Project description:Neurospora crassa is a reference organism to study carotene biosynthesis and light regulation for decades. However, there is no evidence of its capacity to produce secondary metabolites, a characteristic of many filamentous fungi. In this work, we report the role of the fungal specific velvet regulator complex in development and secondary metabolism in N. crassa. Four velvet genes were found in the genome. Deletion of ve-1 or ve-2 affects asexual and sexual development, secondary metabolism and light-dependent carotene biosynthesis. Deletion of vos-1 did not show significant differences in comparison to wild type. Deletion of lae-1 resulted in reduced protoperithecia formation and affected secondary metabolism. VE-1, VE-2, LAE-1 and VOS-1 showed nucleo-cytoplasmic localization, which was independent of light input. Two distinct velvet complexes were found in vivo: a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 complex, respectively. The heterotrimer-complex positively regulated sexual development and repressed asexual spore formation. Moreover, it repressed siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controlled the production of carotene pigments. VE-1 regulated the expression of more than 15% of the whole genome, which corresponded mainly to regulatory, developmental and redox proteins. We also studied intergenera functions of the velvet complex through complementation of A. nidulans veA, velB, laeA, vosA mutants with their Neurospora crassa orthologues ve-1, ve-2, lae-1 and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substituted the developmental and secondary metabolite functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. The N. crassa lae-1 and vos-1 genes did not complement respective A. nidulans mutants and failed to form corresponding complexes. Reciprocally, expression of veA restored the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans were predominantly localized to nuclear fraction independent of light signal. These data highlight the conservation of the complex formation potential in N. crassa and A. nidulans. However, they also underline the similarities and differences of the velvet roles across genera according to the different life styles, niches and ontogenetic processes.

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: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.

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Genome-wide analyses for the WOPR regulator osaA and the velvet regulator veA

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