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. 2000 Nov;20(22):8364-72.
doi: 10.1128/MCB.20.22.8364-8372.2000.

Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion

X Pan  1 , J Heitman
Affiliations

Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion

X Pan et al. Mol Cell Biol. 2000 Nov.

Abstract

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

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Figures

FIG. 1
FIG. 1
Sok2 represses pseudohyphal growth and cell elongation. A homozygous wild-type strain (MLY61a/α) containing a control plasmid (YEplac195) or a 2μm TPK2 overexpression plasmid (pXP3) and a sok2Δ/sok2Δ mutant diploid strain (XPY80a/α) containing a control plasmid (YEplac195) were incubated on SLAD, SMAD, and SHAD media for 12 h at 30°C. Colonies were photographed at 50× magnification.
FIG. 2
FIG. 2
sok2 mutation enhances expression of the PHD1, ASH1, and SWI5 genes. Isogenic wild-type (MLY61a/α) and sok2Δ/sok2Δ mutant (XPY80a/α) strains were grown in YPD medium to an OD600 of 1.0. Cells were washed twice, transferred to liquid SLAD or SHAD medium, and incubated for 2 h. Total RNA was prepared and fractionated by formaldehyde gel electrophoresis. RNA was transferred to a nylon membrane and probed with portions of the SOK2, ASH1, PHD1, SWI5, and ACT1 genes.
FIG. 3
FIG. 3
Sok2 regulates pseudohyphal differentiation and FLO11 expression via Ash1 and Phd1. (A) Sok2 regulates expression of the FLO11 gene through Ash1 and Phd1. Isogenic wild-type (MLY40α) and ash1Δ (XPY138α), phd1Δ (MLY182α), ash1Δ phd1Δ (XPY192α), sok2Δ (XPY80α), sok2Δ ash1Δ (XPY191α), sok2Δ phd1Δ (XPY83α), and sok2Δ phd1Δ ash1Δ (XPY193α) mutant haploid strains were grown in synthetic glucose minimal medium to an OD600 of 1.0. Total RNA was prepared, fractionated, and probed with portions of the FLO11 and ACT1 genes. (B) Sok2 regulates pseudohyphal growth largely through Ash1 and Phd1. Isogenic wild-type (MLY61a/α) and phd1Δ/phd1Δ (MLY182a/α), ash1Δ/ash1Δ (XPY138a/α), ash1Δ/ash1Δ phd1Δ/phd1Δ (XPY192a/α), sok2Δ/sok2Δ (XPY80a/α), sok2Δ/sok2Δ phd1Δ/phd1Δ (XPY83a/α), sok2Δ sok2Δ ash1Δ/ash1Δ (XPY191a/α), and sok2Δ/sok2Δ phd1Δ/phd1Δ ash1Δ/ash1Δ (XPY193a/α) mutant strains were grown on SLAD medium for 3 days at 30°C. Representative colonies in this and the following figures were photographed at 25× magnification.
FIG. 4
FIG. 4
PHD1 overexpression enhances pseudohyphal differentiation through both Flo11-dependent and -independent mechanisms. (A) PHD1 overexpression enhances FLO11 gene expression. Isogenic wild-type (MLY40α) and ash1Δ (XPY138α), tpk2Δ (XPY5α), flo8Δ (XPY95α), and tec1Δ (MLY183α) mutant haploid strains containing a control plasmid (YEplac195) or a 2μm PHD1 plasmid (pCG38) were grown in SD-Ura to an OD600 of 1.0. Total RNA was prepared, fractionated, and probed with portions of the FLO11 and ACT1 genes. (B) PHD1 overexpression restores pseudohyphal growth in flo8Δ and flo11Δ mutant strains. Isogenic wild-type (MLY61a/α) and ash1Δ/ash1Δ (XPY138a/α), tpk2Δ/tpk2Δ (XPY5a/α), flo8Δ/flo8Δ (XPY95a/α), tec1Δ/tec1Δ (MLY183a/α), and flo11Δ flo11Δ (XPY107a/α) mutant strains containing a control plasmid (YEplac195) or a 2μm PHD1 plasmid (pCG38) were grown on SLAD medium for 3 days at 30°C and photographed.
FIG. 5
FIG. 5
Swi5 activates FLO11 gene expression and pseudohyphal growth via Ash1. (A) Swi5 regulates expression of the FLO11 gene through Ash1. An isogenic wild-type strain (MLY40α) containing a control plasmid (YEplac195), a 2μm plasmid expressing ASH1 (pXP104), a 2μm plasmid expressing ASH1 under control of the ADH1 promoter (pXP159), or a 2μm plasmid expressing SWI5 (pXP114), a swi5Δ mutant strain (XPY194α) containing a control plasmid (YEplac195) or a 2μm plasmid expressing ASH1 under control of the ADH1 promoter (pXP159), and an ash1Δ mutant strain (XPY138α) containing a control plasmid or a 2μm plasmid expressing SWI5 (pXP114) were incubated in synthetic glucose minimal medium to an OD600 of 1.0. Total RNA was prepared, fractionated, and probed with portions of the FLO11 and ACT1 genes. (B) Overexpression of the SWI5 gene enhances pseudohyphal growth. Wild-type strain MLY61a/α containing a control plasmid (YEplac195), a 2μm plasmid expressing ASH1 (pXP104), or a 2μm plasmid expressing SWI5 (pXP114) was incubated on nitrogen limitation media (50 and 200 μM ammonium sulfate) at 30°C for 3 days.
FIG. 6
FIG. 6
swi5, egt2, and cts1 mutations enhance pseudohyphal growth. (A) Swi5 regulates expression of the CTS1, EGT2, and ASH1 genes. Isogenic wild-type (MLY61a/α) and swi5Δ/swi5Δ (XPY194a/α), sok2Δ/sok2Δ (XPY80a/α), swi5Δ/swi5Δ sok2Δ/sok2Δ (XPY203a/α), and ash1Δ/ash1Δ (XPY138a/α) mutant strains were grown in YPD medium to an OD600 of 1.0. Cells were washed twice, transferred to liquid SLAD medium, and incubated for 2 h at 30°C. Total RNA was prepared, fractionated by formaldehyde gel electrophoresis, transferred to a nylon membrane, and probed with portions of the EGT2, CTS1, ASH1, and ACT1 genes. (B) Mutations in the SWI5, CTS1, or EGT2 gene enhance pseudohyphal growth in the absence of the FLO11 gene. Isogenic wild-type (MLY61a/α) and swi5Δ/swi5Δ (XPY194a/α), egt2Δ/egt2Δ (XPY205a/α), cts1Δ/cts1Δ (XPY208a/α), flo11Δ/flo11Δ (XPY107a/α), flo11Δ/flo11Δ swi5Δ/swi5Δ (XPY198a/α), flo11Δ/flo11Δ egt2Δ/egt2Δ (XPY206a/α), and flo11Δ/flo11Δ ets1Δ/ets1Δ (XPY209a/α) mutant strains were incubated on SLAD medium at 30°C for 3 days and photographed. (C) Flo11 is required for agar invasion in wild-type, swi5, egt2, and cts1 mutant strains. Representative colonies of the isogenic diploid strains grown on SLAD medium shown in panel B were washed with running water, and invasive cells that remained in the agar were photographed.
FIG. 7
FIG. 7
Sok2 regulates yeast pseudohyphal differentiation via Phd1, Ash1, and Swi5. In this model, Sok2 normally represses expression of the PHD1, SWI5, and ASH1 genes. The products of these three genes activate FLO11 gene expression, which is required for pseudohyphal differentiation. Swi5 is also required for expression of the EGT2 and CTS1 genes, both of which are required for mother-daughter cell separation after cytokinesis. As a result, swi5, egt2, and cts1 mutations enhance pseudohyphal growth, even in the absence of Flo11.
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