Submitted to: Journal of General Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/19/1999
Publication Date: N/A
Citation: Interpretive Summary: The Luteoviruses, e.g. barley yellow dwarf (BYDV) and potato leafroll (PLRV), are economically important viruses of cereal and vegetable crops. Current control measures are only marginally successful. Understanding the molecular and cellular mechanisms of host infection will allow the development of novel strategies that disrupt these processes and control the viruses. In this manuscript we describe the use of the jellyfish green fluorescent protein (GFP) as a way to tag the luteovirus genome and visualize the initial events of infection. The cells infected by the virus and expressing GFP produce a green glow. Although luteovirus infection is normally confined to vascular tissue aphids, the natural vector of the virus, did inoculate other plant tissues. The virus did replicate in these other tissues, but apparently could not escape from the inoculated cell. Inoculation into vascular tissues resulted in systemic infection of plants. .The inoculated vascular cells expressed the green fluorescence and the fluorescence expanded to surrounding cells as the virus moved. Unfortunately the GFP gene is lost from the virus shortly after the virus begins to move from the inoculated cell. The loss of foreign genes from viruses is a common phenomenon. Continuing work will determine if the GFP gene can be inserted into other regions of the genome. This system is a useful to study the effects of mutations in the virus on inoculation events and tissue specificity.
Technical Abstract: A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthamiana protoplasts electroporated with plasmid DNA containing this cDNA behind the Cauliflower mosaic virus 35S RNA promoter became infected with the recombinant virus (PLRV-GFP). Up to 5% of transfected protoplasts showed GFP-specific fluorescence. Progeny virus particles were morphologically indistinguishable from those of wild type PLRV, but, unlike PLRV particles, they bound to grids coated with antibodies to GFP. Aphids fed on extracts of these protoplasts transmitted PLRV-GFP to test plants, as shown by specific fluorescence in some vascular tissue and epidermal cells and subsequent systemic infection. In plants agroinfected with PLRV- GFP cDNA in pBIN 19, some cells became fluorescent and systemic infections developed. However, after either 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 occurring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stages of PLRV infection.