Fig. 2: RGLG1/2 preferentially associate with the hypo-phosphorylated BIK1. | Nature Communications

Fig. 2: RGLG1/2 preferentially associate with the hypo-phosphorylated BIK1.

From: BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling

Fig. 2

a BIK1 associates with RGLG2 as assayed by split-luciferase complementation. BIK1-CLuc (CL) and RGLG2-NLuc (NL) were expressed in Nicotiana benthamiana. The N. benthamiana leaves were treated with or without 5 μM flg22 for 30 min before harvesting. PUB25-NLuc and empty NLuc vector (EV) were used as positive and negative controls, respectively. Relative luciferase activity was quantified and the values are means ± SD (n = 3 leaves from three independent biological repeats). Different letters denote significance difference (one-way ANOVA, P = 0.0003). Source data are provided as a Source Data file. b, c RGLG1/2 associate with BIK1 as assayed by co-IP. RGLG1/2-FLAG and BIK1-HA were co-expressed in Arabidopsis protoplasts. The protoplasts were treated with or without 2.5 μM flg22 for 30 min. RGLG1/2-FLAG was immunoprecipitated with anti-FLAG antibodies, the associated BIK1-HA proteins were detected by immunoblotting with anti-HA antibodies. d RGLG1 associate with BIK1 as assayed by co-IP. RGLG1-HA and BIK1-FLAG were co-expressed in Arabidopsis protoplasts. The protoplasts were treated with or without 2.5 μM flg22 for 30 min. BIK1-FLAG was immunoprecipitated with anti-FLAG antibodies. e RGLG2 is associated with BIK1 in stable transgenic plants. pRGLG2 (NP)::RGLG2-HA/p35S::BIK1-FLAG and pRGLG2(NP)::RGLG2-HA/Col-0 seedlings were treated with 5 μM flg22 for 30 min. BIK1-FLAG was immunoprecipitated with anti-FLAG antibodies. f RGLG1 associates with BIK1 at the plasma membrane. The indicated BiFC constructs were transfected into Arabidopsis protoplasts, and fluorescence was visualized by confocal microscopy. The scale bar = 10 μm. g RGLG1 directly interacts with BIK1 but not FLS2CD. The recombinant GST-BIK1 or GST-FLS2CD proteins immobilized on GSH beads were incubated with His-FLAG-RGLG1 proteins. GST pull-down was performed and the pulled-down His-FLAG-RGLG1 proteins were detected by immunoblotting with anti-FLAG antibodies. All input proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). h Flg22 treatment does not affect the association between RGLG1 and BIK1K105E. RGLG1-FLAG and BIK1-HA or BIK1K105E-HA were co-expressed in protoplasts. The protoplasts were treated with flg22 and co-IP was performed as described in (b). b–h Images shown are representative of at least two independent experiments.

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