Summary• Proteins belonging to the cerato-platanin family are small proteins with phytotoxic activity. A member of this family, BcSpl1, is one of the most abundant proteins in the Botrytis cinerea secretome.• Expression analysis of the bcspl1 gene revealed that the transcript is present in every condition studied, showing the highest level in planta at the late stages of infection. Expression of a second cerato-platanin gene found in the B. cinerea genome, bcspl2, was not detected in any condition.• Two bcspl1 knock-out mutants were generated and both showed reduced virulence in a variety of hosts.• bcspl1 was expressed in Pichia pastoris and the recombinant protein was able to cause a fast and strong necrosis when infiltrated in tomato, tobacco and Arabidopsis leaves, in a dose-dependent manner. The BcSpl1-treated plant tissues showed symptoms of the hypersensitive response such as induction of reactive oxygen species, electrolyte leakage, cytoplasm shrinkage, and cell autofluorescence, as well as the induction of defense genes considered to be markers of the hypersensitive response. The Arabidopsis bak1 mutation partially prevented the induction of necrosis in this plant by BcSpl1. Two different BcSpl1-derived 40-amino acids peptides were also active in inducing necrosis.

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.

BackgroundThe Botrytis cinerea xylanase Xyn11A has been previously shown to be required for full virulence of this organism despite its poor contribution to the secreted xylanase activity and the low xylan content of B. cinerea hosts. Intriguingly, xylanases from other fungi have been shown to have the property, independent of the xylan degrading activity, to induce necrosis when applied to plant tissues, so we decided to test the hypothesis that secreted Xyn11A contributes to virulence by promoting the necrosis of the plant tissue surrounding the infection, therefore facilitating the growth of this necrotroph.ResultsWe show here that Xyn11A has necrotizing activity on plants and that this capacity is conserved in site-directed mutants of the protein lacking the catalytic activity. Besides, Xyn11A contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity, as the catalytically-impaired Xyn11A variants were able to complement the lower virulence of the xyn11A mutant. The necrotizing activity was mapped to a 30-amino acids peptide in the protein surface, and this region was also shown to mediate binding to tobacco spheroplasts by itself.ConclusionsThe main contribution of the xylanase Xyn11A to the infection process of B. cinerea is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates binding to plant cells.

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low-molecular weight plant compounds, and we have collected the proteins secreted during the first 16 hours by a double precipitation protocol. 2D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by liquid chromatography-tandem mass spectrometry of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

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