Steffenson, B. | Search bioRxiv

bioRxiv2024

Discovery of functional NLRs using expression level, high-throughput transformation, and large-scale phenotyping

Protecting crops from pests and diseases is vital for the sustainable agricultural systems needed for food security. Introducing functional resistance genes to enhance the plant immune system is an effective method of disease control, but identifying new immune receptors is time-consuming and resource intensive. We observed that functional immune receptors of the NLR class show a signature of high expression in uninfected plants across both monocot and dicot species. Here we show that this signature, combined with high throughput crop transformation, can be used to rapidly identify candidate NLRs from diverse plant species and validate pathogen resistance directly in crop plants. As a proof of concept, we generated a wheat transgenic library carrying 995 NLRs from 18 grass species. Screening the collection with the stem rust pathogen Puccinia graminis, which is a major threat to wheat production, we confirm 19 new resistance genes. This pipeline facilitates resistance gene discovery, unlocking a large gene pool of diverse and non-domesticated plant species and providing in-planta gene validation of disease resistance directly in crops.

plant biology↗

bioRxiv2020

rcs5-mediated spot blotch resistance in barley is conferred by wall-associated kinases that resist pathogen manipulation

Plant biotrophic pathogen disease resistances rely on immunity receptor-mediated programmed cell death (PCD) responses, but specialized necrotrophic/hemi-biotrophic pathogens hijack these mechanisms to colonize the resulting dead tissue in their necrotrophic phase. Thus, immunity receptors can become necrotrophic pathogen dominant susceptibility targets but resistance mechanisms that resist necrotroph manipulation are recessive resistance genes. The barley rcs5 QTL imparts recessive resistance against the disease spot blotch caused by the hemi-biotrophic fungal pathogen Bipolaris sorokiniana. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four wall-associated kinase (WAK) genes, designated HvWak2, Sbs1, Sbs2 (susceptibility to Bipolaris sorokiniana 1&2), and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in susceptible barley cultivars resulted in resistance showing dominant susceptibility function. Allele analysis of Sbs1&2 from resistant and susceptible barley cultivars identified sequence polymorphisms associated with phenotypes in their primary coding sequence and promoter regions, suggesting differential transcriptional regulation may contribute to susceptibility. Transcript analysis of Sbs1&2 showed nearly undetectable expression in resistant and susceptible cultivars prior to pathogen challenge; however, upregulation of both genes occurred specifically in susceptible cultivars post-inoculation with a virulent isolate. Apoplastic wash fluids collected from barley infected with a virulent isolate induced Sbs1, suggesting regulation by an apoplastic-secreted effector. Thus, Sbs1&2 function as B. sorokiniana susceptibility targets and non-functional alleles or alleles that resist induction by the pathogen mediate rcs5-recessive resistance. The sbs1&2 alleles underlying the rcs5 QTL that the pathogen is unable to manipulate are the first resistance genes identified against spot blotch. SIGNIFICANCE STATEMENTThe rcs5 locus in barley confers a high level of seedling resistance and a moderate level of adult plant resistance to spot blotch. It is part of a complex that has provided durable spot blotch resistance in many North American barley cultivars (cv) for more than 50 years. Genetic characterization and positional cloning of rcs5 identified the dominant susceptibility genes, Sbs1 and Sbs2 (susceptibility to Bipolaris sorokiniana 1 and 2) as wall-associated kinases. These genes are hijacked by the hemibiotrophic pathogen in its necrotrophic phase to induce programmed cell death, facilitating disease development. We report the first spot blotch resistance/susceptibility genes cloned that function via alleles that cannot be specifically induced and hijacked by virulent isolates of the pathogen.

plant biology↗

bioRxiv2019

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here we show that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis revealed that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or reassortment. Generation of genetic variation by nuclear exchange may favour the evolution of dikaryotism by providing an advantage over diploidy.

genetics↗