In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast-type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation. Important elements of this network have been identified, including nutrient signal receptors, GTP-binding proteins, components of the pheromone-dependent MAP kinase cascade and several transcription factors. However, the structural and functional mapping of these pathways is far from complete. Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11which form an essential part of the signal transduction network establishing invasive growth. Both MSN1 and MSS11 are involved in the co-regulation of starch degradation and invasive growth. Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1. We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth. In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but that Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p. We also show that starch metabolism in S. cerevisiae is subject to regulation by components of the MAP kinase cascade.
Pseudohyphal differentiation and invasive growth of diploid and haploid cells of the yeast Saccharomyces cerevisiae has been described as a cellular adaptation to growth on substrates containing either limiting amounts of, or inefficiently utilized, nutrients (Gimeno et al., 1992; Gimeno and Fink, 1994; Roberts and Fink, 1994; Lambrechts et al., 1996a). Research on the processes responsible for this cellular differentiation has focused on signal transduction mechanisms that transmit information regarding the nutritional status of the substrate and initiate the molecular, morphological and physiological changes observed during the switch from yeast-type unicellular growth to pseudohyphal and invasive growth. These studies revealed a complex network of interacting signal transduction pathways of both an inhibitory and an activating nature and contributed vastly to our knowledge on signal transduction in eukaryotic organisms, as well as to our understanding of cellular differentiation processes. The two phenomena, pseudohyphal differentiation and invasive growth, are closely related and seem to be regulated by the same signal transduction mechanisms. However, they can be separated genetically and could correspond to different implementations of similar developmental pathways (Mösch and Fink, 1997).
One of the most outstanding aspects of signal transduction to emerge from recent data has been the modular nature of the signalling pathways involved (reviewed in Elion, 1995; Herskowitz, 1995; Levin and Errede, 1995; Madhani and Fink, 1998). Modules include small and heterotrimeric G-proteins, MAP kinase cascades, second messengers and transcription factors, with some of these elements playing important roles in several signal transduction events.
Recent data suggest that the mating-specific MAP kinase cascade, comprising the MEKK, Ste11p, the MEK, Ste7p and the MAPK, Fus3p, has an inhibitory effect on establishing an invasive phenotype in haploids (Cook et al., 1997; Madhani et al., 1997). The same MEKK and MEK activate a second MAPK, Kss1p, which induces invasive growth when phosphorylated. However, the absence of this cascade does not eliminate an appropriate regulation of pseudohyphal differentiation, indicating that MAPK-independent pathways play a major part in the process. Elements identified as being involved in MAPK-independent regulation include the small G-protein, Ras2p (Kübler et al., 1997; Lorenz and Heitman, 1997), the α-subunit of a heterotrimeric G-protein, Gpa2p (Kübler et al., 1997; Lorenz and Heitman, 1997), Whi2p, a regulator of cell proliferation under starvation conditions (Radcliffe et al., 1997), and Ash1p, a negative regulator of HO expression in daughter cells (Chandarlapaty and Errede, 1998). In addition, the ammonium-specific receptor, Mep2p, has been shown to signal via MAP kinase-independent pathways (Lorenz and Heitman, 1998).
Furthermore, several regulators of transcription, acting downstream of the elements described above, have been identified. They include proteins such as Ste12p (Liu et al., 1993), which, together with Tec1p (Gavrias et al., 1996), acts downstream of the MAP kinase cascade to activate specific genes involved in the process. Other genes directly or indirectly responsible for the transcriptional regulation of genes involved in the invasive growth response are Phd1p (Gimeno and Fink, 1994) and Flo8p (Liu et al., 1996).
However, whereas the MAP kinase-dependent signal transduction process is relatively well mapped, little data are available regarding the MAP kinase-independent processes. In particular, a very limited amount of information is available about downstream elements responding to Ras2p and Gpa2p.
In this paper, we present data characterizing the role of three previously identified genes, MSN1/MSS10, MSS11 and MUC1/FLO11, in the establishment of the invasive and pseudohyphal growth phenotypes. Two of these genes, MSN1 and MUC1, have been shown previously to be important for invasive and pseudohyphal growth. MSN1 was first identified as a multicopy suppressor of snf1 mutants (MSN1 ) (Estruch and Carlson, 1990). Other authors cloned the same gene as FUP1, an enhancer of iron-limited growth in S. cerevisiae (Eide and Guarente, 1992), as PHD2, a multicopy inducer of pseudohyphal growth (Gimeno and Fink, 1994) and, in our laboratory, as MSS10, a multicopy suppressor of the repression exerted by STA10 on the STA1–3 glucoamylase-encoding genes, involved in starch metabolism in S. cerevisiae (Lambrechts et al., 1994; 1996b). MSN1 has been suggested as encoding a transcriptional activator, as multiple copies of the gene seem to enhance the transcription of several genes, most of which are involved in nutrient utilization. In addition, MSN1 has been shown to activate reporter gene expression if fused to the LexA DNA-binding domain (Estruch and Carlson, 1990).
MSS11, like MSN1, was identified as a suppressor of the STA10-dependent phenotype and was shown to induce STA1–3-encoded glucoamylase expression when present on a 2 μm plasmid (Webber et al., 1997). The protein displays homologies with a number of transcriptional activators and suppressors, such as S. cerevisiae Snf5p, Ssn6p/Cyc8p and Drosophila NTF-1 and, in particular, with Flo8p, a protein activating genes involved in flocculation (Kobayashi et al., 1996). The third gene investigated here, MUC1, cloned in our laboratory (Lambrechts et al., 1996a) and later isolated as FLO11 (Lo and Dranginis, 1996), encodes a cell wall-bound protein with homologies to mammalian membrane-bound mucins and to dominant yeast flocculation genes. MUC1 was shown to be necessary for both invasive growth and filamentation to occur and to induce invasive growth when overexpressed (Lambrechts et al., 1996a). Lo and Dranginis (1998) confirmed these data recently and presented additional evidence showing that MUC1 was regulated by Ste12p and induced in response to nitrogen limitation in diploid cells. Here, we show that Mss11p is an essential factor in the establishment of the invasive and pseudohyphal growth responses. We show further that the two genes MSN1 and MSS11 define a critical part of the signal transduction pathway regulating these adaptive responses, and that this regulation occurs in part through the transcriptional regulation of MUC1. Through genetic analysis, we show that both Msn1p and Mss11p act in a linear pathway downstream of Mep2p and Ras2p. Both genes show complex epistatic interactions with other elements of the signal transduction cascade. Our data show that Mss11p, like Msn1p, regulates the transcription of MUC1. We also show that starch metabolism in S. cerevisiae is regulated by components of the MAP kinase cascade that regulates the invasive growth response.
MSS11 is involved in the regulation of pseudohyphal development and invasive growth
MSS11 was cloned initially as a gene that, when present on a 2 μm plasmid, enhanced starch utilization by S. cerevisiae strains containing the STA1–3 glucoamylase genes (Webber et al., 1997). In these strains, we observed that the presence of MSS11 on a multiple copy plasmid leads, in addition to more effective starch degradation (Webber et al., 1997) and flocculation phenotypes (unpublished results), to strong invasive growth (Fig. 1A and B), including filaments of elongated cells (pseudohyphae) in a haploid background (Fig. 1C). In fact, strains bearing multiple copies of MSS11 grow invasively directly after plating and at the beginning of growth, including in rich YPD medium. This phenotype was verified in several haploid and diploid laboratory strains, including the Σ1278 and S288C genetic backgrounds, as well as on different growth media. MSS11 induced invasive growth in all genetic backgrounds and in all growth conditions tested (data not shown). Invasive growth by strains containing MSS11 on a 2 μm plasmid was directly correlated with colony growth and was clearly visible after only 24 h. Control strains, containing only the plasmid without MSS11, were unable to grow invasively in media containing glucose and showed invasive growth only in media with a limited nitrogen source (SLAD) and media containing starch (SCS) or glycerol/ethanol (SCGE) as carbon sources after prolonged incubation periods. Multiple copies of MSS11, therefore, seem to induce the genes necessary for invasive growth on a permanent and signal-independent basis.
To ascertain whether MSS11 played an important role in the invasive growth process, the gene was disrupted in several genetic backgrounds. The Δmss11 strains were unable to grow invasively (Fig. 1B), even after prolonged incubation periods under all conditions tested. MSS11 therefore seems to encode an important component in the ability of yeast cells to grow invasively. Disruption of the gene, however, did not affect the general growth of the strains in liquid and solid media in any of the growth media tested (with the exception of starch-containing media), the mating ability of the strain, osmosensitivity or heat shock resistance (data not shown), indicating that MSS11 is specifically required for some cellular differentiation processes, but does not affect the general yeast physiology.
Epistatic relationship between Msn1p, Mss11p and Muc1p
To assess whether the two genes MSN1 and MSS11 act in the same pathway, we established their epistatic relationship. Several ISP15 strains, in which either MSN1 or MSS11 or both were deleted, were used for this study. These strains were transformed with 2 μm plasmids bearing MSN1, MSS11 or the vector without any insert as a negative control. The strains were subsequently spotted onto different media to assess the extent of the invasive growth phenotypes, which are shown for nitrogen-limited SLAD medium in Fig. 2(A). If compared with wild-type ISP15 transformed with the vector alone, multiple copies of both MSN1 and MSS11 lead to strongly induced invasive growth phenotypes, whereas the deletions of MSN1 or MSS11 or both lead to strongly reduced or absent invasive growth phenotypes.
Multiple copies of MSN1 are unable to overcome the effect of an MSS11 disruption, as no invasive growth was observed in the corresponding strain. This result suggests that the function of Msn1p depends on Mss11p, or that Msn1p functions upstream of Mss11p in a linear signal transduction pathway. Multiple copies of MSS11, however, are able to overcome the effect of a deletion in MSN1 very efficiently, resulting in very strong invasive growth. We propose, therefore, that Msn1p is situated upstream of Mss11p in a signal transduction pathway resulting in invasive growth. Interestingly, the invasive phenotype of strains carrying multiple copies of MSS11 is significantly stronger in strains with disrupted MSN1 loci than in the wild-type strains.
To assess the relation of Msn1p, Mss11p and Muc1p, further epistasis studies were carried out using strains with either deletions of MUC1 (Fig. 2B) or carrying a plasmid with the MUC1 gene fused to the constitutive PGK1 promoter (Fig. 2C). Multiple copies of MSN1 were unable to overcome the effect of a deletion in MUC1, as no invasive growth could be observed in this case, even after prolonged periods of incubation (Fig. 2B). However, strains carrying multiple copies of MSS11 were able to grow invasively in a Δmuc1 strain, but with greatly reduced efficiency compared with vector-transformed wild-type strains. In the opposite situation (Fig. 2C), strains with disrupted MSN1 or MSS11 loci were able to grow invasively when MUC1 was expressed under the control of the PGK1 promoter. This suggests that both Msn1p and Mss11p act above Muc1p in a linear signal transduction pathway that establishes the invasive growth phenotype. It is also evident that Mss11p does not function through Muc1p alone, as multiple copies of MSS11 were still able to induce invasive growth in strains with deleted MUC1 loci.
Mss11p, like Msn1p, enhances transcription of MUC1
Figure 3 presents the effect of deleted or multiple copies of MSN1 and MSS11 on the transcription levels of MUC1 and STA2 in different strains and growth media. If compared with transcript levels of wild-type ISP15 (lane 1) grown on nitrogen-limited media (SLAD) (Fig. 3A), SCD (Fig. 3B) or media containing starch as the carbon source (SCS) (Fig. 3C), multiple copies of either MSN1 or MSS11 lead to enhanced levels of both STA2 and MUC1 mRNA. In SLAD and SCD media, which both contain glucose as carbon source, transcript levels of MUC1 as well as STA2 are significantly reduced in all strains when compared with media with starch or glycerol/ethanol (data not shown) as carbon sources. As the promoter areas of MUC1 and STA2 are to a large extent homologous (Lambrechts et al., 1996a, b) and as the transcription of STA2 has been shown to be subject to glucose repression (Pretorius et al., 1986b), this phenomenon is probably the result of glucose repression on the transcription of STA2 and MUC1. Strains in which MSN1, MSS11 or both MSN1 and MSS11 were deleted showed a dramatic reduction in transcript levels of both MUC1 and STA2 mRNA, irrespective of the carbon source used. These results, considered together with the increased mRNA levels in strains with multiple copies of MSN1 and MSS11, suggest that MSS11, like MSN1, mediates the transcriptional activation of MUC1.
The results of the invasive growth epistasis analysis were also confirmed by the mRNA levels of STA2 and MUC1. The presence of multiple copies of MSN1 in a strain with a deleted MSS11 locus did result in very low levels of STA2 or MUC1 mRNA. In the reverse situation, however, multiple copies of MSS11 in a strain with a disrupted MSN1 locus resulted in very high mRNA levels of STA2 and MUC1. This again confirmed that Mss11p functions downstream of Msn1p in establishing the transcriptional state of MUC1 and STA2 and correlates with the stronger invasive growth observed in strains carrying multiple copies of MSS11 in a Δmsn1 background.
Msn1p and Mss11p function downstream of Ras2p
To verify whether Msn1p and Mss11p act in a Ras2p-dependent pathway, we transformed ISP15Δmsn1 and ISP15Δmss11 with either the hyperactivated RAS2 allele, RAS2val19 or the wild-type RAS2 allele as a control. The effect on invasive growth can be seen on nitrogen-limited SLAD medium (Fig. 4A). Whereas a hyperactivated Ras2p results in an increased invasive growth response in a wild-type strain, it is unable to do so in the strain with a disrupted MSS11 locus, even after prolonged periods of incubation on all media tested. However, the figure shows that the RAS2val19 allele is able to induce invasive growth weakly in the Δmsn1 strain.
These results were confirmed on media containing starch as the carbon source. In all cases, the strength of the invasive growth response correlated with the efficiency of starch degradation (data not shown).
Both Msn1p and Mss11p act downstream of Mep2p
The MEP2 gene encodes one of several ammonium permeases and was shown to be responsible for ammonium-dependent signalling (Lorenz and Heitman, 1998). This signal is, at least in part, transmitted by Ras2p. We therefore transformed the MSN1 and MSS11 multiple copy plasmids into strains with a MEP2 deletion and into the isogenic wild-type strain. When spotted onto media with glycerol/ethanol as carbon source, the Δmep2 and wild-type strains showed similar invasive behaviour, which was strongly amplified in both cases by the presence of MSN1 or MSS11 on 2 μm plasmids (data not shown). On SLAD medium (Fig. 4B), in which the nitrogen source, ammonium, is limiting, the wild-type strain again showed increased invasive behaviour when MSN1 and MSS11 were present on 2 μm plasmids. The Δmep2 strain transformed with a control plasmid alone did not show any invasive growth on this medium, confirming the results obtained by Lorenz and Heitman (1998). The same strain transformed with either MSN1 or MSS11 on 2 μm plasmids regained the ability to invade, with MSS11 being more efficient than MSN1. The efficiency of invasion in these strains was, however, well below the level of the untransformed, wild-type strain. These data suggest that Msn1p and Mss11p act in a pathway downstream of the Mep2p permease.
Msn1p and Mss11p act independently or downstream of Ste20p, Ste11p and Ste7p
To determine the epistatic relationship between the kinases Ste20p, Ste11p and Ste7p, and Msn1p and Mss11p, 2 μm plasmids bearing MSN1 or MSS11 were transformed into Σ1278 strains in which STE20, STE11 or STE7 were deleted and, as control, the isogenic wild-type strain. The results are shown in Fig. 5. The wild-type strain carrying only the vector was able to form pseudohyphae and grow invasively into the agar after a short (48 h) incubation on nitrogen-limited SLAD medium. The presence of multiple copies of MSN1 and MSS11, as expected, resulted in significantly stronger invasive growth and pseudohyphae formation, similar to the results obtained with the ISP15 strain (Fig. 1). Strains with disrupted STE20, STE11 or STE7 loci transformed with the vector alone showed significantly reduced invasive growth when compared with the wild type. This reduction was most prominent in the case of Δste11, which did not grow invasively even after prolonged incubation, and least pronounced in Δste7, with Δste20 showing an intermediate phenotype. This confirms data obtained previously showing that both STE11 and STE20 are required for additional functions independent of the pheromone/invasive growth MAP kinase cascade (Leberer et al., 1997; Posas and Saito, 1997). Multiple copies of MSN1 and MSS11 re-established the invasive growth phenotype in all the strains to close to, or above, wild-type level. In every case, multiple copies of MSS11 proved more efficient in overcoming the invasive growth defect than multiple copies of MSN1.
The results indicate that both Msn1p and Mss11p act either downstream of the MAP kinase cascade or in a pathway functioning in parallel to this cascade.
Msn1p induces invasive growth independent of Ste12p, whereas Mss11p functions downstream, or in conjunction with, both Ste12p and Msn1p
A putative binding site for Ste12p was identified in the promoter of MUC1, suggesting that it could be the final step in the activation of a gene required for the pseudohyphal or invasive growth response (Lo and Dranginis, 1998). We had to establish, therefore, whether Msn1p and Mss11p function through Ste12p or independent thereof in activating transcription of MUC1. MSN1 and MSS11 present on 2 μm plasmids were transformed into strains with a disrupted STE12 locus to assess the effect thereof on invasive growth on media containing starch as carbon source (SCS) (Fig. 6) or nitrogen-limited SLAD media (data not shown). Whereas the Δste12 strain transformed with the vector alone is unable to invade the substrate, the strains transformed with either 2 μm-MSN1 or 2 μm-MSS11 regained the ability to invade the agar efficiently, indicating that both Msn1p and Mss11p function either downstream of Ste12p or independent thereof in the signalling pathway, resulting in invasive growth. In the reverse experiment, a 2 μm plasmid bearing STE12 was used to transform ISP15 strains with a deletion of either MSN1 or MSS11. The results (Fig. 6) indicate that multiple copies of STE12 result in invasive growth in both the wild-type and the Δmsn1 strain, but not in a Δmss11 strain. This suggests that Msn1p functions independently of Ste12p in establishing the invasive growth phenotype, whereas Mss11p acts either downstream or in combination, but not independently of Ste12p.
Starch metabolism is regulated by the MAP kinase cascade
Some strains of S. cerevisiae carry any one (or more) of three genes, STA1, STA2 or STA3, which encode extracellular glucoamylases (reviewed by Vivier et al., 1997). Once secreted, glucoamylases hydrolyse starch molecules by liberating glucose molecules from the non-reducing end of the molecule, thereby making it available to the yeast cell. This enables the yeast cell to grow on starch as the sole carbon source. MUC1 and the STA1–3 genes have highly homologous promoter areas. As MUC1 has been shown to be an important role-player in pseudohyphal differentiation and invasive growth, both processes under the regulation of the mating pheromone/invasive growth MAP kinase cascade, the question arose whether starch metabolism is also regulated by the same cascade. In addition, a putative Ste12p binding site was identified in the upstream region of MUC1 (Lo and Dranginis, 1998), and the same sequence is also present in the promoter of STA2.
The effect of deletions in two of the MAP kinase modules, STE7 and STE12, as well as the presence of multiple copies thereof, on a yeast strain's ability to degrade starch can be seen in Fig. 7. The sizes of the haloes around these colonies are indicative of the ability of these strains to degrade starch and indicate that multiple copies of both STE7 and STE12 result in enhanced starch utilization. Deletion of either STE7 or STE12 results in a severe decrease in this phenotype, which can be overcome by multiple copies of either MSN1 or MSS11 (Figs 6 and 7).
Effect of growth phase on invasive growth
S. cerevisiae L5366 was inoculated into liquid SCD medium and grown to an optical density at 600 nm (OD600) of 1.0. From this culture, four precultures were inoculated and grown to optical densities of 0.6, 1.2, 2.0 and 3.0 respectively. From these cultures, equal amounts of cells (1.5 × 105) were taken, the volumes adjusted to 20 μl and dropped onto nitrogen-limited SLAD plates. Plates were incubated for 4 days, after which the plates were investigated for invasive growth. Figure 8 clearly shows the effect that the growth phase of the precultures had on the ability of the yeast cells to grow invasively into the agar. Cells taken at later growth phases (OD600 of 3.0) started growing invasively at a much earlier stage than those taken from the mid-log cultures (OD600 of 0.6, 1.2 and 2.0). This was repeated with strains ISP15 and L5366-h1, and the observations confirmed (data not shown). The invasive phenoytype did not increase in a linear manner with corresponding increases in OD600. Indeed, phenotypes were similar for cells taken at an OD600 of 0.6, 1.2 and 2.0, but not for cells taken at an OD600 of 3, suggesting a sudden switch in cell physiology occurring between mid- and late-log phase. The effect of multiple copies of MSN1 or MSS11 was, however, always clearly visible, and even strains spotted at OD600 of 3 showed a marked increase in invasion when transformed with those plasmids (data not shown).
In this paper, we present data positioning three genes, MSN1, MSS11 and MUC1, in a signal transduction pathway downstream of MEP2 and RAS2. As expected for a network of signal transduction cascades, the epistasis analysis reveals complex interactions between the different components. Our genetic data clearly suggest that Msn1p, Mss11p and Muc1p act in this hierarchical order to activate invasive growth in yeast cells. Msn1p has been suggested to act as a transcriptional activator. The position of Mss11p downstream of Msn1p could suggest that either it is itself a target of Msn1p-mediated activation or an interaction between the two proteins is required to allow Msn1p to exert its effects. This second hypothesis is more plausible for several reasons. First, multiple copies of MSS11 are more efficient in inducing invasive growth in a strain deleted for MSN1 than in a wild-type strain, and it is therefore unlikely that Msn1p is required to activate MSS11. Secondly, these same data suggest that a genomic copy of MSN1 somehow attenuates the effect of MSS11 overexpression. This would suggest a more direct interaction between Msn1p and Mss11p.
The expression of MUC1 from a strong, constitutive promoter increases the invasiveness of yeast cells significantly. The Northern blots clearly demonstrate the strong induction of MUC1 by multiple copies of MSN1 and MSS11. The increased invasiveness of these cells is, therefore, at least in part, caused by the transcriptional activation of MUC1. This is confirmed further by a very strong reduction in invasive growth in Δmuc1 strains. MUC1, however, is not the only target gene of Mss11p, as deletion thereof still allows Mss11p to re-establish invasive growth in a MUC1 deletion strain, although at a significantly reduced level. The Northern blot data correlate well with the observed phenotypes described above. Indeed, the effect of MSS11 overexpression on MUC1 and STA1–3 transcription is significantly stronger in strains with disrupted MSN1 loci than in a wild-type strain. In all cases, the level of MUC1 transcription reflects the strength of the invasive growth observed.
The effect of multiple copies of MSN1 and MSS11 or the deletion of genomic copies thereof on the transcription of MUC1 and STA1–3 suggest that both genes either encode transcriptional activators or proteins that directly affect transcription factors. Both genes were shown to induce the transcription of MUC1 as well as the STA1–3 genes and, in all cases, deletions or overexpression had similar effects on invasive growth and starch utilization. This co-regulation of invasive growth and starch metabolism was also confirmed through the deletion or overexpression of genes encoding components of the invasive growth/pheromone response MAP kinase cascade. The ability to degrade starch through activation of the STA1-3 genes is, therefore, an excellent reporter system for invasive growth in strains bearing these genes.
The genes regulated by both Msn1p and Mss11p encode proteins involved in either nutrient utilization or invasive and pseudohyphal growth. The effect of Msn1p and Mss11p on invasive growth could, therefore, be a consequence of increased or decreased efficiency of nutrient utilization, and not of a direct effect of Msn1p or Mss11p on invasive growth genes themselves. However, the data indicate that both proteins have a direct effect on invasive growth. In particular, Muc1p, which is required for invasive and pseudohyphal growth and is not involved in nutrient uptake or utilization, is directly regulated by both Msn1p and Mss11p. In addition, multiple copies of MSN1 or MSS11 result in yeast strains invading the agar directly after plating, even on rich glucose media (YPD). Genes acting indirectly through nutrient utilization would not be expected to show such a behaviour.
Our genetic data suggest that Msn1p and Mss11p act downstream or in parallel with the MAP kinase cascade. Both genes overcome deletions in STE7, STE11 and STE20. Multiple copies of both MSN1 and MSS11 are, however, less efficient in overcoming the invasive growth defect of a Δste20 and a Δste11 strain than of a Δste7 strain. This is in accordance with previous reports indicating that Ste20p has functions independent of the MAP kinase cascade in establishing the invasive growth phenotype (Leberer et al., 1992), and that Ste11p functions independently in several other signal transduction events (Posas and Saito, 1997).
Further data indicate that Msn1p acts independently of Ste12p in a parallel pathway, whereas Mss11p functions downstream, or is required for the activity, of Ste12p. Indeed, multiple copies of MSS11 still result in increased invasive growth in strains with deletions of STE12, whereas the overexpression of STE12 is unable to overcome the effects of a deletion of the MSS11 locus. In all cross-complementation experiments involving MSN1 (disruptions in MSN1 complemented by multiple copies of STE12 or disruptions in genes encoding MAP kinase cascade elements or STE12 complemented by multiple copies of MSN1 ), the invasive phenotypes observed were reduced compared with those induced by multiple copies of MSN1 or multiple copies of STE12 in a wild-type background. This reduction in the ability to invade again suggests that the MAP kinase cascade and Msn1p act in parallel pathways with additive effects on invasiveness. Mss11p seems to be situated at the confluence of the two signalling pathways, one depending on the invasive growth MAP kinase cascade, the other signalling via Msn1p. Both these pathways are Ras2p dependent, explaining the complete block of invasive growth in a Δmss11 strain.
Indeed, the RAS2 gene has already been shown to act via at least two different signal transduction pathways, one of which is MAPK dependent (Mösch et al., 1996; Lorenz and Heitman, 1998). Disruption of MSS11 completely eliminates invasive growth in strains carrying a plasmid encoding the hyperactivated form of Ras2p, Ras2val19p. This clearly places Mss11p downstream of the Ras2p signal. Our data suggest furthermore that the transmission of the signal via Msn1p is under the control of the RAS2-dependent, but MAP kinase-independent, pathway, as the RAS2-dependent signal is partially blocked by a deletion of MSN1. We are currently investigating the relation of Msn1p and Mss11p with Ash1p, a DNA binding protein that was shown to act as an activator of pseudohyphal growth and has similar epistatic relations with RAS2 and the pheromone-associated MAP kinase cascade. Interestingly, the MUC1 gene is activated by both Ras2p-dependent pathways, as we observed a strong induction of the STA1–3 and MUC1 genes in a strain carrying STE12 on a 2 μm plasmid. This suggests that Muc1p plays a role in different events requiring cell–cell or cell–substrate adhesion, during both mating and pseudohyphal differentiation.
Both Msn1p and Mss11p act downstream of the ammonium-specific permease Mep2p which specifically signals ammonium limitation. The ability to invade the agar of Δmep2 strains carrying multiple copies of MSN1 or MSS11 is restored, but at a significantly weaker level than in any of the other investigated genetic backgrounds. This reinforces the idea that Msn1p and Mss11p are situated downstream of Mep2p. Indeed, of all the strains used, the Δmep2 strain is the only one in which the nutritional signal itself is absent. All other mutants used for the epistasis analysis are affected in one of several parallel signal transduction pathways. In those mutants, the signal itself will still be perceived and transmitted via non-affected parallel pathways, if perhaps with reduced efficiency. The fact that multiple copies of MSN1 and MSS11 are able to re-establish invasive growth in a MEP2 deletion strain at very reduced levels indicates that their activity is partly dependent on the presence of the signal itself. This suggests that these proteins not only amplify the signal simply through stoichiometrical effects, as might be suggested by phenotypes generated by multiple copy plasmids, but that some type of signal-dependent modification has to take place in order for them to function efficiently. This signal is specifically Mep2 dependent in ammonium-limited conditions. These genetic data suggest a model, which is summarized in Fig. 9.
The exact role of MSS11 is not yet understood. The data presented here suggest that Mss11p is specifically required for the establishment of the invasive and pseudohyphal growth phenotypes in response to a signal emanating from Ras2p. Data presented elsewhere (M. Gagiano et al., submitted) show that the effect of MSS11 overexpression on MUC1 transcription can be pinpointed to a specific area within the MUC1 promoter. In addition, the sequence homologies of Mss11p with Flo8p and other transcription factors strongly suggest that Mss11p itself could be a transcription factor. The presence of an ATP or GTP binding loop within the protein sequence gives an indication on the possible regulation of this factor. We are currently investigating whether Mss11p is binding ATP or GTP and which proteins might be directly involved in this regulation. In addition, we are establishing the interactions of this protein and of Msn1p with some of the other transcription factors involved in pseudohyphal differentiation, such as Phd1p, Ste12p and Ash1p. We suggest that Mss11p mediates the transcriptional activation specifically of genes required for pseudohyphal and invasive response.
The role for MUC1 in mediating invasive growth is unclear. Overexpression results in increased invasive growth phenotypes, whereas deletion thereof diminishes the invasive growth phenotype strongly. Based on the structure of Muc1p, which resembles the mammalian mucins (Lambrechts et al., 1996a) and yeast flocculins (Lo and Dranginis, 1998), an adhesion function can be suggested for Muc1p. Whether this involves only cell–cell adhesion or cell–substrate adhesion remains to be verified. Adhesion to a specific substrate was shown to be a prerequisite for invasion by Candida albicans, as elimination of the ability to adhere to a substrate also eliminated the ability to invade that substrate (Gale et al., 1998). The close evolutionary relation between the STA1–3 and MUC1 genes could suggest such a role for Muc1p.
Our results regarding the efficiency of invasion of various strains were always obtained in strictly controlled conditions including the growth phase and growth speed of the preculture and cultures from which cells were spotted onto plates, the number of cells spotted and incubation periods. The invasive growth behaviour observed in these conditions is highly reproducible. Comparisons were only made between isogenic strains showing similar growth speeds in the conditions tested.
An important factor for the efficiency of the invasiveness of all the strains proved to be the growth phase, and not the cell concentration, at which strains were spotted from the liquid preculture onto the test plate. When cells of the same strain were sampled at different growth phases and spotted at adjusted cell densities, they showed markedly different invasion efficiencies, a higher OD600 resulting in a more invasive phenotype. This behaviour could be accounted for by a difference in transcription patterns between early-, mid- and late-log phase cells. The last type might have induced genes in response to limited nutrients, including some of the genes responsible for invasion, before being spotted onto the plates. However, more interestingly, the change in OD600 did not only result in a difference in invasive efficiency, but reproducible differences were observed with regard to the behaviour of different strains. The results of epistasis analysis could indeed be different according to the growth phase of the cells used for plating. This might explain some of the differences seen between papers published in the past by different groups. However, the effect of multiple copies of MSN1 and MSS11 was not affected by the growth phase of the culture.
Further considerations concern the genetic background of the strains used in epistasis analysis. In this work, the effects of mutations and overexpression were verified in several strains with different genetic backgrounds. This includes the strain that has been used as the reference strain for most pseudohyphal research work, Σ1278, FY23 (S288C) and yeast strains constructed in our laboratory, i.e. ISP15 and ISP20. In general, results obtained in one of the strains were always reproducible in all the others. However, during epistasis analysis, clear differences in the intensity of responses in the different strains were observed. For example, the increase in invasiveness after transformation with multiple copy plasmids containing MSN1 or MSS11 was significant in all strains investigated. However, the relative strength of the invasion varied. In some strains (ISP15 and ISP20), the efficiency of invasion was increased similarly by multiple copy plasmids carrying either MSN1 or MSS11, whereas in other strains (Σ1278 and FY23), MSS11 was significantly more efficient than MSN1.
Finally, our results were always verified for several types of either nitrogen or carbon limitation. Again, we found that, as a rule, a result obtained on one medium could be reproduced on another. However, as for the different genetic backgrounds, significant differences in the relative strength of the invasive response emerged. Some of the mutants responded more strongly in one medium than in another. We are conducting experiments to verify whether this specificity can be linked to specific genes.
Yeast strains and culture conditions
Yeast strains used in these experiments are listed in Table 1. All strains were grown at 30°C in standard yeast media, prepared according to Sherman et al. (1991). Standard protocols were used in the transformation of yeast strains (Ausubel et al., 1994). Selective media contained 0.67% yeast nitrogen base, the specific amino acids required by each strain, as well as 2% glucose for SCD, 2% starch for SCS or 3% glycerol and 2% ethanol for SCGE. Agar was added to a final concentration of 2% for all plates. SLAD media, which contains 50 μM ammonium sulphate as sole nitrogen source, were prepared as described by Lorenz and Heitman (1997).
S. cerevisiae strains ISP15 and ISP20, both exhibiting the ability to use starch as a carbon source, form pseudohyphae and grow invasively into the agar, were used for strain constructions. Yeast strains of the Σ1278 genetic background, for which the pseudohyphal and invasive phenotypes are well established, were used as control strains. FY23, a standard S288C laboratory strain (Winston et al., 1995) that cannot form pseudohyphae or grow invasively because of a naturally occurring mutation in the FLO8 gene, was transformed with the wild-type FLO8 gene on centromeric plasmids, YCpLac22-FLO8 or pF415-1, and also used as a control strain for the pseudohyphal and invasive growth phenotypes. To create a wild-type haploid Σ1278 strain, L5366 was sporulated and 15 tetrads analysed. A single haploid strain, L5366-h1, was selected and used for these experiments.
An existing Δmss11::LEU2 disruption cassette (Webber et al., 1997) was used to disrupt the MSS11 open reading frame (ORF) in strains ISP20 and FY23 by means of homologous recombination and integration (Ausubel et al., 1994). Disruptions were verified by the polymerase chain reaction (PCR) and Southern blots. The Δste7 ::LEU2 and Δste12 ::URA3 disruption cassettes constructed for this work, pΔste7 and pΔste12, were used to disrupt the STE7 and STE12 loci in strains FY23, ISP15 and ISP20. STE7 and STE12 disruptions were verified by Southern blot analysis and the inability of successfully disrupted strains to mate with strains of opposing mating type (data not shown).
Plasmid construction and recombinant DNA methods
Standard procedures for isolation and manipulation of DNA were used throughout this study (Ausubel et al., 1994). Restriction enzymes, T4 DNA ligase and Expand Hi-Fidelity polymerase used in the enzymatic manipulation of DNA were obtained from Boehringer Mannheim and used according to the specifications of the supplier. Escherichia coli DH5α (Gibco BRL/Life Technologies) was used as host for the construction and propagation of all plasmids.
All plasmids used in or constructed for this study are listed in Table 2. A 1675 bp XhoI–SnaBI fragment containing MSN1 was obtained from the plasmid pMS2A (Lambrechts et al., 1996b) and cloned into the unique Sal I and SmaI sites of plasmids YEpLac112 and YEpLac195 (Gietz and Sugino, 1988) to generate YEpLac112-MSN1 and YEpLac195-MSN1. A 3326 bp EcoRI fragment containing MSS11 was derived from the plasmid pMSS11-g (Webber et al., 1997) and cloned into the unique EcoRI site of plasmids YEpLac112 and YEpLac195 to generate plasmids YEpLac112-MSS11 and YEpLac195-MSS11. STE12 was obtained as a 2889 bp SacI–NarI fragment from plasmid YCp12-3 (Pi et al., 1997) and cloned into the unique SacI and NarI sites of plasmid YEpLac112 to generate plasmid YEpLac112-STE12. A 2094 bp HindIII fragment containing STE7 was obtained from plasmid STE7-1 (Chaleff and Tatchell, 1985) and cloned into the unique HindIII site of plasmid YEpLac112 to generate plasmid YEpLac112-STE7. RAS2 and the mutant allele, RAS2val19, were obtained as 1637 bp StuI–HindIII fragments from pRAS2 and pRAS2val19, respectively, and cloned into the unique SmaI and HindIII sites of plasmid YCpLac22 (Gietz and Sugino, 1988) to generate YCpLac22-RAS2 and YCpLac22-RAS2val19. FLO8 was obtained as a 3252 bp SphI–EcoRV fragment from plasmid pF415-1 (Kobayashi et al., 1996) and cloned into the unique SmaI and SphI sites of plasmid YCpLac22 (Gietz and Sugino, 1988) to generate plasmid YCpLac22-FLO8.
A 1129 bp Bal I–BlnI fragment was deleted from plasmid YEpLac112-STE7, removing most of the STE7 ORF. A 1680 bp SmaI–NheI fragment containing the entire LEU2 gene, obtained from YDp-L (Berben et al., 1991), was subsequently inserted, resulting in plasmid pΔste7. A STE12 disruption construct was created by deleting a 647 bp MluI–XbaI fragment from plasmid YCp12-3, removing the translational start site (ATG) and a large part of the ORF in the process. A 1175 bp fragment containing the URA3 gene from plasmid YDp-U (Berben et al., 1991) was inserted to generate pΔste12.
To create a plasmid for overexpressing MUC1 in the different yeast strains, a 1872 bp HindIII fragment containing the PGK1 promoter and terminator was obtained from plasmid pHVX2 (Volschenk et al., 1997) and inserted into the unique HindIII site of plasmid YEpLac112. A 4101 bp EcoRI fragment containing the entire MUC1 ORF was then obtained from plasmid pADMU (Lambrechts et al., 1996a) and subsequently inserted into the EcoRI site between the PGK1 promoter and terminator, resulting in plasmid YEpLac112-PGK1P-MUC1.
Invasive growth and pseudohyphal development assays
Yeast strains were transformed with plasmids bearing MSN1, MSS11, STE7, STE12 and RAS2val19, as well as all the control plasmids and plated onto selective plates. Three colonies from each transformation were inoculated into SCD and grown to an OD600 of 1.0. To assess the ability of these yeast strains to grow invasively into the agar, 10 μl of this liquid culture suspension was dropped onto SLAD, SCS, SCGE and SCD plates. Plates were incubated at 30°C and investigated for invasive growth at intervals of 2 days. Yeast colonies were washed off the surface of the agar by rubbing the surface of the plates with a gloved finger under running water. Cells that invaded the agar cannot be washed off and are clearly seen below the surface of the agar. Plates were photographed both before and after the washing process. After washing off the cells, each of the colonies was investigated for elongated cells or filaments under the 10 × magnification of a light microscope (Nikon Optiphot-2), and photographs of cells below the agar surface were taken with a Matrox Intellicam 2 (Matrox Electronics).
Plate assays to determine starch utilization
The STA2 gene encodes an extracellular glucoamylase, which hydrolyses starch by liberating glucose molecules from the non-reducing end of the starch molecule (Vivier et al., 1997). The presence of the STA2 gene therefore enables most yeast strains to grow on starch as the sole carbon source. On plates containing starch (SCS), a clear zone is formed around such starch-degrading colonies, and the diameter of the zone is indicative of the amount of glucoamylase secreted (Yamashita et al., 1985; Pretorius et al., 1986a). The expression of STA2 in the different yeast strains, transformed with the plasmids bearing MSN1, MSS11, STE7, STE12 and RAS2val19, as well as all control plasmids, was therefore determined by the size of the clear zone around each of the colonies on the SCS plates.
RNA isolation and Northern blot analysis
Using standard protocols (Ausubel et al., 1994), total RNA was isolated from the wild-type ISP15 strain or ISP15 strains from which MSN1, MSS11 or both were deleted. RNA preparations were also obtained from different ISP15 strains transformed with 2 μm plasmids bearing copies of either MSN1 or MSS11. Cultures were inoculated from an overnight culture and grown to an OD600 of 1.0 in selective SCD, SLAD, SCS and SCGE media. For electrophoresis analysis of the samples, 10 μg of each RNA preparation was subjected to electrophoresis on a formamide gel. The RNA was transferred to MSI Magnacharge membranes, and Northern blotting was performed according to standard procedures (Ausubel et al., 1994). A 777 bp XhoI–BstEII fragment unique to the MUC1 ORF was used to probe for MUC1 transcripts, whereas a Bal I–Sal I fragment from the ORF of STA2 was used as a probe for STA2 transcripts. ACT1 was used as internal control, and a 563 bp ClaI fragment was used to probe for ACT1 transcripts. All probes were labelled radioactively with [32P]-dATP using the Prime-It II random primer labelling kit (Stratagene).
These authors contributed equally to this study
The authors wish to thank Drs K. Tatchell, S. Fields and M. Vanoni for generously providing constructs, Drs G. K. van der Merwe, P. Sudbery and F. Winston for providing yeast strains, and Dr E. Leberer for providing yeast strains and constructs. The authors wish to thank Dr K. J. J. Luyten and H. Swiegers for critical reading of the manuscript. This work was supported by funding from the Foundation for Research Development (FRD) and the South African wine industry (Winetech).