Wheat powdery mildew, caused by the fungus *Blumeria graminis* f. sp. *tritici* (Bgt), significantly impacts global wheat production. The rapid evolution of Bgt and the emergence of virulent isolates have rendered many race-specific resistance genes ineffective, highlighting the urgent need for new resistance sources. While over 100 powdery mildew resistance genes/alleles have been identified, only a few have been cloned and characterized. Most cloned Pm genes encode coiled-coil nucleotide-binding leucine-rich-repeat (NLR) proteins, known for their susceptibility to pathogen evolution. In contrast, some genes conferring durable resistance encode proteins like ABC transporters or hexose transporters. Wheat landraces represent a valuable reservoir of disease resistance genes, including Pm24, Pm24b, and MIHLT, mapped to the same region of chromosome 1DS. This study aimed to map-based clone *Pm24* and characterize the molecular basis of its resistance function.

Existing literature extensively documents powdery mildew resistance genes in wheat, many of which have been mapped to specific loci. However, the molecular mechanisms underpinning resistance remain incompletely understood for many genes. Previous research highlights the success of genes like Pm38/Yr18/Lr34/Sr57 (ABC transporter) and Pm46/Yr46/Lr67/Sr55 (hexose transporter) in providing durable resistance to multiple pathogens. The focus on NLR proteins has shown success in conferring resistance, but often temporary due to the pathogen's adaptability. This study explores an alternative mechanism of resistance, moving beyond the NLR protein class to investigate a tandem kinase.

The study employed a map-based cloning approach using a mapping population derived from a cross between the powdery mildew-resistant landrace Hulutou (HLT) and the susceptible cultivar Shi 4185 (S4185). High-resolution mapping narrowed the *MIHLT* locus to a 532 kb region. Candidate genes within this region were identified through sequence comparison between HLT and S4185. Functional validation involved transgenic assays in the susceptible cultivar Fielder using constructs expressing *WTK3* (the candidate gene) and *CNL* (another candidate) under native and ubiquitin promoters. Ethyl methanesulfonate (EMS) mutagenesis of HLT was used to generate mutant lines, enabling further investigation into the *WTK3* gene's role in resistance. Expression profiling of *WTK3* and pathogenesis-related (PR) genes was conducted using RT-PCR and quantitative real-time RT-PCR. Haplotype analysis was performed using a panel of wheat accessions to assess the prevalence of the identified mutation. Finally, transgenic assays were performed with chimeric WTK3 variants to identify the exact amino acids essential for resistance.

The study successfully cloned *Pm24*, which encodes a tandem kinase protein (WTK3) with two putative kinase domains (Kin I and Kin II). Allelism tests confirmed that *Pm24*, *Pm24b*, and *MIHLT* are allelic. Transgenic assays demonstrated that *WTK3* is sufficient for powdery mildew resistance, while *CNL* is not. EMS mutagenesis identified 11 independent mutants with mutations in *WTK3*, all showing susceptibility to powdery mildew. These mutations affected both Kin I and Kin II domains. Haplotype analysis revealed that a 6-bp deletion in the Kin I domain, leading to a lysine-glycine deletion (K400G401) in the protein, is associated with resistance and is rare among worldwide wheat germplasm, being found primarily in landraces from Shaanxi province, China. Further transgenic assays using chimeric variants of *WTK3* showed that the specific K400G401 deletion is critical for resistance, indicating that the precise amino acid deletion is critical for a gain-of-function effect. The expression of *WTK3* is induced post-inoculation with Bgt and appears to be an upstream regulator of PR genes involved in plant defense.

The identification of *Pm24* as a tandem kinase gene expands the range of resistance mechanisms beyond the common NLR proteins. The finding of a rare 6-bp deletion conferring resistance provides insight into the evolution of disease resistance in wheat and the potential for durable resistance. The specific requirement of the lysine-glycine deletion for resistance suggests a precise interaction between WTK3 and other proteins in the defense signaling pathway. The conservation of WTK3 homologs in other grass species suggests an ancestral role in defense mechanisms. The low prevalence of the resistance allele in global wheat germplasm underscores the importance of preserving landraces for future breeding efforts.

This research successfully identified and characterized *Pm24*, a rare gain-of-function allele encoding a tandem kinase (WTK3) that confers broad-spectrum resistance to powdery mildew in wheat. The 6-bp deletion in the Kin I domain is critical for the resistance function. The findings highlight the potential of tandem kinases in plant innate immunity and the importance of landrace conservation for crop improvement. Future research should focus on elucidating the precise molecular mechanism of WTK3 action and utilizing this knowledge for developing resistant wheat cultivars via MAS and genome editing.

The study focused primarily on a single Bgt isolate (E09) for most experiments. While the resistance allele showed effectiveness against 93 isolates, testing against a broader range of globally diverse Bgt isolates would strengthen the findings. Further investigations into the interaction partners of WTK3 and the downstream signaling pathway are required to fully understand the mechanism of resistance. Finally, the study did not assess the potential pleiotropic effects associated with expressing the *Pm24* gene.