Detection of episomal replicon in N. benthamiana line pOri-2 plants infected with PVX expression vectors. (A) DNA samples were extracted from line pOri-2 plants infected either with PVX/ACm1m4, PVX/AC14, PVX/AC1m4, or PVX/AC14-GFP and were used for PCR amplification of a fragment of the replicon with primers P1 and P2. The positions and sizes of DNA markers (bp) are indicated. (B) The same DNA samples were analyzed by Southern blotting with a GUS-specific probe to detect episomal replicon. The positions of circular single-stranded DNA (ssc) and covalently closed circular (ccc), and open circular (oc) dsDNA forms of the replicon are indicated. (C) Proteins were extracted from line pOri-2 plants infected with either PVX/AC14-GFP or PVX/GFP, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by Western blot analysis by using antiserum raised against GFP. The positions of AC1-GFP and free GFP are indicated.