Blocking tombusvirus replication through the antiviral functions of DDX17-like RH30 DEAD-box helicase
Fig 2
Knockdown of NbRH30 gene expression leads to enhanced tombusvirus replication in N. benthamiana plants.
(A) Top panel: Accumulation of the TBSV genomic (g)RNA and sgRNAs in RH30-silenced N. benthamiana plants 1.5 days post-inoculation (dpi) was measured by Northern blot analysis. Inoculation of TBSV gRNA was done 12 days after silencing of RH30 expression. VIGS was performed via agroinfiltration of tobacco rattle virus (TRV) vector carrying 5’ or 3’-terminal NbRH30 sequences, whereas as a control, 3’-terminal GFP sequences. Second panel: Ribosomal RNA is shown as a loading control in an ethidium-bromide stained agarose gel. Third panel: Northern blot analysis shows the knock-down level of NbRH30 mRNA in the silenced and control plants. Fourth panel: Northern blot analysis shows 18S ribosomal RNA as a loading control. Fifth and seventh panels: RT-PCR analysis of NbRH30 mRNA level in the silenced and control plants. Sixth and eighth panels: RT-PCR analysis of TUBULIN mRNA level in the silenced and control plants. Each experiment was repeated. Bottom panel: Accelerated and more severe TBSV-induced symptom development is observed in RH30-silenced N. benthamiana plants as compared with the control plants. Note the mild growth defect phenotype in RH30-silenced N. benthamiana plants. The picture was taken 5 dpi. (B-C) Top panel: Accumulation of the CNV or CIRV gRNA in RH30-silenced N. benthamiana plants 2 days post-inoculation (dpi) was measured by Northern blot analysis. See further details in panel A.