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 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.

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach

“…Moreover, the genotype Timilia presents a much higher relative abundance of αamylase inhibitor 0.28 in comparison with the other two genotypes investigated here. This finding appears very interesting because this protein represents one of the non-gluten components, listed in the Allergome database (http://www.allergome.org/, accessed on date 8 February 2021), likely to be allergenic, and may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD) [54][55][56].…”

Synthesis and accumulation of amylase-trypsin inhibitors and changes in carbohydrate profile during grain development of bread wheat (Triticum aestivum L.)

“…Although several studies helped to understand the complex gene network that regulates protein expression during grain development, the biochemical mechanisms for ATI synthesis still require further research. The complexity of trypsin inhibition was impressively demonstrated in a recent research study: although total ATI concentrations were reduced by gene silencing of three major ATI genes, TIA was increased [ 39 ].…”

From Genetic Maps to QTL Cloning: An Overview for Durum Wheat

“…However, the research in this field is rapidly increasing. Recently, two independent studies [ 206 , 207 ] reported CRISPR/Cas9 editing of the α-Amylase/Trypsin inhibitor (ATI) genes, reducing the allergen proteins in durum wheat. The ATI subunits WTAI-CM3 and WTAI-CM16 from the durum wheat cultivar Svevo were edited to produce lines with reduced allergens.…”