Cloning and expression of the major allergen genes in apple fruit By G. PAGLIARANI * , R. PARIS, S.TARTARINI and S. SANSAVINI Department of Fruit Tree and Woody Plant Sciences,University of Bologna,Viale Fanin 46, 40127 Bologna, Italy (e-mail: gpagliarani@agrsci.unibo.it) (Accepted 1 September 2009) SUMMARY Apple, the most common fruit in European and North American diets, can cause allergic reactions in susceptible individuals, and genes for four families of apple allergens have been identified to date: Mal d 1, Mal d 2, Mal d 3, and Mal d 4. Our remit was to evaluate the effects of genotype, tissue, and stage of fruit development on the expression of these allergen genes and, hence, on the potential allergenicity of apple fruit.Transcript levels were investigated in fruit of the apple cultivars, ‘Gala’ and ‘Florina’, using quantitative Real-Time PCR. The resulting patterns of allergen gene expression differed, with Mal d 1 and Mal d 2 being the most highly expressed in the skin and flesh of ripe fruit, respectively. Overall, ‘Florina’ fruit showed higher levels of expression than ‘Gala’ fruit for all allergens tested. H igh fruit consumption is reported to be beneficial to human health (Boyer and Liu, 2004). However, like other Rosaceae fruit species, apples can cause severe allergic reactions. The four major gene families of apple allergens reported to date are included in the official list of the World Health Organisation’s allergen nomenclature (http://www.allergen.org): Mal d 1, Mal d 2, Mal d 3 and Mal d 4. Two distinct patterns of apple allergy have been described across Europe. In Northern and Central areas, apple allergy is mainly due to an IgE-mediated cross-reaction between the major birch-pollen allergen, Bet v 1 and the highly homologous apple allergen, Mal d 1 (Son et al., 1999). This class of food allergy is frequent in patients sensitised to birch pollen and provokes mild local symptoms. In contrast, allergies to apple in Mediterranean areas are frequently found in patients without pollen sensitisation (Fernandez-Rivas et al., 2006). This form of allergy can provoke more severe symptoms, and Mal d 3 is the allergen mainly involved (Diaz-Perales et al., 2002). Mal d 1 is a multigene family containing 18 different loci mainly clustered on the homeologous linkage groups (LG) 13 and 16. Four different sub-families (I – IV) have been described in relation to the presence and length of the intron. Comparisons of Mal d 1 gene coding sequences have revealed different levels of identity: 71 – 83% between sub-families; 86 - 98% within a sub- family; and 98 - 100% between alleles of a single gene (Gao et al., 2005a). Mal d 1 genes code for a 17 – 18 kDa protein consisting of 158 – 159 amino acids (aa), classified as a pathogenesis-related (PR) protein 10 (van Loon et al., 2006). High levels of expression of Mal d 1 have been found in ripe apple fruit and in mature leaves (Pühringer et al., 2003). While Mal d 2 was the second apple allergen to be identified (Krebitz et al., 2003), its relevance to allergenicity is still unclear. It is an apoplastic, 31 kDa protein containing 246 aa encoded by 1,119 – 1,121 nucleotides (nt) organised in two exons (61 nt and 680 nt) and one intron (378 – 380 nt). Gao et al. (2005b) assumed that the intron size was locus-specific, and named the two loci Mal d 2.01A and Mal d 2.01B, respectively, and both were mapped on LG 9. The N-terminus of the mature protein is about 50% identical to the superfamily of thaumatin-like proteins (TLPs), also known as PR-5 proteins, with antifungal activity (Kebitz et al., 2003). TLPs contain 16 conserved cysteine residues that form eight disulphide bonds which are essential for the overall folding of the proteins, and possibly for their anti-fungal and allergenic potential (van Loon et al., 2006). Mal d 3 is a non-specific lipid transfer protein (nsLTP) belonging to the PR-14 protein family. It is characterised by having resistance to pepsin hydrolysis and thermal denaturation (van Loon et al., 2006). Gao et al. (2005c) described two Mal d 3 genomic sequences called Mal d 3.01 and Mal d 3.02 that were mapped on LG 12 and LG 4, respectively. Both genes contain a single exon of 348 nt, of which the first 72 nt code for a putative signal peptide (Kader, 1996). The two Mal d 3 consensus sequences share 89% identity in their coding region, although the similarity is very low in the upstream region. LTPs are found mainly in aerial plant organs such as leaves, seeds, flowers, and fruit, with expression levels being low (or even nil) in roots. LTPs accumulate preferentially in exposed surfaces such as fruit skin (Borges et al., 2006). It is known that the Mal d 3.01 gene is expressed in apple fruit (Diaz-Perales et al., 2002). Mal d 4 is a small (12 – 15 kDa) cytosolic protein belonging to the profilin protein family. Profilins are found in all eukaryotic cells, and their allergenic potency has frequently been reported (Asero et al., 2003). Three distinct profilin sequences (Mal d 4.01, Mal d 4.02, and Mal d 4.03) have been reported in apple, with 75 – 80% identity in both their coding and amino acid sequences (Gao et al., 2005b). All apple profilins have a coding sequence of 396 nt and two introns of different size in conserved positions. Mal d 4.01 was mapped on LG 9, Mal d 4.02 on LG 2, and Mal *Author for correspondence. Journal of Horticultural Science & Biotechnology (2009) ISAFRUIT Special Issue 176–181