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Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum
尖孢镰刀菌菌丝体的体内ATP水平的测定   

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Abstract

Glycolysis provides metabolites for energy production via oxidative phosphorylation during vegetative growth of Fusarium oxysporum. Therefore, determination of intracellular ATP levels might be of valuable help to analyze regulation of glycolysis/gluconeogenesis pathways. The protocol described here can be applied to other filamentous fungi.

Keywords: ATP production(ATP的产生), Fusarium(镰刀菌), ATP quantification(ATP定量)

Materials and Reagents

  1. Monodur nylon filters 15 µm diameter (Filtravibracion S.L., Spain, catalog number: Nylon-15 )
  2. Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
  3. Eppendorf tube (2 ml)
  4. 5 mm-diameter glass beads (Sigma-Aldrich, USA, catalog number: 18406-500G )
  5. Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
  6. Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and mutant strains.
    Note: Strains used in this study are wild type strain 4287 (race 2) and Δcon7-1 and cΔcon7-1 mutant strains.
  7. Glycerol (Merck, catalog number: 104092 )
  8. Potato dextrose broth (PDB) (Scharlau, Spain, catalog number: 01483 )
  9. Sterile dH2O
  10. Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
  11. EDTA (Amresco, catalog number: 0322 )
  12. MgSO4·7H2O (Merck, catalog number: 1058865.5000 )
  13. KH2PO4 (Merck, catalog number: 104873.1000 )
  14. KCl (Merck, catalog number: 104933.0500 )
  15. NH4NO3 (Merck, catalog number: 101187.1000 )
  16. FeSO4 (Merck, catalog number: 103965.0500 )
  17. ZnSO4·7H2O (Merck, catalog number: 108883 )
  18. MnSO4 monohydrate (Merck, catalog number: 105941 )
  19. Glucose (Coger SAS, catalog number: 24379.363 )
  20. ATP determination kit (Thermo Fischer Scientific, Molecular Probes®, catalog number: A22066 ).
    Kit contents:
    1. D-Luciferin (Component A, MW 302, blue cap), 5 vials, each containing 3 mg of lyophilized powder
    2. Luciferase, firefly recombinant (Component B, red cap) 40 μl of a 5 mg/ml solution in 25 mMTris-acetate, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v) glycerol and 30% (v/v) ethylene glycol
    3. Dithiothreitol (DTT) (Component C, MW 154, black cap) 25 mg
    4. Adenosine 5’-triphosphate (ATP) (Component D, Green cap), 400 μl of a 5 mM solution in TE buffer
    5. 20x Reaction Buffer (Component E) 10 ml of 500 mM
    6. Tricine buffer, pH 7.8, 100 mM MgSO4, 2 mM EDTA and 2 mM sodium azide
  21. Extraction buffer (see Recipes)
  22. Potato dextrose broth (PDB) medium (see Recipes)

Equipment

  1. Sterile spatula (Fisher Scientific, catalog number: S50821 )
  2. Mini-BeadBeater-16 homogenizer (BioSpec Products)
  3. Hemocytometer Thoma (Marienfeld, Germany, catalog number: 06 407 10 )
  4. Orbital incubator (Infors, Multitron Pro, Switzerland)
  5. Microcentrifuge (Eppendorf, MiniSpin plus) (Fisher Scientific)
  6. Fluorimeter (TECAN, SpectraFluorPlus, catalog number: InfiniteM200PRO )

Procedure

  1. F. oxysporum f.sp. lycopersici wild type strain 4287 (race 2) was obtained from J. Tello, Universidad de Almeria, Spain. Δcon7-1 and cΔcon7-1 strains were described previously (Ruiz-Roldan et al., 20015). Strains are stored at -80 °C with 30 % glycerol as microconidial suspension.
  2. For fresh microconidia production, aliquots of frozen microconidial stocks are inoculated into 100 ml of potato dextrose broth (PDB) and incubated for 3 days at 150 rpm and 28 °C in an orbital incubator. Cultures are then filtered through Monodur nylon filters placed on funnels to separate mycelia (Figure 1A), and centrifuged at 3,020 x g for 5 min to collect microconidia. Finally, pellets containing fresh microconidia are resuspended into 1 ml sterile dH2O and counted using a hemocytometer.


    Figure 1. Filtration and mycelia harvesting procedure. A. Filtration of a fungal culture through a Monodur nylon membrane placed on a funnel. B. Subsequent harvesting of mycelia by scraping a nylon membrane using a spatula.

  3. Aliquots containing 2.5 x 108 fresh microconidia are grown in 100 ml PDB at 170 rpm and 28 °C for 14 h (wild-type and cΔcon7-1 strains) or 24 h (Δcon7-1 mutant).
  4. After harvesting by Monodur filtration, mycelia are washed twice with sterile dH2O, separated from the filter by scraping using a spatula (Figure 1B) and transferred to 2 ml-Eppendorf tubes containing a 5 mm-diameter glass bead.
  5. Cells are resuspended into 1 ml of extraction buffer (380 mM trichloroacetic acid and 12.7 mM EDTA) and disrupted by 3 cycles of 30 sec agitation each using a Mini-BeadBeater homogenizer, followed by incubation at room temperature with shaking at 250 rpm for 15 min.
  6. The supernatant is harvested by centrifugation at 11,336 x g for 15 min at 4 °C. Aliquots (10 μl each) are used for quantification of ATP levels using the ATP determination kit (http://tools.thermofisher.com/content/sfs/manuals/mp22066.pdf) following these instructions of the manufacturer with some modifications (indicated in underlines):
    1. Reagent preparation
      1. Make 1.0 ml of 1x Reaction Buffer by adding 50 μl of 20x Reaction Buffer (Component E) to 950 μl of deionized water (dH2O). This volume will be sufficient to make 1 ml of 10 mM D-luciferin stock solution.
      2. Make 1 ml of a 10 mM D-luciferin stock solution by adding 1 ml of 1x Reaction Buffer (prepared in the previous step) to one vial of D-luciferin (Component A, blue cap). Protect from light until use. The D-luciferin stock solution is reasonably stable for several weeks if stored at ≤ -20 °C, protected from light.
      3. Prepare a 100 mM DTT stock solution by adding 1.62 ml of dH2O to the bottle containing 25 mg of DTT (Component C, black cap). Aliquot into ten 160 μl volumes and store frozen at ≤ -20 °C. Stock solutions of DTT stored properly are stable for six months to one year. Thawed aliquots are kept on ice or at 4°C until use.
      4. Prepare low-concentration ATP standard solutions by diluting the 5 mM ATP solution (Component D, green cap) in dH2O. The concentrations and volumes to make depend upon the sensitivity and the luminometer used. Typically, ATP concentrations ranging from 1 nM to 1 μM are appropriate. These dilute solutions are stable for several weeks when stored at ≤ -20°C.
    2. Standard reaction solution
      1. The manufacturer suggests combining the components of the reaction as follows to make 10 ml of a standard reaction solution. Adjust the volumes according to particular requirements.
        8.9 ml dH2O
        0.5 ml 20x Reaction Buffer (Component E)
        0.1 ml 0.1 M DTT (from the previous step)
        0.5 ml of 10 mM D-luciferin (from the previous step, store the remaining 0.5 ml at ≤ -20 °C for up to several weeks)
        2.5 μl of firefly luciferase 5 mg/ml stock solution
      2. Gently invert the tube to mix, do not vortex; the firefly luciferase enzyme is easily denatured. Keep the reaction solution protected from light until use, it can be stored at 2-6 °C protected from light for several days.
    3. Standard curve
      1. Place 190 µl aliquots of the standard reaction solution (prepared in previous steps) in a microtiter plate and measure the background luminescence using a flurorometer.
      2. Start the reaction by adding 10 µl of diluted ATP standard solutions (prepared in previous steps) and read the luminescence. The volume of the dilute ATP standard solution that is added to the standard assay solution (prepared in the previous step) should be less than 10% of the total assay volume. For example, a 100 μl total assay volume should contain 10 μl or less of the ATP standard solution.
      3. Subtract the background luminescence.
      4. Generate a standard curve for a series of ATP concentrations (Figure 2). Be sure to always add a constant sample volume of the ATP containing solution. R-values of around 0.9 are normally obtained with this kit.
    4. Sample analysis
      1. Follow the directions given in Standard Curve, substituting ATP standard solutions for 10 µl fungal experimental samples. Please note that the total volume of the experimental sample assays should be equal to that of the ATP standard assays, with the amount of sample added amounting to no more than 10% of the total assay volume.
      2. Calculate the amount of ATP in the experimental samples from the standard curve. The assay is repeated three times with independent biological samples.

Representative data

  1. Figure 2 shows a representative standard curve for calculation of ATP concentration.


    Figure 2. A representative standard curve for calculation of ATP concentration. A. Representative Luminescence values of the diluted ATP standard solutions. B. Graphical representation of Luminescence values (Y-axis) vs. nM of ATP of the standards (X-axis).

    In this example, a luminescence value of 20,000 indicates that our test sample contains:

  2. Figure 3 shows a representative example of data obtained following this protocol.


    Figure 3. Intracellular ATP levels present in germlings of the indicated strains were quantified using the ATP determination kit (Molecular Probes)

Recipes

  1. Extraction buffer
    380 mM trichloroacetic acid
    12.7 mM EDTA
    Dissolve in deionized water
  2. Potato dextrose broth medium (PDB)
    Dissolve 24 g Potato dextrose broth in 1 L distilled water and sterilize by autoclaving

Acknowledgments

This research was supported by Junta de Andalucia (Proyecto de Excelencia CVI-7319) and the Spanish Ministerio de Economia y Competitividad (grant BIO2013-47870 and the Ramon y Cajal Program). This protocol was adapted from Ruiz-Roldan et al., 2015.

References

  1. Di Pietro, A. and Roncero, M. I. (1998). Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Mol Plant Microbe Interact 11(2): 91-98.
  2. Herreros, E., Martinez, C. M., Almela, M. J., Marriott, M. S., De Las Heras, F. G. and Gargallo-Viola, D. (1998). Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii, and filamentous fungi. Antimicrob Agents Chemother 42(11): 2863-2869.
  3. Palicz, A., Foubert, T. R., Jesaitis, A. J., Marodi, L. and McPhail, L. C. (2001). Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components. J BiolChem 276(5): 3090-3097.
  4. Ruiz-Roldan, C., Pareja-Jaime, Y., Gonzalez-Reyes, J. A. and MI, G. R. (2015). The transcription factor Con7-1 is a master regulator of morphogenesis and virulence in Fusarium oxysporum. Mol Plant Microbe Interact 28(1): 55-68.

简介

糖酵解在玉蜀黍的营养生长期间通过氧化磷酸化提供能量产生的代谢物。 因此,细胞内ATP水平的测定可能有助于分析糖酵解/糖异生途径的调节。 这里描述的协议可以应用于其他丝状真菌

关键字:ATP的产生, 镰刀菌, ATP定量

材料和试剂

  1. 直径15μm的Monodur尼龙过滤器(Filtravibracion S.L.,Spain,目录号:尼龙-15)
  2. 无菌塑料漏斗(直径80mm)(Tecnylab,目录号:45000150)
  3. Eppendorf管(2ml)
  4. 5mm直径的玻璃珠(Sigma-Aldrich,USA,目录号:18406-500G)
  5. 微量滴定板(TermoFisher Scientific,目录号:2205)
  6. <尖孢镰刀菌 f.sp.来自野生型和突变株的 lycopersici 微结实悬浮液 注意:本研究中使用的菌株是野生型菌株4287(种族2)和Δcon7-1和cΔcon7-1突变株。
  7. 甘油(Merck,目录号:104092)
  8. 马铃薯葡萄糖肉汤(PDB)(Scharlau,Spain,目录号:01483)
  9. 无菌dH 2 O 2/b
  10. 三氯乙酸(Sigma-Aldrich,目录号:T6399)
  11. EDTA(Amresco,目录号:0322)
  12. MgSO 4·7H 2 O(Merck,目录号:1058865.5000)

  13. (Merck,目录号:104873.1000)
  14. KCl(Merck,目录号:104933.0500)

  15. (Merck,目录号:101187.1000)

  16. (Merck,目录号:103965.0500)
  17. ZnSO 4·7H 2 O(Merck,目录号:108883)
  18. MnSO 4·一水合物(Merck,目录号:105941)
  19. 葡萄糖(Coger SAS,目录号:24379.363)
  20. ATP测定试剂盒(Thermo Fischer Scientific,Molecular Probes ,目录号:A22066)。
    套件内容:
    1. D-荧光素(组分A,MW 302,蓝色帽),5个小瓶,每个含有3mg冻干粉末
    2. 萤光素酶,萤火虫重组体(组分B,红帽)40μl在25mM Tris-乙酸盐,pH7.8,0.2M硫酸铵,15%(v/v)甘油和30%(v /乙二醇
    3. 二硫苏糖醇(DTT)(组分C,MW 154,黑帽)25mg
    4. 腺苷5'-三磷酸(ATP)(组分D,Green cap),400μl5mM TE缓冲液溶液
    5. 20x反应缓冲液(组分E)10ml 500mM
    6. 三羟甲基氨基甲烷缓冲液,pH7.8,100mM MgSO 4,2mM EDTA和2mM叠氮化钠
  21. 提取缓冲液(参见配方)
  22. 马铃薯右旋糖肉汤(PDB)培养基(见配方)

设备

  1. 无菌刮刀(Fisher Scientific,目录号:S50821)
  2. Mini-BeadBeater-16匀浆器(BioSpec Products)
  3. 血细胞计数器Thoma(Marienfeld,德国,目录号:06 40710)
  4. 轨道孵化器(Infors,Multitron Pro,瑞士)
  5. 微量离心机(Eppendorf,MiniSpin plus)(Fisher Scientific)
  6. 荧光计(TECAN,SpectraFluorPlus,目录号:InfiniteM200PRO)

程序

  1. F。尖孢镰孢野生型菌株4287(种族2)从J.Tello,Universidad de Almeria,Spain获得。 ( ,<200>)中描述了cΔ con7-1 和cΔ con7-1 将菌株在-80℃下与30%甘油一起储存为微小分枝悬浮液。
  2. 对于新鲜的小分生孢子生产,将冷冻的微小分生孢子的等分试样接种到100ml马铃薯葡萄糖肉汤(PDB)中,并在轨道培养箱中在150rpm和28℃下孵育3天。然后通过置于漏斗上的Monodur尼龙过滤器过滤培养物以分离菌丝体(图1A),并在3,020×g离心5分钟以收集微小分生孢子。最后,将含有新鲜微小分生孢子的沉淀重悬于1ml无菌dH 2 O中,并使用血细胞计数器计数。


    图1.过滤和菌丝体收获程序。A.通过置于漏斗上的Monodur尼龙膜过滤真菌培养物。 B.随后通过使用刮刀刮尼龙膜收获菌丝体
  3. 将含有2.5×10 8个新鲜微小分生孢子的等分试样在100ml PDB中以170rpm和28℃生长14小时(野生型和cΔ con7-1 菌株)或24小时(Δcon7-1突变体)。
  4. 在通过Monodur过滤收获后,用无菌dH 2 O 2洗涤菌丝体两次,使用刮刀通过刮擦从过滤器分离(图1B),并转移至含有5mm直径的2ml eppendorf管玻璃弹珠。
  5. 将细胞重悬浮于1ml提取缓冲液(380mM三氯乙酸和12.7mM EDTA)中,并使用Mini-BeadBeater匀浆器通过3次循环30秒的搅拌进行破碎,随后在室温下以250rpm振荡温育15分钟。
  6. 通过在4℃下以11,336×g离心15分钟收获上清液。使用ATP测定试剂盒( http://tools.thermofisher.com/content/sfs/manuals/mp22066.pdf )按照制造商的这些说明进行一些修改(以下划线表示):
    1. 试剂准备
      1. 通过加入50μl20x反应缓冲液(组分E)到950μl去离子水(dH 2 O)中制备1.0ml 1x反应缓冲液。该体积足以制备1ml 10mM D-荧光素储备溶液
      2. 通过向1瓶D-荧光素(组分A,蓝色帽)中加入1ml 1x反应缓冲液(在前面的步骤中制备)制备1ml 10mM D-荧光素储备溶液。避光使用。如果储存在≤-20°C,避光保存,D-荧光素储备溶液相当稳定几周
      3. 通过将1.62ml dH 2 O添加到含有25mg DTT(组分C,黑帽)的瓶中来制备100mM DTT储备溶液。等分成十个160μl体积,存储在≤-20°C冷冻。储存的DTT储存溶液稳定储存六个月至一年。将解冻的等分试样保存在冰上或4℃直至使用
      4. 通过稀释dH 2 O中的5mM ATP溶液(组分D,绿色帽)制备低浓度ATP标准溶液。所用的浓度和体积取决于所用的灵敏度和光度计。通常,1nM至1μM的ATP浓度是适当的。这些稀释溶液在≤-20℃储存时可稳定数周。
    2. 标准反应溶液
      1. 制造商建议如下组合反应的组分以制备10ml标准反应溶液。根据特定要求调整音量。
        8.9ml dH 2 O 2 / 0.5ml 20x反应缓冲液(组分E)
        0.1ml 0.1M DTT(来自前面的步骤)
        0.5 ml 10 mM D-荧光素(来自上一步,在≤-20°C存储剩余的0.5 ml长达几周)
        2.5μl萤火虫萤光素酶5mg/ml储备液
      2. 轻轻倒转管混合,不要涡旋;萤火虫荧光素酶容易变性。保持反应溶液避光,直到使用,它可以储存在2-6°C避光保存几天。
    3. 标准曲线
      1. 将190μl等分的标准反应溶液(在前面的步骤中制备)置于微量滴定板中,并使用荧光光度计测量背景发光。
      2. 通过加入10μl稀释的ATP标准溶液(在之前的步骤中制备)并读取发光来开始反应。加入到标准测定溶液(在上一步骤中制备)中的稀释的ATP标准溶液的体积应当小于总测定体积的10%。例如,100μl总测定体积应含有10μl或更少的ATP标准溶液
      3. 减去背景发光。
      4. 生成一系列ATP浓度的标准曲线(图2)。确保始终添加恒定的样品体积的含ATP溶液。通常使用该试剂盒获得约0.9的R值。
    4. 样品分析
      1. 遵循标准曲线中给出的指示,用ATP标准溶液代替10μl真菌实验样品。请注意,实验样品测定的总体积应等于ATP标准测定的总体积,加入的样品量不超过总测定体积的10%。
      2. 从标准曲线计算实验样品中的ATP量。用独立的生物样品重复该测定三次

代表数据

  1. 图2显示了用于计算ATP浓度的代表性标准曲线

    图2.用于计算ATP浓度的代表性标准曲线 A.稀释的ATP标准溶液的代表性发光值。 B.标准品的发光值(Y轴)对ATP的nM的图形表示(X轴)
    在这个例子中,发光值为20,000表示我们的测试样品包含:

  2. 图3显示了遵循该协议获得的数据的代表性实例

    图3.使用ATP测定试剂盒(Molecular Probes)对所指示菌株的种系中存在的细胞内ATP 水平进行定量。

食谱

  1. 提取缓冲区
    380mM三氯乙酸 12.7mM EDTA 溶于去离子水中
  2. 马铃薯葡萄糖肉汤培养基(PDB)
    将24g马铃薯葡萄糖肉汤溶解在1L蒸馏水中并通过高压灭菌消毒

致谢

这项研究由Junta de Andalucia(Proyecto de Excelencia CVI-7319)和西班牙经济部长竞争委员会(授予BIO2013-47870和Ramon y Cajal计划)支持。此协议改编自Ruiz-Roldan 。

参考文献

  1. Di Pietro,A。和Roncero,MI(1998)。  克隆,表达和在编码血管枯萎病原体尖孢镰刀菌的主要细胞外内聚半乳糖醛酸酶的pg1的致病性中的作用。 Mol Plant Microbe Interact 11(2) :91-98。
  2. Herreros,E.,Martinez,CM,Almela,MJ,Marriott,MS,De Las Heras,FG and Gargallo-Viola,D.(1998)。  Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii ,和丝状真菌。 42(11):2863-2869。
  3. Palicz,A.,Foubert,TR,Jesaitis,AJ,Marodi,L.and McPhail,LC(2001)。  磷脂酸和二酰基甘油通过与酶组分相互作用直接激活NADPH氧化酶 5):3090-3097。
  4. Ruiz-Roldan,C.,Pareja-Jaime,Y.,Gonzalez-Reyes,JA和MI,GR(2015)。  转录因子Con7-1是形态发生和毒力的主要调节因子,在 303 Mol Plant Microbe Interact 28(1):55-68。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ruiz-Roldan, C. and Roncero, M. I. (2016). Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum. Bio-protocol 6(14): e1869. DOI: 10.21769/BioProtoc.1869.
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