Complexes containing both polarity strands of avocado sunblotch viroid: Identification in chloroplasts and characterization

RNA analysis by nondenaturing polyacrylamide gel electrophoresis and Northe rn blot hybridization of avocado chloroplasts purified from protoplasts of leaves infected by avocado sunblotch viroid (ASBVd) revealed the main ASBVd -specific bands found previously in preparations of total leaf RNA: the mon omeric, dimeric and subgenomic RNAs, and two bands, x and y. After RNase tr eatment in high ionic strength, bands x and y remained resistant, indicatin g a high content of double-stranded RNAs, whereas the other viroid-specific and cellular RNAs were degraded. Analysis by denaturing polyacrylamide gel electrophoresis and Northern blot hybridization showed that the major cons tituents of the purified y and x bands were the monomeric circular and line ar ASBVd forms of both polarities, but band y contained additionally multim eric ASBVd RNAs, also of both polarities, that probably cause its slower mi gration in nondenaturing gels. After RNase treatment, the composition of ba nd y was essentially unaffected, but only the monomeric linear ASBVd RNAs o f both polarities was recovered from band x. However, in the presence of hi gher RNase concentrations, band y was converted into band x, indicating tha t they are closely related. The structure of complexes x and y, containing minus ASBVd strands and particularly the monomeric circular form, supports a role of replicative intermediates in the symmetric rolling circle mechani sm proposed for ASBVd, whereas their localization in the chloroplast is str ong evidence in favor of this organelle as the replication site of ASBVd. ( C) 1999 Academic Press.