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Infection of Nicotiana benthamiana Plants with Potato Virus X (PVX)
使用马铃薯X病毒(PVX)感染本氏烟植物   

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本实验方案简略版
Molecular Plant Microbe Interactions
Dec 2015

Abstract

Potato Virus X (PVX) is the type member of Potexvirus genus, a group of plant viruses with a positive-strand RNA genome (~6.4 kb). PVX is able to establish compatible infections in Nicotiana benthamiana, a commonly used host in plant virology, leading to mild symptoms, such as chlorotic mosaic and mottling. PVX has been widely used as a viral vector for more than two decades (Chapman et al., 1992; Baulcombe et al., 1995; Aguilar et al., 2015). It provides a feasible means for the systemic expression in plants of heterologous proteins, such as avirulence factors, proteins with pharmacological properties, etc., (Hammond-Kosack et al., 1995; Gleba et al., 2014), and also as a tool to help decipher the function of genes in plants by virus-induced gene silencing (VIGS) (Lacomme and Chapman, 2008). Two different protocols, i.e., rubbing (A) and agroinfiltration (B), that allow efficient multiplication and propagation of PVX in N. benthamiana are described here in detail. The rubbing method requires previously infected sap, and infection is achieved by inducing mechanical damages to leaf tissue, allowing viral particles to penetrate the plant surface. Agroinfiltration needs previously modified Agrobacterium to carry and deliver T-DNA with PVX sequences into the plant cell. Agrobacterium is grown until saturation and infection is established by infiltrating it into plant tissue with a syringe. Any of these two methods can be successfully applied, and the choice should be based mainly on the availability of material and time, but it is recommended to use agroinfiltration when chimeric viruses are being used.

Keywords: Potato Virus X (马铃薯X病毒), Inoculation (接种), Agroinfiltration (农杆菌渗入法), Heterologous protein expression (异源蛋白表达), Virus-induced gene silencing (病毒诱导的基因沉默), Gene function (基因功能)

Background

PVX is transmitted by mechanical means, so the easiest and fastest way to infect plants is by rubbing the leaves with sap from infected tissue. However, since RNA viruses have high mutation rates, caution must be taken when rubbing is used as propagation method. In this regard, the number of serial passages between plants should be limited, and the inoculum should be used fresh from original stocks. To solve this inconvenience, an infectious PVX cDNA clone has been introduced into a binary T-DNA vector, which allows its easy delivery into N. benthamiana by Agrobacterium tumefaciens. Agroinfiltration should be considered as preferred method when recombinant PVX is being used, in order to prevent serial propagation of deleted viral forms from a previous experiment to the next one (Chung et al., 2007).

Materials and Reagents

  1. Rubbing
    1. 2 ml safe-lock tubes (Eppendorf, catalog number: 022363352 )
      Note: This product has been discontinued.
    2. Latex powder-free gloves (Staples, Ambitex®, catalog number: SS2072105 )
    3. Gauze (Fisaude. Kinefis, catalog number: 10901 )
    4. N. benthamiana plants at the stage of 4-6 fully expanded true leaves (see Figure 1)
    5. Flash frozen, infected tissue (PVX virus inoculum from DSMZ Plant Virus Collection, Reference No.: 15649, DSMZ No.: PV-0847, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures)
    6. Liquid nitrogen
    7. Ice
    8. Sodium dihydrogen phosphate (NaH2PO4) (EMD Millipore, catalog number: 106346 )
    9. Di-sodium hydrogen phosphate (Na2HPO4) (EMD Millipore, catalog number: 106559 )
    10. Abrasive carborundum powder (CARLO ERBA Reagents, catalog number: 434786 )
    11. Sodium phosphate buffer (see Recipes)

  2. Agroinfiltration
    1. Petri dishes (Gosselin, catalog number: BP93B-102 )
    2. 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
    3. 13 ml centrifuge tubes (SARSTEDT, catalog number: 62.515.006 )
    4. 1 ml syringes, without needle (BD, catalog number: 309659 )
    5. 10 ml sterile pipettes (Corning, Falcon®, catalog number: 357551 )
    6. N. benthamiana plants (at the 4-6 leaves stage, see Figure 1)
    7. Agrobacterium tumefaciens (any disarmed strain, like GV3101) carrying a T-DNA binary vector with a full-length cDNA clone of PVX (Chapman et al., 1992; Lu et al., 2003, see Notes)
    8. Agrobacterium tumefaciens (any disarmed strain, like GV3101) carrying the empty T-DNA binary vector, as control
    9. Magnesium chloride (MgCl2) (EMD Millipore, catalog number: 105833 )
    10. 2-morpholinoethanesulfonic acid (MES) (SERVA Serving Scientists, catalog number: 29834 )
    11. 3’,5’-dimethoxy-4’-hydroxyaceto-phenone (Acetosyringone) (Sigma-Aldrich, catalog number: D134406 )
    12. Dimethyl sulfoxide (DMSO) (EMD Millipore, catalog number: 102952 )
    13. Tryptone (BD, BactoTM, catalog number: 211705 )
    14. Yeast extract (Conda, catalog number: 1702 )
    15. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
    16. American bacteriological agar (Conda, catalog number: 1802 )
    17. Appropriate, selective antibiotics (Kanamycin and tetracycline) (Sigma-Aldrich, catalog numbers: K1377 , 87128 )
    18. Induction buffer (see Recipes)
    19. LB liquid medium supplemented with appropriate antibiotics (see Recipes)
    20. LB/agar medium supplemented with appropriate antibiotics (see Recipes)

  3. Western blot detection
    1. 1.5 ml safe-lock tubes (Eppendorf, catalog number: 0030120086 )
    2. Polyvinyl PVDF membrane (GE Healthcare, catalog number: 10600023 )
    3. Tris-hydroxymethyl-aminomethane (Tris) base (EMD Millipore, catalog number: 108386 )
    4. Hydrochloric acid (HCl) (Hydrochloric acid 37%) (EMD Millipore, catalog number: 100317 )
    5. Ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 324503 )
    6. Lithium chloride (LiCl) (EMD Millipore, catalog number: 105679 )
    7. β-mercaptoethanol solution (EMD Millipore, catalog number: 805740 )
    8. Sodium dodecyl sulfate (SDS) (EMD Millipore, catalog number: 817034 )
    9. Bromophenol Blue (BPB) (Sigma-Aldrich, catalog number: 114391 )
    10. Glycerol (Glycerol 87% solution) (EMD Millipore, catalog number: 104094 )
    11. 3-hydroxy-4-(2-sulfo-4-[4-sulfophenylazo] phenylazo)-2,7-naphthalenedisulfonic acid sodium salt (Ponceau S) (Sigma-Aldrich, catalog number: P3504 )
    12. Acetic acid (Acetic acid glacial 100% solution) (EMD Millipore, catalog number: 100063 )
    13. Sodium chloride (NaCl) (EMD Millipore, catalog number: 116224 )
    14. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 60230 )
    15. Potassium chloride (KCl) (EMD Millipore, catalog number: 104936 )
    16. Acrylamide (30% Acrylamide/Bis solution, 37.5:1) (Bio-Rad Laboratories, catalog number: 1610158 )
    17. Rabbit anti-PVX CP antibody (LOEWE Biochemica, catalog number: 07037 )
    18. Goat anti-rabbit antibody conjugated with AP (Sigma-Aldrich, catalog number: A3687 )
    19. SigmaFastTM BCI/NBT tablets (Sigma-Aldrich, catalog number: B5655 )
    20. Protein extraction buffer for Western blot (see Recipes)
    21. 2x Laemmli solution for western blot (see Recipes)
    22. Ponceau S solution (see Recipes)
    23. Blocking solution (see Recipes)
    24. 10x PBS, pH 7.4 (see Recipes)

Equipment

  1. Common equipment
    1. Plant growth chambers (SANYO Electronic) at 24 °C, 2,500 lux of daylight intensity, 16 h/8 h day/night photoperiod
    2. P1000 , P200 and P20 micropipettes (Gilson, Pipetman ClassicTM)

  2. Rubbing
    1. Mortar and pestle (Silico & Chemico Porcelain SE)
    2. Refrigerated table microcentrifuge (Hettich Lab Technology, model: Mikro 200R )
    3. Pacisa Weighing Balances (Precisa Gravimetrics, model: XB620C-G )

  3. Agroinfiltration
    1. 28 °C incubator for plate culture (JP Selecta, model: 2001258 )
    2. 28 °C refrigerated incubator shaker (Eppendorf, New Brunswick Scientific, model: 4330 )
    3. Refrigerated centrifuge for 13 ml tubes (Hettich Lab Technology, model: Universal 320R )
    4. 600 nm wavelength-sensitive spectrophotometer (Eppendorf, model: Bio Photometer 6131 000.012 )

  4. Western blot detection
    1. Blue polypropylene, pellet pestles for 1.5 Eppendorf tubes (Sigma-Aldrich, catalog number: Z359947 )
    2. Thermomixer (Eppendorf, catalog number: 5384000012 )
    3. Cork-borer set (Sigma-Aldrich, catalog number: Z165220 )

Procedure

  1. Rubbing
    1. A tissue extract must be obtained from infected leaves. It is possible to use commercial viral inoculum as starting point (PVX from DMSZ, see Materials and Reagents). In the same way, PVX-infected tissue derived from plants that have been previously inoculated by mechanical means can be used. Tissue obtained from these plants at 7 days post-infection can efficiently initiate viral propagation. The plant tissue should come from the upper leaves of the plant, which usually show the most evident symptoms. Try to select the proximal part of the leaf because the virus infection front does not always reach the distal part. Discard the largest leaf vein (see Figure 1)


      Figure 1. PVX-infected leaf showing symptoms at 7 days post-infection. Use as source material leaves with evident symptoms of infection (on the left). Try to select the proximal part of the leaf and discard the largest leaf vein (on the right, selected zones in white).

    2. Collect tissue in aliquots of 300-400 mg. Take one aliquot and keep it into liquid nitrogen, and store the rest at -80 °C. Discard tissue that has been previously defrosted.
    3. Grind infected tissue with mortar and pestle refrigerated with liquid nitrogen. Try to obtain a fine powder.
    4. Add 5 equivalents in volume of cold sodium phosphate buffer (see Recipes) to the powder while maintaining the mortar on ice (i.e., mixing 300 mg of powdered tissue with 1.5 ml of buffer). The components usually become frozen, so mix them with the pestle until a homogeneous, liquid mixture is obtained.
    5. Take the extract mixture with a micropipette and put it into 2 ml tubes. Use cut tips. Keep the mixture on ice to prevent degradation.
    6. Centrifuge at 4 °C and 16,000 x g for 5 min.
    7. Take the supernatant and discard the pellet. Put the supernatant into a new 2 ml tube. Keep it on ice (for immediate use) or store at -80 °C (for long term use).
    8. Select appropriate plants grown under long-day conditions. These plants must be at the stage of 4-6 fully expanded true leaves (see Figure 2) in order for the plant to become fully susceptible to virus infection. Dust with carborundum powder the entire adaxial surface of two fully expanded leaves (see Notes) per plant. The amount of carborundum should be enough as to observe it on the leaves. Follow the same procedure with all the selected plants.


      Figure 2. Nicotiana benthamiana plants. These plants must be at the stage of 4-6 fully expanded leaves in order for the plant to become fully susceptible to virus infection. White arrows point out leaves where rubbing is feasible.

    9. Put viral extract on every carborundum-sprayed leaf of the entire pool of plants (see Figure 3). Place it directly with the pipette on the adaxial surface. Use 20 μl of extract per selected leaf, adding 10 μl per each half of leaf. Proceed immediately with the next step to avoid the evaporation of the extract.


      Figure 3. Delivery of viral extracts. Use 20 μl of extract per selected leaf, adding 10 μl per each half of leaf (on the left). Drops of viral extracts are marked with white arrows (on the right).

    10. Every selected leaf must be rubbed carefully using latex gloves (see Figure 4). The leaf can be sustained by one hand while the other one is used for rubbing. Try not to press it, just only run the finger lightly over the surface about three times. Make sure the leaf is completely covered by the viral extract before starting with the next one (see Figure 5).


      Figure 4. Rubbing. Every selected leaf must be rubbed carefully using latex gloves.


      Figure 5. The leaf must be completely covered by the viral extract. The correct way (on the left) was made by rubbing it carefully; the wrong way (on the right, white arrows) produced injuries on the foliar surface after a while.

    11. As a control group, inoculate the same number of plants with a tissue extract made from healthy plants following the same steps as the above. These plants are usually referred to as ‘mock inoculated’. Change your gloves before proceeding. The number of plants depends on the experimental design. Although this protocol is highly successful, it is worth considering ten plants as the minimum representative sample size.

  2. Agroinfiltration
    1. Plate on LB/agar dishes the corresponding Agrobacterium constructs with appropriate selective antibiotics. Try to plate the bacteria 3 days before starting the experiment: this procedure ensures obtaining colonies with the proper age and higher growth rates in liquid medium (see next step).
    2. Pick up a single colony and grow it overnight until saturation. Use 6 ml of LB medium supplemented with appropriate antibiotics, in 50 ml Falcon tubes, and fix one incubator shaker at 230 rpm (revolutions per minute) and 28 °C.
    3. Centrifuge a volume of saturated culture using 13 ml tubes at 1,660 x g for 10 min. Discard the supernatant. This step does not require optical density measurements.
    4. Resuspend the Agrobacterium pellet using induction buffer (see Recipes). Mix gently by pipetting until a homogeneous solution is obtained. It is advisable to use three volumes of induction buffer (i.e., 18-20 ml of induction buffer per pellet obtained from 6 ml overnight culture).
    5. Measure optical density at 600 nm wavelength (OD600). It should be around OD600 ~1.
    6. Different dilution degrees must be tried in order to achieve the correct one for your purpose. Make dilutions adding more induction buffer. An OD600 ~0.2 is enough to infect N. benthamiana.
    7. Incubate the Agrobacterium solution at room temperature, in a dark place, for 3 h.
    8. Use 1 ml syringes without needle to infiltrate the solution through the abaxial surface of the leaf, while pushing with the index finger from the other side (see Figure 6). Infiltrate two fully-expanded leaves per plant, using 0.5 ml per each half of leaf (see Figure 7). This procedure makes possible the easy spread of virus infection from lower parts of the plant to the new emerging tissues. These plants must be at the stage of 4-6 fully expanded true leaves (see Figure 2), and they should be grown under long-day conditions.


      Figure 6. Agroinfiltration. Use 1 ml syringes without needle to infiltrate the solution.


      Figure 7. Infiltrate two fully expanded leaves per plant. Use 0.5 ml per each half of leaf. Infiltrated areas are shown with white circles.

    9. Follow the same procedure to infiltrate the control group. The number of plants depends on the experimental design. Although this protocol is highly successful, it is worth considering ten plants as the minimum representative sample size.

Data analysis

Young plants infected with PVX can be easily distinguished from non-infected ones from 6 days after infection onwards by the naked eyes, but it could be more difficult when plants are infected at older stages. In addition, it would be interesting to check viral accumulation under different experimental conditions. So, it may be worth monitoring PVX through Western blotting, using antibodies against the PVX coat protein (CP). Briefly:

  1. Take 100 mg of infected systemic tissue and put it into 1.5 ml tubes in liquid nitrogen. It is easier to collect four leaf discs using a cork-borer with a 15 mm diameter. Follow the same procedure with healthy controls.
  2. Add 400 μl of protein extraction buffer (see Recipes) and homogenize using blue, polypropylene, pellet pestles.
  3. Take 50 μl of the homogenized solution and mix well with 50 μl of 2x Laemmli solution (see Recipes). Boil samples at 95 °C for 3-5 min using a Thermomixer, and centrifuge them at maximum speed for 5 min.
  4. Use 16 μl of the supernatant to run an SDS-PAGE electrophoresis; any protocol for SDS-PAGE can be successfully applied (Aguilar et al., 2015). Resolve proteins in 15% SDS-polyacrylamide gels, because PVX CP protein is around 25 kDa of molecular weight. Transfer proteins from the gel to Hybond-P PVDF membranes, and stain the blotted membrane with Ponceau S solution (see Recipes) to detect Rubisco protein as loading control. Ponceau S staining can be easily achieved by incubating the membrane with the solution and gentle shaking for five minutes. Before proceeding with the next step, de-stain the membrane by cleaning it with water and gentle shaking for five minutes.
  5. Incubate the membrane for 2 h at room temperature with blocking solution (see Recipes), and subsequently incubate overnight with rabbit anti-PVX CP antibody diluted 1/400 in blocking solution, at room temperature. The next day, incubate with a secondary anti-rabbit AP-conjugated antibody diluted 1/10,000 in blocking solution, for 1 h at room temperature. Finally, develop with BCIP/NBT tablet system based on alkaline phosphatase-mediated chromogenic reaction following manufacturer procedure.


    Figure 8. Western blot of PVX-infected plants at 7 days post-agroinfiltration. This figure shows three non-contiguous lines belonging to the same Western blot, with M: Molecular weight markers (kDa), H: Healthy plant extracts, and PVX: PVX-infected plant extracts. The upper panel shows the result for anti-PVX CP antibody incubation and BCIP/NBT chromogenic reaction. The lower panel shows the blotted, Ponceau S-stained membrane prior to antibody incubation, for Rubisco protein detection as loading control.

Notes

  1. pGR107 (Chapman et al., 1992; Lu et al., 2003) is a commonly used binary vector that originated from Prof. D. C. Baulcombe group, harboring an infectious PVX cDNA clone that contains an additional CP promoter and a polylinker, making it possible to insert foreign sequences.
  2. Viral symptoms start to be noticeable around 4 days after inoculation (dai), reaching a maximum between 6-8 dai. Symptoms start to disappear one week after the first date of appearance, a phenomenon known as ‘recovery’.
  3. To apply the rubbing method, fill a 50 ml tube with carborundum and cover it with 2-3 layers of gauze. This ‘homemade’ system allows for its appropriate delivery.
  4. To apply the rubbing method, it is important to use the same kind of gloves and to follow the same rubbing patterns in order to achieve reproducibility.

Recipes

  1. Sodium phosphate buffer
    20 mM NaH2PO4/Na2HPO4, pH 7.0
  2. Induction buffer (100 ml)
    1 ml MgCl2 stock solution (final: 10 mM MgCl2)
    1 ml MES stock solution (final: 10 mM MES)
    150 μl acetosyringone stock solution (final: 150 μM acetosyringone)
    Complete to a final volume of 100 ml with sterilized double distilled water
    Stock solutions:
    1 M MgCl2 (Magnesium chloride)
    1 M MES (2-morpholinoethanesulfonic acid) buffer, pH 5.7
    100 mM acetosyringone (3’,5’-dimethoxy-4’-hydroxyaceto-phenone), dissolved in DMSO (Dimethyl sulfoxide)
  3. Luria Broth medium (LB medium), pH 7.0
    1% (w/v) tryptone
    0.5% (w/v) yeast extract
    1.0% (w/v) NaCl
  4. LB/agar medium
    LB + 1.5% (w/v) agar
  5. Protein extraction buffer for Western blot
    0.1 M Tris-HCl, pH 8.0
    10 mM EDTA
    0.1 M LiCl
    1% (v/v) β-mercaptoethanol
    1% (w/v) SDS
    Stock solutions:
    1 M Tris-HCl (tris-hydroxymethyl-aminomethane and hydrochloric acid), pH 8.0
    0.5 M EDTA (ethylenediaminetetraacetic acid), pH 8.0
    20% SDS (sodium dodecyl sulfate) solution
  6. 2x Laemmli solution for Western blot
    4% (w/v) SDS
    2% (v/v) β-mercaptoethanol
    0.1 M Tris-HCl, pH 6.8
    0.05% (w/v) BPB powder reagent
    20% (v/v) glycerol
    Stock solution:
    1 M Tris-HCl (tris-hydroxymethyl-aminomethane and hydrochloric acid), pH 6.8
  7. Ponceau S solution
    0.1% w/v Ponceau S powder reagent
    5% v/v acetic acid solution
  8. Blocking solution
    5% w/v skimmed milk powder
    10 mM 1x PBS buffer
    Stock solution:
    10x PBS (phosphate-buffered saline), pH 7.4
  9. 10x PBS, pH 7.4 (1 L)
    80 g NaCl (sodium chloride)
    2.0 g KCl (potassium chloride)
    14.4 g Na2HPO4 (sodium dihydrogen phosphate)
    2.4 g KH2PO4 (potassium dihydrogen phosphate)
    Complete to a final volume of 1 L with sterilized double distilled water

Acknowledgments

This protocol was developed and optimized for the T-DNA binary vector pGR107 by modifying the procedure used for pGR106 (Lu et al., 2003). E.A. is recipient of a FPU fellowship from the former Spanish Ministry of Education and Sport. This work was supported by the Spanish Ministry of Economy and Competitiveness (Grant Ref. BIO2013-47940-R); The Rural Development Administration (RDA) of the Republic of Korea (Grant Refs. PJ00946102; PJ010246).

References

  1. Aguilar, E., Allende, L., Del Toro, F. J., Chung, B. N., Canto, T. and Tenllado, F. (2015). Effects of elevated CO2 and temperature on pathogenicity determinants and virulence of potato virus X/potyvirus-associated synergism. Mol Plant Microbe Interact 28(12): 1364-1373.
  2. Baulcombe, D. C., Chapman, S. and Santa Cruz, S. (1995). Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7:1045-1053.
  3. Chapman, S., Kavanagh, T. A. and Baulcombe, D. C. (1992). Potato virus X as a vector for gene expression in plants. Plant J. 2: 549-557.
  4. Chung, B. N., Canto, T. and Palukaitis, P. (2007). Stability of recombinant plant viruses containing genes of unrelated plant viruses. J Gen Virol 88(Pt 4): 1347-1355.
  5. Gleba, Y. Y., Tusé, D., and Giritch, A. (2014). Plant viral vectors for delivery by Agrobacterium. Curr Top in Microbiol Immunol 375: 155-192.
  6. Hammond-Kosack, K. E., Staskawicz, B. J., Jones, J. D. G. and Baulcombe, D. C. (1995). Functional expression of a fungal avirulence gene from a modified potato virus X genome. Mol Plant-Microbe Interact 8: 181-185.
  7. Lacomme, C. and Chapman, S. (2008). Use of potato virus X (PVX)-based vectors for gene expression and virus-induced gene silencing (VIGS). Curr Protoc Microbiol. Chapter 16: Unit 16I 1.
  8. Lu, R., Malcuit, I., Moffett, P., Ruiz, M. T., Peart, J., Wu, A. J., Rathjen, J. P., Bendahmane, A., Day, L. and Baulcombe, D. C. (2003). High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J 22(21): 5690-5699.

简介

马铃薯病毒X(PVX)是Potexvirus属(一组具有正链RNA基因组(〜6.4kb)的植物病毒)的类型成员。 PVX能够在烟草烟草(Nicotiana benthamiana)中建立相容的感染,这是常用于植物病毒学的宿主,导致轻度症状,如褪色马赛克和斑驳。 PVX已经被广泛用作病毒载体二十多年(Chapman等人,1992; Baulcombe等人,1995; Aguilar等人。它为异源蛋白质植物中的全身表达提供了可行的手段,例如无毒性因子,具有药理学性质的蛋白质,等等(Hammond-Kosack等人, 1995; Gleba等人,2014),并且还作为通过病毒诱导的基因沉默(VACS)(Lacomme和Chapman,2008)帮助解释植物中基因功能的工具。摩擦(A)和农用过滤(B)的两种不同的方案,即允许PVX在N中的有效乘法和传播。本文详细描述本文。摩擦方法需要先前感染的液体,并且通过诱导对叶组织的机械损伤来实现感染,从而允许病毒颗粒穿透植物表面。农杆菌过滤需要先前修饰的农杆菌携带并将PVX序列递送到植物细胞中。将土壤杆菌生长直至饱和,并用注射器将其浸入植物组织中建立感染。这两种方法中的任何一种都可以成功应用,选择应主要基于材料和时间的可用性,但建议在使用嵌合病毒时使用农杆菌滤过。

背景 PVX通过机械手段传播,因此感染植物的最简单和最快捷的方法是通过用感染组织的汁液擦拭叶子。然而,由于RNA病毒具有高突变率,因此当使用摩擦作为传播方法时,必须小心。在这方面,应限制植物之间连续通道的数量,接种物应从原始库存中新鲜使用。为了解决这个不便,传染性PVX cDNA克隆被引入到二元T-DNA载体中,这允许其容易地递送到N中。根癌土壤杆菌 。当使用重组PVX时,应将Agroin过滤视为优选的方法,以防止从先前的实验向下一个实验的连续繁殖缺失的病毒形式(Chung等人,2007)。

关键字:马铃薯X病毒, 接种, 农杆菌渗入法, 异源蛋白表达, 病毒诱导的基因沉默, 基因功能

材料和试剂

    1. 2 ml安全锁管(Eppendorf,目录号:022363352)
      注意:本产品已停产。
    2. 乳胶无粉手套(Staples,Ambitex ®,目录号:SS2072105)
    3. 纱布(Fisaude。Kinefis,目录号:10901)
    4. n。本草本植物在4-6叶充分膨胀的真叶中(见图1)
    5. 快速冷冻的感染组织(来自DSMZ Plant Virus Collection的PVX病毒接种物,参考号:15649,DSMZ编号:PV-0847,莱布尼兹研究所DSMZ-德国微生物和细胞培养物收集物)
    6. 液氮

    7. 磷酸二氢钠(NaH 2 PO 4)(EMD Millipore,目录号:106346)
    8. 磷酸氢二钠(Na 2 HPO 4)(EMD Millipore,目录号:106559)
    9. 磨料碳化硅粉(CARLO ERBA试剂,目录号:434786)
    10. 磷酸钠缓冲液(见食谱)

  1. 农业滤清器
    1. 培养皿(Gosselin,目录号:BP93B-102)
    2. 50ml锥形离心管(Corning,Falcon ®,目录号:352070)
    3. 13ml离心管(SARSTEDT,目录号:62.515.006)
    4. 1毫升注射器,无针(BD,目录号:309659)
    5. 10 ml无菌移液器(Corning,Falcon ®,目录号:357551)
    6. n。本地植物(在4-6叶阶段,见图1)
    7. 携带具有PVX的全长cDNA克隆的T-DNA二元载体的农杆菌(根癌土壤杆菌)(任何解除武装的菌株,如GV3101)(Chapman等人,1992; Lu < em>等人,2003,见注释)
    8. 携带空T-DNA二元载体的根瘤土壤杆菌(Agrobacterium tumefaciens)(任何撤防菌株,如GV3101)作为对照
    9. 氯化镁(MgCl 2)(EMD Millipore,目录号:105833)
    10. 2-吗啉代乙磺酸(MES)(SERVA服务科学家,目录号:29834)
    11. 3',5'-二甲氧基-4'-羟基乙酰苯胺(Acetosyringone)(Sigma-Aldrich,目录号:D134406)
    12. 二甲基亚砜(DMSO)(EMD Millipore,目录号:102952)
    13. Tryptone(BD,Bacto TM ,目录号:211705)
    14. 酵母提取物(Conda,目录号:1702)
    15. 氯化钠(NaCl)(EMD Millipore,目录号:106404)
    16. 美国细菌琼脂(Conda,目录号:1802)
    17. 适当的选择性抗生素(卡那霉素和四环素)(Sigma-Aldrich,目录号:K1377,87128)
    18. 诱导缓冲液(参见食谱)
    19. 补充适量抗生素的LB液体培养基(见食谱)
    20. 补充有适当抗生素的LB /琼脂培养基(参见食谱)

  2. 蛋白质印迹检测
    1. 1.5 ml安全锁管(Eppendorf,目录号:0030120086)
    2. Polyvinil PVDF膜(GE Healthcare,目录号:10600023)
    3. 三羟甲基氨基甲烷(Tris)碱(EMD Millipore,目录号:108386)
    4. 盐酸(HCl)(盐酸37%)(EMD Millipore,目录号:100317)
    5. 乙二胺四乙酸(EDTA)(EMD Millipore,目录号:324503)
    6. 氯化锂(LiCl)(EMD Millipore,目录号:105679)
    7. β-巯基乙醇溶液(EMD Millipore,目录号:805740)
    8. 十二烷基硫酸钠(SDS)(EMD Millipore,目录号:817034)
    9. 溴苯酚蓝(BPB)(Sigma-Aldrich,目录号:114391)
    10. 甘油(甘油87%溶液)(EMD Millipore,目录号:104094)
    11. 3-羟基-4-(2-磺基-4- [4-磺基苯偶氮基]苯偶氮)-2,7-萘二磺酸钠盐(Ponceau S)(Sigma-Aldrich,目录号:P3504)
    12. 乙酸(醋酸冰100%溶液)(EMD Millipore,目录号:100063)
    13. 氯化钠(NaCl)(EMD Millipore,目录号:116224)
    14. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:60230)
    15. 氯化钾(KCl)(EMD Millipore,目录号:104936)
    16. 丙烯酰胺(30%丙烯酰胺/双溶液,37.5:1)(Bio-Rad Laboratories,目录号:1610158)
    17. 兔抗PVX CP抗体(LOEWE Biochemica,目录号:07037)
    18. 与AP缀合的山羊抗兔抗体(Sigma-Aldrich,目录号:A3687)
    19. SigmaFast TM BCI/NBT片剂(Sigma-Aldrich,目录号:B5655)
    20. 用于Western印迹的蛋白质提取缓冲液(参见食谱)
    21. 2x Laemmli溶液用于Western印迹(参见食谱)
    22. Ponceau S解决方案(见配方)
    23. 阻塞解决方案(见配方)
    24. 10倍PBS,pH 7.4(参见食谱)

设备

  1. 普通设备
    1. 植物生长室(三洋电子)在24°C,2500 lux的日光强度,16 h/8 h日/夜光周期
    2. P1000,P200和P20微量移液器(Gilson,Pipetman Classic TM

  2. 摩擦
    1. 砂浆和杵(Silico& Chemico瓷器SE)
    2. 冷藏台式微量离心机(Hettich Lab Technology,型号:Mikro 200R)
    3. Pacisa称重天平(Precisa Gravimetrics,型号:XB620C-G)

  3. 农业滤清器
    1. 28℃用于培养皿的培养箱(JP Selecta,型号:2001258)
    2. 28°C冷藏培养箱振动筛(Eppendorf,New Brunswick Scientific,型号:4330)
    3. 冷冻离心机13 ml管(Hettich Lab Technology,型号:Universal 320R)
    4. 600nm波长敏感分光光度计(Eppendorf,型号:Bio Photometer 6131 000.012)

  4. 蛋白质印迹检测
    1. 蓝色聚丙烯,1.5粒Eppendorf管的颗粒杵(Sigma-Aldrich,目录号:Z359947)
    2. Thermomixer(Eppendorf,目录号:5384000012)
    3. 软木塞(Sigma-Aldrich,目录号:Z165220)

程序

    1. 必须从受感染的叶子获得组织提取物。可以使用商业病毒接种物作为起点(来自DMSZ的PVX,参见材料和试剂)。以相同的方式,可以使用来自植物的PVX感染组织,这些组织先前已通过机械手段接种。在感染后7天从这些植物获得的组织可以有效启动病毒繁殖。植物组织应来自植物的上部叶,通常表现出最明显的症状。尝试选择叶片的近端部分,因为病毒感染前沿并不总是到达远端部分。丢弃最大的叶脉(见图1)


      图1.感染后7天时PVX感染的叶子显示症状。用作具有明显感染症状的源材料叶(左侧)。尝试选择叶片的近端部分并丢弃最大的叶脉(右侧,白色选定的区域)。

    2. 收集300-400 mg等份的组织。取一等份并保存在液氮中,将其余部分储存在-80°C。丢弃已经解冻的组织。
    3. 用用液氮冷冻的研钵和研杵研磨感染组织。尝试获得细粉。
    4. 将冰醋酸缓冲液(参见食谱)的体积为5当量加入到粉末中,同时将冰浆上的砂浆(即,将300mg粉末状组织与1.5ml缓冲液混合)保持在一起。组分通常会冻结,因此将其与杵混合,直到获得均匀的液体混合物
    5. 取出提取物混合物用微量移液管,并将其放入2 ml管中。使用切割技巧。将混合物保持在冰上以防止降解。
    6. 在4℃和16,000×g离心5分钟。
    7. 取上清液弃去沉淀。将上清液放入新的2 ml管中。保存在冰上(供立即使用)或储存于-80°C(长期使用)。
    8. 选择在长日照条件下种植的合适植物。这些植物必须处于4-6完全扩张的真叶的阶段(见图2),以使植物完全易受病毒感染。具有碳化硅粉末的粉尘使两个完全膨胀的叶子的整个正面表面(参见注释)每个植物。金刚砂的量应足以在叶子上观察。按照与所有选定植物相同的步骤。


      图2.本田烟草植物 这些植物必须处于4-6叶充分展开的阶段,以使植物完全易受病毒感染。白色箭头指出可能摩擦的叶子。

    9. 将病毒提取物放在整个植物池的每个金刚砂喷洒叶上(见图3)。直接用移液管放在近轴表面。每个叶子使用20μl提取物,每半叶加10μl。立即进行下一步骤,以避免提取物蒸发。


      图3.病毒提取物的递送。每个选择的叶子使用20μl提取物,每半叶加入10μl(左侧)。一滴病毒提取物用白色箭头标记(右侧)。

    10. 必须使用乳胶手套仔细擦拭每个选定的叶子(见图4)。叶可以用一只手持续,另一只则用于摩擦。尽量不要按下它,只需轻轻地在表面上轻轻地移动手指三次。在开始下一步之前,请确保病毒提取物完全覆盖叶子(见图5)

      图4.摩擦。必须使用乳胶手套仔细擦拭每个选定的叶子。


      图5.叶片必须被病毒提取物完全覆盖。正确的方法(左侧)是通过仔细摩擦来进行的;错误的方法(右侧,白色箭头)在一段时间后在叶面上产生伤害。

    11. 作为对照组,按照与上述相同的步骤,用健康植物制成的组织提取物接种相同数量的植物。这些植物通常被称为"模拟接种"。继续之前更换手套。植物数量取决于实验设计。虽然这个协议非常成功,但值得考虑的是十个工厂作为最小代表性的样本量
  1. 农业滤清器
    1. LB /琼脂上的板用适当的选择性抗生素培养相应的农杆菌构建体。尝试在开始实验前3天对细菌进行平板:该程序确保在液体培养基中获得具有适当年龄和较高生长速率的菌落(见下一步骤)。
    2. 拿起一个殖民地,长到一直饱和。使用6 ml补充有适当抗生素的LB培养基,在50ml Falcon管中,并以230 rpm(每分钟转数)和28°C固定一个孵化器振荡器。
    3. 使用13ml管以1,660×g离心一定体积的饱和培养物10分钟。丢弃上清液。此步骤不需要光密度测量。
    4. 使用诱导缓冲液重悬土壤杆菌沉淀(参见食谱)。通过移液轻轻混合,直到获得均匀的溶液。建议使用三个体积的诱导缓冲液(即,从6ml过夜培养物获得的每粒沉淀18-20ml的诱导缓冲液)。
    5. 测量600nm波长(OD 600)的光密度。它应该在大约600 〜1
      之间
    6. 必须尝试不同的稀释度才能达到正确的目的。使稀释液加入更多的诱导缓冲液。 OD 600至0.2足以感染N。本ham iana
    7. 在室温下,在黑暗的地方孵育农杆菌溶液3小时。
    8. 使用1毫升无针注射器通过叶子的背面渗透溶液,同时用食指从另一侧推(见图6)。每个植物渗透两个完全扩张的叶子,每半叶叶数为0.5ml(见图7)。这个程序使病毒感染从植物下部容易传播到新出现的组织上。这些植物必须处于4-6个完全扩张的真叶的阶段(见图2),它们应该在长的日子条件下生长。


      图6. Agroinfiltration。使用1毫升无针注射器渗透溶液。


      图7.每株植物渗透两片完全膨胀的叶子。每半叶使用0.5毫升。渗透区域以白色圆圈显示。

    9. 按照相同的步骤渗透对照组。植物数量取决于实验设计。虽然这个协议非常成功,但值得考虑的是十个工厂作为最小代表性的样本量

数据分析

感染PVX的年轻植物可以通过肉眼从感染后6天容易地与未感染的植物区分开,但是当植物在较旧的阶段感染时可能更困难。此外,在不同的实验条件下检查病毒积累将是有趣的。因此,使用抗PVX外壳蛋白(CP)的抗体可能通过Western印迹监测PVX。简单地说

  1. 取100毫克感染的全身组织,并放入1.5毫升的液氮管中。使用15毫米直径的软木钻头收集四片盘更容易。按照与健康对照相同的步骤。
  2. 加入400μl蛋白质提取缓冲液(参见食谱),并使用蓝色,聚丙烯,颗粒杵匀浆
  3. 取50μl均质溶液,并与50μl2x Laemmli溶液充分混合(参见食谱)。使用Thermomixer在95℃下煮沸样品3-5分钟,并以最大速度离心5分钟。
  4. 使用16μl上清液进行SDS-PAGE电泳;任何用于SDS-PAGE的方案都可以成功应用(Aguilar等人,2015)。由于PVX CP蛋白质的分子量约为25 kDa,因此在15%SDS-聚丙烯酰胺凝胶中解析蛋白质。将蛋白质从凝胶转移到Hybond-P PVDF膜,并用Ponceau S溶液染色印迹膜(参见食谱)以检测Rubisco蛋白作为加载对照。通过将膜与溶液孵育并轻轻摇动5分钟可以容易地实现Ponceau S染色。在进行下一步骤之前,用水清洗并轻轻晃动5分钟去除膜
  5. 在室温下用封闭溶液孵育膜2小时(参见食谱),然后在室温下,用稀释在1/400封闭溶液中的兔抗PVX CP抗体孵育过夜。第二天,在封闭溶液中用1/10,000稀释的第二抗兔AP缀合抗体孵育1小时。最后,使用基于碱性磷酸酶介导的生成反应的BCIP/NBT片剂系统开发

    图8.在过滤后7天,PVX感染的植物的Western印迹。该图显示属于相同Western印迹的三条不连续的线,M:分子量标记(kDa),H :健康植物提取物和PVX:PVX感染植物提取物。上图显示了抗PVX CP抗体孵育和BCIP/NBT显色反应的结果。下图显示了在抗体孵育之前印迹的Ponceau S染色膜,用于Rubisco蛋白检测作为载体对照。

笔记

  1. pGR107(Chapman等人,1992; Lu等人,2003)是一种常用的二元载体,源自DC Baulcombe教授组,其携带感染性PVX cDNA克隆,其中包含一个额外的CP启动子和一个多接头,使得插入外来序列成为可能
  2. 病毒症状在接种后4天开始显着(dai),达到6-8 d之间。症状在第一次出现之后的一周开始消失,这种现象被称为"恢复"
  3. 应用摩擦方法,用金刚砂填充50ml管,并覆盖2-3层纱布。这种"自制"系统允许其适当的交付。
  4. 要应用摩擦方法,重要的是使用相同种类的手套,并遵循相同的摩擦模式,以达到重现性。

食谱

  1. 磷酸钠缓冲液
    20mM NaH 2 PO 4/Na 2 HPO 4,pH 7.0
  2. 诱导缓冲液(100ml)
    1ml MgCl 2储备溶液(最终:10mM MgCl 2)
    1 ml MES储备溶液(最终:10 mM MES)
    150μl乙酰丁香酮储备溶液(最终:150μM乙酰丁香酮)
    用灭菌的双蒸水完成最终体积为100 ml 库存解决方案:
    1M MgCl 2(氯化镁)
    1 M MES(2-吗啉代乙磺酸)缓冲液,pH 5.7
    溶于DMSO(二甲基亚砜)的100mM乙酰丁香酮(3',5'-二甲氧基-4'-羟基乙酰苯酮)
  3. Luria肉汤培养基(LB培养基),pH 7.0
    1%(w/v)胰蛋白胨
    0.5%(w/v)酵母提取物 1.0%(w/v)NaCl
  4. LB /琼脂培养基 LB + 1.5%(w/v)琼脂
  5. 用于Western blot的蛋白质提取缓冲液 0.1M Tris-HCl,pH8.0
    10 mM EDTA
    0.1 M LiCl
    1%(v/v)β-巯基乙醇
    1%(w/v)SDS
    库存解决方案:
    1M Tris-HCl(三羟甲基 - 氨基甲烷和盐酸),pH8.0
    0.5M EDTA(乙二胺四乙酸),pH8.0
    20%SDS(十二烷基硫酸钠)溶液
  6. 2x Laemmli溶液用于Western blot检测 4%(w/v)SDS
    2%(v/v)β-巯基乙醇
    0.1M Tris-HCl,pH6.8。
    0.05%(w/v)BPB粉末试剂
    20%(v/v)甘油 库存解决方案:
    1M Tris-HCl(三羟甲基 - 氨基甲烷和盐酸),pH6.8
  7. Ponceau S解决方案
    0.1%w/v Ponceau S粉末试剂 5%v/v乙酸溶液
  8. 阻塞解决方案
    5%w/v脱脂奶粉
    10 mM 1x PBS缓冲液 库存解决方案:
    10倍PBS(磷酸缓冲盐水),pH 7.4
  9. 10×PBS,pH 7.4(1L)
    80克NaCl(氯化钠)
    2.0克KCl(氯化钾)
    14.4g Na 2 HPO 4(磷酸二氢钠)
    2.4g KH 2 PO 4(磷酸二氢钾)
    用无菌双蒸水完成1L的最终体积

致谢

通过修改用于pGR106的程序(Lu et al。,2003),为T-DNA二元载体pGR107开发和优化了该方案。大肠杆菌是前西班牙教育和体育部的FPU奖学金的接受者。这项工作得到了西班牙经济与竞争力部(Grant Ref。BIO2013-47940-R)的支持。大韩民国农村发展管理局(RDA)(Grant Ref。PJ00946102; PJ010246)。

参考文献

  1. Aguilar,E.,Allende,L.,Del Toro,FJ,Chung,BN,Canto,T。和Tenllado,F。(2015)。  升高的CO 2 和温度对致病性决定因素和马铃薯病毒X毒力的影响/病毒相关的协同作用。 Mol Plant Microbe Interact 28(12):1364-1373。
  2. Baulcombe,DC,Chapman,S.and Santa Cruz,S。(1995)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/7599646" target ="_ blank">水母绿色荧光蛋白作为病毒感染的记者。植物J 。 7:1045-1053。
  3. Chapman,S.,Kavanagh,TA和Baulcombe,DC(1992)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/1344890"target = "_blank">马铃薯病毒X作为植物中基因表达的载体。植物J 。 2:549-557。
  4. Chung,BN,Canto,T.and Palukaitis,P。(2007)。  含有不相关植物病毒基因的重组植物病毒的稳定性。 J Gen Virol 88(Pt 4):1347-1355。
  5. Gleba,YY,Tusé,D.和Giritch,A.(2014)。  用于农杆菌递送的植物病毒载体 。微量元素免疫中的Curr Top 375:155-192。
  6. Hammond-Kosack,KE,Staskawicz,BJ,Jones,JDG和Baulcombe,DC(1995)。< a class ="ke-insertfile"href ="http://www.apsnet.org/publications/mpmi/backissues Mol Plant-Microbe Interact 8:修饰的马铃薯病毒X基因组中真菌无毒素基因的功能表达。 181-185。
  7. Lacomme,C.和Chapman,S.(2008)。使用马铃薯病毒X (PVX)基因载体进行基因表达和病毒诱导的基因沉默(VIGS)。第36章:Curr Protoc Microbiol。单位16I 1.
  8. Lu,R.,Malcuit,I.,Moffett,P.,Ruiz,MT,Peart,J.,Wu,AJ,Rathjen,JP,Bendahmane,A.,Day,L.and Baulcombe,DC(2003) ; 高通量病毒诱导的基因沉默涉及热休克蛋白90植物抗病性。 EMBO J 22(21):5690-5699。
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引用:Aguilar, E., del Toro, F. J., Chung, B., Canto, T. and Tenllado, F. (2016). Infection of Nicotiana benthamiana Plants with Potato Virus X (PVX). Bio-protocol 6(24): e2063. DOI: 10.21769/BioProtoc.2063.
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