Overexpression of <i>NPR2</i>, a putative urea transporter located in the amplified portion of chromosome 3, in the evolved resistant isolates is sufficient to confer enhanced resistance to azoles in the absence of <i>RGD1</i>.

<p><b>A)</b> The azole-resistant CaCi-17 <i>rgd1Δ/rgd1Δ</i> R1 lineage was passaged without azole and an individual colony with improved fitness in the absence of stress was selected (CaCi-17 <i>rgd1Δ/rgd1Δ</i> R1-A). Copy number variation was analyzed using the Y_MAP pipeline. Loss of amplification of chromosome 7 was observed, however, the unusual chromosome 3 aneuploidy was maintained. <b>B)</b> Chromosome 3 haplotype map analyzed using Y_MAP. For the newly evolved CaCi-17 <i>rgd1Δ/rgd1Δ</i> R1-A isolate, the monosomic region of Chr3L and highly amplified region of Chr3L show similar haplotype composition as the parental CaCi-17 <i>rgd1Δ/rgd1Δ</i> R1 strain: haplotype A (Cyan), and haplotype B (magenta). <b>C)</b> Azole resistance of indicated mutants was evaluated by MIC assay. Cells were grown as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007319#pgen.1007319.g001" target="_blank">Fig 1</a>. Growth was measured after 24 hours and normalized to the no drug control for each strain. See colour bar. <b>D)</b> Replacing the native promoter of one allele with the strong <i>tetO</i> promoter results in elevated expression of <i>NPR2</i> (left panel), <i>orf19</i>.<i>304</i> (middle panel), and <i>orf19</i>.<i>344</i> (right panel). Addition of doxycycline reduced expression of each transcript. Transcript levels were monitored by qRT-PCR and normalized to <i>TEF1</i>. Error bars represent standard error of the mean for triplicate samples. Expression levels of evolved strains were compared to the parental strain using a one-way ANOVA with Bonferroni post-test. Asterisk indicates significant difference in transcript level relative to the parental strain in the absence of doxycycline (* <i>P<</i>0.01). <b>E)</b> Overexpression of <i>NRP2</i> results in enhanced azole susceptibility in the CaCi-17 <i>rgd1Δ/rgd1Δ</i> background. Strains were grown in the absence or presence of 128 μg/mL fluconazole, 0.05 μg/mL doxycycline (DOX), or a combination of fluconazole and DOX as indicated. Growth was assessed by OD₆₀₀ measurements every 15 minutes for 48 hours in a Tecan plate reader.