Studying Cell Wall-Associated Effectors in U. maydis.

Fungal cell wall-binding secreted effector proteins play pivotal roles in plant-microbe interactions and cell wall remodeling in Ustilago maydis. Accurate assessment of their localization and binding mode is essential for understanding their biological function. Here, a protocol combining immunolocalization with fluorescence microscopy and immunoprecipitation (IP) under both native and denaturing (SDS-containing) conditions is described. Immunolocalization provides direct visualization of protein distribution at the cell wall, while comparative IP experiments reveal the strength and nature of protein-cell wall associations. This combined approach offers a reproducible and adaptable framework for analyzing secreted proteins that associate with cell walls.