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A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants

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Abstract

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics.

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Availability of data and material

All data generated or analyzed during this study are included in this published article and its supplementary information files. The vectors generated by the current study are available from the corresponding author on request.

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Acknowledgements

We thank Dr. Tsuyoshi Nakagawa Shimane University for providing the pUGW vectors.

Funding

This study is supported by the National Transgenic Science and Technology Program (2016ZX08010002), the National Natural Science Foundation of China (31622047), and the State Key Laboratory of Hybrid Rice and a startup fund from Huazhong Agricultural University (2015RC009). YY is supported by NSF Plant Genome Research Project Grant (1740874) and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04659 and Accession #1016432. The funding bodies have no roles in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript.

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement and Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan, 430070, China

    Hong Li, Caiyun Wu, Manman Du, Yache Chen & Kabin Xie

  2. State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan, 430072, China

    Xin Hou

  3. Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, State College, PA, 16802, USA

    Yinong Yang

Authors
  1. Hong Li
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  2. Caiyun Wu
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  3. Manman Du
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  4. Yache Chen
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  5. Xin Hou
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  6. Yinong Yang
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  7. Kabin Xie
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Contributions

KX designed the experiments. HL, CW, MD, and YC performed the experiments. HL, XH and KX analyzed the data. HL, XH, YY and KX wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Kabin Xie.

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Li, H., Wu, C., Du, M. et al. A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants. Mol Breeding 41, 13 (2021). https://doi.org/10.1007/s11032-021-01210-7

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