Phenotypic Switching and Mating Assays

White-opaque switching and mating assays were performed as previously described [36] (link). The cells were incubated in air or in 5% CO₂ for 4 to 10 days as indicated in the main text. We examined 350 to 500 colonies for each strain. More were tested for nonswitchable strains or on nonconducive media. To verify the colony phenotype, several randomly selected colonies were examined for the cellular morphology. The dye phloxine B, which exclusively stains opaque colonies red, was added to the media. Scanning electron microscopy (SEM) assay was described as we described previously [22] (link). To observe the mating response, 10⁶ cells of each of the two mating strains indicated in the text were mixed and spotted onto Lee's GlcNAc agar and incubated at 25°C for 4 days. At least 1×10⁷ cells of each mating patch were examined with a light microscopy. Quantitative mating assay was performed as previously described with slight modifications [10] (link). Briefly, the mating experiments were performed on Lee's GlcNAc medium at 25°C. The experimental opaque cell samples were collected from Lee's GlcNAc medium plates. To test the mating ability of the MTLa/α strain (SZ306u), 1×10⁶ of MTLa/a (or MTLα/α) cells and 1×10⁶ of MTLa/α cells were mixed and cultured on Lee's GlcNAc medium plates for 48 hours. The mating mixtures were resuspended, diluted, and plated onto three types of selectable plates (without uridine, or arginine, or both) for prototrophic growth. Mating efficiencies were calculated as previously described [14] (link).
The library of transcription factor mutants contains the TF mutants generated by the Johnson lab [37] (link) and strains collected from Candida community [36] (link). Cells of each mutant were plated onto Lee's GlcNAc plates and incubated at 25°C for 7 to 10 days. Opaque colonies were replated and tested for the MTL genotype with PCR.