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Title: CHARACTERIZATION OF NUCLEAR IMPORT OF POTATO SPINDLE TUBER VIROID RNA IN PERMEABILIZED PROTOPLASTS

Author
item WOO, YOUNG-MIN - OK STATE UNIV STILLWATER
item ITAYA, ASUKA - OK STATE UNIV STILLWATER
item Owens, Robert
item TANG, LI - OK STATE UNIV STILLWATER
item Hammond, Rosemarie
item CHOU, HUEI-CHI - HOWARD HUGHES MED INST CA
item LAI, MICHAEL - HOWARD HUGHES MED INST CA

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are the smallest pathogenic agents yet described small (246-399 nucleotides), circular RNA molecules lacking both the messenger RNA activity and protein coat of conventional viruses. Thus far, viroids are known to infect only plants where they cause a number of economically significant diseases. In this study, potato spindle tuber viroid (PSTVd) was used as a "probe" to characterize the process by which RNA molecules move from the cytoplasm into the nucleus of a plant cell. We have chosen to study this process because RNA transport to the correct cellular compartment is absolutely critical to the proper functioning of both normal and genetically engineered plant genes. To our knowledge, our studies are the first to examine the mechanisms of nuclear transport of RNA in plants. As such, they will be of greatest interest to research scientists and others working in the general area of plant molecular biology.

Technical Abstract: Most viroids replicate in the nuclei of infected plant cells. Nuclear import 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 nuclear 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 replicate 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 control RNAs. Import was not inhibited by pre-incubation with GTP- -S or GDP- -S, however. Disruption of microtubules and actin filaments with oryzalin or cytochalasin D did not inhibit F-PSTVd import. Taken together, our results indicate that (i) PSTVd possesses a sequence and/or structural motif for nuclear import and (ii) the import is a cytoskeleton- independent process that is mediated by a specific and saturable receptor. Insensitivity to GTP- -S and GDP- -S treatment suggests that PSTVd import is not couple 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.