Fig. 5: The E3 ligase MREL57 interacts with and ubiquitinates WDL7.

a Schematic representation of MREL57 truncated proteins. b WDL7 interacts with MREL57 and MREL57 N in a yeast two-hybrid assay. c A semi-in vivo pull-down assay showed an interaction between MREL57 and WDL7. MBP-WDL7-FLAG and MBP were marked with asterisks, respectively. The experiment repeated three times with similar results. d Split-luciferase complementation assay to analyze the interaction between MREL57 and WDL7 in N. benthamiana leaves. MG132 at 50 μM was injected into the leaf tissues 12 h before imaging. The pseudo-color bar shows the range of luminescence. Scale bar = 1 cm. e ABA enhances the interaction between MREL57 and WDL7. MYC-MREL57^C486A-nluc and cluc-WDL7-FLAG were transformed into N. benthamiana leaves. After a 2-day incubation, the leaves were treated with mock buffer or 100 μM ABA for 2 h. Scale bar = 1 cm. f WDL7 and MREL57 protein levels were detected by western blotting using anti-FLAG and anti-MYC antibodies. Actin was used as a control. g Luminescence values were determined using a spectrophotometer. The experiment was repeated three times. Data represent mean ± standard deviation (SD) values. Two-tailed Student’s t test, **p < 0.01. h MREL57 ubiquitinates WDL7 in vitro. Recombinant proteins were purified from E. coli and then incubated at 30 °C for 3 h. The ubiquitination of WDL7 was detected with anti-Ub and anti-FLAG antibodies. The experiment repeated three times with similar results. i MREL57 ubiquitinates WDL7 in vivo. WDL7-GFP and WDL7-GFP/mrel57-1 transgenic seedlings were treated with 0 or 10 μM of ABA for 1 h in the presence of 50 μM MG132. Total proteins were extracted and incubated with anti-GFP mAb-magnetic agarose. Anti-Ub was used to detect the polyubiquitination of WDL7. j Quantitative analysis of the signal intensity in i. Data represent the mean ± SD for three independent experiments. Different letters represent significant differences at p < 0.01 (one-way ANOVA).