Bacterial Growth Inhibition Assay for Xanthomonas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts
检测植物叶提取物抑制稻生黄单胞菌条斑致病变种或大肠埃希杆菌K12生长试验
发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2069 浏览次数: 9680
评审: Marisa RosaAnonymous reviewer(s)
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The authors used this protocol in:
Apr 2016
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Abstract
We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue.
Keywords: Crop plant (农作物)
Rice (水稻)
Pathogen (病原体)
Bacteria (细菌)
Antimicrobial (抗菌)
Jacalin (木菠萝凝集素)
Lectin (凝集素)
Dirigent (Dirigent)
Background
Defense of plants against harmful organisms can be specific against particular pathogen species or groups of pathogens. Aiming at increasing pathogen resistance of crop plants, breeding for resistance against particular diseases can be useful but the ultimate goal is to implement broad-spectrum disease resistance. The rice protein OsJAC1 is a modular protein consisting of a jacalin-related lectin domain predicted to bind to sugar residues and a dirigent domain that might act during coupling of monolignols. This fusion protein is specific to Poaceae and represents a novel type of resistance protein. The protocol described here was used to assess the antimicrobial capabilities of leaf extracts from transgenic rice plants overexpressing the OsJAC1 cDNA (Weidenbach et al., 2016). For this assay, the bacterial lab strain Escherichia coli K12 and the bacterial rice pathogen Xanthomonas oryzae pv. oryzae PXO86, causing bacterial blight, were used.
Materials and Reagents
- Test tubes for bacterial liquid cultures
- Pipettes and sterile tips
- Inoculation loops
- Safe lock reaction tubes (1.5 and 2.0 ml) (e.g., Eppendorf tubes)
- Sterile toothpicks
- 0.2 µm syringe filter (Sartorius, catalog number: 16532 )
- E. coli K12 (standard lab strain)
- Xanthomonas oryzae pv. oryzae PXO86 (received from C.M. Vera Cruz, IRRI, Manila, Philippines)
- Rice plants, approx. 2-3 weeks old (see Weidenbach et al., 2016)
- Sterilized tap water
- Liquid nitrogen
- Peptone (Duchefa Biochemie, catalog number: P1328 )
- Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
- Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
- Agar-agar (Carl Roth, catalog number: 5210 )
- Sucrose (Carl Roth, catalog number: 4621 )
- Trisodium phosphate (Na3PO4) (Carl Roth, catalog number: 8613 )
- Calcium nitrate [Ca(NO3)2] (Carl Roth, catalog number: P740 )
- Ferrous sulphate (FeSO4) (Carl Roth, catalog number: P015 )
- HCl (Carl Roth, catalog number: 2607 )
- LB-medium (see Recipes)
- Modified Wakimoto medium (see Recipes)
- Ferrous sulphate (FeSO4) stock solution (see Recipes)
Equipment
- Laminar flow hood
- 28 °C Thermo constant incubator
- 37 °C Thermo constant incubator
- 37 °C Thermo shaking incubator
- -80 °C low temperature freezer
- Spectrophotometer and respective cuvettes
- Mortar and pestle (e.g., Carl Roth, catalog numbers: 1568 and 3831 )
- Autoclave
- Benchtop centrifuge (cooled at 4 °C)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Loehrer, M., Delventhal, R., Weidenbach, D. and Schaffrath, U. (2016). Bacterial Growth Inhibition Assay for Xanthomonas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts. Bio-protocol 6(24): e2069. DOI: 10.21769/BioProtoc.2069.
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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 > 体内实验模型 > 植物
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