Development of reduced gluten wheat enabled by determination of the genetic basis of thelys3alow hordein barley mutant

Abstract

Celiac disease is the most common food-induced enteropathy in humans with a prevalence of approximately 1% world-wide [1]. It is induced by digestion-resistant, proline-and glutamine-rich seed storage proteins, collectively referred to as "gluten," found in wheat. Related prolamins are present in barley and rye. Both celiac disease and a related condition called non-celiac gluten sensitivity (NCGS) are increasing in incidence [2] [3]. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here we used BSR-seq (Bulked Segregant RNA-seq) and map-based cloning to identify the genetic lesion underlying a recessive, low prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild type barley gene and then used TILLING (Targeting Induced Local Lesions in Genomes)[4] to identify induced SNPs (Single Nucleotide Polymorphisms) in the three homoeologs of the corresponding wheat gene. Combining inactivating mutations in the three sub-genomes of hexaploid bread wheat in a single wheat line lowered gliadin and low molecular weight glutenin accumulation by 50-60% and increased free and protein-bound lysine by 33%. This is the first report of the combination of mutations in homoeologs of a single gene that reduces gluten in wheat.Gluten is the common name for a complex mixture of approximately 100 proline-and glutamine-rich seed storage proteins found in wheat endosperm. In hexaploid bread wheat, in particular, gluten is responsible for the unique viscoelastic properties of dough used for making leavened bread. Related prolamins known as hordeins in barley and secalins in rye, but lacking the viscoelastic properties of wheat gluten, are found in the endosperms of these related cereals. Among gluten proteins, the most important for the elasticity and strength of bread dough are the polymeric high molecular weight glutenins [5]; monomeric gluten proteins, primarily gliadins, which are classified based on size, charge and repetitive amino acid sequences into α, β, γ and ω types, contribute to the viscosity of gluten [6] [7].Gluten is the causative factor of celiac disease, the most common food-induced autoimmune enteropathy in humans [1]. Celiac disease is found primarily in individuals expressing human leukocyte antigen (HLA) alleles DQ2 and/or DQ8. Immunodominant peptide epitopes have been found in α gliadins [8] and in ω gliadins of wheat and in C hordeins of barley [9]. Additional epitopes are also found in LMW and HMW-glutenins, gamma-gliadins from wheat and B and gamma hordeins from barley [10] [9]. Celiac disease patients exhibit a range of intestinal and extra-intestinal symptoms of which the most common is flattening of the villi of the small intestine, which can lead to poor absorption of nutrients and other pathological consequences. The only current treatment is a life-long avoidance of all gluten in the diet,which can be challenging given the prevalent use of gluten in diver...

Gene editing towards hypoimmunogenic gluten proteins in wheat

“…Following the idea of working on the regulatory elements (Putcha, 2016) of gliadin genes rather than the gene themselves, another alternative would be overexpression of a transcription factor (TF) -using acetyltransferase -that represses gliadin expression, or silencing a TF activating their expression. Moehs et al (2018) combined stable mutations in three Lys3a-like transcription factors to reduce gluten gene expression by 50%. However, the transcription factors found so far similarly in uence the expression of both gliadins and glutenins genes and are therefore not ideal candidates since the aim is to keep the glutenins expression -at least HMW-glutenins -and decrease the immunogenic gliadin expression.…”