Candida albicans Rho-Type GTPase-Encoding Genes Required for Polarized Cell Growth and Cell Separation

Abstract

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.

There is a strong interest in studying the molecular basis of cell differentiation in Candida albicans based on the importance of the yeast-to-hypha transition as a virulence trait in this human fungal pathogen (5). Upon induction by environmental cues, C. albicans produces hyphal filaments that enable C. albicans to adhere to and penetrate tissues and thus conquer new territories or niches in the human host or escape from the host's cellular immune defense.

Recent progress has identified GTP-binding proteins of the Ras and Rho superfamilies as being key regulators of morphogenesis in C. albicans. RAS1 is required for hyphal morphogenesis by activating downstream cyclic AMP- and mitogen-activated protein kinase-signaling pathways (21). A dominant active allele of RAS1 [ras1(GV13)] stimulates enhanced/constitutive hyphal growth in C. albicans (12). However, Ras1-induced hyphal morphogenesis requires actin cytoskeletal components that regulate exo- and endocytosis, since a deletion of either the formin BNI1 or the Wiskott-Aldrich syndrome protein homolog WAL1 blocked hyphal morphogenesis (25, 39). The cell polarity establishment Rho protein Cdc42 is essential for viability and is also required for polarized hyphal growth (35, 37). In contrast, the C. albicans RAC1 homolog plays a specialized role in the organization of the actin cytoskeleton and in hyphal morphogenesis under embedded conditions (1). Furthermore, the Ras-related GTPase encoded by BUD1 in C. albicans plays a role in hyphal growth guidance similar to the role of its homolog in the filamentous ascomycete Ashbya gossypii (3, 16). In Saccharomyces cerevisiae, it was shown that the essential function of Rho3 and Rho4 is to activate formin homologs (either Bni1 or Bnr1) (9). In Ashbya gossypi, a deletion of RHO4 did not result in any obvious phenotypes (43). In contrast, deletion of the RHO4 homologs in either Neurospora crassa or Schizosaccharomyces pombe showed an involvement of Rho4 in septation (28, 31, 32). The C. albicans RHO4 (CaRHO4) gene was previously identified in a screen that isolated C. albicans genes that interfere with the S. cerevisiae pheromone response and was termed CRL1 for C. albicans RHO-like protein 1 (45). Given the role of Rho proteins in the organization of the actin cytoskeleton and in morphogenesis, we aimed to elucidate the role of the C. albicans RHO3 and CRL1/RHO4 genes. We show via MET3 promoter shutdown experiments that Rho3 is essential for hyphal morphogenesis. Crl1/Rho4 is apparently not involved in this process. On the other hand, a deletion of CRL1/RHO4 leads to a cell elongation and cell separation defect. This cell separation defect is similar to that observed in a chitinase cht3 mutant. In contrast, the cell elongation phenotype resembles that of bnr1 cells with a deletion of one of the C. albicans formin genes. In C. albicans, specific functions for Rho3 at the hyphal tip and for Rho4 at septal sites were discovered. Due to its role in septation, CaRho4 is functionally more similar to N. crassa and S. pombe than to S. cerevisiae and A. gossypii RHO4 homologs.

MATERIALS AND METHODS

Strains and media.

The C. albicans strains used and generated in this study are listed in Table 1. The strains were grown either in complete YPD medium (1% yeast extract, 2% peptone, 2% glucose) or in complete supplement mixture (CSM) minimal medium (6.7 g/liter yeast nitrogen base with ammonium sulfate and without amino acids, 0.69 g/liter CSM, 20 g/liter glucose) or SD medium (6.7 g/liter yeast nitrogen base with ammonium sulfate and without amino acids, 20 g/liter glucose). Minimal media were supplemented with the strain-specific requirements for amino acids and uridine. Control of regulatable gene expression via the MET3 promoter was exerted by the presence or absence of methionine and cysteine (3.5 mM each) in the medium (7). Induced expression via the MAL2 promoter was achieved by growth on maltose (20 g/liter). Hyphal induction of C. albicans cells was done by adding 10% serum to the growth medium unless stated otherwise. Escherichia coli strain DH5α was used for plasmid propagation. Transformation of E. coli was done by electroporation.

TABLE 1.

Strains used in this study

Straina	Relevant genotype	Reference or source
SC5314	C. albicans wild type	13
BWP17	ura3::λimm34/ura3::imm34 his1::hisG/his1::hisG arg4::hisG/arg4::hisG	46
GC19	bnr1::URA3/bnr1::HIS1 arg4	25
CAA3	cht3::URA3/cht3::HIS1 arg4	10
CAA3MX	cht3::ARG4/cht3::HIS1 ura3	This study
CAA34	RHO4/rho4::HIS1 ura3 arg4	This study
CAA35	RHO4/rho4::ARG4 ura3 his1	This study
CAA41	RHO3/rho3::URA3 his1 arg4	This study
CAA43	RHO3/rho3::ARG4 ura3 his1	This study
CAA49	rho4::HIS1/rho4::ARG4 ura3	This study
CAA50	rho3::URA3/HIS1-MET3p-RHO3 arg4	This study
CAA51	rho3::ARG4/HIS1-MET3p-RHO3 ura3	This study
CAA57	rho4::ARG4/rho4::URA3 his1	This study
CAA57MX	rho4::ARG4/rho4::HIS1 ura3	This study
CAA58	rho4::ARG4/rho4::SAT1 ura3 his1	This study
CAA58MX	rho4::ARG4/rho4::URA3 his1	This study
CAA61	rho4::ARG4/rho4::HIS1 leu2::URA3/LEU2	This study
CAA62	cht3::ARG4/cht3::HIS1 leu2::URA3/LEU2	This study
CAA63	rho3::ARG4/HIS1-MET3p-RHO3 ade2::URA3-MAL2p-ras1(G13V)/ADE2	This study
CAA64	ade2::URA3-MAL2p-ras1(G13V)/ADE2 his1 arg4	This study
CAA66	rho4::ARG4/rho4::URA3 HIS1-AgTEFpENG1/ENG1	This study
CAA67	rho4::ARG4/rho4::URA3 HIS1-AgTEFpCHT3/CHT3	This study
CAA69	rho4::ARG4/rho4::URA3 leu2::HIS1-MAL2p-GFP-RHO4/LEU2	This study
CAA74	bnr1::URA3/bnr1::HIS1 leu2::SAT1-MAL2p-GFP-BNR1/LEU2 arg4	This study

^aAll strains are derivates of BWP17 with the indicated genotypic alterations.

Transformation of C. albicans.

PCR products used in transformation were amplified from pFA cassettes using primers S1 and S2 as described previously (14, 34). Primers (see Table S1 in the supplemental material) were obtained from biomers.net GmbH (Ulm, Germany). Within these primers, approximately 100 nucleotides of the target homology region are incorporated at their 5′ ends. Specific primer sequences will be provided upon request. Transformation was done either by the lithium acetate procedure or by electroporation (19, 38). Incubation periods varied between 3 and 5 days to identify transformants. For each new strain, at least two independent mutants were generated. Verification of correct integration at the target locus and the absence of the target open reading frame (ORF) in homozygous null mutants was done by PCR.

The exchange of marker genes, e.g., to convert a URA3/HIS1 strain into an ARG4/HIS1 strain, was done by transformation of the corresponding PCR products obtained from pFA plasmids (for a list of plasmids used in this study, see Table S2 in the supplemental material) and selection of transformants on the appropriate selective media. Reintegration of the URA3 marker at the CaLEU2 locus was done by using the cloned targeting cassette pSK+-CaURA3 (see below).

Promoter shutdown experiments.

To deplete cells of Rho3, shutdown experiments with MET3 promoter-controlled RHO3 strains were performed. To this end, cultures of the strains were grown overnight in minimal SD medium with the appropriate amino acid supplements. Starter cultures were diluted to an optical density at 600 nm of 0.1 in the same type of fresh medium with the addition of 3.5 mM methionine and cysteine to allow for the shutdown of RHO3 expression and then incubated for 4 h at 30°C (which results in the maximal repression of a gene expressed via a fungal MET3 promoter) (our unpublished observations) prior to further analyses. Cell cultures were then used for staining and imaging, or, in the case of hyphal induction, 10% serum was added to the cultures, which were incubated for an additional 4 to 5 h at 37°C prior to microscopy.

Molecular techniques. (i) Generation of pSK+-CaURA3.

Two PCR fragments derived from the CaLEU2 locus were amplified using primers containing restriction sites at their 5′ ends. This generated a 479-bp SacII/XbaI fragment via primers 1438 and 1439, used as a 5′ homology region, and a 633-bp XhoI/KpnI fragment via primers 1440 and 1994, used as a 3′ homology region. Both fragments were cloned into pBluescript SK(+), generating pHEIb (where HEI is high-efficiency integration). CaURA3 was excised from pFA-URA3 as a BamHI/EcoRV fragment and inserted into pHEIb. The new cassette containing the URA3 marker and the LEU2 flanks was excised via SacII/KpnI and used for transformation.

(ii) Generation of GFP cassettes.

To construct a fusion of green fluorescent protein (GFP) to the 5′ end of RHO4, we amplified the RHO4 ORF with its terminator from a plasmid library (kindly provided by Joachim Ernst, Düsseldorf, Germany), added terminal restriction sites with the primers, and cloned the PCR fragment into plasmid pFA-CaHIS1-MAL2p-GFP(GA)₆ using the unique EcoRI and ClaI restriction sites. The CaHIS1-MAL2p-GFP(GA)₆-CaRHO4 fragment was then excised as a BamHI/SacII fragment, the SacII site was blunted via Klenow treatment, and the fragment was inserted into pHEIb linearized with BamHI/EcoRV. The correct in-frame GFP-RHO4 fusion into the new plasmid 857 pHIEb-CaHIS1-MAL2p-GFP(GA)₆-CaRHO4 was verified by sequencing. The integrative cassette was released by SacI-AatII cleavage and used for the transformation of C. albicans.

To generate a GFP-BNR1 cassette, plasmid 857 was modified in that an EcoRI site in the 3′ LEU2 flank was eliminated. This was done by amplifying a LEU2 fragment using primers 2007 and 2008 and replacing the previous 3′ LEU2 flank of plasmid 857 to generate plasmid 869. Next, the HIS1 marker was replaced by SAT1. To this end, a PCR product was generated from pFA-SAT1 using primers 2013 and 2014, and the resulting fragment was cloned as BamHI-PmeI, generating plasmid 870. CaBNR1 was amplified from a plasmid library with primers 2018 and 2019 and cloned as an EcoRI/PvuII fragment into plasmid 870 cleaved by EcoRI/EcoRV, which generated plasmid 875 pHEIc-SAT1-MAL2p-GFP(GA)₆-CaBNR1. The resulting cassette was excised via SacII-KpnI and transformed into bnr1/bnr1 strain GC19. All newly generated plasmids that carried PCR-amplified inserts were checked for their correct sequences (MWG-Biotech AG, Ebersberg, Germany).

(iii) cDNA synthesis.

Total RNA was prepared from cells by acid phenol extraction. Poly(A)⁺ RNA was purified from the total RNA preparation by binding to oligo(dT) bound to a column according to the manufacturer's protocol (Oligotex; QIAGEN, Hilden, Germany). First-strand synthesis of cDNA was done using SuperScript III and oligo(dT) as a primer (Invitrogen, Karlsruhe, Germany). PCR products were cloned into the pDrive plasmid vector (PCR cloning kit; QIAGEN, Hilden, Germany). All sequencing was carried out by MWG-Biotech AG (Ebersberg, Germany).

Microscopy and staining procedures.

All microscopy was performed using an AxioplanII-Imaging fluorescence microscope (Zeiss, Göttingen, Germany) with the aid of Metamorph software tools (Molecular Devices Corp., Downington, PA) and a MicroMax1024 charge-coupled-device camera (Princeton Instruments, Trenton, NJ) as described previously (39). Fluorescence microscopy was done using the appropriate filter combinations. Chitin staining was done by directly adding calcofluor (1 mg/ml) to the cells. Actin staining with rhodamine-phalloidin was done as described previously (30).

Nucleotide sequence accession number.

The CaRHO3 sequence was deposited in the GenBank database under accession number AY534886.

RESULTS

Sequence analysis of CaRHO3.

The initial sequence information in the Candida genome database did not identify the correct sequence of CaRHO3 at the time that we started this project due to sequencing errors or misaligned contig information at the RHO3 locus. Therefore, we isolated this region from genomic DNA, cloned and resequenced this region, and identified the RHO3 ORF based on amino acid sequence identity to other Rho proteins (GenBank accession number AY534886). This required the placement of one intron in the 5′ region of the gene (see Fig. S1 in the supplemental material). To verify the presence of this intron, we generated RHO3 cDNA from mRNA via reverse transcription-PCR. The sequence of the cloned cDNA corroborates the intron that contains standard intron splice sites as well as a standard “TACTAAC” lariat site. CaRHO3 thus encodes a protein of 210 amino acids with the conserved sequence features of a Rho-type GTPase including conserved GTP-binding domains and a C-terminal CAAX motif for posttranslational truncation and modification. Comparison of CaRho3 with other fungal Rho proteins showed the highest degree of sequence identity (>70%) to the Rho3 proteins of Kluyveromyces lactis, Yarrowia lipolytica, and Ashbya gossypii. Rho proteins of these organisms showed conserved N and C termini indicating the correct length of the CaRho3.

Depletion of CaRHO3 reveals a cell polarity defect.

Deletion of the S. cerevisiae RHO3 homolog has proven difficult to do, and thus, phenotypic analyses relied on the use of temperature-sensitive mutants (17, 27). Also, a number of SRO genes were found to be “suppressors of rho3” (26). To study the effect of the depletion of CaRho3, we placed the single copy of RHO3 in a heterozygous RHO3/rho3 mutant under the control of the regulatable CaMET3 promoter and performed shutdown experiments based on the presence of methionine/cysteine in the medium. Wild-type cells are ellipsoidal in shape, with a length/width ratio of about 1.36. Cells of strain CAA50 in which the single remaining RHO3 allele is expressed under MET3 promoter control are essentially like the wild type, with a length/width ration of 1.41. Shutdown of RHO3 expression produces yeast cells of a round shape with a length/width ratio of 1.1 (Table 2). This is indicative of a cell polarity defect after shutdown resulting in nonpolarized growth.

TABLE 2.

Defects in morphogenesis resulting from RHO3 shutdown^a

Parameter	Value
	Wild type		rho3/MET3p-RHO3
	0 mM Met/Cys	3.5 mMMet/Cys	0 mM Met/Cys	3.5 mM Met/Cys
Cell shape
Length (μm)	5.89 ± 0.5	5.95 ± 0.6	6.02 ± 0.6	6.41 ± 0.7
Width (μm)	4.33 ± 0.4	4.41 ± 0.4	4.26 ± 0.5	5.78 ± 0.6
Length/width ratio	1.36	1.35	1.41	1.10
No. of counted cells	132	155	221	251
Polarized actin (%)	97	95	93	54
Depolarized actin (%)	3	5	7	46
No. of counted hyphae	100	106	111	106

^aLength and width of yeast cells were determined from cells out of exponential-phase cultures. Actin staining was done with germ tubes that were induced by 10% serum at 37°C for 4 h.

To analyze the effect of a RHO3 shutdown under hypha-inducing conditions, the expression of RHO3 was blocked by the addition of methionine/cysteine to the medium prior to the induction of hyphal growth via serum at 37°C. This procedure resulted in the initiation of germ tube formation and polarized hyphal growth with septum formation in the hyphal tube in most of the cells. However, soon after germ tube formation, hyphal tips began to swell, and polarized growth was blocked (Fig. 1A). Analysis of the actin cytoskeleton in Rho3-depleted cells showed that after shutdown, the actin cytoskeleton became depolarized, and cortical actin patches were often not found clustered in the tip (Table 2).

FIG. 1.

Hyphal growth defects of a down-regulated MET3p-RHO3 strain. (A) Wild-type hyphae showing polarized cell growth, septation, and a polarized actin cytoskeleton upon growth in medium supplemented with 10% serum at 37°C for 4 h in either the presence or the absence of methionine/cysteine in the medium. Regulated expression of RHO3 leads to wild-type-like hyphal development under inducing conditions, whereas hyphal growth is blocked and the hyphal tips swell under restrictive conditions. (B) Either strain BWP17 with its endogenous RAS1 or strain CAA64 [BWP17 with an additional ras1(G13V) allele] generates regular hyphae after 4 h in medium with maltose as the sole carbon source and containing 3.5 mM methionine and cysteine at 37°C. Under similar conditions that shut down RHO3, expression in MET3p-RHO3/rho3 strain CAA50 is not able to generate hyphae. Filamentation in strain CAA63 that carries an additional ras1(G13V) allele is also blocked. Cells were stained by calcofluor prior to photography. Bar, 10 μm.

Previously, we showed that the deletion of either the formin BNI1 or the Wiskott-Aldrich syndrome protein homolog WAL1 abolishes hyphal growth in C. albicans (25, 39). This hyphal growth deficiency could not be overcome by the expression of a constitutively active ras1 allele [ras1(G13V)], indicating that both Wal1 and Bni1 are essential components of the actin cytoskeleton downstream of the pathway that triggers hyphal growth. A constitutively active Ras1 triggered hyphal formation in the wild type already at 30°C in the absence of external inducing stimuli. Therefore, we went on to test whether ras1(G13V) could suppress the rho3 shutdown phenotype. To this end, we integrated the ras1(G13V) allele into strain CAA51. Expression of the ras1 allele was under the control of the MAL2 promoter. After the shutdown of MET3 promoter-controlled RHO3 expression, this newly generated strain, even with constitutively active Ras1, also stopped polarized hyphal growth (Fig. 1B). Thus, Rho3 is required for the polarized hyphal growth of C. albicans. Signaling via the Ras pathway is not sufficient to bypass or overcome a block in polarized cell growth posed by the deletion of RHO3. Rho3 is therefore part of a central network that regulates growth decisions.

Sequence comparison of RHO4 genes.

In a recent paper on a RHO4 homolog in N. crassa, two groups of Rho4 proteins within the ascomycete clade were identified (31). One group, which includes the Rho4 homologs of the hemiascomycetes S. cerevisiae and A. gossypii, does not reveal any striking defects upon the deletion of the respective RHO4 genes. The other group includes the archiascomycetes and euascomycetes S. pombe and N. crassa. In this group, deletion of RHO4 homologs yielded septation defects. Phylogenetic tree analyses did not place the C. albicans Crl1/Rho4 (for the sake of convenience, we will refer to CRL1/RHO4 solely as RHO4) clearly into one of these groups. A comparison of Rho4 proteins revealed a slightly higher degree of identity at the amino acid level between CaRho4 and S. pombe Rho4 (41.4%) than between CaRho4 and A. gossypii Rho4 (AgRho4) (37.1%). However, based on these analyses, we were not able to draw any conclusions regarding any functional similarities of CaCrl1/Rho4.

Deletion of CaCRL1/CaRHO4 results in a cell elongation and cell separation defect.

Next, we went on to generate deletion mutants of RHO4. In contrast to the efforts used to delete RHO3, we could readily obtain complete ORF deletions and rho4/rho4 mutant strains. Mutant cells did not show defects in polarized growth that were found in Rho3-depleted cells. Quantification of cell lengths and cell width measurements, however, indicated that in contrast to a ratio of 1.36 in the wild type, rho4 mutant cells showed a length/width ratio of 1.55. This ratio came about because rho4 cells are smaller in width than wild-type cells but more elongated. We also found that the budding pattern in the rho4 mutant was unipolar to a large extent compared to a bipolar budding pattern of the wild type (Table 3). The organization of the actin cytoskeleton was found to be similar to that of the wild type, and filament formation in the rho4 mutant cells occurred in a manner that was not distinguishable from that of the wild type under the conditions tested (Fig. 2). Interestingly, rho4 yeast cells showed a cell separation defect that resulted in the formation of chains of cells (Fig. 2A). This defect is URA3 status independent, as it does not occur in the progenitor strains used. Nuclear distribution between mother and daughter cells was not affected in rho4 strains (our unpublished results). This indicates that a deletion of RHO4 specifically interrupts the ability to finish the last stage of cytokinesis separating mother and daughter cells.

TABLE 3.

Cellular morphologies of rho4 mutant strains

Strain	Cell shape				Budding pattern
	Length (μm)	Width (μm)	Length/width ratio	No. of counted cells	% Bipolar	% Unipolar	% Random	No. of counted cells
Wild type	5.89 ± 0.5	4.33 ± 0.4	1.3	132	66.9	31.7	1.4	326
rho4/rho4 (ura3)	6.22 ± 0.7	3.99 ± 0.5	1.5	178	24.0	68.8	7.2	304
rho4/rho4 (URA3)	6.31 ± 0.7	4.05 ± 0.4	1.5	211	22.3	72.1	5.6	343

FIG. 2.

Growth pattern of Carho4 mutants. Wild-type strain SC5314 and rho4 mutant strains CAA49 and CAA57 were grown in liquid SD medium at 30°C (A) or either on plates or in liquid medium at 37°C supplemented with 10% serum (B). (A) Yeast rho4 cells show a cell separation defect that leads to chains of cells connected at the septum, as seen in the images of calcofluor (ca)-stained cells. The actin cytoskeleton (ac) appeared to be normal in rho4 cells. dic, differential interference contrast. (B) During the hyphal growth phase, no differences between wild-type and rho4 strains could be observed under all conditions tested. The bar for microscopic images is 5 μm.

Cell separation defects coupled with fast growth result in altered colony morphology.

In our efforts to construct rho4 deletion strains, we used different marker combinations. This resulted, for example, in deletion mutants that were genotypically rho4::ARG4/rho4::SAT1 (CAA58) or rho4::ARG4/rho4::URA3 (CAA57). When grown on full-medium YPD plates, these colonies showed different colony morphologies in that CAA58 showed smooth colony surfaces, while CAA57 had a wrinkled colony appearance. Independent of the marker combinations used, all rho4 mutants exhibited an identical cell separation defect (Fig. 3). We therefore analyzed these strains in more detail. The cell cycle durations of rho4 mutants in the BWP17 strain background were found to be dependent on the URA3 status. Strain CAA57, which carries one copy of the URA3 marker, showed faster doubling rates than mutants that were generated using the ARG4/SAT1 markers (Table 4). To demonstrate that the colony phenotype of these strains is dependent on the rho4 phenotype and on the cell cycle length, we exchanged the URA3 marker in CAA57 (rho4::URA3/rho4::ARG4) for HIS1 and the SAT1 marker in CAA58 (rho4::SAT1/rho4::ARG4) for URA3 and compared the growth phenotypes of these mutants. As expected, cell cycle durations changed due to the presence or absence of the URA3 marker. But, strikingly, the rho4 deletion strain that had become ura3 now formed smooth colonies, while the rho4 strain that had gained the URA3 marker could be converted to form wrinkled colonies (Fig. 3).

FIG. 3.

Colony morphology of Carho4 and Cacht3 mutants. Dilutions of the indicated strains were spotted onto either CSM minimal medium or YPD rich medium and grown for 3 days at 30°C. Marker exchange (MX) strains were generated by PCR-based gene targeting in the RHO4 locus. Marker reintegration was done by targeting the URA3 gene into the LEU2 locus of marker exchange strains. Wrinkled colony morphology was dependent on the cell separation defect of either rho4 or cht3 mutants, on the growth medium, and on the URA3 status of the strains (but not on the position of the URA3 marker in the genome), which resulted in different generation times for the mutants. WT, wild type.

TABLE 4.

Colony morphology is influenced by cell cycle lengths and cytokinesis^a

Strain	Relevant genotype	Cell cycle length (min)	Colony morphology
SC5314	URA3/URA3 HIS1/HIS1 ARG4/ARG4	73 ± 8	Smooth
BWP17	ura3/ura3 his1/his1 arg4/arg4	111 ± 10	Smooth
CAA58	rho4::ARG4/rho4::SAT1 ura3/ura3 his1/his1	108 ± 12	Smooth
CAA58MX	rho4::ARG4/rho4::URA3 his1/his1	92 ± 9	Wrinkled
CAA57	rho4::ARG4/rho4::URA3 his1/his1	88 ± 10	Wrinkled
CAA57MX	rho4::ARG4/rho4::HIS1 ura3/ura3	112 ± 15	Smooth
CAA61	rho4::ARG4/rho4::HIS1 leu2::URA3/LEU2	82 ± 13	Wrinkled
CAA3	cht3::URA3/cht3::HIS1 arg4/arg4	90 ± 11	Wrinkled
CAA3MX	cht3::ARG4/cht3::HIS1 ura3/ura3	102 ± 16	Smooth
CAA62	cht3::ARG4/cht3::HIS1 leu2::URA3/LEU2	87 ± 9	Wrinkled

^aAll strains were grown to logarithmic phase in full medium.

Recently, we identified CaCHT3 as encoding the functional homolog of the S. cerevisiae CTS1 chitinase gene (10). Deletion of CaCHT3 resulted in an even stronger cell separation defect than that observed in the Carho4 mutant. We therefore went on to analyze the colony morphology of cht3/cht3 strains of either the URA3 or ura3 genotype. Strikingly, as with the rho4 strains, we observed that cht3/cht3 URA3 strains were able to form wrinkled colonies, while cht3/cht3 ura3 strains formed smooth colonies only. To further corroborate that the change in doubling rates is solely responsible for the colony morphology phenotype in cell separation-defective mutants, we reintegrated the URA3 marker at the LEU2 locus in both rho4/rho4 ura3 and cht3/cht3 ura3 strains. As expected, the formerly smooth-colony-forming strains could be converted to the wrinkled phenotype (Fig. 3).

All of the strains tested exhibit smooth colony surfaces on minimal medium or on rich medium at lower growth temperatures (e.g., 20°C), both of which reduce doubling rates in all strains to a similar level (Fig. 3 and our unpublished results). Thus, we conclude that wrinkled colony morphology phenotypes can arise in mutant strains with delayed or inhibited cell separation when grown under optimal growth conditions allowing for fast cell cycle rates.

The Carho4 cell elongation phenotype can be suppressed by overexpression of CaENG1.

Previously, we had shown that a cht3 mutant, defective in the S. cerevisiae CTS1 homolog, has a strong cell separation defect. We went on to explore whether the rho4 cell separation defect could be suppressed by overexpressing a cell wall-degrading enzyme. To this end, we placed the C. albicans CHT3 gene, coding for a chitinase, and the ENG1 gene, which encodes an endo-1,3-beta-glucanase required for cell separation after division, under the control of the AgTEF1 promoter in the rho4 background (11) (Fig. 4). We then quantified the number of cells in aggregates in the strains used in this study (Fig. 5). Cells of the wild type occur predominantly either as single cells or as mother-daughter cells. Smaller aggregates mostly consisting of three to four cells also occurred. In the rho4 mutant, the majority of cells were in aggregates of 3 to 10 cells. This phenotype could be reversed by reintroducing a copy of RHO4. In this case, a MAL2 promoter-driven GFP-RHO4 construct was used, which was shown to be functional, as the mutant phenotype could be suppressed, depending on the carbon source used, to allow for the expression of RHO4. This also demonstrated that the mutant phenotype observed is in fact due to the deletion of rho4. We found that only the overexpression ENG1 was able to suppress the cell separation defect, while the overexpression of CHT3 did not alter the mutant phenotype (Fig. 5). Interestingly, the bnr1 mutant, which carries a deletion of both alleles of the formin BNR1, also exhibited a cell separation defect similar to that of rho4. Using the same strains, the cellular morphology was analyzed by determining the length/width ratios of the cell populations. The cell elongation phenotype of rho4 could be reversed only by reintroducing a functional RHO4 gene. Neither the overexpression of ENG1 nor that of CHT3 was able to suppress this phenotype, suggesting that Rho4 may act in two separate pathways (Fig. 5).

FIG. 4.

Suppression of the rho4 cell separation defect by ENG1 overexpression. The indicated strains were grown either on YPD plates for 3 days (upper row) or in liquid full medium (bottom row) at 30°C prior to photography. The wrinkled colony appearance of the rho4 mutant strain persists in CAA67 but is reversed to that of the wild type in CAA66, in which ENG1 is overexpressed (upper row). Microscopic images of cultured cells indicate that the rho4 cell separation defect is complemented by ENG1 but not by CHT3 overexpression (bottom row). The bar in the bottom panel is 10 μm.

FIG. 5.

Cell shape and cell aggregation in the strains used in this study. The indicated strains were grown in SM or SD media, and length/width ratios of cells of these strains were measured using Metamorph software. Cell aggregates were counted and classified into three groups as shown. Note that overexpression of ENG1 can suppress the cell aggregation phenotype but not the elongated cell morphology.

CaRho4 and CaBnr1 localize to septal sites.

To study the subcellular localization of Rho4, we generated a cassette in which RHO4 was tagged at the 5′ end with GFP and placed under the control of the MAL2 promoter. This construct was flanked by sequences that allow homologous targeting to the C. albicans LEU2 locus. The construct, although not expressing RHO4 from its endogenous promoter, was able to complement the rho4 phenotypes. This suggests that it is functional and thus may reflect the endogenous situation. Under inducing conditions that allow the expression of GFP-RHO4, we observed a distinct localization of Rho4 to septal sites in both yeast and hyphal stages (Fig. 6). Rho4 was not found to localize to the bud tip or the hyphal tip and thus apparently specifically marks the septum. Rho4 was found to persist at septal sites after septation.

FIG. 6.

Localization of GFP-Rho4 in yeast and hyphae. Cells of C. albicans strain CAA69 were used. (Top) Yeast cells expressing GFP-RHO4 (GFP) were stained with calcofluor (ca), and images of both labels were acquired with the appropriate filter sets and merged. Bright-field images are shown (differential interference contrast [dic]). Rho4-GFP fluorescence can be found at septal sites but not at the hyphal tip. (Bottom) Localization of GFP-Rho4 at septal sites in hyphal cells. Hyphal growth was induced by adding 10% serum to the growth medium. Note that the Rho4 signal persists for more than one round of septation. Bar, 5 μm.

As we reinvestigated the bnr1 mutant phenotype, we found certain similarities to rho4. Thus, we went on to investigate the subcellular localization of GFP-BNR1. Previously, it was reported that Bnr1 can be found at the hyphal tip and thus partially substitute for a loss of the second formin, BNI1. We generated a GFP-BNR1 fusion under the control of the MAL2 promoter and integrated this cassette at the LEU2 locus of a bnr1/bnr1 mutant. We chose to produce an N-terminal GFP fusion since difficulties arose with nonfunctional Bni1 versions to which GFP had been fused to the C-terminal end. The GFP-BNR1 construct appears to be fully functional, as it complements the bnr1 defects. We observed GFP-Bnr1 fluorescence only at septal sites in both yeast and hyphal stages (Fig. 7). GFP-Rho4 localizes to septal sites apparently in both mother and daughter cell. In contrast, GFP-Bnr1 localizes to a septal site from the mother side only, as is apparent by the position of the septum in the calcofluor-costained cells (Fig. 7). Thus, both Rho4 and Bnr1 exhibit a distinct localization pattern at septal sites.

FIG. 7.

Localization of GFP-Bnr1 in yeast and hyphae. Cells of C. albicans strain CAA74 were used. (Top) Yeast cells expressing GFP-BNR1 (GFP) were stained with calcofluor (ca), and images of both labels were acquired with the appropriate filter sets and merged. Bright-field images (differential interference contrast [dic]) are shown. Bnr1-GFP fluorescence can be found at septal sites but not at the hyphal tip. (Bottom) Localization of GFP-Bnr1 at septal sites in hyphal cells. Hyphal growth was induced by adding 10% serum to the growth medium. Bar, 5 μm.

DISCUSSION

C. albicans is a human fungal pathogen that changes growth forms in response to environmental stimuli or host niches (5). Based on the yeast paradigm of polarized cell growth, a number of molecular analyses in recent years have identified GTP-binding proteins as being key regulators of polarized cell growth and/or hyphal morphogenesis in a variety of fungal species including, for example, A. gossypii, C. albicans, Ustilago maydis, and S. pombe (2, 23, 41, 44). In this report, we have analyzed CaRHO3 and CaRHO4 genes. Interestingly, there seems to be little functional overlap between both genes. CaRho3 is required for sustained polarized cell growth. Using DNA array technologies, RHO3 was found to be transiently up-regulated in the initial phase of germ tube induction (29). Our difficulties in obtaining a null deletion mutant suggest that RHO3 is an essential gene. Similar results have been obtained for S. pombe (36). In A. gossypii, Rho3 is required for germ tube formation at elevated temperatures. Freshly germinated spores lyse shortly after germ tube formation at 37°C (44). Agrho3 hyphae grown at 30°C show frequent depolarization at the hyphal tip, which results in swellings similar to those seen in CaRHO3-depleted hyphae. In A. gossypii, the resumption of polarized growth follows the direction of previous cell polarity (44).

Rho3 in S. cerevisiae was shown to activate the formin Bni1 in inducing the polarized assembly of actin filaments, and AgRho3 was found to interact with AgBni1 using a two-hybrid approach (9, 18). We and others previously showed that a deletion of the BNI1 homolog in C. albicans abolishes hyphal growth (8, 22, 25). This block in polarized morphogenesis could not be overcome by constitutively active Ras1 (25). Similarly, a shutdown of RHO3 expression resulted in a block of polarized hyphal growth that could not be suppressed by constitutively activating the Ras pathway. Shutdown of RHO3 expression using the MET3 promoter and on the other hand activating the transcription of the ras1(G13V) allele required a specific nutritional regimen. Due to the leakiness of the regulatable promoters, a terminal phenotype will not be established. Rather, at a given time, a majority of cells will display the shutdown phenotype, while other cells may have a partial phenotype or are already recovering from the block of expression. In particular, this led to the observation that upon Rho3 depletion, a certain amount of cells exhibited a delocalized actin cytoskeleton, while the actin cytoskeleton was apparently normal in others. Various attempts to localize Rho3 in C. albicans using GFP were unsuccessful. The role in tip growth, however, suggests that in C. albicans, Rho3 and the formin Bni1, which is part of the C. albicans Spitzenkörper in hyphal tips, act in a common pathway.

RHO4 deletions in hemiascomycetes did not reveal a unique function for Rho4 in either A. gossypii or S. cerevisiae (26, 43). In contrast, deletion of RHO4 homologs in S. pombe or in the euascomycete N. crassa showed an involvement of Rho4 in septation (28, 31, 32). In N. crassa, deletion of RHO4 abolishes septation, causing a failure in actin ring formation. On the other hand, a dominant active allele of N. crassa RHO4 showed multiple rounds of septation at nearby distances (31). In S. pombe, rho4 cells were found to generate a cell separation defect at elevated temperatures (32). Thus, Rho4 may be involved either in septum construction or in septum degradation to allow cell separation. Consistently, Rho4 was recently shown to be involved in the secretion of the endoglucanase Eng1, and ENG1 overexpression was able to suppress the rho4 defect in S. pombe (33).

Deletion of RHO4 in C. albicans revealed a mutant phenotype of defective cell separation similar to was observed in S. pombe rho4 cells. Thus, the role of Rho4 in cell separation/septation is not confined to archiascomycetes and euascomycetes but can also be observed in hemiascomycetes. In the basidiomycete U. maydis, the activation of the U. maydis Rho protein Cdc42 by the Rho guanine nucleotide exchange factor Don1 is required for cell separation. Deletion of either DON1 or CDC42 results in a defect similar to that with a deletion of the RHO4 homologs in C. albicans and S. pombe (23, 42). This suggests that conserved signaling functions required for cellular morphogenesis were bestowed on specific but different Rho-type GTPases in the fungal kingdom.

The cell separation defect in Carho4 cells could efficiently be suppressed by the overexpression of the CaENG1 gene. Thus, regulatory events requiring Rho4 during cell separation, e.g., at the level of endoglucanase localization, may be conserved between S. pombe and C. albicans. Overexpression of the chitinase gene CHT3 did not suppress the cell separation defect. Interestingly, the overexpression of CHT3 resulted in an increased sensitivity of the cells against cell wall-perturbing agents such as 0.1% sodium dodecyl sulfate, which was not observed in ENG1-overexpressing strains (our unpublished results).

Our studies on the CaCHT3 and CaRHO4 genes revealed a relationship between cellular morphology and colonial growth. With the use of different marker combinations (“URA3 status”), the resulting mutant strains showed differing cell cycle lengths. This indicated that optimal generation times in conjunction with a lack of cell separation lead to a wrinkled colony appearance. This may be due to the larger number of cells generated in URA3 strains than that generated the otherwise isogenic ura3 strains. Due to the failure in cell separation, mother and daughter cells cannot be moved apart from each other to make way for newly emerging buds. Thus, instead of dispersing laterally, an elevated colony morphology arises. Colony morphology, therefore, is an “open” structure that varies in a stochastic manner. This also suggests an explanation for the wrinkled colony appearance during hyphal growth of C. albicans, since the hyphal growth phase also results in nonseparated cells that form filaments. Similarly, in S. cerevisiae, changes in colony morphology were observed between “fluffy” wild S. cerevisiae strains and “smooth” laboratory strains (20). Furthermore, structured colony morphology, i.e., a wrinkled appearance, was found to be linked to incompletely separated yeast cells that showed a monopolar budding pattern. Also, in Cryptococcus neoformans, an analysis of conserved genes in the RAM (regulation of Aces2 activity and morphology) signaling pathway revealed phenotypes similar to those that we observed in the Carho4 mutant strain. In that study, C. neoformans cbk1, kic1, mob2, sog2, or tao3 mutants grown on YPD medium showed cell separation defects and a “crinkled” colony morphology (40). Fungal colony morphology is thus determined in a similar manner by cell separation defects or delays in cytokinesis.

A MET3 promoter-driven RHO3 in a rho4 mutant background was not able to suppress the rho4 cell separation defect, providing further evidence that Rho3 and Rho4 fulfill separate cellular functions. We could localize C. albicans Rho4 at septal sides in both yeast and hyphal stages. Rho4 was found to persist at several septal sites in hyphae. This may allow for additional functions of Rho4 beyond vesicle delivery for septum formation or degradation during one cell cycle. Activated Rho4 could thus act to direct polarized cell growth to septal sites upon lateral branch formation, which regularly occurs at septa in C. albicans (25, 39). This notion may also provide an explanation for the altered budding pattern in rho4 yeast cells, which was found to be largely monopolar. The formin Bnr1 also was found to specifically localize at septal sites. However, the localization appeared to be in spots at the mother side of the neck rather than as a uniform ring as seen in GFP-Rho4. Thus, although mutant rho4 and bnr1 cells were elongated compared to the wild type, both proteins could act in different pathways. Elongated bud morphology has been observed in a variety of mutants in both C. albicans and S. cerevisiae. Different processes can contribute to an elongated cell phenotype: (i) maintained polarized cell growth at the tip of the bud, e.g., as seen in mutants with prolonged G₁ cyclin activity (4), (ii) delays in mitosis/nuclear distribution, e.g., as seen in dynein mutants or in mutants that activated a process collectively described as a “morphogenesis checkpoint” (24), and (iii) maintained polarization of the actin cytoskeleton at the tip of an emerging bud due to the prolonged activation of the cell polarity establishment Cdc42-Rho protein module (15). We hypothesize that cell elongation may also occur in the absence of a transport system that delivers polarity establishment functions to septal sites to redirect growth in preparation for septation. Rho4 could be well suited to elaborate such a mechanism by triggering actin cable formation, generating new tracks within the cell to promote secretion to septal sites. In its absence, this process may be taken over by other Rho proteins and Bni1, which also localizes to septal sites. However, a delay in this mechanism may prolong tip growth and thus lead to cell elongation (Fig. 8). In S. cerevisiae, polarized growth at the bud tip has been shown to depend on both positive and negative feedback loops (6, 15). This involves stabilizing Cdc24 at the sites of polarized growth by Bem1 or destabilizing the Cdc24-Bem1 interaction by phosphorylating Cdc24 via Cla4, which is itself an effector protein of Cdc42. Our model suggests a novel mechanism in which Rho4 helps to establish a new axis of polarity at the septal site. By establishing such a new site of polarity, both bud tip and bud neck may compete for either the same pool of vesicles or cellular components required for keeping up the established axis of polarity at the tip. This may finally result in breaking the tipward axis of polarity and redirecting secretion towards the bud neck. The lack of RHO4 could therefore avoid activation of a morphogenesis checkpoint and thus may not be Swe1 dependent. This model is supported by the localization of CaRho4 solely at septal sites after bud emergence, similar to what was found with S. cerevisiae (36). Furthermore, we could show, by two-hybrid analyses, that Rho4 and Bni1p interact, suggesting that a Rho4-Bni1 complex may compete with Cdc42-Bni1 (S. Seitz and J. Wendland, unpublished data). During hyphal growth, Rho4 was also observed at septal sites, suggesting that similar mechanism should operate. However, the distance between the hyphal tip and the septal site is greater than the distance between the tip of the bud and the septal site in yeast cells, which could allow both systems to operate at the same time in filaments without competing for the same set of polarity establishment proteins or secretory vesicles. Rho4 is not essential for the apical-isotropic switch but may be useful for the timely occurrence and regulation of cellular morphology. Future work will need to clarify the temporal localization pattern of Rho4 at septal sites, the mechanism of Rho4 activation and localization, the dependence of Rho4 on septin localization, and the role of Rho4 in actin filament assembly.

FIG. 8.

Model of Rho4p function during repolarization of growth to septal sites. In the wild type, a cell cycle-regulated switch from polarized apical growth to isotropic growth and repolarized growth to the bud neck occurs. Rho4p may promote the process of repolarization of growth by establishing a new axis of polarity at the bud neck. Deletion of Rho4 may therefore delay this reorientation of growth to the septal site, resulting in an elongated cell morphology.