Species-specific gene regulatory network rewiring mediated by the GATA-type regulator NsdD in Aspergillus

The evolutionarily conserved GATA-type transcription factor (TF) NsdD regulates sexual and asexual development as well as secondary metabolism in various Aspergillus species. Despite its well-known multifunctionality, the mechanisms by which NsdD coordinates such diverse biological processes remain unclear. To address this gap, we have conducted network-based multiomics analyses in two distantly related species, Aspergillus nidulans and Aspergillus flavus. Transcriptomic profiling reveals that NsdD regulates gene expression in a cell type- and species-specific manner. The potential evolutionary conservation of NsdD was tested by a cross-complementation experiment in which the A. nidulans nsdD gene was introduced into the A. flavus ΔnsdD mutant. This partially restored key phenotypes and gene expression profiles but failed to fully recapitulate wild-type regulation, suggesting species-specific functionality. To further dissect NsdD’s roles, we have performed genome-wide ChIP-seq analyses and identifiedidentifiedidentified502 and 674 potential direct targets in A. nidulans and A. flavus, respectively, including major developmental and metabolic regulators such as veA, flbD, brlA, vosA, rosA, and laeA. Motif analysis reveals a conserved NsdD binding site (5′-GATCT-3′), designated as the NsdD response element. Network analyses uncover core regulatory modules and reveal extensive gene regulatory network (GRN) rewiring between the two species. While NsdD governs conserved biological processes, divergence in its direct targets and downstream interactions contributes to species-specific traits, including differences in asexual morphology and production of sterigmatocystin/aflatoxin. This study provides the first genome-wide comparative map of NsdD-mediated GRNs in filamentous fungi and highlights how evolutionary rewiring allows a conserved TF to acquire distinct regulatory functions across species.