Gene cloning in repeat-rich polyploid genomes remains challenging. Here we describe a strategy for overcoming major bottlenecks in the cloning of the powdery mildew (Pm) resistance gene (R-gene)Pm69derived from tetraploid wild emmer wheat (WEW). A conventional positional cloning approach encountered suppressed recombination due to structural variations, while chromosome sorting yielded an insufficient purity level. APm69physical map, constructed by assembling ONT long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster. A single candidate NLR was identified within this cluster by anchoring RNASeq reads of susceptible mutants to ONT contigs and was validated by the virus-induced gene silencing (VIGS) approach.Pm69, comprising Rx_N with RanGAP interaction sites, NB-ARC, and LRR domains, is probably a newly evolved NLR discovered only in one location across the WEW distribution range in the Fertile Crescent.Pm69was successfully introgressed into durum and bread wheat, and a diagnostic molecular marker could be used to accelerate its deployment and pyramiding with other resistance genes.

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 post-infection. The proportion of apoROS accumulation was 70-80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses were detected in 11-15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat (NLR) R genes (e.g. Pm3F, Pm41, TdPm60, MIIW72, Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed very less intraROS responses, while 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 (PR) genes. These results provide new insights on the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.