INSITU LOCALIZATION OF PLANT VIRAL GENE-PRODUCTS

Methods for the in situ localization of both viral nucleic acids and gene products were developed. Well-established systemic infections of two taxonomically distinct viruses (cauliflower mosaic virus [CaMV] and tobacco mosaic virus [TMV]) were used to establish trends in gene product localization that transcend virus group characteristics. An analysis of variance of in situ labeling is combined with a least significance difference determination to quantify results. The in situ localization studies were augmented by in vitro analyses to compare the validity, specificity, and sensitivity of the methods used. In TMV-infected tissue, structural proteins, nonstructural proteins, and ubiquitin (a key element in a protein degradative process in plants) were localized in viral inclusions. Negative sense RNA, a critical component of the replication complex, was not detected in these inclusions. On the basis of these results we conclude that TMV inclusions function in sequestration and degradation of viral products in infected cells. The in situ hybridization technique used in this study detected statistically significant amounts of negative sense RNA between 24 and 72 h after inoculation in the cytoplasm only. This coincided roughly with the only times that the 130K and 180K proteins could be detected in the tissues by in vitro (western blot) techniques. A cytoplasmic site for TMV replication is, therefore, the only conclusion indicated by our studies. The in situ labeling results are discussed against the background of our knowledge of TMV replication and in relation to the in vitro data obtained here.