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

TL;DR

This study shows that phytotoxins botrydial and botcinic acid are crucial for the virulence of the fungus Botrytis cinerea, and their absence can be partially compensated by changing the inoculation medium.

Contribution

The study demonstrates the significant role of botrydial and botcinic acid in B. cinerea virulence and the impact of inoculation conditions on host-pathogen interactions.

Findings

A double mutant lacking botrydial and botcinic acid showed reduced virulence on tomato leaves in minimal medium.

Adding yeast extract to the medium restored the mutant's virulence.

Overexpression of four effector genes did not recover full virulence in the absence of yeast extract.

Abstract

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 ∆bot2∆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, the 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 ∆bot2∆boa6 double mutant. Four genes encoding cell death-inducing effector proteins were upregulated in B. cinerea by the addition of yeast extract, and they were members of a co-expression module that was correlated with the compatible interaction. Functional analyses of these effector genes were performed by overexpressing them individually in the ∆bot2∆boa6 background, followed by disease assays with the Gamborg B5 medium without yeast extract. The overexpression of these four effector genes, individually, did not result in recovery of the expansion of lesions in the absence of yeast extract.

The gray mold 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 using different inoculation media. Supplementation by overexpression of distinct cell death-inducing proteins could not restore the full virulence. This work highlights the pivotal roles of these phytotoxins as compared with other virulence factors, as well as the significant impact of inoculation conditions on compatible and incompatible interactions between the fungus and its hosts.

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