Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice
从带病毒的灰飞虱中分离水稻条纹病毒制备物并机械接种于水稻
发布: 2017年11月05日第7卷第21期 DOI: 10.21769/BioProtoc.2597 浏览次数: 6278
评审: Anonymous reviewer(s)
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Apr 2016
Abstract
Tenuiviruses can infect the plants of the family Poaceae, and cause serious loss of crops, particularly rice and maize, in South-Eastern Asian countries. Tenuiviruses usually depend on insect vectors for their transmission and cannot be transmitted between plants through wounds or abrasions. Rice stripe virus (RSV), a typical member of tenuiviruses, is efficiently transmitted by the small brown planthopper Laodelphax striatellus in a persistent-propagative manner to cause rice stripe disease. Here we presented a convenient method, the midrib micro-injection, to mechanically inoculate insect-derived RSV into rice leaves for conducting pathogenicity assay on rice plants.
Keywords: Rice stripe virus (水稻条纹病毒)
Mechanical inoculation (机械接种)
Micro-injection (微量注射)
Small brown planthopper (灰飞虱)
Background
Tenuiviruses cannot be mechanically inoculated into plants, unless through vascular puncture inoculation with quite different transmission rates ranging from 1% to 90% according to different experimental details (Louie, 1995; Hogenhout et al., 2008). As to RSV, mechanical transmission usually fails or yields a low infectious rate (Ling, 1972). In particular, the transmission rate was only 6% after the injection of the RSV crude extraction from diseased plants (Okuyama and Asuyama, 1959). The midrib micro-injection method mentioned in this work promotes the RSV transmission rate to 17%. Though the incidence of RSV by mechanical transmission is still much lower than that by insect vector transmission (53%), our method provides a convenient way for mechanical inoculation of persistent-propagative plant viruses. Moreover, based on this method, replication and gene expression of a persistent-propagative plant virus can be determined more accurately in infected plant hosts without the interference of insects, i.e., the inoculation doses and the insect proteins.
Materials and Reagents
- 15 ml centrifuge tube, RNase-/DNase-free (Corning, catalog number: 430791 )
- 1.5 ml clear Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
- Plastic tissue grinder pestles (Tiangen Biotech, catalog number: OSE-Y001 )
- Pipettes with tips of 10 μl, 100 μl, and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
- PVDF Western blotting membranes (Sigma-Aldrich, catalog number: 03010040001 )
Note: Currently, it is ‘Merck, catalog number: 03010040001 ’. - Extra thick blot paper filter paper (Bio-Rad Laboratories, catalog number: 1703965 )
- 96-well ELISA Microplates (Greiner Bio One International, catalog number: 650001 )
- Adhesive plastic
- 25 G ⅝ to 30 G ½ gauge needle
- Drummond replacement glass capillaries, 100/vial (Drummond Scientific, catalog number: 3-000-203-G/X )
- Parafilm® M (Sigma-Aldrich, catalog number: P7793 )
Note: Currently, it is ‘Merck, catalog number: P7793 ’. - Insect vectors (Laodelphax striatellusi, Fallen) (collected from a field population in Hai’an, Jiangsu Province, China)
- RSV cp gene was amplified with the primers: 5’-ATGGGTACCAACAAGCCAGC-3’, and 5’-CTAGTCATCTGCACCTTCTG-3’. PCR was run on the ProFlexTM PCR System under cycling conditions of 95 °C for 5 min, followed by 30 cycles of 95 °C for 20 sec, 50 °C for 30 sec and 72 °C for 30 sec. The purified 969 bp of PCR products were cloned into pET-28a (Merck, Novagen, catalog number: 69865-3 ) for Cp protein expression. The recombinant cp plasmid was sent to Beijing Genomics Institute for monoclonal anti-Cp antibody production
- Host plants (Oryza sativa L. spp. japonica var. Nippobare)
Note: Healthy 2-week-old rice leaves with the length of approximately 15 cm were used for microinjection. - Protein loading buffer (5x) (Adipogen International, catalog number: AG-10T-0020-L001 )
- ExpressPlusTM PAGE gel, 4-20% (GenScript, catalog number: M42010 )
- PageRulerTM prestained protein ladder, 10 to 180 kDa (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 ), used for SDS-PAGE
- HEPES (Fisher Scientific, catalog number: BP310-100 ), used for SDS-PAGE
- EMPLURA® methanol (Merck, catalog number: 822283 )
- ELISA coating buffer, 1x (Solarbio, catalog number: C1050 )
- SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34095 )
- Goat anti-mouse IgG (H+L) secondary antibody, HRP (Thermo Fisher Scientific, InvitrogenTM, catalog number: 32430 )
- Anti-Cp antibody (Beijing Genomics Institute)
- 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, catalog number: T0440 )
Note: Currently, it is ‘Merck, catalog number: T0440 ’. - Sulfuric acid (H2SO4) (5 N) (Merck, catalog number: 480364 ), used for ELISA
- Sodium dodecyl sulfate (SDS) (AMRESCO, catalog number: 0227 ), used for ELISA
- Mineral oil (Sigma-Aldrich, catalog number: M8410 )
Note: Currently, it is ‘Merck, catalog number: M8410 ’ . - Tween® 20 (Sigma-Aldrich, catalog number: P1379 )
- Phosphate buffer saline (PBS), pH 7.4, basic (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
- OxoidTM Skim milk power (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0031B )
- Phosphate buffer saline with Tween 20 (1x) (PBST) (see Recipes)
- Transfer buffer (see Recipes)
- Blocking buffer (see Recipes)
- Diluted monoclonal anti-Cp antibody (see Recipes)
- Diluted Goat anti-mouse IgG, HRP-conjugated antibody (see Recipes)
Equipment
- TGrinder High Speed Tissue grinder (Tiangen Biotech, catalog number: OSE-Y30 )
- Centrifuge 5424R (Eppendorf, model: 5424 R )
- Decolorizing Orbital Shaker (T-Bota Scietech Instruments & Equipment, model: TS-200 ), used for antibody incubation in ELISA and Western blots
- Chemiluminescent Imaging System (Tanon, model: Tanon 5200 )
- SpectraMax® Paradigm® Multi-Mode Detection Platform (Molecular Devices, model: SpectraMax Paradigm Multi-Mode )
- Micropipette Puller (Sutter Instrument, model: P-97 )
- ProFlexTM PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: ProFlexTM 3 x 32-Well PCR System, catalog number: 4484073 )
- Glass incubators (Φ55 x 150 mm, Φ110 x 150 mm), used for planthopper incubation
Note: Each glass incubator must be sealed with a nylon mesh in order to protect the cultured plants or insects from other interference sources. The culture condition is 25 °C, with 16 h of light daily (Figure 1).
Figure 1. Plant culture and insect rearing. The small brown planthoppers and rice seedlings are cultured in glass incubators sealed with nylon meshes. The viruliferous or nonviruliferous planthoppers are geographically isolated, which are cultured in different insectaries. - MixMate® , the 3-in-1 mixer (Eppendorf, catalog number: 5353000014 )
- H2O3 dry bath (Coyote Bioscience, model: H2O3-100C )
- PowerPacTM universal power supply (Bio-Rad Laboratories, catalog number: 1645070 )
- Trans-Blot® TurboTM transfer system (Bio-Rad Laboratories, catalog number: 1704150 )
- Nanoject II Programmable Nanoliter injector (World Precision Instruments, model: Nanoliter 2000 )
- Backfilling Needle for Nanoject II Auto-Nanoliter Injector, 2” Length (Drummond Scientific, model: 3-000-027 )
- SZ61 Stereo microscope (Olympus, model: SZ61 ), used for microinjection
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhao, W., Kang, L. and Cui, F. (2017). Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice. Bio-protocol 7(21): e2597. DOI: 10.21769/BioProtoc.2597.
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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 > 植物生理学 > 表型分析
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