βC1 interferes with the interaction between WRKY20 and ERF-ORA59

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Fig. 5 βC1 interferes with the interaction between WRKY20 and ERF-ORA59. βC1 interferes with the interaction between WRKY20 and ERF-ORA59. (A and B) Relative expression level of AtPDF1.2 (A) or AtORA59 (B) in Arabidopsis plants under MeJA treatment. Bars represent means ± SD (n = 3) (*P < 0.05 and **P < 0.01, Student’s t test). (C) GST pull-down assay. Two micrograms of MBP-AtWRKY20 fusion protein was used to pull down 2 μg of GST fusion proteins. WB, Western blot. (D) BiFC analysis of AtWRKY20 interaction only with AtORA59 but not with AtMYC2 or AtERF1. Scale bars, 50 μm. (E) Effects of βC1 on the interaction of WRKY20 with ORA59 by modified BiFC assay. The EYFP fluorescences were detected after coproduction of cEYFP-AtORA59 + nEYFP-AtWRKY20 (control), βC1 + cEYFP-AtORA59 + nEYFP-AtWRKY20 (βC1), or GUS + cEYFP-AtORA59 + nEYFP-AtWRKY20 (GUS). Scale bars, 50 μm. (F) Quantitative data of EYFP fluorescence intensity show effects of βC1 on the interaction of WRKY20 with ORA59. Bars represent means ± SD (n = 20) (**P < 0.01, Student’s t test). (G) Pull-down protein competition assays. The indicated protein amount of His-βC1 or GST was mixed with 2 μg of GST-AtORA59 and pulled down by 2 μg of MBP-AtWRKY20. Immunoblots were performed using anti-GST antibody to detect the associated proteins. (C and G) Membranes were stained with Coomassie brilliant blue to monitor input protein amount. Pingzhi Zhao et al. Sci Adv 2019;5:eaav9801 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).