Biotinylation-dependent near-neighbor analysis for identification of septation regulators in Aspergillus fumigatus

Recent work has established septation as a critical process for virulence in A. fumigatus, as strains lacking septa are avirulent. While our work has focused on the well-studied Septation Initiation Network (SIN), understanding of septation machinery upstream and downstream of SIN remains limited in filamentous fungi. Proximity labeling techniques are powerful tools to study pathway interactions, especially those that are transient. Recent advances have seen TurboID used to study pathways and processes important for fungal pathogenesis. In this study, we adapt TurboID for use in Aspergillus fumigatus and use overlapping datasets of two SIN components, the terminal NDR kinase SidB and its binding partner MobA, to identify putative septation effectors. Many known septation effectors, including septins and cell wall synthesis proteins, were enriched in our datasets, validating the approach. Phenotypic characterization of hits identified by LC-MS/MS revealed several previously uncharacterized gene products involved in septum formation and cell wall stress tolerance. These included the PP2A regulatory subunit PabA and IQGAP SepG, which have been shown to regulate septation in Aspergillus nidulans. Thus, TurboID is a useful tool to study fungal signaling and physiology in A. fumigatus and may be used to improve understanding of pathogenic processes.