Deletion of 29 cell death-inducing proteins and phytotoxin biosynthetic genes does not completely abolish virulence of Botrytis cinerea

Botrytis cinerea is a necrotrophic plant pathogen with an extremely wide host range. During invasion, the fungus induces rapid host cell death and proliferates in the necrotic tissue. The mechanisms of host killing are still incompletely understood, they involve secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs). We have previously shown that the sequential knockout of up to 12 CDIPs leads to a substantial reduction of virulence of B. cinerea mutants. In this study, we have identified additional CDIPs and generated an extended mutant series culminating in a 29x mutant which is deficient in most currently known CDIPs and two phytotoxic metabolites. These mutants are strongly reduced in infection, but still induced necrosis and grey mould symptoms, demonstrating that additional determinants of host killing remain unidentified. Overexpression of the highly phytotoxic Nep1 in a 22-fold CDIP mutant failed to increase its virulence. Reevaluation of several CDIPs previously described as virulence factors revealed for most tested CDIPs no major contribution to pathogenesis. Together with the observation that none of the CDIPs are specific to B. cinerea, our data question a particular role of CDIPs for necrotrophic pathogenesis. In contrast, generation of a mutant lacking all six predicted endo-polygalacturonases confirmed their major but not exclusive role for tissue degradation and infection.