An atypical forkhead-containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum.
ABSTRACT: Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)-box-containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)-based gene silencing was employed to alter the expression of SsFkh1. RNA-silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis-related laccase genes and a polyketide synthase-encoding gene were significantly down-regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi-silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.
Project description:The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum.
Project description:Sclerotinia sclerotiorum is a destructive ascomycete plant pathogen with worldwide distribution. Extensive research on different aspects of this pathogen's capability to cause disease will help to uncover clues about new ways to safely control Sclerotinia diseases. The thioredoxin (Trx) system consists of Trx and thioredoxin reductase (TrxR), which play critical roles in maintenance of cellular redox homeostasis. In this study, we functionally characterized a gene encoding a TrxR (SsTrr1) in S. sclerotiorum. The amino acids of SsTrr1 exhibited high similarity with reported TrxRs in plant pathogens and targeted silencing of SsTrr1 lead to a decrease in TrxR activities of mycelium. SsTrr1 showed high expression levels during hyphae growth, and the levels decreased at the different stages of sclerotial development. SsTrr1 gene-silenced strains produced a smaller number of larger sclerotia on potato dextrose agar medium. The observations were consistent with the inhibitory effects on sclerotial development by the TrxR inhibitor, anrunofin. The expression of SsTrr1 showed a dramatic increase under the oxidative stress and the hyphal growth of gene-silenced strains showed more sensitivity to H2O2. SsTrr1 gene-silenced strains also showed impaired virulence in different hosts. Taken together, our results suggest that SsTrr1 encodes a TrxR that is of great important for oxidative stress tolerance, virulence, and sclerotial development of S. sclerotiorum.
Project description:Sclerotinia sclerotiorum is a plant-pathogenic ascomycete fungus and infects over 400 host plants, including pea (Pisum sativum L.). The fungus causes white mold on pea, and substantial yield loss is attributed to the disease. To improve white mold management, further understanding of S. sclerotiorum pathogenicity is crucial. In this study, 389 transcription factors (TFs) were mined from the complete genome sequence of S. sclerotiorum and their in planta expression patterns were determined in susceptible and partially resistant pea lines and compared to in vitro expression patterns on culture medium. One of the transcription factors was significantly induced in planta at 24 and 48?h postinfection compared to the expression in vitro This putative C6 transcription factor of S. sclerotiorum (SsC6TF1) was knocked down using a gene-silencing approach to investigate its functions in vegetative growth and sclerotial development as well as its virulence and pathogenicity in pea. While the SsC6TF1 knockdown mutants had hyphal growth rates identical to those of the wild-type strain and were capable of infection, the knockdown mutants produced no sclerotia or significantly fewer and smaller sclerotia on the culture medium and exhibited reduced virulence on both pea lines. This study profiled genome-wide expression for S. sclerotiorum transcription factors in planta and in vitro and functionally characterized a novel transcription factor, SsC6TF1, which positively regulates sclerotial development and virulence on pea. The finding provides molecular insights into S. sclerotiorum biology and interaction with pea and other economically important crops.IMPORTANCE White mold, caused by Sclerotinia sclerotiorum, is a destructive disease on important legume species such as soybean, dry bean, and pea. This study investigated expression levels of transcription factors in S. sclerotiorum in planta (pea lines) and in vitro (culture medium). One transcription factor displaying high expression in planta was found to be involved in sclerotial development and virulence on pea. This report provides a new understanding regarding transcription factors of S. sclerotiorum in development and virulence.
Project description:Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S. sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.
Project description:Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S.?sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.
Project description:The Solexa/Illuminaâs digital gene expression (DGE) system was used to gain insight into the broad range of transcriptional responses during the vegetative growth, sclerotial development, myceliogenic germination, carpogenic germination, apothecium formation (stipe) and infection of Sclerotinia sclerotiorum. We obtained a sequencing depth of approximately 3.3 million clean tags per cDNA library. Tag mapping indicated that these six cDNA libraries in total represented more than 66.7% of all of the genes presented in the predicted transcript databases of the Broad Institute. Thouthands of differentially expressed genes were indentified during the various developmental stages compared to the vegetative growth stage. Our results could increase and deepen the understanding of the vegetative and reproductive development as well as the infection of S. sclerotiorum The S. sclerotiorum strain Ep-1PNA367 was grown or treated under different conditions, and samples were collected for RNA extraction for DGE analysis during the following stages: (i) Vegetative stage: activating hyphal agar discs of the Ep-1PNA367 stain were placed on a cellophane membrane overlaid onto PDA medium at 20Â°C; subsequently, the mycelia were collected at 12, 24, 36, 48 and 60 h; (ii) Sclerotial formation stage: the colonies growing on the cellophane membrane overlaid onto PDA medium were further incubated under the same conditions and then the cultures were collected at 84, 96, 108, 120 and 132 h; (iii) Early stages of infection: fresh hyphal fragments of the Ep-1PNA367 strain were overlaid onto sterilized cheese cloth, which was overlaid onto the leaves of the A. thaliana ecotype Columbia-0, followed by inoculation at 20Â°C with 100% relative humility; the cheese cloth with hyphae was then rolled up from the leaves at 9 h and 12 h; (iv) Sclerotial myceliogenic germination stage: sclerotia were surface sterilized with sodium hypochlorite and were sowed on a PDA plate at 20Â°C to induce myceliogenic germination, and when approximately 50% of sclerotia had germinated, the sclerotia were harvested; (v) Sclerotial carpogenic germination stage: sclerotia were dried at room temperature and were pretreated in a freezer (4-6Â°C) for up to one month and then were surface sterilized and sowed on wet sterilized sands in a plate at 15Â°C to induce carpogenic germination; when approximately 50% of the sclerotia germinated (stipes having only emerged from sclerotia), the sclerotia were harvested; and (vi) Early apothecial formation stage: sclerotia were allowed to grow in the same incubator, and the stipes were cut and collected immediately before apothecium formation for RNA extraction. Finally, different time-point samples from the vegetative stage, sclerotial formation stage and infection stage were pooled together respectively according to equal quantities for RNA extraction.
Project description:Stems and pods of hyacinth bean cultivated in a farmer's field in Gazipur District, Bangladesh, were found rotted in nearly 5% hyacinth bean plants. A fungus having fluffy mycelium and large sclerotia was isolated from affected tissues. Combined results of morphological, molecular and pathological analyses identified the fungus as Sclerotinia sclerotiorum (Lib) de Bary. Inoculating the fungus on healthy hyacinth bean plants and pods reproduced the symptoms previously observed in the field. The three isolates obtained from naturally infected plants were cross inoculated in hyacinth bean, okra and African-American marigold and they were pathogenic to these hosts. The optimum temperature and pH for its growth were 20°C and pH 5.0, respectively. Sclerotial development was favored at pH 5.0. Sucrose and mannitol were the best carbon sources to support hyphal growth, while glucose was the most favourable for sclerotial development. The hyacinth bean genotypes, HB-82 (Rupban Sheem) and HB-102 were found highly resistant, while HB-94 (Ashina) was moderate resistant to the fungus. Finally, S. sclerotiorum was sensitive to Bavistin, Dithane M-45 and Rovral fungicides and Ca in the form of CaCl2. This observation could possibly aid in eliminating field loss in hyacinth bean caused by an emerging pathogenic fungus S. sclerotiorum.
Project description:SFH1 (for Snf5 homolog) protein, comprised in the RSC (Remodels Structure of Chromatin) chromatin remodeling complex, functions as a transcription factor (TF) to specifically regulate gene transcription and chromatin remodeling. As one of the well-conserved TFs in eukaryotic organisms, little is known about the roles of SFH1 protein in the filamentous fungi. In Sclerotinia sclerotiorum, one of the notorious plant fungal pathogens, there are nine proteins predicted to contain GATA-box domain according to GATA family TF classification, among which Sssfh1 (SS1G_01151) encodes a protein including a GATA-box domain and a SNF5 domain. Here, we characterized the roles of Sssfh1 in the developmental process and fungal pathogenicity by using RNA interference (RNAi)-based gene silencing in S. sclerotiorum. RNA-silenced strains with significantly reduced Sssfh1 RNA levels exhibited slower hyphal growth and decreased reactive oxygen species (ROS) accumulation in hyphae compared to the wild-type (WT) strain. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that SsSFH1 interacts with SsMSG5, a MAPK phosphatase in S. sclerotiorum. Furthermore, Sssfh1-silenced strains exhibited enhanced tolerance to NaCl and H2O2. Results of infection assays on soybean and common bean (Phaseolus vulgaris) leaves indicated that Sssfh1 is required for full virulence of S. sclerotiorum during infection in the susceptible host plants. Collectively, our results suggest that the TF SsSFH1 is involved in growth, ROS accumulation and virulence in S. sclerotiorum.
Project description:AMS2, a multicopy suppressor for the cpn1 (SpCENP-A) mutant, functions to specifically regulate histone genes transcription and chromosome segregation. As a cell-cycle-regulated GATA transcription factor in eukaryotic organisms, little research has been done on the role of AMS2 protein in pathogenic fungi. In Sclerotinia sclerotiorum, Ssams2 (SS1G_03252) encodes a protein which has been predicted to contain GATA-box domain. Here, Ssams2-silenced strains with significantly reduced Ssams2 gene expression levels exhibited defect in hyphal growth, hyphal branching patterns, compound appressoria differentiation and the oxalic acid production compared to the wild-type (WT) strain. By common bean leaves infection assays, we identified the role of Ssams2 in full virulence. Furthermore, the numbers of cell nucleus in the same length of mycelium in Ssams2-silenced transformants were significantly less than that in the WT strain. The expression levels of histone genes and cell cycle genes in transformants were down-regulated significantly in the RNAi strains. Taken together, our work suggests that the TF SsAMS2 is required for growth, appressoria formation, virulence, and chromosome segregation in S. sclerotiorum.
Project description:Ascomycete Sclerotinia sclerotiorum (Lib.) de Bary is one of the most damaging soilborne fungal pathogens affecting hundreds of plant hosts, including many economically important crops. Its genomic sequence has been available for less than a decade, and it was recently updated with higher completion and better gene annotation. Here, we review key molecular findings on the unique biology and pathogenesis process of S. sclerotiorum, focusing on genes that have been studied in depth using mutant analysis. Analyses of these genes have revealed critical players in the basic biological processes of this unique pathogen, including mycelial growth, appressorium establishment, sclerotial formation, apothecial and ascospore development, and virulence. Additionally, the synthesis has uncovered gaps in the current knowledge regarding this fungus. We hope that this review will serve to build a better current understanding of the biology of this under-studied notorious soilborne pathogenic fungus.