Geneviève Billon-Grand

pH controls both transcription and post-translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis 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.

pH modulation differs during sunflower cotyledon colonization by the two closely related necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum

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.

The Homeobox BcHOX8 Gene in Botrytis Cinerea Regulates Vegetative Growth and Morphology

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.

A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica

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 [Fevre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca2+, Mg2+ and Mn2+, 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.

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.

Nitrite réductase des Saccharomyces (groupe Torulaspora) et des Debaryomyces. implications systématiques

Intracellular nitrite reductase was found in some species of Saccharomyces (Torulaspora group of Van der Walt 1970) and all Debaryomyces (excepted D. castelli and D. coudertii. The taxonomical relationship between the 2 groupes are discussed.

Recherche D'enzymes intracellulaires dans le genre schizosaccharomyces. Implications systématiques

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

Etude de quelques enzymes intracellulaires dans les genres Dekkera et Brettanomyces; consequences systematiques

Intracellular osidases, nitrite reductase, nitrate reductase were found in some species ofDekkera and Brettanomyces. Taxonomical relationships between the 2 genus are discussed. These genus are compared withHanseniaspora andKloeckera.

Osidases, nitrite et nitrate réductases dans les genres Hanseniaspora et Kloeckera

Intracellular osidases, nitrite reductase and nitrate reductase were found in some species of Hanseniaspora and Kloeckera. Taxonomical relationships between the 2 genus are discussed.

Hybridation ADN-ADN chez quelques espèces du genre Hansenula

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%), H. ciferrii (33,1%), H. holstii (37%) belonging to the same line 2, and also with H. beckii (38,3%) 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.