Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus
Fig 3
Knockdown of VPS34 gene expression inhibits tombusvirus replication in N. benthamiana plants.
(A) Top panel: Accumulation of the TBSV genomic (g)RNA in VPS34- silenced N. benthamiana plants 2 days post-inoculation (dpi) was measured by Northern blot analysis. Inoculation of TBSV gRNA was done 12 days after silencing of VPS34 expression. VIGS was performed via agroinfiltration of tobacco rattle virus (TRV) vector carrying NbVps34 or 3’-terminal GFP (as a control) sequences. Second panel: Ribosomal RNA is shown as a loading control in an ethidium-bromide stained agarose gel. Note that the TBSV genomic RNA is also visible in the gel. Third panel: Northern blot with N. benthamiana 18S ribosomal RNA specific probe was used as a loading control. Fourth panel: RT-PCR analysis of NbVps34 mRNA level in the silenced and control plants. Fifth panel: RT-PCR analysis of TUBULIN mRNA level in the silenced and control plants. Each experiment was repeated. (B) Delayed development of TBSV-induced symptoms is observed in VPS34-silenced N. benthamiana plants as compared with the control plants. Note the lack of phenotype in VPS34-silenced N. benthamiana plants. The picture was taken 5 dpi. (C) Top panel: Accumulation of the CIRV genomic (g)RNA in VPS34-silenced N. benthamiana plants 2.5 dpi was measured by Northern blot analysis. See further details in panel A. (D) Delayed development of CIRV-induced symptoms is observed in VPS34-silenced N. benthamiana plants as compared with the control plants. The picture was taken 5 dpi. (E) Western blot analysis of Vps34 level in yeast replicating TBSV repRNA or lacking viral components. The HA-tagged Vps34p was expressed from its natural promoter and original chromosomal location. The 6xHis-tagged p33 was detected by anti-His antibody. (F) Top panel: Induction of VPS34 mRNA expression in N. benthamiana plants infected with TBSV was detected by semi-quantitative RT-PCR. Middle panel: RT-PCR of tubulin mRNA was used as a control. Bottom panel: Ribosomal RNA is shown as a loading control in an ethidium-bromide-stained agarose gel.