Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

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• 作者：Yukichi Fujikawa (1)
  Takahiro Nakanishi (1)
  Hiroko Kawakami (1)
  Kanako Yamasaki (2)
  Masa H Sato (2)
  Hiroyuki Tsuji (3)
  Makoto Matsuoka (4)
  Naohiro Kato (5)
  
  1. Graduate School of Biosphere Science ; Hiroshima University ; 1-4-4 Kagamiyama ; Higashi-Hiroshima ; Hiroshima ; 739-8528 ; Japan
  2. Faculty of Human Environmental Sciences ; Kyoto Prefectural University ; Kyoto ; 606-8522 ; Japan
  3. Department of Plant Biology ; Graduate School of Biological Sciences ; Nara Institute of Science and Technology ; 8916-5 Takayama ; Ikoma ; Nara ; 630-0192 ; Japan
  4. Bioscience and Biotechnology Center ; Nagoya University ; Nagoya ; Aichi 464-8601 ; Japan
  5. Department of Biological Sciences ; Louisiana State University ; 226 Life Sciences Building ; Baton Rouge ; LA ; 70803 ; USA
  
• 关键词：Interactome ; Split luciferase complementation ; Regulated protein ; protein interactions
• 刊名：Rice
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：7
• 期：1
• 全文大小：884 KB
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• 刊物主题：Plant Sciences; Plant Genetics & Genomics; Plant Breeding/Biotechnology; Agriculture; Plant Ecology;
• 出版者：Springer US
• ISSN：1939-8433

文摘

Background The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.