Asexual sporulation is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In Aspergillus, the production of asexual spores is primarily governed by the BrlA→AbaA→WetA regulatory cascade. The final step in this cascade is controlled by the WetA protein and governs not only the morphological differentiation of spores but also the production and deposition of diverse metabolites into spores. While WetA is conserved across the genus Aspergillus, the structure and degree of conservation of the wetA gene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses of comparisons between wetA null mutant and wild-type asexual spores in three representative species spanning the diversity of the genus Aspergillus: A. nidulans, A. flavus, and 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, data from chromatin immunoprecipitation sequencing (ChIP-seq) experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in our data. These results suggest that the BrlA→AbaA→WetA cascade's regulatory role in cellular and chemical asexual spore development is functionally conserved but that the wetA-associated GRN has diverged during Aspergillus evolution.IMPORTANCE The formation of resilient spores is a key factor contributing to the survival and fitness of many microorganisms, including fungi. In the fungal genus Aspergillus, spore formation is controlled by a complex gene regulatory network that also impacts a variety of other processes, including secondary metabolism. To gain mechanistic insights into how fungal spore formation is controlled across Aspergillus, we dissected the gene regulatory network downstream of a major regulator of spore maturation (WetA) in three species that span the diversity of the genus: the genetic model A. nidulans, the human pathogen A. fumigatus, and the aflatoxin producer A. flavus Our data show that WetA regulates asexual sporulation in all three species via a negative-feedback loop and likely binds a novel regulatory element that we term the WetA response element (WRE). These results shed light on how gene regulatory networks in microorganisms control important biological processes and evolve across diverse species.

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