A modified transient gene expression protocol for subcellular protein localization analysis in rice
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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.
KeywordsParticle bombardment Subcellular localization Protein secretion Plant–microbe interactions
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).
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