2. Academic Validation
  3. Rapid and dynamic detection of endogenous proteins through in-locus tagging in rice

Rapid and dynamic detection of endogenous proteins through in-locus tagging in rice

  • Plant Commun. 2024 Jul 11:101040. doi: 10.1016/j.xplc.2024.101040.
Yifu Tian 1,  Dating Zhong 2,  Rundong Shen 3,  Xinhang Tan 4,  Chen Zhu 5,  Kai Li 6,  Qi Yao 2,  Xinbo Li 3,  Xuening Zhang 4,  Xuesong Cao 5,  Pengcheng Wang 5,  Jian-Kang Zhu 7,  Yuming Lu 8
Affiliations

Affiliations

  • 1 Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), Institute of Crop Sciences and National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan 572024, China; Hainan Seed Industry Laboratory, Sanya, Hainan 572024, China.
  • 2 Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China.
  • 3 Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), Institute of Crop Sciences and National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan 572024, China; Hainan Seed Industry Laboratory, Sanya, Hainan 572024, China.
  • 4 Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China.
  • 5 Institute of Advanced Biotechnology, and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China.
  • 6 Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
  • 7 Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), Institute of Crop Sciences and National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan 572024, China; Institute of Advanced Biotechnology, and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: luymin@sjtu.edu.cn.
  • 8 Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: luymin@sjtu.edu.cn.
PMID: 39001607 DOI: 10.1016/j.xplc.2024.101040
Abstract

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in Plants presents substantial challenges. While mammalian studies have leveraged in-locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of Native Proteins, this approach remained unexplored in Plants. Here, we introduce the in-locus HiBiT tagging of rice proteins and demonstrate its feasibility in Plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, which is attributed to the low luminescence activity in Plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence by up to 6.9-fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in Plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in-situ visualization using a cross-breeding strategy, and effective immunoprecipitation for protein interaction analysis. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

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