No abstract

SummaryPlant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato ( Lycopersicon esculentum ) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli , followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1-or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein).The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines.These assays revealed a strong (10-to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts.These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Gene silencing of Lyc e 1 leads to reduced allergenicity of tomato fruits but impaired growth of transgenic tomato plants. The aim of the study was to restore growth of Lyc e 1-deficient tomato plants while retaining reduced allergenicity by simultaneous complementation of profilin deficiency by expression of nonallergenic yeast profilin. Transgenic plants were generated and tested by RT-PCR and immunoblotting; allergenicity of yeast profilin and transgenic fruits was investigated by IgE binding, basophil activation, and skin-prick tests. Lyc e 1 content of transgenic tomato fruits was <5% of that of wild-type plants, causing significantly reduced IgE antibody binding. Simultaneous coexpression of yeast profilin restored growth and biomass production almost to wild-type levels. Yeast profilin, sharing 32.6% amino acid sequence identity with Lyc e 1, displayed low IgE-binding capacity and allergenic potency. Among 16 tomato-allergic patients preselected for sensitization to Lyc e 1, none showed significant reactivity to yeast profilin. Yeast profilin did not induce mediator release, and coexpression of yeast profilin did not enhance the allergenicity of Lyc e 1-reduced fruits. Simultanous coexpression of yeast profilin allows silencing of tomato profilin and generation of viable plants with Lyc e 1-deficient tomato fruits. Therefore, a novel approach to allergen avoidance, genetically modified foods with reduced allergen accumulation, can be generated even if the allergen fulfills an essential cellular function in the plant. In summary, our findings of efficiently complementing profilin-deficient tomato plants by coexpression of low allergenic yeast profilin demonstrate the feasibility of creating low-allergenic food even if the allergen fulfills essential cellular functions.

Gene silencing of Lyc e 1 leads to reduced allergenicity of tomato fruits but impaired growth of transgenic tomato plants. The aim of the study was to restore growth of Lyc e 1‐deficient tomato plants while retaining reduced allergenicity by simultaneous complementation of profilin deficiency by expression of nonallergenic yeast profilin. Transgenic plants were generated and tested by RT‐PCR and immunoblotting;allergenicity of yeast profilin and transgenic fruits was investigated by IgE binding, basophil activation, and skin‐prick tests. Lyc e 1 content of transgenic tomato fruits was <5% of that of wild‐type plants, causing significantly reduced IgE antibody binding. Simultaneous coexpression of yeast profilin restored growth and biomass production almost to wildtype levels. Yeast profilin, sharing 32.6% amino acid sequence identity with Lyc e 1, displayed low IgE‐binding capacity and allergenic potency. Among 16 tomato‐allergic patients preselected for sensitization to Lyc e 1, none showed significant reactivity to yeast profilin. Yeast profilin did not induce mediator release, and coexpression of yeast profilin did not enhance the allergenicity of Lyc e 1‐reduced fruits. Simultanous coexpression of yeast profilin allows silencing of tomato profilin and generation of viable plants with Lyc e 1‐deficient tomato fruits. Therefore, a novel approach to allergen avoidance, genetically modified foods with reduced allergen accumulation, can be generated even if the allergen fulfills an essential cellular function in the plant. In summary, our findings of efficiently complementing profilin‐defi‐cient tomato plants by coexpression of low allergenic yeast profilin demonstrate the feasibility of creating low‐allergenic food even if the allergen fulfills essential cellular functions.—Le, L. Q., Mahler, V., Scheu‐rer, S., Foetisch, K., Braun, Y., Weigand, D., Enrique, E., Lidholm, J., Paulus, K. E., Sonnewald, S., Vieths, S., Sonnewald, U. Yeast profilin complements profilin deficiency in transgenic tomato fruits and allows development of hypoallergenic tomato fruits. FASEB J. 24, 4939–4947 (2010). http://www.fasebj.org