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

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, -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 three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease of 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 changes 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 such pathologies, 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.

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

“…In another paper from our group (Kalunke et al, 2020), the same ATI genes, plus the 0.28 gene, were silenced in the bread wheat cultivar Bobwhite by using RNAi. Different parameters related to yield resulted not affected, although one related to dough quality was strongly affected due to the lower expression of high molecular weight glutenin subunits, as an unpredictable effect likely due to RNAi procedure.…”

Wheat grain proteins: Past, present, and future

“…However, it is difficult to envisage how this understanding can be achieved without reliable in vitro test systems. Understanding structure:function relationships should also facilitate the development of safer wheat by using gene editing to make precise changes to the sequences and structures of ATIs, instead of aiming for complete eliminations as in recent studies using transgenesis (Kalunke et al, 2020) and gene editing (Camerlengo et al, 2020).…”