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Quantification of the Volume and Surface Area of Symbiosomes and Vacuoles of Infected Cells in Root Nodules of Medicago truncatula
根瘤感染的蒺藜苜蓿细胞中共生体和液泡的体积和表面积的定量测定   

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本实验方案简略版
The Plant Cell
Sep 2014

Abstract

Legumes are able to form endosymbiotic interactions with nitrogen-fixing rhizobia. Endosymbiosis takes shape in formation of a symbiotic organ, the root nodule. Medicago truncatula (M. truncatula) nodules contain several zones representing subsequent stages of development. The apical part of the nodule consists of the meristem and the infection zone. At this site, bacteria are released into the host cell from infection threads. Upon release, bacteria are surrounded by a host cell-derived membrane to form symbiosomes. After release, rhizobia grow, divide, and gradually colonize the entire host cell of the fixation zone of root nodules. Therefore, mature infected cells contain thousands of symbiosomes, which remain as individual units among other organelles. Visualization of the organization and dynamics of the symbiosomes as well as other organelles in infected cells of nodules is essential to understand mechanisms regulating the development of endosymbiosis between plants and rhizobia. To examine this highly dynamic developmental process, we designed a useful imaging technique that is based on confocal scanning microscopy combined with different fluorescent dyes and GFP-tagged proteins (Gavrin et al., 2014). Here, we describe a protocol for microscopic observation, 3D rendering, and volume/area measurements of symbiosomes and other organelles in infected cells of M. truncatula root nodules. This protocol can be applied for monitoring the development of different host-microbe interactions whether symbiotic or pathogenic.

Keywords: Symbiosis (公司治理), Cell imaging (细胞成像), 3D rendering (3D渲染), Organelle visualization (细胞器的可视化), Cell measurements (细胞测量)

Materials and Reagents

  1. Microscope cover glasses
  2. Microscope slides
  3. Perlite (Maasmond-Westland, The Netherlands) 
  4. 7 days old seedlings of M. truncatula (seeds can be obtained at The Samuel Roberts Noble Foundation)
  5. Agrobacterium rhizogenes MSU440 strains (can be obtained at the Laboratory of Molecular Biology, Wageningen University, The Netherlands) containing pUB:GFP-MtVTI11 or pLB:GFP-MtVTI11 binary vector (see Note 1)
  6. Sinorhizobium meliloti 2011 (see Note 2)
  7. 0.1 M sodium phosphate (Sigma-Aldrich, catalog numbers: S8282 and S7907 ) buffer (pH 7.2) with 3% sucrose (Sigma-Aldrich, catalog number: S7903). For preparation protocol see Sambrook and Russell (2001)
    Note: Sodium phosphate monobasic (Sigma-Aldrich, catalog number: S8282) and Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S7907)
  8. 10 μg/ml Propidium iodide (store at 4 °C) (Sigma-Aldrich, catalog number: P4170 )
  9. 1% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15700 )
  10. 0.75% glutaraldehyde (Electron Microscopy Sciences, catalog number: 16000 )
  11. 50 mM phosphate buffer (Sambrook and Russell, 2001)
  12. Fixative (see Recipes)

Equipment

  1. Fluorescence stereomicroscope (Leica Microsystems, model: MZFLIII )
  2. Confocal laser scanning microscope with digital camera (ZEISS, model: Axiovert 100M )
  3. Razor blades
  4. Fine tweezers (Structure Probe, Dumont, model: 0T05B-XD )

Software

  1. IMARIS software (Bitplane) with modules for data management, visualization, 3D rendering and analysis
  2. Zeiss LSM Image Browser

Procedure

  1. Generation of transgenic nodules
    1. Generation of pUB:GFP-MtSYP22 or pLB:GFP-MtSYP22 expressing roots by Agrobacterium rhizogenes1-mediated transformation of 7 days old seedlings of M. truncatula. For transformation protocol see Limpens et al. (2004). Plants were grown for 14 days after transformation.
    2. Inoculation of composite M. truncatula plants with pUB:GFP-MtSYP22 or pLB:GFP-MtSYP22 transgenic roots with Sinorhizobium meliloti (S. meliloti) 2011 (Sambrook and Russell, 2001). Composite Medicago plants with transgenic roots are transferred to perlite saturated with Färhaeus medium without Ca(NO3)2 (Limpens et al., 2004). After 2 days, plants are inoculated with 2 ml of the S. meliloti 2011 culture (OD600 nm of 0.1) per plant and grown for 2 weeks (21 °C; 16/8 h light/darkness).

  2. Sample preparation for image acquisition
    1. Transgenic nodules (14 days after inoculation) are selected using a fluorescent stereomicroscope with DsRED1 filter settings (Figure 1A-B).
    2. Harvested nodules are collected with tweezers in an Eppendorf tube with 500 µl of fixative for 1 h fixation at 4 °C.
    3. Fixed nodules are hand-sectioned by razor blades in phosphate buffer (Figure 1C). Try to do sections as thin as possible.
    4. Nodule sections are collected by a tweezer in an Eppendorf tube with propidium iodide solution for 10-20 min staining at room temperature.
    5. Place nodule sections in a drop of phosphate buffer on a microscopic glass slide. Then place an edge of the cover glass over the sample and carefully lower the cover; make sure there is no air between tissue and glass.


      Figure 1. Collection and sectioning of Medicago root nodules. A. Medicago root nodules. B. Medicago root nodules on a transgenic root (selected on the basis of red fluorescence resulting from DsRED1 expression). C. Nodule sectioning.

  3. Image acquisition
    1. Observe the nodule section under the confocal microscope using an oil immersion objective lens (x63 or higher) with 488 nm excitation and 505-530 nm emission wavelengths for GFP and with 543 nm excitation and 636 nm emission wavelengths for propidium iodide. Filters: Ch1: LP560, Ch2: BP505-530. Pinhole: 106 μm.
    2. Find a group of infected cells at the developmental stage of interest and focus on it.
    3. Obtain Z-stack serial images of region of interest. Z-stack size: 512 x 512 x 20-73 pixel. Number of single images in Z-stacks 20-67 depending on the position and size of scanned cells. Using Projection tool you can create a 3D projection image or a rotation in 3D, which can be animated and exported as an AVI movie (Figure 2, Video 1).


      Figure 2. 3D projection of Z-stack serial images of nodule cells expressing GFP-MtSYP22 (green). Bacteria and bacteroids are contrasted by propidium iodide (red). ic, infected cell; it, infection thread; v, vacuole. Scale bar 40 µm.

      Video 1. 3D projection of nodule cell

  4. Image processing: 3D rendering and volume/area measurements of vacuoles and symbiosomes in infected cells
    1. Open Z-stack serial images of the nodule using IMARIS software.
    2. Select a region of interest by 3D Crop operation for further manipulations (Figure 3).
    3. If an image is too dim to view, adjust brightness of channels individually in the Display Adjustment window.
    4. To get rid of out of focus light or other artefacts before measurements, select Smoothing in the Image Processing menu. Select the channels you want to have filtered and filter out noise signal from the 3D image.
    5. Generate surfaces of vacuoles and symbiosomes of infected cells in the Surface generation wizard (select the Source Channel for segmentation: for vacuoles pick GFP channel and for symbiosomes RFP channel). To generate the surface of the whole infected cell, use manual 3D construction mode.
    6. Adjust the threshold (the thresholded regions are shown in grey during adjustment).
    7. Generated and complete surfaces will be present in the image with changed colours (Figure 3B). You can take measurements from your surfaces by pressing the measure icon (Imaris Measurement). If necessary, adjust the measurement parameters and use filters. Using Movie Maker you can create animations with generated surfaces (Video 2).


      Figure 3. 3D rendering of the infected cell at the moment of bacteria release in Medicago root nodules. A. 3D projection of Z-stack serial images of nodule cells expressing at the moment of bacteria release. B. An example of 3D rendering of the infected cell outlined in (A). ic, infected cell; it, infection thread; r, release of bacteria; v, vacuole. Scale bar 20 µm.

      Video 2. Fragmented vacuole of an infected cell

Notes

  1. MtSYP22 is a vacuole SNARE protein, controlling pre-vacuolar compartment-to-vacuole trafficking. GFP-MtSYP22 fusion protein allows visualizing vacuoles in nodule cells. pUB:GFP-MtSYP22 fusion is controlled by a ubiquitin promoter expressed in the apical part of root nodules (meristem and the infection zone). pLB:GFP-MtSYP22 fusion is driven by a leghemoglobin promoter, which is active in mature infected cells of the fixation zone of root nodules. These binary vectors enable the selection of transgenic roots and nodules based on additional DsRED1 (Discosoma sp. red fluorescent protein) expression.
  2. Sinorhizobium meliloti 2011 is a symbiont of M. truncatula (can be obtained at Laboratory of Molecular Biology, Wageningen University).

Recipes

  1. Fixative
    1% paraformaldehyde
    0.75% glutaraldehyde
    50 mM phosphate buffer (Sambrook and Russell, 2001)
    Stored at -20 °C

Acknowledgments

We thank our colleagues Norbert de Ruijter for assistance with confocal imaging and T. W. J. Gadella (University of Amsterdam) for help with the analysis of Imaris 3D reconstructed images. A. G. received a Ph.D. fellowship from the EPS School of Biological Sciences (Wageningen University).

References

  1. Gavrin, A., Kaiser, B. N., Geiger, D., Tyerman, S. D., Wen, Z., Bisseling, T. and Fedorova, E. E. (2014). Adjustment of host cells for accommodation of symbiotic bacteria: Vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago. Plant Cell 26(9): 3809-3822.
  2. Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T. and Geurts, R. (2004). RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55(399): 983-992.
  3. Sambrook, J. and Russell, D. W. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, third edition.

简介

豆类能够与固氮根瘤菌形成内共生相互作用。内共生形成共生器官,根瘤的形成。 Medic虫truncatula ( truncatula )结节包含代表后续发展阶段的几个区域。结节的顶部由分生组织和感染区组成。在这个位置,细菌从感染线程释放到宿主细胞中。释放后,细菌被宿主细胞衍生的膜包围以形成共生体。释放后,根瘤菌生长,分裂,并逐渐定植于根瘤根固定区的整个宿主细胞。因此,成熟的感染细胞含有数千个共生体,其在其他细胞器中保持为单独的单元。可视化的共生体以及受感染的结节细胞中的其他细胞器的组织和动力学是必要的理解机制调节植物和根瘤菌之间的内共生的发展。为了检查这种高度动态的发育过程,我们设计了基于共聚焦扫描显微镜结合不同荧光染料和GFP标记蛋白的有用的成像技术(Gavrin等人,2014)。在这里,我们描述协议的显微镜观察,3D渲染和体积/面积测量的共生体和其他细胞器在感染细胞的M。 truncatula 根瘤。此协议可以应用于监测不同宿主微生物相互作用的发展,无论是共生的还是致病的。

关键字:公司治理, 细胞成像, 3D渲染, 细胞器的可视化, 细胞测量

材料和试剂

  1. 显微镜盖玻璃
  2. 显微镜载玻片
  3. 珍珠岩(Maasmond-Westland,荷兰)
  4. 7天龄的幼苗。 truncatula (种子可以在The Samuel Roberts Noble基金会获得)
  5. 可以包含pUB:GFP-MtVTI11或pLB:GFP-MtVTI11二元载体(参见注释1)
    的毛发土壤杆菌MSU440菌株(可以在荷兰瓦赫宁根大学的分子生物学实验室获得) >
  6. Sinorhizobium meliloti 2011(见注2)
  7. 含有3%蔗糖(Sigma-Aldrich,目录号:S7903)的0.1M磷酸钠(Sigma-Aldrich目录号:S8282和S7907)缓冲液(pH7.2)。有关制备方案,请参见Sambrook和Russell(2001)
    注意:磷酸二氢钠(Sigma-Aldrich,目录号:S8282)和磷酸氢二钠(Sigma-Aldrich,目录号:S7907)
  8. 10μg/ml碘化丙啶(4℃保存)(Sigma-Aldrich,目录号:P4170)
  9. 1%多聚甲醛(Electron Microscopy Sciences,目录号:15700)
  10. 0.75%戊二醛(Electron Microscopy Sciences,目录号:16000)
  11. 50mM磷酸盐缓冲液(Sambrook和Russell,2001)
  12. 固定剂(见配方)

设备

  1. 荧光立体显微镜(Leica Microsystems,型号:MZFLIII)
  2. 具有数码相机的共聚焦激光扫描显微镜(ZEISS,型号:Axiovert 100M)
  3. 剃刀刀片
  4. 精细镊子(结构探针,Dumont,型号:0T05B-XD)

软件

  1. IMARIS软件(Bitplane),带有用于数据管理,可视化,3D渲染和分析的模块
  2. Zeiss LSM图像浏览器

程序

  1. 转基因结节的生成
    1. 通过毛根土壤杆菌 产生

      pUB:GFP-MtSYP22 或

      pLB:GFP-MtSYP22 - 介导的7天龄的转化 M的幼苗。 truncatula 。转化方案参见Limpens等人 et al。 (2004)。转化后植物生长14天

    2. 复合材料的接种。具有 pUB:GFP-MtSYP22 或

      pLB:GFP-MtSYP22 转基因根的转基因植物 meliloti )2011(Sambrook和Russell,2001)。复合苜蓿植物 转基因根转移到用没有Ca(NO 3)2(Limpens等人,2004)的F?rhaeus培养基饱和的珍珠岩。 2天后,用2ml的S接种植物。 meliloti 2011培养物(OD 600nm为0.1),并生长2周 (21℃; 16/8h光/黑暗)。


  2. 图像采集的样品准备
    1. 使用a。选择转基因结节(接种后14天) 荧光立体显微镜,具有DsRED1滤光片设置(图1A-B)
    2. 收获的结节用镊子收集在Eppendorf管中,500μl固定剂在4°C固定1小时。
    3. 在磷酸盐缓冲液中用剃刀刀片对固定的结节进行手工切片(图1C)。尽量做尽可能薄的部分。
    4. 结节切片用镊子在Eppendorf管中收集 ?碘化丙啶溶液在室温下染色10-20分钟
    5. 将结节切片放在一滴磷酸盐缓冲液中的微观上 ?玻璃载玻片。然后将盖玻片的边缘放在样品上 小心降低盖子;确保组织之间没有空气 玻璃

      图1.苜蓿根瘤的收集和切片。A.苜蓿根瘤。 B.转基因根上的苜蓿根瘤 ?(基于从DsRED1产生的红色荧光选择 表达)。 C.结节切片。

  3. 图像获取
    1. 观察在共焦显微镜下使用油的结节部分 浸没物镜(x63或更高),具有488nm激发和 505-530 nm发射波长为GFP和543 nm激发和 636nm发射波长。过滤器:Ch1:LP560, Ch2:BP505-530。针孔:106μm
    2. 在感兴趣的发育阶段找到一组感染的细胞,并专注于它
    3. 获取感兴趣区域的Z堆栈串行图像。 Z堆叠大小: 512 x 512 x 20-73像素。 Z-stacks中的单个图像数量20-67 这取决于被扫描细胞的位置和大小。使用投影 工具,您可以创建3D投影图像或3D中的旋转,这可以 ?动画并导出为AVI电影(图2,视频1)。


      图2.结节细胞的Z-堆叠系列图像的3D投影 表达GFP-MtSYP22 (绿色)。细菌和类菌体形成对照 通过碘化丙锭(红色)。 ic,感染细胞;它,感染线程; v, 液泡。比例尺40μm

      视频1.结节细胞的3D投影
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  4. 图像处理:感染细胞中液泡和共生体的3D渲染和体积/面积测量
    1. 使用IMARIS软件打开Z-stack系列结节的结节。
    2. 通过3D裁剪操作选择感兴趣区域进行进一步操作(图3)。
    3. 如果图像太暗无法查看,请在显示调整窗口中单独调整通道的亮度
    4. 为了摆脱焦点光或其他人工制品之前 测量,请在"图像处理"菜单中选择"平滑"。选择 通道您想要过滤和滤除噪声信号从 3D图像。
    5. 产生液泡和共生体的表面 表面生成向导中的受感染单元格(选择源 通道分割:对于液泡选择GFP通道和 共生体RFP通道)。生成整个被感染的表面 单元格,使用手动3D构造模式
    6. 调整阈值(调整时阈值区域显示为灰色)。
    7. 生成的和完整的表面将存在于图像中 改变颜色(图3B)。你可以从你的测量 按下测量图标(Imaris Measurement)。如果 必要时,调整测量参数并使用滤波器。使用 Movie Maker您可以使用生成的曲面创建动画(视频2)。


      图3.感染细胞在细菌瞬间的3D渲染 在苜蓿根瘤中释放。 A.Z-堆叠系列的3D投影 在细菌释放时表达的结节细胞的图像。乙。 在(A)中概述的感染细胞的3D渲染的实例。我知道了, 感染细胞;它,感染线程; r,细菌释放; v,液泡。 ?比例尺20μm

      视频2.受感染细胞的碎片液泡
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笔记

  1. MtSYP22是一种液泡SNARE蛋白,控制前液泡室到液泡运输。 GFP-MtSYP22融合蛋白允许可视化结节细胞中的空泡。 pUB:GFP-MtSYP22融合由在根瘤根(分生组织和感染区)的顶部表达的泛素启动子控制。 pLB:GFP-MtSYP22融合由leghemoglobin启动子驱动,其在根瘤根的固定区的成熟感染细胞中有活性。这些二元载体使得能够基于另外的DsRED1(Discosoma sp。红色荧光蛋白)表达来选择转基因根和结节。
  2. Sinorhizobium meliloti 2011是 M的共生体。 truncatula (可以在Wageningen大学分子生物学实验室获得)

食谱

  1. 固定剂
    1%多聚甲醛
    0.75%戊二醛
    50mM磷酸盐缓冲液(Sambrook和Russell,2001) 储存于-20°C

致谢

我们感谢我们的同事Norbert de Ruijter帮助共焦成像和T. W. J. Gadella(阿姆斯特丹大学)帮助分析Imaris 3D重建图像。获得博士学位。来自EPS生物科学学院(瓦赫宁根大学)的奖学金。

参考文献

  1. Gavrin,A.,Kaiser,B.N.,Geiger,D.,Tyerman,S.D.,Wen,Z.,Bisseling,T.and Fedorova,E.E。(2014)。 调节宿主细胞以适应共生细菌:真菌去功能化,HOPS抑制和TIP1g再定向, em> Medicago 。植物细胞 26(9):3809-3822。
  2. Limpens,E.,Ramos,J.,Franken,C.,Raz,V.,Compaan,B.,Franssen,H.,Bisseling,T.and Geurts,R。(2004)。 RNA干扰发根农杆菌 - 转化拟南芥根,/em> 55(399):983-992。
  3. Sambrook,J。和Russell,D.W。(2001)。 分子克隆:实验室手册。 Cold Spring Harbor Laboratory Press,第三??版。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gavrin, A. and Fedorova, E. E. (2015). Quantification of the Volume and Surface Area of Symbiosomes and Vacuoles of Infected Cells in Root Nodules of Medicago truncatula. Bio-protocol 5(23): e1665. DOI: 10.21769/BioProtoc.1665.
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