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A modified transient gene expression protocol for subcellular protein localization analysis in rice

  • Yiming Wang
  • Jingni Wu
  • Sang Gon Kim
  • Ju Soon Yoo
  • Ravi Gupta
  • Byeong Il Je
  • Jong-Seong Jeon
  • Ki-Hong Jung
  • Yu-Jin Kim
  • Kyu Young Kang
  • Sun Tae KimEmail author
Technical Report
  • 6 Downloads

Abstract

Transient protein expression is a useful technique for investigating the protein localization and functional analysis of stress responses in rice plants. Currently, available methods for in planta transient expression analysis include Agrobacterium-mediated transformation, protoplast transformation using polyethylene glycol or electroporation, and biolistic bombardment expression which have several disadvantages and are not well suited for the rice. Therefore, development of a method for rapid and efficient analysis of protein expression, subcellular localization, pathogen effector screening, and protein–protein interaction in rice is required. We developed a protocol for in planta gene expression analysis in sliced rice sheath cells by modifying and optimizing the biolistic particle bombardment technique. By obtaining thin sections (~400 μm) of rice sheath cells, auto-fluorescence from chlorophyll was eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and tonoplast. In addition, high transformation efficiency of 30% was achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assay in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is applicable to plant–microbe interaction studies. The transient expression protocol established here is well optimized for protein localization, secretion, and host–pathogen protein interaction studies in rice. A typical experiment can be completed in three days.

Keywords

Particle bombardment Subcellular localization Protein secretion Plant–microbe interactions 

Notes

Acknowledgements

We thank Dr. Woo Sik Chung for supplying the NLS::RFP, H+-ATPase::RFP, and ACA11::RFP plasmids, Dr. Jae-Yean Kim for supplying the mCherry vector and Dr. Randeep Rakwal for reviewing and commenting on the manuscript. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2018R1A4A1025158).

Supplementary material

11816_2019_569_MOESM1_ESM.pptx (1.2 mb)
Bombardment efficiency test. Rice sheath cells were bombarded with tungsten particles carrying ZmUbipro::mCherry, and signals were detected 48 hab. Total cell numbers and mCherry-expressing cells were counted. Bars = 200 μm (PPTX 1262 kb)
11816_2019_569_MOESM2_ESM.pptx (814 kb)
Transient expression of two rice superoxide dismutase (SOD1 and SOD2) in rice leaf sheaths. mCherry signals in rice leaf sheaths were detected at 48 hab (PPTX 814 kb)
11816_2019_569_MOESM3_ESM.docx (13 kb)
Supplementary file3 (DOCX 13 kb)

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Copyright information

© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  • Yiming Wang
    • 1
    • 2
  • Jingni Wu
    • 3
    • 4
  • Sang Gon Kim
    • 5
  • Ju Soon Yoo
    • 6
  • Ravi Gupta
    • 6
  • Byeong Il Je
    • 7
  • Jong-Seong Jeon
    • 8
  • Ki-Hong Jung
    • 8
  • Yu-Jin Kim
    • 8
  • Kyu Young Kang
    • 4
  • Sun Tae Kim
    • 6
    Email author
  1. 1.College of Plant ProtectionNanjing Agriculture UniversityNanjingChina
  2. 2.Department of Plant Microbe InteractionsMax-Planck Institute for Plant Breeding ResearchCologneGermany
  3. 3.National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and EcologyChinese Academy of SciencesShanghaiChina
  4. 4.Division of Applied Life Science (BK21 Program)Gyeongsang National UniversityJinjuSouth Korea
  5. 5.Gyeongnam Oriental Anti-Aging InstituteGyeongnamSouth Korea
  6. 6.Department of Plant BiosciencePusan National UniversityMiryangSouth Korea
  7. 7.Department of Horticultural BiosciencePusan National UniversityMiryangSouth Korea
  8. 8.Graduate School of Biotechnology and Crop Biotech InstituteKyung Hee UniversityYonginSouth Korea

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