Purification, in situ Localization, and Comparative Serological Properties of Passionfruit Woodiness Virus-Encoded Amorphous Inclusion Protein and Two Other Virus Proteins

Abstract

Passionfruit woodiness potyvirus (PWV) is one of the major limiting factors for growing passionfruit in Taiwan. To characterize this virus at the molecular level, a viral protein with M r 51,000 (51 K) was purified by a procedure developed in this study. Extracts from leaf tissues of PWV-infected passionfruit (Passiflora edulis f. flavicarpa) or tobacco (Nicotiana benthamiana) were treated with 5% Triton X-100 and centrifuged at 220 g for 10 min. The resulting pellets were further processed with two cycles of 5% Triton X-100 treatment and low-speed centrifugation (900 g for 10 min). The 51K protein was then purified from the pellets by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and antisera were produced in New Zealand white rabbits. Light microscopic immunostaining with the IgG to the 51K protein and protein A-gold complex demonstrated that the cytoplasmic amorphous inclusions were the major location of the 51K protein. Immunogold labeling with the IgG to the 51K protein in electron microscopy also revealed that gold particles were mainly distributed on the cytoplasmic amorphous inclusions and not on organelles such as nuclei and chloroplasts. The localization of the 51K antigen was distinct from that of the cylindrical inclusion protein (CIP, 66K) and the coat protein (CP, 36K), which were mainly concentrated on cytoplasmic cylindrical inclusions and virions, respectively. These results verified that the 51K protein is the amorphous inclusion protein (AIP) of PWV. Serological analyses by immunodiffusion, indirect enzyme-linked immunosorbent assay (ELISA), and immunoblotting of different viral antigens produced in vivo indicated that the purified AIP is serologically distinct from CP and CIP of PWV. Among the 14 potyviruses tested by SDS-immunodiffusion, antiserum to PWV CP reacted with extracts from plant tissues infected with New York and Florida isolates of watermelon mosaic virus 2 (WMV-2), and black eye cowpea mosaic virus (Florida), showing spur with the homologous antigen. The antiserum to PWV CIP, on the other hand, only spur-reacted with sap from plant tissue infected with Florida isolate of MWV-2 and did not react with any of the other heterologous antigens tested