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An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana
Authors:Lijing Liu  Yiyue Zhang  Sanyuan Tang  Qingzhen Zhao  Zhonghui Zhang  Huawei Zhang  Li Dong  Huishan Guo  Qi Xie
Institution:1. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China;2. School of Life Science, Liaocheng University, Liaocheng 252059, China;3. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
Abstract:The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate‐specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase–substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well‐known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene‐silencing suppressor p19 and by time‐courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.
Keywords:ubiquitination assay  agroinfiltration  E3 ligase  substrate  in vivo
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