Gene expression profiling by RNA seq analysis and qPCR.

<p><b>A</b>) Genes induced by salt, tunicamycin (TUN), or galactose (GAL). All RNA seq comparisons are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004734#pgen.1004734.s007" target="_blank">Table S1</a>. <b>B</b>) Genes induced in a Pbs2p-dependent manner under the indicated conditions. Genes outlined by the dark blue circle (Pbs2p-dependent GAL specific) were functionally annotated in a pie chart in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004734#pgen.1004734.s002" target="_blank">Fig. S2</a></b>. <b>C</b>) Heat map of genes induced by the indicated stresses. Common targets and targets unique to each stimulus is shown. Asterisk, target of ESR. <b>D</b>) qPCR of HOG pathway target mRNAs in wild type and the <i>pbs2</i>Δ mutant grown in glucose (GLU, YEPD) and galactose (GAL, YEP-GAL). Error bars indicate +/−S.E.M. of three independent experiments. Actin (<i>ACT1</i>) mRNA was used as a control. <b>E</b>) Activity of p8X<i>CRE-lacZ</i> in wild-type cells (PC313) and <i>pbs2</i>Δ mutant (PC5035) grown in YEPD (5.5 hr), YEP-GAL (5.5 hr), and YEPD+0.4 M KCl (30 min). <b>F</b>) qPCR of Ste12p target mRNAs in wild type (PC538) and the <i>ste12</i>Δ (PC2382) mutant grown in glucose (YEPD) and galactose (YEP-GAL). See panel D for details.</p

Primers used for qPCR in the study.

<p>Primers used for qPCR in the study.</p

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

<div><p>Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In <i>Saccharomyces cerevisiae</i>, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen <i>Candida albicans</i> showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.</p></div

Comparison of HOG pathway activation by galactose and osmotic stress.

<p>For all phosphoblots involving Hog1p and Kss1p, the sizes of proteins are P∼Hog1p (∼49 kDa), Hog1p (∼49 kDa), P∼Kss1p (∼43 kDa), Kss1p (∼43 kDa), and Pgk1p (∼45 kDa). Pgk1p was used as a loading control. Asterisk (*) refers to a background band detected by the Kss1p antibody. Basal P∼Hog1p and P∼Kss1p showed variable levels under un-inducing conditions. <b>A</b>) Wild type cells (PC538) cells were grown to mid-log phase (∼5.5 hrs) in YEPD (GLU) or YEP-GAL (GAL) media and evaluated by immunoblot analysis for phosphorylation of the MAPKs Hog1p and Kss1p. <b>B</b>) Graph of P∼Hog1p levels under the indicated conditions, as determined by ImageJ analysis. <b>C</b>) Time-course analysis. Wild-type cells (PC538) were grown to mid-log phase and transferred to media containing salt (YEPD+0.4 M KCl) or galactose (YEP-GAL) for the indicated times. <b>D</b>) Extended time course of Hog1∼P during growth in galactose. <b>E</b>) Combinatorial analysis of the response to osmotic stress and galactose. Cells were grown to mid-log phase in YEPD, YEP-GAL, or YEPD+0.4M KCl, which was added to the cells growing in YEPD for 5 min. <b>F</b>) P∼Hog1p levels in cells shifted from galactose (YEP-GAL) to glucose (YEPD) for the indicated time points. Cells in YEP-GAL media were harvested by centrifugation, washed twice in water, and resuspended in YEPD for the indicated time points. <b>G</b>) P∼Hog1p levels during growth in 0.4M KCl and galactose in mutants lacking Ssk1p or Ste11p branches of the HOG pathway. Wild type cells (PC538), and the <i>ssk1</i>Δ (PC1523), <i>ssk2</i>Δ (PC6086), <i>ssk22</i>Δ (PC6085), <i>ssk2</i>Δ <i>ssk22</i>Δ (PC6031), <i>ste11</i>Δ (PC3861), <i>ste11</i>Δ <i>ssk1</i>Δ (PC2061), <i>pbs2</i>Δ (PC2053) and <i>hog1</i>Δ (PC6047) mutants were grown in YEP-GAL medium or YEPD medium containing 0.4M KCl for 5 min.</p

Strains used in this study.

<p>Strains used in this study.</p

Cross-inhibition between the filamentous growth and HOG pathways during growth in galactose.

<p><b>A</b>) Morphology of wild-type cells (PC538) and the <i>pbs2</i>Δ mutant (PC2053), grown on YEPD and YEP-GAL for 24 hrs. Bar, 5 microns. <b>B</b>) p<i>FRE-lacZ</i> reporter activity in wild-type cells (PC313) and the <i>pbs2</i>Δ mutant (PC5035) in YEP-GAL medium. <b>C</b>) Role of protein tyrosine phosphatases in P∼Hog1p activity in galactose. Wild-type cells (PC538), and the <i>ptp2</i>Δ (PC6156), <i>ptp3</i>Δ (PC6157) and <i>ptp2</i>Δ <i>ptp3</i>Δ double mutant (PC6158) were grown in YEPD and YEP-GAL media for 5.5 hrs. <b>D</b>) P∼Kss1p activity in wild-type cells and the <i>pbs2</i>Δ mutant (PC2053) grown in YEP-GAL medium over a time course as indicated. <b>E</b>) P∼Hog1p activity in the <i>kss1</i>Δ mutant (PC620) grown in YEP-GAL medium for the times indicated. <b>F</b>) qPCR showing the relative expression of <i>STE12</i> mRNA in the wild-type (PC538), <i>pbs2</i>Δ (PC2053) and <i>ste12</i>Δ (PC2382) mutant cells. Error bars indicate +/− standard error mean of three independent experiments. Actin (<i>ACT1</i>) mRNA was used as a control. <b>G</b>) Ste12p-HA protein levels in the wild-type and <i>pbs2</i>Δ strains. Hog1p levels by immunoblot analysis are also shown.</p

Role of increased metabolic respiration and Snf1p in activation of the HOG pathway.

<p><b>A</b>) Immunoblot showing P∼Hog1p levels in cells grown in glucose (YEPD), galactose (YEP-GAL) or glucose and galactose (YEPD+2% GAL). <b>B</b>) Wild-type cells (PC6016) and the <i>gal3</i>Δ, <i>gal4</i>Δ, <i>gal7</i>Δ and <i>gal10</i>Δ mutants grown in YEP-GAL. <b>C</b>) P∼Hog1 levels in cells grown under the indicated conditions for 3 h with or without antimycin, ANT. <b>D</b>) Wild type (PC538) and the <i>aco1</i>Δ (PC3912), <i>fum1</i>Δ (PC6152), <i>mdh1</i>Δ (PC6153) and <i>kgd1</i>Δ (PC6155) and <i>idh1</i>Δ (PC6154) mutants were grown in galactose for 5.5 hrs. <b>E</b>) Wild-type cells (PC538), and the <i>snf1</i>Δ (PC560), <i>mig1</i>Δ (PC4843) and <i>snf1</i>Δ <i>mig1</i>Δ (PC6076) mutants were grown in YEP-GAL medium for 5.5 hrs.</p

Role of the HOG and filamentous growth pathways in growth in galactose and effect of the inhibitory role of the HOG pathway on filamentous growth pathway outputs.

<p><b>A</b>) Serial dilutions of wild-type (PC313), <i>ste7</i>Δ (PC4928), <i>pbs2</i>Δ (PC5035) and <i>ste7</i>Δ <i>pbs2</i>Δ (PC6272) cells were spotted on YEPD and YEP-GAL media. <b>B</b>) Morphology of wild-type cells (PC538), the <i>pbs2</i>Δ mutant (PC2053), the <i>ste7</i>Δ mutant (PC4982), and the <i>ste7</i>Δ <i>pbs2</i>Δ double mutant (PC6272) grown YEP-GAL media for 24 hrs. Bar, 5 microns. <b>C</b>) Septin staining of wild-type and <i>pbs2</i>Δ cells harboring the pCdc12p-GFP plasmid. Cells were grown to mid-log phase in YEPD. <b>D</b>) Mat formation in cells lacking the filamentous growth or HOG pathways. Wild-type (PC538), <i>flo11</i>Δ (PC1029), and <i>pbs2</i>Δ (PC2053) strains were grown in YEPD medium for 16 hrs and then spotted onto low agar (0.3%) YEP-GAL medium for 3 d at 30°C. Bar, 1 cm.</p

Bypass of <i>Candida albicans</i> Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1

<div><p>Biofilm formation on implanted medical devices is a major source of lethal invasive infection by <i>Candida albicans</i>. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A <i>cak1</i> <u>d</u>iminished e<u>x</u>pression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the <i>cak1</i> DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one.</p></div

DX mutant isolate comparison.

<p>Multiple isolates of each DX strain indicated were obtained from a single transformation of a heterozygous deletion mutant. RNA was extracted from cells grown for 4 hr at 30°C in YPD and used for nanoString expression analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>). Hierarchal clustering of gene expression data was performed using MeV software. Fold change values were obtained by dividing normalized expression values for each mutant strain by the wild-type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up.) Strains are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>.</p