Extended Data Fig. 10: Monoubiquitination mediates release of BIK1 from the plasma membrane upon ligand detection.
From: Ligand-induced monoubiquitination of BIK1 regulates plant immunity
a, PYR-41 impairs flg22-induced dissociation of BIK1 from FLS2. FLS2–HA was co-expressed with BIK1–FLAG or control in protoplasts. After pretreatment with 50 μM PYR-41 for 30 min, protoplasts were stimulated with 100 nM flg22 for 15 min. Co-IP and immunoblotting were performed as in Fig. 4g. b, A working model of RHA3A/B-mediated BIK1 monoubiquitination in plant immunity. Under non-activated, steady-state conditions (0 min), BIK1 remains hypo-phosphorylated and associates with FLS2 and BAK1. Upon flg22 detection, FLS2 dimerizes with BAK1, which stimulates BIK1 phosphorylation (<1 min). Phosphorylated BIK1 is monoubiquitinated by the E3 ligases RHA3A and RHA3B, leading to dissociation of BIK1 from the FLS2–BAK1 complex, accompanied by endocytosis (10–20 min). Ligand-induced monoubiquitination of BIK1 contributes to the activation of ROS and other defence responses. FLS2 is polyubiquitinated and endocytosed 40 min after detection of flg22 to attenuate signalling. c, BIK1(9KR) shows comparable protein expression to BIK1 in transgenic plants. 35S::BIK1-HA or 35S::BIK19KR-HA transgenic plants in wild-type background were used for immunoblotting to detect BIK1 proteins with anti-HA antibody. Control, empty vector. d, Stability of BIK1 and BIK1(9KR) proteins after treatment with cycloheximide (CHX). BIK1–HA or BIK1(9KR)–HA was expressed in wild-type protoplasts for 12 h followed by treatment with 500 μg/ml CHX for the indicated time. BIK1 or BIK1(9KR) proteins were analysed by immunoblotting with anti-HA antibody. Asterisk indicates that CHX was added immediately after transfection, thus blocking protein synthesis. Experiments were repeated three times with similar results.
