Eisermann, I.; Sahu, N.; Garduno-Rosales, M.; Derbyshire, P.; Menke, F. L. H.; Ma, W.; Talbot, N. J.

Septin GTPases are essential cytoskeletal regulators that organize membranes and scaffold protein complexes to control cytokinesis, polarity, and morphogenesis. How septins execute these functions remains poorly understood, and comprehensive, stage-resolved interaction maps are lacking. Here, we define a quantitative, time-resolved septin interactome in the rice blast fungus Magnaporthe oryzae using immunoprecipitation coupled to mass spectrometry. We map more than 350 interactors of septins Sep3, Sep4, Sep5 and Sep6, revealing a dynamic network required for appressorium-mediated plant infection. Beyond canonical roles in cytoskeletal organisation and polarity, septins associate with proteins linked to membrane remodelling, metabolism, and virulence, deployed during host invasion. Integration with ultra-high-throughput yeast two-hybrid analysis defines a high-confidence septin interactome and identifies previously uncharacterised factors, including Msi1, a BAR domain protein required for invasive growth. Together, these findings establish septins as dynamic organisers of infection-related processes and provide a framework for understanding how cytoskeletal scaffolds coordinate fungal pathogenesis.