Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice

Materials and Reagents

1. 15 ml centrifuge tube, RNase-/DNase-free (Corning, catalog number: 430791 )
   
2. 1.5 ml clear Microtubes (Corning, Axygen^®, catalog number: MCT-150-C )
   
3. Plastic tissue grinder pestles (Tiangen Biotech, catalog number: OSE-Y001 )
   
4. Pipettes with tips of 10 μl, 100 μl, and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
   
5. PVDF Western blotting membranes (Sigma-Aldrich, catalog number: 03010040001 )
   Note: Currently, it is ‘Merck, catalog number: 03010040001 ’.
   
6. Extra thick blot paper filter paper (Bio-Rad Laboratories, catalog number: 1703965 )
   
7. 96-well ELISA Microplates (Greiner Bio One International, catalog number: 650001 )
   
8. Adhesive plastic
   
9. 25 G ⅝ to 30 G ½ gauge needle
   
10. Drummond replacement glass capillaries, 100/vial (Drummond Scientific, catalog number: 3-000-203-G/X )
    
11. Parafilm^® M (Sigma-Aldrich, catalog number: P7793 )
    Note: Currently, it is ‘Merck, catalog number: P7793 ’.
    
12. Insect vectors (Laodelphax striatellusi, Fallen) (collected from a field population in Hai’an, Jiangsu Province, China)
    
13. RSV cp gene was amplified with the primers: 5’-ATGGGTACCAACAAGCCAGC-3’, and 5’-CTAGTCATCTGCACCTTCTG-3’. PCR was run on the ProFlex^TM 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
    
14. 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.
    
15. Protein loading buffer (5x) (Adipogen International, catalog number: AG-10T-0020-L001 )
    
16. ExpressPlus^TM PAGE gel, 4-20% (GenScript, catalog number: M42010 )
    
17. PageRuler^TM prestained protein ladder, 10 to 180 kDa (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 26616 ), used for SDS-PAGE
    
18. HEPES (Fisher Scientific, catalog number: BP310-100 ), used for SDS-PAGE
    
19. EMPLURA^® methanol (Merck, catalog number: 822283 )
    
20. ELISA coating buffer, 1x (Solarbio, catalog number: C1050 )
    
21. SuperSignal^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 34095 )
    
22. Goat anti-mouse IgG (H+L) secondary antibody, HRP (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 32430 )
    
23. Anti-Cp antibody (Beijing Genomics Institute)
    
24. 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, catalog number: T0440 )
    Note: Currently, it is ‘Merck, catalog number: T0440 ’.
    
25. Sulfuric acid (H₂SO₄) (5 N) (Merck, catalog number: 480364 ), used for ELISA
    
26. Sodium dodecyl sulfate (SDS) (AMRESCO, catalog number: 0227 ), used for ELISA
    
27. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
    Note: Currently, it is ‘Merck, catalog number: M8410 ’ .
    
28. Tween^® 20 (Sigma-Aldrich, catalog number: P1379 )
    
29. Phosphate buffer saline (PBS), pH 7.4, basic (1x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010031 )
    
30. Oxoid^TM Skim milk power (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: LP0031B )
    
31. Phosphate buffer saline with Tween 20 (1x) (PBST) (see Recipes)
    
32. Transfer buffer (see Recipes)
    
33. Blocking buffer (see Recipes)
    
34. Diluted monoclonal anti-Cp antibody (see Recipes)
    
35. Diluted Goat anti-mouse IgG, HRP-conjugated antibody (see Recipes)
    

Equipment

1. TGrinder High Speed Tissue grinder (Tiangen Biotech, catalog number: OSE-Y30 )
   
2. Centrifuge 5424R (Eppendorf, model: 5424 R )
   
3. Decolorizing Orbital Shaker (T-Bota Scietech Instruments & Equipment, model: TS-200 ), used for antibody incubation in ELISA and Western blots
   
4. Chemiluminescent Imaging System (Tanon, model: Tanon 5200 )
   
5. SpectraMax^® Paradigm^® Multi-Mode Detection Platform (Molecular Devices, model: SpectraMax Paradigm Multi-Mode )
   
6. Micropipette Puller (Sutter Instrument, model: P-97 )
   
7. ProFlex^TM PCR System (Thermo Fisher Scientific, Applied Biosystems^TM, model: ProFlex^TM 3 x 32-Well PCR System, catalog number: 4484073 )
   
8. 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.
   
   
9. MixMate^® , the 3-in-1 mixer (Eppendorf, catalog number: 5353000014 )
   
10. H₂O³ dry bath (Coyote Bioscience, model: H₂O³-100C )
    
11. PowerPac^TM universal power supply (Bio-Rad Laboratories, catalog number: 1645070 )
    
12. Trans-Blot^® Turbo^TM transfer system (Bio-Rad Laboratories, catalog number: 1704150 )
    
13. Nanoject II Programmable Nanoliter injector (World Precision Instruments, model: Nanoliter 2000 )
    
14. Backfilling Needle for Nanoject II Auto-Nanoliter Injector, 2” Length (Drummond Scientific, model: 3-000-027 )
    
15. SZ61 Stereo microscope (Olympus, model: SZ61 ), used for microinjection
    

Procedure