The 3R-MYB transcription factor VvMYB3R5 regulates Colletotrichum viniferum resistance through affecting proanthocyanidin synthesis in grapevine.

Grape ripe rot (Colletotrichum spp.), a significant fungal disease, causes substantial economic losses. Although multiple MYB transcription factors have been reported to be involved in plant disease resistance against ripe rot, the specific functions and mechanisms of 3R-MYB subfamily members in grape responses to fungal pathogen infection remain unknown. Here, we identified and functionally characterized a 3R-MYB transcription factor, VvMYB3R5, for the first time, revealing its crucial positive regulatory role in grape resistance to C. viniferum and in proanthocyanidin (PA) accumulation. We demonstrated that VvMYB3R5 is specifically induced by C. viniferum and that its overexpression significantly reshapes the defense-related metabolic network. Through the integration of multi-omics analysis, we elucidated that VvMYB3R5 overexpression activated the phenylpropanoid biosynthesis and plant-pathogen interaction pathways. Furthermore, we elucidated a novel regulatory module comprising VvMYB3R5-VvLAR1/VvLAR2. VvMYB3R5 directly binds to and activates the promoters of the key PA synthesis genes, VvLAR1 and VvLAR2, thereby driving the accumulation of PAs with in vitro antifungal activity and enhancing disease resistance. Taken together, these results revealed a previously overlooked 3R-MYB transcription factor disease resistance regulator, but more importantly, systematically clarified the novel mechanism by which it precisely programs the synthesis of defense metabolites through direct regulation of specific downstream genes, thereby enhancing immunity. The discovery of this VvMYB3R5-VvLARs module provides new insights into how plants coordinate metabolic defense through transcriptional reprogramming and offers a distinct genetic resource beyond conventional MYB targets for molecular breeding of disease-resistant grapes and potentially other crops.