title: Ubiquitination of plant immune receptors.
abstract: Ubiquitin is a highly conserved regulatory protein consisting of 76 amino acids and ubiquitously expressed in all eukaryotic cells. The reversible ubiquitin conjugation to a wide variety of target proteins, a process known as ubiquitination or ubiquitylation, serves as one of the most important and prevalent posttranslational modifications to regulate the myriad actions of protein cellular functions, including protein degradation, vesicle trafficking, and subcellular localization. Protein ubiquitination is an ATP-dependent stepwise covalent attachment of one or more ubiquitin molecules to target proteins mediated by a hierarchical enzymatic cascade consisting of an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. The plant plasma membrane resident receptor-like kinase Flagellin Sensing 2 (FLS2) recognizes bacterial flagellin and initiates innate immune signaling to defend against pathogen attacks. We have recently shown that two plant U-box E3 ubiquitin ligases PUB12 and PUB13 directly ubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, which in turn attenuates FLS2 signaling to prevent excessive or prolonged activation of immune responses. Here, we use FLS2 as an example to describe a protocol for detection of protein ubiquitination in plant cells in vivo and in test tubes in vitro. In addition, we elaborate the approach to identify different types of ubiquitin linkages by using various lysine mutants of ubiquitin. The various in vivo and in vitro ubiquitination assays will provide researchers with the tools to address how ubiquitination regulates diverse cellular functions of target proteins.
authors.affiliation: Department of Biochemistry and Biophysics, and Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, 77843, USA.
authors.forename: Jinggeng
authors.initials: J
authors.lastname: Zhou
authors.forename: Ping
authors.initials: P
authors.lastname: He
authors.forename: Libo
authors.initials: L
authors.lastname: Shan
mesh_terms: D029681:Arabidopsis Proteins / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D057865:Plant Immunity / Q000235:genetics; D011494:Protein Kinases / Q000378:metabolism; D059748:Proteolysis; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C558941:PUB12 protein, Arabidopsis; C558942:PUB13 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; C411853:FLS2 protein, Arabidopsis