7 amino 4 chloromethylcoumarin

Hyphae were cultured in liquid CM medium for 24 h at 25°C before staining. For vacuole observation, a final concentration of 10 μM of CMAC (7-amino-4-chloromethylcoumarin, Sigma-Aldrich) was used to stain the hyphae or conidia for 30 min at 37°C as described [5 (link), 58 (link), 71 (link)]. For conidial septum staining, a final concentration of 10 μg/ml of calcofluor white (CFW) was used in the dark at room temperature for 1 min [72 (link)]. Images were taken under Zeiss LSM 710 confocal laser scanning microscope.

To examine endocytosis, strains were cultured in liquid CM medium. After growing at 28°C for 24 h, the hyphae were stained with N‐(3‐triethylammoniumpropyl)‐4‐(p‐diethylamino‐phenyl‐hexatrienyl) pyridinium dibromide (FM4‐64) (Molecular Probes, Eugene, OR, USA). This dye is used for staining the Spitzenkorper, plasma membrane, septum, early endosomes, late endosomes and vacuolar membranes, as well as for the examination of endocytosis, as described previously (Fischer‐Parton et al., 2000). Photographs were taken using a Zeiss LSM 710 confocal microscope with a 63/1.2 NA C‐Apochromat oil immersion objective (Zeiss, Oberkochen, Germany). The relative fluorescent density was analysed using Image‐pro Plus (Media Cybernetics, Shanghai, China). 7‐amino‐4‐chloromethylcoumarin (Sigma‐Aldrich) (10 mM stock solution in dimethyl sulfoxide) was used for vacuole staining, as previously described (Ohneda et al., 2002; Shoji et al., 2006). Photographs were taken using the confocal laser scanning microscope, as described earlier.