Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
Peer-reviewed

Plant Assays for Quantifying Ralstonia solanacearum Virulence

植物实验分析青枯雷尔氏菌的毒力

Devanshi Khokhani Devanshi Khokhani
T Tuan Minh Tran
TL Tiffany M. Lowe-Power
CA Caitilyn Allen

发布: 2018年09月20日第8卷第18期 DOI: 10.21769/BioProtoc.3028 浏览次数: 7734

评审: Modesto Redrejo-RodriguezDavid NormanAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of mBio, featuring study using the protocol.

Sep 2017

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

基于β-葡萄糖醛酸酶 (GUS) 的细菌竞争测定以评估植物感染期间适应性的细微差异

基于β-葡萄糖醛酸酶 (GUS) 的细菌竞争测定以评估植物感染期间适应性的细微差异

Julien S. Luneau [...] Alice Boulanger

2022年07月05日 2256 阅读

根定植细菌种类的测量

根定植细菌种类的测量

Vílchez Juan Ignacio [...] Huiming Zhang

2021年04月05日 5066 阅读

番茄茎杆注射法精确评价青枯菌在植物中的适应性

番茄茎杆注射法精确评价青枯菌在植物中的适应性

Yaru Wang [...] Alberto P. Macho

2021年08月20日 2911 阅读

Abstract

Virulence assays are powerful tools to study microbial pathogenesis in vivo. Good assays track disease development and, coupled with targeted mutagenesis, can identify pathogen virulence factors. Disease development in plants is extremely sensitive to environmental factors such as temperature, atmospheric humidity, and soil water level, so it can be challenging to standardize conditions to achieve consistent results. Here, we present optimized and validated experimental conditions and analysis methods for nine assays that measure specific aspects of virulence in the phytopathogenic bacterium Ralstonia solanacearum, using tomato as the model host plant.

Keywords: Virulence assay (毒力检测) search Bacterial wilt (青枯病) search Vascular plant pathogen (维管植物病原体) search Plant colonization (病毒的植物定植) search Xylem sap (伤流液) search R. solanacearum (青枯雷尔氏菌) search Tomato (番茄) search

Background

Ralstonia solanacearum is a soil-borne bacterium that causes bacterial wilt in a vast range of plants and continues to infect new hosts across the globe (Hayward, 1991; Elphinstone, 2005; Wicker et al., 2007; Genin, 2010; Weibel et al., 2016). As a result, R. solanacearum is among the most intensively studied phytopathogenic bacteria (Mansfield et al., 2012).

R. solanacearum can persist in soil or water reservoirs for long periods (Alvarez et al., 2008), and in the presence of a suitable host, it can enter the plant through wounds or lateral root emergence points (Denny, 2007). Thereafter it colonizes the water-transporting plant xylem vessels and thrives there. Massive production of exopolymeric substances (EPS) likely contributes to the clogging of the xylem channels, leading to blockage of water transport, followed by symptoms like wilting leaves, stunted growth, stem discoloration, and death. Molecular genetic studies revealed a consortium of many virulence factors that are required for pathogenesis and fitness in planta (Genin and Denny, 2012; Tran et al., 2016a and 2016b); recent in silico modeling (Peyraud et al., 2016) and in vivo transcriptomics and metabolomics (Jacobs et al., 2012; Khokhani et al., 2017; Lowe-Power et al., 2018) have further enhanced our understanding of how this bacterium switches from saprophytic to parasitic lifestyle.

To test hypotheses suggested by molecular data, researchers measure virulence on model host plants under controlled conditions. To be useful, such assays must be quantitative, biologically relevant, and replicable. We have developed or adapted several protocols to assess R. solanacearum interactions with tomato, a natural host and economically important crop plant. A naturalistic soil soak assay replicates many aspects of the infection process that occurs in the field. This assay quantifies the defects of mutant strains lacking virulence factors involved in the early phases of the disease such as sensing, invading, and colonizing host roots. For example, mutants lacking chemotaxis, swimming motility, extracellular plant cell wall-degrading enzymes, and type II and III secretion systems are all impaired in virulence following soil soak inoculation.

A petiole inoculation disease assay that introduces the pathogen directly into stem xylem vessels can identify traits that contribute to pathogen success in xylem vessels (Saile et al., 1997). Some mutants that are defective in virulence following soil soak inoculation have full wild-type virulence following cut-petiole inoculation into the stem; examples include mutants lacking motility and chemotaxis (Tans-Kersten et al., 2001; Yao and Allen, 2006). In another case, comparing results of these two assays revealed that extracellular DNA degradation, a trait initially thought to only play a role in interactions with host roots, was also critical for normal biofilm formation inside host xylem later in disease (Tran et al., 2016a and 2016b). When virulence traits are functionally redundant or make small contributions to pathogen success, neither soil soak nor petiole inoculation assays can reveal subtle differences between the wild-type and the target mutant strain (Macho et al., 2010). In those cases, we can use single or co-inoculation experiments to directly compare the growth of competing strains in planta (root and/or shoot colonization) and calculate their relative competitive fitness as a competitive index (CI). We also describe here the protocol to measure bacterial attachment to plant roots. Since R. solanacearum is a xylem-dwelling bacterium, it is important to understand how it affects and is affected by host plant xylem sap. Therefore, we provide a protocol for collection of xylem sap from healthy and infected tomato plants; this ex vivo sap can be used as a medium for bacterial growth curves or for metabolomic analyses.

Materials and Reagents

  1. Sterile conical flask 250 ml (Corning, PYREX®, catalog number: 4980-250 )
  2. Sterile 50 ml conical tubes (Stellar Scientific, catalog number: T50-100 )
  3. Flasks for preparing large volume of cultures (size depends on the experimental goals)
  4. Seedling tray (36-cell insert with holes) (J&P Park Acquisitions, Park Seed, catalog number: 96377 )
  5. Flat trays (greenhouse megastore, 11" W x 21.37" L x 2.44" D, CN-FL)
  6. 1.5 ml microcentrifuge tubes (Sigma-Aldrich, BRAND, catalog number: Z336769 )
  7. 8 cm wide pots (greenhouse megastore)
  8. Planting sticks (greenhouse megastore)
  9. Metal beads (2.4 mm) (OMNI, catalog number: 19-640 )
  10. Bead-beater tubes (USA Scientific, catalog number: 1420-9300 )
  11. Gosselin Square Polystyrene Petri Plate with 4 vents, 120 x 120 x 15.8 mm, Sterile (Corning, catalog number: BP124-05 )
  12. MicroporeTM Surgical Paper Tape (1 inch size) (3M, catalog number: 1530-1 )
  13. Aluminum foil (W.W. Grainger, catalog number: 16W479 )
  14. Paper towel (Singlefold Paper Towel, 9.1 x 10.25) (Cascades Pro, catalog number: H165 )
  15. WhatmanTM paper (Grade 1 Qualitative Filter Paper) (GE Healthcare, Whatman)
  16. Petri dish (Corning, Falcon®, catalog number: 351029 )
  17. 0.22-μm sterile filter (Merck, catalog number: SLGP033RS )
  18. 10 ml pipette (Disposable Polystyrene Serological Pipettes) (Corning, Falcon®, catalog number: 357551 )
  19. 1 ml syringe (New Sterile, Sealed, Tuberculin, Luer slip tube, No Needle, Disposable) (BD, catalog number: 9602 )
  20. 96-well half-area microplates (Corning, catalog number: 3697 )
  21. Soil mix, propagation mix, Sunshine® resilience silicon enriched, Re Plug and Seed Rsi (Sun Gro Horticulture, Lot-code: Q15322; SKU: 7263924)
    Ingredients:
    55-65% Canadian Sphagnum peat moss, vermiculite, dolomite lime, wetting agent;
    0.0001% Silicon dioxide (SiO2) from calcium silicate to increase root growth
  22. Ralstonia solanacearum strain from glycerol or water stock
  23. Bonny best wilt susceptible variety of Tomato seeds (Mountain valley seed) (stored at 4 °C)
  24. Sterile reverse osmolyzed water by Milli-Q system (SMQ)
  25. Agar (Fisher Scientific, catalog number: BP1423-2 )
  26. Bleach (Clorox Performance Bleach with CloroMax) (The Clorox Company, catalog number: 980042447 )
  27. 70% Ethanol (Diluting 100% Ethanol 200 proof) (Decon Labs, catalog number: 2716 )
  28. Glucose (Fisher Scientific, catalog number: D16-1 )
  29. Peptone (Fisher Scientific, catalog number: NC9931583 )
  30. Casamino acids (RPI, catalog number: C45000-5000.0 )
  31. Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
  32. KOH (Fisher Scientific, catalog number: P250 10 )
  33. 1% 2,3,5-triphenyl tetrazolium chloride (TZC) (Sigma-Aldrich, catalog number: T8877 )
  34. KNO3 (Fisher Scientific, catalog number: P383 100 )
  35. KH2PO4 (Merck, Calbiochem, catalog number: 529568 )
  36. MgSO4 (MP biomedicals, catalog number: 150136 )
  37. Ca(NO3)2•4H2O (Fisher Scientific, catalog number: C109 )
  38. H3BO3 (Fisher Scientific, catalog number: A73 1 )
  39. MnCl2•4H2O (Sigma-Aldrich, catalog number: M8054 )
  40. ZnSO4•7H2O (Fisher Scientific, catalog number: Z68 )
  41. CuSO4•5H2O (VWR, BDH, catalog number: BDH9312 )
  42. (NH4)6Mo7O24 (Sigma-Aldrich, catalog number: A1343 )
  43. FeSO4•7H2O (Fisher Scientific, catalog number: I146 3 )
  44. Na2EDTA (Fisher Scientific, catalog number: S311 ) 
  45. Casamino acid-peptone-glucose (CPG) agar (see Recipes)
  46. CPG broth (see Recipes)
  47. Modified Hoagland's solution (see Recipes)

Note: All the chemicals were purchased from Sigma-Aldrich, Fisher Scientific, or other chemical companies.

Equipment

  1. P1000 pipette (Eppendorf, catalog number: 3120000062 )
  2. P200 pipette (Eppendorf, catalog number: 3120000054 )
  3. P10 pipette (Eppendorf, catalog number: 3120000020 )
  4. Forceps (VWR, catalog number: 470018-952)
    Manufacturer: Dunrite Instruments, catalog number: 141001 .
  5. Scalpel (Bard-Parker® SafeSwitchTM Reusable Scalpel Handle, Size 3 L) (Aspen Surgical, catalog number: ST-1013LNS )
  6. Sharp razor blade (Carbon Steel Razor Blades) (Azpack, catalog number: YSJ-762-Q )
  7. Incubator (6M Lab Incubator) (Precision Scientific, catalog number: 31487 )
  8. Benchtop Shaker (Thermo Fisher Scientific, model: MaxQTM 4000 )
  9. Growth chamber with the following conditions:
    Light intensity: 300-500 μmol/m2•sec-1
    12 h, light, 28 °C
    12 h, dark, 28 °C
    50-70% humidity
    ~500 ppm CO2 measured
  10. Powerlyzer® 24 homogenizer (MO BiO Laboratories, catalog number: 13155 )
  11. Centrifuge (15 amp version) (Eppendorf, model: 5810 R )
  12. Centrifuge (Eppendorf, model: 5417 R )
  13. Spectrophotometer (UV/Vis) (Beckman Coulter, model: DU 730 )
  14. Vortex mixer (Vortex-Genie 2) (Scientific industries, catalog number: SI-0246 )
  15. Biosafety cabinet (The Baker, SterilGard®, model: SG403A-HE )
  16. Balance (Roche Diagnostics, model: 05942861001 )
  17. Autoclave (Vacuum Steam Sterilizer) (Getinge, model: 533LS-E )

Software

  1. PRISM Graphpad software

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Khokhani, D., Tuan, T. M., Lowe-Power, T. M. and Allen, C. (2018). Plant Assays for Quantifying Ralstonia solanacearum Virulence . Bio-protocol 8(18): e3028. DOI: 10.21769/BioProtoc.3028.

ris ris Download Citation in RIS Format

分类

微生物学 > 微生物-宿主相互作用 > 细菌

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件