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Mol Cell Biol. Nov 2000; 20(22): 8364–8372.
PMCID: PMC102143

Sok2 Regulates Yeast Pseudohyphal Differentiation via a Transcription Factor Cascade That Regulates Cell-Cell Adhesion


In response to nitrogen limitation, Saccharomyces cerevisiae undergoes a dimorphic transition to filamentous pseudohyphal growth. In previous studies, the transcription factor Sok2 was found to negatively regulate pseudohyphal differentiation. By genome array and Northern analysis, we found that genes encoding the transcription factors Phd1, Ash1, and Swi5 were all induced in sok2/sok2 hyperfilamentous mutants. In accord with previous studies of others, Swi5 was required for ASH1 expression. Phd1 and Ash1 regulated expression of the cell surface protein Flo11, which is required for filamentous growth, and were largely required for filamentation of sok2/sok2 mutant strains. These findings reveal that a complex transcription factor cascade regulates filamentation. These findings also reveal a novel dual role for the transcription factor Swi5 in regulating filamentous growth. Finally, these studies illustrate how mother-daughter cell adhesion can be accomplished by two distinct mechanisms: one involving Flo11 and the other involving regulation of the endochitinase Cts1 and the endoglucanase Egt2 by Swi5.

Nitrogen limitation stimulates diploid cells of Saccharomyces cerevisiae to undergo a dimorphic transition to a filamentous growth form referred to as pseudohyphal differentiation (19, 23). Filamentation represents a dramatic change in cell growth in which the cells elongate, adopt a unipolar budding pattern, remain physically connected in chains, and invade the growth medium (19, 28). Pseudohyphal differentiation may allow diploid cells to forage for limiting nutrients and may also assist haploid cells to locate distant mating partners (50).

Two signaling pathways that regulate yeast filamentous growth have been defined. The first involves components of the mitogen-activated protein (MAP) kinase pathway that also functions during mating and invasive growth in haploid cells (10, 31, 39, 52). This pathway inactivates the repressors Dig1 and Dig2, allowing the transcription factors Ste12 and Tec1 to form heterodimers that regulate expression of Tec1 itself and additional targets, such as the cell surface protein Flo11, which is required for cell adhesion and filamentous growth (2, 9, 16, 33, 37). The upstream components of this pathway include Ras2, Cdc42, and the 14-3-3 proteins Bmh1 and Bmh2 (43, 44, 51), all of which regulate pseudohyphal differentiation, possibly in response to the Sho1 osmosensing receptor (11, 48).

The cyclic AMP (cAMP) signaling pathway functions in parallel with the MAP kinase pathway to regulate pseudohyphal differentiation (34, 49, 54). This pathway involves the G-protein-coupled receptor Gpr1 and the Gα subunit Gpa2, which stimulate cAMP production by adenylyl cyclase in response to fermentable carbon sources (8, 27, 36, 45, 64, 65). Both Gpr1 and Gpa2 are required for pseudohyphal differentiation (29, 34, 36). The target of cAMP in yeast is the cAMP-dependent protein kinase, protein kinase A (PKA), which consists of a regulatory subunit, Bcy1, and three catalytic subunits encoded by the TPK1, TPK2, and TPK3 genes (59, 60). Among the three catalytic subunits, Tpk2 is required for pseudohyphal differentiation, whereas Tpk1 and Tpk3 play negative roles, likely via feedback inhibition of cAMP production (49, 53). Tpk2 activates expression of the FLO11 gene by activating the transcription factor Flo8 and inactivating the repressor Sfl1 (49, 53, 54). Therefore, the MAP kinase and cAMP pathways converge to regulate expression of the FLO11 gene, which is required for the adhesion of mother and daughter cells and the integrity of pseudohyphal filaments.

In addition to Ste12, Tec1, Flo8, and Sfl1, several other transcription factors are known to regulate filamentous growth, including Sok2, Phd1, and Ash1. Sok2 contains a basic helix-loop-helix motif that is highly conserved among a family of transcription factors that regulate fungal cell cycle progression and morphogenesis. Sok2 was originally identified as a suppressor of a temperature-sensitive PKA mutation (62). Interestingly, Sok2 also negatively regulates pseudohyphal differentiation (sok2/sok2 mutants are hyperfilamentous) and has been proposed to be a downstream effector of the PKA pathway (63).

Phd1 is a second transcription factor with a highly conserved helix-loop-helix motif that is related to Sok2 and other transcription factors. Although phd1/phd1 mutant strains do not exhibit obvious defects in pseudohyphal differentiation, overexpression of the PHD1 gene dramatically enhances pseudohyphal growth even on nitrogen-rich medium (18). Moreover, overexpression of PHD1 suppresses the pseudohyphal growth defects of tpk2 and ste12 mutant strains (16, 49), and phd1 mutations exacerbate the filamentation defect of ste12 mutants (32), indicating that Phd1 could act in a pathway distinct from the cAMP and MAP kinase pathways. The Candida albicans homologue of Phd1, Efg1, plays a prominent role in regulating filamentous growth and virulence of this human pathogen (32, 58). However, how Phd1 and Efg1 regulate filamentous differentiation is not understood in molecular detail.

Ash1 is a GATA-type transcription factor that represses expression of the HO gene in daughter cells (3, 56). The ASH1 gene is also required for diploid pseudohyphal differentiation (7). An ash1 mutation blocks pseudohyphal growth, whereas ASH1 overexpression enhances pseudohyphal differentiation and restores filamentation in ste12 mutant strains (7). Ash1 is known to regulate unipolar budding and cell elongation during pseudohyphal growth (7). However, it is not known how Ash1 is regulated during pseudohyphal growth in response to nitrogen starvation.

Swi5 is a zinc finger class transcription factor that is required for cell cycle-specific expression of the HO gene (42, 46, 57). In addition, Swi5 regulates expression of several other genes, including ASH1, SIC1, and EGT2 (3, 25, 26), and plays a minor role in regulating CTS1 expression (12, 41). EGT2 encodes the enzyme endoglucanase, and CTS1 encodes the enzyme endochitinase; both are required for proper separation of mother and daughter cells after cytokinesis (26, 30). Although Ash1 and Cts1 are known to regulate filamentous growth (7, 24), the role of Swi5 and Egt2 in pseudohyphal differentiation had not been previously examined.

Here we report our studies on the role of Sok2 in pseudohyphal growth. First, sok2/sok2 mutant strains undergo pseudohyphal differentiation on nitrogen-rich medium, and cells in these filaments are dramatically elongated. Second, the sok2 mutation enhances expression of the PHD1, ASH1, and SWI5 genes. Phd1, Ash1, and Swi5 all activate FLO11 gene expression independently of the PKA and MAP kinase pathways. Swi5 has a dual role in regulating pseudohyphal growth via Ash1-dependent activation of FLO11 expression and regulation of the expression of EGT2 and CTS1 genes, which encode enzymes required for separation of mother and daughter cells. In summary, we found that Sok2 regulates pseudohyphal differentiation via a complex cascade of transcription factors involving Phd1, Swi5, and Ash1.


Yeast strains and plasmids.

The yeast strains used (Table (Table1)1) are all isogenic with the Σ1278b background. All mutant strains were created by the PCR-mediated gene disruption technique (35, 61), using the G418 resistance cassette from plasmid pFA6-KanMX2 (61) or the hygromycin B resistance cassette from plasmid pGA32 (21). Independently derived haploid strains (created in strains MLY40α and MLY41a [Table 1]) were mated to produce homozygous diploid strains (Table (Table1).1). Haploid strains with single or double gene deletions were crossed, sporulated, and dissected to produce double or triple mutant strains.

Yeast strains used in this study

Plasmids used in this study include YEplac195 (2μm URA3 [17]), pCG38 (2μm URA3 PHD1 [18]), and pXP3 (2μm URA3 TPK2 [49]). Plasmids pXP104, pXP105, pXP114, and pXP159 are derivatives of the 2μm plasmid YEplac195 containing ASH1, SOK2, SWI5, and ASH1 under control of the ADH1 promoter, respectively. Genomic DNA of strain MLY61a/α was completely digested with the restriction enzymes PstI and SalI. DNA fragments of ~4.2 kb were gel purified and cloned into plasmid YEplac195. Clones bearing the ASH1 gene were identified with the ASH1 open reading frame (ORF) (PCR product) as a probe, and a representative ASH1 clone (pXP104) was chosen. A similar procedure was used to clone the wild-type SOK2 gene. Genomic DNA of strain MLY61a/α was completely digested with the restriction enzymes XbaI and HindIII. DNA fragments of ~4.6 kb were cloned into plasmid YEplac195 and screened with the SOK2 ORF (PCR product) as a probe. The resulting clone is pXP105. The SWI5 gene, including the ORF and 500 bp each of the 5′ and 3′ untranslated regions, was PCR amplified with Pfu-Turbo DNA polymerase using genomic DNA of strain MLY61a/α as the template. A PCR product of 5.1 kb was cloned into the 2μm plasmid YEplac195 to yield plasmid pXP114. To put the expression of ASH1 under control of an exogenous promoter, part of the ADH1 promoter (−408 to start codon) was PCR amplified with Pfu-Turbo DNA polymerase using genomic DNA of strain MLY61a/α as the template and cloned into YEplac195 vector with restriction enzymes SphI and SalI. The DNA sequence of the ASH1 ORF and 3′ untranslated region was then PCR amplified and cloned downstream of the ADH1 promoter with restriction enzyme BamHI. The resulting plasmid pXP159 confers a hyperfilamentous phenotype when transformed into wild-type strain MLY61a/α.

Media and growth conditions.

Standard yeast media and genetic manipulations were as described elsewhere (55). Limiting nitrogen medium contains 0.17% yeast nitrogen base without amino acids or ammonium sulfate (34), 2% dextrose, 2% Bacto Agar, and 50 μM (SLAD [19]), 200 μM, 500 μM (SMAD [1]), or 5,000 μM (SHAD [1]) ammonium sulfate. Standard sporulation medium was used to study the effects of the sok2 mutation on sporulation and SPO13 expression (55).


All single-colony photographs were taken directly from petri plates using a Nikon Eclipse E400 microscope with a 10× primary objective and a 2.5× trinocular camera adapter for a final magnification of 25×. With the same adapter, photographs of the doublet colonies in Fig. Fig.11 were taken with a 20× objective.

FIG. 1
Sok2 represses pseudohyphal growth and cell elongation. A homozygous wild-type strain (MLY61a/α) containing a control plasmid (YEplac195) or a 2μm TPK2 overexpression plasmid (pXP3) and a sok2Δ/sok2Δ mutant diploid strain ...

Northern (RNA) analysis.

To analyze FLO11 gene expression, haploid strains were incubated in SD-Ura (synthetic glucose minimal medium lacking uracil) liquid medium overnight, then transferred to fresh SD-Ura medium, and incubated to an optical density at 600 nm (OD600) of 1.0. Cells were washed with ice-cold water, and total RNA was isolated with acid phenol, separated in formaldehyde denaturing agarose gels, and transferred overnight by capillary action to nylon membranes. The FLO11 and ACT1 genes were then used to probe the membranes. RNA was visualized by autoradiography.

For the analysis of expression of all other genes, diploid strains were incubated in SD-Ura liquid medium overnight, transferred to fresh SD-Ura medium, and incubated to an OD600 of 1.0. Cells were washed twice with water, transferred to SLAD or SHAD liquid medium, and incubated for 2 h at 30°C. Cells were then collected and washed with ice-cold water. Total RNA was prepared and analyzed as described above.

Genome array analysis.

Genome analysis was performed as described by Cardenas et al. (6), with minor modification in total RNA isolation. Isogenic diploid wild-type (MLY61a/α) and sok2/sok2 mutant (XPY80a/α) strains, and the wild-type strain containing the 2μm PHD1 plasmid pCG38, were incubated in SD-Ura liquid medium overnight, transferred to fresh SD-Ura medium, and incubated to an OD600 of 1.0. Cells were washed twice with water, transferred to SLAD liquid medium, and incubated for 2 h at 30°C. A total of 200 OD600 units of cells for each strain was collected for total RNA preparation with an RNeasy Midi kit (Qiagen). Poly(A) mRNA was isolated from total RNA with a mini-oligo(dT)-cellulose spin column kit from 5 Prime-3 Prime Inc. (Boulder, Colo.). cDNA was prepared with the Superscript Choice system (GIBCO BRL), and biotinylated cRNA was synthesized with biotin-11-CTP and a Megascript T7 kit (Ambion). Biotinylated cRNA was fragmented by incubation at 94°C for 35 min in 40 mM Tris acetate (pH 8.1)–100 mM potassium acetate–30 mM magnesium acetate. Free unincorporated biotin nucleotides were eliminated with an RNeasy Mini kit (Qiagen). Biotinylated cRNA was hybridized to the Affymetrix yeast genome arrays at 45°C overnight. Hybridization, washing, and streptavidin staining were performed in the Affymetrix Gene Chip fluidics station 400. Gene chips were scanned in a Hewlett-Packard G2500A gene array scanner, and expression data were analyzed with the Affymetrix Gene Chip analysis suite version 3.1.


Sok2 represses pseudohyphal differentiation and sporulation.

sok2 mutations were previously found to enhance pseudohyphal growth, possibly as a downstream target of PKA (63). Several observations suggest that Sok2 may not act solely in the PKA pathway. First, sok2/sok2 mutant strains produce pseudohyphal filaments that consist of chains of highly elongated cells (Fig. (Fig.1).1). In contrast, overexpression of the Tpk2 catalytic subunit of PKA enhances pseudohyphal differentiation, but the filaments produced contain chains of round cells (Fig. (Fig.1).1). tpk1/tpk1 tpk3/tpk3 and bcy1/bcy1 mutant cells also produce hyperfilamentous colonies of round cells (data not shown). Haploid cells overexpressing Tpk2 were found to be more invasive and flocculent than haploid sok2 mutant cells on rich medium (data not shown). These findings suggest that PKA and Sok2 may regulate pseudohyphal growth via different mechanisms.

A second interesting finding was that sok2 mutant cells elongate and form pseudohyphae on nitrogen-rich medium, which completely represses filamentous growth of wild-type cells (Fig. (Fig.1).1). This observation suggests that pseudohyphal growth in sok2/sok2 mutant cells is insensitive to the presence of good nitrogen sources. Interestingly, sporulation of sok2/sok2 mutant cells in the Σ1278b strain background was also accelerated on solid sporulation medium. Within 24 h, 61% of sok2/sok2 mutant cells formed tetrads, whereas less than 1% of the wild-type cells sporulated under the same conditions (data not shown). Sporulation of the sok2/sok2 mutant could occur to a limited extent on nitrogen-rich medium, such as yeast nitrogen broth plus glucose. By Northern blot analysis, expression of the sporulation-specific gene SPO13 was induced to a much greater extent in sok2/sok2 mutant cells than in to isogenic SOK2/SOK2 wild-type cells (data not shown). Taken together, these findings indicate that Sok2 represses both filamentous growth and sporulation.

Sok2 negatively regulates expression of the PHD1, SWI5, and ASH1 genes.

To identify targets of Sok2 that regulate filamentous growth, we used whole genome array analysis. RNA was isolated from isogenic wild-type and sok2/sok2 mutant strains grown in liquid SLAD medium, and hybridization to arrays representing the whole yeast genome was performed. A large number of genes were found to be significantly altered in expression in sok2/sok2 mutant cells compared to wild-type cells. Among these genes, we focused on those that had been previously linked to regulation of filamentous growth or that regulate these genes. Interestingly, this analysis revealed that the genes encoding the Phd1, Ash1, and Swi5 transcription factors were induced 4.1- to 6.6-fold in sok2/sok2 mutant cells compared to the isogenic wild-type strain (Table (Table2).2). In addition, the EGT2 gene encoding an endoglucanase was induced 5.7-fold in the sok2/sok2 mutant strain, and expression of the EGT2 gene is known to be regulated by Swi5 (26). sok2 mutations also enhanced expression of the meiosis-specific B-type cyclin gene CLB1 (22), which is correlated with the finding that sporulation is increased in sok2/sok2 mutant strains. In a similar experiment, we found that expression of none of these genes was changed in tpk2/tpk2 mutant strains (data not shown), further suggesting that Sok2 may not act in the PKA pathway to regulate pseudohyphal growth. (The whole genome data set for the comparison of sok2/sok2 mutant and wild-type cells is available online [see the footnote to Table Table2].)2].)

Gene expression profiles in sok2Δ/sok2Δ mutants compared to the wild-type straina

Gene expression patterns in the sok2/sok2 mutant strains were also examined by Northern blotting. By this approach, expression of the ASH1, PHD1, and SWI5 genes was again found to be increased in the sok2/sok2 mutant strain (Fig. (Fig.2),2), confirming the results obtained by genome array analysis. These findings suggest that Sok2 normally represses expression of the ASH1, PHD1, and SWI5 genes and that the sok2 mutation may enhance filamentous growth by increasing expression of ASH1, PHD1, and SWI5.

FIG. 2
sok2 mutation enhances expression of the PHD1, ASH1, and SWI5 genes. Isogenic wild-type (MLY61a/α) and sok2Δ/sok2Δ mutant (XPY80a/α) strains were grown in YPD medium to an OD600 of 1.0. Cells were washed twice, transferred ...

Because Swi5 is transcriptionally expressed only during the G2/M phase of the cell cycle (47), the effect of sok2 mutation on expression of the SWI5 and ASH1 genes could be indirect if the G2/M stage of the cell cycle were prolonged. To test this, we studied cell cycle progression of sok2/sok2 mutant and wild-type strains grown in SLAD liquid medium. Cells were grown in logarithmic phase and photographed, and the unbudded (G1), small-budded (S), and large-budded (G2/M) cells were counted (>200 total cells for each strain). The sok2 mutation caused only a slight delay in G2/M phase. Considering that the sok2 mutation did not alter expression of the cell cycle-regulated CLN1 and CLB2 genes as detected by Northern analysis (not shown), it is unlikely that the sok2 mutation enhances SWI5 and ASH1 gene expression because of an indirect effect on cell cycle.

Sok2, Phd1, and Ash1 transcription factors regulate FLO11 expression.

Flo11 is normally required for pseudohyphal differentiation, and genome array analysis revealed that PHD1 overexpression enhanced expression of the FLO11 gene 3.2-fold (data not shown). We therefore examined the potential role of the SOK2, ASH1, and PHD1 genes in regulating FLO11 gene expression. Northern blot analysis revealed that the sok2 mutation enhanced FLO11 expression, the ash1 mutation impaired expression of FLO11, and the phd1 mutation had no obvious effect (Fig. (Fig.3A).3A). The sok2 mutation restored FLO11 expression in a sok2 ash1 double mutant strain, indicating that SOK2 must have targets in addition to the ASH1 gene. Consistent with this interpretation, the ability of the sok2 mutation to restore FLO11 expression in an ash1 sok2 double-mutant background was largely abrogated by introduction of a phd1 mutation (sok2 ash1 phd1 triple-mutant strain) (Fig. (Fig.3A).3A). Thus, the sok2 mutation enhances FLO11 expression and also suppresses the FLO11 expression defect of ash1 mutant cells by increasing expression of the PHD1 gene. By Northern blot analysis, neither overexpression nor mutation of PHD1 altered ASH1 expression (not shown). Ash1 also did not regulate PHD1 expression (not shown). Taken together, these findings indicate that Phd1 and Ash1 independently regulate FLO11 gene expression. Consistent with this model, the hyperfilamentous growth of sok2/sok2 mutant strains was markedly reduced by introducing both phd1 and ash1 mutations, whereas the phd1 and ash1 single mutations had only modest effects (Fig. (Fig.3B).3B).

FIG. 3
Sok2 regulates pseudohyphal differentiation and FLO11 expression via Ash1 and Phd1. (A) Sok2 regulates expression of the FLO11 gene through Ash1 and Phd1. Isogenic wild-type (MLY40α) and ash1Δ (XPY138α), phd1Δ (MLY182α), ...

Overexpression of PHD1 enhanced FLO11 expression in both wild-type and ash1 mutant cells (Fig. (Fig.4A).4A). These findings further confirm that Sok2 regulates FLO11 expression through both Phd1 and Ash1. Overexpression of PHD1 also in part restored FLO11 gene expression in tpk2 and tec1 mutant cells but not in flo8 mutant cells (Fig. (Fig.4A).4A). As a result, PHD1 overexpression restored pseudohyphal growth in ash1/ash1, tpk2/tpk2, and tec1/tec1 mutant strains (Fig. (Fig.4B).4B). Interestingly, PHD1 overexpression restored pseudohyphal growth (but not invasive growth) in both flo8/flo8 and flo11/flo11 mutant strains (Fig. (Fig.4B),4B), suggesting that Phd1 must have targets in addition to FLO11 that regulate pseudohyphal growth. In fact, like the sok2 mutation, PHD1 overexpression promoted cell elongation, whereas the phd1 mutation impaired cell elongation (data not shown).

FIG. 4
PHD1 overexpression enhances pseudohyphal differentiation through both Flo11-dependent and -independent mechanisms. (A) PHD1 overexpression enhances FLO11 gene expression. Isogenic wild-type (MLY40α) and ash1Δ (XPY138α), tpk2Δ ...

In accord with a model in which the sok2 mutation enhances PHD1 expression, pseudohyphal growth in ash1/ash1, tpk2/tpk2, tec1/tec1, flo8/flo8, and flo11/flo11 mutant strains was restored by the introduction of a sok2 mutation (data not shown). Again, like PHD1 overexpression, the sok2 mutation also restored filament formation but not invasive growth in the flo8 and flo11 mutants. Considering that enhanced pseudohyphal growth by the activated PKA pathway is largely abrogated by flo8 or flo11 mutations (49), these results again suggest that Sok2 and Phd1 act differently from the PKA pathway to regulate pseudohyphal growth.

Swi5 has a dual role in regulating pseudohyphal growth.

The transcription factor Swi5 has been shown to be required for expression of the ASH1 gene (3). Because Ash1 is required for FLO11 expression and pseudohyphal growth, we tested if Swi5 plays a similar role. As shown in Fig. Fig.5A,5A, a swi5 mutation reduced FLO11 expression, whereas overexpression of SWI5 enhanced FLO11 expression. The expression of the FLO11 gene in these strains was correlated with ASH1 expression, and the defect in FLO11 gene expression in the swi5 mutant was suppressed when ASH1 was overexpressed under control of the ADH1 promoter (Fig. (Fig.5A).5A). On the other hand, overexpression of SWI5 did not overcome the defect in FLO11 gene expression in an ash1 mutant strain. These results suggest that Swi5 indirectly activates the expression of the FLO11 gene via ASH1. In accord with a role for Swi5 in regulating FLO11, overexpression of SWI5 from a multicopy plasmid enhanced pseudohyphal growth (Fig. (Fig.5B).5B).

FIG. 5
Swi5 activates FLO11 gene expression and pseudohyphal growth via Ash1. (A) Swi5 regulates expression of the FLO11 gene through Ash1. An isogenic wild-type strain (MLY40α) containing a control plasmid (YEplac195), a 2μm plasmid expressing ...

Surprisingly, the swi5 mutation also dramatically enhanced filamentous growth (Fig. (Fig.6B).6B). This was unanticipated because the swi5 mutation prevents expression of the ASH1 and FLO11 genes, both of which are required for filamentation. We therefore analyzed how the swi5 mutation increases filamentous growth. In accord with previous studies by others (12, 26, 41), mutations in swi5 blocked EGT2 expression and partially reduced CTS1 expression (Fig. (Fig.6A).6A). Both the endochitinase Cts1 and the endoglucanase Egt2 promote mother-daughter cell separation after cytokinesis. Consistent with this interpretation, the egt2 and cts1 mutations both enhanced pseudohyphal growth and suppressed the filamentation defect of strains lacking Flo11 (Fig. (Fig.6B).6B). However, the filaments in the swi5 flo11, egt2 flo11, and cts1 flo11 mutant strains are largely confined to the surface of the agar and are therefore noninvasive (Fig. (Fig.6C).6C).

FIG. 6
swi5, egt2, and cts1 mutations enhance pseudohyphal growth. (A) Swi5 regulates expression of the CTS1, EGT2, and ASH1 genes. Isogenic wild-type (MLY61a/α) and swi5Δ/swi5Δ (XPY194a/α), sok2Δ/sok2Δ (XPY80 ...

In accord with the finding that sok2 mutations enhance SWI5 gene expression, the sok2 mutation also induced both the EGT2 and CTS1 genes (Table (Table2;2; Fig. Fig.6A).6A). Moreover, the effect of the sok2 mutation on ASH1, CTS1, and EGT2 expression was dependent on the presence of Swi5, because the expression of all three genes was reduced in a sok2 swi5 double mutant (Fig. (Fig.6A).6A). On the other hand, the ASH1 gene was not required for the expression of either the EGT2 or the CTS1 gene (Fig. (Fig.6A).6A). Taken together, these findings reveal that Sok2 negatively regulates the expression of SWI5, which activates the expression of the ASH1, CTS1, and EGT2 genes.


Genome array analysis is proving extremely useful to study signal transduction, especially to dissect complicated signaling pathways (6, 38). Here we applied this technique in conjunction with Northern blot analysis to study signaling pathways that control pseudohyphal differentiation of the yeast S. cerevisiae. Our findings support a model in which the transcription factor Sok2 regulates yeast pseudohyphal differentiation via a complex cascade of transcription factors including Phd1, Ash1, and Swi5 that regulate cell-cell adhesion (Fig. (Fig.7).7).

FIG. 7
Sok2 regulates yeast pseudohyphal differentiation via Phd1, Ash1, and Swi5. In this model, Sok2 normally represses expression of the PHD1, SWI5, and ASH1 genes. The products of these three genes activate FLO11 gene expression, which is required for pseudohyphal ...

Sok2 represses pseudohyphal differentiation by inhibiting expression of the transcription factors Phd1, Swi5, and Ash1, which activate FLO11 gene expression. The simplest hypothesis is that Sok2 directly represses the expression of PHD1 and SWI5. However, further studies are needed to determine if Sok2 directly binds to the PHD1 and SWI5 gene promoters. Swi5 has a dual role in pseudohyphal differentiation. Swi5 activates expression of the ASH1 and FLO11 genes, which are both required for pseudohyphal growth. Swi5 is also required for expression of the EGT2 and CTS1 genes. EGT2 and CTS1 encode the enzymes endoglucanase and endochitinase, which are both involved in mother-daughter cell separation after cytokinesis. As a result, overexpression of SWI5 enhances pseudohyphal growth by activating ASH1 and FLO11 gene expression, and swi5 mutation also enhances pseudohyphal growth by preventing EGT2 and CTS1 expression. In accord with this model, the swi5, cts1, and egt2 mutations all restore pseudohyphal growth in strains lacking Flo11, which is normally required for mother-daughter cell adhesion.

Sok2 may regulate pseudohyphal growth independently of PKA.

Our studies support a model in which Sok2 regulates pseudohyphal growth independently from PKA, and both pathways converge to regulate FLO11 expression (15, 49, 53, 54). In this model, Sok2 represses FLO11 expression by inhibiting expression of genes encoding the transcription factors Phd1, Ash1, and Swi5, whereas the PKA pathway activates FLO11 expression by activating the transcription activator Flo8 and inactivating the transcription repressor Sfl1 (49, 53, 54). Because the effect of PHD1 overexpression on FLO11 expression depends on the presence of Flo8, it is possible that there is cross-talk between the Sok2-regulated pathway and PKA pathway in regulating FLO11 expression. In addition, sok2 mutations promote filamentous growth by enhancing cell elongation, whereas activated PKA does not enhance cell elongation (49). Moreover, although the Sok2 protein has one site matching the PKA consensus phosphorylation site, we have been unable to detect any physical interaction between the Tpk2 catalytic subunit of PKA and Sok2 (X. Pan and J. Heitman, unpublished data). A sok2 mutation exacerbates the growth defect of a PKA-deficient strain (63), suggesting that Sok2 may be involved in a pathway other than PKA that also contributes to vegetative growth.

Sok2 represses filamentation and sporulation.

Although both pseudohyphal differentiation and sporulation occur in response to nitrogen limitation, very few mutations that affect both processes have been identified. The G proteins Gpa2 and Ras2 both activate filamentous growth and inhibit sporulation. Gpa2 stimulates pseudohyphal growth by regulating cAMP production, whereas Gpa2 inhibits sporulation by interacting with and inhibiting the Ime2 kinase (8, 13, 29, 34). Ras2 activates filamentous growth by stimulating both the PKA and MAP kinase signaling pathways, and it inhibits sporulation by increasing cellular cAMP levels (34, 40, 44, 49, 51). The opposing roles of Gpa2 and Ras2 in filamentation versus sporulation are in accord with the known role of these proteins in glucose sensing (27, 36, 65). The presence of glucose activates Gpa2 and Ras2 to promote filamentation and inhibit sporulation, whereas in the absence of fermentable carbon sources Gpa2 and Ras2 are inactive and sporulation ensues. Sok2 is the first protein identified that inhibits both filamentation and sporulation. We propose two possible models to account for this role of Sok2. First, Sok2 may act in a nitrogen-sensing pathway that promotes vegetative growth in the presence of abundant nitrogen. Alternatively, Sok2 may act as a general repressor in the differentiation processes such that, when mutated, both pseudohyphal differentiation and sporulation are enhanced.

Phd1 and Ash1 regulate FLO11 gene expression.

Overexpression of the transcription factor Phd1 enhances pseudohyphal growth and suppresses the pseudohyphal growth defects of tpk2, ash1, and ste12 mutants (7, 18, 49). However, how Phd1 regulates pseudohyphal growth was not known in molecular detail. Our studies reveal that Phd1 activates FLO11 expression and cell elongation. Although phd1 mutants have no defect in FLO11 expression, overexpression of Phd1 enhances FLO11 expression and restores FLO11 expression in tpk2 and tec1 mutant strains. Therefore, Phd1 regulates FLO11 expression independently of both the PKA and MAP kinase pathways.

Our studies also reveal that Ash1, a GATA family transcription factor, is also required for expression of the FLO11 gene. The defect in FLO11 expression and pseudohyphal growth in ash1 mutant strains is suppressed by TEC1 or TPK2 overexpression, whereas ASH1 overexpression restores FLO11 expression and pseudohyphal growth in mutant strains lacking either Tec1 or Tpk2 (Pan and Heitman, unpublished). Thus, Ash1 positively regulates FLO11 expression and pseudohyphal growth independently of the MAP kinase and PKA pathways.

Why is Ash1 required for FLO11 gene expression and pseudohyphal growth? Ash1 is localized in daughter cells in both haploid and diploid cells (7, 56). In haploid cells, Ash1 restricts mating type switching to mother cells by inhibiting HO expression in daughter cells (3, 56). In diploid cells, localized Ash1 might activate Flo11 expression in daughter cells to promote cell-cell adhesion. To test if Ash1 localization is important, we mutated the SHE2 gene required for the daughter cell localization of Ash1 mRNA in haploid vegetative cells (3). she2 mutants exhibited reduced pseudohyphal growth and FLO11 expression, although the defects were not as severe as those in ash1 mutants (data not shown). Further studies will be required to address a role for Ash1 localization in regulating filamentous growth.

Taken together, our studies reveal that Sok2 regulates FLO11 gene expression via Phd1 and Ash1. In addition to the PKA and MAP kinase pathways, Sok2, Phd1, and Ash1 constitute a third pathway that regulates FLO11 gene expression. That yeast cells employ multiple distinct pathways to regulate FLO11 gene expression further underscores the important role of cell-cell adhesion in filamentous growth.

Flo11 promotes cell-cell adhesion; glucanase and chitinase promote cell-cell separation.

Flo11 is critical for the integrity and formation of pseudohyphal filaments (33). Mutations in the TPK2, FLO8, STE12, TEC1, and ASH1 genes reduce FLO11 expression and confer defects in filamentous growth (37, 49, 54). By comparison, overexpression of FLO11 enhances pseudohyphal formation (33, 49, 53). A previous study showed that mutation of the ACE2 gene restored pseudohyphal growth in a flo8-1 mutant strain, which normally does not express Flo11 (24). Our studies also reveal that yeast cells can adhere by Flo11-independent mechanisms. One of these mechanisms involves glucan and chitin, which are both polysaccharides in the yeast cell wall that are cleaved to separate mother and daughter cells. The EGT2 gene encodes an endoglucanase, and the CTS1 gene encodes an endochitinase. egt2 and cts1 mutations impair cell-cell separation and enhance pseudohyphal growth, even in the absence of Flo11. cts1 flo11, egt2 flo11, sok2 flo8, and sok2 flo11 mutant strains form noninvasive filaments that lie largely on the surface of the agar, suggesting that Flo11 is required for agar invasion. That yeast cells can employ two distinct mechanisms to promote cell-cell adhesion during filamentous growth suggests that Flo11-independent, Swi5/Egt2/Cts1-dependent mechanisms may operate under certain physiological conditions or in other dimorphic fungi.

Relevance to pathogenic fungi.

The dimorphic transition to filamentous growth is linked to virulence in both human and plant fungal pathogens. In the corn smut Ustilago maydis, mutations in the PKA pathway confer constitutive filamentous growth and impair virulence (14, 20). Similarly, mutation of the Tup1 repressor causes constitutive filamentation in the human fungal pathogen C. albicans and attenuates virulence (4, 5). The hyperfilamentous phenotype of S. cerevisiae sok2 mutants is analogous to constitutive filamentous growth in U. maydis gpa3, uac1, and adr1 mutants and C. albicans tup1 mutants which may involve transcription factor regulatory cascades similar to the Sok2, Phd1, Ash1, and Swi5 network defined here.


We thank Maria Cardenas, Daniel Lew, John McCusker, Robin Wharton, Chris Counter, Rey Sia, and John Rhode for advice and discussions; Helena Abushamaa and Shane Cutler for assistance with genome array analysis; Miguel Arevalo-Rodriguez for experimental advice; Mike Lorenz for strains; and Steve Garrett and David Stillman for constructive criticism.

Joseph Heitman is a Burroughs Welcome Scholar in Molecular Pathogenic Mycology and an associate investigator of the Howard Hughes Medical Institute.


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