Feruloyl-CoA 6′-hydroxylase-mediated scopoletin accumulation enhances cotton resistance to <i>Verticillium dahliae</i>

Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and substantially reduce cotton (Gossypium hirsutum) yield and quality. Scopoletin, a natural coumarin, exhibits antifungal activity against V. dahliae; however, the mechanisms of action remain unclear. In this study, we reveal the regulatory activities of feruloyl-CoA 6'-hydroxylase 1 (GhF6'H1) in enhancing V. dahliae resistance by modulating scopoletin accumulation. Silencing GhF6'H1, encoding the pivotal enzyme in scopoletin biosynthesis, through virus-induced silencing resulted in increased susceptibility to V. dahliae and decreased scopoletin accumulation. In transgenic cotton plants expressing GhF6'H1 under the CaMV 35S promoter, GhF6'H1 modulated scopoletin accumulation, affecting cotton resistance to V. dahliae, with increased resistance associated with increased scopoletin accumulation. GhF6'H1 has been identified as a direct target of the transcription factor GhWRKY33-like, indicating that GhWRKY33-like can bind to and activate the GhF6'H1 promoter. Moreover, GhWRKY33-like overexpression in cotton-enhanced resistance to V. dahliae through scopoletin accumulation, phenylpropanoid pathway activation, and upregulation of defense response genes. Ectopic expression of GhF6'H1 resulted in effective catalysis of scopoletin synthesis in enzyme assays using substrates like feruloyl coenzyme A, while molecular docking analysis revealed specific amino acid residues playing crucial roles in establishing salt-bridge interactions with the substrate. These findings suggest that GhF6'H1, regulated by GhWRKY33-like, plays a crucial role in enhancing cotton resistance to V. dahliae by modulating scopoletin accumulation.