Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.

Crop wild relatives offer natural variations of disease resistance for crop improvement. Here, we report the isolation of broad-spectrum powdery mildew resistance gene Pm36, originated from wild emmer wheat, that encodes a tandem kinase with a transmembrane domain (WTK7-TM) through the combination of map-based cloning, PacBio SMRT long-read genome sequencing, mutagenesis, and transformation. Mutagenesis assay reveals that the two kinase domains and the transmembrane domain of WTK7-TM are critical for the powdery mildew resistance function. Consistently, in vitro phosphorylation assay shows that two kinase domains are indispensable for the kinase activity of WTK7-TM. Haplotype analysis uncovers that Pm36 is an orphan gene only present in a few wild emmer wheat, indicating its single ancient origin and potential contribution to the current wheat gene pool. Overall, our findings not only provide a powdery mildew resistance gene with great potential in wheat breeding but also sheds light into the mechanism underlying broad-spectrum resistance.

Tandem kinase proteins underlie the innate immune systems of cereal plants, but how they initiate plant immune responses remains unclear. This report identifies wheat protein wheat tandem NBD 1 (WTN1), a noncanonical nucleotide-binding leucine-rich repeat (NLR) receptor featuring tandem nucleotide binding adaptor shared by APAF-1, plant R proteins, and CED-4 (NB-ARC) domains, required for WTK3-mediated disease resistance. Both WTK3 and its allelic variant Rwt4—known for conferring resistance to wheat powdery mildew and blast, respectively—are capable of recognizing the blast effector PWT4. They activate WTN1 to form calcium-permeable channels, akin to ZAR1 and Sr35. Thus, tandem kinase proteins and their associated NLRs operate as “sensor-executor” pairs against fungal pathogens. Additionally, evolutionary analyses reveal a coevolutionary trajectory of the tandem kinase-NLR module, highlighting their cooperative role in triggering plant immunity.