Glucose depletion enables Candida albicans mating independently of the epigenetic white-opaque switch

Guan, Guobo; Tao, Li; Li, Chao; Xu, Ming; Liu, Ling; Bennett, Richard J.; Huang, Guanghua

doi:10.1038/s41467-023-37755-8

Download PDF

Article
Open access
Published: 12 April 2023

Glucose depletion enables Candida albicans mating independently of the epigenetic white-opaque switch

Nature Communications volume 14, Article number: 2067 (2023) Cite this article

2569 Accesses
10 Altmetric
Metrics details

Subjects

Abstract

The human fungal pathogen Candida albicans can switch stochastically and heritably between a “white” phase and an “opaque” phase. Opaque cells are the mating-competent form of the species, whereas white cells are thought to be essentially “sterile”. Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFα1, FIG1, FUS1, and CEK1/2) are increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 is decreased. Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promotes white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increases white cell mating ~4000 fold in glucose-depleted medium relative to that in the presence of glucose. Our findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature.

Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice

Article Open access 15 April 2024

Streptomyces umbrella toxin particles block hyphal growth of competing species

Article Open access 17 April 2024

Single-cell multiplex chromatin and RNA interactions in ageing human brain

Article Open access 27 March 2024

Introduction

Sexual reproduction is ubiquitous across eukaryotic organisms. In fungi, sexual reproduction drives the evolution of new traits and generates diversity that can enable adaptation to environmental changes^1,2,3. Although certain aspects of sexual reproduction are conserved in fungi, such as pheromone signaling via a MAPK transduction pathway, there are also important differences in the regulation of this program between different species¹. A unique regulatory mechanism is observed in the human pathogenic fungus Candida albicans and its closely related species Candida tropicalis and Candida dubliniensis, where sexual mating is regulated by an epigenetic switch^4,5,6,7. In each of these species, cells must switch from the “sterile” white state to the mating-competent opaque state to undergo efficient sexual mating. Besides mating competence, C. albicans white and opaque cells differ in a number of phenotypic aspects including colony and cellular morphologies (Fig. 1a) and virulence in different infection models^4,8,9.

**Fig. 1: Glucose depletion induces the expression of mating-associated genes in *C. albicans* white cells.**

The white-opaque phenotypic switch occurs ectopically at low frequency under standard culture conditions but can be induced by a variety of environmental cues such as high levels of CO₂ or N-acetylglucosamine (GLcNAc), as well as in response to changes in pH or temperature⁸. This phenotypic transition is operated by a transcriptional circuit centered on the key regulators Wor1 and Efg1 in C. albicans^10,11,12,13. Wor1 is a fungal-specific transcription factor that is considered the master regulator of C. albicans white-opaque switching and is essential for the opaque cell type^10,11,12.

C. albicans opaque cells mate approximately one million times more efficiently than white cells⁴. It has long been thought, therefore, that switching from the white to the opaque state is essentially a prerequisite for mating. However, several studies have indicated that white cells are able to respond to pheromone signals and may play a critical role in promoting sexual reproduction^14,15. When mixed with opaque cells or exposed to pheromone, C. albicans white cells exhibited increased expression levels of a subset of mating-associated genes and formed “sexual biofilms” that facilitated opaque cell mating¹⁶. In a related study, we showed that C. albicans white cells secrete pheromone and promote opaque cell mating in a mixed culture system containing both cell types¹⁵. These studies indicate that although white cells are unable to mate efficiently under standard culture conditions, the major components of the mating response MAPK signaling pathway can be activated and can generate a conducive environment for opaque cell mating.

Although C. albicans white cells can exhibit mating-related responses under certain conditions, cell–cell conjugation between white cells is limited due to the low expression of pheromone MAPK signaling genes such as STE4, CST5, and CEK1/CEK2 (encoding the β subunit of the G-protein complex, the MAPK scaffold, and the terminal MAP kinases, respectively)¹⁷. Indeed, ectopic expression of STE4, CST5, and CEK2, both individually and in combination, increases mating between white cells, demonstrating that the white-opaque switch controls mating through regulation of the pheromone MAPK signaling pathway¹⁷.

Nutrient limitation is a common stress encountered by C. albicans cells in its natural niches, and we recently reported that glucose depletion activates the pheromone signaling MAPK pathway and promotes efficient same-sex (homothallic) mating by C. albicans opaque cells¹⁸. The Hsf1/Hsp90-mediated stress response pathway plays a central role in this regulation¹⁸. These findings suggested that it may be possible to alleviate bottlenecks in the expression of mating-associated genes and enable sexual mating in white cells through similar perturbations in nutrients.

In this study, we demonstrate that depletion of glucose activates the pheromone MAPK pathway in C. albicans white cells, and thereby promotes their ability to undergo sexual mating. The MAPK Cek2 and transcription factor Cph1 function as positive regulators of white cell mating, whereas the transcription factors Tec1 and Dig1 inhibit white cell mating. Given glucose limitation is a common stress encountered by C. albicans in its mammalian host, our findings indicate that the white-opaque switch may not be essential for efficient mating in nature. Thus, this epigenetic switch, which is a relatively newly evolved regulatory mechanism in C. albicans and closely associated species, can be bypassed under certain environmental conditions.

Results

Glucose depletion induces the expression of mating-associated genes in C. albicans white cells

Most media used for culturing C. albicans contains 1–2% of glucose (w/v), which is much higher than the level of glucose in environmental niches (e.g., the lower animal gut, mucosal surfaces, and soil environments)^19,20. Glucose depletion has multiple effects on the life cycle of microbes^18,21,22. We previously found that glucose depletion induces the expression of mating-associated genes and promotes same-sex mating in opaque cells of C. albicans¹⁸. To test the effect of glucose depletion on white cells, we examined the relative expression levels of mating-associated genes in C. albicans that are homozygous for the MTLa mating type locus. Media contained either no glucose (YP-K) or 2% glucose (YPD-K) in these tests. As shown in Fig. 1b, the relative expression levels of several pheromone MAPK genes were significantly increased in white cells grown in YP-K medium compared to those grown in YPD-K medium. These genes included MFA1, MFα1, STE2, and STE3 encoding the a- and α-pheromones and their receptors, respectively, CEK1 and CEK2 encoding the terminal MAPKs of the pheromone signaling pathway, and STE4 encoding the ortholog of the S. cerevisiae G protein β-subunit Ste4^17,23. Two additional genes, FIG1 and FUS1^{17,23,24,25,26}, were also transcriptionally upregulated in the YP-K medium relative to the YPD-K medium. Both of these genes are integral to mating in S. cerevisiae and Fig. 1 protein is localized to mating projections in C. albicans^26,27.

Since white cells can switch to the opaque phase at low frequency which would increase the expression of mating-associated genes, we also performed qRT-PCR assays in a white-locked wor1/wor1 mutant. A similar expression pattern was observed in the wor1/wor1 mutant to that of the WT strain, with higher expression of the set of mating-related genes in YP-K medium than in YPD-K medium (Fig. 1c). In the YP-K and YPD-K media, K₂HPO₄ was used as a pH buffer. These results reveal that glucose depletion induces the expression of mating-associated genes in C. albicans white cells.

Effect of different culture media on white cell mating

To further investigate the effect of nutrient states on C. albicans white cell mating, we performed quantitative mating assays with the white-locked strains GH1248 and GBS2347 on several culture media (Table S1 and Fig. 2a). First, we found that there was a dosage effect of glucose on white cell mating. The presence of 0.5% of glucose dramatically decreased the mating efficiency and 1.0% of glucose completely blocked mating. Second, compared to that on YP-K medium, the mating efficiency was slightly increased on Y-K (10 g/L yeast extract, 2.5 g/L K₂HPO₄, 20 g/L agar) and P-K (20 g/L peptone, 2.5 g/L K₂HPO₄, 20 g/L agar) media. However, it was increased 571 times on agar medium (2.5 g/L K₂HPO₄, 20 g/L agar), which represents an extremely poor nutrient condition.

**Fig. 2: Mating frequencies of the WT and mutated strains of *C. albicans*.**

To examine whether altered glucose utilization leads to mating competency, we performed mating assays using white cells of the gpr1/gpr1 and hgt12/hgt12 mutants, we found that deletion of these two genes caused a slight increase of mating competency on both YP-K and YPD-K media (Table S2, crosses 32 and 33). This limited effect on mating could be due to the fact that there are multiple pathways involved in the regulation of glucose and nutrient utilization and there are multiple glucose transporters in C. albicans. These pathways and transporters play redundant roles in glucose utilization. To further examine the effect of altered glycolysis, we added the competitive inhibitor of glycolysis, 2-deoxy-D-glucose (2-DG), to the medium and found that the presence of low levels of 2-DG promoted white cell mating (Table S1, crosses 7 and 9). These results suggest that depletion of glucose plays a major role in the induction of white cell mating and that yeast extract and peptone have an additive effect.

We also examined the impact of different carbon sources on white cell mating (Table S1, crosses 10 to 15). Oleinic acid (added to YP-K medium) had no significant effect on mating; galactose and glycerol reduced the mating efficiency; and mannitol, sodium acetate, or succinic acid completely blocked white cell mating. These results were reasonable because C. albicans can utilize mannitol but not glycerol and oleinic acid efficiently as a carbon source in the absence of glucose. Sodium acetate and succinic acid could affect medium pH after a period of cell growth and thus cause cellular stress to C. albicans.

Glucose depletion sensitizes the pheromone response and promotes the development of mating projections in white cells

We predicted that increased expression of mating-associated genes in glucose-depleted medium would promote the pheromone response in white cells. To test this hypothesis, we treated WT white and opaque MTLa cells and white-locked wor1/wor1 MTLa cells with α-pheromone. The expression of MFA1 was used as a reporter of the pheromone response as this gene is not expressed in either white or opaque cells in the absence of α-pheromone²⁸. As shown in Fig. 3a, pheromone treatment increased the expression levels of MFA1 about tenfold in WT and wor1/wor1 white cells on YPD-K medium. In contrast, MFA1 expression levels increased more than 531 times in pheromone-treated white cells grown on YP-K medium, comparable to pheromone-induced levels in opaque cells. These findings indicate that glucose depletion sensitizes white cells to pheromone treatment.

**Fig. 3: Glucose depletion sensitizes the pheromone response pathway and promotes the development of mating projection in *C. albicans* white cells.**

To verify that white cells respond more strongly to pheromone under glucose depletion conditions, microscopy was performed using a set of reporter strains in which GFP was under the control of the MFA1 promoter. Consistent with the qRT-PCR results, GFP signal was observed in white cells when treated with α-pheromone in YP-K medium but not in YPD-K medium (Fig. 3b). Under the same conditions, we also observed the development of mating projections in both WT and wor1/wor1 white cells in response to pheromone (Fig. 3b). To further verify the cell identity, we examined the relative expression levels of mating-associated, white-specific, and opaque-specific gene expression using qRT-PCR assays. As shown in Fig. S1, the expression of white-specific genes was increased in white cells and the expression of mating-specific genes was increased in both opaque and white cells. Our findings reveal that glucose depletion promotes the mating response, including the development of mating projections, in C. albicans white cells.

To characterize protein levels in white cells, we performed a comparative analysis of the proteome in a white-locked wor1/wor1 strain grown on YP-K and YPD-K media (Fig. S2 and Dataset S1). We found that the expression profile on YP-K medium was different from the expression profile of opaque cells^29,30. The differentially expressed proteins included both phase-dependent and phase-independent ones. As expected, a large set of metabolism-associated genes were differentially expressed under the two culture conditions. These results indicate that C. albicans white cells grown on YP-K medium did not switch to the opaque phenotype. Taken together with the transcriptional expression profile, the proteomic results confirm that cells in the white state are responding to pheromone without undergoing switching.

Glucose depletion promotes efficient mating in white cells

To test whether glucose depletion would facilitate sexual mating by white cells, we performed quantitative mating assays as reported in Fig. 2 and Tables 1 and S2. When both partners were white cells (cross 1), C. albicans mated at least >391 times more efficiently on YP-K medium than on the glucose-containing YPD-K medium (with no mating progeny obtained on YPD-K medium). When one mating partner was white and the other was opaque (crosses 2 and 16), C. albicans mated 298 and 163 times more efficiently on YP-K medium than on YPD-K medium. Although depletion of glucose had no notable effect on white-to-opaque switching¹⁸, to verify that glucose depletion promotes white cell mating, we also examined the mating competency of white-locked wor1/wor1 cells (crosses 4, 5, and 17). As shown in Fig. 2 and Tables 1 and S2, when WOR1 was deleted from both mating partners, C. albicans mated on YP-K medium at least >467 times more efficiently than on YPD-K medium (no mating obtained on YPD-K medium, Cross 5). When one mating partner was white wor1/wor1 cells and the other was WT opaque cells (crosses 4 and 17), mating efficiencies increased 1375 and 324 times on YP-K medium relative to YPD-K medium. Moreover, mating between two opaque partners on YP-K medium was five times higher than that on YPD-K medium (cross 3). These results demonstrate that depletion of glucose has a remarkable effect in enabling C. albicans white cell mating but only a relatively small effect on increasing opaque cell mating.

Table 1 Mating frequencies of the WT and mutated strains of C. albicans on YP-K and YPD-K media

Full size table

Roles of the pheromone MAPK signaling pathway and transcription factors Cph1, Tec1, and Dig1 in white cell mating

We next tested the role of the conserved pheromone MAPK signaling pathway in supporting C. albicans white cell mating. As expected, deletion of the α-pheromone receptor-encoding gene STE2 blocked white cell mating (Table S2, cross 31). Deletion of either one or both of the CEK1/2 MAPK genes significantly decreased the efficiency of white cell mating on YP-K medium (crosses 6, 7, 8, 29, and 30). Consistently, two previous reports also found that inactivation of CEK1 or CEK2 decreases the mating efficiency in C. albicans^23,31. However, ectopic expression of CEK2 but not CEK1 in the white-locked wor1/wor1 mutant highly enhanced mating on both YP-K and YPD-K media (cross 11, 12, and 13). Mating of the strain ectopically expressing CEK2 was (1.5 ± 0.1) × 10⁻² on YP-K medium, which was 600 times higher than that on YPD-K medium (cross 13). These results suggest that both Cek1 and Cek2 play roles in mating of WT cells on regular media, while white cell mating in glucose-depleted conditions was highly sensitive to Cek2 expression levels.

The pheromone MAPK signaling pathway activates the Cph1 transcription factor which is essential for C. albicans mating²³. We found that ectopic expression of CPH1 in white cells significantly increased mating on both YP-K and YPD-K media (crosses 11 and 14). The conserved TEA/ATTS transcription factor Tec1 is a target of the Cek2 kinase and involved in the regulation of the white cell pheromone response and development of “sexual biofilms” in C. albicans^32,33,34. As shown in Fig. 2b and Table 1 (crosses 5 and 9), deletion of TEC1 in the wor1/wor1 mutant promoted white cell mating on both YP-K and YPD-K media. Ectopic expression of CPH1 or deletion of TEC1 increased mating frequencies ~941- or 333-fold, respectively, on YPD-K medium (crosses 5, 9, 11, and 14), suggesting that these genetic modifications at least partially override the repressing effect of glucose. Consistent with Tec1 negatively impacting mating, ectopic TEC1 expression completely blocked white cell mating on both YP-K and YPD-K media (crosses 19, 20, 21, and 22) and reduced the efficiency of opaque cell mating (crosses 23 and 24). These findings indicate that Cph1 and Tec1 play critical, but opposing, roles in C. albicans white cell mating.

C. albicans Dig1 is the single ortholog of S. cerevisiae Dig1 and Dig2, which are MAPK-responsive inhibitors of Ste12, the S. cerevisiae ortholog of C. albicans Cph1³⁵. Given the critical function of Dig1 in regulating S. cerevisiae Ste12 and C. albicans Cph1 expression^35,36, we examined the role of Dig1 in C. albicans white cell mating. As shown in Fig. 2b and Table 1, deletion of DIG1 in the wor1/wor1 mutant increased mating 400 fold on YPD-K medium, whereas the efficiency was increased 3357 fold on YP-K medium compared to the control expressing DIG1 (crosses 5 and 10). Of note, the mating efficiency of the wor1Δ/Δ dig1Δ/Δ double mutant (cross 10) on YP-K medium was ~4000 fold higher than that on YPD-K medium. Conversely, ectopic expression of DIG1 blocked white cell mating on both YP-K and YPD-K media (crosses 11 vs. 15, and 21 vs. 26) and decreased the efficiency of opaque cell mating (crosses 19 vs. 25, and 23 vs. 27). These results establish that Dig1 functions as a key negative regulator of glucose depletion-induced white cell mating in C. albicans.

Expression and roles of Cek2, Cph1, Tec1, and Dig1 in white cell mating under glucose depletion conditions

To define the differential roles of these MAPK regulators in the presence and absence of glucose, we examined their RNA or protein levels in white cells (Figs. 1 and S3). Both the RNA and protein expression levels of CEK2 were increased in YP-K medium compared to those in YPD-K medium (Figs. 1b, c, and S3), whereas the expression levels of Tec1 were decreased in YP-K medium (Fig. 4a and Fig. S3). The protein expression levels of Dig1 were decreased in both “a” and “α” cells in YP-K medium compared to that in YPD-K medium (Fig. 4c). As aforementioned, Cek2 plays a positive role, whereas Tec1 and Dig1 function as negative regulators in the control of white cell mating. Therefore, these expression differences were in line with their roles in promoting/inhibiting white cell mating in C. albicans.

**Fig. 4: Relationships among the Cph1, Tec1, and Dig1 transcription factors.**

The relative transcriptional expression levels of CPH1 were comparable in YP-K and YPD-K media in the single strain cultures (“a” or “α”, Fig. 4a). However, the expression level of CPH1 in YP-K medium was much higher than that in YPD-K medium in the “a” and “α” cell mixed cultures (Fig. 4a). Deletion of DIG1 also increased the transcriptional expression of CPH1 and, consistently, ectopic expression of DIG1 reduced the expression level of Cph1 (Fig. 4b and d). Ectopic expression of CPH1 repressed the expression of TEC1 (Fig. 4b).

These results highlight the regulatory interplay between Cph1, Tec1, and Dig1 to control white cell mating under glucose depletion conditions. Based on the regulatory relationships found here and previous publications^17,32,33,36, we propose that glucose depletion promotes white cell mating through the control of the expression of these key regulators and activation of the pheromone MAPK pathway in C. albicans. A model is shown for how Cek2, Cph1, Tec1, and Dig1 may coordinately regulate white cell mating (Fig. 4e).

Discussion

There are advantages and disadvantages associated with undergoing sexual reproduction in eukaryotic organisms^37,38. Although sexual reproduction creates genetic and phenotypic diversity enabling rapid adaptation to new environments, its costs are also significant (e.g., it takes additional energy, typically produces fewer offspring and can result in the loss of favored genetic configurations compared to asexual reproduction). In fungal species, an enormous diversity of regulatory mechanisms has evolved to control sexual reproduction^1,39. In the pathobiont C. albicans, the white-opaque switch provides a stringent additional tier of control for sexual reproduction⁴, which may represent a balancing strategy between sexual and asexual life cycles. However, it is still unclear as to what benefits the white-opaque switch provides to C. albicans, or why mating gained this extra level of regulatory control in this species and two close relatives.

In this study, we demonstrate that glucose depletion alleviates the bottlenecks on sexual mating in C. albicans white cells due to activation of the pheromone MAPK pathway and downregulation of mating repressors such as Tec1 and Dig1 (Fig. 4e). These expression changes act in combination to elevate the mating competency of WT white cells by more than two orders of magnitude. Given that the white-opaque switch evolved relatively recently in C. albicans and closely related species, this mating strategy could represent a holdover from a more ancestral mode of sexual reproduction.

We previously reported that glucose depletion supports the development of mating projections and same-sex mating in opaque cells of C. albicans through the regulation of the pheromone signaling MAPK and stress-response Hsf1-Hsp90 signaling pathways¹⁸. In haploid cells of S. cerevisiae, depletion of glucose promotes an elongated morphology and invasive growth through activation of the Ras GTPase controlled cAMP/PKA signaling and Ste11-Ste7 MAPK pathways²², while MAPK signaling also regulates filamentous growth in C. albicans⁴⁰. Thus, in both yeasts, components of the pheromone signaling MAPK pathway regulate both filamentous transitions and sexual mating. Interestingly, glucose depletion has also been linked to mating in S. pombe and Cryptococcus neoformans, where a drop in glucose levels leads to increased mating efficiencies^41,42,43. Together, these investigations further link glucose starvation to a conserved role in the regulation of fungal signaling pathways, including enabling sexual reproduction in multiple fungal species.

The terminal MAP kinases Cek1/2 have been shown to have overlapping roles in C. albicans mating, as deletion of both factors was necessary to block mating²³. Scaduto et al. demonstrated that CEK2 expression is lower in white cells than in opaque cells and that ectopic expression of CEK2 (but not CEK1) dramatically increased the mating efficiency of white cells¹⁷. Here, we show that both Cek1 and Cek2 are required for glucose depletion-induced white cell mating (Fig. 2b and Table 1, crosses 1, 6, and 7), and again that ectopic expression of CEK2 but not CEK1 promotes this mating (Fig. 2b and Table 1, crosses 11, 12, and 13). Consistent with these observations, Cek2 protein levels (but not Cek1 levels) were increased under glucose-depleted conditions (Fig. S3), indicating that Cek2 contributes to mating in the absence of glucose. Cph1 (ortholog of S. cerevisiae Ste12) and Tec1 are transcription factors that act downstream of the pheromone MAPK pathway in C. albicans and S. cerevisiae^23,32,33,34. In response to pheromone, S. cerevisiae Tec1 is phosphorylated by Fus3 (C. albicans Cek2 ortholog) and degraded through SCFCdc4. This pheromone-induced destruction of Tec1 ensures that MAPK signaling specifically promotes mating^32,33. Interestingly, pheromone induces the expression of TEC1 and enhances the formation of biofilms in white cells of C. albicans³⁴, although the relative impact of TEC1 differed in two independent studies^34,44. We demonstrate that TEC1 expression is reduced upon glucose depletion and that deletion of TEC1 promotes white cell mating in C. albicans (Fig. 4a and Table 1). Conversely, ectopic expression of Tec1 repressed white cell mating (Table S2). These findings indicate that Tec1 functions as a key repressor of mating in C. albicans white cells.

The Dig1 transcription factor is involved in invasive/filamentous growth and mating, and functions as an inhibitor of Cph1 in C. albicans³⁶. Although Tec1 and Efg1 are not essential for mating in C. albicans, they coordinate with Dig1 to regulate mating efficiency³⁶. Here, we identified Dig1 as a key regulator of Cph1 and white cell mating (Figs. 2 and 4, Tables 1 and S2). Therefore, together with Cek2, Tec1, and Cph1, Dig1 functions as a key regulator of glucose depletion-induced white cell mating in C. albicans (Fig. 4e).

In addition to regulating mating, the white-opaque switch impacts many other aspects of C. albicans biology including interactions with host cells⁸. Under certain culture conditions, mating-incompetent MTLa/α heterozygous cells are also capable of undergoing white-opaque switching⁴⁵, suggesting that this epigenetic switch could have evolved for purposes other than mating. Given the remarkable differences in gene expression and metabolic profiles between white and opaque cells, this “division of labor” may benefit C. albicans adaptation to different host niches. Given that the white-cell type of C. albicans is more common than the opaque-cell type, white-cell mating could also be more frequent in nature than that of opaque cells. Thus, white cell mating may represent a more ancient mode of sexual reproduction that shows closer parallels to that of other yeasts such as S. pombe and S. cerevisiae. Our study therefore further demonstrates the diverse mating strategies of fungal species and sheds new light on the regulation of sex in C. albicans.

Methods

Strains and growth conditions

Yeast strains used in the study are listed in Table S3. C. albicans strains were stored at −80 °C in yeast extract-peptone-dextrose (YPD, 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) medium plus 25% glycerol. To revive the strains from the frozen stocks, C. albicans cells were scratched with a sterile toothpick and streaked and grown on YPD medium plates at 30 °C. YPD and modified Lee’s glucose medium supplemented with 5 μg/mL phloxine B were used for routine growth of C. albicans.

YPD-K (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose, 2.5 g/L K₂HPO₄, 20 g/L agar, pH 7.3) and YP-K (20 g/L peptone, 10 g/L yeast extract, 2.5 g/L K₂HPO₄, 20 g/L agar, pH 7.3) medium plates were used for the induction of mating projections and quantitative mating assays of C. albicans¹⁸. Phloxine B and K₂HPO₄ were purchased from Sigma-Aldrich (Cat. Nos., P2759 and P9666; St. Louis, MO, USA). Synthetic complete dextrose (SCD) amino acid dropout media were used for selectable growth in quantitative mating assays.

Plasmid construction

Primers used for PCRs in the study are listed in Table S4. To construct plasmids pACTS-CEK1, pACTS-CEK2, pACTS-CPH1, and pACTS-DIG1, the CEK1, CEK2, CPH1, and DIG1 open reading frame (ORF) fragments were PCR amplified from C. albicans SC5314 genomic DNA and subcloned into the EcoRV/HindIII site of plasmid pACTS¹⁸. To construct plasmid pACT1-TEC1, the TEC1 open reading frame (ORF) fragments were PCR amplified from C. albicans SC5314 genomic DNA and subcloned into the EcoRV/HindIII site of plasmid pACT1¹⁰. To construct the reconstituted plasmid pSFS2a-TEC1p-TEC1, the 3′-UTR fragment and 5′-UTR plus ORF region of TEC1 were amplified from C. albicans SC5314 genome and sequentially inserted into the SacII/SacI and ApaI/XhoI sites of the plasmid pSFS2a⁴⁶.

Strain construction

To construct C. albicans strain GH1350α (GGB1418), the ApaI/SacI linearized plasmid L23.14 (also named plasmid T2A-MTL, a pSFS2A-based MTL locus deletion plasmid) was transformed into strain SN250 to replace the MTLa allele⁴⁵. The resulting strain was then grown in YPmal medium (20 g/L peptone, 10 g/L yeast extract, 20 g/L maltose) for FLP-mediated excision of the SAT1/flipper cassette. Using a similar strategy, the MTLa or MTLα locus was disrupted in the corresponding MTL heterozygous strains with ApaI/SacI linearized plasmid L23.14, generating strains SN152αu, CCJS837 (wor1Δ/Δα), GBS2775 (cek1Δ/Δa), GBS2776 (cek2Δ/Δa), GBS2783 (cek1Δ/Δα), and GBS2784 (cek2Δ/Δα).

Strain GBS2347

The URA3-IRO1 cassette was amplified from C. albicans SC5314 genomic DNA and integrated into the original locus of strain CCJS837¹⁵, generating strain GBS2347.

Strains GBS2824 (cek1Δ/Δ ura3Δ/Δa) and GBS2826 (cek2Δ/Δ ura3Δ/Δa)

Stains GBS2775 and GBS2776 were streaked onto solid SCD medium containing 5-fluorooroic acid (5-FOA, A601555, Sangon Biotech, Shanghai, China) and incubated at 37 °C for 7 days for the selection of ura3Δ/Δ mutant. Colonies of uridine auxotrophy were confirmed by growing on solid SCD medium lacking uridine. The resulting ura3Δ/Δ strains were then transformed with the ARG4 cassette amplified from SC5314 genomic DNA, generating GBS2824 and GBS2826.

GBS2799 (wor1 Δ/Δa pACTS-CEK1), GBS2809 (wor1 Δ/Δα pACTS-CEK1), GBS2802 (wor1 Δ/Δa pACTS-CEK2), GBS2810 (wor1 Δ/Δα pACTS-CEK2), GBS2682 (wor1 Δ/Δa pACTS-CPH1) and GBS2779 (wor1 Δ/Δα pACTS-CPH1)

To construct strains ectopically expressing CEK1, CEK2, or CPH1, the AscI-linearized pACTS-CEK1, CEK2, or CPH1 plasmid, respectively, was transformed into strains GH1608 and CAY3336u.

GBS2654 (wor1 Δ/Δα pACT1-TEC1) and GBS2763 (WTα pACT1-TEC1)

To construct strains ectopically expressing TEC1, the AscI-linearized pACT1-TEC1 plasmid was transformed into strains CCJS837 and GH1605.

GBS2937 (wor1 Δ/Δa pACTS-DIG1), GBS2931 (wor1 Δ/Δα pACTS-DIG1), GBS2946 (wor1 Δ/Δα pACTS-DIG1), and GBS2963 (WTα pACTS-DIG1)

To construct strains ectopically expressing DIG1, the AscI-linearized pACTS-DIG1 plasmid was transformed into strains GH1608, CAY3336u, CCJS837, and GH1350α.

GBS2891 (tec1Δ/Δa + TEC1p-TEC1) and GBS2897 (tec1Δ/Δα + TEC1p-TEC1)

To generate the TEC1 reconstituted strain, the plasmid pSFS2a-TEC1p-TEC1 was linearized with ApaI/SacI and transformed into strains GH2783 and JSM115.

GBS2828 (wor1Δ/Δa MFA1p-GFP)

To construct the MFA1p-GFP reporter strain, the GFP-SAT1 cassette was amplified from the plasmid pNIM1⁴⁶ and then transformed into strain GH1248.

GBS2604 (tec1 Δ/Δ wor1 Δ/Δα) and GBS2792 (tec1 Δ/Δ wor1 Δ/Δa)

To construct the tec1Δ/Δ wor1Δ/Δ double mutant, the first copy of WOR1 gene in JSM115 (tec1Δ/Δα)⁴⁷ was disrupted with the ApaI/SacI linearized plasmid pSFS2a-WOR1 KO⁴⁵. The resulting strain was subject to the FLP-mediated excision of the SAT1/flipper cassette by growing on YPmal medium, generating strain tec1Δ/Δα WOR1/wor1::FRT. To disrupt the second allele of WOR1, the ApaI/SacI linearized plasmid pSFS2a-WOR1 KO was transformed into strain tec1Δ/Δα WOR1/wor1::FRT, generating the double mutant GBS2604. A similar strategy was used to delete the first allele of WOR1 in strain GH2783 (tec1Δ/Δa). The resulting strain tec1Δ/Δa WOR1/wor1::FRT was then transformed with the PCR product of SAT1 cassette flanked by 60-bp 5′- and 3′-homologous sequences of WOR1, generating the double mutant GBS2792.

GBS3005 (dig1Δ/Δa), GBS3009 (dig1Δ/Δα), GBS2874 (wor1Δ/Δ dig1Δ/Δa), and GBS2877 (wor1Δ/Δ dig1Δ/Δα)

To delete both alleles of DIG1 in the WT and wor1Δ/Δ mutant, fusion PCR reactions were performed⁴⁸. PCR products for targeting the DIG1 locus (5′- and 3′-flank regions of DIG1) were amplified from C. albicans SC5314 genomic DNA with primers GGB1289/GGB1290 and GGB1291/GGB1292, respectively. Selectable marker genes CaURA3, CaHIS1, CmLEU2, and caSAT1 were PCR amplified from plasmids pGEM-URA3, pGEM-HIS1, pSN40, and pNIM1, respectively^46,48,49. Fusion PCR assays were then performed using the DIG1 5′- and 3′-flank fragments and selectable marker cassettes as templates. To delete the first allele of DIG1, fusion PCR products containing the CaURA3 marker were transformed into the WT strains GH1013 (WTa) and SN152αu (WTα). To delete the second allele of DIG1, fusion PCR products containing the CaHIS1 or CmLEU2 marker were transformed into heterozygous mutants. To generate the dig1Δ/Δ wor1Δ/Δ double mutant, fusion PCR products containing the CaHIS1 or CmLEU2 marker were transformed into strain CAY3339 (wor1Δ/Δa) or CAY3336 (wor1Δ/Δα), respectively. Then, the second allele of DIG1 was deleted using fusion PCR products containing the caSAT1 marker.

LTS1161 (WTa Cph1-TAP), LTS1172 (wor1Δ/Δa Cph1-TAP), LTS1338 (WTα Cph1-TAP), and LTS1209 (wor1Δ/Δα Cph1-TAP)

To construct the TAP-tagged Cph1 strains, one allele of CPH1 was first deleted using a CaURA3 cassette flanked by 5′- and 3′-fragments of CPH1 amplified with the fusion PCR assay as earlier described. The CPH1-CaURA3 disruption cassette was transformed into strains GH1013, GH1608, SN152αu, and CCJS837, generating corresponding cph1::CaURA3/CPH1 heterozygous strains. Then, the TAP-ARG4 cassette flanked by approximately 60 bp 5′- and 3′-homologous sequences of CPH1 was PCR amplified from strain CaLC2993⁵⁰ with primers LTG01/LTG02 and was transformed into the cph1::CaURA3/CPH1 heterozygous strains, generating strains in which the endogenous Cph1 protein was TAP-tagged: LTS1161, LTS1172, LTS1338, and LTS1209.

A similar strategy was used to construct the TAP-tagged Cek2, Tec1, Cek1, and Dig1 expression strains: LTS1164 (WTa Cek2-TAP), LTS1176 (wor1Δ/Δa Cek2-TAP), LTS1343 (WTα Cek2-TAP), and LTS1221 (wor1Δ/Δα Cek2-TAP); LTS1186 (WTa Tec1-TAP), LTS1183 (wor1Δ/Δa Tec1-TAP), LTS1336 (WTα Tec1-TAP), and LTS1213 (wor1Δ/Δα Tec1-TAP); LTS1302 (WTa Cek1-TAP), LTS1179 (wor1Δ/Δa Cek1-TAP), LTS1341 (WTα Cek1-TAP), LTS1217 (wor1Δ/Δα Cek1-TAP), LTS1483 (wor1Δ/Δa Dig1-TAP) and LTS1480 (wor1Δ/Δα Dig1-TAP).

LTS1463 (wor1Δ/Δa Cph1-TAP pACTS-DIG1) and LTS1345 (wor1Δ/Δa, Cph1-TAP pACTS)

The plasmids pACTS-DIG1 and pACTS were linearized with AscI and transformed into the TAP-tagged Cph1 expression strain LTS1172, generating LTS1463 and LTS1345, respectively.

LTS1452 (gpr1Δ/Δ arg4Δ/Δa) and FSR320 (hgt12Δ/Δ ura3Δ/Δa)

To construct the gpr1Δ/Δa mutant, one allele of GPR1 was deleted using a CdHIS1 cassette flanked by 5′- and 3′-fragments of GPR1 amplified with the fusion PCR assay as earlier described. The GPR1-CdHIS1 disruption cassette was transformed into the WT strain SN152a (WTa), generating corresponding gpr1::CdHIS1/GPR1 heterozygous strains. To delete the second allele of GPR1, fusion PCR products containing the CmLEU2 marker were transformed into the heterozygous mutant. Similarly, to generate the hgt12Δ/Δa mutant, fusion PCR products containing the CaARG4 marker were transformed into the WT strains GH1013 (WTa) to generate the heterozygous mutant and the second allele of HGT12 was then deleted using fusion PCR products containing the cdHIS1 marker.

Mating assays

C. albicans mating assays were performed as described in previous publications with slight modifications¹⁵. Briefly, C. albicans cells were first grown on solid Lee’s glucose medium at 25 °C for 5 days. Homogeneous white or opaque cells were collected and resuspended in water at a concentration of 1 × 10¹⁰ cells/mL. C. albicans cells of the two mating partners (3 μl for each) were mixed and spotted on YP-K and YPD-K media. After 7 days of incubation at 25 °C, mating mixtures were then replated onto three types of selective SCD media lacking amino acids depending on the mating partner auxotrophies. Mating frequency = conjugants / (limiting parents + conjugants)¹⁵. Three biological replicates were performed for each mating cross.

RNA extraction and quantitative real-time PCR assays

RNA extraction and quantitative real-time PCR (qRT-PCR) assays were performed according to our previous publication with slight modifications¹⁵. C. albicans strains were grown overnight in SCD liquid medium at 25 °C. Cells were washed and resuspended in ddH₂O at a concentration of 3.3 × 10⁹ cells/mL. Three microliters of fungal cells were spotted on YP-K or YPD-K media. After 3 days of incubation at 25 °C, cells were harvested and washed with 1 × PBS. Total RNA was then extracted using RNA purification kit (Thermo scientific, K0732, Thermo scientific, Waltham, USA). For qRT-PCR assays, 1 μg of total RNA of each sample was used to synthesize cDNA with ReverAid H Minus Reverse Transcriptase (EP0451, Thermo Scientific, Waltham, USA). Quantification of transcripts was performed in Bio-Rad CFX96 real time detection system (Bio-Rad, Hercules, USA) using SYBR green master mix (QPS-201, TOYOBO, Osaka, Japan) under the condition: 95 °C for 1 min, followed by 95 °C for 15 s, 57 °C for 20 s, and 72 °C for 50, for 40 cycles. The signal of each sample was normalized to the expression level of C. albicans ACT1. Data were analyzed using Bio-Rad CFX Manager 3.1 for Bio-Rad Real-Time PCR system and presented as the average ± standard error (SEM). Three biological replicates were performed.

Statistics and reproducibility

All data presented in quantitative mating and qRT-PCR assays were performed at least three independent biological replicates and analyzed using GraphPad Prism 9.0 software. For quantitative mating analyses, data were analyzed using two-sided Student’s t-tests. For qRT-PCR assays, data were analyzed using two-sided Student’s t-tests. Methods for statistical analyses are specified in the legend of every individual figure or table. Colony and cellular morphologies presented in the figures were representative images of three independent experiments.

Proteomic assay

The wor1Δ/Δa strains were initially grown in YPD-K medium at 25 °C for 2 days. Approximately 1 × 10⁷ cells were spotted on YP-K and YPD-K media and cultured for 3 days at 25 °C. Cells were harvested and protein extracts were prepared as described below for the western blot analyses. For LC-MS/MS analysis, proteins were digested via FASP method using Nanosep 10k filters (Pall Life Sciences, USA)⁵¹. After three-time buffer displacement with 8 M urea in 25 mM NH₄HCO₃, proteins were reduced with 10 mM DTT at 37 °C for 30 min, and followed by alkylation with 30 mM iodoacetamide at 25 °C for 45 min in dark. Then, the filter was washed with 20% acetonitrile (ACN) once followed by digestion buffer (30 mM NH₄HCO₃) three times. Overnight digestion was carried out using trypsin (enzyme/protein (w/w) ratio as 1:50) at 37 °C for 12 h. Then, the solution was filtrated out and the filter was washed twice with 15% ACN. All the filtrates were pooled and vacuum-dried. LC-MS/MS analysis was performed using an EASY-nLC 1200 system (Thermo Fisher Scientific, USA) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, USA). A one-column system was adopted for all analyses. Samples were analyzed on a home-packed C18 column (75 µm i.d. × 25 cm; ReproSil-Pur 120 C18-AQ, 1.9 µm (Dr. Maisch GmbH, Germany))⁵². The mobile phases consisted of Solvent A (0.1% formic acid) and Solvent B (0.1% formic acid in 80% ACN). The eluted peptides were sprayed directly into Orbitrap Fusion Lumos mass spectrometer. Data-dependent analysis was employed in MS analysis: The MS1 scans were acquired in Orbitrap (resolution of 60,000, 350-1600 m/z, and auto maximum ion injection time). The cycle time between master scans was set as 3 s, and the precursor ions were fragmented in HCD mode and analyzed using Orbitrap analyzer with a resolution of 15,000 and normalized collision energy (NCE) of 30%. The raw data were processed using Proteome Discoverer software (version 2.4, Thermo Fisher Scientific) with in-house Mascot search engine (version 2.7.0, Matrix Science). The data search was performed against the C. albicans protein database (19401, 20211129) downloaded from UniProt. Trypsin/P was chosen as the enzyme, and up to two missed cleavages were allowed. The precursor mass tolerance was set as 10 ppm and fragment mass tolerance was set as 0.05 Da. Carbamidomethylation on cysteine as a fixed modification, N-acetylation in the protein N-terminal and oxidation on methionine as variable modifications. The false discovery rate threshold was set at 0.05 and 0.01 for proteins and peptides, respectively. Three replicates were performed. Differentially enriched or expressed proteins were tested using the DEP package version 3.16.

Western blotting

Total protein was extracted for western blot analysis according to a published protocol¹⁸. C. albicans strains were grown in YPD-K medium at 25 °C for 2 days. Approximately 1 × 10⁷ cells of each experimental strain were then spotted on YP-K and YPD-K media and cultured for 3 days at 25 °C. Cells were harvested, rinsed twice with 1 × PBS, and then resuspended in 200 μL lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% (w/w) SDS, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF) containing the protease inhibitor mix (Cat. No. 11873580001 Roche Diagnostics, Mannheim, Germany). C. albicans cells were lysed with a bead beating instrument (40 s beating followed by 1 min cooling on ice for 5 cycles). The supernatant of cell lysates was collected by centrifugation at 12,000 rpm for 15 min and the protein concentration was determined by Bradford assays (Cat. No. B6916, Sigma-Aldrich, Inc). Total protein of each sample was separated by 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membranes were washed twice for 10 min with TBS buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) and blocked in 5% milk in TBS buffer containing 0.05% Tween-20 (TBST) at room temperature for 2 h with gentle shaking. For anti-TAP blots, a horseradish peroxidase (HRP)-conjugated monoclonal anti-TAP antibody (1:1000 dilution, P1291, Sigma-Aldrich, Inc) was used. For anti-Cdc28 blots, a monoclonal anti-Cdc28 antibody (1:1000 dilution, sc-515762, Santa Cruz Biotechnology, Inc) was used, followed by a HRP-conjugated secondary antibody (1:2000 dilution, 7076 S, Cell Signaling Technology Inc). The immunopositive bands were visualized using an enhanced chemiluminescent substrate (32209 Thermo Scientific, USA) and analyzed using Image Lab 3.0 for a Chemidoc XRS + system (Bio-Rad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD040559. Source data are provided in this paper. Source data are provided with this paper.

References

Heitman, J. Sexual reproduction and the evolution of microbial pathogens. Curr. Biol. 16, R711–R725 (2006).
Article CAS PubMed Google Scholar
Feretzaki, M. & Heitman, J. Unisexual reproduction drives evolution of eukaryotic microbial pathogens. PLoS Pathog. 9, e1003674 (2013).
Article PubMed PubMed Central Google Scholar
Ene, I. V. & Bennett, R. J. The cryptic sexual strategies of human fungal pathogens. Nat. Rev. Microbiol. 12, 239–251 (2014).
Article CAS PubMed PubMed Central Google Scholar
Miller, M. G. & Johnson, A. D. White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating. Cell 110, 293–302 (2002).
Article CAS PubMed Google Scholar
Pujol, C. et al. The closely related species Candida albicans and Candida dubliniensis can mate. Eukaryot. Cell 3, 1015–1027 (2004).
Article CAS PubMed PubMed Central Google Scholar
Porman, A. M., Alby, K., Hirakawa, M. P. & Bennett, R. J. Discovery of a phenotypic switch regulating sexual mating in the opportunistic fungal pathogen Candida tropicalis. Proc. Natl Acad. Sci. USA 108, 21158–21163 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Xie, J. et al. N-Acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution. Eukaryot. Cell 11, 773–782 (2012).
Article CAS PubMed PubMed Central Google Scholar
Soll, D. R. Why does Candida albicans switch? FEMS Yeast Res. 9, 973–989 (2009).
Article CAS PubMed Google Scholar
Anderson, J. M. & Soll, D. R. Unique phenotype of opaque cells in the white-opaque transition of Candida albicans. J. Bacteriol. 169, 5579–5588 (1987).
Article CAS PubMed PubMed Central Google Scholar
Huang, G. et al. Bistable expression of WOR1, a master regulator of white-opaque switching in Candida albicans. Proc. Natl Acad. Sci. USA 103, 12813–12818 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Srikantha, T. et al. TOS9 regulates white-opaque switching in Candida albicans. Eukaryot. Cell 5, 1674–1687 (2006).
Article CAS PubMed PubMed Central Google Scholar
Zordan, R. E., Galgoczy, D. J. & Johnson, A. D. Epigenetic properties of white-opaque switching in Candida albicans are based on a self-sustaining transcriptional feedback loop. Proc. Natl Acad. Sci. USA 103, 12807–12812 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Zordan, R. E., Miller, M. G., Galgoczy, D. J., Tuch, B. B. & Johnson, A. D. Interlocking transcriptional feedback loops control white-opaque switching in Candida albicans. PLoS Biol. 5, e256 (2007).
Article PubMed PubMed Central Google Scholar
Yi, S. et al. Self-induction of a/a or alpha/alpha biofilms in Candida albicans is a pheromone-based paracrine system requiring switching. Eukaryot. Cell 10, 753–760 (2011).
Article CAS PubMed PubMed Central Google Scholar
Tao, L. et al. White cells facilitate opposite- and same-sex mating of opaque cells in Candida albicans. PLoS Genet. 10, e1004737 (2014).
Article PubMed PubMed Central Google Scholar
Daniels, K. J., Srikantha, T., Lockhart, S. R., Pujol, C. & Soll, D. R. Opaque cells signal white cells to form biofilms in Candida albicans. EMBO J. 25, 2240–2252 (2006).
Article CAS PubMed PubMed Central Google Scholar
Scaduto, C. M. et al. Epigenetic control of pheromone MAPK signaling determines sexual fecundity in Candida albicans. Proc. Natl Acad. Sci. USA 114, 13780–13785 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Guan, G. et al. Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway. PLoS Biol. 17, e2006966 (2019).
Article CAS PubMed PubMed Central Google Scholar
Chang, D. E. et al. Carbon nutrition of Escherichia coli in the mouse intestine. Proc. Natl Acad. Sci. USA 101, 7427–7432 (2004).
Article ADS CAS PubMed PubMed Central Google Scholar
Opulente, D. A. et al. Pathogenic budding yeasts isolated outside of clinical settings. FEMS Yeast Res. 19, foz032 (2019).
Article CAS PubMed PubMed Central Google Scholar
Granot, D. & Snyder, M. Glucose induces cAMP-independent growth-related changes in stationary-phase cells of Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 88, 5724–5728 (1991).
Article ADS CAS PubMed PubMed Central Google Scholar
Cullen, P. J. & Sprague, G. F. Jr. Glucose depletion causes haploid invasive growth in yeast. Proc. Natl Acad. Sci. USA 97, 13619–13624 (2000).
Article ADS CAS PubMed PubMed Central Google Scholar
Chen, J., Lane, S. & Liu, H. A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans. Mol. Microbiol. 46, 1335–1344 (2002).
Article CAS PubMed Google Scholar
Erdman, S., Lin, L., Malczynski, M. & Snyder, M. Pheromone-regulated genes required for yeast mating differentiation. J. Cell Biol. 140, 461–483 (1998).
Article CAS PubMed PubMed Central Google Scholar
Trueheart, J., Boeke, J. D. & Fink, G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol. Cell Biol. 7, 2316–2328 (1987).
CAS PubMed PubMed Central Google Scholar
Alby, K., Schaefer, D., Sherwood, R. K., Jones, S. K. Jr. & Bennett, R. J. Identification of a cell death pathway in Candida albicans during the response to pheromone. Eukaryot. Cell 9, 1690–1701 (2010).
Article CAS PubMed PubMed Central Google Scholar
Yang, M. et al. Fig1 facilitates calcium influx and localizes to membranes destined to undergo fusion during mating in Candida albicans. Eukaryot. Cell 10, 435–444 (2011).
Article CAS PubMed PubMed Central Google Scholar
Dignard, D., El-Naggar, A. L., Logue, M. E., Butler, G. & Whiteway, M. Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone. Eukaryot. Cell 6, 487–494 (2007).
Article CAS PubMed PubMed Central Google Scholar
Lan, C. Y. et al. Metabolic specialization associated with phenotypic switching in Candidaalbicans. Proc. Natl Acad. Sci. USA 99, 14907–14912 (2002).
Article ADS CAS PubMed PubMed Central Google Scholar
Tuch, B. B. et al. The transcriptomes of two heritable cell types illuminate the circuit governing their differentiation. PLoS Genet. 6, e1001070 (2010).
Article PubMed PubMed Central Google Scholar
Magee, B. B., Legrand, M., Alarco, A. M., Raymond, M. & Magee, P. T. Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans. Mol. Microbiol. 46, 1345–1351 (2002).
Article CAS PubMed Google Scholar
Bao, M. Z., Schwartz, M. A., Cantin, G. T., Yates, J. R. 3rd & Madhani, H. D. Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast. Cell 119, 991–1000 (2004).
Article CAS PubMed Google Scholar
Chou, S., Huang, L. & Liu, H. Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast. Cell 119, 981–990 (2004).
Article CAS PubMed Google Scholar
Sahni, N. et al. Tec1 mediates the pheromone response of the white phenotype of Candida albicans: insights into the evolution of new signal transduction pathways. PLoS Biol. 8, e1000363 (2010).
Article PubMed PubMed Central Google Scholar
Olson, K. A. et al. Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms. Mol. Cell Biol. 20, 4199–4209 (2000).
Article CAS PubMed PubMed Central Google Scholar
Regan, H. et al. Negative regulation of filamentous growth in Candida albicans by Dig1p. Mol. Microbiol. 105, 810–824 (2017).
Article CAS PubMed PubMed Central Google Scholar
Annila, A. & Annila, E. The significance of sex. Biosystems 110, 156–161 (2012).
Article PubMed MATH Google Scholar
Wiener, P., Feldman, M. W. & Otto, S. P. On genetic segregation and the evolution of sex. Evol. Int J. Org. Evol. 46, 775–782 (1992).
Article Google Scholar
Ni, M., Feretzaki, M., Sun, S., Wang, X. & Heitman, J. Sex in fungi. Annu. Rev. Genet. 45, 405–430 (2011).
Article CAS PubMed PubMed Central Google Scholar
Kohler, J. R. & Fink, G. R. Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc. Natl Acad. Sci. USA 93, 13223–13228 (1996).
Article ADS CAS PubMed PubMed Central Google Scholar
Joyner, RP, et al. A glucose-starvation response regulates the diffusion of macromolecules. Elife 5, e09376 (2016).
Lengeler, K. B., Wang, P., Cox, G. M., Perfect, J. R. & Heitman, J. Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct. Proc. Natl Acad. Sci. USA 97, 14455–14460 (2000).
Article ADS CAS PubMed PubMed Central Google Scholar
Tian, X. Y. et al. Cryptococcus neoformans sexual reproduction is controlled by a quorum-sensing peptide. Nat. Microbiol. 3, 698–707 (2018).
Article CAS PubMed Google Scholar
Lin, C. H. et al. Genetic control of conventional and pheromone-stimulated biofilm formation in Candida albicans. PLoS Pathog. 9, e1003305 (2013).
Article CAS PubMed PubMed Central Google Scholar
Xie, J. et al. White-opaque switching in natural MTLa/alpha isolates of Candida albicans: evolutionary implications for roles in host adaptation, pathogenesis, and sex. PLoS Biol. 11, e1001525 (2013).
Article CAS PubMed PubMed Central Google Scholar
Park, Y. N. & Morschhauser, J. Tetracycline-inducible gene expression and gene deletion in Candida albicans. Eukaryot. Cell 4, 1328–1342 (2005).
Article CAS PubMed PubMed Central Google Scholar
Du, H. et al. The transcription factor Flo8 mediates CO₂ sensing in the human fungal pathogen Candida albicans. Mol. Biol. Cell 23, 2692–2701 (2012).
Article CAS PubMed PubMed Central Google Scholar
Noble, S. M. & Johnson, A. D. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot. Cell 4, 298–309 (2005).
Article CAS PubMed PubMed Central Google Scholar
Wilson, R. B., Davis, D. & Mitchell, A. P. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J. Bacteriol. 181, 1868–1874 (1999).
Article CAS PubMed PubMed Central Google Scholar
Leach, M. D. et al. Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans. Nat. Commun. 7, 11704 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Wu, Z., Huang, J., Huang, J., Li, Q. & Zhang, X. Lys-C/Arg-C, a more specific and efficient digestion approach for proteomics studies. Anal Chem 90, 9700–9707 (2018).
Article CAS PubMed Google Scholar
Kovalchuk, S. I., Jensen, O. N. & Rogowska-Wrzesinska, A. FlashPack: fast and simple preparation of ultrahigh-performance capillary columns for LC-MS. Mol. Cell. Proteom. 18, 383–390 (2019).
Article CAS Google Scholar

Download references

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2021YFC2300400 to G.H.), National Natural Science Foundation of China awards (31930005 and 82272359 to G.H., 32170194 to L.T. and 32022002 to L.L.), and Shanghai Municipal Science and Technology Major Project (HS2021SHZX001 to G.H.).

Author information

These authors contributed equally: Guobo Guan, Li Tao, Chao Li.

Authors and Affiliations

Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Shanghai Institute of Infectious Disease and Biosecurity and State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200438, China
Guobo Guan, Li Tao, Chao Li, Ming Xu & Guanghua Huang
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China
Guobo Guan & Ling Liu
Molecular Microbiology and Immunology Department, Brown University, Providence, RI, 02912, USA
Richard J. Bennett
Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai, 200438, China
Guanghua Huang
Shanghai Huashen Institute of Microbes and Infections, Shanghai, 200052, China
Guanghua Huang

Authors

Guobo Guan
View author publications
You can also search for this author in PubMed Google Scholar
Li Tao
View author publications
You can also search for this author in PubMed Google Scholar
Chao Li
View author publications
You can also search for this author in PubMed Google Scholar
Ming Xu
View author publications
You can also search for this author in PubMed Google Scholar
Ling Liu
View author publications
You can also search for this author in PubMed Google Scholar
Richard J. Bennett
View author publications
You can also search for this author in PubMed Google Scholar
Guanghua Huang
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

G.G. contributed conceptualization, methodology, resources, formal analysis, writing-original and writing-review/editing, validation. L.T. contributed methodology, resources, formal analysis, writing-original draft, validation, funding acquisition. C.L. contributed methodology, formal analysis, validation. M.X. contributed methodology, formal analysis. L.L. contributed validation, funding acquisition. R.J.B. contributed conceptualisation, formal analysis, writing—review & editing. G.H. contributed conceptualisation, formal analysis, writing-original and writing-review/editing, project administration, supervision, funding acquisition.

Corresponding author

Correspondence to Guanghua Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Dataset 1

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Guan, G., Tao, L., Li, C. et al. Glucose depletion enables Candida albicans mating independently of the epigenetic white-opaque switch. Nat Commun 14, 2067 (2023). https://doi.org/10.1038/s41467-023-37755-8

Download citation

Received: 19 September 2022
Accepted: 30 March 2023
Published: 12 April 2023
DOI: https://doi.org/10.1038/s41467-023-37755-8

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.