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Sep 2016

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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   

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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

  1. Pipette tips (RNase free 1 ml, 0.2 ml and 0.02 ml, Corning, Axygen®, catalog numbers: T-1000 , T-200 , TF-20 )
  2. 1.5 ml clear Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
  3. Clip-cage (Home-made, 40 x 30 x 35 cm, Figure 1A)
  4. Artificial suction-implement (Home-made, Figure 1B)
  5. Ceramic mortar (external diameter = 120 mm)
  6. PVDF membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88585 )
  7. 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.

  8. X-ray film
  9. RRSV-infected rice plants (at vegetative growth phase)
  10. Host plants (Oryza sativa L. spp. japonica var. Nippobare) (all the transgenic lines are Nippobare background)
  11. 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. 
  12. Primers
    For β-Actin gene:
    5'-CAGCCACACTGTCCCCATCTA-3’
    5'-AGCAAGGTCGAGACGAAGGA-3’
    For RRSV CP:
    5'-GAGCAAACTTGAGGCGTA-3’
    5'-AAGCTACCGTGTAGGTGGCG-3’
  13. Plant RNA Kit (Omega Bio-Tek, catalog number: R6827 )
  14. DNase I (Omega Bio-Tek, catalog number: E1091 )
  15. ReverTra Ace qPCR RT Kit (TOYOBO, catalog number: FSQ-301 )
  16. THUNDERBIRD qPCR Mix (TOYOBO, catalog number: QPS-201 )
  17. Liquid nitrogen
  18. Agarose
  19. A homemade monoclonal antibody against RRSV-encoded CP
  20. ProteinFind Anti-β-Tubulin Mouse Monoclonal Antibody (TransGen Biotech, catalog number: HC201-02 )
  21. ProteinFind Goat Anti-Mouse IgG(H+L), HRP Conjugate (TransGen Biotech, catalog number: HS201-01 )
  22. Chemiluminescent HRP substrate (Merck, catalog number: WBKLS0100 )
  23. Prestained marker
  24. Ethanol (ALADDIN, catalog number: E111963 )
  25. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 75746-1KG )
  26. Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
  27. β-mercaptoethanol (Sigma-Aldrich, catalog number: 97622 )
  28. Glycerinum (Sigma-Aldrich, catalog number: G5516 )
  29. NaCl (Sigma-Aldrich, catalog number: S7653 )
  30. KCl (Sigma-Aldrich, catalog number: P9333 )
  31. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
  32. Na2HPO4•12H2O (Sinopharm Chemical Reagent, CAS number: 10039-32-4)
  33. Skim milk powder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0031B )
  34. 30% acrylamide solution 
  35. (NH4)2S2O8 (Sigma-Aldrich, catalog number: A3678-25G )
  36. TEMED (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
  37. Tween 20 (Xilong Scientific, CAS number: 9005-64-5)
  38. Glycine (Solarbio, catalog number: G8200 )
  39. Methanol (Sinopharm Chemical Reagent, catalog number: 10014108 )
  40. 10% PAGE gel (see Recipes)
  41. Plant total protein extraction buffer (see Recipes)
  42. 10x PBS buffer (see Recipes)
  43. PBS-T (see Recipes)
  44. Blocking solution (see Recipes)
  45. Transferring buffer (see Recipes)
  46. Monoclonal anti-CP antibody solution (see Recipes)

Equipment

  1. Eppendorf pipettes suite (1 ml, 0.2 ml, 0.02 ml, 0.01 ml and 0.0025 ml)
  2. Ceramic mortar
  3. Centrifuge (Eppendorf, models: 5424 R and 5427 R )
  4. Vortex (Kylin-Bell Lab Instruments, model: VORTEX-5 )
  5. PCR amplifier (Bio-Rad Laboratories, model: T100 )
  6. Fluorescence quantitative PCR detection system (Bio-Rad Laboratories, model: CFX ConnectTM )
  7. Gel Imaging System (Bio-Rad Laboratories, model: GelDocTM XR+ )
  8. Basic electrophoresis apparatus (Bio-Rad Laboratories, model: PowerPacTM Basic )
  9. Mini-Protean Tetra (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell )
  10. Semi-dry transfer slot (Bio-Rad Laboratories, model: Trans-Blot SD )
  11. Ultra-micro UV spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 2000 )
  12. Developing machine (Carestream Health, model: 102 )
  13. Decolorizing orbital shaker (Kylin-Bell Lab instruments, catalog number: TS-1000 )
  14. Black box
  15. Film Cassette (Guangdong YueHua Medical Instrument Factory, model: AX-II )

Software

  1. SPSS for Windows version 11.5 (IBM, Armonk, NY, USA)
  2. Gel imaging software (Bio-Rad Laboratories, model: GelDocTM XR+ )
  3. Bio-Rad qPCR software (Bio-Rad Laboratories, model: CFX ConnectTM )

Procedure

  1. Detection of RRSV-diseased rice plants through RT-PCR and Western blots
    The source of RRSV-diseased rice plants, exhibiting typical symptoms including stuntedness, excess tillering, and leaves that are dark green and/or have serrated edges or twisted tips (Zhang et al., 2016), are collected from the field (Fuzhou City, Fujian Province). The presence of RRSV is confirmed by RT-PCR and Western blot.
    1. RT-PCR
      1. Extract total RNA from 0.1 g leaves with disease symptoms using a Plant RNA kit (OMEGA Bio-Tek Inc.) following the manufacturer’s instructions. 
      2. Reverse transcribe 1 μg total RNA by using the ReverTra Ace qPCR RT Kit (TOYOBO), following the manufacturer’s manuals. 
      3. Then amplify the RRSV CP gene with the primers: 5’-GAGCAAACTTGAGGCGTA-3’, and 5’-AAGCTACCGTGTAGGTGGCG-3’.
      4. Run PCR on the Bio-Rad CFX ConnectTM PCR System under the cycling conditions of 95 °C for 5 min, followed by 25 cycles of 95 °C for 20 sec, 55 °C for 30 sec and 72 °C for 5 sec.
      5. Using rice β-Actin gene amplified with the primers: 5’-CAGCCACACTGTCCCCATCTA-3’, and 5’-AGCAAGGTCGAGACGAAGGA-3’ as a control. 
      6. Perform Agarose gel electrophoresis and then visualize the amplification products of RRSV CP gene using Gel Imaging System was used to.
    2. Western blots
      Total protein is extracted from 0.1 g RRSV-infected leaves, which were grounded into powder with liquid nitrogen in ceramic mortar and then transferred into a 1.5 ml pre-cooled clear Microtubes, followed by adding 100 μl plant protein extraction buffer. Make the sample mix evenly by using a vortex. Then centrifuge at 6,010 x g 4 °C for 10 min. For RRSV detection, 20 µl of the prepared total protein is used to conduct polyacrylamide gel electrophoresis and Western blot. The detailed steps are as follows:
      1. Load 20 µl of the prepared total protein in each well of the PAGE gel, along with prestained marker, and run the gel for 90 min at 120 V.
      2. Prepare a PVDF membrane which is slightly bigger than the size of the gel. Prepare 2 pieces of filter paper which is as big as PVDF membrane. Place the PVDF membrane, gel and filter papers in 1x transferring buffer for 10 min. Prepare the transfer sandwich (filter paper-PVDF membrane-gel-filter paper) and place it in a Semi-dry transfer slot with 3.0-5.5 mA/cm2 for 30 min.
      3. Block the membrane in 5% skimmed milk in PBS-T (see Recipes) at room temperature (RT) for 0.5-1 h.
      4. Wash the membrane with PBS-T three times for 5 min each time.
      5. Incubate the membrane in the monoclonal anti-CP antibody solution (see Recipes) by using a decolorizing orbital shaker at RT for 2 h or at 4 °C overnight.
      6. Wash the membrane with PBS-T three times for 10 min each time.
      7. Incubate in the HRP-conjugated antibody solution by using Decolorizing Orbital Shaker at RT for 1 h. Wash the membrane with PBS-T five times for 5 min each time.
      8. Use the Chemiluminescent HRP substrate to blot according to the manufacturer’s instruction.
      9. Put a single piece of x-ray film into a film cassette in a black box.
      10. Use the developing machine to automatically develop, fix, wash, and dry exposed film.

  2. Vector virus-acquisition
    1. Wash RRSV-infected rice plants confirmed as described in Procedure A with water and plant them in a square plastic box, then put the box into a clip-cage (Figure 2).


      Figure 2. The instar nymphs acquire virus on RRSV-infected rice plants. A. An overview of the feeding virus process of insect vector in RRSV-infected rice plants in the clip-cage. B. The insect vector on the diseased rice plants. C. The morphology of female BPH before (upside) and after (bottom) mating process.

    2. To prepare viruliferous BPH population with high virus-carrying rate and overcome the limited lifespan of BPH, we allow 20 female BPH adults finished in mating to be fed on RRSV-diseased rice plants, and 3-5 days later, they lay eggs on RRSV-infected rice plants. About 7 days later, nymphs began to hatch, feed on infected rice and acquire virus as early as they can.
    3. About 12 days later, after nymphs hatching, all the nymphs go through a 9-day latent period (Jia et al., 2012), and then they can be used for virus transmission.
    4. Simultaneously, we put 20 female BPH adults finished in mating to be fed on healthy rice plants to generate non-viruliferous BPH population as control vector.

  3. Preparation of rice seedlings for virus transmission
    1. Add 150-200 ml of fine soil to clean square plastic boxes and moisten the soil with 100 ml water added along the box wall.
    2. Scatter about 100 pre-germinated seeds on the soil and then sprinkle with a thin layer of soil. Put the square plastic boxes into a clip-cage (Figures 3A and 3B) and place on the tissue culture frame in a growth room at 25 °C with a photoperiod of 16 h/8 h (light/dark) with intensity 5,000-7,000 Lux until the rice seedlings have grown to 2 leaf stage, when they can be used for virus transmission.

  4. Virus transmission
    1. Allow the nymphs to feed at test seedlings for 3 days on the tissue culture frame in the growth room.
    2. Suck up viruliferous and non-viruliferous BPH nymphs by using an artificial suction-implement to test rice seedlings at three-leaf stage. The ratio of the number of nymphs and the number of seedlings is 2:1, owing to the virus carrying rate of BPH nymphs is approximately 70% (Zhang et al., 2013). 

  5. Transplanting of RRSV-inoculated rice seedlings
    Once rice seedlings inoculated with viruliferous and non-viruliferous BPH nymphs, remove the vector thoroughly from the test seedlings and then grow the rice seedlings in the greenhouse for symptom observation. As shown in Figure 3C, usually sixteen rice seedlings are transplanted into one basin evenly (Figure 3C).


    Figure 3. Preparation of test rice seedlings and transplanting of rice plants that finished virus transmission. A. Preparation of test rice seedlings in a clean square plastic box containing 150-200 ml of humid fine soil. B. The rice seedlings grown in a clip-cage to avoid vector feeding. C. Virus-inoculated rice seedlings transplanted into a basin grown in the greenhouse.

Data analysis

  1. RT-PCR and Western blot identify the RRSV-infected source plants
    RRSV-infected source plants are identified by RT-PCR with 236 bp PCR products of gene RRSV CP (Figures 4A and 4B). The expression of RRSV CP is further verified by Western blots (Figure 4C) with β-Tubulin as a loading control. These results indicate that these plants used for virus inoculation were infected by RRSV.


    Figure 4. RRSV-diseased rice plants were identified by RT-PCR and Western blots. A. RRSV-diseased rice plants with typical symptoms including stuntedness, increased tillering number and leaves that are dark green and/or have serrated edges or twisted tips. B. RT-PCR detection of RRSV CP gene for identification of RRSV-infected source plants. C. Identification of RRSV-infected source plants by Western blots. Rice actin1 and β-Tubulin were conducted as loading controls.

  2. Observation of disease symptoms
    The disease symptoms of test rice plants emerge from 20 days after inoculation. The differences of disease symptoms of test rice samples can be observed from plant height and tillering number. We usually take pictures of each rice sample at 45 days after inoculation.
  3. Incidence statistics
    The number of RRSV-diseased rice plants is counted at 28 days after inoculation. Virus inoculation is repeated three times. The RRSV-infected rice plants are identified by RT-PCR (Figure 5A). The incidence of all the test samples is compared by column diagram (Figure 5B) and significant difference is analyzed by Student's t-test. A P-value of ≤ 0.05 was considered statistically significant.


    Figure 5. Incidence statistics of RRSV-infected samples and comparison of virus content in different samples. A. RRSV-diseased rice plants were detected by RT-PCR. M, DNA marker; -, negative control detected from cDNA of healthy rice plants; +, positively control, this band was amplified from a plasmid containing RRSV CP gene. B. The disease incidence of RRSV-inoculated rice plants at 4 weeks post inoculation. C. RT-qPCR was performed to indicate the relative expression level of RRSV CP gene in different RRSV-inoculated rice plants.

  4. Make a comparison of virus content by qRT-PCR
    The severity degree of disease symptoms is always positively correlated with the virus accumulation in a virus-infected rice plant. We compare the different virus content by detecting RRSV CP gene relative expression level through qRT-PCR (Figure 5C).
    To integrally describe the whole procedure of virus inoculation assay, a process diagram is illustrated in Figure 6.


    Figure 6. An overview of RRSV propagation and infection on rice plants

Notes

  1. Finish virus inoculation before viruliferous insect vector spawning.
  2. Rice seedlings with two leaves are suitable for virus inoculation.
  3. All the diluted antibody solution can be stored at 4 °C for no longer than 1 day.

Recipes

  1. 10% PAGE gel (5 ml)
    1.9 ml ddH2O
    1.7 ml 30% acrylamide solution
    1.3 ml 1.5 M Tris-HCl (pH = 8.8)
    0.05 ml 10% (NH4)2S2O8
    0.002 ml TEMED
    Prepare 10% (NH4)2S2O8 solution by adding 100 mg (NH4)2S2O8 into 1 ml deionized water
  2. Plant total protein extraction buffer
    0.25 M Tris-HCl (pH = 6.8)
    8% β-mercaptoethanol
    20% Glycerinum
    8% SDS
  3. 10x PBS buffer
    16 g NaCl
    4 g KCl
    4 g KH2PO4
    6 g Na2HPO4•12H2O
    Dissolve in 900 ml of distilled water, make the final volume to 1,000 ml (pH = 7.5)
  4. PBS-T
    10x phosphate-buffered saline (PBS) diluted by 1:9 volume ratio, added 0.1% Tween 20
  5. Blocking solution
    5% (w/v) skimmed milk powder in PBS-T
  6. Transferring buffer
    48 mM Tris
    39 mM glycine
    20% methanol
    0.04% SDS
  7. Monoclonal anti-CP antibody solution
    Add 5 μl monoclonal anti-CP antibodies into 10 ml blocking solution

Acknowledgments

This work was supported by grants to C. Zhang and J. G. Wu from the National Natural Science Foundation of China (Nos. 31722045; 31772128; 31701757 and 31201491); the National Basic Research Program 973 (2014CB138400); and the Natural Science Foundation of Fujian Province of China, Outstanding Young Scientific Research Plan and Excellent Talent Plan in the New Century of Fujian Province (JA3091 and 2014J06011). We also appreciate the previous work related to virus propagation and infection on rice plants conducted by others.

Competing interests

There are no any conflicts of interest or competing interests.

References

  1. Hibino, H. (1979). Rice ragged stunt, a new virus disease occurring in tropical Asia. Rev Plant Prot Res 12: 98-110.
  2. Jia, D., Guo, N., Chen, H., Akita, F., Xie, L., Omura, T. and Wei, T. (2012). Assembly of the viroplasm by viral non-structural protein Pns10 is essential for persistent infection of rice ragged stunt virus in its insect vector. J Gen Virol 93(Pt 10): 2299-2309.
  3. Ling, K. C., Tiongco, E. R., and Aguiero, V. M. (1978). Rice ragged stunt, a new virus disease. Plant Dis Rep 62(8): 701-705.
  4. Zhang, C., Ding, Z., Wu, K., Yang, L., Li, Y., Yang, Z., Shi, S., Liu, X., Zhao, S., Yang, Z., Wang, Y., Zheng, L., Wei, J., Du, Z., Zhang, A., Miao, H., Li, Y., Wu, Z. and Wu, J. (2016). Suppression of jasmonic acid-mediated defense by viral-inducible microRNA319 facilitates virus infection in rice. Mol Plant 9(9): 1302-1314.
  5. Zhang, S. B., Song, G. W., Yang, L., Wu, Z. J. and Xie, L. H. (2013). Determination of Rice ragged stunt virus and vector transmission characteristics. J Fujian Agric For Uni (in Chinese).

简介

病毒接种是评估水稻品种或转基因材料对病毒感染的抗性的基本实验程序。 我们最近证明,由褐飞虱(BPH)传播的 Rice Ragged Stunt Virus >(RRSV)是一种 oryzavirus >,可以通过诱导抑制茉莉酸介导的抗病毒防御 microRNA319并促进水稻中的病毒感染。 为了验证这一点,我们通过改良的组接种方法对野生型水稻植物和miR319-TCP21相关的转基因水稻植物进行了病毒接种实验。 在这里,我们介绍了水稻植物RRSV繁殖和感染过程的详细程序。
【背景】研究病毒发病机制和筛选抗病毒水稻品种已经在克服水稻病毒病和保持粮食安全方面做了大量工作。 在该领域中,病毒接种是评估转基因材料或水稻品种的抗性的必要且可靠的方法。 水稻粗糙特技病毒>(RRSV)可以在许多亚洲国家以持续繁殖的方式通过褐飞虱传播水稻褴褛特技病(Ling et al。>,1978; Hibino,1979)。 两种经典方法,包括单苗接种和组接种,已用于进行病毒接种实验(Zhang et al。>,2013)。 通过修改传统的群体接种方法,我们提供了一种与自然感染条件非常相似的便捷方法。

关键字:水稻齿叶矮缩病毒, RRSV, 褐飞虱, 病毒接种

材料和试剂

  1. 移液器吸头(无RNase 1 ml,0.2 ml和0.02 ml,Corning,Axygen ®,产品目录号:T-1000,T-200,TF-20)
  2. 1.5毫升透明微管(Corning,Axygen ®,目录号:MCT-150-C)
  3. 夹笼(自制,40 x 30 x 35 cm,图1A)
  4. 人工吸尘器(国产,图1B)
  5. 陶瓷砂浆(外径= 120毫米)
  6. PVDF膜(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88585)
  7. 滤纸(Bio-Rad Laboratories,目录号:1703967)


    图1.本研究中使用的自制夹笼和人工吸痰器。 A.用于培育昆虫载体和病毒传播的夹笼。 B.用于转移昆虫载体的人工吸痰器。

  8. X光片
  9. RRSV感染的水稻植株(在营养生长期)
  10. 寄主植物( Oryza sativa > L. spp.japonica var.Nippobare)(所有转基因品系均为Nippobare背景)
  11. 昆虫载体( Nilaparvate lugens > Stal)(收集自中国福建省福州市的田间种群,在25°C下培养16 h / 8 h [光照/黑暗]的光周期组织培养框架);昆虫载体会在栽培过程中自然交配。 
  12. 引物
    对于β-肌动蛋白>基因:
    5'-CAGCCACACTGTCCCCATCTA-3'
    5'-AGCAAGGTCGAGACGAAGGA-3'
    对于RRSV CP >:
    5'-GAGCAAACTTGAGGCGTA-3'
    5'-AAGCTACCGTGTAGGTGGCG-3’
  13. 植物RNA试剂盒(Omega Bio-Tek,目录号:R6827)
  14. DNase I(Omega Bio-Tek,目录号:E1091)
  15. ReverTra Ace qPCR RT Kit(TOYOBO,目录号:FSQ-301)
  16. THUNDERBIRD qPCR Mix(TOYOBO,目录号:QPS-201)
  17. 液氮
  18. 琼脂糖
  19. 针对RRSV编码的CP的自制单克隆抗体
  20. ProteinFind抗β-微管蛋白小鼠单克隆抗体(TransGen Biotech,目录号:HC201-02)
  21. ProteinFind Goat Anti-Mouse IgG(H + L),HRP Conjugate(TransGen Biotech,目录号:HS201-01)
  22. 化学发光HRP底物(默克,目录号:WBKLS0100)
  23. 预染色标记
  24. 乙醇(ALADDIN,目录号:E111963)
  25. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:75746-1KG)
  26. Tris-HCl(Sigma-Aldrich,目录号:V900312)
  27. β-巯基乙醇(Sigma-Aldrich,目录号:97622)
  28. Glycerinum(Sigma-Aldrich,目录号:G5516)
  29. NaCl(Sigma-Aldrich,目录号:S7653)
  30. KCl(Sigma-Aldrich,目录号:P9333)
  31. KH 2 PO 4 (Sigma-Aldrich,目录号:P0662)
  32. Na 2 HPO 4 •12H 2 O(国药化学试剂,CAS号:10039-32-4)
  33. 脱脂奶粉(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:LP0031B)
  34. 30%丙烯酰胺溶液 
  35. (NH 4 ) 2 S 2 O 8 (Sigma-Aldrich,目录号:A3678-25G)
  36. TEMED(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:17919)
  37. Tween 20(Xilong Scientific,CAS号:9005-64-5)
  38. 甘氨酸(Solarbio,目录号:G8200)
  39. 甲醇(国药化学试剂,目录号:10014108)
  40. 10%PAGE凝胶(参见食谱)
  41. 植物总蛋白质提取缓冲液(见食谱)
  42. 10x PBS缓冲液(参见食谱)
  43. PBS-T(见食谱)
  44. 阻止解决方案(见食谱)
  45. 转移缓冲液(见食谱)
  46. 单克隆抗CP抗体溶液(见食谱)

设备

  1. Eppendorf移液器套件(1毫升,0.2毫升,0.02毫升,0.01毫升和0.0025毫升)
  2. 陶瓷砂浆
  3. 离心机(Eppendorf,型号:5424 R和5427 R)
  4. Vortex(Kylin-Bell实验室仪器,型号:VORTEX-5)
  5. PCR放大器(Bio-Rad Laboratories,型号:T100)
  6. 荧光定量PCR检测系统(Bio-Rad Laboratories,型号:CFX Connect TM )
  7. 凝胶成像系统(Bio-Rad Laboratories,型号:GelDoc TM XR +)
  8. 碱性电泳仪(Bio-Rad Laboratories,型号:PowerPac TM Basic)
  9. Mini-Protean Tetra(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Cell)
  10. 半干转移槽(Bio-Rad Laboratories,型号:Trans-Blot SD)
  11. 超微紫外分光光度计(Thermo Fisher Scientific,型号:NanoDrop TM 2000)
  12. 开发机器(Carestream Health,型号:102)
  13. 脱色轨道振动筛(Kylin-Bell Lab仪器,目录号:TS-1000)
  14. 黑盒子
  15. 胶片盒(广东粤华医疗器械厂,型号:AX-II)

软件

  1. SPSS for Windows 11.5版(IBM,Armonk,NY,USA)
  2. 凝胶成像软件(Bio-Rad Laboratories,型号:GelDoc TM XR +)
  3. Bio-Rad qPCR软件(Bio-Rad Laboratories,型号:CFX Connect TM )

程序

  1. 通过RT-PCR和Western印迹检测RRSV病株的水稻植物
    RRSV病害水稻植物的来源,表现出典型的症状,包括发育不良,分蘖过度,以及深绿色和/或有锯齿状边缘或扭曲尖端的叶子(Zhang et al。>,2016),从田间采集(福建省福州市)。通过RT-PCR和Western印迹确认RRSV的存在。
    1. RT-PCR
      1. 使用Plant RNA试剂盒(OMEGA Bio-Tek Inc.)按照制造商的说明从具有疾病症状的0.1g叶中提取总RNA。 
      2. 按照制造商的说明书,使用ReverTra Ace qPCR RT试剂盒(TOYOBO)逆转录1μg总RNA。 
      3. 然后用引物扩增RRSV CP >基因:5'-GAGCAAACTTGAGGCGTA-3'和5'-AAGCTACCGTGTAGGTGGCG-3'。
      4. 在Bio-Rad CFX Connect TM PCR系统上,在95°C的循环条件下运行PCR 5分钟,然后进行25个循环,95°C,20秒,55°C,30秒, 72°C,5秒。
      5. 用引物扩增水稻β-肌动蛋白>基因:5'-CAGCCACACTGTCCCCATCTA-3'和5'-AGCAAGGTCGAGACGAAGGA-3'作为对照。 
      6. 进行琼脂糖凝胶电泳,然后使用凝胶成像系统观察RRSV CP >基因的扩增产物。
    2. 西方印迹
      从0.1g RRSV感染的叶中提取总蛋白,将其用液氮在陶瓷研钵中研磨成粉末,然后转移到1.5ml预冷的透明微管中,然后加入100μl植物蛋白提取缓冲液。使用涡旋使样品混合均匀。然后在6,010 x g > 4℃下离心10分钟。对于RRSV检测,使用20μl制备的总蛋白进行聚丙烯酰胺凝胶电泳和Western印迹。具体步骤如下:
      1. 在PAGE凝胶的每个孔中加载20μl制备的总蛋白质以及预先染色的标记物,并在120V下运行凝胶90分钟。
      2. 准备一个略大于凝胶大小的PVDF膜。准备2张与PVDF膜一样大的滤纸。将PVDF膜,凝胶和滤纸置于1x转移缓冲液中10分钟。准备转移夹层(滤纸-PVDF膜 - 凝胶 - 滤纸)并将其置于具有3.0-5.5mA / cm 2 2 的半干转移槽中30分钟。
      3. 在室温(RT)下在PBS-T(参见配方)中用5%脱脂乳封闭膜0.5-1小时。
      4. 用PBS-T洗涤膜三次,每次5分钟。
      5. 通过在室温下使用脱色轨道振荡器将膜在单克隆抗CP抗体溶液(参见配方)中孵育2小时或在4℃过夜。
      6. 每次用PBS-T洗涤膜三次,每次10分钟。
      7. 通过在室温下使用脱色轨道振荡器在HRP缀合的抗体溶液中孵育1小时。用PBS-T洗涤膜5次,每次5分钟。
      8. 根据制造商的说明使用化学发光HRP底物进行印迹。
      9. 将一片X光胶片放入黑盒子中的胶片盒中。
      10. 使用显影机自动显影,固定,清洗和干燥曝光的胶片。

  2. 矢量病毒获取
    1. 洗涤RRSV感染的水稻植物,如程序A中所述用水确认并将其置于方形塑料盒中,然后将盒子放入夹笼中(图2)。


      图2.幼虫若虫在RRSV感染的水稻植物上获得病毒。 A.夹子笼中RRSV感染的水稻植物中昆虫载体的摄食病毒过程概述。 B.患病水稻植物上的昆虫载体。 C.雌性BPH在交配过程之前(上部)和之后(下部)的形态。

    2. 为了制备具有高病毒携带率的含毒BPH群体并克服BPH的有限寿命,我们允许20名在交配中完成的雌性BPH成年人用RRSV患病的水稻植物喂养,3-5天后,他们在RRSV上产卵感染的水稻。大约7天后,若虫开始孵化,以感染的大米为食,并尽早获得病毒。
    3. 大约12天后,在若虫孵化后,所有的若虫都经历了9天的潜伏期(贾等。>,2012),然后它们可用于病毒传播。
    4. 同时,我们将20只交配完成的雌性BPH成虫喂养健康水稻植株,以产生非毒性BPH群体作为对照载体。

  3. 用于病毒传播的水稻幼苗的制备
    1. 加入150-200毫升的细土以清洁方形塑料盒,并在箱壁上加入100毫升水润湿土壤。
    2. 在土壤上散布约100粒预先发芽的种子,然后撒上一层薄薄的土壤。将方形塑料盒放入夹笼中(图3A和3B),放置在25℃的生长室中的组织培养框架上,光周期为16小时/ 8小时(亮/暗),强度为5,000-7,000 Lux直到水稻幼苗已经长到2叶期,才可以用于病毒传播。

  4. 病毒传播
    1. 让若虫在生长室中的组织培养框架上在试验幼苗中饲养3天。
    2. 通过使用人工抽吸工具在三叶期测试水稻幼苗来吸收含有毒性和非含毒性的BPH若虫。由于BPH若虫的病毒携带率约为70%,若虫数量与幼苗数量之比为2:1(Zhang et al。>,2013)。 

  5. 移栽RRSV接种水稻幼苗
    一旦水稻幼苗接种含有毒性和非含毒性的BPH若虫,从试验幼苗中彻底除去载体,然后在温室中种植水稻幼苗用于症状观察。如图3C所示,通常将16个水稻幼苗均匀地移植到一个盆中(图3C)。


    图3.测试水稻幼苗的制备和完成病毒传播的水稻植株的移植。 :一种。在含有150-200毫升潮湿细土的干净方形塑料盒中制备试验稻苗。 B.在夹子笼中生长的水稻幼苗以避免载体摄食。 C.将接种病毒的水稻幼苗移植到温室中生长的盆中。

数据分析

  1. RT-PCR和Western印迹鉴定RRSV感染的来源植物
    通过RT-PCR用基因RRSV CP >的236bp PCR产物鉴定RRSV感染的来源植物(图4A和4B)。通过Western印迹进一步验证RRSV CP的表达(图4C),其中β-微管蛋白作为上样对照。这些结果表明用于病毒接种的这些植物被RRSV感染。


    图4.通过RT-PCR和蛋白质印迹鉴定RRSV病株。 A.具有典型症状的RRSV病株,包括矮小,分蘖数增加和深绿色叶片和/或有锯齿状边缘或扭曲尖端。 B.RTSV CP >基因的RT-PCR检测,用于鉴定RRSV感染的来源植物。 C.通过蛋白质印迹鉴定RRSV感染的来源植物。水稻 actin1 >和β-微管蛋白作为上样对照进行。

  2. 观察疾病症状
    试验稻植物的疾病症状在接种后20天出现。从植物高度和分蘖数可以观察到试验稻样品的疾病症状的差异。我们通常在接种后45天拍摄每个大米样本的照片。
  3. 发病率统计
    在接种后28天计数RRSV患病水稻植物的数量。病毒接种重复三次。通过RT-PCR鉴定RRSV感染的水稻植物(图5A)。通过柱图比较所有测试样品的发生率(图5B),并通过Student's t > -test分析显着差异。 P > - 值≤0.05被认为具有统计学意义。


    图5. RRSV感染样本的发病率统计数据和不同样本中病毒含量的比较。 A.通过RT-PCR检测RRSV病株。 M,DNA标记; - ,从健康水稻植物的cDNA中检测到阴性对照; +,阳性对照,从含有RRSV CP >基因的质粒扩增该条带。 B.接种后4周RRSV接种的水稻植物的发病率。 C.进行RT-qPCR以指示RRSV CP >基因在不同RRSV接种水稻植物中的相对表达水平。

  4. 通过qRT-PCR比较病毒含量
    疾病症状的严重程度始终与病毒感染的水稻植物中的病毒积累正相关。我们通过qRT-PCR检测RRSV CP >基因相对表达水平来比较不同的病毒含量(图5C)。
    为了整体描述病毒接种分析的整个过程,过程图如图6所示。


    图6. RRSV在水稻上的繁殖和感染概述

笔记

  1. 在有毒昆虫载体产卵之前完成病毒接种。
  2. 具有两片叶子的水稻幼苗适合于病毒接种。
  3. 所有稀释的抗体溶液均可在4°C下保存不超过1天。

食谱

  1. 10%PAGE凝胶(5毫升)
    1.9毫升ddH 2 O
    1.7毫升30%丙烯酰胺溶液
    1.3ml 1.5M Tris-HCl(pH = 8.8)
    0.05毫升10%(NH 4 ) 2 S 2 O 8
    0.002毫升TEMED
    通过加入100mg(NH )制备10%(NH 4 ) 2 S 2 O 8 溶液。 4 ) 2 S 2 O 8 加入1 ml去离子水
  2. 植物总蛋白提取缓冲液
    0.25M Tris-HCl(pH = 6.8)
    8%β-巯基乙醇
    20%甘油
    8%SDS
  3. 10x PBS缓冲液
    16克NaCl
    4克KCl
    4 g KH 2 PO 4
    6 g Na 2 HPO 4 •12H 2 O
    溶于900毫升蒸馏水中,最终体积为1000毫升(pH = 7.5)
  4. PBS-T
    以1:9的体积比稀释10倍磷酸盐缓冲盐水(PBS),加入0.1%吐温20
  5. 阻止解决方案
    PBS-T中的5%(w / v)脱脂奶粉
  6. 转移缓冲区
    48 mM Tris
    39 mM甘氨酸
    20%甲醇
    0.04%SDS
  7. 单克隆抗CP抗体溶液
    将5μl单克隆抗CP抗体加入10 ml封闭液中

致谢

这项工作得到了国家自然科学基金委员会的C. Zhang和J. G. Wu的资助(第31722045号; 31772128; 31701757和31201491);国家基础研究计划973(2014CB138400);和福建省自然科学基金,福建省新世纪杰出青年科研计划和优秀人才计划(JA3091和2014J06011)。我们还赞赏以前与其他人进行的水稻病毒繁殖和感染有关的工作。

利益争夺

没有任何利益冲突或竞争利益。

参考

  1. Hibino,H。(1979)。 稻米褴褛特技,一种在亚洲热带地区发生的新病毒病。 Rev Plant Prot Res > 12:98-110。
  2. Jia,D.,Guo,N.,Chen,H.,Akita,F.,Xie,L.,Omura,T。和Wei,T。(2012)。 通过病毒非结构蛋白Pns10装配病毒对于水稻粗糙特技病毒的持续感染至关重要在昆虫载体中。 J Gen Virol > 93(Pt 10):2299-2309。
  3. Ling,K.C.,Tiongco,E.R。和Aguiero,V。M.(1978)。 稻米褴褛特技,一种新的病毒病。 Plant Dis Rep > 62(8):701-705。
  4. Zhang,C.,Ding,Z.,Wu,K.,Yang,L.,Li,Y.,Yang,Z.,Shi,S.,Liu,X.,Zhao,S.,Yang,Z。, Wang,Y.,Zheng,L.,Wei,J.,Du,Z.,Zhang,A.,Miao,H.,Li,Y.,Wu,Z。和Wu,J。(2016)。 通过病毒诱导的microRNA319抑制茉莉酸介导的防御有助于水稻中的病毒感染。 Mol Plant > 9(9):1302-1314。
  5. Zhang,S。B.,Song,G。W.,Yang,L.,Wu,Z。J. and Xie,L。H.(2013)。 确定水稻粗糙特技病毒和病媒传播特征。 J福建农业大学>(中文)。
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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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