mitigating allergenicity of crops (advances in agronomy, volume 107)

C H A P T E R T H R E E

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Mitigating Allergenicity of Crops

Peggy Ozias-Akins, Ye Chu, Joseph Knoll,1 and

Anjanabha Bhattacharya2

Contents

1. In

s in

065

enntnt

troduction

Agronomy, Volume 107 # 2010

-2113, DOI: 10.1016/S0065-2113(10)07003-3 All rig

t of Horticulture, University of Georgia Tifton Campus, Tifton, Georgia, USAaddress: USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GAaddress: Bench Biotechnology, Vapi, Gujarat, India

Else

hts

93

1
.1. A llergens and allergenicity 94
1
.2. A llergen protein families 95
1
.3. C rop-specific allergens 96
2. M
ethods to Alter Allergen Content of Crops 97
2
.1. N atural variation 97
2
.2. In duced variation: Mutagenesis 102
2
.3. In duced variation: Transgenics 107
3. C
onclusions 113
Refe
rences 114
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 prote-

omics. 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 entirelyfeasible given the roles that allergenic proteins play in plant growth anddevelopment; nevertheless, a substantial body of information has accumulated

vier Inc.

reserved.

93

94 Peggy Ozias-Akins et al.

on the consequences of protein modification, which suggests that at leastmitigation is attainable. Crop plants are cultivated for food, feed, fiber, andfuel, and their increased production in recent history has been significantlydependent on genetic gains. Domesticated plants have been undergoinghuman selection for thousands of years, but intensive genetic enhancementthrough breeding has occurred only within about a century (Duvick, 1996).While our existence is dependent on crop plants, certain of their biochemicalcomponents can invoke an immune response in humans upon oral or inhala-tion exposure that results in negative health consequences. Artificial selectionpracticed during plant breeding usually narrows the germplasm base for a cropand may or may not have an associated effect on allergen content or composi-tion depending on linkage of allergen genes with selected traits or pleiotropiceffects. Since many allergens are seed storage proteins, and artificial selectionfor seed characteristics is routine, associated changes in seed protein content orcomposition are inevitable. Only recently has artificial selection been con-ducted to intentionally alter composition or content of an allergenic protein ina crop. Herein, we review attempts to reduce or eliminate pollen and foodallergens from crops using germplasm resources, mutagenesis, and geneticengineering (GE).

1.1. Allergens and allergenicity

An allergen is a substance that triggers a misguided human immune responseand usually is found in pollen, mold, dander, and food. The intricacies ofinteractions among components of the human immune system and allergensstill are not fully understood (Shreffler, 2009). Food and pollen allergenstypically induce an IgE response from the immune system during sensitiza-tion and trigger an IgE-mediated reaction upon subsequent exposure. Pollenallergic reactions present as mucosal and respiratory symptoms (allergicrhinitis, better known as hay fever, to asthma). Food allergic reactionspresent as symptoms ranging from skin reactions (urticaria, or hives, andangioedema) and gastrointestinal symptoms (nausea, abdominal pain, diar-rhea, vomiting) to life-threatening anaphylaxis. In the latter case, timelyintervention with administration of epinephrine is essential (Simons, 2008;Young et al., 2009). Food allergy (food hypersensitivity) is not to be confusedwith food intolerance, which is a nonimmunologic reaction, although bothfood allergy and food intolerance are considered adverse food reactions (Leeand Burks, 2006; Perry et al., 2006).

Themost 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 tomilk, egg, soy, and wheat; but others, particularly peanut, tree nuts, fish, andshellfish, often persist to adulthood. Allergy diagnosis is much easier thanmanagement, and recommended therapy usually means avoiding the food.

Mitigating Allergenicity of Crops 95

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 inprocessed food products. Progress in developing immunotherapies for foodallergies has been made but none are yet approved or recommended forstandard treatment. Use of injection immunotherapy, while common forinhaled (including pollen) allergens, is not generally recommended for foodallergies because the potential for serious adverse reactions is high (Burkset al., 2001). A number of novel immunomodulating therapies are underinvestigation, including peptide immunotherapy, DNA immunization,herbal remedies, and anti-IgE immunotherapy (Burks et al., 2006; Wangand Sicherer, 2009). Pollen–food allergies also have been documentedwhere sensitization to inhaled allergens results in cross-reactivity to certainfood allergens. The best characterized examples of pollen–food allergysyndrome (also known as oral allergy syndrome) are sensitization to birch,ragweed, grass, and mugwort pollen resulting in allergic reactions to certainraw vegetables and fruits. Pollen allergens from noncrop species implicatedin pollen–food allergy syndrome cannot easily be avoided in certain geo-graphic areas. As with treatment of other food allergies, the recommendationis to avoid the associated allergenic foods even though injection immuno-therapy 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 exposureis via inhalation or ingestion, respectively. While many of these proteins areglycosylated, and cross-reactive carbohydrate determinants are recognizedby IgE, the carbohydrate side-chains have minimal allergenic activity(Altmann, 2007; Mari and Scala, 2006). Protein allergens are named accord-ing to the rules established by the World Health Organization and Interna-tional Union of Immunological Societies (WHO/IUIS) and included anabbreviation of the taxonomic name (first three letters of the genus followedby a space and the first letter of the species) plus an Arabic numeral that isassigned in the order that an allergen is identified (http://www.allergen.org/Allergen.aspx; Larsen and Lowenstein, 1996). The number of IUIS recog-nized allergens is less than the number actually described in the literature anddatabases, and nonconventional names persist. Currently, 208 food allergensare distributed among 40 protein families and 204 pollen allergens fall into 52allergen 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 hasbeen reviewed (Radauer and Breiteneder, 2006, 2007). Protein allergens

http://www.allergen.org/Allergen.aspx



http://www.meduniwien.ac.at/allergens/allfam/




are represented by relatively few protein classes, that is, only 2% of the 9318protein families in the Pfam database (Finn et al., 2008) are known tocontain allergenic proteins (Radauer et al., 2008). Some of these proteinfamilies are essential to metabolic function, such as profilins, which areactin-binding proteins important for cytoskeleton organization. The prola-min superfamily contains the largest number of food allergens (27%) andcomprises seed storage proteins, prominent components of legume seedcotyledons and cereal endosperm, as well as protease inhibitors and lipidtransfer proteins. The most prevalent protein families for pollen allergens areprofilin (12%) and expansin C-terminal domain (10%), allergenic forms ofwhich are confined to the grass family. Allergenic profilins are distributedacross 10 plant families and they rank third (12%), behind prolamins andcupins (17%), among food allergens. Cupins have conserved barrel domainsand include 7S and 11S seed storage proteins, also known as vicilins andglycinins (legumins), respectively. Other protein families containing signif-icant 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 inpollen–food allergy syndrome while EF-hand domain proteins are calciumbinding and form helix-loop-helix motifs.

While no individual structural features of a protein can be used to predictallergenicity, particularly for ingested proteins, some common properties ofallergens are resistance to degradation in the gastrointestinal tract or uponexposure 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 predictingallergenicity (Thomas et al., 2004) that has been validated in a mouse model(Bowman and Selgrade, 2008). Of the crops among the ‘‘Big 8’’, soybeanand peanut each contain allergenic members of the prolamin, cupin, profilin,and Bet v 1-like protein families among others. Soybean has been the mostintensively studied allergenic crop with significant advances toward allergenreduction through genetic means (L’Hocine and Boye, 2007).

1.3. Crop-specific allergens

Major crops represented among the ‘‘Big 8’’ allergens are soybean (Glycinemax), peanut (Arachis hypogaea), and wheat (Triticum aestivum). Multiple seedproteins within each of these species are food allergens. A major allergen isdefined as one that reacts with serum IgE from >50% of allergic individualstested. To be classified as an allergen by the IUIS Allergen NomenclatureSubcommittee, binding of IgE from serum of at least five patients or 5% ofthe population tested that are allergic to the respective allergen source mustbe demonstrated (http://www.allergen.org/Allergen.aspx). A database ofnamed allergens is maintained at this website. Other databases with links




to more extensive information about named allergens and their relatedproteins can be found at http://www.allergome.org and http://www.allergenonline.com. For peanut, there are 11 named allergens, 6 for soy-bean, and 10 for wheat. The classification of food allergens for these crops,in terms of their protein families and levels of allergenicity, is shown inTable 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. Oneinvolves different forms of an allergenic protein encoded by different genes,either members of a multigene family and/or homeologous genes asencountered in polyploids. The second source of variation is allelic andderives from different forms of a gene among individuals in a population orspecies. Protein isoforms can show small variations in amino acid sequencesand in posttranslational processing, thus potentially can be distinguished bymolecular weight, isoelectric point, and peptide signatures. Such variationmay affect allergenicity as has been demonstrated for Bet v 1-like isoforms inbirch and apple (Vieths et al., 1994; Wagner et al., 2008). Within a class ofproteins, some members may be highly allergenic, while others invoke littleresponse from the immune system. This is particularly true for profilinswhere the only allergenic profilins are found in flowering plants, althoughprofilins 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, althoughthis 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 complexmultigene family in Betula verrucosa (European white birch, syn. B. pendula)and its relatives. Sensitization to this pollen allergen has been implicated as amajor factor in pollen–food allergy syndrome. While European white birchis not endemic to North America, sensitization to birch pollen is neverthe-less prevalent and attributed to Bet v 1 homologs from other birch species.The Bet v 1 gene family has been extensively characterized in eight Betulaspecies at the nucleotide and predicted protein sequence levels where multipleexpressed isoforms as well as pseudogenes were identified (Schenk et al., 2006,2009). One hundred twelve unique genomic sequences were predicted toencode 80 distinct protein isoforms. Members of only two out of the fivesubfamilies, however, were expressed in pollen andwould be likely to provideexposure via inhalation. While some of these isoforms have been shown to behypoallergenic in that they have low IgE reactivity (Ferreira et al., 1996;Wagner et al., 2008), the potential for producing or identifying a

http://www.allergome.org


http://www.allergenonline.com



Table 1 Classification and function of food allergens in major crops among the ‘‘Big 8’’ (soybean, peanut, and wheat)

Protein

superfamily

Allergen

name

Biological function

according to Uniprot

(www.uniprot.org) Alias Species of origin

Major versus

minor allergen

classification

according to IUIS

(www.allergen.

org)a

Prolamin Ara h 2 Nutrient reservoir

activity

Serine-type endo-

peptidase inhibitor

activity

Conglutin-7

2S albumin

Arachis hypogaea Major

Ara h 6 Nutrient reservoir

activity

2S albumin A. hypogaea Minorb


activity

2S albumin A. hypogaea Minor

Ara h 9 Lipid binding/

transport

Nonspecific lipid

transfer protein

A. hypogaea Minorc

Gly m 1 Seed protein Hydrophobic

seed protein

Glycine max Major

Tri a 14 Lipid binding/

transport

Nonspecific lipid

transfer protein

Triticum aestivum Majord

Tri a 19 Nutrient reservoir

activity

Tri a gliadin

Omega-gliadin

Gluten

T. aestivum Major

Cupin (vicilin,

7S globulin)


activity

Conarachin A. hypogaea Major

http://www.uniprot.org

http://www.allergen.org)

Gly m 5 Nutrient reservoir

activity

b-Conglycinin G. max Minor

Cupin

(glycinin,

11S globulin)


activity

Arachin, Legumin A. hypogaea Minor


activity

Arachin, Legumin A. hypogaea Major (but near

50%)

Gly m 6 Nutrient reservoir

activity

Legumin G. max Minor

Profilins Ara h 5 Actin binding A. hypogaea Minor

Gly m 3 Actin binding Profilin-1 G. max Majord

Tri a 12 Actin binding T. aestivum Major; minord

Bet v 1 related Ara h 8 Plant defense PR-10 protein A. hypogaea Major

Gly m 4 Plant defense Stress-induced

protein SAM-

22

G. max Major

Papain-like

cysteine

protease

Gly m

Bd 30K

Proteolysis P34 G. max Majord

Oleosins Ara h 10 Lipid storage 16 kDa oleosin

Oleosin 2

A. hypogaea Minore

Ara h 11 Lipid storage 14 kDa oleosin

Oleosin 1

A. hypogaea Minore

Hevein like Tri a 18 Agglutinin Wheat germ

agglutinin

T. aestivum Minor

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-likeproteins.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.



hypoallergenic birch tree is limited by the complexity of the gene familycontributing to Bet v 1 expression. Furthermore, birch is a native treewhose gene diversity would not be manipulated to the same level as adomesticated crop.

Even in a domesticated crop such as maize, the primary pollen allergen,b-expansin is encoded by a complex multigene family. Among these group1 allergens, 15 genes and seven pseudogenes have been identified, and 13/15genes were expressed in pollen since their sequences were identified frompollen-specific EST libraries (Valdivia et al., 2007). Phylogenetic analysisrevealed that group 1 allergens could be divided into two groups, A and Bhaving�60% amino acid similarity, that probably diverged subsequent to thewhole-genome duplication event shared by grass family members (Valdiviaet 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 highlevel of duplication is displayed by one B-group subfamily (EXPB11) inmaize, which contains five expressed members that produce identical matureproteins, 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 cross-reactivity between Bet v 1 and Mal d 1, another PR-10 protein. Applecultivar-dependent reactions have been described suggesting either quanti-tative or perhaps qualitative differences in apple PR-10 proteins (Marzbanet al., 2005; Vieths et al., 1994). An in-depth analysis of Mal d 1 sequences inapple established that 18 genes mapping to three chromosomes were presentin the genome (Gao et al., 2005). Two clusters contained 16 of the geneswhich was consistent with the duplicated genome origin of apple. Eight ofthe genes are known to be expressed in fruit (Beuning et al., 2004). Onegroup of seven intron-containing genes was investigated for allelic diversityamong 10 cultivars with known high or low allergenicity (Gao et al., 2008).Forty-six nucleotide sequences were predicted to encode 25 Mal d 1 iso-forms, and alleles of two genes were found to be associated with the level ofallergenicity. Further investigation will be required to distinguish the roles ofquantitative versus qualitative differences for fruit-expressedMal d 1 proteinson 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 nonpollenplant particulates containing allergens, is a significant route of sensitization.Studies involving workers in bakeries and soybean mills with respiratoryallergies caused by soy flour have implicated the Kunitz trypsin inhibitor asan airborne allergen (reviewed by L’Hocine and Boye, 2007). Several germ-plasm lines are available which lack this protein, and a cultivar named‘‘Kunitz’’ has been released (Bernard et al., 1991). Because of the antinutri-tional properties of the Kunitz trypsin inhibitor, these soybean lines were


initially developed for use in livestock feed, but the trait could be easilyintroduced into soybean varieties used in baking to reduce occupationalexposure to sensitive individuals.

Natural diversity has allowed isolation of potentially hypoallergenicvariants 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 theseed (Ogawa et al., 1991). A wild soybean line QT2 (Glycine soja) was foundto lack all three subunits (a, a0, and b) of the major allergen b-conglycinin.Subsequent studies determined that a single dominant gene (Scg-1) wasresponsible for the lack of b-conglycinin (Hajika et al., 1998; Teraishiet al., 2001). The discovery of a simply inherited gene has facilitated theintrogression of this trait into breeding lines and cultivars (Tsubokura et al.,2006). P34 is a papain-family protease comprising <1% of total soybeanseed protein that may play a role in disease resistance. Multiple efforts toidentify P34-null soybean genotypes (Joseph et al., 2006; Ogawa et al.,2000; Yaklich et al., 1999) ultimately resulted in success upon screening ofthe entire USDA soybean germplasm collection that consisted of 16,266accessions ofG. max,G. soja, and wild relatives. Only twoG. max P34 nullswere obtained from this herculean effort (Joseph et al., 2006), both of whichcontain the same transcribed P34 gene sequence. Six nucleotide differencesbetween mutant and wild-type alleles were predicted to result in fouraltered amino acid residues, one of which changed cysteine to serine andthereby was presumed to interfere with the formation of disulfide bonds.Subsequent analysis of these accessions, however, established that they werenot true nulls but that translation of the mutant alleles was severely con-strained by a 4-bp insertion at the start codon resulting in an eightfoldreduction in P34 protein accumulation (Bilyeu et al., 2009).

Variation for allergen gene expression levels, and not just structure, alsohas been documented. For example, Kang et al. (2007a) surveyed 60accessions of peanut assembled as part of a minicore collection (Holbrookand Dong, 2005) and found 2–2.6-fold variation in protein amounts ofthree major allergens, Ara h 1, Ara h 2, and Ara h 3, in mature seeds. Theaccession with the highest level of Ara h 1 (max. 18.5%, ave. �12%) alsoshowed the lowest amount of Ara h 2 (min. 6.2%, ave. �11%), and lowerthan average Ara h 3 (27.3%, ave. �30.5%). It is likely that seed storageprotein fractions are adjusted to compensate for higher or lower levels ofone while maintaining a relatively constant total protein amount (Hartweckand Osborn, 1997; Tada et al., 2003b). Geographic origin of an accessionwas not a contributing factor to allergen protein differences. Null alleleswere 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 showingreduced recognition by peanut-sensitive patient IgE (Kang and Gallo,2007). Differences in the regulation of gene expression could account inpart for the resulting variation in accumulation of allergenic proteins during


seed development. Delayed transcript expression for all three allergen geneswas observed in two out of twelve cultivars surveyed over four seeddevelopmental stages (Kang et al., 2007b).

Another example of gene expression differences is for the ripening inhibi-tor gene of tomato that was first reported by Robinson et al. (1968). Plantshomozygous for this recessive gene (rin/rin) do not ripen fruit, but fruits ofhybrids (RIN/rin) do turn red and have extended shelf life (Kitagawa et al.,2006). Kitagawa et al. (2006) discovered that the hybrids showed reducedexpression of two allergen genes (b-fructofuranosidase and polygalacturonase2A), had reduced accumulation of the allergenic proteins, and showed reducedIgE reactivity to extracts of the hybrid fruit. The actual mutation is in a genethat codes for a MADS-box transcription factor (Vrebalov et al., 2002), whichcontrols expression of multiple genes. As with the soybean Scg-1 gene, thisexample demonstrates that mutations need not be within the allergen genesthemselves to have an effect on reducing allergenicity.

Minor sequence variation has been documented among alleles of somepeanut allergen genes, most of which is not expected to alter IgE-bindingcapacity. Empirical determination of IgE binding can lead to unexpectedresults, however, as in the case of a natural allele of Ara h 2 discoveredthrough EcoTILLING of Arachis duranensis, the putative A-genome donorof polyploid peanut (Ramos et al., 2009). EcoTILLING is a sequence-basedassay for allelic differences of a target gene in natural populations (Comaiet al., 2004). Ramos et al. (2009) showed the presence of several variants ofAra d 2.01, the A. duranensis ortholog of Ara h 2.01, one of which caused anamino acid change (S73T) in an immunodominant epitope and displayedsignificantly decreased IgE binding.

2.2. Induced variation: Mutagenesis

While natural allelic variation has been identified for some allergens, thisvariation does not always reveal hypoallergenic isoforms or null mutants thatwould be of value for crop modification. Induced variation through delib-erate mutagenesis relies on the same DNA modifying mechanisms thatgenerate spontaneous mutations albeit at an accelerated pace. Methods toinduce mutations have been extensively reviewed (Malmberg, 1993; Redeiand Koncz, 1992; Walbot, 1992); therefore, we only briefly address thistopic and focus on relevant discoveries of interesting mutants.

2.2.1. Methods for inducing genetic variation in allergensIn some crop species where significant natural variation is extremely limited,mutagenesis can be employed to create variation. Multiple means of gen-erating mutations have been developed, each with advantages and disad-vantages. Various forms of ionizing radiation, such as X-rays and g-rays,have been used successfully to generate genetic variation in plants.


Radiation tends to induce moderate to large-scale chromosomal changes,often resulting in deletion or rearrangement of large portions of thegenome. An advantage of this technique is that gene knockouts are oftenrecovered, but frequently more than one gene is affected, having a deleteri-ous effect on the overall phenotype of the plant. Optimizing the dosage ofradiation is crucial in order to recover a significant number of mutationswithout 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 deletionmutations (Balyan et al., 2008; Li et al., 2002; Yu, 2006).

Alteration of DNA sequences via chemical mutagens is also commonlypracticed. Chemicals most frequently used for mutagenesis include ethylmethanesulfonate (EMS), diethyl sulfate (DES), andN-nitroso-N-methylurea(NMU), among others. As with radiation, the dosage of mutagen must beoptimized to maximize the rate of mutation recovery while minimizing thedirect toxic effects of the chemicals. Compared to radiation, chemicalmutagens tend to induce very small changes to DNA sequences: single-nucleotide polymorphisms (SNPs) or very small insertions and deletions(indels). Thus, generating knockouts via chemical mutagenesis is less likelyunless an SNP creates a premature stop codon or alters the start codon of agene, or if an indel results in a frameshift. However, slight alterations to genesof interest, such as missense mutations, are commonly obtained. Conserva-tive 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 adramatic effect on IgE recognition (Burks et al., 1999). Recently a differentclass of chemical mutagens, termed deletogens (Balyan et al., 2008), has beendescribed. These chemicals induce larger deletions than typical chemicalmutagens, but generally smaller than those arising from irradiation. For themodel nematode Caenorhabditis elegans an average deletion size of 1400 bpwas reported by Liu et al. (1999). Deletogens include diepoxybutane (DEB),diepoxyoctane (DEO), and trimethylpsoralen (TMP). TMP is applied in thepresence of ultraviolet light to induce deletions (UV–TMP). EMS andethlynitrosourea (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 randomlythroughout the genome, and in the case of altered allergenicity a physicalphenotype is not easily observable. Thus, a high-throughput screeningtechnique 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 individualscarrying mutations in the genes of interest. This reverse genetics approach iseven more relevant for polyploids where duplicate genes frequently maskmutant phenotypes.


TILLING is a PCR-based approach which utilizes mismatch cleavage todetect mutations on polyacrylamide gels. Target gene sequence is thusrequired for this type of mutant screen. For TILLING, equal amounts ofDNA from several mutagenized individuals are pooled up to eightfold,depending on the size and complexity of the genome. The target gene isamplified by PCR using IRDye end-labeled primers, and then theproduct is denatured and allowed to reanneal. During reannealing, mutantstrands may pair with wild-type strands, creating heteroduplexes. Thesemismatches can be detected and cleaved by several nucleases. The mostcommonly used of these is CEL1 nuclease, which can be extracted fromcelery. Cleaved products are visualized by electrophoresis in polyacrylamideslab gels in a Li-Cor DNA Analyzer which detects the fluorescent signalsfrom 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 befeasible. An amplicon is designed to span several kilobases including thegene of interest, and the PCR products are visualized by standard agarosegel electrophoresis. Products of smaller size indicate possible deletions.Smaller products tend to be preferentially amplified in PCR with shorterextension times, thus facilitating their detection in highly pooled samples.Analogous to the pooling strategy used in TILLING, DNA from multipleindividuals can be combined, but at an even higher throughput. For thistype of screen in rice, one deletion variant can be detected in a pool of asmany as 200 individuals (Wu et al., 2005). This strategy of identifyingdeletion mutations has been termed DEALING (detecting adduct lesionsin genomes; Balyan et al., 2008). In Arabidopsis a similar strategy, termedDeleteageneTM, has been used to detect deletions caused by fast neutronmutagenesis, with detection possible in a pool of 1000 individuals (Li et al.,2002). These mutagenesis systems which generate or allow selection ofsmall scale deletions could be easily transferred to important crop speciesfor elimination of allergen-producing genes. The pool size and rate ofmutation would need to be determined empirically, especially for specieslike peanut with large complex genomes.

Insertional mutagenesis using T-DNA from Agrobacterium has beenextensively utilized in Arabidopsis as a forward genetics tool to generateknockout mutations by disrupting the genes into which the T-DNAs insert.Also, the extreme stresses brought about during tissue culture have beenshown to induce mutations by the mobilization of endogenous transposableelements in rice; examples include the retrotransposon Tos17 (Hirochikaet 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 bemobilized by subjecting intact rice seeds to high hydrostatic pressure. It hasbeen proposed that some of the mutations resulting from irradiation or


chemical mutagenesis are caused by the activation of transposable elements.For any insertionmutant where the sequence of an activated mobile elementor T-DNA is known, it should be possible to develop a reverse-geneticscreen to identify individuals with insertions in genes of interest, includingknown allergen genes. However, little is known about mobile DNA ele-ments in species other thanArabidopsis, rice, andmaize, and high-throughputtransformation and T-DNA tagging systems are lacking for most cropspecies. Also, with the currently available methods, the probability ofobtaining an insertion in a specific gene is quite low. In addition, the long-term stability of insertional mutations over many generations of breeding isunknown. Although insertional mutagenesis has been very useful for geneticstudies, its use in developing variation in allergens in crop species may belimited, at least in the near future.

2.2.2. Examples of allergen content changes resultingfrom mutagenesis

Mutagenesis has been used extensively to create novel variation in orna-mental plant species, to improve agronomic characteristics of various plantspecies, and for modification of food quality traits in crops (Ahloowalia andMaluszynski, 2001). For example, gamma radiation was used to inducemutations for improved tuber quality (Love et al., 1996a) and reducedglycoalkaloid content (Love et al., 1996b) in potato. However, the use ofmutagenesis in generating variation in allergens has not been extensivelyapplied, though several examples of induced allergen variation have beenreported. These examples highlight the potential of mutagenesis for alter-ation or elimination of allergens in crops.

Nishio and Iida (1993) reported the reduction of allergenic proteins inseeds 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 traceamounts of these proteins. Of the latter, one was an M2 derived fromgamma-irradiated material, and the other was derived from a spontaneouswhite-panicle mutant that was subjected to EMS treatment. The mutantswith the lowest levels of these proteins always had floury endosperms, sotheir use as food would be limited. Furthermore, they were sterile. Thosewith reduced levels of the allergen also had floury endosperms, but only inthe very center of the kernel. The authors noted that the quality of thesemutants would be acceptable as cooked rice, but the reduction in allergencontent may not be significant enough to be promoted as reduced-allergenrice. In a subsequent paper, however, Iida et al. (1993) found that a reduced-allergen mutant could facilitate the production of hypoallergenic processedrice by reducing the cost of removing the allergenic proteins. Given that thelevel of flouriness in the endosperm seems to correlate with the level of


reduction in 16- and 26-kDa proteins, and that this was observed in fourindependent mutants, there probably is a pleiotropic effect of theseproteins on endosperm texture rather than a linked mutation resultingfrom deletion of a large piece of the genome, as may happen withgamma-irradiation induced mutations. Also, if this protein is indeed anamylase inhibitor, changes to the endosperm would be expected when theprotein is reduced or eliminated.

Several soybean lines lacking specific seed proteins, including severalallergens, have been derived from g-ray mutagenesis. The breeding lineEnB1 lacks all five subunits of the seed storage protein glycinin (Odanakaand Kaizuma, 1989; Teraishi et al., 2001), which comprise the named allergenGly m 6. The mutant breeding line must therefore either contain severalindependent glycinin gene mutations (glycinin genes are known to locate tomultiple chromosomes) or more likely a single mutation in a trans-actingmodifier gene (Beilinson et al., 2002). Takahashi et al. (1994) subjected thebreeding line ‘‘Kari-kei 434,’’ a spontaneous mutant already lacking the a0-subunit of b-conglycinin, to gamma radiation, and from this experiment theyderived ‘‘Tohoku 124’’ (also known as ‘‘Yumeminori’’), a linewhich lacks thea- and a0-subunits of the allergen b-conglycinin, and has reduced levels of theb subunit (collectively Gly m 5, a vicilin). A PCR-based assay has beendeveloped to screen for the mutant alleles of both a- and a0-subunits of b-conglycinin that allows testing for seed purity (Ishikawa et al., 2006). ‘‘Tohuko124’’ 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 toremove 99.8% of another allergen Gly mBd 30K (P34) from soymilk throughprocessing. Nakamura et al. (1989) and Phan et al. (1996) also have reportedreduction or elimination of b-conglycinin subunits in soybean resulting fromgamma irradiation. Phan et al. (1996) noted that there is a lethal chlorosisassociated with the deletion of both subunits and suggested that their mutationresults from a large chromosomal deletion, which is one of the drawbacks of g-raymutagenesis.More recentlyManjaya et al. (2007) subjected soybean varietyVLSoy-2 to gamma radiation. From this treatment three mutants were iden-tified which lacked the A3 subunit of glycinin. Of these, one also had reducedlevels of the a- and a0-subunits of b-conglycinin, and two of the mutantslacked these two subunits altogether.

Induced mutation also may be a successful approach to eliminate aller-gens from peanut. A preliminary report described peanut mutants missingisoforms of Ara h 2 and Ara h 3 (Perkins et al., 2006). Currently, a peanutTILLING population is being developed and screened for variations in themajor allergens Ara h 1 and Ara h 2 (Knoll et al., unpublished). It has beenshown that both of these allergens are present in two isoforms (Knoll et al.,unpublished; Ramos et al., 2006), as peanut is an allotetraploid and oneisoform is derived from each of the two subgenomes. Possible allergenvariants have been found for both isoforms of Ara h 2, and for Ara h 1a.


A truncation mutation has been identified for ara h 1b and a disrupted startcodon has been identified in ara h 2.02. TILLING populations are valuablegenetic resources that can be tapped for allergen gene variation. SeveralTILLING populations have already been developed for soybean (Cooperet al., 2008), with mutation rates as high as 1 SNP/140 kb, but no data havebeen published on screening for allergen gene mutations although otherseed traits have been targeted (Dierking and Bilyeu, 2009).

The identification of less allergenic isoforms of a protein or gene knock-outs, either as natural variants or induced mutations, may facilitate conven-tional and molecular breeding strategies toward the development ofhypoallergenic food crops, particularly where the number of genes encodinga protein is small. This approach becomes more difficult where large multi-gene families are responsible for allergenic protein expression as is the casefor group 1 and Bet v 1 pollen allergens. For these situations, homology-dependent gene silencing, as reviewed below, may be the most feasiblemeans for allergen reduction.

2.3. Induced variation: Transgenics

Most crop allergens can be effectively reduced through genetic transforma-tion for gene silencing. All of the current published allergen silencing workis based on the mechanism of posttranscriptional gene silencing (PTGS).PTGS is a naturally evolved pathway in plants that serves multiple biologicalfunctions such as suppression of viral infection and developmental generegulation (Baulcombe, 2004; Chapman and Carrington, 2007). To silencespecific allergen proteins, a transgene can be introduced into the plantthrough genetic transformation in the sense, antisense, or inverted-repeatorientation. Aberrant transcripts of sense and antisense transgenes such asthose missing the 50 cap structure are recognized by the endogenous RNA-dependent RNA polymerase (RDRP) (Gazzani et al., 2004). The RDRPsubsequently synthesizes the complementary strand of the transgene to formdouble-stranded RNA (dsRNA). If the transgene is introduced as aninverted repeat, the transcript forms a dsRNA hairpin due to self comple-mentarity. These transgene dsRNAs are further recruited by a dicer-likecomplex and sliced into 21–24 bp short interfering RNAs (siRNA)(Hamilton and Baulcombe, 1999; Schauer et al., 2002). RNA-inducedsilencing complex (RISC) in the cytoplasm binds to the siRNA andunwinds the double strand. The siRNA guide strand base-pairs with com-plementary mRNA (e.g., targeted allergen RNAs) and confers sequencespecificity to silencing. Argonaute proteins within the RISC cleave themRNA and downregulate its expression (Song et al., 2004).

The effectiveness of allergen silencing depends on several factors includ-ing complexity of allergen families, arrangement and length of transgenearms, transgene copy number, promoter specificity and strength, allergen


turnover rate, spatial and temporal expression pattern of allergens, etc.(Kerschen et al., 2004). Selection of an allergen to be targeted for silencingdepends on the availability of the allergen sequence, its clinical importance,and biological function in the plant. Frequently, allergen proteins areencoded by multiple gene family members as well as multiple alleles foreach gene. For example, Ara h 2 is a major peanut allergen that has twogenomic copies, Ara h 2.01 and Ara h 2.02. It also shares 63% homology toAra h 6, another peanut allergen in the conglutin family (Ramos et al.,2006). Since PTGS is homology dependent, selection of a sequence regionthat is highly conserved between these two peanut allergens during designof a silencing construct resulted in effective silencing of both peanut aller-gens (Chu et al., 2008). On the other hand, if a specific allergen needs to besilenced with minimum collateral effect, a unique sequence fragment inthe 30- or 50-untranslated regions with least homology to other genes canbe selected. For crops that producemultiple allergenic proteins, it is also possibleto construct a silencing vector to target several allergens simultaneously. Thepotential downside of this strategy is that the viability of the transgenic crop canbe severely compromised due to the knockdown of multiple functional pro-teins. It is therefore important to prioritize the list of allergens to be silencedbased on their clinical relevance and biological function.

A number of studies on the arrangement of transgene arms showed thatthe most effective silencing constructs are intron-spliced hairpin structures(ihpRNA) or inverted repeats separated by a nonintron spacer where theoptimum length of the transgene direct or inverted-repeat unit ranges from�100 to 850 bp (Chuang and Meyerowitz, 2000; Hirai et al., 2007; Smithet al., 2000; Waterhouse et al., 1998; Wesley et al., 2001). Various genericsilencing constructs that accommodate inverted repeats are available (http://www.pi.csiro.au/rnai/; http://www.chromdb.org/) and strategies forsilencing including the use of artificial microRNAs have recently beenreviewed (Ossowski et al., 2008). In general, upon transcription, an invertedrepeat forms a hairpin dsRNA and the intron/spacer forms a loop structure.It is suggested that this panhandle structure stabilizes the dsRNA andincreases the silencing efficiency. Ninety to 100% of transgenic lines pro-ducing ihpRNA show PTGS whereas only an average of 12% of transgeniclines from sense and antisense constructs have a silencing effect. Introns asspacers between inverted repeats may or may not be more effective thannonintron sequences (Hirai et al., 2007). Other factors that affect silencingefficiency include level and location of transcription; strong promoters arecorrelated with stronger silencing effect than weak promoters (Chuang andMeyerowitz, 2000; Hirai et al., 2007), and tissue specific expression can beutilized for some allergens since those such as seed storage proteins oftenare specifically expressed in seeds. Other allergens may be expressed in morethan one type of plant tissue, such as the tomato allergen profilin and appleallergen Mal d 1, which can be found not only in fruits but also in vegetative

http://www.pi.csiro.au/rnai/



http://www.chromdb.org/

http://www.chromdb.org/


tissues. Even in these cases, selection of a fruit specific promoter may bebeneficial to plant health since the allergen would be reduced only in theedible part of the plant and its function maintained in other parts of theplant.

Single-copy, homozygous transgenic lines are desired since the silencingeffect is more likely to be stable across generations. Inserts at multiplegenomic loci are expected to segregate among progenies and degree ofsilencing may vary among events. While Agrobacterium-mediated transfor-mation is considered to more frequently result in single-copy insertions thanmicroprojectile bombardment, the literature provides a significant body ofevidence to question this dogma (Altpeter et al., 2005). Regardless oftransgene delivery method, variation among transgenic lines generatedwith a single silencing construct is inevitable, requiring that degree andstability of silencing be determined empirically. Since allergens are largelyproteins, methods for detecting changes in protein expression include 1Dand 2D protein gels, Western blots, ELISA assays, and more sophisticatedproteomic techniques (Stevenson et al., 2009; Thelen, 2009).

Evaluation of the effect of allergen silencing on allergenicity involves IgEbinding using sera from allergic patients and in vitro histamine release fromsensitized human or humanized basophils (Palmer et al., 2005). Even morerelevant allergenicity data can be collected in a clinical setting where skinprick testing and double-blind placebo-controlled food challenge(DBPCFC) can be carried out (Peeters et al., 2007). However, as mentionedearlier, the presence of multiple allergens in one food source makes theDBPCFC test risky for allergic patients. Therefore, it has not been used forevaluation of any allergen reduced transgenic lines.

2.3.1. Allergens silenced in cropsBoth food and pollen allergens from rice, ryegrass, apple, soybean, peanut,and tomato have been successfully silenced by genetic transformation. In1996, a group of 14–16 kDa rice allergens (RAs) was discovered byscreening fractionated rice proteins with serum IgE from rice-allergicpatients. These proteins were highly reactive to all 31 patient sera screenedby a radio allergo-sorbent test (RAST) defining them as major RAs(Nakamura and Matsuda, 1996). Two antisense constructs, each containingtwo copies of the antisense gene driven by seed-specific promoters and aconserved region of the RA genes, were used to knockdown expression ofthis allergen family. One construct had the antisense gene pair controlled byglutelin and prolamin promoters and oriented as tandem repeats while thesecond had the antisense genes in inverted orientation and under control ofRA gene 1 and starch branching enzyme I gene promoters. The rice waxyterminator was common to all four chimeric genes. Using monoclonalantibody cross-reacting with the majority of the gene family members, itwas found that the allergen content was reduced by 80% (Tada et al., 1996).


The incomplete suppression of this allergen family by antisense constructswas further investigated in T3 and T4 progenies of transgenic lines (Tadaet al., 2003a), where it was found that members of the RA family with lowerlevels of homology to the antisense sequence were expressed at normallevels in the transgenic lines, whereas the expression of RAs sharing highhomology to the antisense sequence was greatly reduced. Therefore, thedata provided evidence that the degree of silencing was correlated with theextent of sequence homology.

Ryegrass pollen allergen Lol p 5, a 31-kDa protein, is a major ryegrassallergen recognized by 90% of patients allergic to this grass pollen. Anantisense construct driven by a pollen specific promoter from Ory s1 wastransformed into ryegrass callus tissue via particle bombardment, and trans-genic lines showed significantly reduced accumulation of Lol p 5 protein(Bhalla et al., 1999, 2001). IgE binding from patient sera also demonstratedless reactivity by transgenic lines. Plant growth and pollen viability wereunaffected; therefore, the putative roles of Lol p 5 in self-incompatibilityand pollen germination were excluded. Similarly, antisense technologycombined with pollen-specific expression conferred by the maize Zm13promoter was used to significantly reduce expression of ryegrass pollenallergens Lol p 1 and Lol p 2 (Petrovska et al., 2004).

A soybean major allergen, Gly m Bd 30K also known as P34, is amember of the papain superfamily of cysteine proteases (Kalinski et al.,1992). It induces allergic reactions in 65% of soy-sensitive patients yetcomprises less than 1% of soy total seed protein (Herman et al., 2003;Ogawa et al., 1991). Cosuppression was achieved in transgenic soybean byseed-specific expression of the allergen gene under control of a b-congly-cinin promoter (Herman et al., 2003). The reduction of Gly m Bd 30Kexpression in one transgenic line was confirmed by allergen specific andhuman IgE immunoblots. No collateral effects on other proteins weredetected in the silenced line. Seed size, shape, protein composition, andoil content in the silenced line were comparable to that of the nontransgeniccontrol. Comparative 2D gel electrophoresis/mass spectrometry data alsoshowed no further protein composition change other than the downregula-tion of Gly m Bd 30K. In contrast, a significant change in protein profile,including enhanced expression of Gly m Bd 30K, was observed in anotherstudy that used cosuppression to reduce expression of the a- and a0-subunitsof b-conglycinin, components of Gly m 5, a minor soybean allergen(Kinney et al., 2001). The increases in Gly m Bd 30K and unprocessedglycinin (Gly m 6) were attributed to altered protein trafficking that resultedin accumulation of novel protein bodies. The frequency of silenced linesrecovered using antisense or cosuppression constructs in the studiesdescribed above was not reported. However, since the inverted-repeatRNAi construct design has proved to be very effective for transgene-


induced silencing, subsequent studies on allergen silencing have utilized thisstrategy.

Two minor tomato allergens, Lyc e 1 and Lyc e 3, have been indepen-dently silenced. Lyc e 1 is a profilin that binds to actin to regulate cellelongation, cell shape maintenance, and flowering in plants. It is recognizedby IgE from 22% to 26% of tomato allergic patients. Two Lyc e 1 isoformssharing 88.1% identity at the nucleotide level were silenced simultaneouslyby an RNAi construct driven by the CaMV 35S promoter (Le et al., 2006a).Transgenic plants displayed reduced Lyc e 1 expression at both RNA andprotein levels. A reduction in IgE binding also was observed. Patientssensitized to only Lyc e 1 showed 65–100% reduction in wheal reactionto transgenic fruit in a skin prick test whereas multiallergen sensitizedpatients had only 16–25% reduction in wheal reaction. Elimination ofmultiple allergens from tomato fruit would be necessary in order to sub-stantially alter the immune reaction in patients showing the latter response.Not unexpectedly, Lyc e 1-silenced plants demonstrated severe growthretardation and reduced fruit and seed set because profilin plays an essentialrole in cell structure and cell growth. Silencing this allergen without repla-cing it with a nonallergic variant was proven to be detrimental to planthealth and production. It is therefore important to take the function of anallergen into consideration before silencing. Lyc e 3, a nonspecific lipidtransfer protein (nsLTP) and a member of a multigene family with a highlyconserved cysteine-rich structure, has potential biological functions in plantdefense and calcium metabolism. The expression of Lyc e 3 is mainly in thepeel of tomato fruit. This protein is recognized by 29% of tomato allergicpatients (Le et al., 2006b). Two isoforms Lyc e 3.01 and Lyc e 3.02 sharing76.5% identity at the nucleotide level were silenced by a single RNAiconstruct (Lorenz et al., 2006). Silencing of both isoforms was confirmedbyWestern blot. Competitive ELISA showed that the level of Lyc e 3 in thetransgenic line was below 0.5% of that of the wild-type. Basophil histaminerelease and skin prick tests showed reduced allergenic potency of transgeniclines. Contrary to Lyc e 1, no phenotypic differences were observed in Lyce 3 transgenic and transgene null lines found among T1 progenies.

Apple allergen Mal d 1 has cross-reactivity to birch pollen allergenBet v 1, as described in Section 2.1, and is considered a major allergenwith 18 family members belonging to the PR protein PR10 group. Aconstitutively expressed inverted repeat of Mal d 1b 50-untranslated regionand first exon was introduced into apple via Agrobacterium-mediated trans-formation (Gilissen et al., 2005). Since Mal d 1 is expressed in leaves as wellas fruit, protein extracted from leaf tissue was analyzed to assess the effec-tiveness of silencing before fruit production. Reduced skin prick test reac-tion and IgE binding were demonstrated with the silenced transgenic lines.These results need to be further confirmed by testing of fruit tissue which isthe allergen source for affected patients.


More recently, Ara h 2, the most potent peanut allergen, was silenced byan RNAi construct in two independent studies (Chu et al., 2008; Dodoet al., 2008). Ara h 2 is one of eleven identified peanut allergens, but it isconsidered one of the major allergens because it can be recognized by serafrom more than 90% of peanut allergic patients (Burks et al., 1995). There isa 63% nucleotide sequence similarity between Ara h 2 and Ara h 6, anotherpeanut allergen recognized by 80% of peanut allergic patients (Flintermanet 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 peanutembryogenic cultures via microprojectile bombardment led to recovery ofthree independent transgenic lines, all of which showed significant reduc-tion 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 2.01and less similar to Ara h 6, the homology-dependent silencing effect wasmore variable for Ara h 6 than Ara h 2, analogous to the RA silencingresults. Human IgE immunodetection of proteins from one of the threesilenced lines showed less signal from Ara h 2 than Ara h 6, although Ara h 6was 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 Arah 6; therefore, the immune reaction to peanuts probably would not beeliminated upon silencing of only these two allergens. The transgenic plantswere phenotypically normal, and in spite of demonstrated trypsin inhibitoractivity for Ara h 2 (Maleki et al., 2003), transgenic lines did not show anyincreased susceptibility to Aspergillus flavus fungal infection compared tonontransgenic lines. Ara h 2- and Ara h 6-silenced lines had largely normalglobal protein profiles with some minor collateral changes such as anelevation in Ara h 10 (oleosin), 13-lipoxygenase and Ahy-3 (arachin) anda decrease in conarachin (Stevenson et al., 2009). Independently generatedAra h 2-silenced lines showed surprisingly large variations in protein profilesin addition to reduced Ara h 2 expression (Dodo et al., 2008).

Silencing of crop allergens by PTGS has been successful but holds somerisk for commercialization due to potential instability (Ozias-Akins et al.,2009). Certain plant viruses can produce silencing suppressor proteinswhich negatively affect the stability of silencing (Li and Ding, 2001). Apotential solution to this problem is heritable transcriptional gene silencing(TGS). While both PTGS and TGS are epigenetic phenomena, TGS resultsfrom DNA modifications. Both processes are initiated by the RNAimachinery and similar transformation construct design is effective. Ratherthan expressing an inverted-repeat transcript containing gene codingsequence, the promoter sequence of a target gene is transcribed to generatedsRNA. The siRNA from the promoter sequence can be transported backinto the nucleus to induce homology-dependent promoter methylationand silence expression of the downstream open reading frames (Matzkeand Birchler, 2005; Mette et al., 2000; Wang et al., 2001). Maintenance of


DNA methylation after TGS is dependent upon a DNA methyltransferaseand independent of the presence of siRNA ( Jones et al., 2001). Therefore,TGS should not be susceptible to suppression of silencing upon viralinfection, although it requires prior knowledge of allergen promotersequences.

2.3.2. Allergenicity of transgenic cropsA contrasting aspect of crop allergen silencing is associated with the poten-tial increase in allergenicity of genetically modified plants. In the early1990s, Brazil nut protein Ber e 1 was introduced into soybean to enhanceits sulfur amino acid content. Since allergic reactions to Brazil nut wereknown, the transgenic soybean was tested for reactivity with human IgE andwas shown to present a positive reaction (Nordlee et al., 1996). This workprovided additional evidence that Ber e 1 was a major Brazil nut allergen.Commercialization of such a transgenic product was never considered orattempted; however, allergenicity data are now a standard component ofsafety assessments required by federal agencies in the United States and othercountries prior to deregulation of a GE food or feed crop (Ladics, 2008).The weight-of-evidence approach currently is used and is based on priorknowledge of characteristics of a transgenic protein, structural comparisonof the transgenic protein to other allergens, and digestive stability.

3. Conclusions

Multiple approaches to the reduction in allergenicity of crops havebeen described including the exploitation of natural variation for quantita-tive or sequence differences, the induction of variation through mutagene-sis, and the suppression of allergen expression using GE. While spontaneousand natural mutants are largely unregulated, public opinion toward GEproducts and deregulation of transgenic crops continues to be an issuedespite more than a decade of safe deployment and consumption of trans-genic crops in the United States. A comprehensive review of the factsrelated to GE products draws from an extensive body of peer-reviewedliterature that addresses perceived and actual risks (Lemaux, 2008, 2009).While the science typically is overlooked in anti-GE platforms with under-lying sociological or religious convictions, individual consumers are morelikely to judge a product according to its perceived personal benefit (Schenket al., 2008). In some cases, the benefits of a hypoallergenic GE crop may beperceived by the general public as greater than the risks, increasing accep-tance over first-generation GE crops with solely crop protection traits. It islikely that both mutation and GE approaches will contribute to the futurerelease of crop varieties with the output trait of reduced allergenicity.


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