Figure 9.

IOS1 Associates with CERK1.

(A) Coimmunoprecipitation of IOS1 with CERK1 proteins in Arabidopsis protoplasts. Arabidopsis protoplasts expressing CERK1-GFP and CERK1-HA₃ (lanes 1 and 2), IOS1-GFP and CERK1-HA₃ (lanes 3 and 4), EV-GFP and CERK1-HA₃ (lane 5), or LTI6b-GFP and CERK1-HA₃ (lane 6) constructs were treated with (+) or without (−) 0.2 mg/mL chitin for 10 min. Total proteins (input) were subjected to immunoprecipitation (IP) with GFP trap beads followed by immunoblot analysis with anti-HA antibodies to detect CERK1-HA₃. EV-GFP and LTI6b-GFP, a known plasma membrane protein, were used as controls to illustrate that CERK1-HA₃ does not stick to GFP beads or associate with GFP at the plasma membrane, respectively. This experiment was repeated twice with similar results.

(B) BiFC analyses of IOS1 interactions with CERK1. Arabidopsis protoplasts were cotransfected with CERK1-YFP^N + CERK1-YFP^C and CERK1-YFP^N + IOS1-YFP^C, and treated with (+) or without (−) 0.2 mg/mL chitin 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. At least two independent experiments were performed with similar results. Bars = 10 μm.