Reduced TBSV repRNA accumulation in yeast with depleted Pgk1 level.

Abstract

<p>(A) Northern blot analysis shows decreased TBSV repRNA accumulation in a yeast strain (GALS::PGK1) when Pgk1 was depleted. To launch TBSV repRNA replication, we expressed His₆-p33 and His₆-p92^pol from the copper-inducible <i>CUP1</i> promoter, and DI-72(+) repRNA from the <i>ADH1</i> promoter in the parental (BY4741) and in GALS::PGK1 yeast strains. Note that GALS::PGK1 yeast strain expresses HA-tagged Pgk1 from the galactose-inducible <i>GALS</i> promoter from chromosomal location (i.e., HA-Pgk1 replaces the wt Pgk1 in this haploid yeast strain). The yeast cells were cultured for 16 hours at 29°C in either 2% galactose +2% raffinose [(Raf+Gal), inducing condition for HA-Pgk1] or 2% raffinose (lack of induction of HA-Pgk1 expression) SC minimal media supplemented with 50 μM CuSO₄. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels (second panel from top). Middle panel: Northern blot analysis of <i>PGK1</i> mRNA levels in total RNA samples. Bottom two panels: Western blot analysis of the accumulation level of HA-tagged Pgk1, His₆-tagged p33, His₆-p92^pol proteins using anti-HA and anti-His antibodies, respectively. Each experiment was performed three times. (B) Complementation assay with plasmid-borne expression of His₆-Pgk1 in GALS::PGK1 yeast strain. His₆-Pgk1 was expressed from the <i>TEF1</i> constitutive promoter, whereas the expression of the chromosomal HA-tagged Pgk1 was suppressed via 2% glucose in the growth media. Top panels: Northern blot analysis of repRNA level, middle panel: ethidium-bromide stained gel with ribosomal RNA, as a loading control, whereas bottom panels show Western blot analysis using anti-His antibody. Panels on the right represent samples obtained from yeast grown on the nonfermentable glycerol media. (C) The effect of over-expression of yeast Pgk1 on TBSV repRNA accumulation. The plasmid-borne His₆-Pgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B. (D) The effect of heterologous expression of NbPgk1 on TBSV repRNA accumulation in yeast. The plasmid-borne His₆-NbPgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B.</p