination of the sequence databases reveals that the occurrence of THN domain proteins and C1q domain proteins in organisms is usually mutually exclusive (Table IV). Notable exceptions are Dictyostelium discoideum (amoeba), Phytophthora sojae (fungus), Acyrthosiphon pisum (pea aphid), Caenorhabditis elegans (nematode), and Tr. castaneum (beetle), all having both C1q-TNF and THN domain proteins (Table IV). However, a cautionary note should be added here. Annotations change over time as sequence databases receive new data and are revised accordingly. For example, a Tr. castaneum TNF family protein is annotated only in the Superfamily 1.69 database, but not in the Genbank or the Pfam 2.3 databases (Table IV). Similarly, an Ac. pisum C1q domain protein is annotated only in the Genbank, but not in the Superfamily 1.69 or the Pfam 2.3 databases. Overall, there seems to be an overlap between occurrence of THN domain proteins and C1q-TNF domain proteins only in lower life forms. Thus it is worthwhile considering whether the THN domain could be the equivalent of the C1q-TNF domain. This would imply that THN domain proteins, beside their already described pathogen defense functions, have additional functions that remain to be discovered and characterized.

IV. DIRECTIONS IN WHICH CURRENT CLASSIFICATION OR DEFINITION OF PR-PROTEINS MAY CHANGE IN THE COMING YEARS AS ADVANCED FUNCTIONAL STUDIES PROGRESS Despite extensive research in the past few decades, the biological functions of many PR-proteins, notably the most studied of all, PR-1, are still not established. Genetic studies using loss of function alleles would elucidate the function of PR-proteins, but such analyses have been hampered by the redundancy in the PR gene families. It is often unclear which gene within a PR-protein family corresponds to which isolated protein. As such, the current classification of PR-proteins is likely to change dramatically in the coming years as advanced functional studies progress. If a more general definition of ‘‘pathogenesisrelated protein’’ is applied that considers all proteins induced by microbes as PR-proteins (regardless of whether they have been shown to have measured antimicrobial properties or not), then recognition of a much more comprehensive list of PR-proteins and PR-protein families is warranted. We illustrate here

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that PAMP and pathogen-encoded elicitor recognition in innate immunity is manifested by a complex but hierarchical array of not only R gene-mediated control of antimicrobial processes, but also by PR-proteins, some of which can exhibit remarkable properties of signal recognition and function themselves, probably including PAMP recognition (Veronese et al., 2003). The classification of PR-proteins began based on the properties of the induced proteins and the inducibility of their genes. Gradually it was modified and extended based on functional information and ultimately on sequence similarities. Although sequence is often closely related to function because of evolutionary expansion of gene families, ultimately function does not rely on or correlate strictly with sequence. Protein domain structure, although not abundantly known, is probably the most reliable trait upon which to base protein/gene function. The strong similarity between THN domain proteins and Clq-TNF superfamily proteins—in subcellular location, existence of membrane-anchored and soluble forms, defense, and energy metabolism—suggests that the study of PR-5 proteins may reveal how plant defense responses are spatially integrated and set an example for elucidating the functional role of other PR-proteins. The innate immune signaling pathways in plants and animals have common features (Ausubel, 2005; Staal and Dixelius, 2007). They respond to similar stimuli, include extra- and intracellular receptors for microbeassociated PAMPs, MAPK modules, activation of transcription factors, and the production of antimicrobial peptides. In addition, the innate immune signaling pathways of animals produce cytokines which are polypeptides involved in local and systemic communication between cells. Some cytokines are pro-inflammatory and others are anti-inflammatory. They serve as an alarm system for future insults and also coordinate immune response with energy metabolism. Of the cytokines, Interleukin-6 and TNF are connected to energy, carbohydrate and/or lipid metabolism (Hoene and Weigert, 2008; Hotamisligil, 2006). More interestingly, many individual components of the innate immune signaling pathways in plants and animals share recognizable protein domains. LRRs occur in the extracellular regions of transmembrane PRRs such as Toll-like receptors of animals and RLKs of plants, which detect PAMPs (Ausubel, 2005; Staal and Dixelius, 2007). Intracellular PRRs in plants and animals typically contain three domains, of which LRRs and nucleotide binding domains (NBDs) are common to both plant and animal intracellular PRRs. These intracellular PRRs detect PAMP degradation products in animals and pathogen effector proteins in plants. Induced immune effectors, such as defensins of plants and animals, also have similar structure. We have shown that the PR-5 proteins (THN domain) share a common domain with TNF (C1q-TNF domain) and can affect

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energy balance signaling in heterologous systems (Table III; Narasimhan et al., 2005). Whereas a role for THN domain proteins in plant energy metabolism remains to be proven, the data suggest that some PR-proteins may have cytokine-like function and that induced expression of at least some PR-proteins in response to various stresses may constitute an inflammatory response in plants. That is, the end products of innate immune signaling in animals and plants may have the same underlying logic, a possibility that has not yet been fully appreciated.

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Role of Iron in Plant–Microbe Interactions

P. LEMANCEAU,*,1 D. EXPERT,{ F. GAYMARD,{ P. A. H. M. BAKKER} AND J.-F. BRIAT{

*INRA, Universite´ de Bourgogne, UMR1229, Microbiologie du Sol et de l’Environnement, CMSE, 17 rue Sully, BV 86510, F-21034 Dijon cedex, France { INRA, AgroParisTech, Universite´ Paris 6, UMR217, Interactions Plantes Pathoge`nes, 16 rue Claude Bernard, F-75005 Paris, France { CNRS, Universite´ Montpellier II, SupAgro, INRA, UMR5004, Biochimie et Physiologie Mole´culaire des Plantes, Place Pierre Viala, F-34060 Montpellier cedex I, France } Plant–Microbe Interactions, Institute of Environmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Strategies of Iron Acquisition and Homeostasis by Plants and Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plants ............................................................................. B. Microorganisms................................................................. III. Reciprocal Interactions Between Plants and Microorganisms During Their Saprophytic Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Impact of Plant Iron Acquisition on Associated Microbes .............. B. Impact of Microbial Iron Acquisition on the Host Plant ................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51012-9

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IV. Reciprocal Interactions Between Plants and Microorganisms During Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Iron And Microbial Virulence: Role of High-Affinity Iron Assimilation Systems .................................................... B. Iron and Plant Defense ........................................................ V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Iron is an essential micronutrient for plants and associated microorganisms. Iron nutrition of these organisms relies on the soil supply. However, bioavailability of iron in cultivated soils is low. Plants and microorganisms have thus evolved active strategies of iron uptake based on acidification, chelation, and/or reduction processes. Iron acquisition by these organisms leads to complex interactions ranging from mutualism to competition. In the rhizosphere, plants support abundant and active microbial communities through the release of rhizodeposits. Iron uptake by these microorganisms and by the host plant decrease even more the concentration of iron in solution. Therefore, there is an intense competition for iron among rhizosphere microorganisms, favoring those with the most efficient iron uptake strategy. This is the case for fluorescent Pseudomonas bacteria that synthesize siderophores, called pyoverdines or pseudobactines, which have a high affinity for iron and suppress fungal phytopathogens and deleterious microorganisms. Pyoverdines also elicit plant defense reactions and contribute to plant iron acquisition. Taken together, these mutual effects promote plant growth and health. However, competition for iron may also occur between plants and microbes during pathogenesis. Siderophores contribute to the iron uptake of the host plant and to the virulence of pathogens; conversely, host plants activate mechanisms aimed at depriving pathogens of nutritional iron. The iron-withholding mechanisms of the host plant rely on controlling its iron homeostasis. In this chapter, we describe the strategies of iron uptake of plants and microorganisms, the resulting complex interactions between them, and the challenges represented by their monitoring in agroecology.

I. INTRODUCTION Ferric iron is essential for plants and microorganisms. Indeed, iron is involved in major metabolic processes such as reduction of ribonucleotides and molecular nitrogen, and the energy-yielding electron transfer reactions of respiration and photosynthesis (Guerinot and Yi, 1994). These major functions are related to the electronic structure of iron, making it capable of reversible changes in oxidation state over a wide range of redox potentials. For plants and their associated microorganisms, iron nutrition relies on the soil supply. Iron is the fourth most abundant element of the earth’s crust. However, in cultivated soils, iron is mostly oxidized. Fe(III) species, including the free metal ion Fe3þ, are precipitated as hydroxides, oxyhydroxides, and oxides at pH values compatible with plant growth, the more so with increasing pH. These oxides have a very

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poor solubility. Therefore, the concentration of Fe3þ is extremely low in cultivated soils (Lindsay, 1979; Marschner, 1995). To acquire iron for their physiology and growth, plants have evolved active uptake mechanisms. These mechanisms rely on a range of chemical processes occurring at the root level. They include acidification of the soil solution through the excretion of protons or organic acids, the chelation of Fe(III) by ligands, and the reduction of Fe3þ to the more soluble Fe2þ by reductases and reducing compounds. By these strategies, plants modify the physicochemical properties of the soil surrounding their roots (Hinsinger, 1998; Hinsinger et al., in press), which is called the rhizosphere (Hiltner, 1904). More generally, plants release a significant part of their photosynthates at the root level through a process called rhizodeposition. These products, that is, the rhizodeposits, consist of exudates, lysates, mucilage, secretions, and dead cell material, as well as gases including respiratory CO2 and ethylene (ET) (Jones et al., in press). Depending on plant species, age, and environmental conditions, rhizodeposits can account for up to 40% of net fixed carbon (Lynch and Whipps, 1990) with an average of 17% (Nguyen, 2003). This carbon release has a major impact on soil microorganisms, which are mainly in stasis (fungistasis/bacteriostasis) (Lockwood, 1977). This stasis is related to the fact that soil‐borne microorganisms are mostly heterotrophic and soils are mostly oligotrophic environments (Wardle, 1992). More specifically, rhizodeposition promotes microbial population density (Clark, 1949; Rovira, 1965), biomass (Barber and Lynch, 1977), and activity (So¨derberg and Ba˚a˚th, 1998). Some microbial groups and populations appear to be preferentially adapted and favored in the rhizosphere. This leads to variations of the structure and diversity of the microbial community in the rhizosphere compared to that from the surrounding soil (Berg and Smalla, 2009; Edel et al., 1997; Lemanceau et al., 1995; Mougel et al., 2006). Energy metabolism of heterotrophic microorganisms is based on electron donors (organic compounds) and electron acceptors (ferric iron, oxygen, and oxidized nitrogen species). Bacteria are usually able to use a wide range of organic compounds as shown for fluorescent pseudomonads (Lemanceau et al., 1995; Stanier et al., 1966). Consequently, depending on the organic compounds present, different catabolic activities can be induced to take advantage of the nutrients available in the rhizosphere (Cheng et al., 1996). In contrast, the range of possible electron acceptors is limited (Latour and Lemanceau, 1997), making the competition for those extremely intense. This is the case for Fe(III) species, including the free metal ion Fe3þ. As for plants, microorganisms have evolved active strategies to acquire iron. These strategies, based on the synthesis of siderophores and Fe-siderophore membrane receptors, are described in Section II.B. The active iron uptake by both roots and microbes decreases the already low soil concentrations of Fe(III) species. As a result, concentrations in the

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rhizosphere are generally far below those required for optimal growth of microbes and plants, 105 to 107 M for microbes and 104 to 109 M for plants (Guerinot and Yi, 1994; Loper and Buyer, 1991). The low concentration of Fe(III) in the soil solution (low supply) together with the requirements of aerobic organisms (plants and microorganisms) (high demand) lead to a considerable level of competition for Fe(III) in the rhizosphere (Guerinot and Yi, 1994; Loper and Buyer, 1991). Plant iron acquisition leading to decreased iron availability together with the release of rhizodeposits impact the associated microflora in the rhizosphere, and favor the populations with efficient iron uptake strategies (see Section III.A). These populations decrease the iron availability even more for other organisms that have a less efficient strategy of uptake, for example, phytopathogenic fungi (see Section III.B). This iron deprivation leads to a reduced saprophytic growth of these pathogens (microbial antagonism), and therefore to a decreased rate of root infections and a lower number of diseased plants. Siderophore synthesis by rhizosphere bacteria may also promote plant defense reactions and iron nutrition (see Section III.B). Plants not only host microorganisms in their rhizosphere as saprophytes, but may also harbor microorganisms in their tissues as parasites or symbionts. These airborne or soilborne microorganisms rely, during their parasitic and symbiotic life, on the iron supply from the host plant. This chapter focuses on the pathogen–plant interactions with (i) on the pathogen side illustrations of the role of their high-affinity iron assimilation systems in their virulence (see Section IV.A), and (ii) on the host side illustrations of the role of their iron homeostasis in pathogen control by decreasing iron availability for the pathogens and elicitation of plant defense reactions (see Section IV.B). There is an increasing demand of the public for an agroecology aiming both at providing high-quality products and protecting the quality of the environment. In this context, we will finally discuss the relevance of studies on plant– microbe interactions in relation with iron in order to attempt to monitor these interactions and ultimately to promote plant health and iron content.

II. STRATEGIES OF IRON ACQUISITION AND HOMEOSTASIS BY PLANTS AND MICROORGANISMS A. PLANTS

1. Iron uptake by plant roots a. Iron uptake in grasses (Strategy II). In grasses, iron uptake is based on chelation of Fe(III) to strong ligands belonging to the mugineic acids (MAs) family (Fig. 1). These amino acid derivatives, resulting from the condensation of three S-adenosyl methionine molecules, are secreted into the rhizosphere,

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PM Root Grasses only

PS

PS NA

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All plants

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Grasses

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Fig. 1. Iron uptake systems of plant roots. Grass and non‐grass species acquire iron from the soil through the plasma membrane (PM) of their root epidermal cells by two different strategies (Strategy II and Strategy I, respectively). Nicotianamine (NA) is synthesized from methionine (Met) in all plants, but NA is used as a precursor for phytosiderophore (PS) synthesis only in grasses. PS are small organic molecules belonging to the mugineic acid family, and having a strong affinity for metals, in particular for Fe3þ. PS are secreted from the roots by an uncharacterized mechanism into the rhizosphere where they chelate Fe3þ. The Fe3þ–PS complex is then transported into the epidermal cells of the roots by the Yellow stripe 1 (YS1) transporter. In non‐grass species, acidification of the rhizosphere occurs in part through the activity of a plasma membrane (PM) Hþ-ATPase that has not yet been described molecularly. This Hþ excretion contributes to the solubilization of Fe3þ, which is reduced to Fe2þ by the FRO2 ferric chelate reductase, transferring electrons (e) from NADPH to Fe3þ. Fe2þ is then transported through the plasma membrane of root epidermal cells by the Iron-regulated transporter 1 (IRT1).

the resulting ferric iron chelates being taken up by a specific transporter (Ma et al., 1995; Shojima et al., 1990; Takagi, 1976; Von Wire´n et al., 1994). The Fe(III)–MA transport system has been characterized by using maize as a model grass species. The maize yellow stripe 1 (ys1) mutant carries a recessive mutation responsible for a defect in the transport of the Fe(III)–MA complex through the root plasma membrane. In the mutant, MA synthesis and

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secretion are normal, as is iron translocation from roots to shoots (Von Wire´n et al., 1994). The maize YS1 gene has been cloned (Curie et al., 2001). It encodes a transmembrane protein bearing a Glu-rich N-terminal region containing an Arg-Glu-Gly-Leu-Glu (REGLE) domain known for interacting with Fe(III). Electrophysiological analysis of ZmYS1 expressed in Xenopus oocytes demonstrated that Fe(III)-phytosiderophore transport depends on proton co‐transport and on the membrane potential (Schaaf et al., 2004). This can explain why grasses are less susceptible to chlorosis than non‐grasses, ZmYS1-mediated transport occurring even at alkaline pH. Recently, the orthologous YS1 gene from barley (HvYS1) was cloned and the protein it encodes has a metal specificity restricted to Fe(III). Consistent with a role in iron uptake, the HvYS1 mRNA was localized in root epidermal cells by in situ hybridization analysis of iron-deficient barley roots, and the corresponding protein was observed at the plasmalemma by immunohistological staining (Murata et al., 2006). Information on phytosiderophore synthesis may be found in previous papers (Curie et al., 2009; Haydon and Cobbett, 2007; Mori, 1999).

b. Iron uptake in the non‐grass model plant Arabidopsis thaliana (Strategy I). In non‐grass plants, iron uptake under conditions of iron deficiency relies on acidification of the rhizosphere and a Fe(III) reduction system coupled to Fe(II) transport across the plasmalemma of root epidermal cells (Marschner, 1995) (Fig. 1). The Arabidopsis FERRIC REDUCTASE OXIDASE 2 (FRO2) gene encodes the ferric chelate reductase protein (Robinson et al., 1999), a 725-amino acid protein with eight putative transmembrane domains that shares similarities with human phagocytic NADPH gp91phox oxydoreductase and with the yeast ferric chelate reductases. The FRO2-generated Fe(II) is taken up by Iron-regulated transporter 1 (IRT1), which is the major root Fe(II) transporter activated under iron-deficient conditions (Eide et al., 1996; Guerinot 2000; Rogers et al., 2000). It is located at the plasmalemma of root epidermal cells and was shown to be essential for plant growth and development in Arabidopsis by the characterization of mutant irt1 knockout lines (Vert et al., 2002). The FRO2/IRT1 iron uptake system, originally described for the model plant Arabidopsis, is a general system which occurs also in tomato (Bereczky et al., 2003; Eckhardt et al., 2001; Li et al., 2004) and legume plants (Lo´pez-Milla´n et al., 2004; Waters et al., 2002).

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2. Organic compound exudates from roots and iron uptake In addition to the well-known iron uptake mechanisms mentioned above, iron deficiency is also responsible for excretion of organic compounds by roots, and therefore impacts rhizodeposition and the associated microbial abundance, diversity and activity. Among these organic compounds, two major types of molecules have been reported: the phenolics (Jin et al., 2007), and the flavins (Susı´n et al., 1993). Phenolics have been implicated in many chemical and biological functions, including chelation and reduction of iron, free radical scavenging, antimicrobial activity and as a carbon source for microbial growth. Indeed, recent reports have established that phenolics secreted in response to iron deficiency promote iron uptake in two ways. First, phenolics secreted from red clover roots altered the soil microbial community, which favored plant Fe acquisition through microbial release of auxins and siderophores (Jin et al., 2006). Second, these red clover phenolics improved plant Fe nutrition by enhancing reutilization of apoplastic iron, thereby enhancing Fe nutrition of the shoot (Jin et al., 2007). Flavin secretion by iron-deficient plants has been documented in sugar beet, and is characterized by an important natural variation between species (Susı´n et al., 1993). The best characterized molecular species are riboflavin sulfates. They are secreted in the root zone where the ferric chelate reductase is known to be active. Their putative role is thus to serve as an electron transfer shuttle between the reductase and Fe(III), leading to an increased rate of iron reduction in the vicinity of the root. 3. Regulation of high-affinity iron-transport systems a. Transcriptional and translational regulation. The major level of regulation of plant iron uptake systems occurs at the transcriptional level (Walker and Connolly, 2008). In tomato, the orthologs of the Arabidopsis IRT1 and FRO2 genes have been cloned, and their expression is upregulated in response to iron deficiency. However, this expression does not occur in the tomato Fe-regulated (fer) mutant (Bereczky et al., 2003; Li et al., 2004). The FER gene was cloned by map-based cloning (Ling et al., 2002) and found to encode a protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. At the same time, and independently, various laboratories have identified a unique FER gene homolog in the Arabidopsis genome, which was shown to complement the fer mutation when expressed in the tomato fer background (Yuan et al., 2005). This gene received three different names: FER-LIKE REGULATOR OF IRON UPTAKE (FRU) (Jakoby et al., 2004), FE-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1 (FIT1) (Colangelo and Guerinot, 2004), and BASIC HELIX-LOOP-HELIX 29 (bHLH29) (Yuan et al., 2005). Afterwards it was decided to call this

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transcriptional regulator FIT1, which is now widely used by the scientific community. FIT1 is required for regulating the Fe(III) chelate reductase FRO2 at the level of steady-state mRNA accumulation, and for controlling protein and mRNA accumulation of the Fe(II) transporter IRT1. Microarray analysis of the fit1 mutant reveals that it is involved in the regulation of many genes implicated in iron homeostasis. The FIT1 gene is itself controlled by the iron status of the plant (Brumbarova and Bauer, 2005; Colangelo and Guerinot, 2004). This indicates that regulators act upstream of, or interact with the FER gene to regulate the plant response to iron deficiency. Indeed, transcriptomic analysis has revealed four other bHLH transcription factors that are involved in the regulation of the iron-deficiency response of non‐grasses (Wang et al., 2007; Yuan et al., 2005, 2008). It has been demonstrated that two of those (bHLH38 and bHLH39) physically interact with FIT, and that transgenic plants constitutively co‐expressing either bHLH38 or bHLH39 with FIT exhibit ironindependent high-level expression of FRO2 and IRT1 (Yuan et al., 2008). A bHLH transcription factor involved in the iron-regulated expression of genes from a grass plant has also been recently documented (Ogo et al., 2006). In rice, IRON-RELATED TRANSCRIPTION FACTOR 2 (OsIRO2) is strongly expressed in both roots and shoots, specifically in response to Fe-deficiency stress, and it has numerous homologs in other Gramineae. The cis-acting sequence bound by IRO2 was determined, and is found upstream of several genes known to be involved in iron acquisition by grasses. The barley IRON DEFICIENCY-SPECIFIC CLONE 2 (IDS2) gene encodes a 2-oxoglutarate-dependent dioxygenase required for deoxymugineic acid synthesis, a key molecule for iron uptake, and is upregulated in response to iron deficiency (Nakanishi et al., 2000). The IDS2 promoter was analyzed in transgenic tobacco enabling the identification of two cis-acting elements, named iron-deficiency-responsive elements 1 and 2 (IDE1 and IDE2), which act synergistically in inducing Fe-deficiency specific expression in tobacco roots (Kobayashi et al., 2003). Sequences homologous to IDE1 and IDE2 are present in many promoters of genes regulated in response to iron deficiency. Recently, trans-acting factors interacting with the IDE1 and IDE2 cis-elements have been characterized (Kobayashi et al., 2007; Ogo et al., 2008). The rice IDEF1 protein belongs to the ABI3/VP1 family of transcription factors and binds specifically to the IDE1 element. Its own expression is not regulated by iron. In rice, IDEF1 overexpression causes upregulation of OsIRO2, thereby revealing a potential network of transcription factors for the regulation of the iron-deficiency response in grasses. The IDEF2 protein binds to the IDE2 cis-element, and is a member of the NAC family of transcription factors.

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Transgenic Arabidopsis plants containing a p35SCaMV::IRT1 or a p35SCaMV:: FRO2 construct overexpress IRT1 and FRO2 transcripts independent of the iron nutritional status of the plants, whereas the corresponding proteins are accumulated only under iron deficiency. Therefore, an additional control of the steady‐state amount of the proteins encoded by the IRT1 and FRO2 genes could occur at the protein stability level, and not only at the transcriptional level (Connolly et al., 2002, 2003). b. Hormonal regulation. Long-distance signals from shoot to root are likely to participate in the regulation of the root iron uptake system (Vert et al., 2003). In this context, the role of hormones in the regulation of the iron transport system has been investigated. The use of ET precursors and inhibitors has demonstrated that this hormone is involved in the regulation of expression and activity of the FRO/IRT system in Arabidopsis and tomato by affecting the FIT transcriptional regulator (Lucena et al., 2006). Another gaseous hormone, nitric oxide (NO), has also been implicated in the upregulation of the tomato FIT gene in response to iron deficiency, and consequently in the regulation of the root iron uptake system (Graziano and Lamattina, 2007). In Arabidopsis, iron deprivation caused by the toxic metal cadmium was shown to promote NO synthesis in roots which, in turn, positively regulated the genes encoding FIT, IRT1, and FRO2. Taken together, these data enlighten a role for NO in mediating the plant adaptive responses to iron deficiency (Besson-Bard et al., 2009). Finally, in Arabidopsis, IRT1, FRO2, and FIT have been shown to be repressed at the transcript abundance level by exogenous addition of cytokinins (CKs). However, the CKs and iron-deficiency signals act through distinct pathways to regulate the iron uptake genes. It was hypothesized that CKs control the root iron uptake machinery through a root growthdependent pathway in order to adapt nutrient uptake to the demand of the plant (Se´gue´la et al., 2008). B. MICROORGANISMS

1. Siderophore-mediated iron uptake The major active strategy of iron uptake by microbes relies on the synthesis of low-molecular-weight (generally less than 1000 Da) molecules called siderophores (‘‘iron carrier’’ in Greek). They represent a remarkable group of microbial iron-binding molecules secreted in response to iron deficiency and since the first isolation and characterization in 1952 of ferrichrome (Neilands, 1952), a fungal siderophore from Ustilago sphaerogena, they became the focus of great interest from a biological and structural

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standpoint. Siderophores show a high affinity for ferric iron (Guerinot, 1994) and ferric siderophores are transported into cells via specific Fe-siderophore membrane receptors (Neilands, 1981). There is a high diversity among siderophores, with more than 500 having been characterized so far (Boukhalfa and Crumbliss, 2002). Most siderophores form six-coordinate octahedral complexes with Fe(III) (Guerinot, 1994) and are classified according to the functional groups acting as ligands: catecholates, hydroxamates, hydroxypyridonates, hydroxy- or amino-carboxylates (Bossier et al., 1988; Winkelmann, 2001, 2002, 2007), some siderophores including mixed functional compounds (Mossialos and Amoutzias, 2007). The affinity for iron differs depending on the type of siderophore, with K values ranging between 1023 and 1025 for carboxylates, 1029 and 1032 for trihydroxamates, and up to 1052 for the catecholate siderophore, enterobactin (Drechsel and Jung, 1998). Fungal siderophores belong to the hydroxamate group (Winkelmann, 2001). The ability of microorganisms to produce siderophores may be assessed in vitro using the Chrome Azurol S (CAS) assay (Schwyn and Neilands, 1987). Siderophores produced by the microorganisms induce a change of the blue color of the CAS medium to orange. We refer the reader to a number of excellent review papers for further information on siderophore biosynthesis (Cornelis et al., 2008; Guerinot, 1994; Haas, 2003; Mossialos and Amoutzias, 2007; Neilands, 1981; Visca et al., 2007; Winkelmann, 2001). a. Iron uptake by fluorescent pseudomonads. Among bacteria, a lot of attention has been dedicated to the siderophore-mediated iron uptake of fluorescent pseudomonads. This group of Gram-negative bacteria belongs to the genus Pseudomonas sensu stricto, and includes several species which are either human–animal pathogens (Ps. aeruginosa), phytopathogens (Ps. cichorii, Ps. marginalis, Ps. syringae, Ps. tolaasii, Ps. viridiflava), or nonpathogens (Ps. aureofaciens, Ps. chlororaphis, Ps. fluorescens, Ps. putida). All these species share the property of fluorescing under UV light when grown under iron-stress conditions (Bossis et al., 2000). This fluorescence results from the synthesis of pyoverdines, which are the major class of siderophores produced by fluorescent pseudomonads and show a high affinity for Fe(III) (Fe-pyoverdine, K ¼ 1032) (Meyer and Abdallah, 1978). Active iron uptake by fluorescent pseudomonads also relies on the synthesis of membrane protein receptors that are usually specific for the cognate siderophore (Hohnadel and Meyer, 1988). Pyoverdines consist of three parts: a conserved dihydroxyquinoline chromophore, responsible for their fluorescence, a peptide chain of from six to 12 amino acids, and a small dicarboxylic acid (or its monoamide) connected amidically to the NH2-group of the chromophore. Pyoverdines have molecular masses ranging from 1000 to

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1800 Da (1764 for the biggest one reported so far by Meyer et al., 2008). They contain one catechol group included in the chromophore and two hydroxamate groups included in the peptide chain (Budzikiewicz, 1997). More than 20 genes are required for pyoverdine synthesis (Visca et al., 2007). The chromophore and the peptide chain are synthesized by non‐ ribosomal peptide synthases (NRPS) (Mossialos et al., 2002). The first steps of chromophore formation are performed by a conserved NRPS (pvdL), whereas the synthesis of the peptide chain is achieved by other nonconserved NRPS. This explains why the peptide chains differ among fluorescent pseudomonad siderophores. Indeed, there is a high diversity among these siderophores, with more than 100 different pyoverdines having been characterized so far (Budzikiewicz, 2004; Meyer et al., 2008). Once synthesized, pyoverdines are transported out of the cell and sequester Fe3þ from the environment. The resulting complexes, Fe-pyoverdines, are selectively recognized by and bound to TonB-dependent receptors—a family of -barrel proteins from the outer membrane of Gram-negative bacteria—and then transported into the cell (Faraldo-Go´mez and Sansom, 2003). The selective recognition of the pyoverdine relies on the diversity of their peptide chain (Meyer et al., 1987). However, there are exceptions: some strains have been reported to use siderophores produced by other fluorescent pseudomonads (Mirleau et al., 2000) or even by other microbial groups (Poole, 2004). Fluorescent pseudomonads may also produce additional siderophores such as pyochelin, pseudomonine, quinolobactin/thioquinolobactin, and pyridine-2,6-dithiocarboxylic acid (PDTC) (Cornelis and Matthijs, 2007). These siderophores usually show a lower affinity for Fe3þ than pyoverdine and their synthesis is repressed by this major siderophore. Further information on the iron uptake by fluorescent pseudomonads can be found in the recent reviews of Visca et al. (2007) and Cornelis et al. (2008). Different methods have been proposed to characterize pyoverdines. The so-called siderotyping is based on analytical and biological methods. As an example of an analytical method, isoelectrofocusing (IEF) is based on the physicochemical properties of the molecules. Indeed, pyoverdines differ in net electric charge, allowing their characterization based on their isoelectric pH values (pHi or pI). IEF can be followed by UV illumination for pyoverdine detection or more generally, for all siderophores, by an overlay of the gel with the CAS medium (Koedam et al., 1994). This characterization can be refined by biological methods based on the usual specificity of the uptake of Fe-pyoverdines as tested by cross‐feeding (Cornelis et al., 1989) and 59Fe-siderophore-mediated uptake experiments (Meyer et al., 1997). Promotion of bacterial growth under iron-stress conditions or increased 59 Fe bacterial content in the presence of a pyoverdine chelated with Fe,

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labeled or not, means that the bacterial strain is able to incorporate the tested pyoverdine, and therefore the structure of this pyoverdine corresponds to its own. However, as indicated above, there are exceptions with bacterial strains being able to incorporate so-called heterologous or xenopyoverdines produced by other strains. These exceptions appear to be due to the ability of multivalent pyoverdines to recognize several receptors or to the presence of multiple receptors for different pyoverdines in a single strain (Ghysels et al., 2004; Meyer et al., 2002; Mirleau et al., 2000). Siderotyping has been proposed as a taxonomic tool for fluorescent and nonfluorescent pseudomonads (Meyer et al., 2002). Characterization of the structure and identification of the mass of the pyoverdine may finally be accomplished by high-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (Kilz et al., 1999). As indicated above, several siderophores may be produced by the same organism. Notably the synthesis of pyoverdine may mask the ability of fluorescent pseudomonads to synthesize other siderophores. The strategy described by Cornelis and Matthijs (2007) consists of testing the remaining ability of a pyoverdine-minus (pvd) mutant to still discolor CAS medium as an indication of their ability to produce at least one additional siderophore (Mirleau et al., 2000). b. Iron uptake in Erwinia. The importance of iron in plant pathogenesis has been thoroughly investigated in interactions between Erwinia spp. and their plant hosts (Expert, 1999). Such investigations were based on the genetic and biochemical characterization of the high-affinity iron uptake systems produced by these bacteria in iron-limited environments. Erwinia chrysanthemi strain 3937 has emerged as a model to study the role of iron in plant–pathogen interactions (see Section IV). In this strain two iron uptake systems mediated by two structurally unrelated siderophores, achromobactin and chrysobactin, have been characterized (Expert et al., 2004; Franza et al., 2005). These siderophores are produced in a sequential manner in culture supernatants of bacterial cells grown under iron limitation. Achromobactin can be detected during the mid-exponential phase of growth, while chysobactin appears much later. As a catecholate readily identifiable using the Arnow test, chrysobactin was the first siderophore of strain 3937 to be characterized (Persmark et al., 1989). Achromobactin is a hydroxycarboxylate siderophore that does not contain any catecholate or hydroxamate group (Mu¨nzinger et al., 2000). This siderophore was discovered in chrysobactin-deficient mutants, which are still able to form a halo of discoloration on CAS agar medium (Schwyn and Neilands, 1987). Chrysobactin-deficient mutants fail to grow in the presence of the ferric iron chelator

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ethylenediamine-N,N0 -bis(2-hydroxyphenylacetic acid) (EDDHA), but the production of achromobactin enables them to thrive on a medium containing the ferrous iron chelator 2,20 dipyridyl. A compound identical to chrysobactin has been identified in low-iron cultures of Erwinia carotovora subsp. carotovora W3C105 (Barnes and Ishimaru, 1999). Structural properties of chrysobactin have been studied in detail. Chrysobactin has been identified as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine and belongs to a class of siderophores that are basically dihydroxybenzoic acid (DHB) derivatives of amino acids or peptides. Unlike the tricatecholate siderophore enterobactin and other hexadentate ligands that are strong iron binders, chrysobactin possesses only three potential coordination sites for complexing ferric iron, two hydroxyl groups on the catechol moiety and the terminal carboxylate group of serine. Persmark and Neilands (1992) have shown that only catecholate hydroxyl groups are involved in Fe chelation, suggesting that chrysobactin is a bidentate, and thus a relatively weak, ligand. However, depending on the pH and the metal/ligand concentration ratio, chrysobactin forms ferric complexes of different stoichiometries, from 1:1 to 1:3 (Fe:Cb). When the ligand is present four or more times in excess, there is a mixture of bis and tris complexes in solution at physiological pH values. By contrast, the siderophores produced by various strains of Erwinia amylovora belong to the same class of hydroxamate molecules, the desferrioxamines (DFO), which contain repeating units of 1-amino-w-N-hydroxy-amino alkane alternated with succinic or acetic acids as acyl functions (Feistner, 1995; Kachadourian et al., 1996). Both linear and cyclic members are known. Among microbial siderophores, DFO are particularly interesting molecules. DFO B, available commercially under the trade name Desferal, is widely used since 1962 in the treatment of human overload and iron poisoning.

2. Regulation of the high-affinity iron-transport systems The ability of microbial strains to synthesize a siderophore in vitro, such as on CAS medium, does not imply that this siderophore is actually produced in situ. Indeed, because of the toxicity of iron at high concentration and the cost for the cell represented by the synthesis of siderophores and related membrane protein receptors, siderophore-mediated iron uptake is strictly regulated and expressed only when required. In Gram-negative bacteria, iron uptake (siderophore synthesis, receptors) is accurately controlled by the sensory and regulatory ferric uptake regulator (Fur) protein. The Fur protein acts as a dimer, each monomer containing a non‐heme ferrous ironbinding site (Hantke, 1981). If the cellular iron level becomes too low, the

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active Fur repressor loses Fe2þ, its co‐repressor, and is no longer able to bind to its operator sites (Escolar et al., 1999). For fluorescent pseudomonads, Fur iron uptake regulation has been explored mostly in Ps. aeruginosa (Cornelis et al., 2009). Fur controls iron uptake through extracytoplasmic sigma factors (ECF ) as well as other regulators, including two-component systems. The genome of Ps. aeruginosa PAO1 contains 19 genes encoding ECF (Potvin et al., 2008), of which 10 are predicted to be Fur regulated (Van Oeffelen et al., 2008). Among those, pvdS controls the transcription of genes encoding pyoverdine, and fpvI transcription of the Fe-pyoverdine receptor gene fpvA (Visca et al., 2007). PvdS-dependent gene expression occurs through binding to a promoter sequence of 30–40 bp from the transcription start site, called the iron-starvation box (Wilson et al., 2001). Pyoverdine synthesis is regulated further by quorum sensing through the production of N-acyl homoserine lactones (AHLs) when the bacterial density, and hence iron demand, are high (Stintzi et al., 1998; Whiteley et al., 1999). In the rhizosphere, the high density of fluorescent pseudomonads and frequency of AHL producers (Elasri et al., 2001), together with the low iron bioavailability, are expected to favor siderophore synthesis. However, the commonly reported strategy for demonstrating siderophore synthesis relies on indirect methods. Pyoverdine synthesis was shown to occur in the rhizosphere by the use of (i) monoclonal antibodies raised against Fe-pyoverdine (Buyer et al., 1990), and of (ii) the ICENUCLEATION ACTIVITY reporter gene inaZ (Duijff et al., 1999; Loper and Henkels, 1997). The reporter gene is under the control of the promoters of ironregulated genes (Loper and Lindow, 1997). Such constructs were made in Ps. fluorescens Pf-5 (Loper and Lindow, 1994) and in Ps. putida WCS358 (Duijff et al., 1994a) by fusing a promoterless inaZ gene to an iron-regulated promoter regulating the production of fluorescent siderophores. In Er. chrysanthemi, the fur gene was cloned by functional complementation of an Escherichia coli fur mutant. The fur gene encodes a protein highly similar to Es. coli Fur (Franza et al., 1999). Analysis of transcriptional fusions to several genes of the chrysobactin and achromobactin systems in fur-proficient and -deficient backgrounds established that Fur is the main actor in the regulation by iron in Er. chrysanthemi. Monitoring the expression of -GALACTOSIDASE (lacZ) fused to the FERRIC CHRYSOBACTIN RECEPTOR gene (fct-lacZ), representative of the chrysobactin gene system in planta, provided direct evidence of in vivo regulation of an ironcontrolled function associated with phytopathogenicity and of low iron availability in the plant apoplasm (Masclaux and Expert, 1995). The Er. chrysanthemi Fur protein was purified and band shift assays demonstrated that in vitro Fur specifically binds the regulatory regions of genes

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involved in chrysobactin- and achromobactin-mediated iron transport, indicating that this metalloregulation is direct (Franza et al., 2002, 2005). The determination of the Fur-binding sites by DNA footprinting experiments supports the model initially described in Es. coli, in which the regulatory process involves direct competition between the RNA polymerase and Fur. Iron availability was also shown to regulate transcription of PECTATE LYASE (pel) genes (Masclaux et al., 1996; Sauvage and Expert, 1994), the major virulence factors in Er. chrysanthemi. Inspection of the promoter regions of the pelD and pelE genes encoding the isoenzymes PelD and PelE, revealed two conserved Fur boxes located 30 and 50 bp, respectively, upstream of the -35 promoter site of these two genes. Besides their unusual position, these Fur boxes overlap the binding sites for the cAMP-cAMP receptor protein (CRP) complex that activates transcription of the pel genes (Franza et al., 2002). In this case, Fur acts as a transcriptional repressor by blocking the action of CRP. These molecular studies indicate that low iron availability is a triggering signal for coordinated expression of the genes involved in the pathogenicity of Er. chrysanthemi.

III. RECIPROCAL INTERACTIONS BETWEEN PLANTS AND MICROORGANISMS DURING THEIR SAPROPHYTIC LIFE A. IMPACT OF PLANT IRON ACQUISITION ON ASSOCIATED MICROBES

1. Diversity The low available Fe(III) concentration in cultivated soils may be even lower in the rhizosphere because of the iron uptake by plant roots and microorganisms, whose growth and activity are promoted by plant rhizodeposits (Fig. 2A). The resulting competition is thus expected to favor microorganisms with the most efficient siderophore-mediated iron uptake (competitive advantage). The further iron depletion by the efficient iron competitors is then expected to reduce saprophytic growth of microorganisms with a less efficient iron uptake system through iron starvation (microbial antagonism). Therefore, variations of iron availability in the rhizosphere should lead to shifts in the microbial community. Such shifts have indeed been demonstrated by comparing the susceptibility to iron starvation of a large collection of fluorescent pseudomonads isolated from the rhizosphere of flax cultivated in two soils (Lemanceau et al., 1988b). The strategy followed consisted of determining, for each isolate, the minimal inhibitory concentration (MIC) of 8-hydroxyquinoline (8HQ), a strong iron chelator (Geels et al., 1985). The higher the

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A

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Fig. 2. (A) Schematic representation of reciprocal interactions between plants and microbes during their saprophytic life. (1) Fe(III) bioavailability is low in cultivated soils as influenced by their physicochemical properties. (2) Plants release a significant part of their photosynthates as rhizodeposits, which promote microbial abundance and activity. (3) The concentration of Fe(III) in solution is decreased in the rhizosphere due to uptake by roots and microbes. (4) As a result, active iron acquisition strategies are activated. The iron-stress conditions together with the release of rhizodeposits lead to the selection of microbial populations producing siderophores with higher affinities for iron than those of phytopathogens or deleterious microorganisms,

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MIC, the stronger the ability of an isolate is to grow under iron-stress conditions, and the lower is its susceptibility to iron starvation. The distributions of the soil and rhizosphere isolates in the different MIC classes were significantly different. In the classes with high MIC values, more rhizosphere than soil isolates were found. These data indicate that populations from the rhizosphere are less susceptible to iron starvation than those from bulk soil, suggesting that they benefit from a more efficient iron uptake system, which contributes to their rhizosphere adaptation (Lemanceau et al., 1988b). The stimulation of populations less susceptible to iron starvation in the rhizosphere as a result of decreased iron availability due to plant nutrition was confirmed and extended by an innovative approach consisting of comparing microbial communities in the rhizosphere of a tobacco wild type (WT) and a transgenic line that overexpresses ferritin (OV) (Robin et al., 2006a,b, 2007). Ferritins are multimeric proteins that store iron in a soluble and biologically available form, thereby contributing to iron homeostasis (Briat et al., 1999). Upregulation of their synthesis in the transgenic line was shown to activate iron uptake genes, leading to iron overaccumulation in the plant (Van Wuytswinkel et al., 1999). Bacterial populations associated with the OV transgenic line, cultivated in a soil with a low iron content and a high pH (Oudun, France), were significantly less susceptible to iron starvation than those associated with the tobacco WT, as indicated by the greater densities of bacteria able to grow in vitro at a high concentration of 8HQ (Robin et al., 2006a). These data also indicated that, as expected, iron bioavailability was lower in the rhizosphere of the transgenic line overaccumulating iron than in that of the tobacco WT. Since the carbon content and microbial carrying capacity of both rhizospheres were similar (Robin et al., 2007), these differences could only be ascribed to the higher phytoextraction of iron by the OV transgenic line and not to differences in microbial density and activity. The impact of the differential iron nutrition between the WT and OV transgenic

which are suppressed (microbial antagonism). (5) Microbial siderophores may also elicit defense reactions in the host plant and promote plant iron nutrition. Together, these actions promote plant growth and health. (B) Schematic representation of reciprocal interactions between plants and microbes during pathogenesis. (1) Phytopathogens triggers iron depletion in leaves, (2) leading to a mobilization of vacuolar iron mediated by AtNRAMP3 and AtNRAMP4. (3) The reactive iron released into the cytosol first contributes to amplify the production of ROS, (4) resulting in the inhibition of the bacterial growth, and then (5) induces AtFER1 synthesis which deprives the bacteria of iron. (6) Moreover, the changes in leaf iron status lead to iron mobilization in the roots from both the vacuole and the soil. (7) The effect of this iron mobilization on plant susceptibility to soilborne phytopathogens and plant–microbe interactions during saprophytic life (Part A) remains to be explored. C: chloroplast. V: vacuole. (Adapted from Segond et al., 2009.)

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line on the rhizosphere bacteria was characterized further by automated ribosomal intergenic spacer analysis (ARISA) of DNA directly extracted from the rhizospheres. The data indicated that the structure of the bacterial community was significantly different in the rhizosphere of the two tobacco genotypes, confirming the impact of plant iron acquisition on the bacterial microflora (Robin et al., 2006b). Variations in microflora composition were also reported previously by Yang and Crowley (2000) in the rhizosphere of barley, a strategy II species, when the iron-nutritional status of plants cultivated under iron-limiting conditions was varied by spraying the leaves with either water or iron citrate. Because of their known ability to be competitive for iron, the community of pseudomonads was more specifically examined within the total bacterial community by PCR-restriction fragment length polymorphism (RFLP) of DNA that was extracted from the rhizosphere of the tobacco WT and OV transgenic line and amplified with specific primers (Locatelli et al., 2002). The structure of the pseudomonad community in the root samples differed between the two plant genotypes increasingly with their successive cultures in the Oudun soil (Robin et al., 2006b). This observation suggests that the structuration of the pseudomonad community was determined by the progressive iron depletion as a result of the repetitive cultivation of the overaccumulating transgenic line in the same soil. In order to assess their phenotypic diversity, a collection of fluorescent pseudomonads was established from the rhizosphere of the WT tobacco and its OV transgenic line (Robin et al., 2007). Overall, isolates taken from the iron-stressed rhizosphere differed from those of the WT rhizosphere by (i) their lower susceptibility to iron starvation, as indicated by their higher 8HQ MIC, (ii) their ability to produce specific siderophores, as indicated by their IEF types, and (iii) their different genetic backgrounds, as indicated by their random amplified polymorphism DNA (RAPD) patterns. This differential effect was mostly expressed in the vicinity of the roots. Altogether, these data clearly show the impact of plant iron acquisition on the rhizosphere pseudomonads, which leads to the selection of populations that have a low susceptibility to iron starvation and produce specific siderophores. This conclusion suggests that siderophore synthesis contributes to the adaptation of fluorescent pseudomonads to the rhizosphere as an iron-stressed environment. This hypothesis was tested further by studying the rhizosphere adaptation of model strains. As an example, the survival kinetics of Ps. fluorescens C7R12 and its pvd mutant were compared in competition, in the absence (gnotobiotic conditions) and in the presence of the indigenous microflora, in the rhizosphere of flax (Mirleau et al., 2000, 2001). In the absence of the indigenous microflora, the competition favored the pyoverdine-producing WT, whereas in

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non‐gnotobiotic conditions the survival of both the WT and the mutant strain was similar. Similarly, as compared to the WT strain the ability of pvd mutants of Ps. putida WCS358 to colonize the rhizospheres of several plant species was shown not to be impaired under non‐gnotobiotic conditions (Bakker et al., 1986; Duijff et al., 1999; Lemanceau et al., 1992; Raaijmakers et al., 1995a). The fitness of the pvd mutants in competition with the corresponding WT strains was assumed to be related to their ability to take up pyoverdines of foreign origin (Bakker et al., 1988, Bitter et al., 1991; Jurkevitch et al., 1992; Koster et al., 1993; Loper and Henkels 1999; Mirleau et al., 2000; Raaijmakers et al., 1995b). Indeed, Ps. fluorescens C7R12 and Ps. putida WCS358 have the ability to take up several heterologous pyoverdines (Loper and Henkels, 1999; Mirleau et al., 2000) and the presence of these pyoverdines in the rhizosphere was shown to enhance iron availability to Ps. putida WCS358 (Loper and Henkels, 1999). The role of the siderophore receptor in rhizosphere competence was further supported by experiments performed with a mutant of Ps. fluorescens WCS374 that harbored the siderophore receptor PupA from Ps. putida WCS358 and was then able to utilize Fe-pyoverdine from WCS358. This ability was shown to provide the WCS374 transformant with a competitive advantage over the corresponding WT strain when co‐inoculated with WCS358 (Raaijmakers et al., 1995b). Because of the cost represented by pyoverdine synthesis, it has been suggested that receptors and pyoverdines would co‐evolve to maintain mutual specificity (Smith et al., 2005), and that the high diversity of the peptide chain in pyoverdines would result from an evolutionary pressure to prevent acquisition of siderophores by other strains or species (Visca et al., 2007). The conclusions drawn from these experiments with model strains were confirmed and extended by a population approach. The survival rates of 21 strains representative of the diversity of a larger collection (340) characterized previously (Latour et al., 1996; Lemanceau et al., 1995), were measured in the rhizosphere of tomato grown in soil in non‐gnotobiotic conditions. Survival rates varied from 61.4% for the most competitive down to 0.11% for the least competitive, indicating the high diversity of the strains tested. Nine different siderotypes were identified, but only one of those was shared by eight of the nine most competitive in the rhizosphere, suggesting again the importance of the pyoverdine-mediated iron uptake in rhizosphere competence (S. Delorme et al., unpublished data). 2. Activity Bacterial isolates representative of the diversity on roots of the tobacco WT and OV transgenic line were studied further in vitro under iron-stressed conditions for their antagonistic activity against the pathogenic oomycete

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Pythium aphanidermatum. None of the isolates from the tobacco WT suppressed growth of the oomycete, whereas all those from the OV transgenic line did (Robin et al., 2007). Since siderophores are known to suppress saprophytic growth of some phytopathogens through iron competition and to promote plant iron nutrition (see Section III.B), this striking difference prompted us to further test the contribution of the siderophores produced by these isolates to the antagonism observed. Purified siderophores of the isolates from the OV transgenic line largely appeared to be more antagonistic than those from the tobacco WT, since a significantly lower concentration was required to suppress saprophytic growth of the pathogen. These siderophores also provided better iron nutrition to the plant (P. Lemanceau et al., unpublished data). They were also better able to extract iron from the iron oxides ferrihydrite and goethite, and can thus be expected to increase iron availability for plants in soils (P. Lemanceau et al., unpublished data). Taken together, these data indicate that the increased plant iron nutrition in the tobacco OV transgenic line promotes pseudomonad populations which produce specific siderophores (Robin et al., 2007) and are more active in suppressing phytopathogens and promoting plant iron nutrition. B. IMPACT OF MICROBIAL IRON ACQUISITION ON THE HOST PLANT

The impact of siderophore-mediated microbial iron acquisition on plants has been studied in detail for the so-called plant growth-promoting rhizobacteria (PGPR) (Bakker et al., 2007b; Lemanceau, 1992) and is summarized in Fig. 2A. The ability of specific PGPR, especially fluorescent Pseudomonas spp., to improve plant health by suppressing soil‐borne plant pathogens has been attributed to the production of siderophores (Kloepper et al., 1980a). Siderophore-mediated competition for ferric iron between PGPR and plant deleterious microorganisms also results in plant growth promotion (Schippers et al., 1987). Moreover, plant growth promotion can also be ascribed to a direct contribution of bacterial siderophores to plant iron nutrition (Vansuyt et al., 2007). 1. Plant health Experimental evidence for the involvement of siderophores was obtained in studies where mutants defective in siderophore biosynthesis were less or no longer effective in disease suppression when compared to their parental strains. Microbial antagonism through competition for iron can only be effective when populations of the PGPR and the pathogen are in each other’s vicinity. However, in some cases PGPR effectively controlled disease even when the populations of the antagonistic bacteria and the pathogen were

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applied, and remained, spatially separated on the plant surface, for instance the PGPR on the roots and the pathogen on the leaves, or the PGPR on one part of a split‐root system and the pathogen on the other part. In such cases, the mechanism of disease suppression is plant mediated and this is referred to as induced systemic resistance (ISR) (Van Loon et al., 1998). Iron-regulated metabolites, including siderophores, have been implicated as rhizobacterial elicitors of ISR (Bakker et al., 2007a; Ho¨fte and Bakker, 2007). We will discuss the importance of iron-regulated metabolites in suppression of plant pathogens during both their saprophytic (microbial antagonism) and parasitic (ISR) lives, focusing on fluorescent Pseudomonas spp. a. Microbial antagonism. Studies on a possible involvement of siderophores in microbial antagonism against soil‐borne pathogens were stimulated by an early report of Kloepper et al. (1980a) describing that addition of Pseudomonas sp. B10 or its pyoverdine to soils conducive to Fusarium wilts and to take-all disease, caused by the fungal pathogen Gaeumannomyces graminis f.sp. tritici, rendered them disease suppressive. They further showed that supplementation of the soils with iron overcame the positive effects of the bacterial inoculation and pyoverdine addition. Possible involvement of pyoverdine in microbial antagonism was then supported by a series of observations: (i) in vitro antagonism by some fluorescent pseudomonads against specific pathogens occurring only under iron-stress conditions when pyoverdines were produced (Geels and Schippers, 1983; Kloepper et al., 1980a; Misaghi et al., 1982), (ii) positive correlations between the levels of siderophore synthesis in vitro by different Pseudomonas isolates and their ability to reduce chlamydospore germination of pathogenic Fusarium oxysporum in soil (Elad and Baker, 1985b; Sneh et al., 1984), (iii) in vitro antagonistic activity of purified pyoverdines against Pythium tolaasi (Meyer et al., 1987) and F. oxysporum (Lemanceau et al., 1992), and (iv) reduced chlamydospore germination of pathogenic F. oxysporum in soil upon application of pyoverdine (Elad and Baker, 1985a). The demonstration of the involvement of pyoverdines in the antagonism achieved by some fluorescent pseudomonads against plant pathogens such as F. oxysporum and Pythium spp., as well as so-called deleterious microorganisms (see Section III.B.2), was borne out by the use of pvd mutants (Bakker et al., 1986, 1987; Becker and Cook, 1988; Buysens et al., 1996; De Boer et al., 2003; Duijff et al., 1993, 1994a; Leeman et al., 1996a; Loper, 1988; Raaijmakers et al., 1995a). The pyoverdine-mediated antagonism against plant pathogens leads to an improvement of plant health (Loper and Buyer, 1991). For example, Ps. putida WCS358, but not its pvd mutant, was able to improve the control of Fusarium wilt determined by

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nonpathogenic F. oxysporum Fo47 (Duijff et al., 1999; Leeman et al., 1996a; Lemanceau et al., 1992). Iron availability to Ps. putida WCS358 was shown to be low enough in the rhizosphere to allow pyoverdine production, as assessed by the ice-nucleation reporter gene construct pvd-inaZ. The pyoverdinemediated improvement of the control by Ps. putida WCS358 was related to a reduced saprophytic density and activity of the pathogenic F. oxysporum as assessed by the -GLUCURONIDASE (gus) reporter gene in a gusA-marked derivative of the pathogen (Duijff et al., 1999). Competition for iron as the mechanism of pyoverdine-mediated antagonism was first suggested upon the use of EDDHA to mimic the effect of the bacterial siderophore on iron availability (Elad and Baker, 1985b; Scher and Baker, 1982). FeEDDHA shows an affinity constant (K ¼ 1033.9) (Lindsay, 1979) close to that of Fe-pyoverdines (K ¼ 1032) (Meyer and Abdallah, 1978) and significantly higher than that of the siderophores of Fusarium, called fusarinines (Emery, 1965; Lemanceau et al., 1986), chelated with iron (K ¼ 1029) (Scher and Baker, 1982). As expected from the comparison of the K values, the synthetic ligand decreased the iron availability to F. oxysporum, as reflected by the reduced germination of chlamydospores and germ tube length in vitro and in soil, whereas the FeEDDHA complex did not. Lemanceau et al. (1993) further showed that the antagonistic activity of increasing concentrations of purified pyoverdine from Ps. putida WCS358 was due to iron competition, since the same concentrations of Fe-pyoverdine did not have any deleterious effect. The highest concentrations even had a promoting effect on fungal growth, probably as a result of dissociation of the chelate leading to iron release. These data are in agreement with the proposal made by Loper and Buyer (1991) that pyoverdine may make Fe(III) unavailable to target pathogens. The importance of iron competition in microbial antagonism was supported further by studies on natural soil suppressiveness to Fusarium wilts, which was ascribed at least partly to fluorescent pseudomonads (Lemanceau et al., 1988b; Scher and Baker, 1980). In these suppressive soils, introduction of FeEDTA increased the availability of Fe(III) to pathogenic F. oxysporum, resulting in less competition for iron and consequently a higher severity of Fusarium wilt. Conversely, introduction of EDDHA lowered the availability of Fe(III) to pathogenic F. oxysporum and resulted in more intense competition for iron and consequently lower severity of Fusarium wilt (Lemanceau et al., 1988a; Scher and Baker, 1982). Pyoverdines do not have a general efficacy (Gutterson, 1990; Loper and Buyer, 1991) and their role in microbial antagonism and disease suppression varies according to (i) the target pathogens, (ii) the bacterial mode of action to which the pathogen is susceptible, and (iii) the soil type.

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Variations with regard to the target pathogen may be related to the endogenous iron content of the pathogenic fungal spores. It has been suggested that the lower susceptibility of Fusarium solani to iron-mediated antagonism compared to that of F. oxysporum can be ascribed to the higher iron content of F. solani chlamydospores, making them less susceptible to iron deprivation (Baker et al., 1986). In contrast with soils naturally suppressive to Fusarium wilts, those suppressive to black root rot of tobacco, caused by Thielavopsis basicola, contain more iron than conducive soils (Stutz et al., 1986). Suppression of the latter disease by the model strain Ps. fluorescens CHA0 requires sufficient iron to allow expression of the bacterial mode of action (Keel et al., 1989), which is ascribed to the production of toxic hydrogen cyanide (Voisard et al., 1980). Absence of pyoverdine-mediated iron competition in this suppression was shown using a pvd mutant of CHA0 which suppressed black root rot as effectively as the WT strain (Keel et al., 1989); even more so, high concentrations of Fe-siderophores appeared to antagonize disease suppression (Ahl et al., 1986). For other target pathogens, such as G. graminis f.sp. tritici, pyoverdine-mediated iron competition seems to play only a minor role compared to other bacterial traits. Although early work had suggested or indicated involvement of this mode of action for some fluorescent pseudomonads (Kloepper et al., 1980a; Weller, 1988; Wong and Baker, 1984), it was then nicely demonstrated that its relative contribution to the antagonism and disease suppression by Ps. fluorescens strains 2‐79 and M4-80R was minor or even null compared to that of the production of the antibiotic phenazine (Hamdan et al., 1991). Thomashow and Weller (1990) suggested that the antagonistic effects against G. graminis f.sp. tritici attributed in the early studies to pyoverdine could be related to an artifact linked to the production of iron-regulated antibiotics, this type of compound having already been described by Gill and Warren (1988). Even when the target pathogen is susceptible to pyoverdine-mediated iron competition, this competition is only expected to occur when the physicochemical properties of the soil contribute to low iron bioavailability. This is the case in soils that are naturally suppressive to Fusarium wilts. These soils are characterized by a high pH and CaCO3 content, resulting in a very low solubility of ferric iron, allowing microbial competition for iron to occur (Alabouvette et al., 1996). Simeoni et al. (1987) reported that the critical concentration of Fe(III) in nutrient solution below which germination of F. oxysporum chlamydospores was suppressed, was between 1019 and 1022 M, and that optimal suppression took place between Fe(III) activities of 1022 and 1027 M. Duijff et al. (1994a) further showed that Ps. putida WCS358 decreased Fusarium wilt incidence only when the iron availability of the nutrient solution was reduced by EDDHA supplementation. On top of

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the low Fe(III) solubility, suppressive soils are characterized by a high biomass, exerting a high demand for this ion and enhancing the intensity of iron competition. This may explain why the suppression of Fusarium wilt based on pyoverdine-mediated iron competition was reported to be most effective when microbial biomass is high (Duijff et al., 1991). A different interpretation of the latter observation concerns the interaction between competition for carbon and for iron as described for the suppressive soil of Chaˆteaurenard (Lemanceau, 1989) and in the synergistic interaction between Ps. putida WCS358 and nonpathogenic F. oxysporum Fo47 in suppressing Fusarium wilt in carnation (Lemanceau et al., 1992, 1993). Interaction between competition for carbon and iron was evidenced during experiments with combined supplementation of the Chaˆteaurenard suppressive soil with glucose and EDDHA. Addition of glucose reduced carbon competition, promoting saprophytic growth of pathogenic F. oxysporum and ultimately disease expression, whereas addition of EDDHA, together with glucose, enhanced iron competition, and decreased the disease promotion induced by the glucose supply (Lemanceau, 1989). This type of interaction was elucidated further by studying the modes of action involved in the protection by combined inoculation with WCS358 and Fo47. The enhanced efficacy of this protection was ascribed to a synergistic effect between (i) carbon competition by Fo47, reducing the amount of carbohydrates available to the pathogen, and (ii) pyoverdine-mediated iron competition by WCS358, reducing the efficacy of the energy metabolism of the pathogen and making it more susceptible to carbon competition (Lemanceau et al., 1993). b. Induced systemic resistance. Upon appropriate stimulation, plants can develop a state of enhanced defensive capacity that is called induced resistance (Van Loon et al., 1998). In the early 1990s, ISR was reported to be another mode of action of disease suppression by PGPR Pseudomonas spp. (Van Peer et al., 1991; Wei et al., 1991). The bacterial elicitors of ISR include cell envelope components, that is, flagella and lipopolysaccharides (LPS), antibiotics such as 2,4-diacetylphloroglucinol and pyocyanin, and ironregulated metabolites, including an N-alkylated benzylamine derivative, pyoverdine, salicylic acid (SA), and pyochelin (Bakker et al., 2007a). We focus here on the involvement of the iron-regulated metabolites in ISR. Leeman et al. (1996b) observed that lowering iron availability for the ISReliciting strains Ps. fluorescens WCS374 and WCS417 increased the level of ISR-mediated suppression of Fusarium wilt in radish. It was hypothesized that siderophores produced by these PGPR can effectively elicit ISR. To test this hypothesis, mutants lacking fluorescent siderophore production were compared to the WT strains for elicitation of ISR and effects of purified

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pyoverdine siderophores were investigated. For both WCS374 and WCS417, the mutants were as effective as the WT, but the purified pyoverdine of WCS374 did induce ISR in radish, whereas the pyoverdine of WCS417 did not (Leeman et al., 1996b). Thus, the fluorescent siderophore of WCS374 can elicit ISR, but is not necessary for elicitation, suggesting that also other traits of this strain are important. Indeed, LPS of the strains can elicit ISR in radish (Leeman et al., 1995), but a role for the iron-regulated metabolite SA was also suggested (Leeman et al., 1996b). It should be noted here that although Ps. putida WCS358 suppresses Fusarium wilt in radish, it cannot elicit ISR in this crop species (Leeman et al., 1995). However, Ps. putida WCS358 can suppress bacterial wilt caused by Ralstonia solanacearum in Eucalyptus urophylla and in this plant species the mode of action is ISR (Ran et al., 2005a). In this case, involvement of the fluorescent siderophore in ISR was more clear. Treatment with Ps. putida WCS358 or its purified pyoverdine resulted in a significant reduction of bacterial wilt, whereas the pvd mutant was not effective. In young Eucalyptus seedlings, redundancy of ISR-triggering traits in Ps. fluorescens WCS374 was again observed: the WT strain, the pvd mutant and the purified pyoverdine were all effective in suppressing bacterial wilt (Ran et al., 2005a). WCS374 produces at least three iron-regulated metabolites: pyoverdine, SA, and pseudomonine, a siderophore containing a SA-moiety (Djavaheri, 2007; Mercado-Blanco et al., 2001). When applied exogenously, SA elicits systemic resistance in many plant species, including radish (Leeman et al., 1996b) and Arabidopsis (Pieterse et al., 1996). It was shown that WCS374 can trigger ISR in radish (Leeman et al., 1996b), but not in Arabidopsis (Van Wees et al., 1997), despite the fact that WCS374 produces large amounts of SA in vitro. These findings are explained by assuming that in the rhizosphere strain WCS374 does not excrete SA but all SA is channeled into the production of pseudomonine. Indeed, most studies that investigated a role of bacterially produced SA in induced resistance conclude that it is not SA itself that is the microbial signal (Audenaert et al., 2002; Press et al., 1997; Ran et al., 2005b). To add to the confusion, Djavaheri (2007) recently showed that WCS374 can elicit ISR against Pseudomonas syringae pv. tomato in Arabidopsis, but only when applied at a much lower cell density (103 cells/g of soil) than used in previous studies (5 107 cells/g of soil). Moreover, none of the ironregulated metabolites appear to be involved in this low WCS374 cell density mediated ISR (Djavaheri, 2007). A possible involvement of the three ironregulated metabolites of WCS374 in ISR in rice against Magnaporthe oryzae was also investigated (De Vleesschauwer et al., 2008). In this monocot system the pyoverdine siderophore of WCS374 turned out to be the crucial

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determinant responsible for ISR elicitation. Thus, it can be concluded that WCS374-mediated ISR depends on different bacterial determinants in different plant species. Whereas in Ps. putida WCS358-elicited ISR in Arabidopsis a role of pyoverdine production is uncertain, in tomato WCS358 does trigger ISR and a pvd mutant is not effective (Meziane et al., 2005). In bean there appears again to be redundancy in ISR-eliciting traits, since pyoverdine triggers ISR but the pvd mutant is as effective as the WT strain (Meziane et al., 2005). In tobacco, ISR mediated by Ps. fluorescens CHA0 was found to be effective against tobacco necrosis virus (TNV). The fluorescent siderophore played a major role here because a pvd mutant was less effective in reducing numbers of viral lesions and lesion diameter than the parental strain (Maurhofer et al., 1994). Similarly, Ps. putida BTP1 induces ISR in bean against Botrytis cinerea, but only when the bacteria are grown under iron-limited conditions (Ongena et al., 2002). In this case, an N-alkylated benzylamine derivative was characterized as the iron-regulated elicitor of ISR (Ongena et al., 2005). The role of siderophores produced by fluorescent Pseudomonas spp. in the biological control of soil‐borne diseases by these PGPR may not be attributed simply to either microbial antagonism or ISR. It seems more likely that a combination of the two modes of action of siderophores contributes to the control of disease. 2. Plant growth and nutrition Plant-growth promotion by rhizosphere bacteria mediated by siderophore synthesis is related to both indirect (microbial antagonism) and direct (iron nutrition) effects. The possible contribution of microbial antagonism in plant-growth promotion by PGPR was first observed by Kloepper and Schroth (1981). These authors showed that growth promotion occurred only when the PGPR were applied to plants cultivated in nonsterilized soil and not when the plants were cultivated in sterilized soil, suggesting that the resident microflora was reducing plant growth. These authors further showed that only antagonistic bacterial strains were able to promote plant growth (Kloepper et al., 1980b). The indigenous microflora was shown to include deleterious microorganisms that induce growth and yield depression without any obvious symptoms, in contrast to classic plant pathogens (Bakker et al., 1986, 1987; Moulin et al., 1994; Schippers et al., 1987; Suslow and Schroth, 1982). This type of deleterious effect was clearly evident in yield depression of potato in the Netherlands (Bakker and Schippers, 1987), where population densities of deleterious pseudomonads producing harmful levels of cyanide were significantly increased (Bakker and Schippers, 1987). This growth depression could be overcome by cultivating potato in

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long rotation or by inoculation with antagonistic fluorescent pseudomonads (Bakker et al., 1986, 1987). Similarly, depression of cucumber growth and yield by deleterious Pythium spp. in soil-less culture could be overcome by inoculation with antagonistic fluorescent pseudomonads (Moulin et al., 1996). Involvement of pyoverdine-mediated iron competition in the antagonism against deleterious microorganisms, ultimately promoting plant growth, was shown by the use of siderophore-minus mutants (Bakker et al., 1986, 1987; Kloepper et al., 1980b). Despite their high affinity for iron, pyoverdines were shown to improve iron nutrition in both the strategy I plant species Arabidopsis and tobacco (Vansuyt et al., 2007; G. Vansuyt et al., unpublished data) and the strategy II grasses barley, fescue, rye-grass and wheat (G. Vansuyt et al., unpublished data). In Arabidopsis, the improvement of plant iron nutrition in the presence of Fe-pyoverdine led to increased plant growth compared to the unamended control (Vansuyt et al., 2007). Other microbial siderophores, such as ferrioxamine and rhodothorulic acid, were similarly shown to improve iron nutrition of various strategy I and II species (Crowley and Gries, 1994; Crowley et al., 1988, 1992; Ho¨rdt et al., 2000; Siebner-Freibach et al., 2003; Yehuda et al., 1996, 2000, 2003). The improvement of iron nutrition in Arabidopsis recorded by Vansuyt et al. (2007) does not seem to be related to the mechanisms described for strategy I plants, as indicated by the incorporation of iron from Fe-pyoverdine in knockout mutants in the major iron transporter IRT1 or the FRO2 reductase. In agreement with these observations, Fe-pyoverdine itself seems to be taken up by Arabidopsis, as shown by the presence of pyoverdine in planta as measured by an enzyme-linked immunosorbent assay (ELISA) using an antibody raised against the pyoverdine, and by isotope measurements of 15N-labelled pyoverdine (Vansuyt et al., 2007). Additionally, pyoverdine was observed in planta by confocal laser scanning microscopy and transmission electron microscopy (L. Avoscan et al., unpublished data). The way by which Fe-pyoverdine is taken up by Arabidopsis remains to be clarified. Mechanisms by which strategy II plants may draw benefit for their nutrition from iron chelated to siderophores remain to be explored. Indirect mechanisms are expected to account for improved plant nutrition by pyoverdines due to their significantly higher affinity for iron compared to phytosiderophores (Meyer and Abdallah, 1978; Sugiura et al., 1981). Duijff et al. (1994b) proposed that possible degradation of Fe-pyoverdines by microbes could lead to the release of iron that would then be available for phytosiderophores. Whether this is plausible depends on their concentration, location, and release kinetics. The diurnal production cycle of phytosiderophores

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results in pulses (Crowley and Gries, 1994; Reichman and Parker, 2005; Takagi et al., 1984), during which their concentration in the rhizosphere might be higher than that of microbial siderophores, thereby affecting the Fe exchange in favor of phytosiderophores (Jurkevitch et al., 1993; Yehuda et al., 1996). However, one should be cautious about this hypothesis because of the thermodynamic and kinetic constraints involved in such ligand exchange processes. Even if they do occur in the rhizosphere, they are probably too slow to contribute significantly to plant iron nutrition. Besides these temporal aspects, the iron-chelating efficiency of phytosiderophores also depends on their spatial location. Their release occurs at a greater rate in apical root zones (Marschner et al., 1987), where rhizodeposition is maximal (Nguyen, 2003). Despite this observation, interactions between phytosiderophores and the resident microflora are likely to be minor since the rhizosphere effect on microorganisms occurs mostly at some distance from the root apex (Gamalero et al., 2002; Yang and Crowley, 2000) because of the longer time required for root growth than for microbial growth upon root exudation.

IV. RECIPROCAL INTERACTIONS BETWEEN PLANTS AND MICROORGANISMS DURING PATHOGENESIS During the parasitic life of the pathogen, its interaction with the host organism leads to a competitive relationship where both protagonists try to defeat each other. In plants, as in animal hosts, a successful pathogen produces virulence factors enabling it to gain access to essential nutrients, to rapidly adapt to new environmental conditions, and to overwhelm host defense. As a growth-limiting factor for most living organisms, iron plays a critical role in this competition. This metal almost never exists in a free form in living tissues. Bound to proteins and nonproteinaceous ligands, it may be difficult to capture at infection sites, including extracellular and intracellular compartments (Sutak et al., 2008; Weinberg, 2008). Bacterial and fungal pathogens display a variety of mechanisms to acquire iron from host cells, based on secretion of siderophores which can remove the metal from almost any mineral or organic substrate, expression of ferric chelate reductases located at the plasma membrane, or production of specific membrane transporters allowing the direct utilization of heme and binding of host-derived iron. Besides high-affinity iron transport per se, toxins or hydrolytic enzymes that are produced in response to low iron availability can also greatly benefit the pathogen (Litwin and Calderwood, 1993;

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Van Baarlen et al., 2007). Conversely, upon pathogen attack, hosts can activate various mechanisms aimed at depriving the invader of nutritional iron (Fig. 2B). Major advances have improved our understanding of the ironwithholding mechanisms involved in human and animal hosts (Schaible and Kaufmann, 2004). By complexing iron to proteins of the transferrin family and scavenging it into ferritins, the vertebrate host inhibits the growth of certain bacteria and fungi. The ferrous iron transporter Natural resistanceassociated macrophage protein 1 (NRAMP1) (syn.: Solute carrier family 11 (proton-coupled divalent metal ion transporters) member 1 (SLC11A1)) (Vidal et al., 1993) confers resistance to intracellular bacterial parasites, possibly by extruding iron from the phagosomal vacuole to the cytoplasm. Increased expression of the iron regulatory peptide hepcidin during infection and inflammation leads to iron sequestration in enterocytes and macrophages and interrupts the delivery of iron in plasma (Ong et al., 2006). NGAL (Neutrophil gelatinase-associated lipocalin) is induced in epithelial cells during inflammation and binds catecholate type siderophores, thus preventing pathogenic bacteria from acquisition of siderophore-bound iron (Flo et al., 2004; Nelson et al., 2005). These antimicrobial mechanisms are considered to be part of the vertebrate innate immune system. Although there is still little information on how plants control their iron homeostasis under conditions of infection (Table I), such antimicrobial mechanisms in plant hosts could exist, and deciphering them is a matter of recent investigations. In addition, the participation of iron in ‘‘Fenton-type’’ redox chemistry makes this metal particularly important in the modulation of the oxidative burst, which in plants has a dual role: (i) antimicrobial action, and (ii) construction of barriers by cross-linking of cell-wall components. It may also be possible that interconnections between pathogen-triggered resistance responses and control of iron homeostasis occur in plants. In mammals, there is a concerted action of interferon- , a key cytokine mediating cell immune responses in the macrophage and iron regulation to enhance host defense (Nairz et al., 2008). The following section describes the various disease situations in which iron represents a stake in microbial virulence and plant defense. A. IRON AND MICROBIAL VIRULENCE: ROLE OF HIGH-AFFINITY IRON ASSIMILATION SYSTEMS

In pioneer studies, attention has been drawn to siderophores as potential pathogenicity determinants. Discovered in animal infections, the role of siderophores has then been addressed in microbial pathogenesis of plants by investigating the production of these compounds by diverse plant

TABLE I Plant–Microbe Interactions Involving Control of Iron Homeostasis During Pathogenesis Fe transport/storage function/gene,a involved in: Pathogen—host

Disease

Bacterial pathogens Er. chrysanthemi—Arabidopsis

Soft rot

Er. Amylovora—apple flower

Fire blight

Fungal pathogens C. heterostrophus—maize Al. brassicicola—Arabidopsis F. graminearum—wheat U. maydis—maize Bl. graminis—wheat Ph. infestans—potato

Microbial virulence Siderophores: chrysobactin achromobactin Siderophores: desferrioxamine E

Leaf blight

Siderophores: coprogen

Smut disease Powdery Mildew

Ferric permease/Ferroxidase

Late blight

Plant reaction/defense AtFer1, Nramp3-4 gene upregulation/resistance IRT1-FRO2 upregulation

TmNAS1, TmFER1 gene downregulation Cell wall appositions StF1 gene upregulation

Genes AtFer1, TmFER1, and StF1 encode ferritins, genes Nramp3 and Nramp4 encode vacuolar metal transporters, and genes IRT1 and FRO2 encode the root ferrous transporter and ferric reductase, respectively; TmNAS1 encodes a nicotianamine synthase.

a

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pathogens. These studies were completed by genetic approaches aimed at analyzing the virulence properties of mutants deficient in siderophore biosynthesis and transport functions. The role of siderophores (or their uptake systems) in bacterial diseases was assessed in crown gall caused by Agrobacterium tumefaciens (Leong and Neilands, 1981), fruit spots caused by Ps. syringae (Cody and Gross, 1987), soft rot caused by pectinolytic Erwinia species (Expert, 1999), black rot of crucifers caused by Xanthomonas campestris pv. campestris (Wiggerich and Pu¨hler, 2000), and fire blight caused by Er. amylovora (Dellagi et al., 1998). The role of ferrichrome in smut disease of maize caused by the fungus Ustilago maydis has also been the subject of investigations (Mei et al., 1993). The assumption that iron assimilation via siderophores could play a role in plant infection was experimentally shown by work on the pathogenicity of Er. chrysanthemi and Er. amylovora. Despite the apparent similarity in symptoms elicited by Er. chrysanthemi and Er. carotovora (syn.: Pectobacterium carotovorum), the latter does not require the production of siderophores for successful infection. More recently, Oide et al. (2006) demonstrated that biosynthesis of siderophores is a virulence determinant of plant-pathogenic ascomycetes. Yet, a mutant of the fungus Microbotryum violaceum that lacks the ability to synthesize the siderophore rhodotorulic acid, shows wild-type virulence on its host Silene latifolia (Birch and Ruddat, 2005). A study of the possible role in virulence on tomato of the siderophores produced by Ps. syringae pv. tomato DC3000, yersiniabactin and pyoverdine, was also reported (Jones et al., 2007). Although yersiniabactin can be produced in planta, no differences in either bacterial growth or disease symptoms were observed between mutant and WT strains inoculated on tomato and Arabidopsis. On the other hand, whereas iron assimilation via ferrichrome is not essential for U. maydis virulence, recent work indicates involvement of a permease-based high-affinity iron uptake system for biotrophic development of this fungus in maize (Eichhorn et al., 2006). Thus, it appears that a role of high-affinity iron uptake systems in the virulence of plant pathogenic microorganisms is intimately associated with the life of the pathogen within its host. We present below a detailed picture of the models where iron acts as a pathogenicity factor. 1. Siderophore-controlled iron acquisition and Er. chrysanthemi pathogenicity Er. chrysanthemi is a typical member of this pectinolytic genus, which was recently reclassified as Dickeya. The species D. dadantii causes soft rot of economically important plants, including vegetables and ornamentals. Strain 3937 produces a systemic disease in African violets as well as in Arabidopsis (Fagard et al., 2007; Murdoch et al., 1999). Symptoms consist of tissue

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disorganization due to the release of a set of bacterial pectinolytic enzymes that degrade plant cell walls. Pectin hydrolysis gives rise to oligogalacturonides that are used by the bacteria as a carbon source. Cell wall deconstruction weakens plant cells and thus allows bacteria free access to cellular nutrients. Colonization of plant tissues begins with a symptom-less phase that may last several days, during which bacterial cells remain clustered in intercellular spaces of the cortical parenchyma and then migrate intercellularly without causing severe injury of cellular structures. Bacterial cells do not migrate into the vascular tissues. Successful infection throughout aerial parts of the plant depends on the bacterial production of chrysobactin and achromobactin (Dellagi et al., 2005; Franza et al., 2005). Chrysobactin-defective mutants produce only localized symptoms on saintpaulia plants and compared to a chrysobactinproficient strain their growth is reduced. This decline coincides with the emergence of a necrotic border surrounding the lesion initiated by the mutant (Masclaux and Expert, 1995). Chrysobactin was detected in leaf intercellular fluids from plants inoculated with the WT strain, suggesting that this compound can sequester the iron present in colonized tissues, while at the same time inducing a plant reaction to deprive the bacterial cells of essential iron (Neema et al., 1993). To further understand the biological function of chrysobactin, Anne-Marie Albrecht-Gary and coworkers (Tomisˇic´ et al., 2008) have investigated the coordination properties of the different ferric complexes of this siderophore. They found that chrysobactin is a less effective ferric chelator than hexadentate siderophores, such as enterobactin or ferrioxamine B. However, chrysobactin exhibits a higher pFe value than citrate or malate (pFe of chrysobactin ¼ 17.1 vs. pFe of citrate ¼ 14.8), which are known to be major ferric ion carriers in plants, and can effectively sequester the iron from their ferric complexes. Achromobactin-deficient mutants are also affected in their virulence but are more aggressive than the chrysobactin nonproducers (Franza et al., 2005). On the other hand, double mutants deficient in both achromobactin and chrysobactin production are impaired in symptom initiation, indicating that these two siderophores act in a complementary way to satisfy the bacterial need for iron during their passage within the plant. Although the coordination chemistry of achromobactin has not yet been investigated, it can be predicted that this ligand is less competitive than chrysobactin. It is noteworthy that the WT cells, but not the double mutants, can survive for several days in intercellular spaces of host tissues without multiplying substantially. In contrast, during the symptomatic phase, the number of viable cells appears to increase strongly, whereas the proliferation of the double

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mutant is tenfold lower than that of the WT strain. Therefore, both siderophores contribute to successful infection. Initiation of the symptomatic phase of the disease appears to be critical if no siderophore is produced. The low availability of iron in the apoplast acts as a signal that turns on the transcription of the genes that encode the major pectin-degrading enzymes and the genes that belong to the chrysobactin and achromobactin iron uptake systems (Franza et al., 1999, 2002). This metalloregulation is mediated by the transcriptional repressor Fur, which controls intracellular iron concentration and is involved in the pathogenicity of strain 3937. Otherwise, during infection bacterial cells have to cope with the production of reactive oxygen species (ROS) by plant cells (Fagard et al., 2007; Santos et al., 2001). Several studies have demonstrated the importance of a perfect control of iron homeostasis involving a connection between iron metabolism and tolerance to oxidative stress. In particular, there was the discovery that the Suf (Mobilization of sulfur) machinery, encoded by the sufABCDSE operon, participates in the formation of Fe–S clusters under iron starvation and oxidative conditions and is necessary for full virulence (Nachin et al., 2001). Microarray profiling of bacterial genes that are specifically up- or downregulated in saintpaulia leaves, as well as in vivo expression analysis of promoter–GFP (Green fluorescent protein) reporter constructs in leaves of spinach allowed the identification of Er. chrysanthemi genes that are regulated during plant infection (Okinaka et al., 2002; Yang et al., 2004). Upregulation of genes involved in iron uptake and in stress responses to ROS was observed. Er. chrysanthemi possesses three ferritins, of which the heme-free ferritin FtnA and the heme-containing bacterioferritin Bfr play differential roles in virulence, depending on the host (Boughammoura et al., 2008). FtnA constitutes the main iron storage protein. Indeed, a ftnA mutant that lacks a functional FtnA ferritin has increased sensitivity to iron deficiency compared to the WT. In addition, this mutant has increased sensitivity to oxidative and nitrosative stresses, as well as an increased content in ferrous iron. By limiting the concentration of reactive iron, FtnA reduces the cytotoxic effect of the Fenton reaction, and thus confers tolerance to oxidative stress. Bacterioferritin acts as a transient iron store which plays an important role in distribution of iron between essential metalloproteins, particularly under conditions of iron deficiency (Expert et al., 2008). 2. Siderophore-controlled iron acquisition and Er. amylovora pathogenicity Er. amylovora is an enterobacterium that causes fire blight, a disease of members of the Maloideae characterized by a progressive necrosis of tissues of infected aerial parts of the plant and often associated with ozone

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production. Natural infections mainly occur through wounds and natural openings, especially on flowers. On susceptible hosts, bacteria first move through the intercellular spaces of the parenchyma and at a later stage into the xylem vessels, provoking extensive lesions and sometimes complete dieback of the tree. The strategy of infection of Er. amylovora differs from that of Er. chrysanthemi in that the two main pathogenicity factors for this bacterium are the exopolysaccharide amylovoran and the secretion of effectors through the type three secretion system (T3SS) (Venisse et al., 2003). The T3SS is required during early infection steps that lead to local necrosis, whereas amylovoran is required during later stages which result in bacterial progression in planta. Er. amylovora effector proteins transiting through the T3SS contribute to pathogenesis by controlling plant defenses and triggering cell death (Degrave et al., 2008). In iron-limited environments, Er. amylovora produces cyclic trihydroxamate siderophores belonging to the DFO family, which were first isolated from actinomycetes and are also found in other bacteria such as Erwinia herbicola (Kachadourian et al., 1996). DFO E appears to be predominant. Unlike nonpathogenic mutants lacking a functional T3SS, mutants affected in DFOmediated iron transport still cause fire blight (Dellagi et al., 1998, 1999). The lack of DFO-dependent iron uptake has no effect on pathogenicity when tested on apple seedlings. However, the mutants are less able to colonize floral tissues and to initiate necrosis on apple flowers, indicating that the production of DFO is critical at the onset of infection. This observation led the authors to speculate that DFO is involved not only in iron acquisition but also in oxidative stress responses of the pathogen. Indeed, Er. amylovora WT strains induce electrolyte leakage from host plant cells as a result of cell death. The ability of DFO biosynthetic mutants to induce this reaction is severely reduced and this defect is rescued by the addition of exogenous DFO. As DFO alone does not induce electrolyte leakage, it was proposed that this compound, by inhibiting the generation of toxic radicals via the Fenton-type redox chemistry, protects the bacterial cells against the toxic effects of ROS produced at the onset of infection. The reduced ability of the DFO biosynthetic mutants to cause electrolyte leakage would result from the transient decrease in bacterial population size following the initial oxidative burst. In support of this interpretation is the increased survival of Er. amylovora cells treated with 1 mM H2O2 in the presence of DFO B, which is known to protect animal tissues from damage by ROS. By forming high-stability iron complexes, compounds of the DFO family inhibit the generation of hydroxyl radicals via the Fenton reaction. Interestingly, Zhao et al. (2005) found that the gene encoding the ferritin Ftn is induced during infection in pear tissue.

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3. Siderophore-mediated iron acquisition and pathogenicity of ascomycete fungi The filamentous ascomycete Cochliobolus heterostrophus is representative of a genus that attacks monocots, including all major cereal crops, worldwide. This fungus was known as a mild pathogen of corn until 1970, when a highly virulent race caused the Southern corn leaf blight, which devastated the US corn crop along the eastern seaboard. Thus, C. heterostrophus emerged as a model necrotrophic fungus for the study of plant pathogenesis (Turgeon and Baker, 2007). Its life cycle includes both asexual and sexual stages. The fungus overwinters as conidia and mycelia on debris of dead corn plants. Sexual reproduction is easily induced in the laboratory and controlled by a single mating-type locus (MAT1). Conidia infect corn leaves by direct penetration and cause small lesions, which under epidemic conditions can cover the entire leaf, thereby killing the plant. C. heterostrophus and related taxa are known for their ability to produce host-specific toxins that serve as pathogenicity factors. Synthesized by NRPS, each toxin appears to be necessary for development of a particular disease. However, in a number of physiological and genetic studies, they were found to be insufficient to explain pathogenicity entirely. Looking for general pathogenicity factors, Lee et al. (2005) undertook a genome-wide search for NPRS-encoding genes in C. heterostrophus. Twelve loci predicted to encode NPRS were deleted singly, and only one, NPS6, appeared to be involved in virulence on maize (Oide et al., 2006). Interestingly, NPS6 is the only gene that has an ortholog in all other ascomycetes examined. To examine the functional conservation of NPS6, NPS6 orthologs were deleted in the rice pathogen Cochliobolus miyabeanus, the Arabidopsis pathogen Alternaria brassicicola, and the wheat/maize/barley pathogen Fusarium graminearum. Lesions caused by C. heterostrophus nps6 mutants are smaller compared to those of the WT. Conidia of both the WT and the nps6 mutant germinate normally, form appressoria and penetrate successfully into the host, indicating that deletion of NPS6 does not cause a defect in pre-penetration growth or penetration efficiency. However, the extent of colonization by the nps6 mutant was less than that of the WT. Lesion sizes upon infection with the nps6 mutants of C. miyabeanus and Al. brassicicola were also decreased. Similarly, symptom development on wheat spikes inoculated with the nps6 mutants of F. graminearum was much delayed compared with the WT strain. Besides their reduced virulence, nps6 mutants have increased sensitivity to iron depletion and increased sensitivity to oxidative stress. Indeed, C. heterostrophus, Al. brassicicola, and F. graminearum NPS6 genes are upregulated under iron-depleted conditions. These observations suggested that the NPRS encoded by NPS6 are involved in siderophore biosynthesis. HPLC analyses

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of the culture filtrate and mycelial fractions of C. heterostrophus WT and nps6 mutants confirmed this assumption. Further analyses demonstrated that the siderophores produced are coprogens, which are hydroxamates widely distributed among fungal species, including the saprophyte Neurospora crassa (Winkelmann, 2007). The role of these siderophores in iron nutrition of these fungi in planta was supported by the observation that exogenous application of iron enhances the virulence of nps6 mutants. The application of the extracellular siderophore of Al. brassicicola restored wild-type virulence to the nps6 mutant on Arabidopsis. Whether these siderophores play a role in virulence through the protection of fungal pathogens against ROS generated in planta requires further investigation. 4. High-affinity iron acquisition and Ustilago maydis pathogenicity The plant-pathogenic basidiomycete U. maydis causes smut disease of maize. The main symptoms are tumors which can develop on all green parts of the plant (Bo¨lker, 2001). The fungal hyphae proliferate in these tumors and differentiate into diploid spores. Spores are distributed by air and can germinate after landing on plant surfaces. During germination, meiosis occurs and results in the production of nonpathogenic haploid cells. Fusion of compatible haploid cells is required to generate a dikaryotic mycelium which is infectious. The fusion of cells and the development of the pathogenic dikaryon are governed by the a and b mating-type loci. During mating, cells secrete specific pheromones and respond to the presence of cells of the opposite mating type. This process involves coordinated cAMP-dependent protein kinase A (PKA), as well as mitogen-activated protein kinase (MAPK) signaling. Transcriptome analysis using whole genome microarrays to identify putative targets of the PKA catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity uptake systems, located in three gene clusters on chromosomes 1, 2, and 4 of the U. maydis genome (Eichhorn et al., 2006). The cluster on chromosome 1 contains the siderophore biosynthetic genes sid1 and sid2 previously shown to be involved in the production of ferrichrome (Mei et al., 1993). Chromosome 2 harbors a cluster of Fe-regulated genes, fer3–fer10, several of which were predicted to encode the enzymes catalyzing the biosynthesis of ferrichrome A, the second siderophore released by U. maydis, as well as the corresponding transporters. The two genes found on chromosome 4, fer1 and fer2, encode a putative ferroxidase and a highaffinity ferric permease, respectively. The fer2 gene is able to complement the growth defect of an FTR1 mutant of Saccharomyces cerevisiae affected in high-affinity iron uptake due to a lack of functional ferric iron permease. It is plausible that, like in Sa. cerevisiae, the U. maydis iron permease and oxidase

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are part of a ferrous uptake system in which a plasma-membrane reductase reduces ferric ions present in the medium. The resulting ferrous ions are then reoxidized by the multicopper oxidase before transport by the permease. Like the sid1 and sid2 genes, the fer genes were found to be negatively controlled by the GATA transcription factor Ustilago regulator of biosynthesis of siderophores 1 (Urbs1) in the presence of iron. As these genes are also regulated through a cAMP-dependent pathway, it was postulated that the regulatory activity of Urbs1, which has eight putative PKA phosphorylation sites, could depend on phosphorylation. When maize plants are infected with a compatible combination of WT strains, tumors develop in most infected plants and a high proportion of these plants die. Upon infection with either fer1 or fer2 mutants, fewer plants develop tumors and these tumors remain small. As a consequence, plant death is only rarely observed. These mutants are not affected in early development on the leaf surface: they form appressoria and penetrate normally. However, proliferation of the fungal cells in the plant is not observed. Interestingly, the fer2 mutant is more virulent on a WT host than on the maize ys1 mutant, which has a defect in ferric phytosiderophore uptake (see Section II.A.1.a). Thus, the ferroxidation/permeation system is decisive for iron acquisition during plant colonization by U. maydis. As ferrichrome biosynthetic mutants are not affected in virulence, an intriguing question is why phytopathogenic fungi use different strategies for iron acquisition during plant colonization. It is possible that the lifestyle of the fungus during infection, that is, growing as a biotroph or a necrotroph, may determine in which form the iron present in the host plant can be acquired. At which stages of its life cycle U. maydis makes use of the ferrichrome-mediated iron uptake system is another remaining question. B. IRON AND PLANT DEFENSE

1. Iron homeostasis in wheat upon infection by Blumeria graminis The powdery mildew fungus Bl. graminis is a biotrophic pathogen that requires successful host cell wall penetration, development of a functional haustorium and maintenance of host cell integrity to establish a compatible interaction with its host. Resistance to this pathogen is expressed by prevention of penetration through localized cell wall strengthening. Cell wall strengthening by wall appositions (papillae) is observed in nonspecific resistance responses. During Bl. graminis attack, ROS accumulate in epidermal and mesophyll cells close to the infection sites. In an expressed sequence tag (EST) library developed from wheat epidermis challenged with the wheat powdery mildew fungus Bl. graminis f.sp. tritici, Liu et al. (2007) noted a high

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occurrence of Fe-related transcripts, which prompted these authors to investigate changes in iron homeostasis in wheat leaves during the fungal attack. Using inductively coupled plasma mass spectrometry (ICPMS) to track changes in metal concentrations in the epidermis and mesophyll of wheat (Triticum monococcum) leaves infected with Bl. graminis, Liu et al. (2007) found that ferric ions accumulate in the infected epidermis but not in the mesophyll. This accumulation takes place when cell wall appositions are mature and the success or failure of the infection is distinguishable. Further analysis indicated that the accumulated iron enhances the production of ROS triggered during pathogen attack. Indeed, a pretreatment of the inoculated leaves with the ferric iron chelator DFO decreased the oxidative burst detected by diaminobenzidine (DAB) staining. The iron-regulated genes TmNAS1 and TmFER1, encoding a nicotianamine synthase and a ferritin isoform, respectively, are induced by iron treatment of wheat leaves but appeared to be downregulated during pathogen attack, suggesting cytosolic iron depletion in the infected tissues. In addition, a correlation was established between cytosolic iron depletion and activation of the pathogenesis-related gene TmPR-1b. Otherwise, using the membrane-permeable fluorescent chelator calcein, the authors showed that a treatment of wheat suspension cells with H2O2 promoted active cytosolic Fe efflux. Collectively, these findings led to the proposal of a model of the involvement of iron in wheat leaves during Bl. graminis attack, where the redistribution of this metal and the oxidative burst triggered after infection contribute to the amplification of a redox signal involved in the defense response. 2. Involvement of ferritin in the response of potato to Phytophthora infestans Oomycetes in the genus Phytophthora are fungus-like plant pathogens that are devastating for virtually all dicots (Attard et al., 2008). The species Ph. infestans causes potato late blight and this disease is initiated when wind-blown sporangia release zoospores onto the plant surface. The zoospores encyst to form appressoria, after which hyphae develop and can spread throughout the plant. When these structures penetrate plant cells, they remain enveloped by a lipid membrane derived from the plant plasma membrane. Treatment of potato leaves with DFO before spraying a suspension of sporangia resulted in a reduction of lesion development and a decrease in the production of ROS (Garcı´a Mata et al., 2001). Upon infection with Ph. infestans expression of the StF1 potato ferritin gene was increased, suggesting that ferritin may be a protective molecule for plant cells. By scavenging the intracellular iron, ferritin can limit the generation of ROS. Although increased ferritin gene expression does not suffice to confer resistance to Ph. infestans, a previous study indicated that transgenic tobacco

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plants ectopically expressing an alfalfa ferritin gene had increased tolerance to viral (TNV) and fungal (Alternaria alternata, Bo. cinerea) infections (Dea´k et al., 1999). These reports support the idea that ferritin can be part of host defense responses triggered during infection. 3. Iron homeostasis and resistance of Arabidopsis to Er. chrysanthemi As efficient iron uptake/storage mechanisms are of critical importance to the survival of the bacterium Er. chrysanthemi during infection, the question of whether this pathogen could induce iron-withholding reactions in the plant has been addressed. A first observation made by Neema et al. (1993) indicated that iron incorporated into plant ferritins drastically decreased in soybean suspension cells challenged with Er. chrysanthemi. This effect was also observed during treatment of the cells with chrysobactin. The possibility of a competition for iron between the pathogen and the host was also illustrated by the observation that accumulation of polyphenols in plant tissues inhibits growth of mutants of Er. chrysanthemi affected in their siderophore-mediated iron transport pathway (Mila et al., 1996). Polyphenols have iron-chelating properties and could play the role fulfilled by iron-binding proteins, such as transferrin in animal immunity. In order to identify plant genes that are regulated in response to infection by Er. chrysanthemi, Dellagi et al. (2005) differentially screened cDNA libraries from Arabidopsis and found that the gene encoding the ferritin AtFer1 is upregulated in infected plants. These authors established that accumulation of AtFer1 transcripts and production of ferritins during infection is a defense reaction against proliferation of the pathogen. The siderophore chrysobactin, as well as DFO are elicitors of this response. As only the iron-free siderophores induce this reaction, it was suggested that these iron-sequestering molecules could cause severe iron depletion in Arabidopsis leaf tissues, resulting in the redistribution of intracellular iron stores and/or the activation of iron acquisition systems of the cell. Intracellular redistribution could involve remobilization of vacuolar iron by the specific metal transporters NRAMP3 and NRAMP4. Indeed, among the six NRAMP genes present in Arabidopsis, AtNRAMP3 was found to be strongly upregulated in response to several biotic stresses. Because there is a functional redundancy between AtNRAMP3 and AtNRAMP4 in seed germination and the encoded proteins share 50% sequence identity with the mouse NRAMP1 metal ion transporter, the role of these genes in resistance to Er. chrysanthemi was investigated further (Segond et al., 2009). AtNRAMP3 is upregulated in leaves challenged with Er. chrysanthemi, while AtNRAMP4 expression does not change. Using simple and double nramp3 and nramp4 mutants, as well as lines

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ectopically expressing either of these genes, Segond et al. (2009) showed that AtNRAMP3, and to a lesser extent AtNRAMP4, are involved in the resistance of Arabidopsis against this bacterium. The susceptibility of the nramp3 nramp4 double mutant is associated with a reduced accumulation of ROS and AtFER1, which are effective defense components against Er. chrysanthemi. By promoting an efflux of iron and possibly other metals from the vacuole to the cytosol, the activity of NRAMP3 and NRAMP4 proteins may contribute to exacerbation of the oxidative stress generated during infection and be responsible for basal resistance to this pathogen. Both increased oxidative stress and efflux of iron could be at the origin of ferritin upregulation in infected leaves. In addition, roots from Er. chrysanthemi-challenged plants accumulate transcripts of AtNRAMP3 as well as the iron deficiency markers IRT1 and FRO2 (see Section II.A.1.b). This finding suggests the existence of a shoot to root signal similar to the systemic iron-deficiency signal that is activated by pathogen infection. Whether the redistribution of iron in Er. chrysanthemi-challenged leaves and uptake of iron by the roots are physiologically linked is worth further consideration. Whether the siderophores produced by the bacterium during infection are the main contributors to these reactions is also an appealing question. Collectively, these data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants. However, the contribution of these proteins to host resistance seems to differ as, for instance, the growth of Ps. syringae pv. tomato DC3000 is not affected in the nramp3 nramp4 double mutant (Segond et al., 2009).

V. CONCLUSIONS Despite the abundance of iron in soils, its availability for plants and microbes is low. Because of the major importance of this element for essential physiological processes, including respiration and photosynthesis, plants and microorganisms have evolved active strategies of iron uptake. These strategies are subject to complex regulations in order to be expressed only when required because of their cost and of the toxicity of iron when present in excess. It has been proposed that mutualistic interactions in the rhizosphere between plants and microorganisms have contributed to plant adaptation in soils with low fertility and with soil‐borne pathogens (Cook et al., 1995). Indeed, plants release a significant part of their photosynthates as rhizodeposits, which supports and selects specific microbial populations that, in return, favor plant growth and health. This notion is well illustrated by plant–microbe interactions resulting from the low iron availability in most

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soils. The promotion of microbial communities in the rhizosphere, which require iron for their metabolism, together with iron acquisition by the plant result in a strong competition for iron in the rhizosphere. On this battleground, populations with the most efficient iron uptake strategies benefit from a competitive advantage. Benefiting from plant rhizodeposits, these populations produce very efficient siderophores that deprive plant pathogens and deleterious microorganisms from iron, leading to the suppression of their saprophytic growth, and ultimately to reduction of root infections (microbial antagonism). These microbial siderophores do not seem to compete with the eucaryotic host plant; plants even seem to have evolved strategies to take advantage of these siderophores for their iron nutrition. In fact, these siderophores appear to be able to extract iron from soils and to increase its availability for the host plant. Bacterial siderophores may also act as signal molecules that elicit ISR in the host plant. Taken together, these interactions can be considered as a feedback loop in which plants invest a significant part of their photosynthates in their rhizosphere microflora, and select specific populations that promote plant growth and health in return. These interactions are continuously adapting and contribute to plant–microbe co‐evolution. They are under the control of the physicochemical properties of the surrounding soil, which impact iron availability. This has been particularly well demonstrated in soils that are naturally suppressive to soilborne diseases as a result of iron competition. A better knowledge of these complex interactions and of their monitoring to improve plant growth and health is a major challenge. Indeed, increased biomass productivity and quality of the products, within the frame of an environmentally friendly and sustainable agriculture on reduced acreages, will be a major task for the years to come. A lot of data have been obtained in the last few years on the biomass production by the ocean in relation with iron fertilization (Blain et al., 2007; Pollard et al., 2009). Iron fertilization of terrestrial plants and its impact on yield and quality have received far less attention (Fan et al., 2008; Ravet et al., 2009), but deserve to be considered as a potential parameter for improving plant yield and product quality (Briat et al., 2007). Similarly, the impact of rhizosphere microbes on iron nutrition of plants is largely underestimated. Therefore, there is an urgent need for defining innovative agricultural practices that will take advantage of interactions between biotic (plants— microorganisms) and abiotic (soil physicochemical properties) components in cropping. Such practices should include plant breeding programs aimed at integrating, in plant genotypes, traits involved in the selection of microbial populations favorable to the host plant. Attempts have already been made in this direction to identify plant genetic traits that promote the survival of

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antagonistic bacteria in the rhizosphere (Smith et al., 1999). As reported in this chapter, there are now examples which clearly demonstrate that plant genotypes engineered in traits modifying plant iron homeostasis influence rhizosphere microbial populations in a soil-dependent manner. Possible consequences of these practices on plant iron homeostasis and allocation, and ultimately on seed iron content, as well as on the virulence of pathogens during their parasitic life will have to be explored. Such research provides an attractive challenge to ecologically engineer plant–soil–microbe interactions in order to improve both plant nutrition and defense against pathogens.

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Adaptive Defense Responses to Pathogens and Insects

LINDA L. WALLING1

Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Co‐evolution of Defense Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Portals of Entry and Activation of Defenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Microbial Invasion Strategies: From Accessing the Apoplast to Haustoria Formation .................................... B. Defense Pathway Activation by Microbes................................ C. Herbivore-Feeding Guilds: Mechanisms to Violate the Integrity of Plant Cells .................................................. IV. Perceiving Pathogen and Pest Visitations: The Role of Microbial and Herbivore Elicitors and Molecular Patterns . . . . . . . . . . . . . . . . . . . . . . . . . A. PAMPs, Pattern-Recognition Receptors, and BAK1................... B. Elicitors of Plant Origin ..................................................... C. Herbivore Elicitors ........................................................... V. Integrating Signals and Activating Defenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mitogen-Activated Protein Kinase Signaling Cascades ................ B. Linking PAMPs to SA-, JA-, and ET-Regulated Defense Responses ........................................................ VI. Adaptations to Unfriendly Hosts: Effectors and Evasion Tactics . . . . . . . . A. Microbial Effectors........................................................... B. Herbivore Effectors .......................................................... VII. Effector-Triggered Immunity: Resistance to Pathogens and Pests . . . . . . . A. The Guard and Decoy Models ............................................. B. Plant–Herbivore Gene-for-Gene Interactions............................

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

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VIII. Summary and Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

ABSTRACT The relative resistance of a plant to pathogens is determined by preformed, constitutive defenses, and the quality and diversity of the induced defenses deployed upon attack. Pathogens have evolved strategies to breach structural barriers and avoid or counter preformed and induced chemical defenses of their host plants. Plants have evolved sensitive mechanisms to perceive bioagressors and innate immune responses are activated when plants encounter non‐adapted microbes, virulent microbes and insects, or when plants engage in a gene-for-gene resistance response. The earliest events in innate immunity are triggered by the perception of the mechanical and chemical signals associated with injury and highly conserved molecules called pathogen-associated molecular patterns (PAMPs), which signal the presence of a nonself organism (i.e., a microbial or animal aggressor). Using mitogen-activated protein kinase (MAPK) cascades, PAMP-triggered immunity integrates with the salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene (ET)-regulated defense pathways. This complex and dynamic network provides effective, inducible defenses to resist pathogens and herbivores. Consistent with the defense arms race of plants and pathogens, these intruders attempt to derail defenses by the strategic deployment of virulence factors or effectors. In this chapter, the similarities and distinctions of the adaptive defense strategies used by pathogens and insect herbivores to recover nutrients and promote their establishment and proliferation on host plants will be emphasized. The role of PAMPs and the distinctive chemical nature of herbivore elicitors and effectors in plant–herbivore interactions are discussed.

I. INTRODUCTION Plants encounter continuous challenges from bacteria, fungi, oomycetes, viruses, and herbivores that inhabit their environment. The relative resistance of a plant to a specific invader is determined by preformed, constitutive defenses, and the quality and diversity of the defense arsenal that is deployed upon attack (induced defenses). In most of these interactions, the plant is able to prevent microbe/arthropod colonization and, therefore, avoid extensive tissue damage. This is considered a nonhost interaction and the microbes/pests are considered non‐adapted organisms. Some microbes and arthropod herbivores have acquired genetic adaptations to circumvent or tolerate the constitutive and induced plant defenses. These adapted organisms are pathogens and pests and can establish residency causing disease/ damage on their host plants. To limit pathogen and pest damage, plants have evolved mechanisms to rapidly perceive a bioaggressor. Plants integrate the mechanical and chemical cues associated with insect and microbial pathogen attack and orchestrate

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defense responses that are tailored to their trespasser (De Vos et al., 2005; Glazebrook, 2005; Walling, 2000). The biochemical, physiological, and molecular changes that occur in response to an invader are dictated by the mechanisms used for nutrient retrieval, amount of physical damage that occurs, and chemical elicitors and effectors that are provided or generated in planta by the attacker. Despite the different lifestyles and mechanisms for attaining nutrients from plants, the innate immune responses activated by microbes and insects display a remarkable conservation. The transcriptional reprogramming and metabolic adjustments that occur are designed to preserve vegetative growth and reproduction, deter pathogen/pest colonization, and limit cellular and tissue damage. Innate immunity, also called basal resistance, was first recognized for its role in limiting the growth of virulent pathogens. It is now widely accepted that innate immunity is at the core of plant responses to both adapted and non‐adapted pathogens. Innate immunity is activated with different intensities and timing when plants encounter non‐adapted microbes, are attacked by virulent microbial pathogens or phytophagous arthropods, or display gene-for-gene resistance to a pathogen. The earliest events in innate immunity are triggered by the perception of highly conserved molecules called pathogen-associated molecular patterns (PAMPs) that signal injury and the presence of a nonself organism (i.e., a microbial or animal aggressor). PAMPtriggered immunity is the first line of defense and must integrate with the well-characterized defense pathways that are regulated by salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) (Glazebrook, 2005; Wang et al., 2008a). These major defense pathways are intricately connected by MAPkinase cascades and must also integrate abscisic acid (ABA)-, auxin-, gibberellic acid (GA)-, and reactive oxygen species (ROS)-mediated signaling (Lo´pez et al., 2008; Robert-Seilaniantz et al., 2007; Spoel and Dong, 2008). This complex and dynamic network, along with less-well-understood signal transduction pathways, provides effective, inducible defenses to resist pathogens and pests. To assure their success, microbial pathogens and herbivores seek to derail effective defenses by the strategic use of virulence factors or effectors. In this chapter, the similarities and distinctions of the adaptive defense strategies used by pathogens and insect herbivores are discussed. The mechanisms of nutrient acquisition employed by pathogens and pests determine chemical and physical defenses that must be surmounted to establish residency and proliferate and, therefore, have guided the co‐evolution of the defenses and counter-defenses in the plant–pathogen and plant–herbivore arms race (Berenbaum and Zangerl, 2008; Jones and Dangl, 2006). The oscillations in host and bioaggressor genotypes and their role in triggering or suppressing

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innate immunity and resistance to intruders will be discussed. The emerging data linking PAMP-triggered immunity and SA, JA, and ET signaling will be highlighted. Finally, the role of PAMPs, elicitors, effectors, and Resistance (R) gene proteins in the adaptive strategies displayed in plant–herbivore interactions will be emphasized and placed in the context of plant–pathogen interactions.

II. CO‐EVOLUTION OF DEFENSE STRATEGIES The mechanisms used by plant cells to perceive intruders, and pathogens to avoid defenses have co‐evolved and determine the outcome of microbe and insect interactions with their host plant (Berenbaum and Zangerl, 2008; Chisholm et al., 2006; Jones and Dangl, 2006). At the core of these relationships are the constitutive defenses (physical barriers and stored allelochemicals) (Conn, 1981) and the innate immunity system of plants, which can be rapidly activated or manipulated by an invader (Bittel and Robatzek, 2007; Nu¨rnberger et al., 2004). The chemical constituents of the preformed and induced defenses that are deployed determine the success of the attacker on its host. In addition, the timing and intensity of responses and the subcellular location of defensive chemicals, proteins, and physical barriers are critical components in these interactions, for pathogens and pests must contact defense compounds and/or barriers for defenses to be effectual. The armsrace model predicts that plants expressing effective defenses will create selective pressure for their pathogens/herbivores to evolve new genetic traits to circumvent plant defenses (Berenbaum and Zangerl, 2008; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004). Reciprocally, when plants encounter virulent pathogens/herbivores, there will be selection for valuable countermeasures to constrain the pathogen/herbivore. Evaluations of the transitions between pathogen lifestyles indicate that single-gene mutations can convert an endophyte to pathogen status, suggesting that the origins of plant innate immunity may have evolved from mutualistic or commensalistic interactions of microbes and plants (Kogel et al., 2006). Five phases in defense evolution have been proposed to explain these transitions. The ZigZag scheme depicts the requisite oscillations of genetic variation in the host and pathogen (Jones and Dangl, 2006) (Fig. 1). Phase one reflects the vast majority of plant–organism interactions, where the two-pronged approach to defense (constitutive defenses and innate immunity) is effective. Most microbes and insects are deterred by preformed defenses, since most plant–intruder interactions do not result in disease or herbivore damage. Plants recognize these non‐adapted microbes by their microbe-associated

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Effectortriggered immunity

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Fig. 1. The ZigZag model of innate immunity evolution. The ZigZag Model is redrawn and legend adapted from Jones and Dangl (2006). In Phase 1, plants are presented with an array of elicitors (small blue and green circles), including the highly conserved pathogen-associated molecular patterns (PAMPs). PAMPs (small green circles) are recognized by pattern-recognition receptors (PRRs; big green circle) and activate PAMP-triggered immunity. During this phase, a modest resistance is developed, which is sufficient to relegate most pathogens to nonhost status. In Phase 2, successful pathogens deliver virulence factors (effectors, triangles) to disrupt PAMP-triggered immunity and induce a phase of susceptibility. In Phase 3, a race-specific effector (RSE1, orange triangle) or effector activity is recognized by a Resistance gene protein (R1, orange square) to activate effectortriggered immunity. This rapid and amplified defense response (a gene-for-gene response) often passes the physiological threshold for the hypersensitive response (HR), causing cell death locally and microlesions systemically. In phase 4, new pathogen isolates are selected that are not suppressed by RSE1-triggered immunity. Some of these isolates may lose the effector and/or evolve new RSEs (RSE2, red triangle) to antagonize effector-triggered immunity. In Phase 5, new plant R gene alleles (red squares) are selected to recognize RSE2 and restore efficacious defenses (effector-triggered immunity). Further development of new effectors and R gene products can give rise to additional susceptibility/resistance specificities (gene-for-gene model).

molecular patterns (MAMPs) (also called PAMPs) that include highly conserved proteins, glycolipids, and polysaccharides (see Section IV). Microbial invaders are thereby perceived as ‘‘nonself’’ by plants (Bittel and Robatzek, 2007; Jones and Dangl, 2006; Nu¨rnberger et al., 2004). This recognition

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activates PAMP-triggered immunity (Fig. 1). In this manner, the attacker is rapidly and efficiently relegated to nonhost status. In the second phase of invader–plant interaction evolution, plant-pathogenic microbes and insects (adapted pathogens/pests) evolved mechanical or chemical strategies (called virulence factors or effectors) to breach physical barriers, detoxify chemical constituents, and antagonize defense gene expression (Fig. 1). These innovations allow pests and pathogens to evade and/or manipulate innate immunity, allowing successful colonization of their host plant (see Section VI). The classical term for this type of interaction is a compatible interaction, where defenses are activated but cannot adequately constrain the pathogen/pest. In these circumstances, pathogens cause disease and herbivores cause damage. To address these biotic challenges, plants entered phase three in the co‐evolutionary arms race (Fig. 1). Plant resistance (R) gene proteins were evolved; R proteins perceive specific microbial/pest effectors or effector end products. R proteins enabled plants to abolish or circumvent pathogen effector (virulence factor or race-specific effector, RSE) action and thereby activate the innate immune system (effector-triggered immunity) to curtail pathogen or insect attack and limit symptoms and damage. Traditionally, this rapid perception and defense activation is associated with localized cell death (a hypersensitive response, HR) and is often called an incompatible interaction. This game of co‐evolutionary tag continues in phase four, where pathogens restore virulence by losing or altering the RSE that was recognized and/or recruiting new effectors to action. In phase five, plants counter with the generation of new R gene alleles to perceive new RSEs. The cycling between phases four and five is continuous and reflects the ongoing arms race between plants and their bioagressors.

III. PORTALS OF ENTRY AND ACTIVATION OF DEFENSES Many pests and pathogens acquire their nutrients from foliage. Plants have adapted their leaf surface to make it a formidable physical barrier to most herbivores and pathogens. The hydrophobic cuticular waxes and trichomes that cover plant surfaces harbor a diverse array of defensive chemicals that directly antagonize development, reproduction, and/or survival of pathogens and pests (antibiosis effect), and/or deter pests from choosing the plant as a host for colonization (antixenosis effect) (Pollard et al., 2008; Wagner et al., 2004). The second physical barrier is the primary cell wall, which is composed of complex intertwined polysaccharides interdigitated with proteins, ions,

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and phenolic compounds (Carpita and McCann, 2000). The cell wall must be solubilized or punctured by attackers to allow ingress. The polysaccharide composition of the wall and species-specific components that reinforce the wall (lignin, tannins, silicon, and suberin) make tissue difficult to penetrate, tear, or chew. After attack, the cell wall is fortified by H2O2-mediated protein cross-linking and lignification, increased suberization, as well as the addition of defense-induced structural proteins (Hu¨ckelhoven, 2007). Some microbes tolerate the hostile phylloplane and have evolved an epiphytic lifestyle allowing them to complete all, or a portion of their life cycle on the leaf surface without causing damage to the host plant (Lindow and Brandl, 2003). In fact, most herbivores and microbes are effectively deterred by the preformed and induced structural and chemical barriers at the plant surface and are consigned to nonhost status. However, pathogenic microbes and phytophagous arthropods have adapted to this environment and developed chemical and mechanical mechanisms to breach these physical barriers, establish residence, and cause disease and damage to host plants. These invasion strategies determine pest/pathogen exposure to toxins and deterrents and influence the co‐evolution of plant–pathogen defense strategies. A. MICROBIAL INVASION STRATEGIES: FROM ACCESSING THE APOPLAST TO HAUSTORIA FORMATION

Except for some of the microbial pathogens that are vectored by insects, the apoplastic space between plant cells is the site of residence for pathogenic bacteria. These pathogens must gain access to this internal domain, recover nutrients, and tolerate the preformed and induced defenses in this realm. Most bacterial pathogens enter the leaf interior via wounds and natural openings, such as stomata and hydathodes (Melotto et al., 2008). Plants limit access to the leaf interior by closing stomata in response to PAMPs, oligosaccharides, and defense signals (nitric oxide (NO), SA, and ABA) (Melotto et al., 2008). To counter this defense, some pathogens have evolved effectors and toxins to promote stomatal opening (Melotto et al., 2006). Other pathogens produce toxins that stimulate stomatal closure (Bender et al., 1999), perhaps to provide a humid and protected environment that promotes pathogen proliferation. While providing a more nutrient-rich environment than the leaf surface (Lindow and Brandl, 2003; Solomon and Oliver, 2001), it is clear that the apoplast does not provide pathogens a ‘‘friendly’’ chemical environment, since many PAMP-triggered defenses include secreted proteins and chemicals with antimicrobial activities (Nu¨rnberger et al., 2004; Van Loon et al., 2006). Foliar pathogens have adapted to this nutrient-rich but chemically

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daunting environment by introducing effectors to promote virulence. Pathogenesis strategies include evading detection by the plant or, if detected, neutralizing or avoiding these antimicrobial activities (Section VI). Fungi and oomycetes that cause foliar diseases initiate host interactions by adherence to, and germination of their spores on the plant leaf surface (Hu¨ckelhoven, 2005; O’Connell and Panstruga, 2006; Tucker and Talbot, 2001). Hyphal paths may traverse over or under the cuticle, between plant cells, or within host cells with or without differentiation of appressoria (penetration structures). Thus, these pathogens will primarily encounter the chemical and physical deterrents of the cuticle and cell wall. Appressoria use a combination of turgor pressure and lytic enzymes to breach the plant cell wall (Kars and Van Kan, 2004; Pryce-Jones et al., 1999), releasing oligogalacturonides (OGAs) to activate PAMP-triggered immunity and provoke stomatal closure. Nutrient-recovery strategies for necrotrophs are straightforward: kill the cell recover nutrients. This life strategy is enabled by toxins and virulence factors that push plant cells beyond the HR threshold (Fig. 1) and cause massive cell death (Glazebrook, 2005; Kliebenstein and Rowe, 2008). Necrotrophic pathogens that induce tissue collapse will encounter the stored chemical defenses of trichomes and vacuoles, as well as apoplastic and cuticular phytochemicals (Morant et al., 2008; Wagner et al., 2004). In contrast, biotrophic fungi and oomycetes use a different strategy: extract nutrients from viable cells. To this end, biotrophic pathogens induce a complex reorganization of plant and pathogen cellular membranes to establish haustoria within plant cells, which allows nutrient recovery (Hu¨ckelhoven, 2005, 2007; O’Connell and Panstruga, 2006; Panstruga, 2003). Biotrophs primarily contact the secreted defenses that reside in the apoplast. In accordance, biotrophs deliver effectors into the apoplast and/or across the extrahaustorial host cell membrane to suppress or counter these plant defenses (Hogenhout et al., 2009). B. DEFENSE PATHWAY ACTIVATION BY MICROBES

Virulent pathogens present PAMPs, elicitors, and virulence factors that are perceived by plant cells. These cues are used to gauge the amplitude and nature of the defense pathways that are activated (Glazebrook, 2005). Necrotrophs primarily activate JA- and ET-regulated defense/wound response pathways. These pathways have been demonstrated to be important in the resistance to necrotrophic pathogens; plant mutants in which the JA- or ETmediated defense-signaling pathways are abolished or enhanced, are more susceptible or resistant, respectively, to necrotrophic pathogens. In contrast,

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biotrophs primarily activate defenses that are SA responsive (Glazebrook, 2005). Plant mutants with abolished or enhanced SA-mediated defense signaling are more susceptible or resistant, respectively, to biotrophic pathogens. However, if JA-dependent defenses are activated, they can also deter biotrophic pathogens. The SA- and JA/ET-defense response pathways crosstalk and can act antagonistically, additively, or even synergistically depending on the intensity and duration of the signals provided to the host plant (Mur et al., 2006). Furthermore, these pathways are interacting with other defense signals (ABA, auxin, GA, H2O2, and NO) known to enhance or antagonize SA- and/or JA-defense signaling (Lo´pez et al., 2008; RobertSeilaniantz et al., 2007; Spoel and Dong, 2008). Several pathogens and herbivores have leveraged this hormonal cross-talk to avoid effective host defenses (see Section VI). Recent microarray studies have evaluated the changes in gene expression after Pseudomonas syringae pv. maculicola ES4326 (Psm) infection. By determining the cadre of genes activated or suppressed in wild-type Arabidopsis thaliana and 11 Arabidopsis defense-signaling mutants, the interactions between the SA-, JA-, and/or ET-signaling pathways are being revealed (Wang et al., 2008a). While a majority of the Psm-responsive genes are controlled by these major defense-signaling pathways, a substantial number of Psm-regulated genes are regulated in a SA-, JA-, and ET-independent manner, suggesting that new defense pathways are to be revealed. The integration of the SA, JA, and ET pathways with PAMP-triggered immunity appears in Section V.B. C. HERBIVORE-FEEDING GUILDS: MECHANISMS TO VIOLATE THE INTEGRITY OF PLANT CELLS

Unlike bacterial, fungal, and oomycete pathogens, most arthropods that feed on plants have a mobile life stage that allows them to evaluate different plant hosts. Insects use tactile, visual, olfactory, and gustatory cues to assess the suitability of their potential host. If the plant is an unacceptable host for the herbivore’s progeny (i.e., poor plant nutritional quality, high levels of allelochemicals, or heavily infested with conspecifics or natural enemies), the herbivore can depart and find a more suitable leaf or host plant. This assessment is commonly accomplished by the herbivore sampling its diet or by sensing volatile cues emanating from the host plant (Paschold et al., 2007; Pieterse and Dicke, 2007; Powell et al., 2006; Turlings and Ton, 2006). The decision to ‘‘stay or go’’ is a critical one, because some insects have immobile immature stages and prolonged interactions with host plants (Chen, 2008; Walling, 2008).

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Herbivores use a wide variety of mechanisms to recover nutrients from plants. Feeding mode and life style strategies categorize insects into feeding guilds, which include chewers, sap suckers, cell-content feeders, gall formers, and borers (Hawkins and MacMahon, 1989). Depending on their guild, wound responses are variable, ranging from barely perceptible (whiteflies) to extensive (caterpillars). Given the different mechanisms of recovering nutrients, the preformed and induced defenses that these phytophages encounter are distinctive from each other and from microbial pathogens. These defenses drive the chemical co‐evolution of plants and their arthropod herbivores and have given rise to host‐plant specialists and generalists (Berenbaum and Zangerl, 2008) (see Section VI.B.2.e). 1. Tissue-damaging herbivores: Modes of feeding Chewing insects, such as caterpillars and beetles, use strong mandibles to excise leaf segments and mechanical forces to demolish plant cuticles and cell walls, and liberate cell contents. Folivores that chew encounter defenses located throughout the entire leaf (the cuticles, cell walls, and apoplast) and in all cell types (epidermis, mesophyll, and vascular bundle). Since cell and tissue integrity are demolished, chewing insects also encounter the stored defenses in vacuoles and trichomes (Morant et al., 2008; Wagner et al., 2004). Mandibular and labial salivary gland secretions are delivered into the feeding site on the host and into the insect’s oral cavity to lubricate the food and aid in solubilizing the leaf nutrients (Elzinga, 1987). In addition, saliva may provide antimicrobial defenses. It is estimated that the corn earworm, Helicoverpa zea, may secrete 15 g of salivary protein in 24 h (Peiffer and Felton, 2005). In addition, these insects often regurgitate contents of the mid-gut (called regurgitant) into the feeding site to predigest the food extraorally. Oral secretions (insect saliva plus regurgitant) are potent sources of elicitors, molecular patterns, and effectors that influence both direct and indirect defenses (see Sections IV.C and VI.B) (Felton and Tumlinson, 2008; Frost et al., 2008; Mitho¨fer and Boland, 2008; Turlings and Ton, 2006). Cell-content feeders, such as spider mites and leaf miners, and piercing/ sucking insects of the hemipteran suborders Heteroptera (mirid bugs, pyrrhocorids, and lygaeids) and Auchenorrhyncha (planthoppers and leafhoppers) also cause extensive damage. Spider mites and leaf miners use their mouthparts to rasp or lacerate cells (Needham et al., 1928; Zhang, 2003). While the destructive hemipterans insert their mouthparts (stylets) intracellularly to consume cell contents, they lacerate epidermal and/or mesophyll cells or penetrate many cell layers causing channels of dead cells (Backus, 1988; Backus et al., 2005; Miles, 1999). All of these phytophages secrete hydrolytic enzymes and/or toxins into plant cells to solubilize their contents;

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they then consume the cell ‘‘soup.’’ These herbivores contact apoplastic defenses and stored cell-type specific defenses. 2. Tissue-damaging herbivores: Defense signaling and discerning the role of injury The destructive feeding habits of phytophagous arthropods such as beetles, caterpillars, spider mites, fungal gnats, thrips, mirid bugs, planthoppers, and leafhoppers cause a profound reprogramming of plant gene expression and metabolism (Bodenhausen and Reymond, 2007; De Vos et al., 2005; Heidel and Baldwin, 2004; Major and Constabel, 2006; Mozoruk et al., 2006; Ralph et al., 2008; Reymond et al., 2000, 2004; Schmidt et al., 2005; Voelckel and Baldwin, 2004a; Vogel et al., 2007). Consonant with the breach of tissue and cellular integrity that accompanies feeding by these herbivores, it is not surprising to find that herbivory provokes changes that substantially overlap with the molecular, physiological, and biochemical responses to mechanical wounding. The degree of similarity in the responses to herbivory and mechanical injury is hard to assess for several reasons (Mitho¨fer et al., 2005). Herbivores introduce oral secretions containing elicitors and/or effectors, which have an important role in early events of plant perception of, and responses to, the herbivore (see Sections IV.B and IV.C). In addition, the frequency and patterning of damage-inducing events and the amount of total damage caused by different herbivores are distinct. Furthermore, herbivoreinduced damage is poorly mimicked by single wounding events inflicted by laboratory tools (hemostats, pliers, pattern wheels, razor blades, or pins), which are often used for comparisons to herbivore feeding. Studies with a mechanical chewer Mecworm, which punches holes in leaves and approximates the size and frequency of bites of Spodoptera littoralis (Egyptian cotton worm) larvae, have shown that repeated physical injuries can accurately mimic responses to caterpillar feeding. Mecworm causes a rise in JA levels and release of a volatile blend (monoterpenes, sesquiterpenes, homoterpenes, and C6-volatiles) that is qualitatively similar to the volatiles released by Sp. littoralis larvae (Arimura et al., 2008; Mitho¨fer et al., 2005). The employment of Mecworm on model plants, such as tomato (Solanum lycopersicum) or Arabidopsis, where wound-signaling pathways have been well-studied, could allow a more accurate assessment of the contributions of injury versus oral secretions to arthropod feeding. Studies, such as those in Nicotiana attenuata (wild tobacco) with Manduca sexta (tobacco hornworm) and poplar with Malacosoma disstria (forest tent caterpillar), have compared responses to single wounding events and to wounds plus insect regurgitant, providing estimates of the responses to oral secretions (Halitschke et al., 2001; Major and Constabel, 2006). However, recent results

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indicate that the quantities of regurgitant introduced into feeding sites may be variable and the ratio of regurgitant versus saliva introduced is not understood at this time (Peiffer and Felton, 2009); the ratios of these secretions could influence results obtained. Herbivory causes immediate physiological responses in plants (Mitho¨fer and Boland, 2008). Some of these responses are injury related, while others are clearly attributable to perception of elicitors in oral secretions. The disruption of the plant cell liberates cell-wall fragments and fatty acids, and allows mixing of enzymes and substrates from different cellular compartments, which alerts the plant of a possible biotic attack (Morant et al., 2008). For example, OGAs, which are released from cell wall pectin, are potent elicitors of PAMP-triggered immunity (see Section IV.B). The linolenic acid released from membranes can be used for JA synthesis and is immediately used by stored volatile biosynthesis enzymes, resulting in a rapid burst of fatty acid-derived C6-volatiles that influence insect behavior, are antimicrobial, and activate and prime defense gene expression (Howe and Jander, 2008; Turlings and Ton, 2006; Walling, 2000). In addition, enzymes and substrates stored in different subcellular compartments are intermingled to give rise to toxic metabolites that deter herbivores and have antimicrobial activity (Morant et al., 2008). While the magnitude of changes in gene expression profiles attributed to wounding versus insect elicitors is not yet resolved, it is clear from microarray studies that JA-regulated gene expression changes predominate after tissue damage resulting from caterpillar feeding in Arabidopsis, poplar, N. attenuata, Solanum nigrum (black nightshade), and Boechera divaricarpa (purple rock cress) (Bodenhausen and Reymond, 2007; De Vos et al., 2005; Heidel and Baldwin, 2004; Major and Constabel, 2006; Ralph et al., 2008; Reymond et al., 2000, 2004; Schmidt et al., 2005; Voelckel and Baldwin, 2004a; Vogel et al., 2007). Jasmonates, which include JA and its conjugate JA-isoleucine, regulate a multidimensional defense network to herbivores by increasing secondary metabolites, enhancing emission of volatile blends to attract natural enemies and prime defense, stimulating wound signaling and perception, and increasing the levels of proteins that inhibit or deter insect feeding or growth (Frost et al., 2008; Howe and Jander, 2008). JA biosynthesis and mechanisms of perception have been recently reviewed (Browse and Howe, 2008; Wasternack, 2007). Analysis of herbivore performance on mutants with impaired biosynthesis or perception of JA, shows that jasmonates have a prominent role in controlling the defenses that deter herbivory and influence host/nonhost status in the lab and field (Kessler et al., 2004; Li et al., 2004; McConn et al., 1997; Paschold et al., 2007).

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3. Phloem-feeding hemipterans Phloem-feeding hemipterans (i.e., whiteflies, aphids, psyllids) have more refined feeding strategies. These insects use highly specialized mouthparts called stylets that take an intercellular path to the phloem. The primary force for stylet penetration and movement is mechanical, but saliva constituents may also enable this process (Miles, 1999) (see Section IV.C.5.b). Stylets have an alimentary canal to ingest large quantities of fluids from phloem and a canal to deliver watery and gelling salivas into the plant (Tjallingii, 2006). The watery saliva is egested at the leaf surface, along the stylet path, and upon cell punctures. This secreted saliva is resorbed via the food canal to allow ‘‘tasting’’ of the cellular environment prior to establishing a feeding site (Powell et al., 2006; Walling, 2008). Watery saliva has important roles in maintaining feeding site function and is a source of elicitors and effectors that activate or manipulate host defenses (see Sections IV.C.5 and VI.B.2). The gelling saliva forms a sheath around the stylets, insulates the stylets from apoplastic defenses, and serves as a track along which stylets can travel (Backus, 1988; Miles, 1999; Walling, 2008). The degree of tissue damage imparted by phloem-feeding insects varies and this influences the gene expression programs that have been elucidated using sentinel genes, gene chips, customized cDNA-microarrays, or sequencing of clones from high-throughput comparative transcripts analyses (Broekgaarden et al., 2008; Couldridge et al., 2007; Divol et al., 2005; Kaloshian and Walling, 2005; Kempema et al., 2007; Kus´nierczyk et al., 2007, 2008; Li et al., 2008; Moran et al., 2002; Voelckel and Baldwin, 2004b; Zhu-Salzman et al., 2004). As with tissue-damaging herbivores, molecular responses to hemipteran feeding are complex. Changes in levels of RNAs encoding proteins involved with wound/defense responses, ROS generation or scavenging, synthesis of secondary metabolites, and well as primary metabolism are often observed. Changes in a variety of transcription factors, kinases and phosphatases, calcium signaling, and leucine-rich-repeat receptor-like kinases (LRR-RLKs) have also been noted. Most noteworthy are the distinctions in defense/wound-response gene expression patterns when different hemiptera are examined. For example, these responses range from extensive overlap with wounding (tomato and the potato aphid (Macrosiphum euphorbiae)) to evasion of wound responses (Arabidopsis and the silverleaf whitefly (Bemisia tabaci biotype B)) (Kempema et al., 2007; Martinez de Ilarduya et al., 2003). Aphids puncture virtually all mesophyll cells on their path to a major vein of the phloem, but the integrity of the tonoplast (central vacuole) is not violated in these probings (Powell et al., 2006; Tjallingii and Hogen Esch, 1993). These punctures are accompanied by watery saliva egestion into

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mesophyll cells, provoking defenses and often substantial amounts of cellular damage (Edwards and Singh, 2006; Kaloshian and Walling, 2005). In accordance, tomato responses to the potato aphid and the green peach aphid (Myzus persicae) are biphasic with JA-regulated wound-response genes being expressed early and genes encoding pathogenesis-related (PR) proteins expressed at later times (Martinez de Ilarduya et al., 2003). Co‐expression of JA- and SA-responsive genes was also noted in My. persicae and Brevicoryne brassicae (cabbage aphid) infestations of Arabidopsis (Kus´nierczyk et al., 2007, 2008; Moran and Thompson, 2001). In contrast, in the greenbug (Shizaphis graminum)-sorghum and silverleaf whitefly-Arabidopsis interactions, SA-responsive genes are primarily expressed (Kempema et al., 2007; Zhu-Salzman et al., 2004). The silverleaf whitefly, which causes barely perceptible damage, is perhaps the most delicate feeder studied at the molecular level to date (see Section VI.B.2.b).

IV. PERCEIVING PATHOGEN AND PEST VISITATIONS: THE ROLE OF MICROBIAL AND HERBIVORE ELICITORS AND MOLECULAR PATTERNS A. PAMPs, PATTERN-RECOGNITION RECEPTORS, AND BAK1

While microbes and herbivores have distinctive mechanisms of establishing residency and recovering nutrients, these attackers all present chemical and protein elicitors that are recognized by the plant. Elicitors may be general or specific to a taxon, species, or biotype. PAMPs/MAMPs are highly conserved proteins, glycolipids and polysaccharides of microbes that contain motifs (or epitopes) that are perceived to activate innate immunity in plants and animals (Altenbach and Robatzek, 2007; Bittel and Robatzek, 2007; Nu¨rnberger et al., 2004). PAMPs are found in pathogens and nonpathogenic microbes and are essential for microbial life and, therefore, are difficult to mutate without compromising microorganism vitality. For this reason, PAMPs serve as unwavering beacons signaling a bacterial, fungal, or oomycete invader. By definition, PAMPs are not present in plants or animals. Therefore, PAMPs are red-flags signaling the presence of ‘‘nonself’’ molecules and are used to activate PAMP-triggered immunity. PAMPs activate cellular MAPK-signaling cascades, cause rapid influx of Hþ and Ca2þ, stimulate production of ROS and emission of ET, cause rapid changes in defense-related gene expression profiles, and induce resistance to microbial pathogens.

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Bacterial PAMPs include flagellin, elongation factor-Tu, cold-shock protein, and lipopolysaccharide (LPS) (Nu¨rnberger and Kemmerling, 2009). Fungi and oomycetes are also rich sources of PAMPs, including cellwall- or plasma-membrane-derived molecules such as chitin and chitosan (Fig. 2), -1,3-glucan, heptaglucoside, xylanase, and ergosterol, as well as necrosis-inducing peptides (Nu¨rnberger and Kemmerling, 2009). Some PAMPs (i.e., flagellin, LPS, -glucans, and chitin) are perceived by a broad range of plants and animals, while others have more limited ranges. For example, flagellin perception has been reported in seven diverse plant species and in animals (Nu¨rnberger and Kemmerling, 2009). In contrast, perception of EF-Tu is restricted to members of the Brassicaceae (Kunze et al., 2004), and perception of cold-shock protein is limited to members of the Solanaceae (Felix and Boller, 2003). PAMPs are recognized by pattern-recognition receptors, which are members of the nucleotide-binding, leucine-rich repeat (NB-LRR) family of proteins (Altenbach and Robatzek, 2007). Most pattern-recognition receptors are integral membrane proteins and are LRR-RLKs. Other pattern-recognition receptors are receptor-like proteins (RLPs), which are LRRRLKs that lack the cytosolic kinase domain (Altenbach and Robatzek, 2007). Recently, the Arabidopsis BAK1 (BRI1-associated kinase 1), which is a co‐receptor of the brassinosteroid receptor (BRI1), was identified to be also a co‐receptor for PAMP perception, with a role in pathogen-triggered cell death (Chinchilla et al., 2007; Heese et al., 2007; Kemmerling et al., 2007). The events in PAMP signaling are best elucidated for the archetypal PAMP flagellin. In Arabidopsis, the ligand flg22 binds to the pattern-recognition receptor FLS2 (Flagellin-sensing 2) (Go´mez-Go´mez and Boller, 2000). After binding flg22, FLS2 and BAK1 rapidly form a heterodimer and BAK1 phosphorylates FLS2 (Chinchilla et al., 2007; Robatzek et al., 2006; Wang et al., 2008b). This initiates endocytosis of the flg22-FLS2 complex (Robatzek et al., 2006). After endocytosis, FLS2 is degraded via the proteasome, presumably to reduce PAMP-triggered signaling pathways. As reflected by its status as a key regulator of PAMP-triggered immunity and negative regulator of pathogen-induced cell death, BAK1 is a target of the Ps. syringae pv. tomato virulence factors AvrPto and AvrPtoB (Shan et al., 2008). B. ELICITORS OF PLANT ORIGIN

The OGAs derived from plant cell-wall pectin are potent injury signals and strong activators of PAMP-triggered immunity (Fig. 2). OGAs cause rapid ion fluxes, production of ROS, activation of MAPKs, and massive changes in gene expression profiles that are similar to, and yet distinct from, responses

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Fig. 2. Structure of chitin, chitosan, and oligogalacturonides (OGAs). Chitin is found in fungi, nematodes and insects, and consists of repeating units of acetylD-glucosamine. Bioactive chitin oligomers are typically eight monomers in length. Chitosan is found in some fungi. It is a (largely) deacetylated chitin with a repeating unit of D-glucosamine. OGAs are fragments from plant cell‐wall pectin that are released after injury or by pectinolytic enzymes, such as endopolygalacturonases. The OGA repeating unit is galacturonic acid.

to well-characterized PAMPs, such as flg22 and chitin (Denoux et al., 2008; Ferrari et al., 2007; Moscatiello et al., 2006) (see Section V.B). In Arabidopsis, both flg22 and OGAs induce a resistance to the necrotrophic fungus Botrytis

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cinerea that is SA-, JA- and ET-independent and PHYTOALEXIN DEFICIENT 3 (PAD3)-dependent (Ferrari et al., 2007). The Arabidopsis wall-associated kinase WAK1 binds OGA dimers that assume an ‘‘egg-box’’ conformation (Cabrera et al., 2008). WAK1 appears to activate SA-regulated defenses but it is not clear if WAK1 or another receptor mediates OGAmediated immunity and the PAD3-mediated resistance to Bo. cinerea. In tomato, OGAs have a well-established role in stimulating the JA-regulated wound response, which promotes wound healing and production of defense proteins and compounds that deter herbivory (Ryan, 2000; Walling, 2000). OGAs are a general ‘‘injury’’ signal released from wounded or necrotic cells. Cell damage can be caused by adverse environmental conditions, or feeding by tissue-damaging herbivores. OGAs are also released in response to pathogen- and herbivore-encoded pectinases. Endopolygalacturonases (PGs) are important virulence factors for bacterial soft rot pathogens and fungal necrotrophs (Abbott and Boraston, 2008; Choquer et al., 2007), and oomycetes use PGs to loosen plant cell walls to allow tissue penetration (Pryce-Jones et al., 1999). To limit damage to cell walls, plants express PG-inhibiting proteins (PGIPs) to inactivate pathogen-produced PGs (D’Ovidio et al., 2004). The importance of PGIP in plant defense is heralded by the fact that an Arabidopsis pgip mutant is more susceptible to the necrotroph Bo. cinerea (Ferrari et al., 2006). Pectin-hydrolyzing enzymes are also found in the saliva of hemipterans (aphids, flea hoppers, and mirid bugs) (Agusti and Cohen, 2000; Cohen and Wheeler, 1998; Frati et al., 2006; Miles, 1999). Like microbial pathogens, aphids are strongly influenced by the polysaccharide composition of the cell wall. For example, in sorghum, cultivar resistance to the greenbug Sh. graminum and greenbug probing behavior are correlated with the degree of pectin methylation and the level of pectinases in the saliva of greenbug biotypes (Dreyer and Campbell, 1984). In addition, heteroptera secrete copious amounts of watery saliva rich in oxidoreductases and hydrolytic enzymes, including PGs and -amylase (a starch-degrading enzyme) into cells to dissolve cellular contents and matrices. The PGs of heteroptera are important virulence factors, are correlated with cellular damage, and are inhibited by plant PGIPs (Cantu et al., 2008; D’Ovidio et al., 2004). C. HERBIVORE ELICITORS

Over the past decade an understanding of the herbivore elicitors that are introduced by saliva and oral secretions has exploded (Felton and Tumlinson, 2008; Mitho¨fer and Boland, 2008). There may be several sources of insect elicitors (Felton and Tumlinson, 2008; Walling, 2000). Elicitors may

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be insect-encoded and delivered directly to the feeding site. In some instances, elicitors are plant-derived molecules that are consumed and modified by insect enzymes prior to delivery into the feeding site. The microbes residing on insect surfaces are also a potential sources of elicitors. Finally, many insects harbor endosymbiotic microbes (Baumann, 2005) and while the role of endosymbionts in insect nutrition is well established, the potential role of endosymbionts as a source of elicitors remains to be explored. While the presence of herbivore elicitors is indisputable, at the present time, it is unclear if any of these elicitors should be called a herbivoreassociated molecular pattern (HAMP). Using PAMP criteria, HAMPs should (1) be proteins, lipids or polymers that are highly conserved in structure and indispensible for herbivore viability; (2) be introduced into the plant during herbivore infestation and elicit rapid defense responses, including MAPK activation and reprogramming of gene expression; (3) be recognized as nonself and therefore not have functional homologues in plants; and (4) bind to a cognate receptor. A number of bona fide herbivore elicitors that stimulate plant defenses have been purified. However, with one exception, their requirement for insect vitality has not been established. Receptors have yet to be identified for any insect elicitor. Therefore, it might be argued that a ‘‘true’’ HAMP has yet to be discovered. The best-characterized insect elicitors stimulate volatile synthesis and emission. Volatile blends serve as an indirect defense by attracting natural enemies to herbivore-infested plants, have important roles in defense gene activation and priming, and are antimicrobial (Frost et al., 2008; Turlings and Ton, 2006). It should be noted that the role of elicitors in plant– herbivore interactions has yet to be tested genetically, since the genes that control the biosynthesis of herbivore elicitors have not been cloned and transformation methods for herbivores are only emerging. It is clear that with the development of RNA-silencing technologies as an effective mechanism to abolish or downregulate insect gene expression, the importance of elicitors can be tested in the future (Mutti et al., 2008). 1. Lipid signals without receptors: Volicitin and caeliferin Volicitin is the best candidate for a HAMP (Fig. 3). Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] is a fatty acid conjugate (FAC) that was first purified from regurgitant of the beet armyworm Spodoptera exigua (Alborn et al., 1997). When applied to plant wounds, purified volicitin induces the synthesis of a volatile blend similar to that induced by Sp. exigua feeding or regurgitant (Turlings et al., 1993). Volicitin is synthesized by the concerted efforts of the plant and insect (Pare´ et al., 1998). Volicitin’s backbone is linolenic acid, which must be acquired from the insect’s diet (Canavoso et al.,

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Fig. 3. Herbivore elicitors of volatile emission. (A) Structures of three fatty-acid conjugates (FACs). Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-linolenoyl-Lglutamine, and N-linolenoyl-L-glutamate. Other FACs have different moieties at C17 (Mitho¨fer and Boland, 2008). (B) Structures for caeliferins A and B. Caeliferin A16:1 ¼ (E)-2,16-disulfooxy-6-hexadecenoic acid; caeliferin A16:0 ¼ 2,16-disulfooxyhexadecanoic acid; caeliferin B16:1 ¼ N-[(E)-15-sulfooxy,15-carboxy-10-pentadecenoyl] glycine; and caeliferin B16:0 ¼ N-(15-sulfooxy,15-carboxy pentadecanoyl) glycine. C. Inceptin peptides. Inceptin peptides are derived from a chloroplast ATPase

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2001). Within the insect mid-gut, linolenic acid is conjugated to glutamine and a hydroxyl moiety to form volicitin (Lait et al., 2003; Yoshinaga et al., 2005). Since volicitin’s discovery, a series of volicitin-related FACs were discovered in lepidopteran larvae (Halitschke et al., 2001; Mori et al., 2003; Pohnert et al., 1999). Linolenic and linoleic acid are the core of these FACs, although oleic acid and other fatty acids are occasionally detected. All FACs are conjugated to glutamine or glutamic acid. For example, in the tobacco hornworm, N-linolenoyl-L-glutamic acid and N-linolenoyl-L-glutamine are the primary FACs in oral secretions and volicitin is not detected (Fig. 3) (Halitschke et al., 2001). The responses of maize and N. attenuata to FACs have been extensively studied (Kessler and Baldwin, 2002; Turlings and Ton, 2006). Like PAMPs, FACs activate the SA-induced protein kinase, SIPK, and the Wound-induced protein kinase, WIPK, in N. attenuata, which regulate ET, JA, and SA accumulation (Meldau et al., 2009; Wu et al., 2007). FACs cause JA levels to rise transiently in N. attenuata, which in turn causes changes in defense gene expression and accumulation of proteins and chemicals that antagonize insect feeding, reduce the quality of the diet (i.e., proteinase inhibitors, germins, nicotine, etc.), and induce synthesis of volatiles that have roles in attracting predators or parasitoids to insect-infested plants (Halitschke et al., 2001). FACs were also recently discovered in crickets (Teleogryllus taiwanemma and Tel. emma) (Orthoptera), Drosophila melanogaster (Diptera) larvae, and katydids (Alborn et al., 2007; Yoshinaga et al., 2007). However, while broadly distributed, volicitin and its related FACs are not ubiquitous in the insect kingdom (Alborn et al., 2007; Yoshinaga et al., 2007). The surprising conservation of FAC structures in three insect orders suggests that FACs provide an important physiological role in insects. FACs may serve as biosurfactants or oil emulsifiers within the insect midgut to enable nutrient recovery (Halitschke et al., 2001). Recently, FACs were demonstrated to be a storage form of nitrogen (Gln) in Spodoptera litura (tobacco cutworm) (Yoshinaga et al., 2008). FACs are conjugated to Gln in the mid-gut and FAC-Gln is hydrolyzed in the gut lumen to provide fatty acids and Gln for fat bodies and hemolymph, respectively. The central role of FACs in insect nutrition and nitrogen metabolism suggests that volicitin and related FACs meet an important HAMP criterion. Several

subunit (cAPTC). Four inceptin peptides were isolated from Spodoptera frugiperda oral secretions after feeding on Vigna unguiculata. Vu-In has a Cys disulfide bridge, which does not appear to be essential for activity; the other inceptin-like peptides are linear. Vu-In, Vu-GEIn and Vu-EIn elicit ethylene production and volatile release. Vu-INA, which lacks the C-terminal Ala residue, is biologically inactive.

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other criteria also implicate volicitin and other FACs as HAMPs. A highaffinity voliticin-binding protein was identified in maize (Truitt et al., 2004); however, the volicitin receptor has eluded further biochemical characterization and cloning. The major elicitor of volatiles from the American bird grasshopper (Schistocerca americana) oral secretions is not volicitin, but caeliferin (Alborn et al., 2007). Caeliferins are a new class of lipid-based elicitors, which are restricted to the suborder Caelifera. The core structure of caeliferins consists of C16 to C19 fatty-acid chains (saturated or unsaturated), with the C16 caeliferins being the most abundant (Fig. 3). Caeliferin A (16:1 and 16:0) contains two sulfate moieties, on the - and !-carbons, while caeliferin B (16:1 and 16:0) contains one sulfate (-carbon) and a terminal glycine (!-carbon). The significance of the major and minor forms of caeliferin, modes of caeliferin synthesis, presence of caeliferin binding protein(s), and changes in defense gene expression in response to caeliferins have yet to be investigated. While it is not clear if caeliferins are perceived by plants other than maize (Alborn et al., 2007), given the broad host range of Sc. americana, it is possible that caeliferins are widely recognized in plants and a contender for the title of HAMP. 2. Peptide elicitors of volatile emissions: Inceptin and -glucosidase -Glucosidase was the first elicitor of volatile release that was identified (Hopke et al., 1994; Mattiacci et al., 1995). Treatment of Lima bean and cabbage with -glucosidase from almond stimulates the release of volatile blends that are similar to those emitted after Tetranychus urticae (twospotted spider mite) and Pieris brassicae (large cabbage white) caterpillar infestations, respectively. The detection of a -glucosidase in Pi. brassicae oral secretions suggests that the -glucosidase is an elicitor. The substrate of the insect -glucosidase has not been identified but could be a glucoseconjugated metabolite of plant or insect mid-gut origin. Inceptin is an 11-residue peptide isolated from oral secretions of Spodoptera frugiperda (fall armyworm) raised on cowpea (Vigna unguiculata) (Schmelz et al., 2006). Inceptin is a potent inducer of defense signals (i.e., ET, JA, and SA) and volatile release from volicitin‐nonresponding plants such as cotton, Lima bean, and cowpea. Inceptin (Vu-In) has a disulfide bridge between residues Cys2 and Cys8, while other bioactive inceptin-like peptides (Vu-GEIn, VU-EIn) are linear (Fig. 3). Residues critical for inceptin activity have been identified and the disulfide bond is not essential for inceptin perception. An inceptin binding-protein or receptor has not been identified at this time. However, if acting similarly to other bioactive peptides of plant or microbial (i.e., flg22) origin

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(Farrokhi et al., 2008; Nu¨rnberger et al., 2004), inceptin may bind a receptor in the NB-LRR family. Inceptin is a peptide that is derived from a highly conserved, redox-sensing regulatory domain of the chloroplast ATP synthase subunit (cATPC) of cowpea. Sp. frugiperda regurgitant contains proteases that process a GSTcATPC fusion protein to inceptin and inceptin-related peptides. Since inceptin is derived from a plant protein, it is technically not a HAMP. For this reason, inceptin is more analogous to OGAs. It is not clear if inceptin peptides are generated at the site of damage by proteases in insect oral secretions or if cATPC, like linolenic acid, is modified within the insect mid-gut and subsequently inceptin peptides are delivered into the feeding site for perception. 3. Oviduct secretions: Elicitors of defense gene expression and volatile emissions When insect eggs are laid on plant surfaces, small incisions are often (but not always) made and oviduct and accessory gland secretions accompany egg deposition. Relatively little is known about the chemical composition of these secretions (Hilker and Meiners, 2006). However, there is ample evidence that egg deposition and oviduct secretions stimulate volatile release, decrease ET production, and induce genes important in terpenoid volatile biosynthesis (Ko¨pke et al., 2008; Schro¨der et al., 2007). Pieris rapae egg deposition on Arabidopsis also causes reprogramming of plant RNA profiles (Little et al., 2007). The patterns of gene expression changes in response to egg deposition and larval feeding were surprisingly dissimilar, suggesting that plant responses to different insect life stages can be distinctive. In contrast, over 50% of the egg-induced and 69% of the egg-repressed genes were also differentially regulated in the incompatible interaction between Arabidopsis RPM1 and Ps. syringae (avrRPM1) or in the Arabidopsis accelerated-cell-death mutant acd2–2. These data suggest that egg deposition provokes a general stress response. Analysis of the egg-induced changes in gene expression in the defense mutants coronatine insensitive 1-1 (coi1-1) and SA induction deficient 2-1 (sid2-1) indicate that the response to egg deposition is largely independent of the JA- and SA-signaling pathways. The chemical signals important in mediating egg-induced signaling in Arabidopsis are not yet known; the involvement of PAMP-triggered immunity will be interesting to determine. A lipid elicitor is important in the perception of eggs and the elicitation of direct defenses in pea. When pea weevil (Bruchus pisorum) and cowpea weevil (Callosobruchus maculatus) eggs are deposited on seed pods from of the pea genotype Neoplastic pod (Np), neoplasm-like growths are produced beneath

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the weevil egg. The neoplasm deters larval penetration of the pod, prominently displays the egg and emerging larva to natural enemies, and may promote egg desiccation. The chemicals recognized by Np cells are called bruchins (Doss et al., 2000). Bruchins are active in the fmol range and have C22 and C24 backbones. They are ,!-diols and are esterified at one or both oxygens with 3-hydroxypropanoic acid (Fig. 4). While the bruchin receptor(s) and signaling pathways have not been identified, bruchin B has been shown to alter pea defense gene expression and phytoalexin accumulation (Cooper et al., 2005). Fatouros et al. (2008) have recently shown that the chemical cues associated with egg deposition may be more complex than originally imagined. When Pi. brassicae females oviposit on cabbage (Brassica oleracea) plants, after three days a volatile blend is emitted that arrests the egg parasitoid wasp Trichogramma brassicae (Fatouros et al., 2005). The elicitor of volatile synthesis and parasitoid arrest was present in accessory reproductive gland secretions from mated female butterflies but, surprisingly, not from virgin

A

Bruchin A

O O

HO

Bruchin B

O

O O

O

HO

OH

Bruchin C

O

O

O

HO

O

OH

O

OH

Bruchin D

O HO

OH

O

O

B

N

Benzyl cyanide

Fig. 4. Oviposition elicitors. (A) Structures of bruchins. Bruchin A ¼ (Z)-9docosene-1,22-diol, 1-(3-hydroxypropanoate)ester, bruchin B ¼ (Z)-9-docosene-1, 22-diol bis-(3-hydroxypropanoate) ester, bruchin C ¼ (Z)-9-tetracosene-1,24-diol bis-(3-hydroxypropanoate) ester, and bruchin D ¼ (Z)-7,15-tetracosadiene-1,24-diol bis-(3-hydroxypropanoate) ester. (B) Benzyl cyanide or 2-phenylacetonitrile.

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females. The elicitor is the anti-aphrodisiac benzyl cyanide (Fig. 4), which was transmitted from males to females in the spermatophore during mating. Using Arabidopsis 70-mer Gene-chip arrays, changes in Brassica RNA profiles in response to egg deposition and benzyl cyanide were evaluated. Both treatments provoke a relatively small number of changes in Brassica gene expression and these responses were largely non‐overlapping (Fatouros et al., 2008). It is likely that the Arabidopsis array underestimates the magnitude of changes by eggs and benzyl cyanide in Brassica. 4. Chitin Chitin is a biopolymer of N-acetyl--D-glucosamine that is found primarily in fungal cell walls, nematode egg shells and arthropod exoskeletons (Fig. 2) (Merzendorfer and Zimoch, 2003). Plants have pattern-recognition receptors that detect chitin fragments (N-acetylchitooligosaccharides) and chitosan (N-chitooligosaccharides) (Kaku et al., 2006; Miya et al., 2007) (Fig. 2). Chitin oligosaccharides are PAMPs released during fungal infections. However, it is not clear if chitin oligosaccharides are important in plant–insect interactions. Chitin is an essential component of insect cuticles associated with the epidermis, trachea, and the peritrophic matrices (a permeability barrier) of the gut epithelium (Merzendorfer and Zimoch, 2003). Chitin synthesis and degradation are developmentally controlled in arthropods. It is possible that chitin oligomers from the peritrophic matrices are present in the oral secretions that are delivered to the site of caterpillar feeding and serve as PAMPs. To support this idea, chitosan has been detected within Solanum dulcamara (bittersweet nightshade) epidermal cells after gall mite (Eriophyes cladophthirus) punctures (Bronner et al., 1989). Three additional sources of chitin oligomers need to be considered. First, plants express chitinases in response to pathogen and insect attack (Van Loon et al., 2006). Therefore, it is possible that plant chitinases can use insect chitin as a substrate and thereby elicit PAMP-triggered immunity. Second, since plant and insect surfaces have variable but complex microbial populations, it is possible that resident fungi may inadvertently enter the herbivore feeding site and contact insect- or plant-encoded chitinases to release bioactive chitin oligomers. Finally, some of the microbes that reside on plant surfaces may secrete lytic enzymes, including chitinases, which may release chitin oligomers upon contact with insects. 5. Salivary proteins as elicitors a. Lysozyme. As tissue-damaging caterpillars feed on plant foliage, mandibular and labial saliva and mid-gut regurgitant are delivered to the feeding site. At present, there is a limited knowledge on the composition of

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mandibular and labial gland salivas (Eichenseer et al., 1999). Helico. zea saliva and regurgitant are complex and chemically distinct. One of the most abundant proteins in the labial saliva, which is absent in regurgitant, is lysozyme (Liu et al., 2004). Low levels of lysozyme mRNAs have also been detected in labial salivary glands in Man. sexta (Mulnix and Dunn, 1994) and in the guts of many insect species (Fujita, 2004). Lysozyme hydrolyzes the peptidoglycan of bacterial cell walls. Lysozyme is thought to have a role in an insect’s defense against its pathogens (Liu et al., 2004). However, it is possible that the salivary lysozyme would contact and hydrolyze the cell walls of the epiphytic bacterial microflora and pathogens that reside on plant foliage and release peptidoglycans which act as PAMPs (Gust et al., 2007). It is not clear if the bacterial community on the leaf surface or associated with insects is large enough to make peptidoglycan perception a component of plant responses to herbivore feeding. b. Hemipteran saliva. Watery and gelling salivas have been characterized enzymatically in a small number of hemipterans and were extensively reviewed by Miles (1999). Some hemipteran insects produce salivary sheaths that surround their flexible stylets. This rapidly gelling saliva is composed primarily of protein, phospholipids and conjugated carbohydrates (Miles, 1999). It is not known if unpolymerized sheath materials are elicitors. The watery saliva of phloem feeders is better characterized. The composition of this digestive saliva is dependent on the insect species and the plant host (Habibi et al., 2001; Miles, 1999). A wide array of activities have been detected, including Ca2þ-binding proteins, pectinesterases, PGs, lipases, peroxidases, phenoloxidases, amylases, cellulases, sucrases, proteases, and alkaline and acid phosphatases (Funk, 2001; Miles, 1999; Will et al., 2007). While the role of many of the salivary proteins as elicitors or effectors is unknown, several proteins have implicated roles in defense. Ca2þ-binding proteins and oxidases are potential effectors and are discussed in Section VI. B.2. Pectin-degrading enzymes may facilitate cell wall dissolution, stylet penetration, and release OGAs (Section IV.B). While proteases may aid hemipteran digestion, salivary proteases could release peptides that elicit defense signaling, as noted for inceptin (Section IV.C.2). It is anticipated that biotype/race-specific salivary factors will be identified. Some of these factors will be recognized by cognate R gene products important in gene-for-gene defense responses (see Section VII.B). Other factors may be toxins or effectors that enhance virulence on a host plant, and evade R protein detection. Some salivary factors will explain the ability of closely related whitefly biotypes to induce different sets of defense genes in squash, or cause biotype-specific developmental disorders in a variety of horticultural

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and crop plants (Van de Ven et al., 2000; Walling, 2008). Species-specific salivary factors are also likely explanations of the ability of some aphid species to cause distinctive infestation symptoms in alfalfa (Medicago sativa) and Medicago truncatula (barrel medic) (Edwards and Singh, 2006). Isolation and biochemical characterization of salivary protein extracts are already proving to be effective strategies to identify symptom-inducing elicitors. For example, salivary extracts from the Russian wheat aphid (Diuraphis noxia) mimic the symptoms of infested susceptible wheat (Triticum aestivum) plants (Lapitan et al., 2007).

V. INTEGRATING SIGNALS AND ACTIVATING DEFENSES Microbes and herbivores present a diverse array of protein and chemical elicitors and virulence factors that are perceived and provoke changes in plant cell integrity, physiology, metabolism, and gene expression. While many of the elicitors from herbivore saliva and oral secretions have yet to be identified, those discovered indicate that many novel chemical motifs without counterparts in microbial pathogenesis have evolved. Understanding the circuitry that connects the microbial and herbivore elicitors and their receptors to regulatory hubs for integration and orchestration of the SA, JA, ET, and novel defense pathways will continue to be an important focus for future research. There are striking similarities in the early events of microbial PAMPtriggered immunity and plant cell responses to herbivore-derived elicitors (Mitho¨fer and Boland, 2008; Zipfel et al., 2004). These events include rapid ion fluxes across plasma membranes, cytosolic/nuclear calcium fluxes, production of ROS, generation of ET, MAP kinase cascade activation, callose deposition, and massive reprogramming of gene expression. The expectation is that the magnitude and timing of these responses will be dependent on the quantity, potency, and persistence of the microbial or herbivore elicitor(s) and the nature of the host plant (Denoux et al., 2008). Different microbial elicitors are known to provoke responses that are qualitatively similar but quantitatively distinct (Lecourieux et al., 2005). Two PAMPs, chitin (a sentinel of nonself) and OGAs (a beacon of injury), and possibly LPS (a sentinel of bacterial pathogens), are presented when either microbes or herbivores interact with plants, suggesting that PAMP-triggered immunity may be at the core of the earliest responses to both herbivores and microbes. Other elicitors are likely to be discovered and join this group of nonself and injury signals shared by animal and microbial pathogens.

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Current data suggest that at the earliest times, PAMPs activate similar downstream events. They utilize MAP kinase cascades that communicate with each other (Fig. 5) (see Section V.A) and result in changes in RNA profiles that have substantial overlap (Denoux et al., 2008). These data suggest that there is considerable redundancy in responses to PAMPs, perhaps ensuring activation of these essential immunity functions. However, there are unique features to each PAMP response. It appears that these idiosyncratic attributes are the ‘‘specificity’’ in PAMP responses but they may also reflect a PAMP’s stability or potency as a trigger of innate immunity (Denoux et al., 2008; Lecourieux et al., 2005). Species-specific elicitors or virulence factors are likely to tailor the spatial and temporal facets of defense responses to provide an apparently unique response to each attacker (De Vos et al., 2005; Glazebrook, 2005). A. MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING CASCADES

PAMP perception is translated into changes in gene expression and defense compound synthesis by the activation of MAPK signaling cascades (Colcombet and Hirt, 2008; Schweighofer and Meskiene, 2008). MAP kinases and their regulatory phosphatases are widely used to integrate biotic and abiotic stress signals, as well as developmental cues. All MAP kinase cascades utilize a hierarchy of MAP3Ks (MAP2K kinases or MEKKs), MAP2Ks (MAPK kinases or MKKs), and MAPKs (MAP kinases or MPKs). Individual MAPKs, MAP2Ks, and MAP3Ks can participate in more than one MAP-kinase cascade, providing flexibility for signaling pathways. In Arabidopsis, MKK1/2 and MKK4/5 cascades regulate PAMP-triggered immunity (Fig. 5). The MEKK1, MKK4/5 and MPK3/6 signaling cascade is activated within 5 min of PAMP perception (Asai et al., 2002) (Fig. 5). MPK3 phosphorylates VIP1, causing phospho-VIP1 translocation from the cytosol to the nucleus, and transcription of PR-1 (Djamei et al., 2007); the full complement of genes regulated by VIP1 has yet to be identified. MPK6 regulates ET biosynthesis by phosphorylating ACS6 (1-Amino cyclopropane-1-carboxylic acid synthase 6), which catalyzes the rate-limiting step in ET biosynthesis (Joo et al., 2008). Phospho-ACS6 evades degradation by the 26S proteasome and thereby stimulates evolution of ET (a hallmark of PAMP-triggered defenses). A protein microarray-based screen has further identified a variety of potential MPK3 and/or MPK6 substrates (Feilner et al., 2005). In addition, MPK3 and MPK6 phosphorylation results in the expression of WRKY transcription factors that activate SA-responsive gene expression and resistance to biotrophs.

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RLK

RLP

PAMP BAK

HAMP receptor HAMP

PAMP ?

? ?

?

Stabilize ACS6 Ethylene biosynthesis

MEKK1

MEKK1

MKK4/5

MKK1/2

MPK6 MPK3

AP2C1

MPK4 MKS1

???? WRKY33/WRKY25

VIP1 PR gene transcription

WRKY29/22 SA-regulated defense responses Resistance to biotrophs

JA-/ET-regulated defense responses Camalexin Resistance to necrotrophs

Fig. 5. MAP-kinase cascades in plant innate immunity. The Arabidopsis MKK4/5 and MKK1/2 MAP kinase cascades that are involved in PAMP-signal transduction are illustrated. Based on flg22 signaling, pattern-recognition receptors (RLK; green rectangle) require the co‐receptor BAK1 (blue square) for signaling after ligand binding (small circles). It is not clear if RLPs (purple rectangle) require BAK1 and what cytosolic partners are needed for signal transduction (brown circle). It is not clear if herbivore elicitor or HAMP receptors are LRKs, RLPs or have novel structures (orange oval) and whether or not co‐receptors (not shown) will be needed for their function. In addition, it is not clear if HAMPs will activate one or both PAMPresponsive cascades (dotted arrows). Upon PAMP perception, MEKK1 phosphorylation activates both the MKK4/5 and MKK1/2 cascades (solid arrows). MKK1/2 phosphorylates MPK4, which subsequently phosphorylates MKS1. MPK4 dissociates from the MKS1–WRKY33 complex (not shown) and allows WRKY33 to activate camalexin biosynthesis (in a SA- and JA-independent manner). While the molecular mechanism for WRKY25 activation is not known at this time, WRKY transcription factors also activate JA/ET-regulated defenses, conferring resistance to necrotrophs. Phosphorylated MKK4/5 phosphorylates MPK3 and/or MPK6. By phosphorylating and stabilizing VIP1 (VirE2-interacting protein1) and ACS6 (1-Amino cyclopropane-1-carboxylic acid synthase 6), MPK3 and MPK6 activate transcription factors involved in SA-responsive gene expression and ethylene biosynthesis, respectively. In addition, both MPK3 and MPK6 activate SA-responsive gene expression to confer resistance to biotrophs. The MKK4/5 and MKK1/2 MAP kinase cascades are negatively regulated by the phosphatase AP2C1. In Arabidopsis, the importance of the MKK4/5 and MKK1/2 cascades in herbivore defense has only been tested using ap2c1 mutants at this time. However, there is substantial evidence for the importance of the MAP kinase cascades in herbivore resistance in tobacco, tomato and Nicotiana attenuata.

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MEKK1, MKK1/2, and MPK4 constitute the second MAP kinase cascade important in PAMP-triggered immunity (Gao et al., 2008b; Qiu et al., 2008; Suarez-Rodriguez et al., 2007). Nuclear-localized MPK4 is a negative regulator of SA-regulated defenses and promotes ET- and JA-mediated defenses and resistance to necrotrophs (Brodersen et al., 2006; Petersen et al., 2000) (Fig. 5). In nonstimulated cells, MPK4 forms a complex with MKS1 (MAP kinase substrate 1) and the transcription factor WRKY33 in the nucleus, thereby sequestering WRKY33. Upon PAMP treatment or infections with virulent or avirulent pathogens, MPK4 phosphorylates MKS1 and MPK4 is released from the MPK4-MKS1-WRKY33 complex. WRKY33 can bind to, and activate the PAD3 promoter, which is involved in the synthesis of camalexin, a potent phytoalexin and controversial aphid deterrent (Kus´nierczyk et al., 2008; Pegadaraju et al., 2005). Presumably, MPK4 sequestration of transcription factors in nonstimulated cells enables the negative regulation of SA-responsive defense traits. The molecular mechanisms for activation of other WRKY factors that are important for resistance to necrotrophs have not been elucidated at this time. Both of the PAMP-triggered MAP kinase cascades are downregulated by the MAP-kinase phosphatase AP2C1 (Arabidopsis Ser/Thr phosphatase type 2C) (Schweighofer et al., 2007). AP2C1 dephosphorylates both MPK6 and MPK4 and provides a link between the two PAMP-responsive MAPK cascades. Consistent with this role, transgenic Arabidopsis that ectopically expresses AP2C1 has decreased ET production and compromised innate immunity to necrotrophs. There are three additional MKK cascades (MKK3, MKK7, and MKK9) that contribute to Arabidopsis defense against pathogens (Do´czi et al., 2007; Takahashi et al., 2007; Xu et al., 2008; Yoo et al., 2008; Zhang et al., 2007). While the MKK3 and MKK9 cascades influence JA- and ET-regulated signaling, respectively, the MKK7 cascade regulates SA-responsive defenses and systemic acquired resistance. At present it is not clear how the MKK3, MMK7, and MKK9 cascades communicate with the cascades that are used for PAMP-triggered immunity. However, MPK6 is involved with both the MKK3, MKK9 and the MKK4/5 cascades suggesting that additional linkages may be revealed in the future (Colcombet and Hirt, 2008; Schweighofer and Meskiene, 2008; Takahashi et al., 2007). MAPK cascades are also important in defense responses to herbivores. For example, in the Arabidopis ap2c1 mutant, which cannot downregulate MPK6 or MPK4, two-spotted spider mite oviposition is deterred (Schweighofer et al., 2007). Also, the WIPKs and SIPKs of N. attenuata, tobacco and tomato are important regulators of defense, wounding, herbivory, and responses to insect oral secretions (Halitschke et al., 2001; Kandoth

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et al., 2007; Li et al., 2006; Seo et al., 2007; Stulemeijer et al., 2007) (Section IV.C.1). Expression of some MAP kinase gene mRNA levels increase in Arabidopsis upon infestation with the silverleaf whitefly, rice attacked by the brown planthopper Nilaparvata lugens, and N. attenuata assaulted by the tobacco hornworm (Kempema et al., 2007; Wu et al., 2007; You et al., 2007). Since there is a good correlation between MAPK RNA levels and MAPK activity in Arabidopsis, the herbivore-regulated MAPKs identified in silico may have important roles in modulating induced defenses upon insect feeding (Menges et al., 2008). B. LINKING PAMPs TO SA-, JA-, AND ET-REGULATED DEFENSE RESPONSES

Examination of the global changes in transcript levels at early times after PAMP treatment and microbe interactions in Arabidopsis is elucidating the overlap in PAMP-triggered immunity, effector-triggered immunity, and nonhost responses. Studies of plants responding to (a) different PAMPs (e.g., OGAs, flg22, and chitin), (b) effector-deficient pathogen mutants, (c) nonhost microbes, (d) virulent biotrophic and necrotrophic pathogens, and (e) avirulent pathogens have provided an overwhelming amount of data regarding changes in gene expression in response to biotic challenges (Bae et al., 2006; De Torres et al., 2003; Denoux et al., 2008; Menges et al., 2008; Moscatiello et al., 2006; Navarro et al., 2004; Qutob et al., 2006; Ramonell et al., 2005; Truman et al., 2006; Tsuda et al., 2008; Wang et al., 2008a; Zipfel et al., 2004, 2006). Integral to these studies and others is the strategic utilization of mutants that impact biosynthesis of defense regulators or block signal perception or transduction by defense signaling compounds (Wang et al., 2008a). The gene expression changes that occur within 30 min to 1 h after flg22, chitin, and OGAs treatments overlap extensively with each other (Denoux et al., 2008; Navarro et al., 2004; Ramonell et al., 2005). The genes that are upregulated encode transcription factors (such as members of the WRKY transcription factor family), RLKs, kinases and phosphatases, and enzymes involved in ET or phenylpropanoid biosynthesis (Asai et al., 2002). SA- and JA-biosynthesis genes are also transiently (between 30 min and 1 h) upregulated by PAMPs (Denoux et al., 2008). However, two to three hours after treatment, distinctions in PAMP-triggered responses are revealed (Denoux et al., 2008; Moscatiello et al., 2006; Tsuda et al., 2008). For example, after 2 h, OGAs activate genes that are involved in biosynthesis of JA and C6 volatiles. A majority of the OGA-responsive genes are activated due to the Ca2þ transients that occur after OGA treatment (Moscatiello et al., 2006). In contrast, while flg22 and chitin also enhance the synthesis of phenolic compounds, these PAMPs prolong expression of SA-biosynthesis

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and -responsive genes, such as PR genes and genes associated with NONEXPRESSOR OF PR GENES-1 (NPR1)-regulated changes in secretory processes (Tsuda et al., 2008). OGAs and flg22 are known to confer a JA-, SA- and ET-independent resistance to pathogens (Denoux et al., 2008; Zipfel et al., 2004). Therefore, it was anticipated that PAMP-triggered changes in gene expression would be largely distinct from pathogen-induced defenses. However, in Arabidopsis it is clear that not only are the different PAMP-triggered gene expression profiles similar to each other (Denoux et al., 2008; Navarro et al., 2004; Ramonell et al., 2005), they also have a large overlap with responses to non‐adapted and adapted pathogens (Navarro et al., 2004; Tao et al., 2003). Using the sid2-2 and pad4-1 mutants or applications of treatments with the SA analog BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; synonym: acibenzolar S-methyl, ASM), Tsuda et al. (2008) showed that flg22 causes increases in SA levels and induces both SAdependent and SA-independent gene sets.

VI. ADAPTATIONS TO UNFRIENDLY HOSTS: EFFECTORS AND EVASION TACTICS A. MICROBIAL EFFECTORS

To ensure their success on host plants, microbial pathogens introduce a bevy of virulence factors (effectors) into plant cells to antagonize PAMP-triggered immunity (Fig. 1) and to create a more favorable environment for microbial growth (Bent and Mackey, 2007; Da Cunha et al., 2007; Hogenhout et al., 2009; Me´traux et al., 2009; O’Connell and Panstruga, 2006). The number and diversity of effectors employed by microbial pathogens has been revealed by large-scale screens designed to identify factors that antagonize plant defense. In bacteria, a type III protein secretion system is used to translocate virulence factors (type III effectors) from bacteria into plant cells. The importance of these effectors in pathogenesis is evidenced by the reduced virulence of bacteria with dysfunctional type III protein secretion systems. Similarly, fungi and oomycetes enhance their success on a host by secreting a multitude of effectors into the plant apoplast or across the extrahaustorial membrane to reach the interior of plant cells (Hogenhout et al., 2009; O’Connell and Panstruga, 2006). Microbial effectors alter a wide variety of cellular activities to impair innate immunity and promote virulence. For example, effectors block stomatal closure, disrupt MAPK signaling, inactivate transcription factors, block perception of PAMPs by targeting pattern-recognition receptors or

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BAK1, and prevent delivery of vesicles with their defensive cargos to the plasma membrane. Effectors cause these disruptions by manipulating protein turnover, causing posttranslational modifications of proteins (phosphorylation, dephosphorylation, and ubiquitinylation), and altering gene transcription and RNA stability. In addition virulence factors, such as coronatine, are thought to leverage cross-talk between defense signaling networks to suppress PR‐gene expression in tomato (Zhao et al., 2003). These elegant manipulations of plant defense are provided in detail in recent reviews (Bent and Mackey, 2007; Da Cunha et al., 2007; Hogenhout et al., 2009; Me´traux et al., 2009; O’Connell and Panstruga, 2006). Finally, RSEs are rapidly perceived by cognate R proteins to activate effector-triggered immunity (Fig. 1) (see Section VII.B). B. HERBIVORE EFFECTORS

While the numerous microbial effectors and RSEs and their modes of action are known, only one herbivore effector has been biochemically characterized to date. However, there is ample evidence for the co‐evolution of herbivore effectors and plant defenses and the subversion of host defenses in plant– insect interactions (Berenbaum and Zangerl, 2008; Walling, 2008). Both saliva and regurgitant are sources of the effectors that make a plant host less formidable to attack. The adaptive mechanisms used by insects to cope with or evade defenses are more diverse than those of microbes spanning coopting of defense cross-talk to sequestration and/or inactivation of chemical deterrents. Specialist insects have adapted to, and actually prefer, certain phytochemically challenging hosts, and some insects leverage these toxic chemicals for their own defense. Insects have also adapted to their hosts to avoid or minimize the release of volatiles that attract the insect’s natural enemies (Rodriguez-Saona et al., 2003; Tooker and De Moraes, 2007); some of these adaptations may be due to feeding behaviors and/or effectors that suppress this important indirect defense. 1. Effectors in regurgitant While numerous studies have identified elicitors of volatile biosynthesis in the regurgitant of insects (Section IV.C), regurgitant may also be a source of effectors. Application of regurgitant from the Colorado potato beetle (Leptinotarsa decemlineata) to wounds in tomato and potato leaves suppresses the wound-induced increase in proteinase-inhibitor mRNAs (Lawrence et al., 2007, 2008). Proteinase inhibitors inactivate digestive proteases and are therefore potent resistance factors against insects (Zhu-Salzman et al., 2008). Therefore, this heat-labile, 10–30 kDa regurgitant factor could be considered an insect effector.

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2. Effectors in saliva a. Glucose oxidase: A salivary effector that suppresses wound signaling. By ablating caterpillar spinnerets, labial salivary gland secretions cannot be delivered to their feeding sites. Comparisons of plant defense responses to untreated and spinneret-ablated lepidopteran caterpillars has allowed the identification of salivary elicitors (Section IV.C.5) and the first herbivore effector—glucose oxidase (GOX). GOX is an abundant protein in labial salivary gland secretions of several Helicoverpa spp. (Eichenseer et al., 1999; Zong and Wang, 2004). GOX oxidizes D-glucose to D-gluconic acid and H2O2. GOX is not transported through the plant vascular system and, therefore, must act locally (Peiffer and Felton, 2005). Its product H2O2, not GOX, is the bioactive signal in planta. The bactericidal activity of H2O2 is well established. Therefore, GOX is proposed to a have role in an insect’s defense repertoire (Musser et al., 2005b). GOX-derived H2O2 could also kill adapted or non‐adapted plant microbes that enter the insect’s feeding site; it is possible that microbe demise causes the release of PAMPs that activate PAMP-triggered immunity. Insect-derived GOX suppresses the JA-regulated defenses of tobacco (Nicotiana tabacum) that antagonize insect growth (Musser et al., 2002, 2005a). Using Helico. zea caterpillars with intact or ablated spinnerets and applications of purified GOX to wounds, GOX was shown to suppress JAregulated nicotine production and to increase SA-regulated PR-1 protein levels. While these data suggest that GOX leverages SA–JA cross-talk to modulate nicotine and PR-1 levels in tobacco, an examination of the nicotine levels in NahG tobacco, that is unable to accumulate SA, shows that GOX effects are independent of SA-regulated signaling (Musser et al., 2005a). It is possible that ET is the regulatory signal, since herbivore-induced ET is known to antagonize nicotine production in N. attenuata (Von Dahl and Baldwin, 2007). Sp. exigua salivary effectors are also implicated in regulating the levels of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and D-1-deoxyxylulose 5-phosphate reductoisomerase (DXPR) transcripts in Med. truncatula (Bede et al., 2006). HMGR and DXPR are early enzymes in the mevalonate (MVA) and 2C-methyl erythritol 4-phosphate (MEP) pathways, which synthesize volatile terpenoids. Salivary GOX and its product H2O2 suppress DXPR mRNA accumulation; other salivary factors appear to modulate HMGR mRNAs. Collectively, the data above indicate that salivary GOX is an effector that suppresses both plant direct defenses (nicotine) and indirect defenses (volatiles that attract natural enemies) in Med. truncatula and tobacco. GOX activity has been detected in the labial salivary glands of many, but not all,

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noctuid species tested (Eichenseer et al., 1999; Hu et al., 2008; MerkxJacques and Bede, 2004; Zong and Wang, 2004). GOX was also recently discovered in the salivary glands of the phloem-feeding aphid My. persicae (Harmel et al., 2008). Salivary GOX activity is controlled developmentally and influenced by the insect’s diet (Hu et al., 2008; Merkx-Jacques and Bede, 2004; Peiffer and Felton, 2005). For example, while GOX is detected in the Helico. zea midgut, the gut epithelium produces little GOX. Therefore, it appears that the salivary GOX may contribute to mid-gut physiology. Within the mid-gut, GOX may serve as an effector by inhibiting ingested plant oxidative enzymes (such as polyphenol oxidase, peroxidases, and lipoxygenase) that decrease the digestibility of plant proteins (Eichenseer et al., 1999). GOX-derived H2O2 could also damage arginase, which catabolizes Arg and limits the availability of this essential amino acid (Chen et al., 2005). It is also possible that GOX could scavenge molecular oxygen and limit its availability to the array of plant-derived oxidative enzymes in the insect diet. Finally, GOX may alter the balance of polyphenol oxidase and peroxidase activity within the mid-gut by providing the rate-limiting substrate H2O2 for peroxidases, thereby enhancing peroxidation of selected substrates. b. Decoy defenses: Suppressing effective defenses using SA–JA cross-talk. Like microbial biotrophs, some hemipterans establish long-term and intimate interactions with their hosts and, therefore, evasion, tolerance, or suppression of resistance to plant defenses is a necessity for pathogen/pest success (Glazebrook, 2005; Walling, 2008). The silverleaf whitefly (SLWF) interaction with Arabidopsis provides one of the best examples of insect evasion of plant defenses. For example, SLWF stylets rarely puncture cells other than the sieve element chosen as a feeding site and, therefore, SLWFs avoid wound responses and activation of JA-responsive genes and volatile release (Freeman et al., 2001; Kempema et al., 2007; Rodriguez-Saona et al., 2003; Zarate et al., 2007). In addition, SLWF nymphs are immobile and feed from a single sieve element for more than 28 days; therefore, no further physical damage occurs. During the prolonged interaction with its host, SLWFs secrete watery and gelling salivas that introduce elicitors and effectors into sieve elements. Little is known about the composition of SLWF saliva or its salivary secretome (Funk, 2001; Leshkowitz et al., 2006). Therefore, the origin (insect or endosymbiont) and biochemical identity of the SLWF elicitors and effectors are unknown (Rosell et al., 2008; Walling, 2000). By using Affymetrix Gene Chips, and leveraging Arabidopsis defense mutants to investigate sentinel defense-response genes in the SLWF–Arabidopsis interaction, it became clear that SLWFs deploy decoy defenses and

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leverage SA–JA cross-talk to enhance the rate of SLWF nymph development (Kempema et al., 2007; Zarate et al., 2007). After 21 days of SLWF feeding, JA- and ET-responsive gene mRNAs decrease or remain unchanged, while SA-responsive gene mRNAs increase locally and systemically. To determine if the induced SA-regulated defenses antagonize SLWF development, the rates of nymph development in four mutants (cev1, npr1, cim10, and coi1) and one dominant transgenic line (NahG) that alter defense signaling, were compared with that in wild-type plants (Zarate et al., 2007). In cev1, NahG, and npr1 plants, where JA-responsive gene expression is elevated and SA-responsive gene expression is suppressed, SLWF nymph development is protracted. Reciprocally, in cim10 and coi1 plants, where JA-regulated defenses are impaired and SA-regulated defenses are enhanced, SLWF nymph development is accelerated. In addition, SLWF nymph development is severely delayed in methyl jasmonate (MeJA)-treated npr1 plants. Because npr1 plants have impaired SA-regulated defenses and almost no SA–JA cross-talk (Spoel et al. 2003), these experiments show that JA controls defenses that actively antagonize SLWF nymph development. At this time, the identities of the JA-regulated resistance traits are unknown. Despite the fact that SLWFs are biotrophs and both SLWFs and biotrophic pathogens induce SA-regulated defenses, the SLWF–Arabidopsis interaction is distinct. Biotrophic pathogens are commonly antagonized by SA-responsive defenses (Glazebrook, 2005). In contrast, the SLWF-induced SA-regulated defenses are a ruse to suppress the JA-regulated defenses that thwart nymph development. The operational mechanism for the SLWF subterfuge is not known. SLWF salivary effectors could directly antagonize activation of JA-regulated defenses and, due to SA–JA cross-talk, allow enhanced expression of the noneffective SA defenses. Alternatively, SLWF saliva could contain elicitors that trigger SA biosynthesis, stimulate SA-responsive defense traits, and exploit SA–JA cross-talk to prevent activation of JA-regulated defenses. Recent experiments show that SLWFs increase SA levels both locally and systemically (S. I. Zarate, D. A. Navarre and L. L. Walling unpublished data). As reflected by their broad host range, refined and stealthy behaviors, and ability to suppress the effective JA-regulated defenses, SLWFs are well adapted to their hosts. Similar gene expression trends may occur in aphidArabidopsis, -sorghum, and -Med. truncatula interactions. For example, SA-regulated mRNAs increase substantially after aphid feeding and more modest changes in JA-responsive mRNA levels are noted (De Vos et al., 2005; Ellis et al., 2002; Gao et al., 2007; Moran and Thompson, 2001; Zhu-Salzman et al., 2004). Although neither SA, nor JA levels are elevated after aphid infestation of Arabidopsis (De Vos et al., 2007; Thompson and Goggin, 2006), recent RNA-profiling studies suggest that both SA- and

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JA-responsive genes are substantially induced by Bre. brassicae and My. persicae, and these changes vary in different ecotypes (Cvi, Ws, and Ler) (Kus´nierczyk et al., 2007, 2008). Analysis of Bre. brassicae and My. persicae performance on SA-, JA- and ET-defense mutants has yielded variable results (De Vos et al., 2007). In contrast, MeJA treatments clearly indicate that JA-regulated defenses can suppress aphid population expansion in Arabidopsis, Med. truncatula and sorghum (Ellis et al., 2002; Gao et al., 2007; Mewis et al., 2006; Moran and Thompson, 2001; Zhu-Salzman et al., 2004). Collectively, these data suggest that aphids, like whiteflies, express ‘‘decoy’’ defenses and suppress or avoid the JA-regulated defenses that antagonize insect performance (Thompson and Goggin, 2006; Walling, 2008; Zhu-Salzman et al., 2006). Since the decoy defense trend is observed in Med. truncatula and sorghum, it is possible that the principles established with aphid and whitefly interactions in Arabidopsis will translate to other monocot and dicot crop plants. However, some aphid–plant interactions will not fit this model, as seen in the tomato–Mac. euphorbiae interaction. In this system, both SA and JA are important in basal resistance (Bhattarai et al., 2007b). The importance of specific defense pathways against hemipterans can only be effectively tested when a comprehensive set of mutants that inactivate or enhance branches of the JA-, SA- and ET-signaling networks are developed, unambiguous sentinel genes for each defense pathway identified, and insect performance assays performed. c. Antagonizing wound healing with calcium-binding proteins. Hemipteran stylets penetrate the cell wall and plasma membrane of phloem sieve elements to establish a feeding site. These wounds need to be sealed rapidly to prevent leakage of phloem sap into the apoplast. The insect’s rapidly gelling salivary sheath cements the stylet sheath to the puncture site and apoplastic callose depositions reinforce this seal (Kempema et al., 2007; Saheed et al., 2007; Tjallingii and Hogen Esch, 1993). Within the cytosol, protein complexes accumulate at the wound site to seal the sieve element lesion. In the Fabaceae, these protein complexes are called forisomes; forisomes alter their conformation in response to changes in redox state or free calcium to rapidly occlude membrane lesions (Will and Van Bel, 2006). While the sealing of stylet-induced wounds is critical for sieve element viability, forisomes can accidentally block the alimentary canal of a hemipteran stylet. Therefore, aphids antagonize cytosolic wound-healing events by limiting increases in cytosolic Ca2þ. By sealing the wound in the apoplast, the salivary sheath prevents a rapid influx of Ca2þ. In addition, hemiptera egest watery saliva into the sieve element prior to the consumption of sap

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(Tjallingii, 2006). Vetch aphid (Megoura viciae) watery saliva contains Ca2þ-binding proteins that prevent the Ca2þ-dependent structural changes to forisomes in vitro (Will et al., 2007). It is presumed that in planta these Ca2þ-binding proteins chelate Ca2þ to prevent the forisome coagulation and blockage of the aphid’s food canal. These salivary Ca2þ-binding proteins are effectors, since they antagonize wound healing to make the plant a more suitable host for aphids. It appears that salivary Ca2þ-binding proteins are one of the defenses that have co‐evolved with host defenses, since the melon Vat (VIRUS APHID TRANSMISSION RESISTANCE) gene, which confers resistance to the melon or cotton aphid (Aphis gossypii), enhances protein deposition and sealing of the sieve element puncture (Martin et al., 2003a).

d. Salivary oxidases and the Redox Hypothesis. Elicitors and PAMPs stimulate the synthesis of phenolic compounds and their oxidases, which can deter insects and pathogens in two ways. First, monomers are polymerized to increase lignification of the cell wall (Hu¨ckelhoven, 2007). Second, phenolic monomers are oxidized to quinones, which are highly reactive compounds that covalently modify proteins and make proteins less digestible and toxic (Felton et al., 1992). The reactive quinones polymerize with other nonoxidized phenolics to form nontoxic oligomers. A plant cell under attack attempts to shift cellular and apoplastic redox balance to promote the toxic effects of phenolics. Not surprisingly, it appears that insects attempt to counter this activity with salivary effectors. Aphid and heteropteran salivas contain a variety of oxidases, including peroxidases and phenoloxidases (Miles, 1999). Peroxidases may polymerize the sheath saliva, providing the stylet a physical barrier from apoplastic defenses. Salivary oxidases might also alter the redox state of plant cells (Miles and Oertli, 1993). This redox state could promote phenolic monomer polymerization into inert oligomers, and thereby provide a less toxic environment that promotes aphid success. Consistent with the Redox Hypothesis, when aphid-infested plants were treated with ascorbate and glutathione (antioxidants), which should promote the persistence of monomeric phenolics, aphid mortality increased (Miles and Oertli, 1993). These data support the idea that the salivary oxidases should be viewed as insect-encoded effectors that perturb the redox balance in a cell to promote insect performance. With emerging aphid genomics resources and the efficacy of RNA-silencing strategies in aphids (Mutti et al., 2008; Ramsey et al., 2007; Tagu et al., 2008), the importance of salivary oxidases in plant cell redox status is ready to be rigorously tested.

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e. Specialist insects: Tolerating toxic phytochemicals. Phylogenetic studies of insects and their hosts have supported the concept of chemical co‐evolution (Berenbaum and Zangerl, 2008). Plants produce and store a diverse array of toxic secondary metabolites to defend themselves against herbivore feeding. These phytochemicals can act as toxins or antifeedants and include: alkaloids, cyanogenic glycosides, furanocoumarins, glucosinolates, hydroxamic acids, saponins, tannins, and terpenoids (Chen, 2008; Howe and Jander, 2008). Despite these deterrents and toxins, there are numerous examples of insect specialists that have co‐evolved with their toxic hosts and display adaptive traits to allow them to feed preferentially on these plants. Glucosinolates will be used as an example. Glucosinolates, sulfur-containing amino acid derivatives, are a wellstudied example of mediators of chemical co‐evolution between the Brassicaceae and specialist insect herbivores belonging to different orders (Grubb and Abel, 2006; Halkier and Gershenzon, 2006; Hopkins et al., 2009; Kliebenstein et al., 2001). Glucosinolates and their hydrolyzing enzymes (myrosinases) are preformed defenses that are developmentally regulated (spatially and temporally) and can increase in response to herbivory. Glucosinolates and myrosinases are stored in separate cell types (Andre´asson et al., 2001). Upon injury, highly toxic hydrolysis products are formed, including nitriles, isothiocyanates, thiocyanates, and epithionitriles. Generalist insects are repelled by these compounds. In contrast, specialist insects use glucosinolates or their breakdown products as phagostimulants, oviposition stimulants, or attractants (Hopkins et al., 2009). Given the different preferences of specialist and generalist insects, it was initially speculated that specialists might provide effectors to subdue, or lack elicitors that trigger, increases in glucosinolates, thus avoiding activation of host defenses. However, when Arabidopsis gene expression programs were compared with specialist and generalist Lepidoptera (Pi. rapae and Sp. littoralis, respectively) or specialist and generalist aphids (Bre. brassicae and My. persicae, respectively), no or small differences in plant gene expression were noted (De Vos et al., 2005; Kus´nierczyk et al., 2007; Mewis et al., 2006; Moran et al., 2002; Reymond et al., 2004). In contrast, when changes in N. attenuata gene expression in response to the oligophagous Man. sexta (a nicotine specialist) and polyphagous generalists Heliothis virescens (tobacco budworm) and Sp. exigua were compared, changes in gene expression were noted and correlated with FACs in their oral secretions (Voelckel and Baldwin, 2004a). The co‐evolutionary adaptations to glucosinolates are displayed primarily by specialist insects (Hopkins et al., 2009). Both generalists and specialist insects detoxify glucosinolates using general detoxification enzymes

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(oxidases) and glutathione transferase. However, specialist insects, such as Plutella xylostella (diamondback moth) and Pi. rapae, have novel enzymatic adaptations (glucosinolate sulfatase and nitrile-specifier proteins, respectively) to hydrolyze glucosinolates to less toxic products (Ratzka et al., 2002; Wittstock et al., 2004). Some specialist insects sequester glucosinolates and have co-opted these molecules for their own defense. For example, Bre. brassicae and Lipaphis erysimi (turnip aphid) sequester thioglucosinolates in their hemolymph and aphid-encoded myrosinases in crystalline microbodies. When the insect is injured, cellular compartments are mixed and the glucosinolate is hydrolyzed to its toxic form (Bridges et al., 2002). The released isothiocyanates and the aphid alarm pheromone E--farnesene act synergistically to signal ‘‘danger’’ to conspecifics (Dawson et al., 1987). In addition, predators do not survive on Bre. brassicae that have been raised on a glucosinolate-rich diet; these data indicate that the stored glucosinolates provide an effective defense against natural enemies (Kazana et al., 2007).

VII. EFFECTOR-TRIGGERED IMMUNITY: RESISTANCE TO PATHOGENS AND PESTS Deployment of R genes to control attack by a specific pathogen race or insect biotype is an effective mechanism of control (Kaloshian and Walling, 2005; Van der Hoorn and Kamoun, 2008). R proteins scrutinize the cellular milieu for direct or indirect evidence of an invading pest or pathogen. The Guard Model explains the ability of R proteins to recognize one or multiple RSEs or product(s) of RSE action (Fig. 1). Over the past decade substantial support for the Guard Model has been garnered (Van der Biezen and Jones, 1998). The principles established for plant–pathogen R-RSE interactions appear to resonate with the data accumulating for plant–herbivore interactions (Chen, 2008; Kaloshian, 2004). However, to date, only two R genes for herbivory has been cloned and the identities of the RSEs that trigger gene-for-gene resistance have not been determined. A. THE GUARD AND DECOY MODELS

The Guard Model for effector-triggered immunity has dominated the literature given its congruence with a multitude of pathogen/plant gene-for-gene interactions (Van der Biezen and Jones, 1998). In this model, a RSE (virulence factor) binds or modifies a specific plant cell target, which is critical for an effective defense. By altering the conformation or structural features of this target, the RSE short-circuits the defense network and compromises

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plant defenses (Fig. 1). As a result, disease ensues or herbivore infestations are promoted. R gene products combat the suppression of host defenses that are caused by effectors. R proteins recognize one or a small number of RSEs (Fig. 1). In a few cases, R proteins directly bind the RSE (Deslandes et al., 2003; Dodds et al., 2006; Jia et al., 2000; Ueda et al., 2006). However, in most cases, R proteins recognize a RSE indirectly by monitoring the RSE’s target. In this context, the R protein guards the target. R proteins often recognize changes in target protein conformation, protein posttranslational modifications, or enzymatic activity that are provoked by an attacker’s RSE. By surveillance of the RSE’s target, R proteins perceive a specific invader and effector-triggered immunity is rapidly activated to suppress pathogen/herbivore establishment and growth. More recently, the Decoy Model has been proposed to explain R protein surveillance and RSE–target interactions (Van der Hoorn and Kamoun, 2008). This model discriminates between the operative target (the RSEbinding protein that alters defense) and decoy RSE-binding proteins with no role in defense. The Decoy Model proposes that R proteins monitor RSE– decoy protein interactions or RSE modifications of the decoy protein. The Decoy Model accounts for the selective pressures on R proteins and target– RSE interactions. To decrease pathogen virulence, it is advantageous to decrease target-RSE binding affinities or increase the number of proteins that can scavenge the RSE (decoy proteins). Decoy proteins can be generated by duplication of RSE target genes and subsequent genetic drift. Alternatively, structurally distinct proteins can evolve and acquire the ability to bind an RSE. The Decoy Model proposes that the R protein recognizes a high affinity decoy–RSE interaction. This model can readily account for the ability of a limited number of R proteins to recognize myriad RSEs encountered in plant–pathogen and plant–pest gene-for-gene interactions. The Decoy Model may change our perception of effector target proteins and their role in the evolution of effector-triggered immunity. As plant–herbivore R protein-effector interactions are elucidated, both models should be rigorously evaluated. B. PLANT–HERBIVORE GENE-FOR-GENE INTERACTIONS

1. Resistance genes A substantial number of genes that confer resistance to viruses, bacteria, fungi, oomycetes, and nematodes have been cloned (Martin et al., 2003b; Van Ooijen et al., 2007). Most R proteins are cytosolic and are classified by their N-terminal domains, which may include a coiled-coiled, ARC or TIR

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domain. The ARC domain is named for a conserved region found in the human apoptotic protease-activating factor, R proteins, and the Caenorhabditus elegans CED-4 protein. The TIR domain was originally identified in Drosophila’s Toll and human Interleukin-1 Receptors. A smaller number of R proteins are RLKs or RLPs, with structures similar to pattern-recognition receptors. Two genes conferring resistance to insects have been cloned: Mi1.2 from tomato and Vat from melon; both are NBS-LRR proteins (Dogimont et al., 2008; Milligan et al., 1998). Many other monocot and dicot R loci that confer resistance to herbivores have been fine-mapped and molecular markers are being used to identify these genes (Kaloshian, 2004). For example, two R genes in Med. truncatula that confer resistance to the blue-green aphid Acyrthosiphon kondoi (AKR) and the spotted alfalfa aphid Therioaphis trifolii f. maculata (TTR) are linked to NBS-LRR gene loci (Klingler et al., 2005). Recent data suggest that these R gene–insect interactions have all the hallmarks of gene-for-gene interactions, since R genes specifically recognize selected insect biotypes. The evidence for gene-for-gene interactions is particularly strong in the Hessian fly (Mayetiola destructor)–cereal interactions. Coupled with intensive breeding initiatives in wheat and barley, there are over 32 resistance genes (Hessian fly resistance 1 (H1)-H32) that recognize a Hessian fly biotype. Hessian fly biotypes that are virulent on plants harboring H genes evolve rapidly (Lobo et al., 2006). Five virulence genes have been mapped on the May. destructor genome and BACs spanning the region of vH13 (virulence Hessian fly 13) were recently reported (Lobo et al., 2006). While considerable progress has been made and the secretome for May. destructor is being defined, neither wheat R or insect virulence genes have been identified to date. 2. Mi1.2: One gene—many herbivores The Mi1.2 gene that confers resistance to herbivores in tomato is the best characterized. Mi1.2 encodes a NB-LRR protein with a coiled-coil domain and was first identified based on its role in conferring resistance to three rootknot nematode species, (Meloidogyne incognita, Mel. arenaria and Mel. javanica) (Milligan et al., 1998). Mi-1.2 also confers resistance to the potato aphid Mac. euphorbiae, two whitefly biotypes (the SLWF and Be. tabaci biotype Q), and the potato psyllid (Bactericerca cockerelli). The mechanisms for Mi-1.2-mediated resistance to these taxa are distinct. This is based on four observations: (1) only nematode resistance is accompanied by an HR (Martinez de Ilarduya et al., 2003; Milligan et al., 1998); (2) nematode resistance can be transferred to other plants but aphid resistance cannot (Goggin et al., 2006); (3) resistance to psyllids and whiteflies appears

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antixenotic (Casteel et al., 2006; Nombela et al., 2001); and (4) resistance to the potato aphid is phloem-based (Kaloshian et al., 2000). Despite these distinctions, the Rme locus, which is proposed to be the effector target, is required for resistance to nematodes, aphids, and whiteflies (Martinez de Ilarduya et al., 2001, 2004). The biochemical basis for the Mi-1.2-mediated resistance to four animal taxa and the pest effectors in these interactions are not presently known. However, the evidence supporting the Guard Model has also been derived from this system, since Mi-1.2 resistance requires HSP90 and SGT1, two conserved components in several R protein complexes (Bhattarai et al., 2007a; Van Ooijen et al., 2007). The unusual ability of Mi1.2 to confer resistance to three nematode species and four hempiterans suggests that there are complexities in R protein-effector interactions that have yet to be revealed. As with many biotrophic pathogens, SA-responsive defenses are required for Mi-1.2-mediated resistance to the Mac. euphorbiae and PR mRNAs accumulate more rapidly in resistant than in susceptible plants (Bhattarai et al., 2007b; Li et al., 2006; Martinez de Ilarduya et al., 2003). Increases in SA and/or PR gene expression have also been noted for R gene-mediated responses to aphids in wheat and barley (Forslund et al., 2000; Mohase and Van der Westhuizen, 2002). While HR is not detected in the potato aphid–Mi1.2 interaction, at least six plant-insect resistance responses do display an HR (Chen, 2008). 3. Medicago resistance to aphids: A JA-dependent event Responses to the blue-green aphid Ac. kondoi, spotted alfalfa aphid Th. trifolii f. maculata, and pea aphid Acyrthosiphon pisum are well characterized in susceptible and resistant Med. truncatula plants. Three R genes confer resistance to these three aphid species and all R genes map to Med. truncatula chromosome 3 (Gao et al., 2008a; Klingler et al., 2007). Clear distinctions in resistance mechanisms, aphid feeding behaviors and defense gene expression have been noted. AKR (ACYRTHOSIPHON KONDOI RESISTANCE)-based resistance to the blue-green aphid is phloemmediated, induces a systemic resistance to further aphid feeding, and is correlated with enhanced JA-defense gene expression (Gao et al., 2007). In contrast, although pea aphid resistance is also phloem mediated, JA-responsive mRNAs accumulate to low levels and are not correlated with resistance (Gao et al., 2008a). Moreover, the pea aphid resistance does not provide systemic resistance to aphids. The resistance gene for the bluegreen aphid and the pea aphid map to a similar region and it is not clear if a single gene or tightly linked genes confer resistance to these two insects. If resistance is mediated by the single gene AKR, AKR could monitor the status

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of two distinct targets; one being the target of the pea aphid effector and the second being the target of the blue-green aphid effector. Alternatively, if the pea and blue-green aphid effectors interact with the same target, AKR must be able to activate distinctive resistance pathways.

VIII. SUMMARY AND FUTURE PROSPECTS Our understanding of the co‐evolutionary adaptations of plants and their interacting herbivores/microbes is at an interesting junction. The innovative use of plant and microbe genetics in conjunction with the immense data sets being generated from microarray studies, high-throughput screens for effectors, and microbial genome sequences have propelled the field of plant–pathogen interactions to new heights. Within the last decade, PAMP-triggered immunity has risen as the core of basal resistance to adapted and non‐adapted pathogens, and the interconnections to major defense-signaling pathways regulated by SA, JA, and ET, MAP kinase cascades, and other known modulators are being elucidated. Due to these initiatives, the identity of many PAMPs that activate innate immune responses, the effectors that surgically inactivate plant defenses, and the RSEs and their targets that allow R proteins to rapidly respond to attack are known. The field of molecular plant–herbivore interactions is approaching this same status and findings are being integrated into a holistic understanding of responses to bioagressors. Due to their multicellular nature, complex physiologies and broad range of feeding behaviors, herbivores have evolved strategies that are not available to microbes to avoid and adapt to the daunting chemical and physical challenges imposed by plant preformed and induced defenses (locomotion/flight, sensing the plant’s chemical composition using gustatory and olfactory cues, and stealthy or destructive feeding behaviors). However, at the molecular level, herbivores appear to leverage the same signaling pathways as used by microbial pathogens to activate or manipulate host defenses. Gene expression profiles after herbivore feeding and egg deposition indicate that plants have evolved sensitive surveillance systems to identify herbivore elicitors and effectors. The signaling pathways activated and suppressed by insect feeding are providing insights into evasive defense manipulations used by phloem-feeding insects and the importance of injury in herbivore– plant interactions. Injury-induced responses can be a dominant or minute component of plant defense. The sentinel of injury, OGA, and PAMPtriggered immunity appear to be a critical element in plant responses to most herbivores. While there is ample physiological and molecular evidence

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to support this conclusion, the critical role of OGA perception has yet to be genetically tested. In addition, it is not clear at this time if other PAMPs (insect chitin, fungal chitin and bacterial LPS), which are speculated to contribute to plant perception of attack, contribute to the complex signals perceived in plant–insect interactions. The foundational studies of plant– herbivore interactions in Arabidopsis have set the groundwork to allow these principles to be genetically dissected in this model plant. Virus-induced gene silencing can be used to test these principles in plants with less well-developed genetic resources. While injury-associated signals are important in plant–herbivore interactions, herbivore elicitors and effectors profoundly influence the specificity and magnitude, as well as the timing and spatial distribution, of induced defenses. Current studies indicate that herbivore elicitors are biochemically distinct from microbial elicitors. At least one class of these elicitors (FACs) performs an important role in nutrition and is presumed to be essential for herbivore viability. FACs may deserve the moniker of HAMP, the herbivore equivalent of a PAMP. In the future, it will be of interest to determine if potential HAMPs and other herbivore elicitors serve essential biological functions. In addition, it will be critical to understand if pattern-recognition receptors are also utilized for recognizing HAMPs, or if novel receptor– ligand interactions will be revealed. Like microbial pathogens, herbivores have evolved biochemical mechanisms to evade effective defenses and enhance their success on their hosts. Herbivores sequester or use effectors to detoxify allelochemicals. In addition, herbivore effectors actively antagonize gene expression programs and prevent activation of effective defenses. While the identity of only one herbivore effector (GOX) is firmly established at this time, there is ample evidence that oxidases and Ca2þ-binding proteins will emerge as effectors in hemipteran saliva to influence redox status and wound healing, respectively. While the chemical identity and mechanisms of herbivore effector action are not known, it is likely that herbivores will utilize the strategies employed by microbial pathogens and deploy a wide array of salivary and mid-gut effectors, including biotype-specific effectors, to manipulate different steps of elicitor and HAMP perception, MAP kinase cascades, and plant defensesignaling pathways. With the current and emerging crop herbivore genome projects, mapbased cloning efforts, and the success of RNA-silencing strategies in testing gene function in herbivores (Grimmelikhuijzen et al., 2007; Lobo et al., 2006; Mutti et al., 2008; Ramsey et al., 2007; Tagu et al., 2008), the secretomes of salivary glands and mid-guts will be established and the identity of new elicitors, general and biotype-specific effectors should emerge rapidly in the

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future. Based on the diversity of RSEs in microbes and the salivary signals from tissue-damaging herbivores that stimulate volatile production, the insect effectors may be a chemical, protein or peptide. These advances in conjunction with the cloning of R genes and elicitor receptors should reveal the secrets of plant surveillance for bioagressors and herbivore adaptive strategies to evade detection.

ACKNOWLEDGEMENTS I thank L.C. van Loon and an anonymous reviewer for insightful suggestions and Bob Doss for aide with the naming of bruchins.

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Plant Volatiles in Defence

MERIJN R. KANT,*,1 PETRA M. BLEEKER,{ MICHIEL VAN WIJK,* ROBERT C. SCHUURINK{ AND MICHEL A. HARING{

*Department of Population Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands { Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

I. Introduction to Volatile Organic Compounds (VOCs) From Plants . . . . . II. Herbivore-Produced Elicitors and Suppressors of Plant VOC Emission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Biosynthesis of Plant VOCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Linoleic Acid/Octadecanoid Pathway-Related Compounds ........... B. Phenylalanine-Derived Volatiles ........................................... C. Terpenoids ..................................................................... D. Methanol....................................................................... E. Ethylene ........................................................................ IV. Volatile Metabolism in Plant Trichomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Trichome Function and Occurrence ....................................... B. Trichome Metabolomics and Transcriptomics........................... V. Volatile Defence Hormones MeJA, MeSA and Ethylene. . . . . . . . . . . . . . . . . VI. VOC Signals Are Influenced by Abiotic Factors and Plant Developmental Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Natural Variation in VOC Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. VOC-Mediated Specificity of Indirect Defences . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51014-2

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IX. VOCs as Alarm Signals for Neighbouring Plants. . . . . . . . . . . . . . . . . . . . . . . . . A. Transcriptional Responses to VOC Exposure ........................... B. Priming of Plant Defences by Volatiles ................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Plant odours are the most ubiquitous volatiles in nature. This chapter deals with the biochemistry and molecular biology of plant volatiles that are emitted from vegetative tissues during pathogen- or herbivore-induced stress and the contribution of these volatiles to plant defences. While mechanical tissue wounding causes the non-specific release of volatiles, herbivore-specific elicitors from their saliva distinctly alter the volatile signature. These volatiles acquired diverse roles in ecological interactions. Firstly, they can be toxic to, or repel conspecific or other herbivores from already infested plants. This function is referred to as ‘direct defence’. Secondly, volatiles function as prey-associated signals for foraging carnivorous arthropods. This is referred to as ‘indirect defence’. Finally, volatiles elicit metabolic changes in unattacked neighbouring plants resulting in priming and induction of defences already before herbivores have arrived. This is referred to as ‘plant–plant communication’. Although volatile production by native plants and crops is highly variable under different growth conditions and during development, its function in direct and indirect defences is remarkably robust. With the current state of knowledge, it is now possible to manipulate these direct and indirect defences through breeding or transgenic approaches. Taken together, plant volatiles play profound roles in plant– herbivore and plant–pathogen interactions and are promising targets for improved crop protection.

I. INTRODUCTION TO VOLATILE ORGANIC COMPOUNDS (VOCs) FROM PLANTS Plants secrete organic and inorganic compounds during growth, development and reproduction from most of their tissues into the environment. For example, plant leaves secrete specific waxes and proteins onto their surface as a protective coating against pathogens (Shepherd and Wagner, 2007), leaf hairs secrete sticky substances that make it hard for small insects to move around on the plant surface (Wagner, 1991), and plant roots secrete a wide range of small molecules that act as signals for rhizobacteria and mycorrhizal fungi (Bertin et al., 2003). Many plant secretions are volatile and diffuse rapidly through air or soil. Volatile products often have been considered as waste or metabolic overflow, since secretion implies that these products will, in most cases, be lost (Harrewijn et al., 1995). Although many of them might once have occurred as waste products during evolution, they have clearly acquired novel functions in the plant’s physiology and ecology and likely have evolved these functions from there on.

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Plant-secreted volatile organic compounds (VOCs) create the plant’s ‘headspace’, the blend of volatiles filling the arbitrary space surrounding a plant, after they have evaporated from the plant surface. It has been estimated that up to 36% of the assimilated carbon is released back into the atmosphere (Kesselmeier et al., 2002) in the form of volatile products, although Halitschke et al. (2000) concluded that the resource requirements for the volatile release are minor. VOCs can be small and highly volatile molecules (e.g. ethylene (ET), methanol, isoprene), but also be compounds with moderate volatility (‘oils’), such as mono- and sesquiterpenes or aromatics such as methyl salicylate (MeSA) or lipoxygenase (LOX)-derived C6-volatiles such as hexenal, often collectively referred to as ‘green-leaf volatiles’ (GLVs), although some authors only consider the latter to be true GLVs. Many of these VOCs are released during the primary processes of growth and development. In addition, emission of VOCs is influenced by abiotic factors, such as nutrient availability, temperature or the spectral composition of light, especially photosynthetically active radiation (Pen˜uelas and Llusia`, 2001, 2004). Although most plants release VOCs constitutively, the composition and quantity of the emitted blend changes markedly in response to biotic stresses, especially after herbivore and pathogen attack. Plants display a wide variety of mechanisms to resist being consumed by pathogens or herbivores. Some of these mechanisms provide direct protection against these attackers, such as the production of toxins, feeding deterrents or repellents, many of which can be volatile. These defences are targeted directly at the herbivores or pathogens. In addition, plants have evolved mechanisms that recruit natural enemies of herbivores, and this attraction is often mediated by induced VOCs that function as prey-associated signals for experienced foraging carnivores. This phenomenon is referred to as indirect defence. Currently, it is widely acknowledged that such tritrophic interactions between plant, herbivore and carnivore occur throughout the plant kingdom (Dicke et al., 2003). The observation that a plant’s metabolic status may be encrypted in the VOCs it releases led, more than two decades ago, to the hypothesis that those volatiles might also facilitate plant–plant information exchange and alert neighbouring plants that herbivores or pathogens are in the area, making it worthwhile to initiate defences prior to actual infection or infestation (Baldwin et al., 2006). In this chapter, we will elaborate on the role of induced VOCs in plant defences, that is, on their role as toxins, repellents and attractors of ‘bodyguards’, and also on their properties to change the metabolism and defence status of neighbouring plants. We will first discuss the biochemical processes involved in the formation of various VOCs and the within-plant spatial organisation of their production and storage. Next, we will discuss the

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hormonal control of their production and release by individual plants and the herbivore- and pathogen-derived signals—elicitors—that initiate production and release during stress. We will then discuss the factors that cause VOC release to be variable and focus on the impact of this variation on responses of nearby herbivores and predators. Finally, we will elaborate on plant–plant eavesdropping, that is, the priming and induction of defence responses in plants that perceive VOCs from induced neighbouring plants.

II. HERBIVORE-PRODUCED ELICITORS AND SUPPRESSORS OF PLANT VOC EMISSION Plants produce a wide variety of organic compounds that are in principle volatile if they are exposed to the open air. Many of these are intermediates in biosynthetic pathways of those secondary metabolites that actually are released, in small amounts, into the headspace, and many of these have no known primary function and are often stored, for example in trichomes, to serve a defensive function (Gershenzon and Dudareva, 2007). A relatively small, but biologically significant portion of these VOCs is aerially released, possibly because of metabolic overflow, although autotrophic organisms, unlike heterotrophic organisms, are not known to make use of these types of catabolic processes that deliver much waste. Emission of volatiles from vegetative tissue is in some cases associated with heat regulation or scavenging of radicals (Chanjirakul et al., 2007; Vallat et al., 2005), but it remains to be seen whether these functions are common. Although plants emit volatiles already under their normal growth conditions, emission is elevated notably during herbivory and pathogenesis, as well as in response to abiotic stress, such as drought, and altered in response to, for example, decreased nutrient status and increased UV light. Together, these factors can lead to a dramatic increase in the amount of volatiles (quantitative) and/or alteration of the types of volatiles (qualitative) in the plant’s headspace. Herbivores rupture cells and membranes during feeding. The degree and type of damage can differ highly among different feeding styles. Chewing herbivores, such as caterpillars, miners and borers, cause more loss of tissue and damage to walls and membranes than stylet feeders, such as phloemfeeding aphids. Mesophyll-feeding stylet feeders, such as mites and thrips, leave cell walls and plasma membranes largely intact, but can empty cell contents. Phloem-feeding stylet feeders, such as aphids and whiteflies, cause marginal damage to mesophyll cells and walls, but cause a shift in a plant’s source–sink flow.

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Mechanical wounding has been shown to induce accumulation of jasmonic acid (JA) and to elicit the release of volatiles, but these represent only part of the volatile mixture characteristic of different plant–herbivore interactions. Thus, herbivore-specific signals give rise to the release of additional volatiles. The fact that treatment of wounded plant tissue with herbivore oral secretions, that is, regurgitant, is sufficient to mimic herbivore-induced JA accumulation and defence gene expression, confirmed this assumption. In addition, some of the active components (elicitors) have been identified and in some cases turned out to be inducers of plant volatiles as well (Gaquerel et al., 2009). The first identified herbivore-derived elicitor was the large cabbage white (Pieris brassicae)-derived enzyme -glucosidase, which induced sufficient volatiles in cabbage to attract the parasitic wasp Cotesia glomerata (Mattiacci et al., 1995). The second elicitor described was a fattyacid conjugate, N-(17-hydroxylinolenoyl)-L-glutamine, named volicitin (Alborn et al., 1997), which is formed in the gut of beet armyworm (Spodoptera exigua) larvae after ingestion of plant material via a 17-hydroxylation reaction of plant fatty acids and subsequent conjugation with insect-derived glutamine. Volicitin has also been found in secretions of tobacco budworm (Heliothis virescens) and corn earworm (Helicoverpa zea) (Mori et al., 2001). Additional volatile-inducing fatty-acid conjugates were found to be produced by the tobacco hornworm (Manduca sexta) (Halitschke et al., 2001) and sulphooxy fatty acids, such as caeliferin A16:0, by the American grasshopper (Schistocerca americana) (Alborn et al., 2007). These fatty-acid conjugates come into contact with ruptured plant tissue during insect regurgitation. Apparently, plants possess membrane receptors that recognise such fatty-acid conjugates to trigger defence responses and volatile production, as suggested for maize (Zea mays) (Truitt and Pare´, 2004). In wild tobacco (Nicotiana attenuata, also called coyote tobacco), the Ma. sexta regurgitant compound 2-hydroxyoctadecatrienoic acid (2-HOT), derived from plant linolenic acid by the action of plant -dioxygenase proteins in the alkaline insect mid-gut, is responsible for induction of the major volatile sesquiterpene trans--bergamotene (Gaquerel et al., 2009). Moreover, a peptide fragment derived from cowpea (Vigna unguiculata) chloroplastic ATP-synthase, called inceptin (Schmelz et al., 2007), has been identified from fall armyworm (Spodoptera frugiperda) secretions. It induces several herbivore-induced plant volatiles, resulting in attraction of conspecific neonates that use these compounds as host plant location and recognition cues (Carroll et al., 2008). When comparing the activity of these elicitors across plant species, Schmelz et al. (2009) found that volicitin exhibited the widest range of phytohormone- and volatile-inducing activity, spanning maize, soybean (Glycine max) and eggplant (Solanum melongena), whereas the

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action of inceptin was limited to cowpea and that of caeliferin to Arabidopsis thaliana. These findings suggest receptor-mediated elicitor specificity among plant species. Finally, oviposition by Pi. brassicae on Brussels sprouts (Brassica oleracea) induced metabolic changes that arrested the egg parasitoid Trichogramma brassicae. It appeared that the anti-aphrodisiac benzyl cyanide, released by mated female butterflies during egg deposition, accounted for this induction (Fatouros et al., 2008). The notion that herbivores produce specific elicitors of VOC emission indicated that plants can evolve to recognise a pest and to initiate indirect defences to augment direct defences. Indirect defences comprise the attraction to, and/or arrest of foraging carnivores to plants via, for example, emission of volatiles that signal the presence of prey, and also by providing shelter or alternative food sources such as extra-floral nectar. Hence, it is not surprising that there are indications that some herbivores, possibly due to selective pressure caused by predation, can suppress indirect defences (Bede et al., 2006; Kant et al., 2008). Takabayashi et al. (2000) found distinct intra-specific variability in the generalist two-spotted spider mite (Tetranychus urticae) in the sense that of two genetically distinct lines of the herbivore (a ‘red’ tomato line and a ‘green’ cucumber line) only one induced indirect defence by feeding on tomato. Subsequently, individuals of the Kanzawa spider mite (Tetranychus kanzawai) were found to differentially affect induced volatiles in Lima bean (Phaseolus lunatus) (Matsushima et al., 2006), while different individuals of Te. urticae collected from the same natural population either induced or repressed emission of volatiles from tomato (Solanum lycopersicum) (Kant et al., 2008). Moreover, in Canadian goldenrod (Solidago altissima) plants, the generalist caterpillar Heliot. virescens elicited strong indirect defensive responses, whereas the gall-inducing tephritid fly Eurosta solidaginis did not. The suppression by the tephritid fly appeared sufficient to repress volatiles induced by the generalist caterpillar as well (Tooker et al., 2008). Since direct and indirect defences are metabolically tightly linked, it is well possible that adaptations that lead to suppression of indirect defences might be the same as those that lead to suppression of the direct ones. While volatile-mediated attraction of natural enemies of herbivores clearly benefits plants, it is much harder to imagine that indirect defences also operate against pathogenic micro-organisms. Nevertheless, pathogen infection also often appears to be accompanied by changes in the plant’s headspace. Several bacterial pathogens, for example, Pseudomonas syringae and Xanthomonas campestris, differentially induce volatile emission upon infection of Arabidopsis and tobacco (Nicotiana tabacum), but there is no evidence that specific elicitors other than known avirulence factors are involved

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(Cardoza et al., 2002; Huang et al., 2003, 2005). Yet, also -glucans, chitin and nodulation (Nod)-factors were shown to induce sesquiterpene emission in Medicago trunculata, with characteristic volatile bouquets for each elicitor (Leitner et al., 2008). Although the function of these volatiles remains to be elucidated, it is clear that a secondary infection by a pathogen following herbivory might have an influence on indirect defences against the primary insect attacker. For experimental purposes, herbivory is often simulated. Roughly, two types of induction are used in experiments: (a) a single artificial stimulus, for example, a wound inflicted with scissors or a haemostat with or without addition of purified or chemically synthesised elicitors or insect saliva, and (b) a continuous stimulus, for example, repetitive artificial stimuli (Mitho¨fer et al., 2005) as a mimic of actual herbivore feeding. A single stimulus usually results in a ‘burst’ of volatiles, reminiscent of the burst of JA (Paschold et al., 2006), which fades with time. This method is typically used in experiments to study the impact of an external treatment or transgene on the qualitative composition of the induced volatile blend. Alternatively, repetitive or continuous stimuli are usually applied when the temporal dynamics of emission or behavioural responses of insects are studied. Experimentally mimicking the feeding rhythm and pattern of insects is hampered by the fact that different insects have very different feeding strategies and adjust these depending on cues that emanate from their food source: absence or presence of such cues, for example, due to a transgene, can alter such behaviour dramatically (Halitschke et al., 2008; Kessler et al., 2004).

III. BIOSYNTHESIS OF PLANT VOCs The majority of the different volatile organic molecules that are released by plants are (1) linoleic acid/octadecanoid pathway-derived jasmonates and C6-compounds; (2) phenylalanine (Phe)-derived aromatic products; (3) isoprene-derived products such as terpenoids (Fig. 1); (4) methanol; and (5) ET. A. LINOLEIC ACID/OCTADECANOID PATHWAY-RELATED COMPOUNDS

The octadecanoid (C18) pathway starts with linolenic acid and proceeds through dioxygenation in the plastids by C13-LOX via dehydration by allene oxide synthase (AOS) and cyclisation by allene oxide cyclase (AOC) to form oxophytodienoic acid (OPDA), the precursor of JA (Fig. 1). Multiple -oxidation cycles in the peroxisomes result in the formation of JA. JA,

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MeSA MeJA cis-jasmone

SAMT Sesquiterpenes

JA GLVs e.g. hexenal

OPDA

STS MTS

DTS GGPP

FPP

PAL Shikimate pathway

GPP

Erythrose phosphate

AOS IPP Lipoxygenase (LOX) Lipase (DAD)

Benzenoids

Monoterpenes Diterpenes

AOC HPL

SA Indole

Mevalonate pathway

Fatty acids Acetyl CoA

Non-mevalonate pathway Pyruvate

Primary metabolism

Fig. 1. Biosynthesis of plant volatiles. Metabolic pathways leading to the formation of fatty-acid derivatives, terpenoids via the mevalonic or non-mevalonic pathways, and Phe-derived volatiles via the shikimate pathway. Abbreviations: AOC: ALLENE OXIDE CYCLASE; AOS: ALLENE OXIDE SYNTHASE, DAD: DEFENDER AGAINST APOPTOTIC DEATH (a phospholipase); DTS: DITERPENE SYNTHASE; FPP: farnesyldiphosphate; GGPP: geranylgeranyldiphosphate; GLVs: green-leaf volatiles; GPP: geranyldiphosphate; HPL: HYDROPEROXIDE LYASE; IPP: isopentenyl pyrophosphate; JA: Jasmonic acid; LOX: LIPOXYGENASE; MeJA: methyl jasmonate; MeSA: methyl salicylate; MTS: MONOTERPENE SYNTHASE; OPDA: oxophytodienoic acid; PAL: L-PHENYLALANINE AMMONIA-LYASE; SA: salicylic acid; SAMT: SALICYLIC ACID METHYLTRANSFERASE; STS: SESQUITERPENE SYNTHASE.

and some of its amino acid derivatives, particularly JA-isoleucine, are key signalling compounds in plant defence (Howe et al., 1996; McConn et al., 1997). Alternatively, JA-methyltransferase (JMT) can catalyse the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of JA. Methylated JA (MeJA) is volatile. MeJA, and the decarboxylated JA-derivative cis-jasmone, occur widely in diverse plant species and their endogenous levels increase dramatically during insect attack, in parallel with those of other JA derivatives (Katsir et al., 2008). Pure MeJA has been shown to act as an elicitor of defences in undamaged plants, as it activates the expression of defensive genes, such as proteinase inhibitors (PIs) (Farmer and Ryan, 1990), and elicits VOC emissions (Kessler et al., 2006) after adsorption and subsequent conversion to endogenous JA-amino acid conjugates (Tamogami et al., 2008).

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Biosynthesis of C6-volatiles is closely associated with that of the C12 stress hormone traumatin (Zimmerman and Coudron, 1979) and probably makes use of the same linolenic acid pools as the octadecanoid pathway. C6-aldehydes and C6-alcohols and their esters are commonly emitted by green plant tissues (Pare´ and Tumlinson, 1999). C6-volatiles are produced in relatively low amounts by undamaged plants, but their emission increases in response to wounding and herbivory (Hatanaka, 1993; Matsui, 2006; Turlings et al., 1995). Upon tissue disruption, hydrolysis of galactolipids from chloroplast membranes provides free fatty acids necessary for the formation of C6-volatiles (Matsui et al., 2000). Hence, C6-volatiles are derived from fatty acid metabolism and are synthesised usually from C18 -linolenic and linoleic acids through dioxygenation by a C13-LOX and subsequent cleavage by a hydroperoxide lyase (HPL), a member of the cytochrome P450 family. C6-aldehydes can be metabolised further by alcohol dehydrogenase (ADH) possibly in conjunction with an isomerisation factor (IF) (Hatanaka et al., 1992) to form the corresponding C6-alcohols. These can then be further modified by acyltransferases or alkenal reductases (D’Auria et al., 2002; Mano et al., 2002). There are indications that C6-volatiles determine host plant acceptance or rejection by feeding or ovipositing herbivores (Halitschke et al., 2008). The group of Junji Takabayashi dedicated a lot of time to study the inducing effects of C6-volatiles on resistance in Arabidopsis against grey mould caused by the fungus Botrytis cinerea. They found that C6-volatiles have bactericidal and fungicidal properties, as reviewed by Matsui (Matsui, 2006), and also against B. cinerea (Kishimoto et al., 2008; Matsui et al., 2006). Thus, C6-volatiles also have anti-microbial properties against fungi and bacteria (Croft et al., 1993; Nakamura and Hatanaka, 2002; Ongena et al., 2004). B. PHENYLALANINE-DERIVED VOLATILES

Biosynthesis of aromatic volatiles relies on precursors from primary processes involved in the production of aromatic amino acids, which are also the precursors of lignins and anthocyanins. Volatile phenylpropanoids and C6-benzenoids are the most abundant constituents of floral scents and play an important signalling role in attracting pollinators (Knudsen and Tollsten, 1993; Negre et al., 2003). Most research on phenylpropanoid-derived volatiles has been carried out with petunia (Schuurink et al., 2006), roses (Guterman et al., 2002) and snapdragon (Dudareva and Pichersky, 2000). The synthesis of phenylpropanoid-derived compounds proceeds from chorismate and isochorismate by the shikimate pathway (Catinot et al., 2008). Chorismate is subsequently converted into the amino acid Phe, the universal

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building block for all phenylpropanoids and benzenoids, while isochorismate is converted into salicylate (SA) (Ament et al., 2004; D’Auria et al., 2003; Vlot et al., 2008). Many benzenoids originate from the trans-cinnamic acid (t-CA) route, where the enzyme L-phenylalanine ammonia-lyase (PAL) deaminates Phe to produce t-CA (Fig. 1). This part of the biosynthetic pathway is shared with the production of anthocyanins and lignins. Volatile benzenoids, such as eugenol, methyleugenol, chavicol and methylchavicol, are formed from p-coumaric acid. Phenylacetealdehyde is produced directly from Phe by phenyl-acetealdehyde synthase and can subsequently be converted to 2-phenylethanol. Benzoic acid (BA), a key component in benzenoid formation, is made from t-CA via multiple pathway branches: the CoA-dependent -oxidative branch (analogous to the -oxidation giving rise to fatty acidderived JA), the CoA-independent non- -oxidative pathway branch, or a combination of the two (Boatright et al., 2004). Both BA and SA can be methylated by the BA/SA methyltransferases (called BAMT and SAMT, respectively) into volatile MeBA and MeSA. The enzymes responsible for the methylation of JA, SA and BA belong to the SABATH family, named after the first three genes identified: SAMT, BAMT and THEOBROMINE SYNTHASE (D’Auria et al., 2003). However, when BAMT is silenced in petunia, production of other benzenoids does not increase, indicating a more complex network of regulation than currently known. The petunia benzenoid pathway is regulated by the transcription factor ODORANT1, which regulates expression of genes early in the shikimate pathway, that is, 5-enol-pyruvylshikimate-3-phosphate synthase (Verdonk et al., 2005). C. TERPENOIDS

Terpenes are composed of isoprenes, and while many terpenoids are precursors of primary plant products such as plant hormones, photosynthetic pigments as well as electron carriers and structural components of membranes, several were found to function as defensive metabolites (Logan et al., 2000). The majority of herbivore-induced terpenoids are monoterpenes (C10 compounds), sesquiterpenes (C15), diterpenes (C20) and homoterpenoids (C11 or C16), many of which can be found in the plant headspace as well as within plant tissues (Ament et al., 2004; Boatright et al., 2004; Leitner et al., 2005; Logan et al., 2000; Verdonk et al., 2005). Terpene biosynthesis takes places via two compartmentalised pathways (Fig. 1). The cytosolic mevalonate (MVA) pathway produces sterols and predominantly sesquiterpenoids, whereas the plastidial 2-C-methyl-Derythritol 4-phosphate (MEP or non-MVA) pathway is involved in the

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biosynthesis of chlorophyll, tocopherols, vitamins, hormones such as gibberellins (GAs) and abscisic acid (ABA), and of mono- and diterpenoids. All terpenes are generated through condensation of universal C5compounds: isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In the cytosolic MVA pathway, isoprene units are generated from acetyl-CoA and sesquiterpenes are produced from C15farnesyldiphosphate (FPP) by the enzyme FPSynthase. In the MEP pathway, isoprenes are produced from pyruvate and glyceraldehyde-3-phosphate via several enzymatic steps in the plastid. Subsequently, C10-geranyldiphosphate (GPP) and C20-geranylgeranyldiphosphate (GGPP) are the precursors for monoterpenes and diterpenes, respectively, produced via the enzymes GPSynthase or GGPSynthase. Although the MVA and MEP pathways are separated by the plastidial membranes, cross-talk between the two pathways via precursor exchange has been suggested (Hemmerlin et al., 2003; Lichtenthaler, 1999). Moreover in basil and snapdragon, it appears that the production of terpenes depends almost exclusively on the MEP pathway (Dudareva et al., 2004; Xie et al., 2008). The diversity of volatile terpenes found in nature originates from the activity of terpene synthases (TPSs) and modifying cytochrome P450 enzymes. TPSs have differentiated into multi-gene families and can catalyse the synthesis of single terpenoids, or of multiple terpene products in specific ratios (Bohlmann et al., 1998; Cheng et al., 2007; Segura et al., 2003). As a consequence, TPSs are responsible for the formation of the most abundant and structurally diverse group of plant metabolites. To date, hundreds of TPSs have been functionally characterised, several of which are induced by biotic stress conditions (Herde et al., 2008; Lin et al., 2008; Van Schie et al., 2007). D. METHANOL

Plants produce and emit methanol (MeOH), which impacts OH˙ radical concentrations and photochemical ozone. Emission of MeOH is associated with growth, ageing of plant tissues and leaf abscission (Huve et al., 2007), as well as tissue damage. Wound-induced release of MeOH increased dramatically when larvae of Ma. sexta attacked N. attenuata plants. MeOH emission was sustained for 24 h after herbivore feeding and was substantially higher than the release of E-2-hexenal. It appeared that herbivore-induced MeOH results from a pH shift at the wound site due to larval oral secretions (pH 8.5–9.5). It coincided with increased transcript accumulation and activity of leaf pectin methylesterases. It has been suggested that

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herbivore-induced MeOH is actually beneficial to herbivores, since it has a negative effect on the induction of some well-known defence marker genes and increases the total weight gain of the attacking larvae (Von Dahl et al., 2006). E. ETHYLENE

Ethylene is a volatile plant hormone. The biology and genetics of ET responses were primarily dissected by studying the lack of effect of ET on dark-grown mutant seedlings (the ‘triple response’) (Guzman and Ecker, 1990). Active in minute amounts, ET is essential for plant development, fruit ripening and senescence. Most plants induce ET emission in response to herbivory or in response to pathogen infection. The biosynthesis of ET starts with the conversion of methionine to SAM (Ado-Met), which is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by the rate-limiting ET-biosynthetic enzyme ACC synthase (ACS). The enzyme ACC oxidase (ACO) finally produces ET. ET biosynthesis can be induced by exogenous ET, auxins or cytokinins. ET receptors are encoded by multiple genes and are located in the endoplasmic reticulum (Hall et al., 2007). ET is known to modulate various defence responses against pathogens and insects (Van Loon et al., 2006).

IV. VOLATILE METABOLISM IN PLANT TRICHOMES A. TRICHOME FUNCTION AND OCCURRENCE

Production of aromatics and C6-volatiles occurs mostly in plastids, while terpenoids are produced in both plastids and the cytosol. How these compounds are transported from there to their site of release is unknown. C6-volatiles in the plant headspace are likely to result from de novo synthesis. In contrast, terpenoids and aromatics after synthesis are often stored in specialised organs. Storage organs can be oil ducts or glandular trichomes, which are distinct and relatively autonomous cellular structures largely separated from the rest of the plant, possibly because of the phytotoxic properties of high levels of secondary metabolites, only to be released in response to biotic stresses (Wagner, 1991). In general, trichomes are small, single-celled or large, multi-cellular structures with diverse morphology: nonglandular trichomes are simply leaf hairs without specialised storage function (Werker, 2000), whereas glandular trichomes consist of cells equipped to store volatile products and phenolics. It is suggested that these cells might often contain plastids, fix carbon themselves and function relatively independently from the rest of the plant (Wagner, 1991).

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Glandular trichomes are present on leaf and stem surfaces of many plant species, but can also be found on petioles, seed coats, flower petals and pedicels. In many plant species, trichome development starts early during leaf differentiation and trichome density decreases with leaf expansion. The sub-cuticular trichome storage cavities at the tip are loaded with metabolites from secretory cells that lie below. How volatile components are released from trichomes is not known. Possibly, they evaporate when trichomes are ruptured, for example, by crawling insects. However, there are also likely active mechanisms for release, since induced emission of volatiles often occurs systemically, that is, also from healthy undamaged tissues. Per gram fresh weight or unit area, young leaves are far more active in the release of volatiles than older leaves, although this could very well be the result of differences in trichome density. Also, the qualitative composition of exudates produced by glandular trichomes may be different in old as compared to young leaves (Maffei et al., 1989), as described in section VI. Trichome densities are not simply genetically determined and depending solely on developmental stage, since in wild tomato (Lycopersicum hirsutum) they can fluctuate over the seasons, consequently influencing the production of specific exudates (Antonious et al., 2005). Whereas trichomes are found on plant above-ground surfaces, root hairs can be considered a type of below-ground ‘root epidermal trichomes’ (Werker, 2000). The development of root hairs and trichomes has been shown to be under the same genetic control and regulation (Kellogg, 2001). Like trichomes, root hairs exude secondary metabolites, for instance carotenoid-derived strigolactones, which are involved in host recognition in plant– mycorrhizal and plant–parasite interactions (Lopez-Raez et al., 2008), and fatty acid-derived lipid resorcinols and benzoquinones in sorghum, which are proposed to suppress growth of competing seedlings (Dayan et al., 2007). B. TRICHOME METABOLOMICS AND TRANSCRIPTOMICS

To elucidate the molecular genetic basis of trichome developmental processes, such as cell differentiation, the focus has been mainly on the unicellular and non-glandular trichomes of Arabidopsis. These likely have a primary function in focussing light onto the leaf surface and act as structural barriers against small herbivores (Kryvych et al., 2008; Szymanski et al., 2000). However, in general, glandular trichomes function as chemical barriers since they synthesise, store and secrete high concentrations of secondary metabolites and defensive proteins. As a target for metabolic engineering, the glandular trichomes of species like basil (Ocimum basilicum) and mint (Mentha x piperita) have been studied intensively (Aziz et al., 2005;

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Gang et al., 2001; Wagner et al., 2004). Glandular trichomes are characterised by a glandular head, but come in different types, shapes and sizes, some of which can co-occur on a single plant or on a single organ (Simmons and Gurr, 2005). The collection of metabolites stored in the trichome glandular head can vary greatly among plant species. For example, tomato trichomes contain mostly acyl sugars, fatty acid-derived methylketones and sesquiterpenes (Fridman et al., 2005; Van der Hoeven et al., 2000), whereas the glandular trichomes of mint contain predominantly monoterpenes such as p-methane (Alonso et al., 1992; Lange et al., 2000) and N. tabacum trichomes contain predominantly diterpenes (Amme et al., 2005). In contrast, sweet basil trichomes produce phenylpropanoids abundantly (Gang et al., 2001), whereas in the trichomes of hop cones, the female inflorescences, different terpenophenolic metabolites and prenylflavonoids are present (Stevens and Page, 2004). The genes involved in the development of non-glandular trichomes and those playing a role in the biosynthetic pathways that load the extra-cellular cavity of glandular heads with secondary metabolites, have been identified primarily through mutant screening and analyses of expressed sequence tags (ESTs). For many plant species, cDNA libraries of glandular trichomes have been constructed, which were used to analyse the correlation between the presence or abundance of transcripts and the production of specific compounds in order to link genes to metabolites. For instance, Nagel et al. (2008) analysed hop trichomes and found 100 ESTs representing all seven enzymes of the plastidial MEP pathway, whereas only 10 ESTs matched six genes related to the cytosolic MVA pathway. Through analysis of the mint ESTdatabase of Lange et al. (2000), it was found that 25% of the total ESTs were associated with essential oil metabolism, including all genes of the monoterpene biosynthetic pathway, and these data were in concurrence with earlier findings that the MVA biosynthetic route in mint is blocked (McCaskill and Croteau, 1995). Moreover, sweet basil produces aromatic phenylpropenes in addition to terpenes, and this correlated very well with the abundances of ESTs associated with specific enzymes involved in the production of precursors for the phenylpropanoid and terpenoid pathways isolated from sweet basil trichomes (Gang et al., 2001; Xie et al., 2008). Finally, Aziz et al. (2005) confirmed the presence of ESTs corresponding to enzymes for all steps in the biosynthesis of flavonoids in alfalfa trichomes. Taken together, in many cases glandular trichomes were found to be equipped well enough to support independent production of many secondary metabolites, although clear-cut evidence for such independence is still lacking (Wagner, 1991). The increasing knowledge of the genes involved in the production of volatiles in trichomes has opened up new possibilities for crop improvement

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as well as for applications in biotechnology through pathway engineering. Since it appears that trichomes are relatively independent from the rest of the plant, and can be used to produce and store secondary metabolites in otherwise toxic concentrations, they provide an ideal environment for such genetic metabolite engineering. This was advocated by Wang et al. (2001), who demonstrated that it is possible to enhance insect resistance in plants by down-regulation of a trichome-specific cytochrome P450 hydroxylase gene responsible for cembratriene-diol production, thereby decreasing the product and increasing the concentration of its specific diterpene precursor cembratriene-ol. Hence, targeted trichome-specific pathway engineering is essentially possible. Less specific, but also promising, is engineering trichomes via manipulations of a plant’s responsiveness to phytohormones. The tomato protein Coronatin insensitive 1 (COI1), which is required for plant responsiveness to JA, was shown to be essential for the production of glandular trichomes on immature fruit and to modulate the density of type-VI trichomes, the most common glandular trichome morphotype, on leaves and sepals. Consequently, mutations in COI1 altered the biosynthetic capacity of glandular trichomes and decreased the plant’s resistance to herbivores (Li et al., 2004). Taken together, engineering the trichome appears a realistic goal and may have many advantages over engineering green tissues, since the metabolic changes can essentially be localised to the trichome tissue only.

V. VOLATILE DEFENCE HORMONES MeJA, MeSA AND ETHYLENE The two plant volatiles MeJA and MeSA are particularly well-documented because they are believed to act as volatile stress hormones. MeJA is a derivative of the stress hormone JA. Therefore, its formation is closely associated with JA-dependent stress responses. Its endogenous occurrence has been described in detail (Baldwin et al., 1997; Seo et al., 2001; Von Dahl and Baldwin, 2004), but MeJA is found only occasionally in the headspace of (induced) vegetative tissues (Farmer and Ryan, 1990; Hopke et al., 1994; Meyer et al., 2003). Notably sagebrush, Artemisia tridentata, is known for its constitutive aerial release of MeJA, which increases upon wounding. Farmer and Ryan (1990) discovered that like pure MeJA, naturally released MeJA from sagebrush was sufficient to induce accumulation of PIs in down-wind tomato leaves. This prompted the question whether volatile MeJA might play a role in plant–plant interactions, since unharmed plants may benefit by building up defences when their neighbours are attacked by herbivores before they are attacked by these herbivores themselves. Although

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plant–plant communication mediated by volatiles still receives a lot of attention, it is unlikely that MeJA plays a general role in this phenomenon simply because, despite its endogenous accumulation, it is rarely emitted by green plant tissues. Also for sagebrush–neighbour interactions under natural conditions, a role for volatile MeJA is doubtful (Karban 2007a,b; Preston et al., 2001, 2002, 2004). However, for artificial induction of JA-dependent defences, that is, to simulate herbivory under experimental conditions, the compound MeJA has proven to be highly suitable (Wu et al., 2008). Simulated herbivory and the use of mutant plants have shown beyond doubt that JA and/ or its derivatives are essential signalling molecules for the production and release of herbivore-induced volatiles such as terpenoids. For example, the tomato JA biosynthesis mutant defenceless 1 (def1) has normal housekeeping levels of JA and normal trichome densities, but is deficient in the emission of JA-induced terpenoids and MeSA (Ament et al., 2004; Halitschke and Baldwin, 2003; Li et al., 2004) (Table I). For plant–plant interactions, another volatile JA-derivative, cis-jasmone, might be a more realistic mediator, since its occurrence as a herbivore-induced volatile is more general, while it has inducing properties in exposed plants similar to those of MeJA (Birkett et al., 2000; Bruce et al., 2008; Pickett et al., 2007). Whereas MeJA and cis-jasmone elicit JA-dependent responses, MeSA elicits SA-specific responses. SA is well-known for its role in inducing the accumulation of pathogenesis-related (PR) proteins and the induction of systemic acquired resistance (SAR) during pathogenesis (Glazebrook, 2005). Reminiscent of MeJA, whose activity is most likely due to its conversion to endogenous JA (Wu et al., 2008), the action of MeSA is probably due to its conversion to SA after uptake by the plant (Chen et al., 2003; Park et al., 2007; Seskar et al., 1998; Shulaev et al., 1997). However, unlike MeJA, MeSA is commonly found in herbivore-induced plant headspaces. Whereas SA is essential as a precursor (Ross et al., 1999), JA signalling is essential for the induction of the SA-methylating enzyme SAMT (Ament et al., 2004). MeSA has been shown to be a critical not only in planta mobile signal for the establishment of SAR (Vlot et al., 2008), but is also released into the air by stressed vegetative tissues. Synthetic MeSA was found to be a potent inducer of the SA-related defence marker gene PR-1 (Shulaev et al., 1997) and, as an odour, to act as an elicitor of foraging responses in predatory mites (De Boer and Dicke, 2004). The previous implies that treating a plant with MeJA or MeSA is equivalent to treating it with JA or SA. It is well-known that SA/MeSA-signalling and JA/MeJA-signalling have antagonistic properties, since a response induced by one of the hormones is often associated with loss of responsiveness to the other. Intra-cellularly, this antagonistic cross-talk is mediated by the

TABLE I Mutant and Genetically Modified Plants with Altered Volatile Production and Associated Biological Responses Receiver Plant

Gene or mutant line

Arabidopsis

Type

Details

Volatile

all84

Knockout mutant

Octadecanoid pathway mutant

Decreased Pierisinduced (Z)-3hexenal

Arabidopsis

S-12

Co-suppressed 35S::Lox2

JA-biosynthesis mutant

Arabidopsis

NahG

NahG overexpresser

SA-hydroxylase converts SA to catechol

Nicotiana attenuata

NaNPR1

Knock-down Mediator in SA/ ´ cross-talk (inverted repeat) JA

Response

Decreased preyfinding success of parasitoid Cotesia glomerata Unknown Decreased attractiveness of Pieris rapaeinfested Arabidopsis to parasitoid Cotesia rubecula Unknown Decreased attractiveness of Pieris rapaeinfested Arabidopsis to parasitoid Cotesia rubecula Decreased induced Decreased cis-abundance of, bergamotene and predation by, emission predator Geocoris pallens

References Shiojiri et al., 2006a Van Poecke and Dicke, 2002

Van Poecke and Dicke, 2002

Rayapuram and Baldwin, 2007

(continues)

TABLE I Receiver Plant Nicotiana attenuata Nicotiana attenuata

Gene or mutant line NaWRKY3, NaWRKY 6 and NaWRKY3/6 NaSIPK or NaWIPK

Type

(continued)

Details

Knock-down Transcription (inverted repeat) factor in JA signalling Gene silencing

Early defence signalling

Nicotiana attenuata

NaLOX3

Knock-down (anti-sense)

JA biosynthesis mutant

Nicotiana attenuata

NaHPL

Knock-down (anti-sense)

GLV biosynthesis mutant

Nicotiana attenuata

NaHPL

Knock-down (anti-sense)

GLV biosynthesis mutant

Volatile

Response

References

Decreased induced cis-bergamotene emission Decreased cis-3hexenol, terpineol, trans-bergamotene and duprezianene Decreased Manducainduced cis-bergamotene emission Depleted in Manducainduced C6volatiles Depleted in Manducainduced C6volatiles

Decreased Manduca sexta egg predation

Skibbe et al., 2008

Decreased Manduca sexta egg predation

Meldau et al., 2009

Decreased abundance of, and predation by predator Geocoris pallens Decreased abundance of herbivore Epitrix hirtipennis Manduca sexta larval development slowed

Halitschke et al., 2008

Halitschke et al., 2008 Halitschke et al., 2008

Nicotiana attenuata

NaHPL x NaLOX3

Heterozygous Terpene and GLV cross of as-Lox3 biosynthesis and as-Hpl mutant

Potato

Hpl

Tomato

def1

C6-volatiles biosynthesis mutant Knock-out mutant JA biosynthesis mutant

Tomato

def1

Knock-out mutant JA biosynthesis mutant

Tomato

spr2

Knock-out mutant JA biosynthesis mutant

Knock-down (anti-sense)

Depleted in Manducainduced C6volatiles and terpenoids

Reducing GLV emissions in asLox3 plants by as-Hpl ‘rescued’ these plants from being heavily damaged by Manduca sexta Decreased hexanal Increased fecundity and 3-hexenal of aphid Myzus accumulation persicae Depleted in several Decreased Tetranychus attraction urticae-induced of predator terpenes Phytoseiulis persimilis Depleted in several Decreased Spodopteraattraction induced of predator terpenes Phytoseiulis persimilis Depleted in Increased Manducaoviposition of induced Manduca sexta terpenes

Meldau et al., 2009

Vancanneyt et al., 2001 Ament et al., 2004

Thaler et al., 2002

Sa´nchezHerna´ndez et al., 2006

Abbreviations: all84, aldehyde-less 84; def1, defenceless1; hpl, hydroperoxide lyase; LOX3, LIPOXYGENASE 3; NahG, Pseudomonas putida PpG7 salicylate hydroxylase gene; NPR1, NONEXPRESSOR OF PR GENES 1; SIPK, SALICYLIC ACID-INDUCED PROTEIN KINASE; spr2, suppressor of prosysteminmediated responses 2; WIPK, WOUND-INDUCED PROTEIN KINASE.

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ankyrin-repeat protein Non-expressor of PR genes 1 (NPR1), but its mode of operation in not identical between plant species. Silencing NPR1 in Arabidopsis resulted in loss of the antagonism (Beckers and Spoel, 2006), whereas in N. attenuata NPR1 silencing antagonised JA accumulation and JA-related defences, making the plant more susceptible to Sp. exigua larvae (Rayapuram and Baldwin, 2007). Although the biological necessity for the metabolic trade-off between JA and SA is not clear, the general consensus is that it reflects the plant’s need to time, prioritise and fine-tune poorly compatible processes that may consume considerable amounts of energy (Kahl et al., 2000) and are embedded in tailored adaptive responses that sometimes involve the action of a herbivore’s natural enemies. In N. attenuata, ET mediates a switch from direct to indirect defence during feeding of direct-defence insensitive Ma. sexta caterpillars (Kahl et al., 2000). Upon herbivory, N. attenuata rapidly accumulates the alkaloid nicotine and starts emitting terpenes somewhat later in time. It was found that ET repressed the accumulation of nicotine shortly after the emission of terpenes had started. It was suggested that the plant used this ET-mediated switch to facilitate nicotine-sensitive parasitoids of the herbivore by preventing the nicotine-tolerant prey to protect itself by sequestering nicotine.

VI. VOC SIGNALS ARE INFLUENCED BY ABIOTIC FACTORS AND PLANT DEVELOPMENTAL STAGE The release of induced plant volatiles can be highly variable in time and space. This variation is roughly the resultant of the kinetics of the responses, diurnal rhythms, growth conditions, age and type of emitting tissue and the type of stress that elicits the emission. The time of day is relevant for not only the quantitative, but also the qualitative emission pattern, since emission of volatiles exhibits diurnal photoperiodicity (Loughrin et al., 1994; Turlings et al., 1995). In tomato, emission of most terpenes is positively dependent on light (Maes and Debergh, 2003) and JA (Ament et al., 2004), the sesquiterpene -copaene being a notable exception. Loughrin et al. (1994) observed that Sp. exigua on cotton (Gossypium hirsutum) induced C6-volatiles and cyclic and acyclic terpenes in a diurnal rhythm of higher emission during the day than during the night. After removal of the caterpillar, the emission of the cyclic terpenes ceased, but the C6-volatiles and acyclic terpenes kept their rhythm of emission, albeit that they were released in smaller amounts. Similarly, the larvae of Heliot. virescens, Ma. sexta and Helico. zea induced release of several C6-volatiles in N. tabacum, that is, (Z)-3-hexenyl butyrate, (Z)-3-hexenyl isobutyrate,

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(Z)-3-hexenyl acetate and (Z)-3-hexenyl tiglate, exclusively during the dark phase, and (E)-2-hexenal in higher amounts than during the day. The nocturnal volatiles were used by adult Heliot. virescens to avoid plants already containing feeding larvae (De Moraes et al., 2001). Schmelz et al. (2001) showed that the same treatments, that is, wounding with or without application of JA or volicitin, applied at different times of the day on excised maize leaves or intact plants, resulted in differential volatile emission during the subsequent light period. Excised leaves emitted three to eight times more sesquiterpenes than leaves on intact plants, and in different ratios. Induction had the strongest effect on excised leaves treated in the middle of the dark phase, whereas intact plants displayed little or no response to volicitin when they had been treated at the beginning of the light cycle. Tissue type and age influence particularly quantitative emission profiles of induced volatiles. As a rule, on a weight basis, young tissue is more active than older tissue. Accordingly, the predatory mite Phytoseiulus persimilis preferred volatiles from young leaves infested with the spider mite Te. urticae over those emitted by old infested leaves, while their qualitative composition was similar (Takabayashi et al., 1994). When older leaves of cotton were damaged by Sp. exigua caterpillars and started to produce volatiles, the undamaged younger leaves started to release some of these volatiles as well, showing systemic induction (Ro¨se et al., 1996). In sweet-scented tobacco (Nicotiana suaveolens), the quality and quantity of the floral volatile blend was altered by Ma. sexta larvae feeding on the leaves (Effmert et al., 2008). Moreover, in a comprehensive analysis of the kinetics of Sp. frugiperdainduced volatiles in soybean, it was found that plants in the vegetative stage emitted 10-fold more volatiles per biomass than reproductive plants, that young leaves emitted three times more volatiles than old leaves and also that systemic induction in single leaves was stronger and faster (after one day) in the acropetal than in the basipetal direction (Rosta´s and Eggert, 2008). Hence, local responses affect the metabolism in distal tissues as well, but the magnitude of the response depends on tissue-specific parameters such as age and relative position. Considering the nature of the plant’s ‘indirect’ defences, it is clear that the receiving party, that is, a foraging carnivore or a host-searching parasitoid, must be able to discriminate volatile signals from background noise and to decipher the qualitative and quantitative information in the volatile blend to the best of their abilities. In principle, the concentration of volatiles decreases with increasing distance from the odour source, but evaporation gradients can be asymmetric due to air currents. When studying the impact of volatiles on insects, or on plants, working with realistic concentrations is not trivial. In insects behavioural assays, concentrations that are too high might elicit

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avoidance or escape or, alternatively, evoke behaviour where ‘something’ is preferred over ‘nothing’. Similarly, when studying induction of defences by volatiles in neighbouring plants, high concentrations might cause a general ‘stress’ reaction, which often will be difficult to separate from ‘defence’ since the two have much in common. Finally, when working with high amounts of volatile hormones, such as MeSA and MeJA, one might easily elicit responses that do not occur under natural conditions. To study the impact of different amounts of MeJA on the defence chemistry of N. attenuata, Preston et al. (2004) determined that the concentration of the naturally constitutively released MeJA from neighbouring sagebrush plants remained constant within an area of 40 cm, but decreased rapidly over a distance of 1.5 m. They reasoned that the entire amount of MeJA released from one plant will not all be deposited onto a single neighbouring tobacco plant, and at realistic concentrations of synthetic MeJA (1–3 mg released per hour) they did not find evidence that MeJA elicits resistance or resistance-related metabolic changes. Still, volatile-mediated priming of resistance between sagebrush and N. attentuata does occur in nature, although its relevance to the plant’s survival strategy is still under debate. Co-occurrence of N. attentuata and sagebrush is less common in nature than one would expect if their relationship would have evolved to be truly beneficial for the tobacco plants (Karban 2007a; Kessler et al., 2006). Changes in the plant’s induced volatile metabolism depend not only on tissue type, age and time of day, but also on growth conditions. How abiotic factors influence the profile of herbivore-induced volatiles has been only marginally investigated. Gouinguene´ and Turlings (2002) tested the effect of soil humidity, air humidity, temperature, light and fertilisation (complete nutrient solution vs. demineralised water) on the induced terpene, indole and C6-volatiles emission of maize plants. They concluded that plants standing in dry soil released more volatiles overall than in wet soil, and emission was optimal at 60% relative air humidity and a temperature between 22 8C and 27 8C. Emission appeared fully photophase dependent and fertilisation had a strong overall positive effect on the quantities released. Takabayashi et al. (1994) tested the impact of light conditions, time of year and water stress on the relative composition of the spider mite-induced volatile blend of Lima beans. While the blend from low-light plants consisted of 5% (E)- -ocimene, in high light this increased to 21%, and predatory mites were attracted more towards high-light plants in a choice test. The attractiveness of high-light plants compared to low-light plants was clearest from April until September, and absent in winter. Plants grown under water stress conditions (3–3.5 pFsoil) compared to plants grown under normal moisture levels (1.5–1.8 pFsoil), but both at 60–70% relative air humidity, produced higher

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amounts of linalool, (E)- -ocimene, 4,8-dimethyl-1,3,7-nonatriene (DMNT) and MeSA, and were more attractive to predatory mites. Induction of these volatiles in Lima bean (Gols et al., 2003) and tomato (Ament et al., 2004) depends on JA, which is produced by the 13-LOX pathway (Halitschke and Baldwin, 2003). Although poorly documented and largely inferred from transcript analyses, most results indicate that the activity of the 13-LOX pathway and JA accumulation are indeed induced by abiotic stress (Fujita et al., 2006; Nemchenko et al., 2006; Walia et al., 2007). Ozone exposure triggered DMNT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl acetate emission in Lima beans to a sufficient extent to attract predatory mites when unexposed clean plants were the alternative. Also, diamondback moth (Plutella xylostella)-infested ozone-exposed Br. oleracea plants attracted more predatory mites than un-infested ozone-exposed plants (Vuorinen et al., 2004). The preference of the parasitoid Cotesia plutellae for diamondback moth-infested Br. oleracea in the presence of ozone remained unaffected, although some terpenes and C6-volatiles were oxidised by ozone (Pinto et al., 2007). This suggested that tritrophic interactions are not significantly affected by ozone and that herbivore-induced terpenes might function to quench ozone and reactive oxygen species (ROS) (Holopainen, 2004). Taken together, growth conditions and abiotic stress clearly alter induced volatile emission, but the volatile-mediated indirect defences appear quite robust.

VII. NATURAL VARIATION IN VOC PRODUCTION The spectrum of herbivore-induced volatiles can vary across species, within species across cultivars and even between individuals within cultivars. Here, we will limit ourselves to studies where plants were compared directly within one study, since differences between methods/sampling techniques hamper making a sensible meta-analysis of the available literature. Variation in the production of secondary metabolites across plant species is well-known and well-studied (Bennett and Wallsgrove, 1994) and could reflect diversification of plant defences, possibly as a result of ongoing arms races (Aubourg et al., 2002; Benderoth et al., 2006). Plants of different families produce different volatiles when infested with the same herbivore species. Van den Boom et al. (2004) undertook a comprehensive across-species analysis, collecting the induced volatiles of 11 plant species during infestation with the generalist spider mite Te. urticae and comparing those to volatiles from mechanically damaged control plants. They concluded that almost all species produce novel compounds upon

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infestation, including MeSA, terpenes, oximes and nitriles, and that only two species, tobacco and eggplant, alter their emission only quantitatively after induction. Nevertheless, when seven of these species, including eggplant, were tested for attractiveness to Te. urticae’s natural enemy Ph. persimilis, all elicited a positive response to this predatory mite. In addition, when comparing the relative attractiveness of four Te. urticae-infested gerbera varieties to Ph. persimilis, this response appeared to be positively correlated with the amount of terpenes each produced and with the level of infestation (Krips et al., 2001). Hence, although volatile production may be variable, the outcome of the tritrophic interaction apparently is not. If variation in induced volatile production during crop breeding is generated by coincidence—since there is no targeted selection for it—one would expect that the degree of variability between cultivars of a crop is different from that between ecotypes of wild plants that are under natural selection. For example, when Takabayashi et al. (1991) measured the emission of volatiles from two commercial apple varieties, ‘Summer Red’ and ‘Cox Orange Pippin’, after infestation by two different species of spider mites, that is, Te. urticae and Panonychus ulmi, they found that differences between the two apple varieties infested by the same mite species were bigger than of a single apple variety infested by the two different mite species. This indicated that genetic differences between races of the same plant species might translate into markedly different induced volatiles. Turlings et al. (1998) observed considerable differences in the timing of volatile emission from two maize cultivars after induction with Spodoptera littoralis oral secretions, since one of the cultivars produced several monoterpenes and sesquiterpenes, which the other did not at all. Subsequently, Gouinguene´ et al. (2001) compared the induced volatile emissions among seven maize cultivars and five of their wild ancestors, including a comparative analysis of eight individuals from a single natural population of wild teosinte. They observed considerable differences, for example, absence or presence of the sesquiterpene -caryophyllene, and quantitative differences in ratios for all groups except among the eight individuals of the same wild ancestor. In a follow-up study, Degen et al. (2004) compared the induced volatiles of 31 maize inbred lines, representing a large portion of the genetic diversity used by breeders, by a principal component analysis. The genotypes showed highly variable odour profiles and, again, -caryophyllene stood out as being typical for European, but not American, varieties. There appeared to be no relation between the genetic distances of the lines and their odour-profile distances. Different natural populations of N. attenuata growing in the field appeared variable in their production of volatiles as well (Halitschke et al., 2000) and, similarly, within natural populations of sacred thornapple (Datura wrightii) considerable

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heritable variation in the production of -caryophyllene as well as trichome type was shown, the latter under control of a single gene (Hare, 2007). Taken together, variation in induced volatiles is common both between ecotypes in nature and between varieties of crops, suggesting that insects using such information for finding prey must cope with this variability irrespective of the circumstances. To appreciate the role of herbivore-induced volatiles in tritrophic interactions, it is essential to understand how insects perceive and process volatile information. Electrophysiological studies indicate that VOCs are perceived by a wide variety of insect taxa. High sensitivities to C6-volatiles have, for example, been reported in beetles (Blight et al., 1995), moths (Anderson et al., 1995), flies (De Bruyne et al., 2001) and parasitoids (Smid et al., 2002). Terpenoids such as linalool, -caryophyllene, -ocimene and geraniol are also well-perceived by moths (Anderson et al., 1995; Rostelien et al., 2005; Shields and Hildebrand, 2000), flies (De Bruyne et al., 2001) and parasitoids (Smid et al., 2002). Phenylpropanoids such as MeSA have been reported to be well perceived by flies (De Bruyne et al., 2001), moths (Shields and Hildebrand, 2000), parasitoids (Smid et al., 2002) and predatory mites (De Bruyne et al., 1991). The parasitic wasp Cotesia marginiventris was used to test whether quantitative or rather qualitative variability in beet armyworm-induced volatile production of maize plants impacts its success in finding caterpillars. Cowpea and 13 different Mexican maize varieties, producing quantitatively and qualitatively significantly different volatile blends, were tested in all combinations using dual choice tests. While naı¨ve wasps preferred induced cowpea odours to maize odours, they did not discriminate between most of the maize varieties, even though these produced markedly different blends (Hoballah et al., 2002). The absence of clear-cut fixed preferences raises an important question: how do foraging predators solve the problem of qualitative and quantitative variation in herbivore-induced VOCs? Although some insects possess special sensitivities for only a sub-set of specific host plant volatiles, such as isothiocyanates for cruciferous plants in the cabbage seed weevil (Ceuthorhyncus assimilis) (Blight et al., 1995), perception of a wide variety of VOCs appears to be common. Comparative electrophysiological studies in closely related species of Drosophila (Stensmyr et al., 2003), Heliotis (Rostelien et al., 2005) and Rhagoletis (Olsson et al., 2006, 2009) all suggest that although behaviour towards odours among these sister species is different, their olfactory sensitivities hardly vary. Possibly, the stringent expression pattern of insect olfactory receptors allows for less genomic drift of olfactory receptor genes than the more random selection of vertebrate olfactory receptors (Nei et al., 2008). Compared to vertebrates,

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the low rate of evolutionary change, combined with the variation in VOC emissions by host plants, may explain why insects perceive a wide variety of VOCs. In theory, predatory arthropods might solve the problem of qualitative and quantitative variation in herbivore-induced VOC mixtures in two ways. Different individuals of the same species could vary in the expression of different olfactory receptors. Within the population, this variation might be maintained as a balanced polymorphism in the presence of spatial or temporal heterogeneity of different profitable VOC mixtures in the environment. Alternatively, all individuals may possess the same ‘nose’ but ‘learn’ about the relevance of specific VOC mixtures. Whereas in humans polymorphisms in odour perception are common (Nei et al., 2008), we are not aware of such examples for insects. This could be a consequence of the relatively low rate of evolutionary change of insect olfactory receptors. Predatory arthropods have been reported to readily learn to distinguish herbivore-induced VOCs. For example, the parasitoid Co. glomerata learns so fast that it already develops a long-term memory for VOC mixtures after a single rewarding oviposition experience (Smid et al., 2007). Herbivoreinduced VOCs as such are not required to elicit attraction of predators as is evident from the observation that the predatory mite Ph. persimilis develops a preference for the odour of herbivore-infested plants over uninfested plants, not only when fed in the presence of the infested-plant odours, but also vice versa, when fed in the presence of uninfested plants (Drukker et al., 2000; Van Wijk et al., 2008). These results indicate that learning by association plays an important role in shaping the behavioural response elicited by VOC mixtures in predatory arthropods. However, it follows that predators, in order to learn, can be misled by volatile signals that resemble those associated with another learning experience. Indeed, Shimoda and Dicke (2000) observed that the blind predatory mite Ph. persimilis can have difficulties in discriminating plants with prey, Te. urticae, or non-prey, Sp. exigua, on beans or cucumber. It was found that for predators such as predatory mites, their genetic background (Maeda et al., 2001) and rearing history, that is, prior experiences (Maeda et al., 2000), strongly determine their ability to make proper decisions under experimental circumstances. Hence, for optimal foraging of predators that use prey-induced plant volatiles for finding plants carrying the prey, the clarity of such signals above the background noise might be more important than long-term signal stability. This implies that temporal and spatial variation in induced plant volatiles might not be a big obstacle for smart carnivores.

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VIII. VOC-MEDIATED SPECIFICITY OF INDIRECT DEFENCES If blends of volatiles elicit specific foraging responses, do single volatiles have the same effect? If so, manipulating plants to produce a novel compound might increase their attractiveness for carnivores and enhance protection. For this purpose, transgenic plants over-expressing specific biosynthetic enzymes have been created. Table II lists transgenic lines that have an altered attractiveness for herbivores or carnivorous bodyguards as a result of manipulated VOC emission. Constitutive expression of several TPSs, that is, FaNES1, MxpSS2 and ZmTPS10, altered the attractiveness of Arabidopsis plants in the desired fashion, except for the isoprene gene PcISPS, which led to decreased attractiveness to the parasitoid wasp Anagrus nilaparvatae. Larger amounts of released VOCs through over-expression of HPL, which increases release of C6-volatiles, or by constitutive activation of JA signalling, increased the attractiveness of Arabidopsis and tomato to parasitoids as well (Table II). The complementary approach, using mutant and knock-out lines, has also been taken. Table II lists mutants and transgenic lines in which expression of key genes of the JA biosynthesis and response pathways or the C6-volatile pathway has been knocked out by point mutations or knock-down RNA interference. In most cases, these genetic alterations resulted in a reduction in VOCs release, which in some cases dramatically altered the interaction with herbivorous and carnivorous bodyguards. These results show that it is well possible to alter the behaviour of herbivores and carnivores by manipulating the plant’s headspace. However, this leaves the question whether such manipulations can suffice to establish enhanced biological control. Given the evidence that many insects rapidly learn to avoid unrewarding stimuli, enhancing attraction will only work if alternative rewards are offered in the absence of prey. Hence, constitutive expression might not be the ultimate solution, and optimising the ‘natural’ process by engineering herbivoreinducible enhancers of indirect defences may be far more sustainable. However, besides the intriguing possibilities for improving crop protection, plants with altered inducible VOCs are also extremely useful for testing the effect of induced defences under more natural conditions. Turlings et al. (1998) found differences in the volatile emissions induced in maize plants by a folivorous caterpillar (Sp. littoralis), a stemborer (Ostrinia nubilalis) and the maize aphid (Rhopalosiphum maidis), which prompted the question if specialist parasitoids and predators can discriminate between plants with and without prey, irrespective of the plant species. Indeed, the parasitic wasp Cardiochiles nigriceps discriminates between prey-induced volatiles in cotton, tobacco or maize, and non-prey-induced volatiles

TABLE II Transgenic Plants with Modified Volatile Production and Associated Biological Responses Receiver Plant

Donor plant

Transgene linalool/nerolidol synthase FaNES1 linalool/nerolidol synthase FaNES1

Promoter

Details

Volatile

Arabidopsis

Strawberry

35S

Targeted to plastids

Linalool and derivatives

Arabidopsis

Strawberry

35S

Targeted to mitochondria

(E)-DMNT; (3S)(E)-nerolidol

Arabidopsis

Gray poplar

Isoprene synthase PcISPS

35S

Isoprene

Arabidopsis

Mentha x piperita

Modified MxpSS2

35S

(E)- -Farnesene

Arabidopsis

Maize

ZmTPS10

35S

Sesquiterpenes, predominantly (E)- -farnesene and (E)-bergamotene

Response

Reference

Enhanced repellence of aphid Myzus persicae Enhanced attraction of predator Phytoseiulus persimilis Loss of attraction of parasitic wasp Diadegma semiclausum Enhanced repellence of aphid Myzus persicae; enhanced arrestment of parasitoid Diaeretiella rapae Enhanced preference of parasitoid Cotesia marginiventris

Aharoni et al., 2003 Kappers et al., 2005 Loivama¨ki et al., 2008 Beale et al., 2006; Prosser et al., 2006

Schnee et al., 2006

Arabidopsis

Rice

OsBSMT1

35S

Most of the ‘volatiles’ accumulated endogenously

Arabidopsis

Arabidopsis

AtHPL

35S

Also compared to C6-aldehydes knock-out

Arabidopsis

Arabidopsis

AtHPL

35S

C6-aldehydes

Arabidopsis

Arabidopsis

AtJMT

35S

Rice

Rice

OsTPS3

35S

Tomato

Tomato

PROSYSTEMIN

35S

Constitutive high MeJA production

MeSA and MeBA

Constitutive high endogenous MeJA accumulation Increase in (E) -caryophyllene; decrease in -humulene Constitutive high Higher release of JA biosynthesis -Pinene, and -myrcene, responsiveness 3-carene, limonene and -ocimene

Enhanced susceptibility to pathogens Pseudomonas syringae and Golovinomyces orontii Enhanced resistance to pathogenic fungus Botrytis cinerea Increased attractiveness to parasitoid Cotesia glomerata; increased parasitation and mortality of Pieris rapae Enhanced resistance to fungus Botrytis cinerea Increased attraction of parasitoid wasp Anagrus nilaparvatae Increased attractiveness to parasitoid wasp Aphidius ervi

Koo et al., 2007

Kishimoto et al., 2008 Shiojiri et al., 2006b

Seo et al., 2001

Cheng et al., 2007 Corrado et al., 2007

Abbreviations: BSMT, S-ADENOSYL-L-METHIONINE:BENZOIC ACID CARBOXYL METHYLTRANSFERASE; HPL, HYDROPEROXIDE LYASE; ISPS, ISOPRENE SYNTHASE 1; JMT, S-ADENOSYL-L-METHIONINE:JA-CARBOXYL METHYLTRANSFERASE; MxpSS2, SESQUITERPENE SYNTHASE 2; NES1, NEROLIDOL SYNTHASE 1; TPS, TERPENE SYNTHASE.

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(De Moraes et al., 1998). Moreover, herbivore foraging behaviour is also influenced by such volatile information. Herbivores with different feeding habits, that is, the piercing–sucking western flower thrips (Frankliniella occidentalis), the chewing herbivore Heliot. virescens, or both simultaneously, elicited emission of different blends of VOCs in N. tabacum. In choice tests, herbivorous thrips consistently preferred uninduced plants over all other treatments and, hence, possibly use the same herbivore-induced volatiles that natural enemies use to find prey, to avoid competition with other herbivores (Delphia et al., 2007). Evidence that, despite all variation, herbivore-induced volatiles indeed mediate indirect defences under natural circumstances has been accumulating slowly. Drukker et al. (1995) observed that predatory anthocorid bugs aggregated near cages containing pear trees infested with pear psyllids (Cacopsylla spp.), and in laboratory olfactory choice assays Scutareanu et al. (1997) showed that the same bugs were attracted to the induced volatiles of Cacopsylla-infested pear leaves. Field application of synthetic herbivoreinduced volatiles as a mimic of plant ‘alarm’ calls is currently being explored for its effectiveness to recruit and retain beneficial insects in the field (James, 2003; James and Grasswitz, 2005; James and Price, 2004). However, without testing this hypothesis under field conditions, it should not be assumed that predators that are attracted by herbivore-induced volatiles necessarily benefit the plant (Karban, 2007b). In a landmark study, Kessler and Baldwin (2001) showed that mimicking the natural emissions induced by leaf-feeding herbivores from N. attenuata in the field, that is, by application of cis-3-hexen-1-ol, linalool and cis--bergamotene, egg predation rate by the generalist predator Geocoris pallens was increased, while application of linalool also decreased lepidopteran oviposition rates. The authors estimated that herbivore-induced volatiles reduced the number of herbivores by more than 90%. Fieldplanted N. attenuata silenced for LOX3 or HPL genes and deficient in -bergamotene and GLV emission, respectively, not only were more vulnerable to N. attenuata’s adapted herbivores, but also attracted novel herbivore species, which fed and reproduced successfully (Kessler et al., 2004). In a follow-up study, Halitschke et al. (2008) showed that predatory bugs use terpenoids and C6-volatiles to locate their prey on herbivore-attacked plants, and thereby reduce herbivory, and also that plants producing C6-volatiles are damaged more by flea beetles. Yet, although not obtained from field experiments, there are some indications that indirect defences can really increase a plant’s fitness. Van Loon et al. (2000) showed that both Arabidopsis accessions Ler and Col-0 infested with unparasitised larvae of Pi. rapae produced less seeds than when infested with parasitised larvae. Similar results were obtained using maize plants under attack by Sp. littoralis: these plants

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showed increased attractiveness to endoparasitoids, that is, Co. marginiventris and Campoletis sonorensis. Not only did parasitism significantly reduce feeding and weight gain of the host larvae, but also plants attacked by a single parasitised larva produced about 30% more seed than plants that were attacked by an unparasitised larva (Hoballah and Turlings, 2001). Finally, release of (E)- -caryophyllene induced by Diabrotica virgifera virgifera beetles from the roots of Z. mays into the soil appeared to attract the beetle’s entomopathogenic nematode Heterorhabditis megidis and decreased emergence of adult beetles to less than half (Rasmann et al., 2005). Taken together, herbivore-induced volatiles are highly suitable cues for predatory insects to locate prey. Under many circumstances, this may increase hostplant fitness through reduced herbivory.

IX. VOCs AS ALARM SIGNALS FOR NEIGHBOURING PLANTS Plant volatiles diffuse freely through the surrounding air and hence reach not only foraging insects, but also neighbouring plants. Rhoades (1983) found that field-grown willows next to herbivore-attacked conspecifics were less palatable to larvae than were unattacked trees growing next to unattacked willows. This prompted the question whether exudates of the attacked willows and possibly volatiles from leaves or roots, had ‘induced’ their unattacked neighbours. Baldwin and Schultz (1983) discovered that exposing undamaged, individually potted sugar maples and poplars to the VOCs of mechanically damaged relatives increased the trees’ levels of tannins and phenolics. Since in this study the plants did not have root contacts, the observed effects could only have been mediated by above-ground airborne VOCs. These two studies initiated a whole new field of research on plant– plant interactions via induced volatiles and there is now a long list of researchers who have tried to discern which VOCs elicit such effects. In numerous follow-up studies in the laboratory and in the field, additional evidence for the occurrence of VOC-mediated defence activation among plants has been found (Baldwin et al., 2006; Dicke et al., 2003). A. TRANSCRIPTIONAL RESPONSES TO VOC EXPOSURE

In the first two decades after the publications of Baldwin and Schultz (1983) and of Rhoades (1983) the field of VOCs-mediated plant–plant interactions was led primarily by the working hypothesis that herbivore-induced volatiles induce defences in neighbouring plants, and focussed more on the

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consequences, for example, for herbivore performance on receiver plants than on the mechanism of the response. Unbiased methods to study such mechanisms in receiver plants became available in the genomics era, especially after the invention of gene chip micro-arrays. The ease of collecting gene expression data has boosted our knowledge of the transcriptional changes that determine the downstream changes in protein and enzyme activity, herbivore performance and VOC-induced volatile production. For example, from field experiments it was already known that airborne molecules released from clipped sagebrush induced polyphenol oxidase activity in wild tobacco (Karban and Baxter, 2001; Karban et al., 2000, 2003), but Kessler et al. (2006) showed that, both in the glasshouse and in nature, clipping the sagebrush led to considerable transcriptional changes in the wild tobacco. When micro-arrays are available, one can obtain a fairly good overview of the transcriptome upon plant exposure to a volatile without having to deal with responses at the phenotypic level (Kant and Baldwin, 2007). The goals of these analyses are sometimes straightforward, that is, to identify a marker for the response to a particular volatile, but often complex, such as prediction of the phenotype based on the response at the transcriptional level. The number of studies analysing transcriptional responses of plants to volatiles has increased rapidly since the discovery that MeJA can induce PI expression in tomato and tobacco (Farmer and Ryan, 1990; Farmer et al., 1992). MeJA is likely converted to JA in planta (Tamogami et al., 2008). Interest in volatile signalling was boosted when it was discovered that tobacco plants reacting hypersensitively to tobacco mosaic virus (TMV) could induce PR-1 expression in neighbouring uninfected tobacco plants through volatile MeSA, as described in section IV (Shulaev et al., 1997). However, in planta MeSA is almost completely converted to SA, which acts as the endogenous signalling compound in inducing PR-1 transcripts (Chen et al., 2003; Park et al., 2007; Seskar et al., 1998; Shulaev et al., 1997). Attention has therefore deviated from MeSA to other volatiles. The only other benzenoid tested for its ability to bring about a transcriptional change in plants is methylbenzoate (MeBA), of which 10 M was applied to germinating Arabidopsis seeds for 8 h (Horiuchi et al., 2007). Although considered phytotoxic at this concentration because of its inhibition of seed germination, MeBA changed the expression of more than 1% of the genes significantly. Several of these genes are related to the phytohormones auxin, cytokinin, ABA and ET. Interestingly, the germination of the auxin-resistant mutant axr1 and cytokinin response mutant cre1 was less affected by MeBA than that of the wild type, suggesting that some of the responses to MeBA are mediated by these hormones (Horiuchi et al., 2007). Since MeBA is predominantly a floral volatile, it can be speculated that floral volatiles can have allelopathic activity.

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In addition, a lot of attention has been paid to the most abundant class of plant volatiles, the terpenoids. In an influential paper Arimura et al. (2000) showed that -ocimene, DMNT and TMTT, which are emitted in higher amounts by Lima bean leaves upon herbivory by spider mites, are capable of inducing the transcription of PR, LOX, PAL and FPS genes. Linalool did not induce transcription of these genes, indicative of specificity of the response. Moreover, the authors also showed that these genes were induced either transiently or at different time points. In an additional experiment with custom-made cDNA micro-arrays, the transcriptional response of uninfested, excised Lima bean leaves on the complete (predominantly terpenoid) bouquet of volatiles released by spider mite-infested excised leaves was investigated (Arimura et al., 2000). The finding that ET biosynthetic genes were up-regulated in the detached receiver leaves led to the discovery that these genes were also up-regulated in intact receiver plants (Arimura et al., 2002). Moreover, in leaves of intact receiver plants ET production, as well as JA production, increased upon exposure to volatiles from intact Lima bean plants infested with Te. urticae. Clearly, micro-array experiments paid off here in the discovery of the regulation of ET biosynthesis by a mixture of volatiles. Moreover, each individual volatile (terpenoid or C6) of this mix could also induce expression of the ET biosynthetic genes. The logical followup studies with ET mutants were done, for some unknown reason, with allo-ocimene (Kishimoto et al., 2005). Induction of a selected set of genes by allo-ocimene was similar in mutant ET-insensitive etr1-1 and in wild-type Arabidopsis. This volatile also enhanced resistance against the fungus Bo. cinerea equally in the etr1-1 mutant and in wild-type plants (Kishimoto et al., 2006b). Thus, a role for ET in these processes has yet to be determined. Most attention has been paid to the transcriptional response upon perception of C6-volatiles. Bate and Rothstein (1998) investigated the response of Arabidopsis plants, using different C6 volatiles and selected marker genes associated with defence. They demonstrated that various C6-volatiles had differential effects on the expression of LOX2, albeit that a high concentration (10 M) was used. These distinct effects of different C6-volatiles on gene expression were confirmed by Arimura et al. (2002). With only four marker genes—PR-2, SAMS, SAMDC and ACO—they clearly demonstrated that Z-3-hexenol, E-2-hexenal and Z-3-hexenyl acetate had differential effects on their expression after 3 and 24 h. Kishimoto et al. (2006a) concluded that transcriptional responses of JA- and SA-related marker genes such as AOS, LOX2, HPL, VEGETATIVE STORAGE PROTEIN 1 (VSP1), PLANT DEFENSIN 1.2 (PDF1.2) and various PRs to the C6 aldehydes E-2-hexenal or Z-3-hexenal were significantly reduced in the JA-response mutant jar1–1, the ET-response mutant etr1–1 and the phytoalexin-defective pad2–1, but

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not in the SA-insensitive npr1–1 mutant (Table III). Their study highlights an essential problem with this and other studies, that is, the lack of specific gene markers for C6-volatiles. Conversely, these studies also raise the question how specific the traditional JA, ET and SA markers are. Studies in other plant species have confirmed the capability of C6-volatiles to induce gene expression. Z-3-hexenol induces MAIZE PROTEINASE INHIBITOR (MPI), PAL and LOX in maize (Farag et al., 2005) but Z-3-hexenol is rapidly converted to Z-3-hexenyl acetate that by itself does not induce PAL expression. Another study with maize showed that Z-3-hexenyl acetate induced the expression of several OPDA reductases (Frost et al., 2008b). A recent study using N. attenuata plants genetically engineered to be ‘mute’ in different aspects of their volatile vocabulary revealed that the transcriptional responses of neighbouring ‘eavesdropping’ plants were not elicited by the presence of specific C6-volatiles in the volatile bouquet but, rather, by their absence (Paschold et al., 2006). This study underscores the need to keep an open mind about the nature of the information encrypted in the VOC bouquet. Although the notion that pure VOCs can elicit specific responses in plants is now generally accepted, it remains unclear how such VOCs are perceived by plants. The volatiles, ET, MeSA and MeJA, may simply adhere to the leaf and diffuse from the surface into the epidermal cells, where upon additional modification or not, they may play direct signalling roles. However, for most induced plant volatiles conversion into known phytoactive substances seems unlikely. VOC receptors have not yet been identified, with the notable exception of the ET receptors. In an attempt to identify components of C6volatile perception and signalling pathways, a mutagenesis screen was performed on Arabidopsis using a root elongation assay, as E-2-hexenal inhibits root elongation in wild-type plants. Analysis of the first mutant found, her-1, revealed a link between E-2-hexenal-induced responses and -aminobutyric acid (GABA) metabolism (Mirabella et al., 2008).

B. PRIMING OF PLANT DEFENCES BY VOLATILES

The phenomenon of VOC-mediated defence activation among plants raises intriguing questions about how plants discriminate between signals and noise. The activation of defence responses requires energy and activation of defences is therefore, by definition, undesirable in the absence of herbivory. Solutions to this problem have been proposed; for example, it would make sense if plants did not activate their complete defence arsenal after the first signs of trouble but were to ‘prime’ their defence metabolisms so as to be in a

TABLE III Transcriptional Responses Elicited by Volatiles Mutants/ transgenics

Species

Volatile

Receiver

Arabidopsis

-

MeBA

Arabidopsis

jar1-1

C6-volatiles

Arabidopsis

etr1-1

Terpenoids, C6-volatiles

Plants

Arabidopsis

etr1-1, jar1-1, npr1-1, pad2-1 –

C6-volatiles

Plants

Terpenoids

Excised leaves

Plant volatiles

Excised leaves Plants

Lima bean Lima bean

Germinating seeds Plants

Lima bean

–

Lima bean

–

Terpenoids, plant volatiles C6-volatiles

Maize

–

C6-volatiles

Plants

Maize

–

Plant volatiles

Plants

Plants

Read-out 26k Micro-array CHS, PAL, PRs, LOX AOS, HPL, PRs, VSP1,CHS, COMT, DGK1, GST1, LOX2, PR-2 AOS, LOX2, HPL, VSP1, PDF1.2, PRs PRs, LOX, PAL, FPS 2k cDNA-microarray Ethylene biosynthetic genes SAMS, ACO, SAMDC, PR-2 HPL, FPS, PAL, LOX, IGL, MPI Differential cDNAs

Reference Horiuchi et al., 2007 Bate and Rothstein, 1998 Kishimoto et al., 2005, 2006a,b Kishimoto et al., 2006a,b Arimura et al., 2000 Arimura et al., 2000 Arimura et al., 2002 Arimura et al., 2002 Farag et al., 2005 Ton et al., 2007 (continues)

TABLE III Mutants/ transgenics

Species

Volatile

(continued) Receiver

Read-out

Reference Paschold et al., 2006 Kessler et al., 2006

Plant volatiles

Plants

1k oligo-micro-array

Sagebrush volatiles

Plants

–

C6-volatiles

Plants

Tobacco

–

MeSA

Plants

PI accumulation upon Manduca sexta feeding; feeding damage and mortality rate 5.4 k cDNA-microarray PR-1

Tobacco, Tomato

PI-reporter line

MeJA

Plants

PI

Nicotiana attenuata Nicotiana attenuata

hpl, lox3

Poplar

Frost et al., 2008a,b Shulaev et al., 1997 Farmer et al., 1992

Abbreviations: ACO: 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE; AOS: ALLENE OXIDE SYNTHASE; CHS: CHALCONE SYNTHASE; COMT: CAFFEIC ACID O-METHYLTRANSFERASE; DGK1: DIACYLGLYCEROL KINASE 1; etr1: ethylene resistant 1; FPS: FARNESYL PYROPHOSPHATE SYNTHETASE; GST1: GLUTATHIONE S-TRANSFERASE 1; HPL: HYDROPEROXIDE LYASE; IGL: INDOLE-3-GLYCEROL PHOSPHATE LYASE; jar1: jasmonate resistant 1; LOX: LIPOXYGENASE; MPI: MAIZE PROTEINASE INHIBITOR; npr1: non-expressor of PR genes 1; pad2: phytoalexin deficient 2; PAL: PHENYLALANINE AMMONIA-LYASE; PDF1.2: PLANT DEFENSIN 1.2; PI: PROTEINASE INHIBITOR; PR: PATHOGENESIS-RELATED; SAMDC: S-ADENOSYL METHIONINE DECARBOXYLASE; SAMS: S-ADENOSYL METHIONINE SYNTHASE; VSP1: VEGETATIVE STORAGE PROTEIN 1.

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temporal state of ‘enhanced alertness’ (Conrath et al., 2006). Then, the induction of defences, observed under many experimental conditions, might in some cases have been an artefact caused by using unrealistically high concentrations of pure volatiles (Preston et al., 2001, 2004). Priming is thought to entail low-cost metabolic changes (Van Hulten et al., 2006). These may be small and therefore difficult to detect experimentally, but could enable a plant to launch defence responses more rapidly and more strongly when attacked. By allowing themselves to be primed after eavesdropping on a neighbour, plants might very well prevent resources from being wasted on unnecessary induction of responses, and thereby realise an overall fitness benefit (Kessler et al., 2006; Ton et al., 2007). A specific function for C6-volatiles in priming defences was demonstrated by Engelberth et al. (2004), who showed that upon herbivore-regurgitant treatment both JA production and volatile release were higher in maize plants that had been previously exposed to C6-volatiles. Within 30 min, C6volatile perception led to a transient increase in JA levels, which were fully reset after 3 h. Since this effect occurred with all three C6-volatiles tested, that is, Z-3-hexenal, Z-3-hexen-1-ol and Z-3-hexenyl acetate, the specificity of this priming response needs further testing. A follow-up study (Ton et al., 2007) confirmed that volatiles released from Sp. littoralis-infested maize plants could indeed prime receiver maize plants. This study showed that this occurred not only at the level of gene activation, but also at the level of herbivore performance: caterpillars remained smaller on primed maize plants. These primed maize plants also emitted more volatiles, leading to greater attraction of the parasitic wasp Co. marginiventris. Although this result is not directly related to priming, this is clearly an example of increased indirect defences induced by volatiles. Since the complete headspace of caterpillar-infested maize plants were used, specific roles of individual volatiles still need to be assessed in order to investigate if there are specific components that establish priming. However, this will not always be easy to address since some volatiles can be converted by the plant to other volatiles, as is the case for Z-3-hexenol into Z-3-hexenyl acetate (D’Auria et al., 2007; Farag et al., 2005; Mirabella et al., 2008; Pare´ et al., 2005). Still, Frost et al. (2008a) clearly showed that Z-3-hexenyl acetate, in turn, not only primed expression of genes mediating oxylipin signalling and direct defences induced upon feeding by the gypsy moth (Lymantria dispar), but also JA levels and volatile emission in hybrid poplar. In Arabidopsis, the terpenoid ocimene could prime for enhanced lignification and production of the phytoalexin camalexin upon infection with Bo. cinerea (Kishimoto et al., 2006b). Additional studies in priming by volatiles were done in Lima bean plants. Here the priming effect on induced

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indirect defences, that is, the production of extra-floral nectar as an alternative food source for predatory arthropods, was determined under laboratory (Choh and Takabayashi, 2006a,b) as well as field conditions (Heil and Kost, 2006). Although plants were exposed to an artificial blend of volatiles they clearly showed that extra-floral nectar production increased much more rapidly in wounded than in non-wounded plants. Another field study that focused on the role of volatiles in within-plant signalling in Lima bean plants led to similar results (Heil and Silva Bueno, 2007). Apparently, within-plant priming by volatiles can overcome vascular constraints, as also indicated by Frost et al. (2007) for hybrid poplar. Although in the latter study tubing was used to transport volatiles from one leaf to another, it clearly demonstrated that leaves can respond to naturally occurring, very low concentrations of volatiles by becoming primed for their inducible volatile production. Interbranch signalling could be a feature of perennial shrubs with newly developed lateral branches that have little or no vascular connections, such as in blueberries (Rodriguez-Saona et al., 2003) and sagebrush (Karban et al., 2006). Sagebrush can be primed by volatiles released from neighbouring Art. tridentata both in a laboratory setting and in field-based experiments (Kessler et al., 2006), resulting in an accelerated production of trypsininhibiting PIs and lower total herbivore damage. It is likely that the low doses of volatiles required for priming will entail at most a small fitness cost, but this still needs to be investigated. In conclusion, herbivore-induced plant volatiles play a significant role in complex ecological interactions, and function as signals that alert preysearching carnivores to the presence of food. Such foraging carnivores might, in turn, contribute positively to the plant’s defensive state and thereby to the plant’s fitness. However, these volatiles also serve other functions that benefit the plant, that is, as scavengers of free radicals, to dispose of metabolic wastes, as toxins to pathogens or repellents of herbivores. For the purpose of enhancing crop protection through improvement of volatile-mediated biological control, genetic engineering of plants to manipulate volatile production is a promising approach. However, for designing efficient pest control strategies it will be essential to understand what motivates a predator to use a particular odour during foraging, that is, whether it is an innate preference or an association with its prey after a positive experience. Finally, plant defences in undamaged plants can be activated by exposure to herbivore-induced volatiles or synthetic mimics, but how plants regulate such responses and to what extent they depend on the ability to recognise volatile signals from neighbours when coping simultaneously with competitors, herbivores, pathogens and abiotic stresses, are questions still open for the future.

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identify a key enzyme in the biosynthesis of methylketones. The Plant Cell 17, 1252–1267. Frost, C. J., Appel, H. M., Carlson, J. E., De Moraes, C. M., Mescher, M. C. and Schultz, J. C. (2007). Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecology Letters 10, 490–498. Frost, C. J., Mescher, M. C., Carlson, J. E. and De Moraes, C. M. (2008a). Plant defense priming against herbivores: Getting ready for a different battle. Plant Physiology 146, 818–824. Frost, C. J., Mescher, M. C., Dervinis, C., Davis, J. M., Carlson, J. E. and De Moraes, C. M. (2008b). Priming defense genes and metabolites in hybrid poplar by the green leaf volatile cis-3-hexenyl acetate. New Phytologist 180, 722–733. Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., YamaguchiShinozaki, K. and Shinozaki, K. (2006). Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current Opinion in Plant Biology 9, 436–442. Gang, D. R., Wang, J. H., Dudareva, N., Nam, K. H., Simon, J. E., Lewinsohn, E. and Pichersky, E. (2001). An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiology 125, 539–555. Gaquerel, E., Weinhold, A. and Baldwin, I. T. (2009). Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera; Sphigidae) and its natural host Nicotiana attenuata. VIII. An unbiased GCxGC-ToFMS analysis of the plant’s elicited volatile emissions. Plant Physiology 149, 1408–1423. Gershenzon, J. and Dudareva, N. (2007). The function of terpene natural products in the natural world. Nature Chemical Biology 3, 408–414. Glazebrook, J. (2005). Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology 43, 205–227. Gols, R., Roosjen, M., Dijkman, H. and Dicke, M. (2003). Induction of direct and indirect plant responses by jasmonic acid, low spider mite densities, or a combination of jasmonic acid treatment and spider mite infestation. Journal of Chemical Ecology 29, 2651–2666. Gouinguene´, S. P. and Turlings, T. C. J. (2002). The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiology 129, 1296–1307. Gouinguene´, S., Degen, T. and Turlings, T. C. J. (2001). Variability in herbivoreinduced odour emissions among maize cultivars and their wild ancestors (teosinte). Chemoecology 11, 9–16. Guterman, I., Shalit, M., Menda, N., Piestun, D., Dafny-Yelin, M., Shalev, G., Bar, E., Davydov, O., Ovadis, M., Emanuel, M., Wang, J., Adam, Z. et al. (2002). Rose scent: Genomics approach to discovering novel floral fragrance-related genes. The Plant Cell 14, 2325–2338. Guzman, P. and Ecker, J. R. (1990). Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. The Plant Cell 2, 513–523. Halitschke, R. and Baldwin, I. T. (2003). Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growthrelated transcriptional reorganization in Nicotiana attenuata. The Plant Journal 36, 794–807. Halitschke, R., Kessler, A., Kahl, J., Lorenz, A. and Baldwin, I. T. (2000). Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata. Oecologia 124, 408–417. Halitschke, R., Schittko, U., Pohnert, G., Boland, W. and Baldwin, I. T. (2001). Molecular interactions between the specialist herbivore Manduca sexta

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Ecological Consequences of Plant Defence Signalling

MARTIN HEIL*,1 AND DALE R. WALTERS{

*Departamento de Ingenierı´a Gene´tica, CINVESTAV—Irapuato. Km. 9.6 Libramiento Norte, 36821 Irapuato, Guanajuato, Me´xico { Crop & Soil Systems Research Group, Scottish Agricultural College, King’s Buildings, Edinburgh EH9 3JG, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Signalling at Three Different Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Local Signalling................................................................. B. Systemic Within-Plant Signalling ............................................ C. Airborne Plant–Plant Communication...................................... III. Costs of Induced Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Allocation Costs ................................................................ B. Ecological Costs ................................................................ IV. Resistance Induced by Mutualistic Micro-organisms. . . . . . . . . . . . . . . . . . . . . . . A. Resistance Mediated by Plant Growth-Promoting Rhizobacteria (PGPR)......................................................... B. Resistance Induction by Mycorrhiza ........................................ V. Defence Signalling at the Level of Plant Individual, Community and Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Variable Resistance at the Genetic Level ................................... B. Variable Resistance at the Phenotypic Level ............................... C. Plant–Plant Communication at the Community Level? .................. D. Evolutionary Considerations ................................................. E. Predicting Patterns of Induced Resistance Responses .................... VI. Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51015-4

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ABSTRACT Plants respond to local attack by pathogens and herbivores with systemic resistance expression. Much has been learned on major signalling cascades underlying local enemy perception, on systemic signals such as plant hormones and small RNAs, and on genes involved in local and systemic resistance expression. Induced resistance offers exciting prospects for using the plant’s own defences as environmentally friendly means of protecting crops from pests and pathogens. However, many questions still need to be answered to understand the ecology of induced resistance and before artificially induced resistance can serve as a reliable crop protection strategy. Direct activation of defences is costly and possibly causing yield reductions, particularly if enemy pressure is low. Such costs likely make directly induced resistance unpopular. In contrast, priming triggers defences only following pest or pathogen challenge and is, thus, a less wasteful use of resources. Induced resistance to herbivores and pathogens may benefit crops in the field. Much more has to be done, however, to understand how abiotic and biotic factors such as soil nutrients and the presence of enemies and of mutualistic micro-organisms affect the consequences of a certain resistance induction for plant health, growth, and yield.

I. INTRODUCTION Plants are sessile organisms and cannot move away from their enemies when under attack. Plant resistance depends, therefore, mainly on physical and chemical properties that make feeding by herbivores or infection by pathogens less efficient, difficult, or—ideally—impossible. Traits that mediate plant defence to herbivores comprise anatomical structures such as hairs, spines and thorns, morphological characteristics such as fibres, and compounds that make the tissue difficult to digest, unattractive, or toxic (Karban and Baldwin, 1997; Walling, 2000). Plants may also establish mutualisms with the third trophic level for their defence, since the presence of predators and parasitoids reduces herbivore pressure. Common traits by which such an ‘indirect defence’ is achieved are herbivore-induced volatile organic compounds (VOCs) and extra-floral nectar (EFN) (Heil, 2008). Resistance to pathogens includes strong, heavily reinforced cell walls aimed at preventing penetration by infecting pathogens, toxic secondary compounds termed phytoalexins, and the so-called pathogenesis-related (PR) proteins, which may have, among other characteristics, activities as chitinases and glucanases, capable of actively hydrolysing pathogen cell walls (Jones and Dangl, 2006; Paiva, 2000; Van Loon, 1999). However, resistance expression requires limited resources and thus is costly (Bergelson and Purrington, 1996; Cipollini et al., 2003; Heil and Baldwin, 2002; see Section III). Many, if not most, resistance traits are, therefore, inducible: an initial attack changes gene expression patterns,

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which finally enhances plant resistance (Karban and Baldwin, 1997; Kessler and Baldwin, 2002; Walling, 2000). We can distinguish three major levels at which signalling takes place: (i) local or intra-cellular signalling cascades that mediate the recognition of the attacker and trigger resistance in the affected organ, (ii) long-distance (systemic) signalling, which enhances resistance expression in distant, so far uninfected organs of the attacked plant, and (iii) plant–plant signalling: the response of neighbouring, yet unaffected plant individuals to cues that are released from an attacked plant. The present chapter focuses on induced systemic and plant–plant signalling and presents the mobile signals involved and the little knowledge that exists on its ecological consequences. In particular, we will highlight the questions that are still open for discussion: Why do plants use both internal and external signals for systemic resistance induction? How do plants balance their resistance to multiple enemies and the need to induce resistance while growing or reproducing? How far do these signals travel within and among plants? Are these phenomena ecologically and evolutionary relevant? Finally, we will discuss whether airborne resistance induction likely can be used to control pathogens and herbivores in agronomic systems.

II. SIGNALLING AT THREE DIFFERENT LEVELS A. LOCAL SIGNALLING

1. Pathogen recognition Because plants lack the adaptive immune system of vertebrate animals, they rely on innate immune responses for defence against pathogens. Once a pathogen has breached the outer layers of the plant (e.g., cuticle and cell wall), it is subject to molecular recognition by individual plant cells. Plants have evolved two classes of immune receptors to detect non-self molecules. The first of these are pattern-recognition receptors (PRRs), which are located on the cell surface. These sense microbes by perception of pathogenassociated molecular patterns (PAMPs; also referred to as MAMPs for microbe-associated molecular patterns, since they are not restricted to pathogenic microbes). MAMPs are conserved microbial molecules that include components of fungal cell walls such as chitin, lipopolysaccharides (LPS) from Gram-negative bacteria, and short peptides derived from bacterial flagellin (Zipfel, 2008). This first level of immunity represents a basal resistance and is known as PAMP-triggered immunity (PTI) (Chisholm et al., 2006). It is associated with a range of intra-cellular responses, including changes in ion fluxes across the plasma membrane, production of reactive

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oxygen species (ROS), transcriptional activation of early defence-response genes and mitogen-activated protein (MAP) kinase signalling (Shen and Schulze-Lefert, 2007). Virulent pathogens can successfully infect host plants by evading recognition or suppressing MAMP-triggered signalling, the latter probably mediated by secretion of virulence effectors (e.g., De Torres-Zabala et al., 2007). In turn, some plants have evolved resistance (R) proteins capable of recognising these effector proteins, resulting in a second line of defence known as effector-triggered immunity (ETI) (Pieterse and Dicke, 2007). ETI is also associated with ROS accumulation and defence gene activation, and is often accompanied by local cell death, the hypersensitive response (HR). In an ongoing evolutionary arms race, pathogens have evolved effectors capable of suppressing ETI (Zipfel, 2008). 2. Resistance hormones Various studies have shown that salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are signals in the activation of defence genes (Heil and Ton, 2008; Thomma et al., 2001). However, these hormones serve multiple roles in plants and have important functions also in the regulation of plant growth and development. For example, SA is involved in the regulation of cell growth (Vanacker et al., 2001), flowering, and thermogenesis (Malamy and Klessig, 1992; Raskin, 1992; Shah and Klessig, 1999), and is also known to induce protection against a variety of abiotic stresses (Yuan and Lin, 2008). Equally, JA plays an important role in a range of developmental processes, including seed germination, tuber formation, senescence, and flower development (for a recent review, see Wasternack, 2007), while ET has an essential role in flowering, fruit ripening and abscission, and leaf senescence (Dugardeyn and Van der Straeten, 2008). Given the importance of SA, JA and ET in host development, it should come as no surprise that plant defence and plant growth and development are interconnected (see Sections II.A.4 and V.E.1). In the context of induced resistance to pathogens, the SA pathway is linked mainly to resistance to biotrophic pathogens (Glazebrook, 2005) and is involved in the expression of PR genes encoding proteins with antimicrobial or other defensive functions during basal resistance, as well as in the production of ROS and the induction of the HR in gene-for-gene resistance (Ton et al., 2006). In contrast, work using Arabidopsis thaliana mutants affected in the biosynthesis of, or responsiveness to, JA has shown that JA and ET are involved in resistance to necrotrophic pathogens, at least in this plant species (Glazebrook, 2005). JA-dependent defences contribute to basal resistance to a range of pathogens, including Alternaria brassicicola, Botrytis cinerea and Erwinia carotovora pv. carotovora (Ton et al., 2006). Similarly, mutants of

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Arabidopsis have been used to demonstrate that ET-dependent defences contribute to basal resistance to, for example, Pseudomonas syringae pv. syringae and Xanthomonas campestris pv. vesicatoria (Ton et al., 2006). Importantly, there is considerable cross-talk between these pathways and although pathway interactions vary between plant species and type of pathogen, it is clear that cross-talk is necessary for the plant to fine-tune its defence responses (Bostock, 2005; Pieterse et al., 2006; see Section III.B.1). 3. Further plant hormones Which hormones besides JA, SA and ET are involved in the modulation of plant defence responses? It is well known that plant growth and development are regulated through signalling pathways governed by hormones such as auxins, cytokinins and abscisic acid (ABA), and it should be no surprise therefore that the regulation of these pathways can help to determine the outcome of a plant–pathogen interaction. For example, evidence suggests that part of the invading strategy of pathogens might be the stimulation of auxin signalling. Thus, Chen et al. (2007) showed that Ps. syringae uses virulence factors such as AvrRpt2 to increase auxin levels during infection in order to promote disease. Increased auxin levels could suppress plant defences (Chen et al., 2007), and at the same time alter other aspects of host physiology to favour pathogen growth and establishment (Lopez et al., 2008). Interestingly, plants can repress auxin signalling as a component of basal resistance. Thus, Navarro et al. (2006) have shown that downregulation of auxin responses is the result, in part, of activation of microRNAs (miRNAs) which repress auxin signalling. Remarkably, SA has been reported to be involved in repression of auxin signalling via stabilisation of the Aux/IAA repressor proteins (Wang et al., 2007), while a novel gene, GH3, has been shown to be involved in the stress adaptation response through auxin homeostasis (e.g., Ding et al., 2008). Although less information is available, cytokinins have also been implicated in the outcome of host–pathogen interactions. For example, infection by biotrophic pathogens such as rusts and powdery mildews often leads to the formation of green islands, where the fungal pustule sits in an island of green, photosynthetically active tissue, surrounded by senescing tissue (Walters and McRoberts, 2006; Walters et al., 2008). Green islands are thought to provide a source of nutrients for pathogen growth and development and evidence supports a role for cytokinins in their formation (Walters and McRoberts, 2006; Walters et al., 2008). Indeed, early events in the host–pathogen interaction include enhanced expression of genes encoding an extra-cellular invertase and a hexose transporter, both of which are known to be increased by cytokinin and both of

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which would supply carbohydrates to the area (Roitsch and Ehness, 2000; Walters and McRoberts, 2006; Walters et al., 2008). However, to think that cytokinins are only involved in helping pathogen establishment in host tissues would be wrong. Numerous reports suggest that cytokinins can increase disease resistance. Thus, cytokinins have been shown to induce programmed cell death and expression of PR-1, contributing to resistance against biotrophic pathogens (Carimi et al., 2003; Mlejnek and Prochazka, 2002; Sano et al., 1994). An increasing number of recent publications have reported altered ABA levels in plant–pathogen interactions, although information regarding the functions of ABA in response to pathogens is still fragmentary (Asselbergh et al., 2008a; Lopez et al., 2008). ABA acts as both a positive and negative regulator of disease resistance and a number of mechanisms have been proposed to underpin its action. These include stimulation of callose deposition (e.g., Ton and Mauch-Mani, 2004), induction of stomatal closure (Melotto et al., 2006), suppression of ROS generation (e.g., Asselbergh et al., 2007, 2008b) and suppression of SA- and JA/ET-dependent basal defences (Asselbergh et al., 2008a). Depending on the pathogen, ABA can enhance resistance or enhance susceptibility (Mauch-Mani and Mauch, 2005). For example, ABA enhances resistance of Arabidopsis to the oomycete Pythium irregulare, as ABAdeficient and ABA-insensitive mutants are more susceptible to the pathogen (Adie et al., 2007). ABA has also been shown to protect Arabidopsis against the necrotrophic fungi Alt. brassicicola and Plectosphaerella cucumerina, while the ABA-deficient and ABA-insensitive mutants display enhanced susceptibility to these pathogens (Adie et al., 2007; Ton and Mauch-Mani, 2004). More recently, ABA signalling was shown to regulate resistance to the powdery mildew pathogen Golovinomyces cichoracearum in Arabidopsis edr1 mutants (Wawrzynska et al., 2008). However, ABA can also enhance susceptibility. Thus, ABA application enhances susceptibility of Arabidopsis to Fusarium oxysporum (Anderson et al., 2004) and susceptibility of tomato (Solanum lycopersicum) to Bo. cineria (Audenaert et al., 2002). Similarly, ABA-deficient Arabidopsis mutants were more resistant to F. oxysporum (Anderson et al., 2004) and Bo. cinerea (Adie et al., 2007). It appears therefore that the outcome of any changes in ABA levels or signalling in plants is dependent on the particular plant–pathogen interaction, rather than pathogen lifestyle (e.g., whether it is a biotroph or a necrotroph), although the timing of infection appears to be crucial in its role in defence against pathogens (Asselbergh et al., 2008a). The relative paucity of knowledge of the multifarious regulatory effects of ABA on plant defence precludes any clear-cut model of its effects on

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resistance. Nevertheless, Asselbergh et al. (2008a) suggest that ABA might be involved in controlling a global shift between plant responses to abiotic and biotic stresses. They suggest that the increase in ABA levels in response to, for example, water stress, shifts the priority away from pathogen resistance towards abiotic stress tolerance. This would be consistent with ABA acting as a pathogen virulence factor, suppressing SA- and JA/ET-dependent defence responses (Asselbergh et al., 2008a). Equally, a reduction in ABA levels, as in ABA deficiency, leads to reduced tolerance of abiotic stress and greatly increased disease resistance (Asselbergh et al., 2008a). 4. Cross-talk between growth and defence Plant survival depends on both normal growth and development, and on an adequate defence against attackers (Chung et al., 2008). Indeed, in many plants, the expression of disease resistance is dependent on the developmental stage at which the plant–pathogen encounter occurs (Whalen, 2005). Although considered historically as separate in the plant life cycle, much evidence now suggests that growth and defence are tightly linked. In some interesting recent work, a novel R protein in Arabidopsis was shown to mediate cross-talk between developmental processes and defence responses (Chung et al., 2008; Igari et al., 2008). In this work, the altered morphological phenotype of a semi-dominant Arabidopsis mutant, uni-1D, was found to be due to a gene encoding a coiled coil— nucleotide binding site—leucine-rich repeat (CC-NBS-LRR)-type protein belonging to the R gene family (Igari et al., 2008). The mutant displays alterations in both SA and cytokinin signalling and the plants exhibit up-regulated expression of PR-1 and PR-5, as well as genes involved in cytokinin biosynthesis. The elevated cytokinin levels detected in these plants were responsible for both the altered phenotype and PR gene expression, demonstrating for the first time that a stress-independent signalling pathway can act as a positive upstream signal for a stress-related response under an R-protein-mediated pathway (Chung et al., 2008). B. SYSTEMIC WITHIN-PLANT SIGNALLING

As discussed in the previous sections, plants respond to pathogen infection or herbivore feeding with extensive changes in gene expression patterns (Durrant and Dong, 2004; Karban and Baldwin, 1997; Sticher et al., 1997; Walling, 2000). As both pathogens and herbivores can spread from the initial site of attack to other organs, induced resistance is usually not restricted to the damaged tissue itself but expressed systemically, hence also in yet undamaged parts of the plant. The second level of defence signalling that we discuss here concerns, therefore, the transmission of signals from the local

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(infected) to the systemic (still uninfected) organs. To enable this systemic response, hormones such as JA and SA and their derivatives, are produced at the site of attack and spread throughout the plant. Plant vascular bundles represent a highly sophisticated system for longdistance transport of water, nutrients and assimilates (Le Hir et al., 2008). Early research on the mobile defence signals has therefore searched for signalling compounds in the phloem and the xylem (Constabel et al., 1995; Dicke and Dijkman, 1992; Me´traux et al., 1990; Thorpe et al., 2007; Zhang and Baldwin, 1997; see Heil and Ton, 2008; Starck, 2006; Wasternack, 2007 for reviews). Systemic signals may, however, also move through the plant’s headspace (see Section II.B.4). 1. Systemic signals triggering herbivore resistance A well-studied example of systemic induction of plant resistance to herbivores is the synthesis of proteinase inhibitors (PIs) in tomato, compounds that inhibit protein digestion in insects and thereby diminish the nutritive value of the plant tissue (Green and Ryan, 1972). An 18-amino acid peptide, systemin, is released upon feeding by chewing herbivores and processing from the 200-amino acid precursor protein prosystemin. 14C labelling studies showed that systemin is mobile within the plant (Narva´ez-Va´squez et al., 1995). However, grafting experiments—particularly with spr1, a mutant that is defective in the perception of systemin (Lee and Howe, 2003)—revealed that jasmonates, rather than systemin, are responsible for the systemic wound response of tomato (see Heil and Ton, 2008 for a review). According to current knowledge, JA and its precursors and derivatives (collectively termed jasmonates) represent not only hormones triggering resistance of Arabidopsis to nectrotrophic pathogens, but they form also the most common signals in the wound response of many plant species (Chini et al., 2007; Schilmiller and Howe, 2005; Thines et al., 2007; Wasternack, 2007; Wasternack and Parthier, 1997). Jasmonates induce a broad spectrum of defensive responses such as PIs, the release of VOCs, nicotine production and the secretion of EFN. Biosynthesis of JA is required at the site of damage, whereas its perception is required in the distal plant parts for a systemic induction of PIs (Schilmiller and Howe, 2005), and labelling studies demonstrated a transport of JA through the plant (Thorpe et al., 2007; Zhang and Baldwin, 1997). In the phloem, expression of several genes even amplifies the signal during its transport (Stenzel et al., 2003). The synthesis of JA starts with the liberation of linolenic acid from membranes followed by a multi-enzyme conversion (Wasternack, 2007). JA, however, does not directly induce gene activity. In fact, the JA–amino acid conjugate jasmonoyl–isoleucine binds to the COI1 unit of an E3

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ubiquitin ligase complex termed SCFCOI1 (for Skip/Cullin/Fbox-COI1), thereby stabilising a COI1–JAZ complex (Thines et al., 2007). The formation of this complex allows the ubiquitination of JAZ-proteins and thus their rapid degradation. Since JAZ-proteins are repressors of MYC2 and related transcription factors, their degradation liberates these transcription factors and thus allows gene activation (Chini et al., 2007; Farmer, 2007). 2. Systemic signals triggering pathogen resistance The most common hormone mediating pathogen resistance is SA. First reports of a long-distance induction of pathogen resistance were published by A. Frank Ross, who found that inoculating the lower leaves of tobacco with tobacco mosaic virus (TMV) enhanced resistance to a second infection in the upper leaves (Ross, 1961). This phenomenon has been termed systemic acquired resistance (SAR). In the 1970s, Leendert C. van Loon reported that both locally and systemically induced resistance to pathogens is associated with the induction of PR-proteins (Van Loon, 1997). Since SA treatment of tobacco enhanced resistance to TMV and triggered the accumulation of PR-proteins (Van Loon and Antoniw, 1982; White, 1979), it was generally assumed that SA might function as the mobile signal. Indeed, SA levels increase in the petioles of pathogen-infected tobacco (Malamy et al., 1990) and cucumber (Me´traux et al., 1990) leaves before the onset of SAR. However, removal of pathogen-inoculated leaves before the rise of SA levels in the petioles did not impair the induction of SAR in upper leaves of cucumber (Rasmussen et al., 1991), and SA hydroxylase-expressing, and thus SAdeficient, tobacco rootstocks were still capable of exporting the signal to wild-type scions (Vernooij et al., 1994). Apparently, SAR does not require SA accumulation in the attacked tissue and SA is not the long-distance signal of SAR, even though it is transported throughout the plant and activates SAR. Recent evidence makes it likely that SA is (mainly) transported as its methyl ester, methyl salicylate (MeSA). In tobacco, SA-binding protein 2 (SABP2) converts the biologically inactive MeSA into active SA (Forouhar et al., 2005), and SA methyltransferase 1 (SAMT1) catalyses the formation of MeSA from SA (Ross et al., 1999). Using grafts of the respective mutants, Park et al. (2007) demonstrated that MeSA functions as the critical long-distance SAR signal in tobacco. It remains uncertain, however, whether MeSA plays a similar role in other plant species (Farmer et al., 2003; Pen˜a-Corte´s et al., 2005). For instance, JA can induce the production of phytoalexins in rice cells (Nojiri et al., 1996) and the resistance of tomato to infection by Phytophthora (Cohen et al., 1993), lipid-derived molecules may function as critical long-distance signals in the activation of SAR in Arabidopsis (Maldonado et al., 2002; Truman et al., 2007), and oxylipins enhance the resistance of common bean to the bean rust

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pathogen Uromyces fabae (Walters et al., 2006; see also Champigny and Cameron, 2009). 3. Small RNA signalling In recent years it has become apparent that small non-protein coding RNAs (20–24 nucleotides in length) play important roles in various phenomena such as RNA interference (RNAi), co-suppression and gene silencing (Hamilton and Baulcombe, 1999; Lu et al., 2008). Major classes of small RNAs include miRNAs and small interfering RNAs (siRNAs). Recently, miRNAs have been shown to be important regulators of gene expression in both plants and animals (Jones-Rhoades et al., 2006), both of which employ small RNAmediated gene silencing as an important mechanism for host immunity against bacterial, viral and fungal pathogens. Small RNAs can be up- or downregulated in response to pathogen challenges and subsequently contribute to gene expression fine-tuning and reprogramming by silencing negative regulators and inducing positive regulators of immune responses. Arabidopsis miR393 was the first miRNA identified to play a role in plant basal resistance to bacterial infection, which it did by regulating the auxin signalling pathway (Navarro et al., 2006). Apparently, miR393 targets auxin receptor mRNAs and subsequently represses auxin signalling in Arabidopsis (Navarro et al., 2006). While plants utilise silencing machinery for defence against pathogens, pathogens are likely to develop countermeasures to fight against silencing. Some authors speculate that bacterial and fungal pathogens may have evolved silencing suppressors, as in the case of many RNA viruses (Jin, 2008). 4. Airborne systemic signals Although JA, SA and small RNAs move through the vascular system and can function as long-distance signals, systemic resistance can also be mediated by volatile compounds that move in the headspace outside plants (Heil and Ton, 2008). VOCs are mainly known to attract parasitoids as a means of indirect defence (Dicke and Van Loon, 2000; Heil, 2008; Turlings and Wa¨ckers, 2004), but they also have direct inhibitory effects on microbes (Fernando et al., 2005; Matsui, 2006; Nakamura and Hatanaka, 2002; Shiojiri et al., 2006). Most recently, VOCs have been brought forward as hormone-like signals. Airborne within-plant signalling has now been demonstrated for sagebrush (Artemisia tridentata), poplar (Populus deltoides x nigra) and Lima bean (Phaseolus lunatus) (Frost et al., 2007; Heil and Silva Bueno, 2007; Karban et al., 2006). However, although airborne MeSA-triggered induction of resistance to plant viruses in tobacco has been reported more than ten years ago (Shulaev et al., 1997; see Section II.C.2), airborne within-plant signalling has so far only been reported in the context of herbivore defence.

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C. AIRBORNE PLANT–PLANT COMMUNICATION

Volatile compounds such as VOCs, methyl jasmonate (MeJA) and methyl salicylate (MeSA) are common and highly active means of systemic signal transduction in plants. Since such volatiles are released from the plant surface and then move freely in the air, they can also affect neighbouring plants and then mediate a resistance response in intact plants (Farmer, 2001; Heil and Ton, 2008; Pickett and Poppy, 2001). Our third level of defence signalling concerns, therefore, the signalling among different plant individuals, hence ‘plant–plant communication’. The first reports on ‘talking trees’ date back to the early eighties. In the same year, Rhoades (1983) reported increased levels of anti-herbivore resistance in undamaged Sitka willow trees that grew close to herbivore-infested conspecific plants, and Baldwin and Schultz (1983) found increased chemical defence levels in poplar and sugar maple saplings sharing the same air with damaged plants. Apparently, the attacked plants had ‘warned’ their neighbours. 1. Does ‘plant–plant communication’ exist? Does the above phenomenon represent a true ‘communication’ among plants? Richard Karban suggested to apply the term ‘plant communication’ to situations where cues that are emitted from plants cause rapid responses in a receiver organism and where the emission of these cues is plastic and conditional (Karban, 2008). As we explain below, damaged plants release cues that elicit specific responses in neighbouring intact plants and whose emission is subject to a phenotypic plasticity: following Karban’s definition, plants indeed do communicate! 2. Airborne resistance induction to herbivores and pathogens Since 1983, airborne induction of plant anti-herbivore resistance has been detected in Arabidopsis (Brassicaceae), black alder (Alnus glutinosa, Betulaceae), barley (Hordeum vulgare) and corn (Zea mays, both Poaceae), cotton (Gossypium hirsutum, Malvaceae), the legumes Lima bean (Pha. lunatus) and broad bean (Vicia faba), poplar (Populus euroamericana) and Sitka willow (Salix sitchensis, both Salicaceae), sagebrush (Artemisia tridentata, Asteraceae), sugar maple (Acer saccharum, Aceraceae), and for the solanaceous species wild and cultivated tobacco (Nicotiana attenuata and Nicotiana tabacum) and tomato (Solanum lycopersicum) (Baldwin and Schultz, 1983; Birkett et al., 2000; Bruin et al., 1992; Choh et al., 2006; Dolch and Tscharntke, 2000; Engelberth et al., 2004; Farmer and Ryan, 1990; Glinwood et al., 2004; Godard et al., 2008; Heil and Silva Bueno, 2007;

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Karban et al., 2000, 2006; Kost and Heil, 2006; Rhoades, 1983; Ruther and Fu¨rstenau, 2005; Ton et al., 2007). The first reports on plant–plant communication were criticised for their lack of ecological realism (Baldwin and Schultz, 1983) or of true controls (Rhoades, 1983). Later observations on black alder trees confirmed, however, that individuals growing downwind of manually clipped plants became more resistant to future herbivore attack (Dolch and Tscharntke, 2000; Tscharntke et al., 2001). Plant–plant communication even takes place between species: clipping of sagebrush could induce resistance in neighbouring tobacco plants (Karban, 2001; Karban et al., 2000), and barley exposed to air coming from Cirsium plants exhibited an increased resistance to aphids (Glinwood et al., 2004). However, manual clipping might release unrealistically high amounts of VOCs or even compounds that are not released after herbivore feeding. Wild tobacco is an early pioneer that seldom grows in the immediate vicinity of sagebrush, and cultivated barley does not normally grow close to thistles. While many of the above-mentioned articles thus could (and have been) criticised for a lack of ecological realism, field studies on Lima bean at the plant’s natural growing site revealed that otherwise untreated plants suffered less from herbivory when they were exposed to the air that came from beetledamaged emitters: plant–plant communication indeed takes place under ecologically realistic conditions (Heil and Silva Bueno, 2007). Most reported cases of airborne resistance induction concern herbivore resistance. Are similar phenomena also involved in pathogen resistance? In tobacco, MeSA is enzymatically converted back to SA by SABP2, and SA then forms the active resistance-inducing compound (Forouhar et al., 2005; Kumar and Klessig, 2003; Park et al., 2007). In principle, this opens the possibility of airborne signalling also in the context of pathogen resistance, and expression of resistance to viruses has indeed been reported in tobacco plants that were exposed to the MeSA-rich air coming from infected plants (Shulaev et al., 1997). Moreover, many aspects of pathogen resistance, particularly to necrotrophic pathogens, appear to depend on oxylipins rather than SA (Pieterse et al., 1998; Truman et al., 2007; Walters et al., 2006). Oxylipinderived green-leaf volatiles (GLVs) might, therewith, also mediate pathogen resistance: exposure to GLVs such as trans-2-hexenal, cis-3-hexenal or cis-3hexenol enhanced resistance of Arabidopsis against the fungal pathogen Bo. cinerea (Kishimoto et al., 2005), and VOCs released from resistanceexpressing seedlings enhanced resistance of Lima bean to the biotrophic bacterial pathogen Ps. syringae (H. S. Yi, M. Heil and C.-M. Ryu unpublished data). Although generalisations are still difficult, it appears, therefore, that airborne plant–plant communication commonly triggers resistance against both herbivores and pathogens.

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3. Mechanisms of airborne plant–plant communication Studies aimed at a mechanistic understanding of VOC-mediated resistance induction reported changes in the expression of defence-related genes (Arimura et al., 2000; Farag et al., 2005; Godard et al., 2008; Paschold et al., 2006; Ton et al., 2007), increased production rates of MeJA (Godard et al., 2008), JA or of defensive compounds (Baldwin and Schultz, 1983; Engelberth et al., 2004; Farmer and Ryan, 1990; Ruther and Fu¨rstenau, 2005), and even the increased production of indirect defences such as VOCs (Ton et al., 2007) and EFN (Choh et al., 2006; Kost and Heil, 2006). Plants smell, and the components of their odour are well investigated, but how do plants scent? Compounds that are released from damaged plants and that trigger defensive responses in yet undamaged plants are still being discovered, but most of the volatiles that so far have been identified in this context were either gaseous derivatives of JA and SA (MeJA and MeSA) or GLVs (Arimura et al., 2000; Engelberth et al., 2004; Kost and Heil, 2006; Ruther and Fu¨rstenau, 2005; Ruther and Kleier, 2005). Both MeSA and MeJA can be converted back into the respective plant hormone, a mechanism that provides an obvious explanation for the action of these two particular volatiles. In contrast, little is known about the signalling responses to GLVs. Although it has been suggested that GLVs with an , -unsaturated carbonyl group can trigger defence through their activity as reactive electrophile species (Alme´ras et al., 2003), other biologically active GLVs lack this property (M. Heil et al., 2008; Kost and Heil, 2006; Ruther and Kleier, 2005). Changes in transmembrane potentials are involved in early signalling events in the cellular response to stress (Maffei et al., 2007), and exposure to GLVs such as cis-hexenyl acetate changed membrane potentials in intact Lima bean leaves (M. Maffei, personal communication). It is therefore tempting to speculate that dissolving of volatiles in the membranes leads to changes in transmembrane potentials or somehow disintegrates the membrane and thereby induces gene activity. Apart from MeSA, MeJA and GLVs, cis-jasmone can trigger defensive responses via airborne transport. However, this herbivore-induced volatile activates different sets of genes compared to MeJA (Birkett et al., 2000; Bruce et al., 2007), which suggests a different mode of action. Correspondingly, cis-jasmone failed to induce EFN secretion in Lima bean, a JA-responsive trait that can be elicited by cis-3-hexenyl acetate (Kost and Heil, 2006). In summary, much more research will be required before we understand the mechanisms by which plants perceive, and respond to, resistance-inducing odours.

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4. Priming by VOCs Exposure to lower concentrations of volatiles, which failed to activate defence to its full extent, often did affect plant resistance via a sensitisation process (Choh and Takabayashi, 2006; Engelberth et al., 2004; Heil and Kost, 2006; Heil and Silva Bueno, 2007; Kessler et al., 2006; Ton et al., 2007). This so-called priming prepares the plant to respond more rapidly and/or effectively to subsequent attack and normally is activated at much lower concentrations of the resistance-inducing signal than full resistance induction (Bruce et al., 2007; Van Hulten et al., 2006). Priming by local application of chemicals such as ASM (acibenzolar-S-methyl, CGA 245704, also known as BTH for benzothiadiazole-7-carbothioic acid S-methyl ester) (Cools and Ishii, 2002; Kohler et al., 2002) or -aminobutyric acid (Zimmerli et al., 2000) or by initial infections is best investigated in the context of pathogen resistance (Conrath et al., 2006; Goellner and Conrath, 2008). In contrast, airborne priming has been reported for corn (Engelberth et al., 2004; Ton et al., 2007), Lima bean (Choh and Takabayashi, 2006; Heil and Kost, 2006; Heil and Silva Bueno, 2007), tobacco (Kessler et al., 2006) and poplar (Frost et al., 2007), but so far only in the context of direct and indirect defence against herbivores (Frost et al., 2008), although it apparently plays a role also in airborne disease resistance (H. S. Yi et al., unpublished data).

III. COSTS OF INDUCED RESISTANCE Plants are under continual attack from microbial pathogens and herbivorous insects. Surviving this interminable onslaught requires the ability to escape attack, to resist attack or to tolerate a certain level of damage. Resisting attack in turn requires adequate defences and plants possess a battery of mechanisms to keep attackers at bay. However, activation of defences requires energy and resources, resulting in the need to distribute these resources among growth, development and defence (Herms and Mattson, 1992). Plant defence can therefore be costly and such costs can be separated into direct and indirect costs (Fig. 1). Direct costs include autotoxicity, allocation and opportunity costs, and are defined as those responses and their consequences which are a direct result of the plant response to a single attacker (Heil, 2002; Kliebenstein and Rowe, 2008; Korves and Bergelson, 2004). Indirect costs, in contrast, occur in the presence of other species, with, for example, resistance to one species associated with increased susceptibility to another species (Strauss et al., 2002). Indirect costs can be considered as ecological costs. The diversion of limited resources away from plant growth and development towards defence represents an allocation cost (Gomez

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Resistance induction

Direct costs

Autotoxicity

Allocation costs

Opportunity costs

Indirect costs

Effects on other pathogens

Effects on beneficial microbes Effects on insects

Fig. 1. Direct and indirect costs of plant resistance to pathogen infection. Autotoxicity results from negative effects of a resistance or defence trait on the plant’s own metabolism, while allocation costs result from the allocation of limited resources to resistance or defence rather than to growth or reproduction. Short-term reductions in growth resulting from the synthesis of resistance compounds are not important costs in their own right. Nevertheless, if they lead to a reduced ability to compete for soil nutrients or light, thereby giving neighbouring plants an advantage, this represents an opportunity cost. Indirect costs can include effects on other pathogens, effects on insect pests, and effects on beneficial microbes such as mycorrhizal fungi and nitrogen-fixing symbioses.

et al., 2007; Walters and Heil, 2007). In contrast, ecological costs arise when the expression of a defence trait negatively interacts with one of the other ecological interactions that a plant has with its environment (Walters and Heil, 2007). A. ALLOCATION COSTS

There is now considerable evidence that induced resistance to insects incurs costs (e.g., Zavala et al., 2004). In contrast, the situation with respect to pathogens is less clear (Walters and Heil, 2007). In some pioneering work more than 25 years ago, Smedegaard-Petersen and Stølen (1981) observed a 7% reduction in grain yield in barley plants inoculated with the powdery mildew fungus Blumeria graminis f.sp. hordei compared with uninoculated control plants. They suggested that the reduction in grain yield was the result of increased dark respiration, required to provide resistance to pathogen infection (Smedegaard-Petersen and Stølen, 1981). However, these studies were conducted in the presence of the pathogen and as a result, cannot be

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used to quantify the costs associated with induced resistance. Later work by Heil et al. (2000) showed that the chemical inducer ASM applied to wheat in the absence of pathogen pressure, reduced plant growth and yield and provided a clear indication that use of ASM incurred allocation costs. Since then similar results have been reported in other crop plants, including sunflower (Prats et al., 2002), tobacco (Csinos et al., 2001), cauliflower (Ziadi et al., 2001), strawberry (Hukkanen et al., 2007), melon (Buzi et al., 2004) and cowpea (Latunde-Dada and Lucas, 2001). Further evidence that SAR incurs costs comes from studies on Arabidopsis mutants that overexpress SAR; such transformants usually have stunted growth and reduced seed yields (Bowling et al., 1994; Greenberg et al., 2000; Jirage et al., 2001; Mauch et al., 2001; see Heil and Baldwin, 2002 for an overview). In contrast to these data, work by Verberne et al. (2000) showed that transgenic tobacco expressing up to a 1000fold increase in levels of SA and SA-glycoside were not altered in growth or phenotype. These discrepancies between the different SAR mutants in terms of plant growth and development remain unexplained. Yet, application of JA to induce resistance to herbivorous insects often led to reduced seed production and leaf growth (Alves et al., 2007; Baldwin, 1998; Redman et al., 2001). Perhaps this should not be surprising, since JA is known to be associated with accelerated senescence (Wasternack, 2007). As for the mechanisms by which SAR-associated costs are incurred, resistance induction often has negative effects on the amount or activity of proteins functioning in plant carbon or nitrogen assimilation (Jain and Srivastava, 1981; Logemann et al., 1995; Reinbothe et al., 1993), which can reduce net photosynthetic rates (Pancheva et al., 1996; Scharte et al., 2005). These results have been confirmed by gene array studies, which also indicate that genes involved in photosynthesis and growth are down-regulated during the expression of induced resistances to pathogens or herbivores (Heidel and Baldwin, 2004; Scheideler et al., 2002). It is notable that plants may compensate for this effect, at least in uninfected leaves (Roberts and Walters, 1986; Rooney and Hoad, 1989; Williams and Ayres, 1981). For instance, inoculation of the lower leaves of broad bean with rust led to increases in both photosynthesis and resistance to rust infection in upper leaves (Murray and Walters, 1992) and the authors suggested that without the increased rates of photosynthesis, assimilates to fund defence responses would need to be found from existing resources, diverting them away from plant growth and development (Murray and Walters, 1992). Work on coffee treated with ASM found differences in gene expression in roots and leaves (De Nardi et al., 2006). Here, the main response in leaves was an increase in physical and chemical barriers, together with repression of genes involved in photosynthesis and general cell metabolism. In roots on the

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other hand, genes responsible for cell wall reinforcement were up-regulated, accompanied by down-regulation of glutathione-S-transferase and superoxide dismutase, both of which were up-regulated in leaves (De Nardi et al., 2006). Perhaps unsurprisingly, genes involved in energy metabolism are up-regulated in plants expressing systemic resistance, highlighting the need to provide energy for resource-demanding defence responses (De Nardi et al., 2006; Schenk et al., 2003). It seems likely therefore that a ‘switch from housekeeping to pathogen defence metabolism’ may be a prerequisite for the full commitment of a plant to transcriptional activation of resistance pathways (Logemann et al., 1995; Scheideler et al., 2002). During SAR, whether costs are incurred and the magnitude of such costs will depend on environmental factors, both abiotic and biotic. It is also likely to be genotype dependent, although this has received little attention to date. Whether or not costs were incurred in wheat and Arabidopsis following induction of resistance depended on nitrogen supply (Dietrich et al., 2005; Heil et al., 2000). Similarly, successful induction of resistance by ASM in barley was dependent on establishment of a mycorrhizal association (Sonnemann et al., 2005). The importance of the rhizosphere community in the ability of plants to express induced resistance is becoming increasingly evident. For example, the root-colonising basidiomycete Piriformospora indica has been shown to induce resistance in barley to powdery mildew and to root rot caused by Fusarium culmorum (Waller et al., 2005). Moreover, there was a significant increase in grain yield, thus challenging the idea that enhanced resistance imposes a cost. Further, a recent report by Nair et al. (2007) suggests that the presence of certain rhizobacteria may be able to compensate for some of the costs associated with induced resistance. In this work, growth of Amaranthus was retarded upon treatment with ASM, but not when plants were treated with a combination of ASM and the rhizobacterial strain Pseudomonas fluorescens PN026R. The workers suggested that the growth retardation effect of ASM was compensated for by the growthpromoting properties of the rhizobacteria. B. ECOLOGICAL COSTS

1. Trade-offs with resistance to insects Insect damage leads to the induction of a series of events, including the generation and release of specific signals, the subsequent perception and transduction of those signals and, finally, activation of wound-related defences (Leo´n et al., 2001). This is followed by the generation of secondary signals, leading to the further activation of local and systemic defences (Heil and Ton, 2008; Leo´n et al., 2001). Included among the secondary

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signals are oxylipins (oxygenated fatty acids) such as JA and its volatile methyl ester (MeJA), both of which play an important role in regulating induced resistance to insect attack (Bostock, 2005; Walters et al., 2006; see Sections II.A.2 and II.B.1). The situation is however, considerably more complex than the apparently simple split between SA regulation of pathogen defence and JA regulation of defence against insects. Thus, it is known that the nature of the activated signalling pathway depends on the particular plant/insect combination (Bostock, 2005), since both SA- and JA-responsive gene expression can be elicited by aphids and whiteflies, while MeSA is induced by aphid attack in maize (Bernasconi et al., 1998; Walling, 2000). Nevertheless, there are several examples of negative cross-talk between the JA and SA signalling systems, especially for SA-mediated suppression of JA-inducible gene expression (Glazebrook et al., 2003; Heil and Bostock, 2002; Koornneef and Pieterse, 2008; Van Wees, 1999). For example, activation of SA-dependent SAR has been shown to suppress JA signalling, thereby compromising the plant’s ability to induce defences to insect attack (Stout et al., 1999; Thaler et al., 1999, 2002). Indeed, tobacco plants expressing TMV-induced SAR were more susceptible to grazing by the tobacco hornworm Manduca sexta than non-induced plants (Preston et al., 1999), while application of ASM to fieldgrown tomato plants reduced resistance to the beet armyworm Spodoptera exigua (Thaler et al., 1999). Recent work on Arabidopsis demonstrated that SA-mediated defences triggered by a virulent strain of the biotrophic pathogen Ps. syringae increased susceptibility of infected tissues to infection by the necrotroph Alt. brassicicola by suppressing JA signalling (Spoel et al., 2007). Interestingly, SA-induced responses have also been shown to be suppressed by JA, although reports of this phenomenon are few (Bostock, 2005; Glazebrook et al., 2003; Niki et al., 1998). It is important to note that although many trade-offs between induced resistance to pathogens and insect pests are negative (Heil and Bostock, 2002), there are reports of no effect of induced resistance to pathogens on resistance to insects (Ajlan and Potter, 1992; Inbar et al., 1998) and even reports of positive effects. For example, Stout et al. (1999) found that inoculation of tomato leaves with Ps. syringae pv. tomato induced resistance against both Ps. syringae pv. tomato and the corn earworm Helicoverpa zea, while grazing of Rumex obtusifolius by the beetle Gastrophysa viridula reduced infection by a number of fungal pathogens (Hatcher and Paul, 2000). This cross-talk between the JA and SA signalling pathways highlights the complex nature of signalling for disease and pest resistance. An interesting insight into how plants integrate insect- and pathogen-induced signals into specific defence responses was provided by De Vos et al. (2005). Working on

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Arabidopsis, they tracked the dynamics of SA, JA and ET signalling following attack by pathogens and insect pests. When global gene expression profiles were compared, the workers found considerable overlap in the changes induced by pathogens and insects. Thus, all of the different pathogen and insect attackers stimulated JA biosynthesis, although most of the changes in JA-responsive gene expression were attacker-specific (De Vos et al., 2005). It was suggested that although SA, JA and ET play a primary role in orchestrating plant defence, the final defence response is shaped by other regulatory mechanisms, for example, cross-talk between different pathways. Indeed, ET produced by Arabidopsis following damage by Pieris rapae was found to prime the plant for enhanced SA-mediated defences activated following infection by Turnip crinkle virus (De Vos et al., 2006). As indicated above, signalling cross-talk is thought to help plants to decide on the most appropriate defensive strategy to employ, depending on the lifestyle of the attacker. In an interesting twist, attackers appear to have evolved the ability to manipulate plants for their own benefit, by suppressing induced defences or modulating the defence signalling network (Pieterse and Dicke, 2007). For example, a recent study indicates that herbivorous nymphs of the silverleaf whitefly (Bemisia tabaci) may activate the SA signalling pathway as a decoy strategy to sabotage JA-dependent defences and so enhance insect performance (Zarate et al., 2007). Plants, however, might fight back and suppress specific direct defences in the favour of more general indirect defence strategies when fed upon by specialist herbivores, which are resistant to their host plant’s direct, chemical defences (Kahl et al., 2000). Meanwhile, pathogens can trick plants into mounting inappropriate defences by producing hormones or their functional mimics to manipulate the plant’s signalling network (Robert-Seilaniantz et al., 2007). A good example is the production of coronatine, a functional mimic of JA, by virulent Ps. syringae (Krumm et al., 1995; Nomura et al., 2005). 2. Trade-offs with mutualistic symbioses Because induced resistance is a broad-spectrum resistance against microorganisms, it is likely to affect plant interactions with mutualistic symbionts, such as mycorrhizal fungi and nitrogen-fixing bacteria (Heil, 2002). Unfortunately, this area of research has received little attention to date. However, some studies of the legume-Rhizobium symbiosis have shown that application of SA to the rooting substrate exerted a negative effect on nodule formation and/or functioning (Lian et al., 2000; Martı´nez-Abarca et al., 1998; Ramanujam et al., 1998), while leaf treatment of Vicia faba plants with ASM led to a reduction in the number and size of nodules compared with untreated controls (Heil, 2001). Although some studies have reported

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negative effects on colonisation of tobacco roots by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae in plants constitutively expressing -1,3-glucanase (Vierheilig et al., 1994), other workers found no effect of ASM treatment on mycorrhizal infection of barley roots (Sonnemann et al., 2002). 3. Does induced resistance alter phytobacterial communities? Because induced resistance generates defence against a broad spectrum of microbial pathogens, it seems reasonable to assume that it would affect a much wider range of microbes, for example, phytobacterial communities. Although any effects of induced resistance are likely to be greatest for endophytic bacterial communities, it might also affect epiphytic communities and rhizosphere bacterial communities. In some interesting recent work, a comparison was made of endophytic and epiphytic bacterial communities on two mutants of Arabidopsis deficient in SA and JA signalling pathways (Kniskern et al., 2007). The results revealed that induction of SA-mediated defences reduced endophytic bacterial community diversity, whereas epiphytic bacterial diversity was greater in plants deficient in JA-mediated defences. As a result, it is possible that natural variation in the induction of systemic defences among plants within a population or between populations might generate spatial and temporal variation in the community dynamics of microbes that depend on plants for survival (Kniskern et al., 2007). But, what about effects on the rhizosphere bacterial community? Rhizosphere bacterial communities can also be affected, as demonstrated by Hein et al. (2008) who found clear differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants, although there was no apparent decrease in rhizosphere microbial diversity due to constitutive SAR expression. 4. Ecological costs of resistance to biotrophic versus necrotrophic pathogens Although direct and indirect costs have been well studied in plant–insect interactions, much less is known about these costs in interactions between plants and pathogens. Nevertheless, evidence suggests that necrotrophic plant–pathogen interactions impart an ecological cost on plant resistance to biotrophic pathogens. Kliebenstein and Rowe (2008) define this ecological cost as resulting from any host mechanisms which provide resistance to biotrophic pathogens, but increase susceptibility to necrotrophs. For example, although a HR is frequently associated with resistance to biotrophic pathogens, it can provide potential entry points for necrotrophic pathogens. Thus, when leaves were pre-treated with avirulent biotrophic bacteria capable of inducing a HR, and the necrotrophic pathogen Bo. cinerea was placed in the middle of the pre-treated zone, the resulting lesions were much larger than those produced

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by Bo. cinerea in the absence of bacterial pre-treatment (Govrin and Levine, 2000). Interestingly, the timing of the HR with regard to pathogen inoculation is important for determining the ecological risk. For example, mutants of Arabidopsis with a thinner cuticle produce antifungal toxins more rapidly following germination of fungal necrotrophs on the leaf surface. This more rapid response resulted in increased resistance to fungal necrotrophs even in the presence of a biotroph-induced HR (Chassot et al., 2007; Tang et al., 2007). Impressively, Bo. cinerea can delay this rapid host response by producing 2-methyl succinate, which can delay the triggering of plant defences, possibly by modulation of host ABA signalling (Asselbergh et al., 2007). Kliebenstein and Rowe (2008) also suggest that there is a cost to the organism of possessing the machinery for programmed (or organised) cell death. They suggest that the very existence of plant signalling pathways that initiate a HR provides molecular targets that a necrotrophic pathogen could use to facilitate its infection and colonisation by stimulating host cells to kill themselves. This is supported by evidence showing that the virulence of necrotrophic plant pathogens is increased by stimulation of the HR (Govrin and Levine, 2000; Van Baarlen et al., 2007). These data suggest that plants which have evolved in an environment biased towards resistance to biotrophic pathogens, may suffer from the inability to effectively resist necrotrophs. It also raises important questions about how the potential pathogen community encountered by a crop should influence plant breeding strategies (Kliebenstein and Rowe, 2008).

IV. RESISTANCE INDUCED BY MUTUALISTIC MICRO-ORGANISMS Sections III.B.2 and III.B.3 have dealt with the impact of induced resistance on mutualistic symbioses and rhizosphere bacterial communities. However, this interaction functions in both directions and plant resistance can also be induced by non-pathogenic bacteria and mycorrhizal fungi that establish a mutualistic interaction with plant roots. What impact can mutualistic symbioses have on the ability of plants to express induced resistance? A. RESISTANCE MEDIATED BY PLANT GROWTH-PROMOTING RHIZOBACTERIA (PGPR)

Since the presence of many strains of bacteria in the rhizosphere can increase plant growth rates, at least under laboratory conditions, they are collectively termed ‘plant growth-promoting rhizobacteria’ (PGPR). The first reports on

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this phenomenon date back to 1980 (Kloepper et al., 1980), and plant-growth promotion by PGPR is now a generally accepted phenomenon with a wide application even covering restoration ecology (Bacilio et al., 2006; Rodrı´guez et al., 2006). Many of these bacteria also induce a systemic resistance to pathogen infection, a phenomenon that has been termed induced systemic resistance (ISR) and that—in contrast to SAR—depends on JA signalling rather than SA signalling, at least in Arabidopsis (Kloepper et al., 1992; Pieterse et al., 1996, 2001; Ryu et al., 2004a). The bacteria-derived volatile, 2,3-butanediol, has been reported to cause the resistance-enhancing effect which is achieved with PGPR strains Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a (Ryu et al., 2004b) but has not been detected in other plant–PGPR systems. According to Glick (2005), the mechanisms postulated to explain the growth-promoting effects of PGPR can be categorised as direct and indirect. Direct mechanisms include the production of plant hormones, nitrogen fixation and phosphate solubilisation by the bacteria, while indirect mechanisms result from suppressing growth of plant pathogens and harmful rhizosphere microbes, thereby releasing the plant from the growth limitations associated with the presence of such microbes (Kloepper, 1992; Van Loon, 2007). Many species of bacteria are known to produce auxin, and indeed the growthpromoting effects of the PGPR Azospirillum brasilense on wheat and pearl millet have been shown to be due to auxin production by the bacterium (Barbieri and Galli, 1993). In this work, bacterial mutants that had lost most of their capacity to produce indole-acetic acid had lost their growthpromoting activity. Modulation of plant growth by PGPR can also involve other hormones. For instance, ET is known to reduce root growth at moderate levels (Abeles et al., 1992). The ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which is exuded from roots along with other amino acids, can be utilised by rhizobacteria as a carbon source following breakdown by ACC deaminase. This bacterial catabolism of ACC prevents re-uptake of ACC by the roots, lowering its level in the root and reducing ET production. This in turn relieves the inhibition of root growth by ET and represents a mechanism by which ACC deaminase-containing rhizobacteria can increase root growth (Glick, 2005). In some interesting work using Arabidopsis mutants, Ryu et al. (2005) found that PGPR-induced growth promotion under gnobiotic conditions in vitro involved brassinosteroid, SA and gibberellin signalling, while growth promotion in vivo involved ET signalling. Besides promoting growth, nitrogen-fixing Rhizobia can also induce resistance following infection and colonisation of legume roots. For example, Arfaoui et al. (2006) identified a number of isolates of Rhizobia which reduced the incidence of infection of chickpea by F. oxysporum f.sp. ciceris,

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although this effect was genotype-dependent, being more effective in moderately resistant varieties than in susceptible ones. Subsequent work showed that treatment of chickpea plants with Rhizobia isolates led to enhanced expression of genes encoding phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase, and accumulation of phenolic compounds. Similarly, colonisation of pea roots by Rhizobium leguminosarum resulted in accumulation of phenolics and pisatin and induced resistance to the parasitic plant Orobanche crenata (Mabrouk et al., 2007). Interestingly, a suppression of SA responses is required for the establishment of a functional Rhizobium-legume association (Stacey et al., 2006), although whether this is associated with an enhancement of JA responses is not known. B. RESISTANCE INDUCTION BY MYCORRHIZA

Arbuscular mycorrhizas (AM) are found in the vast majority of terrestrial plant species. AM fungi (AMF) are obligate biotrophs since they are dependent on the host plant for reproduction and survival (Smith and Read, 1997). The AM symbiosis is well known to provide benefits to the host plant in terms of fitness, for example through enhanced mineral nutrition as well as increased ability to cope with biotic and abiotic stresses (Pozo and Azco´nAguilar, 2007). AM can provide growth benefits to plants, particularly under nutrient-limiting conditions, although it is well established that plants vary in their responsivness to AMF and many plant and environmental factors can influence the response (Jakobsen et al., 2002; Smith and Read, 1997). There are a number of reports indicating an effect of mycorrhizal infection on plant defence against pathogens (Pozo and Azco´n-Aguilar, 2007). For example, colonisation of tomato roots by Gl. mosseae induced cell defence responses and local and systemic protection against Phytophthora parasitica, while infection of barley roots with Gl. mosseae induced systemic protection against the take-all fungus Gaeumannomyces graminis f.sp. tritici (Cordier et al., 1998; Khaosaad et al., 2007). AMF have also been reported to protect chilli plants against infection by Phytophthora capsici (Alejo-Iturvide et al., 2008), tomato against infection by Alternaria solani (Fritz et al., 2006), Medicago truncatula against the bacterial pathogen X. campestris (Liu et al., 2007), and Musa sp. against infection by plant-parasitic nematodes (Elsen et al., 2008). Mycorrhizal infection and colonisation have also been shown to modify the effectiveness of induced resistance. Thus, Sonnemann et al. (2005) found that at low and medium levels of colonisation of barley roots by Glomus etunicatum, ASM had either no effect or decreased foliar infection by powdery mildew, while high levels of mycorrhizal colonisation increased mildew infection. According to Pozo and Azco´n-Aguilar (2007), although infection by AMF usually leads to

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reduced infection by soilborne pathogens, effects on shoot pathogens are dependent on the lifestyle of the attacker. Thus, resistance induced by AMF in shoots appears to be effective against necrotrophic pathogens and generalist chewing insects, but less so against biotrophic pathogens. It has been proposed that the establishment of a functional mycorrhizal symbiosis requires a partial suppression of SA-dependent responses in the plant, compensated for by an enhancement of JA-regulated responses (Pozo and Azco´n-Aguilar, 2007). Therefore, the resistance induced by AMF correlates with a potentiation of JA-dependent defences and the priming of tissues for effective defence activation following challenge (Pozo and Azco´n-Aguilar, 2007). The mechanistic basis of most of these effects remains, however, to be investigated. AMF and Rhizobia positively affect their host plant’s nutritional status, and host resistance has repeatedly been shown to be limited be resource supply (Section III.A). Mutualistic micro-organisms thus might enhance host resistance by direct resistance induction or by reducing resource limitations and, thus, allocation costs of resistance. Future studies will be needed to disentangle these two possible mechanisms.

V. DEFENCE SIGNALLING AT THE LEVEL OF PLANT INDIVIDUAL, COMMUNITY AND EVOLUTION A variable host resistance phenotype makes phenotypic and evolutionary adaptations of the pathogens more difficult. The variability per se that results from signalling is, thus, beneficial as compared to constitutive, static resistance strategies (Agrawal and Rutter, 1998). Moreover, resistance traits are costly (see above), and plants therefore suffer from a ‘growth-differentiation’ dilemma, that is, they can allocate their limited resources to growth or defence, but not both (Herms and Mattson, 1992). Defence signalling allows expression of resistance traits only when they are required and might, thus, also present simply a ‘cost-saving’ strategy (Heil and Baldwin, 2002). However, variation in the level and expression of defensive traits occurs, and this variation occurs at the level of species, phenotype and even among different organs of the same individual. The following paragraphs try to summarise the little knowledge that we have so far on patterns of—and reasons for— varying defence signalling at these different levels. A. VARIABLE RESISTANCE AT THE GENETIC LEVEL

Systemic resistance to pathogens is assumed to be a general phenomenon but in fact has been demonstrated only for a few plant species, most of which are crop plants (Heil, 1999). In fact, different plant species respond differently to

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the same type of infection or induction. For example, a screening of species with different life histories indicated that plants flowering early in spring tend to express little SAR, most probably because they generally develop and reproduce under low pathogen pressure (Heil and Ploss, 2006). In Arabidopsis, the PGPR strain Ps. fluorescens WCS417r elicited ISR on all ecotypes examined apart from two, Wassilewskija (Ws-0) and RLD (Ton et al., 1999; Van Wees et al., 1997). Later work showed the presence of a locus (ISR1) involved in the ethylene signalling pathway in Arabidopsis. The ecotypes Ws-0 and RLD carried a recessive trait that affected ISR by perturbing ethylene signalling, although these plants could still express SAR (Ton et al., 2001). Clearly therefore, in ecotypes of Arabidopsis, allelic variability exists in genes that exert an influence on ISR pathways. Similarly, different tobacco cultivars differed in their resistance expression in response to PGPR, although not to treatment with ASM (Ryu et al., 2007). B. VARIABLE RESISTANCE AT THE PHENOTYPIC LEVEL

Different species respond differently to the same elicitor, but even individuals of the same species do not necessarily behave in the same way. For a certain genotype, both the net costs of resistance induction (Cipollini, 2002; Cipollini et al., 2003; Heil et al., 2000) and the level of resistance that is expressed in response to a standardised induction event (Cipollini and Bergelson, 2001; Dietrich et al., 2004) can be affected by environmental factors such as resource availability and competition. Plants might, therefore, obtain fitness benefits by reducing their defence investments in situations where future competition is likely (Cipollini, 2004). Surprisingly enough, a connection between far-red sensing and defence induction has been found in a South American native tobacco species, Nicotiana longiflora (Izaguirre et al., 2006). Plants that were exposed to far-red-supplemented lateral light to mimic the presence of competitors were less resistant to specialist herbivores and showed impaired defence induction when actually being damaged. Plants use far-red sensing to monitor the presence of other plants (Ballare´, 1999), and they are able to adjust their actual defensive efforts according to the presence or absence of competitors (Izaguirre et al., 2006). C. PLANT–PLANT COMMUNICATION AT THE COMMUNITY LEVEL?

One of the major arguments against airborne plant–plant communication concerned the possibility of a ‘runaway’ process: if VOCs induce herbivore resistance in neighbouring plants, and if a part of this resistance response consists of the release of VOCs, should not all plants in nature be fully

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induced? However, several researchers found induction of herbivore resistance in nature (Baldwin, 1998; Heil, 2004; Heil et al., 2001), and ASM treatment induced the activity of at least one class of PR-proteins in 17 out of 18 wild species, which represented twelve plant families (Heil and Ploss, 2006). In addition, exposure to beetle-damaged Lima bean shoots reduced herbivore damage on receivers at the plant’s natural growing site in the coastal area of Southern Me´xico (Heil and Silva Bueno, 2007). Plants growing under natural conditions are not necessarily fully induced, and plant–plant communication can indeed occur under ecologically realistic conditions! How is, then, the ‘runaway’ of defences in natural plant communities avoided? A major limiting factor in this context appears to be distance: most studies on plant–plant communication found effects in plants that were growing very close to the emitter, such as wild tobacco plants growing 15 cm downwind from clipped sagebrushes (Karban et al., 2000) and in black alders growing at a distance of 1 m from clipped trees (Tscharntke et al., 2001). Likewise, in their experiments referred to above, Heil and Silva Bueno (2007) had intertwined senders and receivers to mimic the natural growth of Lima bean, a liana. Although generalisations are so far impossible, it seems safe to assume that VOC-mediated plant–plant communication takes place only over short distances. Due to their rapid diffusion under natural conditions it is plausible that resistance-inducing volatiles are normally diluted to priming or—at larger distances—completely inactive concentrations. Systematically investigating the maximum distance over which cues can be exchanged would be important but will be difficult: volatiles diffuse in the air and move by eddy current dispersal; the distances over which they can affect other plants thus depend strongly on wind speed, air humidity and temperature. Much more has to be done before we can determine how common plant–plant communication is under realistic conditions and how it acts at the level of plant communities. Plant–plant communication offers considerable potential for use in protecting crops against pests and possibly also pathogens. However, research on plant–plant communication in crop situations lags behind work on natural systems, with precious little information available on inter-plant communication in relation to pathogens. A good example of the use to which knowledge of signalling in plant–biotic interactions can be put, is the work on the push-pull approach for controlling insect pests and weeds (Hassanali et al., 2008). The push–pull effect exploits semiochemicals to repel insect pests from the crop (push) and to attract them into trap plants (pull). This has been shown to work particularly well against lepidopterous pests on maize and other cereals, and also against the parasitic plant Striga (Hassanali et al., 2008).

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D. EVOLUTIONARY CONSIDERATIONS

Monitoring enemy pressure and inducing costly resistance traits only when they are actually required appears to represent a cost-saving means of enabling beneficial phenotypic plasticity, and the evolutionary fixture of induced resistance strategies is thus easy to understand (Agrawal, 1999). In fact, beneficial effects of induced defences on fitness-relevant traits have been reported for various plant species, and both in the context of resistance to herbivores (Agrawal, 1998; Baldwin, 1998; Heil, 2004) and pathogens (Heidel and Dong, 2006; Traw et al., 2007). An evolutionary problem arises, however, when we consider plant–plant communication. First, our knowledge on the fitness effects on both partners is surprisingly restricted. Communication requires an emitter and a receiver, but the physiological, ecological and—ultimately—the fitness effects of this interaction might differ among the partners. While communication among animals often benefits sender and receiver, plant–plant communication appears much more unidirectional: in fact, most published examples on VOC-mediated plant–plant communication changed the receiver’s resistance level without having apparent direct effects on the emitter. Why should plants warn their neighbours that enemies are around? Using information on the status of attack of a neighbour benefits the receiver at the cost of the— already damaged (!)—emitter, which competes with the receiver for space, light, water and nutrients. How can such a signal be evolutionarily stable? One explanation appears to lie in our second level of defence-signalling: as predicted by Farmer (2001) and Orians (2005), VOCs can mediate signalling among different parts of the same plant individual (Frost et al., 2007; Heil and Silva Bueno, 2007; Karban et al., 2006). Airborne systemic signalling, instead of signalling via the vascular system, is faster, independent of orthostichy and allows a priming of distant plant parts in order to achieve an optimised systemic defence expression (Heil and Ton, 2008). Plants, therefore, do need the capacities to emit and perceive VOCs and cannot completely avoid the danger of ‘eavesdropping’ by identically equipped neighbours. E. PREDICTING PATTERNS OF INDUCED RESISTANCE RESPONSES

Environmental factors such as nutrient availability and the presence of competitors mediate resistance induction, and not all parts of an infected plant respond equally. Moreover, plants that have higher amounts of resources at their disposal can invest more in their resistance, but at the same time they might become more attractive and/or suitable for infecting pathogens. The interplay among plant species, pathogen species, plant nutrition and

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resistance (or susceptibility, respectively) is, therefore, complex and tends not to yield linear responses (Walters and Bingham, 2007). Much more research will be required before we can understand why a plant protects which parts, at what intensity and by which means. This knowledge is crucial when induced pathogen resistance is to be used as a means of crop protection. To give just one example, ASM-mediated resistance induction in wheat cultivated under agricultural field conditions resulted in no positive effect on yield, most probably because the benefits of reduced infection rates were counterbalanced by the costs of SAR induction (Stadnik and Buchenauer, 1999). Although few data exist on how environmental conditions affect the expression or efficacy of SAR, several hypotheses have been published that deal with the interplay of life history, growth rate, environmental conditions and plant defence. Although these hypotheses have been formulated in the context of anti-herbivore defence, they may help to predict—and understand—the variation of pathogen resistance within an individual, among individuals of the same species and among species. 1. Growth-differentiation balance hypothesis (GDBH) and optimal defence hypothesis (ODH) Based on original ideas presented by Loomis (1953), Herms and Mattson (1992) suggested that the synthesis of defensive compounds presents a differentiation process and thus is subject to a general physiological trade-off between growth and differentiation. At the cellular level, both growth and differentiation processes make use of the cellular machinery for transcription and translation and compete for common precursors such as acetyl CoA and amino acids. This trade-off can lead to reduced overall growth rates in plants that express pathogen resistance (see Section III.A), but it restricts particularly the defence of young, fast growing organs, in which cell division and enlargement use up most, if not all, available resources. Moreover, inherently fast growing species (growth-dominated plants) should have lower levels of defence than inherently slow growing species (differentiation-dominated plants). The optimal defence hypothesis (ODH) was originally formulated by McKey (1974) and was later extended (McKey, 1979; Rhoades, 1979) to answer the main question—‘in which regions of the plant should the limited quantity of defensive compounds be concentrated?’ Enemy pressure and fitness consequences of damage are assumed to constitute important evolutionary forces that vary among different parts of a plant, and the defence needs of any part of the plant are assumed to be determined by two factors: (1) ‘value’, or the cost to the plant of damage or loss of the part in question, and (2) ‘vulnerability’, the probability that the part in question would be attacked if not protected.

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The ODH is focussed on what a plant ‘should’ do, while the growthdifferentiation balance hypothesis (GDBH) points out what a plant ‘can’ do; the two hypotheses lead, therefore, partly to contradictory results, particularly with respect to the defence of developing organs. Recent studies demonstrated that indirect defence traits against herbivores (VOCs and EFN) can indeed be expressed according to ODH requirements (Radhika et al., 2008; Rosta´s and Eggert, 2008), and labelling experiments demonstrated that the conflict between ODH and GDBH in this case can be solved by a transport of defence compounds from mature to the younger leaves (Radhika et al., 2008). Future studies are, however, required to unravel whether an active transport of phytoalexins or PR-proteins can also help to protect young organs from pathogen infection. Few studies have focussed on potential trade-offs between pathogen resistance and the growth rates of certain organs, but a general pattern appears that induction of pathogen resistance under enemy-free conditions has a negative effect on the growth rate of entire plants, particularly when occurring under limiting nutrient conditions (see Section III.A). Most intriguingly, inducing resistance in wheat by treatment with ASM caused strongest negative effects on growth when conducted during the tillering stage, hence during the phase in which meristems required for branching are particularly active (Fig. 2). This observation clearly demonstrates a competition between growth and resistance expression. Even the few studies reporting compensatory increases of photosynthetic rates in yet uninfected leaves of locally infected plants are underlining the importance of inherent growth rates. When slow-growing leeks were infected locally with rust, photosynthetic rates in younger, uninfected leaves strongly increased as a compensatory response (Roberts and Walters, 1986). Such effects were, however, quantitatively much smaller in fast-growing species such as barley (Roberts and Walters, 1986; Walters and Ayres, 1983). Other reports are also in line with the GDBH. For example, induction of PR genes in tobacco by exogenous sugars indeed depended on leaf age, since young leaves hardly responded to elicitor concentrations that readily induced mature leaves (Herbers et al., 1996). Similarly, the resistance to long-distance movement of Cauliflower mosaic virus in leaves of turnip and Arabidopsis was impaired in younger leaves (Leisner et al., 1992, 1993). Although generalisations are impossible so far, it appears that induced pathogen resistance can be limited by growth—differentiation trade-offs (Heil and Bostock, 2002). 2. Carbon/nutrient balance hypothesis (CNBH) and resource availability hypothesis (RAH) Both these hypotheses deal with the strategies plants may use to keep their resistance costs low. However, the carbon/nutrient balance hypothesis (CNBH) is formulated at the phenotypic level while the resource availability

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ASM

A Control

B After

C

D During

Lateral shoot production

Fig. 2. Phenological effects on costs of resistance induction. Resistance to pathogens of wheat was induced at different developmental stages by application of the chemical inducer, acibenzolar-S-methyl (ASM). Negative effects on numbers of shoots produced and, thus, seed production were observed only when resistance was induced during the tillering stage (lateral shoot production) and not when ASM was applied later during plant development. This result illustrates the importance of trade-offs between growth and defence induction, as expressed in the ‘growthdifferentiation balance hypothesis’ (GDBH).

hypothesis (RAH) deals with evolved differences among species. Bryant et al. (1983) asked how plants of a given genotype should respond to certain growing conditions and argued that costs of a defence compound depend on the ratio of N and C that are available in the environment in relation to the chemical composition of the compound in question. If, for example, availability of nitrogen limits growth more than photosynthesis, allocating carbon to strictly ‘carbon-based’ defences such as phenolics might cause low (if any) costs, and plants therefore should defend themselves with such compounds. If, however, nutrients are readily available and photosynthesis and growth are limited by other factors such as light availability, allocating nutrients to defence becomes cheap and plants might synthesise ‘N-based’ defences such as alkaloids, non-proteinogenic amino acids, PR-proteins and so on. Indeed, nitrogen availability limited the synthesis of PR-proteins in Arabidopsis (Dietrich et al., 2004), and increased N-supply enhanced

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resistance of potato to the necrotrophic pathogen, Alt. solani (Mittelstrass et al., 2006). In another study, potato plants that were exposed to elevated CO2 became more resistant to late blight caused by Phytophthora infestans, and this increase was correlated with higher activities of the PR-protein, -1,3-glucanase (Plessl et al., 2007). Although we are far from a general understanding, these studies demonstrate that the supply of elements such as N and C indeed can limit a plant’s capacity to induce resistance against pathogen infection. A factor that has not been considered, however, in the original formulation of the CNBH is that the performance of biotrophic pathogens might be more dependent on the nutritive quality of the living plant tissue than the performance of nectrotrophic pathogens. As a consequence, a general pattern appears that N supply or enhanced CO2 increases the resistance to necrotrophic pathogens but increase the susceptibility to biotrophic pathogens (Mittelstrass et al., 2006; Plessl et al., 2007). Coley et al. (1985) wanted to understand ‘why plant species differ in their commitment to defences and hence in their susceptibility to herbivores’ and formulated a theory that, like ODH, deals with optimal strategies, hence discussing what a plant species ‘should’ do. The RAH (Bryant et al., 1985; Coley et al., 1985) predicts type and amounts of defence based on inherent growth rates and leaf life times that have evolved in adaptation to a species’ typical growing sites. Species that are adapted to resource-poor sites are characterised by inherently slow growth rates, low maximum photosynthetic rates and low turnover rates (e.g., long-lived leaves), while the opposite is true for species at resource-rich sites which, moreover, should respond flexibly to pulses in resources and thus should exhibit a high morphological and biochemical flexibility. The optimal level of defence investment is, therefore, assumed to increase as the potential growth rate of the plant decreases (a prediction in line with the GDBH, although derived from a different theoretical framework), since (i) replacement of plant parts lost to herbivores is more costly when nutrients are limiting future growth, (ii) the relative impact of tissue losses increases with decreasing inherent growth rate and (iii) a certain reduction in growth rate due to the cost of producing defences represents a greater absolute growth reduction for fast-growing species than for slow-growing ones. We are not aware of any empirical study that specifically allows the testing of predictions of the RAH in the context of pathogen resistance. However, most of our crop plants, as well as Arabidopsis, are fast-growing species from nutrientrich soils, which exhibit low levels of constitutive resistance, while apparently all crop plants that have been investigated so far showed strong quantitative changes in their overall resistance level following infection. These general observations are thus in line with the predictions of the RAH and GDBH.

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VI. CONCLUSIONS AND OUTLOOK Much has been learned since the earliest discoveries of a plant-wide induced resistance following local virus infection of tobacco leaves (Ross, 1961) or after local herbivore feeding on tomato plants (Green and Ryan, 1972). We now know some major signalling cascades underlying the local perception of pathogens and herbivore attack, we have discovered important systemic signals, and we have characterised hundreds of genes involved in local and systemic resistance expression. Since the earliest discoveries of ‘talking trees’ (Baldwin and Schultz, 1983; Rhoades, 1983), it also became increasingly accepted that systemic signalling does not stop at the plant surface, but even can affect neighbouring plants. The topic, which so far has been mainly investigated under artificial laboratory conditions, appears ready to move to the field: to the agronomic field, since induced resistance offers exciting prospects for using the plant’s own defences for protection against pests and pathogens, and to the natural field, where we wish to understand the importance of induced resistance for the survival of individual plants, for shaping plant community structure and, ultimately, for plant evolution. However, as discussed above, many questions still need to be answered before we can understand the ecology and evolution of induced resistance, and before resistance induced biologically or by means of specific chemical elicitors can serve as a reliable, environmentally friendly strategy for crop protection. Focussing major research efforts on a few selected model species that are regarded as representative of other species has resulted in partly contradictory results, particularly concerning the hormones regulating resistance induction to herbivores and/or to nectrotrophic versus biotrophic pathogens. A considerable part of these apparent contradictions might, however, just be a consequence of different plant species using different signals for mounting phenotypically similar responses. Also, intense, direct activation of defences can be costly (see Sections III.A and III.B), particularly if enemy pressure is low or non-existent. From a crop protection perspective, incurring costs, with possible associated yield reductions, are likely to make direct induced resistance unpopular. In contrast, priming, with its triggering of defences only following pest or pathogen challenge, is a less wasteful use of resources and indeed has been shown to provide benefits to plants under the pressure of pathogens (Van Hulten et al., 2006) or herbivores (Heil and Silva Bueno, 2007). If priming is to find a place in practical crop protection, it will be necessary to increase our understanding of the molecular, physiological and ecological aspects of the phenomenon. The ecology of priming is particularly important, since failure to

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understand priming in crops under field conditions is likely to result in inappropriate and ineffective use of the technology in practice. First important steps towards an application of induced resistance have been made in the field of herbivore resistance and have shown that induced release of VOCs from above-ground parts or certain intercropping strategies can enhance predation and parasitation pressure on leaf herbivores (Kessler and Baldwin, 2001; Khan et al., 1997; Thaler, 1999), while -caryophyllene release from maize roots could guide entomopathogenic nematodes towards root-feeding beetle larvae (Rasmann et al., 2005). Much more has to be done, however, to disentangle the complex interplay of abiotic factors such as soil nutrients, biotic factors such as the presence of pathogenic and herbivorous enemies, and beneficial micro-organisms, which all affect the net outcome of a given resistance induction treatment, on plant health, growth and yield.

ACKNOWLEDGMENTS D.R.W. receives funding from the Rural Environment Research and Analysis Directorate of the Scottish Government, M.H. gratefully acknowledges financial support from the Consejo Nacional de Ciencia y Tecnologı´a de Me´xico (CONACyT) and from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG).

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AUTHOR INDEX

Numbers in bold refer to pages on which full references are listed.

A Aarts, N., 96, 112, 404, 426 Abbott, D.W., 569, 595 Abdallah, M.A., 247, 273, 500, 511–512, 516–517, 542 Abdin, M.Z., 451, 480 Abe, H., 373, 384 Abeles, F.B., 293, 310, 688, 699 Abel, P.P., 65, 74 Abel, S., 588, 600 Abe, M., 129, 131, 156 Abramovitch, R.B., 54, 74 AbuQamar, S., 447, 474 Aderem, A., 5, 9, 15, 28 Adie, B.A., 672, 699 Afzal, A.J., 458, 474 Agorio, A., 104, 112 Agrawal, A.A., 379–380, 384, 690, 693, 699 Agrios, G.N., 148, 157 Agusti, N., 569, 595 Aharoni, A., 640, 651 Ahl, P., 513, 532 Ahn, I.P., 149, 152, 157, 231, 246, 262, 266, 363, 371, 384 Aist, J.R., 190, 209 Ajlan, A.M., 684, 699 Akira, S., 4, 9, 15–16, 20, 28 Akiyoshi, D.E., 292, 310 Akram, A., 231, 267 Alabouvette, C., 342, 356, 514, 532 Alborn, H.T., 132, 138, 157, 571–573, 595, 617, 619, 651 Aldwin, I.T., 629, 651 Alejo-Iturvide, F., 689, 699 Alfano, G., 340, 350 Alfano, J.R., 4, 10, 27, 28, 441, 474 Allen, M.D., 409, 496 Allis, C.D., 420–421, 436 Alme´ras, E., 679, 700 Alonso, J.M., 236, 267 Alonso, W.R., 626, 651 Alstro¨m, S., 226, 267 Altamura, M.M., 452, 476 Altenbach, D., 93, 112, 564–565, 595 Altincicek, B., 469, 474 Altomare, C., 335, 350

Alvarez, M.E., 140, 157, 363, 385 Alvarez-Venegas, R., 422–423, 425–426, 426 Alves, M.N., 682, 700 Amasino, R.M., 422, 430 Ament, K., 622, 628, 630–632, 635, 651 Amme, S., 628, 651 Amoutzias, G.D., 500, 542 Anand, A., 452, 474 Anandalakshmi, R., 70, 74 Ananieva, E.A., 468, 474 Anderson, A.J., 325, 327, 351 Anderson, J.P., 674, 700 Anderson, P., 639, 651 Andre´asson, E., 416, 426, 588, 595 Anfoka, G., 195, 209 An, H., 128–129, 157 Antonious, G.F., 627, 651 Antoniw, J.F., 182, 186, 202, 221, 675, 714 Antnez-Lamas, M., 50, 74 Arfaoui, A., 690, 700 Argueso, C.T., 294, 310 Arimura, G.-I., 562, 595, 645, 647, 651, 679, 700 Arnold, D.L., 50, 59, 63, 74, 106, 113 Arshad, M., 291, 293, 313 Asai, T., 12, 23, 28, 404, 416, 426, 445–446, 474, 577, 580, 595 Ashfield, T., 57, 74, 106, 113 Aslam, S.N., 41, 51, 64, 74, 257, 267 Assaad, F.F., 101–102, 108, 113 Asselbergh, B., 43–44, 75, 674–675, 687, 700 Atchley, W.R., 412, 427 Atreya, C.D., 70, 75 Attaran, E., 406, 426 Attard, A., 528, 532 Aubourg, S., 637, 651 Audenaert, K., 229, 252, 254–255, 263, 267, 515, 532, 672, 700 Ausubel, F.M., 2–5, 18, 27–28, 237, 270, 308, 310, 405, 435, 441, 468, 473–474 Avni, A., 7, 20, 36, 93, 119 Axtell, M.J., 56, 75 Ayre, B.G., 129, 157 Ayres, P.G., 682, 695, 715

718

AUTHOR INDEX

Azco´n-Aguilar, C., 266, 276, 322, 324, 326–328, 356, 364, 392, 689–690, 710 Azco´n, R., 325, 350 Azevedo, C., 97, 113 Aziz, A., 7, 11, 28 Aziz, N., 625–626, 651 B Baath, E., 323, 350 Bacilio, M., 690, 700 Backus, E.A., 560, 563, 595–596 Badel, J.L., 58, 60, 75 Bae, H., 12, 28, 580, 596 Baena-Gonza´lez, E., 449, 474 Bailey, B.A., 6, 28 Bakan, B., 153, 157 Baker, R., 342, 355, 511–512, 514, 536, 545, 548 Baker, S.C., 525, 547 Bakker, A.W., 511, 516–518, 532 Bakker, P.A.H.M., 151, 157, 227, 233, 247, 271, 279, 301, 315, 319, 491–532, 532, 538 Balague´, C., 108, 113 Balbi, V., 134, 157 Baldwin, I.T., 136–137, 162, 168, 366, 377, 379–380, 385, 393–394, 561–563, 570, 583, 585, 588, 601–602, 610, 615, 627–629, 632, 635, 637, 642–645, 651, 655, 657–658, 662, 665, 668–670, 673–674, 676–682, 684, 690, 692–695, 698–699, 700, 704–706, 715 Ballare´, C.L., 693, 700 Bandurski, R.S., 288, 311 Barash, I., 291, 310 Barber, D.A., 494, 533 Barbieri, I., 289–290, 310 Barbieri, P., 289, 310, 688, 700 Barea, J.M., 286, 291, 310 Barna, B., 200–201, 210 Barnes, H.H., 504, 533 Barriuso, J., 228, 233, 263–264, 267 Bartel, B., 287, 320 Bartel, D.P., 69, 75 Barthakur, S., 452, 474 Barton, G.M., 22, 28 Bartsch, M., 98, 113 Bashan, B., 190–191, 210 Basse, C.W., 7, 9, 28, 43, 75 Bassler, B.L., 245, 274 Bastow, R., 422, 427 Batalia, M.A., 450, 465, 474 Bateman, A., 19, 29 Bate, N.J., 647, 652

Bauer, D.W., 10, 29 Bauer, P., 498, 533 Bauer, W.D., 299, 310 Bauer, Z., 16, 29 Baulcombe, D.C., 65–67, 75, 80, 676, 704 Baumann, P., 568, 596 Baureithel, K., 7, 9, 29 Baxter, K.J, 644, 657 Bayer, E.M., 458, 463, 474 Bayliss, C., 294, 310 Beale, M.H., 640, 652 Becker, J.O., 511, 533 Beckers, G.J.M., 227, 267, 632, 652 Beckers, G.M.J., 371, 385 Be´clin, C., 70, 75 Bede, J.C., 583–584, 596, 604, 618, 652 BeVa, R.S., 453, 475 Belaish, R., 469, 475 Be´langer, R.R., 191, 196, 210 Belkhadir, Y., 56, 75 Bender, C.L., 52, 76, 557, 596 Benderoth, M., 635, 652 Benfey, P.N., 414, 427 Benhamou, N., 151, 157, 191, 196, 210, 342–343, 346, 350, 372, 385 Benitez, T., 334–336, 350 Bennett, R.N., 637, 652 Ben Rebah, F., 306–307, 310 Benson, D.M., 323, 331–332, 354, 356 Bent, A.F., 141, 157, 581–582, 596 Bent, E., 227, 233, 267 Bentley, W.E., 299, 314 Bereczky, Z., 496–497, 533 Berenbaum, M.R., 553–554, 560, 582, 588, 596 Bergelson, J., 379, 390, 668, 691, 700, 702 Berger, S.L., 373, 385, 421, 427 Berg, G., 493, 533 Bergstrom, G.C., 202, 210 Berkhout, B., 73, 76 Berkowitz, G.A., 51, 83 Berna, A., 444, 475 Bernasconi, M.L., 684, 700 Bernier, F., 444, 475 Bernier, G., 125–128, 157, 166 Bernstein, E., 67, 76 Bertin, C., 614, 652 Besson-Bard, A., 499, 533 Bhat, R.A., 102, 113 Bhattarai, K.K., 586, 592, 596 Bigirimana, J., 229, 252, 267, 336, 350 Biles, C.L., 343, 351 Binder, B.M., 149, 157, 403, 427 Bingham, I.J., 694, 715 Birch, L.E., 522, 533 Birch, P.R.J., 52, 87 Birkett, M.A., 628, 652, 677, 679, 701 Bisaro, D.M., 70, 76 Bisgrove, S.R., 95, 113

AUTHOR INDEX Bittel, P., 4, 19–20, 24, 27, 29, 441, 475, 555–556, 564, 596 Bitter, W., 509, 533 Blaha, D., 294, 310 Blain, S., 531, 533 Blanco, F., 414, 427 Bleecker, A.B., 19, 36, 236, 267, 403, 427, 458, 485 Blee, K.A., 325, 327, 351 Bleeker, P.M., 613–650 Blight, M.M., 637, 652 Blilou, I., 326–327, 351 Block, A., 95, 113, 199–200, 210 Bloemberg, G.V., 301–302, 310, 312 Boatright, J., 622, 652 Boch, J., 370, 385 Bodenhausen, N., 561–562, 596 Bogdanove, A.J., 58, 76 Bo¨hlenius, H., 129, 131, 157 Bohlmann, J., 623, 652 Boiero, L., 295, 310 Boland, A., 560, 562, 597 Boland, W., 567, 576, 605 Bo¨lker, M., 526, 533 Boller, T., 5–6, 8–9, 16, 18, 20, 23, 29–31, 41, 76, 94, 115, 234, 237, 270, 565, 599–600 Bonanomi, A., 327, 351 Bonello, P., 177, 210 Bonfante, P., 324, 351 Bonhomme, F., 127, 157 Boraston, A.B., 569, 595 Borevitz, J.O., 411, 427 Bo¨rger, H., 41, 84 Borowicz, V.A., 322, 326, 351 Bos, J.I.B., 48, 76 Bossier, P., 500, 533 Bossis, E., 500, 533 Bostock, R.M., 175, 181, 210, 449, 479, 671, 684, 695, 701, 705 Boter, M., 413, 427 Bottini, R., 293, 315 Bouarab, K., 44, 76 Boughammoura, A., 523, 533 Boukhalfa, H., 500, 533 Bowling, S.A., 108, 113, 236, 267, 370, 380, 385, 456, 475 Boyle, C., 185, 198, 208, 210, 222 Bozarth, R.F., 186, 210 Brading, P.A., 226, 238, 267 Bradley, D., 126, 158 Brandl, M.T., 289, 310, 557, 603 Brazelton, J.N., 253, 267 Bressan, R.A., 439–474 Bretz, J.R., 62, 76 Briat, J.-F., 491–532 Bridges, M., 589, 596 Brigneti, G., 66, 70, 76 Brishammar, S., 195, 220

719

Brisset, M.N., 183–184, 210 Britigan, B.E., 254–255, 267 Brodersen, P., 98, 108, 113, 416, 427, 579, 596 Broekaert, W.F., 442, 475 Broekgaarden, C., 563, 596 Bronner, R., 574, 596 Brotman, Y., 334, 351 Browse, J., 237, 273, 400, 433 Bruce, T.J.A., 628, 652, 679–680, 701 Bruin, J., 677, 701 Brumbarova, T., 498, 533 Brunner, F., 7–8, 29 Bryant, J.P., 697, 701 Bublin, M., 465, 475 Buchel, A.S., 424, 427 Buchenauer, H., 195, 209, 694, 712 Buck, M.J., 412, 427 Budzikiewicz, H., 501, 534 Bufe, A., 442, 475 Buhot, N., 145, 158 Burch-Smith, T.M., 111, 113 Burdman, S., 284–285, 305–306, 310–311 Butterbrodt, T., 424, 427 Bu¨ttner, M., 410, 427 Buyer, J.S., 494, 504, 512, 534, 541 Buysens, S., 511, 534 Buzi, A., 682, 701 Bycroft, M., 19, 29 C Cabrera, J.C., 567, 597 Cahill, D.M., 206, 210 Cairns, B.R., 423, 428 Calderwood, S.B., 518, 540 Callebaut, I., 467, 475 Calvert, O.H., 107, 118 Cameron, R.K., 123–156, 158, 178, 188, 198–199, 210, 363, 385, 676, 701 Campbell, B.C., 567, 599 Campelo, A.B., 296, 312 Canavoso, L.E., 568, 597 Cancel, J.D., 403, 427 Cantu, D., 567, 597 Cao, H., 110, 113, 142, 146, 158, 225, 237, 267, 370, 385, 406, 427 Cardoza, Y.J., 619, 652 Carimi, F., 672, 701 Carlsen, H., 449, 475 Carmichael, N.M., 449, 475 Carmona, M.J., 131, 158 Carpita, N., 557, 597 Carreno-Lopez, R., 290, 311 Carrington, J.C., 66, 70, 82 Carr, J.P., 202–203, 211, 217 Carroll, M.J., 617, 652 Carthew, R.W., 66, 73, 76, 83, 87

720

AUTHOR INDEX

Cartieaux, F., 363, 385 Cartwright, K., 440, 476 Caruso, F.L., 178–179, 198–199, 210 Casacuberta, J., 379, 385 Casteel, C.L., 592, 597 Catala, R., 132, 158 Catanzariti, A.M., 94, 114 Catinot, J., 621, 653 Celenza, J.L., 411, 427 Century, K.S., 96, 148, 158, 237, 268 Cessna, S.G., 44, 76 Chae, H.S., 294, 311 Chai, J., 95, 121 Chailakhyan, M.K., 125, 127, 158 Chakrabarti, C., 450, 478 Chakravarthy, S., 410, 427 Champigny, M.J., 123–156, 188, 210, 363, 385, 676, 701 Chandanie, W.A., 323, 345, 351 Chang, C., 236, 268, 403, 428 Chang, P.F.L., 341, 351 Chang, S., 423, 428 Chanjirakul, K., 616, 653 Chapman, E.J., 72, 76 Charmont, S., 457, 463, 476 Chassot, C., 365, 377, 385, 687, 701 Chaturvedi, R., 144–145, 148, 150, 153, 158 Che, F.S., 10, 29 Chellappan, P., 67, 76 Chen, C.C., 462, 476 Chen, F., 628, 644, 653 Cheng, A.X., 623, 640, 653 Cheng, C.S., 456, 476 Cheng, W., 493, 534 Chen, H., 584, 597 Chen, M.S., 561, 589–590, 594, 597 Chen, W.P., 451, 476 Chen, X., 69, 77, 452, 454, 476 Chen, Z.Y., 55–56, 77, 254, 268, 290, 311, 456, 476, 671, 673, 701 Cheong, J.J., 136, 158 Cheong, Y.H., 410, 428 Chester, K.S., 139, 158 Chet, I., 333, 340, 351, 358 Chin-A-Woeng, T.F.C., 302, 304, 311 Chinchilla, D., 21–22, 25, 29, 234, 268, 404, 428, 445, 476, 565, 597 Chini, A., 402, 428, 674–675, 701 Chisholm, S.T., 2–4, 27, 29, 41, 77, 93–95, 114, 224, 268, 554, 597, 669, 701 Chivasa, S., 202–204, 211 Choh, Y., 650, 653, 679–680, 701 Choi, Y.D., 136, 158 Choquer, M., 567, 597 Cho, S.M., 256, 268, 300, 311 Christians, M.J., 403, 428 Chrousos, G.P., 449, 477 Chu, K.T., 450, 476

Chung, H.Y., 449, 476 Chung, K.M., 673, 701 Cintas, N.A., 52, 77 Cipollini, D.F., 379, 668, 691, 702 Clapier, C.R., 423, 428 Clark, E., 288, 311 Clarke, A., 21, 29 Clark, F.E., 493, 534 Clark, K.L., 403, 428 Clay, N.K., 42, 77 Cloud, A.M.E., 193, 211 Clough, S.J., 108, 114 Coaker, G., 26, 29 Cobbett, C.S., 496, 538 Cody, Y.S., 521, 534 Cogoni, C., 65, 77 Cohen, A.C., 295, 311, 567, 569, 595, 597 Cohen, J.D., 288, 311 Cohen, Y.R., 178, 181, 184, 190–191, 194, 210–211, 365, 380, 382, 386, 677, 702 Cohn, J.R., 55, 77 Colangelo, E.P., 251, 268, 497–498, 534 Colcombet, J., 577, 579, 597 Coley, P.D., 697, 702 Collins, N.C., 92, 101–102, 114, 375, 386 Collmer, A., 4, 28, 58, 84, 441, 474 Conconi, A., 138, 158 Conn, E.E., 554, 597 Connolly, E.L., 497, 499, 534, 548 Conn, V.M., 232–233, 264, 268 Conrath, C.P., 367–369, 386 Conrath, U., 148, 151, 158, 176, 188, 206, 211, 227, 260, 267–268, 328, 351, 361–384, 386, 406, 428, 649, 653, 680, 702, 704 Consonni, C., 102, 114 Constabel, C.P., 366, 386, 561–562, 604, 674, 702 Contesto, C., 294, 311 Conti, G.G., 190–191, 211 Cook, D.N., 15, 29 Cook, R.J., 302, 311, 511, 531, 533 Cools, H.J., 191, 211, 371, 386, 680, 702 Cooper, A.J., 208, 211 Cooper, L.D., 573, 597 Coppinger, P., 148, 158 Corbesier, L., 127–131, 159 Cordier, C., 151, 159, 328, 352, 373, 386, 689, 702 Cornelis, P., 299, 317, 501–502, 504, 534 Cornelissen, B.J., 450, 476 Corrado, G., 640, 653 Cosma, M.P., 421, 428 Costacurta, A., 289, 291, 311 Costa, R., 304, 311 Coudron, C.A., 621, 666 Couldridge, C., 563, 597 Coupland, G., 128, 131, 159

AUTHOR INDEX Cournoyer, B., 59, 77 Covey, S.N., 67, 77 Crespi, M., 292, 311 Creus, C.M., 300, 312 Croft, K.P.C., 621, 653 Croteau, R., 626, 660 Crowley, D.E., 508, 517–518, 534, 548 Cruickshank, I.A.M., 41, 77, 178, 194, 211 Crumbliss, A.L., 500, 533 Csinos, A.S., 682, 702 Cui, J., 94, 145, 150, 159 Curie, C., 496, 535 Custers, J.H., 444, 476 D Da Cunha, L., 42, 45, 77, 94–95, 114, 581–582, 597 Dalisay, R.F., 188, 206, 211 Dalmay, T., 66, 77 D’Angeli, S., 452, 476 Dangl, J.L., 2–4, 10, 13, 27, 29, 32, 41, 45, 49, 56, 58, 64, 77, 81, 85, 95, 114, 174, 215, 224, 265, 272, 404, 431, 442, 445, 480, 553–555, 601 Danie, X., 411, 428 Dann, E.K., 177, 182, 194, 208, 212 Dardanelli, M.S., 305, 312 Dardick, C., 468, 476 Darvill, A., 7, 11, 29 Datta, K., 452, 476 Datta, S.K., 442, 476 D’Auria, J.C., 621–622, 649, 653 Davies, P.J., 286, 312 Davis, R.M., 324–325, 352 Dawson, G.W., 589, 597 Dayan, F.E., 625, 653 De A Campos, M., 453, 466, 477 Dea´k, M., 529, 535 Dean, R.A., 179, 189, 207–208, 212 Debener, T., 58, 77 Debergh, P.C., 632, 659 De Boer, J.G., 628, 653 De Boer, M., 511, 535 Debro, P., 440, 477 DebRoy, S., 58, 60, 78, 84 De Bruyne, M., 637, 653–654 De Cal, A., 323, 344, 352 Deepak, S.A., 192, 211 De´fago, G., 299, 313 Degen, T., 636, 654 Degrave, A., 524, 535 De Groot, A., 234, 268 Dehne, H.W., 327–328, 352 Delaney, T.P., 139–140, 142, 156, 159, 167, 175, 196, 205, 212, 225, 237, 268, 363, 370, 386 Dellagi, A., 521–522, 524, 529, 535

721

Delledonne, M., 22, 29 Delphia, C.M., 642, 654 De Lucca, A.J., 442, 477 De Meyer, G., 229, 252, 263, 268, 333, 352 De Moraes, C.M., 582, 609, 633, 642, 654 Dempsey, D.A., 446, 477 De Nardi, B., 682–683, 702 Denny, T.P., 257, 269 Denoux, C., 235, 240, 269, 566, 577–578, 580–581, 598 De Salamone, I.E.G., 291, 312 Deshmukh, S., 329, 352 Desikan, R., 9, 29 Deslandes, L., 95, 114, 417, 428, 590, 598 De Souza, J.T., 244–245, 269 Despre´s, C., 99, 114, 141, 146, 155, 159, 407, 414, 428–429 Desveaux, D., 57, 78, 265, 277 De Torres, M., 55, 78, 580, 598 De Torres-Zabala, M., 43, 55, 78, 670, 702 Deverall, B.J., 176–177, 182, 184, 192–194, 211–212, 220 De Vleesschauwer, D., 149, 151, 159, 176, 212, 223–266, 269, 301, 312, 363, 386, 515, 535 De Vos, A.M., 450, 477 De Vos, M., 412, 429, 553, 561–562, 577, 585–586, 588, 598, 684–685, 702 Devoto, A., 134, 157, 426, 429 De Weert, S., 301–302, 312 De Weger, L.A., 234, 239, 242–243, 269 De Wit, P.J.G.M., 41, 45, 78, 82 D’Haeze, W., 286, 312 Dhawan, R., 448, 477 Dicke, M., 559, 607, 615, 626, 628, 638, 643, 653–654, 663, 665, 670, 674, 676, 685, 702–703, 709 Dietrich, R.A., 108, 114, 683, 691, 696, 703 Diez, J.J., 463, 477 Dijkman, H., 674, 702 Dimond, A.E., 366, 378, 388 Ding, S.-W., 70, 72, 78, 82 Ding, X., 671, 703 Divol, F., 563, 598 Dixelius, C., 473, 486 Dixon, M.S., 45, 78 Dixon, R., 327, 353 Djamei, A., 579, 598 Djavaheri, M., 230, 239–240, 248, 250, 264, 269, 515, 535 Djonovic´, S., 333, 340–341, 352 Doares, S.H., 138, 159, 445, 477 Dobbelaere, S., 284–285, 289, 296, 298, 304–305, 307, 309, 312 Dobereiner, J., 296, 312 Dobert, R.C., 293, 312 Do´czi, R., 579, 598 Dodds, P.N., 95, 114, 590, 598

722

AUTHOR INDEX

Doehlemann, G., 47, 78 Dogimont, C., 591, 598 Doke, N., 42, 78 Dolch, R., 677–678, 703 Dolgov, S.V., 451, 485 Doliana, R., 465, 477 Dombrecht, B., 413, 429 Dombrowski, J.E., 133, 159 Domenech, J., 228, 232–233, 263, 269 Donaldson, P.A., 444, 477 Dong, H., 184, 212 Dong, X., 55, 78, 99, 114–115, 141, 149, 159, 175, 213, 225, 250, 254, 269, 278, 362–363, 367, 386, 398, 414, 429, 436, 446, 477, 553, 559, 609, 675, 693, 703–704 Donofrio, N.M., 196, 212 Dore, I., 457, 465, 477 Do¨rZing, K., 43, 78 Doss, M., 198–200, 212 Doss, R.P., 573, 598 Dostert, C., 14, 30 Douchkov, D., 102, 115 Dou, D., 94, 115 D’Ovidio, R., 567, 598 Dow, M., 241, 269 Downie, J.A., 286, 316 Drechsel, H., 500, 535 Dreyer, D.L., 567, 599 Drukker, B., 638, 642, 654 Druzhinina, I.S., 333, 352 Dubuis, C., 302, 304, 313 Dudareva, N., 616, 621, 623, 654–655 DuVy, B.K., 299, 313 Dugardeyn, J., 670, 703 DuijV, B.J., 266, 269, 344, 352, 504, 509, 512–514, 516, 535–536 Du, L., 405, 429 Duman, J.G., 469, 488 Dunning, F.M., 234, 269 Dunn, P.E., 575, 606 Dunoyer, P., 67, 69, 72, 79 Du, Q.S., 67, 79 Durner, J., 21–22, 30 Durrant, W.E., 99, 115, 175, 213, 225, 250, 269, 362–363, 367, 386, 446, 477, 675, 703 D’Urzo, M.P., 439–474 Duus, K., 465, 477 Dwivedi, D., 302, 304, 313 E Ebel, J., 41, 79 Ebhardt, H.A., 70, 79 Ecker, J.R., 236, 270, 403, 430, 624, 655 Eckhardt, U., 496, 536 Edel, V., 493, 536 Edwards, O., 564, 576, 599

EVmert, U., 633, 654 Eggert, K., 633, 662, 695, 711 Ehness, R., 672, 711 Eichenseer, H., 575, 583–584, 599 Eichhorn, H., 521, 526, 536 Eide, D., 496, 536 Elad, Y., 511–512, 536 Elasri, M., 504, 536 Elbashir, S.M., 66, 79 Elenkov, I.J., 449, 477 Ellis, C., 236, 269, 401, 429 Ellis, J.G., 585–586, 598 Elliston, J., 192–193, 213 Elmerich, C., 296, 313 Elsen, A., 689, 703 Elzinga, R.J., 560, 599 Emery, T., 512, 536 Engelberth, J., 14, 30, 377, 387, 649, 654, 677, 679–680, 703 Engelhardt, S., 10–11, 30 Enkerli, J., 7, 30 Erbs, G., 5–6, 30, 374, 387 Eriksson, S., 128, 131, 159 Escolar, L., 504, 536 Espinosa, A., 62, 79 Eulgem, T., 23, 30, 99, 115, 224, 270, 411, 415, 429 Evans, L.T., 127, 162 Expert, D., 491–532, 536 F Fabritius, A.-L., 8, 30 Fagard, M., 521, 524, 536 Fagoaga, C., 451, 478 Faize, M., 187, 197, 208, 213 Falk, A., 143, 159, 236, 270 Fallik, E., 307, 313 Fang, X., 463, 467 Fan, M,-S., 531, 536 Fan, W., 414, 429 Farag, M.A., 646–647, 649, 654, 679, 703 Faraldo-Go´mez, J.D., 501, 537 Farmer, E.E., 133–134, 136, 159, 398, 435, 620, 627, 644, 648, 654, 675, 677, 679, 693, 703 Farrokhi, N., 572, 599 Fatouros, N.E., 573–574, 599, 618, 654 Fauth, M., 7, 11, 30 Feilner, T., 577, 599 Feil, S.C., 13, 35 Feistner, G.J., 503, 537 Felix, G., 3, 5–6, 8–9, 20–21, 24, 27, 30, 38, 41, 76, 234–235, 265, 270, 281, 565, 599 Fellbrich, G., 6, 9, 12–14, 30 Felton, G.W., 560, 562, 567, 583–584, 587, 599, 607 Feng, S., 402, 429

AUTHOR INDEX Ferguson, B.J., 293, 313 Fernando, W.G.D., 676, 703 Ferrandon, D., 4–5, 15–16, 20, 30 Ferrari, S., 445, 478, 567, 599 Feys, B.J.F., 236, 270, 401, 405, 429 Fierens, E., 453, 465, 478 Finlay, R.D., 322, 324, 352 Finn, R.D., 450, 459, 461, 463, 478 Fire, A., 66, 73, 79 Fliegmann, J., 7, 16, 30 Flores, T., 443, 478 Flor, H.H., 95, 115, 445, 478 Flo, T.H., 519, 537 Fobert, P.R., 141, 146, 155, 159 Fodor, J., 201, 213 Follett, B.K., 125, 160 Forde, B.G., 125, 160 Forouhar, F., 145, 160, 675, 678, 703 Forslund, K., 592, 599 Fowler, S., 128, 160 Foyer, C.H., 255, 270 Fraga, R., 298, 317 Francia, D., 365, 387 Frankenberger, W.T., 291, 293, 313 Franken, P., 322, 324, 352–353 Franza, T., 502, 504–505, 523–524, 537 Frati, F., 567, 600 Fravel, D.R., 343, 353, 355 Freche, B., 449, 478 Freeman, T.P., 584, 600 Freialdenhoven, A., 102, 115 Fridman, E., 626, 654 Fridovich, I., 255, 271 Friedrich, L., 178, 183, 213, 364, 383, 387 Fritz, M., 689, 703 Frost, C.J., 378, 387, 560, 562, 568, 600, 646, 648–650, 655, 676, 680, 693, 703–704 Frye, C.A., 148, 160, 369, 387 Fuchs, J.G., 343, 353 Fu, D.L., 451, 478 Fujimoto, S.Y., 410, 429 Fujita, A.I., 575, 600 Fujita, M., 635, 655 Fulchieri, M., 293, 313 Funk, C.J., 577, 584, 600 Fu¨rstenau, B., 678–679, 711 Fuster, M.D., 380, 387 Fu, Z.Q., 56, 79 G GaVney, T., 141, 160, 175, 183, 213, 226, 238, 270, 363, 387, 446, 478 Gaille, C., 405, 429 Galli, E., 289, 310 Gallucci, S., 11, 31 Gamalero, E., 518, 537 Gang, D.R., 626, 655

723

Gan, L., 468, 478 Gao, L.-L., 332, 353, 403, 430, 585–586, 592, 600 Gao, M.H., 579, 600 Gaquerel, E., 617, 655 Garand, C., 342, 350 Garcı´a-Garrido, J.M., 322, 324–327, 353 Garcı´a-Lepe, R., 344, 353 Garcı´a Mata, C., 528, 537 Garcı´a-Rodrı´guez, S., 325, 353 Garcion, C., 97, 115 Gasciolli, V., 67, 79 Gasque, P., 468, 478 Gatz, C., 397–426 Gaudriault, S., 58, 79 Gaulin, E., 7, 31 Ga¨umann, E., 42, 79 Gaymard, F., 491–532 Gechev, T.S., 12, 31 Geelen, J.L.M.C., 186, 201, 221 Geels, F.P., 505, 511, 537 Geigenberger, P., 366, 387 Gerhenzon, J., 588, 601 Germeier, C., 44, 79 Gershenzon, J., 616, 655 Gessler, C., 190, 220 Geurts, R., 286, 313 Ghai, R., 465, 478 Ghosh, R., 450, 478 Ghysels, B., 502, 537 Gibbs, A., 72, 82 Gijzen, M., 12–13, 31 Gilliland, A., 204, 213 Gill, P.R. Jr., 513, 537 Gimenez-Ibanez, S., 55, 79 Girardin, S.E., 4–5, 15, 31 Glazebrook, J., 12, 31, 97–98, 105, 113, 115, 120, 175–176, 205, 208, 213, 225, 236–237, 259, 270, 553, 558–559, 577, 584–585, 600, 628, 655, 670, 684, 704 Glick, B.R., 288–289, 294–295, 313, 316, 688, 704 Glickmann, E., 288, 313 Glinwood, R., 677–678, 704 Godard, K.A., 677, 679, 704 Godiard, L., 22, 31 Godoy, G., 44, 79 Goel, A.K., 59, 79 Goellner, K., 680, 704 Goggin, F.L., 585–586, 591, 600, 609 Go¨hre, V., 25, 31, 55, 80, 224, 270 Goldbach, R.W., 39–74 Golden, H.B., 449, 479 Go¨llner, K., 367, 386 Gols, R., 635, 655 Go´mez-Ariza, J., 379, 387 Go´mez-Go´mez, L., 16, 18, 21–23, 31, 94, 115, 234, 237, 270, 565, 600

724

AUTHOR INDEX

Gomez-Roldan, V., 286, 313 Gomez, S., 680, 704 Goodger, J.Q.D., 125, 167 Goodman, H.M., 462, 479 Goodman, R.M., 175, 207, 221, 324, 357 Gophna, U., 53, 80 Goritschnig, S., 111, 115 Go¨rlach, J., 183, 213, 364, 387 Goto, K., 71, 80, 129, 168 Gough, J., 461, 479 Gouinguene´, S.P., 634, 636, 655 Govrin, E.M., 180, 213, 687, 704 Graham, J.H., 325, 353 Graham, M.Y., 449, 479 Graham, T.L., 200, 214 Granado, J., 7, 9, 31 Grant, M.R., 58, 80, 141, 145, 160, 250, 273, 363, 387 Grassmann, R., 73, 80 Grasswitz, T.R, 642, 657 Graziano, M., 499, 537 Greenberg, J.T., 225, 270, 448, 479, 682, 704 Green, T.R., 132–133, 160, 365, 376–377, 387, 674, 698, 704 Grenier, J., 465, 479 Gries, D., 518–519, 534 GriVth, M., 454, 465, 479 Grimmelikhuijzen, C.J.P., 594, 600 Gro¨nberg, H., 331, 353 Gross, A., 242, 265, 270 Gross, D.C., 521, 534 Grubb, C.D., 588, 600 Grumet, R., 65, 80 Gru¨ner, R., 424, 430 Gudesblat, G.E., 52, 80 Guedes, M.E.M., 140, 142, 160 Guerinot, M.L., 251, 268, 493–494, 496–498, 500, 534, 536–537 Guo, A., 408, 430 Guo, F.-Q., 22, 31 Guo, H., 403, 430 Guo, Z., 454, 458, 468, 476, 479 Guo, Z.J., 454, 479 Gurr, G.M., 626, 663 Gust, A.A., 5–6, 20, 31, 575, 600 Guterman, I., 621, 655 Gutterson, N., 512, 537 Guttman, D.S., 42, 49, 83 Gu, Y.-Q., 410, 430 Guzma¨n, P., 236, 270, 403, 430, 624, 655 Guzzo, S.D., 257, 270 Gyaneshwar, P., 298, 313 H Haas, D., 302, 304, 313 Haas, H., 500, 538 Haasnoot, J., 73, 80 Habibi, J., 575, 600

Hahlbrock, K., 326, 357 Hahn, M.G., 41, 80 Halim, V.A., 8, 10, 31, 195, 214 Halitschke, R., 561, 570, 579, 600, 615, 617, 619, 621, 628, 630, 635–636, 642, 655–656 Halkier, B.A., 588, 601 Hall, B.P., 624, 656 Hamdan, H., 513, 537 Hamiduzzaman, M.M., 376, 388 Hamilton, A.J., 66–67, 80, 676, 704 Ham, J.H., 59–60, 80, 105, 115 Hammerschmidt, R., 173–209, 214, 362, 372, 388 Hammond, S.M., 66, 80 Hann, D.R., 10, 32, 53–55, 80, 234, 271 Han, S.H., 229, 256, 258, 271 Hantke, K., 503, 538 Harari, A., 289, 314 Hardoim, P.R., 285, 314 Hare, J.D., 637, 656 Haring, M.A., 613–650 Harman, G.E., 323, 333, 335–337, 339, 353, 357 Harmel, N., 584, 601 Harrewijn, P., 614, 656 Harrison, M., 327, 353 Hartmann, A., 284, 289–290, 296, 301, 314, 318 Hase, S., 260, 271, 323, 346, 353, 372, 388 Hassanali, A., 692, 706 Hassan, H.M., 255, 271 Hastings, M.H., 125, 160 Hatanaka, A., 621, 656, 660, 676, 709 Hatcher, P.E., 684, 704 Hauck, P., 25, 32, 53, 80 Hause, B., 136, 160 Hawkins, C.P., 560, 601 Hayama, R., 131, 160 Hayashi, F., 17–18, 32 Haydon, M.J., 496, 538 Hayman, D.S., 323, 350 Heath, M.C., 42, 80 Heck, S., 98, 115 He, C.Y., 371, 373, 388 Heese, A., 565, 601 Heidel, A.J., 379–380, 388, 561–562, 601, 693, 704 Heil, M., 136, 160, 208, 214, 222, 226, 256, 271, 363, 377–379, 388, 449, 479, 650, 656, 667–699, 704–705, 707, 715 Hemmerlin, A., 623, 656 Hemmes, H., 72, 80–81 Hempel, F.D., 126, 160 Henkels, M.D., 509, 541 He, P., 25, 32, 53–54, 80, 417, 430 Herbers, K., 325, 354, 366, 378, 388, 695, 705

AUTHOR INDEX Herde, O., 138, 160 Herms, D.A., 690, 694, 705 Herms, S., 185, 214, 382, 388 He, S.Y., 6, 9, 25, 32 Hevisi, M., 198–200, 212 He, Y., 422, 430 Hietala, A.M., 331, 354 Higashi, K., 416, 430 Hildebrand, J.G., 637, 663 Hilker, M., 572, 601 Hill, L.M., 199, 219 Hiltner, L., 284, 308, 314, 493, 538 Himber, C., 69, 81 Hinsch, M., 61, 81 Hinsinger, P., 284, 314, 493, 538 Hirai, M.Y., 411, 430 Hirt, H., 577, 579, 597 Hissen, A.H.T., 250, 271 Hoad, G.V., 682, 711 Hoballah, M.E.F., 637, 643, 656 Hodge, S., 365, 388 Hoene, M., 473, 479 HoZand, E., 251, 258, 271 Ho¨fte, M., 149, 151, 159, 176, 212, 223–266, 271, 301, 312, 363, 386, 511, 538 Hogen Esch, T., 563, 586, 609 Hogenhout, S.A., 558, 581–582, 601 Hohnadel, D., 500, 538 Holland, M.A., 292, 314 Holopainen, J.K., 635, 656 Holsters, M., 286, 312 Holt, B.F., 97, 115 Honeycutt, E.W., 331, 354 Hon, W.C., 444, 454, 479 Hooshangi, S., 299, 314 Hopke, J., 571, 601, 627, 656 Hopkins, R.J., 588, 601 Ho¨rdt, W., 517, 538 Horiuchi, J., 644, 647, 656 Hornsperger, J.M., 247, 273 Horsfall, J.G., 366, 378, 388 Horz, W., 425, 435 Hossain, M.M., 152, 160, 262, 271, 323, 344–345, 354 Hotamisligil, G.S., 449, 473, 479 Hotson, A., 56, 81 Houterman, P.M., 47, 81 Ho, V.S., 450, 453, 466, 479 Howe, G.A., 134–135, 137, 161, 163, 167, 238, 271, 365–366, 389–390, 398, 448, 479, 562, 588, 601, 620, 657, 674, 712 Howell, C.R., 323, 333, 336, 341, 354 Howles, P., 95, 115 Hrde, M., 623, 656 Hruz, T., 408, 430 Hsieh, M.H., 462, 479 Hsu, C.-Y., 131, 161 Hua, J., 236, 271, 403, 430

725

Huang, Y., 129, 161, 183, 214, 619, 657 Hu¨ckelhoven, R., 174, 197, 214, 557–558, 587, 601 Hudson, A.O., 98, 115 Hug, C., 465, 479 Hughes, D.T., 299, 314 Hukkanen, A.T., 682, 705 Hunter, S., 459, 461, 480 Husaini, A.M., 451, 480 Hussey, R.S., 325, 354 Huve, K., 623, 657 Hu, X., 444, 456, 479 Hu,Y.-H., 584, 601 Hwang, J., 331, 354 I Iavicoli, A., 151, 161, 230, 253, 262, 271 Ibeas, J.I., 465, 480 Ichimura, K., 23, 32, 400, 431 Igarashi, D., 454, 465, 480 Igari, K., 673, 705 Iglesias, P., 463, 477 Imaizumi, T., 128, 161 Inbar, M., 684, 705 Ingle, R.A., 93, 115 Innamorati, G., 465–469, 480 Innes, R.W., 55, 57, 81, 85, 369, 387 Ipcho, S.V.S., 176, 218 Ipper, N.S., 232, 257, 271 Ishiguro, S., 415, 431 Ishihara, T., 406, 431 Ishii, H., 184, 191, 197, 211, 215, 371, 386, 680, 702 Ishii, K.J., 15, 32 Ishimaru, C.A., 504, 533 Ito, Y., 7, 19, 32 Izaguirre, M.M., 691, 705 J Jabs, T., 14, 32 Jacinto, T., 136, 161 Jackson, R.W., 39–74, 81 Jacqmard, A., 127, 161 Jacques, P., 243–244, 274 Jaeger, K.E., 130, 161 Jagadeeswaran, G., 98, 116, 406, 431 Jahn, C.E., 50, 81 Jakab, G., 365, 376, 382, 389 Jakobsen, I., 689, 705 Jakoby, M., 413, 431, 497, 538 James, D.G., 642, 657 Jameson, P.E., 291, 314 Jamet, E., 458, 480 Jami, S.K., 450, 453, 466, 480 Janda, T., 376, 389 Jander, G., 448, 479, 562, 588, 601 Janeway, C.A. Jr., 4–5, 8, 34

726

AUTHOR INDEX

Janjusevic, R., 54, 81 Jeang, K.-T., 73, 76, 80 Jeblick, W., 368, 389 Jeimy, S.B., 466, 480 Jenks, M.A., 439–474 Jenns, A.E., 140, 142, 161, 178–179, 189, 207, 215 Jeun, Y.C., 195, 215 Jiang, S., 197, 215 Jia, Y., 95, 116, 590, 601 Jih, P.-J., 137, 161 Jin, C.W., 497, 538 Jin, H.L., 676, 706 Jin, J.B., 132, 161 Jin, Q., 52, 81 Jin, R., 462, 480 Jirage, D., 143, 161, 237, 271, 682, 706 Jofuku, K.D., 408, 431 Johannes, E., 134, 165 Johnson, C., 414, 431 Johnson, R., 366, 378, 389 Johri, B.N., 302, 304, 313 Jonak, C., 22–24, 32 Jones, A.M.E., 58, 81, 521, 538 Jones, D.L., 493, 538 Jones, J.D.G., 2–4, 10, 13, 27, 32, 41, 45, 49, 58, 64, 77, 81, 88, 95, 114, 120, 174, 215, 224, 265, 272, 290, 314, 404, 416, 431, 435, 442, 445–446, 480, 487, 553–555, 589, 601, 610, 668, 706 Jones, K.M., 257, 272 Jones, L.R., 40, 81 Jones-Rhoades, M.W., 676, 706 Joo, S., 577, 601 Jørgensen, J.H., 102, 161 Journot-Catalino, N., 416, 431 Judelson, H.S., 8, 30 Jung, G., 500, 535 Jung, H.W., 363, 389, 406, 431 Jun, S.-H., 238, 272 Jurkevitch, E., 509, 518, 538 Jurkowski, G.I., 108, 116 K Kachadourian, R., 503, 524, 538 Kachroo, P., 110, 116, 204, 215 Kadouri, D., 307, 314 KaVarnik, F.A.R., 58, 81 Kagaya, Y., 409, 431 Kahl, J., 632, 657, 685, 706 Kakimoto, T., 292, 314 Kaku, H., 19, 32, 93, 116, 445, 480, 574, 601 Kalde, M., 418, 431 Kaloshian, I., 564, 589, 591–592, 602 Kalpana, K., 452, 480

Kaminaka, H., 413, 431 Kamoun, S., 9, 13, 32, 46, 88, 94–95, 117, 120, 589–590, 610 Kandoth, P.K., 134, 161, 579, 602 Kang, B.R., 229, 272 Kang, J.-H., 136, 161 Kang, L., 103, 105, 116 Kant, M.R., 613–650, 657 Kaparakis, G., 331, 354 Kappers, I.F., 640, 657 Kapulnik, Y., 297, 306, 314 Karban, R., 136, 161, 628, 634, 642, 644, 650, 657, 668–669, 673, 676–678, 692–693, 706 Kars, I., 558, 602 Kasschau, K.D., 66, 70, 82 Katagiri, F., 141, 163, 414, 431 Katiyar-Agarwal, S., 104, 116 Katsir, L., 138, 144, 150, 155, 162, 620, 658 Katz, V.A., 367–369, 389 Kaufmann, S.H.E., 250, 277, 519, 545 Kaur, R., 344, 354 Kauss, H., 186, 190, 219, 368, 377, 382, 389 Kay, S.A., 128, 161 Kazana, E., 589, 602 Kazan, K., 138, 162 Keel, C., 302, 313, 513, 539 Keen, N.T., 41, 58, 82–83 Keese, P.K., 72, 82 Kellogg, E.A., 358, 625 Kemen, E., 94, 116 Kemmerling, B., 1–28, 41, 84, 180, 218 Kempema, L.A., 563–564, 580, 584–586, 602 Kesarwani, M., 414–415, 431 Keshavarzi, M., 41, 82 Kesselmeier, J., 615, 658 Kessler, A., 136, 162, 378, 389, 562, 570, 602, 619–620, 634, 642, 644, 648–650, 658, 669, 680, 699, 706 Kessmann, H., 181, 184, 215, 364, 382, 389 Khalid, A., 288, 314 Khan, Z.R., 699, 706 Kieber, J.J., 236, 272, 294, 311, 403, 431 Kiefer, I.W., 140, 142, 146, 162 Kilic-Ekici, O., 233, 272 Kilz, S., 502, 539 Kim, H.K., 371, 389 Kim, H.-S., 56, 82 Kim, J.-G., 56, 82 Kim, K.-C., 418, 423, 426, 432 Kim, M.G., 25, 33, 200, 215 Kim, M.J., 58, 82, 453, 465, 480 Kim, M.S., 229, 272 Kim, Y.J., 54, 82 Kim, Y.S., 456, 480 King, R.W., 126–127, 162 Kinkema, M., 406, 432

AUTHOR INDEX Kishimoto, K., 621, 640, 645, 647, 649, 658, 678, 706 Kishore, U., 463, 465–469, 481 Klarzynski, O., 7, 33 Kleier, S., 679, 711 Klessig, D.F., 22–24, 38, 145, 163, 449, 488, 670, 678, 707–708, 712 Kliebenstein, D.J., 558, 588, 602, 680, 686–687, 706 Klingler, J.P., 591–592, 602 Kloepper, J.W., 226–229, 272, 275, 363, 389, 510–511, 513, 516–517, 539, 688, 706–707 Kniskern, J.M., 686, 707 Knoester, M., 149, 153, 162, 231, 238, 259, 272 Knogge, W., 12, 37 Knott, J.E., 125, 162 Knudsen, J.T., 621, 658 Kobayashi, D.Y., 106, 116 Kobayashi, K., 454, 465, 481 Kobayashi, M., 289, 314 Kobayashi, T., 249, 272, 498, 539 Kobayashi, Y., 128, 155, 162 Koch, M., 98, 116 Kocsis, M., 382, 389 Koedam, N., 501, 539 Koehle, H., 383, 389 Koga, J., 7, 33, 289, 315 Koganezawa, H., 383, 390 Kogel, K.-H., 197, 215, 554, 602 Kohler, A., 367, 369–370, 376, 390, 680, 707 Koike, N., 345, 354 Koiwa, H., 450, 454, 456, 481 Kojima, S., 131, 162 Komiya, R., 131, 162 Konishi, M., 404, 432 Koornneef, A., 398, 424, 432 Koo, Y.J., 145, 162, 640, 658 Ko¨pke, D., 572, 602 Korneeva, I.V., 452, 481 Korolev, N., 333, 342, 355 Korves, T., 379, 390 Kost, C., 377–378, 388, 650, 656, 678–680, 705, 707 Koster, M., 509, 539 Kovats, K., 190, 195, 215 Krebitz, M., 450, 453, 465–466, 481 Krips, O.E., 636, 660 Kroj, T., 23, 33 Kru¨ger, J., 45, 82 Krumm, T., 685, 707 Kryvych, S., 625, 658 Kuhlmann, M., 413, 432 Kuc´, J., 139–142, 161–162, 169, 175–179, 188–189, 194–195, 198–199, 206–208, 210–212, 214–216, 221, 364–365, 367, 388, 390

727

Kunierczyk, A., 563–564, 579, 586, 588, 603 Kumar, D., 145, 163, 678, 707 Kunkel, B.N., 56, 83 Kunze, G., 6, 8, 18, 33, 404, 432, 565, 603 Kunz, W., 183, 216 Kuo, M.H., 421, 432 Kupchak, B.R., 462, 467, 481 Kvitko, B.H., 10, 33 Kwon, C., 101–102, 116 L Lafontan, M., 463, 481 Lait, C.G., 570, 603 Lakatos, L., 70–71, 82 Lamattina, L., 499, 537 Lamb, C., 141, 145, 160, 363, 387 Lambert, D.H., 325, 355 Lam, E., 94, 116 LaMondia, J.A., 183, 216 Lanahan, M.B., 238, 273 Lanfranco, L., 327, 355 Lang, A., 127, 163 Lange, B.M., 626, 658 Langen, G., 197, 215 Lapitan, N.L.V., 576, 603 Larkin, R.P., 343, 355 Larsen, P.B., 403, 427 Lascombe, M.-B., 143, 163 Latour, X., 493, 499, 509, 539 Latunde-Dada, A.O., 193–194, 216, 371, 390, 682, 707 Lauge´, R., 45, 82 Laurie-Berry, N., 94, 116, 144, 163 Lawrence, S.D., 582, 603 Lawton, K.A., 139–140, 149, 163, 183, 216, 225, 273, 364, 390, 456, 481 Lazarovits, G., 205, 216 Leadbeater, A., 183–185, 216 Lebeer, S., 308, 315 Lebel, E., 414–415, 422, 432 Lecourieux, D., 576–577, 603 Lee, B.-N., 525, 539 Lee, G.I., 134–135, 366, 390, 674, 707 Lee, J., 6, 9–11, 23, 33, 132, 163 Leeman, M., 226, 230–231, 242, 249–251, 273, 374, 383, 390, 510, 512, 514–515, 539 Lee, M.W., 98, 117, 148, 163 Lee, S., 134, 163 Le Hir, R., 674, 707 Leisner, S.M., 695, 707 Leister, R.T., 141, 163 Leitner, M., 619, 622, 659 Lemaitre, B., 22, 33 Lemanceau, P., 491–532, 540 Lenk, A., 91–112 Leone, P., 450, 481

728

AUTHOR INDEX

Leong, S.A., 521, 540 Leo´n, J., 683, 707 Le´on-Kloosterziel, K.M., 372, 390, 457, 481 Leon-Reyes, A., 398, 432 Lerouge, I., 240, 273 Leshkowitz, D., 584, 603 Levine, A., 687, 704 Leyman, B., 376, 390 Lian, B., 685, 707 Li, B., 420, 432 Li, C., 134–135, 164 Lichtenthaler, H.K., 623, 659 Lichter, A., 292, 315 Li, F., 70, 82 Lifschitz, E., 129, 131, 164 Lightner, J., 134, 164 Ligon, J.M., 304, 319 Ligterink, W., 23, 33 Li, J., 21, 33, 70, 73, 82, 134, 163, 418, 423, 432 Li, L., 135, 163, 468, 478, 496–497, 540, 562, 603, 627–628, 659 Lilja, A., 331, 355 Lim, M.T.S., 56, 83 Lin, C.F., 623, 659 Lindbo, J.A., 65, 83 Lindeberg, M., 96, 106, 117 Lindermayr, C., 23, 33 Lindow, S.E., 289, 310, 504, 541, 557, 603 Lindsay, W.L., 493, 540 Ling, H.-Q., 497, 540 Lin, H.H., 670, 715 Linke, C., 366, 379, 390 Lin, M.-K., 129–131, 164 Lin, N.-C., 96, 106, 117 Linthorst, H.J.M., 397–426 Lin, W., 296–297, 315 Lipka, V., 4, 8, 35, 96, 101–103, 106, 117, 375, 390 Li, Q., 561, 580, 592, 603 Li, R., 452, 482 Little, D., 572, 603 Litwin, C.M., 518, 540 Liu, F., 575, 604 Liu, G., 250, 273, 527–528, 540 Liu, H., 63, 83 Liu, J., 328, 355, 364, 390 Liu, J.Y., 689, 707 Liu, Y., 24, 34, 403, 432 Li, X., 53, 61–62, 82, 103, 105, 111, 117, 120, 407, 432, 449, 481 Llorente, F., 22, 34 Llusi, J., 615, 661 Loake, G., 250, 273 Lobo, N.F., 591, 594, 604 Locatelli, L., 508, 540 Lockwood, L., 493, 540 Loeve, K., 450, 487 Logan, B.A., 622, 659

Logemann, E., 682–683, 707 Loivama¨ki, M., 640, 659 Loomis, W.E., 694, 708 Loper, J.E., 494, 504, 509, 511–512, 541 Lo´pez-Fraga, M., 73, 83 Lo´pez, M.A., 553, 559, 604, 671–672, 707 Lo´pez-Milla´n, A.-F., 496, 541 Lopez-Raez, J.A., 625, 659 Lorang, J.M., 58, 83 Lorenzo, O., 144, 164, 237, 273, 401–402, 409, 413, 432–433 Lorito, M., 333, 355 Lorrain, S., 107, 117 Lotze, M.T., 449, 482 Loughrin, J.H., 632, 659 Lough, T.J., 129–130, 147, 164 Lovrekovich, L., 198–199, 216 Lozano, J.C., 198–199, 216 Lucangeli, C., 293, 315 Lucas, J.A., 193–194, 216, 371, 390 Lucas, W.J., 129–130, 147, 164 Lucena, C., 499, 541 Lucy, M., 284–285, 315 Lugtenberg, B.J., 301, 310 Lu, H., 108, 117 Luo, H., 454, 482 Lu, R., 70, 83 Luschnig, C., 236, 253, 273 Lusso, M., 195, 216 Lu, Y.-D., 676, 708 Lynch, J.M., 493, 533 Lyon, G., 181, 184, 216 Lyons, T.J., 462, 482 M Mabrouk, Y., 689, 708 Mach, J.M., 108, 117 Macino, G., 65, 77 Mackey, D., 5, 26, 34, 45, 57–58, 83, 404, 433, 581–582, 596 Mackintosh, C.A., 452, 482 MacMahon, J.A., 560, 601 Maeda, T., 638, 659 MaVei, M.E., 625, 659, 679, 708 Maizel, A., 131, 164 Major, I.T., 561–562, 604 Malamy, J., 139, 164, 182, 187, 216, 670, 675, 708 Maldonado, A.M., 99, 117, 143–144, 153, 164, 363, 391, 675, 708 Maleck, K., 99, 117, 380, 391 Malone, M., 137, 164 Mandeel, Q., 342, 355 Mandryk, M., 178, 194, 211 Manners, J.M., 138, 162 Mano, J., 621, 659 Manulis, S., 288, 290, 315 Manulis-Sasson, S., 291, 310

AUTHOR INDEX Mao, P., 418, 433 Marchler-Bauer, A., 461, 482 Mariathasan, S., 14, 34 Marques, J.T., 73, 83 Marra, R., 339, 355 Marschner, H., 299, 315, 493, 496, 518, 541 Marsh, J.F., 327, 355 Martı´nez-Abarca, F., 685, 708 Martinez, C., 131, 164 Martinez de Ilarduya, O., 563–564, 591–592, 604 Martinez-Romero, E., 285, 317 Martin, G.B., 55, 77, 94–96, 117, 445, 482, 587, 590, 604 Martin-Hernandez, A.M., 44, 83 Martinon, F., 14, 34 Martyn, R.D., 343, 351 Maruthasalam, S., 452, 482 Masaie, H., 465, 482 Masclaux, C., 504–505, 522, 541 Mathesius, U., 293, 299, 310, 313 Mathieu, J., 129–130, 164 Matsui, K., 621, 660, 676, 708 Matsumara, K., 106, 117 Matsushima, R., 618, 660 Matthijs, S., 501–502, 534 Mattiacci, L., 571, 604, 617, 660 Mattinen, L., 12, 34 Mattoo, A.K., 293, 315 Mattson, W.J, 690, 694, 705 Matzinger, P., 11, 14, 31, 34, 36 Mauch, F., 379, 391, 672, 682, 708 Mauch-Mani, B., 184, 196, 216, 221, 365, 375, 394, 672, 708, 713 Maurhofer, M., 230, 249, 252, 273, 516, 541 Mavrodi, D.V., 304, 315 Ma, W., 42, 51, 83 Maxson-Stein, K., 183–184, 216 Mayer, A.M., 42, 87 Mayers, C.N., 202–203, 216 Mazzola, M., 302, 315 McCann, H.C., 49, 83 McCann, M., 557, 597 McConn, M., 237, 273, 400, 433, 562, 606, 620, 660 McFall, A.J., 5, 34, 404, 433 McGrath, K.C., 409, 433 McGurl, B., 133, 164, 366, 391 McIntire, C.R., 449, 482 McKey, D., 694, 708 McRoberts, N., 671–672, 715 Medzhitov, R., 3–5, 8–9, 15–16, 22, 27, 28, 34 Meera, M.S., 345, 355 Mei, B., 521, 526, 541 Meijer, A.H., 409, 433 Meiners, T., 572, 601 Melchers, L.S., 457, 463, 482 Meldau, S., 570, 604, 630–631, 660

729

Mellor, J., 421, 433 Melotto, M., 26–27, 34, 41, 52, 83, 150, 164, 557, 604, 672, 708 Memelink, J., 409, 437 Menge, J.A., 324–325, 352–353 Menges, M., 580, 604 Mengiste, T., 439–474, 483 Menke, F.L.H., 23, 34, 409, 433 Menu-Bouaouiche, L., 450, 453, 483 Me´rai, Z., 70–71, 83 Mercado-Blanco, J., 301, 315, 515, 542 Merkx-Jacques, M., 584, 604 Merzendorfer, H., 574, 605 Meskiene, I., 577, 579, 608 MetchnikoV, E., 308, 316 Me´traux, J.-P., 27, 34, 39–74, 97, 118, 140, 165, 182, 187–188, 216–217, 236, 274, 405, 434, 446, 483, 581–582, 605, 674–675, 708 Metz, M., 62, 84 Meuwly, P., 187, 217, 363, 391 Mewis, I., 586, 588, 605 Meyer, A., 6, 34 Meyer, J.-M., 247, 273, 500–502, 511–512, 516–517, 542 Meyer, R., 627, 660 Meziane, H., 151, 165, 231, 235, 239, 242, 247, 273, 516, 542 Michiels, K.W., 301, 316 Miernyk, J.A., 295, 316 Mila, I., 529, 541 Miles, P.W., 560, 563, 575, 587, 605 Miller, L.D., 301, 316 Miller, M.B., 245, 274 Milligan, S.B., 591, 605 Mills, P.R., 182, 217 Minardi, P., 201, 217 Mindrinos, M., 141, 165 Min, K., 450, 465, 483 Mirabella, R., 646, 660 Mirleau, P., 501–502, 508–509, 542 Misaghi, I.J., 511, 542 Mishina, T.E., 98, 105, 117, 140, 148, 165, 180–181, 217, 225, 235, 237, 240, 242, 265, 274, 446, 483 Mitchell, R.E., 52, 84, 401, 433 Mitho¨fer, A., 9, 15–16, 34, 41, 79, 560–562, 567, 569, 576, 605, 619, 660 Mittelstrass, K., 697, 708 Miya, A., 19, 34, 445, 483, 574, 605 Mizoguchi, T., 128, 165 Mlejnek, P., 672, 708 Mohase, L., 592, 605 Moissiard, G., 67, 84 Mo¨lders, W., 187, 217 Molina, A., 208, 217, 383, 391 Molina-Favero, C., 300–301, 316 Molna´r, A., 67, 84 Montesano, M., 15, 35

730

AUTHOR INDEX

Montoya, T., 134, 165 Moon, J., 131, 165 Morandi, D., 326, 355 Moran, P.J., 563–564, 585–586, 588, 605 Morant, A.V., 558, 560, 562, 605 Morel, J.-B., 66, 84 Morgan, W., 94, 117 Mor, H., 58, 84 Morillo, S.A., 22, 35 Mori, N., 570, 605, 617, 660 Mori, S., 496, 543 Morris, R.O., 288, 316 Morrone, D., 293, 316 Moscatiello, R., 566, 580, 605 Mosher, R.A., 407, 433 Mosher, S.L., 422, 433 Mossialos, D., 500–501, 542 Mougel, C., 493, 543 Mougous, J.D., 63, 84 Moulin, F., 516, 543 Mourrain, P., 66, 84 Mou, Z., 99, 118, 406, 433 Moyen, C., 134, 165 Mozoruk, J., 561, 605 Muchovej, J.J., 325, 355 Mu¨ler, K.O., 180, 217 Mulnix, A.B., 575, 606 Munro, J., 180, 217 Mu¨nzinger, M., 502, 543 Murakami, Y., 73, 84 Murata, Y., 496, 543 Murdoch, L., 521, 543 Mur, L.A.J., 369, 391, 559, 606 Murphy, A.M., 202–204, 217 Murphy, K.M., 441, 483 Murre, C., 412, 433 Muslim, A., 331, 355 Musser, R.O., 583, 606 Muthukrishnan, S., 442, 452, 476, 487 Mutti, N.S., 568, 587, 594, 606 Mysore, K.S., 92, 118 N Nachin, L., 523, 543 Nagasawa, T., 289, 316 Nagel, J., 626, 660 Nahalkova, J., 342, 355 Nairz, M., 519, 543 Naito, K., 8, 10, 35, 50, 84 Nakagami, H., 23, 35 Nakamura, K., 415, 431 Nakamura, S., 621, 660, 676, 709 Nakanishi, H., 498, 543 Nakano, T., 408, 433 Nakashita, H., 185, 217 Nakazato, Y., 250, 274 Nandi, A.N., 95, 110–111, 118, 142, 144, 165, 363, 391

Napoli, C., 66, 84 Narasimhan, M.L., 439–474, 483 Narlikar, G.J., 421, 433 Narusaka, Y., 182, 217 Narva´ez-Va´squez, J., 134, 136–137, 165, 366, 391 Navarro, L., 18, 24, 35, 54, 57, 84, 96, 104, 118, 254, 274, 290, 316, 417, 433, 580–581, 606, 671, 676, 709 Nawrath, C., 97, 118, 236–237, 274, 405–406, 434, 446, 483 Naylor, M., 202–203, 217 Ndamukong, I., 415, 434 Needham, J.G., 560, 606 Neema, C., 522, 529, 543 Negre, F., 621, 660 Neilands, J.B., 499–500, 502–503, 521, 540, 543–545 Nei, M., 637–638, 661 Nelson, A.L., 519, 543 Nelson, C.E., 137, 165 Nemchenko, A., 635, 661 NeuVer, M.G., 107, 118 Newman, M.-A., 5–6, 9, 35, 181, 200, 218, 241, 243, 265, 274, 374, 391 Ng, D.W.-K., 421–422, 434 Ng, T.B., 450, 453, 483 Nguyen, C., 493, 518, 543 Nicholson, R.L., 174, 205, 214 Nickstadt, A., 144, 166 Niki, T., 684, 709 Nishitami, K., 444, 488 Nobuta, K., 98, 118, 406, 417, 434 Noctor, G., 255, 270 Noel, K.D., 243, 274 Nojiri, H., 675, 709 Nombela, G., 592, 606 Nomura, K., 60, 84, 685, 709 Noori, S.A.S., 452, 483 Novina, C.D., 104, 118 Nu¨rnberger, T., 1–28, 35, 41, 84, 180, 218, 265, 274, 554–555, 557, 564–565, 572, 606 O Oberhansli, T., 289, 316 Ocampo, J.A., 322, 325–327, 350, 353 O’Connell, R.J., 558, 581–582, 606 O’Donnell, P.J., 138, 166, 447, 483 Oecking, C., 12, 36 Oertli, J.J., 587, 605 Ogo, Y., 498, 543 Oh, H.-S., 58–60, 62, 84 Ohme-Takagi, M., 408, 434 Ohto, M.-A., 127, 166 Oide, S., 521, 525, 544 Oka, Y., 365, 391 Okinaka, Y., 206, 218, 523, 544

AUTHOR INDEX Okon, Y., 283–309, 316 Okuda, S., 444, 483 Oku, H., 42, 85 Okuno, T., 191, 218 Oldroyd, G.E., 286, 316 Olivain, C., 342, 356 Oliver, R.P., 176, 208, 218, 557, 609 Olmedo, G., 404, 434 Olson, H.A., 323, 331–332, 335, 356 Olsson, S.B., 637, 661 Olszak, B., 98. 118 Ona, O., 290, 316 On˜ate-Sa´nchez, L., 410, 434 Ongena, M., 228, 231, 243–244, 246, 265, 274–275, 304, 316, 516, 549, 621, 661 Ong, L.E., 57, 85 Ong, S.T., 519, 544 Onishi, M., 456, 483 Oostendorp, M., 183, 218 Orians, C.M., 137, 166, 693, 709 Orozco-Ca´rdenas, M.L., 133, 137–138, 156, 166 Ortı´z-Castro, R., 300, 316 Ortmann, I., 257, 275 Ortun˜o, A., 380, 391 Osman, H., 7, 35 Ouchi, S., 42, 85 Ouyang, B., 452, 484 Ozinsky, A., 15, 37, 241, 279 P Page, J.E., 626, 663 Paiva, N.L., 668, 709 Pallas, J.A, 140–141, 156, 166 Palma, K., 111, 118 Pancheva, T.V., 682, 709 Panstruga, R., 102, 558, 581–582, 606 Pare´, P.W., 255–256, 275, 377, 392, 568, 606, 617, 621, 649, 661, 664 Parisy, V., 237, 275 Park, C.-J., 442, 484 Park, D.H., 128, 166 Parker, D.R., 518, 545 Parker, J.E., 236, 275 Parker, M.W., 13, 35 Parkhi, V., 451, 484 Park, K.S., 228–229, 258, 275 Park, M.R., 229, 257, 275 Park, S.-W., 99, 118, 145–146, 166, 226, 275, 363, 392, 406, 434, 644, 661, 675, 678, 709 Parthier, B., 674, 715 Paschold, A., 559, 562, 606, 619, 648, 661, 679, 709 Passardi, F., 442, 484 Patten, C.L., 288–289, 316 Paul, N.D, 684, 704

731

Peake, P.W., 465, 484 Pearce, G., 133–134, 166, 365, 392 Pegadaraju, V., 579, 607 PeiVer, M., 562, 583–584, 607 Pemberton, C.L., 6, 12, 35 Pen˜a-Corte´s, H, 675, 709 Peng, J.-L., 180, 185, 218 Penmetsa, R.V., 338, 356 Penninckx, I.A.M.A, 447, 484 Pen˜uelas, J., 615, 661 Pe´rilleux, C., 125–127, 166 Perrin, D.R., 41, 77 Persmark, M., 502–503, 544 Pertry, I., 292, 317 Peskan-Bergho¨fer, T., 329–330, 356 Petersen, M., 108, 118, 579, 607 PfeVer, S., 73, 85 Pfitzner, U.M., 424, 430 Picard, K., 346, 356 Piccoli, P.N., 293, 317 Pichersky, E., 621, 654 Pickett, J.A., 628, 661, 677, 709 Pierson, E.A., 300, 317 Pierson, L.S., 300, 317 Pieterse, C.M.J., 151–153, 166, 175–176, 199, 218, 227, 231, 240, 258–262, 275, 364, 372–373, 392, 398, 407, 432, 434, 515, 544, 559, 607, 670–671, 678, 688, 709–710 Pikaard, C.S., 423, 428 Pinto, D.M., 635, 661 Pirone, T.P., 70, 75 Pitman, A.R., 50, 85 Plessl, M., 697, 710 Ploss, K., 691, 705 Pohnert, G., 570, 607 Pollard, M., 556, 607 Pollard, R.T., 531, 544 Pollmann, S., 288, 317 Pont, M., 178, 194, 218 Poole, K., 501, 544 Popowich, E.A., 451, 484 Poppy, G.M., 677, 709 Poromarto, S.H., 331, 356 Potter, D.A., 684, 699 Potuschak, T., 236, 275, 403, 434 Potvin, E., 504, 544 Pouteau, S., 126, 167 Powell, G., 559, 563, 607 Powell, P.A., 65, 85 Pozo, M.J., 153, 167, 231, 260, 266, 276, 322, 324, 326–328, 356, 364, 373, 392, 413, 434, 689–690, 710 Prats, E., 371, 392, 682, 710 Pre´, M.R., 401, 409–410, 434 Press, C.M., 232, 251, 276, 515, 544 Preston, C.A., 136, 167, 628, 634, 649, 661, 684, 710

732

AUTHOR INDEX

Price, T.S., 642, 657 Prinsen, E., 290, 292, 317 Prins, M., 65, 85 Prochazka, S., 672, 708 Prosser, I.M., 640, 661 Pryce-Jones, E., 558, 567, 607 Puckhaber, L.S., 341, 354 Pu¨hler, A., 521, 548 Pukatzki, S., 63, 85 Punja, Z.K., 451, 476 Purrington, C.B., 668, 700 Q Qian, S., 73, 85 Qin, X.-F., 414, 435 Qiu, J.-L., 416, 435, 579, 607 Quevillon, E., 458, 461, 484 Quilis, J., 454, 484 Quist, T.M., 468, 484 Qutob, D., 6, 9, 12–13, 35, 580, 607 R Raaijmakers, J.M., 253, 276, 302, 317, 399, 509, 511, 544 Racape´, J., 10, 36 Rademacher, W., 293, 317 Radhika, V., 695, 710 Radutoiu, S., 20, 36 RaVaele, S., 411, 435 Raggi, V., 186–187, 218 Rahe, J.E., 176, 192, 218 Rai, M., 384, 392 Rairdan, G.J., 142, 167 Rajam, M.V., 452, 484 Ralph, S.G., 561–562, 607 Ramanujam, M.P., 685, 710 Ramonell, K., 580–581, 607 Ramos, H.C., 234, 276 Ramos Solano, B., 233, 276 Ramsey, J.S., 587, 594, 607 Ran, L.X., 229–231, 239–240, 247, 251, 276, 515, 544–545 Rask, H.M., 306–307, 319 Raskin, I., 203, 218, 670, 710 Rasmann, S., 643, 662, 699, 710 Rasmussen, J.B., 140–142, 167, 175, 179, 186–187, 191, 219, 363, 392, 675, 710 RatcliV, F., 67, 85 Rathjen, J.P., 10, 32 Rathmell, W.G., 200, 219 Ratzka, A., 589, 607 Ravel, J., 299, 317 Ravet, K., 531, 545 Rayapuram, C., 629, 632, 662 Read, D.J., 689, 712 Rea, S., 423, 435

Reddy, A.S.N., 456, 479 Redman, A.M., 682, 710 Reglinski, T., 181, 184, 209, 219 Reichman, S.M., 518, 545 Reignault, P., 181, 184, 219 Reinbothe, S., 682, 710 Reinke, H., 425, 435 Reiss, E., 451, 456, 484–485 Reitz, M., 231, 243, 276 Remans, R., 291, 305–306, 317 Ren, C.-M., 423, 435 Ren, D., 24, 36 Rep, M., 47, 85, 457, 463, 485 Reymond, P., 365, 392, 398, 413, 435, 561–562, 588, 608 Rey, P., 346, 356 Rhoades, D.F., 643, 662, 677–678, 694, 698, 710 Richmond, S., 178, 189, 207–208, 215, 219 Ride, J.P., 207, 219 Riemann, M., 238, 276 Ritter, C., 56, 58, 85 Robatzek, S., 4, 16, 19–22, 24, 27, 29, 36, 93–94, 112, 119, 224, 234, 239, 270, 276, 564–565, 596, 608 Roberts, A.M., 682, 695, 711 Roberts, D.A., 179, 219 Robert-Seilaniantz, A., 266, 277, 553, 559, 608, 685, 710 Robin, A., 299, 317, 507–508, 510, 545 Robinson, N.J., 496, 545 Rocher, F., 155–156, 167 Rochon, A., 407, 414, 435 Rodelas, B., 306, 317 Rodrı´guez, H., 298, 317, 688, 711 Rodriguez-Saona, C., 582, 584, 608, 650, 662 Rogers, E.E., 405, 435, 496, 545 Rohmer, L., 58, 85 Roitsch, T., 672 Roman, G., 236, 253, 277, 403, 435 Romeis, T., 449, 485 Ro¨mheld, V., 299, 315 Ronald, P., 468, 476 Ronald, P.C., 53, 85 Roncadori, R.W, 325, 354 Rondinone, C.M., 467, 485 Ron, M., 7, 20, 36, 93, 119 Rooney, H.C.E., 45, 86 Rooney, J.M., 682, 711 Rosebrock, T.R., 54, 86 Rose, J.K.C., 43, 86, 143, 171 Rosell, R.C., 584, 608 Rosenblueth, M., 285, 317 Ro¨se, U.S.R., 633, 662 Ross, A.F., 139, 142, 167, 175, 177–178, 186, 201, 204, 206, 210, 219–220,

AUTHOR INDEX 362, 393, 405, 435, 628, 662, 675, 698, 711 Rossi, M., 95, 119 Rosta´s, M., 633, 662, 695, 711 Rostelien, T., 637, 662 Rotblat, B., 10, 36 Roth, B.M., 70, 86 Rothstein, S.J., 647, 652 RoubenoV, R., 449, 485 Rovira, A.D., 493, 545 Rowe, H.C., 558, 680, 686–687, 706 Rowland, O., 416, 435 Ruddat, M., 521, 533 Ruegger, M., 402 Ruess, W., 382, 393 Rushton, P.J., 415, 429 Ruther, J., 678–679, 711 Rutter, M.T., 690, 699 Ryals, J.A., 362–364, 367, 382, 393, 446, 485 Ryan, C.A., 132–134, 136, 138, 159–161, 165–168, 285, 318, 365–366, 376–378, 387–389, 567, 608, 620, 627, 644, 654, 674, 677, 679, 698, 703–704 Ryu, C.-M., 92, 118, 227–228, 230, 232, 256, 262–263, 277, 300, 318, 373, 393, 688, 691, 711 S Sabuquillo, P., 344, 356 Saheed, S.A., 586, 608 Sakai, H., 403, 435 Sakamoto, K., 187, 208, 219, 469, 485 Saleem, M., 294–295, 318 Salmond, G.P.C., 6, 35 Salzman, R.A., 465, 485 Samuels, G.J., 333, 356 Sa´nchez-Herna´ndez, C., 631, 662 Sano, H., 672, 711 Sansom, M.S., 501, 537 Santner, A., 286, 318 Santos, R., 523, 545 Sarig, S., 306, 318 Satyanarayana, T., 70, 86 Sauter, H., 382, 393 Sauvage, C., 505, 545 Schaaf, G., 496, 545 Schachtman, D.P., 125, 167 Scha¨fer, P., 329, 356 Schaible, U.E., 250, 277, 519, 545 Schaller, A., 12, 36 Scharte, J., 682, 711 Schechter, L.M., 94, 119 Scheer, J.M., 134, 167 ScheVer, R.P., 40, 86 Scheideler, M., 682–683, 711 Schenk, P.M., 683, 712 Scher, F.M., 512, 545

733

Schestibratov, K.A., 451, 485 Schilmiller, A.L., 134, 137, 167, 674, 712 Schindler, U., 413, 436 Schippers, B., 226, 231, 277, 280, 301, 318, 510–511, 516, 532, 537, 545 Schirmbo¨ck, M., 333, 357 Schittko, U., 137, 168 Schlaich, N.L., 98, 119 Schmele, I., 186, 190, 219 Schmelz, E.A., 571, 608, 617, 633, 662 Schmid, M., 296, 318 Schmidt, D.D., 43, 86, 561–562, 608 Schnee, C., 640, 662 Schnettler, E., 39–74, 86 Scho¨nbeck, F., 327–328, 352 Schoonbeek, H.-J., 44, 86 Schreiber, K., 265, 277 Schro¨der, R., 572, 608 Schroth, M.N., 513, 516, 539, 546 Schuhegger, R., 232, 237, 246, 277, 300, 318 Schuler, M.A., 468, 485 Schultze, M., 327, 355, 662 Schultz, J.C., 377, 385, 643, 645, 651, 677–681, 698, 700 Schulze-Lefert, P., 670, 712 Schuurink, R.C., 613–650, 662 Schwachtje, J., 366, 393 Schweighofer, A., 577, 579, 608 Schwyn, B., 500, 502, 545 Scutareanu, P., 642, 662 Seet, B.T., 420, 436 Segarra, G., 262, 277, 321–350, 357, 364, 393 Segond, D., 507, 529–530, 546 Seguchi, K., 249, 277 Se´gue´la, M., 499, 546 Sekine, M., 288, 318 Sels, J., 42, 86, 442, 444, 485 Senaratna, T., 376, 393 Sen, R., 331, 354 Seo, H.S., 640, 663 Seong, S.-Y., 11, 36 Seo, S., 400, 436, 580, 608, 627, 641, 663 Sequeira, L., 198–200, 216, 219 Seskar, M., 628, 644, 663 Sessa, G., 410, 436 Shabab, M., 46, 86 Shah, J., 108, 110, 119, 156, 168, 225, 277, 370, 393, 670, 712 Shang, Y., 57, 86 Shan, L., 16, 25, 36, 54–55, 86, 565, 608 Shapiro, A.D., 148, 171 Sharon, A., 43, 88 Sharp, P.A., 67, 86, 104, 118 Shatters, R.G., 454, 458, 466, 469, 485 Shaukat, S.S., 229–230, 253, 278 Shen, Q.-H., 28, 36, 96, 99, 111, 119, 417, 436, 670, 712 Shepherd, R.W., 614, 663

734

AUTHOR INDEX

Shields, V.D.C., 637, 663 Shimoda, T., 638, 663 Shimono, M., 250, 277 Shinshi, H., 408, 434 Shiojiri, K., 629–630, 640, 663, 676, 712 Shiraishi, T., 42, 86–87 Shirano, Y., 95, 119 Shirasu, K., 371, 393 Shiu, S.-H., 18–19, 36, 458, 485 Shojima, S., 495, 546 Shoresh, M., 262, 278, 323, 333, 336–340, 357, 373, 393 Shulaev, V., 140, 145, 168, 175, 187, 219, 363, 378, 393, 628, 644, 648, 663, 676, 678, 712 Siddiqui, I.A., 229, 278 Siddiqui, M.A., 230, 253, 278 Sieberer, T., 253, 278 Siebner-Freibach, H., 517, 546 Siegrist, J, 141, 168, 184, 219, 383, 393 Siewers, V., 43, 87 Sijen, T., 67, 87 Silhavy, D., 72, 87 Silipo, A., 241, 278 Silva Bueno, J.C., 136, 160, 377, 388, 650, 656, 676–678, 680, 692–693, 698, 705 Simeoni, L.A., 513, 546 Simmons, A.T., 626, 663 Simons, T.J., 186, 201, 219–220 Singh, D.P., 176, 204, 220 Singh, K.B., 410, 434, 564, 566, 576, 578, 599 Singh, N.K., 457, 485 Singh, R.S., 344, 354 Skibbe, M., 630, 663 Skipp, R.A., 177, 192, 220 Slusarenko, A.J., 140, 142, 146, 162, 196, 216 Smalla, K., 284, 318, 493, 533 Smedegaard-Petersen, V., 681, 712 Smid, H.M., 637–638, 663 Smith-Becker, J., 183, 188, 203, 220, 363, 393 Smith, E.E., 509, 532, 546 Smith, G.S., 322, 325, 357 Smith, J.A., 178–181, 183, 190, 220 Smith, J.L., 462, 485 Smith, K.P., 324, 357 Smith, S.E., 689, 712 Sneh, B., 511, 546 So¨derberg, K.H., 493, 546 Sohn, K.H., 61, 87 Sokhansanj, A., 452, 483, 485 Solano, R., 229, 233, 236, 278, 402, 436 Solomon, P.S., 557, 609 Soman, K.V., 465, 486 Sommer-Knudsen, J., 187, 220

Somssich, I.E., 99, 115, 224, 270, 326, 357 Song, J.T., 46, 87, 97, 119, 148, 168 Sonnemann, I., 683, 686, 689, 712 Sontheimer, E.J., 66, 73, 76, 87 Spaepen, S., 266, 278, 283–309, 318 Spencer, M., 229, 256, 278 Sperandio, V., 299, 314 Speth, E.B., 150, 168 Spikman, G., 45, 78 Spoel, S.H., 254, 278, 398, 407, 417, 436, 553, 559, 585, 609, 632, 652, 684, 712 Sreekantan, L., 131, 168 Srivastava, A., 14, 36 Staal, J., 473, 486 Stacey, G., 689, 712 Stadnik, M.J., 694, 712 Stanier, R.Y., 493, 546 Staples, R.C., 42, 87 Starck, Z., 674, 712 Stark, G.R., 449, 481 Staskawicz, B.J., 56–57, 61, 75, 81, 87, 445, 486 Staswick, P.E., 135, 168, 237, 238, 400, 402, 436 Staub, T., 183–185, 216 Steenhoudt, O., 285, 297–298, 319 Stein, B.D., 190, 220 Stein, M., 99, 106, 119 Stennis, M.J., 377, 394 Stensmyr, M.C., 637, 663 Stenzel, I., 134, 136, 168, 674, 712 Stepanova, A.N., 288, 319 Stephens, J.H.G., 306–307, 319 Stermer, B.A., 186, 220 Stevens, J.F., 626, 663 Sticher, L., 175, 177, 220, 446, 486, 673, 712 Stintzi, A., 456, 486, 504, 546 Stølen, O., 681, 712 Stolle, K., 195, 220 Stone, J.M., 12, 37 Stout, M.J., 684, 712 Stracke, R., 411, 436 Strahl, B.D., 420–421, 436 Stratmann, J.W., 133–134, 168 Strauss, S.Y., 680, 713 Strawn, M.A., 97, 119 Strobel, N.E., 180, 220 Stromberg, A., 195, 220 Strompen, G., 414, 424, 436 Stulemeijer, I.J.E., 580, 609 Stumm, D., 190, 220 Stutz, E.W., 513, 546 Sua´rez-Lo´pez, P., 126–128, 168 Suarez-Rodriguez, M.C., 23–24, 37, 579, 609 Sugiura, Y., 517, 546 Summermatter, K., 199–200, 220 Sun, C., 416, 436

AUTHOR INDEX Susı´n, S., 497, 546 Suslow, T.V., 516, 546 Sutak, R., 518, 546 Suttle, J.C., 293, 315 Sutton, D.C., 193, 221 Suzuki, H., 146, 168 Sweeney, G., 463, 467, 478 Szymanski, D.B., 625, 664 T Tachi, H., 456, 486 Tada, Y., 12, 23, 37 Tagu, D., 587, 594, 609 Takabayashi, J., 618, 633–634, 636, 650, 653, 664, 680, 701 Takada, S., 129, 168 Takagi, S.I., 495, 518, 546 Takahashi, F., 579, 609 Takai, R., 234, 239, 278 Takeda, A., 70–71, 87 Takemoto, D., 454, 465, 486 Takemura, Y., 465, 486 Takenaka, S., 346, 357 Takken, F.L.W., 95, 119 Talbot, N.J., 558, 610 Tally, A., 183, 221 Tamaki, S., 130, 168 Tamietti, G., 343, 357 Tamogami, S., 136, 169, 620, 644, 664 Tanaka Hall, T.M., 67, 87 Tang, D., 687, 713 Tang, X., 410, 436 Tao, Y., 100, 119, 288, 319, 442, 486, 581, 609 Tax, F.E., 22, 35 Taylor, I.B., 295, 319 Teale, W.D., 286, 319 Temussi, P.A., 462, 465, 486 Teplitski, M., 300, 319 Thaler, J.S., 631, 664, 684, 699, 713 Thatcher, L.F., 401, 436 Theunis, M., 288, 319 Thielert, W., 383, 394 Thilmony, R., 18, 37 Thines, B., 402, 437, 674–675, 713 Thomashow, L.S., 513, 547 Thomas, J.F., 126, 170 Thomas, L., 325, 357 Thomas, M.R., 131, 168 Thomma, B.P.H.J., 205, 221, 447, 486, 670, 713 Thompson, G.A., 564, 585–586, 605, 609 Thordal-Christensen, H., 91–112, 119 Thorpe, M.R., 136, 169, 674, 713 Thulke, O.U., 367–369, 394 Tian, D., 379, 394 Tian, M., 46, 87 Tien, T.M., 291, 319 Timmusk, S., 229, 278

735

Tiryaki, I., 135, 168, 237, 238, 400, 402, 436 Tjaden, J., 366, 394 Tjallingii, W.F., 563, 586–587, 609 Tjamos, S.E., 152, 169, 229, 233, 263, 278 Toledo-Ortiz, G., 412, 437 Tolleson, W.H., 12, 37 Tollsten, L., 621, 658 Tomari, Y., 67, 73, 87 Tomas, E., 463, 486 Tomiic´, V., 522, 547 Ton, J., 136, 152, 160, 169, 184, 206, 221, 226, 235–236, 256, 259, 278–279, 363, 365, 374–680, 394, 559–560, 562, 568, 570, 647, 649, 664, 670–672, 683, 691, 693, 705, 713 Tooke, F., 126, 169 Tooker, J.F., 582, 609, 618, 664 Tosa, Y., 106, 117 Toth, I.K., 52, 87 Toth, R., 325, 357 Tran, H., 151, 169, 230, 245, 279 Traw, M.B., 693, 713 Triboulet, R., 73, 88 Trillas, I., 321–350 Trotel-Aziz, P., 233, 279 Trudel, J., 453, 486 Truitt, C.L., 571, 609, 617, 664 Trujillo, M., 102, 120 Truman, W., 99–100, 120, 143–145, 169, 260, 279, 363, 394, 580, 609, 675, 678, 713 Tsavkelova, E.A., 295, 319 Tscharntke, T., 677–678, 703 Tsuda, K., 404, 437, 580–581, 609 Tucker, S.L., 558, 610 Tudzynski, B., 43, 88 Tumlinson, J.H., 132, 169, 377, 392, 560, 567, 599, 621, 661 Tung, C.-W., 458, 487 Turgeon, B.G., 525, 547 Turgeon, R., 129, 157 Turlings, T.C.J., 132, 169, 559–560, 562, 568, 570, 610, 621, 632, 634, 636, 639, 643, 651, 655–656, 664, 676, 714 Turner, J.G., 236, 269, 402, 404, 437 Tuzun, S., 140, 142, 169, 194, 221 U Ueda, H., 95, 120, 590, 610 Uknes, S., 139, 169, 182, 196, 198–199, 221, 424, 437, 456–457, 463, 487 Ulevitch, R., 5, 15, 28 Umehara, M., 286, 319 Umemoto, N., 15, 37 Underhill, D.M., 15, 37, 241, 279 Underwood, W., 62, 88 Usami, S., 134, 169

736

AUTHOR INDEX

V Vaast, P., 324, 357 Vailleau, F., 411, 437 Vaistij, F.E., 67, 88 Vallad, G.E., 175, 207, 221 Vallat, A., 616, 664 Valverde, F., 128, 169 Vanacker, H., 670, 714 Van Baarlen, P., 519, 547, 687, 714 Van Bel, A.J.E., 586, 611 Vancanneyt, G., 631, 664 Van Dam, N.M., 379, 394 Vande Broek, A., 290, 301, 319–320 Van de Mortel, J.E., 251, 279 Van den Boom, C.E., 635, 664 Van den Burg, H.A., 46, 88 Van der Biezen, E.A., 95, 120, 445, 487, 589, 610 Van der Ent, S., 152, 169, 231, 237, 251, 261, 279, 340, 342, 357, 373, 394, 412, 437 Van der Fits, L., 409, 437 Van der Hoeven, R.S., 626, 664 Van der Hoorn, R.A.L., 45–46, 88, 95, 120, 589–590, 610 Van der Krol, A.R., 66, 88 Vanderleyden, J., 240, 273, 283–309 Van der Straeten, D., 670, 703 Van der Wel, H., 450, 487 Van der Westhuizen, A.J., 592, 605 Van de Ven, W.T.G., 576, 610 Van Esse, H.P., 46–47, 88 Van Hulten, M., 227, 279, 380, 394, 649, 665, 680, 698, 714 Van Kammen, A., 185–186, 221, 441–442, 487 Van Kan, J.A., 558, 602 Van Laer, S., 292, 319 Van Loon, J.J.A., 642, 665, 676, 703 Van Loon, L.C., 7, 37, 42, 88, 139, 151, 169–170, 174–176, 181–182, 185–186, 188, 201–202, 205, 218, 221–222, 225–227, 231, 233, 247, 249, 262–263, 279–280, 284, 295, 301, 319, 367, 394, 441–442, 444, 450, 453, 487, 511, 514, 547, 557, 574, 610, 624, 665, 668, 675, 688, 714 Van OeVelen, L., 504, 547 Van Ooijen, G., 590, 592, 610 Van Oosten, V.R., 152, 170 Van Pee, K.H., 304, 319 Van Peer, R., 151, 170, 226, 231, 280, 372, 394, 514, 547 Van Poecke, R.M.P., 626, 665 Van Rhijn, P., 325, 358 Van Schie, C.C.N., 623, 665 Van Strien, E.A., 175, 221, 444, 487 Vansuyt, G., 249, 280, 510, 517, 547

Van’t Slot, K.A.E., 12, 37 Van Verk, M.C., 397–426, 437 Van Wees, S.C.M., 98, 105, 120, 151–153, 170, 231, 239, 242, 251, 258, 260–262, 280, 369, 372–373, 395, 515, 547, 684, 691, 714 Van Wijk, M., 613–650, 665 Van Wuytswinkel, O., 507, 547 Varma, A., 329, 358 Veit, S., 6, 9, 12, 37 Velazhahan, R., 452, 487 Venisse, J.-S., 524, 547 Vera, P., 104, 112 Verberne, M.C., 139, 149, 170, 405, 437, 682, 714 Verdonk, J.C., 622, 665 Verhagen, B.W.M., 152, 170, 260, 280, 330, 358, 372, 395 Vernooij, B., 140, 142, 156, 170, 175, 187, 205, 222, 363, 395, 675, 714 Veronese, P., 442–443, 447, 450, 453, 466, 473, 487 Vert, G., 496, 499, 547 Vial, L., 299, 320 Vidal, S.M., 519, 547 Vierheilig, H., 325, 358, 686, 714 Viguerie, N., 463, 481 Villa, N.Y., 462, 465, 487 Visca, P., 247, 280, 500–501, 504, 509, 547 Vitali, A., 450, 453, 456, 466, 487 Viterbo, A., 14, 37, 334, 340–341, 358 Vleeshouwers, V.G.A.A., 48, 88 Vlot, A.C., 146, 150, 170, 226, 280, 363, 395, 622, 628, 665 Voelckel, C., 561–563, 588, 610 Vogel, G., 291, 320 Vogel, H., 561–562, 610 Voinnet, O., 67, 69–70, 73, 84, 88 Voisard, C., 513, 547 Volksch, B., 295, 320 Volpin, H., 322, 326–327, 358 Von Dahl, C.C., 583, 610, 624, 627, 665 Von Wire´n, N., 495–496, 547 Vorwerk, S., 5, 11, 37 Vuorinen, T., 635, 665 W Wagner, E.G.H., 229, 278 Wagner, G.J., 556, 558, 560, 610, 614, 624, 626, 663, 665 Walia, H., 635, 665 Walker, E.L., 497, 548 Waller, F., 323, 329–330, 358, 364, 395, 683, 714 Walley, J. W., 423, 425–426, 437 Walling, L.L., 551–595, 610, 668–669, 673, 684, 715

AUTHOR INDEX Wallsgrove, R.M., 637, 652 Walsh, U.F., 309, 320 Walters, D.R., 175, 177, 181, 184–185, 198, 208, 210, 219, 222, 667–699, 715 Walton, J.D., 40, 89 Wang, C.Z., 583–584, 612 Wang, D., 226, 254, 261, 266, 281, 417–418, 437, 446, 488, 671, 715 Wang, E., 627, 665 Wang, G., 20, 38 Wang, H., 12, 37 Wang, H.-Y., 498, 548 Wang, K.L.-C., 236, 281, 403, 437 Wang, L., 415, 438, 469, 488, 553, 559, 580, 610 Wang, X., 458, 466, 488, 565, 611 Wang, Y., 465, 488 Wang, Y.-Q., 23, 38 Wang, Z.-X., 187, 208, 222 Wang, Z.-Y., 21, 38 Wan, J., 93, 120, 445, 487 Ward, E.R., 139, 170, 182, 222 Ward, E.W.B., 205–206, 210, 216 Wardle, D.A., 493, 548 Warren, G.J., 513, 537 Washburn, C.F., 126, 170 Wasternack, C., 134, 170, 400, 438, 562, 611, 670, 674, 682, 715 Waters, B.M., 496, 548 Wawrzynska, A., 672, 715 Wei, G., 226, 281, 514, 548 Weigel, D., 155, 162 Weigert, C., 473, 479 Weinberg, E.D., 518, 548 Weingart, H., 295, 320 Wei, Z.-M., 6, 9, 38 Weller, D.M., 230, 253, 276, 281, 302–305, 320, 513, 547–548 Werck-Reichhart, D., 468, 485 Werker, E., 624–625, 665 Whalen, M.C., 55, 89, 673, 715 Wheeler, A.G., 567, 597 Whipps, J.M., 299, 320, 493, 541 Whisson, S.C., 94, 120 Whitehead, J.P., 467, 488 Whiteley, M., 504, 548 White, R.F., 182, 186, 202, 222, 675, 715 Wiermer, M., 96, 106, 111, 120 Wigge, P.A., 128–130, 161, 170 Wiggerich, H.-G., 521, 548 Wildermuth, M.C., 97, 120, 237, 281, 405, 438 Wildon, D.C., 137, 171 Williams,G.M., 682, 715 Will, T., 575, 586–587, 611 Wilson, M.J., 504, 548 Winkelmann, G., 500, 526, 548

737

Winter, D., 456, 459 Wirthmueller, L., 405, 438 Wittstock, U., 589, 611 Wolfert, M.A., 9, 38 Wolpert, T.J., 12, 38 Wolyn, D.J., 371, 388 Wong, C.E., 202, 204, 222 Wong, P.T.W., 513, 548 Wood, R.K.S., 182, 217 Woodward, A.W., 287, 320 Wright, K.M., 202, 222 Wroblewski, T., 154, 171 Wu, J., 570, 580, 611 Wu, J.Q., 628, 666 Wu, K., 410, 425, 438 X Xiang, C., 414, 438 Xiang, T., 54, 89 Xiao, F., 55, 89 Xia, Y., 146, 171, 363, 395 Xie, D.-X., 236, 281, 402, 438 Xie, Z., 623, 626, 666 Xing, W., 25, 38 Xiong, L., 468, 488 Xue, L., 332, 358 Xu, J., 579, 611 Xu, R., 250, 281 Xu, X., 418, 438 Y Yaish, M.W.F., 454, 479 Yalpani, N., 140, 171, 363, 395 Yamada, T., 43, 89 Yamaguchi, A., 131, 171 Yamaguchi, S., 292, 320 Yamaguchi, T., 7, 38 Yanagisawa, S., 404, 432 Yang-Cashman, P., 189, 214 Yang, C.-H., 508, 518, 548 Yang, S., 523, 548 Yang, Y., 226, 238, 281 Yang, Z., 409–410, 438 Yan, L., 131, 171 Yan, Z., 228, 230, 262, 281 Yao, N., 225, 270 Yasuda, M., 184, 222 Yeats, T. H., 143, 171 Yedidia, I., 333, 335–338, 340, 358–459 Yehuda, Z., 517–518, 548–549 Ye, X.S., 141, 171, 186, 195, 220, 222 Yi, Y., 492–494, 537 Yoder, O.C., 41, 89 Yokoyama, R., 444, 488 Yoo, B.-C., 69, 89 Yoo, S.-D., 404, 438, 579, 611 Yoshikawa, M., 41, 89

738

AUTHOR INDEX

Yoshinaga, N., 570, 611 Yoshioka, K., 108, 120, 185, 222, 383, 395, 449, 488 You, M.K., 580, 611 Young, H., 401, 433 Yuan, S., 670, 715 Yuan, Y.X., 497–498, 549 Yuen, G.Y., 233, 272 Yun, B.-W., 96, 101, 106, 120 Yu, X.M., 465, 488 Yuzaki, M., 463, 465–467, 488 Z Zamore, P.D., 66–67, 73, 86–87, 89 Zangerl, A.R., 553–554, 560, 582, 588, 596 Zarate, S.I., 584–585, 611, 685, 715 Zavala, J.A., 681, 715 Zeevaart, J.A.D., 126, 128, 131, 171 Zeidler, D., 6, 22, 38, 241–242, 281 Zeier, J., 98, 105, 117, 140, 148, 165, 180–181, 217, 225, 235, 237, 240, 242, 265, 274, 445–446, 483 Zemojtel, T., 23, 38 Zhang, B., 18, 38 Zhang, C., 142, 148, 171 Zhang, H., 256, 281, 300, 320 Zhang, J., 61, 89 Zhang, S., 22–24, 34, 38, 228, 230, 232, 281, 403, 432, 449, 488

Zhang, X., 71, 89, 579, 611 Zhang, Y., 95, 108, 111, 120–121, 414–415, 438 Zhang, Z., 98, 100–102, 108–110, 121 Zhang, Z.-P., 674, 715 Zhang, Z.-Q., 560, 611 Zhao, J., 404, 438 Zhao, Y., 94, 106–107, 121, 524, 549, 582, 612 Zheng, Z., 447, 489 Zhou, C., 425, 438 Zhou, J.M., 95, 121, 410, 438 Zhou, L., 325, 359 Zhu, B., 453, 489 Zhu, J., 376, 395 Zhu-Salzman, K., 563–564, 582, 585–586, 612 Ziadi, S., 682, 715 Zimand, G., 333, 359 Zimmerli, L., 96, 101, 106, 121, 365, 374–376, 395, 680, 716 Zimmerman, D.C., 621, 666 Zimmer, W., 289, 298, 301, 314, 320 Zimoch, L., 574, 605 Zipfel, C., 3, 5, 8–9, 16, 18–19, 24, 27, 38, 94, 121, 234–235, 265, 281, 576, 580–581, 612, 669–670, 716 Zong, N., 583–584, 612 Zuker, A., 451, 489

SUBJECT INDEX

A Abiotic stress, 376 Abscisic acid (ABA), 43, 295, 365 Achromobactin, 502 N-Acyl-L-homoserine lactone (AHL), 245–246 Adaptive defense responses, pathogens and insects bioaggressor, 552–553 co-evolution arms-race model, 554 invader-plant interaction, 556 microbial invaders, 555 PAMP-triggered immunity, 555–556 plant-organism interactions, 554 resistance (R) gene proteins, 556 ZigZag model, 554–555 guard and decoy models, 589–590 herbivore eVectors decoy defenses, 584–586 glucose oxidase, 583–584 regurgitant, 582 salivary oxidases and redox hypothesis, 587 specialist insects, toxic phytochemicals, 588–589 wound healing antagonism, 586–587 herbivore elicitors chitin, 574 endosymbiotic microbes, 568 hemipteran saliva, 575–576 herbivore associated molecular pattern (HAMP), 568 inceptin and -glucosidase, 571–572 lysozyme, 574–575 oviduct secretions, 572–574 saliva and oral secretions, 567 volicitin and caeliferin, 568–571 herbivore-feeding guilds defense signaling, 561–562 feeding modes, 560–561 phloem-feeding hemipterans, 563–564 plant host, 559–560 hostile phylloplane, 557 integrating signals AP2C1, 579 Arabidopis, 579–580 MAP3Ks, MAP2Ks and MAPKs, 577 microbial elicitors, 576

MKK1/2, and MPK4, 578–579 PAMPs to SA-, JA-, and ET-regulated defense responses, 580–581 plant innate immunity, 577–578 species-specific elicitors, 577 leaf surface, 556 microbe/arthropod colonization, 552 microbial eVectors, 581–582 microbial invasion strategies, 557–558 nutrient acquisition, 553 PAMPs, PRR and BAK1, 564–565 pathway activation, microbes, 558–559 plant–herbivore gene-for-gene interactions Medicago resistance, aphids, 592–593 Mi1.2 gene, 591–592 resistance genes, 590–591 plant origin elicitors Arabidopsis, 566–567 flg22 and chitin, 566 PAMP-triggered immunity, 565–566 PG-inhibiting proteins (PGIPs), 567 wall-associated kinase (WAK1), 567 primary cell wall, 556–557 Agastache rugosa, 371 Agrobacterium tumefaciens, 521 Alternaria alternata (AAL) toxin, 12 Alternaria brassicicola, 375, 445, 525, 684 Alternaria solani, 366, 689 Amaranthus caudatus, 442 1-Aminocyclopropane-1-carboxylic acid (ACC), 403 Antibiotics 2,4-diacetylphloroglucinol, 253–254 pyocyanin, 254–255 APETALA2/ethylene-response factor (AP2/ERF) transcription factor AtERF, 410 defense gene regulation, 410 JA-inducible gene expression, 409 ORA factors, 409–410 structural and functional characteristics, 408–409 Arabidopsis thaliana adaptive defense responses, 566–567 Arabidopsis–Pseudomonas fluorescens WCS417r system, 262–263 helix loop-helix transcription factor MYC2, 260

740

SUBJECT INDEX

Arabidopsis thaliana (cont.) JA- and ET-response mutant, 259–260 NPR1, 260–261 Ps. syringae pv. tomato, 261–262 R2R3-MYB-like transcription factor gene MYB72, 261 SA, 258–259 Arabidopsis Ser/Thr phosphatase type 2C (AP2C1), 579 decoy defenses, 584–585 fungal and oomycete suppression, 196 fungi and plant interactions, 329–330 LMMs enhanced disease susceptibility 1 (EDS1) protein, 96–97 phytoalexin deficient 4 (PAD4 ) protein, 96–97 nonhost resistance response, bacteria, 200 priming constitutive expresser of PR genes-1(cpr1) mutant, 370 enhanced disease resistance-1 (EDR1) protein, 369–370 MPK3 and MPK6 activity, 370–371 nonexpresser of PR genes-1 (npr1) mutant, 370 pseudobactin (PSB), 251 Arbuscular mycorrhizal (AM) fungi biotrophic pathogens, 323–324 functions, 323 induced resistance, 689–690 plants nutrition improvement, 324–325 resistance induction, 326–328 root colonization, 324 symbiosis, 689 Ascomycete fungi, 525–526 Asparagus oYcinalis, 371 Aspergillus nidulans, 469 AtMYC2 factor, 401, 413 atx1 mutant, 423 Automated ribosomal intergenic spacer analysis (ARISA), 508 Auxins, 286–291. See also Indole-3-acetic acid (IAA) vs. cytokinins, 291–292 indole-3-acetic acid (IAA) Azospirillum brasilense, 289–290 biosynthesis, 288–290 information-processing system, 290–291 Avian myeloblastosis virus, 410 Azelaic acid, 363, 406 B BABA-IR, 184, 374–376 Bacillus amyloliquefaciens, 688 Bacillus subtilis, 688

fengycins, 243–244 surfactins, 243 Bacterial suppression flagellum filament, 50 PAMP-triggered mmunity (PTI) avrPto, 53–54 avrPtoB (hopAB2), 54–55 avrRps4, 61–62 avrRpt2, 55–56 complexity and evolution, 63–64 coronatine toxin suppression, 52 extracellular polysaccharides (EPS), 51–52 hopAM1 eVector, 59 hopAN, 58–59 hopAO1, 62 MAP kinase signaling, 61 negative regulators, 57–58 RNA and RNA-binding protein targeting, 56–57 suppressed NHO1 expression, 62–63 Type three protein secretion eVectors (T3SE), 52–53 Type VI secretion system (T6SS), 63 vesicle traYcking disruption, 59–60 xopD, 56 systemic acquired resistance (SAR), 201 Arabidopsis, nonhost resistance response, 200 cell wall modifications, 200 disease tolerance, 199 induced response, 198 symptom suppression, inducer nature, 199–200 Type three protein secretion eVectors (T3SE), 48, 52–53 ZigZag model, 48, 49 -Aminobutyric acid (BABA), 365 abiotic stress, 376 biotic stress callose deposition, 375 indications, 376 PR-1 gene induction kinetics, 374–375 SNARE genes, 375–376 Basal resistance, 553, 586, 593. See also Pathogen derived molecular pattern (PAMP)-triggered immunity (PTI) AtWhy1 protein, 407 Bo. cinerea, 341 dye accumulation, 60 fungicides, 208 JA spraying, 144 NahG eVect, 195 pathogen defense, 41 TGA1 and TGA4 transcription factor, 415 WRKY factor, negative regulator, 416 Beneficial microorganism induced resistance

SUBJECT INDEX induced systemic resistance (ISR), 363–364 symbiotic fungi, 364 Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 364 Biological nitrogen fixation (BNF), 296 Biosurfactants Bacillus subtilis fengycins, 243–244 surfactins, 243 Pseudomonas spp. classification, 244 massitolide A, 244–245 Blumeria graminis, 364, 375, 377, 527–528, 681 Botrytis cinerea, 409, 446, 447, 516, 566–567 Brevicoryne brassicae, 586, 588, 589 BZIP transcription factor AtbZIP10, 413 TGA factor basal resistance, 415 NPR1 interaction, 414 PR-1 gene expression, 414–415 C Cacopsylla, 642 Caeliferin, 571, 617 Calcium signaling suppression, 51–52 Carbon/nutrient balance hypothesis (CNBH), 695–697 Cardiochiles nigriceps, 639 Catharanthus roseus, 409 Cercosporidium personatum, 380 Chemical induction chemical induced resistance -aminobutyric acid (BABA), 365 synthetic SA analogs, 364 SAR 2,6-dichloroisonicotinic acid, 182 acibenzolar-S-methyl, 183–184 -aminobutyricacid (BABA), 184 harpin protein, 184–185 probenazole, 185 pyraclostrobin, 185 saccharin, 185 salicylic acid, 182 tiadinil, 184 Chloroplast ATP synthase subunit (cATPC), 572 Chromatin modification gene expression, 420–421 innate immunity JA pathway, 424–426 SA pathway, 422–424, 425 plants, 421–422 Chrysobactin, 502–503 Cirsium, 678 Cochliobolus heterostrophus, 525 Cochliobolus miyabeanus, 525

741

Colletotrichum destructivum, 371 Colletotrichum lagenarium, 372 Colletotrichum orbiculare, 371 Constitutive triple response 1 (CTR1), 403–404 Coronatine coronatine insensitive 1 (coi1) mutant, 109, 401 long-distance signal, 150 PAMP, 64 stomatal closure, 52 Coronatine toxin suppression, 52 Cotesia marginiventris, 377 Cytokinins vs. auxins, 291–292 local signalling, 671–672 long-distance signaling progress, 127 D Damage-associated molecular patterns (DAMPs), 11, 41 Dendroides canadensis, 469 Denitrification, 297–298 Desferrioxamines (DFO), 503, 524 Diabrotica virgifera virgifera, 643 2,4-Diacetylphloroglucinol, 253–254 E EVector-triggered immunity (ETI), 4, 670 NO production, 22 vs. PAMPs, 10 phytohormones, 24 R-protein-mediated sensing, 26 Elicitins, 148 Endopolygalacturonase (PG)-inhibiting proteins (PGIPs), 567 Enhanced disease susceptibility 1 (EDS1) protein, LMMs ALD1 and AGD2 gene mutation, 97–98 Arabidopsis, 96–97 enhanced disease susceptibility 5 (eds5) mutant, 97 flavin-dependent monooxygenase 1 (FMO1), 98 nonexpressor of PR genes 1 (NPR1), 99 SA induction deficient 2 (SID2) gene, 97 systemic acquired resistance (SAR), 99 W-box, 99 Enhanced disease susceptibility 5 (eds5) mutant, 97 Erwinia amylovora, 503, 523–524, 524 Erwinia chrysanthemi, 502 microbial virulence, 521–523 plant defence, 529–530 Erwinia herbicola, 524 Erysiphe cichoracearum, 445 Erysiphe pisi, 375

742

SUBJECT INDEX

Ethylene biosynthesis 1-aminocyclopropane-1-carboxylate deaminase (AcdS), 294–295 bacteria, 295 regulation, 294 S-adenosylmethionine (SAM), 293–294 characteristics, 293 defense responses, 295 signal transduction biosynthesis, 402–403 histidine kinases, 403–404 phosphorylation, 404 Ethylenediamine-N,N’-bis(2hydroxyphenylacetic acid) (EDDHA), 503, 512, 514 Ethylene-response factor 1 (ERF1), 403, 408 Eucalyptus urophylla, 515 Exopolysaccharides, 257 Extracellular proteinase inhibitors (EPIs), 46 Extracytoplasmic sigma factors (ECF s), 504 F Fatty acid conjugate (FAC), 568, 570–571 Fatty acid desaturase-7 (FAD7) gene, 144 Fengycins, 243–244 Fenton-type redox chemistry, 519 fer mutation, 497 Ferric reductase oxidase 2 (FRO2) gene, 496 Ferric siderophores, 500 Ferric uptake regulator (Fur) gene, 503–504 Ferritin FtnA, 523 Flagellin-induced immune responses, 16–18 Flavin, 497 Flavin-dependent monooxygenase 1 (FMO1), 98, 148 Flowering locus C (FLC ) expression, 421–422 Fumonisin B1 (FB1), 12 Fungal and oomycete suppression abscisic acid (ABA), 43 BvPAL and cinnamic acid 4-hydroxylase (BvC4H) genes, 43 glucanase-inhibiting protein (GIP), 43 mechanisms, 49 oxalic acid, 44 race-specific elicitors, 44–48 supprescins A and B, 42–43 systemic acquired resistance (SAR) Arabidopsis, 196 cereals, 197–198 Cucurbits, 188–192 Japanese pear, 197 legumes, 192–194

Solanaceous species, 194–196 tomatinase, 43–44 yeast, plant–fungus interaction, 43 Fungal siderophores, 500 Fungi and plant interactions, 348–347 arbuscular mycorrhizal (AM) fungi functions, 323 pathogens, 323–324 plant nutrition improvement, 324–325 plant resistance induction, 326–328 plant root colonization, 324 binucleate Rhizoctonia, 331–332 biological control agents (BCAs) nonspecificity, 350 plant response patterns, 347, 350 F. oxysporum, 342–344 mycorrhiza biological control agents (BCAs), 323 categories, 322 Penicillium spp. Penicillium oxalicum, 344 Penicillium simplicissimum, 344–345 Phoma spp., 345 Piriformospora indica antioxidants, 330 Arabidopsis plants, 329–330 gene markers indication, 330 growth-promoting eVects, 329 induced systemic resistance (ISR), 330 root cell colonization, 329 Pythium oligandrum, 345–346 Trichoderma species plant nutrition improvement, 335–336 plant resistance induction, 336–342 plant root colonization, 333–335 Fusarinines, 512 Fusarium culmorum, 364, 469, 683 Fusarium graminearum, 525 Fusarium oxysporum, 371, 373, 511–512, 688 Fusarium solani, 513 G Gaeumannomyces graminis, 511, 689 Gastrophysa viridula, 684 GCC box, 409–410 Gibberellins long-distance signaling, 127–128 plant growth promoting rhizobacteria (PGPR), 292–293 Glomus etunicatum, 689 Glomus intraradices, 364 Glomus mosseae, 686 Glucanase-inhibiting protein (GIP), 43 Glucose oxidase (GOX), 583–584 -Glucuronidase (GUS ) gene, 369 Greenhouse and field, priming beneficial microbes, 383–384

SUBJECT INDEX Brotomax, 380, 382 BTH, 382 fungicide-mediated resistance, 383 INA, 382 OryzemateÒ , 383 Pyraclostrobin, 382–383 stress shield eVect, 383 Green-leaf volatiles (GLVs), 678–679 Growth-diVerentiation balance hypothesis (GDBH), 694–695 H Harpins (HrpZ), 10 Helianthus annuus, 442 Helicoverpa zea, 560, 617 Heliothis virescens, 588, 617 Heptaglucoside-binding protein (HGP), 15–16 Herbivore associated molecular pattern (HAMP), 568–569, 594 Herbivore eVectors decoy defenses AVymetrix Gene Chips, 584 biotrophic pathogens, 585 Bre. brassicae, 586 My. persicae, 586 SA-JA cross-talk, 585–586 SLWF-Arabidopsis interaction, 584–585 glucose oxidase, 583–584 regurgitant, 582 salivary oxidases and redox hypothesis, 587 specialist insects, toxic phytochemicals, 588–589 wound healing antagonism, 586–587 Herbivore elicitors -glucosidase, 571 caeliferin, 571 chitin, 574 endosymbiotic microbes, 568 hemipteran saliva, 575–576 herbivore associated molecular pattern (HAMP), 568 inceptin, 571–572 lysozyme, 574–575 oviduct secretions anti-aphrodisiac benzyl cyanide, 573–574 bruchins, 573 lipid elicitor, 572 weevil egg, 572–573 saliva and oral secretions, 567 volicitin emission, 568–569 fatty acid conjugate (FAC), 568, 570–571

743

linolenic acid, 568, 570 N. attenuata, 570 Spodoptera exigua, 568 Heterorhabditis megidis, 643 High-sugar resistance, 366 Histone code, 420–421 Homeostasis and iron acquisition strategy microorganism high-aYnity iron-transport system regulation, 503–505 siderophore, 499–503 plant grasses, 494–496 hormonal regulation, 499 non-grass model, 496 organic compound and iron uptake, 497 transcriptional and translational regulation, 497–499 Host defense suppressors. See also RNA silencing, viruses bacterial pathogens coronatine toxin suppression, 52 extracellular polysaccharides (EPS), 51–52 flagellum filament, 50 multifunctional eVectors, 53–56 RNA and RNA-binding protein targeting, 56–57 Type three protein secretion eVectors (T3SE), 48, 52–53 ZigZag model, 48, 49 cell wall-degrading enzymes and toxins, 40–41 fungal and oomycete pathogens abscisic acid (ABA), 43 BvPAL and cinnamic acid 4-hydroxylase (BvC4H) genes, 43 glucanase-inhibiting protein (GIP), 43 mechanisms, 49 oxalic acid, 44 race-specific elicitors, 44–48 supprescins A and B, 42–43 tomatinase, 43–44 yeast, plant–fungus interaction, 43 phytoalexins, 41 RNA silencing suppressor (RSS) protein genome organization, Tombusviridae, 70–71 HC-Pro expression, 70 mode of action, 71 RSS gene introduction, 72 sequence homology, 71–72 viral replication, 72 Hyaloperonospora arabidopsidis, 97, 149, 182, 184, 196, 253, 260, 342, 365, 375

744

SUBJECT INDEX

I Inceptin, 617 Indole-3-acetic acid (IAA) Azospirillum brasilense, 289–290 biosynthesis, 288 gene mutants, 290 indole-3-acetamide (IAM), 288 indole-3-acetonitrile (IAN), 289 indole-3-pyruvate (IPyA), 288–289 tryptamine (TAM), 289 tryptophan side-chain oxidase (TSO), 288 information-processing system, 290–291 Induced plant defense response induced resistance (IR) beneficial microorganisms, 363–364 chemicals, 364–365 primary metabolism modifications, 366 systemic acquired resistance (SAR), 362–363 wounding, 365–366 priming mechanism, 367 BABA-IR, 374–376 beneficial microorganisms, 372–374 features, 384 parsley cell culture, 367–369 primary metabolism modifications, 378–379 wound-induced resistance, 376–378 relevance, plant production cost and benefits, 379–380 greenhouse and field, 380–384 Induced systemic resistance (ISR), 174–175 long-distance signaling induction, 151–152 priming, 153 signal perception, 152–153 nonpathogenic fungi and plants, interactions, 330 rhizobacteria (see Rhizobacteria-induced systemic resistance (ISR)) Trichoderma species T. asperellum strain T34-mediated ISR, 342 T. harzianum strain T39-mediated ISR, 314–342 Iron acquistion strategy (see Homeostatis and iron acquistion strategy) Ascomycete fungi, 525–526 Er. amylovora pathogenicity, 523–524 Er. chrysanthemi pathogenicity achrysobactin-defective mutants, 522–523 chrysobactin-defective mutants, 522 gene transcription, 523 symptoms, 521 plant defense

Blumeria graminis infected wheat, 527–528 Er. chrysanthemi infected Arabidopsis, 529–530 Er. chrysanthemi infected Arabidopsis, 529–530 Phytophthora infestans infected potato, 528–529 siderophore biosynthesis, 519, 521 Ustilago maydis, 526–527 Iron-regulated transporter 1 (IRT1), 496 Isochorismate synthase (ICS), 404–405 J Jasmonic acid (JA) signal transduction biosynthesis, 400 coronatine, 401–402 growth inhibition, 401 mutant characteristics, 400–401 a-linolenic acid, 400 wound-induced resistance, 400 wound response eVector genes, 139 grafting experiments, 135 methyl-JA (MeJA) and JA-isoleucine (JA-Ile), 135–136 modulation, 138–139 mutants, 134 sieve element/companion cell complex, 136–137 L Lentinula edodes, 469 Lesion-mimic mutants (LMMs) defense-signaling genes, mutant screening MOS mutants, 111 SFD mutants, 110–111 SSD mutants, 108–110 examples, 108 host and nonhost pathogens, similarities nonhost resistance, bacteria, 103–104 penetration resistance, Arabidopsis, 100–103 pathogen-associated molecular patterns (PAMPs) avirulence (Avr) proteins, 94 bacterial PAMP receptors, 93–94 decoy model, 95 eVector proteins, 94 fungus PAMP receptors, 93 guard model, 95 microbes, 93 R gene-mediated resistance, 94–95 transcription profiling, 96

SUBJECT INDEX phenotype, 107–108 SA signaling pathways, EDS1/PAD4 regulation ALD1 and AGD2 gene mutation, 97–98 Arabidopsis, 96–97 enhanced disease susceptibility 5 (eds5) mutant, 97 flavin-dependent monooxygenase 1 (FMO1), 98 nonexpressor of PR genes 1 (NPR1), 99 SA induction deficient 2 (SID2) gene, 97 systemic acquired resistance (SAR), 99S W-box, 99 Lipopolysaccharides bacterial and plant interactions, 241 functions, 240–241 O-antigen, 242 Ps. fluorescens WCS374 vs. Ps. putida WCS358, 242–243 Rhizobium etli, 243 Localized acquired resistance (LAR), 362 Long-distance signaling flowering time cytokinins, 127 FT, universal floral stimulus, 130–131 gibberellins, 127–128 phloem mobile Flowering locus T (FT) protein, 129–130 regulation, gene characterization, 128–129 sucrose, 127 ISR induction, 151–152 priming, 153 signal perception, 152–153 SAR constitutive in disease resistance-1 (CDR1), 146–147 cucumber–Arabidopsis model system, 150 defective in induced resistance (DIR1), 143–144, 147–148 detachment 9 (dth9) mutant, 148 enhanced disease resistance-1 (EDR1) gene, 148 establishment stage, 140 ET, 149 fatty acid desaturase-7 (FAD7) gene, 144 flavin-dependent monooxygenase (FMO1), 148 induction, 139–140 JA, 144–145 long-distance signal movement, 140

745

manifestation stage, 141 MeSA, 145–146 SA and NPR1 role, 141–142 signal transport, 142–143 suppressor of fatty acid desaturase deficiency 1 (sfd1) mutant, 144 tobacco and Arabidopsis, 149–150 WIN3 expression, 148 techniques, 153–154 vegetative to flowering transition, flowering time florigen concept, 125 flowering control, 125–126 phloem mobile signal, 126–127 salicylic acid (SA), 131–132 wound response JA (see Jasmonic acid (JA)) systemin (see Systemin, wound response) M Magnaporthe oryzae, 515 Maloideae, 523 Manduca sexta, 684 Medicago truncatula, 364 MEKK1 signaling cascade, 577–579 Mi1.2 gene, 591–592 Microbe-associated molecular patterns (MAMPs), 42, 367, 554–555. See also Pathogen derived molecular pattern (PAMP)triggered immunity (PTI) Microbial iron acquisition impact induced systemic resistance WCS374 elicitors, 515–516 WCS417 mutant, 515 microbial antagonism carbon-iron interaction, 514 naturally suppressive soil, 512–513 pyoverdine, 511–512 plant growth and nutrition deleterious pseudomonads inoculation, 516–517 Fe-pyoverdines, 517 phytosiderophores, 517–518 plant growth-promoting rhizobacteria (PGPR), 510–511 Mitogen-activated protein kinase (MAPK) signaling cascades, 23–24 AP2C1, 579 Arabidopis, 579–580 MAP3Ks, MAP2Ks and MAPKs, 577 MKK1/2, 578–579 MKK4/5, 577–578 MPK3/6, 577–578 MPK4, 578–579 plant innate immunity, 577–578 Modifier of snc1 (MOS) mutants, 111

746

SUBJECT INDEX

Monosporascus cannonballus, 380 Mugineic acids (MAs) synthesis, 496 Multidrug and toxin extrusion (MATE) antiporters, 405 MYB transcription factor defense response, 412 hypersensitive response, bacterial pathogen, 411–412 metabolite biosynthesis regulation, 411 protein characteristics, 410–411 Mycorrhiza infection eVects, 689–690 MYC transcription factor pathogen defense response, 412–413 structural and functional characteristics, 412 Myzus persicae, 564, 584, 586, 588 N Natural resistance-associated macrophage protein 1 (NRAMP1), 519 Necrosis and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs) cellular ion homeostasis, 14 Erwinia spp., 12 Phytophthora sojae, 13 vs. phytotoxins, 13–14 Pythium aphanidermatum, 13 Neutrophil gelatinase-associated lipocalin (NGAL), 519 Nicotiana attenuata defense signaling, 561–562 FAC, 570 MAPK cascades, 578–580 Nicotiana longiflora, 691 Nitric oxide (NO), 300–301 Nitrogen transformations, PGPR biological nitrogen fixation (BNF), 296 denitrification, 297–298 nitrogen uptake, 296–297 Nonexpressor of PR genes 1 (npr1) mutant, 406–407, 414 Nonpathogenic plant growth-promoting rhizobacteria (PGPR), 363 Non-ribosomal peptide synthases (NRPS), 501 NRAMP3 and NRAMP4 mutants, 529–530 Nucleotide binding domains (NBDs), 473 Nutrient acquisition strategy disease resistance, 447–448 NPR1, 446 PDF1.2 gene, 447 PR-1 gene expression, 446–447 signaling pathways, 448–449 WRKY33 gene, 447

O Octadecanoid pathway, 365 Oligogalacturonides (OGAs), 565–567 Oomycete and fungal suppression abscisic acid (ABA), 43 BvPAL and cinnamic acid 4-hydroxylase (BvC4H) genes, 43 glucanase-inhibiting protein (GIP), 43 mechanisms, 49 oxalic acid, 44 race-specific elicitors, 44–48 supprescins A and B, 42–43 systemic acquired resistance (SAR) Arabidopsis, 196 cereals, 197–198 Cucurbits, 188–192 Japanese pear, 197 legumes, 192–194 Solanaceous species, 194–196 tomatinase, 43–44 yeast, plant–fungus interaction, 43 Optimal defence hypothesis (ODH), 694–695 Orobanche crenata, 689 Oxidative burst, 368 12-Oxo-phytodienoic acid (OPDA), 400–401 Oxylipins, 684 P Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), 224, 669 bacterial suppression avrB, 57–58 avrPto, 53–54 avrPtoB (hopAB2), 54–55 avrRpm1, 58 avrRps4, 61–62 avrRpt2, 55–56 complexity and evolution, 63–64 coronatine toxin suppression, 52 extracellular polysaccharides (EPS), 51–52 hopAI1, 61 hopAM1(avrPpiB), 59 hopAN (avrE1/wtsE/ dspA/dspE), 58–59 hopAO1 (hopPtoD2), 62 hopM1 (hopPtoM), 59–60 hopT1-1, 57 hopU1 (hopPtoS2), 56–57 MAP kinase signaling, 61 negative regulators, 57–58 RNA and RNA-binding protein targeting, 56–57 suppressed NHO1 expression, 62–63

SUBJECT INDEX Type three protein secretion eVectors (T3SE), 52–53 Type VI secretion system (T6SS), 63 vesicle traYcking disruption, 59–60 xopD, 56 definition, 553 integrating signals, 580–581 lesion-mimic mutants (LMMs) avirulence (Avr) proteins, 94 bacterial PAMP receptors, 93–94 decoy model, 95 eVector proteins, 94 fungus PAMP receptors, 93 guard model, 95 microbes, 93 R gene-mediated resistance, 94–95 transcription profiling study, 96 pathogen-associated molecular patterns (PAMPS) harpins (HrpZ), 10 hypersensitive response, 10 inducers, 5, 6–7 insects, 4 jawed vertebrates, 4 Pep-13 motif, 8 recognition systems, 8–9 structure, 5 vertebrate and nonvertebrate organisms, 5 pathogen-derived toxins Alternaria alternata (AAL) toxin, 12 fumonisin B1 (FB1), 12 necrosis and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs), 12–14 peptaibols, 14–15 pattern-recognition receptors and BAK1, 564–565 plant origin elicitors, 565–566 signal transduction BRI1-associated receptor kinase 1 (BAK1), 21–22 FLS2, 21 kinase-associated protein phosphatase (KAPP), 21 mitogen-activated protein kinase (MAPK) cascades, 23–24 NO production, 22–23 plant hormones, 24 suppression abscisic acid-dependent stomatal closure, 26–27 FLS2 activity interference, 25, 26 PPR activity suppression, 25, 26 RPM1-interacting protein 4 (RIN4), 25–26 ZigZag model, 555–556 Pathogen-derived toxins Alternaria alternata (AAL) toxin, 12

747

fumonisin B1 (FB1), 12 necrosis and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs) cellular ion homeostasis, 14 Erwinia spp., 12 Phytophthora sojae, 13 vs. phytotoxins, 13–14 Pythium aphanidermatum, 13 peptaibols, 14–15 Pathogenesis-related (PR) proteins, 362 biotic and abiotic stress and hormone signaling PAMP-FLS2 protein interaction, 445 pathogen recognition, 444–446 SAR, 446 classification, 442–444 definition, 442 energy balance and immunity, 449–450 gene set and nutrient acquisition strategy disease resistance, 447–448 NPR1, 446 PDF1.2 gene, 447 PR-1 gene expression, 446–447 signaling pathways, 448–449, 473 WRKY33 gene, 447 immunity system, 440–441 plant immunity, 441–442 PR-5 function antifungal activity, 452 binding activity, 453 co-expression, 453 fungal membrane permeabilization, 452 genomic databases, 454–462 overexpression, 450–452 stress tolerance, 451–452 structural feature, 450 thaumatin (THN)-C1q-TNF domain, 464–465 adiponectin, 463 binding domain, 468–469 fusion protein, 467–468 intracellular mechanism, 463, 466 phyla, 469–472 Pattern-recognition receptors (PRRs), 669 Pectobacterium atrosepticum, 366, 378–379 Penicillium spp. Penicillium oxalicum, 344 Penicillium simplicissimum, 344–345 Peptaibols, 14–15 -Phaseolin (PHAS) promoter, 422 Phenolics, 497 Phenylalanine ammonia-lyase (PAL) gene expression, 367 Phosphates, 298 Phospho-ACS6, 577 Phytoalexin deficient 4 (PAD4) protein, 96 Phytoalexins, 41 Phytohormones

748

SUBJECT INDEX

Phytohormones (cont.) abscisic acid (ABA), 295 auxins, 286–291 (see also Indole-3-acetic acid (IAA)) cytokinins, 291–292 ethylene, 293–295 functions, 286 gibberellins, 292–293 strigolactone, 286 Phytophthora infestans, 366, 379, 528–529, 697 Phytophthora parasitica, 373, 689 Pieris rapae, 412, 685 Piriformospora indica, 364, 384, 683 antioxidants, 330 Arabidopsis plants, 329–330 gene markers indication, 330 growth-promoting eVects, 329 induced systemic resistance (ISR), 330 root cell colonization, 329 Plant defense response. See also Induced plant defense response airborne plant-plant communication community level, 691–692 herbivore and pathogen resistance, 677–678 mechanisms, 679 VOC priming, 680 chromatin modifications gene expression, 420–421 innate immunity, 422–426 plants, 421–422 defense signaling regulatory compounds ethylene signal transduction, 402–404 jasmonate signal transduction, 400–402 SA signal transduction, 404–407 evolutionary considerations, 693 immune signaling pathways, 398–399 induced resistance (see Resistance induction) local signalling abscisic acid (ABA), 672–673 auxin signalling, 671 cytokinins, 671–672 pathogen recognition, 669–670 plant growth-defence cross-talk, 673 resistance hormones, 670–671 resistance traits, 668–669 systemic signalling, 673–674 airborne systemic signals, 676 herbivore resistance, 674–675 pathogen resistance, 675–676 RNA signaling, 676 transcriptional activation, 419 transcription factors AP2/ERF, 408–410 BZIP, 413–415

MYB, 410–412 MYC, 412–413 Venn diagram, 407–408 WRKY, 415–418 variable resistance genetic level, 690–691 phenotypic level, 691 Plant growth-promoting rhizobacteria (PGPR). See also Rhizobacteriainduced systemic resistance (ISR) agricultural aspects and relevance endophytes, 304–305 grain filling, 306 inoculant technology, 306–308 probiotics, 308 symbiotic systems, 304–306 vegetative growth, 306 infection and colonisation resistance, 688–689 iron, 298–299 mechanism, 688 nitric oxide (NO), 300–301 nitrogen transformations biological nitrogen fixation (BNF), 296 denitrification, 297–298 nitrogen uptake, 296–297 phenomenon, 687–688 phosphate, 298 phytohormones, 287 auxins, 286–291 cytokinins, 291–292 ethylene, 293–295 gibberellins, 292–293 strigolactone, 286 quorum-sensing (QS) mechanism, 299–300 rhizosphere antibiosis, 302–304 competition, 301–302 rhizodeposition, 284–285 siderophores, 299 types, 285 vitamins, 298 volatile compounds, 300 Plant immunity damage-associated molecular patterns (DAMPs), 11 eVector-triggered immunity (ETI), 4 NO production, 22 vs. PAMPs, 10 phytohormones, 24 R-protein-mediated sensing, 26 iron acquisition impact, 506 antagonistic activity, 509–510 ferritin, 507–508 minimal inhibitory concentration (MIC), 505, 507 pseudomonad community, 508

SUBJECT INDEX rhizosphere adaptation model strain, 508–509 microbial colonization, 3 molecular organization antimicrobial defense systems, 2–3 nonself recognition, 2 PAMP-triggered immunity (PTI) (see Pathogen associated molecular pattern (PAMP)-triggered immunity (PTI)) pattern-recognition receptors (PRRs), 2 receptors mediating pattern recognition elongation factor receptor (EFR), 18–19 flagellin-induced immune responses, 16–18 flagellin sensing 2 (FLS2) gene, 16 heptaglucoside-binding protein (HGP), 15–16 leucine-rich repeat (LRR) protein (LRR-P), 20 lysine motif (LysM) receptor, 19–20 Toll-like receptors (TLRs), 15 Plant–microbe interactions, iron uptake. See also Siderophores acquisition strategy and homeostasis microorganisms, 499–505 plants, 494–499 biomass productivity and quality, 531 biotic–abiotic interactions, 531–532 metabolic process, 492–493 pathogenesis homeostasis control, 519, 520 microbial virulence, 519–527 plant defence, 527–530 rhizodeposits, 530–531 saprophytic life microbial iron acquisition impact, 510–518 plant iron acquisition impact, 505–510 soil iron concentration, 493–494 Plant-plant communication, 677, 691 Runaway process, 691–692 Plant-plant interaction, VOC priming defence C6-volatiles, 649 response, 646, 649 within-plant signalling, 649–650 transcriptional response, 647–648 C6-volatiles, 645–646 herbivore performance, 643–644 MeJA and MeBA, 644 terpenoids, 645 Plant volatiles. See Volatile organic compounds (VOCs) Plectosphaerella cucumerina, 365, 375 Posttranscriptional gene silencing (PTGS). See RNA silencing, viruses

749

PR-5 protein. See also Thaumatin (THN)-C1q-TNF domain function antifungal activity, 452 binding activity, 453 co-expression, 453 fungal membrane permeabilization, 452 overexpression, 450–452 stress tolerance, 451–452 genomic databases acidic, basic, and neutral protein, 454, 456 characteristics and tissue specific expression, 454–458 energy balance, 462 kinase domain, 458–459 stress, senescence, and nutrients response, 454, 457, 458 THN domain fusions, 458, 460–462 Primary metabolism induced resistance, 366 Priming induced resistance BABA-IR abiotic stress, 376 biotic stress, 374–376 beneficial microorganisms bacterial lipopolysaccharides, 374 ISR, 372–373 defensive capacity, IR, 367, 368 features, 384 parsley cell culture, 367–369 primary metabolism modifications, 378–379 relevance, plant production cost and benefits, 379–380 greenhouse and field, 380–384 SAR Arabidopsis, 369–371 Asparagus oYcinalis, 371 cowpea plants, 371 cucumber plant, 371–372 soybean cell suspension cultures, 371 sunflower plants, 371 tobacco, 369 wound-induced resistance herbivores, 376–378 plant species, 378 Prosystemin gene, 366 Pseudobactin (PSB) Arabidopsis, 251 functions, 247 Ps. fluorescens WCS374r, 247–249, 250 Ps. putida WCS358, 247 S-adenosyl-L-methionine (SAM), 249–250 Pseudomonas fluorescens, 508–509 Pseudomonas putida, 509, 511–512 Pseudomonas syringae, 365, 423, 445, 448, 671 Pseudomonine, 515

750

SUBJECT INDEX

Puccinia helianthi, 371 Pyocyanin, 254–255 Pyoverdine characterization, 501–502 iron uptake regulation, quorum sensing, 504 microbial antagonism, 511–512 parts, 500–501 plant growth and nutrition, 517 synthesis, 501 Pythium aphanidermatum, 510 Pythium irregulare, 672 Pythium oligandrum, 345–346 Pythium tolaasi, 511 Q Quorum-sensing (QS) mechanism iron uptake, 504 plant growth-promoting rhizobacteria (PGPR), 299–300 R Race-specific eVector (RSE), 556 Race-specific elicitors AVR3aKI protein, 47–48 cognate Avr gene product, 45–46 extracellular proteinase inhibitors (EPIs), 46 INF1, 48 PEP1 gene, 47 Phytophthora-inhibited protease (PIP1), 46 SIX1 gene, 47 Ralstonia solanacearum, 515 Random amplified polymorphic DNA (RAPD), 508 Receptors mediating pattern recognition elongation factor receptor (EFR), 18–19 flagellin-induced immune responses, 16–18 heptaglucoside-binding protein (HGP), 15–16 leucine-rich repeat (LRR) protein (LRR-P), 20 lysine motif (LysM) receptor, 19–20 Toll-like receptors (TLRs), 15 Resistance induction allocation impact, 681–683 ASM inducer, 682–683 SAR-associated cost, 682, 683 direct and indirect impact, 680–681 ecological impact biotrophic and necrotrophic pathogens, 686–687 insect, 683–685 mutualistic symbioses, 685–686 phytobacterial communities, 686

mutualistic micro-organisms mycorrhiza, 689–690 plant growth-promoting rhizobacteria (PGPR), 687–689 response patterns carbon/nutrient balance hypothesis (CNBH), 695–697 growth-diVerentiation balance hypothesis (GDBH), 694–695 optimal defence hypothesis (ODH), 694–695 plant nutrition and resistance, 693–694 resource availability hypothesis (RAH), 697 Resource availability hypothesis (RAH), 695–697, 697 Rhizobacteria-induced systemic resistance (ISR), 226–227. See also Plant growth-promoting rhizobacteria (PGPR) Arabidopsis–Pseudomonas fluorescens WCS417r system, 262–263 helix-loop-helix transcription factor MYC2, 260 JA- and ET-response mutants, 259–260 NPR1, 260–261 Ps. syringae pv. tomato, 261–262 R2R3-MYB-like transcription factor gene MYB72, 261 SA, 258–259 ISR, 227, 233 vs. plant–pathogen system, 228–232 resistance-inducing molecules 4-(aminocarbonyl) phenylacetate, 258 antibiotics, 253–255 biosurfactants, 243–245 exopolysaccharides, 257 flagella, 234–240 lipopolysaccharides, 240–243 N-acyl-L-homoserine lactone (AHL), 245–246 N-alkylated benzylamine, 246 siderophores, 247–252 volatiles, 255–256 SA-dependent and SA-independent signaling, 264 SA-dependent ISR signaling, 263–264 Rhizobium leguminosarum, 689 Ripening hormone. See Ethylene RNA silencing, viruses discovery co-suppression, 66 pathogen-derived resistance (PDR) concept, 65 RNA-mediated resistance, 65 RNA silencing suppressor (RSS), 66 virus-specific siRNAs, 66

SUBJECT INDEX miRNA pathway antiviral mechanism, plants, 69 viral RSS protein, 72–73 RNA silencing suppressor (RSS) protein genome organization, Tombusviridae, 70–71 HC-Pro expression, 70 mode of action, 71 RSS gene introduction, 72 sequence homology, 71–72 viral replication, 72 siRNA pathway antiviral mechanism, plants, 67–69 insects, 73 RPM1-interacting protein 4 (RIN4), 25–26 Rumex obtusifolius, 684 S Saccharomyces cerevisiae, 453, 462, 526 S-adenosylmethionine (SAM), 402–403 Salicylic acid (SA), 251–252 lesion-mimic mutants (LMMs) ALD1 and AGD2 gene mutation, 97–98 Arabidopsis, 96–97 enhanced disease susceptibility 5 (eds5) mutant, 97 flavin-dependent monooxygenase 1 (FMO1), 98 nonexpressor of PR genes 1 (NPR1), 99 SA induction deficient 2 (SID2) gene, 97 systemic acquired resistance (SAR), 99 W-box, 99 long-distance signaling progress, 131–132 rhizobacteria-induced systemic resistance (ISR) siderophores, 251–252 signaling mechanism, 263–264 signal transduction EDS1 promoter, 405 EDS5 protein, 405–406 NPR1 protein, 406–407 pbs3 mutant, 406 phylloquinone synthesis, 404–405 protein–protein interactions, 406 Salivary proteins hemipteran saliva, 575–576 lysozyme, 574–575 Sclerotinia homeocarpa, 458 Sesquiterpene trans-a-bergamotene, 617 Siderophores Erwinia spp. achromobactin, 502 chrysobactin, 502–503 iron uptake regulation, 504–505

751

fluorescent pseudomonads (see also Pyoverdine) membrane protein synthesis, 500–501 siderotyping, 501–502 functional group, 499–500 pseudobactin (PSB) Arabidopsis, 251 functions, 247 Ps. fluorescens WCS374r, 247–249, 250 Ps. putida WCS358, 247 S-adenosyl-L-methionine (SAM), 249–250 salicylic acid, 251–252 Silverleaf whitefly (SLWF), 584–585 Solanaceae sps., 365–366 Spilanthes calva, 384 Spodoptera exigua, 568, 684 Spodoptera frugiperda, 571–572 Spodoptera littoralis, 561 Stomatal closure, 52 Stress hormone. See Ethylene Strictosidine synthase (STR) gene expression, 409 Strigolactone, 286 Supprescins, 42–43 Suppressed in prosystemin-mediated responses-1 (spr1) mutant, 366 Suppressor of fatty acid desaturase deficiency (SFD) mutants, 110–111 Suppressors of syntaxin-related death (SSD) mutants, 108–110 Systemic acquired resistance (SAR), 225–226 bacteria, 201 Arabidopsis, nonhost resistance response, 200 cell wall modifications, 200 disease tolerance, 199 induced response, 198 symptom suppression, 199–200 biochemical changes gene expression, 187 hydroxyproline-rich glycoproteins (HRGP), 186–187 pathogenesis-related proteins, 185–186 SA accumulation, 187–188 biological spectrum, 177 characteristics, 175–176 fungi and oomycetes Arabidopsis, 196 cereals, 197–198 Cucurbits, 188–192 Japanese pear, 197 legumes, 192–194 Solanaceous species, 194–196 induction chemical induction, 181–185

752

SUBJECT INDEX

Systemic acquired resistance (SAR) (cont.) hypersensitive response, 178 necrotizing pathogens, 177–178 pathogen-produced inducers, 181 long-distance signaling constitutive in disease resistance-1 (CDR1), 146–147 cucumber–Arabidopsis model system, 150 defective in induced resistance (DIR1), 143–144, 147–148 detachment 9 (dth9) mutant, 148 enhanced disease resistance-1 (EDR1) gene, 148 establishment stage, 140 ET, 149 fatty acid desaturase-7 (FAD7) gene, 144 flavin-dependent monooxygenase (FMO1), 148 induction, 139–140 JA, 144–145 long-distance signal movement, 140 manifestation stage, 141 MeSA, 145–146 SA and NPR1 role, 141–142 signal transport, 142–143 suppressor of fatty acid desaturase deficiency 1 (sfd1) mutant, 144 tobacco and Arabidopsis, 149–150 WIN3 expression, 148 nonexpressor of PR genes 1 (NPR1) protein, 176 priming, 188 priming induced resistance Arabidopsis, 369–371 Asparagus oYcinalis, 371 cowpea plants, 371 cucumber plant, 371–372 soybean cell suspension cultures, 371 sunflower plants, 371 tobacco, 369 PR-proteins, 362 salicylic acid (SA) signalling, 363 synthetic SA analogs, 364 viruses cell-to-cell movement inhibition, 202 lesion size and number, 201–202 SA-induced resistance, 203–204 systemic movement inhibition, 202–203 virus replication inhition, 202 Systemic induced susceptibility (SIS), 150 Systemin, wound response, 365–366 formation prosystemin, 133–134 systemin receptor SR160, 134 spr1 mutant, 135 wound-response mutants, 134

T Thaumatin (THN)-C1q-TNF domain, 464–465. See also PR-5 protein adiponectin, 463 binding domain, 468–469 fusion protein, 467–468 intracellular mechanism, 463, 466 phyla, 469–472 Thaumatococcus danielli, 450 Tissue-damaging herbivores defense signaling, 561–562 feeding mode, 560–561 Toll-like receptors (TLRs), 15 Tomatinase, 43–44 Transcriptional and translational iron uptake regulation Fe-deficiency induced transcription factor 1 (FIT1), 497–498 iron-deficiency specific clone 2 (IDS2) gene, 498 iron-related transcription factor 2 (IRO2), 498 p35S CaMV promoter, 499 Tribolium castaneum, 469 Trichoderma asperellum, 364, 373 Trichoderma species plant nutrition improvement, 335–336 plant resistance induction, 336–342 mitogen-activated protein kinase (MAPK), 340 T. asperellum strain T34-mediated ISR, 342 T. asperellum strain T203, 336–337 T. asperellum T203–cucumber– Ps. syringae pv. lachrymans system, 337–338 T. atroviride strain P1, 339 T. hamatum strain 382, 339–340 T. harzianum strain T39, 336 T. harzianum strain T39-mediated ISR, 314–342 T. longibrachiatum, 341 T. virens strain Gv29–8, 340–341 plant root colonization mycelial extracts, 335 T. asperellum strain T34, 334–335 T. asperellum strain T-203, 333–334 Turnip crinkle virus, 685 Type three protein secretion eVectors (T3SE), 52–53 Type three secretion system (T3SS), 524 U Uromyces fabae, 676 Ustilago maydis, 526–527 Ustilago sphaerogena, 499

SUBJECT INDEX V Venn diagram, transcription factor-encoding genes, 407–408 Vicia faba, 685 Viruses RNA silencing suppressor (RSS) protein (see also RNA silencing, viruses) genome organization, Tombusviridae, 70–71 HC-Pro expression, 70 mode of action, 71 RSS gene introduction, 72 sequence homology, 71–72 viral replication, 72 systemic acquired resistance (SAR) cell-to-cell movement inhibition, 202 lesion size and number, 201–202 SA-induced resistance, 203–204 systemic movement inhibition, 202–203 virus replication inhibition, 202 Volatile organic compounds (VOCs), 255–256, 300 biosynthesis ethylene, 624 linoleic acid/octadecanoid pathway, 619–621 methanol, 623–624 phenylalanine-derived volatiles, 621–622 terpenoids, 622–623 defence hormones ethylene, 632 MeJA, 627–628 MeSA, 628, 632 mutant and genetically modified plants, 629–631 emission abiotic stress, 635 concentration, 633–634 defence chemistry, 634 diurnal photoperiodicity, 632–633 plant growth conditions, 634–635 tissue type and age, 633 function, 614 growth and development, 614–615 herbivore-derived elicitor indirect defence, 618–619 plant damage, feeding style, 616 stimulation, 619 types, 617–618 indirect defence endoparasitoids, 643 herbivore foraging behaviour, 639, 642 host-plant fitness, 642–643

753

synthetic volatile, 642 transgenic plants, 639, 640–641 plant-plant interaction priming defence, 646, 649–650 transcriptional response, 643–646, 647–648 production variation cross-species analysis, 636–637 infestation, 635–636 olfactory sensitivity, 637–638 predatory arthropods, 638 quantitative and qualitative variability, 637 tritrophic interactions, 637 resistance mechanism, 615–616 trichome metabolism function and occurrence, 624–625 metabolomics and transcriptomics, 625–627 Volicitin emission, 568–569 fatty acid conjugate (FAC), 568, 570–571 linolenic acid, 568, 570 N. attenuata, 570 Spodoptera exigua W Wall-associated kinase (WAK1), 567 W-box, 99 Withania somnifera, 384 Wound-induced resistance (WIR), 376–378 Wound response jasmonic acid (JA) eVector genes, 139 grafting experiments, 135 methyl-JA (MeJA) and JA-isoleucine (JA-Ile), 135–136 modulation, 138–139 mutants, 134 sieve element/companion cell complex, 136–137 systemin prosystemin, 133–134 spr1 mutant, 135 systemin receptor SR160, 134 wound-response mutants, 134 wound-induced resistance (WIR) glutathione and camalexin role, 376–377 plant species, 378 prosystemin gene, 366 protease inhibitor (PIN), 365 VOC-induced priming, 377–378 wound-inducible protein kinase (WIPK), 400

754

SUBJECT INDEX

WRKY transcription factors, 577–579 avirulence eVector recognition, 417 ICS1 gene expression, 416–417 NtWRKY12, 416, 418 pathogen resistance, 417–418 structural and functional characteristics, 415–416 transcriptional activators, 416

X Xanthomonas campestris, 364, 374, 521, 671 Xenopyoverdines, 502 Y Yellow stripe 1 (ys1) mutant, 495–496