采集活细菌进入花椰菜泌水孔的Z-栈共聚焦图像

Aude Cerutti; Alain Jauneau

doi:10.21769/BioProtoc.2451

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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

AC Aude Cerutti

Alain Jauneau email

发布: 2017年10月20日第7卷第20期 DOI: 10.21769/BioProtoc.2451 浏览次数: 8547

评审: Anonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Jun 2017

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

玉米根系相关简化细菌群落组成动力学定量及其生物防治效果评价

Ben Niu and Roberto Kolter

2018年06月20日 9314 阅读

根相互作用系统的示踪实验和质量控制

Hassan Massalha [...] Asaph Aharoni

2019年04月20日 6361 阅读

细菌分离物对番茄青枯病病原菌青枯雷尔氏菌生防效果的快速筛选方法

Heena Agarwal [...] Niraj Agarwala

2020年11月20日 4632 阅读

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

Razor blade
Microscope glass slides (Thermo Fisher Scientific, Superfrost, catalog number: 10143560WCUT )
Cover slip 24 x 60 mm (Thermo Fisher Scientific, catalog number: 15747592 )
Compost (Proveen, catalog number: 14926 )
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.
Xcc strain 8004::GUS-GFP (Xanthomonas campestris pv. campestris (Xcc); Cerutti et al., 2017)
Magnesium chloride (MgCl₂) (Merck, catalog number: 814733 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
Casamino acids (BD, BD Biosciences, catalog number: 223050 )
Potassium phosphate dibasic (K₂HPO₄) (Merck, catalog number: 105101 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Merck, catalog number: 105886 )
Silwet L-77 (CAAHMRO, catalog number: 115950H )
Calcofluor (Fluorescent Brightener 28) (Sigma-Aldrich, catalog number: F3543 )
MOKA medium (see Recipes)

Equipment

Miniature greenhouse purchased from a garden center (Nortene, catalog number: ME673714 )
Hollow punch (Harris, Uni-Core) (Electron Microscopy Sciences, catalog number: 69039-70 ) or razor blades
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
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

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.