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Rice Black-streaked Dwarf Virus Preparation and Infection on Rice
水稻黑条矮缩病病毒的制备及其对水稻的感染   

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本实验方案简略版
Plant & Cell Physiology
Apr 2015

Abstract

Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.

Keywords: Rice black-streaked dwarf virus (水稻黑条矮缩病毒), Small brown planthopper (灰飞虱), Virus inoculation (病毒接种), RT-PCR (RT-PCR), DIBA detection (DIBA检测)

Background

Rice black-streaked dwarf virus (RBSDV), a recognized member of the second group within the genus Fijivirus, family Reoviridae, seriously affects the production of rice and maize in East Asia (Shikata and Kitagawa, 1977; Zhang et al., 2001a). RBSDV is propagatively transmitted to rice, maize, barley and wheat by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner (Shikata and Kitagawa, 1977). SBPH can acquire RBSDV from fresh or frozen infected rice leaves and transmit it to healthy plants (Shikata and Kitagawa, 1977; Li et al., 2010). Once SBPH has acquired the virus, the virus can multiply in the insect vector and can be transmitted to host plants throughout the life of the insect. However, SBPH cannot transmit the virus transovarially (Boccardo and Milne, 1984). RBSDV has icosahedral, double-layered particles about 70-75 nm in diameter and 10 linear genomic segments of double-stranded RNA (dsRNA), ranging in size from approximately 1.8 to 4.5 kb (Marzachi et al., 1995; Zhang et al., 2001b). Our previous studies have shown the complexity of the interaction between RBSDV and host plants (Sun et al., 2015; He et al., 2017). Unfortunately, the numerous genomic segments make it difficult to construct infectious cDNA clones for reverse genetics studies. To facilitate the investigation of the interaction between RBSDV and its host plants, we describe a simple and efficient method for maintaining RBSDV infection in a greenhouse, including the propagation of the healthy planthopper population, preparation of rice plants, RBSDV inoculations and finally the evaluation of the rice disease symptom after inoculation.

Materials and Reagents

  1. Filter paper (Sangon Biotech, catalog number: F503311 )
  2. Fine soil
  3. Gauze and rubber band
  4. RNase-free tube (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12400 )
  5. RNase-free tip of pipettes (Sangon Biotech, catalog number: F611216 )
  6. Brush
  7. Washing bottle (Sangon Biotech, catalog number: F505001-001 )
  8. Pooter
  9. Black cloth
  10. Toothpick
  11. Fresh leaves (collected from 30-day-old rice)
  12. RNA extraction reagent
    1. Trizol reagent kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
    2. Chloroform (ShangHai LingFeng Chemical Reagent, catalog number: 1519021)
    3. 75% ethanol (Singpharm Chemical Chemical Reagent, catalog number: 10009257 )
    4. RNase-free double distilled H2O
    Note: All stored at 4 °C.
  13. Reverse transcription kit (Vazyme Biotech, catalog number: R223-01 , stored at -20 °C)
  14. Sterilized beads (Sangon Biotech, catalog number: B529319-0025 )
  15. TAE buffer (Sangon Biotech, catalog number: B548101-0500 )
  16. Agarose (YEASEN, catalog number: 10208ES60 )
  17. DNA maker 2,000 bp (Takara Bio, catalog number: 3427A )
  18. Ethidium bromide (Sangon Biotech, catalog number: A600195 )
  19. Anti-P10
  20. Horseradish peroxidase-labeled goat antimouse immunoglobulin G (YEASEN, catalog number: 33201ES60 )
  21. TMB color-substrate solution (Sangon Biotech, catalog number: C510025-005 )
  22. Sodium carbonate (Na2CO3)
  23. Sodium bicarbonate (NaHCO3)
  24. Sodium chloride (NaCl)
  25. Potassium chloride (KCl)
  26. Potassium phosphate monobasic (KH2PO4)
  27. Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O)
  28. 5% skimmed milk powder (Sangon Biotech, catalog number: A600669 )
  29. 4-Chloro-1-naphthol
  30. 30% H2O2
  31. Fertilizer solution (containing 5% carbamide)
  32. Carbonate coating solution (see Recipes)
  33. 10x phosphate-buffered saline (PBS) (see Recipes)
  34. PBS-T (see Recipes)
  35. Blocking solution (see Recipes)
  36. TMB color-substrate solution (see Recipes)

Equipment

  1. 1 L beakers (Sangon Biotech, catalog number: F606544-0001 )
  2. PCR machine (Thermo Fisher Scientific, Applied BiosystemsTM, model: VeritiTM 96-Well Thermal Cycler )
  3. Pipettes
  4. Glass bottle with blue cap (Sangon Biotech, catalog number: F505041 )
  5. Microwave oven (Sangon Biotech, catalog number: G003408-0001 )
  6. Electrophoresis tank (Sangon Biotech, catalog number: G500310-0001 )
  7. Gel imaging system
  8. Eppendorf centrifuge (Thermo Fisher Scientific, for use at room temperature and 4 °C)
  9. Micro vacuum concentrator (Gene Company Limited)
  10. Automatic sample grinding machine (Shanghai Jingxin Industrial Development, model: Tissuelyser-96 )

Software

  1. Gel imaging software

Procedure

  1. Preparation of seedlings for raising insects
    1. Prior to inoculation of RBSDV, 80-100 rice seedlings are needed to obtain a big population of the small brown planthoppers. Plump seeds of the rice variety ‘Wuyujing 3’ are pre-germinated by immersing them in water for 2 days, rinsing thoroughly with water and then allowing them to germinate on water-soaked filter paper for 1 day.
    2. 150-200 ml of fine soil is added to clean 1 L beakers and moistened with 100 ml water added along the beaker wall.
    3. About 100 pre-germinated seeds are then scattered on the soil and sprinkled with a thin layer of soil. The beakers are covered with gauze (Figure 1) and placed on the shelf in a growth room at 25-27 °C, relative humidity 50%-70%, 14/24 h light with intensity 5,000-7,000 lux until the rice seedlings have grown to 6 cm (about 10-day-old seedlings), when they can be used for feeding the small brown planthoppers (Figure 2).


      Figure 1. Cultivating rice seedlings in beakers


      Figure 2. Planted rice seedlings indoors

  2. The source of RBSDV infected rice plants RBSDV-infected rice plants (Figure 3) are collected from the field (Jining city, Shandong Province) and the presence of RBSDV is confirmed by RT-PCR. Total RNA is extracted from leaves with symptoms using Trizol Reagent. Reverse transcription is performed using the reverse transcription kit with random primers, following the instructions supplied (Vazyme). The primers used were designed from the published sequence of S10 of RBSDV (S10-Forward: AAC AAC CGA CCA ACA ATC AC and S10-Reverse: GAG CAG GAA CTT CAC GAC AG). The detailed steps are as follows:
    1. Reverse transcription (10 μl), the aim of this step is to reverse transcribe the RNA into cDNA.
      1. Remove Genomic DNA (the reaction in the RNase-free tube) using the following reaction:
        RNase-free ddH2O
        to 8 μl
        4x gDNA mix (containing DNase)
        2 μl
        Total RNA
        1-500 ng
        Mix the solution using pipettes and then heat at 42 °C for 2 min.
      2. Conduct the reverse transcription reaction as follows:
        5x HiScript II Q Select RT SuperMix
        2 μl
        Mixed solution from step B1a
        8 μl
        Mix the solution with pipettes and then place in the PCR machine for the following procedure:
        25 °C
        10 min
        50 °C
        30 min
        85 °C
        5 min
    2. PCR amplification
      1. Prepare a mixture as follows:
        2x HieffTM PCR Master Mix
        5 μl
        Forward primer
        0.3 μl
        Reverse primer
        0.3 μl
        cDNA from reaction above
        0.5 μl
        RNase-free ddH2
        To 10 μl
        Mix the solution fully, put it into the PCR machine and set up the following amplification
        94 °C
        3 min


        94 °C
        30 sec


        58 °C
        30 sec
        35 cycles
        72 °C
        30 sec

        72 °C
        10 min


        4 °C
        End


      2. Agarose gel electrophoresis
        1. Preparation of gels: Firstly, weigh 1 g of agarose into a glass bottle with blue cap. Secondly, add 100 ml TAE buffer, and heat in a microwave oven until completely dissolved.
        2. Production of plastic sheet: After the above-mentioned agarose gel has cooled to about 65 °C, add the right amount of ethidium bromide dye and then pour into a transparent plate with a pre-inserted comb.
        3. Add sample: Firstly, put the agarose gel plate into an electrophoresis tank and gently move the plate to squeeze out the bubbles within the discharge hole. Then, add 5 μl of DNA marker to the first well followed by the PCR products in turn to the following wells. To avoid cross-contamination between samples, use a new pipette tip for each sample. Finally, connect to the power supply and adjust the voltage to 150-180 V.
        4. Observation: Turn off the power when the front of the band has reached about halfway down the gel, remove the plate and gently dry the electrophoresis buffer. Then put the gel into a gel imaging system to observe.


          Figure 3. RBSDV infected rice plants

  3. Propagation of the healthy planthopper population
    It takes about a month for planthoppers to complete their life cycle which consists of 5 instar nymph stages (Figure 4A), and 1 adult stage. The fifth instar nymphs (recognized through the short black line on the abdomen of the female) have the ability to spawn. The adults are transferred to fresh ‘Wuyujing 3’ rice seedlings (100-150 planthoppers/beaker) with the pooter (Figure 4B), allowed to spawn for 48 h (Figure 4C), and the adults are then removed with a brush. Generally, small white hatched nymphs will be seen on the rice seedlings 7-10 days later (Figure 4D). The nymphs on the seedlings can be used to acquire RBSDV. It is advised to restrict the watering of rice seedlings before collecting nymphs.


    Figure 4. The adult planthoppers spawned on ‘Wuyujing 3’ rice seedlings. A. The different stage of planthoppers; B. The pooter used in this study; C. The adult planthoppers were spawning; D. Hatched out the first and second instar nymphs.

  4. Rice black-streaked dwarf virus inoculations
    1. Several RBSDV-infected rice plants prepared as described in Procedure B are washed with water and planted in a 3 L beaker.
    2. First to second instar nymphs prepared as described in Procedure C are collected from the rice plants on which they have been reared, and released onto the RBSDV-infected rice plants (about 150-200 planthoppers/plant) for 3-5 days to acquire virus (Figure 5).
    3. Subsequently, the planthoppers are transferred to healthy ‘Wuyujing 3’ rice seedlings for an incubation period of 10-12 days. The proportion of viruliferous planthoppers is then assessed by using a dot immunobinding assay (DIBA) with antibodies against the viral structural proteins (Zhou and Liu, 2004; Yang et al., 2007) (see Procedure E).
    4. For RBSDV inoculation, planthoppers are transferred by pooter to test seedlings at the 2-3 leaf stage and allowed to feed for 3 days. The numbers transferred are calculated to ensure that three viruliferous adult planthoppers are present on each seedling. The planthoppers are then removed and the seedlings are grown in the greenhouse for symptom observation.


      Figure 5. The instar nymphs acquire virus on RBSDV infected rice plants

  5. Dot Immunobinding Assay (DIBA) To determine the viruliferous rate of the SBPH population, the method for DIBA is used for the detection of RBSDV in single SBPH. The detailed steps are as follows:
    1. Put a single SBPH into a fresh centrifuge tube (500 μl) and add 80 μl carbonate coating solution. Then use a toothpick to mash the insect and pipette 2 μl supernatant onto the NC membrane. Dry it at room temperature.
    2. Immerse the NC membrane into PBS-T blocking solution containing 5% skimmed milk in the condition (37 °C, 50 rpm, shake 1 h).
    3. Transfer the NC membrane into blocking solution containing anti-P10 (1:1,000) at the condition (37 °C, 50 rpm, shake 2 h). Wash the membrane 3 times with PBS-T for 10 min each time.
    4. Transfer the NC membrane into blocking solution containing horseradish peroxidase-labeled goat antimouse immunoglobulin G (1:5,000) at the condition (37 °C, 50 rpm, shake 1.5 h). Wash the membrane 3 times with PBS-T for 10 min each time.
    5. Remove the PBS-T solution from the NC membrane with filter paper and cover the NC membrane with TMB color-substrate solution. React for 5-15 min in the dark until the positive control is conspicuous.

  6. Symptom evaluation after RBSDV inoculation Symptoms on the inoculated rice seedlings (Procedure D) are assessed 30 days after transfer to the glasshouse by measuring the plant height and by RT-PCR (see Procedure B).

Data analysis

  1. The RBSDV infected rice plants used as source plants are identified by RT-PCR. Figure 6 shows an example of the 517 bp PCR products amplified by the S10 primers. These results suggest that most plants collected from fields were infected by RBSDV.


    Figure 6. Bands amplified by RT-PCR from ten separate rice plants collected from the field as potential sources of RBSDV for inoculation experiments. M: Marker; 1-9: RBSDV infected rice plants; +: Positive control; -: Negative control.

  2. After a 10-12 days incubation period, DIBA is used to assess the proportion of viruliferous planthoppers (Figure 7). In this example, 60 planthoppers were tested, of which 21 (35%) were infected with RBSDV. Based on these results, eight planthoppers were used to inoculate each of the test rice seedlings.


    Figure 7. DIBA assay to determine the proportion of viruliferous planthoppers. +: Positive control; -: Negative control; Black spots, viruliferous planthoppers.

  3. RBSDV infected rice plants show significant dwarfing symptoms compared with the control (Figure 8A). RT-PCR is used to confirm whether the dwarf rice is infected by RBSDV (Figure 8B). In this example, all 22 inoculated rice seedlings were tested and the 17 dwarfed plants were all shown to be infected by RBSDV, while the other plants were healthy. This infection rate of 77% indicates that the inoculation of RBSDV on rice through planthoppers in the greenhouse is feasible and efficient. The overall process of RBSDV inoculation by SBPH is illustrated in Figure 9.
     

    Figure 8. The symptoms and detection of RBSDV 30 days after inoculation using viruliferous insect vectors. A. Healthy (CK) and RBSDV-infected (RB) plants; B. RT-PCR results showing the detection of RBSDV in the 22 inoculated rice seedlings. M: Marker; +: Positive control; -: Negative control.
     

    Figure 9. The overall process of RBSDV inoculation by SBPH

Notes

Keep the soil of growing rice seedlings dry when collecting the nymphs.

Recipes

  1. Carbonate coating solution
    1.59 g Na2CO3
    2.93 g NaHCO3
    Dissolve in 900 ml of distilled water, make the constant volume to 1,000 ml (pH = 9.6)
  2. 10x phosphate-buffered saline (PBS)
    16 g NaCl
    4 g KCl
    4 g KH2PO4
    6 g Na2HPO4·12H2O
    Dissolve in 900 ml of distilled water, make the constant volume to 1,000 ml (pH = 7.5)
  3. PBS-T
    10x PBS diluted by 1:9 volume ratio, added 0.1% Tween 20
  4. Blocking solution
    5% (w/v) skimmed milk powder in PBS-T
  5. TMB color-substrate solution
    3 mg 4-chloro-1-naphthol
    2 ml ethanol
    7 μl 30% H2O2
    10 ml PBS
    Adjust pH to 7.5

Acknowledgments

We thank Professor Yijun Zhou (Jiangsu Academy of Agricultural Sciences, Nanjing, China) and his team for providing us with the healthy planthoppers, and Jianxiang Wu (Zhejiang University) for providing antibody. We thank Professor M. J. Adams, Stevenage, UK for the help in correcting the English of the manuscript. The protocol was modified from previous works, including Zhang et al. (2001a), Sun et al. (2015), and He et al. (2017) and has been successfully used for studies on viral characterization and virus-plant interaction. All authors declare no any conflicts of interest.

References

  1. Boccardo, G. and Milne, R. G. (1984). Plant reovirus group. CMI/AAB, Descriptions of Plant Viruses No.294, Commonwealth Microbiology Institute and Association of Applied Biology. United Kingdom.
  2. He, Y., Zhang, H., Sun, Z., Li, J., Hong, G., Zhu, Q., Zhou, X., MacFarlane, S., Yan, F. and Chen, J. (2017). Jasmonic acid-mediated defense suppresses brassinosteroid-mediated susceptibility to rice black streaked dwarf virus infection in rice. New Phytol 214(1): 388-399.
  3. Li, L., Li, H. W., Dong, H. B., Wang, X. F. and Zhou, G. H. (2010). Transmission by Laodelphax striatellus fallen of rice black streaked dwarf virus from frozen infected rice leaves to healthy plants of rice and maize. J Phytopathol 159: 1-5.
  4. Marzachi, C., Boccardo, G., Milne, R., Isogai, M. and Uyeda, I. (1995). Genome structure and variability of fijiviruses. Seminars in Virology 6: 103-108.
  5. Shikata, E. and Kitagawa, Y. (1977). Rice black-streaked dwarf virus: its properties, morphology and intracellular localization. Virology 77(2): 826-842.
  6. Sun, Z., He, Y., Li, J., Wang, X. and Chen, J. (2015). Genome-wide characterization of rice black streaked dwarf virus-responsive microRNAs in rice leaves and roots by small RNA and degradome sequencing. Plant Cell Physiol 56(4): 688-699.
  7. Yang, J., Zhang, H. M., Chen, J. P. and Dai, L. Y. (2007). Prekaryotic expression antiserum preparation and some properties of p8 protein of rice black-streaked dwarf fijivirus. Acta Phytophylacica Sinica 34(3): 252-256.
  8. Zhang, H. M., Chen, J. P. and Adams, M. J. (2001a). Molecular characterization of segments 1 to 6 of rice black-streaked dwarf virus from China provides the complete genome. Arch Virol 146: 2331-2339.
  9. Zhang, H. M., Chen, J. P., Lei, J. L. and Adams, M. J. (2001b). Sequence analysis shows that a dwarfing disease on rice, wheat and maize in china is caused by rice black-streaked dwarf virus. Eur J Plant Pathol 107: 563-567.
  10. Zhou, Y. J. and Liu, H. J. (2004). Immunoassay of rice stripe virus carried by Laodelphax striatellus. Jiangsu Agricultural Sciences 1: 50-51.

简介

水稻黑条矮缩病毒(RBSDV)属于呼肠孤病毒科的Fijivirus属的成员,感染水稻,玉米,大麦和小麦,可严重影响作物产量。 RBSDV以持久的方式由小型褐飞虱( Ladellphax striatellus ,SBPH)传播。 RBSDV具有10个线性dsRNA基因组区段,使得构建用于植物功能研究的感染性克隆变得困难。 在这里我们介绍一种在温室中用于在实验室研究中使用的RBSDV接种和维持方法。 该协议使用在实验室饲养的大量的SBPHs。 我们还详细描述了一个健康的稻飞虱种群的繁殖,植物材料的制备,RBSDV接种和接种后水稻的评价。

【背景】水稻黑条矮缩病毒(RBSDV)是Fijivirus属家族中的第二个家族中的公认成员,严重影响东亚水稻和玉米的生产。 (Shikata和Kitagawa,1977; Zhang等人,2001a)。 RBSDV以持续的方式(Shikata和Kitagawa,1977)被小型褐飞虱(灰飞虱,SBPH)传播传播到水稻,玉米,大麦和小麦。 SBPH可从新鲜或冷冻感染的水稻叶片获得RBSDV并将其传递至健康植物(Shikata和Kitagawa,1977; Li等人,2010)。一旦SBPH已经获得病毒,病毒可以在昆虫载体中繁殖,并且可以在昆虫的整个生命周期中传播给宿主植物。然而,SBPH不能透传病毒(Boccardo and Milne,1984)。 RBSDV具有直径约70-75nm的二十面体,双层颗粒和10个双链RNA(dsRNA)的线性基因组区段,大小范围从约1.8至4.5kb(Marzachi等人)。 ,1995; Zhang等人,2001b)。我们之前的研究已经显示了RBSDV与寄主植物之间相互作用的复杂性(Sun等人,2015; He等人,2017)。遗憾的是,众多的基因组片段使得难以构建用于反向遗传学研究的感染性cDNA克隆。为了便于调查RBSDV与其寄主植物之间的相互作用,我们描述了一种简单而有效的方法,用于维持温室中的RBSDV感染,包括健康的稻飞虱种群的繁殖,水稻的制备,RBSDV接种以及最终的接种后的稻病症状。

关键字:水稻黑条矮缩病毒, 灰飞虱, 病毒接种, RT-PCR, DIBA检测

材料和试剂

  1. 滤纸(Sangon Biotech,目录号:F503311)
  2. 细土
  3. 纱布和橡皮筋
  4. 无RNA酶管(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12400)
  5. 无RNA酶的吸头(Sangon Biotech,目录号:F611216)
  6. 刷子
  7. 洗瓶(Sangon Biotech,产品目录号:F505001-001)
  8. Pooter
  9. 黑布
  10. 牙签
  11. 新鲜的叶子(从30天的大米收集)
  12. RNA提取试剂
    1. Trizol试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:15596018)
    2. 氯仿(上海凌峰化学试剂有限公司CAS:67-66-3)
    3. 75%乙醇(Singpharm Chemical Chemical Reagent,目录号:10009257)
    4. 无RNase双蒸H 2 O
    注意:所有储存在4°C。
  13. 逆转录试剂盒(Vazyme Biotech,目录号:R223-01,保存于-20°C)
  14. 灭菌珠(Sangon Biotech,目录号:B529319-0025)
  15. TAE缓冲液(Sangon Biotech,目录号:B548101-0500)
  16. 琼脂糖(YEASEN,目录号:10208ES60)
  17. DNA生产商2,000 bp(Takara Bio,目录号:3427A)
  18. 溴化乙锭(Sangon Biotech,目录号:A600195)
  19. 反P10
  20. 辣根过氧化物酶标记的山羊抗小鼠免疫球蛋白G(YEASEN,目录号:33201ES60)
  21. TMB彩色底物溶液(Sangon Biotech,目录号:C510025-005)
  22. 碳酸钠(Na 2 CO 3)
  23. 碳酸氢钠(NaHCO 3)
  24. 氯化钠(NaCl)
  25. 氯化钾(KCl)
  26. 磷酸二氢钾(KH 2 PO 4)
  27. 磷酸氢二钠十二水合物(Na 2 HPO 4•12H 2 O)
  28. 5%脱脂奶粉(Sangon Biotech,目录编号:A600669)
  29. 4-氯-1-萘酚
  30. 30%H 2 O 2
  31. 肥料溶液(含5%尿素)
  32. 碳酸盐涂层溶液(见食谱)
  33. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  34. PBS-T(见食谱)
  35. 阻止解决方案(见食谱)
  36. TMB彩色基板解决方案(见食谱)

设备

  1. 1升烧杯(Sangon Biotech,产品目录号:F606544-0001)
  2. PCR仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Veriti TM 96-Well热循环仪)
  3. 移液器
  4. 带蓝色瓶盖的玻璃瓶(Sangon Biotech,目录号:F505041)
  5. 微波炉(Sangon Biotech,目录号:G003408-0001)

软件

  1. 凝胶成像软件

程序

  1. 准备饲养昆虫的幼苗
    1. 在接种RBSDV之前,需要80-100个水稻幼苗以获得大量的小褐飞虱。水稻品种“武育粳3号”的丰满种子通过将其浸入水中预发芽2天,用水彻底冲洗,然后使其在水浸滤纸上发芽1天。
    2. 加入150-200毫升细土,清洗1升烧杯,沿烧杯壁加100毫升水。
    3. 然后将约100个预发芽的种子散布在土壤上,撒上一层薄薄的土壤。用纱布覆盖烧杯(图1),置于25-27℃,相对湿度50%-70%,14/24小时光照强度为5,000-7,000勒克斯的生长室的架子上,直到水稻幼苗具有长到6厘米(约10日龄的幼苗),当它们可以用于饲养小褐飞虱(图2)。


      图1.在烧杯中培育水稻幼苗


      图2.种植在室内的秧苗

  2. RBSDV感染的稻株的来源 RBSDV感染的稻株(图3)来自山东省济宁市,通过RT-PCR确认RBSDV的存在。使用Trizol试剂从具有症状的叶中提取总RNA。按照提供的说明(Vazyme),使用带有随机引物的逆转录试剂盒进行逆转录。所使用的引物根据RBSDV(S10-Forward:AAC AAC CGA CCA ACA ATC AC和S10-Reverse:GAG CAG GAA CTT CAC GAC AG)的公开的S10的序列设计。具体步骤如下:
    1. 逆转录(10μl),此步骤的目的是将RNA逆转录成cDNA。
      1. 使用以下反应去除基因组DNA(在无RNA酶的管中的反应):
        RNase-free ddH2O
        to 8 μl
        4x gDNA mix (containing DNase)
        2 μl
        Total RNA
        1-500 ng
        使用移液器混合溶液,然后在42°C加热2分钟。
      2. 进行如下反转录反应:
        5x HiScript II Q Select RT SuperMix
        2 μl
        Mixed solution from step B1a
        8 μl
        用移液器混合溶液,然后放入PCR仪进行以下操作:
        25 °C
        10 min
        50 °C
        30 min
        85 °C
        5 min
    2. PCR扩增
      1. 准备一个混合物如下:
        2x HieffTM PCR Master Mix
        5 μl
        Forward primer
        0.3 μl
        Reverse primer
        0.3 μl
        cDNA from reaction above
        0.5 μl
        RNase-free ddH2O 
        To 10 μl
        Mix the solution fully, put it into the PCR machine and set up the following amplification
        94 °C
        3 min


        94 °C
        30 sec


        58 °C
        30 sec
        35 cycles
        72 °C
        30 sec

        72 °C
        10 min


        4 °C
        End


      2. 琼脂糖凝胶电泳
        1. 凝胶的制备:首先,将1克琼脂糖称重到带有蓝色盖的玻璃瓶中。其次,加入100毫升TAE缓冲液,并在微波炉中加热直至完全溶解。
        2. 生产塑料片:将上述琼脂糖凝胶冷却至约65℃后,加入适量的溴化乙锭染料,然后倒入预先插入梳子的透明板中。
        3. 添加样品:首先,将琼脂糖凝胶板放入电泳槽,轻轻移动,排出孔内的气泡。然后,向第一个孔中加入5μlDNA标记,接着将PCR产物依次加入下列孔中。为避免样品之间的交叉污染,请为每个样品使用新的移液器吸头。最后,连接电源并将电压调整到150-180 V.
        4. 观察:当条带正面到达凝胶的一半时,关掉电源,取下平板,轻轻地将电泳缓冲液干燥。然后将凝胶放入凝胶成像系统观察。


          图3. RBSDV感染的稻株

  3. 繁殖健康的飞虱种群
    飞虱完成其生活周期大约需要一个月,其中包括5龄若虫阶段(图4A)和1个成年阶段。第五龄若虫(通过雌性腹部的短黑线识别)具有产卵能力。将成虫转移到新鲜的“武育粳3号”稻苗(100-150只飞虱/烧杯)(图4B),产卵48小时(图4C),然后用刷子除去成虫。一般来说,7-10天后在稻苗上会看到小的白色若虫(图4D)。幼苗上的若虫可以用来获得RBSDV。建议在收集若虫前限制水稻幼苗的浇水。


    图4“武育粳3号”水稻幼苗产卵后的成虫飞虱A.飞虱的不同阶段; B.本研究中使用的小猪C.成虫是产卵的; D.孵出第一和第二龄若虫。

  4. 水稻黑条矮缩病毒接种
    1. 将如程序B所述制备的几种RBSDV感染的稻植物用水洗涤并种植在3L烧杯中。
    2. 按照程序C所述制备的第一至第二龄若虫从其饲养的稻植物中收集,并释放到RBSDV感染的稻植物(约150-200株飞虱/植物)上3-5天以获得病毒(图5)。
    3. 随后,将飞虱转移至健康的“武育粳3号”水稻幼苗,培养期为10-12天。然后通过使用针对病毒结构蛋白的抗体的斑点免疫结合分析(DIBA)来评估病毒性飞虱的比例(Zhou和Liu,2004; Yang等人,2007)(参见程序E) 。
    4. 对于RBSDV接种,通过小叶蝉转移飞蛾以测试2-3叶期的幼苗并允许饲养3天。计算转移的数量,以确保每个苗上存在三个毒性成虫。然后将飞虱取出,并在温室中种植幼苗以进行症状观察。


      图5.若虫若虫在感染RBSDV的水稻上获得病毒

  5. 斑点免疫结合分析(DIBA) 为了确定SBPH群体的病毒率,采用DIBA方法检测单个SBPH中的RBSDV。具体步骤如下:
    1. 将一个单一的SBPH放入一个新鲜的离心管(500μl)中并加入80μl碳酸盐涂层溶液。然后用牙签捣碎昆虫,吸取2μl上清液到NC膜上。在室温下干燥。
    2. 在37℃,50rpm,摇动1小时的条件下,将NC膜浸入含有5%脱脂乳的PBS-T封闭溶液中。
    3. 在37°C,50 rpm,摇动2 h的条件下,将NC膜转移至含有抗P10(1:1,000)的封闭溶液中。每次用PBS-T清洗膜3次,每次10分钟。
    4. 将NC膜转移至含有辣根过氧化物酶标记的山羊抗小鼠免疫球蛋白G(1:5,000)的封闭液中(37℃,50rpm,振荡1.5h)。每次用PBS-T清洗膜3次,每次10分钟。
    5. 用滤纸从NC膜上取下PBS-T溶液,用TMB色底物溶液覆盖NC膜。在黑暗中反应5-15分钟,直到阳性对照显着。

  6. RBSDV接种后的症状评估 通过测量植物高度并通过RT-PCR(参见程序B)在转移至温室后30天评估接种的稻秧(步骤D)上的症状。

数据分析

  1. 通过RT-PCR鉴定用作来源植物的RBSDV感染的稻植物。图6显示了由S10引物扩增的517bp PCR产物的实例。这些结果表明大多数从田间收集的植物都被RBSDV感染。



    图6.从田间采集的十个单独的稻植物通过RT-PCR扩增的带作为用于接种实验的RBSDV的潜在来源。 1-9:RBSDV感染的稻植物; +:阳性对照; - :负面控制。

  2. 经过10-12天的潜伏期后,用DIBA评估病毒性飞虱的比例(图7)。在这个例子中,测试了60只飞虱,其中21只(35%)感染了RBSDV。根据这些结果,八个飞虱被用来接种每个测试水稻幼苗。


    图7.用DIBA法测定病毒飞虱的比例。+:阳性对照; - :负面控制;黑斑,带毒的飞虱。

  3. 与对照相比,RBSDV感染的稻植物显示显着的矮化症状(图8A)。用RT-PCR来确认矮化的稻是否被RBSDV感染(图8B)。在这个例子中,测试了所有22个接种的水稻幼苗,并且17个矮化的植物都显示出受RBSDV感染,而其他植物是健康的。 77%的感染率表明,通过温室中的稻飞虱接种RBSDV是可行和有效的。图9说明了SBPH接种RBSDV的整个过程。
     

    A.健康的(CK)和RBSDV感染的(RB)植物; B. B.RT-PCR结果显示在22个接种的稻苗中检测到RBSDV。 M:标记; +:阳性对照; - :负面控制。
     

    图9. SBPH接种RBSDV的总体过程

笔记

收集若虫时,要保持水稻幼苗的土壤干燥。

食谱

  1. 碳酸盐涂料解决方案
    1.59克Na 2 CO 3 3 2.93g NaHCO 3•/ 2 溶于900毫升蒸馏水中,定容至1000毫升(pH = 9.6)
  2. 10倍磷酸盐缓冲盐水(PBS)
    16克NaCl
    4克KCl
    4克KH 2 PO 4 4克/克 6克Na 2 HPO 4•12H 2 O O 溶于900毫升蒸馏水中,定容至1000毫升(pH = 7.5)
  3. PBS-T
    用1:9体积比稀释的10倍PBS,加入0.1%Tween 20
  4. 阻止解决方案
    5%(w / v)脱脂奶粉PBS-T
  5. TMB彩色基板解决方案
    3毫克4-氯-1-萘酚
    2毫升乙醇
    7μl30%H 2 O 2 2/2 10毫升PBS
    调整pH值到7.5

致谢

我们感谢江苏省农业科学院的周宜军教授和他的团队为我们提供了健康的飞虱,吴建祥(浙江大学)为我们提供了抗体。我们感谢英国Stevenage教授M.J.Adams帮助纠正稿件英文。该协议是从以前的作品,包括张等人的修改。 (2001a),Sun等人。 (2015年),和他等人。 (2017),并已成功用于病毒表征和病毒 - 植物相互作用的研究。所有作者声明没有任何利益冲突。

参考

  1. Boccardo,G。和Milne,R.G。(1984)。植物呼肠孤病毒组。 CMI / AAB,植物病毒描述294号,英联邦微生物研究所和应用生物学协会。英国。
  2. He,Y.,Zhang,H.,Sun,Z.,Li,J.,Hong,G.,Zhu,Q.,Zhou,X.,MacFarlane,S.,Yan,F.and Chen,J.( 2017年)。 茉莉酸介导的防御抑制水稻对水稻黑条矮缩病毒感染的油菜素内酯介导的易感性< / New Phytol 214(1):388-399。
  3. Li,L.,Li,H.W.,Dong,H.B。,Wang,X.F。和Zhou,G.H。(2010)。 由 Laodelphax striatellus 传播水稻黑条纹病毒从冷冻感染的水稻叶片到水稻和玉米的健康植物。 J Phytopathol 159:1-5。
  4. Marzachi,C.,Boccardo,G.,Milne,R.,Isogai,M.和Uyeda,I.(1995)。 基因组结构和病毒的变异性 病毒学研讨会 6:103-108。
  5. Shikata,E.和Kitagawa,Y。(1977)。 水稻黑条矮缩病毒:其性质,形态和细胞内定位。 >病毒学 77(2):826-842。
  6. Sun,Z.,He,Y.,Li,J.,Wang,X.和Chen,J。(2015)。 小RNA对水稻叶和根中水稻黑条矮缩病毒反应性microRNA的全基因组表征和降解组测序。植物细胞生理学 56(4):688-699。
  7. Yang,J.,Zhang,H.M.,Chen,J.P.and Dai,L.Y。(2007)。 水稻黑条矮缩病毒fijivirus p8蛋白原核表达抗血清制备及其性质。 Acta Phyophylacica Sinica 34(3):252-256。
  8. Zhang,H.M.,Chen,J.P。和Adams,M.J。(2001a)。 来自中国的水稻黑条矮缩病毒的片段1至6的分子表征提供了完整的基因组。 Arch Virol 146:2331-2339。
  9. Zhang,H.M.,Chen,J.P.,Lei,J.L。和Adams,M.J。(2001b)。 序列分析显示,水稻,小麦和玉米在中国的矮化病是由水稻黑条矮缩病毒引起的。 Eur J Plant Pathol 107:563-567。
  10. Zhou,Y.J。和Liu,H.J。(2004)。由灰飞虱携带的水稻条纹病毒的免疫测定。江苏农业科学1:50-51。
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引用:Sun, Z., Zhang, H., Xie, K., Tan, X., Zhang, H. and Chen, J. (2017). Rice Black-streaked Dwarf Virus Preparation and Infection on Rice. Bio-protocol 7(24): e2651. DOI: 10.21769/BioProtoc.2651.
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