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.

FIG. 1

Schematic representation of the mechanism of viroid rolling circle replication by either the symmetric (A) or the asymmetric (B) mode. The polarity of the strands is indicated in parentheses, and the small circle on the strands denotes the cleavage site. The process begins with the infecting circular (+) strand.

FIG. 2

Detection of PLMVd by in situ hybridizations with DIG-labeled riboprobes. (A to E) Observations of the DIG-labeled hybrids by light microscopy. The micrographs show sections of PLMVd-infected leaves hybridized in either the absence (A) or the presence (B) of DIG-PSTVd riboprobe, a section of a healthy peach leaf probed with a minus-polarity PLMVd riboprobe (C), and sections of PLMVd-infected peach leaves probed with either the plus (D) or minus (E) PLMVd riboprobes. Control panels (A to C) were overstained to ensure the detection of trace amounts of PLMVd strands. (F) Typical electron micrograph of the hybridization of PLMVd-infected peach leaves with the minus-strand PLMVd riboprobe. The arrows point to clusters of grains representing PLMVd accumulated strands.

FIG. 3

Autoradiograms of Northern blot hybridizations of RNA samples isolated from both healthy and PLMVd-infected peach leaves. RNA samples were fractionated on either agarose (A to C) or polyacrylamide (D and E) gels and then blotted onto nylon filters. The polarity of the PLMVd riboprobe is indicated by the symbol (+) and (−) at the top of the panel. (A) RNA samples were isolated by various extraction procedures: RNeasy Plant mini kit (Qiagen) in the presence of EDTA (lanes 3 to 5); Tris-EDTA isolation (lanes 6 and 7); RNeasy Plant mini kit in the absence of additional EDTA (lane 8); and the PEG precipitation procedure (lanes 9 to 12). In lanes 1 and 2, nonradioactive synthetic PLMVd transcripts of plus (761 and 588 nt) and minus (745 and 462 nt) polarity, respectively, were loaded as controls. Lane 3 contains a sample of healthy GF-305 peach cultivar; lanes 4, 6, 8, 9, 11, and 12 contain RNA samples isolated from a PLMVd-infected RedGold peach cultivar. The samples in lanes 11 and 12 were subjected to either DNase treatment or alkaline hydrolysis prior electrophoresis. Lanes 5 and 7 contain to samples from a PLMVd-infected Redhaven cultivar, while lane 10 contains a sample from the leaves of the Agua cultivar. (B and C) The same filter following hybridization with either the plus- or minus-polarity riboprobe. All samples were isolated by the PEG precipitation procedure. Lanes 1 and 2 contain samples isolated from different leaves of a Redhaven peach. Lane 3 contains an RNA sample from a healthy Bailey cultivar. Lanes 4 and 5 contain RNA samples of PLMVd-infected Redhaven and Siberian C cultivars. (D and E) Filter following PAGE and blotting that was probed with both the plus- and minus-polarity riboprobes, respectively. In panel D, lanes 1 and 2 contain synthetic circular and linear PLMVd controls, while lanes 3 to 5 (which correspond to lanes 1 to 3 of panel E) contain samples from PLMVd-infected Redgold, Hardired, and Siberian C cultivars and lanes 6 and 7 (which correspond to lanes 4 and 5 of panel E) contain samples from healthy GF-305 and Bailey cultivars. Adjacent to the gel, the positions of several PLMVd transcripts are indicated as size references including both the circular (338C) and linear (338L) strands and the mixture of circular and linear molecules (PLMVd). The position of the unknown ∼1-kb species is also indicated in panels A and B.

FIG. 4

Schematic phylogenetic reconstruction of viroids and viroid-like satellite RNAs based on physical characteristics. The dotted lines for the satellite RNAs indicate their precise relationship to viroid group A and B.