White-Opaque Switching in Natural MTLa/α Isolates of Candida albicans: Evolutionary Implications for Roles in Host Adaptation, Pathogenesis, and Sex

All Mating Type Locus strain types of Candida albicans show white-opaque switching competency, not just MTL homozygotes, which allows them to adapt better to environmental changes.


Introduction
Phenotypic plasticity is critical for microorganisms to survive under fluctuating environments. For fungal pathogens, phenotypic switching is a common strategy to rapidly adapt to different host niches and facilitate colonization and infection [1]. A specific phenotype can also confer the fungus a growth advantage over competing microorganisms in a specific environment or host niche. Candida albicans, the major causative agent of fungal infections in humans, can switch between two different visible cell types: white and opaque [2]. The two cell types differ in a number of biological aspects including morphology, virulence, and mating competence [3][4][5]. White cells are small and round and form ''white,'' dome-shaped colonies on solid media, while opaque cells are large and elongated and form darker and flatter colonies [6]. White cells are more virulent than opaque cells in systemic infections, whereas opaque cells appear more suited to cutaneous colonization [7,8]. Opaque cells possess pimples on the cell wall and exhibit unique antigenicity, which may help the pathogen in evading the host immune system [3][4][5]. Moreover, opaque cells are significantly less susceptible to phagocytosis by cells of the fly and mouse innate immune systems than white cells [9]. Perhaps the best studied feature of opaque cells is their mating competency. Opaque cells mate ,10 6 times more efficiently than white cells [10]. It has recently been shown that Candida tropicalis, another important human fungal pathogen, can also undergo whiteopaque switching and parasexual mating [11,12].
Despite the importance of white-opaque switching in host adaptation, pathogenesis, and parasexual reproduction in C. albicans, only a minority (,10%) of natural strains have been reported to undergo white-opaque switching in vitro [13]. It has been shown that the mating-type locus homeodomain proteins (MTLa1/a2) inhibit white-opaque switching via controlling the expression of the master regulator WOR1 [14][15][16]. Consistent with this, the minority of natural isolates capable of white-opaque switching in vitro are homozygous at the MTL locus; this relieves the block of the mating locus proteins. The majority (.90%) of C. albicans MTLa/a isolates in nature were thought to be incapable of white-opaque switching unless they underwent homozygosis of the mating type locus [13]. These ideas raised a fundamental question. Given the importance of white-opaque switching in host adaptation and pathogenesis, why do the majority of a/a natural isolates of C. albicans not undergo white-opaque switching unless they undergo a genetic rearrangement?
In this study, we provide reasonable answers to this basic question. We show that naturally occurring a/a isolates of C. albicans can indeed undergo white-opaque switching under a specialized set of environmental conditions. Previous studies typically used glucose as the sole carbon source and grew cells in ambient CO 2 . We show here that a/a strains can undergo whiteopaque switching in 5% CO 2 when N-acetylglucosamine (GlcNAc) is used as the sole carbon source. GlcNAc and CO 2 , primarily produced by bacterial commensals, are abundant in the gut and have synergistic effects on the induction of the opaque cell phenotype in C. albicans [17]. Therefore, this culture condition likely mimics certain aspects of host niches, such as those in the gut. Opaque cells of a/a strains exhibit similar phenotypes of typical MTL homozygous opaque cells, except they lack the ability to mate. Further experiments demonstrate that three transcription factors Rfg1, Brg1, and Efg1 are involved in the regulation of white-opaque switching in a/a C. albicans strains. This study indicates that there is an alternative gene circuit, which can bypass the a1/a2 block to switching, and promote white-opaque switching in C. albicans under certain environmental conditions that are reminiscent of niches of the host. We propose that whiteopaque switching is not limited to the minority of MTL homozygotes, but rather is a general characteristic of natural C. albicans strains.

Demonstration of White-Opaque Switching in Natural
MTLa/a Strains of C. albicans There are three MTL types of natural C. albicans isolates (a/a, a/a, and a/a). Under normal conditions, MTL heterozygotes (a/ a) are blocked for switching and ''locked'' in the white phase in vitro [10,13]. Since a/a strains are more competitive than their a/ a or a/a derivatives (at least in some in vivo assays) and carry the entire set of opaque-specific genes essential for switching [18], we suspected that the a/a isolates of C. albicans could also undergo white-opaque switching in their natural niches. We also reasoned that routine laboratory media and culture conditions were totally different from conditions in natural niches and might not be conducive for the transition in a/a strains of C. albicans. To test our hypothesis, we took advantage of the synergistic effects of two host environmental cues, GlcNAc and CO 2 , on the induction of the opaque cell phenotype [17]. We grew 94 natural isolates of C. albicans on Lee's GlcNAc medium in 5% CO 2 . We found that 34 strains (36%) formed opaque colonies under this condition. We then examined the MTL genotype of all 94 tested strains. Of them, 92 were a/a, one was a/a, and one was a/a. The two MTL homozygotes (one a/a and one a/a) were identified as switching to opaque, along with the 32 a/a strains in the switchable strain list (Table S1).
An example of an a/a clinical strain that could undergo whiteopaque switching (SZ306) is shown in Figure 1. We noticed that SZ306 could also form opaque colonies on rich medium (YPD) when cultured for an extended time period ( Figure 1A); some other a/a strains also exhibited this behavior. The white and opaque cells of a/a strains were similar to their counterparts of MTL homozygotes in the size and shape of cells ( Figure 1B): white cells of a/a strains were small and round with no pimples on their cell wall surface, while opaque cells were elongated and possessed obvious opaque-specific pimples ( Figure 1C). Northern blot analysis demonstrated that two opaque-enriched genes, OP4 and the master regulator WOR1, were expressed in opaque cells of a/a strains but not in white cells ( Figure 1D). Conversely, the expression levels of the white-enriched genes WH11, EFG1, and RFG1 were significantly higher in white cells than in opaque cells of a/a strains. These results suggest that opaque cells of a/a strains exhibit similar characteristics of colony and cellular morphology and gene expression profile to the opaque cells of MTL homozygotes.
To exclude the possibility of homozygosis of a/a cells during growth, we re-plated several opaque colonies of each switchable a/a strain onto Lee's GlcNAc medium and incubated them in ambient CO 2 for 5 days. Three single opaque colonies of each replated culture were examined for the MTL configuration, and we verified that all remained heterozygous at the MTL locus. An example of this analysis is given in Figure 1E. These results demonstrate that C. albicans a/a isolates can indeed undergo white-opaque switching.
MTLa/a White-Opaque Switchable Strains of C. albicans

Are Genetically Diverse
Additional examples of white-opaque switching in natural a/a strains of C. albicans are shown in Figure S1 and Table S1. The white colonies of different a/a strains showed variability in their abilities to filament on Lee's GlcNAc medium in 5% CO 2 at 25uC, indicating that the white-opaque switchable strains are genetically diverse and probably not derived from a single strain with a specific genetic background. To characterize the genetic background of these natural strains, we sequenced their CAI

Author Summary
Phenotypic transitions enable fungal pathogens to better adapt to their ever-changing environments. Approximately 10% of natural Candida albicans strains, which are homozygous at the mating type locus (MTL, a/a and a/ a), can switch between two distinguishable morphological forms: white and opaque. The two cell types differ in a number of biological aspects including virulence, susceptibility to host immune attacks, and mating competency. Here, we demonstrate that white-opaque switching competency is not restricted to the MTL homozygous strains, but is a general characteristic of all MTL strain types of C. albicans (a/a, a/a, and a/a). Two host environmental cues, N-acetylglucosamine and CO 2 , promote white-toopaque switching and stabilize the opaque phenotype. Thus, although switching is normally blocked in a/a cells, this block can be overcome through specific environmental changes. We further show that three transcriptional regulators (Rfg1, Brg1, and Efg1) help to regulate whiteopaque switching in MTL heterozygotes of C. albicans. This study generalizes white-opaque switching to strains with all mating-type configurations and emphasizes its importance in host adaptation, pathogenesis, and parasexual reproduction.
microsatellite loci by using a reported assay [19]. As shown in Table S1 (Column D), these strains exhibited several distinct patterns of the CAI genotype, demonstrating their genetic diversity.
The strains listed in Table S1 were all isolated in China. To exclude the possibility of geographical specificity, we tested the white-to-opaque switching ability in 29 clinical strains of C. albicans isolated from different countries. These strains, which were demonstrated incapable of switching on glucose-containing media, were all originally heterozygous at the MTL locus (a/a) and belonged to five different genetic clades [13]. We found that 15 of them (52%) underwent the white-to-opaque transition on Lee's GlcNAc medium in 5% CO 2 at 25uC (Table S2). Two opaque colonies of each switchable strain were examined for the MTL configurations. Twelve of the 15 strains were a/a heterozygotes, two (P75010, P22095) a/a, and one a/a (P78042, perhaps due to spontaneous loss of the MTLa locus, Pujol and Soll, unpublished data). These results further indicate that the white-opaque switchable a/a strains of C. albicans are genetically and geographically diverse.
Opaque Cells of C. albicans MTLa/a Strains Are Mating-Incompetent White-opaque switching and mating are two coupled biological processes that are both controlled by the MTLa1/a2 complex in C. albicans [10]. One possibility that could explain how a/a isolates could undergo white-opaque switching is that the a1/a2 complex might not function properly; thus, cells could behave as though they were a or a cells. Although our DNA sequencing analysis showed that the MTL locus of the switchable a/a isolates were normal and with no obvious defects, the expression of MTLa1 or MTLa2 could, in principle, be defective. To exclude this possibility, we performed a mating experiment with opaque cells from three independent a/a strains. As shown in Figure 2A, these cells showed no mating response, whereas a/a and a/a opaque cell controls mated normally (Figure 2Ba, b, c, e, f, and g). Quantitative mating assay demonstrated that the mating efficiencies of the MTLa/a x MTLa or a cells crosses were undetectable (,1610 27 ). The mating efficiency of the MTL a/D x a/a crosscontrol was (2.360.8)610 22 , at least 1610 5 times higher than that of the MTLa/a crosses ( Figure 2C). These results demonstrate that a/a opaque cells cannot mate with either a/a or a/a opaque cells, suggesting that the white-opaque switchable a/a strains are mating-incompetent. However, once the opaque cells of these a/a strains were converted to a/D or D/a strains by deletion of one allele of the MTL locus, they acquired mating competence and mated as efficiently as the WT a/a or a/a controls (Figure 2Ah). We conclude from these experiments that the a1/a2 complex is functional in the regulation of mating, and that the white-opaque  Table S6. The previously characterized strains (SC5314, WUM5A, and GH1013) served as MTLa/a, a/a, and a/a controls, respectively. Three independent opaque colonies of SZ306 were tested. doi:10.1371/journal.pbio.1001525.g001 switching in these strains is not due to the inactivation of a1 or a2 proteins.
Induction of White-to-Opaque Switching by GlcNAc and CO 2 in C. albicans MTLa/a Strains As described in the introduction, GlcNAc and CO 2 are two potent inducers of white-to-opaque switching and are believed to be characteristic of host niches such as the gastrointestinal (GI) tract [17]. As shown in Figure 3, the frequencies of white-toopaque switching in the a/a strain SZ306 was extremely low on Lee's glucose (,0.6%) or GlcNAc (0.5%) medium in ambient CO 2 . CO 2 alone also had little effect on the induction of opaque phenotype on Lee's glucose medium in this a/a strain (switching frequency,0.4%). However, the switching frequency of white-toopaque in SZ306 was increased to 7.563.1% when cultured on Lee's GlcNAc medium in 5% CO 2 , indicating that GlcNAc and CO 2 had a synergistic effect on the induction of the opaque cell phenotype. To compare the switching features of a/a strains and MTL homozygous ''a'' or ''a'' strains, we converted SZ306 (a/a) to an MTLa/D strain, namely SZ306a, and RVVC10 (a/a) to an MTLD/a strain, namely RVVC10a, by deletion of one allele of the MTL locus. As shown in Figure 3, although the frequency of white-to-opaque switching in SZ306a was only 0.4% on Lee's glucose medium in ambient CO 2 , GlcNAc, or 5% CO 2 alone increased the switching frequencies to 3.062.7% and 34.460.9%, respectively. Notably, SZ306a underwent a mass conversion (switching frequency = 100%) on Lee's GlcNAc medium in 5% CO 2 , consistent with our previous study of the synergistic effect of GlcNAc and CO 2 on white-to-opaque switching in MTL homozygotes [17]. As in SZ306 and SZ306a, GlcNAc and CO 2 had a similar effect on the induction of the opaque cell phenotype in RVVC10 and its derivative, RVVC10a (unpublished data). These results indicate that a/a strains are less sensitive than their ''a/D'' or ''D/a'' derivatives to GlcNAc and CO 2 , but that whiteto-opaque switching is stimulated by GlcNAc and CO 2 in all three MTL configurations.
GlcNAc and CO 2 can also stabilize the opaque phenotype in MTL homozygotes of C. albicans. We next tested whether this was also the case in heterozygous a/a strains. As shown in Figure S2 and Table S3, the opaque phenotype of a/a strains was extremely unstable in Lee's glucose medium when cultured in ambient CO 2 at 25uC (switching frequency to white was 100%). The switching frequencies were 38.667.7, 34.665.3, and 18.767.1 on Lee's GlcNAc medium in ambient CO 2 , on Lee's glucose in 5% CO 2 , and on Lee's GlcNAc in 5% CO 2 , respectively ( Figure S2 and Table S3). These results suggest that GlcNAc and CO 2 stabilize the opaque phenotype of a/a strains. For the ''a/D'' and ''D/a'' strains, SZ306a and RVVC10a, the opaque phenotype was very stable on both Lee's glucose and GlcNAc, irrespective of whether the cells were cultured in air or 5% CO 2 . Under the four conditions tested (Lee's glucose in air, Lee's GlcNAc in air, Lee's glucose in 5% CO 2 , and Lee's GlcNAc in 5% CO 2 ), the opaqueto-white switching frequencies of SZ306a and RVVC10a were all less than 1% ( Figure S2 and Table S3). We sequenced the WOR1 promoter of several switchable a/a strains and found what is believed to be the major a1/a2 cis-regulatory sequence site was intact. These results indicate that although the a1/a2 complex does not provide an absolute block to white-to-opaque switching in these a/a strains, it reduces switching to favor white cells, likely by turning down (but not off) the expression of WOR1.

C. albicans MTLa/a Strains Undergo White-to-Opaque Switching at the Host Physiological Temperature
Since the physiological temperature of human hosts is 37uC, we therefore examined whether MTLa/a strains can undergo whiteto-opaque switching under this temperature. White cells of CY110 and RVVC10 (two MTLa/a strains) were plated onto Lee's glucose and Lee's GlcNAc medium plates and cultured at 37uC for 3 to 4 days. The cells of both strains were locked in white phase on Lee's glucose medium in air or in 5% CO 2 , whereas they formed opaque, opaque-sectored, or mixed colonies on Lee's GlcNAc medium ( Figure 4A and 4B). The switching frequencies of CY110 and RVVC10 on Lee's GlcNAc medium in 5% CO 2 were as high as 60.6610.3% and 100% (mass conversion), respectively. The cellular morphologies demonstrated that opaque or mixed colonies contained typical opaque cells ( Figure 4A and 4B). WOR1 is an opaque phase-specific gene, while WH11 and EFG1 are white phase-specific genes [5]. To further verify their cell identities, we constructed WOR1, WH11, and EFG1 promoters-controlled GFP reporter strains in the MTLa/a strain CY110. As shown in Figure 4C, GFP fluorescence was only observed in opaque cells of the WOR1/WOR1::WOR1p-GFP strain, but not in opaque cells of the EFG1/EFG1::EFG1p-GFP and WH11/WH11::WH11p-GFP strains. As expected, GFP fluorescence was observed in white cells of the EFG1/EFG1::EFG1p-GFP and WH11/WH11::WH11p-GFP strains. These results indicated that the opaque cells formed at 37uC were genetically opaque.
We next tested the stability of opaque cells of MTLa/a strains under host physiological temperature. Opaque cells of three a/a strains (SZ306, RVVC10, and CY110) were plated onto Lee's glucose and Lee's GlcNAc medium and incubated in air at 37uC for 3 days. On Lee's glucose medium, opaque cells underwent a mass conversion to the white cell phase (switching frequency = 100%). On Lee's GlcNAc medium, most colonies (.95%) remained in the opaque phase. The cellular morphology of representative colonies is shown in Figure S3, indicating that GlcNAc can stabilize the opaque phenotype of a/a strains at 37uC.

White and Opaque Cells of C. albicans MTLa/a Strains Differ in Fungal Burden in Systemic and Cutaneous Infections
In MTL homozygous strains of C. albicans, white and opaque cells show differences in their behaviors in systemic and skin infection models [7,8]. White cells are more virulent in systemic mouse model than opaque cells, while opaque cells are better at cutaneous infections. We then tested whether white and opaque cells of C. albicans MTLa/a strains also differed in virulence in different infection models. As shown in Figure 5A, in a systemic mouse infection system, burdens of opaque cells in the liver were notably less than those of white cells of RVVC10 and SZ306 (Student's t test p value,0.05), suggesting opaque cells of C. albicans a/a strains proliferated or colonized less well than their white cell counterparts. This was also the case for colonization of the kidney for RVVC10, although the difference of fungal cell burden between white and opaque cells of SZ306 was not significant. This result is consistent with previous studies; the fungal burdens of opaque cells of the MTL homozygous reference strain WO-1 in both the kidney and liver were less than those of white cells of WO-1 [7], and the fungal burdens of opaque cells of the MTL homozygous reference strain WO-1 in both the kidney and liver were less than those of white cells of WO-1. To test whether opaque cells of a/a strains were better at cutaneous infections, newborn mice were used and the fungal colonization of the skin was assessed by scanning electron microscopy as described  previously [8]. Compared to white cells, opaque cells of both SZ306 and the reference strain WO-1 showed increased colonization in a cutaneous mouse model ( Figure 5B). The number of opaque cells that colonized the skin was significantly higher than that of white cells (Student's t test p value,0.002) ( Figure 5B). These results indicate that the different behaviors documented for white and opaque cells in the systemic and cutaneous mouse models also apply to white and opaque cells of the a/a strains described here.

Global Transcriptional Profiles of White and Opaque Cells of the MTLa/a Strain CY110
To characterize the genome-wide transcriptional profiles of white and opaque cells of the MTLa/a strains, we performed RNA-Seq analysis of CY110, a clinical isolate of MTLa/a genotype. As shown in Table S4 (Sheet 1), the expression levels of 1,631 genes demonstrated a greater than twofold change in white and opaque cells. As expected, previously characterized white cell-enriched genes, such as WH11 and EFG1, were upregulated in white cells, while opaque cell-enriched genes, such as WOR1 and OP4, were strongly up-regulated in opaque cells. A total of 838 genes demonstrated a greater than 3-fold change in our RNA-Seq analysis. Of them, 459 were previously reported as white (205) or opaque (254) cell-enriched genes [20,21], and 379 were only found in our analysis, which could be MTL genotypedependent phase-specific genes. As shown in Table 1, of the highly differentially expressed genes, the ratio of potential MTL genotype-dependent genes remarkably decreased, suggesting that highly differentially expressed genes are less MTL genotypedependent. Interestingly, many cell wall protein and biofilminduced genes were among the MTLa/a-specific genes (Table S4, sheet 3). Of note, the MTLa/a-specific genes may contain a proportion of genes specific to the strain background, especially for those with lower fold-change of expression levels.
Similar to the MTL homozygous strains, opaque and white cells of the MTLa/a strain CY110 specialized in their metabolic  (Table S4, sheet 2). Fermentative metabolism-associated genes were highly expressed in white cells of CY110 (e.g., glucose transporter genes HGT6, HGT7, and HGT8), while oxidative metabolism-associated genes were highly expressed in opaque cells (e.g., isocitrate dehydrogenase IDP2, malate synthase MLS1, acyl-CoA oxidase POX1, and 3-hydroxyacyl-CoA epimerase genes FOX2 and FOX3). Moreover, the differentially expressed genes in white and opaque cells of CY110, which were also found in their MTL homozygous counterparts, included genes associated with the metabolism of other nutrients (such as nitrogen and phosphate), cell wall components, stress response, and transcription factors (Table S4, sheet 2).
In MTL homozygous strains, only the opaque cell type is mating-competent [10]. Consistently, it has been demonstrated that mating-related genes MFa (a-pheromone) and STE2 (apheromone receptor) are highly enriched in opaque cells of WO-1, an MTLa/a isolate of C. albicans [20]. However, the expression levels of either MFa or MFa were not detectable in white and opaque cells of CY110. The expression levels of their receptors STE2 and STE3 in opaque cells of CY110 were very low and similar to that of white cells (Table S4, sheet 4). Additionally, the transcriptional expression of the four genes at the MTL loci (a1, a2, a1 and a2) was all detected. These results served to validate the a/a cell identity of CY110 and its mating incompetence.
Wor1, Rfg1, Brg1, and Efg1 Are Involved in the Regulation of White-Opaque Switching in C. albicans MTLa/a Strains WOR1 is the master regulator of white-opaque switching in MTL homozygotes of C. albicans and is extensively up-regulated in opaque cells of both MTL homozygotes and heterozygotes ( Figure 1D and Table S4) [14][15][16]. Deletion of WOR1 in an MTLa/a strain SZ306u, a derivative of SZ306, blocked white to opaque switching on all media tested including Lee's GlcNAc medium in 5% CO 2 (switching frequency,0.03%) ( Figure S4). Under this culture condition, the white-to-opaque switching frequency of the wild-type SZ306u (WOR1/WOR1) and the single copy mutant (WOR1/wor1) were 4.361.0% and 0.560.3%, respectively, suggesting that the copy number of WOR1 could affect its own expression and the white-to-opaque switching frequency. Therefore, Wor1 is also essential for the induction of opaque phenotype in MTLa/a strains.
The a1/a2 complex inhibits the expression of WOR1 and thus controls white-to-opaque switching in SC5314 background strains [14][15][16]. The promoter region of WOR1 is extremely long (.10 kb), indicating the regulation of WOR1 expression could be very complex. Two facts imply that the a1/a2 complex does not work alone to control the expression of WOR1. First, even in the MTL homozygous strains (that therefore lack the a1/a2 complex), the default cell type is the white form, at least in typical laboratory media, indicating some other regulators must repress the expression of WOR1. Second, there appears to be only a single binding site of the a1/a2 complex on the long promoter region of WOR1. To find the regulators coordinately working with the a1/ a2 complex in repressing WOR1 expression, we screened a library of ,160 transcription factor null mutants (of the MTLa/a genotype) of SC5314 background [22]. The library was suitable for the screening because SC5314 and its derivatives (a/a) used for making the mutants are nonswitchable on the Lee's GlcNAc medium. We predicted that inactivating the transcription factors involved in inhibiting WOR1 expression would lead to the opaque phenotype. And we found three a/a mutants (rfg1/rfg1, brg1/brg1, and efg1/efg1) could undergo white-to-opaque switching on the Lee's GlcNAc medium in 5% CO 2 at 25uC, suggesting the transcription factors Rfg1, Brg1, and Efg1 are involved in the regulation of white-opaque transition in MTLa/a strains of C. albicans. PCR analysis was conducted to confirm that the MTL genotype of the rfg1/rfg1, brg1/brg1, and efg1/efg1mutants were a/a ( Figure 6A). Rfg1 is a member of the HMG domain family of sequence-specific DNA-binding proteins that has been shown to be a regulator of filamentous growth and virulence in C. albicans [23,24]. We observed that the rfg1/rfg1 mutant (a/a) could also form opaque colonies or sectors in Lee's glucose and YPD media when cultured at 25uC for an extended time period (unpublished data). Consistent with the phenotype of rfg1/rfg1 mutant in whiteopaque switching, Northern blots showed that the expression of RFG1 was enriched in white cells, relative to opaque cells in C. albicans MTLa/a strains ( Figure 1D). Brg1, a GATA-type zinc finger transcription factor, has been characterized as a regulator of filamentous growth, biofilm formation, and virulence [22,25]. Efg1 is a bHLH domain containing transcription factor required for maintaining the white cell phenotype of C. albicans MTL homozygotes [26]. The efg1/efg1 null mutants of MTLa/a strains could not switch to opaque in glucose containing medium [27]. However, both brg1/brg1 and efg1/efg1 mutants of MTLa/a strains could indeed undergo white-to-opaque switching on Lee's GlcNAc medium ( Figure 6B). Our findings indicate that numerous environmental signals converge on Wor1 and regulate the ability of C. albicans cells to undergo white-opaque switching.

Discussion
For decades, white-opaque switching was observed in only a minority (,10%) of natural C. albicans isolates: those that were homozygous at the mating-type locus [2,4]. How does this species maintain such a complex switching system if the majority of strains (which are a/a) do not do it? One possibility is that white-opaque Table 1. RNA-Seq analysis of the differentially expressed ORFs in white and opaque cells of CY110. Wh(up), up-regulated in white cells; Op(up), up-regulated in opaque cells. Total, total number of differential expressed ORFs in white or opaque cells of CY110; potential a/a strain specific ORFs, number of differential expressed ORFs only found in white or opaque cells of CY110; MTL-independent ORFs, number of differential expressed ORFs found in white or opaque cells both of CY110 and of MTL homozygous strains reported previously by Lan et al. (2002) and Tuch et al. (2010) [20,21]. Percentages are shown in the brackets. doi:10.1371/journal.pbio.1001525.t001 switching in C. albicans has been maintained as a means to attain mating competency [10]. However, C. albicans populations in the host are primarily clonal, indicating that, if parasexual mating actually occurs in nature, its role may not be to generate genetic diversity [28]. In this study, we have generated evidence for a different explanation for the widespread maintenance of whiteopaque switching in C. albicans clinical isolates. We show that many naturally occurring MTLa/a strains of C. albicans can indeed undergo white-opaque switching, with the opaque phenotype of MTLa/a strains of C. albicans being largely similar to that of MTL homozygotes, except that they do not mate. Although such switching of a/a strains does not readily occur under typical laboratory conditions, we show that the combination of GlcNAc and CO 2 are strong inducers of switching in a/a strains. Importantly, some a/a strains can undergo white-to-opaque switching at 37uC, the physiological temperature of the human host. These conditions are believed to be present in host niches such as the gut, where glucose is limiting and the carbon sources are largely from GI mucus and cell debris of microbes [29]. Together with our recent discovery of white-opaque switching in MTLa/a heterozygotes of C. tropicalis [12], our findings thus generalize white-opaque switching to strains with all mating-type configurations and suggest that the ability to switch is conserved in C. albicans and C. tropicalis.
We have shown that opaque cells of MTLa/a isolates of C. albicans share many features with opaque cells of MTL homozygotes. However, there are some important differences. For example, opaque cells of MTLa/a isolates undergo mass conversion to white cells on glucose containing media, while opaque cells of MTL homozygotes are very stable. Thus, a/a opaque cells are not as stable as opaque a or a cells and require the continuous presence of the environmental signals. Secondly, opaque cells of MTLa/a isolates are mating-incompetent.
The MTLa1/a2 complex inhibits the expression of the master regulator WOR1, thereby blocking white-opaque switching in the laboratory strain SC5314 [14][15][16], which is an a/a strain. However, in the a/a strains described here, white-opaque switching is permitted; the a1/a2 complex ''turns it down'' but does not completely block white-opaque switching. The long upstream region of WOR1 implies that multiple environmental signals and transcriptional regulators feed into it, and thus it is easy to imagine that strains could vary in the precise response of Wor1 to environmental signals. We have demonstrated that more than one third of natural isolates of MTLa/a C. albicans strains tested in this study formed opaque or opaque-sectored colonies on Lee's GlcNAc plates in 5% CO 2 . We propose that the white-opaque phenotypic transition itself is a general feature of C. albicans, but the quantitative response of the switch to features of the environment and to the mating type configuration differs from strain to strain.
The regulation of white-opaque switching in MTL homozygotes involves an interlocking transcriptional circuit, in which Wor1 occupies the central position [27]. We propose that Wor1 also acts as a master regulator in the process of white-opaque switching in MTL heterozygotes of C. albicans. Ectopic expression of WOR1 in the ''non-switchable'' MTL a/a strain CAI4, a derivative of SC5314, induces white-to-opaque switching on glucose-containing laboratory media, suggesting that Wor1, if ectopically expressed, can override the repressing effect of the a1/a2 complex on the white-to-opaque transition [14,16]. By screening a deletion mutant library of C. albicans, we have identified three transcription factors, Rfg1, Brg1, and Efg1, involved in the regulation of white-opaque switching in MTLa/a strains. These three transcription factors inhibit opaque cell formation in MTLa/a strains since their null mutants are capable of switching between white and opaque cell types. Consistent with the phenotype of their null mutants, the transcriptional expression of RFG1 and EFG1 was enriched in white cells of MTLa/a strains ( Figure 1). Nobile et al. have recently demonstrated that Brg1 and Efg1 bind to nearly the entire 10 kb intergenic region between WOR1 and its adjacent divergent gene ORF19.4883 [25]. However, the location of the peaks of Brg1 and Efg1 binding were distant from the putative a1/a2 cisregulatory sequence. These results not only provide direct evidence of Brg1 and Efg1 binding to the promoter of WOR1, but also indicate that they may work together with the a1/a2 complex to reduce the expression of Wor1 in white cells and prevent switching to opaque cells. The transcriptional repressor Rfg1 may work in a similar manner as Efg1 and Brg1. We propose that inactivation of any of these three regulators would lead to increased expression of WOR1, which then initiates a self-positive feedback loop to induce the opaque cell phenotype ( Figure 7A). Together with Wor1, additional transcriptional regulators, such as the positive regulators Wor2 and Czf1, coordinately regulate the expression of WOR1 by binding directly to the WOR1 upstream intergenic region (A.D.H., C.J.N., and A.D.J. unpublished data), and maintain the cells in the opaque phase ( Figure 7B). Consistent with the model in Figure 7B, deletion of WOR2 or CZF1 results in increased opaque-to-white switching frequencies [27]. In summary, we have shown that, in many naturally occurring C. albicans strains, the a1/a2 repressor is not an absolute block to white-opaque switching as it is in the standard laboratory strain SC5314. Rather, the a1/a2 activity reduces switching frequency (and renders the opaque form less stable) in these newly described strains, but this reduction can be partially overcome by the addition of GlcNAc and CO 2 to the growth medium. We propose that the a1/a2 repressor and other regulators (including Efg1, Brg1, and Rfg1) as well as these environmental signals all impinge on the long regulatory region of Wor1, the master regulator of white-opaque switching. This information is somehow integrated by the Wor1 regulatory region, and the level of Wor1 transcription is set accordingly. Because Wor1 appears to be the major determinant of the white-opaque switch frequency [30], the model can account for nearly all the observations in this article.
The most important implication of the work is that all strains of C. albicans (not just strains that are homozygous at the mating type locus, as previously believed) can undergo white-opaque switching if the appropriate signals are present in the growth medium. Thus, we propose that multiple environmental inputs combined with internal transcriptional regulators can activate white-opaque switching in virtually all C. albicans strains. White-opaque switching, in essence, produces two radically different types of cells from the same genome, thereby explaining the ability of C. albicans to occupy different niches in the host. We believe that the discovery of white-opaque switching in naturally occurring a/a strains accounts for the widespread conservation of the whiteopaque switching machinery.

Culture Conditions, Strains, and Plasmids
The strains used in this study are listed in Table S5. YPD (20 g/ L glucose, 20 g/L peptone. 10 g/L yeast extract) was used for routine growth. Lee's + glucose and Lee's + GlcNAc media were used for mating and white-opaque switching assays [17].
The plasmid pSFS2A-URA3 was generated by inserting two DNA fragments containing sequences homologous to the 59-and 3-terminals of C. albicans URA3 gene into the ApaI/XhoI and SacII/ SacI sites of pSFS2A [31]. The auxotrophic strain SZ306u for uridine was constructed by disruption of one copy of URA3 with the linearized plasmid pSFS2A-URA3 and then grown on 5fluoroorotic acid (5-FOA) containing medium. The white-opaque switching-competence of SZ306u was then confirmed. SZ306 and RVVC10 were converted to SZ306a and RVVC10a by deletion of one MTL allele with the plasmid T2A-MTL (Srikantha and Soll, unpublished). The first copy of WOR1 was deleted with the PCR product of pGEM-URA3 with the primers of WOR1-5DR and WOR1-3DR in SZ306u [32]. The second copy of WOR1 was then deleted with the linearized plasmid T2A-WOR1 [33]. A couple of primer sets were used to confirm the correct disruption of WOR1 in SZ306u.
To construct the WOR1/WOR1::WOR1p-GFP, EFG1/EF-G1::EFG1p-GFP, and WH11/WH11::WH11p-GFP strains, CY110 was transformed with PCR products of the GFP-caSAT1 fragment (amplified from the template plasmid pNIM1 with GFP reporter primers, Table S6) [34]. The forward primers contained 60 bp of hanging homology to the promoter region of WOR1, WH11, or EFG1, while the reverse primers contained 60 bp of hanging homology to the 39-UTR of WOR1, WH11, or EFG1. Correct integration of the transformations was verified by genomic DNA PCR with checking primers. All primers used in this study are listed in Table S6.

Microsatellite CAI Genotyping
The CAI genotypes of C. albicans isolates were determined as described by Sampaio et al. (2003) [35]. Briefly, the microsatellite locus CAI was amplified by PCR using a pair of primers (forward,  59-ATG CCA TTG AGT GGA ATT GG -39; reverse, 59-AGT  GGC TTG TGT TGG GTT TT -39). The forward primer was 59 fluorescently labeled with 6-carboxyfluorescein. The sizes of the amplicons were determined by GeneScan analysis using a DNA sequencer, and the number of trinucleotide repeat units in each fragment was calculated. Because of the diploid nature of C. albicans, the CAI genotype of a strain is determined by the repeat number in both alleles of the locus. For example, a strain with a genotype CAI 17-21 means that one allele of the locus contains 17 trinucleotide repeats and the other 21.

White-Opaque Switching and Mating Assays
White-opaque switching and mating assays were performed as previously described [36]. The cells were incubated in air or in 5% CO 2 for 4 to 10 days as indicated in the main text. We examined 350 to 500 colonies for each strain. More were tested for nonswitchable strains or on nonconducive media. To verify the colony phenotype, several randomly selected colonies were examined for the cellular morphology. The dye phloxine B, which exclusively stains opaque colonies red, was added to the media. Scanning electron microscopy (SEM) assay was described as we described previously [22]. To observe the mating response, 10 6 cells of each of the two mating strains indicated in the text were mixed and spotted onto Lee's GlcNAc agar and incubated at 25uC for 4 days. At least 1610 7 cells of each mating patch were examined with a light microscopy. Quantitative mating assay was performed as previously described with slight modifications [10]. Briefly, the mating experiments were performed on Lee's GlcNAc medium at 25uC. The experimental opaque cell samples were collected from Lee's GlcNAc medium plates. To test the mating ability of the MTLa/a strain (SZ306u), 1610 6 of MTLa/a (or MTLa/a) cells and 1610 6 of MTLa/a cells were mixed and cultured on Lee's GlcNAc medium plates for 48 hours. The mating mixtures were resuspended, diluted, and plated onto three types of selectable plates (without uridine, or arginine, or both) for prototrophic growth. Mating efficiencies were calculated as previously described [14].
The library of transcription factor mutants contains the TF mutants generated by the Johnson lab [37] and strains collected from Candida community [36]. Cells of each mutant were plated onto Lee's GlcNAc plates and incubated at 25uC for 7 to 10 days. Opaque colonies were replated and tested for the MTL genotype with PCR.

RNA Extraction, Northern Blot Analysis, and RNA-Seq
White and opaque cells were cultured on Lee's glucose and Lee's GlcNAc plates at 25uC for 4 days, and then inoculated in Lee's glucose and Lee's GlcNAc liquid media, respectively. Cells were collected from the cultures in exponential phase for RNA extraction. Purified PCR products of WH11, OP4, EFG1, WOR1, and RFG1 genes were used to make probes for Northern blot hybridization. Primers used for the PCR reactions are listed in Table S6. RNA-Seq analysis was performed by the company BGI-Shenzhen.

Animal Infections
Systemic infection of mice was performed elsewhere [22]. Male ICR mice (18-22 g) were used in this study. Each male mouse was intravenously injected with 200 ml 16 PBS containing 2610 6 cells via the tail vein. Three to four mice per strain were used for the injections. Mice were sacrificed on the 3 rd day postinfection. The fungal burdens of kidneys and livers were tested. The systemic infection experiments were performed for four indepen-dent times. Newborn ICR mice (2 to 4 days) were used for cutaneous infection. The experiments were performed according the protocol reported by Kvaal et al. [8]. The skin colonization by C. albicans cells was assessed by scanning electron microscopy. The skin infection experiments were performed for three independent trials. All animal experiments were performed according to the guidelines approved by the Animal Care and Use Committee of the Institute of Microbiology, Chinese Academy of Sciences. The present study was approved by the Committee.

Accession Number
The RNA-Seq data have been deposited into the NCBI Gene Expression Omnibus (GEO) portal under the accession number GSE43938. Figure S1 White-opaque switching in six natural MTLa/a strains of C. albicans. Cells were first patched on YPD plates and incubated at 37uC for 2 days. Then, the cells were replated onto Lee's GlcNAc plates and incubated at 25uC in 5% CO 2 for 6 days. Partial opaque colonies were indicated with white arrows. (JPG) Figure S2 Opaque-to-white switching in SZ306 (a/a) and its derivative, SZ306a (a/D). Opaque cells from Lee's GlcNAc plates were plated and incubated under four conditions indicated at 25uC. Lee's glucose or GlcNAc medium was used for cell growth. Switching frequencies are shown below the images. (JPG) Figure S3 Opaque cells of MTLa/a strains of C. albicans are stable in Lee's GlcNAc medium at 37uC. Opaque cells of three natural MTLa/a strains were plated onto Lee's glucose or GlcNAc plates and incubated at 37uC for 3 days. The cellular morphology of a representative colony is shown. (JPG) Figure S4 Deletion of WOR1 blocks GlcNAc and CO 2 induced white-to-opaque switching in MTL heterozygotes of C. albicans. White cells were plated onto Lee's GlcNAc plates and incubated in 5% CO 2 for 5 days at 25uC. White arrows indicated opaque colonies. Switching frequencies (Swit. freq.) are shown below the images. (JPG)