Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell-free system from infected avocado leaves.

Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell-free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200 micrograms/ml of the drug, which typically inhibit RNA polymerase II and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polycrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis.