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A Highly Efficient Method for Measuring Oxygen Consumption Rate in Fusarium graminearum
禾谷镰刀菌中耗氧率的高效测量法   

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Abstract

The filamentous ascomycete Fusarium graminearum is the causal agent of Fusarium head blight, a devastating disease of cereals with a worldwide distribution. Fusarium graminearum infections result in a quantitative yield reduction by impairing the growth of the kernels, and a qualitative reduction by poisoning the remaining kernels with mycotoxins toxic to animals and humans. The colonization of wheat florets by phytopathogenic fungus requires high-efficiency energy generation in the mitochondria (Bönnighausen et al., 2015). Mitochondrial activity in microorganisms can be measured using the oxygen consumption rate (OCR) method. Here we describe a method for the assessment of fungal respiration using an XF24 extracellular flux analyzer. The Seahorse XF Analyzer is a microplate-based respirometer which measures oxygen consumption by changes in the fluorescence of immobilized fluorophores (Gerencser et al., 2009). Multiple mitochondrial parameters can be measured by the application of mitochondrial substrates and inhibitors which are injected automatically during the assays via ports (Divakaruni et al., 2014). The experimental work-flow involves the inoculation with conidia and the application of specific inhibitors of mitochondrial functions. The analysis of fungal respiration represents a valuable tool that complements classical phenotypic screenings.

Materials and Reagents

  1. Pipette tips
  2. Conidia of F. graminearum (preferably fresh, not frozen)
  3. Seahorse XF Cell Mito Stress Test Kit including oligomycin, rotenone, antimycin A and Cyanide-p-trifluoromethoxyphenyl hydrazone (Seahorse Bioscience, catalog number: 103015-100 )
  4. Seahorse XF24 Islet FluxPak containing XF24 cartridges and Islet capture microplates (Seahorse Bioscience, catalog number: 101174-100 )
  5. Calibrant solution (Seahorse Bioscience, catalog number: 103059-000 )
  6. Ca(NO3)2·4H2O (Carl Roth, catalog number: X886.1 )
  7. KH2PO4 (Carl Roth, catalog number: P018.1 )
  8. MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391-500G )
  9. NaCl (Carl Roth, catalog number: 9265.1 )
  10. Sucrose
  11. H3BO3 (Carl Roth, catalog number: 6943.1 )
  12. CuSO4·5H2O (Sigma-Aldrich, catalog number: 209198-250G )
  13. KI (Carl Roth, catalog number: 6750.1 )
  14. MnSO4·H2O (Carl Roth, catalog number: 7347.2 )
  15. (NH4)6Mo7O24·4H2O (Sigma-Aldrich, catalog number: 09880-100G )
  16. ZnSO4·7H2O (Carl Roth, catalog number: 7316.1 )
  17. FeCl3·6H2O (Carl Roth, catalog number: 7119.1 )
  18. Solution A (see Recipes)
  19. Solution B (see Recipes)
  20. Suspension D (see Recipes)
  21. Minimal medium (see Recipes)

Equipment

  1. Multi-channel pipettes (Sigma-Aldrich, catalog number: Z683930-1EA )
  2. XF24 extracellular flux analyzer (Seahorse Bioscience, model: Seahorse XFe24 )
  3. Incubator at 28 °C (Heraeus B20/UB20) (Thermo Fisher Scientific, catalog number: 50061005 )
  4. Microplate centrifuge (Eppendorf, catalog number: 022620509 )

Software

  1. XF24 Extracellular Flux Analyzer Software
  2. Spreadsheet software program [e.g., Excel (Microsoft)]

Procedure

All steps of OCR measurement are summarized in Figure 1. This section describes the setup of an appropriate program on the Seahorse controller. The injection compounds will be injected subsequently in all wells by air pressure.

  1. Program an equilibration step of 35 min.
  2. Measure OCRs
    1. Program three measuring cycles: 2 min mixing, 2 min delay, 3 min measurement.
    2. Optional: use injection port A to inject 50 µl of a test substance, e.g., gamma amino (5 mM final concentration) (Bönnighausen et al., 2015).
    3. After injection of test substance perform 20 measuring cycles (2 min mixing, 2 min delay, 3 min measurement).
    4. Use injection port B to inject oligomycin (55 μl, 4 μM final concentration). Oligomycin is used to estimate the amount of oxygen that is consumed for ATP synthesis.
    5. Immediately perform 3-6 measuring cycles (2 min mixing, 2 min delay, 3 min measurement).
    6. Use injection port C to inject Cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) (60 μl, 4 μM final concentration). FCCP uncouples the respiratory chain. Its application allows estimation of the spare respiratory capacity.
    7. Immediately perform 3-6 measuring cycles (2 min mixing, 2 min delay, 3 min measurement)
    8. Use injection port D to inject rotenone and antimycin A (65 μl, 4 μM final concentration). Rotenone and antimycin A inhibit complex I and II of the mitochondrial electron transport chain, respectively, and, thereby, completely deplete mitochondrial respiration.
    9. Immediately perform 3-6 measuring cycles (2 min mixing, 2 min delay, 3 min measurement).


    Figure 1. Workflow for oxygen consumption rate measurement in Fusarium graminearum

    Preparations on the day before the assay
  3. Hydrate the cartridges according to the manufacturer’s instructions with calibrant 24 h prior to assay in an incubator at 28 °C, turn the XF24 analyzer on, start the XF24 software, and allow the instrument to get a stable temperature of 28 °C.
  4. Insert the capture screens with the capture screen insert tool into the XF24 Islet capture microplate.
  5. Add 450 µl freshly prepared minimal medium into each well of the XF24 Islet capture microplate.
  6. Add 5,000 conidia (50 µl of a 100 conidia µl-1 suspension in water) into an XF24 Islet capture microplate, leave two wells blank for background subtraction.
  7. Centrifuge the plate for 2 min at 1,000 x g at room temperature to get all conidia below the capture screen.
  8. Incubate for 24 h at 28 °C (optimal growth temperature for F. graminearum).

    Workflow on the day of the assay
  9. Check for conidia germination under the dissecting microscope (Figure 2).
  10. Exchange the media with fresh minimal medium by taking up the liquid above the capture screen with a pipette and replacing it with fresh minimal medium (500 µl at room temperature).
  11. Incubate the plate for 1 h at 28 °C to allow the mycelia adjust to the new medium. Longer incubation might lead to nitrogen depletion in the medium due to uptake by the growing fungus.
  12. Load the injection ports of the cartridge as predefined in the program section and then follow the instructions of the XF24 controller to start the calibration of the hydrated cartridge.
  13. Insert the Islet microplate into XF24 extracellular flux analyzer and start the run.
  14. Analyze data according to the XF24 Seahorse Software and Operation Manual and a spreadsheet software program.


    Figure 2. Fungal mycelia of Fusarium graminearum. An XF24 Islet capture microplate filled with 450 µl minimal medium was inoculated with 5,000 conidia and incubated for 24 h at 28 °C. Scale bar: 100 µm

Representative data

A measurement of fungal respiration according to the procedures described above should typically result in an OCR as depicted in Figure 3. We recommend 5 replicates per experimental group.


Figure 3. Oxygen consumption rate of mycelia of Fusarium graminearum grown in liquid minimal medium

Notes

The measurement of oxygen consumption rates in axenic culture described here was highly reproducible.

Recipes

  1. Solution A
    100 g/L Ca(NO3)2·4H2O
  2. Solution B
    20 g/L KH2PO4
    25 g/L MgSO4·7H2O
    10 g/L NaCl, sterilized by filtration
  3. Suspension D
    60 g/L H3BO3
    390 mg/L CuSO4·5H2O
    13 mg/L KI
    60 mg/L MnSO4·H2O
    51 mg/L (NH4)6Mo7O24·4H2O
    5.48 g/L ZnSO4·7H2O
    932 mg/L FeCl3·6H2O
  4. Minimal medium (1 L)
    10 ml of solution A
    10 ml of solution B
    10 g sucrose
    1 ml of suspension D  

Acknowledgments

The authors thank the “Kompetenzzentrum Nachhaltige Universität” of the University Hamburg for funding.

References

  1. Bönnighausen, J., Gebhard, D., Kröger, C., Hadeler, B., Tumforde, T., Lieberei, R., Bergemann, J., Schäfer, W. and Bormann, J. (2015). Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum. Molecular Microbiology 98(6): 1115-1132.
  2. Gerencser, A. A., Neilson, A., Choi, S. W., Edman, U., Yadava, N., Oh, R. J., Ferrick, D. A., Nicholls, D. G. and Brand, M. D. (2009). Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal Chem 81(16): 6868-6878.
  3. Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N. and Jastroch, M. (2014). Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol 547: 309-354.

简介

丝状子囊菌禾谷镰刀菌是镰刀菌的致病剂,其是具有全世界分布的谷物的毁灭性疾病。禾谷镰孢菌感染通过损害谷粒的生长导致定量的产量降低,并且通过用对动物和人有毒的霉菌毒素中毒剩余的谷粒来定性降低。植物病原真菌对小麦的定居需要线粒体中的高效能量产生(Bönighausen等人,2015)。微生物中的线粒体活性可以使用氧消耗速率(OCR)方法测量。在这里我们描述使用XF24细胞外通量分析仪评估真菌呼吸的方法。 Seahorse XF分析仪是基于微孔板的呼吸仪,其通过固定荧光团的荧光变化来测量氧消耗(Gerencser等人,2009)。多种线粒体参数可以通过应用线粒体底物和抑制剂来测量,所述线粒体底物和抑制剂在测定期间通过端口自动注射(Divakaruni等人,2014)。实验工作流程涉及接种分生孢子和应用线粒体功能的特异性抑制剂。真菌呼吸的分析代表了补充经典表型筛选的有价值的工具。

材料和试剂

  1. 移液器提示
  2. 分生孢子。禾本科(最好是新鲜,不冻)
  3. Seahorse XF细胞水分胁迫测试试剂盒,包括寡霉素,鱼藤酮,抗霉素A和氰化物 - 对三氟甲氧基苯基腙(Seahorse Bioscience,目录号:103015-100)
  4. 包含XF24盒和Islet捕获微板的Seahorse XF24 Islet FluxPak(Seahorse Bioscience,目录号:101174-100)
  5. 校准物溶液(Seahorse Bioscience,目录号:103059-000)
  6. (Carl Roth,目录号:X886.1)的紫外线吸收剂。

  7. (Carl Roth,目录号:P018.1)
  8. MgSO 4·7H 2 O(Sigma-Aldrich,目录号:230391-500G)
  9. NaCl(Carl Roth,目录号:9265.1)
  10. 蔗糖

  11. (Carl Roth,目录号:6943.1)
  12. CuSO 4·5H 2 O(Sigma-Aldrich,目录号:209198-250G)
  13. KI(Carl Roth,目录号:6750.1)
  14. MnSO 4 H·H 2 O(Carl Roth,目录号:7347.2)
  15. (NH 4)6 Mo 7 Mo 24 SO 4·4H 2 O(Sigma公司) -Aldrich,目录号:09880-100G)
  16. ZnSO 4 7HH 2 O(Carl Roth,目录号:7316.1)
  17. FeCl 3·6H 2 O(Carl Roth,目录号:7119.1)
  18. 解决方案A(参见配方)
  19. 解决方案B(参见配方)
  20. 暂停D(参见配方)
  21. 最小培养基(见配方)

设备

  1. 多通道移液管(Sigma-Aldrich,目录号:Z683930-1EA)
  2. XF24胞外通量分析仪(Seahorse Bioscience,型号:Seahorse XFe24)
  3. 28℃培养箱(Heraeus B20/UB20)(Thermo Fisher Scientific,目录号:50061005)
  4. 微孔板离心机(Eppendorf,目录号:022620509)

软件

  1. XF24细胞外通量分析仪软件
  2. 电子表格软件程序[例如。,Excel(Microsoft)]

程序

OCR测量的所有步骤总结在图1中。本节介绍在海马控制器上设置适当的程序。注射化合物随后将通过空气压力注射到所有孔中。

  1. 编程35分钟的平衡步骤。
  2. 测量OCR
    1. 编程三个测量循环:2分钟混合,2分钟延迟,3分钟测量
    2. 任选:使用注射口A注射50μl测试物质,例如,γ氨基(5mM终浓度)(B?nighausen等人,2015)。
    3. 注射测试物质后进行20个测量循环(2分钟混合,2分钟延迟,3分钟测量)
    4. 使用注射端口B注射寡霉素(55μl,4μM终浓度)。寡霉素用于估计ATP合成所消耗的氧气量。
    5. 立即执行3-6次测量循环(2分钟混合,2分钟延迟,3分钟测量)
    6. 使用注射端口C注射氰化物 - 对三氟甲氧基苯基腙(FCCP)(60μl,4μM终浓度)。 FCCP解耦呼吸链。其应用允许估计备用呼吸能力。
    7. 立即执行3-6次测量循环(2分钟混合,2分钟延迟,3分钟测量)
    8. 使用注射端口D注射鱼藤酮和抗霉素A(65μl,4μM终浓度)。鱼藤酮和抗霉素A分别抑制线粒体电子传递链的复合物I和II,从而完全消除线粒体呼吸。
    9. 立即执行3-6次测量循环(2分钟混合,2分钟延迟,3分钟测量)。


    图1.禾本科镰刀菌 的耗氧量测量工作流程

    前一天的准备工作
  3. 根据制造商的说明,在测定前24小时,在校准液中在28℃的培养箱中水化盒,打开XF24分析仪,启动XF24软件,并允许仪器获得28℃的稳定温度。 />
  4. 将捕获屏幕与捕获屏幕插入工具插入XF24 Islet捕获微板
  5. 向XF24胰岛捕获微板的每个孔中加入450μl新鲜制备的基本培养基
  6. 将5,000个分生孢子(50μl的100分生孢子悬浮液在水中)加入到XF24岛捕获微板中,将两个孔空白用于背景扣除。
  7. 在室温下将平板在1,000×g下离心2分钟,得到捕获屏幕下方的所有分生孢子。
  8. 在28℃孵育24小时(禾谷镰孢的最适生长温度)。

  9. 检查解剖显微镜下的分生孢子发芽(图2)
  10. 用新鲜的基本培养基更换培养基,用移液管吸取捕获屏幕上方的液体,并用新鲜的基本培养基(室温下为500μl)替换。
  11. 孵育板在28℃下1小时,以允许菌丝体调整到新的媒体。更长的孵育可能导致培养基中由于生长的真菌的摄取而导致的氮耗竭。
  12. 按照程序部分中预定义的方式装载墨盒的进样口,然后按照XF24控制器的说明开始校准水化墨盒。
  13. 将胰岛微孔板插入XF24细胞外通量分析仪,开始运行
  14. 根据XF24 Seahorse软件和操作手册和电子表格软件程序分析数据。


    图2.禾本科镰孢菌的真菌菌丝体。用5,000个分生孢子接种填充有450μl基本培养基的XF24岛捕获微板,并在28℃温育24小时。比例尺:100μm

代表数据

根据上述程序的真菌呼吸的测量通常应当导致如图3所示的OCR。我们建议每个实验组重复5次。


图3.在液体基本培养基中生长的禾生镰孢菌的菌丝体的氧消耗速率

笔记

这里描述的无菌培养中的氧消耗速率的测量是高度可重复的。

食谱

  1. 解决方案A
    100g/L Ca(NO 3)2 H 4·4H 2 O·
  2. 解决方案B
    20g/L KH 2 PO 4
    MgSO 4/7H 2 O 25/g/L MgSO 4/7H 2 O 10g/L NaCl,通过过滤灭菌
  3. 暂停D
    60g/L H sub 3 BO 3 sub
    390mg/L CuSO 4·5H 2 O·dm / 13 mg/L KI
    60mg/L MnSO 4 H·H 2 O·m / 51mg/L(NH 4)6 Mo 7 Mo 24 SO 4·4H 2 > O
    5.48g/L ZnSO 4·7H 2 O·h/v 932mg/L FeCl 3·6H 2 O·h/v
  4. 最小培养基(1L)
    10ml溶液A
    10ml溶液B
    10g蔗糖 1 ml的悬浮液D 

致谢

作者感谢汉堡大学的"Kompetenzzentrum NachhaltigeUniversit?t"资助。

参考文献

  1. Bemann,J.,Gebhard,D.,Kr?ger,C.,Hadeler,B.,Tumforde,T.,Lieberei,R.,Bergemann,J.,Sch?fer,W。和Bormann,J。 中断GABA分流会影响线粒体呼吸和毒力谷类病原体禾谷镰孢。 分子微生物学 98(6):1115-1132。
  2. Gerencser,AA,Neilson,A.,Choi,SW,Edman,U.,Yadava,N.,Oh,RJ,Ferrick,DA,Nicholls,DGand Brand,MD(2009)。< a class ="ke -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19555051"target ="_ blank">基于定量微孔板的肺活量测定法与氧扩散校正 Anal Chem 81(16):6868-6878。
  3. Divakaruni,AS,Paradyse,A.,Ferrick,DA,Murphy,AN and Jastroch,M.(2014)。  基于微孔板的氧消耗和pH数据的分析和解释方法Enzymol 547:309-354。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gebhard, D., Bönnighausen, J., Bergemann, J., Schäfer, W. and Bormann, J. (2016). A Highly Efficient Method for Measuring Oxygen Consumption Rate in Fusarium graminearum. Bio-protocol 6(15): e1887. DOI: 10.21769/BioProtoc.1887.
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