Abstract
Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious, but infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB-1). By deletion and over-expression of bcltf1, we confirmed the predicted role of the transcription factor in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 light-responsive genes, and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. In addition, the deletion of bcltf1 affects the expression of 1,539 genes irrespective of the light conditions, including the overexpression of known and so far uncharacterized secondary metabolism-related genes. Increased expression of genes encoding alternative respiration enzymes, such as the alternative oxidase (AOX), suggest a mitochondrial dysfunction in the absence of bcltf1. The hypersensitivity of Δbctlf1 mutants to exogenously applied oxidative stress - even in the absence of light - and the restoration of virulence and growth rates in continuous light by antioxidants, indicate that BcLTF1 is required to cope with oxidative stress that is caused either by exposure to light or arising during host infection.
Highlights
Botrytis cinerea Persoon: Fries (teleomorph Botryotinia fuckeliana Whetzel) is a necrotrophic pathogen with a broad host range causing gray mold disease in several economically important plants including grape vine and strawberry [1,2]
We provide evidence that an unbalanced reactive oxygen species (ROS) homoeostasis caused by the deletion of the lightresponsive transcription factor BcLTF1 impairs the ability of the necrotrophic pathogen Botrytis cinerea to grow in the presence of additional oxidative stress arising during illumination or during infection of the host
To identify genes in B. cinerea that are associated with the infection process, 2,367 hygromycin-resistant mutants obtained through A. tumefaciens-mediated transformation (ATMT) were screened for virulence on detached tomato leaves
Summary
Botrytis cinerea Persoon: Fries (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a necrotrophic pathogen with a broad host range causing gray mold disease in several economically important plants including grape vine and strawberry [1,2]. Sources of infection by B. cinerea are the conidia that are ubiquitously distributed in the air. After landing on the plant surface, the conidia germinate and form short germ tubes which directly penetrate. Infection may occur from already established mycelia, in this particular case, multicellular infection structures (infection cushions) are formed. After penetration the epidermal and underlying cells die and B. cinerea establishes a primary (restricted) infection. The fungus starts a massive outgrowth (spreading) that results in the maceration of the plant tissue (soft rot) and formation of conidia for new infections. The fungus produces phytotoxic metabolites e.g. botrydial (BOT) and botcinic acid (BOA), necrosisinducing proteins, reactive oxygen species (ROS), cell walldegrading enzymes, peptidases and phytohormones [1,3,4,5]. Gene replacement approaches have identified only a few virulence factors to date, probably because of their redundancy [6,2]
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