Characterisation of Mal d 1-related genes in Malus

Abstract

It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus x domestica Borkh). We sequenced cDNA libraries of cultivar 'Royal Gala' and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis -a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in 'Royal Gala', particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica)

“…By Clustal W alignment, all Mal d 1 coding sequences in the database could be classified to these four subfamilies and members (Table 2). Subfamily I includes reference sequences that were formerly classified as Mal d 1a, Mal d 1b, Mal d 1c (Son et al 1999), and Mal d 1 d (Beuning et al 2004) isoforms or as PR-10a and PR-10c proteins (Ziadi et al 2001). The new sequences of this subfamily were derived from the PCR primer pairs 1 and 2 (Table 1) (Table 1), which not only represent all the reference sequences, but also include some new members.…”

“…The presence of Mal d 1.05 on LG 6 is unexpected, but its mapping is based on two reliable SNAP markers for the specific allele of FS. Two additional Mal d 1-related genes, Mal d 1m (AY428588) and Mal d 1n (AY428589) described by Beuning et al (2004), were not covered, because these sequences were not available at the time of our study. A DNA phylogenetic tree analysis indicated that Mal d 1m belongs to subfamily II, but is distinct from both including the residue S112, which is essential for IgE binding and cross-reactivity (Son et al 1999).…”

“…PR-10c (Mal d 1.01), and PR-10a (Mal d 1.02) will express much more strongly than PR-10d (Mal d 1.03C) and PR-10b (Mal d 1.03D) upon a salicylic acid analogue induction (Ziadi et al 2001), whereas Mal d 1.06A, Mal d 1.03E, and Mal d 1.03F are auxin responsive (Kodde and Van der Geest, unpublished). A recent comprehensive expression study on Mal d 1-related genes in apple demonstrated that 8 out of 12 genes were expressed in tree-ripened fruit, and most of these were also expressed in leaves in response to a challenge with Venturia inaequalis-a fungus causing apple scab (Beuning et al 2004).…”

“…Their phylogenetic tree indicated four distinct groups with seven members (Puehringer et al 2003). Recently, Beuning et al (2004) sequenced cDNA libraries of cultivar Royal Gala and identified 12 Mal d 1-related genes, five of which were new. The Mal d 1 family seemed to consist of at least 15 members (Atkinson et al 1996), which means that at least three Mal d 1 homologous genes have not been cloned and sequenced.…”

Investigation of Mal d 1 Allelic Variants and Phylogenetic Diversity in Contemporary and Historical Polish Apple Cultivars

“…Not all of these members are likely to be involved in allergenicity since only a limited number of different Mal d 1 proteins and mRNAs have been traced back in apple fruit so far (Helsper et al, 2002;Puehringer et al, 2003;Beuning et al, 2004).…”