Overrepresented functional categories in Sfl1p and Sfl2p ChIP-Seq data.

a<p>Grouping of the Sfl1p and/or Sfl2p targets identified in ChIP-Seq data according to GO terminology determined by using the online CGD GO Term Finder tool (<a href="http://www.candidagenome.org/cgi-bin/GO/goTermFinder" target="_blank">http://www.candidagenome.org/cgi-bin/GO/goTermFinder</a>). Analysis conducted in October 2012 (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#s4" target="_blank">Materials and Methods</a>).</p>b<p>Ontology classification according to the three GO terminologies (biological process, cellular component and molecular function).</p>c<p>Percentages were calculated based on the number of genes in each GO category divided by the total number (110 genes for Sfl1p and Sfl2p common targets, 73 genes for Sfl2p specific targets, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#s4" target="_blank">Materials and Methods</a> for details).</p>d<p>Percentages were calculated based on the number of genes in each category divided by the total number of annotated genes of the <i>C. albicans</i> genome, according to CGD (6,513 genes).</p>e<p><i>P</i> values for the overrepresented categories were calculated using a hypergeometric distribution with multiple hypothesis correction (i.e., Bonferroni's correction) as described in the GO Term Finder tool website (<a href="http://www.candidagenome.org/help/goTermFinder.shtml" target="_blank">http://www.candidagenome.org/help/goTermFinder.shtml</a>). The <i>P</i> value cutoff used was ≤0.05.</p>f<p>Gene name or orf19 nomenclature according to CGD. Some genes were attributed to more than one GO term.</p

Genetic interactions of <i>SFL1</i> and <i>SFL2</i> with their transcriptional target genes encoding key regulators of hyphal development.

<p>(<b>A</b>) The wild-type SC5314 (WT) together with the homozygous <i>sfl1</i> (<i>sfl1</i>ΔΔ, CEC2001), <i>sfl2</i> (<i>sfl2</i>ΔΔ,CEC1535), <i>brg1</i> (<i>brg1</i>ΔΔ, CEC2058), the double homozygous <i>sfl1</i>, <i>sfl2</i> (<i>sfl1</i>ΔΔ <i>sfl2</i>ΔΔ, CEC2658) and <i>sfl1</i>, <i>brg1</i> (<i>sfl1</i>ΔΔ <i>brg1</i>ΔΔ, CEC2840) mutants were grown in yeast-promoting (SD at 30°C for 6 h30 min) or sub-hypha-inducing (YP 10% FBS at 30°C for 6 h30 min) conditions and observed microscopically. Scale bar = 10 µm. The detailed cell morphology of each strain grown in YP 10% FBS are shown (Morphological details, bottom panel) (<b>B</b>) The pNIMX expression system <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#ppat.1003519-Chauvel1" target="_blank">[41]</a> was used to drive anhydrotetracycline-dependent overexpression of <i>SFL2</i> (P<i>_TET</i>-<i>SFL2</i>) in a wild-type (WT, BWP17AH complemented for uracil auxotrophy) or in different homozygous mutant backgrounds, including <i>sfl1</i>Δ/<i>sfl1</i>Δ (<i>sfl1</i>ΔΔ), <i>sfl2</i>Δ/<i>sfl2</i>Δ (<i>sfl2</i>ΔΔ), <i>ume6</i>Δ/<i>ume</i>6Δ (<i>ume6</i>ΔΔ), <i>tec1</i>Δ/<i>tec1</i>Δ (<i>tec1</i>ΔΔ), <i>brg1</i>Δ/<i>brg1</i>Δ (<i>brg1</i>ΔΔ) and <i>efg1</i>Δ/<i>efg1</i>Δ (<i>efg1</i>ΔΔ) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#ppat-1003519-t001" target="_blank">Table 1</a>). All strains were grown in YPD medium at 30°C during 18 hours in the presence of 3 µg/ml of anhydrotetracycline before microscopic examination. As a control, the same growth conditions were also used with all strain backgrounds carrying the empty plasmid (CIp10, Control). Two different fields with detailed cell morphology of each strain overexpressing <i>SFL2</i> are shown (Morphological details, right panels).</p

Strategy for tagging Sfl1p and Sfl2p with a triple hemagglutinin (3×HA) epitope tag and characterization of the tagged strains.

<p>(<b>A</b>) Schematic representation of the <i>SFL1-HA₃</i> or <i>SFL2-HA₃</i> tagging cassette allowing expression of the Sfl1p-HA₃ or Sfl2p-HA₃ fusion proteins following a <i>Stu</i>I digestion (<i>Stu</i>I) and integration at the <i>RPS1</i> locus (<i>RPS1</i>, black rectangles) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#ppat.1003519-Care1" target="_blank">[42]</a>. A triple HA tag (dark grey box) was inserted in frame with the <i>SFL1</i> or <i>SFL2</i> coding sequences (<i>SFL1</i> or <i>SFL2</i>; black arrowed rectangle) in plasmid pCaEXP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#ppat.1003519-Care1" target="_blank">[42]</a>. The tagged alleles are placed under the control of the <i>MET3</i> promoter (<i>MET3</i>p; ligh grey rectangle), which is induced in the absence of methionine and cysteine, and are followed by the <i>C. albicans URA3</i> marker (open rectangle). (<b>B</b>) Western blot analysis of homozygous <i>sfl1</i> or <i>sfl2</i> mutants (<i>sfl1</i>Δ/<i>sfl1</i>Δ or <i>sfl2</i>Δ/<i>sfl2</i>Δ) expressing HA₃-tagged versions of the <i>SFL1</i> or <i>SFL2</i> genes, respectively (<i>SFL1-HA₃</i> or <i>SFL2- HA₃</i>) together with the corresponding empty vector controls (Vector). The SGY243 strain expressing the <i>CAP1-HA₃</i> (<i>CAP1-HA₃</i>) or carrying the empty vector (Vector) were used as a positive control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003519#ppat.1003519-Znaidi1" target="_blank">[43]</a>. Strains were grown overnight in SD medium (P<i>_MET3</i>-inducing conditions) and total protein extracts were prepared then subjected to SDS-PAGE. Western blotting was performed using an anti-HA antibody. Positions of the molecular mass standards are indicated on the left (kDa). Immunopositive signals from the Sfl1p-HA₃ and Sfl2p-HA₃ fusions are indicated with black arrows (<b>C</b>) Phenotypic analysis of the strains expressing the HA₃-tagged <i>SFL1</i> or <i>SFL2</i> alleles. Strain SC5314 (control) together with the homozygous <i>sfl1</i> or <i>sfl2</i> mutants expressing the <i>SFL1-HA₃</i> or <i>SFL2-HA₃</i> alleles (<i>SFL1-HA₃</i>, <i>SFL2-HA₃</i>), respectively, or carrying the empty vector (Vector) were grown overnight in YPD at 30°C then transferred to Lee's medium lacking methionine and cysteine and allowed to grow during 4 h at 37°C before being examined microscopically (40× magnification).</p

Efg1p binds to the promoter of many Sfl1p and Sfl2p targets and co-immunoprecipitates with Sfl1p and Sfl2p, <i>in vivo</i>.

<p>(<b>A</b>) ChIP-PCR assay of selected Sfl1p and Sfl2p target promoters. Strains SFL1-TAP (CEC1922), SFL2-TAP (CEC1918) and EFG1-HA (HLCEEFG1) were grown in SC medium at 30°C (30°C) or in Lee's medium at 37°C (37°C) together with the SC5314 control strain (Control) during 4 h before being subjected to chromatin immunoprecipitation (Anti-TAP, Anti-HA) followed by PCR using primers specific to the indicated promoter regions. The <i>URA3</i> and <i>YAK1</i> genes were used as negative controls for ChIP enrichment. (<b>B</b>) Co-Immunoprecipitation of Efg1p with Sfl1p and Sfl2p. Strains coexpressing <i>SFL1-TAP</i> and <i>EFG1-HA</i> (Lanes 2 and 3) or <i>SFL2-TAP</i> and <i>EFG1-HA</i> (Lanes 7 and 8) or controls (Lanes 1 and 6, <i>EFG1-HA</i> only; lanes 4 and 9, <i>SFL1-TAP</i> only; lanes 5 and 10, <i>SFL2-TAP</i> only) were cultivated in SC medium at 30°C or in Lee's medium at 37°C before crosslinking with formaldehyde. Total extracts were incubated with Dynal PanMouse IgG beads directed against TAP epitope tag prior to washing and Western blotting using anti-TAP (IP Anti-TAP, 10% of the beads/total extracts mixture) and anti-HA (Co-IP Anti-HA) antibodies. A portion of the total cell extracts (∼2%) was included to verify the presence of the Efg1p-HA fusion (Total extracts Anti-HA).</p

Genome-wide location of <i>Candida albicans</i> Sfl1p and Sfl2p, <i>in vivo</i>, at a single-nucleotide resolution.

<p>(<b>A</b>) Venn diagram of the overlap between Sfl1p and Sfl2p binding targets. All 113 Sfl1p targets are also bound by Sfl2p, while 75 target promoters are Sfl2p-specific. The total number of Sfl1p or Sfl2p target promoters are indicated between parentheses. Target promoters include those that are clearly associated with given ORFs as well as those that are shared by two ORFs in opposite orientations. (<b>B</b>) A single-nucleotide resolution of Sfl1p and Sfl2p binding at selected <i>C. albicans</i> genomic regions <i>in vivo</i>. Plotted are read-count signal intensities of HA₃-tagged <i>SFL1</i>- (<i>sfl1</i>-CaEXP-<i>SFL1-HA₃</i>) or <i>SFL2</i>- (<i>sfl2</i>-CaEXP-<i>SFL2-HA₃</i>) coimmunoprecipitated DNA and the corresponding empty-vector control signals (<i>sfl1</i>-CaEXP, <i>sfl2</i>-CaEXP, respectively) from merged BAM files of two independent biological replicates. Some read-count signals extend beyond the maximum graduation (not shown) that ranges between 0–500 reads for Sfl1 data (<i>sfl1</i>-CaEXP and <i>sfl1</i>-CaEXP-<i>SFL1-HA₃</i>) and 0–1000 reads for Sfl2 data (<i>sfl2</i>-CaEXP and <i>sfl2</i>-CaEXP-<i>SFL2-HA₃</i>). The position of each signal in selected <i>C. albicans</i> genomic regions from assembly 21 is shown on the <i>x</i>-axis. The location of each selected region from the corresponding chromosome (Chr) is indicated at the top of each panel (limits are shown between parentheses in base pairs). The orientation of each ORF is depicted by the arrowed black rectangle. (<b>C</b>) Enrichment scores of the Gene Ontology (GO) terms to which are assigned Sfl1p and Sfl2p common (shaded area) or Sfl2p-specific (unshaded area) binding targets. GO term enrichment scores are calculated as the negative value of the log₁₀-transformed <i>P</i>-value. The number of genes of each category is shown at the right of each horizontal bar.</p

P<i>_TET</i>-driven OE of 20 genes results in decreased or increased growth rate. A. Impact of OE on the growth rates of 257 OE strains.

<p>Genes whose OE decreases or increases growth rate (≥2 fold and >2 fold, respectively) upon growth in liquid medium are listed in red or green boxes respectively. Genes are classified based on their OE phenotype from the more affected to the less affected. Genes in orange correspond to those whose OE decreased growth with a fold equal to 2. Data are mean and SD of 3 experiments. <b>B. P</b><b><i>_TET</i></b><b>-driven OE of </b><b><i>YCK2</i></b><b>, </b><b><i>BEM1</i></b><b> and </b><b><i>EFH1</i></b><b> results in cell lysis. </b><i>C. albicans</i> strains with integrated CIp10-P<i>_TET</i>-GTW derivatives harbouring the <i>GFP</i>, <i>YCK2</i>, <i>BEM1</i> or <i>EFH1</i> ORFs were grown in YPD or YPD supplemented with 3 µg.mL<b>⁻</b>¹ ATc for 18 h and observed microscopically. Cell lysis is indicated by arrows. Scale bar = 5 µm. <b>C. P</b><b><i>_TET</i></b><b>-driven OE of </b><b><i>YCK2</i></b><b> and </b><b><i>EFH1</i></b><b> reduces growth rate on solid medium.</b> Serial dilutions of cultures of <i>C. albicans</i> strains with integrated CIp10-P<i>_TET</i>-GTW derivatives harbouring the <i>GFP</i>, <i>UME6</i>, <i>YCK2</i>, <i>BEM1</i> or <i>EFH1</i> ORFs were spotted on SD or SD supplemented with 3 µg.mL<b>⁻</b>¹ ATc and observed after 3 days of growth at 30°C.</p

Functionality of the Gateway® OE systems. A. P<i>_PCK1</i>-driven and P<i>_TET</i>-driven OE of <i>UME6</i> but not GFP triggers morphogenesis.

<p><i>C. albicans</i> strains with integrated CIp10-P<i>_PCK1</i>-GTW-TAPtag or CIp10-P<i>_TET</i>-GTW derivatives harbouring the GFP (CEC2407 or CEC2992, respectively) or <i>UME6</i> (CEC1097 or CEC2994, respectively) ORFs were observed microscopically upon growth in gluconeogenic conditions or YPD supplemented with 50 µg.mL<b>⁻</b>¹ Dox at 30°C for 18 h. Scale bar = 5 µm. <b>B. Production of TAPtagged proteins.. </b><i>C. albicans</i> strains with integrated CIp10-P<i>_PCK1</i>-GTW-TAPtag derivatives harbouring the GFP (CEC2407) or <i>UME6</i> (CEC1097) ORFs were grown in SD (−) or YNB 2% casamino acids (+) for 6 h. Whole cell extracts were separated by SDS-PAGE and probed with a peroxidase-coupled antibody allowing the detection of TAPtagged proteins in gluconeogenic conditions only. Proteins of interest are indicated by an arrow along with their deduced size. <b>C. Kinetics of expression from the </b><b><i>PCK1</i></b><b> or </b><b><i>TET</i></b><b> promoters.. </b><i>C. albicans</i> strains with integrated CIp10-P<i>_PCK1</i>-GTW-TAPtag or CIp10-P<i>_TET</i>-GTW derivatives harbouring the GFP (CEC2407 or CEC2992, respectively) or <i>gLUC59</i> (CEC1906 or CEC3083, respectively) ORFs were grown in YNB 2% casamino acids or YPD supplemented with 3 µg.mL<b>⁻</b>¹ ATc for 18 h at 30°C. Data represent luciferase specific activity detected from the different strains at the indicated time points of growth under inducing conditions. Assays were performed in duplicate and means and SD are shown.</p