Fig 1.
Phosphate starvation induces strong acquired resistance for H2O2 in C. glabrata and not in S. cerevisiae.
(A) Experimental design for quantifying Acquired Stress Resistance (ASR): cells were first treated with phosphate starvation (-Pi, or P) or mock-treated (M), then exposed to a secondary H2O2 (O) stress or a mock treatment (M). The resulting treatment regimens were referred to as PO, MO, PM and MM. After each treatment regimen, C. glabrata (B) and S. cerevisiae (C) cells were spotted on rich solid media and imaged after 24–48 hrs. Different H2O2 concentrations were used for the two species to achieve a similar survival rate without the primary stress. (D) Survival rates (%) after the secondary H2O2 challenge were quantified using Colony Forming Units either with (r’) or without (r) phosphate starvation as a primary stress. The biological replicates (n = 8) were shown as dots and their means as bars. Basal survival rates (r) were not different between species (P = 0.38), while there was a significant increase in survival due to the primary stress in C. glabrata but not in S. cerevisiae (P-values = 0.0039 and 0.37, respectively).
Fig 2.
Phosphate starvation strongly induces many oxidative stress related genes in C. glabrata but not in S. cerevisiae.
(A) Proposed transcriptional basis for the acquired stress resistance in C. glabrata: phosphate starvation induces both canonical phosphate homeostasis genes and also oxidative stress response genes, providing protection for the secondary H2O2 challenge. (B-E) Comparison of transcriptional induction of genes known to be involved in OSR in S. cerevisiae. Log2 fold changes after 1 hour of phosphate starvation were shown as the mean (bar) of 3 biological replicates (dots) for genes encoding antioxidants (B), protease components (C), molecular chaperones (D) and TFs involved in the OSR (E). The dotted lines indicate a 2 fold induction. Gene names were based on S. cerevisiae except for CTA1, which had two paralogs in S. cerevisiae and only one in C. glabrata. S. cerevisiae CTT1 is involved in OSR and its fold change is shown in (B). Asterisks above a gene name indicate that the gene was significantly induced in that species at an FDR of 0.05.
Fig 3.
C. glabrata CTA1 is induced during phosphate starvation and is required for the acquired resistance to severe H2O2 stress.
(A) CTA1 mRNA levels were assayed using qRT-PCR following 45 minutes of either 1.5 mM H2O2, phosphate starvation or mock treatment. Fold changes over the mock treated samples were shown using ACT1 as a reference. Two isogenic lab strains of C. glabrata were each assayed in triplicates. Bars represent the mean of each strain. An unpaired Student’s t-test comparing the ΔΔCT values between both treatments with the mock found both to be significantly elevated (P < 0.01, strain was included as a covariate and found to be not significant). (B) Cta1 protein levels were monitored in strains carrying a genomic CTA1-GFP fusion using flow cytometry for 4 hours, during which cells were exposed to either mild H2O2 stress, phosphate starvation or mock treatment. The y-axis values are the median fluorescence intensities (a.u. = arbitrary unit). The dots show the mean of >3 biological replicates; the error bars show 95% confidence intervals based on bootstrapping and the lines are LOESS curves fitted to the data. (C) ASR spotting assays for wild type and cta1Δ C. glabrata strains. 2.5 mM H2O2 was used as the secondary stress for cta1Δ, which resulted in a similar basal survival rate as 100 mM H2O2 for the wild type. (D) ASR effects were quantified by comparing the survival rates after H2O2 treatments either with (solid circles) or without (open circles) the primary stress using a colony forming unit (CFU) assay. The difference in basal survival rates was not statistically significant between the wild type and the cta1Δ strain (P = 0.39). Phosphate starvation significantly increased the survival of both strains during the secondary challenge (raw P = 0.016 in both), but the ASR effect size was much smaller in cta1Δ (ASR-score = 9.8 and 1.5 in wild type and cta1Δ, respectively; Mann-Whitney U test P = 0.001 between the two).
Fig 4.
TFs CgMsn4 and CgSkn7 jointly contribute to CTA1 induction during phosphate starvation in C. glabrata.
(A) Cta1-GFP levels over 4 hours of phosphate starvation for wild type (WT) and TF deletion mutants. The dots represent the mean of > 3 biological replicates and the error bars indicate the 95% confidence interval based on 1000 bootstrap replicates. The lines are LOESS fit to the data. (B) A schematic showing the predicted binding sites (BS) for Skn7 and Msn2/Msn4 in C. glabrata (Materials and Methods). Based on the prediction, two types of cis-mutants were created: the internal deletions (4OL, 8OL) each removed 200 bp containing either the predicted Msn2/Msn4 BS or both Skn7 BS; four point mutation alleles each targeted either the single Msn2/Msn4 BS or one of the Skn7 BS predicted to have a higher affinity. (C, D) Comparing the Cta1-GFP induction driven by each of the mutant promoters in (B) compared with the WT and the corresponding TFΔ mutant.
Fig 5.
C. glabrata Msn4 (CgMsn4) translocates into the nucleus upon phosphate starvation but not its ortholog in S. cerevisiae (ScMsn4).
Cellular localization of CgMsn4 (A) and ScMsn4 (B) under phosphate starvation (0 mM Pi, -Pi), glucose starvation (0.02% glucose, -Glu), and no stress conditions. All treatments were for 45 minutes. From left to right: i. CgMsn4-yeGFP and ScMsn4-mCitrine; ii. nucleus staining with DAPI in C. glabrata or labeled with Nh6a-iRFP in S. cerevisiae; iii. merge; iv. bright field. The percent of cells with nuclear-localized Msn4 and the total number of cells examined by microscopy were shown on the right. Scale bars are 5 μm in length.
Fig 6.
The Greatwall kinase homolog Rim15 mediates phosphate starvation-induced ASR in C. glabrata.
(A) Proposed model for phosphate starvation-induced ASR in C. glabrata: phosphate starvation (-Pi) activates Rim15, which goes on to regulate Msn4’s activity and contributes to ASR. Dashed arrows show proposed connections; solid arrows are regulations supported by previous results; red question marks indicate specific downstream effects to be tested. (B) CgMsn4 nuclear localization in WT and rim15Δ strains in -Pi or rich media. Left: CgMsn4-GFP, % of cells with CgMsn4nuc labeled on the lower left; right: bright field (BF) showing cells. Fisher’s Exact Tests (FET) were performed to compare the two strains under each condition. Raw, two-sided P-values were reported on the side. (C) Cta1-GFP induction during -Pi. Dots are the means of >3 biological replicates and the error bars the 95% CI based on 1000 bootstraps. (D) ASR assay for the wild type (WT) and rim15Δ. Shown are survival rates of the two strains at an equivalent H2O2 strength, either with (-Pi) or without (Mock) a primary stress. Each dot is an independent biological replicate (n = 7) and the red bar represents the mean. Basal survival rates were not different between the two strains (P = 0.5). The ASR effect is significant in both (raw, one-sided P = 0.008 for both), but is significantly lower in rim15Δ (mean ASR-score = 6.29 in WT vs 3.45 in rim15Δ, Mann-Whitney U test, two-sided P = 0.04).
Fig 7.
TORC1 is strongly inhibited by phosphate starvation in C. glabrata, likely contributing to the ASR via its proximal kinase, Sch9.
(A) A representative Western Blot for phosphorylated Rps6 (P-Rps6) and total Rps6 in both species under rich media, nitrogen starvation (-N) or phosphate starvation (-Pi) conditions. Red arrows point to the loss vs presence of the P-Rps6 band under -Pi in C. glabrata and S. cerevisiae, respectively. (B) Quantification of %P-Rps6 over total Rps6 (n = 3). *Bonferroni corrected P-values from Student’s t-tests were shown. (C) Inhibiting TORC1 by rapamycin induces Cta1-GFP in a dose-dependent manner. The dots, error bars and lines have the same meaning as before. (D) ASR for H2O2 with rapamycin as the primary treatment. Plotted are survival rates with or without rapamycin treatment (n = 12 for C. glabrata, n = 8 for S. cerevisiae). 60 mM and 6 mM of H2O2 were used as the secondary stress for the two species, resulting in a similar basal survival rate (P = 0.6). A Wilcoxon signed-rank test was used to compare the paired experiments with or without the primary treatment for each species. The raw, one-sided P-values were shown on the top. (E) Same plot as C, comparing Cta1-GFP induction in a phosphomimetic mutant of Sch9, a key proximal effector of TORC1, and a matching wild type Sch9 strain. (F) Same as D but with phosphate starvation (-Pi) as the primary stress, comparing the Sch9-3E mutant (n = 4) and the matching Sch9-wt control (n = 4). 100 mM and 40 mM of H2O2 were used as the secondary stress for the two genotypes, resulting in a similar basal survival rate (P = 0.7).
Fig 8.
ASR divergence and rewiring of the underlying regulatory network.
(A) Phosphate starvation elicits a fast and strong induction of oxidative stress response (OSR) genes in C. glabrata in addition to the canonical phosphate starvation response (right), providing strong acquired resistance for a secondary H2O2 challenge; in the related S. cerevisiae, the induction of OSR genes is much weaker and slower, explaining its lack of ASR. The orange arrows in the right diagram indicate evolutionary rewiring in part of the response to phosphate limitation between the two species. (B) Evolutionary rewiring at the transcriptional level. Regulation of OSR genes such as CTA1 involves some of the same transcription factors (TFs) during both oxidative stress and phosphate starvation, but with different combinatorial logic. Width of the arrow indicates the importance of the TF; the wide arrows to the right of the TFs indicate their combinatorial logic. Asterisks indicate potential evolutionary events, both at the trans- and cis- (promoter) levels. Combined, they lead to distinct induction kinetics under the two stimuli (right). (C) Proposed rewiring in the Target-of-Rapamycin Complex 1 (TORC1). Nitrogen-sensing is conserved between species and strongly activate two of TORC1’s downstream branches. By contrast, sensing of phosphate is evolutionarily labile; it strongly activates the stress response branch but very weakly affects the nitrogen catabolism branch. The question mark indicates we still lack direct evidence for TORC1 being responsible for the stress gene induction in the ASR. This model suggests that flexibility in connecting individual stimulus with specific downstream branch(es) allows TORC1 to contribute to the ASR evolution by avoiding pleiotropic effects.
Table 1.
Yeast strains used in this study.
Table 2.
Yeast expression plasmids used in this study.
Table 3.
qRT-PCR Primers used in this study.