ABSTRACT
The PVX coat protein (CP) is involved in many aspects of plant-virus interaction (virion morphology, plant symptoms, viral pathogenesis and virulence, and genomic RNA accumulation). Different virus strains have been distinguished according to their compatibility with the host resistance genes Nx, Nb, and Rx. Substitution of the Thr 122 on the CP with a Lys in PVX strain HB has been shown to affect the response of potato cultivars with the Rx resistance gene. In PVX DX the avirulence determinant for the Nx gene has been localized in the Gln 78 of the coat. PVX strain MS, like PVX HB, is able to overcome the Rx, Nx, and Nb genes. Sequencing of the CP gene of PVX MS (EMBL accession number Z34261) shows that it has a Thr in codon 122 and a Gln in codon 78. These results suggest that, in addition to the coat protein gene, other regions of the viral genome are involved in the pathogenicity.
Subject(s)
Capsid Proteins , Capsid/genetics , Potexvirus/genetics , Amino Acid Sequence , Argentina , Base Sequence , Molecular Sequence Data , Potexvirus/isolation & purification , Potexvirus/pathogenicity , Sequence Homology, Amino Acid , Solanum tuberosum/virologyABSTRACT
The coat protein (CP) genes of all the strains of potato virus X (PVX) code for a protein of 25 kDa. Analysis of the CP by SDS-PAGE shows a migration mobility of 27 to 29 kDa, depending of the strain. Amino acid identity between some strains is too high to explain such abnormal migration by differences in primary structure of the protein. Periodate oxidation demonstrated the presence of carbohydrate moieties in purified CP of six PVX strains (cp, CP4, HB, MS, DX, CS35), and trifluoromethanesulfonic acid treatment identified glycosylated and nonglycosylated bands in SDS-PAGE complex patterns of CP and in Western blots. Digestion with glycosidases, recognition by lectins, and mild alkali treatment (beta-elimination) of PVX CP indicated the presence of an O-linked sugar.
Subject(s)
Capsid/chemistry , Glycoproteins/chemistry , Potexvirus/chemistry , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/pharmacology , Hydrogen-Ion Concentration , Lectins/metabolism , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.
