The roles of Med31 in adhesion are different between <i>S. cerevisiae</i> and <i>C. albicans</i>.

<p>A) Colonies of wild type <i>S. cerevisiae</i> Σ1278b and the <i>med31Δ</i> mutant were grown on YPD plates at 30°C and photographed. The <i>flo11Δ</i> strain was used as the negative control, to show smooth colony morphology in the absence of <i>FLO11</i>. B) Expression of <i>FLO11</i> was tested by qPCR after 90 min in 0.2% glucose synthetic complete media, the condition used for biofilm formation in C). Levels of <i>FLO11</i> were normalized to <i>ACT1</i>, and expressed related to the wild type, which was set to 1. Shown are averages from at least three independent biological repeats and the standard error. C) The ability of the <i>S. cerevisiae med31Δ</i> mutant to adhere to polystyrene was assessed in 0.2% glucose synthetic complete media as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#s4" target="_blank">Materials and Methods</a>. Quantification was performed by crystal violet staining. At least three independent cultures were used, assayed in quadruplicates. For the mutant, two independently constructed deletion strains were used and gave equivalent results. The <i>flo11Δ</i> mutant was assayed in parallel as a negative control and showed no adherence at any of the time points (not shown). p&lt;0.001.</p

Cell wall–related genes differentially expressed in the <i>C. albicans med31ΔΔ</i> mutant.

<p>Cell wall–related genes differentially expressed in the <i>C. albicans med31ΔΔ</i> mutant.</p

Med31 is required for cytokinesis and filamentous growth of <i>C. albicans</i>.

<p>A) Cultures of wild type C. <i>albicans</i>, the <i>med31ΔΔ</i> mutant and the complemented <i>med31ΔΔ</i>+<i>MED31</i> strain were grown to log phase and cells were observed by microscopy using DIC for bright field (left panel) or through the DAPI filter for calcofluor white staining (right panel). Strains lacking Med31 display a cell separation defect, forming cell chains with the cells attached at the mother-daughter junction, as judged by staining with calcofluor white. B) To determine the proportion of cell chains at least 200 cells were counted for each of the strains. Cell counts were performed after a brief 1 s sonication to disperse cell aggregates. Shown are averages of three independent experiments and the standard error. C) Wild type or mutant strains were streaked on plates containing filamentation-inducing media and incubated at 37°C for 4–5 days. The colonies were photographed using a stereo dissecting microscope. The <i>med31ΔΔ</i> mutant was unable to undergo filamentous differentiation on plates in all media tested. The mutant was also compromised for filamentation in liquid media (data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s007" target="_blank">Figure S5</a>).</p

Regulation of adhesins and Ace2-dependent genes by <i>MED20</i> and <i>SRB9/MED13</i> in <i>C. albicans</i>.

<p>A) Cells from the indicated strains were grown in yeast form (YPD) and relative mRNA levels for the indicated genes determined by qPCR. <i>ACT1</i> was used for normalization. Similar results were obtained when <i>THD3</i> was used as the normalization control (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s005" target="_blank">Figure S3</a>). Shown are averages for three independent biological repeats and the standard error. B) Yeast cell morphology of the wild type and Mediator mutants was assessed in YPD at 30°C. The scale bar is 20 µm.</p

The <i>med31ΔΔ</i> mutant is defective for filamentation and virulence in an animal host.

<p>A) The worm <i>C. elegans</i> was infected by wild type <i>C. albicans</i>, the <i>med31ΔΔ</i> mutant and the <i>med31ΔΔ</i>+<i>MED31</i> complemented strain and the appearance of penetrative filamentation was monitored daily over a period of seven days. The worms were photographed with a 40× magnification objective. B) The percentage of worm filamentation was determined after three days of infection. Shown are averages of 4 experiments and the standard deviation. The p value was &lt;0.002 for both the comparison of the mutant with the wild type, and the mutant with the complemented strain. C) The ability of the <i>med31ΔΔ</i> mutant to kill worms was determined in the first 80 h post infection, when most of the worm death due to penetrative filamentation occurs. Three independent experiments were performed and equivalent results obtained. A representative experiment is shown. The <i>med31ΔΔ</i> mutant killed the <i>C. elegans</i> host with delayed kinetics compared to the wild type and the reconstituted strains (blue line- <i>med31ΔΔ</i>, grey line- WT, red line- <i>med31ΔΔ</i>+<i>MED31</i>). The log-rank test used for statistical analysis returned a p value of 0.0032 for the wild type versus <i>med31ΔΔ</i> mutant comparison, while there was no significant difference between the wild type and the <i>med31ΔΔ</i>+<i>MED31</i> complemented strain p = 0.5572).</p

Med31 is required for the expression of Ace2-dependent genes and adhesins in yeast and hyphal growth.

<p>A) Cells from <i>med31ΔΔ</i> or <i>ace2ΔΔ</i> mutants were grown in YPD at 30°C (yeast morphology) or in Spider media at 37°C to induce filamentous growth, and levels of the indicated genes determined by quantitative PCR. The expression of the indicated genes was normalised to <i>ACT1</i> and expressed relative to wild type levels, which were set to 1. Equivalent results were obtained when the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene <i>TDH3</i> was used for normalization (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s005" target="_blank">Figure S3</a>). Shown are the averages of at least three independent experiments and the standard error. B) The promoter regions (2 kb) of <i>ALS1</i>, <i>ALS3</i> and <i>EAP1</i> were searched for putative Ace2 binding motifs from <i>S. cerevisiae</i> (RRCCAGC) or <i>C. albicans</i> (MMCCASC) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613-Mulhern1" target="_blank">[57]</a>. The three motifs conforming to <i>C. albicans</i> consensus sequences were found for the <i>ALS3</i> promoter, whereas none were found for the other two genes. The activating regions A1 and A2 in the <i>ALS3</i> promoter were mapped by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613-Argimon1" target="_blank">[59]</a>: A1: from −321 bp to −471 bp (upstream of the START codon); A2: from −1049 to −1438 bp.</p