Expression data from Saccharomyces cerevisiae expressing Candida albicans Nik1p comparing osmotic stress and fludioxonil treatment
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ABSTRACT: Fungal group III histidine kinases are the molecular targets of some classes of fungicides. In contrast to the yeast Saccharomyces cerevisiae, the fungal pathogen Candida albicans possesses a group III histidine kinase, CaNik1p, also called Cos1p. To investigate the function of CaNIK1, the gene was expressed in S. cerevisiae. The transformants became susceptible to antifungal compounds to which the wild-type strain is resistant. The susceptibility was related to the activation of the MAP kinase Hog1p of the osmotic stress response pathway. Gene expression analysis revealed a strong overlap of the responses to osmotic stress and to fludioxonil at early time points. While the response to fludioxonil persisted, the response to osmotic stress was diminished with time.
Project description:Fungal group III histidine kinases are the molecular targets of some classes of fungicides. In contrast to the yeast Saccharomyces cerevisiae, the fungal pathogen Candida albicans possesses a group III histidine kinase, CaNik1p, also called Cos1p. To investigate the function of CaNIK1, the gene was expressed in S. cerevisiae. The transformants became susceptible to antifungal compounds to which the wild-type strain is resistant. The susceptibility was related to the activation of the MAP kinase Hog1p of the osmotic stress response pathway. Gene expression analysis revealed a strong overlap of the responses to osmotic stress and to fludioxonil at early time points. While the response to fludioxonil persisted, the response to osmotic stress was diminished with time. S. cerevisiae expressing Candida albicans Nik1p were treated with 10 µg/ml fludioxonil. As a comparison, another culture of S. cerevisiae expressing Candida albicans Nik1p was treated with 1 M sorbitol to induce osmotic stress response. One culture remained untreated as a control. From all cultures, samples were taken after a duration of 15, 30 and 60 min.
Project description:The antifungal compound fludioxonil targets the fungal specific group III hybrid histidin kinaseTcsC of A. fumigatus, which in turn activate the High Osmolarity Glycerol (HOG) pathway and leads to drastic swelling of the fungal cells. We investigated the different responses of the AfS35 (wild type), the completely resistant mutant strain DtcsC and the partial resistant mutant strain Dskn7 to fludioxonil.
Project description:Microarray analysis was used to identify the fludioxonil-responsive genes dependent on SskA, SrrA, HogA, and AtfA in the filamentous fungus Aspergillus nidulans. In order to identify such genes, we conducted the several types of experiment. One was a comparison between wild type treated with fludioxonil and without the treatment (Exp.1). Others were comparison between wild type treated with fludioxonil and each mutant (sskA, Exp.2; srrA, Exp.3; hogA, Exp.4; atfA, Exp.5) treated with fludioxonil. Compared the result of Exp.1 with that of other experiments, we could identify the genes whose expression was induced or repressed in response to fludioxonil in a manner dependent on SskA, SrrA, HogA, or AtfA. KEY WORD; Aspergillus nidulans, fludioxonil, SskA, SrrA, HogA, AtfA
Project description:Comparison of Candida albicans SC5314 treated with fludioxonil for 30 min and untreated controls under hyphae-inducing conditions 3 biologically independent replicates of a reference sample (U30) and fludioxonil treatment (F30) each
Project description:Botrytis cinerea (gray mold) is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protec-tion and is essential for controlling tomato gray mold. The emergence of fungicide-resistant strains has made the control of Botrytis cinerea more difficult. While the genome of Botrytis cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such flu-dioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) li-braries for three Botrytis cinerea strains [two highly resistant (LR and FR) versus one highly sen-sitive (S) to fludioxonil], with and without fludioxonil treatment, to identify fludioxonil responsive genes that facilitate fungicide resistance. Functional enrichment analysis identified nine resistant related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up regulated, and seven resistant related DEGs down regulated. These included adenosine tri-phosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator super-family (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxo-nil-responsive genes, obtained from the RNA-sequence data sets were validated using quantita-tive real-time PCR (qRT-PCR). Based on RNA-sequence analysis it was found that fugal HHKs, like BOS1, BcHHK2, and Bchhk17, were in some way involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8 were differen-tially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes played a crucial role in the fludioxonil resistant mechanism of B. cinerea. These lines of evidence together allowed us to draw a general portrait of the anti-fludioxonil mechanisms for Botrytis cinerea, and the assembled and annotated transcrip-tome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.
Project description:Ssk1-type response regulator proteins are the core elements of histidine-to-aspartate systems that mediate fungal stress tolerance, a determinant to the biocontrol potential of fungal entomopathogens. We characterized for the first time the functions of Beauveria bassiana Ssk1 (Bbssk1) by analyzing multi-phenotypic changes in DBbssk1 and differentially expressed genes (DEGs) in the digital gene expression (DGE) libraries of DBbssk1 and wild-type constructed under osmotic stress. The results revealed 1003 DEGs, of which many associated with conidiation, xenotics transport, cell wall integrity, and protein and carbohydrate metabolism were greatly down-regulated.
Project description:Ssk1-type response regulator proteins are the core elements of histidine-to-aspartate systems that mediate fungal stress tolerance, a determinant to the biocontrol potential of fungal entomopathogens. We characterized for the first time the functions of Beauveria bassiana Ssk1 (Bbssk1) by analyzing multi-phenotypic changes in DBbssk1 and differentially expressed genes (DEGs) in the digital gene expression (DGE) libraries of DBbssk1 and wild-type constructed under osmotic stress. The results revealed 1003 DEGs, of which many associated with conidiation, xenotics transport, cell wall integrity, and protein and carbohydrate metabolism were greatly down-regulated. Total RNA obtained from Bbssk1 disruption mutant subjected to 0.5 M NaCl for 30 minutes compared to the wild type strain under the same stress treatment.
Project description:Microarray analysis was used to identify the fludioxonil-responsive genes dependent on SskA, SrrA, HogA, and AtfA in the filamentous fungus Aspergillus nidulans. In order to identify such genes, we conducted the several types of experiment. One was a comparison between wild type treated with fludioxonil and without the treatment (Exp.1). Others were comparison between wild type treated with fludioxonil and each mutant (sskA, Exp.2; srrA, Exp.3; hogA, Exp.4; atfA, Exp.5) treated with fludioxonil. Compared the result of Exp.1 with that of other experiments, we could identify the genes whose expression was induced or repressed in response to fludioxonil in a manner dependent on SskA, SrrA, HogA, or AtfA. KEY WORD; Aspergillus nidulans, fludioxonil, SskA, SrrA, HogA, AtfA Conidia of wild type or each mutant were cultured at 37C in 100ml CD medium containing 2% glucose for 18h and treated with fludioxonil (final concentration; 10 ug/ml) or DMSO (as a solvent control) for 15 min. The mycelia were harvested and frozen in liquid nitrogen, ground to powder, and used for RNA preparation. mRNA was purified and used for hybridization experiments. A total of 2 hybridizations were performed for each microarray experiment described in the summary. The following replicates were carried out: 1. In-slide replicates were carried out for each analysis. 2. Dye swap replicates were carried out for each experiment. The slides were scanned with an Axon GenePix 4000B scanner (Molecular Devices). The resulting TIFF images were imported into GenePix Pro and fluorescent intensity of spots were calculated for each of the Cy3 and Cy5 channels. Global normalization was applied to all analyses. Following normalization, spots whose Cy3 or Cy5 intensity was less than 0 were removed from the data set (the exceptional case was that intensity of the other channel was more than 100). The dye-swap replicates and in-slide replicates were subjected to all analyses. Finally, gene expression ratios (test/reference) were calculated for each replicates. Gene expression was considered to be significantly higher or lower whenever the spot intensity changed by at least 3-fold in all four replicates for each experiment.