Coat protein-mediated protection (CPMP) was used to obtain transgenic tobacco (KY14, NC3433-33, TN90, and K326) with resistance to various isolates of potato virus Y (PVY). The tobacco cultivars were transformed with a chimeric gene encoding the coat protein (CP gene) of the 'Chilean' isolate of PVY, via Agrobacterium-mediated transformation. CP expression among Ro plant was generally low or undetectable by indirect ELISA and Western blot. Transgenic R(0) plants which carried from one to five neomycinphosphotransferase II (nptII) transgene loci were identified. There is no correlation among the number of functional nptII or the PVY CP transgenes and the resistance performance of the regenerated transgenic lines. The presence of functional PVY CP transgene was determined by inoculation of the transgenic tobacco with PVY 'Chilean' or 'VAM B'. Inoculation of 41 independent transgenic KY14 with PVY 'Chilean' yielded eight PVY resistant lines, while inoculation with the same isolate of transgenic K326 yielded six resistant lines. Inoculation of 17 transgenic NC3433-33 lines with PVY 'Chilean' yielded six resistant lines and inoculation of 50 transgenic TN90 with PVY 'VAM B' yielded nine resistant lines. Progenies derived from these transgenic plants retain the PVY resistance characteristic of the parental transgenic lines, which demonstrated sexual transmission of the PVY resistance trait.

Transgenic burley (cultivars KY14, NC3433-33, and TN90) and flue-cured (cultivar K326) tobacco with resistance to four necrotic isolates of potato virus Y (PVY) were constructed. These tobacco cultivars were transformed with a chimeric gene designed to express the coat protein (CP) from the necrotic Chilean isolate of PVY CP expression among R(0) plants was undetectable by indirect enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Transgenic R(0) plants that harbored from one to five neomycin phosphotransferase II (NPT II) transgene loci were identified. Although the NPT II and the PVY CP chimeric genes were linked within a common T-DNA, no correlation was found between the number of NPT II and CP transgenes. Inoculation of 41 independent transgenic KY14 R(0) plants with PVY-Chilean identified eight resistant plants, while inoculation of 30 transgenic K326 Ro plants identified six resistant plants. Inoculation of 17 transgenic NC3433-33 R(0) plants with PVY-Chilean identified six resistant plants, while inoculation of 50 transgenic TN90 R(0) plants with PVY isolate VAM B resulted in nine resistant plants. Progeny derived from PVY-inoculated but symptomless Ro plants were also resistant to the Chilean, Europe H, M(s)N(r), N-Canada, and VAM B isolates of PVY, demonstrating sexual transmission of the transgenes that condition resistance. We discuss the ability of the PVY-Chilean CP gene to protect transgenic tobacco K326, KY14, NC3433-33, and TN90 from infection by four necrotic isolates of PVY. The flue-cured tobacco cultivar K326, which carries a gene simultaneously conferring resistance against root-knot nematode and susceptibility to PVY strain M(S)N(R), is protected against this strain when transformed with the PVY-Chilean CP gene.

Complementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate 'Chilean', 'Hungarian', MsNr, NsNr, O, and 'Potato US' were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY 'Chilean' antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated M(r) ranging from 29,799 to 29,980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.