Yeast profilin complements profilin deficiency in transgenic tomato fruits and allows development of hypoallergenic tomato fruits

Abstract

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

Proteomic analysis of apricot fruit during ripening

“…In plant cells, responses to hormones or environmental stresses, along with cell morphogenesis pathways, induce signaling cascades that correlate with the rearrangement/ turnover of actin-based structures [58][59][60]. In this context, ADF and profilin are small actin-binding proteins that control actin dynamics [61,62]; TCTP also interacts with cytoskeletal actin and plays essential roles in cell protection against various stresses [63,64]. Annexins are Ca 2+ -dependent, membranebinding proteins that fasten and possibly hold together biological structures [65]; their expression is under developmental and environmental control.…”

Hypoallergenic profilin – a new way to identify allergenic determinants

“…To compensate for the loss of profilin in Lyc e 1 silenced plants, profilin-deficient transgenic tomato plants were complemented with yeast profilin. Analysis of the resulting transgenic plants revealed an almost complete restoration of plant growth and biomass production [18]. Although these experiments demonstrate the feasibility of generating hypoallergenic food even if essential proteins are targeted, consumer acceptance is likely to be low because there are concerns that introducing foreign genes into crop plants may have an unexpected and negative impact on human health.…”

“…An aliquot of the supernatant was immediately mixed with SDS/PAGE loading buffer, denatured at 95 °C for 10 min and subjected to a 12.5% SDS/PAGE under reducing conditions. Recombinant Lyc e 1 was expressed and purified as described previously [18]. Separated proteins were blotted onto porablot NCL nitrocellulose membrane (Macherey‐Nagel, Düren, Germany).…”

“…We were able to restore the cellular function of profilin in transgenic Lyc e 1 tomato plants by simultaneous expression of yeast profilin (PFY1), which does not possess any identified allergenic properties, demonstrating the feasibility of creating low-allergenic food even if the allergen fulfils essential cellular functions. The co-expression of PFY1 restored growth and biomass production to almost wild-type levels [18].…”

The allergenicity of genetically modified foods from genetically engineered crops

“…12,13 In these situations, there was no evidence to support that GM products are more allergenic than their conventional counterparts; in fact, 7 of these studies suggested that the IgE binding properties of the GM products were actually weaker than those of their conventional counterparts, suggesting that the GM products in those studies could be viewed as potentially hypoallergenic. 69,73,91,93,96,103,109,110 We did identify 2 studies involving human ingestion of GM products, but neither demonstrated that ingestion of the GM food was associated with allergy (to Starlink GM corn, disproven by double-blinded, placebo-controlled, food challenge; or to apple with decreased Mal d 1, which noted significantly less symptom development at ingestion by patients allergic to apple). 58,96 Regarding Starlink corn, this product was approved for animal use in the late 1990s, but then somehow made its way into the human food chain and was detected in some products.…”