Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Intracellular Reactive Oxygen Species-Aided Localized Cell Death Contributing to Immune Responses Against Wheat Powdery Mildew Pathogen.

Reactive oxygen species (ROS)- and hypersensitive response (HR)-mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat pathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes (R genes) at a series of time points postinfection. The proportion of apoROS accumulation was 70 to 80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses was detected in 11 to 15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat R genes (e.g., Pm3F, Pm41, TdPm60, MIIW72, and Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed fewer intraROS responses, whereas 11% of Pm24 line-infected epidermis cells still showed HR cell death, suggesting that different resistance pathways are activated there. Here, we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat, although it induced the expression of pathogenesis-related genes. These results provide new insights into the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.

TdPm60 identified in wild emmer wheat is an ortholog of Pm60 and constitutes a strong candidate for PmG16 powdery mildew resistance.

KEY MESSAGE: We identified TdPm60 alleles from wild emmer wheat (WEW), an ortholog of Pm60 from T. urartu, which constitutes a strong candidate for PmG16 mildew resistance. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta improved the resistance to several local Bgt isolates. Wild emmer wheat (WEW), the tetraploid progenitor of durum and bread wheat, is a valuable genetic resource for resistance to powdery mildew fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). PmG16 gene, derived from WEW, confers high resistance to most tested Bgt isolates. We mapped PmG16 to a 1.4-cM interval between the flanking markers uhw386 and uhw390 on Chromosome 7AL. Based on gene annotation of WEW reference genome Zavitan_V1, 34 predicted genes were identified within the ~ 3.48-Mb target region. Six genes were annotated as associated with disease resistance, of which TRIDC7AG077150.1 was found to be highly similar to Pm60, previously cloned from Triticum urartu, and resides in the same syntenic region. The functional molecular marker (FMM) for Pm60 (M-Pm60-S1) co-segregated with PmG16, suggesting the Pm60 ortholog from WEW (designated here as TdPm60) as a strong candidate for PmG16. Sequence alignment identified only eight SNPs that differentiate between TdPm60 and TuPm60. Furthermore, TdPm60 was found to be present also in the WEW donor lines of the powdery mildew resistance genes MlIW172 and MlIW72, mapped to the same region of Chromosome 7AL as PmG16, suggesting that TdPm60 constitutes a candidate also for these genes. Furthermore, screening of additional 230 WEW accessions with Pm60 specific markers revealed 58 resistant accessions from the Southern Levant that harbored TdPm60, while none of the susceptible accessions showed the presence of this gene. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta conferred resistance against several local Bgt isolates.

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks.