Targeting of prolamins by RNAi in bread wheat: Effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from a-, c-, x-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/ MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only c- and a-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of x-gliadins and c-gliadins, and three of these also silenced a-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic a- and x-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars