Plants activate defense responses against a possible pathogen once pattern-recognition receptors (PRRs) perceive the presence of pathogen-associated molecular patterns (PAMPs). Glycosyl hydrolase family 11 (GH11) endoxylanases from Trichoderma, Fusarium and Botrytis species have been described as being able to induce the defense response in plants, in a way that is independent of its enzymatic activity. However, until now, it has not been possible to establish with certainty which regions of these enzymes are recognized by plants as PAMPs. We show here for the first time that a short 25-residue peptide (named Xyn25) from the Botrytis cinerea xylanase BcXyn11A can reproduce by itself all the effects observed for the treatment of plants with the whole BcXyn11A protein. These include necrosis on leaves, seedling growth inhibition, induction of a ROS burst, electrolyte leakage, cytoplasm shrinkage, autofluorescence, cell death, and induction of defense genes. Two highly conserved four-amino acid regions within Xyn25 were shown to be necessary for the elicitation activity by substituting them with tracts of four alanine residues.

BcSUN1 is a glycoprotein secreted by Botrytis cinerea, an important plant pathogen that causes severe losses in agriculture worldwide. In this work, the role of BcSUN1 in different aspects of the B. cinerea biology was studied by phenotypic analysis of Bcsun1 knockout strains. We identified BcSUN1 as the only member of the Group-I SUN family of proteins encoded in the B. cinerea genome, which is expressed both in axenic culture and during infection. BcSUN1 is also weakly attached to the cellular surface and is involved in maintaining the structure of the cell wall and/or the extracellular matrix. Disruption of the Bcsun1 gene produces different cell surface alterations affecting the production of reproductive structures and adhesion to plant surface, therefore reducing B. cinerea virulence. BcSUN1 is the first member of the SUN family reported to be involved in the pathogenesis of a filamentous fungus.

The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B) were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.