Rice Ragged Stunt Virus Propagation and Infection on Rice Plants
稻株中水稻齿叶矮缩病毒的传播和感染
发布: 2018年10月20日第8卷第20期 DOI: 10.21769/BioProtoc.3060 浏览次数: 5577
评审: Feng LiSonali ChaturvediAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2016
Abstract
Virus inoculation is a basic experimental procedure to evaluate the resistance of a rice variety or a transgenic material upon virus infection. We recently demonstrated that Rice Ragged Stunt Virus (RRSV), an oryzavirus that is transmitted by brown planthopper (BPH), can suppress jasmonic acid-mediated antiviral defense through the induction of microRNA319 and facilitate virus infection in rice. To verify this, we performed virus inoculation experiments on wild-type rice plants and miR319-TCP21-associated transgenic rice plants through a modified group inoculation method. Here, we presented the detailed procedure of RRSV propagation and infection process on rice plants.
Keywords: Rice ragged stunt virus (水稻齿叶矮缩病毒)
RRSV (RRSV)
Brown planthopper (褐飞虱)
Virus inoculation (病毒接种)
Background
Studying mechanism of viral pathogenesis and screening virus-resistant rice varieties have been doing to overcome rice virus diseases and keep food security. In this field, virus inoculation is the essential and reliable method to evaluate the resistance of a transgenic material or a rice variety. Rice Ragged Stunt Virus (RRSV) can transmit rice ragged stunt disease by brown planthopper in a persistent propagative manner in many Asian countries (Ling et al., 1978; Hibino, 1979). Two classical methods, including single seedling inoculation and group inoculation, had been applied to conduct virus inoculation experiments (Zhang et al., 2013). By modifying traditional group inoculation method, we provided a convenient approach which closely resembles the natural infection condition.
Materials and Reagents
- Pipette tips (RNase free 1 ml, 0.2 ml and 0.02 ml, Corning, Axygen®, catalog numbers: T-1000 , T-200 , TF-20 )
- 1.5 ml clear Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
- Clip-cage (Home-made, 40 x 30 x 35 cm, Figure 1A)
- Artificial suction-implement (Home-made, Figure 1B)
- Ceramic mortar (external diameter = 120 mm)
- PVDF membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88585 )
- Filter paper (Bio-Rad Laboratories, catalog number: 1703967 )
Figure 1. Home-made clip-cage and artificial suction-implement used in this study. A. Clip-cage used for raising insect vectors and virus transmission. B. An artificial suction-implement used for transferring insect vectors. - X-ray film
- RRSV-infected rice plants (at vegetative growth phase)
- Host plants (Oryza sativa L. spp. japonica var. Nippobare) (all the transgenic lines are Nippobare background)
- Insect vector (Nilaparvate lugens Stal) (collected from a field population in Fuzhou, Fujian Province, China, and cultured at 25 °C with a photoperiod of 16 h/8 h [light/dark] in a tissue culture frame); the insect vectors will mate during cultivation naturally.
- Primers
For β-Actin gene:
5'-CAGCCACACTGTCCCCATCTA-3’
5'-AGCAAGGTCGAGACGAAGGA-3’
For RRSV CP:
5'-GAGCAAACTTGAGGCGTA-3’
5'-AAGCTACCGTGTAGGTGGCG-3’ - Plant RNA Kit (Omega Bio-Tek, catalog number: R6827 )
- DNase I (Omega Bio-Tek, catalog number: E1091 )
- ReverTra Ace qPCR RT Kit (TOYOBO, catalog number: FSQ-301 )
- THUNDERBIRD qPCR Mix (TOYOBO, catalog number: QPS-201 )
- Liquid nitrogen
- Agarose
- A homemade monoclonal antibody against RRSV-encoded CP
- ProteinFind Anti-β-Tubulin Mouse Monoclonal Antibody (TransGen Biotech, catalog number: HC201-02 )
- ProteinFind Goat Anti-Mouse IgG(H+L), HRP Conjugate (TransGen Biotech, catalog number: HS201-01 )
- Chemiluminescent HRP substrate (Merck, catalog number: WBKLS0100 )
- Prestained marker
- Ethanol (ALADDIN, catalog number: E111963 )
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 75746-1KG )
- Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: 97622 )
- Glycerinum (Sigma-Aldrich, catalog number: G5516 )
- NaCl (Sigma-Aldrich, catalog number: S7653 )
- KCl (Sigma-Aldrich, catalog number: P9333 )
- KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
- Na2HPO4•12H2O (Sinopharm Chemical Reagent, CAS number: 10039-32-4)
- Skim milk powder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0031B )
- 30% acrylamide solution
- (NH4)2S2O8 (Sigma-Aldrich, catalog number: A3678-25G )
- TEMED (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
- Tween 20 (Xilong Scientific, CAS number: 9005-64-5)
- Glycine (Solarbio, catalog number: G8200 )
- Methanol (Sinopharm Chemical Reagent, catalog number: 10014108 )
- 10% PAGE gel (see Recipes)
- Plant total protein extraction buffer (see Recipes)
- 10x PBS buffer (see Recipes)
- PBS-T (see Recipes)
- Blocking solution (see Recipes)
- Transferring buffer (see Recipes)
- Monoclonal anti-CP antibody solution (see Recipes)
Equipment
- Eppendorf pipettes suite (1 ml, 0.2 ml, 0.02 ml, 0.01 ml and 0.0025 ml)
- Ceramic mortar
- Centrifuge (Eppendorf, models: 5424 R and 5427 R )
- Vortex (Kylin-Bell Lab Instruments, model: VORTEX-5 )
- PCR amplifier (Bio-Rad Laboratories, model: T100 )
- Fluorescence quantitative PCR detection system (Bio-Rad Laboratories, model: CFX ConnectTM )
- Gel Imaging System (Bio-Rad Laboratories, model: GelDocTM XR+ )
- Basic electrophoresis apparatus (Bio-Rad Laboratories, model: PowerPacTM Basic )
- Mini-Protean Tetra (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell )
- Semi-dry transfer slot (Bio-Rad Laboratories, model: Trans-Blot SD )
- Ultra-micro UV spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 2000 )
- Developing machine (Carestream Health, model: 102 )
- Decolorizing orbital shaker (Kylin-Bell Lab instruments, catalog number: TS-1000 )
- Black box
- Film Cassette (Guangdong YueHua Medical Instrument Factory, model: AX-II )
Software
- SPSS for Windows version 11.5 (IBM, Armonk, NY, USA)
- Gel imaging software (Bio-Rad Laboratories, model: GelDocTM XR+ )
- Bio-Rad qPCR software (Bio-Rad Laboratories, model: CFX ConnectTM )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhang, C., Shi, C., Chen, D. and Wu, J. (2018). Rice Ragged Stunt Virus Propagation and Infection on Rice Plants. Bio-protocol 8(20): e3060. DOI: 10.21769/BioProtoc.3060.
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分类
微生物学 > 微生物-宿主相互作用 > 病毒
植物科学 > 植物免疫 > 病害生物测定
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