Nélida Brito

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed–Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

The genome sequencing of Botrytis cinerea has supplied a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data have revealed that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) have been characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we have undertaken its functional characterization in the life cycle of the fungus. A null mutant ΔBcstc3 and an overexpressed–Bcstc3 transformant (OvBcstc3) were generated, and both strains show deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5, Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. Phenotypic characterization of both transformants revealed that BcStc3 is involved in oxidative stress tolerance, production of reactive oxygen species and virulence. The metabolomic analysis allowed the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described were identified.

BcIEB is a small protein secreted by the phytopathogenic fungus Botrytis cinerea that is recognized as a pathogen‐associated molecular pattern (PAMP) by plants. This activity is mapped to a highly conserved region of 35 amino acids, the peptide ieb35. Moreover, it has been shown that the protein and the peptide induce systemic resistance to B . cinerea after their infiltration into tobacco leaves. In this work, the possible use of BcIBE1 or ieb35 as plant protective agents has been tested. Tobacco plants were sprayed, infiltrated, or treated at the seedling stage with the protein or the peptide, and plant susceptibility to pathogens with different lifestyles was then studied at various times after treatment. The results showed that both BcIEB1 and ieb35 caused a similar reduction in the lesion sizes caused by B . cinerea and in colonization by Pseudomonas syringae pv. tabaci (reduced by c. 30% and c. 40%, respectively), independently of the treatment type. In addition, the number of B . cinerea inoculations leading to successful infections was also reduced in plants infiltrated or sprayed with BcIEB1/ieb35. The defence‐related genes PR1a , NPR1 , and osmotin ap24 were all induced by the protein and the peptide. BcIEB1, and especially ieb35, may be considered as a potential environmentally friendly treatment to increase plant resistance to pathogens.

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.

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.

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 generation of knock-out mutants in fungal pathogens by gene replacement and insertional mutagenesis is the classical method to validate virulence factors. An alternative strategy consists of silencing the candidate virulence gene by making use of the phenomenon of RNA interference (RNAi), adding features such as the possibility of generating knock-down mutants with variable expression levels of the target gene or the ability to simultaneously target multiple genes. Two different approaches have been assayed to generate knock-down mutants by RNAi in the phytopathogenic fungus Botrytis cinerea. In the first one, the single nitrate reductase gene in the B. cinerea genome, niaD, was silenced by transformation with a construct containing a 400-bp niaD fragment between two opposing promoters, so that a dsRNA fragment was generated. As an alternative approach, the mgfp4 gene coding for the green fluorescent protein (GFP) was silenced by transforming two different GFP-expressing strains of B. cinerea with a hairpin RNA (hpRNA)-expressing vector, containing two inverted copies of a 300-bp mgfp4 fragment separated by a spacer DNA. While the opposing dual-promoter strategy produced gene silencing in about half of the transformants assayed, the efficiency of the hpRNA-expressing vector was higher, inducing a decrease in GFP levels in more than 90 % of transformants. The degree of silencing achieved was high with both methods, but the hpRNA strategy resulted in a higher proportion of strongly silenced transformants.