Rapid demonstration of infectivity of a hybrid strain of potato virus Y occurring in India through overlapping extension PCR

An Indian isolate of Potato virus Y (PVY-Del-66) associated with the mild mottling symptoms in potato was characterized as a N:O type strain, which induced necrosis in tobacco and reacted strongly with the monoclonal antibody to PVYO strain. The complete genome of Del-66 sequence (9674 nucleotides) shared 96.7-99.3% similarities with the N:O strain occurring in USA and Germany. The lack of success in amplification and cloning of the full-length genome of PVY in E. coli was the major limiting factor for demonstrating infectivity of the PVY genome. The overlapping-extension PCR (OE-PCR) was successfully employed in this study for the rapid demonstration of infectivity of PVY. The Del-66 genome was amplified as 4.5 kb and 5.1 kb fragments with 30 nucleotide complementary ends. The mixture of these two fragments in the successive PCR steps with the terminal genome primers containing T7 promoter and poly-A tail resulted in a full-length construct. Rub inoculation of in vitro transcripts from this construct resulted in expression of systematic necrosis as well as the formation of typical filamentous virions in tobacco. This study for the first time characterized a hybrid strain (N:O type) of PVY occurring in India and showed a simple and rapid method of successful production of infectious in vitro transcripts of PVY by OE-PCR.