Regulation of the Ypr10 gene family and identification of the biological role of its gene product (PR-10 protein) on Rosaceous species

The major apple allergen Mal d 1 has been shown to be a pathogenesis-related protein 10 (PR- 10) due to the high sequence similarity and pathogen- and stress-inducibility. In most plant species PR-10 proteins are encoded by multiple genes, so called Ypr10 genes. Despite the ubiquitous occurrence in the plant kingdom the biological role of these proteins remains to be identified. Although there is considerable knowledge about the genomic structure and expression, patterns of Ypr10 genes, there is little information on gene regulation and the involvement of the gene product (PR- 10 protein) in plant defense. Mal d 1, as many other PR-10s, is not only expressed upon challenge with a broad range of pathogen- and stress factors, but also in certain devolopmental stages. It is conceivable, that PR-10 proteins do not directly affect pathogens but rather interact with endogenous components to activate a stress response, also known to be a feature of normal development. The aim of this project is the identification of components i) interacting with Mal d 1, and ii) regulating Ypr10 gene expression. This would be a major step towards the elucidation of the biological function of PR-10 and moreover, would give insight into the mechanism of Ypr10 gene activation.

Mal d1, a 18 kD protein in apple, belongs to the `pathogenesis-related` proteins of family 10 (PR-10), which are distributed throughout the plant kingdom. As the name suggests their expression is associated with pathogen attack, but in various plants they were also found during normal developmental processes. PR-10s are mostly encoded by multiple copies of the so called Ypr10 genes. However, their function remains still unknown. Interestingly many of the allergens found in fruits (apple, apricot, peach, plum, cherry), in vegetables (carrot, celery, parsley) and tree pollen (birch, alder, hazel) are PR-10 proteins. They all show a similar protein sequence and -structure and this high homology makes them cross reactive with allergic patient`s serum containing IgE antibodies primarily directed to the sensitising agent. In the frame of this project a protein was discovered in apple, which constitute a ligand of Mal d1, the major allergen in apple fruits. This Mal d1-Interacting-Protein (MdIP) was isolated from an expression-cDNA-library by using a genetic system derived from yeast. Searches in databases did not reveal any significant homology of MdIP with already known proteins. However, a 45 % identity over the entire length was found with a protein sequence of Arabidopsis thaliana, which was annotated in the database as `hypothetical` protein based on the analysis of the sequenced Arabidopsis genome by gene prediction programs. This finding let suggest that MdIP is indeed a novel protein identified in plants. Furthermore, for the first time a complex formation of Mal d1 (=PR-10) with a hitherto unknown protein could be demonstrated. Although the function of both proteins is still unclear, the Mal d1-MdIP- interaction underlines the hypothesis, that PR-10 proteins are probably not involved directly in pathogen defence. More likely, they may be seen as part of a multimeric complex (e.g. for protein stabilisation or as a carrier or shuttle protein), which might be involved in stress alleviation or in the regulation of developmental processes. Transformed tobacco was used as a model system to study regulatory sequences (promoters) of Ypr10 genes in fruit trees. Promoters in varying length were set in front of a marker gene (ß-glucuronidase) and these constructs were transformed in tobacco. This allowed the identification of DNA regions and sequence motifs essential for function and inducibility of Ypr10-promoters.