Inhibition of tombusvirus and RCNMV RNA accumulation in plants by over-expression of AtPah2p in <i>N. benthamiana</i>.

<p>(A) Expression of the yeast <i>PAH1</i> homolog AtPah2p (lanes 5–8) was done in <i>N. benthamiana</i> leaves by agroinfiltration. Two days later, the same leaves were infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 1–4). Total RNA was extracted from leaves 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 (Second panel). (B) Over-expression of AtPah2p in <i>N. benthamiana</i> protects the plant from rapid necrosis caused by systemic CNV infection. The pictures were taken 12 days after agroinfiltration. (C) Inhibition of the peroxisomal TBSV replication by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with TBSV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of TBSV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A. (D) The lack of inhibition of the mitochondrial CIRV tombusvirus by over-expression of AtPah2p in <i>N. benthamiana</i>. See additional details in panel A. (E) Inhibition of the distantly related RCNMV (which uses ER membranes for replication) by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with RCNMV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of RCNMV RNA1 in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A.