MSc/MRes Bursary Report: Obtaining loss-of-function mutants in a candidate barley NLR conferring resistance to wheat yellow rust

This is the report from a BSPP MSc/MRes Bursary.
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I completed a 10-month MBio placement with the Moscou Lab in The Sainsbury Laboratory and wrote my master’s thesis on my work during this time. The supportive academic environment allowed me to gain experience in qualitative and quantitative genetics, molecular biology and bioinformatics, whilst being exposed to interesting research.

Puccinia striiformis is the causal fungal agent of yellow rust (=stripe rust), a devastating disease in several key crop species. While Puccinia striiformis sensu lato have a wide host range within the grasses (Poaceae), individual adapted lineages (forma specialis) show limited capability for pathogenicity outside of their adapted host. Previous work has identified barley accessions susceptible to the wheat-adapted subspecies, Puccinia striiformis f. sp. tritici (Pst); uncovering the existence of intermediate hosts. The Reaction to Puccinia striiformis 6 (Rps6) locus in barley confers resistance to the wheat yellow rust pathogen and has been uncoupled from resistance to barley yellow rust, Puccinia striiformis f. sp. hordei (Psh). While multiple NLRs are located within the Rps6 locus, numerous lines of experimental evidence have highlighted a specific NLR necessary for Rps6-mediated resistance. This work aimed to obtain putative loss-of-function mutations of this NLR gene through a Cas9-mediated reverse genetic approach. The outcomes of this project were to facilitate future marker-assisted backcrossing and functional studies of the target NLR for validation of the gene conferring Rps6-mediated resistance.

Reported yield losses due to yellow rust reach up to 70% in the worst cases, with the quality of the infected crop greatly diminished. Consequently, yellow rust poses a significant threat to the biosecurity of key crop varieties, such as wheat and barley. With global population projected to reach over 9 billion by 2050, robust strategies that prevent plant diseases will be vital to sustain food provision for present and future generations. This work contributes to a long-term goal of developing R gene stacks for crop improvement.

Previous inoculation screens had revealed three major loci associated with resistance to wheat yellow rust in barley: Rps6, Rps7 and Rps8. While susceptible barley accessions like SusPtrit are void of all three, others like Golden Promise retain the complete roster. The resistance conferred respectively must therefore be disentangled. This negated the possibility of an inoculation screen of Pst against transgenic T₁ barley lines to reveal susceptible individuals, which have undergone Cas9-mediated gene editing. Instead, genotypic analysis via PCR screening, restriction digest assays and sanger sequencing of PCR amplicons was used as a selection process in the T₁ progeny. A T₁F₁ generation was produced from the selected mutant T₁ lines, by crossing with a susceptible cultivar lacking Rps6, Rps7, and Rps8. The selection process was repeated, with convergence of all these data and parental lineage considered in parallel to facilitate full analysis of T₁F₁ progeny. This promoted drafting of NLR mutants highly suspected of having lost functionality pertinent for resistance to wheat yellow rust. A wide variety of mutants were generated through use of a dual guide RNA CRISPR-Cas9 construct targeting a specific NLR in the Rps6 locus.

Loss of half the first exon or premature stop codons are likely to induce loss-of-function in many of the mutants derived, due to N-terminal truncation of the NLR. Mutations eliminating the translational start were also common, abolishing the open reading frame. The high occurrence of double mutants, where both targeted sites were disrupted, indicated high efficiency of the construct and robust sgRNA design. Three T₁F₁ lines null for the Cas9 T-DNA and hemizygous for a mutation, as well as several GMO knockout mutants, were obtained. Future efforts will involve inoculation screens of a T₁F₁BC₁ population against Pst to confirm loss of functionality in the NLR and susceptibility to wheat yellow rust.

I am truly grateful for this bursary. The opportunity to be trained as a plant pathologist was life-changing. Thank you to the BSPP community for supporting others to join and contribute to the world of plant research.

Ifeoluwa Ifekoya

University of Warwick