Subcellular localization and rolling circle replication of peach latent mosaic viroid: Hallmarks of group A viroids

We characterized the peach latent mosaic viroid (PLMVd) replication interme diates that accumulate in infected peach leaves and determined the tissue a nd subcellular localization of the RNA species. Using in situ hybridization , we showed that PLMVd strands of both plus and minus polarities concentrat e in the cells forming the palisade parenchyma. At the cellular level, PLMV d was found to accumulate predominantly in chloroplasts. Northern blot anal yses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polariti es. No multimeric conformer was detected, indicating that both strands self -cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultiv ars. Taken together these results establish two hallmarks for the classific ation of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead s tructures, replicate in the chloroplasts via the symmetric mode. By contras t, group B viroids, which share a conserved central region, replicate in th e nucleus via an asymmetric mechanism. This is an important difference betw een self-cleaving and non-self-cleaving viroids, and the implications for t he evolutionary origin and replication are discussed.