The same GFP ORF with its 35SCaMV promoter and Nos terminator was also cloned into the pJet2.1 vector to make a 4.8 kb fragment. pJL-TRBO-G was used to express GFP from the TMV plasmid. For BFP, the GFP in pJL-TRBO-G was replaced with BFP. For the VIGS assay, a short sequence of the Nicotiana benthamiana PDS gene was cloned into the RNA2 genome of TRV and used with RNA1 for PDS gene silencing. For systemic GFP assays, PVX-GFP was used. For viral symptomatic analysis, a binary vector containing TYLCV2.3 was used. For in vitro transcription and translation assays, the pQ70-deGFP vector (3.2 kb) and a plasmid of 12.3 kb was made by cloning the same GFP expression cassette with its Q70 promoter and terminator. Maps of the plasmids used are provided in the supplementary information.