Project description:Asexual development is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In the filamentous fungal genus Aspergillus, the production of asexual spores is primarily governed by the BrlA-AbaA-WetA central regulatory cascade. The final step in this cascade, which is controlled by the WetA protein, not only governs cellular development (i.e., the morphological differentiation of spores) but also ensures its coupling with chemical development (i.e., the coordinated production and deposition of diverse secondary metabolites, such as aflatoxins, into spores). While the wetA gene is conserved across the genus Aspergillus, the structure and degree of conservation of the BrlA-AbaA-WetA regulatory cascade and the broader wetA gene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses between wetA null mutant and wild type (WT) asexual spores in three representative species spanning the diversity of the genus Aspergillus: the genetic model A. nidulans, the agricultural pest A. flavus, and the human pathogen A. fumigatus. We discovered that WetA regulates asexual sporulation in all three species via a negative feedback loop that represses BrlA, the cascade’s first step. Furthermore, ChIP-seq experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory element, which we term the WetA Response Element (WRE). Interestingly, the WRE is found completely conserved in the non-coding region upstream of the wetA translation start site of many diverse Aspergillus genomes. In contrast, several global transcriptional regulators, most notably those in the velvet complex (veA, velB, and laeA) known to regulate the coupling between asexual development and production of secondary metabolites, show species-specific regulatory patterns. These results suggest that the BrlA-AbaA-WetA cascade’s regulatory role in cellular and chemical development of asexual spores is functionally conserved, but that the WetA-associated GRN has diverged during Aspergillus evolution. This entry is for the ChIP-seq data.

Project description:Asexual development is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In the filamentous fungal genus Aspergillus, the production of asexual spores is primarily governed by the BrlA-AbaA-WetA central regulatory cascade. The final step in this cascade, which is controlled by the WetA protein, not only governs cellular development (i.e., the morphological differentiation of spores) but also ensures its coupling with chemical development (i.e., the coordinated production and deposition of diverse secondary metabolites, such as aflatoxins, into spores). While the wetA gene is conserved across the genus Aspergillus, the structure and degree of conservation of the BrlA-AbaA-WetA regulatory cascade and the broader wetA gene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses between wetA null mutant and wild type (WT) asexual spores in three representative species spanning the diversity of the genus Aspergillus: the genetic model A. nidulans, the agricultural pest A. flavus, and the human pathogen A. fumigatus. We discovered that WetA regulates asexual sporulation in all three species via a negative feedback loop that represses BrlA, the cascade’s first step. Furthermore, ChIP-seq experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory element, which we term the WetA Response Element (WRE). Interestingly, the WRE is found completely conserved in the non-coding region upstream of the wetA translation start site of many diverse Aspergillus genomes. In contrast, several global transcriptional regulators, most notably those in the velvet complex (veA, velB, and laeA) known to regulate the coupling between asexual development and production of secondary metabolites, show species-specific regulatory patterns. These results suggest that the BrlA-AbaA-WetA cascade’s regulatory role in cellular and chemical development of asexual spores is functionally conserved, but that the WetA-associated GRN has diverged during Aspergillus evolution. This entry is for the RNA-seq data.

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.

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