Aspects of the molecular biology of potato virus Y

Aspects from the coat protein from Potato Virus Y (PVY) were investigated. Polyadenylated, full length RNA was isolated from purified preparations of two strains of the virus, PVYN and PVYC. The RNA was used as a template to produce double stranded complementary DNA (cDNA) which was subsequently cloned into the plasmid vector pUC19. Of the resultant PVYC –derived clones, two recombinant plasmids (pVYC5 and pVYC11) were analysed by DNA hybridlsation and DNA sequencing. PVYC11 contained a viral cDNA insert, while pVYC5 did not. A recombinant clone containing PVYN cDNA sequences (pVYN27) was characterised by DNA sequencing. The 3'-terminal 1134 nucleotide sequence coded for the coat protein gene and contained one large open reading frame capable of encoding a protein of 264 amino acid residues with a combined molecular weight of 29 631. The ten amino-terminal amino acids of the protein were confirmed by amino acid sequencing. Transcriptional fusions encoding the PVYN coat protein gene and either the CaMV 35S promoter or the mannopine synthase promoter were inserted into an Agrobacterium binary vector encoding the NPT II gene. These were mobilised into two species of Agrobacterium (A. tumefaciens, LBA4404 and A. rhizogenes, A4T) and transformed into the genomes of Nicotiana plumbaginifolia and Solanum tuberosum. The transgenicity of regenerated tobacco plants was confirmed by 1. the presence of the chimaeric PVYN coat protein gene as demonstrated by DNA hybridisation, 2. expression of the NPT II gene was demonstrated by the regeneration of plants on media containing kanamycin at normally inhibitory concentrations, and 3. demonstration that progeny from transformed plants inherited the NPT II gene in a Mendelian manner. No accumulation of the coat protein by transformants was detected by either Western blots or protein slot blots