breeding for fruit quality (jenks/breeding) || breeding of hypoallergenic fruits

105

Introduction to Fruit Allergy

The food allergy epidemic is increasing worldwide, especially in westernized, developed countries and in economically fast-developing regions. This is largely caused by dramatic changes in food chain supply, life style, diverse environmental factors, and social activities. Most people know what an allergy is, but making it linked to fruit (the predominantly considered healthy food) seems ridic-ulous and unbelievable at first. Fruits may be one of the first edible raw foods for modern human beings before using fire. Fruit is strongly recommended to improve and maintain health. However, several fruits can affect a part of the population, and therefore, the social attention and scientific interests to this old problem are increasing (Marzban et al., 2005b). Recently, species and variety differences in allergenicity were observed at least in apple fruits, which encouraged fruit geneticists to uncover the causes and to apply their results in selection and breeding programs. Also, the new biotechnology approaches to silence specific allergen genes expressed in fruit demonstrated the great potentials for the development of hypoallergenic cultivars specifically for the benefit of patients allergic to fruit.

Prevalence of Fruit Allergy

Allergy to various fresh fruits has been recognized as an increasing problem, and a range of fruit allergens has been characterized in the past 15 years. Overall, fruit allergy appears to be a multifac-torial immunological disease with highly individual features (Andersen et al., 2010). Fruit allergy was first recorded in the clinic as early as in 1942 (Tuft & Blumenstein, 1942). They found the relationship between hay fever and the oral allergy syndrome (OAS). In 1982, apple (Malus domes-tica Bordh) allergy and its association with birch pollen allergy, especially occurring in Central and Northern Europe, was extensively studied (Eriksson et al., 1982). Fruit allergy incidences were scarce or neglected for a long time. The first reports about fruit allergies appeared in the clinical journals in the early 1980s and 1990s. The number of reports increased explosively during the past 20 years, and allergenic fruit species are not limited to a few, but involve now almost all fruits in the

5 Breeding of Hypoallergenic Fruits Zhong-shan Gao and Luud J.W.J. Gilissen*

Breeding for Fruit Quality, First Edition. Edited by Matthew A. Jenks and Penelope J. Bebeli. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

* This work was supported by Natural Science Foundation of China (30971970) and 111 project of State Education Ministry, China (B06014).

106 STRATEGIES FOR IMPROVING SPECIFIC FRUIT QUALITY TRAITS

world, although with different allergenic profiles and relevance. Food allergy symptoms are usually mild local reactions in the oral region (e.g., itching and swelling in the oral cavity, throat and inner ear, running nose and eyes, and coughing)—the so-called OAS. Fresh fruits and vegetables are especially causing such allergy problems. Other symptoms are also observed in organs such as the skin (e.g., local or general urticaria, atopic eczema), the gastrointestinal tract (e.g., cramps, diarrhea, vomiting), nose and lungs (e.g., rhinitis and asthma), and the cardiovascular system (e.g., anaphy-lactic shock). Anaphylactic reactions to fruit do occur, but only in rare cases; systemic reactions were observed without related, cross-reactive pollen allergy. The current literature includes more than 20 fruit genera (Table 5.1). The prevalence of apple allergy was estimated at 2% in the European countries (Hoffmann-Sommergruber, 2005). The prevalence of fruit allergies in young adults in Denmark (where the load of birch pollen is high in the spring season) showed kiwi (Actinidia deli-ciosa; 7.8%), pineapple (Ananas comosus; 4.4%), apple (4.3%), orange (Citrus sinensis; 4.2%), tomato (Solanum lycopersicum Mill.; 3.8%), and peach (Prunus persica L.; 3.0%) as the main offenders (Osterballe et al., 2009).

Most fruit allergy and allergen researches were done in West-European countries, especially Austria, Spain, the Netherlands, and Italy. In China, fruit is one of the 10 major causes of food allergy (Bai, 2005), with the most frequently reported allergenic fruits being peach, mango (Mangiferaindica), and pineapple. The increasing prevalence of peach allergy may follow the dramatic

Table 5.1 Reported allergenic fruit crops in the literature.

Genus Common names References

Malus Apple Ebner et al., 1991Pyrus Pear Karamloo et al., 2001Prunus Peach, plum, apricot,

cherry, almondPastorello et al., 1994; 2000; Scheurer et al., 1997; Pastorello et al., 2000; Reuter et al., 2005; Tawde et al., 2006

Fragaria Strawberry Karlsson et al., 2004Rubus Raspberry Sherson et al., 2003Eriobotrya Loquat Hajime et al., 2002Citrus Mandarin orange Ahrazem et al., 2005; Ebo et al., 2007Litchi Lychee Song et al., 2007Musa Banana Sanchez-Monge et al., 1999; Saraswat & Kumar, 2005Carica Papaya Tamburrini et al., 2005Persea Avocado Blanco et al., 1994; Diaz-Perales et al., 2003Mangifera Mango Duque et al., 1999; Hegde & Venkatesh, 2007Actinidia Kiwi Garcia et al., 1989; Lucas et al., 2003Vitis Grape Pastorello et al., 2003Diospyros Persimmon Bolhaar et al., 2004Lycopersicon Tomato Caballero and Martin-Esteban, 1998; Reche et al., 2001;

Pravettoni et al., 2009Cucumis Melon, cucumber Rodriguez et al., 2000; Caballero and Martin-Esteban, 1998Citrullus Watermelon Jordanwagner et al., 1993; Hoffmann-Sommergruber

and Bruckmuller, 2009Phoenix Date Kwaasi et al., 1999Hylocereus Dragon fruit Kleinheinz et al., 2009Ficus Fig Gandolfo et al., 2001; Focke et al., 2003Artocarpus Jackfruit Wuthrich et al., 1997; Bolhaar et al., 2004Morus Mulberry Caiaffa et al., 2003Punicia Pomegranate Gaig et al., 1999; Zoccatelli et al., 2007Castanea Chestnut Sanchez-Monge et al., 2006

BREEDING OF HYPOLLERGENIC FRUITS 107

increases in peach production and consumption (Yang et al., unpublished). With the increase of fruit consumption worldwide, fresh fruits have already become the most common cause of food allergy in patients over 5 years of age in Europe (Fernandez-Rivas et al., 2008). Mild OAS does not com-pletely deprive patients from fruit consumption and does not require medical treatment, but it does make fruit less attractive. Intense OAS will have direct impact on patients through fruit avoidance, which results in the reduction of actual fruit consumption by the patients and their family members. This will further decrease fruit intake in Europe, where fruit consumption is already below the rec-ommended level (Andersen et al., 2010)

Because most fruit crops in temperate zones belong to the Rosaceae family, fruits from the Maloideae, such as apple (Hoffmann-Sommergruber, 2005; Fernandez-Rivas et al., 2006), pear (Pyrus communis; Karamloo et al., 2001), and Prunoideae, such as peach (Fernandez-Rivas et al., 1997; Pastorello et al., 1999), sweet cherry (Prunus avium; Scheurer et al., 1997; 2004), plum (Prunus domestica; Pastorello et al., 2001), apricot (Prunus persica; Pastorello et al., 2000; Brenna et al., 2004), and almond (Prunus dulcis; Tawde et al., 2006) have been reported to cause allergic reactions in Europe. Research on fruit allergy in this family is broad and intensive. These fruits are common in most markets in the world. In addition, proceeding globalization of the world market leads to an increased supply of foods, including fruits. The consumption of tropical fruits has risen substantially during the last few years, and allergies to tropical fruits have also increased; these include kiwifruit (Fine 1981; Palacin et al., 2008), mango (Paschke et al., 2001b), and lychee (Litchi chinensis; Song et al., 2007). Contact allergies have also increased in recent years in orchard workers because of direct contact with fruit or pollens or during pruning (Chatzi et al., 2006).

In Europe, fruit allergy has become a major research theme and in an extended research program of the EU-SAFE project (granted by the European Commission and run from 2001–2003), apple allergy was chosen as a model to further explore fruit allergies (Hoffmann-Sommergruber, 2005). A follow-up research on apple and peach allergies was carried out as one of the issues within the EU-ISAFRUIT project (Chen et al., 2008; Botton et al., 2008; 2009a; 2009b; Krath et al., 2009), aiming at the increase of fruit consumption in Europe.

Biomedical Mechanisms Related to Fruit Allergy and Cross-Activity

General knowledge on food allergy will help us to understand fruit allergy matters. Generally, an allergy is defined as a hypersensitive reaction of the immune system to normally harmless com-pounds (mostly proteins) after inhalation, ingestion, or skin contact. Most common are the so-called “type-1 allergies”, which are characterized by immunoglobulin E (IgE) mediation (with E derived from erythema, which means red in Greek) (Bohle, 2004). The etiology of allergy distinguishes two phases: (1) The sensitization phase, without symptoms during which the immune system is primed. A potential allergenic protein is taken up by an antigen presenting cell, partly exposed on its surface and recognized by T-cells that further differentiate into Th2 cells, which in turn stimulate B-cells to produce antigen (allergen)-specific IgE. These IgEs bind to high-affinity receptors on the surface of mast cells and basophill cells. (2) The clinical response phase, during which symptoms occur as a result of reexposure to the allergen that is recognized by the IgEs. Bridging of two IgE molecules by a single allergen on the mast cells activates these cells to release mediators, such as histamine, tryptase, and tumor necrosis factor, causing the acute phase of the allergic immediate reaction (Bohle, 2004). Clinically, the IgE-mediated hypersensitive reaction occurs within minutes to 2 h after ingestion (Fernandez-Rives & Miles, 2004). Food allergies are considered type 1 allergies


and within that type, there are two classes. In class-I food allergies, the immune reaction takes place in the gastrointestinal tract. The allergens involved in this class are stable and resistant to heat and proteolytic activity. In class-II food allergies, the responsible proteins come into contact with the immune system through inhalation (e.g., the inhalation of pollen from several tree species and grasses), which leads to the development of food allergy in a multistep process and requires a repeti-tive challenge with a particular food allergen that is recognized by the same IgE as the inhaled allergen. This phenomenon is known as allergenic cross-reactivity.

Fruit allergy is often the result of a cross-reaction with a pollen allergen and is a commonly occurring phenomenon. Some examples of cross-reactivity include apple, peach, kiwi, jackfruit (Artocarpus heterophyllus), mango, and persimmon (Diospyros virginiana) with birch pollen (Voitenko et al., 1997; Paschke et al. 2001a; Bolhaar et al., 2004; Oberhuber et al., 2008), peach and kiwi with mugwort pollen (Blanusa et al., 2007).

The occurrence of fruit allergy shows geographic differences that are related to the local environ-ments (pollen conditions), geographic region, population type (urban, rural), fruit accessibility, and consumption habits. Peach allergy occurs more often in southern European countries (i.e., Spain, Italy, Portugal), where two different peach allergy profiles can be observed. One of these is the OAS caused by the allergens Pru p1 and Pru p 4, and the other is the systemic gastrointestinal symptom caused by Pru p 3 (Gamboa et al., 2007). In China, both profiles have also been reported after eating fresh peach. In addition, some cases are known in which contact with peach fruit hair or even with peach pollen during the blooming season may cause adverse reactions.

The biological basis of cross-reactivity between fruits and pollens among diverse fruit species, genera, and families is now better understood by comparing the responsible allergenic molecules. These allergens have a similar structure and biochemical and physiological property (Breiteneder & Mills, 2006). A nice review by Marzban et al. (2005a) summarized cross-reactive PR-10, nsLTP, and profilin allergens and their classification in Rosaceae fruits.

Feasibility of Hypoallergenic Fruit Cultivars

Currently, there is no other cure of fruit allergy than avoidance of the specific fruits. However, avoiding fruits from the diet may have a negative effect on health in allergic patients and also may affect their quality of life. Therefore, the development of low or nonallergic fruits would be good news to those fruit allergy sufferers. There are two approaches. One is by conventional selection and breeding as long as there is sufficient genetic variation controlling the hypoallergenic traits. Natural variations among the cultivars and preselected breeding materials can be tested by clinical skin prick tests (SPTs) and verified by double-blind placebo controlled food (fruit) challenges (DBPCFCs). In a multidisciplinary approach, as was the case in the EU-SAFE project (Hoffmann-Sommergruber, 2005), medical doctors and fruit geneticists identified the apple cultivar ‘Santana’ as low allergenic in the majority of patients allergic to Dutch apples (Kootstra et al., 2007). The other approach uses the application of advanced molecular breeding technologies, such as silencing of the specific allergen genes (Gilissen et al., 2006; Le et al 2006a) or targeting induced local lesions in genomes (TILLING).

The allergic potency of fruits is determined by several factors, including the geographic factor (i.e., the human population and its environmental allergen exposure), the fruit species and cultivar, the pre- and postharvest practices (i.e., disease and pest management, storage and transport condi-tions), and the manner of consumption (i.e., peeling, juiced, or processed). This became espe-cially clear in apple cultivars. In clinical investigations, similar to the SPT, apple cultivars showed


large differences in their allergenicity. Also, the apple allergen contents with regard to Mal d 1 and Mal d 3 showed great variation between cultivars (Zuidmeer et al., 2006; Sancho et al., 2008). Apple allergenicity was influenced by ripening stage and storage (Hsieh et al., 1995; Brenna et al. 2004, Bolhaar et al., 2005) and cultivation system (Zuidmeer et al., 2006; Matthes & Schmitz-Eiberger, 2009). Similarly, in the content of the main allergens of kiwifruit, the ratio of different proteins and even isoforms of the same allergens (Act c 2) appeared to change during the ripening process (Gavrovic-Jankulovic et al., 2005). And the white strawberry usually has a lower Fra a 1 content than the red ones (Alm et al., 2007). On the other hand, no significant difference in the allergenic potency during fruit ripening was found in mango (Paschke et al., 2001b), and no par-ticular differences in the allergenicity pattern were detected in six different cherry cultivars (Pravettoni et al., 2007).

Fruit Allergens

Fruit Allergen Identification and Classification

Fruit allergens are generally identified by western blotting using the extracted proteins from fruits and clinically confirmed sera from allergic patients to the specific fruit. Routine methods to obtain molecular information of the allergen include estimation of molecular weight of aller-gen proteins, N-terminal sequencing, and the design of degenerate primers to amplify the coding sequences on the cDNA templates. Currently, many expressed sequence tag (EST) data of puta-tive allergen genes are stored in the National Center for Biotechnology Information (NCBI) database for apple, peach, tomato, kiwi, grape and citrus fruits, and many of the proteins are further identified by two-dimensional and mass spectrography (Helsper et al., 2002; Reuter et al. 2005; Herndl et al., 2007). A variety of allergens from different fruits have been identified by means of experimental immunology and molecular biology, in particular, by protein and gene identification and sequencing (Breiteneder & Ebner, 2000). Major and minor allergens have been defined according to the percentage of positive western blot data from certain geographical and cultural populations.

In view of the accumulation of molecular data, an official systematic allergen nomenclature was published (King et al., 1995). Allergens are designated by the first three letters of the name of the genus of origin, followed by a space and the first one or two letters of the species name. These let-ters are usually followed by a space and an Arabic number according to the order in which the allergens or isoallergens were identified, wherein the same number is used to designate homologous allergens of related species. Additional letters can be added to distinguish different isoallergens and variants of more than 95% sequence identity to extend the current nomenclature for the purpose of precise gene identity and genetic analysis (Gao et al., 2005a; 2005c). The official allergen database can be found at www.allergen.org. New allergen databases are emerging and contain huge numbers of nucleotide and deduced amino acid sequences and their structures; these include Allergome (www.allergome.org/), AllFam (www.meduniwien.ac.at/allergens/allfam), and Structural Database of Allergenic Proteins (http://fermi.utmb.edu/SDAP). From these databases and the GenBank, one can find the information about the nucleotide and protein sequences of many allergens. By analyz-ing a large data set of the reported allergen protein sequences, researchers recently found common structures and properties of protein families over a wide range of plant species, genera, and even families (Breiteneder & Mills, 2005; Radauer et al., 2008), including fruit allergen protein families (Table 5.2). More details for the most important protein families are described herein.

Tabl

e 5.

2 M

ajor

fru

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en f

amili

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ed f

ruits

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d 8

Act

d 2

Act

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9A

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Act

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nas

com

osus

Ana

c 1

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c 2

Cit

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us la

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sC

it la

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Cit

rus

sine

nsis

Cit

s 3

Cit

s 2

Cit

rus

reti

cula

taC

it r

3C

itru

s li

mon

Cit

l 3C

ucum

is m

elo

Cuc

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Cuc

m 2

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gari

a ×

an

anas

saFr

a a

1Fr

a a

3Fr

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Lit

chi c

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Lit

c 1

Lyco

pers

icon

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cule

ntum

Lyc

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Lyc

e 1

Lyc

e G

luca

nase

*

Mal

us d

omes

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Mal

d 1

Mal

d 2

Mal

d 3

Mal

d 4

Man

gife

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Man

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Man

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Mus

a ac

umin

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Mus

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Mus

a 2

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Pers

ea a

mer

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s ar

men

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Pru

ar 1

Pru

ar 3

Pru

nus

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mPr

u av

1Pr

u av

2Pr

u av

3Pr

u av

4P

runu

s do

mes

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Pru

d 1*

Pru

d 3

Pru

d 4*

Pru

nus

dulc

isPr

u du

1*

Pru

du 2

*Pr

u du

8*

Pru

du 4

Pru

nus

pers

ica

Pru

p 1

Pru

p 2*

Pru

p 3

Pru

p 4

Pyr

us c

omm

unis

Pyr

c 1

Pyr

c 3

Pyr

c 4

Rub

us id

aeus

Rub

i 1

Rub

iVi

tis

vini

fera

Vit

v T

LP*

Vit

v 1

Vit

v 4*

Vit

v E

ndoc

hitin

ase*

Vit

v G

luca

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izip

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mau

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llerg

ens

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om th

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fici

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f In

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nion

of

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ties

(IU

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om th

e A

llFam

and

Alle

rgom

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taba

se (

adop

ted

from

Yan

g et

al.

2010

, Tab

le 1

).

110


● PR10: Pathogenesis Related family 10 proteins are small, acidic, intracellular proteins with molecular masses ranging from 15 to 18 kDa (Van Loon & Van Strien, 1999). These genes appear in duplicated regions in plants (Gao et al., 2005a; Liu & Ekramoddoullah, 2006). Expression of these genes is induced by stress, such as attack by plant pathogens, and occurs during apple ripening (Atkinson et al., 1996; Liu & Ekramoddoullah, 2006). Regarding the bio-logical function of PR10 proteins, it was recently found that Pru p 1 isoallergens in peach fruit have RNA hydrolysis and cytokine binding activities (Zubini et al., 2009).

● nsLTP: nonspecific Lipid Transfer Protein belongs to pathogenesis-related protein family 14. nsLTP was discovered about 35 years ago as a multigene family encoding 9kDa proteins (90–95 amino acids) that are distributed throughout the entire plant kingdom (Kader, 1996). The protein is stable because of four internal disulfide bonds. nsLTP lacks any specificity for fatty acids, phospholipids, or cutin monomers (Douliez et al., 2001). It can participate in plant defense reactions through antifungal and antibacterial activities. Prunus fruit nsLTPs were the first discovered as allergens in southern Europe and were recognized as a pan-allergen.

● Profilins are small (12–15 kDa) cytosolic proteins that are found in all eukaryotic cells. In 1991, birch pollen profilin was identified as a relevant allergen (Valenta et al., 1991). In the meantime, profilin cDNAs from numerous plant species have been cloned, and their deduced amino acids are typically 70 to 80% similar. They display striking protein features of con-served length (i.e., most consist of 131–134 amino acids), domains, and structure (Radauer & Hoffmann-Sommergruber, 2004). Large profilin multigene families can be grouped into two classes: those constitutively expressed in vegetative tissues and those expressed mainly in reproductive tissue. Multiple profilin isoforms can be expressed in individual tissues. The basic biological functions of profilins have been found in cell elongation, cell shape mainte-nance, and flowering (Ramachandran et al., 2000) and pollen tube growth (McKenna et al., 2004). Apart from pollen profilin allergy, many fruits contain profilins and their allergenic potency has been reported frequently as a result of cross-reactivity with birch Bet v 2 (see Table 5.2).

● Thaumatin like protein (TLP) is one of the major protein constituents of many mature edible fruits (Menu-Bouaouiche et al., 2003). It is homologous to an intensely sweet-tasting protein, isolated from the fruit of Thaumatococcus daniellii. Most TLP amino acid sequences have 16 conserved cysteines that form eight disulfide bonds contributing to the protein’s resistance to proteolysis and heat (Breiteneder, 2004). TLPs belong to the PR-5 family of pathogenesis-related proteins (Van Loon & Van Strien, 1999). Several researchers provided evidence that TLPs play a role in plant defense against pathogens (Ibeas et al., 2000; Venisse et al., 2002; Velazhahan & Muthurishnan, 2004). TLPs are now suggested to be a new class of pan-allergens in food as well as in pollen (Breiteneder, 2004), although their clinical relevance is not well confirmed. Apple Mal d 2 allergen (Krebitz et al., 2003) and kiwifruit TLP (Gavrovic-Jankulovic et al., 2008) are two examples.

● Chitinases are digestive enzymes that break down glycosidic bonds in chitin. Some plant chiti-nases are classified as pathogenesis related (PR) proteins (PR-3) that are induced after systemic acquired resistance induction. Chestnut, avocado, banana, and India date (Diaz-Perales et al., 1998; Sanchez-Monge et al., 1999; Lee et al., 2006) have this type of allergic protein.

● Cysteine proteases have a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad. Cysteine proteases are commonly encountered in fruits including papaya (Carica papaya), pineapple, and kiwifruit. The amount of protease tends to be higher when the fruit is unripe (Tamburrini et al., 2005).


Other allergenic plant fruit allergens include germin-like protein in citrus fruits (Cit s 1, Cit r 1) (Poltl et al., 2007), cucumisin, a subtilisin-like endopeptidase (Cuesta-Herranz et al., 2003), endo-β-1,3-glucanase of banana (Receveur-Brechot et al., 2006), actinidin in kiwifruits (Palacin et al., 2008), and a PR-1 protein in melon (Asensio et al., 2004).

Genomics of Putative Allergen Genes

As the information of the allergen-encoding mRNA sequences and fruit molecular linkage maps were available, localization of these allergen genes in established linkage maps were started in apple in 2001. For example in apple, genetic characterization and mapping was carried out for the currently known apple allergens: Mal d 1 (a PR-10 protein), Mal d 2 (a thaumatin-like protein, TLP, a PR-5 protein), Mal d 3 (nsLTP, a PR-14 protein), and Mal d 4 (profilin). The genes of the indi-vidual protein family members were mapped on the reference map developed from the cross ‘Prima’ × ‘Fiesta’ (Maliepaard et al., 1998) and two alternative maps from ‘Fiesta’ × ‘Discovery’ and ‘Jonathan’ × ‘Prima’, which were all constructed by Plant Research International, WUR. The basic strategies employed here included four consecutive processes: (1) Search for the apple allergen sequences in the DNA databases (GenBank/EMBL) or other published databases and analyze these sequences to design polymerase chain reaction (PCR) primers; (2) clone and sequence the target genes on gDNA templates of ‘Prima’ and ‘Fiesta’ by PCR; (3) analyze the polymorphisms of allelic sequences from the two parents and design primers to create markers; (4) test these markers on the mapping population to obtain segregating data, which enable mapping of these new markers onto the already known linkage maps. Figure 5.1 outlines this process.

Linkage Mapping of Putative Allergen Genes in Representative Rosaceae Fruit CropsThe putative fruit allergen genes of apple were first mapped on linkage maps in 2005 (Gao et al., 2005a; 2005b; 2005c) and updated by adding two Mal d 4 isoallergen/variant genes in 2008 (Chen et al., 2008). Mal d 1 genomic sequences appeared to represent 18 Mal d 1 genes of which 16 genes

Figure 5.1 The process of genomic PCR cloning and linkage mapping of apple and peach allergen genes. EMBL, European Molecular Biology Laboratory; PCR, polymerase chain reaction. Adapted from Gao, Z.S. (2005) Localization of candidate aller-gen genes on the apple (Malus domestica) genome and their putative allergenicity. PhD diss., Wageningen University.

PCR

Primer designPolymorphisms

Segregatin

Allergen sequencescDNA/gDNA

DNA DatabasesGenBank/EMBL

PCROptimization

Cloning onvector

Segregatingpopulations

Position onlinkage groups

AmplificationPfu & Taq

Sequencing

Genomewalking


were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG16. One gene was mapped on LG 6, and one remained unmapped. According to DNA sequence identity and intron sizes, these 18 genes could be subdivided into four subfamilies. Subfamilies I to III had an intron of different size that was subfamily- and gene- specific. Subfamily IV included 11 intronless genes. The deduced amino acid sequence identity of Mal d 1 varied from 65 to 81% among subfamilies, from 82 to 100% among genes within a subfamily, and from 97.5 to 100% among alleles of one gene. Two copies of the Mal d 2 (TLP) gene with more than 95% sequence identity were identified and mapped at identical position on LG9. Other putative TLP sequences in apple were found in fruit ESTs, which have not yet been mapped. Two apple Mal d 3 (LTP) genes, obtained from a pair of cloning primers based on a reference sequence, have been named Mal d 3.01 and Mal d 3.02. These two genes are highly conserved in the coding region; thus, a genome walking approach to obtain upstream sequences was applied and employed in two mapping populations. Finally these two genes were mapped in LG4 and LG12 on homoeologous segments (Figure 5.2). Three different Mal d 4 isoallergens (Mal d 4.01, −02, and −03) corresponding to genomic sequences for each of these three isoallergens led to the identification of six Mal d 4 genes (Mal d 4.01A and −B, Mal d 4.02A and −B and Mal d 4.03A and −B). Their complete genomic DNA sequences varied because of its two introns of different sizes but three conserved exons of 123, 138, and 135 nucleotide lengths. Mal d 4.01 was located on LG9. Mal d 4.02A and Mal d 4.03A were located on LG2 and LG8, respectively, Mal d 4.02B and Mal d 4.03B were both mapped on LG15.

Similar genomic cloning was done in a hybrid (F1) that gave rise to the well-known TxE reference

map for Prunus species (Howad et al., 2005), in which allele specific markers were developed for two patents of T (Texas almond) and E (Earlygold peach). The bin-mapping approach was employed that uses only eight plants (Howad et al., 2005). Eighteen putative peach allergen genes of four families similar to apple were mapped later on five of the TxE reference map (Chen et al., 2008). Apple and peach putative allergen genes demonstrate the expected syntenic and colinear regions (see Figure 5.2): the Pru p 1 gene cluster on LG1 that corresponds to regions with two clusters of homologous Mal d 1genes on apple LG13 and LG16, Pru p/du 4.01 on the lower part of G1 and Mal d 4.03 on LG8, and Pru p 2.01A/B on G3 and Mal d 2.01A/B on LG9. Three more syntenic regions were detected in this study: Pru p 4.02 on G7 and the region of LG2 where Mal d 4.02 maps, Pru p 3.01 to .03 on G6 cor-responding to Mal d 3.01 on LG12 and Mal d 3.02 on LG4; and Pru p 4.01 in the region of LG8, is syntenic with the upper part of LG15 where Mal d 4.02B and Mal d 4.03B mapped.

Many fruit linkage maps are available and updated in recent years, such as apple (Silfverberg-Dilworth et al., 2006), kiwi (Fraser et al., 2009), pear (Yamamoto et al., 2007), raspberry (Stafne et al., 2005), strawberry (Sargent et al., 2009), and loquat (Eriobotrya japonica [Thunb.] Lindl.; Gisbert et al., 2009). The interested allergen genes can be located in the same way. Certainly, recent advances in whole genome sequencing of fruits will provide much more sequence information and physical map position.

Comparative Allergen Genes among Rosaceae MapsThe major temperate fruit crops are apple, pear, peach, cherry, plum, apricot, quince (Cydoniaoblonga), and strawberry, which all belong to the Rosaceae family. Allergen gene mapping results will be comparable in the various subfamilies. Apple, pear, and loquat belong to the Maloideae subfamily and have a similar genome structure with comparable LGs. So the position of apple and peach allergen genes can be used as references to closely related fruits in the Maloideae and Prudeae subfamily. The peach genome sequence was available from 2010 April. Previously filed genomic sequences can be BLASTed for physical position and neighbor regions, which would be informa-tive regarding gene expression and gene diversity surveys among germplasms.


Expression of Putative Allergen Genes

Molecular cloning on a genomic template can result in many homologous putative isoallergen sequences, but their expression profile can be diverse. For a specific tissue at a certain time or devel-opmental stage, several proteins may simultaneously be present. Their individual quantity and

Figure 5.2 Linkage map positions of the four allergen gene families in Prunus (T × E) reference map and alignment with those in apple (Malus domestica) reference map. Relevant linkage map information was derived from Prunus T × E updated maps. Linkage maps taken from Maliepaard et al. (1998) TAG, 97, 60–73; Gao et al. (2005) TAG, 110, 479–491; 111, 171–183 and 1087–1097. An initial reference for alignment is based on Dirlewanger et al. (2004) PNAS, 101, 9891–9896. A bar indicates map position range of a bin. Homologous allergen genes and common RFLP probes are underlined to show their synteny. Pru p, peach; Pru du, almond; RFLP, restriction fragment length polymorphism. Adapted from Chen, L., Zhang, S.M., Illa, E. et al. (2008) Genomic characterization of puta-tive allergen genes in peach/almond and their synteny with apple. BMC Genomics, 9, 543. For color detail, please see color plate.

G1-Prunus

MC041b2.0Mal d 1.06A-C10.6Mal d 1.02/04/07/0811.0CH05a0411.4Mal d 1.0912.5

MC001b32.7

Ma47.0

LG16-AppleCH03h030.0

MC041a8.0

Mal d 1.01/03A-C22.3Mal d 1.03D-F23.1

MC001a42.8

MC001c73.8

LG13-Apple

LY21-E12.0

AAT-213.9

MC022-H23.5

Mal d 4.03A24.0

OPAC-11-078046.0

LG8-Apple

LY37a0.0Mal d 4.03B0.5

Mal d 4.02B31.0

110.0

LG15-Apple

Pru p/du 1.02-05

Pru p/du 2.04

Pru p/du 4.01

CH02C09

AA08B0.0

pchcms413.6

MC04436.0MC00137.5LY2143.7TSA249.8Lap-151.6LY3754.2MC02255.2

FG8A73.1

AG36B87.0

G3-Prunus

MC115b0.0Mal d 2.01A/B6.5MC038a8.8

Mal d 4.01A/B22.0

MC007a48.1

E36M51-19869.1

LG9-Apple

MC115a0.0

MC03811.0

MC201a34.0

LG17-Apple

Pru p/du 2.01A/B

MC1153.3

AB10A24.8MC038A28.4MC00733.0UPD96-00836.4

AF008B48.4

MC032b2.2

CH01g1219.3

Mal d 3.0137.8

E34M57-8849.5

LG12-AppleMC0130.0

Mal d 3.0242.5

SOD-351.2

LG4-Apple

G6-Prunus

Pru p/du 3.01Pru p/du 3.02Pru p/du 3.03

AB06B

A

B

D

E

C

0.0

UPD98-41272.0Pgl174.3

CPPCT02183.7

MC064b0.0

MC116b12.1

MC003e50.2

Mal d 4.02A64.0

71.1

LG2-Apple LG7-Apple

Pru p/du 4.02

Pru p/du 2.02

E34M 48-171

MC064a1.0

MC116a13.0

MS06c0332.0

G7-Prunus

AA12C0.0

PC1224.7

MC003A46.8PS8e849.0

FG4256.1

PS5c370.6

pADH32a0.0

LY29b14.0

MC028b65.0

pAP4a90.0

LG5-Apple

pADH32b9.0

MC024a23.0LY29a27.0

MC02867.0

pAP4b88.0

LG10-AppleG8-Prunus

Pru p/du 2.03

CPSCT0180.0

LY2920.8

AG4A30.1

FG3740.9

PC03659.7

Pru p/du 1.06A-C


allergenicity can, however, be different. The amount of mRNA present in the tissue may not linearly relate to the protein quantity. In this regard, apple and peach are good examples to address this issue for the various gene family representatives. Gene expression using RT-PCR in apple and peach (Botton et al., 2008; 2009; Yang et al., unpublished), comprehensive immunology tests for apple (Herndl et al., 2007), and synergistic combination of molecular biology and proteomics in cherry (Reuter et al., 2005) have been carried out to identify novel allergenic proteins. In apple fruit, a recent comprehensive gene expression study on four classes of putative allergens has gained insight about the genetic and environmental factors affecting the allergenic potential (Botton et al., 2008; 2009b). They showed that transcripts of some allergen gene members were largely accumu-lated in ripe fruit, whereas others were undetectable.

As presented in the previous section, 18 putative apple Mal d 1 genes were identified at genomic level. Until now, mRNA expression for five Mal d 1 allergen genes was observed in mature fruit through real-time PCR and EST sequence analysis. These sequences represented two genes (Mal d 1.01 and −03E) on LG 13 and three genes (Mal d 1.02, −06A, and −06B) on LG 16. Regarding other Mal d 1 genes, mRNA based EST sequences of Mal d 1.04 were only found in mature leaves (Botton et al., 2008; Beuning et al., 2004; Gao et al., 2008). To confirm evidence at the protein level, only three Mal d 1 isoallergen proteins were found to be present in apple fruit, the majority being Mal d 1.02 (Mal d 1b) (Helsper et al. 2002; Herndl et al., 2007) and to a minor extent the isoaller-gens Mal d 1.06 (Helsper et al., 2002; Beuning et al., 2004; Gao et al., 2005a) and Mal d 1.03 (Zheng et al., 2007). Interestingly, the majority of the Mal d 1 isoallergens in the apple fruit are encoded by genes located on LG16.

In peach, as tested by means of real-time PCR, the most abundantly expressed member is Pru p 1.01, followed by Pru p 1.06 (Chen et al., 2008). These two members’ expression levels increased with progressing fruit maturation and ripening. Differential expression of LTP1 (Pru p 3.01) and LTP2 (Pru p 3.02) was observed (Botton et al., 2002; 2009a). Pru p 3.01 and Mal d 3.01 were domi-nantly presented in peach (Zuidmeer et al., 2005) and apple (Sancho et al., 2005) peels, respectively. Tomato LTPs are present as different isoforms in pulp, peel and seeds (Pravettoni et al., 2009). The common habit of consuming tomato fruits entirely makes this tissue-related allergenicity evaluation complex.

As for TLP genes in apple, the highest expression was found for Mal d 2.01, followed by Mal d 2.02 and Mal 2.03, respectively (Botton et al., 2008). A similar expression pattern was found in peach: Pru p 2.01 and Pru p 2.04 were expressed at a higher level than Pru p 2.02 and Pru p 2.03 (Yang et al., unpublished).

Selection of Hypoallergenic Variety

Allergenicity Evaluation of Different Cultivars

From patients’ experience it is known that the severity of allergic reactions to apple was not only related to the specific sensitivity of the individual, but also largely depended on the apple cultivar. For instance, Mal d 1 from the cultivar ‘Golden Delicious’ was found highly reactive to specific IgE anti-bodies from allergic patients’ sera, whereas Mal d 1 from the cultivar ‘Gloster’ generally showed much less reactivity (Vieths et al., 1994). New experiments, SPTs with 21 different apple cultivars and confirmations for specific cultivars in DBPCFCs and oral challenges of whole apples revealed a wide range of allergenic reactivity from very high to very low (Bolhaar et al., 2005). A new cultivar ‘Santana’ was identified as hypo-allergenic for 75% of the patients with a mild apple Mal d 1 allergy and has recently been marketed as “suited for individuals with mild apple allergy” (Kootstra et al., 2007).


A ranking of 21 cultivars was made on the basis of prick-to-prick tests in nine patients, classified as low, intermediate, and high allergenic, with a significant difference between low and high aller-genicity at the P < 0.001 level (Bolhaar et al., 2005). Figure 5.3 shows the skin prick test procedure for allergenicity assessment of different apple accessions. To test the allergenicity of fruits of apple, peach/nectarine, and kiwifruit, in vitro immunoassays were also applied. Using the major apple allergen Mal d 1 as a model, Zuidmeer et al. (2006) found the reasons why different immunoassays for assessing allergenicity at the molecular level of apple cultivars resulted in conflicting data: variable and poorly controllable major allergen modification in both extracts and standards ham-pered the accurate allergenicity assessment and comparability. The Mal d 1 protein proved to be labile under different experimental conditions.

The differences in allergenicity among apple cultivars raised a question: why does it occur and what does it cause? Allergenicity may depend on the total amount of Mal d 1 proteins, as suggested by Son et al. (1999). However, new evidence did not support this hypothesis because a linear res-ponse between Mal d 1 protein content and allergenicity estimations were lacking and even contro-versial (Zuidmeer et al., 2006). Mal d 1 content measurement on apple cultivars in Germany was varied largely from 1.3 to 20.1 μg/gFW at the time of harvest and then increased during storage (Matthes & Schmitz-Eiberger, 2009). Five cultivars from the Netherlands and Italy showed quite different contents (about 100-fold) for three cultivars, ranging from 6 to 455 μg/g (Zuidmeer et al., 2006). These differences in Mal d 1 content could not readily be associated with the differences in SPT responses. For example, Mal d 1 in ‘Golden Delicious’ (grown in The Netherlands) was 135μg/g determined by Bet v 1-ELISA, which is much lower than 455μg/g of ‘Jonathan’, whereas the SPT showed the former has much higher score (Bolhaar et al., 2005).

Figure 5.3 SPT of apple cultivars and accessions. A, Tested sample; B, A doctor performs the SPT; C, The reaction wheal 20 min later; D, Marked reaction area of different cultivars and accessions. SPT, skin prick test. This study was reviewed and approved by the Ethics Committee of the University Medical Center Utrecht under document number 01–050. All patients provided written informed consent before enrolment in the study. For color detail, please see color Plate.

A

B

C

D


Therefore, it becomes more and more clear that also qualitative characteristics of the Mal d 1 proteins are involved, as can be argued from the differences in binding capacity of two protein vari-ants of Mal d 1 to birch pollen-specific IgE (Ma et al., 2006).

The major apple allergen Mal d 1 is labile upon disruption of fruit tissue and heat treatment. The prick-to-prick SPT method with fresh fruit is, therefore, more reliable in comparison to the application of fruit extracts (Bolhaar et al., 2004). To assess the allergenicity of different apple cultivars, a large number of patients and repeated experiments are necessary. When the allergenicity of individual apple cultivars has been assessed, then the genetic and proteomic analyses of different genotypes will reveal the genes and alleles involved in low allergenic cultivars. For a given apple cultivar, not all the patients have the same SPT response. Separating the patients according to their SPT reaction is necessary for proper genetic analysis.

The contents of Pru p 1 and Pru p 3 of different peach and nectarine cultivars showed large variation by sandwich ELISA (Ahrazem et al., 2007). Pru p 1 in pulp ranged from nondetectable (ND) to 0.68 μg/gFW, in peel from ND to 1.76 μg/gFW. Pru p 3 in pulp ranged from ND to 2.1 μg/gFW, in peel from 53.6-338.45 μg/gFW. Most U.S. cultivars showed higher levels of both allergens than Spanish cultivars did. Clinical evaluation of allergenicity for different peach cultivars is needed for association analysis to identify the low allergenic alleles.

IgE immunobloting showed remarkably different protein profiles and IgE binding pattern for three kiwi species: green kiwi (Actinindia deliciosa), golden kiwi (A. chinensis) and hardy (A. arguta)(Chen et al., 2006; Lucas et al., 2003). For a specific patient group, it might be possible to identify and select low allergenic accessions among diverse kiwi germplasm collections present in China.

Diagnostic tests for food allergy frequently resulted in poor sensitivity and specificity (van Ree, 2002). Therefore, the DBPCFC is generally regarded as the golden standard.

Table 5.3 Associating SPT responses with putative protein variants coded by the Mal d1.04and Mal d 1.06A genes of 14 apple cultivars.

Cultivars1 SPT-response2 Mal d 1.04 Mal d 1.06A

‘Priscilla’ (PS) 30 ps1 ps2 02 02‘Santana’ (ST) 35 ps1 ps1 02 02‘Jonathan’ (JO) 48 ps1 04 01 02‘Ecolette’ 51 ps2 04 01 02‘Prima’ (PM) 61 04 04 01 02‘Elstar’ (ES) 61 ps2 04 01 02‘Fuji’ (FJ) 61 ps1 ps2 03 02‘Gala’ 64 ps1/ps2 04 01 02‘Elise’ 67 ps2 04 01 02‘Bellida’ 72 04 04 01 01‘Fiesta’ (FS) 83 ps1 04 01 01‘Delblush’ 87 ps2 04 01 03‘Pinova’ 89 04 ps2 01 03‘Golden Delicious’ (GD) 100 ps2 04 01 03

Adapted from Gao, Z.S., van de Weg, E.W., Matos, C.I. et al. (2008) Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity. BMCPlant Biology, 8, 116.1 Cultivars in bold were sequenced for the intron containing Mal d 1 genes2 In percentage relative to ‘Golden Delicious’.SPT, skin prick test.


Allelic Diversity Survey and Association Analysis on Apple Allergenicity

After completing the genomic characterization of the putative apple allergen genes, preliminary genetic analysis on specific alleles controlling the low Mal d 1 allergenicity was investigated (Gao et al., 2008). The allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of 14 apple cultivars by sequencing or, indirectly by molecular marker tests. Comparison of Mal d 1allelic composition between the high-allergenic cultivar ‘Golden Delicious’ and the low- allergenic cultivars ‘Santana’ and ‘Priscilla’, which are linked in pedigree, showed an association between the presence of specific alleles (Mal d 1.04 and −1.06A genes, both located on LG16) and low allergenic-ity (Table 5.3). This association was confirmed in 10 other cultivars. Furthermore, allele dosage effects are found to be relevant for Mal d 1.06A. A simple SSR marker of 154 bp developed from Mal 1.06Acan be used now to screen germplasm and selections for potential hypoallergenic source. Our findings indicate the need to reconsider the relevance of merely assessing total amounts of Mal d 1 protein in allergenicity research and diagnostic tests and warrant further research on the association of specific Mal d 1 isoforms and allergenicity among a larger group of cultivars and allergy sufferers.

Allelic diversity of the key allergen genes in other fruit is still scarce. In tomato, the ripening inhibitor (rin) mutant showed reduced allergenicity related to the proteins β-fructofuranosidase and polygalactu-ronase 2A, which are cross-reactive with Japanese cedar pollen allergens. Japan tomato allergic patients showed lower level of reactivity to the extract from this hybrid mutant (Kitagawa et al., 2006).

Pru p 1 and Pru p 3 are the main allergens in peach. The encoding genes are located in clusters on two linkage groups, LG1 and LG6. Based on their bin map positions, it was expected to use SSR markers tightly linked or flanking these two major allergen genes for a diversity survey of a large number of genotypes (Chen et al., 2008). However, a preliminary cloning and sequencing experiment of Pru p 1.01 and Pru p 3.01 in the encoding region for a number of peach cultivars did not show any polymorphism probably because of the low genetic diversity between the chosen peach varieties.

Genetic Modification

Genetic modification (GM), as a supplement to plant breeding, has rapidly expanded the horizon of crop improvement and offers a relatively quick way to introduce novel traits into crops. Major GM crops are soybean (77% of the total world production in 2009 was GM), cotton (49%), maize (26%), rapeseed (21%), and sugarbeet (9%), and the list of crops is growing. In 2009, the total world area planted with GM crops was about 134 million hectares. Major GM traits are input traits such herbi-cide, pest, and disease resistances. Upcoming traits are drought and salt resistance, and output traits such as increased oil production, altered plant composition, synthesis of pharmaceutical proteins, and reduced allergenicity (www.gmo-compass.com; Singh & Bhalla, 2008). Regarding the latter trait, reduced allergenicity, successful approaches have been reported from the food crops rice (Tada et al., 1996), soybean (Herman et al., 2003), and peanut (Dodo et al., 2008), and in the vegetable/fruit crops tomato (Le et al., 2006a; 2006b; Lorenz et al., 2006), and apple (Gilissen et al., 2005), which are all based on gene silencing (antisense or RNA-interference [RNAi]) technologies.

RNAi

Here, we will focus on the RNAi GM strategies applied for allergenicity reduction in apple and tomato. Because most of the allergens in these fruits belong to multigene families, the RNAi


approach is especially suitable to silence the posttranscriptional expression of all gene family mem-bers. Such simultaneous inhibition of all gene family members can be achieved by selecting a highly homologous gene region from the coding sequences. The method is especially efficient when the gene construct consists of an inverted repeat of a fragment of the targeted gene sequence separated by an intron. Such a construct results in the formation of a so-called “intron-spliced hair-pin RNA”.

AppleThe most relevant allergens in apple are Mal d 1 (a pathogenesis-related, PR10, protein, and major allergen in apple especially in North and Middle Europe and especially cross-reactive to the birch Bet v 1 allergen), Mal d 2 (a thaumatin-like protein), Mal d 3 (an nsLTP, similar to Lyc e 3, espe-cially relevant as allergen in Southern-European countries), and Mal d 4 (a profilin, homologous to Lyc e 1, with high-sensitization capacity but low or no clinical relevance (Wensing et al., 2002). Mal d 1 is the major allergen. Approximately 70% of patients allergic to birch pollen have been reported to have OAS to apple as a consequence of cross-reactive IgE antibodies.

In apple, Mal d 1 genes contain a single intron or are intron-free. For the purpose of RNAi, an intron-containing gene was used to build up the construct by linking a first gene fragment (containing exon 1 plus the intron plus a short sequence of exon 2, in all 516 bp long) to the second gene fragment (only containing exon 1 and 276 bp long) in the reversed and upside-down position (Figure 5.4). The hairpin construct was cloned in the pRAP expression vector cassette containing the 35S cauliflower mosaic promoter and cloned into the binary expression vector pBINPLUS, which was introduced in Agrobacterium tumefaciens for transformation. For transformation, in vitro leaf explants of the apple cultivar ‘Elstar’ were used (Gilissen et al., 2005). This hairpin construct will result in a double-stranded RNA (dsRNA). The method of RNAi operates by sequence-specific dsRNA degradation through the Dicer nuclear enzyme complex (Ketting et al., 2001). Broken-down RNA products of approximately 25 nucleotides will act as guide- or short-interference (si) RNA that will recognize and target homologous sequences of the naturally formed mRNA (i.e., the Mal d 1 RNA) for further

Figure 5.4 The construct for gene silencing was built up by linkage of fragment 1 (obtained by polymerase chain reaction using primer 1 [All1HpaI; p1] and primer 2 [All2SmaI; p2]) and fragment 2 (from primer 1 [All1HpaI; p1] and primer 3 [All3SmaI;[p3]) at the SmaI site and insertion into the expression cassette pRAP 37. UTR, untranslated region. Gilissen, L., Bolhaar, S.T.H., Matos, C.I. et al. (2005) Silencing the major apple allergen Mal d 1 by using the RNA interference approach. Journal of Allergy and Clinical Immunology, 115(2), 364–369.

Exon 1

Exon 1 Exon 15‘UTR

Ex 2

Exon 2

Exon 2

Fragment 1

Fragment 2SmaI

5’UTR

5’UTR

AscI

pRAP 37 p 35S ALMV Tnos

PacI

Intron

SmaI

SmaI SstI K pnI SmaI Bg1II

HpaIIntron

Intron

SmaIHairpin construct

HpaI

HpaI

HpaI

p1

p1

p2

p3

Exon 1


destruction. In the end, no intact natural mRNA (Mal d 1 mRNA) will be present to pass on to the ribosomes, and thus no protein (Mal d 1 allergen) will be synthesized.

The cultivars ‘Priscilla’ and its progeny cultivar Santana are known as hypoallergenic. These cul-tivars are selected from the large pool of currently marketed cultivars. However, many cultivars on the market are highly allergenic, such as ‘Golden Delicious’, and to a lesser extent, the cultivar ‘Elstar’ (Bolhaar et al., 2005). Apple is the most extensively produced fruit in Europe, and China is the world’s major apple producer. The production of apple plants with a significant reduction of the overall expression of Mal d 1 from an economically successful cultivar seems an attractive, time-saving (by a factor 2), and simpler alternative because its effect is independent of the individual Mald 1 genes and antibody specificities. Indeed, it has been successfully demonstrated by SPT, immu-noblotting, and in greenhouse-grown flowering plants, by RT-PCR that Mal d 1 expression can be silenced up to a 10,000-fold lower than the wild-type plants (Gilissen et al., 2005; Krath et al., 2009). The first fruits obtained were given a preliminary food challenge test and showed reduced allergenic-ity. However, this result still needs to be confirmed in larger challenge experiments with more fruits, more apple allergic volunteers, and a control group. Later on, when more detailed knowledge is available on the genes and alleles that are specifically responsible for high allergenicity, the strategy of RNAi can be directed to these genes, leaving the other Mal d 1 genes functionally intact.

TomatoThe most relevant allergens in the tomato fruit are Lyc e 1 (profilin), Lyc e 2 (a glycosylated β-fructofuranosidase with potential allergenicity through its glucan structure), and Lyc e 3 (a nsLTP, known as a severe allergen in many fruits). Tomato allergy is especially prevalent in the European Mediterranean region, where this food takes a prominent position in the daily diet.

Lyc e 1 has been identified as a minor allergen. This allergen shows a strong cross-reactivity with profilin from birch pollen (Bet v 2) and has allergenic potency through its capability to trigger the release of inflammatory mediators from human basophil cells in vitro and may therefore be consid-ered as a major contributor to clinical symptoms in tomato allergic patients (Le et al., 2006b). Application of RNAi (based on a 35S promoter-controlled construct built up from a sense 300 bp cDNA fragment linked to an intron and to a second, in antisense, gene fragment according to the Gateway technology) resulted in transgenic tomato plants strongly reduced in Lyc e 1 (profilin). Because profilin has an essential function in cell performance, the profilin-silenced plants were characterized by a dwarf phenotype and a delayed flowering time. ELISA inhibition assays revealed a 10-fold reduction in Lyc e 1 accumulation in transgenic tomato fruits. Despite the negative effects on plant development, transgenic tomato fruits could be obtained and used for SPTs, resulting in marked reduction (up to 100%) of the prick response, especially in patients who were monosensi-tized, whereas the effect was much lower in the patients who were polysensitized. These latter patients have specific IgE to several tomato allergens that collectively can contribute to the prick response and to clinical symptoms in general (Le et al., 2006b). The results demonstrate that an apparent 10-fold reduction in Lyc e 1 in tomato fruits was already sufficient to reach below the threshold level for inducing SPT responses in most of the patients who were monosensitized.

Lyc e 3 is a 9 kD nsLTP protein belonging to a large family of small hydrophyllic proteins that can enhance nonspecific intermembrane lipid transfer in plants. nsLTPs have been identified as potent allergens not only in fruits of the Rosaceae family such as apple, peach, cherry, and plum, but also in a wide variety of other fruits and vegetables such as pepper (Capsicum spp.), eggplant (Solanum melongena L.), potato (Solanum tuberosum), and tomato. Thus, they can be considered a pan-allergen. These proteins are highly conserved and cystein rich. From tomato fruits, two isoforms were detected as coding sequences. To silence both these LTP genes in tomato, the entire coding


regions have been cloned in sense and antisense orientation, separated by a potato oxidase gene intron. The so-obtained “hairpin cassette” was controlled by the 35S promoter and Agrobacterium-mediated transformed into tomato (Le et al., 2006a). Because there appeared to be high sequence similarity between both genes, their silencing could be achieved by introducing the complete coding region of either gene in sense plus antisense orientation. From Western blot analysis, only a 0.5% accumulation in the fruit peel of the transgenic fruits was found as compared to the wild-type fruits. Histamine release of human basophil cells challenged with tomato fruit extracts was much lower than in the challenge with wild-type fruit extracts. In contrast to the Lyc e 1-silenced plants, the Lyc e 3-silenced plants were phenotypically indistinguishable from the wild-type plants including the number and size of the fruits. It is to be expected that these Lyc e 1-silenced tomato fruits will have a strong effect on this type of allergy indicating the power of the RNAi technology to introduce hypoallergenicity in tomato fruit.

Profilin is an essential protein to all the cells of eukaryotic organisms. Therefore, only those transformants with an incomplete silencing pattern of Lyc e 1 are able to survive, be it with a signifi-cant penalty for its growth (dwarfism). Especially in this case, it is of high importance to know exactly which proteins of the profilin family carry the T-cell and B-cell epitopes. Genomics approaches will reveal the total number of profilin sequences in the tomato genome, as similarly carried out for the apple Mal d 4 gene family (Gao et al., 2005b; Chen et al., 2008). In addition, the tertiary protein structure with the amino acid residue regions (epitopes) most likely involved in IgE binding might be elucidated. This knowledge should be applied to make epitope-specific RNAi constructs aiming at the silencing of only those gene members that carry these epitopes, leaving the other family members biologically functional. This might result in normal plants that can produce hypoallergenic fruits for further market introduction.

Other Strategies for Reducing Allergenicity

Recently, a different strategy has been developed, based on the recombination activity of zinc finger nucleases (ZFN) that might be exploited to make specific exchanges at the level of individual base pairs (Kumar et al., 2006). This strategy will leave intact the structure of the protein while only inactivating the allergenic epitope. Another advantage of this ZFN technology is that it can be applied in such a way that no transgenic DNA will be left in the transgenic plant and thus will be functional as a mutagen. This will be helpful considerably in changing consumer’s acceptability of this type of GM in food crops.

TILLING was developed to take advantage of newly obtained DNA sequence information and to investigate the function of specific genes by single nucleotide polymorphism (SNP) introduction and phenotype analysis. This technique shows good promise as a non-GM tool to improve domes-ticated crops by introducing and identifying novel genetic variation in genes that affect key traits, such as allergenicity. TILLING techniques mutate targeted site to reduce allergenicity but keep their natural physiological functions. Many allergen genes in a species are member of more or less extended gene families (Gao, 2005). If the number of homologous allergen genes is limited to a few and well-characterized genes, the effect of TILLING as a result of novel base pair changes due to treatment with a chemical mutagen can be easily detected with molecular biological methods, like PCR for SNP discovery (Slade & Knauf, 2005) and 454-sequencing. So far, TILLING has not yet been applied to reduce the allergenicity of fruit plants. An example of application is known from wheat to reduce the toxicity of gluten genes to make safer food products for individuals with celiac disease, but results are not yet published.


Consumer’s Attitudes toward GM

Consumer’s preferences regarding the development of GM hypoallergenic food products are impor-tant because this will influence acceptance of such products. Current questionnaire-based studies, guiding the introduction of the first (conventionally produced and organically grown) hypoaller-genic apple variety (Schenk et al., 2008), examined the influence of “perceived benefits” on the acceptance of GM foods. A two-dimensional attitude structure toward GM foods was found in which “perceived benefit” and “perceived risk” were influential in determining consumer’s accept-ance. Differences in acceptance of GM hypoallergenic products between allergic and nonallergic individuals were mainly explained by the differences in perceived benefits, whereas the perceived risks remained constant. As long as the risks are not as large as to be completely intolerable, accept-ance of GM food products will be driven by perceptions of personal benefit. Acceptance of hypoal-lergenic GM apples was found to be quite high among consumers allergic to apples, which confirms the strong impact of the “personal benefits”. Nonallergic consumers consider hypoallergenic GM foods especially to have a “social benefit” of which a (possibly future) GM hypoallergenic apple would be a good example.

References

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breeding for fruit quality (jenks/breeding) || breeding of hypoallergenic fruits

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