Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops

Adaptation to pH and role of PacC in the rice blast fungus Magnaporthe oryzae.

Fungi are known to adapt to pH partly via specific activation of the Pal signaling pathway and subsequent gene regulation through the transcription factor PacC. The role of PacC in pathogenic fungi has been explored in few species, and each time its partaking in virulence has been found. We studied the impact of pH and the role of PacC in the biology of the rice pathogen Magnaporthe oryzae. Conidia formation and germination were affected by pH whereas fungal growth and appressorium formation were not. Growth in vitro and in planta was characterized by alkalinization and ammonia accumulation in the surrounding medium. Expression of the MoPACC gene increased when the fungus was placed under alkaline conditions. Except for MoPALF, expression of the MoPAL genes encoding the pH-signaling components was not influenced by pH. Deletion of PACC caused a progressive loss in growth rate from pH 5 to pH 8, a loss in conidia production at pH 8 in vitro, a loss in regulation of the MoPALF gene, a decreased production of secreted lytic enzymes and a partial loss in virulence towards barley and rice. PacC therefore plays a significant role in M. oryzae's biology, and pH is revealed as one component at work during interaction between the fungus and its host plants

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

International audienceDuring 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

Snf1 Kinase Differentially Regulates Botrytis cinerea Pathogenicity according to the Plant Host

The Snf1 kinase of the glucose signaling pathway controls the response to nutritional and environmental stresses. In phytopathogenic fungi, Snf1 acts as a global activator of plant cell wall degrading enzymes that are major virulence factors for plant colonization. To characterize its role in the virulence of the necrotrophic fungus Botrytis cinerea, two independent deletion mutants of the Bcsnf1 gene were obtained and analyzed. Virulence of the &Delta;snf1 mutants was reduced by 59% on a host with acidic pH (apple fruit) and up to 89% on hosts with neutral pH (cucumber cotyledon and French bean leaf). In vitro, &Delta;snf1 mutants grew slower than the wild type strain at both pH 5 and 7, with a reduction of 20&ndash;80% in simple sugars, polysaccharides, and lipidic carbon sources, and these defects were amplified at pH 7. A two-fold reduction in secretion of xylanase activities was observed consequently to the Bcsnf1 gene deletion. Moreover, &Delta;snf1 mutants were altered in their ability to control ambient pH. Finally, &Delta;snf1 mutants were impaired in asexual sporulation and did not produce macroconidia. These results confirm the importance of BcSnf1 in pathogenicity, nutrition, and conidiation, and suggest a role in pH regulation for this global regulator in filamentous fungi

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system

International audienceDickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins are first addressed to the periplasm before their secretion by Out. No secretion signal present on the protein allows the identification of substrates of a T2SS. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified five new Out-secreted proteins: the iron-binding protein IbpS, the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that IbpS, SvfA and SvfB are required for full virulence of D. dadantii. Homologues of IbpS are present in other phytopathogenic microorganisms (fungi and oomycetes) where they also play a role in virulence. svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae

Impact of <i>MoPACC</i> deletion on the fungus biology.

<p>(a) Conidia production in the parental (P), control (C2 (ectopic)) and mutant (T2) strains grown for 14 days on solid rice medium buffered to pH 5 (grey) or to pH 8 (black). (b) Pathogenicity tests on barley leaves infected by conidia from the parent, mutant and complemented strains. Lesions on 10 separate leaves were counted after 5 days of incubation. All experiments were run in triplicates and standard deviations are shown.</p

Evidencing New Roles for the Glycosyl-Transferase Cps1 in the Phytopathogenic Fungus Botrytis cinerea

International audienceThe fungal cell wall occupies a central place in the interaction between fungi and their environment. This study focuses on the role of the putative polysaccharide synthase Cps1 in the physiology, development and virulence of the grey mold-causing agent Botrytis cinerea. Deletion of the Bccps1 gene does not affect the germination of the conidia (asexual spores) or the early mycelial development, but it perturbs hyphal expansion after 24 h, revealing a two-phase hyphal development that has not been reported so far. It causes a severe reduction of mycelial growth in a solid medium and modifies hyphal aggregation into pellets in liquid cultures. It strongly impairs plant penetration, plant colonization and the formation of sclerotia (survival structures). Loss of the BcCps1 protein associates with a decrease in glucans and glycoproteins in the fungus cell wall and the up-accumulation of 132 proteins in the mutant’s exoproteome, among which are fungal cell wall enzymes. This is accompanied by an increased fragility of the mutant mycelium, an increased sensitivity to some environmental stresses and a reduced adhesion to plant surface. Taken together, the results support a significant role of Cps1 in the cell wall biology of B. cinere

Impact of <i>MoPACC</i> deletion on the fungal growth and alkalinization.

<p>(a) Growth of the parental (square) and deletion (triangle) strains on solid medium buffered to pH 5 (grey) or pH 8 (black). (b) Growth rate as a function of pH in the parental (square), control (circle) and mutant (triangle) strains grown on 10 solid media buffered to 10 pH values ranging from 5.5 to 7.9. (c) pH of the culture (big triangles) and ammonia accumulation (small triangles) during growth of the deletion strain in liquid non-buffered TNK-YE medium. (d) Expression of the <i>MoPAL</i> genes in the <i>MoPACC</i> mutant strains. Following 15 minutes transfer of the deletion strain from non-buffered liquid medium to fresh medium buffered to pH 5 (grey) or pH 8 (black), total mRNA was extracted and quantitative PCR analysis was carried out using primers specific of the <i>MoPACC</i> and all the six <i>MoPAL</i> genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone.0069236.s002" target="_blank">Table S1</a>). Three independent biological replicates were analyzed and quantification was based on the 2^ΔCT method using the <i>MoEF1</i>α gene as reference. The experiments were performed in parallel to those reported for the wild type strain in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone-0069236-g003" target="_blank">figure 3</a>. Left panel : Gene expression after transfer to pH 5 or to pH 8. Right panel : Change in expression between pH 5 and pH 8 (ratios are plotted using a logarithmic scale).</p