Differential Subnuclear Localization of the (+)- and (−)-Strands of PSTVd in Infected N. benthamiana Cells.
The (+)- and (−)-strands of PSTVd were detected by Alexa Fluor 488–labeled riboprobes (green fluorescence). The U3 and U14 snoRNAs were detected by biotin-labeled antisense riboprobes that were further detected by streptavidin conjugated to Alexa Flour 594 (red fluorescence). The differential interference contrast (DIC) images ([A], [F], and [K]) show the nucleoli (arrows) in the cells. DAPI stains chromatin in the nucleoplasm (blue fluorescence in [B], [G], and [L]). Unstained nucleoli appear as dark spots in the nuclei. Bars = 2 μm.
(A) to (E) Mock-inoculated cell. No PSTVd hybridization signals were detected.
(F) to (J) Nucleolar as well as nucleoplasmic localization of the (+)-strand of PSTVd. The yellowish ring in the merged image ([J]) indicates overlap between (+)-PSTVd and U3 in the nucleolus.
(K) to (O) Nucleoplasmic localization of the (−)-strand of PSTVd. The merged image ([O]) shows no overlap between (−)-PSTVd and U3 snoRNA localization signals (see text for details).
(P) Serial confocal images showing localization of the (+)-PSTVd RNA in the nucleolus as well as in the nucleoplasm. The viroid signal (green) overlaps with U3 snoRNA signal (red) to produce a yellowish ring within the nucleolus.
(Q) Serial confocal images showing localization of the (−)-PSTVd RNA in the nucleoplasm. Note that there is no overlap between (−)-PSTVd (green) and U3 snoRNA (red) localizations throughout the serial sections.
(R) Serial confocal images showing localization of the (+)-PSTVd RNA in the nucleolus as well as in the nucleoplasm. The viroid signal (green) overlaps with U14 snoRNA signal (red) to produce a yellowish ring within the nucleolus.
(S) Serial confocal images showing localization of the (−)-PSTVd RNA in the nucleoplasm. Note that there is no overlap between (−)-PSTVd (green) and U14 snoRNA (red) localizations throughout the serial sections.

Confocal Images Showing the Spatial Localization Patterns of the (+)-Strand of PSTVd That Are Suggestive of Various Stages of Nucleolus-Bound Trafficking of the RNA from the Nucleoplasm.
Serial images of the same cells are presented in the supplemental data online. At stage 1 (S1), the viroid RNA is localized only in the nucleoplasm, as indicated by the nonoverlapping of PSTVd and U3 signals. At S2, there is concentration of the viroid RNA as a nucleoplasmic ring around the nucleolus. At S3, a ring of concentrated RNA appears within the nucleolus. At S4, the RNA is fully within the nucleolus. The diagrams at bottom summarize these patterns. The percentages of cells showing each RNA localization pattern are shown in parentheses. Bars = 2 μm.

Model of PSTVd Replication Featuring Intranuclear Trafficking of the (+)-Strand PSTVd.
The circular monomeric (+)-PSTVd traffics from the cytoplasm into the nucleoplasm to be used as the initial template to synthesize linear, multimeric (−)-PSTVd by DNA-dependent RNA polymerase II (Pol II). The latter then serves as the template to synthesize linear, multimeric (+)-PSTVd by the same enzyme. Two alternative pathways for the processing and trafficking of the (+)-PSTVd are illustrated in the model. In path A, the linear, multimeric (+)-PSTVd traffics into the nucleolus, where it is cleaved into monomers and then circularized. In path B, the linear, multimeric (+)-PSTVd is cleaved into monomers and circularized in the nucleoplasm. Some circular monomers then traffic into the nucleolus. In either pathway, some circular monomers traffic into the cytoplasm through the nuclear pore complex and further into neighboring cells through the plasmodesmata (not shown).

Differential Subnuclear Localization of the (+)- and (−)-Strands of PSTVd in Infected Tomato Tissues.
(A) to (D) Mock-inoculated cell. No PSTVd hybridization signals were detected.
(E) to (H) Nucleolar as well as nucleoplasmic localization of the (+)-strand of PSTVd. The yellowish ring in the merged image ([H]) indicates overlap between (+)-PSTVd and U3 in the nucleolus.
(I) to (L) Nucleoplasmic localization of the (−)-strand of PSTVd. The merged image ([L]) shows no overlap between (−)-PSTVd and U3 snoRNA localization signals (see text for details).
(M) Serial confocal images showing localization of the (+)-PSTVd RNA in the nucleolus as well as in the nucleoplasm. The viroid signal (green) overlaps with U3 snoRNA signal (red) to produce a yellowish ring within the nucleolus.
(N) Serial confocal images showing localization of the (−)-PSTVd RNA in the nucleoplasm. Note that there is no overlap between (−)-PSTVd (green) and U3 snoRNA (red) localizations throughout the serial sections.
Bars = 4 μm.

Asymmetrical Distribution of U3 snoRNA in the Presence of the (+)-PSTVd within the Nucleolus.
(A) Symmetrical distribution of U3 in the nucleolus of a mock-inoculated plant.
(B) and (C) Two infected cells showing the asymmetrical distribution of U3 (red) and PSTVd (green) in the nucleoli. Note that in each case the two RNAs occupy separate domains within the nucleolus with little overlapping.
(D) to (F) Serial confocal images showing the asymmetrical distribution of PSTVd (green in [D]) and U3 (red in [E]). The merged images ([F]) show little overlapping of the two RNAs.
Bars = 2 μm.