Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.

Hybrid necrosis, a century-old mystery in wheat, is caused by complementary genes Ne1 and Ne2. Ne2, encoding a nucleotide-binding leucine-rich repeat (NLR) immune receptor, has been cloned, yet Ne1 remains elusive. Here, we report that Ne1, which encodes an alpha/beta hydrolase (ABH) protein generated by structural variation, triggers hybrid necrosis with Ne2 by activating autoimmune responses. We further verify that not only allelic variation but also copy number variation (CNV) of Ne1 are pivotal for hybrid necrosis diversity in wheat. Ne1 likely originates from wild emmer wheat, potentially through duplication and ectopic recombination events. Unlike Ne2, which is frequently selected for rust resistance in wheat breeding, the lower prevalence of Ne1 in modern wheat cultivars is attributed to its association with hybrid necrosis. Altogether, these findings illuminate the co-evolution of the NLR/ABH gene pair in plant development and innate immunity, offering potential benefits for wheat breeding.