Divergence of TORC1-mediated stress response leads to novel acquired stress resistance in a pathogenic yeast

Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H2O2 stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae. This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of-Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1’s response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.


31
Acquired stress resistance (ASR) is the phenomenon where organisms exposed to a non-lethal 32 primary stress become more resistant to a secondary severe stress of the same or a different 33 type (Lu et al. 1993; Leroi et al. 1994 shown in S. cerevisiae that stressors occurring early during fermentation, e.g., heat and ethanol, 37 have a strong protective effect against stresses that occur later, such as oxidative stress, while 38 the reverse is not true (Mitchell et al. 2009). This asymmetry in cross-stress protection suggests 39 that ASR is not simply a general stress response but rather represents an adaptive anticipatory 40 response, enabling cells to predict and survive future challenges. A natural prediction is that 41 ASR must be adapted to the specific stress patterns organisms encounter in their environments, 42 leading to divergence between species. 43 Despite the potential role of ASR in adaptation, we know little about its evolution. ASR is 44 known to have biochemical, post-translational or transcriptional mechanisms (Davies 2016; 45 Goulev et al. 2017). At the transcriptional level, studies in S. cerevisiae showed that ASR does 46 not rely on a single, all-purpose program; instead, genes involved in ASR are regulated in a 47 stress-specific manner (Gasch et al. 2000). Furthermore, ASR against the same secondary 48 stress is dependent on distinct genes that are regulated by different types of primary stresses 49 (Berry et al. 2011). Such stress-specific regulation enables mutations to target specific contexts 50 and avoid pleiotropic effects, thereby facilitating the evolution of ASR. However, few studies 51 dissected ASR in closely related species. Previous studies on ASR in yeasts, for example, have 52 primarily focused on comparing S. cerevisiae with distantly related species, such as C. albicans 53 and S. pombe, which are estimated to have diverged from the baker's yeast ~200 and ~500 Brown et al. 2020). These substantial evolutionary distances, coupled with the whole genome 56 duplication (WGD) event specific to S. cerevisiae, significantly hindered our ability to discern the 57 evolutionary alterations behind ASR divergence. 58 In this study, we focused on the more closely related S. cerevisiae and C. glabrata, both 59 of which are post-WGD and share more than 90% of their genes with one-to-one orthologs 60 (Dujon et al. 2004). Even though C. glabrata is evolutionarily close to S. cerevisiae, it is including the induction of oxidative stress response (OSR) genes (Ikeh et 83 We found in this study that a short-term phosphate starvation led to the induction of at 84 least 15 OSR genes and enhanced the survival of C. glabrata against a severe H2O2 stress. The 85 same treatment had little to no effect in the related S. cerevisiae. Using transcriptional profiling, 86 genetic perturbations, reporter and survival assays, we identified key players in the network 87 underlying the ASR, including the catalase gene CTA1, transcription factors (TFs) Msn4 and 88 Skn7 and homolog of the Greatwall kinase, Rim15. We found that phosphate limitation quickly 89 and strongly suppressed the Target-of-Rapamycin Complex 1 (TORC1) in C. glabrata but not in 90 S. cerevisiae; the proximal effector Sch9 was also shown to contribute to the ASR in the former 91 species, strongly implicating TORC1 as a major contributor to the divergence in ASR. In 92 summary, our results identified a key signaling-TF-effector gene subnetwork underlying the 93 divergent ASR phenotype and thereby provides a concrete example of ASR evolution. 94

95
Phosphate starvation provides strong acquired resistance for H2O2 in C. glabrata but not 96 in S. cerevisiae 97 Based on the evidence linking phosphate to oxidative stress and virulence in C. glabrata and C. 98 albicans, we asked whether phosphate starvation could provide acquired resistance for severe 99 H2O2 stress in C. glabrata and whether the behavior is different in the low pathogenic-potential 100 relative, S. cerevisiae. To answer this question, wild type cells from both species were subjected 101 to 45 minutes of phosphate starvation, followed by a severe H2O2 challenge (Fig. 1A). We 102 designed the experiment to be able to compare the ASR effect between species despite 103 differences in their basal resistance level to H2O2, which affects the magnitude of ASR (Berry 104 and Gasch 2008). Through titration, we found that 100 mM and 10mM H2O2 resulted in a similar 105 ~2.5% survival in C. glabrata and S. cerevisiae, respectively ( Fig. S1A, P = 1, Materials and 106 Methods). Using these concentrations as the secondary stress, we found that phosphate 107 starvation greatly enhanced the survival of C. glabrata after the H2O2 stress ( Fig. 1B) but had no 108 detectable effect in S. cerevisiae (Fig. 1C). A quantitative colony forming unit (CFU) assay 109 confirmed this (Fig. 1D, raw P = 0.0039 and 0.37 in C. glabrata and S. cerevisiae, respectively). 110 To quantify the magnitude of ASR, we defined ASR-score as the fold increase in survival due to 111 the primary stress treatment, i.e., the ratio between the survival rates with (r') and without (r) the 112 primary stress. Using this definition, we found a 45-minute phosphate starvation led to an ASR-113 score of 3.4 in C. glabrata  for oxidative stress, we altered both the severity of the secondary stress and the length of the 116 primary stress and asked how this affected ASR in both species. We found that more severe 117 secondary stress (higher [H2O2]) led to stronger ASR in C. glabrata but not in S. cerevisiae at 118 the concentrations tested (Fig. S1B). When we increased the primary phosphate starvation 119 treatment to 90 and 135 minutes, we observed a stronger ASR in C. glabrata (ASR-scores = 120 111 and 170, P < 0.05 after Bonferroni correction, Fig. S1C). A similar trend was observed in S. 121 cerevisiae (ASR-score = 11 and 22) but didn't reach statistical significance (Bonferroni-122 corrected P = 0.7 and 0.5). 123 We also wondered whether the ASR for H2O2 is systematically different between the two 124 species or whether phosphate starvation represents a distinct case of divergence. To answer 125 this question, we tested two other stresses, heat shock and glucose starvation, and found that 126 both led to ASR at a similar level between the two species (Fig. S2). Therefore, we conclude 127 that C. glabrata and S. cerevisiae diverged particularly in the phosphate starvation-induced ASR 128 for H2O2, where C. glabrata showed a strong ASR while the same primary stress elicited a much 129 weaker effect in S. cerevisiae. 130 Lastly, we asked if phosphate starvation provides protection for other oxidative stress 131 agents by replacing the secondary stress with either tert-butyl hydroperoxide (tBOOH), an alkyl 132 hydroperoxide, or menadione (menadione sodium bisulfite, MSB), a superoxide agent. We 133 found that phosphate starvation does not provide ASR for tBOOH, while a moderate ASR effect 134 was observed for menadione compared to H2O2 (Fig. S3). We conclude that the existence and 135 magnitude of phosphate starvation-induced ASR depends on the nature of the ROS. This result 136 supports the prediction that ASR is specific to the primary and secondary stress combinations 137 rather than being a general cross-stress protection mechanism (Mitchell et al. 2009). 138 Oxidative stress response genes were induced under phosphate starvation in C. glabrata 139 One potential mechanism for the observed ASR in C. glabrata is that phosphate starvation led 140 to the induction of oxidative stress response (OSR) genes during the primary stress ( Fig. 2A). 141 To test this, we curated a set of H2O2-response genes in the well-studied S. cerevisiae, which 142 include antioxidants, proteases, chaperones and other genes to scavenge ROS and mitigate 143 ROS-induced damages, as well as the TFs regulating them (Lee et al. 1999;Hasan et al. 2002). 144 Using this reference set (Table S1) (Fig. 3A). We also confirmed that Cta1 protein levels increased during 158 phosphate starvation by endogenously tagging Cta1 with GFP (Fig. 3B). Notably, this strain had 159 the same H2O2 resistance as the wild type (Fig. S4A). To determine if CTA1 is required for the 160 phosphate starvation-induced ASR in C. glabrata, we created a cta1∆ strain and compared its 161 ASR phenotype to the wild type using comparable strengths of H2O2. We found that loss of cta1 162 largely abolished the phosphate starvation-induced ASR for H2O2 (Fig. 3C). Quantitative CFU 163 assays revealed a residual ASR effect in cta1∆ (ASR-score = 1.5, 95% CI [1.2, 1.8]), which was 164 significantly lower than that in the wild type strain (ASR-score = 9.8, 95% CI [5.5, 15], Mann-165 Whitney's U test comparing the two genotypes, P = 0.001) (Fig. 3D). The ASR defect in cta1∆ 166 was rescued by putting CTA1 back into its endogenous locus (Fig. S4B both Msn4 and Skn7 were required for CTA1 induction (Fig. 4A). However, since Cta1-GFP was 194 still induced in the msn4∆ skn7∆ strain, additional TFs must be involved. 195 We also measured Cta1 induction during H2O2 treatment and confirmed that both Yap1 196 and Skn7 were required, while msn4∆ resulted in a slight decrease in induction and msn2∆ had 197 no measurable effect under the H2O2 concentration tested (Fig. S7). This showed that the  198  induction of the same effector gene, CTA1, depended on different TF combinations under  199 different stresses, a result consistent with previous findings in S. cerevisiae (Gasch et al. 2000). 200 To determine if Msn4 and Skn7 directly regulate CTA1 under phosphate starvation, we 201 first predicted their binding sites in the promoter of CTA1 (Fig. 4B). This allowed us to construct 202 a series of promoter mutants, including two internal deletions (200 bp) removing the predicted 203 Msn4 or Skn7 binding sites, as well as point mutations predicted to abrogate Msn4 and Skn7 204 binding (Fig. 4B) corrected P < 0.01). This divergence is condition-specific: glucose starvation led to a similar 218 level of Msn4 nuc in both species (Fig. 5, 76% in C. glabrata vs 73% in S. cerevisiae, raw P = 219 0.7). No significant difference was observed between species under the no-stress condition (raw 220 P = 1). These results further support CgMsn4 as one of the TFs mediating the phosphate 221 starvation induced ASR in C. glabrata and that divergence in how Msn4 responds to phosphate 222 starvation is a major contributor to the species divergence in ASR. In contrast, we found that the 223 paralog Msn2 translocated into the nucleus in a comparable fraction of cells in the two species 224 under phosphate starvation: 52% in C. glabrata and 54% in S. cerevisiae ( Fig. S8, P = 0.8). 225 Combined with the lack of effect of msn2∆ on Cta1 induction, we conclude that Msn2 is not 226 involved in the ASR and doesn't contribute to its divergence. 227 The Greatwall kinase, Rim15, is an important signaling component in the ASR network 228 Given Msn4's role in the phosphate starvation-induced ASR in C. glabrata (Fig. 4, 5 during the primary stress in C. glabrata (Fig. 6A). To test this, we compared the nuclear 235 localization of CgMsn4-GFP in the wild type and rim15∆ backgrounds. We found that ~68% of 236 the wild type cells had nuclear-localized CgMsn4 during phosphate starvation, compared with 237 only 22% in the rim15∆ background ( Fig. 6B, P < 0.001). By contrast, both strain backgrounds 238 showed a low fraction of cells with CgMsn4 nuc under high phosphate conditions ( Fig. 6B, P = 239 0.2). Next, we asked if Rim15 is required for CTA1 induction. We found that rim15∆ reduced 240 Cta1-GFP induction to a similar level as msn4∆, supporting its role as a regulator of the ASR 241 (Fig. 6C). msn4∆ rim15∆ had a more severe effect than either deletion alone, suggesting 242 additional players besides the Rim15-Msn4 pathway (Fig. 6C). Lastly, to assess the role of 243 Rim15 in the phosphate starvation-induced ASR, we performed quantitative CFU assays to 244 compare the wild type and rim15∆ strains at a comparable strength of H2O2 (Fig. 6D) minutes of phosphate starvation did not affect the total level of Rps6 but markedly reduced the 263 abundance of phosphorylated Rps6 (P-Rps6) in C. glabrata (Bonferroni-corrected P = 0.06, Fig.  264 7A, B). The same treatment, however, did not affect the proportion of P-Rps6 in S. cerevisiae (P 265 = 1). By contrast, nitrogen starvation rapidly inhibited phosphorylation of Rps6 in both species 266 (P = 0.06 and 0.07). These results confirmed that inhibition of TORC1 by nitrogen starvation is 267 conserved between the two species while only in C. glabrata is TORC1 strongly inhibited by 268 phosphate starvation at 60 minutes, consistent with their distinct ASR phenotypes (Fig. 1). To 269 determine which of the two species represents the ancestral state, we performed the same P-270 Rps6 assay in two outgroup species, K. lactis and L. walti, which diverged from S. cerevisiae 271 and C. glabrata approximately 120 million years ago (Shen et al. 2018 Next, we followed Cta1-GFP induction in C. glabrata exposed to rapamycin and found a dose-285 dependent response except for the highest dose, which resulted in a lower induction than the 286 next dose (Fig. 7C). Lastly, we found 125 ng/mL rapamycin treatment for 45 minutes modestly 287 but significantly enhanced the survival of C. glabrata following a secondary H2O2 stress ( Fig.  288 7D, mean ASR-score = Using an established DAL80pr-GFP reporter (Neklesa and Davis 2009), we found that nitrogen 310 starvation strongly induced NCR-sensitive genes, but phosphate starvation had minimal effects 311 in both S. cerevisiae and C. glabrata (Fig. S12). This suggests that evolution can wire an input 312 such as phosphate limitation to a specific TORC1 output, e.g., stress response, without strongly 313 impacting other outputs. 314

Discussion 315
Acquired stress resistance (ASR) is suggested to be an adaptive anticipatory response (Mitchell 316 et al. 2009). As such, it is expected to evolve between species. However, both the genetic basis 317 of ASR and its evolution remain poorly characterized. In this study, we discovered a divergent 318 ASR phenotype between two related yeasts: in the opportunistic pathogen, C. glabrata, a short, 319 non-lethal phosphate starvation provides a strong protective effect for a severe H2O2 stress, 320 while in the related baker's yeast, S. cerevisiae, the same treatment has little to no effect ( Fig.  321 1). We identified a core subnetwork behind the ASR in C. glabrata, where phosphate starvation 322 not only induces the canonical phosphate starvation (PHO) response via the TF Pho4, but also 323 induces more than 15 oxidative stress response (OSR) genes, mediated by two TFs involved in 324 the canonical OSR, Msn4 and Skn7 (Fig. 2, 8A). Divergence in ASR between the two species is 325 due in part to the difference in nuclear translocation of Msn4 in response to phosphate 326 limitation. Remarkably, the central regulator TORC1 showed differential response to phosphate 327 in the two species while being similarly affected by nitrogen starvation (Fig. 7). This implicates 328 TORC1 as a key point of divergence behind the ASR evolution; it also shows how a conserved, 329 multifunctional complex can be evolutionarily tuned. 330 We showed that the divergence in the ASR for H2O2 between the two species is specific 331 to phosphate -two other primary stresses tested, glucose starvation and heat shock, elicit 332 similar protective effects in both species (Fig. S2 C. glabrata cells were engulfed by macrophage were also induced during phosphate starvation, 341 including genes involved in autophagy, TCA cycle, amino acid biosynthesis and iron 342 homeostasis (Fig. S14). These results suggest that phosphate limitation is physiologically 343 relevant in the host and may serve as a stimulus to induce ASR for more severe stresses. 344 Does phosphate starvation provide general protection against any stress or is it specific 345 to certain types of stresses? We found that the protective effect of phosphate starvation varies 346 depending on the types of ROS used as the secondary stress ( Fig. S3): the protective effect is 347 the strongest for H2O2, moderate for menadione sodium bisulfite, and non-existent for tBOOH. 348 The lack of ASR for tBOOH, an alkyl hydroperoxide, was initially surprising. However, this can 349 be understood based on its many differences from H2O2, e.g., its main cellular targets are lipids 350 in the plasma membrane; it reacts differently with the cellular redox system, and elicits different demonstrates that ASR is specific to the primary and secondary stress combinations. We further 353 hypothesize that these combinations are frequently encountered in the organism's environment, 354 providing the selective pressure for the ASR. 355 The ASR effect in C. glabrata bears similarity to a previously described stress resistance 356 phenotype following chronic starvation in S. cerevisiae (Gresham et  ASR be attributed to the difference in timing/threshold of the chronic starvation response? We 359 think not. First, the chronic starvation response is associated with severe cell cycle arrests. We 360 found that during a short (< 2hrs) phosphate starvation, both species continue to divide with a 361 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; similar fraction of unbudded cells, suggesting that cell-cycle arrest cannot explain the species 362 difference in ASR (Fig. S15). Moreover, while several OSR genes were induced during chronic 363 phosphate starvation in S. cerevisiae, catalase genes were notably not among them (Petti et al. 364 2011). This is in contrast to the crucial role of CTA1 induction for the ASR in C. glabrata. We 365 therefore believe that the phosphate starvation-induced ASR has distinct transcriptional basis 366 and serves a different purpose than the chronic starvation response. 367 Does ASR involve the same effector and regulator genes as the canonical response to 368 the secondary stress, or is it made up of an entirely different set of genes? We found phosphate 369 starvation-induced ASR in C. glabrata shares many of the TFs and effector genes with the 370 canonical OSR, but differs in the TF combinations required to induce the effector genes such as 371 CTA1, resulting in distinct induction kinetics (Fig. 2, 3, 8B). Such condition-specific regulation of 372 stress response genes has also been observed in S. cerevisiae and may be a hallmark of stress 373 response networks in general (Gasch et al. 2000). In theory, this could facilitate the evolution of 374 stress responses, including the ASR, by allowing for the reuse of existing genes and networks. 375 This likely depends on cis-regulatory mutations that modify the expression pattern of a gene in 376 one context with little or no impact under another, thereby reducing the pleiotropic effects. 377 Another intriguing finding is that the paralogous Msn2 and Msn4 showed divergent functions in 378 ASR and H2O2 stress between species. In S. cerevisiae, the two paralogs have largely identical of CTA1 in C. glabrata both during phosphate starvation and H2O2 stress, while Msn4 plays an 382 important role (Fig. 4, 5, S7). We speculate that the maintenance and evolution of TF paralogs 383 is an important source for ASR and, more generally, stress response evolution. 384 Lastly, given the central role and conservation of TORC1 across species, it is surprising 385 that its response to phosphate differed significantly between the two yeasts, which likely led to 386 their ASR divergence. How can a central, multi-functional regulator like TORC1 evolve? Our 387 results show that phosphate starvation specifically induced the stress response branch of 388 TORC1 while it had minimal effects on another branch associated with nitrogen catabolism (Fig.  389 7, S12). We propose that the ability to rewire specific input and output branches of TORC1 390 allows evolution to fine-tune the regulator's function without having pleiotropic effects (Fig. 8C). 391

Materials and Methods 392
Experimental reproducibility and data availability 393 Each experiment was repeated at least two but mostly three or more times with >2 biological 394 replicates each. Data analysis and visualization were performed in R v4.2. Raw data, R 395 markdown files, and output files are available at https://github.com/binhe-lab/E036-ASR-H2O2. 396 This repository will be version controlled by Zenodo upon publication. 397

Yeast media and growth conditions 398
Yeast cells were grown in the Yeast extract-Peptone Dextrose (YPD) medium or the Synthetic 399 Complete (SC), using Yeast Nitrogen Base without amino acids (Sigma Y0626) supplemented 400 with 2% glucose and amino acid mix. Unless specified, all stress treatments were performed 401 with mid-log phase cells. Briefly, cells were grown overnight in either YPD or SC media, diluted 402 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. in the morning to OD600 = 0.2 and grown to OD600 ~ 1. To apply the treatments, cells were 403 collected by centrifugation at 3,000g for 5 minutes, washed 2-3 times with water and released 404 into the treatment media. Nitrogen starvation medium was made using the Yeast Nitrogen Base 405 without amino acids and without ammonium sulfate (Sigma, Y1251). Glucose starvation medium 406 had 0.02% or 0.05% glucose in the SC medium. Phosphate starvation medium was made using 407 Yeast Nitrogen Base with ammonium sulfate, without phosphates, without sodium chloride (MP 408 Biomedicals, 114027812) and supplemented to a final concentration of 2% glucose, 1.5 mg/ml 409 potassium chloride, 0.1 mg/ml sodium chloride and amino acids, as described previously ( the SC medium at the indicated concentrations immediately before the treatment. 422

Generating yeast strains and plasmids 423
Genetic transformation of both S. cerevisiae and C. glabrata was performed using the LiAc 424 method as described in (Gietz and Schiestl 2007). C. glabrata knockout strains were generated 425 with auxotrophic markers HIS3 and URA3 and the nourseothricin drug marker NAT. ~200 bp of 426 flanking homology sequences were added to either a deletion cassette or a knock-in construct 427 using overlap PCR. Transformants were confirmed by colony PCR on both sides of the gene. 428 To construct CTA1 promoter mutants, a URA3 cassette was used to replace the endogenous 429 promoter, after which the mutant allele constructed by PCR mutagenesis was used as the repair 430 template to swap out the URA3 and selecting with 5-fluoroorotic acid (5-FOA) (GoldBio, F-320). 431 A similar approach was used to replace the endogenous CTA1 promoter with the promoter from 432 the C. glabrata MET3 gene. The CgMET3 promoter was amplified from pCU-MET3 (Maroc and  433 Fairhead 2019) (AddGene #45336 strains, plasmids and qRT-PCR primers used in this study are listed in Table 1, Table 2 and  446  Table 3, respectively. 447

Spotting and Colony Forming Units (CFU) assays 448
A semi-quantitative spotting assay or a quantitative Colony Forming Unit (CFU) assay was used 449 to measure the survival rate after a treatment. For the spotting assay, the treated cells were 10x 450 serially diluted in ddH2O. 5 μl of each dilution was spotted onto an SC plate and allowed to dry 451 before the plate was incubated at 30C for 16-36 hours. An image was taken at the end of the 452 incubation using a homemade imaging hood fitted with an iPad Air. For the CFU assay, post-453 treatment cells were diluted 100 to 1,000-fold and plated on an SC plate. The dilution was 454 chosen to produce roughly 10-500 colonies per plate. The plates were incubated for 48 hours 455 and CFU was manually counted using a lab counter (Fisher Scientific). 456

Calibrating H2O2 concentrations 457
To apply an equivalent strength of H2O2 treatment to species and genotypes with different basal 458 survival rates, we measured the cell survival rates after treatment with different concentrations 459 of H2O2. Specifically, we exposed S. cerevisiae cells to a series of H2O2 concentrations from 460 0mM to 10mM, with 2mM steps. Similarly, we exposed C. glabrata cells H2O2 concentrations 461 from 0mM to 100mM with 20mM steps. CFU assays were performed after 2 hours of treatment 462 to estimate the survival rate. A Mann-Whitney U test was used to determine the significance of 463 differences. Concentrations resulting in similar survival rates across species or genotypes were 464 chosen as the condition for the secondary stress in the ASR experiment. 465

Acquired stress resistance (ASR) assay 466
During the primary stress phase, mid-log phase cells were collected, washed, and released into 467 either the SC medium for a mock treatment or the primary stress media, e.g., -Pi for phosphate 468 starvation. After 45 minutes (or as indicated) of incubation with shaking at 30 C, cells were 469 collected by centrifugation and resuspended at OD600 = 0.2 in the secondary stress treatment 470 media, then incubated the same as before for 2 hours. After the secondary stress, cells were 471 diluted and directly used for the spotting or CFU assay. CFU multiplied by the dilution rate was 472 then used to calculate r (MO/MM), r (PO/PM)' and the ASR-score (r'/r When all samples were in the ethanol-dry ice bath for > 20 minutes, cells were collected by 485 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; centrifugation and quickly washed with ice-cold water to remove the methanol and resuspended 486 in RNAlater solution (Qiagen, 76104) for at least 2 hours. Cells were centrifuged to remove the 487 RNAlater, flash-frozen in liquid nitrogen and stored at -80 C until RNA-extraction. For each 488 sample, ~5x10 7 cells were collected and total RNA was extracted using a MasterPure Yeast 489 RNA purification kit (Biosearch Technologies, MPY03100) following the manufacturer's protocol. phosphate starved sample to a mock-treated sample were extracted (GSM578408, 578424, 512 578440). The log2 ratios were already background-subtracted, normalized, and used directly for 513 comparisons. To compare orthologous gene expression, we first curated a list of oxidative 514 stress response genes based on the literature (Table S1) (Lee et al. 1999;Hasan et al. 2002). 515 To identify their homologs in C. glabrata, we downloaded the orthology and best-hit mapping 516 between the two species from CGD. The best-hit mapping was based on less stringent criteria 517 and only used when an orthology mapping was not available. "added them to the cart", and used the "scan using cart" function. We adopted the default 75% 550 minimum percent of maximum score cutoff, and set the background percent A/T to 0.3, which 551 was based on the mean GC% in 1000 bp upstream sequences in the C. glabrata genome 552 calculated in-house. The motifs and prediction results were available in Supplementary Text S1. 553 in each sample to the corresponding total protein stain to obtain the intensity for P-Rps6 or total 590 Rps6. Finally, we divided the two values to get the P-Rps6/total Rps6 ratio. 591

Assaying cell morphology by light microscopy 592
To determine the percent of unbudded cells during a phosphate starvation time course, wild 593 type C. glabrata and S. cerevisiae cells were grown as described above to mid-log phase, 594 collected by centrifugation, washed once with pre-warmed 0 mM Pi media and diluted to 5x10 6 595 cells/mL in 0 mM Pi media. At 0, 20, 45, 75, 105 and 135 minutes into the time course, an 596 aliquot of cells was taken from each sample, sonicated for 5s to break up any cell clumps, 597 diluted 10-50x and counted on a hemocytometer (Marienfeld, 0640030) under a light 598 microscope. The number of unbudded cells over the total number of cells were recorded by 599 manually examining > 100 cells over four fields of view. The numbers from the four fields of 600 view were summed for downstream analysis. 601

Statistical analyses and tests 602
For quantitative ASR assays using CFU, the data from each replicate experiment was used to 603 calculate survival rates with or without the primary stress, expressed as r = CFUMO / CFUMM and 604 r' = CFUPO / CFUPM. The fold change in survival due to the primary stress, expressed as r'/r, aka 605 ASR-score, was calculated for each experiment. We estimated the mean and 95% confidence 606 interval for the ASR-score with 1000 bootstraps using the smean.cl.boot() function in the Hmisc 607 package. To identify equivalent strengths of ROS for different species and strains, a Mann-608 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.06.20.545716 doi: bioRxiv preprint Whitney U test was used to test for differences in the basal survival rates (r) and a two-sided P-609 value was reported. To assess the statistical significance of the ASR effect in a strain, we used 610 the paired r and r' estimates from each replicate and performed a Wilcoxon signed-rank test if 611 n >= 4. When n < 4, a paired Student's t-test was used since the former test has very low power 612 at such a small sample size. The one-sided P-value, with the alternative hypothesis being r' > r, 613 was reported in the legend and text. To compare the ASR effect sizes between two strains, a 614 Mann-Whitney U test (aka Wilcoxson rank-sum test) was performed for the paired data. A two-615 sided P-value was reported. When more than one comparison was made, a Bonferroni 616 correction was applied. For transcriptomic comparisons, we performed a t-test for the triplicates 617 of the log2 fold changes for 29 genes in both species. The alternative hypothesis was that the 618 true log2 fold change was greater than 0 (induced under phosphate starvation). The resulting P-619 values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure (FDR). 620 Genes with an FDR < 0.05 and a log2 fold change > 2 were considered significantly induced.   An unpaired Student's t-test comparing the ∆∆CT values between both treatments with the mock 915 found both to be significantly elevated (P < 0.01, strain was included as a covariate and found to 916 be not significant). (B) Cta1 protein levels were monitored in strains carrying a genomic CTA1-917 GFP fusion using flow cytometry for 4 hours, during which cells were exposed to either mild H2O2 918 stress, phosphate starvation or mock treatment. significantly increased the survival of both strains during the secondary challenge (raw P = 0.016 928 in both), but the ASR effect size was much smaller in cta1∆ (ASR-score = 9.8 and 1.5 in wild type 929 and cta1∆, respectively; Mann-Whitney U test P = 0.001 between the two). 930 931 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; independent biological replicate (n = 7) and the red bar represents the mean. Basal survival rates 965 were not different between the two strains (P = 0.5). The ASR effect is significant in both (raw, 966 one-sided P = 0.008 for both), but is significantly lower in rim15∆ (mean ASR-score = 6.29 in WT 967 vs 3.45 in rim15∆, Mann-Whitney U test, two-sided P = 0.04). Asterisks indicate potential evolutionary events, both at the trans-and cis-(promoter) 1000 levels. Combined, they lead to distinct induction kinetics under the two stimuli (right). (C) 1001 Proposed rewiring in the Target-of-Rapamycin Complex 1 (TORC1). Nitrogen-sensing is 1002 conserved between species and strongly activate two of TORC1's downstream branches. By 1003 contrast, sensing of phosphate is evolutionarily labile; it strongly activates the stress response 1004 branch but very weakly affects the nitrogen catabolism branch. The question mark indicates we 1005 still lack direct evidence for TORC1 being responsible for the stress gene induction in the ASR. 1006 This model suggests that flexibility in connecting individual stimulus with specific downstream 1007 branch(es) allows TORC1 to contribute to the ASR evolution by avoiding pleiotropic effects. 1008 1009 Tables 1010 Table 1 Fig. 7, S2, S3, S4, S10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Fig. 1, 3, 5, 6, 7, S1, S4, S6, S8, S9, S10, S15 ade2-1 trp1-1 can 1-100  leu2-3,112 his3-11,15 ura3 GAL+ ATCC #200903 Fig. 1, 6, 7, S1, S2, S10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made This study Fig. 3 CgACT1 forward qPCR primer oH1348 5'-GACCAAACTACTTACAACTCC -3' This study Fig. 3 1016 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

S. cerevisiae
The copyright holder for this preprint this version posted September 19, 2023. ; Figure 1. Phosphate starvation induces strong acquired resistance for H 2 O 2 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 H 2 O 2 (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 H 2 O 2 concentrations were used for the two species to achieve a similar survival rate without the primary stress. (D) Survival rates (%) after the secondary H 2 O 2 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 (raw P-values = 0.0039 and 0.37, respectively). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; Figure 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 H 2 O 2 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. Each dot is an independent experiment (n = 6). The difference in basal survival rates was not statistically significant between the wild type and the cta1∆ 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 cta1∆ (mean ASR-scores = 9.8 and 1.5 in wild type and cta1∆, respectively; Mann-Whitney U test two-sided P = 0.001).   ASR assay for the wild type (WT) and rim15∆. Shown are survival rates of the two strains at an equivalent H 2 O 2 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 rim15∆ (mean ASR-score = 6.29 in WT vs 3.45 in rim15∆, Mann-Whitney U test, two-sided P = 0.04).    Figure 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 image 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. The two dotted lines on the right image indicate the regions used for quantifying the total Rps6 amount. (B) Quantification of P-Rps6 / 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 were used as the secondary stress for the two genotypes, resulting in a similar basal survival rate (P = 0.7). Figure 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 H 2 O 2 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 potential pleiotropic effects.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Output . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 1. Basal survival rates and phosphate starvation induced acquired resistance for H 2 O 2 at different primary and secondary stress conditions. (A) Basal survival rates (r) at different H 2 O 2 concentrations in C. glabrata and S. cerevisiae were quantified using Colony Forming Unit (CFU) ratios between cells treated with H 2 O 2 and mock treated ones. The red dots and vertical lines show the mean and 95% confidence intervals based on 1000 bootstrap replicates. Individual data points are shown in different shapes grouped by the date of the experiment. (B) Acquired Stress Resistance (ASR) at different H 2 O 2 secondary stress levels in the two species. ASR-score is defined as the fold increase in survival after the H 2 O 2 treatment as a result of the primary stress (phosphate starvation). r' is the survival rate with the primary stress and r is the same as in (A), i.e., without the primary stress. The red dots and lines have the same meanings as in (A). (C) ASR for H 2 O 2 at different primary stress length. 100 mM and 10 mM of H 2 O 2 were used as the secondary stress for the two species as in Fig. 1. The dotted line shows an ASR score of 1 (i.e., no increase in survival due to the primary stress). A paired t-test was performed on the underlying survival rates (r and r') for each of the six species-by-duration combinations. After Bonferroni correction, all three tests in C. glabrata had P < 0.05, while all three tests in S. cerevisiae yielded P > 0.5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; Supplementary Figure 2. Phosphate starvation-induced ASR for H 2 O 2 diverge between species while other primary stresses show similar effects. ASR experiment was performed as in Figure 1, with the exception of using different primary stresses as indicated on the top: Mock -rich SC medium; HS -Heat shock at 43C; -Glu -0.05% glucose (as opposed to 2% in SC); -Pi -no phosphate SC medium. Images were taken 14 hrs and 21 hrs post spotting for C. glabrata and S. cerevisiae respectively.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  background restored the resistance to H 2 O 2 compared with the wild type strain. Each strain was treated at the indicated concentrations of H 2 O 2 for 2 hours, then spotted onto YPD plates and incubated at 30°C for 48 hours (A) and 20hrs (B). (C) ASR in the wild type, CTA1 complement (cta1::CTA1) and cta1∆ strains. The experiment was conducted similarly as in Fig. 3  H 2 O 2 (mM), 2 hrs . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.06.20.545716 doi: bioRxiv preprint Supplementary Figure 5. Induction of CTA1 provides acquired resistance to H 2 O 2 in C. glabrata. To test if pre-inducing CTA1 is sufficient to provide ASR for H 2 O 2 , we replaced the endogenous CTA1 promoter with the promoter of the C. glabrata MET3 gene. When grown in SC medium lacking methionine and cysteine (-MC), CTA1 was induced to a comparable level as in the endogenous CTA1pr-CTA1 strain under phosphate starvation at 45 minutes (A). Dots represent the mean of at least 3 biological replicates, and the error bars the 95% confidence interval by bootstrapping. The line is the LOESS fit to the data. The endogenous CTA1pr-CTA1 has a basal expression level that is higher than the MET3pr-CTA1 (0 min). We also confirmed that the -MC media itself did not provide ASR in the wild type C. glabrata cells (B, top). Note that in this set of ASR experiments, all SC medium containing H 2 O 2 also lacked methionine and cysteine (C). This allows the MET3pr-CTA1 to be induced during the secondary stress, mimicking what the wild type strain experiences during the H 2 O 2 stress. Using this strain, we found that inducing CTA1 during the primary stress significantly enhanced the survival of C. glabrata cells during the secondary oxidative stress, i.e., providing ASR (B, bottom two rows, -MC vs Mock).
10x serial dilution 10x serial dilution Primary Stress: 45 min -Pi . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023.
Supplementary Figure 6. Cta1 is not required for survival during the primary phosphate starvation, and is not as important for ASR with mild H 2 O 2 used as the primary stress. (A) (Left) Deleting cta1 and the OSR TF, yap1, had no defect on survival under phosphate starvation, while deletion of pho4, which is responsible for the PHO response, showed severe growth defects. Mid-log phase cells of the indicated genotypes were spotted on no phosphate SC plates, incubated at 30°C for 48 hours. (Right) Same as (Left) but spotted onto SC plates with 7.5 mM Pi. All images are representatives of >3 biological replicates. (B) CTA1's importance for ASR is dependent on the primary stress type. ASR for wild type and cta1∆ were tested with various primary stresses, including phosphate starvation (-Pi), glucose starvation (-Glu, 0.02% glucose), mild H 2 O 2 (1.5 mM H 2 O 2 ). ASR experiment was performed as described in the text.  Figure 4A.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.06.20.545716 doi: bioRxiv preprint