Cotton GhXEGIP1 confers resistance to Verticillium dahliae by inhibiting of fungal VdEG1 and the salicylic acid signaling pathway.

Verticillium dahliae is a widespread and destructive soilborne fungus that causes vascular wilt disease, significantly reducing cotton (Gossypium hirsutum) yield and quality. Cotton's xyloglucan-specific endoglucanase inhibitor protein (GhXEGIP1) has demonstrated effectiveness against the fungal glycoside hydrolase VdEG1, a member of the glycoside hydrolase family 12. However, the mechanisms underlying GhXEGIP1's defense against V. dahliae remain unclear. This study provides evidence that GhXEGIP1 are instrumental in plant responses to V. dahliae infection. GhXEGIP1 interacts with and regulates the expression of VdEG1. Docking models and molecular dynamics simulations revealed that a conserved arginine residue in GhXEGIP1 penetrates VdEG1's active site, interacting with its catalytic glutamate, resulting in a stable inhibitor-enzyme complex with lower binding free energy compared to that of the other glycoside hydrolases. Ectopic expression and purification of GhXEGIP1 further revealed its inhibitory effect on VdEG1's hydrolytic activity. GhXEGIP1-silenced cotton exhibited increased fungal biomass accumulation and heightened susceptibility to V. dahliae, along with a disproportionate oxidative burst. Transgenic Arabidopsis plants expressing GhXEGIP1 under the CaMV 35S promoter demonstrated reduced VdEG1 activity, enhanced resistance to V. dahliae, and decreased disease symptoms, including chlorosis and wilting. These transgenic plants also showed enhanced xylem lignification, likely contributing to limiting V. dahliae spread. Further analysis suggested that GhXEGIP1 may be involved in the transcriptional activation of defense-related genes and participate in salicylic acid-mediated defense pathways. These findings suggest that GhXEGIP1 is a potential defense gene against V. dahliae in cotton.