Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

Abstract

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. 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 cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed.

Avsunviroidae family: Viroids containing hammerhead ribozymes

“…In infected tissue, ASBVd (+) strands are more concentrated than (-) strands, and for monomers and dimers the circular forms are more abundant than the linears (Hutchins et al, 1985;Darbs et al, 1994). However, in PLMVdinfected peach both (+) and (-) strands accumulate in vivo at comparable concentrations, but the linear is more abundant than the circular (Bussi~re et al, 1999). An intermediate situation is observed in CCh-MVd: (+) strands accumulate in vivo at higher levels than (-) strands, as in ASBVd; but the linear monomers are more abundant than the corresponding circular forms, as in PLMVd (Navarro and Flores, 1997).…”

“…Therefore, they seem to be composed by the monomeric circular (+) and (-) templates bound to the nascent ASBVd RNAs of the complementary polarity. Detection of the PLMVd monomeric (-) circular RNA in infected tissue does not present technical problems because an important imbalance between (+) and (-) strands does not exist (Bussi~re et al, 1999). This is not the situation with CChMVd in which results indicating the in vivo existence of the viroid monomeric (-) circular forms (Navarro and Flores, 1997) need further confirmation.…”

“…More recent results show that PLMVd accumulates predominantly in the chloroplast (Bussi~re et al, 1999). Finding a viroid in a subcellular compartment does not necessarily imply that replication takes place there, but data on the localization of viroid complementary strands support that PSTVd replicates in the nucleus (Spiesmacher et al, 1983), and ASBVd (Bonfiglioli et al, 1994;Navarro et al, 1999) and PLMVd (Bussi~re et al, 1999) in the chloroplast. Therefore, although still incomplete, the emerging evidence strongly suggests that the replication and accumulation site is another demarcation criterion for members of both viroid families.…”

“…Circumstantial evidence supporting a chloroplastic localization for PLMVd has been obtained because this viroid, unlike PSTVd, is not imported into the nucleus of permeabilized tobacco protoplasts (Woo et al, 1999). More recent results show that PLMVd accumulates predominantly in the chloroplast (Bussi~re et al, 1999). Finding a viroid in a subcellular compartment does not necessarily imply that replication takes place there, but data on the localization of viroid complementary strands support that PSTVd replicates in the nucleus (Spiesmacher et al, 1983), and ASBVd (Bonfiglioli et al, 1994;Navarro et al, 1999) and PLMVd (Bussi~re et al, 1999) in the chloroplast.…”

“…RNA ligase NEP ~ RNA ligase Northern blot analysis of nucleic acids preparations from ASBVdinfected avocado using probes of both polarities showed the existence of a ladder of viroid-specific RNAs of multimeric size, ranging from the monomer to the octamer for the (+) polarity, but only to the monomer and the dimer for the (-) polarity (Hutchins et al, 1985). In PLMVdinfected peach, monomers and dimers of both polarities have been also detected (Bussi~re et al, 1999), but in CChMVd-infected chrysanthemum only monomers have been observed (Navarro and Flores, 1997). The oligomeric RNAs most probably accumulate in vivo owing to their inefficient self-cleavage.…”