植物病原体茄青枯病菌胞外多糖的定量分析

Rémi Peyraud; Timothy P. Denny; Stéphane Genin

doi:10.21769/BioProtoc.2289

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Peer-reviewed

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

TD Timothy P. Denny

SG Stéphane Genin email

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2289 浏览次数: 7979

评审: Arsalan DaudiHiroyuki HiraiKanika Gera

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Cover of PLOS Pathogens, featuring study using the protocol.

Oct 2016

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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)

Exopolysaccharide (胞外多糖)

Virulence factor (毒力因子)

Plant pathogen (植物病原体)

Hexoseamine (氨基己糖)

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

Millex^®-GP 33 mm syringe filter with a polyethersulfone membrane and 0.22 µm pore size (EMD Millipore, catalog number: SLGP033RB )
Polypropylene microcentrifuge tubes of 2.0 ml Eppendorf^® Safe-Lock (Eppendorf, catalog number: 0030120094 )
Paper towel
Aluminum foil
Pipette tips
15-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 188271 )
50-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
Bridges for microcentrifuge tube (Milian, catalog number: 045427 )
Gloves
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)
Acetone > 99.8% AnalaR NORMAPUR^®(VWR, catalog number: 20066.296 )
Milli-Q water obtained at a resistivity of 18.2 MOhm cm at 25 °C
Acetyl acetone > 99% AnalaR NORMAPUR^®(VWR, catalog number: 20092.23 0)
Ethanol, 99.8% (Sigma-Aldrich, catalog number: 02851 )
4-(dimethylamino)benzaldehyde, 98% (Erlich’s reagent) (Sigma-Aldrich, catalog number: 109762 )
Sodium L-glutamic acid monohydrate (Sigma-Aldrich, catalog number: 49621 )
Iron(II) sulfate heptahydrate (FeSO₄·7H₂O) (Sigma-Aldrich, catalog number: F8263 )
Ammonium sulfate, (NH₄)₂SO₄ (Sigma-Aldrich, catalog number: A4418 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: M5921 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
Sodium chloride (NaCl) > 99.5% AnalaR NORMAPUR^® (VWR, catalog number: 27810.295 )
N-acetylgalactosamine, 98% (Sigma-Aldrich, catalog number: A2795 )
Hydrochloric acid (HCl), 37%, AnalaR NORMAPUR^® (VWR, catalog number: 20252.29 0)
Sodium carbonate (Na₂CO₃) > 99.9 AnalaR NOR MAPUR^® (VWR, catalog number: 27771.29 0)
Minimal medium (see Recipes)
5 M NaCl solution (see Recipes)
Standard samples of N-acetyl galactosamine (see Recipes)
N-acetylgalactosamine standard stock solution (see Recipes)
2% acetyl acetone, 1.5 M Na₂CO₃ solution (see Recipes)
Erlich’s reagent (see Recipes)

Equipment

Dry bath heater with dual block Corning^®(Corning, model: Corning^® LSE^TM Digital Dry Bath Heater, catalog number: 6786-DB )
Fume hood
Eppendorf^® microcentrifuge 5415 R (Eppendorf, model: 5415 R )
Mini microcentrifuges Corning^® (Corning, model: Corning^®LSE^TM Mini Microcentrifuge, catalog number: 6766 )
Vortex for microcentrifuge tubes
Ultrospec^TM 2100 pro UV/Visible spectrophotometer (GE Healthcare, model: Ultrospec^TM 2100 pro UV/Visible Spectrophotometer , catalog number: 80211221)
10 ml volumetric flask

Software

Appropriate software (SciDavis, Excel, etc.)

Procedure

English

中文翻译

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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.