For the visualization of secreted proteins in infected tissues, maize plants were harvested at 6–7 dpi and analyzed by confocal microscopy as described above. For plasmolysis experiments, infected maize tissue was infiltrated with 1 M mannitol and incubated at least 30 min prior to observation. Experiments were repeated at least three times.
Secreted proteins from fungal cultures were detected as previously described (Djamei et al., 2011 (link)). U. maydis was grown in Complete Medium (CM) medium to an OD600nm of 0.6–0.8, centrifuged, resuspended in Ammonium Medium (AM) medium, and incubated for 5–6 h to induce filamentation. Cultures were centrifuged, and supernatant proteins were precipitated with 10% trichloroacetic acid and 0.02% sodium deoxycholate and resuspended in 100 mM Tris (pH 8). Proteins from the cell pellets were extracted by adding an SDS loading buffer and a spatula tip of glass beads and vortexing for 10 min. All extracts were subjected to immunoblotting using α-HA (Sigma-Aldrich, St. Louis, MO, USA) for detection of effector proteins and α-actin antibodies (Invitrogen, Waltham, MA, USA) for lysis control. Experiments were repeated two times.
Secreted proteins from fungal cultures were detected as previously described (Djamei et al., 2011 (link)). U. maydis was grown in Complete Medium (CM) medium to an OD600nm of 0.6–0.8, centrifuged, resuspended in Ammonium Medium (AM) medium, and incubated for 5–6 h to induce filamentation. Cultures were centrifuged, and supernatant proteins were precipitated with 10% trichloroacetic acid and 0.02% sodium deoxycholate and resuspended in 100 mM Tris (pH 8). Proteins from the cell pellets were extracted by adding an SDS loading buffer and a spatula tip of glass beads and vortexing for 10 min. All extracts were subjected to immunoblotting using α-HA (Sigma-Aldrich, St. Louis, MO, USA) for detection of effector proteins and α-actin antibodies (Invitrogen, Waltham, MA, USA) for lysis control. Experiments were repeated two times.