Fig. 4: The interplay between RGLG1/2 and PUB25.

a, b PUB25 associates with RGLG1/2 as assayed by co-IP. RGLG1/2-HA and PUB25-FLAG were co-expressed in Arabidopsis protoplasts. The protoplasts were pretreated with 30 μM MG132 for 2 h, and treated with or without 2.5 μM flg22 for 30 min. PUB25-FLAG was immunoprecipitated with anti-FLAG antibodies. Images shown are representative of at least two independent experiments. c PUB25 associates with RGLG2 at the plasma membrane. The indicated BiFC constructs were transfected into Arabidopsis protoplasts, and fluorescence was visualized by confocal microscopy. Scale bars: 10 μm. d RGLG2 directly interacts with PUB25 in vitro. The recombinant GST-RGLG2/GST proteins immobilized on GSH beads were incubated with purified His-FLAG-PUB25 proteins. The pulled-down His-FLAG-PUB25 proteins were detected by immunoblotting with anti-FLAG antibodies. c, d images shown are representative of three independent experiments. e PUB25 mediates RGLG2 degradation. RGLG2-FLAG was co-expressed with or without PUB25-HA in Arabidopsis protoplasts. GFP-FLAG was used as an internal transfection control. The protoplasts were treated with 50 μM CHX, and with or without 30 μM MG132 for 3 h before harvesting. f RGLG1/2 protein levels are higher in pub25 pub26 than in Col-0 protoplasts. RGLG1/2-HA were expressed in pub25 pub26 or Col-0 protoplasts. GFP-HA was used as an internal transfection control. The protoplasts were treated with 50 μM CHX for 3 h. e, f Images shown are representative of at least two independent experiments. g RGLG2 represses the ubiquitin ligase activity of PUB25. PUB25-HA and FLAG-Ub were co-transfected with RGLG2-MYC or RGLG2m-MYC into Arabidopsis protoplasts. The protoplasts were treated with 30 μM MG132 for 2 h before harvesting. IPs were performed with anti-FLAG antibodies. PUB25 autoubiquitination was detected by immunoblotting with anti-HA antibodies. Images shown are representative of three independent experiments.