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The S1 Fig is incorrect. The correct version appears below. The publisher apologizes for the error.
Supporting informationS1 Fig. CRISPR/Cas9-mediated gene inactivation of GDH2 and construction of a GDH2-GFP reporter strain.(A) A purified KpnI/SacI fragment from pFS108, harboring GDH2-specific sgRNA, and PCR generated repair template (RT) were introduced into wildtype strain SC5314 by electroporation. NouR transformants were pre-screened in YNB+Arg medium containing the pH indicator bromocresol purple; the initial pH was 4.0. Three NouR colonies were picked for further analysis. Clones #1 and #2 grew poorly and were unable to alkalinize the media; clone #3 grew and alkalinized the media. (B) Genomic DNA, isolated from the three clones, was used as template for PCR amplification of the targeted GDH2 locus; ddH2O was used as negative control. Restriction of the amplified ≈900 bp fragment by XhoI is diagnostic for successful mutagenesis (primers p5/p6; S2 Table). Strains, clone #1 (CFG277) and clone #2 (CFG278), carry inactivated gdh2-/- alleles. (C) GDH2 is not essential but required for robust growth on glutamate or proline as sole nitrogen source. Five microliters of serially diluted wildtype (SC5314), gdh2-/- NATR (CFG277), gdh2-/- NATS (CFG279), and control (CFG182) cells were spotted on yeast peptone (YP), synthetic glutamate (SE) and synthetic proline (SP) media containing either 2% glucose (YPD, SED, SPD) or 1% glycerol (YPG, SEG, SPG) as carbon source. The plates were incubated for 48 h at 30 °C and photographed. (D) Fresh colonies of SC5314 (PLC005; WT), CFG279 (gdh2-/-), CASJ041 (cph1-/- efg1-/-) and CFG352 (cph1-/- efg1-/- gdh2-/-) were individually resuspended in YNB+CAA medium and incubated for 24 h at 37 °C. (E) The insertion of GFP in strain CFG273 (GDH2-GFP) was verified by PCR, the expected 1695 bp fragment was amplified using primers (p24/p25; S2 Table); strain CAI4 served as untagged control (middle left panel). CFG273 was transformed with the CRISPR/Cas9 cassette to inactivate GDH2. Putative gdh2-/- clones were identified as described and verified by PCR-RD (p13/p6; S2 Table) resulting in strain CFG400. Strains CFG273 (GDH2/GDH2-GFP) and CFG400 (gdh2/gdh2-GFP) were grown at a starting OD600 ≈ 2 in SE medium with 0.2% glucose and 1 mM proline (SED 0.2%+Pro) for 2 h. In contrast to CFG273, strain CFG400 failed to express Gdh2-GFP as assessed by immunoblot (middle right panel) and microscopy (lower panels; Scale bar = 5 μm), demonstrating the specificity of CRISPR/Cas9. https://doi.org/10.1371/journal.ppat.1009877.s001.
https://doi.org/10.1371/journal.ppat.1010008.s001
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References1. Silao FGS, Ryman K, Jiang T, Ward M, Hansmann N, Molenaar C, et al. (2020) Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans. PLoS Pathog 16(9): e1008328. pmid:32936835 2. Silao FGS, Ryman K, Jiang T, Ward M, Hansmann N, Molenaar C, et al. (2021) Correction: Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans. PLoS Pathog 17(8): e1009877. pmid:34460867Citation: The PLOS Pathogens Staff (2021) Correction: Correction: Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans. PLoS Pathog 17(10): e1010008. https://doi.org/10.1371/journal.ppat.1010008
Published: October 19, 2021
Copyright: © 2021 The PLOS Pathogens Staff. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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