Figure 4.
IOS1 Localization, Pull-Down, and BiFC Analyses of IOS1 Interaction with PRRs.
(A) Subcellular localization of IOS1-GFP fusion protein in Arabidopsis mesophyll protoplasts. IOS1-GFP expression was driven by the cauliflower mosaic virus 35S promoter and transiently expressed in Arabidopsis mesophyll protoplasts. The images of the GFP fluorescence (GFP), the chlorophyll autofluorescence (chlorophyll), the bright-field image (bright), the plasma membrane marker (pm-rk CD3-1007)-mCherry fluorescence localization, and the combined images (merged) are shown. Similar observations were made in another independent repeat. Bars = 10 µm.
(B) In vitro MBP pull-down assay of IOS1 interaction with FLS2 and EFR. E. coli expressed MBP (negative control), MBP-FLS2KD, or MBP-EFRKD were incubated with Trx-6xHis-IOS1KD and pulled down with amylose resin beads. Input and bead-bound proteins were analyzed by immunoblotting with specific antibodies. Experiments were repeated three times with similar results.
(C) BiFC analyses of IOS1 interactions with FLS2 and BAK1. Arabidopsis protoplasts were cotransfected with BAK1-YFPN + FLS2-YFPC, IOS1-YFPN + FLS2-YFPC, and IOS1-YFPN + BAK1-YFPC and treated with (+) or without (–) 100 nM flg22 for 10 min. The YFP fluorescence (yellow), chlorophyll autofluorescence (red), bright-field, and the combined images were visualized under a confocal microscope 16 h after transfection. Images are representative of multiple protoplasts. Experiments were repeated at least twice with similar results. Bars = 10 µm.
(D) BiFC of LTI6b and IOS1 interaction. Arabidopsis protoplasts were cotransfected with LTI6b-YFPN + LTI6b-YFPC or IOS1-YFPN + LTI6b-YFPC and treated with (+) or without (−) 100 nM flg22 for 10 min. The YFP fluorescence (yellow), chlorophyll autofluorescence (red), bright-field, and the combined images were visualized under a confocal microscope 16 h after transfection. Images are representative of multiple protoplasts. Experiments were repeated twice with similar results. Bars = 10 µm.