A module with multiple transcription factors positively regulates powdery mildew resistance in grapevine.

Erysiphe necator, the causal agent of powdery mildew (PM), is a fungal pathogen that can severely reduce crop yields. In this study, stable overexpression and silencing demonstrated that the transcription factor VqWRKY46 was identified as a positive regulator of PM resistance in grapevine. This enhanced resistance was associated with increased callose deposition and hypersensitive response cell death. We also demonstrated that VqLIMYB specifically activated VqWRKY46 expression by binding to a CAGTTA motif in its promoter. Moreover, VqWRKY46 interacted with VqNF-YC9 and formed homodimers, positively regulating VqNF-YC9 expression by directly binding to three W-box motifs in its promoter. Additionally, DAP-Seq analysis identified VqDSC1, a TIR-NB-LRR resistance protein, as a downstream target of VqWRKY46 in grapevine. Further analysis indicated that VqWRKY46 directly bound to Wbox1 in the VqDSC1 promoter to upregulate its expression, whereas VqNF-YC9 did not bind. Interestingly, the interaction between VqNF-YC9 and VqWRKY46 enhanced the DNA binding activity of VqWRKY46 to promote the expression of VqDSC1. Furthermore, overexpressing VqLIMYB, VqNF-YC9, and VqDSC1 in Arabidopsis and/or instantaneous transformation in grapes showed that they are positive regulators of PM resistance. Collectively, our findings uncover a novel regulatory module (VqLIMYB-VqWRKY46/VqNF-YC9-VqDSC1) that mediates grapevine resistance to E. necator. This study provides new insights into the molecular basis of grapevine resistance to PM and highlights potential targets for breeding disease-resistant grapevines.