title: Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y.
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.
authors.affiliation: Laboratoire de Génétique, Université de Tunis, Tunisia.
authors.forename: H
authors.initials: H
authors.lastname: Fakhfakh
authors.forename: F
authors.initials: F
authors.lastname: Vilaine
authors.forename: M
authors.initials: M
authors.lastname: Makni
authors.forename: C
authors.initials: C
authors.lastname: Robaglia
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction / Q000379:methods*; D017800:Potyvirus / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D014026:Nicotiana / Q000821:virology
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase