FIG 4.

A pfa4 strain exhibits ras1-independent phenotypes. (A) pfa4 mutant cells exhibit abnormal chitin localization. The wild-type (H99) strain and pfa4Δ (CBN201) and ras1Δ (CBN336) mutant strains were incubated overnight in YPD medium, diluted 10-fold in YPD medium, and then grown for an additional 24 h at 37°C. Cells were stained with calcofluor white prior to imaging (magnification, ×100) by fluorescence microscopy. Scale bar, 5 μm. (B) The pfa4Δ mutant strain is sensitive to cell wall stress. The wild-type (H99) strain and pfa4Δ (CBN201) and ras1Δ (CBN336) mutant strains were grown overnight in YPD medium, serially diluted, spotted onto YPD medium containing 1 mg/ml caffeine, 1 mg/ml calcofluor white, 0.5% Congo red, or 0.02% SDS. Plates were incubated at 30°C for 48 h. (C) Pfa4 is required for mating filament formation and differentiation during sexual development in a dose-dependent manner. Bilateral mating mixtures were prepared by mixing overnight cultures of MATα and MATa wild-type cells (H99 and KN99a) and MATα and MATa pfa4 mutant cells (CBN201 and CBN255). Mixtures were spotted onto MS mating medium and incubated for 5 days in the dark. Images were taken at the periphery of the mating mixtures at magnifications of ×100 (top panels) and ×400 (bottom panels).