Mitigating Allergenicity of Crops (Advances in Agronomy, Volume 107)

C H A P T E R

T H R E E

Mitigating Allergenicity of Crops Peggy Ozias-Akins, Ye Chu, Joseph Knoll,1 and Anjanabha Bhattacharya2 Contents 93 94 95 96 97 97 102 107 113 114

1. Introduction 1.1. Allergens and allergenicity 1.2. Allergen protein families 1.3. Crop-specific allergens 2. Methods to Alter Allergen Content of Crops 2.1. Natural variation 2.2. Induced variation: Mutagenesis 2.3. Induced variation: Transgenics 3. Conclusions References

Abstract Reducing the allergenicity of edible crops may be feasible to some extent through genetic means. Allergenicity of different crops varies widely, and consumed components may present multiple allergenic proteins, some of which play essential roles in growth and development of the plant or seeds. Identifying spontaneous or induced mutations in genes for allergenic proteins is facilitated by technological advancements in DNA sequence analysis and proteomics. Furthermore, genetic engineering provides strategies for altering gene expression to study the effects of allergen reduction. In this review, allergens of most concern from major crops within the ‘‘Big 8’’ allergen group are described and approaches for mitigation of allergenicity in these crops are presented.

1. Introduction Eliminating allergens in crops is a lofty goal that may not be entirely feasible given the roles that allergenic proteins play in plant growth and development; nevertheless, a substantial body of information has accumulated Department of Horticulture, University of Georgia Tifton Campus, Tifton, Georgia, USA Current address: USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA Current address: Bench Biotechnology, Vapi, Gujarat, India

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Advances in Agronomy, Volume 107 ISSN 0065-2113, DOI: 10.1016/S0065-2113(10)07003-3

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2010 Elsevier Inc. All rights reserved.

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on the consequences of protein modification, which suggests that at least mitigation is attainable. Crop plants are cultivated for food, feed, fiber, and fuel, and their increased production in recent history has been significantly dependent on genetic gains. Domesticated plants have been undergoing human selection for thousands of years, but intensive genetic enhancement through breeding has occurred only within about a century (Duvick, 1996). While our existence is dependent on crop plants, certain of their biochemical components can invoke an immune response in humans upon oral or inhalation exposure that results in negative health consequences. Artificial selection practiced during plant breeding usually narrows the germplasm base for a crop and may or may not have an associated effect on allergen content or composition depending on linkage of allergen genes with selected traits or pleiotropic effects. Since many allergens are seed storage proteins, and artificial selection for seed characteristics is routine, associated changes in seed protein content or composition are inevitable. Only recently has artificial selection been conducted to intentionally alter composition or content of an allergenic protein in a crop. Herein, we review attempts to reduce or eliminate pollen and food allergens from crops using germplasm resources, mutagenesis, and genetic engineering (GE).

1.1. Allergens and allergenicity An allergen is a substance that triggers a misguided human immune response and usually is found in pollen, mold, dander, and food. The intricacies of interactions among components of the human immune system and allergens still are not fully understood (Shreffler, 2009). Food and pollen allergens typically induce an IgE response from the immune system during sensitization and trigger an IgE-mediated reaction upon subsequent exposure. Pollen allergic reactions present as mucosal and respiratory symptoms (allergic rhinitis, better known as hay fever, to asthma). Food allergic reactions present as symptoms ranging from skin reactions (urticaria, or hives, and angioedema) and gastrointestinal symptoms (nausea, abdominal pain, diarrhea, vomiting) to life-threatening anaphylaxis. In the latter case, timely intervention with administration of epinephrine is essential (Simons, 2008; Young et al., 2009). Food allergy (food hypersensitivity) is not to be confused with food intolerance, which is a nonimmunologic reaction, although both food allergy and food intolerance are considered adverse food reactions (Lee and Burks, 2006; Perry et al., 2006). The most commonly encountered food allergies are to the ‘‘Big 8’’ foods: milk, egg, fish, shellfish, peanut, tree nuts, soy, and wheat (Teuber et al., 2006). Some of these allergies can be outgrown, for example, allergies due to milk, egg, soy, and wheat; but others, particularly peanut, tree nuts, fish, and shellfish, often persist to adulthood. Allergy diagnosis is much easier than management, and recommended therapy usually means avoiding the food.

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Four of the ‘‘Big 8’’ allergenic foods are plant products and one of these, soy, is particularly difficult to avoid because of its nearly ubiquitous use in processed food products. Progress in developing immunotherapies for food allergies has been made but none are yet approved or recommended for standard treatment. Use of injection immunotherapy, while common for inhaled (including pollen) allergens, is not generally recommended for food allergies because the potential for serious adverse reactions is high (Burks et al., 2001). A number of novel immunomodulating therapies are under investigation, including peptide immunotherapy, DNA immunization, herbal remedies, and anti-IgE immunotherapy (Burks et al., 2006; Wang and Sicherer, 2009). Pollen–food allergies also have been documented where sensitization to inhaled allergens results in cross-reactivity to certain food allergens. The best characterized examples of pollen–food allergy syndrome (also known as oral allergy syndrome) are sensitization to birch, ragweed, grass, and mugwort pollen resulting in allergic reactions to certain raw vegetables and fruits. Pollen allergens from noncrop species implicated in pollen–food allergy syndrome cannot easily be avoided in certain geographic areas. As with treatment of other food allergies, the recommendation is to avoid the associated allergenic foods even though injection immunotherapy has been reportedly used to treat pollen–food allergies (Asero, 1998) yet is not in common practice (Steinman, 2009).

1.2. Allergen protein families Plant allergens are usually proteins found in pollen and food, thus exposure is via inhalation or ingestion, respectively. While many of these proteins are glycosylated, and cross-reactive carbohydrate determinants are recognized by IgE, the carbohydrate side-chains have minimal allergenic activity (Altmann, 2007; Mari and Scala, 2006). Protein allergens are named according to the rules established by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) and included an abbreviation of the taxonomic name (first three letters of the genus followed by a space and the first letter of the species) plus an Arabic numeral that is assigned in the order that an allergen is identified (http://www.allergen.org/ Allergen.aspx; Larsen and Lowenstein, 1996). The number of IUIS recognized allergens is less than the number actually described in the literature and databases, and nonconventional names persist. Currently, 208 food allergens are distributed among 40 protein families and 204 pollen allergens fall into 52 allergen families (according to the AllFam database, http://www. meduniwien.ac.at/allergens/allfam/, as of 07 Dec 2009; Radauer et al., 2008). Single-member protein families comprise 58% (23/40) and 50% (26/52) of all food and pollen allergen protein families, respectively. The evolutionary biology of plant food and pollen allergens recently has been reviewed (Radauer and Breiteneder, 2006, 2007). Protein allergens

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are represented by relatively few protein classes, that is, only 2% of the 9318 protein families in the Pfam database (Finn et al., 2008) are known to contain allergenic proteins (Radauer et al., 2008). Some of these protein families are essential to metabolic function, such as profilins, which are actin-binding proteins important for cytoskeleton organization. The prolamin superfamily contains the largest number of food allergens (27%) and comprises seed storage proteins, prominent components of legume seed cotyledons and cereal endosperm, as well as protease inhibitors and lipid transfer proteins. The most prevalent protein families for pollen allergens are profilin (12%) and expansin C-terminal domain (10%), allergenic forms of which are confined to the grass family. Allergenic profilins are distributed across 10 plant families and they rank third (12%), behind prolamins and cupins (17%), among food allergens. Cupins have conserved barrel domains and include 7S and 11S seed storage proteins, also known as vicilins and glycinins (legumins), respectively. Other protein families containing significant numbers of food and pollen allergens, respectively, are Bet v 1-related (7%) and EF-hand domain (9%). Bet v 1 is a pathogenesis-related (PR) protein with ribonuclease activity from birch that plays a significant role in pollen–food allergy syndrome while EF-hand domain proteins are calcium binding and form helix-loop-helix motifs. While no individual structural features of a protein can be used to predict allergenicity, particularly for ingested proteins, some common properties of allergens are resistance to degradation in the gastrointestinal tract or upon exposure to heat, acid, or proteolytic conditions due to disulfide bonds, oligomeric structure, binding to lipid or metal ions, or repeating units. Protease (pepsin) susceptibility has become a standard assay for predicting allergenicity (Thomas et al., 2004) that has been validated in a mouse model (Bowman and Selgrade, 2008). Of the crops among the ‘‘Big 8’’, soybean and peanut each contain allergenic members of the prolamin, cupin, profilin, and Bet v 1-like protein families among others. Soybean has been the most intensively studied allergenic crop with significant advances toward allergen reduction through genetic means (L’Hocine and Boye, 2007).

1.3. Crop-specific allergens Major crops represented among the ‘‘Big 8’’ allergens are soybean (Glycine max), peanut (Arachis hypogaea), and wheat (Triticum aestivum). Multiple seed proteins within each of these species are food allergens. A major allergen is defined as one that reacts with serum IgE from >50% of allergic individuals tested. To be classified as an allergen by the IUIS Allergen Nomenclature Subcommittee, binding of IgE from serum of at least five patients or 5% of the population tested that are allergic to the respective allergen source must be demonstrated (http://www.allergen.org/Allergen.aspx). A database of named allergens is maintained at this website. Other databases with links

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to more extensive information about named allergens and their related proteins can be found at http://www.allergome.org and http://www. allergenonline.com. For peanut, there are 11 named allergens, 6 for soybean, and 10 for wheat. The classification of food allergens for these crops, in terms of their protein families and levels of allergenicity, is shown in Table 1.

2. Methods to Alter Allergen Content of Crops 2.1. Natural variation Two types of variation exist in crop plants or their wild relatives. One involves different forms of an allergenic protein encoded by different genes, either members of a multigene family and/or homeologous genes as encountered in polyploids. The second source of variation is allelic and derives from different forms of a gene among individuals in a population or species. Protein isoforms can show small variations in amino acid sequences and in posttranslational processing, thus potentially can be distinguished by molecular weight, isoelectric point, and peptide signatures. Such variation may affect allergenicity as has been demonstrated for Bet v 1-like isoforms in birch and apple (Vieths et al., 1994; Wagner et al., 2008). Within a class of proteins, some members may be highly allergenic, while others invoke little response from the immune system. This is particularly true for profilins where the only allergenic profilins are found in flowering plants, although profilins are involved in cytoskeleton regulation in plants, fungi, vertebrates, and invertebrates (Radauer and Breiteneder, 2007). Natural variation has been observed among Bet v 1 isoforms, although this is an example of ortholog and paralog rather than allelic variation. Bet v 1 is a PR protein in the PR-10 group that is expressed from a complex multigene family in Betula verrucosa (European white birch, syn. B. pendula) and its relatives. Sensitization to this pollen allergen has been implicated as a major factor in pollen–food allergy syndrome. While European white birch is not endemic to North America, sensitization to birch pollen is nevertheless prevalent and attributed to Bet v 1 homologs from other birch species. The Bet v 1 gene family has been extensively characterized in eight Betula species at the nucleotide and predicted protein sequence levels where multiple expressed isoforms as well as pseudogenes were identified (Schenk et al., 2006, 2009). One hundred twelve unique genomic sequences were predicted to encode 80 distinct protein isoforms. Members of only two out of the five subfamilies, however, were expressed in pollen and would be likely to provide exposure via inhalation. While some of these isoforms have been shown to be hypoallergenic in that they have low IgE reactivity (Ferreira et al., 1996; Wagner et al., 2008), the potential for producing or identifying a

Table 1

Classification and function of food allergens in major crops among the ‘‘Big 8’’ (soybean, peanut, and wheat)

Protein superfamily

Allergen name

Prolamin

Ara h 2

Ara h 6 Ara h 7 Ara h 9 Gly m 1 Tri a 14 Tri a 19

Cupin (vicilin, 7S globulin)

Ara h 1

Biological function according to Uniprot (www.uniprot.org)

Nutrient reservoir activity Serine-type endopeptidase inhibitor activity Nutrient reservoir activity Nutrient reservoir activity Lipid binding/ transport Seed protein Lipid binding/ transport Nutrient reservoir activity Nutrient reservoir activity

Alias

Species of origin

Major versus minor allergen classification according to IUIS (www.allergen. org)a

Conglutin-7 2S albumin

Arachis hypogaea

Major

2S albumin

A. hypogaea

Minorb

2S albumin

A. hypogaea

Minor

Nonspecific lipid transfer protein Hydrophobic seed protein Nonspecific lipid transfer protein Tri a gliadin Omega-gliadin Gluten Conarachin

A. hypogaea

Minorc

Glycine max

Major

Triticum aestivum

Majord

T. aestivum

Major

A. hypogaea

Major

Gly m 5 Cupin (glycinin, 11S globulin)

Ara h 3 Ara h 4 Gly m 6

Profilins

Bet v 1 related

Papain-like cysteine protease Oleosins

Hevein like

Ara h 5 Gly m 3 Tri a 12 Ara h 8 Gly m 4

Nutrient reservoir activity Nutrient reservoir activity Nutrient reservoir activity Nutrient reservoir activity Actin binding Actin binding Actin binding Plant defense Plant defense

Gly m Bd 30K

Proteolysis

Ara h 10

Lipid storage

Ara h 11

Lipid storage

Tri a 18

Agglutinin

b-Conglycinin

G. max

Minor

Arachin, Legumin

A. hypogaea

Minor

Arachin, Legumin

A. hypogaea

Legumin

G. max

Major (but near 50%) Minor

A. hypogaea G. max T. aestivum A. hypogaea G. max

Minor Majord Major; minord Major Major

G. max

Majord

A. hypogaea

Minore

A. hypogaea

Minore

T. aestivum

Minor

Profilin-1 PR-10 protein Stress-induced protein SAM22 P34

16 kDa oleosin Oleosin 2 14 kDa oleosin Oleosin 1 Wheat germ agglutinin

These three crops are represented in 7 out of the top 10 food allergen protein families. The three excluded families are class I chitinase, b-1,3-glucanase, and thaumatin-like proteins. a Major allergens are those where >50% of allergic patients have IgE that recognizes the allergenic protein. b Later reports (Flinterman et al., 2007; Koppelman et al., 2005) consider Ara h 6 to be a major allergen. c Minor according to Krause et al. (2009), but major in a Mediterranean population (Lauer et al., 2009). d According to www.allergome.org. e Minor according to Pons et al. (2002) for 18 kDa oleosin.

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hypoallergenic birch tree is limited by the complexity of the gene family contributing to Bet v 1 expression. Furthermore, birch is a native tree whose gene diversity would not be manipulated to the same level as a domesticated crop. Even in a domesticated crop such as maize, the primary pollen allergen, b-expansin is encoded by a complex multigene family. Among these group 1 allergens, 15 genes and seven pseudogenes have been identified, and 13/15 genes were expressed in pollen since their sequences were identified from pollen-specific EST libraries (Valdivia et al., 2007). Phylogenetic analysis revealed that group 1 allergens could be divided into two groups, A and B having
60% amino acid similarity, that probably diverged subsequent to the whole-genome duplication event shared by grass family members (Valdivia et al., 2007). Both groups have similar functions in pollen cell wall extension, and this group 1 allergen diversity probably is present in all grasses. A high level of duplication is displayed by one B-group subfamily (EXPB11) in maize, which contains five expressed members that produce identical mature proteins, having only synonymous changes in their nucleotide sequences, evidence of purifying selection. A component of pollen–food allergy syndrome is the reaction to fruits, particularly apple, by birch pollen sensitized individuals due to crossreactivity between Bet v 1 and Mal d 1, another PR-10 protein. Apple cultivar-dependent reactions have been described suggesting either quantitative or perhaps qualitative differences in apple PR-10 proteins (Marzban et al., 2005; Vieths et al., 1994). An in-depth analysis of Mal d 1 sequences in apple established that 18 genes mapping to three chromosomes were present in the genome (Gao et al., 2005). Two clusters contained 16 of the genes which was consistent with the duplicated genome origin of apple. Eight of the genes are known to be expressed in fruit (Beuning et al., 2004). One group of seven intron-containing genes was investigated for allelic diversity among 10 cultivars with known high or low allergenicity (Gao et al., 2008). Forty-six nucleotide sequences were predicted to encode 25 Mal d 1 isoforms, and alleles of two genes were found to be associated with the level of allergenicity. Further investigation will be required to distinguish the roles of quantitative versus qualitative differences for fruit-expressed Mal d 1 proteins on allergic response as well as the hypoallergenicity of specific isoforms. Inhalant allergens typically are recognized as originating in pollen grains, but occupational exposure, particularly of bakers and millers, to nonpollen plant particulates containing allergens, is a significant route of sensitization. Studies involving workers in bakeries and soybean mills with respiratory allergies caused by soy flour have implicated the Kunitz trypsin inhibitor as an airborne allergen (reviewed by L’Hocine and Boye, 2007). Several germplasm lines are available which lack this protein, and a cultivar named ‘‘Kunitz’’ has been released (Bernard et al., 1991). Because of the antinutritional properties of the Kunitz trypsin inhibitor, these soybean lines were

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initially developed for use in livestock feed, but the trait could be easily introduced into soybean varieties used in baking to reduce occupational exposure to sensitive individuals. Natural diversity has allowed isolation of potentially hypoallergenic variants of soybean. Among the multiple soy proteins that are food allergens, P34 (Gly m Bd 30K) and the a-subunit of b-conglycinin accumulate in the seed (Ogawa et al., 1991). A wild soybean line QT2 (Glycine soja) was found to lack all three subunits (a, a0 , and b) of the major allergen b-conglycinin. Subsequent studies determined that a single dominant gene (Scg-1) was responsible for the lack of b-conglycinin (Hajika et al., 1998; Teraishi et al., 2001). The discovery of a simply inherited gene has facilitated the introgression of this trait into breeding lines and cultivars (Tsubokura et al., 2006). P34 is a papain-family protease comprising <1% of total soybean seed protein that may play a role in disease resistance. Multiple efforts to identify P34-null soybean genotypes (Joseph et al., 2006; Ogawa et al., 2000; Yaklich et al., 1999) ultimately resulted in success upon screening of the entire USDA soybean germplasm collection that consisted of 16,266 accessions of G. max, G. soja, and wild relatives. Only two G. max P34 nulls were obtained from this herculean effort (Joseph et al., 2006), both of which contain the same transcribed P34 gene sequence. Six nucleotide differences between mutant and wild-type alleles were predicted to result in four altered amino acid residues, one of which changed cysteine to serine and thereby was presumed to interfere with the formation of disulfide bonds. Subsequent analysis of these accessions, however, established that they were not true nulls but that translation of the mutant alleles was severely constrained by a 4-bp insertion at the start codon resulting in an eightfold reduction in P34 protein accumulation (Bilyeu et al., 2009). Variation for allergen gene expression levels, and not just structure, also has been documented. For example, Kang et al. (2007a) surveyed 60 accessions of peanut assembled as part of a minicore collection (Holbrook and Dong, 2005) and found 2–2.6-fold variation in protein amounts of three major allergens, Ara h 1, Ara h 2, and Ara h 3, in mature seeds. The accession with the highest level of Ara h 1 (max. 18.5%, ave.
12%) also showed the lowest amount of Ara h 2 (min. 6.2%, ave.
11%), and lower than average Ara h 3 (27.3%, ave.
30.5%). It is likely that seed storage protein fractions are adjusted to compensate for higher or lower levels of one while maintaining a relatively constant total protein amount (Hartweck and Osborn, 1997; Tada et al., 2003b). Geographic origin of an accession was not a contributing factor to allergen protein differences. Null alleles were not found for any of these allergens with the exception of Ara h 3-im, an Ara h 3 isoform carrying a novel N-terminal sequence and showing reduced recognition by peanut-sensitive patient IgE (Kang and Gallo, 2007). Differences in the regulation of gene expression could account in part for the resulting variation in accumulation of allergenic proteins during

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seed development. Delayed transcript expression for all three allergen genes was observed in two out of twelve cultivars surveyed over four seed developmental stages (Kang et al., 2007b). Another example of gene expression differences is for the ripening inhibitor gene of tomato that was first reported by Robinson et al. (1968). Plants homozygous for this recessive gene (rin/rin) do not ripen fruit, but fruits of hybrids (RIN/rin) do turn red and have extended shelf life (Kitagawa et al., 2006). Kitagawa et al. (2006) discovered that the hybrids showed reduced expression of two allergen genes (b-fructofuranosidase and polygalacturonase 2A), had reduced accumulation of the allergenic proteins, and showed reduced IgE reactivity to extracts of the hybrid fruit. The actual mutation is in a gene that codes for a MADS-box transcription factor (Vrebalov et al., 2002), which controls expression of multiple genes. As with the soybean Scg-1 gene, this example demonstrates that mutations need not be within the allergen genes themselves to have an effect on reducing allergenicity. Minor sequence variation has been documented among alleles of some peanut allergen genes, most of which is not expected to alter IgE-binding capacity. Empirical determination of IgE binding can lead to unexpected results, however, as in the case of a natural allele of Ara h 2 discovered through EcoTILLING of Arachis duranensis, the putative A-genome donor of polyploid peanut (Ramos et al., 2009). EcoTILLING is a sequence-based assay for allelic differences of a target gene in natural populations (Comai et al., 2004). Ramos et al. (2009) showed the presence of several variants of Ara d 2.01, the A. duranensis ortholog of Ara h 2.01, one of which caused an amino acid change (S73T) in an immunodominant epitope and displayed significantly decreased IgE binding.

2.2. Induced variation: Mutagenesis While natural allelic variation has been identified for some allergens, this variation does not always reveal hypoallergenic isoforms or null mutants that would be of value for crop modification. Induced variation through deliberate mutagenesis relies on the same DNA modifying mechanisms that generate spontaneous mutations albeit at an accelerated pace. Methods to induce mutations have been extensively reviewed (Malmberg, 1993; Redei and Koncz, 1992; Walbot, 1992); therefore, we only briefly address this topic and focus on relevant discoveries of interesting mutants. 2.2.1. Methods for inducing genetic variation in allergens In some crop species where significant natural variation is extremely limited, mutagenesis can be employed to create variation. Multiple means of generating mutations have been developed, each with advantages and disadvantages. Various forms of ionizing radiation, such as X-rays and g-rays, have been used successfully to generate genetic variation in plants.

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Radiation tends to induce moderate to large-scale chromosomal changes, often resulting in deletion or rearrangement of large portions of the genome. An advantage of this technique is that gene knockouts are often recovered, but frequently more than one gene is affected, having a deleterious effect on the overall phenotype of the plant. Optimizing the dosage of radiation is crucial in order to recover a significant number of mutations without completely destroying the starting material, which may be pollen, seeds, explants, or even liquid cell cultures (Ahloowalia and Maluszynski, 2001). Fast neutrons and ion beam can also be used to induce deletion mutations (Balyan et al., 2008; Li et al., 2002; Yu, 2006). Alteration of DNA sequences via chemical mutagens is also commonly practiced. Chemicals most frequently used for mutagenesis include ethyl methanesulfonate (EMS), diethyl sulfate (DES), and N-nitroso-N-methylurea (NMU), among others. As with radiation, the dosage of mutagen must be optimized to maximize the rate of mutation recovery while minimizing the direct toxic effects of the chemicals. Compared to radiation, chemical mutagens tend to induce very small changes to DNA sequences: singlenucleotide polymorphisms (SNPs) or very small insertions and deletions (indels). Thus, generating knockouts via chemical mutagenesis is less likely unless an SNP creates a premature stop codon or alters the start codon of a gene, or if an indel results in a frameshift. However, slight alterations to genes of interest, such as missense mutations, are commonly obtained. Conservative nonsynonymous changes often will leave the functional proteins intact, but could potentially reduce their allergenicity by disrupting key epitopes. Single amino acid changes in characterized allergen epitopes can have a dramatic effect on IgE recognition (Burks et al., 1999). Recently a different class of chemical mutagens, termed deletogens (Balyan et al., 2008), has been described. These chemicals induce larger deletions than typical chemical mutagens, but generally smaller than those arising from irradiation. For the model nematode Caenorhabditis elegans an average deletion size of 1400 bp was reported by Liu et al. (1999). Deletogens include diepoxybutane (DEB), diepoxyoctane (DEO), and trimethylpsoralen (TMP). TMP is applied in the presence of ultraviolet light to induce deletions (UV–TMP). EMS and ethlynitrosourea (ENU) have also been reported to induce deletions in C. elegans, similar in size to deletions caused by DEO and UV–TMP (Liu et al., 1999). Mutations generated by chemicals tend to be distributed randomly throughout the genome, and in the case of altered allergenicity a physical phenotype is not easily observable. Thus, a high-throughput screening technique based on the polymerase chain reaction (PCR) such as TILLING (targeting induced local lesions in genomes; Comai and Henikoff, 2006; Greene et al., 2003; McCallum et al., 2000) is needed to identify individuals carrying mutations in the genes of interest. This reverse genetics approach is even more relevant for polyploids where duplicate genes frequently mask mutant phenotypes.

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TILLING is a PCR-based approach which utilizes mismatch cleavage to detect mutations on polyacrylamide gels. Target gene sequence is thus required for this type of mutant screen. For TILLING, equal amounts of DNA from several mutagenized individuals are pooled up to eightfold, depending on the size and complexity of the genome. The target gene is amplified by PCR using IRDye end-labeled primers, and then the product is denatured and allowed to reanneal. During reannealing, mutant strands may pair with wild-type strands, creating heteroduplexes. These mismatches can be detected and cleaved by several nucleases. The most commonly used of these is CEL1 nuclease, which can be extracted from celery. Cleaved products are visualized by electrophoresis in polyacrylamide slab gels in a Li-Cor DNA Analyzer which detects the fluorescent signals from the labeled primers. An analogous procedure using labeled dCTP, termed EMAIL (endonucleolytic mutation analysis by internal labeling; Cross et al., 2008), detects mutations using capillary sequencers. To screen for deletions in genes of interest, a simple PCR assay may be feasible. An amplicon is designed to span several kilobases including the gene of interest, and the PCR products are visualized by standard agarose gel electrophoresis. Products of smaller size indicate possible deletions. Smaller products tend to be preferentially amplified in PCR with shorter extension times, thus facilitating their detection in highly pooled samples. Analogous to the pooling strategy used in TILLING, DNA from multiple individuals can be combined, but at an even higher throughput. For this type of screen in rice, one deletion variant can be detected in a pool of as many as 200 individuals (Wu et al., 2005). This strategy of identifying deletion mutations has been termed DEALING (detecting adduct lesions in genomes; Balyan et al., 2008). In Arabidopsis a similar strategy, termed DeleteageneTM, has been used to detect deletions caused by fast neutron mutagenesis, with detection possible in a pool of 1000 individuals (Li et al., 2002). These mutagenesis systems which generate or allow selection of small scale deletions could be easily transferred to important crop species for elimination of allergen-producing genes. The pool size and rate of mutation would need to be determined empirically, especially for species like peanut with large complex genomes. Insertional mutagenesis using T-DNA from Agrobacterium has been extensively utilized in Arabidopsis as a forward genetics tool to generate knockout mutations by disrupting the genes into which the T-DNAs insert. Also, the extreme stresses brought about during tissue culture have been shown to induce mutations by the mobilization of endogenous transposable elements in rice; examples include the retrotransposon Tos17 (Hirochika et al., 1996) and the MITE mPing (Kikuchi et al., 2003). Lin et al. (2006) showed that mPing and a transposase-encoding element Pong can also be mobilized by subjecting intact rice seeds to high hydrostatic pressure. It has been proposed that some of the mutations resulting from irradiation or

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chemical mutagenesis are caused by the activation of transposable elements. For any insertion mutant where the sequence of an activated mobile element or T-DNA is known, it should be possible to develop a reverse-genetic screen to identify individuals with insertions in genes of interest, including known allergen genes. However, little is known about mobile DNA elements in species other than Arabidopsis, rice, and maize, and high-throughput transformation and T-DNA tagging systems are lacking for most crop species. Also, with the currently available methods, the probability of obtaining an insertion in a specific gene is quite low. In addition, the longterm stability of insertional mutations over many generations of breeding is unknown. Although insertional mutagenesis has been very useful for genetic studies, its use in developing variation in allergens in crop species may be limited, at least in the near future. 2.2.2. Examples of allergen content changes resulting from mutagenesis Mutagenesis has been used extensively to create novel variation in ornamental plant species, to improve agronomic characteristics of various plant species, and for modification of food quality traits in crops (Ahloowalia and Maluszynski, 2001). For example, gamma radiation was used to induce mutations for improved tuber quality (Love et al., 1996a) and reduced glycoalkaloid content (Love et al., 1996b) in potato. However, the use of mutagenesis in generating variation in allergens has not been extensively applied, though several examples of induced allergen variation have been reported. These examples highlight the potential of mutagenesis for alteration or elimination of allergens in crops. Nishio and Iida (1993) reported the reduction of allergenic proteins in seeds of four rice mutants. Two of these mutants, induced by gamma rays, had reduced levels (
50%) of a 16-kDa allergenic protein (probably RA17/ a-amylase inhibitor/Ory s aA_TI; Izumi et al., 1999; Nakase et al., 1996), and of a 26-kDa protein in the seeds, while two others had only trace amounts of these proteins. Of the latter, one was an M2 derived from gamma-irradiated material, and the other was derived from a spontaneous white-panicle mutant that was subjected to EMS treatment. The mutants with the lowest levels of these proteins always had floury endosperms, so their use as food would be limited. Furthermore, they were sterile. Those with reduced levels of the allergen also had floury endosperms, but only in the very center of the kernel. The authors noted that the quality of these mutants would be acceptable as cooked rice, but the reduction in allergen content may not be significant enough to be promoted as reduced-allergen rice. In a subsequent paper, however, Iida et al. (1993) found that a reducedallergen mutant could facilitate the production of hypoallergenic processed rice by reducing the cost of removing the allergenic proteins. Given that the level of flouriness in the endosperm seems to correlate with the level of

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reduction in 16- and 26-kDa proteins, and that this was observed in four independent mutants, there probably is a pleiotropic effect of these proteins on endosperm texture rather than a linked mutation resulting from deletion of a large piece of the genome, as may happen with gamma-irradiation induced mutations. Also, if this protein is indeed an amylase inhibitor, changes to the endosperm would be expected when the protein is reduced or eliminated. Several soybean lines lacking specific seed proteins, including several allergens, have been derived from g-ray mutagenesis. The breeding line EnB1 lacks all five subunits of the seed storage protein glycinin (Odanaka and Kaizuma, 1989; Teraishi et al., 2001), which comprise the named allergen Gly m 6. The mutant breeding line must therefore either contain several independent glycinin gene mutations (glycinin genes are known to locate to multiple chromosomes) or more likely a single mutation in a trans-acting modifier gene (Beilinson et al., 2002). Takahashi et al. (1994) subjected the breeding line ‘‘Kari-kei 434,’’ a spontaneous mutant already lacking the a0 subunit of b-conglycinin, to gamma radiation, and from this experiment they derived ‘‘Tohoku 124’’ (also known as ‘‘Yumeminori’’), a line which lacks the a- and a0 -subunits of the allergen b-conglycinin, and has reduced levels of the b subunit (collectively Gly m 5, a vicilin). A PCR-based assay has been developed to screen for the mutant alleles of both a- and a0 -subunits of bconglycinin that allows testing for seed purity (Ishikawa et al., 2006). ‘‘Tohuko 124’’ was later found to also lack the allergen Gly m Bd 28K, also a vicilin (Samoto et al., 1997). Using this mutant, Samoto et al. (1997) were also able to remove 99.8% of another allergen Gly m Bd 30K (P34) from soymilk through processing. Nakamura et al. (1989) and Phan et al. (1996) also have reported reduction or elimination of b-conglycinin subunits in soybean resulting from gamma irradiation. Phan et al. (1996) noted that there is a lethal chlorosis associated with the deletion of both subunits and suggested that their mutation results from a large chromosomal deletion, which is one of the drawbacks of gray mutagenesis. More recently Manjaya et al. (2007) subjected soybean variety VLSoy-2 to gamma radiation. From this treatment three mutants were identified which lacked the A3 subunit of glycinin. Of these, one also had reduced levels of the a- and a0 -subunits of b-conglycinin, and two of the mutants lacked these two subunits altogether. Induced mutation also may be a successful approach to eliminate allergens from peanut. A preliminary report described peanut mutants missing isoforms of Ara h 2 and Ara h 3 (Perkins et al., 2006). Currently, a peanut TILLING population is being developed and screened for variations in the major allergens Ara h 1 and Ara h 2 (Knoll et al., unpublished). It has been shown that both of these allergens are present in two isoforms (Knoll et al., unpublished; Ramos et al., 2006), as peanut is an allotetraploid and one isoform is derived from each of the two subgenomes. Possible allergen variants have been found for both isoforms of Ara h 2, and for Ara h 1a.

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A truncation mutation has been identified for ara h 1b and a disrupted start codon has been identified in ara h 2.02. TILLING populations are valuable genetic resources that can be tapped for allergen gene variation. Several TILLING populations have already been developed for soybean (Cooper et al., 2008), with mutation rates as high as 1 SNP/140 kb, but no data have been published on screening for allergen gene mutations although other seed traits have been targeted (Dierking and Bilyeu, 2009). The identification of less allergenic isoforms of a protein or gene knockouts, either as natural variants or induced mutations, may facilitate conventional and molecular breeding strategies toward the development of hypoallergenic food crops, particularly where the number of genes encoding a protein is small. This approach becomes more difficult where large multigene families are responsible for allergenic protein expression as is the case for group 1 and Bet v 1 pollen allergens. For these situations, homologydependent gene silencing, as reviewed below, may be the most feasible means for allergen reduction.

2.3. Induced variation: Transgenics Most crop allergens can be effectively reduced through genetic transformation for gene silencing. All of the current published allergen silencing work is based on the mechanism of posttranscriptional gene silencing (PTGS). PTGS is a naturally evolved pathway in plants that serves multiple biological functions such as suppression of viral infection and developmental gene regulation (Baulcombe, 2004; Chapman and Carrington, 2007). To silence specific allergen proteins, a transgene can be introduced into the plant through genetic transformation in the sense, antisense, or inverted-repeat orientation. Aberrant transcripts of sense and antisense transgenes such as those missing the 50 cap structure are recognized by the endogenous RNAdependent RNA polymerase (RDRP) (Gazzani et al., 2004). The RDRP subsequently synthesizes the complementary strand of the transgene to form double-stranded RNA (dsRNA). If the transgene is introduced as an inverted repeat, the transcript forms a dsRNA hairpin due to self complementarity. These transgene dsRNAs are further recruited by a dicer-like complex and sliced into 21–24 bp short interfering RNAs (siRNA) (Hamilton and Baulcombe, 1999; Schauer et al., 2002). RNA-induced silencing complex (RISC) in the cytoplasm binds to the siRNA and unwinds the double strand. The siRNA guide strand base-pairs with complementary mRNA (e.g., targeted allergen RNAs) and confers sequence specificity to silencing. Argonaute proteins within the RISC cleave the mRNA and downregulate its expression (Song et al., 2004). The effectiveness of allergen silencing depends on several factors including complexity of allergen families, arrangement and length of transgene arms, transgene copy number, promoter specificity and strength, allergen

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turnover rate, spatial and temporal expression pattern of allergens, etc. (Kerschen et al., 2004). Selection of an allergen to be targeted for silencing depends on the availability of the allergen sequence, its clinical importance, and biological function in the plant. Frequently, allergen proteins are encoded by multiple gene family members as well as multiple alleles for each gene. For example, Ara h 2 is a major peanut allergen that has two genomic copies, Ara h 2.01 and Ara h 2.02. It also shares 63% homology to Ara h 6, another peanut allergen in the conglutin family (Ramos et al., 2006). Since PTGS is homology dependent, selection of a sequence region that is highly conserved between these two peanut allergens during design of a silencing construct resulted in effective silencing of both peanut allergens (Chu et al., 2008). On the other hand, if a specific allergen needs to be silenced with minimum collateral effect, a unique sequence fragment in the 30 - or 50 -untranslated regions with least homology to other genes can be selected. For crops that produce multiple allergenic proteins, it is also possible to construct a silencing vector to target several allergens simultaneously. The potential downside of this strategy is that the viability of the transgenic crop can be severely compromised due to the knockdown of multiple functional proteins. It is therefore important to prioritize the list of allergens to be silenced based on their clinical relevance and biological function. A number of studies on the arrangement of transgene arms showed that the most effective silencing constructs are intron-spliced hairpin structures (ihpRNA) or inverted repeats separated by a nonintron spacer where the optimum length of the transgene direct or inverted-repeat unit ranges from
100 to 850 bp (Chuang and Meyerowitz, 2000; Hirai et al., 2007; Smith et al., 2000; Waterhouse et al., 1998; Wesley et al., 2001). Various generic silencing constructs that accommodate inverted repeats are available (http:// www.pi.csiro.au/rnai/; http://www.chromdb.org/) and strategies for silencing including the use of artificial microRNAs have recently been reviewed (Ossowski et al., 2008). In general, upon transcription, an inverted repeat forms a hairpin dsRNA and the intron/spacer forms a loop structure. It is suggested that this panhandle structure stabilizes the dsRNA and increases the silencing efficiency. Ninety to 100% of transgenic lines producing ihpRNA show PTGS whereas only an average of 12% of transgenic lines from sense and antisense constructs have a silencing effect. Introns as spacers between inverted repeats may or may not be more effective than nonintron sequences (Hirai et al., 2007). Other factors that affect silencing efficiency include level and location of transcription; strong promoters are correlated with stronger silencing effect than weak promoters (Chuang and Meyerowitz, 2000; Hirai et al., 2007), and tissue specific expression can be utilized for some allergens since those such as seed storage proteins often are specifically expressed in seeds. Other allergens may be expressed in more than one type of plant tissue, such as the tomato allergen profilin and apple allergen Mal d 1, which can be found not only in fruits but also in vegetative

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tissues. Even in these cases, selection of a fruit specific promoter may be beneficial to plant health since the allergen would be reduced only in the edible part of the plant and its function maintained in other parts of the plant. Single-copy, homozygous transgenic lines are desired since the silencing effect is more likely to be stable across generations. Inserts at multiple genomic loci are expected to segregate among progenies and degree of silencing may vary among events. While Agrobacterium-mediated transformation is considered to more frequently result in single-copy insertions than microprojectile bombardment, the literature provides a significant body of evidence to question this dogma (Altpeter et al., 2005). Regardless of transgene delivery method, variation among transgenic lines generated with a single silencing construct is inevitable, requiring that degree and stability of silencing be determined empirically. Since allergens are largely proteins, methods for detecting changes in protein expression include 1D and 2D protein gels, Western blots, ELISA assays, and more sophisticated proteomic techniques (Stevenson et al., 2009; Thelen, 2009). Evaluation of the effect of allergen silencing on allergenicity involves IgE binding using sera from allergic patients and in vitro histamine release from sensitized human or humanized basophils (Palmer et al., 2005). Even more relevant allergenicity data can be collected in a clinical setting where skin prick testing and double-blind placebo-controlled food challenge (DBPCFC) can be carried out (Peeters et al., 2007). However, as mentioned earlier, the presence of multiple allergens in one food source makes the DBPCFC test risky for allergic patients. Therefore, it has not been used for evaluation of any allergen reduced transgenic lines. 2.3.1. Allergens silenced in crops Both food and pollen allergens from rice, ryegrass, apple, soybean, peanut, and tomato have been successfully silenced by genetic transformation. In 1996, a group of 14–16 kDa rice allergens (RAs) was discovered by screening fractionated rice proteins with serum IgE from rice-allergic patients. These proteins were highly reactive to all 31 patient sera screened by a radio allergo-sorbent test (RAST) defining them as major RAs (Nakamura and Matsuda, 1996). Two antisense constructs, each containing two copies of the antisense gene driven by seed-specific promoters and a conserved region of the RA genes, were used to knockdown expression of this allergen family. One construct had the antisense gene pair controlled by glutelin and prolamin promoters and oriented as tandem repeats while the second had the antisense genes in inverted orientation and under control of RA gene 1 and starch branching enzyme I gene promoters. The rice waxy terminator was common to all four chimeric genes. Using monoclonal antibody cross-reacting with the majority of the gene family members, it was found that the allergen content was reduced by 80% (Tada et al., 1996).

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The incomplete suppression of this allergen family by antisense constructs was further investigated in T3 and T4 progenies of transgenic lines (Tada et al., 2003a), where it was found that members of the RA family with lower levels of homology to the antisense sequence were expressed at normal levels in the transgenic lines, whereas the expression of RAs sharing high homology to the antisense sequence was greatly reduced. Therefore, the data provided evidence that the degree of silencing was correlated with the extent of sequence homology. Ryegrass pollen allergen Lol p 5, a 31-kDa protein, is a major ryegrass allergen recognized by 90% of patients allergic to this grass pollen. An antisense construct driven by a pollen specific promoter from Ory s1 was transformed into ryegrass callus tissue via particle bombardment, and transgenic lines showed significantly reduced accumulation of Lol p 5 protein (Bhalla et al., 1999, 2001). IgE binding from patient sera also demonstrated less reactivity by transgenic lines. Plant growth and pollen viability were unaffected; therefore, the putative roles of Lol p 5 in self-incompatibility and pollen germination were excluded. Similarly, antisense technology combined with pollen-specific expression conferred by the maize Zm13 promoter was used to significantly reduce expression of ryegrass pollen allergens Lol p 1 and Lol p 2 (Petrovska et al., 2004). A soybean major allergen, Gly m Bd 30K also known as P34, is a member of the papain superfamily of cysteine proteases (Kalinski et al., 1992). It induces allergic reactions in 65% of soy-sensitive patients yet comprises less than 1% of soy total seed protein (Herman et al., 2003; Ogawa et al., 1991). Cosuppression was achieved in transgenic soybean by seed-specific expression of the allergen gene under control of a b-conglycinin promoter (Herman et al., 2003). The reduction of Gly m Bd 30K expression in one transgenic line was confirmed by allergen specific and human IgE immunoblots. No collateral effects on other proteins were detected in the silenced line. Seed size, shape, protein composition, and oil content in the silenced line were comparable to that of the nontransgenic control. Comparative 2D gel electrophoresis/mass spectrometry data also showed no further protein composition change other than the downregulation of Gly m Bd 30K. In contrast, a significant change in protein profile, including enhanced expression of Gly m Bd 30K, was observed in another study that used cosuppression to reduce expression of the a- and a0 -subunits of b-conglycinin, components of Gly m 5, a minor soybean allergen (Kinney et al., 2001). The increases in Gly m Bd 30K and unprocessed glycinin (Gly m 6) were attributed to altered protein trafficking that resulted in accumulation of novel protein bodies. The frequency of silenced lines recovered using antisense or cosuppression constructs in the studies described above was not reported. However, since the inverted-repeat RNAi construct design has proved to be very effective for transgene-

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induced silencing, subsequent studies on allergen silencing have utilized this strategy. Two minor tomato allergens, Lyc e 1 and Lyc e 3, have been independently silenced. Lyc e 1 is a profilin that binds to actin to regulate cell elongation, cell shape maintenance, and flowering in plants. It is recognized by IgE from 22% to 26% of tomato allergic patients. Two Lyc e 1 isoforms sharing 88.1% identity at the nucleotide level were silenced simultaneously by an RNAi construct driven by the CaMV 35S promoter (Le et al., 2006a). Transgenic plants displayed reduced Lyc e 1 expression at both RNA and protein levels. A reduction in IgE binding also was observed. Patients sensitized to only Lyc e 1 showed 65–100% reduction in wheal reaction to transgenic fruit in a skin prick test whereas multiallergen sensitized patients had only 16–25% reduction in wheal reaction. Elimination of multiple allergens from tomato fruit would be necessary in order to substantially alter the immune reaction in patients showing the latter response. Not unexpectedly, Lyc e 1-silenced plants demonstrated severe growth retardation and reduced fruit and seed set because profilin plays an essential role in cell structure and cell growth. Silencing this allergen without replacing it with a nonallergic variant was proven to be detrimental to plant health and production. It is therefore important to take the function of an allergen into consideration before silencing. Lyc e 3, a nonspecific lipid transfer protein (nsLTP) and a member of a multigene family with a highly conserved cysteine-rich structure, has potential biological functions in plant defense and calcium metabolism. The expression of Lyc e 3 is mainly in the peel of tomato fruit. This protein is recognized by 29% of tomato allergic patients (Le et al., 2006b). Two isoforms Lyc e 3.01 and Lyc e 3.02 sharing 76.5% identity at the nucleotide level were silenced by a single RNAi construct (Lorenz et al., 2006). Silencing of both isoforms was confirmed by Western blot. Competitive ELISA showed that the level of Lyc e 3 in the transgenic line was below 0.5% of that of the wild-type. Basophil histamine release and skin prick tests showed reduced allergenic potency of transgenic lines. Contrary to Lyc e 1, no phenotypic differences were observed in Lyc e 3 transgenic and transgene null lines found among T1 progenies. Apple allergen Mal d 1 has cross-reactivity to birch pollen allergen Bet v 1, as described in Section 2.1, and is considered a major allergen with 18 family members belonging to the PR protein PR10 group. A constitutively expressed inverted repeat of Mal d 1b 50 -untranslated region and first exon was introduced into apple via Agrobacterium-mediated transformation (Gilissen et al., 2005). Since Mal d 1 is expressed in leaves as well as fruit, protein extracted from leaf tissue was analyzed to assess the effectiveness of silencing before fruit production. Reduced skin prick test reaction and IgE binding were demonstrated with the silenced transgenic lines. These results need to be further confirmed by testing of fruit tissue which is the allergen source for affected patients.

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More recently, Ara h 2, the most potent peanut allergen, was silenced by an RNAi construct in two independent studies (Chu et al., 2008; Dodo et al., 2008). Ara h 2 is one of eleven identified peanut allergens, but it is considered one of the major allergens because it can be recognized by sera from more than 90% of peanut allergic patients (Burks et al., 1995). There is a 63% nucleotide sequence similarity between Ara h 2 and Ara h 6, another peanut allergen recognized by 80% of peanut allergic patients (Flinterman et al., 2007). Our lab selected an Ara h 2 coding region that is 80% homologous to Ara h 6 for the RNAi construct. Transformation of peanut embryogenic cultures via microprojectile bombardment led to recovery of three independent transgenic lines, all of which showed significant reduction in Ara h 2 expression. Two lines also had suppressed Ara h 6 expression. Since the transgene sequence in the construct was identical to Ara h 2.01 and less similar to Ara h 6, the homology-dependent silencing effect was more variable for Ara h 6 than Ara h 2, analogous to the RA silencing results. Human IgE immunodetection of proteins from one of the three silenced lines showed less signal from Ara h 2 than Ara h 6, although Ara h 6 was as effectively silenced as Ara h 2 in another of the transgenic lines. Patient sera used in this study detected allergens other than Ara h 2 and Ara h 6; therefore, the immune reaction to peanuts probably would not be eliminated upon silencing of only these two allergens. The transgenic plants were phenotypically normal, and in spite of demonstrated trypsin inhibitor activity for Ara h 2 (Maleki et al., 2003), transgenic lines did not show any increased susceptibility to Aspergillus flavus fungal infection compared to nontransgenic lines. Ara h 2- and Ara h 6-silenced lines had largely normal global protein profiles with some minor collateral changes such as an elevation in Ara h 10 (oleosin), 13-lipoxygenase and Ahy-3 (arachin) and a decrease in conarachin (Stevenson et al., 2009). Independently generated Ara h 2-silenced lines showed surprisingly large variations in protein profiles in addition to reduced Ara h 2 expression (Dodo et al., 2008). Silencing of crop allergens by PTGS has been successful but holds some risk for commercialization due to potential instability (Ozias-Akins et al., 2009). Certain plant viruses can produce silencing suppressor proteins which negatively affect the stability of silencing (Li and Ding, 2001). A potential solution to this problem is heritable transcriptional gene silencing (TGS). While both PTGS and TGS are epigenetic phenomena, TGS results from DNA modifications. Both processes are initiated by the RNAi machinery and similar transformation construct design is effective. Rather than expressing an inverted-repeat transcript containing gene coding sequence, the promoter sequence of a target gene is transcribed to generate dsRNA. The siRNA from the promoter sequence can be transported back into the nucleus to induce homology-dependent promoter methylation and silence expression of the downstream open reading frames (Matzke and Birchler, 2005; Mette et al., 2000; Wang et al., 2001). Maintenance of

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DNA methylation after TGS is dependent upon a DNA methyltransferase and independent of the presence of siRNA ( Jones et al., 2001). Therefore, TGS should not be susceptible to suppression of silencing upon viral infection, although it requires prior knowledge of allergen promoter sequences. 2.3.2. Allergenicity of transgenic crops A contrasting aspect of crop allergen silencing is associated with the potential increase in allergenicity of genetically modified plants. In the early 1990s, Brazil nut protein Ber e 1 was introduced into soybean to enhance its sulfur amino acid content. Since allergic reactions to Brazil nut were known, the transgenic soybean was tested for reactivity with human IgE and was shown to present a positive reaction (Nordlee et al., 1996). This work provided additional evidence that Ber e 1 was a major Brazil nut allergen. Commercialization of such a transgenic product was never considered or attempted; however, allergenicity data are now a standard component of safety assessments required by federal agencies in the United States and other countries prior to deregulation of a GE food or feed crop (Ladics, 2008). The weight-of-evidence approach currently is used and is based on prior knowledge of characteristics of a transgenic protein, structural comparison of the transgenic protein to other allergens, and digestive stability.

3. Conclusions Multiple approaches to the reduction in allergenicity of crops have been described including the exploitation of natural variation for quantitative or sequence differences, the induction of variation through mutagenesis, and the suppression of allergen expression using GE. While spontaneous and natural mutants are largely unregulated, public opinion toward GE products and deregulation of transgenic crops continues to be an issue despite more than a decade of safe deployment and consumption of transgenic crops in the United States. A comprehensive review of the facts related to GE products draws from an extensive body of peer-reviewed literature that addresses perceived and actual risks (Lemaux, 2008, 2009). While the science typically is overlooked in anti-GE platforms with underlying sociological or religious convictions, individual consumers are more likely to judge a product according to its perceived personal benefit (Schenk et al., 2008). In some cases, the benefits of a hypoallergenic GE crop may be perceived by the general public as greater than the risks, increasing acceptance over first-generation GE crops with solely crop protection traits. It is likely that both mutation and GE approaches will contribute to the future release of crop varieties with the output trait of reduced allergenicity.

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