Characterization of nuclear import of potato spindle tuber viroid RNA in permeabilized protoplasts

Most viroids replicate in the nuclei of infected plant cells. Nuclear impor t of the incoming RNA thus represents a key control point for establishment of a systemic infection. However, little is known about the mechanisms by which viroids are transported into the nucleus. We have characterized nucle ar import of infectious, fluorescein-labeled potato spindle tuber viroid (F -PSTVd) in permeabilized tobacco BY2 cells. Import was observed for F-PSTVd but not for mRNA fragments of the same size or two viroids believed to rep licate in the chloroplasts. Import of F-PSTVd was inhibited by addition of a 10-fold excess of nonfluorescent PSTVd but not by similar amounts of cont rol RNAs. Import was not inhibited by pre-incubation with GTP-gamma S or GD P-beta-S, however. Disruption of microtubules and actin filaments with oryz alin or cytochalasin D did not inhibit F-PSTVd import. Taken together, our results indicate that (i) PSTVd possesses a sequence and/or structural moti f for nuclear import and (ii) the import is a cytoskeleton-independent proc ess that is mediated by a specific and saturable receptor. Insensitivity to GTP-gamma-S and GDP-beta-S treatment suggests that PSTVd import is not cou pled to Ran GTPase cycle, which mediates nuclear transport of many proteins and nucleic acids. To our knowledge, our studies are the first to examine the mechanisms of nuclear transport of RNA in plants.