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.     

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.     

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.     

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.     

Saccharomyces Cerevisiae S288c Has a Mutation in Flo8, a Gene Required for Filamentous Growth

Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae. This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen. Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen. Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene. This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation. Since feral strains of S. cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog.

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.     

The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophan availability

Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants ( elm ) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1-15 , has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS /cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A 'signal integration' model for the complex relationship of Elm1 and the RAS/ cAMP pathway in controlling morphogenesis in response to nutrients is proposed.     

Pichia fermentans dimorphic changes depend on the nitrogen source

Pichia fermentans DiSAABA 726 is a biofilm-forming yeast that undergoes dimorphic transition. Under yeast-like morphology it controls brown rot caused by Monilia spp. on apple fruit, while under pseudohyphal form, it shows pathogenic behaviour itself on peach fruit. The present study investigates the nutritional factors that induce and separate yeast-like and pseudohyphal morphologies under laboratory conditions. We show that P. fermentans DiSAABA 726 produces mainly yeast-like cells on media containing millimolar concentrations of urea and diammonium phosphate, and forms pseudohyphae at micromolar concentrations of these two salts. With ammonium sulphate, yeast-like or pseudohyphal morphology depends on the N concentration and the pH of the culture media. Amino acids such as methionine, valine, and phenylalanine invariably induce pseudohyphal morphology irrespective of the N concentration and the pH of the culture media. Methionol, 1-butanol, isobutanol, and isopropanol induce pseudohyphal growth, while phenylethanol and isoamyl alcohol fail to induce the formation of filaments. Thus, the morphogenesis of P. fermentans DiSAABA 726 depends more on the nitrogen source than on the N concentration, and is regulated by the quorum-sensing molecules that are generally produced from amino-acid assimilation under nitrogen starvation.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Delta mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.     

RAM pathway contributes to Rpb4 dependent pseudohyphal differentiation in Saccharomyces cerevisiae

Rpb4, a subunit of RNA Polymerase II plays an important role in various stress responses in budding yeast, Saccharomyces cerevisiae. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathway. In the present study, we show that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4Delta/rpb4Delta cells exhibit pseudohyphae even in the absence of functional MAP kinase and cAMP-PKA pathways. Genome-wide expression profiling showed that in the absence of Rpb4 several genes controlling mother daughter cell separation are down regulated. Our genetic studies also provide evidence for involvement of RNA Pol II subunit Rpb4 in the expression of genes downstream of the RAM pathway. Finally, we show that this effect on expression of RAM pathway may at least be partially responsible for the pseudohyphal phenotype of rpb4Delta/rpb4Delta cells.     

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex

BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.     

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.     

白色念珠菌CaBEM1基因的克隆及其在酿酒酵母丝状生长中的作用

白色念珠菌在不同的生长条件下能发生显著的形态变化 ,这种变化由多种调控因子与信号转导途径所调控。酿酒酵母的G1期细胞周期蛋白Cln1和Cln2参与其形态发生 ,cln1/cln1、cln2 /cln2双缺失株不能形成菌丝。把白色念珠菌基因组文库导入cln1/cln1、cln2 /cln2缺失株 ,筛选能校正菌丝形成缺陷的基因 ,分离得到白色念珠菌中的CaBEM 1基因。从核苷酸序列推导 ,CaBEM1编码一种 6 32个氨基酸的蛋白质 ,氨基酸序列分析表明在其N端有 2个SH3结构域 ,中部有 1个PX结构域 ,C端有 1个PB1结构域 ;CaBem1的氨基酸序列与酿酒酵母的Bem1同源性达 38% ,与裂殖酵母的Scd2同源性达 32 %。在酿酒酵母的缺失株中异源表达CaBEM1,能够部分校正它们在氮源缺乏条件下的菌丝形成缺陷。这种菌丝形成的校正作用绕过MAPK途径和cAMP/PKA途径 ,表明CaBem1在菌丝形成中的作用可能位于这两条信号转导途径的下游     

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

Background

Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi.

Methods

Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.

Results

Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.

Conclusion

Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.

General significance

Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.