Fig. 3.

RNA1 encodes a fully competent cis- and trans-acting replicase. (A) Northern blot analysis of total RNA extracted from agroinfiltrated areas 3 days postinfiltration (dpi). The top panel shows viral plus-strand RNA evaluated with a 2-h exposure to the X-ray film; the middle panel shows the same blot exposed for 4 days to evaluate accumulation of the deleted RNA1 transcript (arrow); the bottom panels show the minus-strand RNA accumulation with a 4-day exposure and the rRNA, respectively. Each agroinfiltrated clone is detailed at the top of each lane and graphically described in Fig. 2. (B) Northern blot analysis of RNA extracted at 3 dpi with a combination of agroclones specified above the panels (except for the samples corresponding to the lanes marked by asterisks, which were instead processed 2 dpi). From top to bottom, each of the four panels shows plus-strand RNA1, plus-strand RNA3, minus-strand RNA1, and minus-strand RNA3. The bottom panel shows rRNA stained with methylene blue on the membrane. The inset replaces the original 2-day exposure with a 6-day exposure. The arrows indicate the two primary transcripts from pGC-RNA1Δ3′ (ribozyme processed and full-length). The RNA ladder bands are those from BrightStar Biotinylated RNA Millennium Markers (Ambion), and the sizes are expressed in kb.