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

Authors

  • Young-Min Woo,

    1. Department of Botany, Oklahoma State University, Stillwater, OK 74078, USA,
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  • Asuka Itaya,

    1. Department of Botany, Oklahoma State University, Stillwater, OK 74078, USA,
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  • Robert A. Owens,

    1. Molecular Plant Pathology Laboratory, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD 20705, USA, and
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  • Li Tang,

    1. Department of Botany, Oklahoma State University, Stillwater, OK 74078, USA,
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  • Rosemarie W. Hammond,

    1. Molecular Plant Pathology Laboratory, Agricultural Research Services, United States Department of Agriculture, Beltsville, MD 20705, USA, and
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  • Huei-Chi Chou,

    1. Howard Hughes Medical Institute and Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA
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  • Michael M. C. Lai,

    1. Howard Hughes Medical Institute and Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA
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  • Biao Ding

    1. Department of Botany, Oklahoma State University, Stillwater, OK 74078, USA,
    2. Howard Hughes Medical Institute and Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA
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*For correspondence (fax +405 744 7074;
e-mail bxding@osuunx.ucc.okstate.edu ).

Summary

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 non-fluorescent PSTVd but not by similar amounts of control RNAs. Import was not inhibited by pre-incubation with GTP-γ-S or GDP-β-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-γ-S and GDP-β-S treatment suggests that PSTVd import is not coupled 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.

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