The strains used in this study are listed in Table S1. YPD medium (2% glucose, 2% peptone, 1% yeast extract) and modified Lee's glucose medium (Huang et al., 2010) were used for routine culture of C. albicans. The red dye phloxine B (5 μg/mL) was added to the solid medium for the filamentation and white-opaque switching assays. Media used for spot serial dilution growth assays: YPD, Lee's (Lee's medium without sugar) (Lee et al., 1975), Lee's media with different carbon sources (replacement of glucose with 1.25% fructose or 3% ethanol plus 2% glycerol), YNB media [yeast nitrogen base with 0.5% ammonium sulfate and 3 amino acids (0.13 g/L leucine, 0.03 g/L histidine, and 0.04 g/L arginine) and different carbon sources (2% glucose, 2% fructose, 2% mannitol, or 4% glycerol)].

BCY1 were deleted in two WT strains (SN152 and SN152a) using the same strategy as described below. The two alleles of BCY1 were deleted using the fusion PCR strategy (Noble and Johnson, 2005). The first allele of BCY1 was replaced with the fusion PCR products of the CdHIS1 marker amplified from plasmid pSN52. The second allele of BCY1 was deleted with the fusion PCR products of the CmLEU2 marker amplified from pSN40. The primers used for the PCR reactions are listed in Table S2. To construct the BCY1-reconstituted strain, the fusion PCR product of three fragments (the CdARG4 marker amplified from plasmid pSN69, and fragments of BCY1 3′-UTR and BCY1 ORF plus 5′-UTR) was used for transformation of the bcy1/bcy1 mutant. The two BCY1-related fragments were amplified from genomic DNA of C. albicans (SC5314) with primer pairs (BCY1-5F-COM plus BCY1-5R-COM and BCY1-3R-COM plus BCY1-3R-COM, respectively).

Plasmid pACT1 was used to construct the TPK1 and TPK2-overexpressing plasmids (Huang et al., 2010). The PCR products of TPK1 were digested with EcoRV and HindIII and inserted into the EcoRV/HindIII site of pACT1 to generate plasmid pACT-TPK1. The PCR products of TPK2 were digested with StuI and HindIII and inserted into the EcoRI/HindIII site of pACT1, to generate plasmid pACT-TPK2. The AscI-linearized overexpressing plasmids were used for transformation of the WT and bcy1/bcy1 mutant.

White-opaque switching assays were performed as described previously (Xie et al., 2013). Lee's glucose and Lee's GlcNAc media were used for the quantitative switching assays. The cells were cultured on the plates at 25°C for 5 days. The cell identity was assessed by cellular morphology and verified by cell type-specific genes (data not shown).

Lee's glucose, Lee's GlcNAc, and YPD media were used for the filamentation assays. The cells were cultured at 25° and 37°C as indicated in the figure legends. For quantitative filamentous-smooth colony type switching assays (Figure 4), cells from a filamentous or smooth colony grown on Lee's glucose medium for 3 days were resuspended and cultured in liquid Lee's glucose for 24–96 h at 25°C. A small aliquot of cells was collected at the time point indicated and replated on YPD plates. After 5 days of growth at 30°C, colonies of the smooth and filamentous types were counted. The switching frequency = (number of colonies with alternative phenotype/total colony number) × 100%.

The cells were grown in liquid Lee's glucose medium for 48 h at 25°C and collected for propidium iodide (PI) and 4′-6-diamidino-2-phenylindole (DAPI) staining assays as described previously (Du et al., 2015). The cells were washed with 1 × phosphate-buffered saline (PBS) and resuspended in 1 × PBS. PI was added to the cells at a concentration of 2 μg/mL. The cells were stained for 15 min at room temperature in the dark with slight shaking and used for microscopy assays. For the DAPI staining assays, cells collected from liquid Lee's glucose medium were first fixed in 70% ethanol for 20 min and then stained with 1 μg/mL of DAPI. The cells were then washed with 1 × PBS and used for microscopy assays.

Cells were grown Lee's GlcNAc plates at 25°C for 3 days and collected for total RNA extraction. The qRT-PCR assay was performed as described in our previous report (Tao et al., 2014a). Total RNA was used to synthesize cDNA with RevertAid H Minus reverse transcriptase (Thermo Scientific). Quantification of transcripts was performed in Bio-Rad CFX96 real-time PCR detection system using SYBR green. The relative expression level of each gene was normalized to that of C. albicans ACT1.

Although the PKA regulatory subunit plays critical roles in a variety of biological processes in fungal species, the gene encoding this subunit is not essential for cell growth in many fungi including the model organisms, Saccharomyces cerevisiae (Cannon and Tatchell, 1987; Toda et al., 1987), Schizosaccharomyces pombe (DeVoti et al., 1991), and Neurospora crassa (Bruno et al., 1996), and the human fungal pathogens, Aspergillus fumigates (Zhao et al., 2006) and Cryptococcus neoformans (D'Souza et al., 2001). However, it has been thought that BCY1, the sole gene encoding the PKA regulatory subunit in C. albicans, is an essential gene (Cassola et al., 2004). Considering the conserved feature of the cAMP signaling pathway, we suspected that BCY1 might not be an essential gene in C. albicans, and thus the failure to obtain its null mutant in a previous report (Cassola et al., 2004) could be due to technical reasons. Using a fusion PCR deletion and prototrophic selection strategy (Noble and Johnson, 2005), we successfully deleted the two alleles of BCY1 in a WT strain of C. albicans (SN152, Figure 1). Correct integration of the CdHIS1 and CmLEU2 markers into the BCY1 locus was confirmed using PCR with two sets of flanking checking primers (Figure 1B, lanes 1–4). Moreover, one set of ORF primers was used to verify the absence of the BCY1 ORF region in the genome (Figure 1B, lanes 5 and 6). These results indicate that the two alleles of BCY1 were successfully deleted and replaced by the CdHIS1 and CmLEU2 markers, respectively. Therefore, as in other previously described fungi (Cannon and Tatchell, 1987; Toda et al., 1987; DeVoti et al., 1991; Bruno et al., 1996; D'Souza et al., 2001 and Zhao et al., 2006), the conserved PKA regulatory subunit Bcy1 is also not required for cell viability in C. albicans.

To evaluate the function of Bcy1 during filamentous development of C. albicans, we cultured the cells of WT, BCY1/bcy1, bcy1/bcy1, and BCY1-reconstituted strains on three different media (Lee's glucose, Lee's GlcNAc, and YPD) at two temperatures (25 and 37°C). These culture conditions were used because the three media exhibit different levels of filamentation induction in C. albicans. A high temperature (37°C) promotes filamentation, whereas a low temperature (25°C) favors yeast cell growth. Therefore, a combination of these conditions would facilitate the discrimination of the filamentation ability of different strains. At 25°C (Figure 2), deletion of one allele of BCY1 had no obvious effect on filamentous growth. Consistently, the BCY1-reconstituted strain also exhibited a similar phenotype to that of the WT control. However, the bcy1/bcy1 null mutant displayed a serious growth defect and had two distinct colony phenotypes (smooth and filamentous) on all three media. Cells of the smooth colonies were swollen and looked unhealthy, whereas filamentous cells had a much healthier appearance. On Lee's GlcNAc medium, a portion of the bcy1/bcy1 mutant cells exhibited an opaque-like phenotype (Figure 2). At 37°C (Figure 3), two colony types of the bcy1/bcy1 mutant were observed on three media. On Lee's glucose medium, filamentous colonies of the bcy1/bcy1 mutant underwent more robust filamentation than the WT, BCY1/bcy1, and BCY1-reconstituted strains, whereas on Lee's GlcNAc medium, all four strains exhibited strong filamentation at 37°C. These results are consistent with previous reports showing that GlcNAc is a potent yeast-to-filamentous growth inducer (Simonetti et al., 1974). The bcy1/bcy1 mutant showed the most robust filametation on YPD medium, while the BCY1/bcy1 and BCY1-reconstituted strains exhibited an intermediate level of filamentation at 37°C. The WT control maintained the yeast form on YPD medium at 37°C. These results suggest that Bcy1 plays a critical role in the regulation of filamentation and that the dosage of Bcy1 also affects this biological process.

The bcy1/bcy1 mutant has two colony phenotypes: smooth and filamentous (Figures 2, 3, 4A). To test whether the two phenotypes could switch between each other, we cultured the two cell types on YPD medium and calculated the switching frequencies of the original to alternative cell type after 3, 5, and 8 days at 30°C (Figure 4B). Extension of incubation on solid medium promoted the filamentous phenotype. To further characterize the switching feature of the bcy1/bcy1 mutant, the smooth and filamentous colonies were cultured in liquid Lee's glucose medium for 0–96 h at 25°C (Figure 4C). The cells were then replated onto YPD medium and cultured at 30°C for 5 days. As shown in Figure 4C, the filamentous-to-smooth (F-to-S) switching frequencies at different time points were similar (20–40%), whereas the smooth-to-filamentous (S-to-F) switching frequencies increased dramatically with extension of the initial culture time.

Deletion of BCY1 causes constitutive activation of the PKA kinase. We examined the effect of overexpression of TPK1 and TPK2, which encode the two isoforms of the catalytic subunit, in the bcy1/bcy1 null mutant. As shown in Figure 5, overexpression of TPK1 in the bcy1/bcy1 null mutant resulted in hyper-filamentation at 25°C, while overexpression of TPK2 did not promote filamentation but led to the formation of two types of colonies: filamentous and opaque-like. On Lee's GlcNAc medium, one colony type was similar to the opaque phenotype, while the other underwent hyper filamentation. Of note, it was very difficult to obtain TPK2-overexpressing transformants in the bcy1/bcy1 null mutant, suggesting that overexpression of TPK2 may cause rapid cell death.

The cAMP signaling pathway regulates cell death in C. albicans (Phillips et al., 2006). Because the deletion of BCY1 results in constitutive activation of this pathway, we next tested the viability of bcy1/bcy1 mutant cells during incubation in Lee's glucose medium. Cells of the WT, smooth, and filamentous types of the bcy1/bcy1 null mutant were grown in liquid Lee's glucose medium for 48 h at 25°C. The cells were then collected and stained with DAPI and PI. As shown in Figure 6, cells of the WT and filamentous form of the bcy1/bcy1 mutant had intact DAPI-stained nuclei, while most cells of the smooth type of the bcy1/bcy1 mutant had a fragmented nucleus or had no intact nuclei. PI staining verified that most cells of the smooth form of the bcy1/bcy1 mutant underwent cell death. Quantitative assays demonstrated that more than 95% of the smooth cells and approximately 70% of filamentous cells of the bcy1/bcy1 mutant were dead after 48 h of incubation in Lee's glucose medium at 25°C. Of note, more than 99% the WT control cells remained viable under the same culture conditions.

Huang et al. (2010) have demonstrated that activation of the cAMP signaling pathway promotes white-opaque switching in C. albicans (Huang et al., 2010). As shown in Figure 2, deletion of BCY1 in the WT strain (MTLa/α) induced the formation of an opaque-like cell type on Lee's GlcNAc medium. Therefore, we deleted BCY1 in a white-opaque switchable MTLa/Δ strain (SN152a Tao et al., 2014a) to generate the BCY1/bcy1 and bcy1/bcy1 mutants (MTLa/Δ). The colony and cellular morphologies of the WT, BCY1/bcy1, and bcy1/bcy1 mutants (MTLa/Δ) are shown in Figure 7A. On both Lee's GlcNAc and Lee's glucose media, the BCY1/bcy1 mutant exhibited similar colony and cellular phenotypes to those of the WT strain. Both the smooth and filamentous types of the bcy1/bcy1 mutant could undergo white-opaque switching on Lee's glucose medium. However, both types underwent hyperfilamentation on Lee's GlcNAc medium. Replating and cell type-specific gene expression assays indicated that on Lee's GlcNAc medium, the filamentous cells had an opaque identity (Figure S1). Quantitative switching assays demonstrated that deletion of both alleles of BCY1 caused a mass conversion to the opaque phenotype on Lee's GlcNAc medium (Figure 7B). These results suggest that Bcy1 plays a negative role in the regulation of the white-to-opaque transition under this culture condition.

Next, we tested whether the regulatory subunit Bcy1 was involved in the regulation of carbon source utilization in C. albicans. As shown in Figure 8, nine media (including rich YPD medium, four Lee's media, and four YNB media containing different types of carbon sources) were used for this assay. The filamentous form of the bcy1/bcy1 mutant grew well on all media, although its growth rate was slower than that of the WT. The smooth form of the bcy1/bcy1 mutant grew well on YPD medium. However, the cells of this form showed a serious growth defect on both Lee's and YNB media. This defect did not appear to be related to the fermentative or non-fermentative features of the carbon sources.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.