通过免疫共沉淀法检测植物中双链RNA结合蛋白、病毒蛋白和初级miRNA转录产物的交互作用

Lijia Zheng; Chao Zhang; Jianguo  Wu; Yi Li

doi:10.21769/BioProtoc.2840

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Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta

通过免疫共沉淀法检测植物中双链RNA结合蛋白、病毒蛋白和初级miRNA转录产物的交互作用

LZ Lijia Zheng

CZ Chao Zhang

Jianguo Wu email

YL Yi Li

发布: 2018年05月05日第8卷第9期 DOI: 10.21769/BioProtoc.2840 浏览次数: 6004

评审: Arsalan DaudiAswad KhadilkarAnonymous reviewer(s)

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Abstract

MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript.

Keywords: Rice stripe virus (水稻条纹病毒)

NS3 (NS3)

Double-stranded RNA binding protein (双链RNA结合蛋白)

Primary-miRNA (初级miRNA)

miRNA (miRNA)

Plant-microbe interaction (植物 - 微生物交互作用)

Background

MicroRNAs (miRNAs) are processed from their primary transcripts (pri-miRNAs) by the RNase III enzyme DICER-LIKE 1 (DCL1) with the help of the double-stranded RNA (dsRNA) binding protein HYPONASTIC LEAVES1 (DRB1/HYL1) and the zinc finger protein SERRATE (SE). Rice stripe virus (RSV) infection broadly perturbs miRNA accumulation. We found that RSV-encoding nonstructural protein 3 (NS3) promotes miRNA accumulation by downregulating pri-miRNAs through interaction with DRB1 in rice (Zheng et al., 2017). To reveal how NS3 enhances pri-miRNA processing, we used co-immunoprecipitation (Co-IP) to illustrate the relationship of NS3, DRB1 and pri-miRNA in vivo. This protocol contributes to understand association patterns between two proteins and one RNA transcript.

Materials and Reagents

Pipette (RNase free 1 ml, 0.2 ml and 0.02 ml, Axygen)
Miracloth (Merck, Calbiochem, catalog number: 475855 )
Centrifuge tube (1.5 ml, 2 ml and 50 ml) (Corning)
4- to 6- weeks old Nicotiana benthamiana (leaves, grow in green house)
Agrobacterium tumefaciens (EHA105 strain, preserved in our lab)
Expression plasmids: pEarleyGate202-DRB1, -mutant DRB1, pEarleyGate203-NS3, -mutant NS3 and pCAMBIA-artificial primary miRNA transcript, -mutant primary miRNA transcript (Constructed by ourselves)
Double-distilled or MilliQ water (ddH₂O)
Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
PBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 70011069 )
Glycine (Sigma-Aldrich, catalog number: V900144 )
Liquid nitrogen
Anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: M4439 ) and mouse IgG control (Thermo Fisher Scientific, Invitrogen, catalog number: 02-6502 )
Protein G-agarose (Roche Diagnostics, catalog number: 11243233001 )
Trizol
Chloroform (Sigma-Aldrich, catalog number: 613304 )
GlycoBlue^TM coprecipitant (15 mg/ml) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9516 )
Ethanol (Sigma-Aldrich, catalog number: E7023 )
Ethanol (Aladdin, catalog number: E111963 )
DNase I (Promega, catalog number: M6101 )
Sucrose (Sigma-Aldrich, catalog number: V900116 )
Ficoll 400 (Sigma-Aldrich, catalog number: F9378 )
Dextran T40 (Sigma-Aldrich, catalog number: 1179708 )
HEPES (Sigma-Aldrich, catalog number: RDD002 )
KOH
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 05892953001 )
DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: 43817 )
Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
NP-40 (Sigma Aldrich, catalog number: NP40S )
RNase inhibitor (RNaseOUT) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10777019 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: V900312 )
Diethyl pyrocarbonate (Sigma-Aldrich, catalog number: 40718 )
Honda buffer (see Recipes)
High salt nuclear lysis buffer (see Recipes)
Dilution buffer (see Recipes)
IP buffer (see Recipes)

Equipment

Eppendorf pipettes suite (1 ml, 0.2 ml, 0.02 ml, 0.01 ml and 0.0025 ml)
Thermomixer C (Eppendorf, model: Thermomixer^® C , catalog number: 5382000023)
Vacuum pump (FJC, model: 6912 )
Rotator (Glas-Col, model: 099A MR1512 )
Centrifuge (Eppendorf, models: 5424 R and 5804 R )
Pico Ultrasonicator (Diagenode, model: Bioruptor^® Pico, model: 4486126 )
Vortex (Kylin-Bell Lab Instruments, model: VORTEX-5 )
Pestle

Procedure

English

中文翻译

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如何引用

Zheng, L., Zhang, C., Wu, J. and Li, Y. (2018). Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta. Bio-protocol 8(9): e2840. DOI: 10.21769/BioProtoc.2840.