The <i>CdTLO2</i> overexpression embedded agar filamentation phenotype observed in wild type <i>C</i>. <i>dubliniensis</i> is not observed in a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain.

<p>Embedded agar filamentation in wild type (yLM339) and <i>med3Δ/Δ</i> (yLM340) <i>C</i>. <i>dubliniensis</i> strains overexpressing <i>CdTLO2</i> from a <i>TDH3</i> promoter is analyzed by diluting cells in YPS agar and inspecting the ‘colony’ growth in the agar after 48 and 72 hours using a stereoscope.</p

Non-Mediator associated CdTlo1p is rapidly degraded by the proteasome.

<p><b>(A)</b> Schematic of treatment cells with cycloheximide (CHX) and the MG132 proteasome inhibitor to determine whether a Tlo protein is subject to proteasome dependent degradation. <b>(B)</b> Immunoblot of endogenous HA-tagged CdTlo1p in a wild type a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain (yLM308) after treatment with cycloheximide, in the absence and presence of the proteasome inhibitor MG132. Coomassie blue staining (CBS) was used as a loading control.</p

Nuclear localization of the overexpressed transcriptional activation domain of CdTlo2 in a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> by fusion of a NLS to its N-terminus facilitates embedded agar and agar invasion phenotypes.

<p><b>(A)</b><i>TLO</i> constructs, which were N-terminally fused with NLS-GFP coding sequence and C-terminally HA-tagged, were overexpressed from a <i>TDH3</i> promoter in a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain (yLM377 for over-expression of <i>NLS-GFP-3HA</i>, yLM378 for <i>CaTLOα12</i>, yLM380 for <i>CdTLO1</i> and yLM382 for <i>CdTLO2</i>). The <i>CaTLOα12TAD</i> strain (yLM379) contained residues 164–252 of <i>CaTLOα12</i>. The <i>CdTLO1TAD</i> strain (yLM381) contained residues 199–320 of <i>CdTLO1</i>. The <i>CdTLO2TAD</i> strain (yLM383) contained residues 255–367 of <i>CdTLO2</i>. Differential contrast (DIC) and fluorescence microscopy were used to visualize GFP localization, while Hoechst staining was used to stain the nuclei. All cells were grown in synthetic complete media overnight, diluted into the same media and grown for 5–6 hours before visualization. <b>(B)</b> Embedded agar filamentation (two left columns) and agar invasion (three right columns) phenotype analysis with NLS-GFP <i>C</i>. <i>dubliniensis</i> strains described in A.</p

CdTlo1 and CaTlos differ in their ability to be stably expressed in a non-Mediator associated form.

<p><b>(A)</b> Immunoblot shows that over-expression of one (1X) and two (2x) copies of HA-tagged <i>CaTLOα12</i> (from strains yLM303 and yLM305), but not C<i>dTLO1</i> (from strains yLM302 and yLM304), from the <i>TDH3</i> promoter results in increased protein levels, compared to a HA-tagged endogenous copy of <i>CdTLO1</i> (yLM301) in <i>C</i>. <i>dubliniensis</i>. Two independent transformants (‘1’ and ‘2’) were tested. Coomassie blue staining (CBS) was used as a loading control. <b>(B)</b> RT-qPCR analysis shows that over-expression of one (1X) and two (2x) copies of HA-tagged <i>CaTLOα12</i> (from strains yLM303 and yLM305) and C<i>dTLO1</i> (from strains yLM302 and yLM304) from the <i>TDH3</i> promoter both result in increased mRNA levels, compared to an endogenous copy of <i>CdTLO1</i> (yLM301) in <i>C</i>. <i>dubliniensis</i>. The error bars represent the technical deviation of two sets of qPCR measurements on a given sample. Two independent transformants (‘1’ and ‘2’) were tested. <b>(C)</b> Plot of fold-changes of <i>CdTLO1</i> and <i>CaTLOα12</i> protein versus mRNA (from Fig 1A and 1B) reveals that CdTlo1 protein levels plateau compared to CaTloα12p. The dashed lines represent an idealized slope of 1 in which the fold-change in protein is equal to the fold-change in mRNA, and an idealized slope of zero in which there is no increase in protein with increasing mRNA. <b>(D)</b> Immunoblot shows that endogenous HA-tagged CdTlo1 protein level drops in <i>med3Δ/Δ</i> (yLM308) versus a wild type (yLM301) <i>C</i>. <i>dubliniensis</i> strain. Two independent transformants (‘1’ and ‘2’) were tested. An anti-tubulin antibody was used as a loading control. <b>(E)</b> Immunoblot shows endogenous protein levels of HA-tagged Tloα34 (yLM391) are not affected upon <i>med3</i> deletion (yLM392) in <i>C</i>. <i>albicans</i>. An anti-tubulin antibody was used as a loading control.</p

A <i>C</i>. <i>albicans</i> med3 null strain has attenuated virulence in a murine model for disseminated Candidiasis.

<p>Equal innocula of a <i>med3</i> heterozygous null strain (yLM116), homozygous null strain (yLM119) and complemented strain (yLM120) were injected into mouse tail veins and the outcome score is calculated from fungal kidney burden and body weight [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006373#pgen.1006373.ref042" target="_blank">42</a>]. The outcome score correlates inversely with 28 day survival. The outcome score in the null mutant was compared to a heterozygous null and a complemented null mutant using a t test. Bars represent means ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. The kidney burden and weight change used to calculate the outcome scores were <i>Δ</i>/<i>MED3</i>(-1.9±3.7%, 4.5±0.6 log₁₀ CFU/g), <i>med3Δ/Δ</i> (3.8±2.6%, 2.2±0.7 log₁₀ CFU/g), and <i>med3Δ/Δ</i>+<i>MED3</i> (-0.9±3.6%, 4.5±0.8 log₁₀ CFU/g).</p

Over-expression of <i>CdTLO2-3HA</i> in <i>C</i>. <i>dubliniensis</i> leads to increased agar invasion and ‘embedded agar’ filamentation.

<p>Phenotypic analysis of one (1X) and two (2x) copies of HA-tagged <i>CdTLO2</i> expressed from the <i>TDH3</i> promoter in a wild type Wü284 <i>C</i>. <i>dubliniensis</i> strain (yLM339 and yLM344 respectively). A fraction of <i>CdTLO2-3HA</i>_<i>1X</i> and <i>CdTLO2-3HA</i>_<i>2X</i> overexpression construct transformants resulted in colonies with wrinkled surfaces, instead of smooth. These super wrinkled (SW) transformants (yLM343 and yLM345, derived from <i>CdTLO2-3HA</i>_<i>1X</i> and <i>CdTLO2-3HA</i>_<i>2X</i> transformation respectively) were also analyzed. Cells grown in YPD liquid media were analyzed by differential contrast (DIC) microscopy (left column, and bottom left panel) and fluorescent microscopy with the cell wall stained with Blankophor (second column from left, and bottom right panel). Agar invasion is analyzed by growing colonies on YPD/2% agar plates, washing the plates with running water, cutting a cross section through the residual colony and inspecting the cross-section on a stereoscope. Embedded agar filamentation is analyzed by diluting cells in YPS agar (2%) and inspecting the ‘colony’ growth in the agar after 48 and 72 hours using a stereoscope. At least two distinct colony morphologies, which are stably inherited, for SW colonies (<i>CdTLO2-3HA</i>_<i>2X</i> (SW) upper row and lower row) are observed.</p

The <i>CdTLO2</i> overexpression embedded agar filamentation phenotype observed in wild type <i>C</i>. <i>dubliniensis</i> is not observed in a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain.

<p>Embedded agar filamentation in wild type (yLM339) and <i>med3Δ/Δ</i> (yLM340) <i>C</i>. <i>dubliniensis</i> strains overexpressing <i>CdTLO2</i> from a <i>TDH3</i> promoter is analyzed by diluting cells in YPS agar and inspecting the ‘colony’ growth in the agar after 48 and 72 hours using a stereoscope.</p

‘Free’ CdTlo2 protein has enhanced stability compared to CdTlo1 in <i>C</i>. <i>dubliniensis</i> after treatment with cycloheximide (CHX).

<p>Immunoblot of overexpressed HA-tagged CdTlo1p, CdTlo2p, CaTloα12p and HyNT1Cp in a wild type Wü284 <i>C</i>. <i>dubliniensis</i> strain (yLM337, yLM339, yLM335 and yLM341 respectively) and <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain (yLM338, yLM340, yLM336 and yLM342 respectively) after treatment with cycloheximide over a time course (minutes). Coomassie blue staining (CBS) was used as a loading control.</p

Non-Mediator associated CdTlo1p is rapidly degraded by the proteasome.

<p><b>(A)</b> Schematic of treatment cells with cycloheximide (CHX) and the MG132 proteasome inhibitor to determine whether a Tlo protein is subject to proteasome dependent degradation. <b>(B)</b> Immunoblot of endogenous HA-tagged CdTlo1p in a wild type a <i>med3Δ/Δ C</i>. <i>dubliniensis</i> strain (yLM308) after treatment with cycloheximide, in the absence and presence of the proteasome inhibitor MG132. Coomassie blue staining (CBS) was used as a loading control.</p

Overexpression of <i>CdTLO2</i> in smooth and ‘SW’ cells show increasing amounts of protein and mRNA.

<p><b>(A)</b> Immunoblot showing protein overexpression of <i>CdTLO2-3HA</i>_<i>1X</i> (yLM339) and <i>CdTLO2-3HA</i>_<i>2X</i> (yLM344) in <i>C</i>. <i>dubliniensis</i> compared to endogenous expression of <i>CdTLO1</i> (yLM301). Coomassie blue staining (CBS) was used as a loading control. The panels are from different lanes of the same blot/gel. <b>(B)</b> Immunoblot showing that multiple isolates of spontaneous SW cells derived from <i>CdTLO2-3HA</i>_<i>1X</i> (yLM343) and <i>CdTLO2-3HA</i>_<i>2X</i> (yLM345) over-expression cassette transformation have higher protein levels than their smooth <i>CdTLO2-3HA</i>_<i>1X</i> (yLM339) and <i>CdTLO2-3HA</i>_<i>2X</i> (yLM344) over-expression counterparts in <i>C</i>. <i>dubliniensis</i>. Numbering of individual isolates corresponds to numbering in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006373#pgen.1006373.s016" target="_blank">S16 Fig</a>. Coomassie blue staining (CBS) was used as a loading control. <b>(C)</b> RT-qPCR analysis showing that multiple isolates of spontaneous SW cells derived from <i>CdTLO2-3HA</i>_<i>1X</i> (yLM343) and <i>CdTLO2-3HA</i>_<i>2X</i> (yLM345) over-expression cassette transformation have higher mRNA levels than their smooth <i>CdTLO2-3HA</i>_<i>1X</i> (yLM339) and <i>CdTLO2-3HA</i>_<i>2X</i> (yLM344) over-expression counterparts in <i>C</i>. <i>dubliniensis</i>. Numbering of individual isolates corresponds to numbering in above. <i>CdTLO2</i> mRNA levels are normalized against <i>TEF1</i> mRNA. The error bars represent the technical deviation of two sets of qPCR measurements on a given sample. <b>(D)</b> Immunoblot showing immunodepletion of Tlo and Med1 from a crude extract and purified CdMediator sample. A crude sample from a strain in which CdTlo2-6His-3Flag was overexpressed, and a purified intact Mediator sample from a CdTlo1-6His-3Flag strain were used for pull-down experiments. An anti-Med1 antibody was used to deplete Med1 from the two samples. Immunodepletion using anti-HA antibody was used a control. In the purified Mediator sample the CdTlo1-6His-3Flag protein was depleted to the same degree as the Med1p, while in the extract the overexpressed CdTlo2-6His-3Flag protein was largely unaffected by the anti-Med1 depletion.</p