A Conserved Transcription Factor Domain Drives Necrotrophic Effector-Mediated Virulence and Putative Protein...

The fungal pathogen Parastagonospora nodorum utilizes necrotrophic effectors (NEs) to cause chlorosis and necrosis on wheat. NE expression is mediated by an assortment of transcription factors (TFs), the most well-characterized of which is PnPf2. Orthologs of PnPf2 regulate virulence in phytopathogenic fungi across the Ascomycete fungal lineage, yet their protein architecture remains functionally uncharacterized. These orthologs are characterized by the archetypal N-terminal Zn₂Cys₆ zinc-finger DNA-binding domain, a conserved yet poorly characterized middle homology region (MHR), and a C-terminal disordered region. We investigated the role of each of the three domains through PnPf2 truncation mutants in situ. Variable deletions of the C-terminal disordered region resulted in a reduction of SnToxA and SnTox3 expression in planta. The MHR domain is indispensable for host virulence and SnToxA and SnTox3 regulation and also facilitates PnPf2 homodimerization. Using the homodimer-mediating MHR as a bait in library-scale protein-protein interaction yeast two-hybrid assays, we identified a putative COP9-signalosome protein, PnCsn6, as a likely interacting partner. PnCsn6 is essential for disease symptoms during P. nodorum infection on wheat, and we observed NE dysregulation in PnCsn6-deletion mutants. This study provides the first domain-level functional characterization of the virulence-regulating TF ortholog Pf2. Our results demonstrate the essential role of the MHR domain in mediating protein-protein interactions and effector regulation. We have also unveiled evidence that suggests that PnCsn6 contributes to pathogenicity and may point to an important signaling pathway that could work in tandem with PnPf2 for fungal pathogenesis. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.