Figure S3.
FIT NBs did not colocalize with Cajal body components (designated type I), and type II and III NB markers and PB markers are similarly localized in single expression as in coexpression with FIT, except PININ (supportsFigs. 5,6, and7). (A–J) Confocal images showing localization of FIT-GFP and NB markers (type I) upon coexpression in the nucleus at t = 5 min, and of NB markers (type II and III) and PB markers upon their single expression in the nucleus at t = 0 and t = 5 min/15 min, in A, coilin-mRFP, B, U2B″-mRFP, C, SR45-mRFP, D, SRm102-mRFP, E, UAP56H2-mRFP, F, P15H1-mRFP, G, PININ-mRFP, H, PIF3-mCherry, and I and J, PIF4-mCherry in two different patterns. (A and B) In the coexpression of FIT-GFP with coilin-mRFP and U2B″-mRFP, FIT-GFP NBs were present at t = 5 min and did not colocalize with NBs of the two markers. (C and D) Single SR45-mRFP and SRm102-RFP localized in NBs similar to the colocalization with FIT-GFP at t = 0 and t = 5 min (compare with Fig. 5). (E and F) Single UAP56H2-mRFP and P15H1-mRFP did not localize in NBs and were uniformly distributed, similar to the colocalization with FIT-GFP at t = 0 (compare with Fig. 6, A and B). (G) Only a single PININ-mRFP showed a different localization pattern between its single expression versus coexpression with FIT-GFP. Upon single expression, it localized in NBs at t = 0 and t = 5 min, while in coexpression with FIT-GFP, it showed no NBs at t = 0 but followed the FIT-GFP NB pattern at t = 5 min (compared with Fig. 6 C). (H and J) Single PIF3-mCherry localized to a very large PB at t = 0 and t = 15 min. Single PIF4-mCherry localized either in a uniform manner in the nucleus as seen in I or in several PBs as seen in J. Hence, PIF3-mCherry and PIF4-mCherry were similarly localized in single expression as in coexpression with FIT-GFP (compare with Fig. 7). Scale bar: 2 µm. Empty arrowheads indicate non-colocalizing NBs, filled arrowheads indicate NBs/PBs in single expression. G = GFP; R = mRFP; C = mCherry. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure. Representative images from three to five independent experiments.

FIT NBs did not colocalize with Cajal body components (designated type I), and type II and III NB markers and PB markers are similarly localized in single expression as in coexpression with FIT, except PININ (supports Figs. 5 , 6 , and 7 ). (A–J) Confocal images showing localization of FIT-GFP and NB markers (type I) upon coexpression in the nucleus at t = 5 min, and of NB markers (type II and III) and PB markers upon their single expression in the nucleus at t = 0 and t = 5 min/15 min, in A, coilin-mRFP, B, U2B″-mRFP, C, SR45-mRFP, D, SRm102-mRFP, E, UAP56H2-mRFP, F, P15H1-mRFP, G, PININ-mRFP, H, PIF3-mCherry, and I and J, PIF4-mCherry in two different patterns. (A and B) In the coexpression of FIT-GFP with coilin-mRFP and U2B″-mRFP, FIT-GFP NBs were present at t = 5 min and did not colocalize with NBs of the two markers. (C and D) Single SR45-mRFP and SRm102-RFP localized in NBs similar to the colocalization with FIT-GFP at t = 0 and t = 5 min (compare with Fig. 5). (E and F) Single UAP56H2-mRFP and P15H1-mRFP did not localize in NBs and were uniformly distributed, similar to the colocalization with FIT-GFP at t = 0 (compare with Fig. 6, A and B). (G) Only a single PININ-mRFP showed a different localization pattern between its single expression versus coexpression with FIT-GFP. Upon single expression, it localized in NBs at t = 0 and t = 5 min, while in coexpression with FIT-GFP, it showed no NBs at t = 0 but followed the FIT-GFP NB pattern at t = 5 min (compared with Fig. 6 C). (H and J) Single PIF3-mCherry localized to a very large PB at t = 0 and t = 15 min. Single PIF4-mCherry localized either in a uniform manner in the nucleus as seen in I or in several PBs as seen in J. Hence, PIF3-mCherry and PIF4-mCherry were similarly localized in single expression as in coexpression with FIT-GFP (compare with Fig. 7). Scale bar: 2 µm. Empty arrowheads indicate non-colocalizing NBs, filled arrowheads indicate NBs/PBs in single expression. G = GFP; R = mRFP; C = mCherry. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure. Representative images from three to five independent experiments.

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