Detection of the subcellular localization of melanin synthesis enzymes.

Conidia of the test strains were suspended in GB5 liquid medium and adjusted to 10⁵ spores/ml. An aliquot (20 μl) of the spore suspension was dropped onto a glass slide and then placed in a moistened box for germination. Spore germination was observed by using a fluorescence microscope (Axio Imager Z2; Zeiss, Germany), using its light, fluorescence, and light/fluorescence-merged fields. Neutral-density filter set D/A (d = 25) was used during microscopic analysis. The excitation/emission wavelengths used were 488 nm/500 to 550 nm for GFP and 561 nm/570 to 620 nm for RFP. Moreover, the endosomes were stained with FM4-64 (Molecular Probes, AAT Bioquest, USA) and visualized under a microscope via excitation/emission wavelengths of 561 nm/570 to 620 nm according to a method described previously (54). In order to track vacuoles, the specific dye neutral red (CAS no. 553-24-2; Life Science Products & Services, China) was applied, and the stained samples were analyzed under a light microscope (55).