Volume 23, Issue 8, Pages 2292-2298 (May 2018) Functional Genomic Screening Reveals Core Modulators of Echinocandin Stress Responses in Candida albicans  Tavia Caplan, Elizabeth J. Polvi, Jinglin L. Xie, Shoshana Buckhalter, Michelle D. Leach, Nicole Robbins, Leah E. Cowen  Cell Reports  Volume 23, Issue 8, Pages 2292-2298 (May 2018) DOI: 10.1016/j.celrep.2018.04.084 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 23, 2292-2298DOI: (10.1016/j.celrep.2018.04.084) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Functional Genomic Screening Identifies Modulators of Echinocandin Tolerance (A) Schematic of screening pipeline to identify modulators of caspofungin tolerance. (B) GRACE strains (left and middle panels) and homozygous deletion mutants (right panel) were grown in YPD in the presence or absence of 31.25 ng/mL caspofungin (CF) or 62.5 ng/mL CF, respectively, and in the presence of 0.05 μg/mL DOX as indicated. Wild-type (WT) represents CaSS1 for GRACE strains and SN250 for homozygous deletion mutants. OD600 was measured after 48 hr, and values were normalized to the wild-type no-drug no-DOX controls. Data are mean ± SD of technical replicates. Growth of each strain was compared to the wild-type in the presence of caspofungin using one-way ANOVA (∗p < 0.0001). (C) Strains were grown as described in (B) in the presence or absence of 7.81 ng/mL micafungin (MF). Growth was measured after 24 hr and analyzed as in (B). (D) Strains were grown as described in (B) in the presence or absence of 0.5 μg/mL fluconazole (FL). The tetO-PKC1/pkc1Δ strain and wild-type strain were grown in the presence or absence of 20 μg/mL DOX. Growth was measured after 48 hr and analyzed as in (B). See also Figure S1 and Tables S1 and S2. Cell Reports 2018 23, 2292-2298DOI: (10.1016/j.celrep.2018.04.084) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Identification of Genes Implicated in Echinocandin Resistance (A) MIC assays were performed in YPD in the presence or absence of 0.1 μg/mL DOX (tetO-HSP90/tetO-HSP90), 20 μg/mL DOX (tetO-PKC1/tetO-PKC1, tetO-GIN4/tetO-GIN4, and tetO-TSC11/tsc11Δ), or 0.05 μg/mL DOX (remaining strains). OD600 was measured after 24 hr, and values were normalized to the no-drug controls. Data were displayed using Java TreeView 1.1.6; see color bar. (B) Assays were performed on homozygous deletion mutants as described in (A). See also Figure S1. Cell Reports 2018 23, 2292-2298DOI: (10.1016/j.celrep.2018.04.084) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Pkc1 and Bck1 Are Hsp90 Clients (A) Cells were grown in YPD with 10 μg/mL DOX as indicated. Total protein was resolved by SDS-PAGE, and blots were hybridized with α-Hsp90, α-TAP to monitor Pkc1 levels, and α-H3 as a loading control. (B) Cells were grown overnight in YPD with 0.5 μg/mL DOX and subsequently subcultured into YPD with 5 μg/mL DOX. Total protein was resolved by SDS-PAGE, and blots were hybridized with α-Hsp90, α-TAP to monitor Bck1 levels, and α-tubulin as a loading control. (C) Immunoprecipitation (IP) of Pkc1-TAP with IgG agarose co-purifies Hsp90, as detected by hybridization with α-Hsp90 and α-TAP. Cells were grown in the presence or absence of 125 ng/mL caspofungin. (D) Cells were grown in YPM (maltose) or YPD (dextrose). Total protein was resolved by SDS-PAGE, and blots were hybridized with α-TAP to monitor Pkc1 levels and α-tubulin as a loading control. Pkc1 levels were normalized to tubulin, and relative levels, compared to the wild-type strain grown in the same medium, were calculated using ImageJ and are indicated below. See also Figure S2. Cell Reports 2018 23, 2292-2298DOI: (10.1016/j.celrep.2018.04.084) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Depletion of the CCT Complex Activates Cell Wall Integrity Signaling and Results in Mislocalized Septins (A) MIC assays were performed with wild-type (WT, CaSS1) and tetO-CCT8/cct8Δ strains in YPD in the presence or absence of 0.05 μg/mL DOX, 12.5 μg/mL cytochalasin A, and 200 μg/mL benomyl. OD600 was measured after 48 hr, and values were normalized relative to the no-drug controls. Data were visualized as described in Figure 2. (B) Assays were performed and analyzed as in (A). (C) Wild-type (WT, CaSS1) and the tetO-CCT8/cct8Δ strains were grown in the presence or absence of 0.05 μg/mL DOX, 1 M sorbitol, and 25 μg/mL cytochalasin A. Cells were stained with 25 μg/mL calcofluor white and imaged by DIC and fluorescence microscopy. Scale bar indicates 20 μm. (D) Wild-type (WT, CaSS1) and tetO-CCT8/cct8Δ strains were grown in the presence or absence of 0.05 μg/mL DOX. Caspofungin was added for 1 hr at 125 ng/mL where indicated. Protein was resolved by SDS-PAGE, and the blot was hybridized with ⍺-phospho-p44-p42 (phospho Mkc1) or ⍺-PSTAIR as a loading control. (E) Wild-type (WT, CaSS1) and tetO-CCT8/tetO-CCT8 strains were grown in the presence or absence of 0.05 μg/mL DOX. Shown are cells expressing GFP-tagged Cdc10 to visualize septin (green) overlaid with DIC microscopy images. Scale bar indicates 5 μm. (F) Quantification of the microscopy described in (E). Cells were treated with 1 M sorbitol as indicated. Data are mean ± SD of biological replicates. See also Figures S3 and S4. Cell Reports 2018 23, 2292-2298DOI: (10.1016/j.celrep.2018.04.084) Copyright © 2018 The Authors Terms and Conditions