Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues.

A combination of native electrophoresis and immunodetection (Western blot) was used for the characterization of nucleoprotein particles of the potyvirus Plum pox virus (PPV). Virus particles were electrophoresed directly from plant extracts in agarose or mixed acrylamide-agarose gels under native conditions, blotted on nitrocellulose membranes, and characterized with the aid of a coat protein-specific antibody. Using this combined methodology, called NEWeB (native electrophoresis and Western blotting), we could show that a population of particles that differ in their electrophoretic mobility can be detected in extracts of Nicotiana benthamiana, that two different strains of PPV can be distinguished in double infections of the same plant and that virus particles from leaves contain detectable levels of helper component proteinase molecules. The potential of the NEWeB method for the study of structure and function of virus particles and similar nucleoprotein complexes in single and mixed infections is discussed.

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.