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Synthesis of RNAs Specific to Citrus Exocortis Viroid by a Fraction Rich in Nuclei from Infected Gynura aurantiaca: Examination of the Nature of the Products and Solubilization of the Polymerase-Template Complex

Unidad de Biologia Molecular y Celular de Plantas, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Calle Jaime Roig 11, 46010 Valencia, Spain

Viroid-specific polymerase activity was detected in preparations rich in nuclei from Gynura aurantiaca infected with citrus exocortis viroid (CEV). The polymerase catalysed the synthesis of several RNAs, shown to be viroid-specific since they could not be observed in control experiments with healthy plants, and they contained CEV-specific sequences most of which were of the same polarity as the viroid RNA. The synthesis of the CEV-specific RNA species was greatly reduced in the presence of 1 µM--amanitin, suggesting the involvement of RNA polymerase II in this process. The structure of the viroid-specific RNA species was studied by chromatography on non-ionic cellulose, digestion with RNase under low and high ionic strength conditions, and analysis by polyacrylamide gel electrophoresis in non-denaturing and denaturing systems. The results showed that these RNAs synthesized in vitro contain unit and longer than unit length linear viroid strands forming multistranded complexes with single- and double-stranded regions. The RNAs therefore have the same structural properties as deduced for RNAs isolated from viroid-infected tissues which are the presumed replicative intermediates of the rolling circle mechanism proposed for viroid synthesis. A soluble fraction containing the polymerase-template complex responsible for the synthesis of the CEV-specific RNAs was isolated by treatment of the nuclei-rich preparation with heparin and DNase. This soluble fraction could be of interest in further studies to characterize the components of the polymerase-template complex involved in CEV replication.

Keywords: CEV, replicative intermediates, G. aurantiaca

Received 14 March 1989; accepted 13 June 1989.