GhLYK5-mediated GhNIT4B activates asparagine and ROS accumulation enhancing cotton Verticillium wilt resistance

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive disease that significantly impairs cotton yield and fiber quality. Lysin motif (LysM) receptor-like kinases (LYKs), a subclass of plant pattern recognition receptors, are crucial for perceiving pathogen-derived molecules and activating immune responses. Here, we show that the transcription of GhLYK5, a gene encoding LYK protein in cotton, was rapidly upregulated following V. dahliae infection. Virus-induced gene silencing (VIGS) of GhLYK5 heightened cotton susceptibility to V. dahliae, whereas heterologous overexpression in Arabidopsis thaliana conferred enhanced resistance. RNA-seq analysis revealed that GhLYK5 silencing down-regulated defense-related genes and altered the expression of secondary metabolic pathways. Using yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), luciferase complementation imaging (LCI), and co-inmunoprecipitation (Co-IP) assays, we demonstrated that GhLYK5 physically interacted with the nitrilase GhNIT4B, with both proteins localizing to the plasma membrane. Silencing of GhNIT4B reduced asparagine accumulation, decreased ROS (H2O2, NO) levels, suppressed defense gene expression, and increased plant susceptibility to V. dahliae, indicating that GhNIT4B acted as a positive immune regulator. In addition, exogenous asparagine significantly inhibited the infection of V. dahliae by activating the expression of defense genes (GhBAK1, GhNPR1, GhPR1, GhLac1). Our findings identified the GhLYK5-GhNIT4B module as a key signaling node in cotton immune responses and provide genetic targets for improving Verticillium wilt resistance in upland cotton.