Regulators of Pseudohyphal Differentiation in Saccharomyces cerevisiae Identified Through Multicopy Suppressor Analysis in Ammonium Permease Mutant Strains

Corresponding author: Joseph Heitman, Department of Genetics, Box 3546, Duke University Medical Center, Durham, NC 27710., heitm001{at}mc.duke.edu (E-mail).

Communicating editor: M. D. ROSE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the G subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of mep2/mep2 and gpa2/gpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in mep1/mep1 mep2/mep2 and mep1/mep1 gpa2/gpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the mep1/mep1 mep2/mep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a mep1/mep1 mep2/mep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the mep1/mep1 mep2/mep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth.

WHEN diploid cells of the budding yeast Saccharomyces cerevisiae are deprived of nitrogen, they differentiate into a filamentous, pseudohyphal form. Pseudohyphal cells are characterized by an elongated morphology, altered budding pattern and cell cycle, and the ability to invade the growth substrate (GIMENO et al. 1992 ; KRON et al. 1994 ). Pseudohyphal differentiation shares both regulatory and morphological similarities with filamentous growth forms of other fungi, including the maize fungus Ustilago maydis and the animal pathogen Candida albicans. The filamentous forms of these organisms have been associated with tissue invasion and dissemination, and they are critical for virulence (BANUETT 1991 ; LO et al. 1997 ). Similarly, pseudohyphal differentiation may serve to disperse yeast cells under adverse conditions, particularly nutrient stresses.

The regulation of pseudohyphal differentiation involves at least two signaling pathways. One includes Gpa2p, the subunit of a heterotrimeric guanine nucleotide-binding protein (KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ). Genetic evidence indicates that Gpa2p regulates cAMP metabolism coordinately with Ras2p: the pseudohyphal defect of gpa2/gpa2 strains and the synthetic growth defect of gpa2 ras2 strains are both suppressed by increasing cAMP concentrations (KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ; XUE et al. 1998 ). In addition, either addition of exogenous cAMP or expression of a dominant-active GPA2 allele induces filamentous growth in nitrogen-rich conditions that normally repress this differentiation pathway (LORENZ and HEITMAN 1997 ). Activated RAS2 alleles, elevated cAMP levels, or expression of a dominant-active GPA2 allele confers sensitivity to heat shock and alters glycogen accumulation (TODA et al. 1985 ; XUE et al. 1998 ). Genetic evidence suggests that some of these phenotypes are mediated by Sch9p, a protein kinase related to protein kinase A (PKA; XUE et al. 1998 ). Overexpression of SCH9 can suppress the lethality of deletions of both RAS genes or of the sole adenylyl cyclase gene CYR1 (TODA et al. 1988 ), and Sch9p has been proposed to act in a pathway that is parallel to PKA. The role of Sch9p in filamentous growth is presently unknown.

A second signaling pathway regulating pseudohyphal differentiation includes elements of the haploid pheromone-responsive MAP kinase cascade, including the protein kinases Ste20p, Ste11p, Ste7p, and Kss1p and the transcriptional activator Ste12p (LIU et al. 1993 ; COOK et al. 1997 ; MADHANI et al. 1997 ). Addition of exogenous cAMP or expression of the dominant GPA2-2 allele suppresses the pseudohyphal defect of strains lacking MAP kinase elements (LORENZ and HEITMAN 1997 ). How this pathway is activated to effect filamentous growth is not understood, though the pheromone receptors and the Gpa1p/Ste4p/Ste18p heterotrimeric G protein are not involved (LIU et al. 1993 ). Ras2p likely plays a dual role in regulating both the MAP kinase pathway and cAMP metabolism (GIMENO et al. 1992 ; MOSCH et al. 1996 ; ROBERTS et al. 1997 ).

A characteristic of nitrogen-starvation-induced filamentation is the ability of cells to invade the agar substrate. A similar phenomenon has been described in haploid cells grown on rich medium (ROBERTS and FINK 1994 ). Haploid-invasive growth is not a response to nitrogen deprivation, though it shares many similarities with pseudohyphal differentiation, including changes in cell polarity and filament formation (ROBERTS and FINK 1994 ). In addition, the same MAP kinase elements that regulate diploid filamentation also regulate haploid invasion (ROBERTS and FINK 1994 ; COOK et al. 1997 ; MADHANI et al. 1997 ). The mechanism by which signal specificity between invasion and pheromone response is achieved in haploid cells is not fully understood, but it involves the Ste12p-binding proteins Dig1p and Dig2p (COOK et al. 1996 ) and specialization of the MAP kinases Fus3p and Kss1p for mating and filamentous/invasive growth (COOK et al. 1997 ; MADHANI et al. 1997 ).

The high-affinity ammonium permease Mep2p is required for pseudohyphal differentiation in response to ammonium limitation and may serve as a sensor of nitrogen starvation (LORENZ and HEITMAN 1998 ). mep2/mep2 mutant strains do not exhibit a filamentation defect in the presence of other nitrogen sources, such as glutamine or proline, indicating that the function of Mep2p is ligand specific. Mutations in the lower-affinity ammonium permeases Mep1p and Mep3p do not confer defects in pseudohyphal differentiation. Because mep2/mep2 strains have no discernible alterations in ammonium uptake or growth rates, we have proposed that Mep2p has a signaling role to regulate filamentation (LORENZ and HEITMAN 1998 ). Activation of the Gpa2p/cAMP-signaling pathway restores pseudohyphal growth in mep2/mep2 strains (LORENZ and HEITMAN 1998 ), suggesting that Mep2p may function upstream in this signaling cascade.

To address the mechanisms by which Mep2p senses ammonium, we further examined the role of the three ammonium permeases and uncovered a potential role for Mep1p in the regulation of pseudohyphal differentiation. Activation of the MAP kinase pathway is more effective in restoring filamentation to mep1/mep1 mep2/mep2 and mep1/mep1 gpa2/gpa2 strains than in the corresponding MEP1⁺ parent strains. Thus, the Mep1p ammonium permease inhibits the filamentous growth response under some conditions.

We took advantage of the unique phenotype of the mep1/mep1 mep2/mep2 double-mutant strain to isolate high copy suppressors of the filamentation defect. Overexpression of 17 genes suppressed either the growth or pseudohyphal defects of the mep1/mep1 mep2/mep2 strain on low-ammonium media. Among these are the known pseudohyphal regulators Phd1p, Phd2p (Mss10p, Msn1p, and Fup4p), and Tec1p (GIMENO and FINK 1994 ; GAVRIAS et al. 1996 ; LAMBRECHTS et al. 1996 ), as well as proteins not previously known to affect filamentous growth, including Cdc6p, Mss11p, Mga1p, Skn7p, Msn5p, Dot6p, and two uncharacterized open reading frames that we have named HMS1 and HMS2 (for high-copy MEP suppressor). Several of these proteins have been previously implicated in the cellular response to changes in carbon source (Phd2p, Mss11p, and Mga1p) or oxidative damage (Skn7p), suggesting that nitrogen limitation may not be the only environmental signal that can trigger pseudohyphal differentiation. Epistasis analysis between these suppressors and previously characterized pseudohyphal regulators illustrates the complexity of filamentous growth and suggests that the cell integrates multiple signals via several pathways to control differentiation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and genetic methods:
Standard yeast media and genetic methods were used (SHERMAN 1991 ). SLAD media, for induction of filamentous growth, contains 0.17% yeast nitrogen base, 50 µM (NH₄)₂SO₄, 2% Bacto-agar, and 2% glucose (GIMENO et al. 1992 ; LORENZ and HEITMAN 1997 ). SLADG, to induce the pGAL-STE12 allele, contains 2% galactose and 0.13% glucose (LIU et al. 1993 ). SLARG, to induce the pGAL-GPA2 alleles, contains 2% raffinose and 0.5% galactose (LORENZ and HEITMAN 1997 ). Epistasis experiments with mep2 and mep1 mep2 strains (Figure 1) were performed on media containing 100 µM ammonium sulfate and 3% Noble agar, as Bacto-agar contains low concentrations of nonammonium nitrogen sources that can complicate analysis of Mep2p function.

View larger version (133K): In this window In a new window Download PPT slide   Figure 1. mep1 mutations alter epistasis of mep2 or gpa2 mutations with the MAP kinase cascade. Homozygous diploids of the indicated genotype (wild type, MLY61; gpa2/gpa2, MLY132a/; mep1/mep1 gpa2/gpa2, MLY135a/) expressing the indicated alleles (vector, YEplac195; STE11-4, pSL1509; TEC1, p2.5-2; PHD1, pCG38) were incubated on SLAD medium for 4 days at 30°. Strains expressing the pGAL-STE12 (pNC252) allele were incubated on inducing SLADG medium for 6 days at 30°. mep2/mep2 (MLY108a/) and mep1/mep1 mep2/mep2 (MLY115a/) strains were incubated on media containing 3% Noble agar instead of Bacto-agar (see MATERIALS AND METHODS) for 4 days (6 days for the pGal-STE12 allele) at 30°.

Yeast strains are listed in Table 1 and plasmids in Table 2. Strains deleted for each of the multicopy suppressors were constructed in strains MLY40 and MLY41 by replacing the coding sequences with a gene encoding G418 resistance using the PCR-mediated disruption technique of WACH et al. 1994 . MLY40 and MLY41 were derived from strains 1278b and 3962c (BECHET et al. 1970 ), respectively. Candidate disruptants in MLY40 and MLY41 were confirmed by PCR and crossed to construct the homozygous diploid strains.

Multicopy suppressor screen:
The mep1/mep1 mep2/mep2 strain (MLY115a/) was transformed with a library made from strain CAY1 in the 2µ-URA3 vector pRS426 (provided by C. Alarcon). Transformants were selected on SD-Ura medium, pooled, diluted, and replated to SLAD at a density of 1000 cells per plate. Plates were screened microscopically after 4–6 days at 30° for filamentous colonies. Plasmid loss and rescue/retransformation tests demonstrated that the filamentous phenotype was plasmid dependent. Twenty-seven unique plasmids were characterized via sequencing and restriction mapping to identify the genomic region carried by the plasmid. Where necessary, plasmids were subcloned to identify the specific open reading frame responsible for the phenotype.

Phenotypic analysis of suppressor deletion strains:
Strains deleted for each of the suppressor genes were analyzed for their effect on filamentous growth. We assayed the morphological response (see Figure 3) and the three activities that comprise pseudohyphal differentiation, cell polarity, cell elongation, and invasive growth (see Table 7), as defined by MOSCH and FINK 1997 . Previous work had found that CDC6 is essential (HARTWELL 1976 ; ZHOU et al. 1989 ), and it was not analyzed in these experiments. In addition, SRK1 is polymorphic in various yeast strains, has pleiotropic effects, and, as such, was excluded from further analysis.

View larger version (55K): In this window In a new window Download PPT slide   Figure 2. Pseudohyphal phenotypes conferred by the mep1 mep2 multicopy suppressors. The mep1/mep1 mep2/mep2 strain MLY115a/ expressing each of the genes listed from a multicopy plasmid were incubated on SLAD medium for 6 days at 30°.

View larger version (70K): In this window In a new window Download PPT slide   Figure 3. Pseudohyphal phenotypes of the suppressors in wild-type strains. Wild-type strains (MLY61) expressing each of the multicopy suppressors indicated were incubated on SLAD medium for 4 days at 30°.

View this table: In this window In a new window   Table 3. Identities of the mep1/mep1 mep2/mep2 suppressors

Invasive growth assays: Nitrogen-starvation-induced invasive growth was assayed as described by MOSCH and FINK 1997 by streaking diploid strains to SLAD medium and incubating at 30° for 3 days. The colony positions were marked, and the surface cells were washed off the plate. Invasive colonies were identified by microscopic analysis. Invasive growth was quantified using the system of MOSCH and FINK 1997 : ++++, >90% of colonies invaded; +++, 70–90%; ++, 30–70%; +, 5–30%; -, <5%. At least 50 colonies were assayed for each strain.

Haploid-invasive growth was assayed as described (ROBERTS and FINK 1994 ; COOK et al. 1997 ). Haploid strains (MATa) were grown on YPD medium at 30° for 3 days. Surface cells were washed off, and the plate was incubated for 30 hr at 30° to better visualize invaded cells.

Cell morphology assays: Cell elongation was assayed as described by MOSCH and FINK 1997 . Diploid strains were grown on SLAD medium for 3 days at 30°. Surface cells were washed off the plate as in the invasion assay, and cells that had invaded the substrate were scraped out of the agar and analyzed microscopically for the proportion of elongated pseudohyphal cells vs. ovate yeast form cells. At least 200 cells were counted for each strain.

Bud site determination assays: Cell polarity and budding pattern were analyzed as described by MOSCH and FINK 1997 . Diploid strains were grown in YPD medium to an OD₆₀₀ ~0.4–0.6 at 30° and then fixed for 2 hr in 3.7% formaldehyde at room temperature. Cultures were washed three times in water and then incubated in 1 mg/ml Calcofluor (Fluorescent Brightener 28; Sigma, St. Louis) for 10 min in the dark. Cells were washed twice in water and spotted to polylysine-coated microscope slides. Fields of cells were photographed at x100, and these photos were used to identify cells with two or more bud scars as being bipolar (scars on both ends), unipolar/axial (scars on only one end), or random (scars not localized to the ends). At least 100 cells were counted for each strain.

Photomicroscopy:
Whole-colony photographs were taken at x25 using a 35-mm camera connected to the trinocular objective of a Nikon Axiophot-2 microscope. To control the density-dependent variability of filamentous growth, strains were streaked to plates at six sectors per plate. We endeavored to photograph colonies whose phenotype was representative of the strain being assayed.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A role for Mep1p as an inhibitor of pseudohyphal differentiation:
Our previous studies revealed that the ammonium permease Mep2p and the G protein Gpa2p are required for pseudohyphal growth (LORENZ and HEITMAN 1997 , LORENZ and HEITMAN 1998 ). In previous epistasis experiments, the filamentation defect of mep2/mep2 and gpa2/gpa2 mutant strains could be readily suppressed by dominant-active GPA2 or RAS2 mutants and by cAMP, but not by activation of the MAP kinase cascade via the dominant STE11-4 allele. Because of the complexity of the signaling pathways regulating pseudohyphal differentiation, as well as the presence of both multiple branchpoints and parallel signaling pathways, we have further analyzed the epistatic relationships between the Mep2p and Mep1p permeases, Gpa2p, and the MAP kinase cascade. Two important observations emerge from this analysis.

First, as shown in Figure 1, modest activation of the MAP kinase cascade by the dominant-active STE11-4 allele does not suppress the filamentation defect of either mep2/mep2 or gpa2/gpa2 mutant strains. We now find that more marked activation of the MAP kinase cascade by overexpression of STE12 can suppress the pseudohyphal differentiation defects of mep2/mep2 and gpa2/gpa2 mutant strains. Expression of the STE11-4 allele does restore filamentation in mutant strains lacking the upstream MAP kinase component Ste20p (LIU et al. 1993 ); thus, this allele can suppress a mutation in the same pathway, but it cannot suppress mutations in genes in the parallel Mep2p/Gpa2/cAMP pathway. Instead, the relatively stronger phenotype of STE12 overexpression is required to cross-suppress mep2/mep2 or gpa2/gpa2 mutations.

More importantly, this epistasis analysis indicated that the Mep1p ammonium permease can, under some conditions, function to inhibit pseudohyphal differentiation. For example, whereas expression of STE11-4 fails to suppress the filamentation defect of either mep2/mep2 or gpa2/gpa2 strains, STE11-4 does suppress mep2/mep2 mep1/mep1 and gpa2/gpa2 mep1/mep1 double-mutant strains (Figure 1). Deletion of the MEP1 gene in an otherwise wild-type background does not reduce or enhance filamentous growth, even in response to a range of ion concentrations from 5 µM to 5 mM (LORENZ and HEITMAN 1998 ; data not shown); thus, the Mep1p ammonium permease inhibits pseudohyphal differentiation only under certain conditions.

One explanation for the difference in epistasis between mep1 mep2 and MEP1 mep2 strains is that the growth defect of the mep1 mep2 strain, resulting from impaired ammonium uptake, predisposes the cell to nitrogen-dependent differentiation. Two lines of evidence indicate that this is not the case. First, lowering the ammonium concentration in the medium to a level below that in standard pseudohyphal media (5 µM in this experiment vs. 50 µM standard) does not alter the epistasis behavior of the mep2/mep2 strain. Second, the gpa2/gpa2 mep1/mep1 mutant does not have a growth defect, indicating that ammonium import is not the basis of these differences (data not shown).

In the epistasis experiment shown in Figure 1, we also expressed the transcription factors Phd1p and Tec1p, which strongly enhance pseudohyphal growth when expressed from a multicopy vector (GIMENO and FINK 1994 ; GAVRIAS et al. 1996 ). As with STE11-4 or STE12 overexpression, induction of filamentation by high-copy expression of PHD1 or TEC1 was more dramatic in the mep1 strains relative to the MEP1⁺ strains. This provides further evidence for an inhibitory function of Mep1p in response to genetic alterations in the regulation of pseudohyphal differentiation.

Multicopy suppressor analysis of mep1/mep1 mep2/mep2 mutants:
To address the role of Mep1p and Mep2p in the regulation of pseudohyphal differentiation, we screened for genes that suppress the mep1/mep1 mep2/mep2 pseudohyphal defect when present in high copy number. The mep1/mep1 mep2/mep2 strain (MLY115a/) was transformed with a yeast genomic DNA library in the high-copy (2µ) URA3 vector pRS426. Transformants were selected on synthetic medium lacking uracil, pooled, diluted, and replated at ~1000 cells per plate on SLAD media. Colonies were screened after 4–6 days for filament formation or for significantly enhanced growth under these conditions. After plasmid loss and plasmid rescue/retransformation controls, 27 unique plasmids (with distinct restriction patterns) representing 17 genomic loci, which suppressed the mep1/mep1 mep2/mep2 phenotype were identified.

The identity and phenotypes of these suppressors are shown in Table 3 and Figure 2. The MEP2 gene itself was identified four times. In addition, we isolated both MEP1 (five times) and MEP3 (once); overexpression of either MEP1 or MEP3 mostly suppresses the growth defect of mep1/mep1 mep2/mep2 strains, although a few filaments were present in each case. Several other primarily growth suppressors were identified, including URE2, DAL80, and SRK1. In the presence of favored nitrogen sources, such as ammonium or glutamine, Ure2p inhibits Gln3p, a GATA family transcription factor that regulates the expression of many genes necessary for the assimilation of alternative nitrogen sources (such as proline or urea). Ure2p has weak homology to glutathione S-transferases and is a prion analog, but its mechanism of action is not known (COSCHIGANO and MAGASANIK 1991 ; WICKNER 1994 ; XU et al. 1995 ). ure2/ure2 (and gln3/gln3) mutants also have a filamentation defect (LORENZ and HEITMAN 1998 ). Dal80p is a DNA-binding protein that negatively regulates many of the same pathways as Gln3p and Ure2p (CUNNINGHAM and COOPER 1991 ; DAUGHERTY et al. 1993 ). Srk1p is a protein of unknown function that has pleiotropic effects on many cellular processes, including RNA polymerase function (CHIANNILKULCHAI et al. 1992 ; STETTLER et al. 1993 ), chromosome stability (UESONO et al. 1994 ), phosphatase function (SUTTON et al. 1991 ; WILSON et al. 1991 ), and polarized cell growth and morphology (COSTIGAN and SNYDER 1994 ; COSTIGAN et al. 1992 ; KIM et al. 1994 ).

The other class of genes identified in this screen did not significantly affect the growth rate of the mep1/mep1 mep2/mep2 parent strain, but did suppress the filamentation defects (Table 3). We identified PHD1, PHD2 (also known as MSS10, MSN1, FUP4), and TEC1, each of which are predicted to be transcription factors and are known to regulate filamentous growth (GIMENO and FINK 1994 ; GAVRIAS et al. 1996 ; LAMBRECHTS et al. 1996 ). In addition, several genes not previously implicated in pseudohyphal growth were identified. These include MSS11, whose product, like Mss10p/Phd2p, regulates the STA1, STA2, and STA3 glucoamylase genes on the basis of the carbon source; overexpression of MSS11 can rescue expression of STA2 in mss10 strains (WEBBER et al. 1997 ). MSS11 encodes a protein very similar to the Ssn6p transcriptional repressor. Ssn6p and Tup1p form a heterodimer that mediates glucose response in conjunction with Snf1p signaling (SCHULTZ and CARLSON 1987 ; WILLIAMS et al. 1991 ; KELEHER et al. 1992 ). Tup1p has also been implicated in filamentous growth in both S. cerevisiae and C. albicans (BRAUN and JOHNSON 1997 ). MGA1 encodes a heat shock transcription factor homolog of unknown function that was identified as a multicopy suppressor of a snf2/gam1 mutation (ZHANG et al. 1997 ). MSN5 was isolated in the same snf1 suppressor screen that identified MSN1 (PHD2; ESTRUCH and CARLSON 1990 ); the function of Msn5p is not known.

Other genes identified include SKN7, which encodes a homolog of response regulator proteins of bacterial two-component systems that appears to mediate several cellular processes, including cell wall biosynthesis, cell cycle control, and response to oxidative stress (MOYE-RAWLEY et al. 1989 ; BROWN et al. 1993 ; BROWN and BUSSEY 1993 ; KUGE and JONES 1994 ; MORGAN et al. 1995 ). Overexpression of CDC6, which encodes an essential component of the origin recognition complex, also suppresses the mep1 mep2 pseudohyphal defect. DOT6 was recently identified as a mutation that affects telomeric silencing.

We also identified two genes that had only systematic ORF designations from the yeast genome project, which we have renamed HMS. Both are predicted to be DNA-binding proteins; Hms1p is in the myc family, while Hms2p is similar to heat shock trascription factors.

The rationale behind this screen was to identify genes that may mediate the signaling function of Mep2p, hopefully including direct effectors. We identified each of the MEP genes, as well as the known pseudohyphal regulators PHD1 and TEC1, demonstrating that the screen worked successfully. However, almost all the genes we identified are likely to be general regulators of pseudohyphal differentiation, an idea confirmed by the experiments described below, and are not specific effectors of Mep2p function. The multiplicity of predicted DNA-binding proteins (11 in this screen) was quite surprising and reaffirms the complexity of this differentiation pathway.

Epistasis with other pseudohyphal-deficient mutants:
We expressed each of the genes identified here in a wild-type strain (MLY61) to test if this would enhance filamentation (e.g., overexpression of PHD1, PHD2, or TEC1 is known to stimulate pseudohyphal differentiation in wild-type cells; GAVRIAS et al. 1996 ; GIMENO and FINK 1994 ). As shown in Figure 3 and Table 4, most of these genes conferred a hyperfilamentous phenotype, including MGA1, CDC6, SKN7, MSN5, and HMS1. Strains were classified as hyperfilamentous on the basis of two criteria: first, colony morphology (more vigorous filamentation than wild type on limiting ammonium SLAD medium; see Figure 3) and, second, induction of filamentation on higher ammonium media that normally repress differentiation (10-fold higher than in SLAD; see Table 4). None of these inducers, however, are as potent as the GPA2^Val132 allele or cAMP, both of which allow filamentous growth, even on nitrogen-rich media with 5 mM ammonium (LORENZ and HEITMAN 1997 ). Filamentation was also assayed on medium containing 100 µM glutamine (Mgln; Table 4). The pseudohyphal phenotypes are similar between cells grown in the presence of ammonium or glutamine, indicating that these suppressors are not specific effectors of Mep2p action (which might be ammonium specific), but instead are general regulators of dimorphism. As expected, the growth suppressors (MEP1, MEP3, URE2, DAL80, and SRK1) had no affect on filamentation when expressed in wild-type strains.

We next expressed the suppressor genes in strains lacking MEP2, GPA2, or STE11 to examine the epistasis relationships between these mutations and suppressor function (Table 5). As with the MAP kinase epistasis experiment (Figure 1), filamentous growth induced by most of these suppressors was more vigorous in the mep1/mep1 mep2/mep2 double-mutant strain than in the mep2/mep2 single mutant (Table 5). Similarly, the phenotype conferred by expression of these suppressor genes was weaker in the gpa2/gpa2 single-mutant strain than in the gpa2/gpa2 mep1/mep1 strain (Table 5). Thus, these findings again indicate that Mep1p functions to inhibit filamentous growth. In contrast, all these genes, except for MGA1, suppressed the filamentation defect conferred by the ste11 mutation, and most did this relatively well (Table 5). The finding that multicopy expression of TEC1 suppresses the defect of a ste strain (ste11/ste11 in this case) is in contrast to an earlier report in which TEC1 overexpression did not restore filamentation in a ste12/ste12 strain (GAVRIAS et al. 1996 ). The presence of Ste12p, even in its basal, unactivated state (resulting from the MAP kinase pathway mutation), may be required for suppression of ste11 by TEC1, as Ste12p and Tec1p form a heterodimer thought to be critical for the filamentation response (GAVRIAS et al. 1996 ; MADHANI and FINK 1997 ). In summary, most of the genes identified as suppressors of the mep1/mep1 mep2/mep2 pseudohyphal growth defect appear not to be specific effectors of Mep2p action, but rather, general downstream elements that likely regulate transcriptional responses necessary for pseudohyphal differentiation.

Deletion analysis of the multicopy suppressors:
We constructed an isogenic series of strains lacking each gene through a PCR-mediated disruption protocol (WACH et al. 1994 ). Homozygous diploids were analyzed for pseudohyphal phenotypes, as shown in Figure 4 and Table 6. Several of these mutations were known to confer pseudohyphal defects, including tec1 (GAVRIAS et al. 1996 ), phd2 (LAMBRECHTS et al. 1996 ), and ure2 (LORENZ and HEITMAN 1998 ). In addition to these genes, mss11 and mga1 mutations confer strong pseudohyphal defects; msn5/msn5 mutant strains have a moderate defect in filamentous growth (Figure 4; Table 6). Strains lacking PHD1, SKN7, DAL80, DOT6, HMS1, or HMS2 do not have significant filamentation defects (Figure 4, Table 6; the phd1 phenotype has been reported previously; GIMENO and FINK 1994 ).

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. Pseudohyphal phenotypes of the suppressor deletion strains. An isogenic strain series lacking each of the multicopy suppressor genes was constructed as detailed in MATERIALS AND METHODS (see also Table 7). These strains were incubated on SLAD medium for 4 days at 30°.

We next tested if alleles known to stimulate filamentation, such as GPA2^Val132 (LORENZ and HEITMAN 1997 ), RAS2^Val19 (GIMENO et al. 1992 ), STE11-4 (LIU et al. 1993 ), or overexpression of PHD1 (GIMENO and FINK 1994 ) or TEC1 (GAVRIAS et al. 1996 ), would continue to do so in the context of these deletion strains. The dominant active GPA2^Val132 allele suppressed each of these deletions, with the exception of tec1/tec1 strains (Table 6). This finding was unexpected, as GPA2^Val132 suppresses ste mutations. Because Tec1p is thought to act as a dimer with Ste12p (MADHANI and FINK 1997 ), we anticipated that the phenotype of the activated GPA2 allele would be similar in both ste12/ste12 and tec1/tec1 strains. In combination with the finding presented above that TEC1 overexpression suppresses the filamentous growth defect of ste11/ste11 (Table 4) but not ste12/ste12 mutant strains (GAVRIAS et al. 1996 ), these results indicate that Tec1p may act at multiple steps or that it may have a function that is not shared with Ste12p in the regulation of pseudohyphal differentiation.

The epistasis data summarized in Table 6 presents a complicated picture. Each of the mutants tested had a pseudohyphal defect, indicating that they are all required for full activation of filamentous growth. With the exception of tec1/tec1, all these mutations were suppressed by at least one of the pseudohyphal stimulatory alleles (or by galactose as a carbon source); thus, it appears that none of these pathways are absolutely essential for the dimorphic transition and each can be bypassed under some conditions. This analysis also indicates a central role for the transcription factor Tec1p in the regulation of filamentous growth. While the role of nitrogen starvation in inducing pseudohyphal differentiation is quite clear, combinations of environmental stresses may also serve to activate this developmental fate, similar to the initiation of meiosis in response to the combination of nitrogen starvation and a nonfermentable carbon source. Several parallel pathways may then be coordinately regulated to properly control dimorphism.

Phenotypic analysis of suppressor deleletion strains:
MOSCH and FINK 1997 separated the phenomenon of pseudohyphal differentiation into its constituent activities: cell elongation, budding pattern (cell polarity) changes, and substrate invasion. This analysis identified mutations that affect all these behaviors, as well as mutations that affect only a subset, e.g., blocking the polarity change without affecting morphology or invasion. We examined each of the strains deleted for a given suppressor gene to determine which properties might be altered.

We assayed substrate invasion associated both with nitrogen starvation in diploid cells and on rich media in haploid cells (GIMENO et al. 1992 ; ROBERTS and FINK 1994 ), as described in MATERIALS AND METHODS. tec1, phd2, and mss11 strains have significant defects in both haploid and diploid invasion (Table 7); mep2, mep1 mep2, and msn5 mutations affect only diploid (starvation-induced) invasion.

To assess the ability of the various mutant strains to adopt an elongated shape, we analyzed the morphology of nitrogen-deprived cells, as described by MOSCH and FINK 1997 and in MATERIALS AND METHODS. Of wild-type cells that have invaded the agar substrate, 12.8% have an elongated morphology (Table 7). This is reduced to 2.9% in mep2/mep2 mutants, though interestingly, the mep1/mep1 mep2/mep2 double-mutant strain does not have a defect in cell elongation. This finding may, in part, explain the epistasis difference between the single- and double-mutant strains. tec1/tec1, mss11/mss11, and ure2/ure2 strains each have severe defects in cell elongation, but mga1/mga1 and msn5/msn5 strains have little or no alteration in the number of elongated cells after growth on nitrogen-starvation media (Table 7). phd2/phd2 strains, despite the defect in filamentous growth assayed by colony morphology, actually have an increase in the number of elongated cells (approximately twofold more than wild type, see Table 7). Among mutants with wild-type colony morphology, phd1/phd1, dot6/dot6, and dal80/dal80 strains do have moderate defects in cell elongation. Thus, the colony morphology of these strains does not necessarily reflect the cellular morphology accurately.

To form filaments, cells must be able to adopt a unipolar budding pattern, which is an alternate form of the standard diploid bipolar pattern in which buds can emerge from either end of the cell. In unipolar division, the buds emerge predominantly from the end of the cell opposite its "birth end," the end at which that cell was attached to its mother. Haploid cells bud in an axial pattern, in which all buds form at the birth end, adjacent to previous bud sites. We analyzed budding patterns using the fluorescent dye Calcofluor (Fluorescent Brightener 28; Sigma) to stain the chitinous scars that mark previous bud sites (see MATERIALS AND METHODS). These assays were performed in diploid cells grown in rich (YPD) media; thus, we expected predominantly bipolar budding patterns. In the wild-type strain MLY61, 59.3% of the cells show the bipolar pattern, 27.8% axial or unipolar, and 13.0% random (Table 7). Both mep2/mep2 and mep1/mep1 mep2/mep2 mutants increase the percentage of random bud patterns (to 21.8 and 24.0%, respectively). Likewise, the number of cells budding randomly was significantly increased in phd2/phd2 (49.6%), mss11/mss11 (38.9%), mga1/mga1 (40.7%), and ure2/ure2 (41.7%) mutants; tec1/tec1 strains had only a minor effect on budding pattern, although this strain does have defects in elongation and invasion. Genes that did not confer a colony morphology defect did not alter budding pattern significantly (Table 7).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cross talk between distinct signaling pathways regulates filamentous growth:
Previous work has demonstrated that filamentous growth in yeast is regulated by at least two distinct signaling pathways, one including elements of the pheromone-responsive MAP kinase cascade and the other involving cAMP signaling (LIU et al. 1994 ; KUBLER et al. 1997 ; LORENZ and HEITMAN 1997 ). Genetic evidence is consistent with a model in which the cAMP signaling branch could be activated via a Mep2p-dependent mechanism upon ammonium starvation (LORENZ and HEITMAN 1997 , LORENZ and HEITMAN 1998 ). Several lines of evidence indicate that there may be some cross talk between these two pathways. First, activation of the cAMP signaling pathway, by dominant mutations in RAS2 or GPA2, or by cAMP, suppresses the morphological defects associated with MAP kinase mutant strains (LORENZ and HEITMAN 1997 ). Second, as shown here, the dominant GPA2-2 allele strongly suppresses the pseudohyphal defect of a ste12/ste12 mutant strain, but only weakly suppresses a tec1/tec1 mutant strain. Ste12p and Tec1p heterodimerize to regulate expression of the filamentation response element found in the FG(TyA)::lacZ reporter and in the TEC1 promoter (MADHANI and FINK 1997 ); thus, this difference in epistasis with GPA2 was not expected. Finally, Ras2p has been implicated in both pathways; in addition to its well-characterized role in regulating adenylyl cyclase activity, the RAS2^Val19 mutant stimulates expression of the FG(TyA)::lacZ reporter via the Ste MAP kinase cascade (MOSCH et al. 1996 ; MADHANI and FINK 1997 ).

In this work, we have identified a third example of cross talk between signaling cascades that regulate filamentous growth. The ability of activating alleles of MAP kinase components (particularly STE11-4) to suppress the pseudohyphal defect of mep2/mep2 or gpa2/gpa2 strains is enhanced by deletion of MEP1, implicating an inhibitory role for Mep1p. Ammonium import or growth defects associated with the mep1 mep2 double mutant are not likely to be responsible for this difference in phenotype, as lowering the ammonium concentration 10-fold did not alter the epistasis behavior of the mep2 single-mutant strain. Furthermore, we also observed enhanced suppression by STE11-4 expression in gpa2 mep1 strains, which have no growth defects; thus, this role of Mep1p does not appear to be directly related to its ammonium transport function. It is possible that Mep2p senses low ammonium concentration and Mep1p senses high ammonium, similar to the glucose sensors Rgt2p and Snf3p, which are homologous to glucose transporters. In the presence of low concentrations of glucose, Snf3p induces the transcription of the genes encoding the high-affinity sugar transporters Hxt2p and Hxt6p; in the presense of high concentrations of glucose, Rgt2p induces expression of the gene encoding the low-affinity transporter Hxt1p (LIANG and GABER 1996 ; OZCAN et al. 1996 ).

Identification of high-copy suppressors of the mep1/mep1 mep2/mep2 mutant:
We used the findings summarized above as a starting point for a genetic screen designed to identify effectors of the signaling function of Mep2p using a high-copy-suppression approach. We chose to use the mep1/mep1 mep2/mep2 strain, as the ability to restore filamentous growth in this strain appeared more permissive than in the mep2/mep2 single mutant. In addition, using the double mutant allowed us to identify genes that improved the growth of this strain under ammonium-limiting conditions. We identified 12 genes whose overexpression suppressed the filamentation defect of the mep1/mep1 mep2/mep2 strain and 5 genes that suppressed the growth defect of this strain on ammonium-limiting media (Table 3). Among this set are several genes that are required for filamentous growth, including MEP2, TEC1, PHD2, MSS11, MGA1, and URE2 (see Table 6; GAVRIAS et al. 1996 ; LAMBRECHTS et al. 1996 ; LORENZ and HEITMAN 1998 ). Epistasis tests between strains lacking the suppressor genes and alleles previously found to regulate filamentous growth confirm that the genetic control of this phenomenon is complex, involving several pathways coordinating multiple signals. No single gene was absolutely required, as conditions that restore filamentation were found for each mutant strain.

A potential role for other signaling events in inducing filamentous growth:
Several of the suppressors have been previously identified to regulate cellular response to changes in carbon source, namely PHD2 (also MSN1, MSS10, and FUP4), MSS11, MSN5, and MGA1. PHD2 (MSN1), MSN5, and MGA1 were each identified as multicopy suppressors of the sucrose non-fermenting (SNF) phenotype of either snf1 or snf2/gam1 mutations (ESTRUCH and CARLSON 1990 ; GIMENO and FINK 1994 ; ZHANG et al. 1997 ); Phd2p (Mss10p) and Mss11p regulate expression of the genes that encode the starch-degrading glucoamylases Sta1p, Sta2p, and Sta3p on the basis of carbon availability (LAMBRECHTS et al. 1996 ; WEBBER et al. 1997 ). Poorly used carbon sources, such as the starch amylopectin, have been found to induce filamentous growth, even in nitrogen-rich conditions; phd2 (mss10) mutations block this response (LAMBRECHTS et al. 1996 ). With the exception of MSN5, each of these genes has significant similarity to transcriptional regulatory proteins.

The connection between carbon signaling and pseudohyphal differentiation is intriguing. Diploid cells have two mutually exclusive developmental fates upon nitrogen starvation: pseudohyphal growth in the presence of carbon abundance and meiosis in the presence of a nonfermentable carbon source. The mechanism by which the cell chooses between these two fates (i.e., how the cell recognizes the carbon source) is not understood. These carbon-regulatory proteins may participate in such signaling to control the pseudohyphal/meiosis decision; none of these mutants, however, affect meiotic competence (M. C. LORENZ and J. HEITMAN, unpublished results). The finding that nitrogen-rich, carbon-poor media can also induce filamentous growth (LAMBRECHTS et al. 1996 ) suggests that a reciprocal signaling event may also be present.

Another of the suppressor genes described here, SKN7, is critical for the cellular response to oxidative stress. skn7 strains are sensitive to several oxidizing agents, including hydrogen peroxide and cadmium, a phenotype shared with mutations in YAP1 (Yeast AP-1, a homolog of c-jun), another transcriptional activator (MOYE-RAWLEY et al. 1989 ; KUGE and JONES 1994 ). Skn7p and Yap1p coordinately regulate expression of TRX2, encoding thioredoxin (MORGAN et al. 1997 ). Yap1p has also been proposed as a downstream effector of PKA activity (GOUNALAKAI and THIREOS 1994 ). Thus, oxidative damage may be another stress that can stimulate differentiation via a Skn7p-dependent pathway. Skn7p also has a role in cell wall biosynthesis and cell cycle progression (BROWN and BUSSEY 1993 ; BROWN et al. 1993 ; MORGAN et al. 1995 ).

We found that high-copy expression of CDC6 strongly suppresses the mep1/mep1 mep2/mep2 pseudohyphal defect. Cdc6p is a component of the origin recognition complex and is essential for cells to initiate DNA synthesis. Thus, Cdc6p may be part of a checkpoint sensing DNA damage or blocks to DNA synthesis, another potential stress-responsive pathway that can trigger filamentous growth. However, we favor an alternate explanation: expression of CDC6 is limited to G1, and when this temporal control is distorted by expressing CDC6 from a constitutive promoter, nuclear division is delayed and cells develop highly elongated buds (BUENO and RUSSELL 1992 ; PIATTI et al. 1995 ). Because the elongated buds produced after CDC6 overexpression are reminiscent of pseudohyphae, it is possible that this phenotype is responsible for the mep1/mep1 mep2/mep2 suppression. Recent findings have identified an N-terminal region of Cdc6p that is necessary for its degradation; deletion of this region allows the protein to persist into G2 (DRURY et al. 1997 ). The CDC6 construct described here lacks ~300 bp of coding sequence from the 3' end. It is possible that this region of the protein may also be a stability determinant.

Ironically, though nitrogen starvation is the best-characterized environmental stress that induces pseudohyphal differentiation, the nitrogen regulatory genes (URE2 and DAL80) have among the weakest phenotypes when overexpressed. Other elements of nitrogen regulatory networks are required for pseudohyphal differentiation, including the protein kinase Npr1p and the ubiquitin ligase Npi1p/Rsp5p (LORENZ and HEITMAN 1998 ), but, again, the only characterized role for these proteins is the regulation of the stability of plasma membrane permeases such as Mep2p. Other connections between these proteins and the filamentation response have yet to be elucidated.

Comparisons to development in other fungi:
The study of differentiation pathways, both in yeast and in other fungi, has been motivated by the connection of such events to pathogenicity. Mating and conjugation in U. maydis leads to a filamentous growth state that is essential for infection of the maize host (BANUETT 1991 ). C. albicans mutant strains that are unable to filament are avirulent in vivo (LO et al. 1997 ). Differentiation events have been correlated with infection of plant or animal hosts in many other species, including Uromyces appendilaticus and Cryptococcus neoformans (ZHOU et al. 1991 ; WICKES et al. 1996 ; ALSPAUGH et al. 1997 ). Moreover, differentiation events in various fungi have regulatory similarities to pseudohyphal development in S. cerevisiae. MAP kinase elements with roles in dimorphism have been identified in C. albicans, Schizosaccharomyces pombe, U. maydis, and several others (BANUETT and HERSKOWITZ 1994 ; LIU et al. 1994 ; KOHLER and FINK 1996 ; LEBERER et al. 1996 ). PKA or cAMP signaling have been shown to regulate development in many organisms, including C. neoformans, S. pombe, Neurospora crassa, and U. maydis (SABIE and GADD 1992 ; YARDEN et al. 1992 ; GOLD et al. 1994 ; MAEDA et al. 1994 ; ALSPAUGH et al. 1997 ).

The implication that stimuli other than nitrogen starvation may trigger pseudohyphal growth in yeast is reminiscent of C. albicans, in which many signals, including serum, temperature, CO₂ levels, and nutrient stresses have been shown to induce filamentation. Mutations in the C. albicans MAP kinase pathway inhibit filamentation in response to some but not all of these stimuli (LIU et al. 1994 ; KOHLER and FINK 1996 ; LEBERER et al. 1996 ). Subsequent deletion of the EFG1 gene, which encodes a transcriptional regulator similar to Phd1p (STOLDT et al. 1997 ), eliminates this residual filamentation (LO et al. 1997 ). This is analogous to yeast in which ste mutant strains have a severe but not absolute filamentation defect (LIU et al. 1993 ). Deletion of PHD1, as for EFG1, eliminates this remaining filamentous growth (LO et al. 1997 ). In addition, Tup1p is a global transcriptional repressor in yeast and regulates filamentous growth in both S. cerevisiae and C. albicans, although the tup1 mutant phenotype is opposite: constitutive filamentation in C. albicans and a pseudohyphal deficiency in S. cerevisiae (BRAUN and JOHNSON 1997 ). In S. cerevisiae, Tup1p physically interacts with Ssn6p (WILLIAMS et al. 1991 ), a protein closely related to Mss11p. Understanding filamentous growth control in yeast, therefore, has broad implications for pathogenicity in many fungi.

Complexity of filamentous growth revealed in transcriptional regulatory proteins:
The other surprising finding from this screen, in addition to the potential involvement of other stress-responsive pathways, is the multiplicity of predicted transcriptional regulators that have been identified to affect pseudohyphal differentiation in some manner. Our studies have identified Phd1p, Tec1p, Phd2p, Mss11p, Mga1p, Skn7p, Dot6p, Hms1p, Hms2p, and Dal80p (see also GIMENO and FINK 1994 ; GAVRIAS et al. 1996 ; LAMBRECHTS et al. 1996 ); other studies have found a role for Ste12p, Ash1p, and Gln3p (LIU et al. 1993 ; CHANDARLAPATY and ERREDE 1998 ; LORENZ and HEITMAN 1998 ). Although many laboratory strains of S. cerevisiae are not competent to undergo pseudohyphal growth, the complexity of transcriptional responses and the reglatory conservation between filamentous growth in yeast and differentiation events in diverse fungi highlights the evolutionary importance of this stress-responsive developmental pathway.

	ACKNOWLEDGMENTS

We thank Gerry Fink and Steve Garrett for strains and plasmids, Alan Myers and Danny Lew for helpful discussions, and Scott Muir for technical assistance. J. Heitman is an associate investigator of the Howard Hughes Medical Institute and a Burroughs Wellcome Scholar in Molecular Pathogenic Mycology.

Manuscript received March 19, 1998; Accepted for publication September 14, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS