Inducible deletion of putative essential protein kinase genes

We selected eight protein kinases (Table 1) for which we had not obtained homozygous deletion mutants during the construction of our library [2,3] to firmly establish by inducible gene deletion whether they are essential or not for viability in the wild-type reference strain SC5314. In a previous genome-wide in vivo transposon mutagenesis study with a haploid C. albicans strain, seven of these were deemed essential and only one of them (KSP1) was found to be dispensable [9]. Mutants in which KSP1 was inactivated by gene-specific transposon insertions had also been obtained before [6], but no homozygous ksp1Δ mutants were recovered in another targeted approach [14]. Both of the latter two studies found BUD32 to be nonessential, but conflicting results were obtained for other kinases. CTK1 and RIO1 were considered as putative essential genes because no homozygous mutants were obtained by Blankenship et al. [6], but ctk1Δ and rio1Δ mutants were recently generated by Kramara et al. [14]. Conversely, ptk2 and rio2 transposon insertion mutants were obtained by Blankenship et al. [6] while no homozygous ptk2Δ and rio2Δ mutants could be generated by Kramara et al. [14]. For the two predicted protein kinases encoded by orf19.3456 and orf19.5376 no null mutants were obtained in all three studies. Interestingly, analysis of the GRACE collection of repressible mutants found none of the eight kinases to be essential, and only one of them (the BUD32 knock-down) exhibited a detectable growth defect [15] (see Table 1 for a summary).

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Table 1. Protein kinases analyzed in this study and reported putative essentiality¹.

https://doi.org/10.1371/journal.pgen.1012118.t001

For these eight putative essential kinases (PEKs), we generated inducible deletion mutants containing a single FLP-deletable gene copy (M6 mutants) as well as control strains that additionally retained one of the endogenous alleles (M7 mutants), as illustrated in Fig 1. All conditional mutants grew as well as the wild-type strain SC5314 and control strains, demonstrating that each of the single ectopically integrated gene copies was sufficient for normal growth under standard conditions (S1 Fig). After the induced gene deletion, five of the eight null mutants (bud32Δ, ctk1Δ, ksp1Δ, rio1Δ, rio2Δ) remained viable and produced colonies upon subsequent plating and incubation on YPD medium (Fig 2). In the case of KSP1, this became evident already after the excision of the SAT1 flipper cassette from the second endogenous allele, as some of the analyzed descendants had simultaneously excised the ectopically integrated KSP1 copy and become null mutants (see Fig 1). The ksp1Δ mutants grew as well as the wild-type strain SC5314 and control strains retaining the ectopically integrated KSP1 copy (Figs 2, S2 Fig; the plates shown in Fig 2 contain both types of M4 derivatives), demonstrating that our previous failure to obtain homozygous ksp1Δ mutants had technical reasons. The bud32Δ, rio1Δ, and especially the ctk1Δ and rio2Δ mutants generated by forced deletion of the ectopically integrated last gene copy grew poorly on rich medium, with improved growth at 37°C compared to 30°C, explaining why homozygous mutants were not recovered in our previous efforts (Figs 2, S2 Fig).

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Fig 2. Growth of viable pekΔ null mutants.

The conditional M6 mutants and M7 control strains were passaged overnight in YCB-BSA-YE medium to induce FLP-mediated excison of the ectopically integrated PEK copy. For KSP1 the M4 mutants were used. Appropriate dilutions of the cultures were plated on YPD medium and incubated at 30°C and at 37°C. Photographs were taken after 2, 4, and 6 days. The two independently generated series of strains behaved identically and only one of them is shown in each case.

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In contrast, after deletion of the last copy of PTK2, orf19.3456, and orf19.5376, the null mutants were unable to grow under standard conditions (Fig 3). The induced gene deletion occurred with high efficiency, since <0.1% of the cells grown in the inducing medium produced normal colonies after plating on rich medium, in contrast to the control strains in which the majority of cells yielded colonies (Table 2). However, upon prolonged incubation, the ptk2Δ mutants started to form visible colonies of different sizes, especially at 37°C in a CO₂ incubator, suggesting that the mutants survived the gene deletion and over time accumulated suppressor mutations that enabled growth (Fig 3). When restreaked on fresh medium, cells from large colonies grew well also at 30°C (S3A Fig), indicating that incubation in a CO₂ incubator only facilitated the acquisition of suppressor mutations. We confirmed that the ectopically integrated PTK2 copy had been correctly excised by FLP-mediated recombination in four well-growing ptk2Δ mutants, two each derived from conditional mutants M6A and M6B (S3 Fig). Ptk2 has two largely redundant orthologs in the model yeast Saccharomyces cerevisiae, Ptk1 and Ptk2, which are required for the activity of the essential plasma membrane H⁺-ATPase Pma1 by phosphorylating its autoinhibitory C-terminal domain at S911 and T912 [16]. The corresponding residues S888 and T889 are conserved in C. albicans Pma1 and have been found to be phosphorylated in two phosphoproteome studies [17,18]. Deletion of the autoinhibitory domain, and also a G648S substitution in Pma1 that interferes with the self-inhibition, bypass the requirement for Ptk1/2 in S. cerevisiae and render ptk1Δ ptk2Δ double mutants viable [16]. We therefore sequenced the PMA1 alleles of the four ptk2Δ suppressor mutants shown in S3 Fig. Intriguingly, three of the four clones contained mutations in PMA1. Clone A1 was heterozygous for a T1760C mutation resulting in a G587A substitution in Pma1, A2 was homozygous for a C1709T mutation resulting in an A570V substitution, and B2 was heterozygous for a T2647C mutation resulting in a S883P substitution. Clone B1 did not contain a mutation in the PMA1 coding sequence and retained the wild-type alleles. These results indicate that the essential function of Ptk1/2 in Pma1 activation in S. cerevisiae is conserved in C. albicans Ptk2, and suppressor mutations in Pma1, but also other mutations, enable growth of ptk2Δ mutants. Some slow-growing colonies also appeared after prolonged growth of orf19.3456Δ and orf19.5376Δ mutants, but at a much lower frequency (Fig 3, which also shows a rare normally growing colony from a cell that had escaped FLP-mediated orf19.5376 deletion), indicating that the encoded kinases are essential for viability of the wild-type strain SC5314.

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Table 2. Efficiency of induced gene deletion.

https://doi.org/10.1371/journal.pgen.1012118.t002

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Fig 3. PTK2, orf19.3456, and orf19.5376 are essential.

The conditional M6 mutants and M7 control strains were passaged overnight in YCB-BSA-YE medium to induce FLP-mediated excison of the ectopically integrated PEK copy. Appropriate dilutions of the cultures were plated on YPD medium and incubated at 30°C, 37°, and in a CO₂ incubator at 37°C. Photographs were taken after 2 and 6 days. The two independently generated series of strains behaved identically and only one of them is shown in each case.

https://doi.org/10.1371/journal.pgen.1012118.g003

Phenotype of orf19.3456Δ and orf19.5376Δ mutants

We focused our further efforts on the two uncharacterized essential protein kinases encoded by orf19.3456 and orf19.5376. We first monitored the terminal phenotype of the null mutants by inoculating them onto YPD agar after the induced gene deletion and observing the cells by time-lapse video microscopy. In contrast to the wild-type strain SC5314 and the M7 control strains, which grew as budding yeasts under these conditions, both mutants were unable to maintain the normal yeast morphology after 2–3 initial cell doublings that were likely enabled by remaining gene product (S1-S5 Videos, Figs 4, S4). The orf19.3456Δ mutants first formed aberrantly shaped cells and then often grew as thin hyphae; over time the filaments stopped elongating and the cells eventually lysed. The orf19.5376Δ mutants produced pseudohyphae with constrictions between highly elongated cells, ultimately followed by cell lysis.

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Fig 4. Terminal phenotype of orf19.3456Δ and orf19.5376Δ mutants.

The conditional M6 mutants were passaged overnight in YCB-BSA-YE medium to induce FLP-mediated excison of the ectopically integrated gene copy. The cultures were diluted in water, transferred to a 35 mm culture dish, covered with YPD agar, and incubated at 30°C. Images were taken every 5 min with a DMI6000 Leica inverted microscope (S1-S3 Videos). The figure shows photographs of the cells at the indicated time points. The wild-type strain SC5314 was treated identically and is included for comparison. M7 control strains are shown in S4-S5 Videos and S6 Fig.

https://doi.org/10.1371/journal.pgen.1012118.g004

To further characterize the filamentous cells of the mutants, the strains were grown in YPD liquid medium after the induced gene deletion and then fixed and treated with calcofluor white, to stain chitin in cell walls and septa, or with DAPI, to visualize nuclei (Fig 5). In the orf19.3456Δ mutants, septa were only detectable between yeast cells that remained attached to one another, but no septa were visible in the long filaments (Fig 5A). While aberrantly shaped yeast cells of the orf19.3456Δ mutants often contained multiple nuclei, nuclear staining became diffuse and extended in the long filaments, indicating that the mutants were defective in nuclear segregation and distribution (Fig 5B). The pseudohyphae of the orf19.5376Δ mutants showed no or mislocalized chitin at the constrictions between cells, pointing to a defect in septum formation (Fig 5A) that resulted in some cells having no or multiple nuclei (Fig 5B, compare with serum-induced wild-type hyphae in S5 Fig). The M7 control strains that retained a wild-type allele after excision of the FLP-deletable gene copy behaved like the parental strain SC5314 (S6 Fig). Altogether, these results indicate that both orf19.3456Δ and orf19.5376Δ mutants have severe cytokinesis defects that ultimately result in cell death.

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Fig 5. orf19.3456Δ and orf19.5376Δ mutants have cytokinesis defects.

YCB-BSA-YE overnight cultures of the wild-type strain SC5314 and the conditional M6 mutants were diluted 1:100 in YPD medium and grown at 30°C. Aliquots of the cultures were taken after 12 h and fixed with formaldehyde. Cells were washed with PBS and stained with calcofluor white (A) or DAPI (B). Cells were imaged by DIC and fluorescence microscopy.

https://doi.org/10.1371/journal.pgen.1012118.g005

The kinase activity of the proteins encoded by orf19.3456 and orf19.5376 is essential for viability

To investigate if the essential function of the proteins encoded by orf19.3456 and orf19.5376 depends on their predicted kinase activity, we generated strains that after loss of the FLP-deletable wild-type allele retained a mutated, “kinase-dead” allele at the endogenous locus (see materials and methods and S1 Table). These strains behaved like the conditional null mutants; they could not generate colonies after the deletion of the wild-type allele (S7A Fig) and had the same terminal phenotype as the null mutants (S6-S7 Videos and S7B Fig). We confirmed that the mutated proteins were produced by introducing 3xHA-tagged wild-type and kinase-dead alleles at the endogenous locus into strains with an additional wild-type allele (to ensure viability). Western blotting showed that wild-type and kinase-dead proteins were produced at comparable levels (S7C Fig). Furthermore, HA-tagging did not detectably compromise protein function, since replacement of the remaining wild-type allele in the heterozygous M2 mutants by a 3xHA-tagged allele did not affect growth (S7D Fig). These results provide evidence that the kinase activity of the predicted protein kinases encoded by orf19.3456 and orf19.5376 is essential for viability in the C. albicans wild-type strain SC5314.

orf19.5376 encodes a functional homolog of Saccharomyces cerevisiae Elm1 that regulates septin localization and cytokinesis

The protein kinase encoded by orf19.5376 is the closest C. albicans homolog of S. cerevisiae Elm1, which has a redundant role with two other kinases, Sak1 and its paralog Tos3, in the activation of Snf1, a protein kinase that is required for adaptation to glucose limitation and utilization of alternative carbon sources [19–21]. In C. albicans, Snf1 phosphorylation in its activation loop is largely abolished in sak1Δ mutants, indicating that orf19.5376 cannot compensate for the loss of Sak1 and does not have a significant, if any, role in Snf1 activation [3]. However, Elm1 has additional functions in the yeast cell cycle. It is localized at the bud neck where it controls the assembly of septin filaments into the hourglass structure and their reorganization into the double ring structure at the division site; absence of Elm1 results in a mitotic delay [22–26]. S. cerevisiae elm1Δ mutants display a highly elongated cell morphology and, in some genetic backgrounds, constitutive pseudohyphal growth, because the cells remain attached to each other after cytokinesis [27]. These phenotypes resemble those of C. albicans orf19.5376Δ mutants (Fig 4 and S3 Video). To study orf19.5376 function further, we tested if the orf19.5376Δ mutant phenotype could also be brought about by auxin-inducible protein depletion [28], and possibly faster than by forced gene deletion. To this aim, the remaining wild-type orf19.5376 allele in the two independently generated heterozygous M2 mutants was fused with the AID* cassette (see materials and methods). Log-phase cells of the resulting strains were treated with 1 µM of the auxin analog 5-Ad-IAA and samples were taken at different time points for detection of the tagged proteins by Western blotting. Fig 6A shows that the degron-tagged kinase, which can be detected with an anti-HA antibody, was successfully removed within 15 min of treatment. To determine the phenotypic consequences of protein depletion, cells grown in YPD liquid medium were transferred to YPD agar containing 1 µM 5-Ad-IAA and observed by video microscopy. As can be seen in S8 Video and Fig 6B, the cells exhibited the same pseudohyphal phenotype as the induced gene deletion mutants, only that it was induced more rapidly, presumably because of the more immediate effect of protein depletion (identically treated, untagged control cells are shown in S9 Video). Furthermore, staining of cells grown in YPD liquid medium showed that auxin-induced protein depletion resulted in the same defects in septum formation and nuclear distribution as seen in the gene deletion mutants (S8 Fig). This was further corroborated by monitoring septin localization in cells containing a GFP-tagged CDC3 allele. In contrast to auxin-treated wild-type cells, which showed the typical septin ring between mother and daughter cells as well as the double ring in large-budded cells (S9 Fig), Cdc3 was largely absent at the restrictions between cells and relocalized to the pseudohyphal tip in the auxin-induced mutants (Fig 6C), similar to the mislocalization of septins in S. cerevisiae elm1Δ mutants [22,26,29,30]. For comparison, serum-induced hyphae of the wild-type strain showed normal septa, nuclei distribution, and septin rings (S10 Fig). Together, these observations demonstrate that the kinase encoded by orf19.5376 controls septin localization and cytokinesis in C. albicans, indicating that it is indeed a functional ortholog of S. cerevisiae Elm1. However, while S. cerevisiae elm1Δ mutants are viable, the morphologial defects caused by the absence of this kinase are much more severe and eventually lethal in C. albicans.

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Fig 6. Phenotypic consequences of auxin-induced degradation of the orf19.5376-encoded kinase.

(A) Strains containing a single orf19.5376 copy fused to the AID* cassette were grown to log phase in YPD medium. Samples were taken before and at the indicated time points after addition of 1 µM 5-Ad-IAA and analyzed by Western blotting. The degron-tagged kinase was detected with an anti-HA antibody and the Tir protein with an anti-Myc antibody. (B) YPD-grown cells containing the degron-tagged kinase were transfered to a 35-mm culture dish, covered with YPD + 1 µM 5-Ad-IAA agar, and incubated at 30°C. Images were taken every 5 min for 24 h with a Leica DMI6000 microscope (S8 Video). The figure shows photographs of the cells at the indicated time points. (C) A YPD overnight culture of the auxin-inducible mutants containing a GFP-tagged CDC3 allele was diluted 1:100 in YPD + 1 µM 5-Ad-IAA and grown at 30°C. Aliquots of the culture were taken every 2 h and fixed with paraformaldehyde. Cells were washed with PBS, stained with DAPI, and imaged by DIC (left panels) and fluorescence microscopy (middle panels). The figure shows photographs of the cells at the indicated time points, including overlays of the DIC and fluorescence micrographs (right panels).

https://doi.org/10.1371/journal.pgen.1012118.g006

The protein kinase encoded by orf19.3456 is part of a septation initiation network in C. albicans

The protein kinase encoded by orf19.3456 does not have an ortholog in S. cerevisiae, but one of its homologs in the fission yeast Schizosaccharomyces pombe is the protein serine/threonine kinase Sid1, a component of the septation initiation network (SIN) that coordinates cytokinesis with chromosome segregation [31,32]. The SIN includes a kinase cascade, Cdc7-Sid1-Sid2, that is positioned at the spindle pole body (SPB) by the GTPase Spg1. The SIN becomes active at the new SPB (by the recruitment of Sid1) when the mitotic cyclin-dependent kinase (CDK) is inactivated in late mitosis. Under restrictive conditions, sid1 as well as other sin mutants undergo nuclear division without septation and form elongated, multinucleate cells that eventually lyse [31]. A related signaling pathway in S. cerevisiae is the mitotic exit network (MEN), which promotes exit from mitosis by triggering the release of the phosphatase Cdc14 from the nucleolus to antagonize the mitotic CDKs [33,34]. The MEN contains homologs of many of the SIN components, including the Spg1 homolog Tem1, the Cdc7 homolog Cdc15, and the Sid2 homologs Dbf2/Dbf20, but it lacks a Sid1 homolog and Dbf2 is directly activated by Cdc15 [35]. S. cerevisiae mutants lacking MEN proteins arrest in late anaphase and fail to inactivate CDKs and initiate cytokinesis [34].

Sid1 has comparable homology to several C. albicans kinases, not only that encoded by orf19.3456 (62.1% similarity in the kinase domain), e.g., Kic1 (64.1%), Cla4 (60.6%), Sps1 (58.5%), or Cdc15 (58.1%). However, the orf19.3456Δ mutant phenotype (multinucleate cells, hyphae without septa) suggested that the encoded kinase could be a Sid1 ortholog and that C. albicans, despite being a budding yeast like S. cerevisiae, possesses a signaling pathway that is more related to the SIN of S. pombe than to the MEN of S. cerevisiae. We tested if the orf19.3456Δ mutant phenotype could be reproduced by auxin-induced protein depletion and replaced the remaining wild-type orf19.3456 allele in the heterozygous M2 mutants by an AID*-tagged copy. Fig 7A shows that the degron-tagged protein became undetectable within 15 min after the addition of 1 µM 5-Ad-IAA to log-phase cells. Similarly to the gene deletion mutants, the cells were unable to grow as budding yeasts and instead formed hyphae without constrictions, and this phenotype was rapidly induced upon protein depletion (S10 Video and Fig 7B). The video also shows that the hyphae originated from buds that had become larger than their mother cells (identically treated, untagged control cells are shown in S11 Video). Moreover, the defect in septum formation and a sometimes multinucleate nature of aseptate hyphae were evident in these mutants (S11 Fig). In cells containing a GFP-tagged CDC3 allele, the septin became mislocalized soon after the auxin-induced protein depletion and was either absent in hyphae or found at apparently random sites without forming a ring structure Fig 7C).

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Fig 7. Phenotypic consequences of auxin-induced degradation of the orf19.3456-encoded kinase.

(A) Strains containing a single orf19.3456 copy fused to the AID* cassette were grown to log phase in YPD medium. Samples were taken before and at the indicated time points after addition of 1 µM 5-Ad-IAA and analyzed by Western blotting with anti-HA and anti-Myc antibodies. (B) YPD-grown cells containing the degron-tagged kinase were transfered to a 35-mm culture dish, covered with YPD + 1 µM 5-Ad-IAA agar, and incubated at 30°C. Images were taken every 5 min for 24 h with a Leica DMI6000 microscope (S10 Video). The figure shows photographs of the cells at the indicated time points. (C) A YPD overnight culture of the auxin-inducible mutants containing a GFP-tagged CDC3 allele was diluted 1:100 in YPD + 1 µM 5-Ad-IAA and grown at 30°C. Aliquots of the culture were taken every 2 h and fixed with paraformaldehyde. Cells were washed with PBS, stained with DAPI, and imaged by DIC (left panels) and fluorescence microscopy (middle panels). The figure shows photographs of the cells at the indicated time points, including overlays of the DIC and fluorescence micrographs (right panels). Note that the hyphae were deformed after the fixation and centrifugation steps (compare with the live hyphae in (B).

https://doi.org/10.1371/journal.pgen.1012118.g007

Several components of the MEN/SIN pathway have already been studied in C. albicans and, in analogy to S. cerevisiae, described as parts of the MEN [36–39]. Indeed, the GTPase Tem1, the kinases Cdc15 and Dbf2, and the phosphatase Cdc14 were thought to constitute all key components of the MEN signaling cascade in C. albicans [36]. TEM1, CDC15, and DBF2 are essential genes, and their functions have been investigated by expressing them under the control of repressible promoters. The tem1 and cdc15 knock-down mutants formed hyphae without septa [36,39], similar to our orf19.3456 mutants. In contrast, dbf2 knock-down mutants showed a different phenotype. They were unable to make septa, but instead of forming hyphae produced chains of cells that failed to separate after promoter shut-off [38]. Cells expressing an HA-tagged DBF2, whose function was partially compromised, exhibited an even stronger phenotype under repressive conditions. They arrested as pairs of large budded cells without a septum after DNA replication and displayed a nuclear segregation defect due to improper mitotic spindle organization [38]. To investigate whether the orf19.3456-encoded kinase and the putative downstream kinase Dbf2 indeed have different functions in the C. albicans cell cycle or the different mutant phenotypes were caused by the experimental approaches (gene repression versus gene deletion/protein depletion), we generated auxin-inducible dbf2 mutants of the wild-type strain SC5314. Dbf2-depleted cells exhibited the same phenotype as the orf19.3456 mutants; they formed aseptate hyphae that often contained two elongated nuclei, suggesting a nuclear segregation defect after they had entered a new cell cycle (Fig 8 and S12 Video; auxin-treated, untagged control cells are shown in S13 Video and S12 Fig). These results suggest that Tem1, Cdc15, the orf193456-encoded kinase, and Dbf2 act in the same signaling pathway and their loss results in the same terminal phenotype.

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Fig 8. Phenotypic consequences of auxin-induced Dbf2 depletion.

(A) Strains containing a single DBF2 copy fused to the AID* cassette were grown to log phase in YPD medium. Samples were taken before and at the indicated time points after addition of 1 µM 5-Ad-IAA and analyzed by Western blotting with anti-HA and anti-Myc antibodies. (B) YPD-grown cells containing the degron-tagged Dbf2 were transfered to a 35-mm culture dish, covered with YPD + 1 µM 5-Ad-IAA agar, and incubated at 30°C. Images were taken every 5 min for 24 h with a Leica DMI6000 microscope (S12 Video). The figure shows photographs of the cells at the indicated time points. (C, D) A YPD overnight culture of the auxin-inducible dbf2 mutants was diluted 1:100 in YPD + 1 µM 5-Ad-IAA and grown at 30°C. Aliquots of the culture were taken every 2 h and fixed with formaldehyde. Cells were washed with PBS, stained with calcofluor white (C) or DAPI (D), and imaged by DIC and fluorescence microscopy. The figure shows photographs of the cells at the indicated time points.

https://doi.org/10.1371/journal.pgen.1012118.g008

orf19.193 encodes a putative regulatory subunit of the orf19.3456-encoded kinase

Sid1 of S. pombe is bound to a regulatory subunit that is required for its activity [31]. To identify potential interaction partners of the orf19.3456-encoded kinase, we performed co-immunoprecipitation (Co-IP) experiments with derivatives of strain SC5314 in which both orf19.3456 alleles were HA-tagged. Liquid chromatography-mass spectrometry identified 105 proteins (including the tagged kinase itself) that were significantly enriched in immunoprecipitates of the tagged strains compared to the untagged wild-type control strain (S2 Table; extended dataset provided in S3 Table). Among those, one protein stood out because of its > 1,000-fold increased abundance. It is encoded by the putative essential orf19.193, the function of which is unknown. However, according to the Candida Genome Database, the protein belongs to the same family as Cdc14, the regulatory subunit of Sid1 in S. pombe (not to be confounded with the phosphatase Cdc14 of S. cerevisiae and C. albicans), and a subsequent BLAST search of the C. albicans genome with S. pombe Cdc14 identified orf19.193 as the best hit. We confirmed the binding of the orf19.193-encoded protein to the orf19.3456-encoded kinase in Co-IP experiments with strains containing Myc-tagged orf19.3456 and/or HA-tagged orf19.193 (see S4 Table). Fig 9 shows that the orf19.193-encoded protein was specifically immunoprecipitated by the Myc-tagged kinase (lanes 5–8), but not in strains with the untagged kinase (lanes 1 and 2).

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Fig 9. The orf19.193-encoded protein specifically interacts with the orf19.3456-encoded kinase.

Strains with or without Myc-tagged orf19.3456 and HA-tagged orf19.193 were grown to log phase in YPD at 30°C. Protein extracts were prepared as described in materials and methods and immunoprecipitated with an anti-Myc antibody. The immunoprecipitated orf19.3456-encoded kinase was detected by Western blotting with an anti-Myc antibody, and the co-immunoprecipitated orf19.193-encoded protein with an anti-HA antibody (top panels, IP). Tagged proteins in the input samples were detected with anti-HA and anti-Myc antibodies (bottom panels, input). Detection of tubulin with an anti-tubulin antibody served as loading control for the input samples. Both independently generated series of strains were used for the experiment.

https://doi.org/10.1371/journal.pgen.1012118.g009

We reasoned that, if orf19.193 encodes a regulatory subunit that is essential for the activity of the orf19.3456-encoded kinase, orf19.193 mutants should exhibit the same phenotype as orf19.3456 mutants. Either of the two orf19.193 alleles could be deleted in the wild-type strain SC5314, but no homozygous mutants were obtained among 24 second-round transformants of the two types of heterozygous mutants, providing further evidence that orf19.193 is an essential gene. We therefore generated auxin-inducible conditional mutants in which the remaining wild-type orf19.193 allele was fused with the AID* cassette. Strikingly, following depletion of the degron-tagged protein, the cells grew as aseptate hyphae containing elongated nuclear masses, just like the orf19.3456 mutants (Fig 10 and S14 Video; auxin-treated, untagged control cells are shown in S15 Video and S13 Fig). This phenotype provided additional evidence that C. albicans regulates cytokinesis via a SIN pathway that includes the orf19.3456-encoded kinase and its regulatory subunit in addition to the upstream kinase Cdc15 and the downstream kinase Dbf2.

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Fig 10. Phenotypic consequences of auxin-induced degradation of the orf19.193-encoded protein.

(A) Strains containing a single orf19.193 copy fused to the AID* cassette were grown to log phase in YPD medium. Samples were taken before and at the indicated time points after addition of 1 µM 5-Ad-IAA and analyzed by Western blotting with anti-HA and anti-Myc antibodies. (B) YPD-grown cells containing the degron-tagged orf19.193 were transfered to a 35-mm culture dish, covered with YPD + 1 µM 5-Ad-IAA agar, and incubated at 30°C. Images were taken every 5 min for 24 h with a Leica DMI6000 microscope (S14 Video). The figure shows photographs of the cells at the indicated time points. (C, D) A YPD overnight culture of the auxin-inducible orf19.193 mutants was diluted 1:100 in YPD + 1 µM 5-Ad-IAA and grown at 30°C. Aliquots of the culture were taken every 2 h and fixed with formaldehyde. Cells were washed with PBS, stained with calcofluor white (C) or DAPI (D), and imaged by DIC and fluorescence microscopy. The figure shows photographs of the cells at the indicated time points.

https://doi.org/10.1371/journal.pgen.1012118.g010

Strains and growth conditions

The C. albicans strains used in this study are listed in S1 Table. All strains were stored as frozen stocks with 17.2% (v/v) glycerol at -80°C and subcultured on YPD agar plates (10 g yeast extract, 20 g peptone, 20 g glucose, 15 g agar per liter) at 30°C. Strains were routinely grown in YPD liquid medium at 30°C in a shaking incubator. For the selection of transformants, 200 µg/ml nourseothricin (Werner Bioagents, Jena, Germany) or 1 mg/ml hygromycin B was added to YPD agar plates. To obtain nourseothricin-sensitive derivatives in which the SAT1 flipper cassette was excised by FLP-mediated recombination, transformants were grown overnight in YCB-BSA-YE medium (23.4 g yeast carbon base, 4 g bovine serum albumin, 2 g yeast extract per liter, pH 4.0) without selective pressure to induce the SAP2 promoter controlling caFLP expression. Appropriate dilutions were plated on YPD agar plates and grown for 2 days at 30°C. Individual colonies were picked and streaked on YPD plates as well as on YPD plates with 100 µg/ml nourseothricin to confirm sensitivity. Specific growth conditions for particular experiments are described in the text and figure legends.

Strain constructions

C. albicans strains were transformed by electroporation as described previously [48]. To generate FLP-deletable cassettes containing functional copies of the protein kinase genes, the coding region and flanking sequences of the genes were amplified from genomic DNA of strain SC5314 with the primers listed in S4 Table and substituted for the SNF1 gene in the previously described pSNF1ex3 [11], resulting in plasmids pBUD32ex3, pCTK1ex3, pKSP1ex3, pPTK2ex3, pRIO1ex3, pRIO2ex3, p3456ex3, and p5376ex3. The cassettes were excised from the vector backbone and integrated at the ADH1 locus in the corresponding heterozygous M2 mutants (2) to obtain M3 mutants (see Fig 1 and S1 Table). The previously described gene deletion cassettes (2,3) were then used to delete the second endogenous alleles in these strains to generate M4 mutants. These strains were grown overnight in YCB-BSA-YE medium to induce FLP-mediated excision of the SAT1 flipper cassette, resulting in M5 mutants which contained only the FLP-deletable gene copy. The insert from plasmid pSAP2FL1 [11] was used to integrate the ecaFLP gene under control of the SAP2–1 promoter into the SAP2–1 allele of M5 and M3 mutants to generate the conditional M6 mutants and the M7 control strains, respectively. Induction of ecaFLP expression by passage in YCB-BSA-YE medium resulted in the loss of the FLP-deletable gene copy. When the null mutants were viable (bud32Δ, ctk1Δ, rio1Δ, rio2Δ), corresponding M8 mutants (from M6) and M9 control strains (from M7) were retained. An exception were the KSP1 mutants for which two types of derivatives of the M4 strains were obtained. In some clones, both the SAT1 flipper cassette and the FLP-deletable ectopic KSP1 copy were deleted, and these were kept as ksp1Δ null mutants (SCKSP1M5). Others had excised only the SAT1 flipper cassette but retained the ectopic KSP1 copy, and these were used as control strains (SCKSP1M6).

The kinase-dead orf19.3456^K41R allele was amplified in two parts from genomic DNA of strain SC5314 with the primer pairs 3456.01/3456.07 and 3456.06/3456.05, followed by a fusion PCR with primers 3456.01 and 3456.05; the overlapping primers 3456.07 and 3456.06 changed the lysine codon AAA (positions +121 to +123 in orf19.3456) into the arginine codon AGA. The PCR product was digested with SacI/SacII and substituted for the upstream flanking sequence in the orf19.3456 deletion cassette to obtain p3456^K41R. The kinase-dead orf19.5376^K65R allele was generated in analogous fashion with the primer pairs 5376.01/5376.13 and 5376.12/5376.11, thereby changing the lysine codon AAA (positions +193 to +195 in orf19.5376) into the arginine codon AGA. The PCR product was digested with SacI/SacII and substituted for the upstream flanking sequence in the orf19.5376 deletion cassette to obtain p5376^K65R. The inserts from these plasmids were integrated into one of the inactivated endogenous alleles of the corresponding M5 mutants to generate M8 mutants, followed by recycling of the SAT1 flipper cassette to produce M9 mutants. The insert from plasmid pSAP2FL1 was then used to integrate the ecaFLP gene under control of the SAP2–1 promoter into the SAP2–1 allele to generate M10 mutants.

To construct 3xHA-tagged orf19.3456 and orf19.3456^K41R alleles, their upstream and coding sequences were amplified with primers 3456.01 and 3456.08; the latter primer introduced a KasI site, encoding a Gly-Ala linker, instead of the stop codon. The PCR products were digested with SacI/KasI and cloned together with a KasI-SacII fragment from pMIG1H3 [17], encoding three copies of the HA epitope followed by a stop codon and the ACT1 transcription termination sequence, into the SacI/SacII-digested p3456^K41R to obtain p3456H1 and p3456H^K41R, respectively. 3xHA-tagged orf19.5376 and orf19.5376^K65R alleles were generated in an analogous way using primers 5376.01 and 5376.14 to obtain plasmids p5376H1 and p5376H^K65R. The inserts from these plasmids were integrated into one of the inactivated endogenous alleles of the corresponding M5 mutants to generate M11 and M13 mutants, followed by recycling of the SAT1 flipper cassette to produce M12 and M14 mutants. The inserts from p3456H1 and p5376H1 were also inserted into the remaining wild-type orf19.3456 and orf19.5376 alleles of the M2 mutants, followed by recycling of the SAT1 flipper cassette to generate H2 strains expressing a single 3xHA-tagged copy these genes from their own promoter. Subsequent integration of the 3xHA-tagged orf19.3456 into the already deleted second allele resulted in H4 strains.

To generate an orf19.193 deletion cassette, the orf19.193 upstream and downstream regions were amplified with the primer pairs 193.09/193/10 and 193.11/193.12, respectively, and cloned on both sides of the SAT1 flipper cassette in plasmid pSFS5 [5]. Replacement of one of the orf19.193 alleles in strain SC5314 by the orf19.193 deletion cassette, followed by recycling of the SAT1 flipper, generated heterozygous M2 mutants.

To obtain auxin-inducible orf19.3456 and orf19.5376 mutants, the AID* cassette from plasmid pHLP728 (a kind gift from Mark Hall) was amplified in two parts with the primer pairs AID01/AID02 and AID03/AID04. The PCR products were digested with KasI/NsiI and NsiI/XhoI, respectively, and, after fusion at the introduced NsiI sites, inserted into the KasI/XhoI-digested p3456H1 and p5376H1 to generate p3456AID1 and p5376AID1, respectively. The inserts from these plasmids were used to tag the remaining wild-type orf19.3456 and orf19.5376 alleles in the corresponding M2 mutants with the AID* cassette. The resulting SC3456AID1 and SC5376AID1 strains express single orf19.3456 and orf19.5376 alleles, respectively, containing a C-terminal fusion to the auxin-inducible degron, the OsTIR1 gene under control of the C. albicans ACT1 promoter, and a nourseothricin resistance marker [a modified version of the caSAT1 marker generated in the Hall lab [28]]. Auxin- inducible dbf2 and orf19.193 mutants were generated in an analogous fashion. The 3’ part of the DBF2 coding region was amplified with primers DBF2.12 and DBF2.06 and the PCR product digested with SacI/KasI and cloned together with the KasI-XhoI fragment from p3456AID1 containing the AID* cassette into the SacI/XhoI-digested pDBF2M1 [2] to generate pDBF2AID1. The 3’ part of orf19.193 was amplified with primers 193.13/193.02 and the orf19.193 downstream region with primers 193.11 and 193.12. The PCR products were digested with SacI/KasI and XhoI/ApaI, respectively, and substituted for the orf19.3456 flanking sequences in p3456AID1 to obtain p193AID1. The inserts from the latter plasmids were used to tag the remaining wild-type DBF2 and orf19.193 alleles in the corresponding M2 mutants with the AID* cassette.

To facilitate HA-, Myc- and GFP-tagging in nourseothricin- or hygromycin-resistant strains, the HygB marker from plasmid pGRP2M2 [49] was first amplified in two parts with the primer pairs HygB-1/HygB-8 and HygB-9/HygB-2, followed by a fusion PCR with primers HygB-1 and HygB-2. The complementary primers HygB-8 and HygB-9 introduced a silent T285G substitution that removed an internal SacI site. The PCR product was digested with XhoI/PstI and cloned in the vector pBluescript II KS to obtain plasmid pHygB. A fragment comprising a KasI linker, three tandem copies of the Myc epitope sequence followed by a stop codon, and the ACT1 transcription termination sequence was amplified from plasmid pKIS1Myc3 [50] with primers Myc3KasI and ACT1TSalI, digested with ApaI/SalI, and cloned in the ApaI/XhoI-digested pHygB to generate pMyc3-HygB. Furthermore, a shorter version of the caSAT1 marker (without the ACT1 intron) was obtained by amplifying the caSAT1 gene in two parts with the primer pairs ACT1P1/ACT1P2 and SAT9/SAT10, followed by a fusion PCR with primers ACT1P1 and SAT10. The PCR product was digested with XhoI/PstI and cloned in the vector pBluescript II KS to generate pSAT5 with the modified caSAT2 marker. A fragment comprising a KasI linker, three tandem copies of the Myc epitope sequence followed by a stop codon, and the ACT1 transcription termination sequence was amplified as described above and cloned in the ApaI/XhoI-digested pSAT5 to generate pMyc3-SAT2.

To generate a 3xMyc-tagged orf19.3456, the orf19.3456 downstream region was amplified with primers 3456.09 and 3456.04. The PCR product was digested with PstI/ApaI and cloned together with the KasI/PstI digested 3xMyc-ACT1T-caSAT2 fragment from pMyc3-SAT2 into the KasI/ApaI-digested p3456H1. The insert from this plasmid was used to replace the remaining orf19.3456 wild-type allele in the heterozygous M2 mutants as well as one of the orf19.3456 wild-type alleles in the strains containing a single HA-tagged orf19.193 allele (described below). To generate a 3xMyc-tagged orf19.193, a part of the orf19.193 coding sequence was amplified with the primers 193.01 and 193.02; the latter primer introduced a KasI site, encoding a Gly-Ala linker, instead of the stop codon. The orf19.193 downstream region was amplified with primers 193.03 and 193.04. The PCR products were digested with ApaI/KasI and PstI/SacI, respectively, and cloned together with the KasI-PstI fragment from pMyc3-HygB in the ApaI/SacI-digested vector pBluescript II KS to generate p193Myc3. A 3xHA-ACT1T fragment was amplified from pMIG1H3 with the primers MIG1HAfwd and ACT1TSalI, digested with KasI/SalI, and cloned together with the XhoI-PstI HygB-Fragment from pHygB in the Kas/PstI-digested p193Myc3 to obtain p193H1, thereby exchanging the 3xMyc tag for a 3xHA tag. The insert from p193H1 was used to replace the remaining orf19.193 wild-type allele in the heterozygous M2 mutants as well as one of the orf19.193 wild-type alleles in the strains containing a single Myc-tagged orf19.3456 allele.

For GFP-tagging, a fragment containing GFP and the ACT1 transcription termination sequence was amplified from plasmid pYOR1G1 [51] with the primers GFP28 and ACT1TSalI. The PCR product was digested with KasI/SalI and used to replace the 3xMyc tag in pMyc3-HygB by GFP, yielding pGFP-HygB. To construct a CDC3-GFP fusion, a part of the CDC3 coding sequence was amplified with the primers CDC3.01 and CDC3.02; the latter primer introduced a KasI site, encoding a Gly-Ala linker, instead of the stop codon. The CDC3 downstream region was amplified with primers CDC3.03 and CDC3.04. The PCR products were digested with ApaI/KasI and PstI/SacI, respectively, and cloned together with the KasI-PstI fragment from pGFP-HygB in the vector pBluescript II KS to generate pCDC3G1. The insert from this plasmid was used to replace one of the endogenous CDC3 alleles by a GFP-tagged copy in the wild type and the auxin-inducible orf19.3456 and orf19.5376 mutants. In all cases two independent series of mutants (A and B) were generated (see S1 Table).

Isolation of genomic DNA and Southern hybridization

Genomic DNA from C. albicans strains was isolated as described previously [4]. The DNA was digested with appropriate restriction enzymes, separated on a 1% agarose gel, transferred by vacuum blotting onto a nylon membrane, and fixed by UV crosslinking. Southern hybridization with enhanced chemiluminescence-labeled probes was performed with the Amersham ECL Direct Nucleic Acid Labelling and Detection System (Cytiva) according to the instructions of the manufacturer.

Determination of induced gene deletion efficiency

Conditional deletion mutants and control strains were grown overnight at 30°C in YCB-BSA-YE medium to induce ecaFLP expression from the SAP2 promoter. Tenfold dilution series were prepared and the 10^-2 dilutions used to determine the total number of cells/ml in a counting chamber (Thoma neu). The number of viable cells (CFUs) was determined by plating 100 µl of the 10^-3 dilutions of the conditional mutants and 100 µl of the 10^-6 dilutions of the control strains on YPD plates. The number of colonies was determined after two days of growth at 30°C.

Time lapse microscopy

YPD overnight cultures of the strains were diluted 1:5 in water. Five µl of the diluted cell suspension was transferred to an IBIDI µ-Dish 35 mm and covered with a previously prepared YPD agar pad, without or with 1 µM 5-Ad-IAA (TCI). The µ-Dish was placed in a Leica DMI6000 microscope equipped with a climate chamber, prewarmed to an ambient temperature of 30°C. DIC images were acquired at 5 min intervals for 24 h. Image processing was carried out with FIJI software [52].

Fluorescence microscopy

For cell wall and nuclei staining, cells from liquid cultures were fixed with 4% formaldehyde and washed with PBS. Fixed cells were stained with 1 µg/ml calcofluor white or 5 µg/ml DAPI. For localization of GFP-tagged Cdc3, cells were fixed with 4% paraformaldehyde, washed with PBS, and stained with 1 µg/ml calcofluor white or 5 µg/ml DAPI. Cells were imaged with a Leica DMI6000 microscope using appropriate filters for fluorescence detection. Z-stacks were acquired over 5 µm in 0.5 µm increments and deconvolved using LAS X software. Additional image processing was carried out with FIJI software [52].

Western blotting

Overnight cultures of the strains were diluted to an OD₆₀₀ of 0.4 in fresh YPD and grown for 5 h at 30°C. Cells were collected by centrifugation, washed with ice-cold water, and resuspended in 300 µl breaking buffer (50 mM Tris-HCl pH 8, 250 mM NaCl, 5 mM EDTA, 0.1% [v/v] Triton X-100, cOmplete EDTA-free Protease Inhibitor Cocktail and PhosStop Phosphatase Inhibitor Cocktail [Roche]). An equal volume of 0.5 mm acid-washed glass beads was added to each tube. Cells were mechanically disrupted on a FastPrep-24 cell-homogenizer (MP Biomedicals) with three 40 s pulses, with 5 min on ice between each pulse. Cell lysates were centrifuged at 21,000 x g for 15 min at 4°C, the supernatant was collected, and the protein concentration was quantified using the Bradford protein assay. Equal amounts of protein of each sample were mixed with one volume of 2 x Laemmli buffer, heated for 5 min at 95°C, and separated on an SDS-polyacrylamide (8% or 10%) gel. Separated proteins were transferred onto a nitrocellulose membrane with a mini-Protean System (Bio-Rad). To detect HA-tagged proteins, membranes were blocked with 5% milk in TBST and incubated overnight with rat monoclonal anti-HA-peroxidase antibody, clone 3F10 (Roche). For the detection of tubulin, membranes were blocked with 5% milk in TBST and incubated overnight at 4°C with rat anti-tubulin alpha antibody MCA 78G (Bio-Rad), washed with TBST, and then incubated with rabbit anti-rat HRP-conjugated antibody STAR21B (Bio-Rad). Myc-tagged proteins were detected with anti-Myc (71D10) rabbit mAb (Cell Signaling Technology) and anti-rabbit HRP G-21234 (Invitrogen) as secondary antibody. To reprobe the immunoblots, membranes were incubated in stripping buffer (0.2 M glycine, 0.1% SDS, 1% Tween 20, pH 2.2), and washed in PBS and TBST before blocking with 5% milk. Signals were generated with the ECL chemiluminescence detection system (Cytiva) and captured with the ImageQuant LAS 4000 imaging system (Cytiva).

Co-immunoprecipitation

For Co-IP of proteins with the HA-tagged orf19.3456-encoded kinase, cells were grown to log phase in YPD and treated for 1 h with 1 mM DSP (CAS 57757-57-0) at room temperature. Crosslinking was stopped by adding Tris-HCl pH 7.4 to a final concentration of 50 mM and incubating for additional 15 min. The cells were then washed with ice-cold water and frozen in liquid nitrogen. Cell extracts were obtained by grinding the cells using mortar and pestle under liquid nitrogen. The powdered extracts were mixed with two volumes of IP-Buffer (40 mM Tris-HCl pH 7.4, 250 mM sodium citrate, 150 mM NaCl, 1% Triton X-100) and centrifuged for 15 min (21,000 x g, 4°C).The supernatants were recovered, mixed with 100 µl anti-HA agarose beads (Pierce 26181, Thermo Scientific), and incubated for 1 h at 4°C with end-over-end rotation. The agarose beads were washed with IP-buffer, followed by three washes with PBS and a final wash with ultrapure water. Bound proteins were eluted with 50 µl 0.1% (v/v) TFA in 30% (v/v) aqueous acetonitrile. The elution step was repeated two additional times and the eluates were combined for a final volume of 150 µl. 10 µl of the eluates were dried in a SpeedVac, resuspended in 2x protein sample buffer and analyzed by Western blotting using an anti-HA HRP-conjugated antibody. Three biological replicates of the tagged strains (two from the A strain and one from the B strain) and three biological replicates of the untagged control strain SC5314 were used for proteomic analysis.

For specific Co-IP of the HA-tagged orf19.193-encoded protein with the Myc-tagged orf19.3456-encoded kinase, cell extracts were prepared as described above. Supernatants were mixed with 100 µl anti-c-Myc agarose beads (Pierce 20168, Thermo Scientific) and incubated for 18 h at 4°C with end-over-end rotation. The agarose beads were washed with IP-buffer, followed by three washes with PBS, and bound proteins eluted with 50 µl of 2x protein sample buffer. Immunoprecipitated samples and corresponding input samples were analyzed by Western blotting as described above.

Proteomics