Transient Expression of VFP3 and VirF

The VFP3 and VirF coding sequences were cloned into the EcoRI-BamHI sites of pSAT5-ECFP-C1 or SalI-BamHI sites of pSAT6-MYC-C1 [32 (link)], respectively. These expression cassettes were excised with I-CeuI or PI-PspI, respectively, and inserted separately or together into the binary pPZP-RCS1 vector [51 (link)]. These resulting constructs were transiently expressed for 72 h in N. benthamiana leaves by agroinfiltration, the leaves were then harvested and extracted, and cell-free degradation assay and western blot analysis were performed as described [31 ], using anti-GFP antibody (Clontech) followed by detection with secondary antibody conjugated to horseradish peroxidase (HRP). For loading controls, we used a major band at about 50 kDa, presumably representing the large chain of RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase), detected on Coomassie blue-stained gels. Protein amounts were estimated by scanning densitometry of the corresponding western blot bands using the ImageJ software (version 1.49, NIH).