Genome-wide characterization of the WAK gene family in Triticum aestivum identifies TaWAK116 conferring resistance to powdery mildew

Wall-associated kinases (WAKs) are critical regulators of plant responses to biotic stress; however, the functions of most WAK family genes in wheat immune responses remain poorly characterized. In this study, we systematically identified 449 TaWAK genes in wheat at the genome-wide scale. These genes exhibited uneven chromosomal distribution and were phylogenetically divided into four distinct clades. Synteny analysis indicated that gene duplication events served as the major driving force for the expansion of the TaWAK gene family. Our analysis showed that 114 TaWAK genes were significantly differentially expressed in response to Blumeria graminis f. sp. tritici ( Bgt ) infection. Quantitative real-time PCR (qRT-PCR) analysis validated the transcriptomic results, with TaWAK116 exhibiting the largest fold change in expression upon Bgt infection. Moreover, knockdown of TaWAK116 via barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) significantly compromised wheat resistance to Bgt . Therefore, TaWAK116 was identified as a key candidate gene for functional characterization. Subsequent functional validation using stable overexpression and RNA interference (RNAi) transgenic lines confirmed that TaWAK116 positively regulates wheat resistance to powdery mildew, with pathogenesis-related ( PR ) genes significantly upregulated in TaWAK116-overexpressing plants but downregulated in TaWAK116-silenced lines upon Bgt infection. Yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) assays identified TIFY as an interacting protein of TaWAK116, which is a key negative regulator of JA signaling. Transient silencing of TIFY via spray-induced gene silencing (SIGS) significantly enhanced wheat resistance to powdery mildew. qRT-PCR analysis further revealed that the expression of TIFY and JA biosynthesis-related genes was significantly repressed in TaWAK116 -overexpressing wheat lines. In addition, transient overexpression of TaWAK116 induced hypersensitive response (HR)-like cell death in Nicotiana benthamiana leaves, and this response was suppressed by co-expression of TIFY . Collectively, this study provides a comprehensive genome-wide characterization of the wheat WAK family and demonstrates that TaWAK116 positively modulates wheat powdery mildew resistance by interacting with the JA signaling repressor TIFY. These findings not only provide valuable genetic resources for identifying disease resistance genes in wheat but also offer novel insights into the molecular mechanisms underlying WAK -mediated innate immunity in cereals.