Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes

Raviraj M Kalunke,Shahidul Islam,Stefania Masci,Colette Larré,Roberta Lupi,Domenico Lafiandra,Stefano D’Amico,Francesco Sestili,Wujun Ma,Sandra Denery-Papini,Silvio Tundo,Francesco Camerlengo

doi:10.3390/ijms21165817

Abstract

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker’s asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Highlights

Wheat (Triticum spp.) is one the “big three” cereal crops, together with maize and rice, cultivated worldwide thanks to its adaptability, nutritional value and versatility
Our group [16] has reported the application of CRISPR-Cas9 silencing technology to silence CM3 and CM16 amylase/trypsin inhibitors (ATI) genes in durum wheat, whereas in this paper we report the development and the extensive characterization, including the allergenic potential and qualitative evaluations, of wheat lines derived from the bread wheat cultivar Bobwhite, in which CM3, CM16 and 0.28 ATI genes have been silenced by RNAi
No significant differences in morphology or growth were observed between the “null” genotype that had lost the transgene by segregation, and the untransformed plants, we have used the untransformed plant for the experiments

Summary

Introduction

Wheat (Triticum spp.) is one the “big three” cereal crops, together with maize and rice, cultivated worldwide thanks to its adaptability, nutritional value and versatility This is mostly due to the unique viscoelastic properties of its dough, capable to give rise to a wide range of products, such as bread, pasta, noodles, biscuits, some of which are characteristic of specific geographical areas. Such properties derive from wheat grain proteins, classified into four major groups based on solvent solubility [1]: albumins (water), globulins (dilute salt solution), prolamins including gliadins (alcohol/water mixture), and glutelins, including glutenins (diluted acid or alkaline solutions). Glutenins play the major role and, in particular, their size and amount are major determinants of dough technological quality [2]

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Results

Conclusion