Taq DNA polymerase (New England Biolabs) was used for all PCR reactions, using manufacturers instructions. For production of 5' phosphorylated PCR products, both forward and reverse direction PCR primers were treated with T4 polynucleotide kinase (New England Biolabs) according to manufacturers instructions, prior to use in the PCR. Amplified reaction products were column purified with the DNA clean up and concentrator kit (Zymoresearch) to remove unincorporated dNTPs and primers.
The duplicated CaMV 35S promoter, the Tobacco etch virus (TEV) 5' non-translated leader sequence, a short polylinker containing an XbaI site, and the polyA/terminator sequences from pRTL2  were cloned as a PstI fragment into PstI cut binary vector pCB301  to generate pJL3. Using inverse PCR a PacI restriction endonuclease site was generated immediately downstream of the TEV leader sequence. The gfp or p19 genes were cloned into the PacI and XbaI sites of the vector to generate pCB35SGFP and pJL3:P19, respectively. The "cycle 3" mutant version of gfp  in p30BGFP  was used as the source of the gfp gene. The coding sequence for the p19 gene from TBSV was amplified from a cDNA sample (a kind gift from H. Scholthof).
The NotI restriction endonuclease site in the pCB301 backbone of pCB35S:GFP was destroyed by digesting with NotI, treatment with T4 DNA polymerase and dNTPs followed by religation to generate pCB 35SGFP ΔN. Inverse PCR of pCB 35SGFP ΔN was used to generate a unique StuI restriction site at the 35S promoter transcription start site as described in Dessens and Lomonossoff . The resulting plasmid was named pJL 22.
The TMV expression vector in p30B  contains a T7 driven cDNA of the U1 strain of TMV, an additional viral subgenomic promoter for expression of foreign gene inserts, and a ribozyme sequence following the end of the viral cDNA (Figure 1). Using standard cloning procedures the cDNA version of the TMV expression vector and ribozyme sequence from p30B (obtained from Large Scale Biology Corporation, Vacaville, CA) was cloned into StuI-XbaI cut pJL 22. This plasmid was then modified to generate pJL36 and pJL43. In pJL36 the multiple cloning site (seq ttaattaacggcctagggcggccgc) was inserted downstream of the additional TMV subgenomic promoter. pJL36, (Figure 1) has unique PacI, AvrII and NotI restriction endonuclease sites for cloning.
To construct pJL43 (Figure 1) the ds DNA cassette [top stand seq (CGAGGCCAGAAGAGCAACCTTTACGTACTTGCTCTTCAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAC)] containing the coding sequence for two SapI restriction endonuclease sites (gctcttc), the V5 epitope (amino acid sequence GKPIPNPLLGLD) and a hexa-histidine (His6) tag coding sequence was inserted downstream of the additional TMV subgenomic promoter.
Sticky RICE cloning into pJL 43
Preparation of vector
To prepare pJL-43 for sticky RICE cloning, plasmid DNA was digested with the restriction endonuclease SapI, treated with calf alkaline intestinal phosphatase and column purified.
Preparation of PCR products
For sticky RICE cloning of PCR products into SapI cut pJL 43 the 5' end of the forward PCR direction primer must have the following sequence: 5' GGCCWW. The 5' end of the reverse direction PCR primer should be 5' GCWW. In the primer sequences W = A or T. The T and A residues in the primers will serve as "stop nucleotides" during the sticky RICE reaction, in which PCR products were incubated in the presence of a DNA polymerase with 3' to 5' exonuclease activity and dATP and dTTP. The DNA polymerase removed nucleotides from the 3' ends of each strand until an A or T residue was present in the template stand. This resulted in 5' overhangs on each end of the PCR products (Figure 2).
Using these guidelines for primer design, the gfp gene was amplified by the PCR from a pUC-based plasmid with forward direction primer JAL 286 (GGCCT aaa atggctagcaaaggagaag) and reverse direction primer JAL 287 (GCttatttgtagagctcatccat). The primer nucleotides that were converted to sticky ends by T4 DNA polymerase are in bold. PCR reactions using either non-phosphorylated or 5' phosphorylated primers were performed. PCR products were purified to remove unincorporated dNTPs and primers and eluted in dH20.
Assembly of the Sticky RICE reaction
A three-fold molar excess of phosphorylated gfp gene PCR product was combined with 50 ng SapI cut and phosphatase treated pJL 43 DNA in a 10 ul reaction volume composed of: 1 × ligase buffer, 0.1 mM (each) dATP/dTTP, 0.25 Units T4 DNA Polymerase, and 400 Units T4 DNA ligase. Enzymes, buffers and unit definitions were all from New England Biolabs. The assembled reaction was incubated at room temperature for 30 minutes.
For purposes of comparison, non-phosphorylated gfp gene PCR products were also cloned using the same reaction conditions except that 0.5 Units T4 polynucleotide kinase were also included in the Sticky RICE cloning reaction. The cloning of the gfp gene into pJL 43 resulted in plasmid pJL43:GFP.
Transformation of E. coli with Sticky RICE cloning reactions
Two microliters of a sticky RICE ligation reaction was added to 25 ul of chemically competent E. coli (Bioline, AlphaSelect Gold efficiency). Transformation conditions were essentially as per manufacturers instructions. Transformed cells were plated on LB plates with 50 ug/ml Kanamycin.
Screening plasmids for inserts
Following transformation of the sticky RICE ligations into E. coli, individual colonies were used to inoculate LB broth with 50 ug/ml Kanamycin. Plasmids were purified from overnight liquid cultures. Because the forward-direction primer used for PCR began with the sequence "GGCCT" a StuI restriction enzyme recognition site was generated upon sticky RICE joining of PCR product to pJL 43. Therefore plasmids were screened by digestion with StuI and digested DNA separated on 1% agarose gels to identify clones that had inserts (Table 1).
Plasmids purified from E. coli cultures were transformed into A. tumefaciens GV3101 using the freeze thaw method . Transformed A. tumefaciens were plated on LB plates with 50 ug/ml Kanamycin, 25 ug/ml gentamycin and 10 ug/ml rifampicin for plasmid selection. Individual colonies of A. tumefaciens transformed with a binary plasmid were grown to an OD600 of 1.0 in liquid LB media supplemented with 10 mM MES pH 5.7, 50 ug/ml Kanamycin, 25 ug/ml gentamycin and 20 uM acetosyringone. Cells were collected by centrifugation and resuspended in 10 mM MES, pH 5.7, 10 mM MgCl2, 200 uM acetosyringone . Cells sat at room temperature in induction media for 2–24 hours before infiltration into the abaxial surface of N. benthamiana leaves using a 1 ml syringe with no needle. A. tumefaciens cultures containing an 'empty vector control' plasmid or pJL3:p19 were co-infiltrated with A. tumefaciens/pJL43:GFP cultures. When mixes of A. tumefaciens cultures were infiltrated, bacterial cultures were prepared separately in induction media and were combined immediately before infiltration.
Healthy and TMV:GFP infected plant tissue (from TMV vector launched from the A. tumefaciens-delivered T-DNA of pJL43:GFP) samples were ground in the presence of 4 volumes (per gram fresh weight) 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20, 0.1% beta-mercaptoethanol. Extracts were clarified by centrifuging for 15 minutes at 12–15 K × g. Samples of clarified extract were combined with SDS-PAGE loading dye and analyzed on 4–20% SDS-PAGE gels. Gels were stained with coomassie blue to visualize proteins.
Plant protein extracts (prepared as described above) were diluted in 50 mM Carbonate buffer (pH 9.6). Samples were assayed for GFP activity with a Perkin-Elmer HTS 7000 BioAssay Reader using filters of 405 nm excitation/535 nm emission. A standard curve was prepared from purified His-6 tagged GFP (purified from plants).