The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.

Saccharomyces cerevisiae mutant E124 was selected in a visual screen based on elongated cell shape. Genetic analysis showed that E124 contains two separate mutations, pps1-1 and elm4-1, each causing a distinct phenotype inherited as a single-gene trait. In rich medium, pps1-1 by itself causes increased doubling time but does not affect cell shape, whereas elm4-1 results in a moderate cell elongation phenotype but does not affect growth rate. Reconstructed elm4-1 pps1-1 double mutants display a synthetic phenotype in rich medium including extreme cell elongation and delayed cell separation, both characteristics of pseudohyphal differentiation. The elm4-1 mutation was shown to act as a dominant factor that potentiates pseudohyphal differentiation in response to general nitrogen starvation in a genetic background in which pseudohyphal growth normally does not occur. Thus, elm4-1 allows recognition of, or response to, a pseudohyphal differentiation signal that results from nitrogen limitation. PPS1 was isolated and shown to be a previously undescribed gene coding for a protein similar in amino acid sequence to phosphoribosylpyrophosphate synthase, a rate-limiting enzyme in the biosynthesis of nucleotides, histidine, and tryptophan. Thus, the pps1-1 mutation may generate a nitrogen limitation signal, which when coupled with elm4-1 results in pseudohyphal growth even in rich medium.     

Mutational Analysis of Morphologic Differentiation in Saccharomyces Cerevisiae

A genetic analysis was undertaken to investigate the mechanisms controlling cellular morphogenesis in Saccharomyces cerevisiae. Sixty mutant strains exhibiting abnormally elongated cell morphology were isolated. The cell elongation phenotype in at least 26 of the strains resulted from a single recessive mutation. These mutations, designated generically elm (elongated morphology), defined 14 genes; two of these corresponded to the previously described genes GRR1 and CDC12. Genetic interactions between mutant alleles suggest that several ELM genes play roles in the same physiological process. The cell and colony morphology and growth properties of many elm mutant strains are similar to those of wild-type yeast strains after differentiation in response to nitrogen limitation into the pseudohyphal form. Each elm mutation resulted in multiple characteristics of pseudohyphal cells, including elongated cell shape, delay in cell separation, simultaneous budding of mother and daughter cells, a unipolar budding pattern, and/or the ability to grow invasively beneath the agar surface. Mutations in 11 of the 14 ELM gene loci potentiated pseudohyphal differentiation in nitrogen-limited medium. Thus, a subset of the ELM genes are likely to affect control or execution of a defined morphologic differentiation pathway in S. cerevisiae.     

Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.     

Mos10 (Vps60) is required for normal filament maturation in Saccharomyces cerevisiae

Early pseudohyphal growth of Saccharomyces cerevisiae is well described, and is known to be subject to a complex web of developmental regulation. In maturing filaments, young cells differ significantly from their pseudohyphal progenitors, in their shape, and in their timing and direction of cell division. The changes that occur during filament maturation result in round and oval cells surrounding and covering the pseudohyphal filament. In a screen for mutants that affect this process, a vacuolar protein sorting gene, MOS10 (VPS60), and a gene encoding an alpha subunit of the proteasome core, PRE9, were isolated. Characterization of the mos10/mos10 phenotype showed that the process of filament maturation is regulated differently from early filamentous growth, and that the requirement for Mos10 is limited to the maturation stage of pseudohyphal development. The mos10/mos10 phenotype is unlikely to be an unspecific effect of disruption of endocytosis or vacuolar protein sorting, because it is not recapitulated by mutants in other genes required for these processes. Disruption of homologues of MOS10, which act as components of the ESCRT-III complex in targeting proteins for vacuolar degradation, results in abnormal early pseudohyphal growth, not in the filament maturation defect seen in mos10/mos10. Thus, Mos10 may function in targeting of specific cargo proteins for degradation, under conditions particular to maturing filaments.     

The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis

Saccharomyces cerevisiaepresents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiaeW27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiaeW27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2gene acts as a negative regulator of pseudohyphal growth.     

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.     

Control of nitrogen catabolite repression is not affected by the tRNAGln-CUU mutation, which results in constitutive pseudohyphal growth of Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to nitrogen availability in several ways. (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR). Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell's doubling time. (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth. (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G1. A specific allele of the SUP70/CDC65 tRNAGln gene (sup70-65) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR. Our data confirm that pseudohyphal growth occurs gratuitously in sup70-65 mutants cultured in nitrogen-rich medium at 30 degrees C. However, we find neither any defect in NCR in the sup70-65 mutant nor any alteration in the control of YVH1 expression, which has been previously shown to be specifically induced by nitrogen starvation.     

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.     

Polarized growth in fungi: symmetry breaking and hyphal formation

Cell shape is a critical determinant for function. The baker's yeast Saccharomyces cerevisiae changes shape in response to its environment, growing by budding in rich nutrients, forming invasive pseudohyphal filaments in nutrient poor conditions and pear shaped shmoos for growth towards a partner during mating. The human opportunistic pathogen Candida albicans can switch from budding to hyphal growth, in response to numerous environmental stimuli to colonize and invade its host. Hyphal growth, typical of filamentous fungi, is not observed in S. cerevisiae. A number of internal cues regulate when and where yeast cells break symmetry leading to polarized growth and ultimately distinct cell shapes. This review discusses how cells break symmetry using the yeast S. cerevisiae paradigm and how polarized growth is initiated and maintained to result in dramatic morphological changes during C. albicans hyphal growth.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Pseudohyphal Growth of Cryptococcus neoformans Is a Reversible Dimorphic Transition in Response to Ammonium That Requires Amt1 and Amt2 Ammonium Permeases

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.     

The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development

The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.     

Variation of colony morphology and chromosomal rearrangement in Candida tropicalis pK233

UV irradiation treatment of the asexual yeast Candida tropicalis gave rise to morphological mutants exhibiting at least four different types of abnormal colonies on glucose-containing solid medium. These mutants were named according to their colony morphologies: 'doughnut', 'frilly', 'echinoid' and 'walnut' mutants. The doughnut mutant produced a wrinkled colony with a hollow in its central region that was rich in filamentous pseudohyphal cells. With increased incubation time, the colony gradually changed to a reticulate shape. The parent strain, which normally produced smooth colonies, gave similar colonies to those of the doughnut mutant when grown in medium containing oleic acid as carbon source. Both the frilly and the walnut mutants produced pseudohyphal cells in a similar fashion to the doughnut mutant. The echinoid mutant produced an echinulate colony morphology with aerial hyphae and contained true hyphal cells as well as pseudohyphal ones. Pulsed-field gel electrophoresis showed that the echinoid and frilly mutants had different karyotypes from that of their parent strain, suggesting the occurrence of chromosomal rearrangements associated with these morphological mutations.