Tagging Potato leafroll virus with the jellyfish green fluorescent proteingene

A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green flu orescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthami ana protoplasts electroporated with plasmid DNA containing this cDNA behind the 35S RNA promoter of Cauliflower mosaic virus became infected with the recombinant virus (PLRV-GFP), Up to 5% of transfected protoplasts showed GF P-specific fluorescence. Progeny virus particles were morphologically indis tinguishable from those of wildtype PLRV but, unlike PLRV particles, they b ound to grids coated with antibodies to GFP, Aphids fed on extracts of thes e protoplasts transmitted PLRV-GFP to test plants, as shown by specific flu orescence in some vascular tissue and epidermal cells and subsequent system ic infection. In plants agroinfected with PLRV-GFP cDNA in pBIN19, some cel ls became fluorescent and systemic infections developed. However, after eit her type of inoculation, fluorescence was mostly restricted to single cells and the only PLRV genome detected in systemically infected tissues lacked some or all of the inserted GFP cDNA, apparently because of naturally occur ring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stage s of PLRV infection.