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Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum

植物病原体茄青枯病菌胞外多糖的定量分析

Rémi Peyraud Rémi Peyraud
TD Timothy P. Denny
SG Stéphane Genin

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2289 浏览次数: 8669

评审: Arsalan DaudiHiroyuki HiraiKanika Gera

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Abstract

Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here we describe a colorimetric method for quantitative determination of the soluble exopolysaccharide present in culture supernatant of R. solanacearum.

Keywords: Ralstonia solanacearum (青枯雷尔氏菌) search Exopolysaccharide (胞外多糖) search Virulence factor (毒力因子) search Plant pathogen (植物病原体) search Hexoseamine (氨基己糖) search

Background

The plant pathogen Ralstonia solanacearum produces exopolysaccharide under the control of quorum sensing system, i.e., at high cell density, above 5 x 107 cell ml-1 (Flavier et al., 1997). The sugar content of the exopolysaccharide includes galactosamine, glucose, and rhamnose in the ratio of 10:2.5:1 (Drigues et al., 1985). A protocol for a reliable extraction and quantification of the exopolysaccharide from culture supernatant was initially developed by Brumbley and Denny (1990) and was updated recently by Peyraud et al. (2016). The quantification is based on the determination of hexoseamine content of the macromolecule using an adapted Elson and Morgan assay (Elson and Morgan, 1933; Gatt and Berman, 1966). Exopolysaccharides containing N-acetyl-D-galactosamine are produced by diverse Gram-negative or Gram-positive bacteria (Vaningelgem et al., 2004; Balzaretti et al., 2017) and also some fungi (Lee et al., 2015), so this protocol may also be applicable to such organisms.

Materials and Reagents

  1. Millex®-GP 33 mm syringe filter with a polyethersulfone membrane and 0.22 µm pore size (EMD Millipore, catalog number: SLGP033RB )
  2. Polypropylene microcentrifuge tubes of 2.0 ml Eppendorf® Safe-Lock (Eppendorf, catalog number: 0030120094 )
  3. Paper towel
  4. Aluminum foil
  5. Pipette tips
  6. 15-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 188271 )
  7. 50-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
  8. Bridges for microcentrifuge tube (Milian, catalog number: 045427 )
  9. Gloves
  10. R. solanacearum strain GMI1000 (Salanoubat et al., 2002). This strain can be retrieved from the CIRM Biological Resource Center (http://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria) in the CIRM-BP collection (code CFBP6924)
  11. Acetone > 99.8% AnalaR NORMAPUR® (VWR, catalog number: 20066.296 )
  12. Milli-Q water obtained at a resistivity of 18.2 MOhm cm at 25 °C
  13. Acetyl acetone > 99% AnalaR NORMAPUR® (VWR, catalog number: 20092.23 0)
  14. Ethanol, 99.8% (Sigma-Aldrich, catalog number: 02851 )
  15. 4-(dimethylamino)benzaldehyde, 98% (Erlich’s reagent) (Sigma-Aldrich, catalog number: 109762 )
  16. Sodium L-glutamic acid monohydrate (Sigma-Aldrich, catalog number: 49621 )
  17. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: F8263 )
  18. Ammonium sulfate, (NH4)2SO4 (Sigma-Aldrich, catalog number: A4418 )
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M5921 )
  20. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  21. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
  22. Sodium chloride (NaCl) > 99.5% AnalaR NORMAPUR® (VWR, catalog number: 27810.295 )
  23. N-acetylgalactosamine, 98% (Sigma-Aldrich, catalog number: A2795 )
  24. Hydrochloric acid (HCl), 37%, AnalaR NORMAPUR® (VWR, catalog number: 20252.29 0)
  25. Sodium carbonate (Na2CO3) > 99.9 AnalaR NOR MAPUR® (VWR, catalog number: 27771.29 0)
  26. Minimal medium (see Recipes)
  27. 5 M NaCl solution (see Recipes)
  28. Standard samples of N-acetyl galactosamine (see Recipes)
  29. N-acetylgalactosamine standard stock solution (see Recipes)
  30. 2% acetyl acetone, 1.5 M Na2CO3 solution (see Recipes)
  31. Erlich’s reagent (see Recipes)

Equipment

  1. Dry bath heater with dual block Corning® (Corning, model: Corning® LSETM Digital Dry Bath Heater, catalog number: 6786-DB )
  2. Fume hood
  3. Eppendorf® microcentrifuge 5415 R (Eppendorf, model: 5415 R )
  4. Mini microcentrifuges Corning® (Corning, model: Corning® LSETM Mini Microcentrifuge, catalog number: 6766 )
  5. Vortex for microcentrifuge tubes
  6. UltrospecTM 2100 pro UV/Visible spectrophotometer (GE Healthcare, model: UltrospecTM 2100 pro UV/Visible Spectrophotometer , catalog number: 80211221)
  7. 10 ml volumetric flask

Software

  1. Appropriate software (SciDavis, Excel, etc.)

Procedure

English
中文翻译

文章信息

版权信息

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

如何引用

Peyraud, R., Denny, T. P. and Genin, S. (2017). Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum. Bio-protocol 7(10): e2289. DOI: 10.21769/BioProtoc.2289.

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分类

植物科学 > 植物免疫 > 病害生物测定

植物科学 > 植物免疫 > 宿主-细菌相互作用

细胞生物学 > 细胞分离和培养 > 细胞分化

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