Kazusato Ohshima

Characterization and Strain Identification of a Potato Virus Y Isolate Non-Reactive with Monoclonal Antibodies Specific to the Ordinary and Necrotic Strains

An isolate of potato virus Y, named PVY-36, reacted with polyclonal antibody against PVY-O (an ordinary strain), but not with any of eight monoclonal antibodies (MAbs) specific to PVY-O or with two MAbs specific to PVY-T (a necrotic strain). From its host range and symptomatology, PVY-36 belongs to the PVYO group. The nucleotide sequence of the coat protein (CP) coding region of the PVY-36 genome was determined and the amino acid sequence was predicted. Based on the CP amino acid sequence, PVY-36 is more closely related to PVY-O than to PVY-T. There were eight amino acid differences between the CPs of PVY-36 and PVY-O in the N-terminal 30 amino acids. It is suggested that amino acids 8-15 and/or 26-30 from the N-terminus may determine a PVY-O-specific epitope (or epitopes).

Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y inEscherichia coli

Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T: PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3′-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5′ end and a stop codon at the 3′ end were constructed by PCR amplication and subcloned into anE. coli expression vector, pKK223-3. Five transformedE. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in theE. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP inE. coli was apapproximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producting biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H2N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.

Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene

SummaryComplete nucleotide sequences of cytoplasmic inclusion (CI) genes of two strains of potato virus Y (PVY) were determined from six polymerase chain reaction (PCR)-amplified cDNA clones. The size of the CI genes of both ordinary (PVY-O) and necrotic strains (PVY-T13) was 1902 nucleotides, with a sequence homology of 83.4%. Comparison of the predicted amino acid sequences showed more than 90% homology. When these were compared with those of other potyviruses, the homology ranged from 53 to 61%. cDNAs of all or a part of the PVY-O CI gene containing an additional initiation codon (ATG) at the 5′ end and a stop codon at the 3′ end were constructed by PCR amplification and cloned into anEscherichia coli expression vector, pKK 223-3. Complete and truncated PVY-O CI proteins were successfully produced inE. coli as judged by reactivities with PVY-O CI protein-specific antiserum. To our knowledge, this is the first report on expression of PVY CI proteins inE. coli.