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. 2018 Oct 24;62(11):e00968-18. doi: 10.1128/AAC.00968-18

FIG 3.

FIG 3

Znc1 and Tac1 contribute individually and in tandem to the regulation of specific FOH- and 1-DD-induced promoters. (A and B) RT-qPCR analysis of RTA3 (A) and CDR1 (B) mRNA expression in tac1Δ/Δ and znc1Δ/Δ strains treated with FNZ, FOH, or 1-DD. A wild-type strain (yLM660) and mutants carrying a tac1 deletion (yLM663), znc1 deletion (yLM661), or double deletion (yLM664) were treated with FNZ (25 μM), FOH (50 μM), or 1-DD (50 μM). CDR1 and RTA3 basal expression in the untreated wild-type strain (yLM660) was individually set to 1. (C) Immunoblot analysis of Cdr1 protein levels in tac1Δ/Δ, znc1Δ/Δ, and double deletion strains treated with FNZ or FOH. Wild-type (yLM665), tac1Δ/Δ (yLM666), znc1Δ/Δ (yLM667), and tac1Δ/Δ znc1Δ/Δ (yLM668) strains expressing C-terminally 3×HA-tagged Cdr1 were treated with FNZ (25 μM) or FOH (50 μM). Cell lysates were resolved on 6% SDS-PAGE gels and probed with an anti-HA antibody. Blot images, acquired at two exposure times, are presented, and Coomassie blue staining (CBS) images are presented as a loading control. (D to E) RT-qPCR analysis of CDR1 (D) and CDR2 (E) mRNA expression in tac1Δ/Δ and znc1Δ/Δ strains, and complementation controls, treated with FOH. A tac1Δ/Δ znc1Δ/Δ strain was complemented by ZNC1 (yLM676), TAC1 (yLM677), or both (yLM675) and treated with FOH (50 μM). Parallel experiments were also conducted in wild-type (yLM660), tac1 deletion (yLM663), parental tac1 znc1 double deletion (yLM664), and mock complementation (yLM678) strains for comparison. A plus sign labels a native or restored gene locus, while Δ and V (vector) mark an unrestored gene disruption and a mock complementation by introducing an empty vector. (F and G) RT-qPCR analysis of orf19.320 (F) and IFD1 (G) mRNA expression in the wild-type ZNC1 or znc1 deletion background (yLM660 and yLM661, respectively) treated with FNZ (25 μM), FOH (50 μM), and 1-DD (50 μM) for the indicated period of time. orf19.320 and IFD1 basal expression in the wild-type strain was individually set to 1. (H) qPCR analysis of orf19.320 expression in the complementation strains using cDNA samples tested in panels D and E. Noninduced expression levels in the wild-type strain (yLM660) were set to 1.