Generating bread wheat varieties with lower level of gluten immunogenicity
Researcher / PhD Candidate : Aurelie Jouanin
Wheat, gluten and Celiac disease background
Wheat grains contains gluten, a polymer of glutenin and gliadin proteins whom the variants and ratio impact bread making. These proteins can be responsible for allergic or autoimmune reactions in genetically predisposed human subjects. This is the case for the 1-2% of the population which suffer from Celiac disease (CD). Gluten-free diet becomes then the only alternative, which is very challenging in daily life. To address this gluten issues, conventional wheat breeding has been performed without great success. New Plant Breeding Techniques (NPBT) are therefore being implemented.
Targeted mutagenesis towards reduction of gluten immunogenicity in bread wheat
Targeted mutagenesis represents a precise and quick alternative to knock out the genes coding for immunogenic gluten proteins while retaining genes coding for gliadin important in bread backing process. CRISPR/Cas9, a cutting edge genome editing technology is being used in order to alter and/or remove gene coding for the most immunogenic gluten protein family: the alpha-gliadins. Targeting such a large gene family, with an estimate copy number of over 150 genes in hexaploid bread wheat, represents a great challenge. Large multiple gene sequences alignments were performed manually –due to highly repetitive but slightly different regions- in order to identify conserved region across all homologous genes. CRISPR/Cas9 constructs targeting conserved regions have been synthesised and integrated into wheat plants. Progenies are going to be screened for lower alpha-gliadin gene copy number. The screening will involve genomic and transcriptomic analysis through sequencing. Gluten proteins profile of plants showing the lowest alpha-gliadin expressions would then be studied. Finally, in vitro stimulation of human immune T-cells using grain extracts could be performed, aiming at testing the immunogenicity level of the newly generated plants.
Summary of Project Findings
In bread wheat the three gliadin protein families, α, β and γ harbour immunogenic epitopes which can trigger Coeliac disease (CD) in genetically predisposed humans. In this project, we used two mutagenesis approach to modify immunogenic gliadin epitopes: gamma-irradiation TILLING and CRISPR/Cas9 gene editing. A gamma-irradiated population in the wheat variety Paragon was screened for changes in the gliadin protein profiles. The ESR also performed targeted mutagenesis against the α- and γ-gliadin genes using CRISPR/Cas9, designing five guide RNA, all transformed into the wheat variety Fielder. Four different methods were optimised and implemented to screen gliadin differences in potential mutant plants compared to the original wild type (WT) plants. Paragon gamma-irradiated mutants, and Fielder-CRISPR lines, were found with α- γ- and or ω-gliadins proteins missing or altered compared to their respective wild-type. A reduction in α-gliadin gene copy number was found in both Paragon mutant and Fielder-CRISPR lines. To identify the mutations in gliadin genomic sequences we have developed GlutEnSeq, an exome capture system that enriches for gluten genes.