Cytological evidence for the production of plant virus ribonucleic acid in the nucleus

Abstract

Living cells in hand sections of tobacco plants, recently infected with tobacco mosaic virus (TMV) and tomato spotted wilt virus (TSW), were examined in the living state or fixed and stained to reveal ribonucleic acid (RNA). Ribonuclease was used in conjunction with staining to characterize the RNA. Two forms of TMV and defective mutants of one, the tobacco form, and mild strains of TSW that caused mainly chlorotic symptoms were used. Cycles of change in the nucleolus of infected cells were followed in phase microscopy by the apparent emission of material optically less dense than the nucleoplasm. Images of stained sections indicated that this material was RNA. Emission varied according to the virus isolate examined. The strain TMV-U5 differed from the common tobacco strain, TMV-U1. In recent U5 infections, RNA-staining material was found only in the nucleus, and aggregated whole virus was found outside in the cytoplasm. It was concluded that U5 virus was finally synthesized in the nucleus or near its perimeter. TMV-U1 RNA seemed to originate in the nucleolus and pass into the cytoplasm, where it was found in material from which the X-bodies originated. As these masses circulated in the cytoplasm they acquired in part the staining reactions of whole virus, and small aggregates of virus particles began to appear. Uncombined RNA-containing material came together in X-bodies. The defective mutants of TMV had two kinds of material in the cell, one protein-like that appeared in linear or gel-like aggregates, and the other staining like RNA. The RNA again appeared to arise from the nucleolus and pass into the cytoplasm, sometimes mixing with the virus protein gel. It could be removed by RNase without affecting the underlying structure. TSW also gave images suggesting nucleolar emission of RNA, but all virus material in the cell was RNase sensitive. If whole virus covered by a protein coat was formed, the protein did not protect it from digestion by the enzyme.