Multi-knockout of 29 phytotoxic proteins and metabolites does not completely abolish virulence of the necrotrophic fungus 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. Host killing involves secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs), but their relative contributions are poorly understood. 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 identified additional CDIPs and generated an unprecedented series of multi-gene deletion mutants in a filamentous fungus, culminating in a 29x mutant carrying deletions of 27 CDIP-encoding genes and two genes required for the biosynthesis of the phytotoxins botrydial and botcinic acid. These multi-k.o. mutants were strongly reduced in virulence and almost unable to infect apple fruit tissue, but still induced slowly expanding necrosis on leaves, demonstrating that additional determinants of host killing remain to be identified. 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 of them only small or no significant contributions to pathogenesis. Generation of a mutant lacking all six predicted endo-polygalacturonases confirmed only for PG1 and PG2 a major role for cell wall degradation and infection. Our work demonstrates that necrotrophic pathogenesis in B. cinerea does not depend on a few primary virulence determinants, but rather on a highly redundant network of host damaging factors.