title: An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana.
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.
authors.affiliation: 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.
authors.forename: Lijing
authors.initials: L
authors.lastname: Liu
authors.forename: Yiyue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sanyuan
authors.initials: S
authors.lastname: Tang
authors.forename: Qingzhen
authors.initials: Q
authors.lastname: Zhao
authors.forename: Zhonghui
authors.initials: Z
authors.lastname: Zhang
authors.forename: Huawei
authors.initials: H
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Dong
authors.forename: Huishan
authors.initials: H
authors.lastname: Guo
authors.forename: Qi
authors.initials: Q
authors.lastname: Xie
mesh_terms: D010940:Plant Proteins / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics; D012231:Rhizobium; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D010940:Plant Proteins; D018749:RNA, Plant; D044767:Ubiquitin-Protein Ligases