Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction.

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 Vvpgipl 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.

Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris.

Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V(max,app) ranged from 10 to 900 U mg(-1), while the K(m,app) ranged from 0.16 to 0.6 mg ml(-1). Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.

Functional analysis of Botrytis cinerea pectin methylesterase genes by PCR-based targeted mutagenesis: Bcpme1 and Bcpme2 are dispensable for virulence of strain B05.10.

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.

Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase.

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.

Functional analysis of an extracellular catalase of Botrytis cinerea.

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.