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Virus-induced gene silencing (VIGS) is a powerful method to study gene function in plants. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector so far. This method was originally developed by the Dinesh-Kumar's group (Liu et al., 2002) . Here, we describe a rapid and high efficient TRV-based VIGS method for knocking down genes in Nicotiana benthamiana. For TRV-based VIGS, Agrobacterium culture containing pTRV1 and Agrobacterium culture containing pTRV2 with plant target gene fragment are mixed and infiltrated into the lower leaves of plant. After 2-3 weeks post infiltration, plant target gene will be silenced.

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VIGS Assays
病毒诱导的基因沉默(VIGS)实验

植物科学 > 植物分子生物学 > RNA > 转录
作者: Haili Zhang
Haili ZhangAffiliation: ChengDu Institute of Biology, Chinese Academy of Sciences, Cheng Du, China
Bio-protocol author page: a1189
 and Yule Liu
Yule LiuAffiliation: School of Life Sciences, Tsinghua University, Beijing, China
For correspondence: yuleliu@mail.tsinghua.edu.cn
Bio-protocol author page: a1190
Vol 4, Iss 5, 3/5/2014, 6094 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1057

[Abstract] Virus-induced gene silencing (VIGS) is a powerful method to study gene function in plants. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector so far. This method was originally developed by the Dinesh-Kumar's group (Liu et al., 2002) . Here, we describe a rapid and high efficient TRV-based VIGS method for knocking down genes in Nicotiana benthamiana. For TRV-based VIGS, Agrobacterium culture containing pTRV1 and Agrobacterium culture containing pTRV2 with plant target gene fragment are mixed and infiltrated into the lower leaves of plant. After 2-3 weeks post infiltration, plant target gene will be silenced.

[Abstract]

Materials and Reagents

  1. 6-leaf-stage Nicotiana benthamiana plants 
    Note: Nicotiana benthamiana can be obtained from our lab (Figure 1).


    Figure 1. 6-leaf-stage Nicotiana benthamiana plant

  2. Bacteria strains 
    a.  Escherichia coli strains, such as DH5α 
    b.  Agrobacterium strains, such as GV3101
    Note: All strains were obtained from our lab.
  3. pTRV1 and pTRV2-LIC based expression vectors (Dong et al., 2007)
    1. pTRV1: a T-DNA vector containing duplicated CaMV 35S promoter, NOS terminator and cDNA clone of TRV RNA1 of Ppk20 strain.
    2. pTRV2-LIC: a T-DNA vector containing  duplicated CaMV 35S promoter,  NOS terminator and cDNA clone of TRV RNA2, of which non-structural genes were replaced by a multiple cloning site (MCS).
    3. pTRV2-NbPDS: gene fragement of NbPDS was inserted at MCS into pTRV2-LIC. This construct was usually used as a control to show the successful gene silencing.
    4. pTRV1 (ABRC, catalog number: CD3-1039 ) and pTRV2-LIC (ABRC, catalog number: CD3-1042 ) could be ordered at  http://www.arabidopsis.org/abrc/catalog/vector_3.html.
  4. Media for Agrobacteria  
    1. Liquid Luria-Bertani (LB) medium
    2. Solid LB plates with 0.12% agar
      Note: LB medium is autoclaved under 120 °C for 20 min.
  5. Antibiotics 
    a.  Kanamycin
    b.  Rifampicin
    c.  Gentamicin
  6. Easy Taq DNA polymerase (Beijing TransGen Biotech)
  7. dNTP (Roche)  
  8. TIANprep Mini Plasmid Kit (Beijing TransGen Biotech)
  9. MgCl2 (Sigma-Aldrich)
  10. MES (AMRESCO)
  11. Acetosyringone (Sigma-Aldrich)
  12. DMSO (AMRESCO)
  13. Infiltration buffer (see Recipes)

Equipment

  1. Centrifuge tubes
  2. Plant growth chamber (24 °C, 16 h photoperiod conditions, 50% huminity)
  3. Sterile 1 ml syringe without needle
  4. Sterile bacterial culture tubes
  5. Centrifuge
  6. PCR instrument
  7. 37 °C and 28 °C incubators with shaking

Procedure

  1. Clone plant target gene fragment into pTRV2-LIC as described previously (Liu et al., 2002), and transform it into Escherichia coli DH5α. Positive clones were picked up and the plasmid DNA was amplified by PCR, using specific primer pairs, and then confirmed for correct insertion by DNA sequencing. Grow a positive clone in 5 ml LB liquid medium (containing 50 µg/ml Kanamycin) in 37 °C incubator at 200 rpm shaking overnight. Collect the bacteria by 14,000 x g centrifuging for 1 min at room temperature and then extract plasmids using mini plasmid kit.
  2. Transform pTRV1, pTRV2 or its derivatives into Agrobacterium strain GV3101 respectively. Transformed Agrobacteria were grown for 2 days on LB plates containing 50 µg/ml Kanamycin, 30 µg/ml Rifampicin and 50 µg/ml gentamicin.
  3. Pick several clones and confirm that the grown Agrobacteria contain correct plasmid using PCR with specific primers.
  4. Grow one positive clone from each transformant containing pTRV1, pTRV2 or pTRV2 derivatives in 5 ml liquid LB media (containing 50 µg/ml Kanamycin, 50 µg/ml Rifampicin and 50 µg/ml Gentamicin) in 28 °C incubator shaking at 200 rpm overnight.
    Note: Inoculating Agrobacteria into media for culturing should be done on super-clean bench, all equipments used needs to be sterile.
  5. Take the culture tubes out of the incubators. Adjust all the Agrobacterium culture to OD600=1.0. Taking equal volumes of agrobacterium culture (OD600 = 1.0) with pTRV1 and that with pTRV2 or pTRV2 derivatives. Mix them together and pellet by centrifuging at 3,000 x g for 5 min, at room temperature.
  6. Pour off the supernatant, re-suspend the agrobacterium pellet in infiltration buffer of equal volume to that of Agrobacterium culture (as to keep the OD600 at around 1.0). Keep the re-suspended culture at room temperature for 2-4 h.
  7. Select 6-leaf-stage plants and inflitrate the re-suspended Agrobacterium culture into abaxial side of expanded leaves, using 1 ml-syringe (without needles). 2 or 3 leaves of each plant need to be injected. Plants were grown in a growth room with a 16-h/8-h photoperiod at a light intensity of 10,000 lux at 24 °C. Figure 2 shows the schematic diagram of infiltration. Figure 3 showed the leaf state right afer inoculation.
    Note: Each leaf for inoculation is often injected 2 circles with 1 cm diameters.


    Figure 2. Schematic diagram of infiltration


    Figure 3. The right picture showed the state right after inoculation and the left one showed a non-infected leaf

  8. 2 weeks post inoculation, target gene will be silenced at whole plant level. The new leaves always show the strong silencing phenotype, and are appropriate for following study. Figure 4 shows the upper leaves of plants that PDS gene was silenced by VIGS.


    Figure 4. Successful silencing of NbPDS. Photograph was taken 2 weeks post inoculation.

Recipes

  1. Infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 µM acetosyringone) (100 ml)
    1 M MgCl2: 20.33 g MgCl2 dissolved in 100 ml dH2O, autoclaved by 120 °C, 20 min. 1 M MgCl2 stock was stored at 4 °C.
    1 M MES: 21.325 g MES dissolved in 100 ml dH2O, filter sterilized with 0.22 µm filter membrane
    200 mM acetosyringone: 0.3924 g acetosyringone dissolved in 10 ml DMSO. 1 M MES stock was stored at room temperature. 200 mM acetosyringone was stored at -20 °C.
    100 ml infiltration buffer
    1 ml 1 M MgCl2
    1 ml 1 M MES
    100 μl 200 mM acetosyringone
    Add dH2O to 100 ml

Acknowledgments

This protocol was adapted from the research article: Wang et al. (2013).

References

  1. Dong, Y., Burch-Smith, T. M., Liu, Y., Mamillapalli, P. and Dinesh-Kumar, S. P. (2007). A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145(4): 1161-1170.
  2. Liu, Y., Schiff, M., Marathe, R. and Dinesh-Kumar, S. P. (2002). Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4): 415-429.
  3. Wang, Y., Yu, B., Zhao, J., Guo, J., Li, Y., Han, S., Huang, L., Du, Y., Hong, Y., Tang, D. and Liu, Y. (2013). Autophagy contributes to leaf starch degradation. Plant Cell 25(4): 1383-1399.

材料和试剂

  1. 6叶期的烟草烟草植物</em>
    注意:本田烟草可以从我们的实验室获得(图1)。


    图1. 6叶期 本塞>烟草 植物

  2. 菌株 
    a。 大肠杆菌菌株,例如DH5α
    b。 农杆菌株,例如GV3101
    注意:所有菌株均来自我们的实验室。
  3. 基于pTRV1和pTRV2-LIC的表达载体(Dong等人,2007)
    1. pTRV1:含有重复的CaMV 35S启动子,NOS终止子和Ppk20菌株的TRV RNA1的cDNA克隆的T-DNA载体。
    2. pTRV2-LIC:一种T-DNA载体, 重复的CaMV 35S启动子, NOS终止子和TRV RNA2的cDNA克隆,其中非结构基因被多克隆位点(MCS)取代。
    3. pTRV2-NbPDS:在MCS中将NbPDS的基因条带插入pTRV2-LIC中。 该构建体通常用作对照以显示成功的基因沉默
    4. 可以在pTRV1(ABRC,目录号:CD3-1039)和pTRV2-LIC(ABRC,目录号:CD3-1042) http://www.arabidopsis.org/abrc/catalog/vector_3.html
  4. 土壤杆菌媒体
    1. 液体Luria-Bertani(LB)培养基
    2. 具有0.12%琼脂的固体LB平板
      注意:将LB培养基在120℃下高压灭菌20分钟。
  5. 抗生素
    一个。  卡那霉素
    b。   Rifampicin
    C。  庆大霉素
  6. Easy Taq DNA聚合酶(北京TransGen Biotech)
  7. dNTP(Roche)
  8. TIANprep微量质粒试剂盒(北京TransGen Biotech)
  9. MgCl 2(Sigma-Aldrich)
  10. MES(AMRESCO)
  11. 乙酰丁香酮(Sigma-Aldrich)
  12. DMSO(AMRESCO)
  13. 渗透缓冲液(参见配方)

设备

  1. 离心管
  2. 植物生长室(24℃,16小时光周期条件,50%亮度)
  3. 无针1毫升无针注射器
  4. 无菌细菌培养管
  5. 离心机
  6. PCR仪
  7. 37℃和28℃振荡孵育器

程序

  1. 如先前所述将克隆植物靶基因片段克隆到pTRV2-LIC中(Liu等人,2002),并将其转化入大肠杆菌DH5α。 挑取阳性克隆,并使用特异性引物对通过PCR扩增质粒DNA,然后通过DNA测序确认正确插入。 在37℃培养箱中以200rpm摇动过夜,在5ml LB液体培养基(含有50μg/ml卡那霉素)中培养阳性克隆。 在室温下通过14,000×g离心1分钟收集细菌,然后使用小型质粒试剂盒提取质粒。
  2. 将pTRV1,pTRV2或其衍生物分别转化到土壤杆菌属菌株GV3101中。转化的土壤杆菌在含有50μg/ml卡那霉素,30μg/ml利福平和50μg/ml庆大霉素的LB平板上生长2天。
  3. 挑选几个克隆,并使用特异性引物通过PCR确认所生长的 grobacteria 包含正确的质粒。
  4. 从含有pTRV1,pTRV2或pTRV2衍生物的每个转化体在28℃培养箱中以200rpm振荡,在5ml液体LB培养基(含有50μg/ml卡那霉素,50μg/ml利福平和50μg/ml庆大霉素)中生长一个阳性克隆过夜。
    注意:将农杆菌接种到用于培养的培养基中应该在超净台上进行,所使用的所有设备都需要是无菌的。
  5. 从培养箱中取出培养管。将所有土壤杆菌培养物调整到OD <600> = 1.0。用pTRV1和pTRV2或pTRV2衍生物取等体积的农杆菌培养物(OD 600 = 1.0)。将它们混合在一起并通过在室温下以3000xg离心5分钟沉淀。
  6. 倒出上清液,将农杆菌沉淀重悬在与土壤杆菌培养物相等体积的浸润缓冲液中(以使OD 600保持在约1.0)。将重悬培养物在室温下保持2-4小时
  7. 选择6叶期植物,并使用1ml注射器(无针)使再悬浮的土壤杆菌培养物膨胀到扩展的叶的背侧。每株植物需要注入2或3片叶子。植物在具有16-h/8-h光周期的生长室中在24℃的光强度为10,000勒克斯下生长。图2显示了渗透的示意图。图3显示了正确接种的叶状态 注意:用于接种的每片叶常常注射2个直径为1cm的圆。


    图2.渗透示意图


    图3.右图显示接种后的状态,左图显示未感染的叶

  8. 接种后2周,靶基因将在整株植物水平上沉默。新叶总是表现出强烈的沉默表型,适合后续研究。图4显示PDS基因被VIGS沉默的植物的上叶

    图4.成功沉默Nb 。接种后2周拍摄照片。

食谱

  1. 浸润缓冲液(10mM MgCl 2,10mM MES和200μM乙酰丁香酮)(100ml)
    1M MgCl 2:20.33g MgCl 2溶解在100ml dH 2 O中,通过120℃高压灭菌20分钟。 将1M MgCl 2储备液在4℃下储存 1M MES:21.325g溶解在100ml dH 2 O中的MES,用0.22μm滤膜过滤灭菌
    200mM乙酰丁香酮:0.3924g乙酰丁香酮溶于10ml DMSO中。 将1M MES原液在室温下储存。 将200mM乙酰丁香酮贮存在-20℃ 100 ml浸润缓冲液
    1ml 1M MgCl 2溶液 1 ml 1 M MES
    100μl200mM乙酰丁香酮
    将dH <2> O添加到100 ml

致谢

该方案改编自研究文章:Wang等人(2013)。

参考文献

  1. Dong,Y.,Burch-Smith,T.M.,Liu,Y.,Mamillapalli,P.and Dinesh-Kumar,S.P。(2007)。 用于高通量病毒诱导基因沉默的不依赖连接的克隆烟草rattle病毒载体识别NbMADS4-1和-2在花发育中。植物生理学145(4):1161-1170。
  2. Liu,Y.,Schiff,M.,Marathe,R。和Dinesh-Kumar,S.P。(2002)。 烟草Rar1,EDS1和NPR1/NIM1样基因是N介导的烟草花叶病毒抗性所必需的。 Plant J 30(4):415-429。
  3. Wang,Y.,Yu,B.,Zhao,J.,Guo,J.,Li,Y.,Han,S.,Huang,L.,Du,Y.,Hong,Y.,Tang, Liu,Y。(2013)。 自噬导致叶淀粉 降解。植物细胞 25(4):1383-1399。

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How to cite this protocol: Zhang, H. and Liu, Y. (2014). VIGS Assays. Bio-protocol 4(5): e1057. DOI: 10.21769/BioProtoc.1057; Full Text



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