The transcription factor Ste12(1) from Nakaseomyces glabratus acts pheromone-responsive in S. cerevisiae but seems decoupled from its cognate upstream GPCR in its native host.

G-protein-coupled receptor (GPCR)-mediated signalling pathways govern critical cellular responses in fungi, including mating, biofilm formation, and virulence. The mating pathway of Saccharomyces cerevisiae, in which peptide-sensing GPCRs (peptide GPCRs) activate a MAP kinase cascade and the transcription factor Ste12, has thereby served as the canonical model for fungal peptide GPCR signalling. Homologues of this pathway are conserved across many fungi, including the high-priority pathogen Nakaseomyces glabratus. Yet the function of this pathway in this mating-incompetent pathogen remains unclear. Here, we investigated whether the central transcription factor Ste12 of N. glabratus (NgSte12(1)) can act as a peptide GPCR-coupled transcription factor using a component-swapping strategy in which pathway elements from N. glabratus and S. cerevisiae were reciprocally exchanged and assessed using fluorescent and growth arrest readouts. We show that NgSte12(1) is in principle capable of supporting peptide-GPCR-dependent activation in S. cerevisiae, however, subtle and dependent on promoter identity. Ste12 chimaeras further demonstrate that all three NgSte12(1) subdomains are individually functional and compatible with their S. cerevisiae counterparts. Heterologous expression of ScSte12 in N. glabratus shows constitutive pathway activation, while NgSte12(1) does not show pathway activation in N. glabratus. Peptide-GPCR expression experiments indicate a block in N. glabratus signalling upstream of Ste12 (1): the S. cerevisiae peptide-GPCR Ste2 (ScSte2) activated the N. glabratus Ste12 GPCR-dependent downstream signalling constitutively, while NgSte2 failed to activate signalling in its native environment. Together, these results indicate that NgSte12(1) is a competent pheromone-responsive transcription factor that can couple to a heterologous GPCR in a heterologous context but is unresponsive to its own cognate upstream peptide/GPCR pair, either because it binds a yet unknown ligand or the signalling is rewired in this pathogen.