1a.Objectives (from AD-416)
1. Sequence whole genomes for at least 3 PVY-N:O strains that do not cause tobacco vein necrosis, and analyze their sequences against at least 3 other PVY-N:O strains that do.
2. Sequence whole genomes for at least 3 PVY isolates that cause tuber necrosis in potato and analyze their sequences against at least 3 other PVY strains that do not.
3. Express PVYO and O5 capsid proteins in bacteria and correlate the monoclonal 1F5 reactivity with the point mutation in the PVY-O capsid protein.
1b.Approach (from AD-416)
Potato virus Y isolates collected by State Potato Seed Certification agencies and partially characterized by either State laboratories or the ARS laboratory at Ithaca, NY will be sent to the University of Idaho. Virus genomes will be amplified using a RT-PCR protocol and the products sequenced. Sequence information will be catalogued in a bioinformatics database that will be made available to all collaborators on the project. Virus protein fragments will be expressed in bacteria and used to characterize epitopes that recognize monoclonal antibodies.
A novel Potato virus Y (PVY) isolate, L26, recovered from a Frontier potato line was initially typed as a tuber necrotic isolate using genome and serological assays. However, L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of other tuber necrotic isolates. The whole genome sequence was determined for L26 and two other tuber necrotic isolates, HR1 and N4. The sequences of all three isolates were nearly identical, differing by only 5 nucleotides, and they were all similar to typical European tuber necrotic isolates. Only one of the five nucleotide changes resulted in a corresponding amino acid change, located in the central region of the HC-Pro protein. Two “signature” amino acid residues, previously reported to be involved in induction of the vein necrosis syndrome in tobacco, were present in L26 as well as HR1 and N4. Multiple alignments of the whole genome sequences of L26 and other tuber necrotic isolates whose phenotype in tobacco has been reported, suggests that the single amino acid change in the HC-Pro protein of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Secondary structure modeling of the HC-Pro protein predicts the altered amino acid in L26 as well as the two “signature” amino acids would all be located on the exposed surface of the protein. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and can include multiple amino acids in multiple regions of the HC-Pro protein. Progress was monitored through email and phone conversations.