Systematic Dissection of the Evolutionarily Conserved WetA Developmental Regulator across a Genus of Filamentous Fungi

Abstract

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.

Keywords: Aspergillus; WetA; asexual development; gene regulatory network; sporulation.

FIG 1

The central regulatory pathway of Aspergillus conidiation. (A) A cartoon depiction of genetic interactions of the central regulators in A. nidulans conidiogenesis. The central regulators cooperatively activate the conidiation-specific genes responsible for the morphogenesis of conidiophores. (B) The predicted protein architectures for the three conserved central regulators of conidiation in A. nidulans, A. fumigatus, and A. flavus. The blue box and the red hexagon represent the C2H2 zinc finger domain and TEA/ATTS domain in BrlA and AbaA, respectively, and were identified in a blastP (version 2.6.0) search (71). The red circle represents a putative transcription activation domain (TAD), which was predicted by 9aaTAD using the “Less stringent Pattern” setting (31), and in A. nidulans it has the amino acid sequence SEAALQAVR. The blue diamond represents the nuclear localization signal (NLS) predicted by NLStradamus using the 4 state HMM static model (32), and in A. nidulans it has the amino acid sequence KTKARREQEARDRRRK. The orange rectangle represents the ESC1/WetA-related domain (PTHR22934) predicted by the PANTHER classification system (72) and located at amino acids 497 to 547 in the A. nidulans protein.

FIG 2

Overview of the WetA-regulated orthologs in A. nidulans, A. fumigatus, and A. flavus. The 20,288 genes belonging to 6,566 orthogroups that possessed at least one member from A. nidulans, A. fumigatus, and A. flavus are represented by the black arcs next to their respective species labels. Gray, orthologs whose expression did not change between ΔwetA and WT conidia. Green, orthologs that were differentially expressed in only one species. Blue, genes that showed the same differential expression pattern in two of the three species. Red, genes that showed the same differential expression pattern in all three species. Orange, genes that showed a divergent differential pattern in two or more species. Lines connect expressed genes from the same orthogroup. Percentages represent the fractions of regulated orthologs from that species that belong to each category.

FIG 3

WetA-mediated regulation of asexual development in the three Aspergillus species. A schematic diagram of the WetA-mediated regulatory model of conidiation is shown. Genes with increased, decreased, and unaffected mRNA levels in the ΔwetA conidia are labeled with red (WetA-inhibited), blue (WetA-activated), and gray (not affected by WetA) circles, and the WetA-regulatory effects in the ΔAniwetA, ΔAfuwetA, and ΔAflwetA conidia are listed under the gene names at the left, middle, and right, respectively. There are two orthologs of fphA in A. fumigatus; one is WetA inhibited, and the other is not regulated by WetA.

FIG 4

WetA-regulatory effects on trehalose, chitin, β-(1,3)-glucan, and α-(1,3)-glucan metabolism in Aspergillus species.

FIG 5

WetA-regulatory effects on DHN-melanin, pyomelanin, and hydrophobin biosynthesis in Aspergillus species.

FIG 6

Identification of WetA regulatory element. (A) Diagram of the recognized region of the customized anti-WetA polyclonal antibodies. The recognized anti-WetA region overlaps part of the highly conserved Esc1/WetA-related domain near the WetA C-terminus. (B) Western blot analysis of the crude proteins of conidia from A. nidulans WT, ΔwetA, and TMY3 strains using anti-WetA and anti-FLAG polyclonal antibodies. The TMY3 strain expresses WetA::3xFLAG and can be recognized by both anti-WetA and anti-FLAG polyclonal antibodies. The results validated the specificity of the customized anti-WetA polyclonal antibodies. (C) The predicted WetA response element (WRE) and its WebLogo. (D) ChIP-qPCR analysis demonstrates the AniWetA DNA-associating capability of the upstream regions of AniwetA (AN1937), AN8643, AN0663, and AniacuF (AN1918). Data are presented as fold change compared with rabbit IgG-enriched DNA fragments. Data presented are means ± standard deviations (SD), n = 3.

FIG 7

Overlap between DEGs and WRE-containing genes in three Aspergillus species. The percentages of genes differentially expressed in the ΔwetA conidia (DEG), the percentages of genes that contain predicted WRE sequences in their upstream 1.5-kb regions (WRE), and the DEGs with a WRE in their upstream 1.5-kb regions (DEG w/WRE) are shown. The A. nidulans, A. fumigatus, and A. flavus genes are shown in light green, light blue, and light orange, respectively.

FIG 8

WRE occurrences in the upstream regions of wetA orthologs in representative fungi. WRE occurrences were identified in a series of regions located upstream of wetA orthologs. (A) Numbers to the left of the sequence indicate at what position relative to the translation start site the sequence shown begins. The sequences shown are from 15 bp upstream of the WRE occurrence that was identified by FIMO (40) with the lowest P value to 14 bp downstream of the WRE occurrence. Bases are colored black if they are conserved in at least 60% of the species. Green—Aspergillaceae. Orange—Trichocomaceae. Blue—Onygenales. Purple—Sodariomycete. (RC)—reverse complement. (B) ChIP-qPCR analysis demonstrated that WetA occupies wetA promoters in A. nidulans, A. fumigatus, and A. flavus. Data are presented as fold change compared with rabbit IgG-enriched DNA fragments. Data presented are means ± SD, n = 3.