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Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment

Fig 2

Co-localization of p33 replication protein with Ufe1 and Use1 SNARE proteins.

(A) Confocal laser microscopy analysis of subcellular distribution of GFP-tagged p33 and RFP-Ufe1 in wt yeast cells. Images were taken at two time points (as shown). Single frames are presented. (B) Top image: interaction between TBSV nYFP-p33 replication protein and the Ufe1-cYFP protein was detected by BiFC. Co-localization of Pho86-RFP (an ER membrane marker protein) with the BiFC signal (see merged image) demonstrates that the interaction between p33 replication protein and Ufe1 occurs in subdomains of the ER membrane. Control BiFC experiments included GST-cYFP protein in combination with nYFP-p33 replication protein (middle image) and nYFP-GST protein in combination with Ufe1-cYFP (bottom image). (C) Top image: Confocal microscopic images show co-localization of GFP-p33 with both RFP-Ufe1 and BFP-SKL (peroxisomal luminar marker) in the replication compartment. Bottom image shows both proximal localization and co-localization of GFP-p33 and RFP-Ufe1 with BFP-SKL (pointed at by arrows). (D) Confocal microscopic images show co-localization of TBSV p33-BFP replication protein and the YFP-AtSyp81 syntaxin, the ortholog of the yeast Ufe1 protein, in planta. Expression of the above proteins from the 35S promoter was done after co-agro-infiltration into N. benthamiana leaves. Scale bars represent 2 μm. (E) Confocal microscopic images show co-localization of TBSV p33-GFP replication protein and the RFP-AtSyp81 syntaxin in N. benthamiana protoplasts. (F) Confocal microscopic images show co-localization of TBSV p33-BFP replication protein and the YFP-AtUse1 SNARE protein, the ortholog of the yeast Use1 protein, in planta. Expression of the above proteins from the 35S promoter was done after co-agro-infiltration into N. benthamiana leaves. Scale bars represent 2 μm. (G) Confocal microscopic images show co-localization of TBSV p33-BFP replication protein with AtSyp81 and AtUse1 within the large replication compartment (top images) and smaller replication compartments (depicted with arrows, bottom images) in N. benthamiana cells. Note that the interaction between of nYFP-AtUse1 and the AtSyp81-cYFP proteins was detected by BiFC. Scale bars represent 2 μm.

Fig 2

doi: https://doi.org/10.1371/journal.ppat.1007028.g002