The BaMV strain S-derived infectious cDNA clone, pBS2-8 (EMBL/GenBank accession no. AF018156; Liao and Hsu, unpublished) and pPVX/UTR containing the 3′ UTR of PVX RNA  were used for mutant construction. Six primers Ba3′P (5′CCCGAACCAACATCAGACTAACTACGTCTACATAACCGA3′), Ba/ABCP (5′GAAAGAAAGGTTTACACCTACGTCTACATAACCGA3′), Ba/ABCDP (5′GCCAGCAGAATAAAGACCCTACGTCTACATAACCGA3′), Ba/PABCDE (5′GAATAATATAAATACTAAACGTTGCATGAT3′), Ba/PDE (5′GTATGAATAATATAAATTTTACACGGACTCTGTT3′), and Ba/PE (5′GTATGAATAATATAAATATAAAGACCTTTT3′) in combination with the downstream primer SacI-polyT (5′GAGCTCT40) were used to generate PCR products. Each of the 181- to 115-bp PCR fragments was used subsequently as the megaprimers together with an upstream primer, BaMV6122 (5′GCCAATGACCAGAAAGGGTTCAA3′), to generate the 478- to 452-bp DNA fragments through a second PCR using pBS2-8 as the template .
All the PCR products were cloned into the pGEM® -T Easy vector (Promega, Madison, WI, USA) and verified by DNA sequencing. The SapI6207-to-SacI (located 3′ to the insert) fragment was then subcloned into pBS2-8 to replace the corresponding SapI-to-SacI region. Because pBS2-8 has two SapI sites, mutant constructs were done by three-fragment ligation and confirmed by DNA sequencing. These mutant plasmids were designated as pBaMV-S/P, -/ABCP, -/ABCDP, -/PABCDE, -/PDE, -/PE, and -/ABCPE, respectively (Figure 1).
To construct the mutant pBaMV-S/ABCDEP, the first PCR product (694 bp) generated with the template pBS2-8 and the primers BaMV5768(+) (5′GGCCTCAGTCTCGAAGC TTTCGA3′) and gg15T+PVX3T (5′TTTATTTGTATTATTCATACAATCAAATCAAACCA GAAAATACTATGAAACTGGGGTAGGCGTCGGTTATGTAGACGTAGGTTTTTTTTTTTTTTTGGA3′) was used as the template for the second PCR. The second PCR product (717 bp) amplified with primers BaMV5768(+) and SacI40TPVX(−) (5′GAGCTCTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTATATTATTC3′) was cloned to pGEM® -T Easy vector and sequenced. The insert in the vector was digested with HindIII and SacI and replaced to that of pBS2-B.
In vitro RNA transcription
Before in vitro transcription, all plasmids were linearized with EcoICRI (Promega). The reaction was carried out at 37°C for 2 h in a 50-μl reaction containing 100 U of T7 RNA polymerase, 40 mM Tris–HCl pH 8.0, 8 mM MgCl2, 2 mM spermidine, 10 mM DTT, 0.5 mM GTP, 1 mM ATP, 1 mM UTP, 1 mM CTP, 2 mM m7GpppG cap analogue, and 5 μg of the linearized template. Subsequently, the reaction was supplemented with 5 U of RNase-free DNase I and incubated at 30°C for 20 min to degrade the DNA template. Transcription reactions were brought up to 200 μl with TE and extracted with an equal volume of phenol/chloroform. RNA was precipitated with ammonium acetate and ethanol. The pellet was dissolved in 300 μl of water, and 200 μl of 7.5 M ammonium acetate was added. The mixture was incubated at −80°C for 20 min, thawed at room temperature, and centrifuged at 4°C for 20 min. RNA transcripts were washed with 70% ethanol, dried, dissolved in 20 μl of water, and quantified by running on a 1% agarose gel with control markers.
Protoplast and plant inoculation
Four grams of sliced N. benthamiana leaves were digested with 25 ml of enzyme solution containing 0.55 M Mannitol-MES, pH 5.7, 0.1% bovine serum albumin, 0.6 mg/ml macerase pectinase (Calbiochem, La Jolla, CA), and 12 mg /ml cellulase (Yakult Pharmaceutical,Tokyo, Japan) at 25°C for overnight. The mesophyll cells were spun down at 300 rpm (KUBOTA KS-5000) for 7 min and suspended in 2 ml of 0.55 M Mannitol-MES buffer. Intact protoplasts were collected from the interphase above the 0.55 M sucrose cushion and washed with Mannitol-MES buffer at least two times. A total of 4 × 105 cells were transfected with 5 μg of RNA transcripts with the help of polyethylenglycol. Finally, the transfected protoplasts were suspended in culture medium (1 μM CuSO4, 10 mM MgSO4, 1 μM KI, 0.2 mM KPO4, 10 mM KNO3 pH 6.5, 10 mM CaCl2, 0.03% cephaloridins, 0.001% gentamycin and 0.55 M Mannitol-MES) and incubated under a constant light at 25°C for 48 h.
N. benthamiana seedlings were grown under 16-h illumination at 28°C until plants had about four leaves. Five or 20 μg of the RNA transcripts or lysates of the inoculated protoplasts (about 105 cells) were gently rubbed onto one leaf of each plant. The inoculated and systemically infected leaves were harvested 10 days after inoculation.
Northern blotting analysis
The detection probe was a 32P-labelled 0.6-kb RNA transcript complementary to the 3′-end of BaMV RNA. The 20-μl in vitro transcription reaction contained 2 μg of template DNA (pBaMV-O/SB 2.6 linearized with HindIII ), 40 mM Tris–HCl pH 8.0, 8 mM MgCl2, 2 mM spermidine, 10 mM DTT, 3 mM ATP, 3 mM CTP, 3 mM GTP and 70 μCi[α-32p]UTP (Dupont-NEN, Boston, MA). Total RNA extracted from the inoculated protoplasts were incubated in a buffer containing 50% DMSO, 1 M glyoxal and 10 mM sodium phosphate pH 7.0 at 50°C for 1hr and resolved on a 1% agarose gel in 10 mM sodium phosphate buffer. The RNA was transferred onto a Zeta-Probe® blotting membrane (Bio-Rad Laboratories, Hercules, CA, USA) which was subsequently hybridized with the probe about one million-cpm at 60°C overnight . After hybridization, the membrane was washed, and scanned with a phosphoimager (BAS-1500; Fujifilm, Tokyo, Japan).
Total protein harvested from the RNA-inoculated protoplasts was separated in a 12% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane (PROTRAN® BA 85; Schleicher & Schuell, Germany). A primary rabbit against-BaMV coat protein polyclonal antiserum and a secondary fluorescence-labeled anti-rabbit IgG antibody (H&L) were used to detect BaMV coat protein on the membrane. Data analysis was carried out using the LI-COR Odyssey (LI-COR Biosciences, Lincoln, USA).
In vitro translation and RNA stability assay
In vitro translation was performed in the TnT-coupled transcription/translation system (Promega, Carlsbad, CA, USA) according to the manufacturer. Briefly, the reaction was carried out in a 12.5 μl-reaction mixture containing 1 μg RNA in wheat germ extract supplemented with all amino acids except methionine. Proteins were labeled by the incorporation of 5 μCi of L-[35S] methionine (1,000 Ci/mmol; 10 mCi/ml; Perkin Elmer, Walthman, MA, USA) in the reaction. Incubations were performed at 30°C for 1 h and terminated by the addition of sample buffer. The translation products were analyzed by 10% SDS-polyacrylamide gel electrophoresis. Gels were dried, and analyzed by a phosphorimager (Fujifilm BAS 2500).
For RNA stability assay, RNAs were incubated with wheat germ extract supplemented with all amino acids. At various time points of post incubation, viral RNAs were isolated and analyzed by Northern blotting.
Templates for in vitro replication assay
The RNA transcripts were generated directly from PCR-generated DNA templates using T7 RNA polymerase . The specific primers, EcoRI-T7-6228 (5′GCGAATTCTAATACGACTCACTATAGGGCGTTGCATGATCG3′), T7-PVX (5′TAATACGACTCACTATAGGGTAACTACGTCTACAT3′), and the down stream primers, PVX40T (5′T40ATTTATATTATTCATAC3′) and 40TGG (5′T40GG3′), were used to synthesize the PCR products. The pPVX/UTR, pBaMV40A, pBaMV-S/ABCP, -/ABCDP, -/PDE, -/PE, -/PABCDE, -/ABCPE, and -/ABCDEP were employed as the template to generate rABCP, rABCDP, rPDE, rPE, rPABCDE, rABCPE, and rABCDEP, respectively. The RNA transcripts were gel-purified and quantified by spectrophotometry.
In vitro BaMV RNA replication assay
For exogenous RNA template activity assay, the detergent-solubilized RdRp preparation  was treated with 20 μg/ml micrococcal nuclease (Pharmacia Biotech) in the presence of 2 mM CaCl2 at 30°C for 30 min and the treatment was stopped by adding 25 mM EGTA. The 50-μl BaMV RNA replication reaction contained 25 μl of the RdRp preparation, 10 mM DTT, 3 mM MgCl2, 2 mM of each ATP, CTP, and GTP, 2 μM UTP, 2 μM (α-32P) UTP (3000 Ci/mmol; Dupont-NEN), 4.8 mg/ml bentonite, and 1 μg of RNA template. Reaction was incubated at 30°C for 1 hr and stopped by phenol/chloroform extraction, followed by ethanol precipitation. The pellet was dissolved in 5 μl water and treated with RNase A (32 μg/ml) and T1 (1.6 μg/ml) at 30°C for 30 min in RNase protection buffer (10 mM Tris–HCl, pH 7.5, 5 mM EDTA, 200 mM NaCl and 100 mM KCl) to collect only double-stranded RNA. After incubation at 37°C for 30 min with 66 mg/ml proteinase K , RNA was extracted with phenol/chloroform and precipitated in ethanol. The reaction products were resolved by gel electrophoresis and detected by using a Bio-Imaging analyzer BAS-1500. The kinetic parameters of the rABCDE, rABCP, rP, and rDE templates were determined by taking measurements at six time points (10, 20, 40, 60, 90, and 120 min) in the presence various template concentrations (30 to 1500 nM). The initial rates were plotted onto a Michaelis-Menten graph and the KM and Vmax were calculated by the double-reciprocal Lineweaver-Burk plot.