A Golgi vesicle-membrane-localized cytochrome B561 regulates ascorbic acid regeneration and confers Verticillium wilt resistance in cotton.

Ascorbic acid (AsA) serves as a key antioxidant involved in the various physiological processes and against diverse stresses in plants. Due to the insufficiency of AsA de novo biosynthesis, the AsA regeneration is essential to supplement low AsA synthesis rates. Redox reactions play a crucial role in response to biotic stress in plants; however, how AsA regeneration participates in hydrogen peroxide (HO) homeostasis and plant defense remains largely unknown. Here, we identified a Golgi vesicle-membrane-localized cytochrome B561 (CytB561) encoding gene, GhB561-11, involved in AsA regeneration and plant resistance to Verticillium dahliae in cotton. GhB561-11 was significantly downregulated upon V. dahliae attack. Knocking down GhB561-11 greatly enhanced cotton resistance to V. dahliae. We found that suppressing GhB561-11 inhibited the AsA regeneration, elevated the basal level of HO, and enhanced the plant defense against V. dahliae. Further investigation revealed that GhB561-11 interacted with the lipid droplet-associated protein GhLDAP3 to collectively regulate the AsA regeneration. Simultaneously silencing GhB561-11 and GhLDAP3 significantly elevated the HO contents and dramatically improved the Verticillium wilt resistance in cotton. The study broadens our insights into the functional roles of CytB561 in regulating AsA regeneration and HO homeostasis. It also provides a strategy by downregulating GhB561-11 to enhance Verticillium wilt resistance in cotton breeding programs.