FIG 1 .

C. neoformans Ram1 is required for thermotolerance. (A) Overnight cultures of the wild-type (H99), ram1Δ (SKE1), and ram1Δ + RAM1 reconstituted (SKE39) strains were serially diluted, spotted onto YPD medium, and incubated at 25°C, 30°C, and 37°C for 48 h. (B) The wild-type (H99), ram1Δ (SKE1), and ram1Δ + RAM1 (SKE39) strains were incubated until reaching mid-logarithmic-phase growth at 25°C, diluted, and shifted to either 25°C or 37°C for 4 h. Cells were imaged with DIC optics to assess temperature-dependent alterations in morphology. Arrows indicate evidence of cell lysis. Bar, 5 µm. (C) Quantification of morphological defects after 4 h of incubation at 37°C. Data represent the percentages (as means ± standard errors) of budded cells with morphological defects, which included wide bud necks and/or chains of unbudded cells. At least 400 cells over 3 biological replicates of each strain were counted using ImageJ software (Fiji) (60). P < 0.0001 for ram1Δ mutant versus WT and ram1Δ mutant versus ram1Δ + RAM1 mutant as determined by one-way ANOVA and Tukey’s multiple comparisons test.