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.

Project description:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeast’s “all-purpose” stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfA’s functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans.

Project description:The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of vegetatively grown cells (mycelia) and asexual spores (conidia) obtained from the surface cultures of control, DatfA, DatfB, DatfADatfB mutant strains with/without menadione sodium bisulfite (MSB) treatment were performed. Probable AtfA and AtfB dependent gene sets were determined by comparing transcriptomes of both single gene deletion mutants with the reference strain, and the double gene deletion mutant with the appropriate single gene deletion mutants. As AtfA is the primary bZip TF governing stress-response in A. nidulans, a significantly higher number was differentially expressed genes (DEGs) by DatfA than DatfB in both mycelial and conidial samples, and most of the AtfB dependent genes showed AtfA dependence, too. Moreover, a low number of genes showing AtfB dependence only can be a consequence of that DatfA leading to downregulation of atfB expression. The abundance of atfA and atfB mRNAs and, concurring with it the number of AtfA and AtfB affected genes were much higher in conidial than in mycelial samples. The number of AtfB- (but not of AtfA-) affected DEGs decreased markedly in the presence of MSB, which was accompanied with decreased mRNA levels of atfB in MSB treated mycelial (reference strain) and conidial (DatfA mutant) samples. The overlap between the AtfA dependent DEGs in the case of MSB treated and untreated mycelial samples was low demonstrating that distinct genes can be under AtfA control in different cell types. The AtfA-dependent DEGs were enriched with carbohydrate metabolism genes. Among them, AtfA-dependence of glycolytic genes in the case of the conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, like the Emericellamide cluster also showed AtfA-dependent regulation. The AtfA affected DEGs under all experimental conditions include those encoding catalase and histidine-containing phosphotransfer proteins. The 23 DEGs that solely dependent on AtfB considering all transcriptomics data sets, included a putative a -glucosidase (agdB), a putative a-amylase, calA involved in early conidial germination and an alternative oxidase. In summary, there is a complex interaction between the two b-Zip TFs in which the main function of AtfB is supporting the regulatory role of the primary b-Zip TF AtfA in A. nidulans.

Project description:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeastâs âall-purposeâ stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfAâs functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans. 14 samples, 7 with the control strain and 7 with an DatfA strain (each series contains samples from untreated as well as menadione, low concentration hidrogen-peroxide, high concentration hidrogen-peroxide, tert-butylhydroperoxide, diamide and NaCl treated cultures)

Project description:Microarray analysis was used to identify the osmotic stress-responsive genes dependent on 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 with and without osmotic shock (Exp.6). Others were comparison between wild type with osmotic shock and each mutant (hogA, Exp.7; atfA, Exp.8) with osmotic shock. Compared the result of Exp.6 with other experiments, we could identify the genes whose expression was induced or repressed in response to osmotic stress in a manner dependent on HogA and AtfA. KEY WORD; Aspergillus nidulans, osmotic stress, HogA, AtfA Conidia of wild type or each mutant were cultured at 37C in 100ml CD medium containing 2% glucose for 18h, and 50 ml of 3 M sorbitol was added (final concentration; 1 M) or equivalent volume of water (as a 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 (channel 2/channel 1) were calculated for each replicates. Gene expression was considered to be significantly higher or lower whenever the spot intensity changed by at least 2-fold in all four replicates for each experiment.

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Aspergillus nidulans

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