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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   

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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

  1. Test tubes for bacterial liquid cultures
  2. Pipettes and sterile tips
  3. Inoculation loops
  4. Safe lock reaction tubes (1.5 and 2.0 ml) (e.g., Eppendorf tubes)
  5. Sterile toothpicks
  6. 0.2 µm syringe filter (Sartorius, catalog number: 16532 )
  7. E. coli K12 (standard lab strain)
  8. Xanthomonas oryzae pv. oryzae PXO86 (received from C.M. Vera Cruz, IRRI, Manila, Philippines)
  9. Rice plants, approx. 2-3 weeks old (see Weidenbach et al., 2016)
  10. Sterilized tap water
  11. Liquid nitrogen
  12. Peptone (Duchefa Biochemie, catalog number: P1328 )
  13. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
  14. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  15. Agar-agar (Carl Roth, catalog number: 5210 )
  16. Sucrose (Carl Roth, catalog number: 4621 )
  17. Trisodium phosphate (Na3PO4) (Carl Roth, catalog number: 8613 )
  18. Calcium nitrate [Ca(NO3)2] (Carl Roth, catalog number: P740 )
  19. Ferrous sulphate (FeSO4) (Carl Roth, catalog number: P015 )
  20. HCl (Carl Roth, catalog number: 2607 )
  21. LB-medium (see Recipes)
  22. Modified Wakimoto medium (see Recipes)
  23. Ferrous sulphate (FeSO4) stock solution (see Recipes)

Equipment

  1. Laminar flow hood
  2. 28 °C Thermo constant incubator
  3. 37 °C Thermo constant incubator
  4. 37 °C Thermo shaking incubator
  5. -80 °C low temperature freezer
  6. Spectrophotometer and respective cuvettes
  7. Mortar and pestle (e.g., Carl Roth, catalog numbers: 1568 and 3831 )
  8. Autoclave
  9. Benchtop centrifuge (cooled at 4 °C)

Procedure

Notes:

  1. All work with bacterial cultures is carried out under sterile conditions using a laminar flow hood. Culture medium for E. coli is LB-medium and incubation temperature is 37 °C. For X. oryzae pv. oryzae (Xoo) modified Wakimoto medium is used and bacteria are incubated at 28 °C.
  2. Streak bacterial cells from glycerol stocks (see Note 1) stored at -80 °C on appropriate media agar plates and incubate overnight for E. coli (on LB-medium agar plates; incubation at 37 °C) or until colonies get visible for Xoo (after 2-3 days at 28 °C on modified-Wakimoto medium agar plates).
  1. Day 1
    1. E. coli
      1. Inoculate 3 ml liquid LB-medium in a test tube with a single colony picked with a sterile toothpick or an inoculation loop from the overnight incubation plate. 
      2. Shake culture at 210 rpm and 37 °C.
  2. Day 2
    1. E. coli
      1. Harvest bacteria from overnight culture by centrifugation and re-suspend pellet in sterilized tap water.
      2. Determine the OD600 using a spectrophotometer. Using the measured OD600 value dilute to a calculated OD600 of 1.25 x 10-6 (equivalent to approximately 1 x 103 cells ml-1 when assuming that OD600 = 1.0 is equivalent to approximately 8 x 108 cells ml-1 for E. coli) in sterilized tap water.
    2. Xoo:
      1. Scrape off bacteria from modified Wakimoto-medium plates (see Note 2) using a sterile inoculation loop, re-suspend the bacteria in sterilized tap water and measure OD600 in the linear absorption range of the spectrophotometer.
      2. Dilute to approximately OD600 1.25 x 10-6 in sterilized tap water (see above).
    3. Plant leaf extracts:
      1. Snap freeze 0.3 g leaf material from transgenic or wild-type rice plants in liquid nitrogen and grind to a fine powder using a mortar and pestle.
      2. Transfer the frozen powder to a 1.5 ml safe lock reaction tube and mix with 1 ml sterilized tap water.
      3. After centrifugation at 16,000 x g at 4 °C in a cooled benchtop centrifuge, the supernatant is transferred to a fresh safe lock reaction tube and mixed 1:2 (v/v) with the bacterial suspension (100 µl supernatant + 100 µl diluted bacterial suspension). Controls consist of bacterial suspension and leaf extracts from wild-type plants.
      4. The mixtures (100 µl) are plated in triplicates on agar plates of the appropriate media. Plates with E. coli are incubated overnight at 37 °C. Xoo plates are incubated at 22 °C for 4-5 days (see Note 3).
  3. Day 3
    Count the number of cfu on E. coli plates.
  4. Day 6-7
    Count the number of cfu on Xoo plates.

Data analysis

Calculate mean value and standard deviation of the number of cfu on the triplicate plates. The number of cfu of the wild-type sample is set to 100%. Test for significant reduction of bacterial growth in sample compared to control by applying Student’s t-test.

Notes

  1. Glycerol stocks are prepared by adding 750 µl of bacterial culture to 250 µl of sterile glycerol in a safe lock reaction tube; mix well and snap freeze in liquid nitrogen. Store at -80 °C.
  2. Use freshly streaked out plates (~3 days) to ensure vitality of Xoo.
  3. Incubation for a longer time at lower temperature ensures better growth of well separated colonies for easier counting.

Recipes

  1. LB-medium
    10.0 g L-1 peptone
    10.0 g L-1 sodium chloride (NaCl)
    5.0 g L-1 yeast extract
    15 g L-1 agar-agar
    Dissolve components in distilled water and sterilize by autoclaving
  2. Modified Wakimoto medium
    20 g L-1 sucrose
    5.0 g L-1 peptone
    1.9 g L-1 trisodium phosphate (Na3PO4)
    0.5 g L-1 calcium nitrate [Ca(NO3)2]
    15 g L-1 agar-agar
    Dissolve components in distilled water and sterilize by autoclaving 
    Afterwards add 1 ml L-1 sterile FeSO4 stock solution (see below)
  3. Ferrous sulphate (FeSO4) stock solution for modified Wakimoto medium
    0.5 g L-1 ferrous sulphate (FeSO4)
    3.0 ml 2 N hydrogen chloride (HCl)
    10 ml distilled water
    Sterilize stock solution by using a 0.2 µm syringe filter and store at room temperature in darkness

Acknowledgments

Protocol based on methods described in Weidenbach et al. (2016). DW was funded in the framework of the BMBF funding activity ‘Plant Biotechnology for the future, PLANT 2030’ within the project ‘BarleyFortress’.

References

  1. Weidenbach, D., Esch, L., Moller, C., Hensel, G., Kumlehn, J., Hofle, C., Huckelhoven, R. and Schaffrath, U. (2016). Polarized defense against fungal pathogens is mediated by the jacalin-related lectin domain of modular poaceae-specific proteins. Mol Plant 9(4): 514-527.

简介

我们进行了生长抑制测定,以测试来自转基因水稻植物的叶提取物中的抗菌化合物。该测定是基于相应细菌的确定浓度的集落形成单位(cfu)与地面叶组织的水提取物的过夜共培养。

背景 对抗有害生物体的植物的防御可以针对特定病原体物种或病原体群体。针对增加作物病原体抗性,对抗特定疾病的育种可能是有用的,但最终目标是实施广谱抗病性。大米蛋白OsJAC1是一种模块化蛋白质,其由预测结合糖残基的茉莉酸相关凝集素结构域和可能在单粘菌偶联期间起作用的活性结构域组成。该融合蛋白特异于禾本科,代表一种新型的抗性蛋白。本文描述的方案用于评估过表达OsJAC1 cDNA cDNA(Weidenbach等人,2016)的转基因水稻植物的叶提取物的抗微生物能力。对于该测定,细菌实验室菌株大肠杆菌K12和细菌水稻病原体xanthomonas oryzae pv。使用oryzae 使用PXO86引起细菌性枯萎病。

关键字:农作物, 水稻, 病原体, 细菌, 抗菌, 木菠萝凝集素, 凝集素, Dirigent

材料和试剂

  1. 用于细菌液体培养的试管
  2. 移液器和无菌提示
  3. 接种循环
  4. 安全锁定反应管(1.5和2.0ml)(例如,Eppendorf管)
  5. 无菌牙签
  6. 0.2微米注射器过滤器(Sartorius,目录号:16532)
  7. E。大肠杆菌K12(标准实验室菌株)
  8. 黄单胞菌Xanthomonas oryzae pv。 oryzae PXO86(收到C.M.维拉克鲁兹,IRRI,马尼拉,菲律宾)
  9. 水稻植物,约2-3周龄(见Weidenbach等人,2016)
  10. 无菌自来水
  11. 液氮
  12. 胨(Duchefa Biochemie,目录号:P1328)
  13. 氯化钠(NaCl)(Carl Roth,目录号:3957)
  14. 酵母提取物(Duchefa Biochemie,目录号:Y1333)
  15. 琼脂(Carl Roth,目录号:5210)
  16. 蔗糖(Carl Roth,目录号:4621)
  17. 磷酸三钠(Na 3 PO 4)(Carl Roth,目录号:8613)
  18. 硝酸钙[Ca(NO 3 3)2](Carl Roth,目录号:P740)
  19. 硫酸亚铁(FeSO 4)(Carl Roth,目录号:P015)
  20. HCl(Carl Roth,目录号:2607)
  21. LB-培养基(参见食谱)
  22. 修改的Wakimoto培养基(见食谱)
  23. 硫酸亚铁(FeSO 4)储备溶液(参见食谱)

设备

  1. 层流罩
  2. 28°C恒温培养箱
  3. 37℃恒温培养箱
  4. 37°C热振荡培养箱
  5. -80°C低温冷冻机
  6. 分光光度计和各自的比色皿
  7. 砂浆和杵(例如,,Carl Roth,目录号:1568和3831)
  8. 高压灭菌器
  9. 台式离心机(4℃冷却)

程序

注意:

  1. 所有使用细菌培养的工作都是在无菌条件下使用层流罩进行的。大肠杆菌培养基为LB培养基,培养温度为37℃。对于米曲霉使用米曲霉(Xoo)修饰的Wakimoto培养基,并将细菌在28℃温育
  2. 将储存在-80℃的甘油储备液(见注1)的细菌细菌从合适的培养基琼脂平板上保存并孵育过夜大肠杆菌(在LB-培养基琼脂平板上; 37℃孵育)或直到菌落得到对于Xoo可见(在28℃下在改良的Wakimoto培养基琼脂平板上2-3天后)
  1. 第1天
    1. E。大肠杆菌
      1. 在试管中接种3ml液体LB-培养基,并将其加入到试管中用灭菌牙齿挑选的单个菌落或来自过夜培养板的接种环。 
      2. 在210 rpm和37°C摇动文化。 >
  2. 第2天
    1. E。大肠杆菌
      1. 通过离心收获来自过夜培养物的细菌,并在无菌自来水中重新悬浮沉淀。
      2. 使用分光光度计测定OD 600。使用测量的OD 600值稀释至计算的OD 600(1.25×10 -6)(相当于约1×10 3当假设OD 600 = 1.0时,细胞ml -1 等于大约8×10 8个细胞ml < 1 对于大肠杆菌)在无菌自来水中。
    2. o
      1. 使用无菌接种回路从改良的Wakimoto培养基板(参见注释2)中除去细菌,将细菌重新悬浮在灭菌的自来水中,并在分光光度计的线性吸收范围内测量OD 600。 br />
      2. 在无菌自来水中稀释至大约OD 600(参见上文)的1.25×10 -6。
    3. 植物叶提取物:
      1. 将来自转基因或野生型水稻植物的0.3g叶片快速冻结在液氮中,并用研钵和研杵研磨成细粉。
      2. 将冷冻粉末转移到1.5 ml安全锁定反应管中,并与1 ml无菌自来水混合。
      3. 在冷却的台式离心机中,在4℃下以16,000xg离心,将上清液转移到新鲜的安全锁定反应管中,并与细菌悬浮液(100μl)混合1:2(v / v)上清液+100μl稀释的细菌悬浮液)。对照组由来自野生型植物的细菌悬浮液和叶提取物组成。
      4. 将混合物(100μl)在适当培养基的琼脂平板上一式三份铺板。板与E。大肠杆菌在37℃下孵育过夜。将板在22℃下孵育4-5天(参见注3)。
  3. 第3天
    计算E上的cfu数。大肠杆菌板。
  4. 第6-7天
    计算Xoo 板上的cfu数。

数据分析

计算三重平板上的cfu数的平均值和标准偏差。野生型样本的cfu数设定为100%。通过应用学生测试,与对照相比,测试样品中细菌生长显着减少。

笔记

  1. 通过在安全的锁定反应管中将250μl无菌甘油加入750μl细菌培养物中来制备甘油,在液氮中混合均匀并快速冷冻。储存于-80°C。
  2. 使用新鲜的条纹板(〜3天),以确保"Xoo"的活力。
  3. 在较低温度下孵育较长时间可确保更好分离的菌落的生长,以方便计数。

食谱

  1. LB-medium
    10.0 g L -1 蛋白胨
    10.0克L 氯化钠(NaCl)
    5.0g L -1 酵母提取物
    15g L -1 琼脂 -
    将组分溶于蒸馏水中,并通过高压灭菌消毒
  2. 修改的Wakimoto培养基
    20克L 蔗糖
    5.0g L -1 蛋白胨
    1.9g L -1磷酸三钠(Na 3 PO 4)
    0.5g L
    硝酸钙[Ca(NO 3 3)2]
    15g L -1 琼脂 -
    将组分溶于蒸馏水中,并通过高压灭菌消毒 然后加入1ml L -1无菌FeSO 4 储备溶液(见下文)
  3. 用于改性Wakimoto培养基的硫酸亚铁(FeSO 4)储备液
    0.5g L 硫酸亚铁(FeSO 4)
    3.0ml 2N氯化氢(HCl)
    10ml蒸馏水
    通过使用0.2μm注射器过滤器灭菌储备溶液,并在室温下在黑暗中储存

致谢

基于Weidenbach等人中描述的方法的协议。 (2016)。 DW在"BarleyFortress"项目的BMBF资助活动"植物生物技术未来","2030年植物"项目的框架下资助。

参考文献

  1. Weidenbach,D.,Esch,L.,Moller,C.,Hensel,G.,Kumlehn,J.,Hofle,C.,Huckelhoven,R.and Schaffrath,U.(2016)。&lt; a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26708413"target ="_ blank">针对真菌病原体的极化防御是由模块化的jacalin相关凝集素域介导的波斯科特异性蛋白质。分子植物 9(4):514-527。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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