Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway.

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an "a" mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1-Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans.

Introduction

Sexual reproduction is a driving force for evolution and is prominent in eukaryotic organisms, with fungi adopting highly diverse strategies for sexual mating and reproduction [1, 2]. The human fungal pathogen C. albicans is a leading cause of death due to mycotic infection, with mortality rates approaching 40%, even with current treatments [3]. C. albicans has long been thought to be asexual until the discovery of a highly complex parasexual program. In C. albicans, heterothallic (opposite-sex) mating between diploid a and α cells occurs to generate tetraploid a/α intermediates [4, 5]. These tetraploid mating products undergo a parasexual process of concerted chromosome loss to generate diploid and aneuploid progeny rather than adopting a more traditional meiotic cycle [6, 7]. As an additional layer of complexity, an epigenetic switch from the white cell type to the opaque cell type is required for efficient mating to occur [8]. Opaque a and α cells secrete a sex-specific pheromone and induce the formation of mating projections in cells with an opposite Mating type locus (MTL) type, thus initiating cell fusion and mating [9]. Besides differences in mating competency, white and opaque cells also differ in a number of aspects, including metabolic profiles, filamentation ability, susceptibility to antifungals, interactions with host immune cells, and virulence in different infection models [10-12].

Given the predominately clonal nature of C. albicans [13, 14], the frequency with which heterothallic mating occurs in nature appears remarkably low. In the model yeast S. cerevisiae, most natural isolates are homothallic and able to undergo clonal mating because of the expression of homothallic switching (HO) endonuclease and mating-type switching [1]. However, C. albicans does not have a homolog of HO endonuclease and is unable to undergo mating-type switching and subsequent clonal mating [15]. Although same-sex mating (homothallism) has been reported in C. albicans, this unisexual mating has only been observed between two a cells, when α cells are present and secrete α-pheromone (ménage à trois mating), or in strains in which the BARrier 1 (Bar1) protease that degrades α-pheromone has been inactivated [16]. However, there is no evidence of natural ménage à trois mating, and no natural mutants of BAR1 have been reported. Given the barriers to opposite- and same-sex mating, the biological relevance of sexual reproduction in C. albicans remains elusive.

Although C. albicans mating seems to be rare in nature, environmental stressors have been reported to promote loss of heterozygosity at the MTL, drive white-to-opaque switching, and result in concerted chromosome loss, suggesting that stress may be a trigger for sexual reproduction [17]. Nutrient starvation and oxidative stresses are two of the most common environmental stressors experienced by C. albicans in nature. Here, we establish that glucose starvation and oxidative stress efficiently induce the expression of sexual pheromone precursors, leading to the formation of mating projections and enabling homothallic mating in C. albicans. A core cellular stress-responsive pathway, mediated by the molecular chaperone Hsp90 and the heat-shock transcription factor Hsf1, is involved in this regulation through the downstream regulator Cwt1. Cwt1 functions through the direct control of the master regulator of a-type mating MTLa2, which regulates the expression of pheromone-encoding genes. Our study identifies a mechanism by which mating could occur much more frequently in nature than was originally appreciated, uncovers a core cellular stress-response pathway regulating this response, and sheds new light, to our knowledge, on the biology of C. albicans.

Results

Glucose starvation triggers the development of mating projections in C. albicans

Despite the fact that cellular stressors promote loss of heterozygosity at the MTL and induce the white-to-opaque switch [17, 18], there still remains no evidence to directly support the hypothesis that environmental perturbations regulate sexual mating in C. albicans. In the human host, C. albicans resides on mucosal surfaces, niches that are often glucose limited [19]. Therefore, we cultured C. albicans cells in the absence of glucose but in the presence of 0.25% K2HPO4, which acts as a pH-buffering reagent. We named this modified medium as YP-K (1% yeast extract, 2% peptone, 0.25% K2HPO4, w/v) and the control medium as YPD-K (1% yeast extract, 2% peptone, 2% glucose, 0.25% K2HPO4, w/v). Opaque cells were used for all experiments in this study because they are the mating-competent form in C. albicans [8]. Upon spotting an MTLa/a C. albicans strain (GH1013) [20], we noted a wrinkled colony morphology on YP-K agar after five days of growth (). Surprisingly, a portion of cells underwent polarized cell growth () that was elongated and irregular in shape, closely resembling mating projections as opposed to the typical hyphal morphology [21]. When the same strain was cultured on the glucose-containing YPD-K medium, the colonies remained smooth, and cells were exclusively in the yeast form (). This was a dose-dependent effect because incremental increases in glucose levels resulted in incremental decreases in the number of polarized cells observed (). We also observed the development of polarized cells on YP-K medium for three independent clinical isolates of C. albicans with an MTLa/a genotype (P37005, L26, and SZ306) (), suggesting that this inducing effect of glucose starvation is a general feature in MTLa/a strains. The induction of mating projections was not observed in any MTLa/α and MTLα/α strains under the same culture conditions.

Glucose starvation promotes efficient polarized cell growth and induces the expression of mating-related genes in MTLa/a cells of C. albicans.

(A) Morphologies of the laboratory strain GH1013 (MTLa/a) grown on YPD-K and YP-K media. 1 × 105 cells were spotted on YPD-K and YP-K media and cultured at 25°C for five days. Scale bar for colonies (left panels), 2 mm; scale bar for cells (right panels), 10 μm. (B) Morphologies of three clinical C. albicans strains (MTLa/a) grown on YP-K medium at 30°C for four days. Scale bar for colonies (inset), 2 mm; scale bar for cells, 10 μm. (C) Relative expression levels of mating-related genes normalized to ACT1. Cells of GH1013 were used, and culture conditions were same as described in panel (A). Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in . ACT1, ACTin 1; Bar1, BARrier 1; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; MFA1, Mating type A1; MFα, Mating α factor precursor; MTL, Mating type locus; p, mating projection; STE2, STErile 2; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

To verify that the elongated cells were true mating projections but not pseudohyphae or true hyphae, we examined the relative expression of mating-related genes using quantitative reverse transcription PCR (qRT-PCR) (). Compared to the YPD-K cultures, the relative expression levels of Mating type A1 (MFA1) (a precursor of a-pheromone), MFα (a precursor of α-pheromone), STErile 2 (STE2) (the α-pheromone receptor), and Factor-Induced Gene 1 (FIG1) and cell FUSion 1 (FUS1) (two pheromone-response genes), as well as BAR1 (an endopeptidase that degrades α-pheromone), were all significantly increased in cells grown on YP-K. We also constructed four reporter strains in which MFA1, MFα, FIG1, and FUS1 were fused with a green fluorescent protein (GFP) fluorescent marker to further confirm their increased expression on YP-K medium (). Thus, glucose depletion leads to the induction of mating-related genes and polarized cell growth, indicative of the development of mating projections in C. albicans.

Glucose starvation promotes same-sex mating in C. albicans

The induction of both a-pheromone and α-pheromone precursors in MTLa/a cells under glucose-starvation conditions suggests those cells might have lost their original sexual identity, exhibiting features of both MTLa/a and MTLα/α cells. This, in theory, could bypass the requirement of an MTLα/α cell in close proximity to induce homothallic mating between two MTLa/a partners. To further explore whether the induction of mating-related genes and polarized cell growth could lead to true homothallic mating, we performed quantitative same-sex mating assays. When two MTLa/a strains with different auxotrophic markers (GH1350a and GH1013) were cultured together on YP-K medium, mating projections were observed (), and tetraploid progeny that remained the a mating type were generated (, confirming homothallic mating had occurred. Further, cell fusion between two MTLa/a cells and the generation of daughter cells were also observed on YP-K medium (). In contrast, cells remained in yeast form and no mating progeny were isolated when the two MTLa/a strains were grown on YPD-K (). These results provide the premier example of an environmentally relevant stress, glucose starvation, capable of inducing same-sex mating in C. albicans.

Glucose starvation induces same-sex mating in C. albicans.

(A) Same-sex mating between two “a” strains (GH1013 and GH1350a). 1 × 107 cells of each strain were mixed and cultured on YPD-K and YP-K media at 25°C. After three days of growth, the mating mixture was replated onto SCD-Arg, SCD-His, and SCD-Arg-His dropout plates to assess mating efficiency. The middle panel (up) indicates that two “a” cells underwent cell fusion and a daughter cell grew out from the conjunction tube. Scale bar, 10 μm. (B) PCR verification of the MTL types. Strains used: lane 1, SN152 (a/α); 2, GH1350 (α/α); 3, GH1350a (a/a); 4, GH1013 (a/a); 5–7, progeny strains of the GH1350a × GH1013a cross. (C) FACS analysis of the DNA content of parental and progeny strains. Parental diploids have the standard G1 and G2 cell cycle peaks representing 2C and 4C DNA levels. Mating progeny contain DNA content corresponding to 4C and 8C peaks, confirming their tetraploid nature. Arg, arginine; d, daughter cell; FACS, fluorescence-activated cell sorting; His, histidine; M, DNA ladder; MTL, Mating type locus; p, mating projection; SCD, synthetic complete medium; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

Multiple environmental stressors mediate the development of mating projections in C. albicans

Glucose deprivation leads to a shift in C. albicans metabolism, resulting in the activation of genes involved in the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation [19]. These metabolic changes stimulate the production of reactive oxidative species (ROSs) that cause protein damage, thereby activating the unfolded protein response [22]. The intracellular levels of ROSs in cells of C. albicans grown on YP-K medium were significantly higher than those on YPD-K medium and increased with the extension of culture time (). To assess whether oxidative stress could stimulate homothallic mating, C. albicans cells were treated with hydrogen peroxide (H2O2), a strong oxidative-stress–inducing agent. Compared to the untreated control, H2O2-treated cells underwent obvious polarized cell development on the glucose-containing medium YPD-K (). Mating-related genes (MFA1, MFα, and FIG1) were significantly induced in H2O2-treated cells (), and mating efficiency upon H2O2 treatment was comparable to that observed under glucose-deprivation conditions ().

Oxidative stress promotes the development of mating projections in C. albicans.

(A) Relative ROS levels. 1 × 105 cells of strain GH1013 were spotted on YPD-K and YP-K media and cultured at 25°C for one, two, three, or five days. For each day, 1 × 106 cells were used for ROS level determination. Error bars represent standard deviation of three biological replicates. * indicates significant difference (p < 0.05, two-tailed Student t test). (B) H2O2 induces the development of mating projections in the presence of glucose. 200 μL of H2O or 5 mM H2O2 solution was spread on YPD-K medium plates (90 mm). 1 × 106 cells of strain GH1350a were spotted on the medium and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. (C) Relative expression levels of mating-related genes in response to H2O2 treatments on YPD-K medium. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate, and a representative image is shown. (D) Efficiency of same-sex mating on YPD-K medium with or without H2O2 treatment. The mating mixtures were grown on different media at 25°C for seven days. To make YPD-K + H2O2 plates, 200 μL H2O2 (5 mM) was spread on the medium surface (of a 90-mm plate). The numerical data are presented in . FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; MFA1, Mating type A1; MFα, Mating α factor precursor; p, mating projection; ROS, reactive oxidative species; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

To further test other environmentally relevant stress conditions, we performed same-sex mating assays using three other nutrient-poor media types: 3% agar with no additional nutrients, agar containing 3% mouse feces, and agar containing C. albicans debris. C. albicans underwent same-sex mating when cultured on these media types, with mating on mouse-feces–containing medium being most efficient (). Notably, although mating projections morphologically resemble filaments in C. albicans, same-sex mating did not occur when cells were cultured on sorbitol medium that induces opaque cell filamentation (). Therefore, multiple nutrient-deprivation conditions are capable of inducing homothallic mating in C. albicans.

Glucose starvation induces the expression of mating-related genes, stress-responsive genes, and HSP90 genetic interactors

To explore the mechanism of glucose-induced same-sex mating in C. albicans, we performed global gene-expression–profile analysis. Total RNA was extracted from opaque cells grown on YPD-K or YP-K media at 25°C for 60 hours, and the samples collected were used for RNA sequencing (RNA-Seq) assays. We incubated cells for only 60 hours because mating projections were not induced at this time point, enabling us to minimize indirect effects on gene expression. As demonstrated in and , we found 412 genes up-regulated in YPD-K medium and 408 genes up-regulated in YP-K medium (with a change greater than 1.5-fold). As expected, a number of mating-related genes—including MFA1, BAR1, Candida ERK-family protein kinase (CEK)1, and CEK2—were up-regulated in YP-K medium. Heat-shock-protein–encoding genes and oxidative-stress–responsive genes were also up-regulated in YP-K medium, whereas cell-wall–related and glycosylphosphatidylinisotol (GPI)-anchored-protein–encoding genes were enriched in YPD-K medium. Of the differentially expressed genes, 49 have been reported as HSP90 genetic interactors ( and ) [23, 24]. Among the differentially expressed HSP90 genetic interactors, 34 genes were down-regulated and only 15 genes were up-regulated in YP-K medium. These genes were enriched in gene functions associated with stress response, cell wall, transcriptional regulation, and signaling transduction.

Global gene-expression–profile analysis of C. albicans in the presence and absence of glucose.

C. albicans cells were spotted and grown on YPD-K or YP-K media at 25°C for 60 hours. Total RNA was extracted and used for RNA-Seq assays. To be considered differentially expressed, a gene must satisfy three criteria: (1) an FPKM value higher than or equal to 20 at least in one sample, (2) a fold change value higher than or equal to 1.5, and (3) an adjusted p-value (FDR) lower than 0.05. (A) Venn diagram depicting relationships between differentially expressed genes on YPD-K (412) and YP-K (408) media and HSP90 genetic interactors (indicated in the ellipse). (B) Selected heat-shock-protein–encoding, oxidative-stress–induced, and mating-related genes up-regulated in YP-K medium. AIF1, Apoptosis-Inducing Factor 1; BAR1, BARrier 1; CEK1, Candida ERK-family protein kinase; ERK, ERK-family protein kinase; FDR, false discovery rate; FPKM, fragments per kb per million reads; GPI, glycosylphosphatidylinisotol; GST3, Glutathione S-transferase; HMX1, HeMe oXygenase; Hsp90, Heat shock protein 90; KAR2, KARyogamy; MAP, mitogen-activated protein; MFA1, Mating type A1; NADH, Nicotinamide adenine dinucleotide; orf19.3475, Candida gene orf19.3475; PST2, Protoplasts-SecreTed 1; RNA-Seq, RNA sequencing; SIS1, Slt4 Suppressor; SOD5, SuperOxide Dismutase 5; SSA2, Stress-Seventy subfamily A; STE4, STErile 4; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

Down-regulation of Hsf1–Hsp90 signaling promotes mating-projection formation and same-sex mating

Several observations led us to hypothesize that Hsp90 could play an important role in the regulation of glucose-starvation–induced mating-projection formation and same-sex mating. First, glucose starvation increased the intracellular level of ROSs () that would contribute to increased protein damage, thus likely overwhelming the functional capacity of Hsp90. Second, the treatment of oxidative reagents (e.g., H2O2) that led to the increase of intracellular ROSs induced mating-projection formation and same-sex mating in C. albicans (, and ). Third, a number of heat-shock-protein–encoding genes, including HSP90, were up-regulated in the absence of glucose, further suggesting that Hsp90 functional capacity was overwhelmed ( and ).

The conserved heat-shock transcription factor Hsf1 plays an important role in regulating the expression of heat-shock proteins, including the molecular chaperone Hsp90 in C. albicans and other eukaryotes [25, 26]. Hsf1 has been implicated in regulating transcriptional changes in response to oxidative stresses and glucose starvation in S. cerevisiae [27, 28]. Therefore, we first tested the role of Hsf1 in the development of mating projection and homothallic mating in C. albicans. Since Hsf1 is essential for cell viability, we generated a tetracycline-induced (tetON)-promoter–controlled conditional expression mutant, tetON-HSF1/hsf1, in order to assess how changes in Hsf1 levels influence same-sex mating. In the absence or presence of 40 μg/mL doxycycline, the tetON-HSF1/hsf1 mutant formed wrinkled colonies and underwent robust polarized growth on YP-K medium (). Of note, doxycycline (40 μg/mL) exhibited an inhibitory effect on the induction of mating projection formation in the wild-type (WT) control (). With increased exposure to glucose on YPD-K medium, mating projections were only observed in the absence of doxycycline after five days but not upon the addition of 40 μg/mL doxycycline nor in the WT control ().

Role of Hsf1 in the induction of mating projections in C. albicans.

(A) Morphologies of the control and tetON-HSF1/hsf1 mutant. 1 × 105 cells were spotted on YPD-K and YP-K media without or with 40 μg/mL Dox and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. Control, GH1013cartTA. (B) Relative expression levels of mating-related genes. 1 × 105 cells of each strain were cultured on YPD-K medium (for six days) or on YP-K medium (for three days) without or with 40 μg/mL Dox at 25°C. Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in . Dox, doxycycline; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; Hsf1, Heat Shock transcription Factor 1; MFA1, Mating type A1; MFα, Mating α factor precursor; p, mating projection; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; WT, wild type; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

Consistent with the morphological changes, mating-related genes—including MFA1, MFα, FIG1, and FUS1—were induced in the tetON-HSF1/hsf1 mutant on both YP-K and YPD-K media relative to the WT strain (). Finally, we tested the effect of HSF1 depletion on homothallic mating. In the absence of doxycycline, the tetON-HSF1/hsf1 mutant underwent same-sex mating with the WT partner even on the glucose-containing YPD-K medium, albeit at a lower frequency than that observed in glucose-limiting conditions ( and ). However, in the presence of 40 μg/mL doxycycline, neither the tetON-HSF1/hsf1 mutant nor the WT control could undergo homothallic mating on YPD-K medium. Taken together, our results indicate that down-regulation of Hsf1 promotes mating-projection formation and same-sex mating in C. albicans, bypassing the requirement of glucose starvation.

Next, we wanted to evaluate whether the impact of Hsf1 on homothallic mating might be mediated through its regulatory effects on HSP90. We constructed a tetON-HSP90/hsp90 strain using an analogous tetON-promoter–controlled conditional expression strategy. As shown in , culturing this strain in media containing 40 μg/mL doxycycline was required to bypass a substantial growth defect of the strain. Further, opaque cells of the conditional tetON-HSP90/hsp90 strain were not stable, and therefore an ACTin 1 (ACT1) promoter controlling White–Opaque Regulator 1 (WOR1), the master regulator of the opaque phenotype, was introduced in the mutant to maintain the opaque state. Similar to the tetON-HSF1/hsf1 mutant, the tetON-HSP90/hsp90 strain underwent the development of mating projections on both YP-K and YPD-K media containing 40 μg/mL doxycycline (). However, the WT control was unable to form mating projections under the same culture conditions (). In the presence of 100 μg/mL doxycycline, the ratio of projected cells on YPD-K medium was remarkably reduced (). As expected, mating-related genes FIG1 and FUS1 were induced on YP-K medium, while MFA1 and MFα were induced in the tetON-HSP90/hsp90 mutant on both media relative to the WT strain in the presence of 40 μg/mL doxycycline (). As demonstrated in , the relative transcript levels of HSF1 or HSP90 in the tetON-HSF1/hsf1 or tetON-HSP90/hsP90 mutant were lower than that in the WT strain even in the presence of 40 μg/mL doxycycline. Together, these data suggest that reduction of HSF1 or HSP90 levels leads to the induction of a homothallic mating program and bypasses the requirement for glucose depletion.

Down-regulation of Hsp90 promotes the development of mating projections.

(A) Morphologies of the control and tetON-HSP90/hsp90 mutant. Control, GH1013 + pACT1-WOR1; tetON-HSP90/hsp90, a tetON-promoter–controlled conditional expression strain of HSP90 with ectopically expressed WOR1 (+ pACT1-WOR1). 1 × 105 cells were spotted on YPD-K and YP-K media with or without Dox as indicated and cultured at 25°C for three, five, or seven days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (B) Relative expression of mating-related genes. 1 × 105 cells of each strain were cultured on YPD-K medium (for seven days) or on YP-K medium (for three days) with 40 μg/mL Dox at 25°C. Relative expression levels were not tested on YP-K medium without Dox since cell viability of the tetON-HSP90/hsp90 mutant was severely impaired. Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in . Dox, doxycycline; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; Hsp90, Heat shock protein 90; MFA1, Mating type A1; MFα, Mating α factor precursor; NA, not analyzed; p, mating projection; pACT1, plasmid pACT1; tetON, tetracycline-induced; WOR1, White–Opaque Regulator 1; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

Transcription factors Cta4 and Cwt1 regulate the development of mating projections and same-sex mating

To further characterize the regulatory mechanism of glucose starvation-induced mating, we screened a transcription factor homozygous deletion library (in an MTLa/a background) [29] to look for mutants capable of enhanced mating projection formation. Through this functional genomic screening approach, we identified the transcription factors Cwt1 and Cta4 that function as negative and positive regulators of the development of mating projections in C. albicans, respectively ( and ). Homozygous deletion of CWT1, a gene involved in the nitrosative stress response [30], resulted in an increase in mating-projection formation on YP-K medium at three days compared to the WT control. Unlike the WT control that grew exclusively as yeast on YPD-K, cwt1/cwt1 mutants also showed polarized growth (). As expected, mating-related genes MFA1, MFα, FIG1, and FUS1 were significantly increased in the cwt1/cwt1 mutant on YPD-K relative to a WT control (). To verify the function of Cwt1 in regulating homothallic mating, we generated an additional homozygous CWT1 deletion mutant in the GH1013 background. Similar to the library mutant, the newly generated cwt1/cwt1 strain exhibited a more robust development of mating projections on both YP-K and YPD-K than the WT control (), and also promoted same-sex mating on YPD-K medium (). Thus, Cwt1 represses homothallic mating in C. albicans.

Role of the Cwt1 transcription factor in the induction of mating projections.

(A and B) Morphologies of the control (GH1013 + ARG4 + HIS1) and cwt1/cwt1 mutant on YP-K (A) or YPD-K (B) medium. 1 × 105 cells of each strain were spotted on YPD-K and YP-K media and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Relative expression levels of CWT1 in YPD-K and YP-K media. Cells of C. albicans were spotted on YPD-K and YP-K media and cultured at 25°C for five days. (D) Relative expression levels of CWT1 in the control (GH1013cartTA) and tetON-HSF1/hsf1 on YP-K medium. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Percentages indicate the ratio of gene expression in tetON-HSF1/hsf1 mutant relative to gene expression in control. The numerical data are presented in . (E) Physical interaction of Cwt1 and Hsp90. Co-IP assays were performed using a strain with 13× Myc-tagged Cwt1 and GFP-tagged Hsp90. Lanes 1–4, samples co-immunoprecipitated by the GFP antibody Sepharose and analyzed by immunoblotting with the anti-Myc antibody. Lanes 5–8, whole-cell extracts analyzed by immunoblotting with the anti-Myc antibody. Experiment was performed in biological replicate, and a representative image is shown. Arg, arginine; Cwt1, Cell Wall Transcription factor 1; Dox, doxycycline; GFP, green fluorescent protein; His, histidine; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; IP, immunoprecipitation; Myc, Myc epitope tag; p, mating projection; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

In contrast, deletion of CTA4, a previously identified genetic interactor of Hsp90 [23, 24] and a gene induced by nitric oxide, significantly repressed the development of mating projections in YP-K medium in C. albicans (). Consistently, the expression of mating-related genes was not induced in the cta4/cta4 mutant in YP-K medium, unlike in the WT control (). However, the relative expression levels of CWT1 were significantly increased in the cta4/cta4 mutant, and chromatin immunoprecipitation (ChIP) assays demonstrated that Cta4 bound to the promoter regions of CWT1 on YP-K medium (). Thus, Cta4 may provide a functional connection between Hsp90 and Cwt1 and directly regulate the transcriptional expression of CWT1.

Consistently, we found that the transcription level of CWT1 was down-regulated in YP-K medium compared to the level observed in YPD-K medium (). Moreover, in the tetON-HSF1/hsf1 background in which levels of HSF1 were reduced relative to a WT control, we also observed a significant reduction in CWT1 transcript levels (), suggesting that the transcriptional expression of CWT1 is directly or indirectly regulated by Hsf1–Hsp90 signaling.

Protein sequence analysis indicated that Cwt1 contains a Zn2Cys6 motif at the carboxyl terminus and a conserved Per-Arnt-Sim (PAS) domain at the amino terminus. Since the PAS domain often interacts with Hsp90 in eukaryotic organisms [31, 32], we next tested whether Hsp90 was able to bind to Cwt1 and regulate its activity in C. albicans. As shown in , co-immunoprecipitation (IP) assays indicated that Hsp90 and Cwt1 physically interact on YP-K, but not YPD-K, medium. Therefore, Hsf1–Hsp90 signaling may regulate the activity of Cwt1 at post-transcriptional levels as well as at the transcriptional level via Cta4 in C. albicans.

Cwt1 regulates the master regulator of a-type mating, MTLa2, to control same-sex mating

MTLa2 is required for the maintenance of a-cell identity [33]. Inactivation of MTLa2 induces the expression of α-pheromone in MTLa/a cells of C. albicans [33]. Given our observation that glucose starvation induced the expression of both pheromone precursors MFA1 and MFα, we assessed whether homothallic mating induced in glucose-limiting conditions was governed by changes in MTL2 levels. To test this, we overexpressed MTL2 and observed a suppression in the development of mating projections under glucose-limiting conditions (). Next, to test whether compromise of Hsf1–Hsp90–Cwt1 signaling impaired MTL2 expression, we monitored MTL2 levels in our tetON-HSF1/hsf1 and tetON-HSP90/hsp90 mutants. Down-regulation of HSF1 or HSP90 or deletion of CWT1 led to significantly decreased expression of MTL2 on YPD-K medium (), implicating this stress-response signaling in the regulation of MTL2 expression. It has been indicated that Cwt1 has potential binding sites in the promoter regions of MTL2 and MFα genes [34]. Using ChIP assays, we observed that Cwt1 directly binds to the promoter regions of both MTL2 and MFα on YPD-K medium in the one-day cultures, and this binding activity was observed on both on YP-K and YPD-K media in the three-day cultures (). These results suggest that Cwt1 directly regulates mating-related gene expression.

MTLa2 regulates the development of mating projections in C. albicans.

(A) Relative expression levels of MTL2 in the corresponding controls, tetON-HSF1/hsf1, tetON-HSP90/hsp90, and cwt1/cwt1 mutants, and MTLa2-overexpressing strain on YPD-K medium. Transcript levels were normalized to ACT1. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate, and a representative image is shown. (B) 1 × 105 cells of each strain were spotted on YP-K medium and cultured at 25°C for five days. Morphologies of the control (WT + pACTS) and MTL2-overexpressing strains (WT + pACTS-MTL2) on YP-K medium. WT, GH1350a. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Cwt1 binds to the promoters of MTL2 and MFα. ChIP assays were performed in TAP-tagged Cwt1 strains. Percentages of input genomic DNA are indicated. 1 × 105 cells of each strain were spotted on YP-K or YPD-K media and cultured at 25°C for one or three days. Dark arrows indicate detected promoter regions. d1, d2, and d3, three detected sites of MFα. Error bars represent standard error of two technical replicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate with a representative image shown. The data for the untagged control correspond to samples harvested at day 1. The numerical data are presented in . (D) Regulatory model of glucose-starvation–or oxidative-stress–induced same-sex mating in C. albicans. Glucose starvation or oxidative stresses cause the overwhelming of the Hsf1/Hsp90 functional capacity that regulates the transcriptional expression and activity of CWT1 in both direct and indirect manners. Cwt1 regulates same-sex mating through the control of MFα or MTLa2. ACT1, ACTin 1; ChIP, chromatin immunoprecipitation; Cta4, Candida TransActivating protein 4; Cwt1, Cell Wall Transcription factor 1; Dox, doxycycline; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; MFα, Mating α factor precursor; MTL, Mating type locus; pACTS, plasmid pACTS; TAP, Tandem affinity purification; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; WT, wild type; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

Overall, our data suggest a model in which Hsf1–Hsp90 signaling controls the expression of Cwt1 through Hsp90 genetic interactors such as Cta4 as well as the activity of Cwt1 post-translationally through a physical interaction with Hsp90. Cwt1 is a dimeric Zn2Cys6 zinc-finger transcription factor. The physical interaction between Hsp90 and Cwt1 could inhibit the dimerization of Cwt1 through the PAS domain in C. albicans, as observed in other eukaryotic organisms [32]. Cwt1 binds to the promoters of MFα or MTLa2 and regulates their transcriptional expression (). It has been demonstrated that same-sex mating in C. albicans is induced by the autocrine and/or paracrine pheromone response [16]. Therefore, the activated pheromone signaling by the environmental cues could then promote the development of mating projections and same-sex mating, possibly through these two response modes ().

Potential autocrine (A) and paracrine (B) pheromone response models for stress-induced mating-projection formation and same-sex mating. Neither α-pheromone nor a-pheromone is constitutively expressed in “a” cells of C. albicans. Glucose starvation or oxidative stress first induces α-pheromone and its receptor Ste2 expression in “a” cells. α-pheromone binds to Ste2 and activates the pheromone-response pathway, which subsequently induces the expression of a-pheromone. “a” cells then become mating competent as both “a” and “α” types. The activation of the pheromone-response pathway promotes mating projection formation and same-sex mating. Autocrine pheromone response, self-activation (A); paracrine pheromone response, activation of neighbor cells (B). MFA1, Mating type A1; STE2, STErile 2.

Discussion

The predominantly clonal nature of C. albicans greatly limits the occurrence of heterothallic mating between two competent cells with opposite mating types in its natural environment. It would be difficult for a mating-competent cell of C. albicans to find a competent cell with an opposite mating type to mate. Although the efficiency of same-sex mating is much lower than that of opposite-sex mating under laboratory conditions in C. albicans [16], same-sex mating involving cells of a single type would lower the barrier to finding a suitable partner [35]. However, the requirement of α-pheromone production in close proximity to drive homothallic mating between two MTL cells or the need to inactivate the Bar1 protease challenges the relevance of same-sex mating in nature [16]. This is especially true given that neither natural C. albicans mutants with loss of Bar1 function nor environmental conditions suppressing Bar1 activity have been discovered. Thus, the question remains: can C. albicans undergo frequent same-sex mating in natural environments?

In our current study, we find that glucose starvation and oxidative stress induce same-sex mating in MTL cells of C. albicans, providing an environmentally relevant means to drive sexual reproduction in this species. C. albicans is commonly exposed to glucose starvation because glucose is limited in its natural niches such as the mouth, lower gut, and environments contaminated with human or animal excreta. However, under the same conditions, we did not observe same-sex mating in opaque Mating type α (MATα) cells, perhaps because of MTLa2, a key regulator in this induction in MTLa cells. MATα/α cells may use a different environmental cue for the induction of same-sex mating. Similar to reports highlighting that the fungal pathogen Cryptococcus neoformans undergoes mating on pigeon guano [36], we found that animal feces promoted same-sex mating in C. albicans (). Given that the gut of human or warm-blooded animals is a major natural niche for C. albicans [12], animal feces would be a major source for most nutritional components. Furthermore, growth on C. albicans debris medium containing no additional nutrients also induced same-sex mating (). When yeast grows on agar-only medium, a portion of cells undergo cell death and release nutrients for the growth and survival of neighboring cells [37]. Under these glucose-limiting conditions, the frequency of same-sex mating in C. albicans ranged from 1 × 10−7 to 3 × 10−5 (), suggesting that the average-aged colony (containing approximately 1 × 108 cells) can produce tens to hundreds of mating progeny. Therefore, the occurrence of same-sex mating under glucose starvation conditions could be considerably more frequent in nature than was originally thought.

The notion that environmental stress may serve as a trigger to induce more frequent sexual reproduction in C. albicans has been previously proposed based on a series of observations [38]. Poor nutritional conditions increase the frequency of opposite-sex mating [39]. Oxidative stress promotes the induction of the white-to-opaque switch [40], and recombination occurs more frequently following exposure to several types of stress, which would provide a critical step for the homozygosis of the MTL locus. Despite these lines of evidence, our work provides the seminal example of stressful conditions governing homothallic mating in C. albicans. In C. neoformans, sexual mating is stimulated by stresses such as nitrogen starvation, desiccation, and darkness [41]. Moreover, treatment of S. pombe with the oxidative agent H2O2 promotes the generation of meiotic spores [42]. Therefore, exposure to harsh environmental conditions could be a general signal for diverse fungal species to integrate environmental response pathways with increased sexual reproduction.

The evolutionarily conserved regulators Hsf1 and Hsp90 play a primary and global role in orchestrating stress responses in eukaryotic organisms [43]. Glucose starvation results in the production of ROSs () that would likely cause protein damage in C. albicans. Under these situations, the functional capacity of Hsp90 could be overwhelmed, causing it to be titrated away from its basal client proteins while it deals with more global problems of protein misfolding. Consistently, a number of heat-shock-protein–encoding genes, including HSP90, were up-regulated in response to glucose deprivation ( and ).

In our study, we implicate the Cta4 and Cwt1 transcription factors, which regulate the nitrosative or nitric oxide stress response [30], as downstream effectors of the Hsf1–Hsp90 signaling and mating-response pathways (). Cta4 has been previously reported as a genetic interactor of Hsp90 [23, 24] and represses the transcriptional expression of CWT1. Moreover, we demonstrate that Hsp90 directly binds to Cwt1 on YP-K but not YPD-K medium, perhaps through the conserved PAS motif of Cwt1. To our knowledge, this is the first time that Hsf1 or Hsp90 have been implicated in sexual mating in fungi, and our results suggest that diverse cellular stresses capable of overwhelming the function of these regulators, including elevated temperature, may also promote sexual reproduction in C. albicans.

Inactivation of the Bar1 protease or the Dipeptidyl aminopeptidase YC1 (Yci1) domain protein Opaque Formation Regulator 1 (Ofr1) has been shown to induce same-sex mating in C. albicans [16, 44]. We observed that BAR1 was not down-regulated but rather highly induced upon glucose starvation (). Further, inactivation of Ofr1 allows MTLa/α cells to undergo same-sex mating as the “a” mating type [44]. Although glucose starvation can only induce same-sex mating in MTLa/a cells but not in MTLa/α or MTLα/α cells, other unidentified environmental conditions may allow MTLa/α or MTLα/α cells to undergo homothallic mating. Therefore, glucose-starvation–induced same-sex mating appears to be independent of Bar1 and Ofr1.

In summary, we uncover a novel, to our knowledge, environmental trigger, glucose depletion, capable of acting as a signal for sexual mating in C. albicans, which not only sheds light on the biology of this pathogen but also expands the diverse repertoire of sexual reproduction modes in fungi. This strategy is different from that used by S. cerevisiae, S. pombe, and C. neoformans, despite the fact that the evolutionarily diverse yeast species achieve a same output for homothallism. Unisexual reproduction generates aneuploidy and de novo phenotypic diversity in fungi [45, 46], thus providing a selective advantage under stressful conditions over those organisms propagating exclusively in an asexual manner. C. albicans exists as an obligate diploid in nature, with many heteryozygous loci between homologous chromosomes. Therefore, when tetraploid intermediate cells generated from same-sex mating return to a lower ploidy state, there is substantial opportunity for the generation of genetically diverse progeny. These tetraploid strains could serve as a capacitor for generating distinct aneuploidies and randomly combined chromosome sets in order to facilitate the evolution of new traits to adapt to changing environments.

Materials and methods

Strains and culture conditions

The strains used in this study are listed in supplementary . YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) and modified Lee’s glucose medium supplemented with 5 μg/ml phloxine B [47] were used for routine growth of C. albicans. Solid YPD-K (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose, 2.5 g/L K2HPO4, 20 g/L agar) and YP-K (20 g/L peptone, 10 g/L yeast extract, 2.5 g/L K2HPO4, 20 g/L agar) media were used for mating-projection induction assays. Peptone, yeast extract, and agar were purchased from BD Biosciences (BD Bacto, Cat. Nos., 211677, 212750, G8270, and 214010; BD Biosciences, Sparks, MD, USA). Glucose, phloxine B, and K2HPO4 were purchased from Sigma-Aldrich (Cat. Nos., G8270, P2759, and P9666; Sigma-Aldrich, St. Louis, MO, USA). The pH of YPD-K and YP-K media were about 7.3. Opaque cells were used for all mating and mating projection formation assays.

Sorbitol medium was made according to a previous study [48]. YPD-K, YP-K, agar-only (3%), agar + C. albicans debris, or agar + 3% mouse feces media were used for quantitative mating assays. To make the C. albicans debris medium, approximately 1 × 1010 cells of SC5314 were collected from an overnight YPD culture, washed with ddH2O, frozen at −80°C, and ground with glass beads. Cell debris and 2% agar were mixed and resuspended in 100 mL ddH2O for autoclaving. To make the mouse feces medium, 2% agar and 3% mouse feces (w/v) were mixed and resuspended in ddH2O. Before subjecting to autoclaving, 0.25% K2HPO4 was added to the agar-only (3%), agar + C. albicans debris, or agar + 3% mouse feces media for pH buffering. Synthetic complete medium (SCD) lacking corresponding nutrients (uridine, histidine [His], and/or arginine [Arg]) was used for selectable growth in quantitative mating assays.

To make H2O2-containing YPD-K medium, 200 μl of 5 mM H2O2 was spread onto YPD-K medium plates. Opaque cells of GH1350a were first grown on Lee’s glucose medium at 25°C for three days. 1 × 106 cells in 10 μL of ddH2O were spotted onto different H2O2-containing YPD-K media and cultured at 25°C for three to five days.

Construction of plasmids

To inactivate the SacII site of tetON-promoter–containing plasmid pNIM1 [49], the plasmid was first digested with SacII and then filled in with the Klenow fragment of DNA polymerase I. The blunt ends were ligated to generate the SacII-free plasmid pNIMsx. To construct the plasmids pNIMsx-HSP90con and pNIMsx-HSF1con for conditional knockout of HSP90 and HSF1, respectively, one fragment of partial ORF region of HSP90 or HSF1 (with SalI and SacII sites) and another fragment of the corresponding 5′-UTR region (with SacII and BglII sites) were simultaneously subcloned into the SalI and BglII sites and replaced the GFP cassette.

To construct the nourseothricin-resistant plasmid pACTS, a caSAT1 fragment was amplified from pNIM1 [49] and subcloned into the HindIII/KpnI site of pACT1 [50]. A fragment containing the MTL2 ORF region was then subcloned into the EcoRV/HindIII site of pACTS, generating the overexpressing plasmid pACTS-MTLa2.

To create a Myc-tagged Cwt1 plasmid, the CdHIS1 cassette was amplified from plasmid pSN52 and inserted into plasmid pACT1 at the ClaI site, generating plasmid pACT1-HIS1. A fusion PCR product containing the CWT1 ORF region and a C-terminal 13× Myc tag were prepared and subcloned into the EcoRV/KpnI site of pACT1-HIS1, yielding plasmid pACT1-CWT1-Myc-HIS1.

A TAP-ARG4 cassette flanked by approximately 70-bp-5′– and 3′–homologous sequences of CWT1 was amplified from strain CaLC2993 with primer pair LT1266 and LT1267 and was then transformed into the WT strain SN95 (CaLC239) to create TAP-tagged strain LTS1036 [51, 52]. The fragment containing the CWT1 ORF region and a C-terminal TAP-ARG4 tag was amplified from strain LTS1036 with oligonucleotides LT1271/LT1272 and then subcloned into the EcoRV/KpnI site of pACT1 [50], generating plasmid pACT-CWT1-TAP-ARG4.

A TAP-ARG4 cassette flanked by approximately 70-bp-5′– and 3′–homologous sequences of CTA4 was amplified from strain CaLC2993 with primer pair LT1459 and LT1460 and was then transformed into the WT strain SN95 (CaLC239) to create TAP-tagged strain LTS1071 [52]. The fragment containing the CTA4 ORF region and a C-terminal TAP-ARG4 tag were amplified from strain LTS1071 with oligonucleotides LT1271/LT1462 and then subcloned into the EcoRV/KpnI site of pACT1, generating plasmid pACT-CTA4-TAP-ARG4.

Construction of C. albicans strains

To construct the conditional knockout mutants of HSP90 in strain GH1013, we first replaced the promoter of one allele with the tetON promoter using SacII-digested plasmid pNIMsx-HSP90con, generating mutant tetON-HSP90/HSP90. The other allele of HSP90 was then replaced with the ARG4 cassette amplified from pRS-ARG4ΔSpeI [53] with oligonucleotides HSP90-5DR/HSP90-3DR, generating the conditional mutant tetON-HSP90/hsp90. Since opaque cells of the tetON-HSP90/hsp90 mutant were not stable, the master regulator WOR1 was overexpressed in this mutant using plasmid pACT1-WOR1 [50]. To construct the conditional knockout mutant of HSF1, we deleted the first allele in strain GH1013 using the URA3 cassette amplified from pGEM-URA3 [53] with oligonucleotides HSF1-5DR/HSF1-3DR, generating the mutant hsf1::URA3/HSF1. Then, we replaced the promoter region of the second allele of HSF1 with the tetON promoter using SacII-digested plasmid pNIMsx-HSF1con, generating the mutant tetON-HSF1/hsf1. The cartTA cassette (reverse tet repressor) is under the control of the white-cell–specific ADH1 promoter in the plasmid pNIM1 [49]. To increase the expression level of cartTA in opaque cells, the cassette was integrated into the opaque-specific OP4 locus in the tetON-HSF1/hsf1 and tetON-HSP90/hsp90 mutants by transformation with fusion PCR products of cartTA-ARG4. pNIM1 and pRS-ARG4ΔSpeI were used as the primary template.

To delete both alleles of CWT1, the HIS1 and ARG4 markers flanked by CWT1 gene 5′- and 3′-fragments were amplified with fusion PCR assays and sequentially transformed into strain GH1013 as described previously [53]. The plasmids pGEM-HIS1 and pRS-ARG4ΔSpeI were used as the PCR templates. All oligonucleotides used are listed in .

To determine Cwt1-binding targets, a TAP-tagged Cwt1-ecotopic strain was constructed. The plasmid pACT-CWT1-TAP-ARG4 was linearized with AscI and transformed into strain SN95 [54] to create a TAP-tagged Cwt1-overexpressing strain (LTS1039). The function of TAP-tagged Cwt1 was verified by mating projection formation assays. To construct the MTL-overexpressing strain, plasmid pACTS-MTLa2 was linearized with AscI and transformed into strain GH1350a.

To construct a TAP-tagged Cta4-ecotopic expression strain, the plasmid pACT-CTA4-TAP-ARG4 was linearized with AscI and transformed into strain SN95 to create TAP-tagged CTA4-ecotopic expression strain (LTS1079). To construct a GFP-tagged Hsp90 strain, a HSP90-GFP-SAT1 fusion fragment into the pACT1 plasmid [50], generating plasmid pACT1-HSP90-GFP-SAT1. The plasmids pACT1-HSP90-GFP-SAT1 and pACT1-CWT1-Myc-HIS1 were linearized with AscI and subsequently transformed into strain SN95 [54], yielding strain LTS1062 with a GFP-tagged HSP90 and 13× Myc-tagged CWT1 allele.

Construction of GFP-reporter strains

To construct the MFa1p-GFP, MFαp-GFP, FIG1p-GFP, and FUS1p-GFP reporter strains, GH1013 was transformed with PCR products of the GFP-caSAT1 cassette amplified from plasmid pNIM1 with corresponding primers. The forward and reverse primers contain a 60-bp flanking sequence homologous to the promoter and 3′-UTR regions of MF1, MFα, FIG1, or FUS1, respectively. Correct integration of the transformations was verified with PCR assays.

Quantitative mating assay

Same-sex mating assays were performed according to our previous publications with slight modifications [55]. Briefly, opaque cells of two “a” strains (1 × 107 for each) were mixed, spotted onto different media, and cultured at 25°C for three to seven days as indicated in the main text. Mating mixtures were then replated onto SCD-His, -Arg, -uridine, -His-Arg, or -Arg-uridine dropout media for prototrophic selection growth. Colonies grown out on the three types of plates were counted, and mating efficiency was calculated.

Flow cytometry analysis

C. albicans cells were incubated in liquid SCD medium with shaking at 30°C overnight, harvested, washed, and resuspended in 1× TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]). Cells were then fixed with 70% ethanol for two hours at room temperature and washed with 1× TE buffer before treating with 1 mg/mL RNase A and 5 mg/ml proteinase K. Propidium iodide (PI, 25 μg/ml) staining assays were then performed. Stained cells were washed and resuspended in 1× TE buffer for DNA content analysis. A total of approximately 30,000 cells of each sample were used for flow cytometry assays, and the results were analyzed using software FlowJo 7.6.1.

Quantitative real-time PCR and RNA-Seq assays

Quantitative real-time PCR assays were performed according to our previous publications with modifications [56]. Cells were collected from cultures grown on solid plates as described in the main text. One μg of total RNA per sample was used to synthesize cDNA with RevertAid H Minus Reverse Transcriptase (Thermo Scientific, Waltham, MA, USA). Quantification of transcripts was performed in Bio-Rad CFX96 real-time PCR detection system using SYBR green. The signal from each experimental sample was normalized to expression of the ACT1 gene.

For RNA-Seq assays, opaque cells of C. albicans were grown to stationary phase on Lee’s glucose medium at 25°C for five days and then spotted onto YPD-K and YP-K medium at 25°C for 60 hours of incubation. Two biological repeats were performed for each condition. Cells were harvested, and total RNA was extracted. RNA-Seq analysis was performed by the company Berry Genomics (Beijing, China) as described previously [57]. Briefly, approximately 10 million (M) reads were sequenced in each library of the samples. The library products were then sequenced using an Illumina HiSeq 2500 V4 (Illumina, San Diego, CA, USA). Illumina software OLB_1.9.4 was used for base calling. The raw reads were filtered by removing the adapter and low-quality reads (the percentage of low-quality bases with a quality value ≤3 was >50% in a read). Clean reads were mapped to the genome of C. albicans SC5314 using TopHat (version 2.1.1) and Cufflinks (version 2.2.1) software [58]. Relative gene expression levels were calculated using the fragments per kb per million reads (FPKM) method. To be considered significantly differentially expressed, a gene must satisfy three criteria: (1) an FPKM value higher than or equal to 20 at least in one sample, (2) a fold change value higher than or equal to 1.5 (except for the Functional categories sheet of , in which a 2-fold change cutoff was used), and (3) an adjusted p-value (false discovery rate [FDR]) lower than 0.05.

Chromatin IP and co-IP assays

ChIP assays were performed as described previously [52, 59]. Briefly, untagged (CaLC239, SN95) and pACT1-Cwt1-TAP–tagged (LTS1039) C. albicans strains were spotted on YP-K medium and incubated at 25°C for one and three days. Cells were collected and fixed in 1× PBS containing 1% formaldehyde and incubated with gentle rocking for 20 min at room temperature. The crosslinking reaction was quenched by adding 2.5 M glycine to a final concentration of 125 mM, and cells were mixed for 5 min at room temperature. Cells were harvested, washed with 1× PBS, and homogenized in ice-cold lysis buffer using a bead beater. Sonication was performed with a Diagenode Bioruptor (Diagenode, Denville, NJ, USA) (12 min, high setting, 30 s on, 1 min off) to obtain chromatin fragments of an average size of 250–1,000 bp. The chromatin was immunoprecipitated with 50 μl packed IgG Sepharose 6 Fast Flow matrix (GE Healthcare, Chicago, IL, USA). The Sepharose matrix was washed, and immunoprecipitated chromatin DNA was eluted and de-crosslinked at 65°C overnight. Quantitative real-time PCR assays were performed to determine Cwt1 targets.

Cells grown on YP-K and YPD-K media were harvested and suspended in lysis buffer containing 50 mM Na-HEPES (pH 7.5), 450 Mm NaOAc (pH 7.5), 1 mM EDTA, 1 mM EGTA, 5 Mm MgOAc, 5% glycerol, 0.25% NP-40, 3 mM DTT, 1 mM PMSF, and EDTA-free protease inhibitor mix (Cat. No.,11873580001; Roche Diagnostics, Mannheim, Germany). Cells were lysed using a beadbeating instrument by five rounds of beating (50 s beating plus 1 min cooling process on ice for each round). The supernatant of cell lysates was collected and incubated with Sepharose beads conjugated anti-GFP monoclonal antibody at 4°C for three hours. The beads were washed for four times with the lysis buffer and then boiled for 10 min in 1× sodium dodecyl sulfate (SDS) sample buffer. Immunoprecipitated proteins were separated through an SDS-10% polyacrylamide gel and used for western blotting analysis using anti-Myc monoclonal antibodies (Cat. No., OP10; MilliporeSigma, Billerica, MA, USA).

Intracellular ROS determination

Cells were cultured for one to five days on YP-K or YPD-K medium at 25°C. Cells were then harvested and washed in 1 × PBS and incubated with DCFDA (Beyotime, Shanghai, China) for 30 min at 37°C. After washing, fluorescence intensity reflecting the ROS level was measured at 488 nm using ELISA and was normalized according to the cell numbers.

Development of polarized growth under different glucose concentrations.

1 × 107 cells of strain GH1350a were spotted on media containing different levels of glucose and cultured at 25°C for three or seven days. Percentages of projected cells are indicated in the corresponding images. The percentage of projected cells decreases with the increase of glucose level.

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Expression of mating-related genes in GFP-reporter strains under glucose starvation conditions.

1 × 105 cells of each GFP-tagged strain (GH1013 background) were spotted on YP-K or YPD-K medium and cultured at 25°C for five days. Scale bar, 10 μm. DIC, differential interference contrast; GFP, green fluorescent protein; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

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Same-sex mating of C. albicans under conditions mimicking natural environments.

(A) Diagram of experimental procedures. (B, C, and D) Mating efficiency on 3% agar without additional nutrients (B), on agar containing 3% mouse feces (C), and agar containing C. albicans debris (D). 1 × 107 cells of GH1013 and 1 × 107 cells of GH1350a were mixed and cultured on different medium plates at 25°C for three to seven days. Mating mixtures were replated onto SCD-Arg, SCD-His, and both dropout plates for selectable growth and mating efficiency calculation. For mating on agar without additional nutrients (B), a portion of cells underwent cell death and released nutrients for the survived cells. (E) Mating on sorbitol medium (opaque filamentation inducing medium). The numerical data are presented in . Arg, arginine; His, histidine; SCD, synthetic complete medium.

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Relative expression levels of HSF1 and HSP90 and FACS analysis of mating progeny.

(A) Relative transcriptional expression levels of HSF1 or HSP90 in the control and tetON-HSF1/hsf1 or tetON-HSP90/hsp90 mutant on YP-K and YPD-K media with or without doxycycline (40 μg/mL). 1 × 105 cells were spotted on YP-K or YPD-K medium and cultured at 25°C for three days. Error bars, standard errors of technical duplicates *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate and representative image is shown. (B) FACS analysis of the DNA content of progeny strains. Parental strain GH1350a used as a diploid control. Mating progeny contain DNA content corresponding to 4C and 8C peaks confirming their tetraploid nature. This figure is related to the quantitative results presented in supplementary S1 Table. The numerical data are presented in . FACS, Fluorescence-activated cell sorting; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

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Identification of the cwt1/cwt1 mutant by screening a transcription factor mutant library of C. albicans.

(A) Formation mating projections in the cwt1/cwt1 mutant on YPD-K and YP-K media. 1 × 105 cells of each strain were spotted on different media and cultured at 25°C for three or five days. Scale bar for colonies, 2 mm; scale bar for cells, 10 μm. (B) Relative expression levels of mating-related genes in the control (GH1350a) and cwt1/cwt1 mutant on YPD-K and YP-K media. Error bars, standard errors. *p < 0.05, two-tailed Student t test. Two biological and two technical repeats were performed, respectively. The numerical data are presented in . Cwt1, Cell Wall Transcription factor 1; p, mating projection; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

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Identification of the cta4/cta4 mutant by screening a transcription factor mutant library of C. albicans.

(A) Colony and cellular morphologies of the control (WT, GH1350a) and cta4/cta4 mutant on YP-K medium. 1 × 105 cells of each strain were spotted on different media and cultured at 25°C for five days. Scale bar for colonies, 2 mm; scale bar for cells, 10 μm. (B) Relative expression levels of CWT1 and mating-related genes in the control (GH1350a) and cta4/cta4 mutant on YPD-K and YP-K media. Cells of C. albicans used for qRT-PCR assays were cultured at 25°C for five days. Error bars, standard errors. *p < 0.05, two-tailed Student t test. Two biological and two technical repeats were performed, respectively. (C) Cta4 binds to the promoters of CWT1. ChIP assays were performed in TAP-tagged Cta4 strains. Cells of C. albicans used for ChIP assays were grown on YP-K or YPD-K medium at 25°C for 24 hours. Percentages of input genomic DNA are indicated. Dark arrows indicate detected promoter regions. d1, d2, and d3, three detected sites of CWT1. Error bars represent standard error of two technical replicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate with a representative image shown. The numerical data are presented in S3 Data. ChIP, chromatin immunoprecipitation; Cta4, Candida TransActivating protein 4; Cwt1, Cell Wall Transcription factor 1; p, mating projection; qRT-PCR, quantitative reverse transcription PCR; WT, wild type; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.

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Efficiency of same-sex mating in the tetON-HSF1/hsf1, tetON-HSP90/hsp90 and cwt1/cwt1 mutants.

Cwt1, Cell Wall Transcription factor 1; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1.

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Strains used in this study.

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Primers used in this study.

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RNA-Seq dataset.

RNA-Seq, RNA sequencing

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HSP90 genetic interactors that differentially expressed in YP-K and YPD-K media.

Hsp90, Heat shock protein 90; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4

(XLSX)

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Excel files containing the underlying numerical data for Figs 1C, 3A, 3C, 3D, 5B, 6B, 7C, 7D, 8AC, S3B, S3C, S3D, S3E, S4A, S5B, S6B and S6C.

(XLSX)

Click here for additional data file.