Binding of recombinant PACC to the <i>gsn</i> promoter.

<p>(A) Upper panel, schematic representation of the <i>pacC</i> probe and the specific competitor oligo <i>pacC</i>. Lower panels, gel shift analysis using increasing amounts of recombinant PACC in the presence of specific competitors and polyclonal anti-PACC antibody. Lanes 1, 5 and 9, <i>pacC</i> probe, no protein added. Lanes 2, 6 and 10, gel shift analysis using 1.0, 2.0 and 5.0 µg of recombinant PACC. Lanes 3 and 7, gel shift analysis in the presence of the 146 bp specific competitor. Lanes 4 and 8, gel shift analysis using the specific competitor oligo <i>pacC</i>. Lanes 11 to 13, supershift assay using 5.0, 10.0 and 20.0 µL of anti-PACC antibody (1∶500). (B) Upper panel, schematic representation of the mutated m<i>pacC</i> probe. Lower panel, gel shift with wild-type and mutated probes. Analysis using different concentrations of recombinant PACC in the presence and in the absence of competitors. Lane 1 and 3, <i>pacC</i> and m<i>pacC</i> probes, respectively, no protein added. Lanes O, gel origin; SC, specific competitor; FP, free probe.</p

<i>pacC</i> gene knockout and phenotypic analysis of the mutant strain.

<p>(A) Schematic illustration of the <i>pacC</i> gene knockout strategy. (B) Diagnostic PCR for validation of the <i>pacC</i> knockout was performed by using 0090-F1 and 0090-R4 oligonucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#pone-0044258-t001" target="_blank">Table 1</a>). (C) Linear growth analysis. Apical extension of basal hyphae was determined in race tubes, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#s4" target="_blank">Material and Methods</a>. The results shown are the average of three independent experiments. (D) Melanization. Strains were cultured in 250 mL flasks containing VM medium for 10 days (3 days at 30°C in the dark and 7 days at room temperature in ambient light/dark). Melanization can be visualized as brown pigment formation in the <i>pacC^KO</i> strain. (E) Radial growth analysis. Basal hyphae growth was examined after cultivating the strains on plates containing solid VM medium, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#s4" target="_blank">Material and Methods</a>. Growth was expressed as colony diameter.</p

PACC binds specifically to the <i>gsn</i> promoter region in an alkaline pH-independent manner.

<p>(A) Gel shift analysis using crude cellular extracts fractionated on a Heparin-Sepharose column. Crude cellular extracts (CCE) from mycelia submitted or not to alkaline pH stress (pH 7.8) were fractionated by affinity chromatography. Left panel, a protein fraction (35 µg) exhibiting DNA-binding activity was assayed in the presence of specific competitors. Lane 1, <i>pacC</i> probe, no protein added. Lanes 2 and 7, proteins from pH 5.8 and pH 7.8 samples, respectively, in the absence of competitors. Lanes 3 and 8, DNA band shift in the presence of the 146 bp <i>pacC</i> specific competitor. Lanes 4 to 6 and 9 to 11, DNA band shifts in the presence of increasing amounts of the 27 bp DNA oligo <i>pacC</i> as a specific competitor. Right panel, a protein fraction (35 µg) from knocked-out strain crude cellular extract was assayed. Lane 12, <i>pacC</i> probe, no protein added. O, gel origin; SC, specific competitor; FP, free probe. (B) PACC shows the same molecular mass at pH 5.8 and pH 7.8. Crude cellular extracts prepared from wild-type <i>N. crassa</i> submitted or not to alkaline pH stress were analyzed by Western blotting using a polyclonal anti-PACC antibody. The protein α-tubulin (theoretical molecular mass 50 kD) was used as a loading control. (C) Chromatin immunoprecipitation assay using the polyclonal anti-PACC antibody. Genomic DNA samples from wild-type <i>N. crassa</i> submitted or not to pH stress were immunoprecipitated with the anti-PACC antibody and subjected to PCR to amplify a 146 bp DNA fragment of the <i>gsn</i> promoter containing the <i>pacC</i> motif. A plasmid construction containing the entire sequence of the <i>gsn</i> gene, including its 5′- and 3′-flanking regions, was used as a positive control. As a negative control, the immunoprecipitation reactions were performed without the anti-PACC antibody. L, 1 kb DNA ladder.</p

<i>gsn, gpn</i> and <i>pacC</i> gene expression during acid and alkaline pH stress.

<p>Cells from the wild-type and <i>pacC^KO</i> strains were cultivated at pH 5.8 for 24 h and shifted to pH 4.2 and pH 7.8. Samples were collected and used to extract total RNA. Total RNA (15 µg) was separated by electrophoresis in a denaturing formaldehyde gel, transferred to nylon membrane and probed with α-³²P-radiolabeled 678 bp <i>gsn</i> cDNA, or 798 bp <i>gpn</i> cDNA or 639 bp <i>pacC</i> cDNA fragments (gel autoradiographies). The 28 S rRNA was used as a loading control after ethidium bromide staining. The results shown are the average of at least three independent experiments. (A) Analysis of the <i>gsn, gpn</i> and <i>pacC</i> genes in the wild-type strain at different times after pH shifting. After pH stress the remaining cultures were transferred back to physiological conditions (RE, recuperation, pH 5.8) and samples were collected. (B) Analysis of the <i>gsn</i> and <i>gpn</i> genes in the <i>pacC^KO</i> strain compared to the wild-type strain at different times after pH shifting. 0, cell samples before pH shifting (control).</p

Glycogen accumulation and gene expression during combined pH and heat shock stress.

<p>Glycogen and total RNA were extracted from wild-type mycelia cultivated at pH 5.8 and 30°C for 24 h and then shifted to pH 5.8, 4.2 and 7.8 at 45°C for 30 min. After 30 min, the remaining samples were transferred back to the physiological temperature (30°C) at the three pH conditions and incubated for different times (RE, recuperation). (A) Accumulation of glycogen. (B) <i>gsn</i> and <i>pacC</i> gene expression. Total RNA (15 µg) was separated by electrophoresis in a denaturing formaldehyde gel, transferred to nylon membrane and probed with the α-³²P radiolabeled 678 bp <i>gsn</i> cDNA and 639 bp <i>pacC</i> cDNA fragments (gel autoradiographies). The 28 S rRNA was used as a loading control after ethidium bromide staining. The results shown are the average of at least three independent experiments. 0, cell samples before pH shifting (control).</p

Glycogen accumulation during acid and alkaline pH stress.

<p>(A) Glycogen content in the wild-type strain. Glycogen was extracted from mycelia grown under physiological conditions (pH 5.8, control) and under acid (pH 4.2) and alkaline (pH 7.8) stress. After 120 min, the remaining cultures were transferred back to physiological conditions (pH 5.8, RE, recuperation). The results shown are the average of at least three independent experiments. (B) Glycogen content in the <i>pacC^KO</i> strain compared to the wild-type strain. 0, cell samples before the pH shift (control).</p