Multi-target silencing, genome editing and epitope mapping of tomato allergens

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The proposed research project aims at (i) providing proof-of-principle that simultaneous down regulation of several allergens in plant-derived food is possible, (ii) developing and applying novel genome editing strategies to modulate the allergenic potential of tomato and (iii) to improve the molecular understanding of allergenic structures of the panallergen profilin. Simultaneous down-regulation of known IgE-binding proteins of tomato will be achieved by breeding available RNAi-mediated mono-, double- and triple-knock-down tomato lines. These lines will be tested for their susceptibility towards common pathogens and for their allergenic potential by IgE immunoblots and skin prick tests. To avoid possible drawbacks of RNAi-mediated down-regulation of gene expression newly developed TAL-nuclease and CRISP/Cas technologies will be applied for genome editing to specifically knock-out allergen encoding genes in tomato. While TAL-nucleases have already successfully been tested in plant systems, RNA-guided genome editing by CRISP/Cas has not been reported for plants. Therefore, available systems will be adapted to plants and suitability of the CRISP/Cas system for genome editing in plants will be tested. The structural elucidation of profilin will be carried out by unbiased selection of profilin variants with altered IgE-binding capacities using our newly developed yeast selection system and by epitope scanning of profilins from different plant sources using sera from tomato allergic patients from north, central and south European countries. Expected outcomes of the proposed research project are: (i) the assessment of molecular strategies to reduce/abolish expression of allergens, (ii) adaptation of genome editing strategies to target plant genes, (iii) knowledge concerning the potential defense function of selected allergens, (iv) molecular insight into structural determinants of profilin and (v) information concerning the potential geographic influence on epitope preferences and their possible cause.