Secreted Glycoside Hydrolase BcGH61 From Botrytis cinerea Induces Cell Death by the Apoplastic Location and Triggers Intracellular Immune Perception.

Cell death-inducing proteins (CDIPs) are critical mediators of infection in the necrotrophic pathogen Botrytis cinerea, enabling host colonisation across its broad plant host range. Here, we identified a novel plant CDIP from B. cinerea, BcGH61, that exhibits dual activity: triggering extensive plant cell death while simultaneously promoting host defence responses. Functional analysis demonstrates that its full cell death-inducing capacity requires its apoplastic compartmentalisation, intact glycoside hydrolase activity, two conserved cysteine residues, and specific structural motifs spanning residues 193-240. Notably, this phytotoxic activity operates through a mechanism independent of the canonical immune coreceptors BAK1 and SOBIR1. During pathogenic invasion, bcgh61 demonstrates upregulated expression. While the bcgh61 gene deletion mutant displays no significant developmental defects, it exhibits severely attenuated virulence, establishing BcGH61 as an essential pathogenicity factor. Furthermore, we identified NbHrBP1, a Nicotiana benthamiana hypersensitive response-binding protein, as a direct interactor of BcGH61 during infection, functioning in intracellular immune perception but not in cell death induction. Taken together, our results provide evidence that BcGH61 is a novel critical virulence factor for establishing infection in host plant by its phytotoxic activity and underscore the role of NbHrBP1-based plant surveillance system in recognising fungal secreted proteins as a pivotal defence strategy against necrotrophic pathogens.