A Conserved Transcription Factor Domain Drives Necrotrophic Effector-Mediated Virulence and Putative Protein Interactions in Parastagonospora nodorum

The fungal pathogen Parastagonospora nodorum utilises necrotrophic effectors (NEs) to cause chlorosis and necrosis on wheat. NE expression is mediated by an assortment of transcription factors (TF), the most well-characterised of which is PnPf2. Orthologues of PnPf2 regulate virulence in phytopathogenic fungi across the Ascomycete fungal lineage, yet their protein architecture remains functionally uncharacterised. These orthologues are characterised by the archetypal N-terminal Zn 2 Cys 6 zinc-finger DNA-binding domain, a conserved yet poorly characterised 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 and loss of virulence on SnTox3-selective wheat. The MHR domain is indispensable for host virulence, SnToxA and SnTox3 regulation, and also facilitates PnPf2 homodimerisation. Using the homodimer-mediating MHR as a bait in library-scale protein-protein interaction yeast-2-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 characterisation of the virulence-regulating TF orthologue 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 PnCsn6 contributes to pathogenicity and may point to an important signalling pathway that could work in tandem with PnPf2 for fungal pathogenesis.