Summary of hypotheses on the possible function(s) of multiple <i>TLO</i> genes in <i>C</i>. <i>albicans</i>.

<p>Tlo proteins are subunits of the tail module of the Mediator complex (Med). (A) Different Tlo proteins could facilitate high-affinity interaction of Mediator with specific promoters or transcription factors, facilitating rapid or high level transcriptional responses. (B) As a consequence of telomere-associated gene expression noise (TAGEN) exhibited by <i>TLO</i> genes, adaptive pressure may select populations of cells expressing specific Tlos. (C) Excess, non-Mediator—associated “free Tlo” may also exhibit regulatory functions, either independently of Mediator or perhaps in an antagonisitic fashion.</p

Current knowledge on structure and function of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>dubliniensis</i> Mediator.

<p>(A) Predicted structure of <i>C</i>. <i>albicans</i> (and <i>C</i>. <i>dubliniensis</i>) Mediator based on structural analysis of <i>S</i>. <i>cerevisiae</i> complex [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref025" target="_blank">25</a>]. Biochemical analyses of Mediator Tail subunits from <i>C</i>. <i>albicans</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref003" target="_blank">3</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref010" target="_blank">10</a>] and <i>C</i>. <i>dubliniensis</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref003" target="_blank">3</a>] supports the proposed structure of this module in the pathogens. Biochemical studies provide direct evidence that Tloβ2, Tloα3, Tloα9, Tloα12, and Tloα34 are mutually exclusive Med2 orthologs of the <i>C</i>. <i>albicans</i> Mediator complex, while not excluding other expressed Tlo paralogs [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref004" target="_blank">4</a>]. Additional biochemical studies show most Mediator in <i>C</i>. <i>dubliniensis</i> incorporates the Tlo1 subunit, but does not rule out the possibility that Tlo2 could associate with the complex under conditions in which its expression is increased [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004614#ppat.1004614.ref003" target="_blank">3</a>]. (B) Summary of virulence related phenotypes associated with <i>C</i>. <i>albicans</i> and <i>C</i>. <i>dubliniensis</i> null mutants of genes encoding individual Mediator subunits. “Filamentation” includes defects in the yeast to hyphae transition. The “White to Opaque” arrows refer specifically to the white to opaque cell phenotypic switching frequency, including also the opaque to white cell switch frequency. “Biofilm” defects specifically refer to the ability to form a structure on a hard solid (i.e., plastic) support. “Stress/Metabolism” is a broad catchall that refers to the cell’s ability to remodel its internal metabolic wiring to respond to environmental stresses such as changes in carbon source, as well as oxidative and heat stress. Detailed information on each of these phenotypes can be found briefly within the body of the text, and in more detail in the references cited.</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

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

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

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

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

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

The CdTlo2p C-terminal TAD, but not the CdTlo1p C-terminal TAD fused to a stabilized CdTlo1 N-terminus is able to confer overexpression filamentation phenotypes similar to CdTlo2p.

<p><b>(A)</b> Schematic of <i>CdTLO1</i>/<i>CdTLO2</i> hybrid constructs for overexpression in <i>C</i>. <i>dubliniensis</i>. Specific amino acid sequences for T1NT2Cp are CdTlo1p (1–198)/CdTlo2p (255–367). Specific amino acid sequences for T2NT1Cp are CdTlo2p (1–254)/CdTlo1p (199–320). The HyNT2Cp construct contains the stabilized CdTlo1/CaTloα12 hybrid N-terminal domain (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006373#pgen.1006373.g003" target="_blank">Fig 3A</a></b>) fused to the CdTlo2p TAD (255–367). The first three amino-acids (MSA or MPE) are denoted at the beginning of each construct. <b>(B)</b> Immunoblot showing overexpression of one copy of HA-tagged C<i>dTLO2</i>(yLM339), <i>HyNT2C</i>(yLM352), <i>T1NT2C</i>(yLM350) and <i>T2NT1C</i>(yLM351) from the <i>TDH3</i> promoter in a wild type <i>C</i>. <i>dubliniensis</i> strain. Two independent transformants (‘1’ and ‘2’) were tested. Coomassie blue staining (CBS) was used as a loading control. <b>(C)</b> Embedded agar filamentation (two left columns) and agar invasion (three right columns) phenotype analysis with one copy of HA-tagged C<i>dTLO2</i>(yLM339), <i>HyNT2C</i>(yLM352), <i>T1NT2C</i>(yLM350) and <i>T2NT1C</i>(yLM351) overexpressed from the <i>CdTDH3</i> promoter in a wild type (Wü284) <i>C</i>. <i>dubliniensis</i> strain. The ‘SW’ strain (yLM343) derived from <i>CdTLO2-3HA</i>_<i>1X</i> transformation was also included in the comparison.</p