Mechanistic analysis of the defence status of low-allergenic carrot and tomato plants

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Carrot allergy is one of the most common types of birch pollen-related food allergy in central Europe. Approximately 24% of food allergic subjects suffer from allergic symptoms after ingestion of carrot. Adverse reactions to carrots are evoked due to cross-reactive IgE epitopes between the major white birch (Betula verrucosa) pollen antigen Bet v 1 and homologous food proteins. Bet v 1 and the major carrot (Daucus carota) allergen Dau c 1 belong to the pathogenesis-related protein-10 (PR10) family. PR10 is a ubiquitous small plant protein induced both by microbial pathogens and abiotic stress, and adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. Two highly similar genes encoding PR10 isoforms have been isolated and designated as carrot allergens Dau c 1.01 and Dau c 1.02.During the second funding period of DFG-PAK132 (please note this is the first for AG Kogel) we created hypoallergenic carrot plants via silencing the Dau c 1 isoforms Dau c 1.01 and Dau c 1.02 using the RNA interference technology (RNAi). Furthermore, we identified a third novel isoform Dau c 1.03 whose allergenic potential was shown by project partner Mahler and Vieths. The decrease of the allergenic potency in Dau c 1.01- and Dau c 1.02 silenced carrot plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests. Assessment of silenced plants for disease resistance showed no difference in susceptibility to the plant pathogens Alternaria dauci, Alternaria radicina, and Botrytis cinerea, as compared with wild type carrots.While Dau c 1 isoforms clearly contribute to allergies, little is known about their mode of action in carrot development and disease resistance. Based on the results obtained from the previous funding period of DFG-PAK132, the proposed research program aims to further characterize transgenic hypoallergenic carrots that will be created by multiple silencing (knockout) of the Dau c 1 isoforms. To obtain double and triple Dau c 1 silenced carrot plants, mutational approaches by RNAi as well as a novel targeted gene knock-out technique using TALE nucleases (TALEs: transcription activator-like effectors) will be employed. Dau c 1-silenced/knockout plants will be characterized and tested for altered resistance responses to common carrot pathogens. Furthermore, selected lines will be provided to our project partner for the assessment of (i) potential compensatory changes in the protein composition, and (ii) allergenic potency by skin prick tests, oral provocation, and bioassays. We will also contribute to DFG-PAK132 by pathotyping transgenic hypoallergenic tomato plants generated in the group of the project partner Sonnewald.