Loss of virulence of Botrytis cinerea mutants defective in phytotoxin production is restored by modifying inoculation medium

Botrydial, botcinic acid and their derivatives are major phytotoxic metabolites produced by the necrotrophic fungal pathogen Botrytis cinerea. These phytotoxins are able to induce programmed cell death in the host and thereby promote plant susceptibility to B. cinerea. We observed that a{Delta} bot2{Delta}boa6 double mutant strain, which synthesizes neither botrydial nor botcinic acid, was almost avirulent on tomato leaves when the disease assay was performed using synthetic minimal Gamborg B5 medium. However, virulence of this mutant was restored when the inoculation medium was supplemented with yeast extract. Further virulence assays which compared the double mutant with other multiple mutants using both inoculation media, revealed a prominent contribution of botrydial and botcinic acid to the full virulence of B. cinerea. Therefore, we performed an RNA-sequencing experiment to identify B. cinerea genes that contribute to the phenotypic switch from an "incompatible" to a "compatible" interaction between tomato and the{Delta} bot2{Delta}boa6 double mutant. Four genes encoding cell death-inducing effector proteins were upregulated in B. cinerea by the addition of yeast extract, and their transcript profiles grouped within a co-expression module that was positively correlated with the compatible interaction. Functional analyses of these effector genes were performed by overexpressing them individually in the{Delta} bot2{Delta}boa6 background, followed by disease assays with the Gamborg B5 medium without yeast extract. ImportanceThe grey mould fungus Botrytis cinerea is a model for necrotrophic plant pathogens due to its wide host range, economic impact, well-assembled genome, and versatile mechanisms for inducing host cell death during colonization. Botrydial and botcinic acid have previously been characterized as major phytotoxins produced by B. cinerea. However, studies from different groups reported variable results regarding the contributions of these phytotoxins to fungal virulence. Here we demonstrate that botrydial and botcinic acid make a prominent contribution to the full virulence of B. cinerea, by performing infection assays with mutants that are defective in phytotoxin production and/or multiple cell death-inducing proteins using different inoculation media. This work highlights the pivotal roles of these phytotoxins as compared to other virulence factors, as well as the significant impact of inoculation conditions on compatible and incompatible interactions between the fungus and its hosts.