Fig 2: Spt4 phosphorylation by Hog1 is required for cell growth in the presence of osmostress.Wild type Spt4 (wt), hog1? and the indicated spt4 mutant strains were grown to mid-log exponential phase in YPD and then diluted 1:10 in YPD, 1.4 M NaCl or 1.4 M KCl before starting the assay. Growth curves over 21 hours were analyzed using a Synergy H1 Hybrid Reader (BioTek). Data are the means ± SD of three technical replicates from a representative experiment. Growth curves are represented on a semi-log plot.

Fig 3: Cbl affected H2S production by promoting the transcription of cysH.(A) H2S production was measured by Pb(Ac)2-soaked paper strips in M9 medium supplemented with five mg/L Na2SO3. The tested strains included WT, ?cbl and the complemented strain (?cbl-Pcbl). Values represented as means ± SD (n = 3). **p < 0.005 was determined by one-way ANOVA and Tukey post-test. (B) Fluorescence intensities were detected by Synergy H1 (BioTek, Winooski, VT, USA) after the tested strains were treated with fluorescent H2S probe in M9 medium. Values represented as means ± SD (n = 3). *p < 0.01 was determined by one-way ANOVA and Tukey post-test. (C) The relative expressions of H2S synthesis genes were detected by qRT-PCR. Values represented as means ± SD (n = 3). *p < 0.01 was determined by one-way ANOVA and Tukey post-test. (D) Fluorescence intensities were detected by Synergy H1 (BioTek). The tested strains expressed PcysIJH only (pTRG+pUC19- PcysIJH -eGFP), and co-expressed both Cbl and PcysIJH (pTRG-Cbl+pUC19- PcysIJH -eGFP), respectively. Values represented means ± SD (n = 3). *p < 0.01 was determined by one-way ANOVA and Tukey post-test.

Fig 4: H4-S47D phosphomimetic mutant exhibits transcriptional and growth defects upon osmostress. (A) Fluorescence of the quadruple-Venus (qV) reporter driven by ALD3 promoter was measured by flow cytometry before (control; gray) and after osmostress (0.4 M NaCl for 45 min, blue) in wild-type (WT) and the mutant H4-S47A and H4-S47D strains. Dotted black lines indicate the level of transcription of the WT in response to stress. Fluorescence intensity under stress was quantified with respect to the WT strain and the percentages are indicated. (B) WT and the indicated histone mutant strains were subjected to osmostress (0.4 M NaCl) for the indicated length of time. Total mRNA was assayed by northern blot with radiolabeled probes for ALD3 and HSP12 (stress-responsive genes) and ENO1 (as loading control). RNA quantification is expressed in percentage as the ratio of mRNA levels normalized by ENO1 and taking the value of maximum gene expression of the WT strain as 100% reference. (C) Volcano plot showing significantly altered gene expression in WT strain upon stress (0.4 M NaCl, 15 min). Dashed vertical lines mark the imposed log2 fold change threshold of 1 (up-/down-regulation), whereas the dashed horizontal line indicates an adjusted P-value of 0.05. (D) Differences in the transcriptional stress-activated and stress-repressed response defined in (C) between H4-S47D mutant and WT strains. Horizontal lines in the boxplots indicate the median, dashed gray lines indicate a relative absolute fold change of 1. The indicated P-value is based on a Wilcoxon-test, while the highlighted numbers represent genes significantly altered by the mutant in response to osmostress compared to non-stress conditions (blue dots). (E, F) Cell growth analysis in response to osmostress. WT and the indicated histone mutant strains were grown to mid-log exponential phase in YPD, serially diluted (2-fold), spotted onto YPD plates with and without 1.2 M NaCl and grown at 30°C for 3–5 days (E). Exponential phase cell cultures were diluted in YPD or YPD 1.2 M NaCl and grown at 30°C with constant agitation for 40 h. Cell density (OD660) was measured using a Synergy H1 Hybrid Reader (BioTek). Growth curve data are the means ± SD of a minimum of three technical replicates from a representative experiment (F).

Fig 5: Analysis of growth rates of PDR1F749I and PDR3T949A single and double mutants. Haploid PDR1F749I and PDR3T949A evolved strains were crossed and sporulated to generate recombinant haploid spores. Spore genotypes were determined by CAPS markers (Supplementary Table 12 in Supplementary File 1, Materials and Methods). a) Segregants were arrayed in a 96-well plate and grown in 9 µM clotrimazole media at 30°C in a Biotek Synergy H1 plate reader that measured growth of each strain by optical density (Supplementary Table 13 in Supplementary File 1). b) Doubling time of each genotype was calculated by linear fit of logarithmic growth phase for each curve in a (Materials and Methods).