Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease that threatens wheat production worldwide. Pm12, which originated from Aegilops speltoides, a wild relative of wheat, confers strong resistance to powdery mildew and therefore has potential use in wheat breeding. Using susceptible mutants induced by gamma irradiation, we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum, also a wild relative of wheat. The resistance function of Pm12 was validated via ethyl methanesulfonate mutagenesis, virus-induced gene silencing, and stable genetic transformation. Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic. Here, we demonstrated that the two orthologous genes, Pm12 and Pm21, possess differential resistance against the same set of Bgt isolates. Overexpression of the coiled-coil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves. However, their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions. Cloning of Pm12 will facilitate its application in wheat breeding programs. This study also gives new insight into two orthologous resistance genes, Pm12 and Pm21, which show different race specificities and intramolecular interaction patterns.

Nucleotide‐binding (NB) leucine‐rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled‐coil (CC) NLR that confers broad‐spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell‐death‐inducing activity, whereas the CC‐containing truncations, including CC‐NB and CC‐NB‐LRR, do not induce cell‐death responses. Coimmunoprecipitation (Co‐IP) assay showed that the eCC domain self‐associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V‐mediated full‐length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell‐death induction. Two mutations (R73Q and E80K) interfered with D491V‐mediated Pm21 autoactivation without affecting the cell‐death‐inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell‐death‐inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell‐death signalling and disease‐resistance signalling, which are potentially independent of each other.

Common wheat and durum wheat are widely cultivated cereal crops, both of which are infected by the obligate biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt), causing powdery mildew. Recently, Pm68 gene was identified on the short arm of chromosome 2B in the Greek durum wheat line TRI 1796, conferring resistance to this disease. Here, we have cloned Pm68 from TRI 1796 using an integrated approach of genetic mapping, association analysis and PacBio sequencing. Transgenic assays demonstrate that Pm68 mediated resistance is controlled by a pair of genetically linked nucleotide-binding leucine-rich repeat (NLR)-encoding genes, Pm68-1 and Pm68-2. Transient expression assays in Nicotiana benthamiana leaves reveal that the activation of Pm68-1 is positively modulated by Pm68-2, likely through its N-terminal coiled-coil (CC)-like domain. Evolutionary analysis traces the origin of Pm68 to a specific wild emmer subpopulation. The introgression and transgenic wheat lines carrying this gene show no significant negative effects on major agronomic traits, highlighting the potential value of Pm68 for disease-resistant breeding programs of both durum wheat and common wheat.

Pm21, derived from wheat wild relative Dasypyrum villosum, is one of the most effective powdery mildew resistance genes and has been widely applied in wheat breeding in China. Mapping and cloning Pm21 are of importance for understanding its resistance mechanism. In the present study, physical mapping was performed using different genetic stocks involving in structural variations of chromosome 6VS carrying Pm21. The data showed that 6VS could be divided into eight distinguishable chromosomal bins, and Pm21 was mapped to the bin FLb4–b5/b6 closely flanked by the markers 6VS-08.6 and 6VS-10.2. Comparative genomic mapping indicated that the orthologous regions of FLb4–b5/b6 carrying Pm21 were narrowed to a 117.7 kb genomic region harboring 19 genes in Brachypodium and a 37.7 kb region harboring 5 genes in rice, respectively. The result was consistent with that given by recent genetic mapping in diploid D. villosum. In conclusion, this study demonstrated that physical mapping based on chromosomal structural variations is an efficient method for locating alien genes in wheat background.