Stimulation of tombusvirus RNA accumulation by over-expression of the eIF4IIIA-like AtRH2 and the DDX5-like AtRH5 DEAD-box helicases in yeast and <i>N. benthamiana</i>.

<p>(A) Expression of AtRH2 and AtRH5 in yeast (BY4741) enhances TBSV repRNA accumulation. Top panel: Replication of the TBSV repRNA was measured by Northern blotting 14 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Middle and bottom panels: The accumulation levels of His₆-AtRH2 and His₆-AtRH5 and FLAG-p33 were tested by Western blotting and total protein loading is shown by SDS-PAGE. Each experiment was repeated twice. (B) Expression of AtRH2 and AtRH5 were done separately or together in <i>N. benthamiana</i> leaves by agroinfiltration. The same leaves were co-infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch <i>Cucumber necrosis virus</i> (CNV, a close relative of TBSV) replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 5–8). Total RNA was extracted from leaves 2.5 days after agroinfiltration that launched CNV replication. The accumulation of CNV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting (Top panel). The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (bottom panel).