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The proteolytic enzymes secreted by Sclerotinia sclerotiorum were characterized using mechanistic inhibitors, affinity chromatography and analytical electrophoresis. The proteolytic system mainly constituted of acidic proteases but a neutral protease (serine protease) was also found at the initial stage of plant infection by the fungus. In vitro and in planta protease profiles were shown to be similar. Properties of the enzymes produced were matched with the ambient pH indicating that the set of enzymes secreted reflected the environmental niche created during mycelial growth. [ABSTRACT FROM AUTHOR]

During pathogenesis on sunflower cotyledons, Botrytis cinerea and Sclerotinia sclerotiorum show a striking resemblance in symptom development. Based on pH change profiles, the colonization process of both fungi can be divided into two stages. The first stage is associated with a pH decrease, resulting from an accumulation of citric and succinic acids. The second stage is correlated with a pH increase, resulting from an accumulation of ammonia. In this article, we also report that oxalic acid is produced at the late stage of the colonization process and that ammonia accumulation is concomitant with a decrease in free amino acids in decaying tissues. Sclerotinia sclerotiorum produces eight-fold more oxalic acid and two-fold less ammonia than B. cinerea. Consequently, during sunflower cotyledon colonization by B. cinerea, pH dynamics differ significantly from those of S. sclerotiorum. In vitro assays support the in planta results and show that decreases in pH are linked to glucose consumption. At different stages of the colonization process, expression profiles of genes encoding secreted proteases were investigated. This analysis highlights that the expression levels of the B. cinerea protease genes are higher than those of S. sclerotiorum. This work suggests that the overt similarities of S. sclerotiorum and B. cinerea symptom development have probably masked our recognition of the dynamic and potentially different metabolic pathways active during host colonization by these two necrotrophic fungi., (© 2011 Bayer Crop Science. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.)

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

In order to redefine the mannitol pathway in the necrotrophic plant pathogen Botrytis cinerea, we used a targeted deletion strategy of genes encoding two proteins of mannitol metabolism, BcMTDH (B. cinerea mannitol dehydrogenase) and BcMPD (B. cinerea mannitol-1-phosphate dehydrogenase). Mobilization of mannitol and quantification of Bcmpd and Bcmtdh gene transcripts during development and osmotic stress confirmed a role for mannitol as a temporary and disposable carbon storage compound. In order to study metabolic fluxes, we followed conversion of labelled hexoses in wild-type and DeltaBcmpd and DeltaBcmtdh mutant strains by in vivo NMR spectroscopy. Our results revealed that glucose and fructose were metabolized via the BcMPD and BcMTDH pathways respectively. The existence of a novel mannitol phosphorylation pathway was also suggested by the NMR investigations. This last finding definitively challenged the existence of the originally postulated mannitol cycle in favour of two simultaneously expressed pathways. Finally, physiological and biochemical studies conducted on double deletion mutants (DeltaBcmpdDeltaBcmtdh) showed that mannitol was still produced despite a complete alteration of both mannitol biosynthesis pathways. This strongly suggests that one or several additional undescribed pathways could participate in mannitol metabolism in B. cinerea.

During pathogenesis, the ascomycete Botrytis cinerea secretes a range of cell-wall-degrading enzymes such as polygalacturonases, glucanases and proteases. We report the identification of a new member of the G1 family of proteases, BcACP1, which is secreted by B. cinerea during infection. The production of BcACP1 correlates with the acidification of the plant tissue, and transcriptional analysis of the Bcacp1 gene showed that it is only expressed under acidic growth conditions. Using a transcriptional reporter system, we showed that pH regulation of Bcacp1 is not mediated by the canonical PacC transcription factor binding site. Like other G1 proteases, BcACP1 is produced as a pro-enzyme. Trapping of the zymogen form allowed investigation of its maturation process. Evidence is presented for an autocatalytic proteolysis of the enzyme that is triggered by acidic pH. Environmental pH therefore controls Bcacp1 production at both the transcriptional and post-translational level.

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.

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.

Penicillium roqueforti secretes an aspartyl protease, ASPA, which represents the main extracellular proteolytic activity. Alkaline pH of the medium plays a major role by inhibiting the enzymatic activity and stopping aspA expression in the presence of casein, an inducing protein. However, casein degradation by the mature enzyme produces peptides which can induce aspA expression at acidic and alkaline pH. ASPA synthesized as a proenzyme is processed at an acidic pH but not at an alkaline pH. The data indicate that, in P. roqueforti, alkaline pH has an indirect repressive effect by inhibiting ASPA maturation and the release of inducers. At an acidic pH, the mature enzyme degrades extracellular proteins and peptides are released to induce aspA. In contrast, at an alkaline pH, the proenzyme remains inactive, the inducing substances are consequently not produced and aspA is no longer expressed.

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.

An apparently novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica has been characterized. The enzyme exhibits properties that differ markedly from those of the enzyme previously described [Fèvre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca 2+ , Mg 2+ and Mn 2+ , and appears to be located mainly in the apical part of the hypha. Taking into consideration the differences in pH optimum and effect of divalent ions, each enzyme activity could be assayed in the presence of the other. The insoluble polymeric product of the enzyme with alkaline pH optimum was characterized as a linear 1,3-β-glucan. Comparisons of the general properties of 1,3-β-glucan synthases suggest that enzymes from the oomycetes are more closely related to enzymes from higher plants than to those of true fungi, reflecting the fact that the oomycetes are highly divergent from chitinous fungi.

The qualitative determination by paper chromatography and the quantitative determination by high performance liquid chromatography (HPLC) of the Coenzyme Q system have been investigated for in 23 species of the genus Pichia. We have adapted HPLC to the quantitative analysis of the Coenzyme Q system. We have found the presence of 2,3 or 4 ubiquinones in the same Coenzyme Q extract.

In the genus Schizosaccharomyces intracellular osidases and nitrite and nitrate reductases are revealed; particularly all the species possessing invertase, alpha-glucosidase and alpha-galactosidase. These characters underline the homogeneity on the genus. On the basis of osidases, nitrite and nitrate reductases results, 2 groups can be distinguished in this genus.

The genus Hansenula was considered a long time ago as a good pattern for phylogenetic research. In 1969, Wickerham proposed an evolutive scheme based upon morphological, physiological and ecological criteria. Recently, relatedness among yeasts were analysed by DNA-DNA hybridization in liquid medium. H. anomala var. anomala (G + C content: 37.1%) was compared with H. anomala var. schneggii (37.6%), H. subpelliculosa (33.8%) line 3, H. sydowiorum (40.1%) and H. muscicola (37.1%). These results showed little relatedness between H. anomala var. anomala/H. ciferrii and H. anomala var. anomala/H. subpelliculosa. On the other hand, H. anomala var. schneggii shared 89.5% of its nucleotide sequences with H. anomala var. anomala. These 2 strains were considered to represent the same species. H. holstii showed 67.1% complementarity with H. anomala var. anomala: this strain is considered to represent valid species, different from H. anomala var. anomala, but H. muscicola with 72.5% relatedness to H. anomala var. anomala could be considered as a 'limit species'. An unexpected finding was that H. beckii was closely related to H. anomala var. anomala (84.8%). These data suggested the inadequacy of current criteria used to establish the phylogenetic lines in genus Hansenula.