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

The Journal of General Virology
H FakhfakhC Robaglia

Abstract

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 PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). 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 immuno-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.

Citations

Sep 29, 2011·Transgenic Research·Lilya KopertekhJoachim Schiemann
Sep 23, 1997·Journal of Virological Methods·A M ChachulskaF Vilaine
Oct 29, 1996·Proceedings of the National Academy of Sciences of the United States of America·I E Johansen
Mar 12, 2015·Molecular Plant-microbe Interactions : MPMI·Rupar MatevzRavnikar Maja
Nov 16, 2004·Journal of Virological Methods·J MatousekD Riesner
Nov 30, 2018·Molecular Biotechnology·C R A Duff-FarrierG D Foster
Mar 5, 2021·Bio-protocol·Katja StareKristina Gruden
Jul 20, 2020··Andreja KežarMagda Tušek Žnidarič

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