Allelic variation in <i>HAP1</i> affects ethanol-induced cross protection against H₂O₂.

<p>(A) Schematic of reciprocal hemizygosity analysis. Each block represents a gene, and each hybrid strain contains a single-copy deletion of <i>hap1</i> or <i>top3</i>, and a single copy of the respective S288c (lab) or YPS163 (oak) allele. (B) Representative acquired H₂O₂ resistance assays for wild-type YPS163, the YPS163-S288c hybrid, and the reciprocal hemizygotes. (C) Each survival score plot shows the mean and standard deviation of biological triplicates. Asterisks represent significant differences in acquired resistance between denoted strains (** <i>P</i> < 0.01, *** <i>P</i> < 0.001, ns = not significant (<i>P</i> > 0.05), <i>t</i>-test).</p

Natural variation in ethanol-induced cross protection against H₂O₂.

<p>(A) A representative acquired H₂O₂ resistance assay is shown. S288c (lab strain–DBY8268) and YPS163 (wild oak strain) were exposed to 5% ethanol or mock (5% water) pretreatment for 60 min, washed, exposed to 11 doses of severe H₂O₂ for 2 hr, and then plated to score viability. (B) A single survival score was calculated from the viability at all H₂O₂ doses (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#sec007" target="_blank">Materials and Methods</a>). Each plot shows the mean and standard deviation of 4 independent biological replicates. The replicates for mock-treated YPS163 all had the same tolerance score and thus zero standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.s006" target="_blank">S1 Table</a> for raw numerical data). Asterisks represent resistance that was significantly different from mock-treated cells (*** <i>P</i> < 0.001, <i>t</i>-test).</p

<i>CTT1</i> function is necessary for ethanol-induced cross protection against H₂O₂.

<p>(A) Representative acquired H₂O₂ resistance assays for wild-type YPS163 and the YPS163 <i>ctt1Δ</i> mutant. (B) Survival score plots indicating the mean and standard deviation of biological triplicates. Asterisks represent significant differences in acquired resistance between denoted strains (*** <i>P</i> < 0.001, <i>t</i>-test).</p

<i>HAP1</i> is required for full induction of <i>CTT1</i> gene expression and cellular peroxidase activity during ethanol stress.

<p>(A) Fold induction of <i>CTT1</i> mRNA in indicated strains following 30 min ethanol stress compared to unstressed cells, assessed by qPCR. (B) Peroxidase activity measured in cell-free extracts in either mock-treated or ethanol-stressed cells. The plots indicate the mean and standard deviation of biological triplicates (mRNA) or quadruplicates (peroxidase activity). Asterisks represent significant differences in <i>CTT1</i> mRNA induction or peroxidase activity between denoted strains (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, paired <i>t</i>-test).</p

Expression variation in Hap1p regulatory targets implicates oxidative stress defense genes as the direct effectors of ethanol-induced cross protection against H₂O₂.

<p>(A) Overlap between genes that were <i>HAP1</i> eQTL hotspot targets from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.ref064" target="_blank">64</a>], genes with defective induction in S288c vs. YPS163 from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.ref064" target="_blank">64</a>], and direct targets of <i>HAP1</i> identified via ChIP experiments compiled from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.ref081" target="_blank">81</a>]. (B) Descriptions of the eight genes that overlapped for all three criteria. (C) Previous eQTL mapping of the yeast ethanol response (newly plotted here using data described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.ref064" target="_blank">64</a>]), implicated <i>HAP1</i> as causative for natural variation in <i>CTT1</i> induction levels during ethanol stress.</p

<i>HAP1</i> is not required for acquired H₂O₂ resistance following mild H₂O₂ or mild NaCl pretreatments.

<p>Cultures of wild-type YPS163 and the YPS163 <i>hap1</i>Δ mutant were split and exposed to either 0.4 mM H₂O₂, 0.4 M NaCl, or a mock (media only) treatment for 60 min, washed, exposed to 11 doses of severe H₂O₂ for 2 hr, and then plated to score viability. The survival scores across each of the 11 doses are plotted as the mean and standard deviation of biological triplicates.</p

Ethanol-induced cross protection against H₂O₂ in YPS163 requires <i>HAP1 and TOP3</i>.

<p>Deletions of all non-essential genes within the 1.5-LOD support interval of the chromosome XII QTL peak were constructed in JL111 (YPS163 <i>MATa</i> haploid) background and tested for defects in acquired H₂O₂ resistance. Each plot shows the mean and standard deviation of 2 independent biological replicates, with the exception of the JL111 control (35 replicates). The replicates for several strains all had the same tolerance score and thus zero standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007335#pgen.1007335.s006" target="_blank">S1 Table</a> for raw numerical data). Asterisks represent acquired H₂O₂ resistance that was significantly lower than wild-type YPS163 (* <i>P</i> < 0.001, one-way ANOVA).</p