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Bean Pod Mottle Virus (BPMV) Viral Inoculation Procedure in Common Bean (Phaseolus vulgaris L.)
菜豆中豆荚斑驳病毒(BPMV)的接种步骤   

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Abstract

Viral vectors derived from the Bean pod mottle virus (BPMV) were shown to be highly efficient tools for functional studies in soybean (Glycine max) and common bean (Phaseolus vulgaris) (Zhang et al., 2010; Diaz-Camino et al., 2011; Pflieger et al., 2013; Pflieger et al., 2014). Indeed, BPMV-derived vectors enable successful foreign gene expression analysis as well as virus-induced gene silencing (VIGS) but the delivery procedure of the viral vector into plants (i.e. primary inoculation) is a critical step. It can be achieved by various techniques such as Agrobacterium-mediated infiltration (agro-inoculation), mechanical inoculation of in vitro transcribed RNA, or biolistic delivery of infectious plasmid DNA (i.e. a DNA plasmid carrying a cDNA copy of the modified viral genome under the control of a 35S promoter). These delivery methods may be incompatible with large-scale functional studies (Pflieger et al., 2013). Here, we present the protocol for rapid, cheap and simple mechanical inoculation of BPMV vectors by direct rubbing of infectious plasmid DNA (direct DNA rubbing). Once infected plants are obtained, we used a classical protocol of mechanical inoculation using infected leaf sap to inoculate new healthy common bean plants (i.e. secondary inoculation).

Materials and Reagents

  1. Phaseolus vulgaris (P. vulgaris) seeds
    Notes:
    1. Seeds of common bean genotypes can be obtained from the Centro Internacional de Agricultura Tropical (CIAT, Colombia).
    2. ‘Black Valentine’ is recommended for primary inoculation to generate infected leaf sap used for further inoculation of any other genotypes. For secondary inoculation, any other genotype of interest can be used.
  2. Vermiculite 1-4 mm (Agrena Rungis, catalog number: VERMOYS100 )
  3. Carborundum 0.037 mm (used as an abrasive) (VWR, catalog number: 22540.298 )
  4. Recombinant BPMV RNA1 plasmid (pBPMV-IA-R1M) (Zhang et al., 2010) (Note 1)
  5. Recombinant BPMV RNA2 plasmid (pBPMV-IA-V1, as a BPMV wild-type control) (Zhang et al., 2010) (Note 1)
  6. Recombinant BPMV RNA2 plasmid (pBPMV-GFP2) (green fluorescence protein) [as a gene expression positive control (Zhang et al., 2010) (Note 1)]
  7. Recombinant BPMV RNA2 plasmid, (pBPMV-PvPDS391bp) (phytoene desaturase) [as a VIGS positive control (Pflieger et al., 2014) (Note 1)]
  8. Miracloth (Calbiochem®, catalog number: 475855-1R )
  9. K2HPO4 (Sigma-Aldrich, catalog number: P3786 )
  10. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
  11. MilliQ water
  12. Absorbant paper 1500F (Argos, catalog number: 106 )
  13. Liquid nitrogen
  14. Qiagen kit “Plasmid Maxi Kit” (QIAGEN, catalog number: 12163 )
  15. Fertilizer Plant-Prod 14-12-32 (Puteaux SA)
  16. Fertilizer Fertiligo L (Fertil International SA)
  17. Nutritive solution (see Recipes)
  18. 0.1 M potassium phosphate buffer (see Recipes)
  19. 50 mM potassium phosphate buffer (see Recipes)

Equipment

  1. Greenhouse or growth chamber for plant growth
  2. Greenhouse or growth chamber for phytopathological tests (Note 2)
  3. Pipetmans (Gilson, model: p1000 , p200 , p100 , p20 and p10 ) and tips
  4. -20 °C refrigerator
  5. -80 °C refrigerator
  6. Vortex
  7. Plastic pots (7 x 7 x 6 cm pots and 20 cm diameter pots of 4 L)
  8. Growth chamber
  9. Latex gloves
  10. Surgical masks
  11. Microcentrifuge
  12. Eppendorf microfuge tube
  13. 1.5 ml microfuge tubes
  14. Mortar (9 cm diameter) and pestle (12 mm diameter)
  15. UV lamp for GFP detection (High intensity 100-Watt long-wave UV lamp, UVP®)
  16. UV face shield (Thermo Fisher Scientific, catalog number: 6355 )

Procedure

  1. Growth of common bean ‘Black Valentine’ plants for primary inoculation by direct DNA rubbing
    1. Sow the common bean ‘Black Valentine’ (Note 3) seeds in plastic pots (7 x 7 x 6 cm) filled with moisted vermiculite. Be careful to place the seed just under the vermiculite surface.
    2. Put the pots in a tub full of tap water and place in a growth chamber at 23 °C under a 16 h light/8 h dark cycle and 75% relative humidity (Note 4).
    3. Water the seedlings by regularly filling the tub with tap water so that watering is done by capillarity (approximately every two days, in our hands).
    4. Let the seedlings grow during 10-12 days (the stage at which primary leaves are fully-expanded).
    5. At this stage of development, the plants are ready to be inoculated (Figure 1A). If you need to maintain the plant for longer than two weeks after inoculation, we recommend to transplant three seedlings in moist vermiculite in a 20 cm diameter pot. Place the pot in a saucer full of water.
    6. Place the plants in a dark room at 19 °C and 75% relative humidity for 24 h prior to inoculation (Note 4).

  2. Mechanical inoculation of BPMV vectors by direct DNA rubbing of BPMV-derived infectious plasmids
    1. For mechanical inoculation of common bean using BPMV-derived infectious plasmids, provide 12 ‘Black Valentine’ plants for each BPMV construct.
    2. To inoculate one plant, mix 5 μg pBPMV-IA-R1M with 5 μg pBPMV-IA-V1 in a final volume of 20 μl of 50 mM potassium phosphate buffer pH 7 in an Eppendorf microfuge tube (Note 5).
    3. To inoculate one plant, mix 5 μg pBPMV-IA-R1M with 5 μg pBPMV-GFP2 or gene expression construct plasmid DNA in a final volume of 20 μl of 50 mM potassium phosphate buffer pH 7 in an Eppendorf microfuge tube (Note 5).
    4. To inoculate one plant, mix 5 μg pBPMV-IA-R1M with 5 μg pBPMV-PvPDS391bp or VIGS construct plasmid DNA in a final volume of 20 μl of 50 mM potassium phosphate buffer pH 7 in an Eppendorf microfuge tube (Note 5).
    5. Vortex briefly and spin down the tubes in a microcentrifuge.
    6. Powder the upper surface of one primary leaf per plant with carborundum (for the procedure see Video 1). The approximate amount of dusted carborundum should be as in Video 1 and Figure 1B. Don’t dust too much carborundum because it can generate undesirable necrotic areas after rubbing.

      Video 1. BPMV primary inoculation in Phaseolus vulgaris (step B6)

    7. Using a pipetman, put 20 μl DNA plasmid mix on the upper leaf surface at the junction between petiole and blade (Figure 1C).
    8. Make a rapid spreading of the DNA plasmid drop all over the leaf surface with a gloved finger (Video 2).

      Video 2. BPMV primary inoculation in P. vulgaris (steps B8-9)

    9. Rub the leaf surface by making 6 passages with a gloved finger. Make this sequential progress twice (Video 2). All the leaf surface should be rubbed. Be careful not to rub too hard.
    10. At ~3-4 min after rubbing, rinse the inoculated leaf with tap water contained in a wash bottle until all carborundum is removed (Figure 1D).
    11. Remove gently the excess of water on the upper side of the leaf using absorbant paper (Figure 1E). As the leaf is weakened, be careful not to tear the leaf (Figure 1F).


      Figure 1. Mechanical inoculation by direct DNA rubbing of BPMV-derived infectious plasmid. A. A fully-expanded primary leaf of ‘Black Valentine’ at 10-12 days. B. One primary leaf per plant powdered with carborundum. C. 20 μl of DNA plasmid mix is put on the upper leaf surface at the junction between petiole and blade. Spreading of the DNA plasmid drop all over the leaf surface is made with a gloved finger as shown in Video 1. D. The inoculated leaf is rinsed intensively with tap water contained in a wash bottle. E. The excess of water is soaked up with absorbant paper. F. The dried inoculated primary leaf.

    12. Place the inoculated plants in a greenhouse or a growth chamber at 19 °C under a 16 h light/8 h dark cycle and 75% relative humidity (Note 4).
    13. Fertilize plants after mechanical inoculation by pouring the nutritive solution directly in the pot saucer (approximately 500 ml in a 20 cm diameter saucer). Fertilize at a 3-4 days interval.
    14. Viral symptoms induced by BPMV-Wt occur at about 3 to 4 weeks post-inoculation on the upper systemic leaves that become mottled and bloated (Figure 2A). Successful infection rate using the BPMV-Wt vector is 92-100% (Pflieger et al., 2014).
    15. Green fluorescence by BPMV-GFP (=pBPMV-IA-R1M + pBPMV-GFP2) can be seen on the primary leaves at about 9 days post-inoculation under UV light and after 17 days post-inoculation on the upper systemic leaves (in Pflieger et al., 2014, see Figure 1). Successful infection rate using the BPMV-GFP vector is 55% (Pflieger et al., 2014).
    16. White photobleaching phenotype corresponding to PDS gene silencing induced by BPMV-PvPDS391bp (=pBPMV-IA-R1M+pBPMV-PvPDS391bp) is not always observed for primary-inoculated plants (Figure 2B). Successful infection rate using the BPMV- PvPDS391bp vector is 58-91% (Pflieger et al., 2014).
    17. For each BPMV construct, harvest systemic infected leaflets at 3 to 4 weeks post-inoculation. Preferably choose young upper leaves. Place one leaflet in aluminum paper and freeze the sample in liquid nitrogen.
    18. Store all the leaflet samples at -80 °C until use for mechanical inoculation of infected leaf tissues (=secondary inoculation).


      Figure 2. Representative symptoms obtained in P. vulgaris cv. ‘Black Valentine’ plants after mechanical inoculation of BPMV vectors by direct DNA rubbing of BPMV-derived infectious plasmids. A. BPMV-Wt, at 4 weeks post-inoculation. B. BPMV-PvPDS391bp, at 3, 5 weeks post-inoculation.

  3. Growth of common bean plants for mechanical inoculation of BPMV vectors using infected leaf tissues (=secondary inoculation)
    1. Follow the same protocol as described in A (Note 6).
    2. When other bean genotypes than ‘Black Valentine’ are analyzed, the growth duration of 10-12 days may be delayed until reaching the full-expanded stage of primary leaves.

  4. Mechanical inoculation of BPMV vectors using infected leaf tissues
    1. Put a fresh or a frozen infected leaflet of common bean ‘Black Valentine’ in a mortar (Figure 3A).
    2. Grind briefly the tissue with a pestle to obtain a green mash (Figure 3B).
    3. Add ~3-4 ml of pH7 50 mM potassium phosphate buffer to make leaf sap.
    4. Grind again with the pestle to obtain a green leaf sap (Figure 3C). As leaf sap is usually heterogeneous, let it settle a few minutes.
    5. Powder the upper surface of one primary leaf per plant with carborundum (Video 1 and Figure 1A). Don’t dust too much carborundum because it can generate undesirable necrotic areas after rubbing (Note 7).
    6. Cut a Miracloth piece of ~8 x 6 cm (Figure 3D) and fold it in four (Figure 3E).
    7. Soak the folded Miracloth in the leaf sap (Figure 3F).


      Figure 3. Mechanical inoculation of BPMV vectors using infected leaf tissues. A-C. Preparation of infected leaf sap by grinding an infected leaflet with a mortar and pestle. D-E. The Miracloth piece is folded in four. F. The folded Miracloth piece is soaked in the leaf sap.

    8. Make a rapid rubbing with the soaked Miracloth all over the leaf surface (Video 3).

      Video 3. BPMV secondary inoculation in P. vulgaris

    9. Rub the leaf surface by making 6 passages with the folded Miracloth (Video 3). Make this sequential progress only once. All the leaf surface should be rubbed.
    10. At ~3-4 min after rubbing, rinse the inoculated leaf with tap water contained in a wash bottle until all carborundum is removed (Figure 1D).
    11. Remove the excess of water using absorbant paper (Figure 1E).
    12. Place the inoculated plants in a growth chamber or greenhouse at 19 °C under a 16 h light/8 h dark cycle and 75% relative humidity.
    13. Fertilize plants after mechanical inoculation by pouring the nutritive solution directly in the pot saucer (approximately 500 ml in a 20 cm diameter saucer). Fertilize at a 3-4 days interval.
    14. Viral symptoms induced by BPMV-Wt occur on the upper systemic leaves at about 2 to 4 weeks post-inoculation depending on the genotype tested (in Pflieger et al., 2014, see Figure 3).
    15. Green fluorescence by BPMV-GFP (=pBPMV-IA-R1M + pBPMV-GFP2) can be seen on the primary leaves at about 4 to 9 days post-inoculation under UV light and after 10 to 17 days post-inoculation on the upper systemic leaves (in Pflieger et al.,  2014, see Figure 1).
    16. White photobleaching phenotype corresponding to PDS gene silencing induced by BPMV-PvPDS391bp (=pBPMV-IA-R1M + pBPMV-PvPDS391bp) is generally observed at 3 to 4 weeks post-inoculation (in Pflieger et al., 2014, see Figure 6), generally on the young upper leaves.
    17. For all BPMV vectors, the successful infection rate of secondary inoculation is close to 100% (Pflieger et al., 2014).

Notes

  1. All recombinant plasmids derived from BPMV RNA1 or BPMV RNA2 are carried by recombinant Escherichia coli strains and plasmids were prepared as concentrated ‘maxi-preps’ using the Qiagen kit “Plasmid Maxi Kit” according to the supplier’s instructions. To construct new BPMV vectors containing foreign fragments of interest, refer back to the detailed protocol in Zhang et al. (2013).
  2. Keep in mind that BPMV is a viral pathogen and that its manipulation must be in accordance with your country legislation in regard to biosafety concern and containment to preclude uncontrolled virus transmission.
  3. We recommend to use the common bean cv. ‘Black Valentine’ for mechanical inoculation by direct DNA rubbing because this genotype is highly susceptible to BPMV thus producing highly concentrated inoculums for secondary inoculation of any other genotype.
  4. Phaseolus vulgaris seed germination and growth are optimum at 23 °C. Viral infection and induction of silencing is better at 19 °C. So 24 h prior to inoculation and once inoculated, the plants were placed in a growth room at 19 °C.
  5. Inoculation of pBPMV-IA-R1M with pBPMV-IA-V1 generates the BPMV-Wt vector, which should be used as a negative control in all experiments. Inoculation of pBPMV-IA-R1M with pBPMV-GFP2 generates the BPMV-GFP vector, which should be used as a positive control in gene expression experiments. Inoculation of pBPMV-IA-R1M with pBPMV-PvPDS391bp generates the BPMV-PDS vector, which should be used as a positive control in VIGS experiments.
  6. For secondary inoculations using infected leaf tissues, the choice of common bean genotypes may depend of your research goals. Keep in mind that the use of BPMV vectors for gene expression or VIGS requires that the common bean genotype is susceptible to BPMV. You can test this by inoculating your genotype of interest with BPMV-GFP vector. However, in our work we showed that in some susceptible bean genotypes (e.g. JaloEEP558), the GFP fluorescence is only visible in the inoculated primary leaf but the viral vector does not move to the upper non-inoculated leaves and thus no GFP fluorescence is observed in these leaves (Pflieger et al., 2014).
  7. For secondary inoculations, don’t dust all the plants with carborundum. After rubbing of 20 leaves with the miracloth it will be soaked of leaf sap and carborundum. As an excess of carborundum generates undesirable necrotic areas after rubbing, we recommend not to dust your additional plants with carborundum or really slightly.

Recipes

  1. Nutritive solution
    Fertilizer Plant-Prod 14-12-32 (final concentration = 0.28 kg/L)
    Fertilizer Fertiligo L (final concentration = 4.35 ml/L)
    Tap water
  2. 0.1 M potassium phosphate buffer (pH 7)
    Prepare a solution of KH2PO4 1 M: 27.2 g in 200 ml of MilliQ water
    Prepare a solution of K2HPO4 1 M: 34.8 g in 200 ml of MilliQ water
    Mix 38.5 ml of KH2PO4 1 M with 61.5 ml of K2HPO4 1 M
    Control pH with a pH paper
    It should be pH 7
  3. 50 mM potassium phosphate buffer (pH 7)
    25 ml 0.1 M potassium phosphate buffer (pH 7)
    25 ml MilliQ water

Acknowledgments

This protocol was developed and optimized for Phaseolus vulgaris by modifying the procedure used for TYMV inoculation in Arabidopsis (Pflieger et al., 2008) and BPMV inoculation in soybean and common bean (Zhang et al., 2010; Zhang et al., 2013). We thank Chunquan Zhang and Steven Whitham at Iowa State University (USA) for sharing the set of BPMV VIGS vectors and providing the common bean cv. ‘Black Valentine’ seeds. CM and MR were supported by fellowships from the French Research Ministry. This work was supported by grants from Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université Paris-Sud, Saclay Plant Sciences LABEX (SPS), the 3P project (Plant Phenotyping Platform), and the PeaMUST project [grant agreement number ANR-11-BTBR-0002].

References

  1. Diaz-Camino, C., Annamalai, P., Sanchez, F., Kachroo, A. and Ghabrial, S. A. (2011). An effective virus-based gene silencing method for functional genomics studies in common bean. Plant Methods 7: 16.
  2. Pflieger, S., Richard, M. M., Blanchet, S., Meziadi, C. and Geffroy, V. (2013). VIGS technology: an attractive tool for functional genomics studies in legumes. Func Plant Biolog 40(12): 1234-1248.
  3. Pflieger, S., Blanchet, S., Camborde, L., Drugeon, G., Rousseau, A., Noizet, M., Planchais, S. and Jupin, I. (2008). Efficient virus-induced gene silencing in Arabidopsis using a 'one-step' TYMV-derived vector. Plant J 56(4): 678-690.
  4. Pflieger, S., Blanchet, S., Meziadi, C., Richard, M. M., Thareau, V., Mary, F., Mazoyer, C. and Geffroy, V. (2014). The "one-step" Bean pod mottle virus (BPMV)-derived vector is a functional genomics tool for efficient overexpression of heterologous protein, virus-induced gene silencing and genetic mapping of BPMV R-gene in common bean (Phaseolus vulgaris L.). BMC Plant Biol 14: 232.
  5. Zhang, C., Bradshaw, J. D., Whitham, S. A. and Hill, J. H. (2010). The development of an efficient multipurpose Bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol 153(1): 52-65.
  6. Zhang, C., Whitham, S. A. and Hill, J. H. (2013). Virus-induced gene silencing in soybean and common bean. Methods Mol Biol 975: 149-156.

简介

源自豆荚斑驳病毒(BPMV)的病毒载体显示为用于在大豆(大豆)和普通豆(大豆)中进行功能研究的高效工具vulgaris )(Zhang等人,2010; Diaz-Camino等人,2011; Pflieger等人,2013年) ; Pflieger等人,,2014)。实际上,BPMV衍生的载体使得能够成功地进行外源基因表达分析以及病毒诱导的基因沉默(VIGS),但是病毒载体到植物中的递送程序(即初次接种)是关键的步骤。它可以通过各种技术实现,例如农杆菌介导的浸润(农业接种),体外转录的RNA的机械接种或感染性质粒DNA(例如,在35S启动子的控制下携带经修饰的病毒基因组的cDNA拷贝的DNA质粒)。这些递送方法可能与大规模功能研究不相容(Pflieger等人,2013)。在这里,我们提出了通过直接摩擦感染性质粒DNA(直接DNA摩擦)快速,便宜和简单机械接种BPMV载体的协议。一旦获得感染的植物,我们使用机械接种的经典方案,使用感染的叶汁接种新的健康普通豆植物(即二次接种)。

新华,D.尿酸标准储备液的制备临床化学2006; v。52,p.2117-2118
  • 来源图1: http://www .taylorscientific.com/taylorscientific /一次性 - PELLET-PESTLES-with-Microtubes-Kimble-Chase-P13260.aspx
  • ... Vermiculite 1-4 mm (Agrena Rungis, catalog number: VERMOYS100)
  • Carborundum 0.037 mm (used as an abrasive) (VWR, catalog number: 22540.298)
  • Recombinant BPMV RNA1 plasmid (pBPMV-IA-R1M) (Zhang et al., 2010) (Note 1)
  • Recombinant BPMV RNA2 plasmid (pBPMV-IA-V1, as a BPMV wild-type control) (Zhang et al., 2010) (Note 1)
  • Recombinant BPMV RNA2 plasmid (pBPMV-GFP2) (green fluorescence protein) [as a gene expression positive control (Zhang et al., 2010) (Note 1)]
  • Recombinant BPMV RNA2 plasmid, (pBPMV-PvPDS391bp) (phytoene desaturase) [as a VIGS positive control (Pflieger et al., 2014) (Note 1)]
  • Miracloth (Calbiochem®, catalog number: 475855-1R)
  • K2HPO4 (Sigma-Aldrich, catalog number: P3786)
  • KH2PO4 (Sigma-Aldrich, catalog number: P0662)
  • MilliQ water
  • Absorbant paper 1500F (Argos, catalog number: 106)
  • Liquid nitrogen
  • Qiagen kit "Plasmid Maxi Kit" (QIAGEN, catalog number: 12163)
  • 肥料厂 - 产品14-12-32(Puteaux SA)
  • Fertilil Fertiligo L(Fertil International SA)
  • 营养解决方案(参见配方)
  • 0.1 M磷酸钾缓冲液(见配方)
  • 50 mM磷酸钾缓冲液(见配方)
  • 设备

    1. 植物生长的温室或生长室
    2. 植物病理学试验的温室或生长室(注2)
    3. Pipetmans(Gilson,型号:p1000,p200,p100,p20和p10)和提示
    4. -20°C冰箱
    5. -80°C冰箱
    6. 涡流
    7. 塑料盆(7×7×6cm盆和20cm直径盆4L)
    8. 生长室
    9. 乳胶手套
    10. 外科口罩
    11. 微量离心机
    12. Eppendorf微量离心管
    13. 1.5 ml微量离心管
    14. 砂浆(直径9cm)和研杵(12mm直径)
    15. 用于GFP检测的UV灯(高强度100瓦长波UV灯,UVP )
    16. UV面罩(Thermo Fisher Scientific,目录号:6355)

    程序

    1. 通过直接DNA摩擦初级接种的普通豆"黑色情人节"植物的生长
      1. 在装满湿蛭石的塑料盆(7×7×6cm)中播种普通豆"黑情人"(注3)种子。小心将种子放在蛭石表面下。
      2. 将花盆放入装有自来水的桶中,并置于23℃,16小时光照/8小时黑暗循环和75%相对湿度的生长室中(注4)。
      3. 通过定期用自来水向盆中灌水来浇灌幼苗,以便通过毛细管作用(大约每两天,在我们手中)进行浇灌。
      4. 让幼苗在10-12天(初级叶完全膨胀的阶段)生长。
      5. 在这个发育阶段,植物准备接种(图1A)。如果您需要在接种后保持植物超过两周,我们建议在20厘米直径的盆中,在潮湿的蛭石中移植三棵幼苗。将锅放在盛满水的托盘上。
      6. 将植物放置在19°C和75%相对湿度的黑暗的房间中24小时,然后接种(注4)。

    2. 通过直接DNA摩擦BPMV衍生的感染性质粒来机械接种BPMV载体
      1. 使用BPMV衍生的感染性质粒机械接种普通豆,为每个BPMV构建体提供12个黑色情人节植物。
      2. 为了接种一株植物,在Eppendorf微量离心管(注释5)中将5μgpBPMV-IA-R1M与5μgpBPMV-IA-V1在终体积为20μl的50mM磷酸钾缓冲液pH 7中混合。
      3. 为了接种一株植物,在Eppendorf微量离心管(注释5)中,在终体积为20μl的50mM磷酸钾缓冲液pH 7中,将5μgpBPMV-IA-R1M与5μgpBPMV-GFP2或基因表达构建体质粒DNA混合。
      4. 为了接种一株植物,在Eppendorf微量离心管(注释5)中,在终体积为20μl的50mM磷酸钾缓冲液pH 7中,将5μgpBPMV-IA-R1M与5μgpBPMV-PvPDS391bp或VIGS构建体质粒DNA混合。 br />
      5. 在微量离心机中短暂涡旋并旋转管
      6. 用金刚砂粉刷每棵植物的一片主叶的上表面(程序参见视频1)。粉尘的金刚砂的大致量应如视频1和图1B所示。不要粉尘太多的碳化硅,因为它可以在摩擦后产生不良的坏死区域。

        视频1.在Phaseolus vulgaris中的BPMV初级接种(步骤B6)
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      7. 使用移液管,将20μlDNA质粒混合物放置在叶柄和叶片之间的连接处的叶上表面(图1C)。
      8. 使用戴手套的手指(视频2)使DNA质粒快速扩散遍及叶子表面。

        视频2.PMP中的BPMV初级接种。 vulgaris (步骤B8-9)
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      9. 用戴手套的手指穿过6次来擦拭叶片表面。 使此顺序进行两次(视频2)。 所有叶面应擦拭。 小心不要过于用力。
      10. 在摩擦后约3-4分钟,用洗瓶中的自来水冲洗接种的叶子,直到所有的金刚砂被去除(图1D)。
      11. 使用吸收纸轻轻地除去叶子上部的过量的水(图1E)。当叶片变弱时,小心不要撕裂叶片(图1F)。


        图1.通过直接DNA摩擦BPMV衍生的感染性质粒进行的机械接种 A.在10-12天的"黑色情人"的完全扩展的初级叶。 B.一棵主要叶每植物用金刚砂粉化。 C.将20μlDNA质粒混合物放在叶柄和叶片之间的接合处的叶上表面上。如图1所示,用戴手套的手指在整个叶子表面上扩散DNA质粒液滴。D.用包含在洗瓶中的自来水充分冲洗接种的叶子。 E.用吸水纸浸泡过量的水。 F.干燥的接种初生叶
      12. 将接种的植物放在温室或生长室中,在19℃,16小时光照/8小时黑暗循环和75%相对湿度下(注4)。
      13. 在机械接种后,通过将营养溶液直接倒入锅碟(在20cm直径的碟中约500ml)来施肥植物。以3-4天的间隔施肥。
      14. BPMV-Wt诱导的病毒症状在接种后约3至4周发生在变成斑驳和膨胀的上部系统叶上(图2A)。使用BPMV-Wt载体的成功感染率为92-100%(Pflieger等人,2014)。
      15. 在紫外光下接种后约9天和接种后17天后,在上部系统叶上(在Pflieger中)可以在初生叶上观察到BPMV-GFP(= pBPMV-IA-R1M + pBPMV-GFP2) et al。,2014,见图1)。使用BPMV-GFP载体的成功感染率为55%(Pflieger等人,2014)。
      16. 对于初次接种的植物,不总是观察到对应于由BPMV-PvPDS391bp(= pBPMV-IA-R1M + pBPMV-PvPDS391bp)诱导的PDS基因沉默的白色漂白表型(图2B)。使用BPMV-PvPDS391bp载体的成功感染率为58-91%(Pflieger等人,2014)。
      17. 对于每个BPMV构建体,在接种后3至4周收获系统感染的小叶。优选选择幼嫩的上叶。将一个小叶放在铝纸中,并将样品冷冻在液氮中
      18. 将所有小叶样品存储在-80℃,直到用于机械接种感染的叶组织(=二次接种)。


        图2.在 P中获得的代表性症状。 vulgaris cv。 通过对BPMV衍生的感染性质粒进行直接DNA摩擦来机械接种BPMV载体后的'Black Valentine'植物。 A.BPMV-Wt,接种后4周。 B.BPMV-PvPDS391bp,接种后3,5周。

    3. 使用感染的叶组织机械接种BPMV载体的常见豆类植物的生长(=二次接种)
      1. 遵循A(注6)中所述的相同协议。
      2. 当分析除"黑色情人"之外的其他豆类基因型时,10-12天的生长持续时间可以延迟,直到达到初级叶的完全扩展阶段。

    4. 使用感染的叶组织机械接种BPMV载体
      1. 将新鲜的或冻结的感染的普通豆"黑色情人节"的传单放在砂浆中(图3A)
      2. 用杵短暂地研磨该组织以获得绿色醪液(图3B)
      3. 加入约3-4ml pH7,50mM磷酸钾缓冲液以制备叶汁
      4. 再次用研杵研磨以获得绿叶汁液(图3C)。 由于叶汁通常是异质的,让它沉淀几分钟。
      5. 粉末每个植物的一个主叶的上表面与金刚砂(视频1和图1A)。 不要粉尘太多的碳化硅,因为它可以在摩擦后产生不良的坏死区域(注7)。
      6. 剪切一张约8 x 6厘米的Miracloth片(图3D),并将其折叠成四片(图3E)。
      7. 将折叠的Miracloth浸泡在叶汁中(图3F)。


        图3.使用感染的叶组织机械接种BPMV载体。 A-C。通过用研钵和研杵研磨感染的叶子来制备受感染的叶汁。 D-E。 Miracloth部分折叠四。 F.将折叠的Miracloth片浸泡在叶汁中。

      8. 与浸泡的Miracloth快速摩擦整个叶面(视频3)。

        视频3. BPMV二次接种in。 vulgaris
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      9. 通过与折叠的Miracloth(视频3)6通过摩擦叶表面。 使此顺序进度只有一次。 所有叶面应擦拭。
      10. 在摩擦后约3-4分钟,用洗瓶中的自来水冲洗接种的叶子,直到所有的金刚砂被去除(图1D)。
      11. 使用吸水纸除去多余的水(图1E)。
      12. 将接种的植物放置在19℃,16小时光照/8小时黑暗循环和75%相对湿度的生长室或温室中。
      13. 在机械接种后,通过将营养溶液直接倒入锅碟(在20cm直径的碟中约500ml)来施肥植物。以3-4天的间隔施肥。
      14. 根据测试的基因型,在接种后约2至4周,在上部系统叶上发生由BPMV-Wt诱导的病毒症状(在Pflieger等人,2014,参见图3) />
      15. 在接种后约4至9天,在UV光下和在接种后10至17天后,在上部系统上可以在初级叶上观察到BPMV-GFP(= pBPMV-IA-R1M + pBPMV-GFP2)叶子(在Pflieger等人,2014年,见图1)。
      16. 通常在接种后3至4周观察到对应于由BPMV-PvPDS391bp(= pBPMV-IA-R1M + pBPMV-PvPDS391bp)诱导的PDS基因沉默的白色漂白表型(在Pflieger等人, 2014,见图6),一般在幼嫩的上叶
      17. 对于所有BPMV载体,二次接种的成功感染率接近100%(Pflieger等人,2014)。

    笔记

    1. 源自BPMV RNA1或BPMV RNA2的所有重组质粒由重组大肠杆菌菌株携带,并使用Qiagen试剂盒"Plasmid Maxi Kit"根据供应商的说明书将质粒制备为浓缩的"大量制备物"。为了构建含有感兴趣的外源片段的新的BPMV载体,参考Zhang等人(2013)中的详细方案。
    2. 请记住,BPMV是一种病毒性病原体,其操作必须符合您的国家立法关于生物安全关注和遏制以排除不受控制的病毒传播。
    3. 我们建议使用common bean cv。用于通过直接DNA摩擦进行机械接种的"黑色情人节",因为该基因型对BPMV高度敏感,因此产生高度浓缩的接种物用于任何其他基因型的二次接种。
    4. <绿豆>种子萌发和生长在23℃是最佳的。病毒感染和沉默诱导在19℃更好。因此,在接种前24小时,一旦接种,将植物放置在19℃的生长室中
    5. pBPMV-IA-R1M与pBPMV-IA-V1的接种产生BPMV-Wt载体,其应在所有实验中用作阴性对照。 pBPMV-IA-R1M与pBPMV-GFP2的接种产生BPMV-GFP载体,其应当在基因表达实验中用作阳性对照。 pBPMV-IA-R1M与pBPMV-PvPDS391bp的接种产生BPMV-PDS载体,其应在VIGS实验中用作阳性对照。
    6. 对于使用感染的叶组织的二次接种,常见的豆类基因型的选择可能取决于您的研究目标。请记住,使用BPMV载体进行基因表达或VIGS需要普通豆基因型对BPMV敏感。你可以通过接种你感兴趣的基因型与BPMV-GFP载体进行测试。然而,在我们的工作中,我们显示在一些易感豆基因型(例如 JaloEEP558)中,GFP荧光仅在接种的原生叶中可见,但病毒载体不移动到上部未接种的叶因此在这些叶子中没有观察到GFP荧光(Pflieger等人,2014)。
    7. 对于二次接种,不要用金刚砂粉末所有植物。用米拉摩擦20片叶子后,它会浸泡叶汁和金刚砂。由于过量的金刚砂摩擦后会产生不需要的坏死区域,我们建议不要用金刚砂或真正轻微地除去其他植物。

    食谱

    1. 营养溶液
      肥料厂 - 产品14-12-32(终浓度= 0.28kg/L) 肥料Fertiligo L(终浓度= 4.35ml/L) 自来水
    2. 0.1M磷酸钾缓冲液(pH7)
      制备KH 2 PO 4 1M的溶液:27.2g在200ml MilliQ水中的溶液
      制备K 2 HPO 4 1M的溶液:34.8g在200ml MilliQ水中的溶液
      将38.5ml的KH 2 PO 4 NH 4 M与61.5ml的K 2 HPO 4 M 1 br /> 用pH试纸控制pH值
      应为pH 7
    3. 50mM磷酸钾缓冲液(pH7) 25ml 0.1M磷酸钾缓冲液(pH7) 25 ml MilliQ水

    致谢

    通过修改用于拟南芥中的TYMV接种的程序(Pflieger等人,2008)和BPMV(Pflieger等人,2008)来开发并优化

    参考文献

    1. Diaz-Camino,C.,Annamalai,P.,Sanchez,F.,Kachroo,A.and Ghabrial,S.A。(2011)。 一种有效的基于病毒的基因沉默方法,用于在普通豆中进行功能性基因组学研究。 em>植物方法 7:16.
    2. Pflieger,S.,Richard,M.M.,Blanchet,S.,Meziadi,C.and Geffroy,V.(2013)。 VIGS技术:用于豆科植物功能基因组学研究的有吸引力的工具。 Func Plant Biolog 40(12):1234-1248。
    3. Pflieger,S.,Blanchet,S.,Camborde,L.,Drugeon,G.,Rousseau,A.,Noizet,M.,Planchais,S.and Jupin,I。(2008)。 使用"一步法"在拟南芥中高效的病毒诱导的基因沉默 'TYMV-derived vector。 Plant J 56(4):678-690。
    4. Pflieger,S.,Blanchet,S.,Meziadi,C.,Richard,M.M.,Thareau,V.,Mary,F.,Mazoyer,C.and Geffroy,V.(2014)。 "一步"的豆荚斑驳病毒(BPMV) - 衍生的载体是用于有效过表达异源蛋白,病毒诱导的基因沉默和在普通豆中的BPMV R基因的遗传作图的功能基因组工具( > BMC Plant Biol 14:232.
    5. Zhang,C.,Bradshaw,J.D.,Whitham,S.A.and Hill,J.H。(2010)。 开发有效的多用途豆荚斑驳病毒病毒载体外源基因表达和RNA沉默。 植物生理学 153(1):52-65。
    6. Zhang,C.,Whitham,S.A。和Hill,J.H。(2013)。 大豆和普通豆中的病毒诱导基因沉默 方法 975:149-156。
    • English
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    Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Pflieger, S., Blanchet, S., Meziadi, C., Richard, M. M. and Geffroy, V. (2015). Bean Pod Mottle Virus (BPMV) Viral Inoculation Procedure in Common Bean (Phaseolus vulgaris L.). Bio-protocol 5(13): e1524. DOI: 10.21769/BioProtoc.1524.
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    Evan LaBrant
    University of Nebraska-Lincoln
    I recently tried this protocol, and the sap-inoculated leaves quickly died after inoculation. I see in the comments this could be cause by left-over carborundum on the leaves, but is some silencing still possible even if the leaves die back?
    2/17/2016 6:18:33 PM Reply
    Stéphanie Pflieger
    Institut of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, France


    Possible explanations :
    1/ Too much carborundum.
    Be carefull not to powder too much carborundum on the leaves before rubbing. If you have many plants to inoculate with the same sap, powder only the first 6-10 plants with carborundum. The miracloth will be charged with carborundum and you can inoculate all the rest of your plants without powdering their leaves. In all cases, always rinse the leaves with tap water and dry them carefully ! The inoculated leaves will appear "crumpled" the first 24 h but should recover their normal shape after.

    2/ Too hard rubbing.
    Did you observe some necrosis areas on the inoculated leaves ? If so, it is possible that you rubbed too hard. The rubbing intensity is also genotype-dependant. Just try to rub more gently.

    The survival of the inoculated leaf is crucial for virus infection, multiplication and spreading in the hole plant and VIGS induction is dependent on these first steps.

    Good luck for your next inoculations.

    2/22/2016 2:11:18 AM


    Evan LaBrant
    University of Nebraska-Lincoln

    Hello Stéphanie, thank you for your reply. I forgot to ask as well, is it possible to save tissue from th secondary inoculation to use as inoculum for further inoculations?

    Thank you,
    Evan

    2/22/2016 5:36:26 PM


    Stéphanie Pflieger
    Institut of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, France

    Yes it is possible.
    However for our own experiments, we never do this because virus genome theoretically accumulates mutations while replicating and that 's why we prefer always use leaf sap derived from leaves of primary inoculated plants.

    3/8/2016 8:36:34 AM


    Claudia Castro
    University of California, Riverside
    Why are leaves always rinsed with water after sap inoculation?
    1/28/2016 4:49:00 PM Reply
    Stéphanie Pflieger
    Institut of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, France

    Leaves must be rinsed with tap water to remove all the carborundum. We tested without rinsing and the leaves died rapidly after inoculation.
    Good luck with your experiments.

    1/29/2016 1:23:20 AM


    Claudia Castro
    University of California, Riverside

    Thank you!

    1/29/2016 9:10:34 AM