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A Protocol to Measure the Extent of Cell-to-cell Movement of RNA Viruses in Planta
一种测定RNA病毒在植株细胞间运动的方法   

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Abstract

Here, we present a simple and rapid protocol to measure the extent of cell-to-cell movement of RNA viruses in planta. To do that, the green fluorescent protein (GFP) gene was incorporated into the genome of Melon necrotic spot virus (MNSV) as a coat protein (CP) fusion protein using the Thosea asigna virus 2A catalytic peptide (TaV 2a) (Serra-Soriano et al., 2014). TaV 2a allows the co-translational cleavage of the fusion protein resulting in the independent expression of both proteins (Kim et al., 2011). Viral infection was initiated by agro-infiltration of Cucumis melo leaves. At 6-7 days post-infiltration, fluorescent infection foci images were taken with a fluorescent stereo microscope and infection areas were measured using FIJI software.

Keywords: Plant virus, Tombusviridae, virus movement , Melon necrotic spot virus , movement proteins

Materials and Reagents

  1. 3-4 weeks old Cucumis melo L. subsp. melo cv. Galia plants
  2. Agrobacterium tumefaciens (A. tumefaciens) strain C58C1, or similar, transformed with the binary vector harboring the GFP-tagged viral genome
    In our case, pMNSV(Al)/GFP encoding GFP-tagged Melon necrotic spot virus (MNSV) genome under the control of the Cauliflower mosaic virus (CaMV) 35S promoter and the potato proteinase inhibitor terminator (PoPit) (Figure 1).


    Figure 1. Schematic representation of the recombinant infectious clone pMNSV(Al)/GFP used in this assay

  3. Yeast extract (Difco, catalog number: 212750 )
  4. Tryptone (Difco, catalog number: 211705 )
  5. Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
  6. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  7. MES (Sigma-Aldrich, catalog number: M8250 )
  8. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M9272 )
  9. Antibiotics
  10. LB medium (see Recipes)
  11. Agrobacterium infiltration buffer (see Recipes)

Equipment

  1. 28 °C growing chamber
  2. 15 ml culture tubes
  3. Swinging centrifuge rotor for 15 ml tubes
  4. 1.5 ml tubes (standard Eppendorf tubes or similar)
  5. BioPhotometer plus (Eppendorf, catalog number: 6132000008 )
  6. 1 ml syringes without needle
  7. Plant growing chamber
  8. Leica MZ12 fluorescent stereo microscope

Software

  1. Adobe Photoshop CS5 or higher
  2. ImageJ, FIJI or similar software
  3. MS Excel

Procedure

  1. Viral inoculation by agro-infiltration
    1. Inoculate a single colony of A. tumefaciens transformed with pMNSV(Al)/GFP in 5 ml of LB medium with Kanamycin (50 µg/ml) and Rifampicin (50 µg/ml). Incubate the culture overnight (about 12-16 h) at 28-30 °C with vigorous shaking.
    2. Measure the A600 of the culture.
    3. Collect the bacteria by slow-speed centrifugation (2,000 x g, 15 min) in a swinging rotor. Remove the supernatant.
    4. Resuspend the pellet with Agrobacterium infiltration buffer. Since some viruses replicate and then move faster than others, the final A600 need to be adjusted depending on the virus. Large inoculum of a fast-moving virus may generate a high number of viral infection foci so close between them that they could be quickly merged. Therefore the resulting average size of the infection foci could be overestimated. If this occurs, reduce the final A600 until you get infection foci physically separated from each other. If, instead, only few infection foci are observed, the final A600 must be increased.
    5. Leave at room temperature for 2 h before infiltration.
    6. Infiltrate the cultures with 1 ml syringe into the lower side of the leaves. Use 3-4 week-old Cucumis melo plants and avoid non fully-expanded leaves. Use your fingertip to apply gentle counter pressure to the other side of the leaf. A fully infiltrated part of the leaf gives a water-soaked appearance.
    7. Label de infiltrated leaves with a ring of adhesive one-sided tape placed around the petiole.
    8. Keep the plants in growth chambers in 16 h light at 25 °C and 8 h dark at 22 °C.

  2. Image acquisition
    1. Several days after infiltration check the appearance of fluorescent infection foci with a Leica MZ12 fluorescent stereo microscope or similar device. The exact time will depend on which virus is assayed (5-8 days after infiltration).
    2. Place the melon leaves beneath the objective (Planachromatic 0.5x) and engage the lowest magnification factor (0.71x)
    3. Select GFP1 filter set [excitation filter 425/60 nm (395-455 nm), barrier filter 480 nm].
    4. Look into the eyepieces and bring the fluorescent foci into focus using the rotatory knob.
    5. Correct the values for "exposure", "gain" and "gamma" until you obtain the desired results for the image and capture it. Don’t forget to add a scale bar.

  3. Image analysis
    1. Open the image with Adobe Photoshop CS5 or higher.
    2. If necessary, enhance the image brightness and contrast until infection foci have well-defined borders.
    3. Use the magic wand selection tool to select the infection foci. This tool selects similarly colored non-contiguous areas. If the magic wand selects less area than you want, increase the tolerance setting.
    4. Fill selection with black color by selecting “Fill” in the “Edit” menu.
    5. Invert selection by choosing “Invert” in the “Select” menu and fill with white color. If you have infection foci that have merged together separate them by drawing a white line were it feels the division should be. Save the resulting black and white image (Figure 2).


      Figure 2. Images of after B and before A the Adobe Photoshop editing

    6. Open the black and white image with ImageJ.
    7. Select “Binary” in the “Process” menu and “Make binary”.
    8. Check the spatial calibration of the image. If the image is calibrated, the number of pixels in each dimension and dimensions with a unit of length will be visible at the top of the image window. If the image is not calibrated, only the number of pixels will be written and you will need to set the dimension of the image manually. Then, open an image with a scale bar and using the "Straight line" tool, draw a line along the scale bar. In the “Analyze” menu select “Set scale”. A dialog box will open and the distance in pixels will be automatically adjusted. By choosing global, you set this calibration for all images you open during the ImageJ session.
    9. Select “Analyze particles” in the “Analyze” menu. In the new dialog box, set size range by trial and error to exclude tiny background particles. Choose “Exclude on edges” option to avoid counting partial infection foci at the edges of the image. Check “Include holes” if you want it to fill in holes and include that area in the measurement. Under “Show” select “Overlay outlines” to number the infection foci that are measured. These numbers correspond to data for individual particles that will be listed in the “Results” window if you check the “Display results” and click “Ok”.
    10. Save results as MS Excel file and calculate the average size of infection foci in MS Excel.

Recipes

  1. LB medium
    Mix 5 g of yeast extract with 10 g of tryptone, 10 g of NaCl
    Add dH2O to 1,000 ml
  2. Agrobacterium infiltration buffer
    10 mM MES (pH 5.6)
    10 mM MgCl2
    150 µM acetosyringone

Acknowledgments

This work was funded by grant BIO2011-25018 from the Spanish Ministerio de Economia y Competitividad and by Prometeo Program GV2011/003 from the Generalitat Valenciana. J.A.N. and M.S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Educacion y Ciencia of Spain.

References

  1. Kim, J. H., Lee, S. R., Li, L. H., Park, H. J., Park, J. H., Lee, K. Y., Kim, M. K., Shin, B. A. and Choi, S. Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6(4): e18556.
  2. Serra-Soriano, M., Pallas, V. and Navarro, J. A. (2014). A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. Plant J 77(6): 863-879.

简介

在这里,我们提出一个简单和快速的协议,以测量RNA病毒在植物中的细胞到细胞运动的程度。 为此,将绿色荧光蛋白(GFP)基因作为外壳蛋白(CP)融合蛋白掺入到Melon坏死斑病毒(MNSV)的基因组中,使用Sucha asigna病毒/Ta> 2A催化肽(TaV 2a)(Serra-Soriano等人,2014)。 TaV 2a允许融合蛋白的共翻译切割,导致两种蛋白的独立表达(Kim等人,2011)。 病毒感染通过对黄瓜叶的农杆菌浸润来启动。 在浸润后6-7天,用荧光立体显微镜拍摄荧光感染灶图像,并使用FIJI软件测量感染面积。

关键字

材料和试剂

  1. 3-4周龄的Cucumis melo L. subsp。 梅洛电视台 Galia 植物
  2. 用含有GFP标记的病毒基因组的二元载体转化的根癌土壤杆菌(根瘤土壤杆菌)菌株C58C1或类似物 在我们的情况下,在花椰菜花叶病毒(CaMV)35S启动子的控制下编码GFP标记的甜瓜坏死斑病毒(MNSV)基因组的pMNSV(A1)/GFP 马铃薯蛋白酶抑制剂终止子(PoPit)(图1)

    图1.本测定中使用的重组感染性克隆pMNSV(A1)/GFP的示意图

  3. 酵母提取物(Difco,目录号:212750)
  4. 胰蛋白胨(Difco,目录号:211705)
  5. 氯化钠(NaCl)(Panreac Applichem,目录号:131659)
  6. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  7. MES(Sigma-Aldrich,目录号:M8250)
  8. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M9272)
  9. 抗生素
  10. LB介质(见配方)
  11. 土壤杆菌渗透缓冲液(参见Recipes)

设备

  1. 28℃生长室
  2. 15 ml培养管
  3. 15 ml离心管旋转离心机转子
  4. 1.5ml管(标准Eppendorf管或类似物)
  5. BioPhotometer plus(Eppendorf,目录号:6132000008)
  6. 1 ml无针头的注射器
  7. 植物生长室
  8. 徕卡MZ12荧光立体显微镜

软件

  1. Adobe Photoshop CS5或更高版本
  2. ImageJ,FIJI或类似软件
  3. MS Excel

程序

  1. 通过农杆菌浸润的病毒接种
    1. 接种单个菌落。用含有卡那霉素(50μg/ml)和利福平(50μg/ml)的5ml LB培养基中的pMNSV(A1)/GFP转化的肿瘤细胞。孵育培养过夜(约12-16小时),在28-30°C,剧烈摇动
    2. 测量培养物的A 600
    3. 在摇摆转子中通过慢速离心(2,000×g/min,15分钟)收集细菌。取出上清液。
    4. 用土壤杆菌浸润缓冲液重悬沉淀。由于一些病毒复制然后比其他病毒移动更快,最终的A 600 需要根据病毒进行调整。快速移动的病毒的大接种物可以产生大量的病毒感染灶,它们之间如此接近,使得它们可以快速合并。因此,感染灶的平均大小可能被高估。如果发生这种情况,减少最后的A 600 ,直到你感染的焦点彼此物理分离。相反,如果观察到仅几个感染灶,则最终的A <600> 必须增加。
    5. 在室温下放置2小时,然后浸润
    6. 用1ml注射器将培养物浸润到叶子的下侧。使用3-4周龄的Cucumis melo 植物,避免未完全展开的叶子。使用指尖应用温柔的计数器 压力到叶片的另一侧。 叶子的完全渗透部分产生水浸泡的外观
    7. 标签浸润的叶子,在叶柄周围放置一圈粘合剂单面胶带
    8. 保持植物在生长室在16小时光照在25°C和8小时黑暗在22°C

  2. 图像采集
    1. 浸润后几天用Leica MZ12荧光立体显微镜或类似装置检查荧光感染灶的外观。 确切的时间将取决于测定的病毒(浸润后5-8天)
    2. 将瓜叶放在物镜下(平面光度0.5x),并使用最低放大系数(0.71x)
    3. 选择GFP1滤光片组[激发滤光片425/60 nm(395-455 nm),屏障滤光片480 nm]。
    4. 查看目镜,并使用旋钮将荧光焦点聚焦
    5. 更正"曝光","增益"和"伽玛"的值,直到获得所需的图像结果并进行捕获。 不要忘记添加比例尺。

  3. 图像分析
    1. 使用Adobe Photoshop CS5或更高版本打开图片。
    2. 如有必要,增强图像亮度和对比度,直到感染灶具有明确界定的边界
    3. 使用魔术棒选择工具选择感染焦点。 此工具选择类似颜色的不连续区域。 如果魔杖选择的面积小于所需的面积,请增加公差设置
    4. 在"编辑"菜单中选择"填充",填充黑色的选择
    5. 通过在"选择"菜单中选择"反转"并使用白色填充来反转选择。 如果你有感染病灶已经合并在一起分开他们通过绘制一条白线,感觉分裂应该是。 保存生成的黑白图像(图2)。


      图2. B之后和Adobe Photoshop编辑之前的图像

    6. 用ImageJ打开黑白图像。
    7. 在"处理"菜单中选择"二进制",然后选择"制作二进制"
    8. 检查图像的空间校准。如果图像被校准,则每个维度中的像素数量和具有长度单位的维度将在图像窗口的顶部可见。如果图像未校准,则只会写入像素数,您需要手动设置图像的尺寸。然后,使用缩放条打开图像,并使用"直线"工具,沿缩放条绘制一条线。在"分析"菜单中选择"设置刻度"。将打开一个对话框,以像素为单位的距离将自动调整。通过选择全局,您可以为在ImageJ会话期间打开的所有图像设置此校准
    9. 在"分析"菜单中选择"分析粒子"。在新对话框中,通过尝试和错误设置大小范围,以排除微小的背景粒子。选择"边距上排除"选项,以避免计数图像边缘的部分感染焦点。如果希望填充孔并在测量中包括该区域,请选中"包括孔"。在"显示"下选择"覆盖轮廓"来计算测量的感染焦点。如果选中"显示结果"并单击"确定",这些数字将对应于将在"结果"窗口中列出的各个粒子的数据。
    10. 将结果保存为MS Excel文件,并计算MS Excel中感染灶的平均大小

食谱

  1. LB培养基
    将5g酵母提取物与10g胰蛋白胨,10g NaCl混合 将dH <2> O添加至1,000 ml
  2. 土壤杆菌浸润缓冲液
    10mM MES(pH 5.6)
    10mM MgCl 2/
    150μM乙酰丁香酮

致谢

这项工作由来自西班牙经济部长竞争力奖项BIO2011-25018和来自Valenciana Generalitat的Prometeo计划GV2011/003资助。 J.A.N.和M.S.是西班牙的教育部长西西亚的博士后合同和博士研究生的接受者。

参考文献

  1. Kim,J.H.,Lee,S.R.,Li,L.H.,Park,H.J.,Park,J.H.,Lee,K.Y.,Kim,M.K.,Shin,B.A。和Choi,S.Y。 人类细胞系,斑马鱼和小鼠中源自猪苔藓病毒-1的2A肽的高切割效率。 PLoS One 6(4):e18556。
  2. Serra-Soriano,M.,Pallas,V。和Navarro,J.A。(2014)。 通过早期分泌途径转运病毒膜蛋白的模型:最小序列和内质网侧向移动性要求。 Plant J 77(6):863-879。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Navarro, J. A., Serra-Soriano, M. and Pallás, V. (2014). A Protocol to Measure the Extent of Cell-to-cell Movement of RNA Viruses in Planta. Bio-protocol 4(20): e1269. DOI: 10.21769/BioProtoc.1269.
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