Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation
Fig 15
Dependence of BaMV replication on Pdc1 and Adh1 proteins in N. benthamiana.
(A) Top panels: semi-quantitative RT-PCR analysis of NbPdc1 and NbAdh1 mRNA levels at 3 dpi in N. benthamiana leaves infected with bamboo mosaic virus (BaMV) or mock-inoculated. Third panel: RT-PCR analysis of tubulin mRNA level in the same plants. Bottom panel: RT-PCR detection of the BaMV CP subgenomic RNA. (B) Semi-quantitative RT-PCR analysis of NbPdc1 and NbAdh1 mRNA levels at 7 dpi in N. benthamiana leaves infected with BaMV or mock-inoculated. Third panel: RT-PCR analysis of tubulin mRNA level in the same plants. Bottom panel: RT-PCR detection of the BaMV CP subgenomic RNA. Each experiment was repeated three times. (C) Knockdown of Pdc1 or Adh1 mRNA levels inhibits BaMV replication in N. benthamiana plants. Top panel: Accumulation of the BaMV genomic (g)RNA in the Pdc1-silenced N. benthamiana plants 2.5 days post-inoculation (dpi) in the inoculated leaves was measured by quantitative RT-PCR. Inoculation of BaMV gRNA was done 12 days after silencing of Pdc1 or Adh1 expression. Agroinfiltration of tobacco rattle virus (TRV) vector carrying NbPdc1 or NbAdh1 or luciferase (LUC, as a control) sequences was used to induce VIGS. Second panel: RT-PCR analysis of tubulin mRNA level in the silenced and control plants. Each experiment was repeated three times. (D-E) The BaMV ORF1-capping-cYFP or ORF1-helicase-cYFP or ORF1-RdRp-cYFP domains of the replicase protein and the AtPdc1-nYFP (panel D) or AtAdh1-nYFP (panel E) proteins were expressed via agroinfiltration. The DIC and the merged images are also shown. The BiFC signals were detected via confocal microscopy 2 days after agroinfiltration to N. benthamiana plants. Scale bars represent 25 μm.