Figure 3. (A) Induction of white-opaque switching by forced expression of CPH1 and STE11 ^ΔN467 in derivatives of the wild-type strain WO-1 and wor1Δ, wor2Δ, and czf1Δ mutants that express GFP or RFP from the opaque-specific OP4 promoter.

White cells of the indicated strains were grown for 18's medium at 30°C in the presence of doxycycline to induce expression of CPH1 or STE11 ^ΔN467, diluted, and plated on Lee's agar plates without doxycycline to determine the percentage of opaque (black bars) and mixed white/opaque colonies (gray bars). In each case, the first columns show the results with the GFP reporter strains and the second columns show the results with the RFP reporter strains; the wild-type parental strains carrying only the reporter fusions, but not the Tet-inducible genes, served as controls. Results are the means and standard deviations from three biological replicates. Only background switching frequencies were observed in all strains when the precultures were grown in the absence of doxycycline (not shown). (B) Top panels: Colony phenotypes of opaque cells obtained after Tet-induced expression of CPH1 or STE11 ^ΔN467 in wild-type and czf1Δ strains containing P_OP4-GFP or P_OP4-RFP reporter fusions. Colonies were photographed after 7 days of growth at room temperature on Lee's agar plates with phloxin B, which stains opaque colonies pink. The GFP and RFP reporter strains had identical colony phenotypes and only one of them is shown in each case; white colonies are shown for comparison. Bottom panels: Microscopic appearance of opaque cells obtained after Tet-induced expression of CPH1 or STE11 ^ΔN467 in wild-type and czf1Δ strains containing the P_OP4-GFP reporter fusion. Cells were taken from opaque colonies grown on plates without phloxin B to avoid fluorescence caused by binding of the dye. The pictures show epifluorescence micrographs of the cells with filter settings for GFP. Analogous results were obtained with the reporter strains expressing RFP instead of GFP from the OP4 promoter (not shown).