Time-Resolved Observation of the Destination of Microinjected Potato Spindle Tuber Viroid (PSTVd) in the Abaxial Leaf Epidermal Cells of Nicotiana benthamiana.

Viroids are single-stranded noncoding RNA molecules of 250-400 nucleotides that cause plant diseases. One of the two families of viroids is Pospiviroidae, the members of which replicate in the nuclei of host cells. To replicate in plants, viroids of Pospiviroidae must enter the nucleus. However, the nuclear import of viroids remains understudied. In this work, we documented the time-dependent characteristics of the changes in microinjected fluorescently labeled potato spindle tuber viroid (PSTVd). The cytoplasmic fluorescence disappeared gradually, with only nuclear fluorescence remaining as the PSTVd injected in the cytoplasm was imported into the nucleus. Through this work, we determined that the time for half-maximal nuclear accumulation of the viroid was about 23 min. Interestingly, we found some cells where the nuclear import did not occur, despite the high level of cytosolic viroid injected. In some cells, the injected viroids disappeared within 10-20 min. The nuclear import of PSTVd is not a simple concentration-dependent process but was probably under the regulation of diverse factors that may be missing from some cells used for our observation.