H. Fakhfakh et al., CELL-FREE CLONING AND BIOLISTIC INOCULATION OF AN INFECTIOUS CDNA OF POTATO-VIRUS-Y, Journal of General Virology, 77, 1996, pp. 519-523
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.