Fig 4. The pro-viral role of cytosolic Pgk1 in tombusvirus replication in N. benthamiana.

(A) Up-regulation of Pgk1 expression in TBSV-infected N. benthamiana leaves. The mRNA levels for the cytosolic Pgk1 were estimated by semi-quantitative RT-PCR (25 and 28 cycles, the latter is shown) in total RNA samples obtained from either TBSV or mock-infected N. benthamiana leaves. Tubulin mRNA was used as a control (second panel). The bottom panel shows an ethidium-bromide stained agarose gel of total RNA samples with ribosomal RNA and TBSV genomic (g)RNA. (B) Western blot analysis of cytosolic Pgk1 level in total protein samples obtained from yeast either supporting TBSV repRNA accumulation or TBSV-free using anti-Pgk1 antibody. (C-E) Knock-down of PGK1 mRNA level by VIGS inhibits the accumulation of tombusvirus RNA in N. benthamiana. Top panels: Total RNA samples obtained from N. benthamiana leaves silenced as shown were analyzed by Northern blotting, which shows the accumulation of TBSV gRNA and sgRNAs in panel C, the closely-related cucumber necrosis virus (CNV) RNAs in panel D and the mitochondria-replicating CIRV RNAs in panel E. Bottom images: ethidium-bromide stained gels show ribosomal RNA level. We chose the 12th day after VIGS to inoculate the upper, systemically-silenced leaves with TBSV virions, or agroinfiltrate with pGD-CNV or pGD-CIRV. Samples for RNA extractions were taken 1 day (TBSV) and 2.5 days (CNV or CIRV) post inoculation from the inoculated leaves. The control experiments included the TRV2-cGFP vector. Each experiment was performed three times. (F) Over-expression of the cytosolic NbPgk1 was done in N. benthamiana leaves by agroinfiltration. The same leaves, which were first agroinfiltrated with pGD-NbPgk1 (expressing NbPgk1 from the 35S promoter), were also inoculated with TBSV virions 2.5 days later. Then, total RNA samples were obtained the subsequent day (1 dpi). The control samples were obtained from leaves agroinfiltrated with pGD empty vector (not expressing proteins) (lanes 1–6). Northern blotting was used to detect the accumulation of TBSV RNAs in total RNA samples obtained from N. benthamiana leaves. The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (bottom panel). The bottom image shows a representative Western blot-based detection of His₆-tagged NbPgk1 using anti-His antibody. Each experiment was performed three times.