Silencing of ATI genes involved in adverse reactions to wheat by RNAi and CRISPR-Cas9 technologies

Although wheat is consumed worldwide as a staple food, it can give rise to different adverse reactions, some of which have not been deeply characterized. They are caused mainly by wheat proteins, both gluten and non-gluten proteins. Structural and metabolic proteins, like \u3b1amylase/trypsin inhibitors (ATI) are involved in the onset of wheat allergies (bakers\u2019 asthma) and probably non-coeliac wheat sensitivity (NCWS). The ATI are encoded by a multigene family dispersed over several. Notably, WTAI-CM3 and WTAI-CM16 subunits are involved in the onset of bakers\u2019 asthma and are likely to contribute to NCWS. In this study we report the RNAi silencing of WTAI-CM3, WTAI-CM16 and WMAI-0.28 genes in the bread wheat cultivar Bobwhite and the CRISPR/Cas9 mediated gene knockout of WTAI-CM3 and WTAI-CM16 in the durum wheat cultivar Svevo. We have obtained different RNAi transgenic lines showing an effective decrease in the expression in the targeted genes. These lines do not show differences in terms of yield, but have unintended effects on the accumulation of the high molecular weight glutenin subunits which play a crucial role in the technological performances of wheat flour. Furthermore, the editing of WTAI-CM3 and WTAI-CM16 genes was obtained through a CRISPR-Cas9 multiplexing strategy in the Italian durum wheat cultivar Svevo with a marker-free approach. The regeneration of plants without selection agents allowed T0 homozygous mutant plants to be obtained without the integration in the wheat genome of CRISPR/Cas9 vectors, demonstrating the capability of CRISPR technology to produce wheat lines in a reduced time compared to conventional breeding approaches. The possibility to develop new wheat genotypes accumulating a lower amount of proteins effectively involved in such pathologies, not only offers the possibility to use them as a basis for the creation of wheat varieties with a lower impact on adverse reactions, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has yet to be established

Applying the adverse outcome pathway (AOP) for food sensitization to support in vitro testing strategies

Background Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches

Wheat ati cm3, cm16 and 0.28 allergens produced in pichia pastoris display a different eliciting potential in food allergy to wheat

International audienceAlthough wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients' sera, with the highest values of beta-hexosaminidase release observed for CM3 protein

4.17. Blé, gluten et pathologies

Les propriétés exceptionnelles de la farine de blé permettent la formation de pains levés et d’une grande diversité de produits constituant une des bases de l’alimentation occidentale. Ces propriétés proviennent du gluten, la fraction protéique principale de la farine de blé (Figure 1). Les protéines du gluten ont la capacité de s’associer les unes aux autres pour former un réseau continu extensible et élastique (Figure 2). Ce réseau emprisonne les granules d’amidon ainsi que le gaz carboniqu..

Les pathologies associées au blé et céréales apparentées

National audienc