Subcellular localization of NtRPT6 and XopJ – NtRPT6 interaction in planta.

(A) Subcellular localization of NtRPT6-GFP and XopJ-mCherry fusions in N. benthamiana leaves transiently transformed by Agro-infiltration. The green fluorescence (GFP), red fluorescence (mCherry) and chlorophyll autofluorescence (Chl) were monitored separately to prevent cross-talk of the fluorescence channels and the resulting fluorescence images were merged. Bars = 20 µm. Pictures show a representative result of at least three repetitions. (B) Visualization of protein interactions in planta by the BiFC assay. YFP confocal microscopy images show tobacco leaf epidermal cells transiently expressing constructs encoding the fusion proteins indicated. Merge indicates an overlay of the YFP and chlorophyll autofluorescence images. A close up of the same cells shows that the YFP fluorescence aligns with the PM. Bars = 20 µm. The experiment has been repeated three times with similar results. (C) Co-immunoprecipitation of NtRPT6-GFP with XopJ-myc and XopJ(C235A)-myc. NtRP6-GFP was transiently co-expressed in leaves of N. benthamiana using Agro-infiltration with either XopJ-myc or XopJ(C235A)-myc. After 48 h, total proteins (Input) were subjected to immunoprecipitation (Eluate) with GFP-Trap beads followed by immunoblot analysis using either anti-GFP or anti-myc antibodies. At least three repetitions with similar result have been conducted. (D) In vitro pull-down assay showing physical interaction of XopJ with RPT6. MBP-XopJ and GST-Rpt6 were expressed in E. coli. Pull down was performed using amylose resin. Proteins were detected in an immunoblot using antibodies as indicated.