Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y

Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7kb PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVY(NTN)). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immune-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.