Plasmids used in this study.

<p>Plasmids used in this study.</p

Defects in filamentation under embedded conditions at 96 hours.

a<p>Allele carried on plasmid, under control of Maltase promoter, integrated at <i>ADE2</i> locus.</p>b<p>Mean±standard deviation; three determinations for untagged <i>CZF1</i> strains; four determinations for tagged <i>CZF1</i> strains.</p>C<p>By two-tailed <i>t</i> test.</p

<i>C. albicans</i> stains used in this study.

<p><i>C. albicans</i> stains used in this study.</p

Primers used in this study.

<p>Primers used in this study.</p

EMSA of wild type and mutant Czf1p binding to the <i>CZF1</i> promoter region.

<p>(Panel A) SDS PAGE analysis of 500 ng of purified wild type and mutant GST-Czf1 fusion proteins stained with Coomassie Blue. (Panel B) Diagram of the <i>CZF1</i> promoter region (not drawn to scale). The DNA fragment used in this study (fragment E, 565 bp) is represented as a black rectangle and is located −3381 to −2816 from the ATG start of the <i>CZF1</i> ORF. The transcriptional start site is represented as an arrow and is located at −2065 from the ATG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039624#pone.0039624-Vinces1" target="_blank">[23]</a>. (Panels C–E) Increasing amounts of GST-Czf1p were incubated with ³²P end-labelled fragment E. The samples were analyzed by electrophoretic mobility shift assay and phosphorimaging. Brackets indicate shifted fragments. All EMSA experiments were repeated at least 3 times and a representative experiment is shown.</p

Wild type and mutant Czf1p interact with Efg1p in a yeast two-hybrid assay.

<p><i>S. cerevisiae</i> strains carrying a <i>lacZ</i> reporter plasmid, a <i>lexA</i> DNA-binding domain bait fusion (indicated below columns) and a Gal4 activation domain prey fusion (black bars, Gal4AD-Efg1; white bars, Gal4AD-Slk19) were grown in synthetic complete medium lacking uracil, leucine and histidine (CM-ULH) and β-galactosidase activity was determined using <i>o</i>-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate. Analysis was performed in triplicate and mean and standard deviation are shown. Differences in activity of Czf1-LexA fusions with Gal4AD-Efg1p and the same fusion with Gal4AD-Slk19 were statistically significant (p<5×10⁻⁴ for all Czf1-LexA fusions; two tailed <i>t</i> test). (Panel B) <i>S. cerevisiae</i> strains were grown in CM-ULH and immunoblot analysis was performed on crude extracts using an anti-LexA antibody to visualize Czf1-lexA protein fusions. Actin was detected as the loading control. Samples from two independent cultures of a given strain are shown. Lanes 1 and 2, strain with <i>lacZ</i> reporter and Gal4AD-Efg1 fusions only; lanes 3 and 4, strains carrying <i>lacZ</i> reporter, Gal4AD-Efg1 and <i>CZF1</i>-lexA fusion; lanes 5 and 6, strains carrying <i>lacZ</i> reporter, Gal4AD-Efg1 and <i>czf1_K322A</i>-lexA fusion; lanes 7 and 8, strains carrying <i>lacZ</i> reporter, Gal4AD-Efg1 and <i>czf1_R321A</i>-lexA fusion. A cross-reacting band, marked with *, is apparent above the Czf1-LexA fusion.</p

Conserved residues of Czf1p.

<p>(Panel A) Diagram of Czf1p. Numbers indicate amino acid residues. Gln, glutamine rich region. ZnF, zinc finger motif. (Panel B) Alignment of the zinc-finger motif amino acid sequence of Czf1p (residues 315–342) and <i>S. cerevisiae</i> Gal4p (residues 11–38). Marked regions A and B, and consensus sequence are from Corton et al. (1998) and are based on amino acids that are conserved in more than 33% of sequences from 79 Cys₆Zn₂ fungal proteins. Lys17 and Lys18 in Gal4p make direct contacts with the target DNA sequence. Underlined residues in the Czf1p amino acid sequence were targeted in this study.</p

Filamentation of <i>czf1</i> mutants during growth under matrix-embedded conditions.

<p>Cells of <i>czf1</i>Δ <i>cph1</i>Δ mutant strains carrying Myc-tagged wild type <i>CZF1</i> under control of the maltase promoter, Myc-tagged mutant <i>CZF1</i> under control of the maltase promoter or vector alone were embedded in YPS + uridine, 1% agar and grown at 25°C. Two independent isolates of each strain were tested in duplicate. (Panel A) Representative colonies were photographed at 10x at the times indicated. (Panel B) The percentage of filamentous colonies for a given strain at various times was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039624#s4" target="_blank">Materials and Methods</a>. Data show the mean of the 4 determinations and the standard deviation. Black bar, <i>czf1</i>Δ (IPC11); white bar, <i>CZF1</i>⁺-myc (IPC22); horizontal lines, <i>CZF1</i>_T328A-myc (IPC51); grey bar, <i>czf1</i>_K322A-myc (IPC31); vertical lines, <i>czf1</i>_R321A-myc (IPC42). (Panel C) Cells were grown in liquid YPS medium at 30°C and 30 µg of total protein was fractionated by SDS PAGE. Immunoblot analysis was performed using an anti-Myc antibody to detect tagged Czf1 proteins. Actin was detected with anti-actin as a loading control.</p

A strain carrying an <i>efg1</i> mutant defective in interaction with Czf1p behaves like a strain carrying one allele of wild-type Efg1p during matrix-embedded growth.

<p>(Panel A) Strains with the genotypes shown at the top of the figure were grown embedded in YPS 1% agar for 72 hours at 23°C. Two independent isolates of each strain were tested in duplicate. Representative colonies were photographed at 10x at the times indicated. (Panel B) Quantitative representation of the percentage of filamentous colonies at various times. Data show the mean of the 4 determinations and the standard deviation. Black bar, 48 hours; grey bar, 72 hours. Differences in percent filamentous colonies between <i>efg1</i>Δ mutant and other strains at 2 days were statistically significant (p<10⁻⁵; two tailed <i>t</i> test). (Panel C) Cells were grown in liquid YPD at 30°C and Efg1p proteins were detected in cell extracts (45 µg total protein) by immunoblotting with anti-HA antibody. Actin was detected with anti-actin antiserum as a loading control.</p

Calmodulin Binding to Dfi1p Promotes Invasiveness of <i>Candida albicans</i>

<div><p><i>Candida albicans</i>, a dimorphic fungus, undergoes hyphal development in response to many different environmental cues, including growth in contact with a semi-solid matrix. <i>C. albicans</i> forms hyphae that invade agar when cells are embedded in or grown on the surface of agar, and the integral membrane protein Dfi1p is required for this activity. In addition, Dfi1p is required for full activation of mitogen activated protein kinase Cek1p during growth on agar. In this study, we identified a putative calmodulin binding motif in the C-terminal tail of Dfi1p. This region of Dfi1p bound to calmodulin <i>in vitro</i>, and mutations that affected this region affected both calmodulin binding <i>in vitro</i> and invasive filamentation when incorporated into the full length Dfi1p protein. Moreover, increasing intracellular calcium levels led to calcium-dependent, Dfi1p-dependent Cek1p activation. We propose that conformational changes in Dfi1p in response to environmental conditions encountered during growth allow the protein to bind calmodulin and initiate a signaling cascade that activates Cek1p.</p></div