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Capturing Z-stacked Confocal Images of Living Bacteria Entering Hydathode Pores of Cauliflower
采集活细菌进入花椰菜泌水孔的Z-栈共聚焦图像   

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Abstract

The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.

Keywords: Cauliflower(花椰菜), Pore(孔), Hydathode(泌水孔), Xanthomonas(黄单胞菌), Confocal microscope(共聚焦显微镜)

Background

Pores of hydathodes and stomata are possible entry points for pathogenic microorganisms to invade plant tissues. In cauliflower, the hydathodes present on leaf margins exhibit large pores, resembling stomata. These pores are routes for the leaf infection by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) (Cerutti et al., 2017). Hydathodes are present on leaves of a wide range of vascular plants. We describe a simple protocol to visualize bacteria at the pore level by confocal microscopy.

Materials and Reagents

  1. Razor blade
  2. Microscope glass slides (Thermo Fisher Scientific, Superfrost, catalog number: 10143560WCUT )
  3. Cover slip 24 x 60 mm (Thermo Fisher Scientific, catalog number: 15747592 )
  4. Compost (Proveen, catalog number: 14926 )
  5. Cauliflower (Brassica oleracea var. botrytis, cultivar Clovis, Vilmorin)
    Note: Plants were grown on compost in a controlled greenhouse (short day conditions 9 h light; temperature 22 °C; relative humidity 70%). After inoculation, they were placed back in the controlled greenhouse for 24 h inside the miniature greenhouse at 100% relative humidity. After 24 h, lid was removed. All the experiments used the second true leaf from four-week-old plants.
  6. Xcc strain 8004::GUS-GFP (Xanthomonas campestris pv. campestris (Xcc); Cerutti et al., 2017)
  7. Magnesium chloride (MgCl2) (Merck, catalog number: 814733 )
  8. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  9. Casamino acids (BD, BD Biosciences, catalog number: 223050 )
  10. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 105101 )
  11. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: 105886 )
  12. Silwet L-77 (CAAHMRO, catalog number: 115950H )
  13. Calcofluor (Fluorescent Brightener 28) (Sigma-Aldrich, catalog number: F3543 )
  14. MOKA medium (see Recipes)

Equipment

  1. Miniature greenhouse purchased from a garden center (Nortene, catalog number: ME673714 )
  2. Hollow punch (Harris, Uni-Core) (Electron Microscopy Sciences, catalog number: 69039-70 ) or razor blades
  3. Confocal microscope (Leica Microsystems, model: Leica TCS SP2 or Leica TCS SP8 ) equipped with an argon laser (ray line at 488 nm), a Helium Neon laser at 633 nm and a diode laser at 405 nm
  4. Water immersion lens 40x with a long working distance (N.A. = 0.8, working distance of 3,300 µm) (Leica Microsystems, catalog number: 50615 5) or water immersion lens 63x with a long working distance (N.A. = 0.9 and a working distance of 2,900 µm) (Leica Microsystems, catalog number: 506148 ) (Leica Mannheim, Germany)

Procedure

  1. Inoculate plants by dipping leaves (the second true leaf from four-weeks-old cauliflower) for 15 sec inside a bacterial suspension (108 cfu/ml) of 1 mM MgCl2 and 0.02% of Silwet L-77 (see Figure 1A). Bacteria are previously grown in MOKA medium (see Recipe 1). Gently move the leaf in the solution to improve leaf wetting.
  2. Place the plants for 24 h in closed miniature greenhouses, watered (1-2 cm of water inside the greenhouse) and keep them at 100% relative humidity (see Figure 1B) in a growth chamber. After 24 h, remove lids and place back the plants in the growth chamber (9 h light; 22 °C and relative humidity, 70%).


    Figure 1. Inoculation procedure of a cauliflower leaf. A. Inoculation by dipping the second true leaf into a bacterial suspension; B. Cauliflower plants are placed after dipping inoculation in miniature greenhouses for 24 h.

  3. At given days post infection (3 and 6 dpi), take pieces (½ leaf or disk ~0.5 cm in diameter, as shown Figure 2) of leaves (margins including hydathodes) and mount the samples in water on a glass slide (Figure 2). Identify the face to be observed (adaxial, the upper side of the leaf, or abaxial, the underside of the leaf), both sides can be observed. Cover the samples with a cover slip and gently add a few drops of an aqueous solution of calcofluor (0.01%).
    Note: Before adding the calcofluor, check the level of autofluorescence at 405 nm. Sometimes the emitted autofluorescence of the epidermal cell wall is sufficient to image the leaf surface.


    Figure 2. Samples of cauliflower leaves mounted on glass slide. A. The abaxial face of ½ leaf of cauliflower simply cut with a razor blade; *: Hydathode. The white circle indicates an area cut for observation as shown in B and C. B and C. Leaf disk samples with the hollow punch, with one hydathode (*) at the leaf margin. (B) Adaxial (the upper side of the leaf) and (C) abaxial (the underside of the leaf) views. Scale bars = 2.5 mm.

  4. To localize regions of interest with bacteria expressing GFP, check the preparation under a microscope (via the eye-pieces) in epifluorescence using a GFP filter cube (excitation in the blue range, and emission filter at 525 ± 25 nm). Use a 10x objective lens to rapidly observe the samples. Then use a 40x or 60x water immersion lenses to acquire images and z-stacks.
  5. In confocal mode, use the diode laser at 405 nm to collect the fluorescence (between 410-470 nm) emitted by the calcofluor, the 488 nm ray line of the argon laser to collect the GFP fluorescence between 500 and 540 nm and the 633 nm ray line of the HeNe laser to collect the autofluorescence of the chlorophyll (between 640 and 680 nm) (Figure 3).
  6. Adjust the settings for each channel of fluorescence (power of the laser used for excitation, and the gain of the photomultiplier (PMT) to avoid overexposure of the PMT detectors (Figure 3).


    Figure 3. Control panel of the SP2 AOBS confocal microscope. The left part corresponds to the settings for the acquisition of the calcofluor and the chlorophyll fluorescences. The 405 nm ray line (white arrow) of a diode laser is used for excitation of the calcofluor (the power of the laser can be modulated from 1 to 100% of the nominal power of the diode, here the value is 99%). The 633 ray line of a helium-neon laser (black arrow) was used for the excitation of the chlorophyll (power laser at 70% of the nominal power of the laser). The Leica confocal is equipped with a spectral module. The fluorescence emitted by the calcofluor is collected between 410 and 470 nm by the PMT-1 (blue channel), and the fluorescence emitted by the chlorophyll is collected between 640 and 680 nm by the PMT-3 (red channel). The right part corresponds to the settings for the acquisition of the GFP alone, using the 488 nm ray line of an argon laser (white arrow) with a power at 48% of the nominal power of the laser and the fluorescence is collected between 500 and 540 nm by the PMT2 (green channel).

  7. Use the sequential mode of the confocal microscope to avoid crosstalk between PMT detectors. To do that collect first the calcofluor and the chlorophyll fluorescences and next, the GFP fluorescence as indicated above (Figure 3). Acquire images (512 x 512 pixels) at 400 Hz with a line averaging of 0 to 8 (depending on the speed of bacterial movements) (see Figure 4).
  8. For z-stack acquisitions, define the top and bottom position of the microscope galvo-stage and the z step close to 0.5 µm. Maximal projection is done (using the Leica Software) from 10 to 25 confocal planes acquired in z-dimension (see Figure 4).


    Figure 4. Confocal images of GFP expressing Xcc at the pore level. A. Maximal projection from 15 confocal planes acquired in z-dimension with a z-step of 0.5 µm and a line-averaging value of 2. B. Maximal projection from 20 confocal planes acquired in z-dimension with a z-step of 0.5 µm and a line-averaging value of 4. C. Maximal projection from 25 confocal planes acquired in z-dimension with a z-step of 0.5 µm and no line-averaging. Arrows indicate the pore. The blue color corresponds to the emitted fluorescence of the calcofluor staining the epidermal cell wall surface, the green color corresponds to the GFP expressing bacteria and the red color corresponds to the chlorophyll. Scale bars =30 µm.

Recipes

  1. MOKA medium
    Note: Bacteria were grown overnight at 30 °C in MOKA medium. They were harvested from the culture medium by centrifugation (4,000 x g, 10 min) and suspended in 1 mM MgCl2 and 0.02% of Silwet L-77 at 108 cfu ml-1.
    In distilled water for 1 L:
    4 g yeast extract
    8 g casamino acids
    2 g K2HPO4
    0.3 g MgSO4·7H2O

Acknowledgments

This work was supported by a PhD grant from the French Ministry of National Education and Research to AC. LIPM is part of the French Laboratory of Excellence project (TULIP ANR-10-LABX-41; ANR-11-IDEX-0002-02). We thank the Région Occitanie for continued financial support of the microscopy platform and Laurent Noël for critically reading the manuscript.

References

  1. Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.

简介

目前在花椰菜水合物的孔隙水平上可视化活细菌的方案简单,共聚焦显微镜下的训练有素的用户可以成功地执行。 它可以容易地适应于在叶表面捕获其他植物 - 微生物相互作用的图像,并且应该有助于获得关于孔隙和气孔生物学的重要信息。 用于观察孔隙中植物 - 微生物相互作用的方法的关键限制是在观察和z堆叠采集期间对样品施加过大的压力。 为了解决这个问题,我们建议使用长工作距离的水浸物镜,即使使用厚样品也能进行观察。
【背景】水合物和气孔的孔隙是病原微生物入侵植物组织的可能入口点。 在花椰菜中,叶片上存在的水合物表现出大孔,类似气孔。 这些毛孔是血管病原菌黄单胞菌(Xanthomonas campestris)pv的叶感染的途径。 ((Cerutti等人,2017)。 水溶性植物存在于各种维管植物的叶上。 我们描述了一个简单的协议,通过共聚焦显微镜在孔隙水平上可视化细菌。

关键字:花椰菜, 孔, 泌水孔, 黄单胞菌, 共聚焦显微镜

材料和试剂

  1. 剃刀刀片
  2. 显微镜玻片(Thermo Fisher Scientific,Superfrost,目录号:10143560WCUT)
  3. 封面24 x 60 mm(Thermo Fisher Scientific,目录号:15747592)
  4. 堆肥(Proveen,目录号:14926)
  5. 花椰菜(Brassica oleracea) var。 botrytis ,品种Clovis,Vilmorin)
    注意:植物在受控温室的堆肥中生长(短日照条件9小时光照;温度22℃;相对湿度70%)。接种后,将它们在100%相对湿度下放置在微型温室内的受控温室中24小时。 24小时后,取下盖子。所有的实验都使用了来自四周龄植物的第二个真叶。
  6. 菌株8004 :: gus-GFP (Xanthomonas campestris pv。 ( Xcc >); Cerutti et al。,2017)
  7. 氯化镁(MgCl 2)(Merck,目录号:814733)
  8. 酵母提取物(Sigma-Aldrich,目录号:Y1625)
  9. Casamino acids(BD,BD Biosciences,目录号:223050)
  10. 磷酸氢二钾(K 2)HPO 4(Merck,目录号:105101)
  11. 七水硫酸镁(MgSO 4•7H 2 O)(Merck,目录号:105886)
  12. Silwet L-77(CAAHMRO,目录号:115950H)
  13. Calcofluor(荧光增白剂28)(Sigma-Aldrich,目录号:F3543)
  14. MOKA中等(见食谱)

设备

  1. 从花园中心购买的微型温室(Nortene,目录号:ME673714)
  2. 空心冲孔机(Harris,Uni-Core)(Electron Microscopy Sciences,目录号:69039-70)或剃刀刀片
  3. 配有氩激光(488nm的射线),633nm的氦氖激光器和405nm的二极管激光器的共聚焦显微镜(Leica Microsystems,型号:Leica TCS SP2或Leica TCS SP8)
  4. 工作距离长(NA = 0.8,工作距离为3,300μm)的水浸透镜40x(Leica Microsystems,目录号:506155)或具有长工作距离(NA = 0.9,工作距离为2900μm)的水浸透镜63x )(Leica Microsystems,目录号:506148)(Leica Mannheim,Germany)

程序

  1. 通过将1ml MgCl 2的细菌悬浮液(10μg/ ml)中的浸泡叶(来自四周花椰菜的第二个真叶)接种15秒钟, >和0.02%Silwet L-77(参见图1A)。细菌先前在MOKA培养基中生长(参见方案1)。轻轻地移动溶液中的叶子,以改善叶子的湿润度
  2. 将植物放置在闭合的微型温室中,浇水(温室内1-2厘米的水),并将其保持在生长室中100%的相对湿度(见图1B)。 24小时后,取出盖子并将植物放回生长室(9小时光照; 22℃,相对湿度70%)。


    图1.花椰菜叶的接种程序。 :一种。通过将第二真叶浸入细菌悬浮液进行接种; B.将花椰菜植物浸泡在微型温室中24小时后放置
  3. 在感染后的给定日期(3和6dpi),取片(1/2叶或直径0.5cm,如图2所示)的叶片(包括水合物的边缘)并将样品安装在玻璃载玻片上的水中(图2 )。识别要观察的面(近轴,叶的上侧,或背面,叶的下侧),可以观察到两侧。用盖子覆盖样品,轻轻地加入几滴calcofluor水溶液(0.01%)。
    注意:在添加calcofluor之前,检查405nm处的自发荧光水平。有时,表皮细胞壁的发射自发荧光足以使叶片成像。


    图2.安装在玻璃载玻片上的花椰菜叶子样品。A.花椰菜叶片的背面仅用刀片切割; *:Hydathode。白色圆圈表示用于观察的区域,如B和C所示。B和C.具有中空冲头的叶盘样品,叶片边缘具有一个阴极(*)。 (B)近轴(叶的上侧)和(C)背面(叶的下侧)视图。比例尺= 2.5毫米。

  4. 为了使表达GFP的细菌定位感兴趣区域,使用GFP滤光片(蓝色范围内激发,以及525±25nm的发射滤光片)检查在荧光下的显微镜(通过眼睛)的制备。使用10x物镜快速观察样品。然后使用40x或60x水浸透镜来获取图像和z叠层。
  5. 在共聚焦模式下,使用405nm处的二极管激光器来收集由calcofluor发射的荧光(在410-470nm之间),即氩激光的488nm线,以收集在500和540nm之间的GFP荧光和633nm HeNe激光的X射线以收集叶绿素的自发荧光(在640和680nm之间)(图3)。
  6. 调整每个荧光通道的设置(用于激发的激光功率)和光电倍增管(PMT)的增益,以避免PMT检测器过度曝光(图3)。


    图3. SP2 AOBS共聚焦显微镜的控制面板。左侧部分对应于采集calcofluor和叶绿素荧光的设置。二极管激光器的405nm线(白色箭头)用于激发calcofluor(激光器的功率可以从二极管的额定功率的1到100%调制,这里的值是99%)。使用氦氖激光器的633线(黑色箭头)来激发叶绿素(功率激光器在激光器的额定功率的70%)。徕卡共焦点配有光谱模块。通过PMT-1(蓝色通道),由calcofluor发出的荧光收集在410和470nm之间,并且由PMT-3(红色通道)收集叶绿素发射的荧光在640和680nm之间。正确的部分对应于单独采集GFP的设置,使用氩激光器的488nm射线(白色箭头),功率为激光器额定功率的48%,荧光收集在500和540nm由PMT2(绿色通道)。

  7. 使用共焦显微镜的顺序模式,以避免PMT检测器之间的串扰。要做到这一点首先收集calcofluor和叶绿素荧光,接下来是GFP荧光,如上所示(图3)。以400 Hz的速度获取图像(512 x 512像素),平均值为0到8(取决于细菌运动的速度)(见图4)。
  8. 对于z-stack采集,定义显微镜galvo阶段的顶部和底部位置,z步骤接近0.5μm。在z维上获得的10到25个共聚焦平台(使用徕卡软件)进行最大投射(见图4)。


    图4.在孔隙水平上表达 Xcc 的GFP的共聚焦图像A.在z-维度上获得的15个共焦面的最大投影,z-步长为0.5μm,线平均值为2. B.从z维度获得的z-step为0.5μm,线平均值为4的20个共聚焦平面的最大投影。C.在z轴上获得的25个共焦面的最大投影,尺寸为z-step为0.5μm,无线平均。箭头表示孔。蓝色对应于表皮细胞壁表面的calcofluor染色的发射荧光,绿色对应于表达GFP的细菌,红色对应于叶绿素。比例尺= 30μm

食谱

  1. MOKA中等
    注意:细菌在30℃下在MOKA培养基中生长过夜。通过离心(4,000×g,10分钟)从培养基中收获并悬浮于1mM MgCl 2和0.02%Silwet L -77 at 108 cfu ml -1 。 在蒸馏水中1升:
    4克酵母提取物 8克酪蛋白氨基酸
    2g K 2 HPO 4
    0.3g MgSO 4•7H 2 O→/ /

致谢

这项工作得到法国国家教育和研究部授予AC的博士资助。 LIPM是法国卓越实验室项目(TULIP ANR-10-LABX-41; ANR-11-IDEX-0002-02)的一部分。我们感谢RégionOccitanie对显微镜平台的持续经济支持,LaurentNoël批评阅读手稿。

参考

  1. Cerutti,A.,Jauneau,A.,Auriac,M.C.,Lauber,E.,Martinez,Y.,Chiarenza,S.,Leonhardt,N.,Berthomé,R.andNoël,L.D。(2017)。 花椰菜水合物的免疫力通过黄单胞菌(Xanthomonas campestris)控制全身感染 pv 。植物生理学 174(2):700-716。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cerutti, A. and Jauneau, A. (2017). Capturing Z-stacked Confocal Images of Living Bacteria Entering Hydathode Pores of Cauliflower. Bio-protocol 7(20): e2451. DOI: 10.21769/BioProtoc.2451.
  2. Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.
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