Microarray analysis of Fkh2 mutants.

<p>Microarray results sorted for the top 30 genes down/up regulated compared to the parental strain in the <i>fkh2(6A)</i> or <i>fkh2ΔΔ</i> mutants as indicated. Note the gene ranked 30 down regulated in the <i>fkh2(6A)</i> mutant (orf19.715 3-fold down) has been replaced with <i>SUN41</i> (2-fold down; adjusted p-value = 5.4 × 10^-5) as indicated by the dotted line. The full microarray of genes up or down regulated in all Fkh2 mutants is available in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.s007" target="_blank">S2 Dataset</a>. Gene annotation was provided from the <i>Candida</i> genome database <a href="http://www.candidagenome.org" target="_blank">http://www.candidagenome.org</a>. Also shown is the presence of a predicted Fkh2 consensus (G/ATAAAC/TAAA) or minimal (AAAT/CAAA) binding site in the upstream 1 kb of each gene.</p

Fkh2 is phosphorylated at serine 533 by Cbk1-Mob2.

<p>A) Autoradiograms from 2D gels and quantitative intensity profiles of the indicated strains at the indicated times and culture conditions. The grey dashed line represents the wild type Fkh2-YFP profile grown in the corresponding condition as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Fig. 1</a>. B) <i>In vitro</i> kinase assay with Mob2-HA purified from a 40 min hyphal culture and <i>E. coli</i> expressed Fkh2 C-terminal fragment used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g002" target="_blank">Fig. 2C</a>. A BWP17 mock lysate controls for the possibility of a co-precipitating kinase from the αHA IP. C) Morphology of <i>fkh2</i> S533A mutants. <i>fkh2/FKH2</i> and <i>fkh2/fkh2(S533A)</i>. Cells were re-inoculated from stationary phase cultures into either yeast or hyphal growth conditions, grown for 6 h and then fixed with 1.5% formaldehyde. Hyphal cells were pepsin treated to remove clumps before imaging at x100 magnification. Scale bars represent 10 μm.</p

Fkh2 is differentially phosphorylated between yeast and hyphal growth.

<p>A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Figs. 1B–D</a> αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Fig. 1E</a> is shown with an independent replicate in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.s002" target="_blank">S2 Fig.</a></p

Biological confirmation of the microarray results—phosphorylation of Fkh2 affects multiple pathogenic processes.

<p>A) Biofilm formation in 12 well plastic plates imaged after 48 h. B) Quantitation shows the average weight for ten individual biofilms. C) Epithelial cell damage measured by LDH release in a TR146 oral epithelial monolayer infection model. D) and E) Immune activation in the infection model was measured by IL-1α (D) or IL-1β (E) production. Error bars are standard deviations. In C-E <i>FKH2/FKH2^‡</i> represents an <i>ARG4⁺ URA3⁺</i> BWP17 strain that has identical auxotrophic requirements to the other strains tested; <i>FKH2/FKH2</i> represents a strain with 2 wild-type alleles of <i>FKH2</i>, one of which is C-terminally tagged with YFP and is prototrophic for <i>URA3</i> only. B-E: Significance of the indicated comparisons was assessed by unpaired, one-tailed t-tests * p ≤ 0.05, ** p ≤ 0.01.</p

Fkh2 is phosphorylated by Cdc28.

<p>A) Schematic showing Cdc28 minimal (circles) and full (diamond) consensus target sites on Fkh2, with those detected by phospho-peptide mapping to be phosphorylated on hyphal induction indicated in blue. B) Phosphorylation of Fkh2 on hyphal induction detected using an antibody that recognises phosphorylated residues in Cdc28 target sites (αP_SER(CDK) (Cell-Signalling 2324S). C) <i>In vitro</i> kinase assay with Cdc28-HA purified from a hyphal lysate and recombinant GST-Fkh2(CT) (aa419–687 intron removed) and GST-Fkh2-A-CT (as GST-Fkh2(CT) fragment with the serine/threonine in the five C-terminal Cdc28 consensus sites mutated to alanine). GST-Fkh2(CT) but not GST-Fkh2-A-CT is phosphorylated <i>in vitro</i> by Cdc28-HA. The parental strain BWP17, in which Cdc28 is not HA-tagged, provided the mock lysate to demonstrate that the activity was not due to a co-purifying kinase. D) Removal of Fkh2’s C-terminus containing the Cdc28 target site cluster abolishes the double band upon hyphal induction. <i>fkh2(1–426)-GFP</i> and <i>fkh2/FKH2-YFP</i> were grown as yeast or hyphae as previously, samples were treated with/without phosphatase and then resolved by SDS-PAGE. E) Autoradiograms from 2D gels and quantitative intensity profiles of the indicated Fkh2 phosphosite mutants at the indicated times and in the indicated culture conditions. The grey dashed line represents the parental Fkh2-YFP profile grown in the corresponding condition as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Fig. 1</a>.</p

The effect of C-terminal deletion or phosphosite mutations on cell cycle progress during hyphal growth.

<p>A) Stationary phase <i>fkh2/FKH2</i> and <i>fkh2(6A)</i> cells (with a single nucleus) were inoculated into hyphal growth conditions. Samples were taken at 30 min intervals from 90 to 180 min, the hyphal cells were fixed with 1.5% formaldehyde and nuclei stained with DAPI. Hyphae were scored according to whether they had a single nucleus in the mother cell, a single migrating nucleus, two or three nuclei. A minimum of 100 cells were counted. B) Nuclear distribution of the indicated genotypes 180 min after hyphal induction. A minimum of 100 cells were counted. The distribution of nuclei in the <i>fkh2∆∆</i> strain was so perturbed that it was not possible to make a meaningful assessment of nuclear content. C) Appearance of cells 180 min after hyphal induction. Images are shown as the merged DAPI (white) and DIC channels, and scale bars are equal to 10 μm. The DIC channel has been darkened for ease of nuclear visualisation.</p

Phosphorylation of Fkh2 does not alter its localisation, but affects its interaction with the chromatin modifier Pob3.

<p>A) Fkh2-YFP/GFP localisation in log phase yeast cells or 40 min after hyphal induction. Images are taken at x100 magnification. Scale bar, 5 μm. B) Co-IP experiments of Fkh2 with Pob3 and Srp1 from samples taken 40 min post hyphal induction. 2 mg of total protein was used for IP and 50 μg for the lysates. IP products were washed twice with lysis buffer containing 150 mM NaCl. C) Interaction of Pob3 with Fkh2 phospho-mutants, performed as above. The co-immunoprecipitation was quantified by normalising the αHA signal (Pob3) against the immunoprecipitated αGFP (Fkh2) for each IP, and then expressing this value as a fraction of the wild type value.</p

GO analysis of <i>fkh2(6A)</i> and <i>fkh2ΔΔ</i> mutants.

<p>A) Venn diagram showing the number of genes down regulated in the <i>fkh2(6A)</i> and <i>fkh2ΔΔ</i> mutants and the overlap in the two data sets. B) Genes significantly more down regulated in <i>fkh2ΔΔ</i> mutant compared to the <i>fkh2(6A)</i> mutant and genes more down regulated in the <i>fkh2(6A)</i> mutant compared to <i>fkh2ΔΔ</i>. Each panel shows the genes down regulated at least two fold at the 5% FDR threshold in <i>fkh2(6A)</i> or <i>fkh2ΔΔ</i> (empirical Bayes moderated t-test). Genes significantly down regulated in <i>fkh2ΔΔ</i> (above) or <i>fkh2(6A)</i> (below) are highlighted in the solid points. Those genes significantly (5% FDR, empirical Bayes moderated t-tests) more down regulated in <i>fkh2ΔΔ</i> than <i>fkh2(6A)</i> are shown in blue (top panel), or more down regulated in <i>fkh2(6A)</i> than <i>fkh2ΔΔ</i> are shown in red (lower panel). Right: GO pathways enriched in genes in each category highlighted on the left. Note for reasons explained in the text the figures in this panel are not consistent with the Venn diagram shown in panel A. C) qPCR comparing the expression of hyphal associated transcripts in the Fkh2 phosphorylation mutants. Expression levels are normalised against <i>ADE2</i> and shown relative to the expression in the <i>fkh2/FKH2</i> strain. Means are from two independent biological repeats, each with three technical replicates. Vertical bars are equal to one standard error.</p

Phosphorylation of Fkh2 affects the long-term maintenance of hyphal growth.

<p>A) Phenotypes of Fkh2 phosphorylation mutants of the indicated genotypes. Left: grown as hyphae for 6 h in GMM with 10% FCS, fixed with 1.5% formaldehyde and pepsin treated. White arrows indicate branching and black arrows indicate constrictions. Right: yeast cells were re-inoculated into GMM pH 4.0 for 4hrs before formaldehyde fixation. Scale bars are equal to 10 μm. B) and C). Quantitation (n = 50) of the hyphal phenotypes was carried out to quantitate hyphal branching (B), and constrictions within a hypha (C). For Figs. B and C, a Z-test was used to compare the proportion of cells displaying the phenotype in the mutants with the wild type. * z < 0.05, ** z < 0.01. D) Long-term effects on hyphal growth observed on solid Spider medium. An overnight culture was diluted to OD₆₀₀ = 1.0 and then serially diluted 10 fold as shown, with 1 μl of each dilution being spotted and the plates left at 37°C for five days. The extent of invasion was observed through washing off the surface colony using deionised water.</p