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Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria
黄单胞菌感染番茄叶片后乙烯产生的定量测定   

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Molecular Plant Microbe Interactions
Feb 2015

Abstract

Ethylene is a gaseous plant hormone controlling fruit ripening, flower opening, leaf senescence as well as abscission, and disease symptom development. Ethylene plays a critical role in the bacterial pathogen Xanthomonas euvesicatoria (X. euvesicatoria)-elicited symptom development in tomato. This protocol describes the measurement of ethylene gas produced by tomato leaves infected with X. euvesicatoria. Infected leaflets are placed in a glass tube for 30 min without sealing. The glass tubes are then capped with a septa stopper, and incubated for an hour. A 1 ml gas sample is removed from the tube using a syringe and then injected into a gas chromatograph to quantify ethylene gas levels. This protocol will be applicable for other plants with other pathogens with modifications.

Materials and Reagents

  1. 1% Ethylene gas can (SCOTT Specialty Gases, catalog number: 01-04-855 )
  2. 1 L Tedlar® PLV Gas Sampling Bag w/Thermogreen® LB-2 Septa (Sigma-Aldrich, Supelco, catalog number: 24633 )
  3. 1.5 ml Microtubes (Corning, Axygen®, catalog number: MTC-150-C )
  4. 1 ml Syringes Without Needles (BD, catalog number: 309659 )
  5. 25 G Needle (BD, catalog number: 305122 )
  6. 15 ml Polypropylene Centrifuge Tubes (Greiner Bio-One GmbH, catalog number: 188271 )
  7. 16 mm diameter x 100 mm Glass tubes (VWR International, catalog number: 47729-576 )
  8. 18 mm diameter x 150 mm Glass tubes (VWR International, catalog number: 47729-583 )
  9. Suba-Seal® septa (Sigma-Aldrich, catalog number: Z124613 )
  10. Tomato plants (4-5 week-old) were grown in a greenhouse or growth chamber (16 h light, 25~28 °C)
  11. Xanthomonas euvesicatoria (Xcv) strain 85-10 wild type and mutants (for example, type III effector deletion mutants)
    Note: Wild type strain is available upon request.
  12. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number: M2393 )
  13. Peptone (BD, catalog number: 211677 )
  14. Yeast extract (BD, catalog number: 212750 )
  15. Agar (BD, catalog number: 214530 )
  16. Glycerol (Certified ACS) (Fisher Chemical, catalog number: G334 )
  17. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465 )
  18. Distilled water
  19. NYGA medium (see Recipes)
  20. 10 mM MgCl2 (see Recipes)

Equipment

  1. 28 ºC Incubator (VWR International, catalog number: 414005-128 )
  2. Vortexer (Scientific Indrustries, model: Vortex-Genie 2 )
  3. Spectrophotometer (Amersham Biosciences, model: Ultrospec 3100 Pro )
  4. Gas chromatograph (GC) (Shimadzu Corporation, model: GC-8A )

Procedure

  1. Bacterial inoculum preparation and inoculation
    1. Streak out each Xcv strain from -80 °C glycerol stocks using a sterilized toothpick onto an independent sterile NYGA agar plate with appropriate antibiotics and grow for 2 d in a 28 °C incubator.
    2. For each strain analyzed, suspend a portion of the bacterial cells from the agar plate using a sterilized toothpick or pipet tip in 1 ml of 10 mM MgCl2 in a 1.5 ml microtube. Vortex the cell suspension well and check its optical density at 600 nm (OD600) by using a spectrophotometer. To prepare the inoculum, dilute the cell suspension with 10 mM MgCl2 to obtain an OD600 = 0.2. (Different inoculum concentrations should be empirically tested for optimal ethylene production.)
    3. Inoculate each leaflet of middle leaves with 10 mM MgCl2 (mock inoculation) or a bacterial inoculum using a 1 ml needleless syringe (Figure 1). Carefully remove excess solution on the surface of the leaflet with tissue paper. Use leaflets on the same branch for comparison to control for leaf age. For statistical analysis, prepare a minimum of three biological replicates.


      Figure 1. Hand infiltration with needless syringe

    4. Move the inoculated plants into a plant growth chamber or greenhouse (~ 25-28 °C, 16 h day/8 h night cycle).

  2. Ethylene gas measurement
    1. At 0, 2, and 3 days post-inoculation (dpi), excise the leaflets (mock or Xcv-infected) and measure their weight.
    2. Roll each leaflet and then place it into a glass tube (16 mm diameter x 100 mm) for 30 min without sealing.
    3. Then cap each glass tube with a Suba-Seal Septa stopper (Figure 2).
      Note: It is important to use the smallest size glass tube to reduce the volume of the gas space to concentrate the ethylene emitted by each leaflet.


      Figure 2. Rolled leaflet in a glass tube capped with a septa stopper

    4. Incubate the glass tubes for 1 h at room temperature.
    5. Using a 25 gauge needle attached to a 1 ml syringe, remove 1 ml of the gas sample from each glass tube (Figure 3) and inject it into a gas chromatograph.


      Figure 3. Gas sampling from a glass tube capped with a septa stopper

    6. Use company instruction manual to detect ethylene [e.g., Shimadzu's GC instruction manual (https://store.shimadzu.com/p-52629-instruction-manualgc-8apfgc-8a.aspx)].

  3. Measuring ethylene using a standard curve
    1. Use the needle attachment on the 1% ethylene can to pump ethylene gas into a Tedlar bag. Label bag as standard 1. Final concentration of standard 1 = 1% = 10,000 nl of ethylene/ml.


      Figure 4. Pumping ethylene gas into a Tedlar bag

    2. Take 1 ml of 1% ethylene from the ethylene-filled Tedlar bag and inject it into a capped glass tube (18 mm diameter x 150 mm glass tube with Suba-seal septa). Label tube as standard 2. Final concentration of standard 2 = 0.0352% or 352 nl of ethylene/ml. (An empty capped tube contains 27.4 ml of air. Ethylene concentration in standard 2 = 1% /1 ml + 27.4 ml.)
    3. Take 1 ml from capped glass tube (standard 2; 18 mm diameter x 150 mm glass tube with Suba-seal septa) and inject it into a capped tube labeled standard 3 (18 mm diameter x 150 mm glass tube with Suba-seal septa). Final concentration of standard 3 = 0.00124% or 12.4 nl of ethylene/ml.
    4. Inject 1 ml of each standard on the GC and determine the peak area.
    5. Construct a standard curve by plotting the peak area (X-axis) by nl of ethylene (Y-axis).
    6. Using the standard curve, determine the level of ethylene in each inoculated leaflet. Ethylene levels are defined as nl of ethylene/gram of fresh weight/hour. Representative results can be found in Lund et al. (1998) (Figure 1C and Figure 2C) and Kim et al. (2013) (Figure 1A-B).

Recipes

  1. NYGA medium (1 L)
    Peptone 5 g
    Yeast extract 3 g
    Agar 15 g
    Glycerol 20 ml
    Add distilled water to make up 1 L
    Adjust pH at 7.0 with 1 N sodium hydroxide
    Sterilize the medium by autoclaving for 20 min
  2. 10 mM MgCl2
    Magnesium chloride hexahydrate 2.03 g
    Add distilled water to make up 1 L
    Sterilize the medium by autoclaving for 20 min

Acknowledgments

This protocol is adopted from Lund et al. (1998). W. Stork was supported by United States Department of Agriculture NIFA Grant 2012-67011-19669. M. B. Mudgett was supported by National Institutes of Health Grant 2 R01 GM068886-06A1.

References

  1. Kim, J. G., Stork, W. and Mudgett, M. B. (2013). Xanthomonas type III effector XopD desumoylates tomato transcription factor SlERF4 to suppress ethylene responses and promote pathogen growth. Cell Host Microbe 13(2): 143-154.
  2. Lund, S. T., Stall, R. E. and Klee, H. J. (1998). Ethylene regulates the susceptible response to pathogen infection in tomato. Plant Cell 10(3): 371-382.
  3. Stork, W., Kim, J. G. and Mudgett, M. B. (2015). Functional analysis of plant defense suppression and activation by the Xanthomonas core Type III effector XopX. Mol Plant Microbe Interact 28(2): 180-194.

简介

乙烯是控制果实成熟,开花,叶衰老以及脱落和疾病症状发展的气态植物激素。 乙烯在细菌病原体黄单胞菌属(Xanthomonas euvesicatoria)( X。euvesicatoria )中起到关键作用 - 引起番茄中的症状发展。 该方案描述了用感染了X的番茄叶产生的乙烯气体的测量。 euvesicatoria 。 将感染的小叶放置在玻璃管中30分钟而不密封。 然后将玻璃管用隔膜塞子盖上,并孵育1小时。 使用注射器从管中移出1ml气体样品,然后注入气相色谱仪中以定量乙烯气体水平。 该协议将适用于其他具有修改的其他病原体的植物。

材料和试剂

  1. 1%乙烯气体(SCOTT Specialty Gases,目录号:01-04-855)
  2. 1L Tedlar PLV气体取样袋w/Thermogreen LB-2 Septa(Sigma-Aldrich,Supelco,目录号:24633)
  3. 1.5ml Microtubes(Corning,Axygen ,目录号:MTC-150-C)
  4. 1ml无针注射器(BD,目录号:309659)
  5. 25 G针(BD,目录号:305122)
  6. 15ml聚丙烯离心管(Greiner Bio-One GmbH,目录号:188271)
  7. 16mm直径×100mm玻璃管(VWR International,目录号:47729-576)
  8. 18mm直径×150mm玻璃管(VWR International,目录号:47729-583)
  9. Suba-Seal septa(Sigma-Aldrich,目录号:Z124613)
  10. 番茄植物(4-5周龄)在温室或生长室(16小时光照,25?28℃)中生长
  11. (Xcv)菌株85-10野生型和突变体(例如,III型效应物缺失突变体)
    注意:野生型菌株可应要求提供。
  12. 氯化镁六水合物(Sigma-Aldrich,目录号:M2393)
  13. 蛋白胨(BD,目录号:211677)
  14. 酵母提取物(BD,目录号:212750)
  15. 琼脂(BD,目录号:214530)
  16. 甘油(Certified ACS)(Fisher Chemical,目录号:G334)
  17. 氢氧化钠(Sigma-Aldrich,目录号:221465)
  18. 蒸馏水
  19. NYGA介质(参见配方)
  20. 10mM MgCl 2(参见配方)

设备

  1. 28oC培养箱(VWR International,目录号:414005-128)
  2. Vortexer(Scientific Indrustries,型号:Vortex-Genie 2)
  3. 分光光度计(Amersham Biosciences,型号:Ultrospec 3100 Pro)
  4. 气相色谱仪(GC)(Shimadzu Corporation,型号:GC-8A)

程序

  1. 细菌接种物制备和接种
    1. 使用a。从-80℃甘油储备液中划出每个Xcv菌株 灭菌的牙签放到独立的无菌NYGA琼脂板上 适当的抗生素,并在28℃的培养箱中生长2天
    2. 对于每种分析的菌株,悬浮一部分细菌细胞 琼脂平板使用消毒的牙签或移液管吸头在1ml的10 mM MgCl 2在1.5ml微管中。涡旋细胞悬浮液和 通过使用a检查其在600nm处的光密度(OD大于600) 分光光度计。为了制备接种物,稀释细胞悬浮液 与10mM MgCl 2以获得OD 600 = 0.2。 (不同接种物 浓度应该根据经验测试最佳乙烯 生产。)
    3. 用10mM接种中间叶的每个小叶 MgCl 2(模拟接种)或使用1ml无针的细菌接种物 ?注射器(图1)。小心地除去表面上的多余溶液 与薄纸的传单。在同一分支上使用传单 与叶龄的对照比较。对于统计分析,准备a 最少三次生物学重复

      图1.用不需要的注射器进行手浸润

    4. 将接种的植物移植到植物生长室或温室中(?25-28℃,16h天/8h夜间循环)。

  2. 乙烯气体测量
    1. 在接种后0,2和3天(dpi),切除小叶(模拟或Xcv感染的)并测量它们的重量。
    2. 卷每个小叶,然后将其放入玻璃管(16毫米直径×100毫米)30分钟,没有密封。
    3. 然后用Suba-Seal Septa塞子盖住每个玻璃管(图2)。
      注意:重要的是使用最小尺寸的玻璃管来减少 ?体积的气体空间以浓缩各自排放的乙烯 传单。


      图2.在带有隔垫的玻璃管中滚动的传单

    4. 在室温下孵育玻璃管1小时。
    5. 使用连接到1ml注射器的25号针头,取出1ml 来自每个玻璃管的气体样品(图3)并将其注入气体中 色谱

      图3.从带有隔垫的玻璃管取样气体

    6. 使用公司使用手册检测乙烯[例如 Shimadzu的 GC使用说明书 ( https://store.shimadzu.com/p-52629- instruction-manualgc-8apfgc-8a.aspx )]。


  3. 使用标准曲线
    测量乙烯
    1. 使用1%乙烯罐上的针头附件泵送乙烯气体 进入Tedlar包。标签袋为标准1.最终浓度 标准1 = 1%= 10,000nl乙烯/ml

      图4.将乙烯气体泵入Tedlar袋

    2. 从填充乙烯的Tedlar袋中取1ml 1%乙烯 将其注入到带盖的玻璃管(18mm直径×150mm玻璃管 带有Suba-seal隔垫)。标签管为最终浓度 标准2 = 0.0352%或352nl乙烯/ml。 (空的加盖管 含有27.4ml空气。标准品2中的乙烯浓度= 1%/1ml ?+ 27.4ml。)
    3. 从盖玻璃管(标准2; 18毫米)取1毫升 直径×150mm玻璃管,具有Suba-seal隔片),并将其注入 (18mm直径×150mm玻璃管) Suba-seal septa)。标准品3的最终浓度= 0.00124%或12.4 nl乙烯/ml
    4. 在GC上注入1 ml的每种标准品并测定峰面积。
    5. 通过绘制峰面积(X轴)由nl乙烯(Y轴)构建标准曲线。
    6. 使用标准曲线,确定每个中的乙烯的水平 接种小叶。乙烯含量定义为nl乙烯/克 的鲜重/小时。代表性的结果可以在Lund等人 al。(1998)(图1C和图2C)和Kim等人(2013)(图 1A-B)。

食谱

  1. NYGA培养基(1L)
    蛋白胨5 g
    酵母提取物3 g
    琼脂15克
    甘油20ml
    加入蒸馏水补足1 L
    用1N氢氧化钠调节pH至7.0 通过高压灭菌20分钟灭菌培养基
  2. 10mM MgCl 2/
    氯化镁六水合物2.03克
    加入蒸馏水补足1 L
    通过高压灭菌20分钟灭菌培养基

致谢

该协议从Lund等人(1998)采用。 W. Stork由美国农业部NIFA拨款2012-67011-19669支持。 M. B. Mudgett得到国立卫生研究院资助2 R01 GM068886-06A1的支持。

参考文献

  1. Kim,J.G.,Stork,W。和Mudgett,M.B。(2013)。 Xanthomonas type III effector XopD desumoylates tomato transcription transcription SlERF4 to suppress ethylene responses and promoting pathogen growth。 a> Cell Host Microbe 13(2):143-154
  2. Lund,S.T.,Stall,R.E.and Klee,H.J。(1998)。 乙烯调节番茄中对病原体感染的敏感反应。 /em> 10(3):371-382。
  3. Stork,W.,Kim,J.G。和Mudgett,M.B。(2015)。 黄单胞菌核心III型效应子XopX对植物防御的抑制和激活的功能分析。 Mol Plant Microbe Interact 28(2):180-194
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kim, J., Stork, W. and Mudgett, M. B. (2016). Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria. Bio-protocol 6(3): e1723. DOI: 10.21769/BioProtoc.1723.
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