INFECTIOUS IN-VIVO AND IN-VITRO TRANSCRIPTS FROM A FULL-LENGTH CDNA CLONE OF PVY-N605, A SWISS NECROTIC ISOLATE OF POTATO-VIRUS-Y

A full-length cDNA clone of the potato virus Y (PVY) genome was obtain ed after cloning difficulties in Escherichia coil were overcome. These difficulties were mainly due to the expression of the CI gene from up stream prokaryotic promoter-like elements within the PW genome. To ove rcome this problem, PW cDNA was maintained in two subclones which were ligated before infection. A plasmid in which these two fragments were contained could be propagated in some E. coil strains but was unstabl e and yielded only low levels of plasmid DNA. Replacement of the 35S p romoter by the SP6 promoter slightly increased the stability of the pl asmid and its RNA transcripts were infectious when capped with m(7)G(5 ')ppp(5')G. Using two inoculation methods (mechanical or biolistic) th e cDNA and its RNA transcript proved infectious on three Nicotiana spe cies and on Solanum tuberosum.