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 9.7 kb PW cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded D NA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promote r and a nopaline synthase terminator) in the final PCR product. Biolis tic bombardment with a helium particle gun was used to inoculate the a mplified product to detached tobacco leaves. Inoculation of tobacco pl ants with ground inoculated leaves followed by northern blot, ELISA an d immune-electron microscopy demonstrated that the DNA was highly infe ctious with up to 90% of bombarded leaves containing the virus. This m ethodology will allow the use of reverse genetics in the study of PVY- plant interactions and will also be useful for obtaining infectious cD NA from other viruses with large RNA genomes.