The Botrytis cinerea transglycosylase BcCrh4 is a cell death-inducing protein with cell death-promoting and -suppressing domains.

Botrytis cinerea is a necrotrophic fungal plant pathogen that causes grey mould and rot diseases in many crops. Here, we show that the B. cinerea BcCrh4 transglycosylase is secreted during plant infection and induces plant cell death and pattern-triggered immunity (PTI), fulfilling the characteristics of a cell death-inducing protein (CDIP). The CDIP activity of BcCrh4 is independent of the transglycosylase enzymatic activity, it takes place in the apoplast and does not involve the receptor-like kinases BAK1 and SOBIR1. During saprophytic growth, BcCrh4 is localized in the endoplasmic reticulum and in vacuoles, but during plant infection, it accumulates in infection cushions (ICs) and is then secreted to the apoplast. Two domains within the BcCrh4 protein determine the CDIP activities: a 20aa domain at the N' end activates intense cell death and PTI, while a stretch of 52aa in the middle of the protein induces a weaker response and suppresses the activity of the 20aa N' domain. Deletion of bccrh4 affected fungal development and IC formation in particular, resulting in reduced virulence. Collectively, our findings demonstrate that BcCrh4 is required for fungal development and pathogenicity, and hint at a dual mechanism that balances the virulence activity of this, and potentially other CDIPs.