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Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.

A gene was cloned from Botrytis cinerea that encodes a protein homologous to glutathione S-transferase (GST). The gene, denominated Bcgst1, is present in a single copy and represents the first example of such a gene from a filamentous fungus. The biochemical function of GSTs is to conjugate toxic compounds to glutathione, thereby detoxifying the compound. In many other organisms, GST plays a role in chemical stress tolerance. We anticipated that GST functions for B. cinerea as a potential virulence factor, enabling the fungus to tolerate fungitoxic plant defence compounds. The expression of Bcgst1 mRNA under various presumably stressful conditions was investigated. Bcgst1 mRNA is expressed at a basal level in liquid cultures and is induced upon addition of hydrogen peroxide to the medium. The level of Bcgst1 mRNA expression during infection of tomato leaves parallels the level of actin mRNA. The role of the Bcgst1 gene in the virulence of Botrytis cinerea was evaluated by constructing gene disruption mutants. Three independent disruption mutants were obtained. The virulence of two mutants on tomato leaves was evaluated. Neither of the mutants showed a decrease in virulence, indicating that the Bcgst1 gene is not essential for virulence on tomato leaves under the conditions tested.

To identify genes of the necrotrophic pathogenic fungus Botrytis cinerea that are expressed during infection of tomato leaves, a differential screening of a genomic library with radioactively labelled cDNA was performed. This resulted in the identification of a B. cinerea gene, denominated Bcubi4, which encodes a precursor protein consisting of four identical head-to-tail repeats of a 76 aa ubiquitin unit. Subsequently a gene denominated Bcubi1CEP79, encoding a single ubiquitin unit joined to a Carboxyl Extension Protein of 79 amino acids, was isolated. The expression of the two ubiquitin genes was studied during pathogenesis of B. cinerea on tomato. Bcubi1CEP79, but not Bcubi4, mRNA was transiently induced at 16 h after inoculation. The increased expression of the Bcubi1CEP79 gene at this stage of pathogenesis might be required for enhanced ribosomal biogenesis.

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgip1 gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.

SUMMARY Botrytis cinerea is a necrotrophic pathogen that produces an array of enzymes capable of attacking the plant cell wall components. We have previously shown that growth of the fungus in planta is accompanied by the degradation of pectin and that endopolygalacturonase (Bcpg) genes are expressed during infection of different plant tissues. It was assumed that pectin demethylation by pectin methylesterases (PME) was essential for the subsequent depolymerization by BcPGs to occur efficiently. We report here on the functional analysis of two Bcpme genes in strain B05.10, using a gene-replacement approach. The method used for the generation of constructs for gene replacement in B. cinerea circumvents the need for cloning and yielded a high proportion of homologous recombinants. Mutants lacking both Bcpme genes are not affected in their growth on highly methylated pectin, nor did they show any reduction in virulence. The results suggest that B. cinerea strain B05.10 can efficiently degrade pectin without prior demethylation.