Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation.

The functional characterization of the FKBP12 encoding gene from the phytopathogenic fungus Botrytis cinerea was carried out. B. cinerea genome sequence owns a single ortholog, named BcFKBP12, encoding a FK506-binding protein of 12kDa. BcFKBP12 mediates rapamycin sensitivity both in B. cinerea and in Saccharomyces cerevisiae, a property unique to FKBP12 proteins, probably via the inhibition of the protein kinase TOR (target of rapamycin). The relative abundance of the prolyl isomerase appeared to be regulated and increased in response to the presence of extracellular nutrients. Surprisingly, the BcFKBP12 deletion did not affect the pathogenic development of the strain B05.10, while it was reported to cause a reduction of the virulence of the strain T4. We report for the first time the BcFKBP12 involvement in the sulfur repression of the synthesis of a secreted serine protease. Rapamycin treatment did not relieve the sulfur repression of the reporter system in the wild-type strain. Thus BcFKBP12 may participate in sulfur regulation and its contribution seems to be independent of TOR.

Characterization of PG2, an early endoPG produced by Sclerotinia sclerotiorum, expressed in yeast.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated. We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons. The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified. Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5. Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer. This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action. These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Involvement of cyclic AMP in the production of the acid protease Acp1 by Sclerotinia sclerotiorum.

The acid protease Acp1 is produced by Sclerotinia sclerotiorum during plant infection. We explored the mechanism involved in the triggering of that production and found that cyclic AMP played a positive role. Acp1 could be produced in the sole presence of exogenous cyclic AMP. The use of molecules known to increase or decrease the intracellular cyclic AMP levels confirmed the impact of this nucleotide on the protease production and suggested its endogenous site of action. Further pharmacological studies showed the specific effect of cyclic AMP on Acp1 production and suggested that protein kinase A would be its likely target. Together, these results provide the first indication that the production of a pathogenesis-related fungal protease could depend on a cyclic AMP/Protein kinase A signalling pathway.

Ambient pH controls the expression of endopolygalacturonase genes in the necrotrophic fungus Sclerotinia sclerotiorum.

In the necrotrophic fungus Sclerotinia sclerotiorum, secretion of polygalacturonases (PGs) and decrease of the environmental pH via oxalic acid production are considered as the main pathogenicity determinants. In order to evaluate the relationship between these two aspects of the infection process, we analyzed the expression of the endoPG-encoding genes pg1-3. Transcription of pg1-3 was not carbon regulated but was strictly controlled by pH and highly favored in a narrow range of acidic pH. During plant infection, a pH gradient was established in relation to oxalic acid secretion. Transcripts of pg1-3 were localized to the zone of colonization of healthy tissues while transcripts of genes encoding other lytic enzymes were restricted to the more acidic zones of the infected tissues. Our results show that progressive acidification of the ambient medium by the fungus is a major strategy for the sequential expression of pathogenicity factors.

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by Sclerotinia sclerotiorum.

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on (13)C- and (31)P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon-nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.

Carbon and pH modulate the expression of the fungal glucose repressor encoding genes.

The phytopathogenic necrotrophic fungus Sclerotinia sclerotiorum secretes large amounts of oxalic acid and cell wall-degrading enzymes (CWDE). Enzyme synthesis is pH dependent and subject to carbon catabolite repression. Because of the possible involvement of the glucose repressor CRE1 in the transcriptional control of the CWDE encoding genes, we studied crel expression with respect to the nature of the extracellular carbon source and the value of the external pH. Comparative analysis of the cre1 homologous gene creA from Aspergillus nidulans was performed with pH-deregulated mutants. The data obtained showed that ambient pH controls the expression level of the carbon repressor genes, and this control does not depend on the pH transcription factor PacC.

Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, secretes multiple pectinolytic enzymes that facilitate penetration, colonization, and maceration of the plant tissues. Molecular analysis has previously revealed that the pectinolytic system of the fungus is organized as a multigene family, among which a subfamily of three members encoding for neutral endopolygalacturonase (endoPG) isoforms has been characterized. Here we describe the isolation and characterization of three additional endoPG-encoding genes ( pg5, pg6, and pg7) that belong to distinct phylogenetic groups. Pairwise sequence comparison between the known endoPGs from S. sclerotiorum revealed 43% to 97% identity, and the genomic organization of the pectinolytic system showed a great similarity to that of the related necrotroph Botrytis cinerea. During plant pathogenesis, a sequential expression of the endoPG-encoding genes was shown.

CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica.

PCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsici and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.

Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences.

The Agrobacterium tumefaciens-mediated transfer of foreign DNA to the phytopathogenic fungus Botrytis cinerea was investigated. Fifteen stable transformants per 10(6) conidia were consistently produced. Monokaryons were purified in a single step and their molecular analysis demonstrated the random integration of predominantly single or tandem copies of the foreign DNA into their genome. Thermal asymmetric interlaced PCR performed directly on conidia led to the rapid identification of the genomic DNA sequences that flanked the integration sites of the transfer-DNA. Transcriptional fusions of green fluorescent protein and beta-glucuronidase-encoding genes to the promoter of the secreted proteolytic enzyme ACP1 were realised to validate the system. We provide herein observations of B. cinerea hyphae producing green fluorescent protein or beta-glucuronidase under growth conditions similar to those known to induce transcription of the acp1 gene.

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags.

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessor's sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5' or 3' flanking regions.

Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum.

When grown in the presence of sunflower cell walls, Sclerotinia sclerotiorum, an ubiquitous necrotrophic fungus, secretes several acid proteases including a non-aspartyl protease. The gene acp1, encoding an acid protease, has been cloned and sequenced. The intronless ORF encodes a preproprotein of 252 aa and a mature protein of 200 residues. In vitro expression of acp1 is subject to several transcriptional regulatory mechanisms. Expression induced by plant cell-wall proteins is controlled by both carbon and nitrogen catabolite repression. Glucose on its own represses acp1 expression while ammonium repression requires the simultaneous presence of a carbon source. Ambient pH higher than pH 5 overrides induction resulting in full repression of acp1. These transcriptional regulatory mechanisms and the presence of several motifs in the promoter of acp1 that may encode binding sites for the regulators CREA, AREA and PacC suggest the involvement of these regulators in the control of acp1 expression. acp1 is expressed in planta during sunflower cotyledon infection. Expression is low at the beginning of infection but increases suddenly at the stage of necrosis spreading. Comparison of in vitro and in planta acp1 expression suggests that glucose and nitrogen starvation together with acidification can be considered as key factors controlling Scl. sclerotiorum gene expression during pathogenesis.

The properties and localization of Saprolegnia monoica chitin synthase differ from those of other fungi.

The presence of non-fibrillar alpha-chitin in cellulosic fungi (class Oomycetes) poses intriguing questions as to its role, subcellular localization and evolutionary significance. Previous studies reported on the similarity of chitin synthase from Saprolegnia monoica with that of other fungi. The present work describes important dissimilarities. There was no evidence that the chitin synthase of S. monoica was present in small low-density vesicles (chitosomes). Chitin synthase sedimented with membranous components of high specific gravity (sp. gr. 1.177) that could be partially but distinctly separated from membranes harbouring most of the 1,3-beta-glucan synthase in the cell (sp. gr. 1.158). In contrast to other fungi, the chitin synthase from S. monoica was greatly stimulated by digitonin: both membrane-bound and dissociated chitin synthase showed little activity in the absence of digitonin. As in other fungi, the chitin synthase from S. monoica was solubilized by digitonin and remained zymogenic after dissociation. However, unlike the enzyme from other fungi, the solubilized chitin synthase of S. monoica had a lower sedimentation coefficient, was not stimulated by phospholipids and was not inhibited by high concentrations of digitonin. Unlike the enzyme from Mucor rouxii, the solubilized chitin synthase from S. monoica did not bind to a cation exchanger. The enzyme was partially purified by four-step scheme that included sucrose density-gradient centrifugation, a single passage through a strong anion exchanger and two consecutive passages through a weak anion exchanger. The final preparation contained five to seven polypeptide bands that cochromatographed with the chitin synthase activity, some of which may be part of a presumed chitin synthase macromolecular complex.