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Peer-reviewed

Pseudomonas syringae Flood-inoculation Method in Arabidopsis

拟南芥的丁香假单胞菌浸没接种法

Yasuhiro Ishiga Yasuhiro Ishiga
Takako Ishiga Takako Ishiga
Yuki Ichinose Yuki Ichinose
KM Kirankumar S. Mysore

发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2106 浏览次数: 12644

评审: Zhaohui LiuBin TianAnonymous reviewer(s)

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Cover of Molecular Plant Microbe Interactions, featuring study using the protocol.

Feb 2016

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Abstract

Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis.

Keywords: Pseudomonas syringae (丁香假单胞菌) search Arabidopsis thaliana (拟南芥) search Plant innate immunity (植物天然免疫) search Pathogenicity (致病性) search

Background

The A. thaliana-Pto DC3000 model pathosystem is widely used to investigate the molecular mechanisms of microbial pathogenesis and plant innate immunity (Ishiga et al., 2012 and 2016; Ishiga and Ichinose, 2016). Although several inoculation methods have been developed to study the interactions in this model system, none of the methods reported to date are similar to those occurring in nature.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Ina-optika, catalog number: LT-0150 )
  2. 1 ml pipette tips (Mettler-Toledo, catalog number: 768/8 RT-1000 )
  3. Inoculating loops (VWR, catalog number: 12000-812 )
  4. 3M Micropore 2.5 cm surgical tape (3M, catalog number: 1530-1 )
  5. Plastic Petri plates (100 x 20 mm) (TrueLine, catalog number: TR4002 )
  6. Plastic Petri plates (100 x 15 mm) (VWR, catalog number: 10053-704 )
  7. Conical tubes (50 ml) (LabPlanet, catalog number: 3181-345-008 )
  8. Plant material: Arabidopsis thaliana ecotype Columbia (Col-0) was used as a wild-type plant
  9. Bacterial strains: P. syringae pv. tomato DC3000 (Pto DC3000) was used as pathogenic strain on A. thaliana-Pto DC3000 was kindly provided by Dr. Fumiaki Katagiri at University of Minnesota
  10. Ethanol (70%) (Wako Pure Chemical Industries, catalog number: 059-07895 )
  11. Sodium hypochlorite (Wako Pure Chemical Industries, catalog number: 197-02206 )
  12. Tween 20 (Wako Pure Chemical Industries, catalog number: 327-32475 )
  13. Sterile distilled water
  14. Rifampicin (50 mg/ml) (Wako Pure Chemical Industries, catalog number: 185-01003 )
  15. Silwet L-77 (BMS, catalog number: BMS-SL7755 )
  16. 5% H2O2 (Wako Pure Chemical Industries, catalog number: 081-04215 )
  17. Mannitol (Wako Pure Chemical Industries, catalog number: 133-00845 )
  18. L-glutamic acid (Wako Pure Chemical Industries, catalog number: 074-00505 )
  19. KH2PO4 (Wako Pure Chemical Industries, catalog number: 169-04245 )
  20. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665 )
  21. MgSO4·7H2O (Wako Pure Chemical Industries, catalog number: 131-00405 )
  22. Agar (Wako Pure Chemical Industries, catalog number: 010-15815 )
  23. NaOH (Wako Pure Chemical Industries, catalog number: 192-15985 )
  24. Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M0404 )
  25. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
  26. PhytagelTM (Sigma-Aldrich, catalog number: P8169 )
  27. KOH (Wako Pure Chemical Industries, catalog number: 165-21825 )
  28. Media
    1. Mannitol-glutamate (MG) medium (see Recipes)
    2. One-half strength Murashige and Skoog (MS) medium (see Recipes)

Equipment

  1. High-speed centrifuge (TOMY, model: MX305 )
  2. Pipette (Mettler-Toledo, model: Pipet-Lite XLS+ )
  3. Labo shaker (TAITEC, model: NR-2 )
  4. Labo incubator (AS ONE, model: ICV-300P )
  5. Clean work station (Hitachi Industrial Equipment Systems, model: CCV-1306E )
  6. Spectrophotometer (JASCO, model: V-630 )
  7. Plant growth chamber (NKsystem, model: LPH-411SP )
  8. Digital scale (AS ONE, model: IB-300 )
  9. Mortars and pestles (AS ONE, catalog number: 6-549-02 , φ90 mm)

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Ishiga, Y., Ishiga, T., Ichinose, Y. and Mysore, K. S. (2017). Pseudomonas syringae Flood-inoculation Method in Arabidopsis. Bio-protocol 7(2): e2106. DOI: 10.21769/BioProtoc.2106.

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分类

植物科学 > 植物免疫 > 病害生物测定

微生物学 > 微生物-宿主相互作用 > 细菌

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