RATIONALE: RNA interference (RNAi) is a powerful method for post-transcriptional down-regulation of protein expression. Our aim was to reduce certain allergens in tomato plants providing a proof of principle for this molecular approach.
METHODS: Expression of tomato (Lycopersicon esculentum) allergens Lyc e 1 (profilin) and Lyc e 3 (non-specific Lipid-Transfer-Protein) were suppressed in a fruit-specific or constitutive manner by RNAi. Transgenic tomato plants were generated by Agrobacterium-mediated gene transfer. Gene silencing was analyzed by Northern - and Western Blotting. In vitro histamine release assays were performed to examine any remaining allergenic potency of tomato fruits.
RESULTS: Fruit-specific and constitutive gene suppression of Lyc e 1 resulted in a great reduction of protein expression in tomato plants; however, best results have been obtained using a constitutive promoter. Constitutive gene silencing was accompanied by impaired plant growth and fruit yield. Therefore, a non-IgE reactive yeast profilin was selected to reconstitute RNAi plants to maintain their physiological function. Fruit specific and constitutive gene silencing of Lyc e 3 resulted in neither IgG binding of an LTP-specific antibody nor IgE binding of Lyc e 3 positive tomato allergic patients` sera. Moreover, histamine release performed with basophils passively sensitized with sera from two LTP-sensitized patients was drastically reduced after stimulation with extracts from transgenic fruits.
CONCLUSIONS: RNAi technology is an effective tool to reduce allergen expression in transgenic tomato fruits and hence to reduce allergenic potency. It might represent a versatile strategy to obtain hypoallergenic food.
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