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Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi
真菌中过氧化氢快速动力学变化的高精度实时测定   

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Abstract

Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique in the filamentous plant pathogen Fusarium graminearum (Mentges and Bormann, 2015). HyPer consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain (RD) of the prokaryotic H2O2-sensing protein, OxyR. The OxyR domain renders the sensor highly specific for H2O2. Oxidation of HyPer increases fluorescence of cpYFP excited at 488 nm and decreases fluorescence excited at 405 nm, thereby facilitating ratiometric readouts (Belousov et al., 2006). HyPer turned out to be pH-sensitive. A single amino acid mutation in the H2O2-sensing domain of HyPer renders the sensor insensitive to H2O2. This reporter is called SypHer and serves as a control for pH changes.

By using the HyPer-imaging technique, we could demonstrate that: i) HyPer imaging enables the specific and accurate detection of rapid changes in the intracellular H2O2 balance, ii) F. graminearum reacts on external stimuli with the transient production of H2O2, and iii) faces increased H2O2 level during initial infection of wheat.

The aim of this protocol is to guide the user through the basic setup of an in vitro HyPer imaging experiment in basically any fungus. It will provide the specific parameter for the fluorescence imaging as well as the construction of customized flow chambers for in vitro applications.

Keywords: Hydrogen peroxide(过氧化氢), Fusarium graminearum(禾谷镰刀菌), Ratiometric(比率计量), Reactive oxygen species(活性氧), Mycelia(菌丝体), Hyphae(菌丝), Filamentous fungi(丝状真菌)

Background

HyPer is a genetically encoded, highly specific H2O2 sensor protein enabling the real-time detection of fluctuations in the intracellular H2O2-level, e.g., in response to external stimuli. Genetically encoded sensors are advantageous over classical staining methods like 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), 3,3’-Diaminobenzidine (DAB), or boronate-based H2O2 probes since they lack the major disadvantages of the latter ones as for example technically sophisticated application, requirement for chemical fixation of cells, insufficient uptake, inadequate intracellular distribution of stains, and occasionally irreversibility (reviewed in Guo et al., 2014). A genetically encoded sensor widely used for in-vivo detection of redox states in a cell is the redox-sensitive GFP (roGFP) system. The roGFP system is, however, not specific to a certain subtype of oxidative agent, i.e., H2O2, but (indirectly) monitors the redox status of a cell.

Materials and Reagents

  1. Microplates, 96 well, black, F-bottom (Greiner Bio One, catalog number: 655076 )
  2. Cover slips (24 x 40 mm) (Carl Roth, catalog number: 1870.2 )
  3. Standard microscope slides (Carl Roth, catalog number: 0656.1 )
  4. 10 ml disposable syringes (Carl Roth, catalog number: 0058.1 )
  5. 0.35 x 25 mm endo needle for root canal rinsing (Vedefar, Dilbeek, catalog number: 99010 )
  6. 92 mm Petri dish (SARSTEDT, catalog number: 82.1473 )
  7. Whatman paper (Sigma-Aldrich, catalog number: WHA10347511 ) (Optional)
  8. Plastic disposal bags (Carl Roth, catalog number: E706.1 )
  9. Double-sided adhesive frame (Gene Frame) (Thermo Fisher Scientific, Thermo Fisher ScientificTM, catalog number AB0576 )
  10. Pipette tips (1,000 µl, 200 µl, 10 µl)
  11. PCR tubes
  12. Conidia of F. graminearum (i.e., of F. graminearum expressing HyPer-2 or SypHer; preferably fresh, not frozen)
  13. pC1-HyPer-2 (Addgene, catalog number: 42211 )
  14. pC1-HyPer-C199S (SypHer) (Addgene, catalog number: 42213 )
  15. PCR primer for HyPer and SypHer amplification: forward primer 5’-ATG GAG ATG GCA AGC CCA GCA GGG CGA GAC GAT GT-3’; reverse primer 5’-GCT TTT AAA CCG CCT GTT-3’
  16. Immersion oil, Immersol W 2010 (Pulch + Lorenz, catalog number: 444969-0000-000 )
  17. Hydrogen peroxide 30% (Carl Roth, catalog number: 9681.4 )
  18. 1,4-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 000000010197777001 )
  19. Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O (Carl Roth, catalog number: X886.1 )
  20. Potassium dihydrogen phosphate, KH2PO4 (Carl Roth, catalog number: P018.1 )
  21. Magnesium sulfate heptahydrate, MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391-500G )
  22. Sodium chloride, NaCl (Carl Roth, catalog number: 9265.1 )
  23. Boric acid, H3BO3 (Carl Roth, catalog number: 6943.1 )
  24. Copper(II) sulfate pentahydrate, CuSO4·5H2O (Sigma-Aldrich, catalog number: 209198-250G )
  25. Potassium iodide, KI (Carl Roth, catalog number: 6750.1 )
  26. Manganese(II) sulphate monohydrate, MnSO4·H2O (Carl Roth, catalog number: 7347.2 )
  27. Ammonium molybdate tetrahydrate, (NH4)6Mo7O24·4H2O (Sigma-Aldrich, catalog number: 09880-100G )
  28. Zinc sulphate heptahydrate, ZnSO4·7H2O (Carl Roth, catalog number: 7316.1 )
  29. Iron(III) chloride hexahydrate, FeCl3·6H2O (Carl Roth, catalog number: 7119.1 )
  30. Chloroform
  31. Sucrose (Carl Roth, catalog number: 9097.2 )
  32. Granulated agar (BD, catalog number: 214530 )
  33. Solution A (see Recipes)
  34. Solution B (see Recipes)
  35. Suspension D (see Recipes)
  36. Minimal medium (see Recipes)

Equipment

  1. Microplate multimode reader (e.g., Berthold Technologies Multimode Microplate Reader Mithras² LB 943, BERTHOLD TECHNOLOGIES, model: Mithras2 LB 943 ), equipped with fluorescence excitation filters (380 x 10 nm and 485 x 14 nm, e.g., BERTHOLD TECHNOLOGIES, catalog numbers: 40087-01 and 40271-01 ), fluorescence emission filter (520 x 10 nm, e.g., BERTHOLD TECHNOLOGIES, catalog number: 38836-01 ), and injectors (e.g., BERTHOLD TECHNOLOGIES, model: 54116 )
  2. Neubauer counting chamber improved (Carl Roth, catalog number: T729.1 )
  3. Confocal laser scanning microscope (e.g., LSM 780 mounted on a Carl Zeiss Axio Imager.Z2 microscope with motorized stage)
  4. 40x objective (e.g., Carl Zeiss C-apochromat Carl Zeiss 40x/1.20 W Korr M27)
  5. Incubator at 28 °C (Thermo Fisher Scientific, model: Heraeus B20/UB20 )
  6. Solid-state laser 405 nm, 50 mW
  7. Argon ion laser 458, 488 and 514 nm, 30 mW
  8. Multi-channel pipette (Eppendorf, catalog number: 3122000035 )
  9. Pipettors: 10-100 µl (Eppendorf, catalog number: 4920000059 ), 100-1,000 µl (Eppendorf, catalog number: 4920000083 )
  10. 300 mm Heidelberger extension (Dezember, Fresenius Kabi Deutschland, catalog number: 2873112 )

Software

  1. Plate reader software (e.g., Berthold MikroWin Lite software [Berthold Technologies])
  2. Spreadsheet software program (e.g., Excel [Microsoft])
  3. ImageJ (Version 1.46r, http://imagej.net/)

Procedure

  1. Generation of HyPer and SypHer reporter mutants
    1. Amplify sequences for HyPer and SypHer from pC1-HyPer-2 and pC1-HyPer-C199S (SypHer) using forward primer 5’-ATG GAG ATG GCA AGC CCA GCA GGG CGA GAC GAT GT-3’ and reverse primer 5’-GCT TTT AAA CCG CCT GTT-3’ (one may add restriction enzyme recognition sites to the 5’ end of the primer to facilitate cloning in the desired binary vector).
    2. Transform the fungus according to standard protocols. For F. graminearum, the authors refer to Maier et al. (2005).
      Note: It is recommended to check HyPer and SypHer expression by fluorescence microscopy or RT-PCR.

  2. 96-well plate assay
    The following paragraph describes an assay using a 96-well plate and multimode reader to analyze the response of F. graminearum mycelia to H2O2, DTT and test substances A and B, which might be any substance of choice at a given concentration to be assayed for its potential to cause an H2O2 burst in F. graminearum.
    1. Fill each well of a black 96-well plate with 100 µl minimal medium and wait 30 min until solidified.
    2. Inoculate the 96-well plate with 200 conidia (i.e., 10 µl of a 20 conidia µl-1 suspension) according to the pipetting scheme shown in Figure 1.


      Figure 1. Scheme for loading and inoculation of a 96-well assay plate to be analyzed in a fluorescence plate reader. Each well is filled with 100 µl minimal medium and inoculated with conidia of the wild type (columns 3 and 4), SypHer (columns 5 and 6), and HyPer (columns 7-12). Columns 1 and 2 remain uninoculated as control.

    3. Cover the 96-well plate, place it into a plastic bag moistened with sterile ddH2O to prevent dehydration and incubate it at 28 °C in permanent darkness for 2 days.
    4. On day 3, the HyPer assay shall be performed. Prolonged incubation leads to an accumulation of aerial hyphae that perturb measurement.
    5. Start the multimode reader and allow for the interior chamber to reach 28 °C.
    6. Prime the injectors according to the manufacturer’s instructions. By way of example, injector 1 could be primed with a 150 mM H2O2 solution, injector 2 with a 150 mM DTT solution, injector 3 with test substance A and injector 4 with test substance B at a concentration threefold the final concentration in the well.
    7. Program the measuring cycles in the plate reader software:
      1. Choose excitation filter ‘380 x 10’ and emission filter ‘520 x 10’ for measuring the fluorescence emitted by HyPer in its reduced state and excitation filter ‘485 x 14’ (emission filter ‘520 x 10’ for the oxidized state).
      2. Configure the injectors. For example, injector 1 shall inject 50 µl of the test substance at cycle 31 and injector 2 shall inject 50 µl of another substance at cycle 61. If possible, choose a slow to medium injection speed in both cases to prevent spilling.
        Note: The user should avoid long measuring cycles because the HyPer response is typically very fast. If a substance is injected into all wells before the measurement of fluorescence resumes, the immediate response might already be over. Yet, the authors recommend to measure one well at a time immediately after injection. 
    8. Add 100 µl ddH2O to all wells using a multi-channel pipette and let plate stand for 30 min at 28 °C.
    9. Insert plate to plate reader and start the measuring cycles.
    10. After the run, export the raw data for further analysis in a spreadsheet software program.
    11. For ratio calculations, divide the fluorescence emission value (FEV) measured after excitation with 485 nm in well A1 by the FEV after excitation with 380 nm in this particular well (equation 1).

      Note: Equation 1 represents the calculation of the ratio of fluorescence intensities measured after excitation with 485 nm and 380 nm, respectively. 
    12. Apply this to all wells. Average the ratios for the biological replicates (i.e., wells A1 to H2 for the media control) for each time point and calculate the standard deviations. Plot the averaged ratios on the y-axis against the time on the x-axis. A measurement of fungal response to H2O2 and DTT according to the procedures described above should typically result in a curve as depicted in Figure 2.


      Figure 2. Ratiometric time course assay of the fungal response to external H2O2 and dithiothreitol (DTT). Timing of H2O2 (50 mM) and DTT (50 mM) induced ratio [485/380 nm] change in hyphae expressing HyPer, SypHer compared to the wild type (no fluorophore) and media control (MM). Mycelia were raised in a 96-well plate as described in the text and analyzed in a fluorometer. Error bars represent the standard deviation.

  3. Confocal laser scanning microscopy assay
    This paragraph describes the construction and usage of a fluidic chamber for in-situ microscopy of fungal response to external H2O2 or any test substance. The assembly of the fluidic chamber is illustrated in Figure 3 and Video 1.
    1. Surface-sterilize microscope slides, e.g., in ethanol or UV light.
    2. Remove the cover foil that covers a gene frame and the window and adhere it to the slide.
    3. Fill 40 µl MM into the frame and immediately cover it with a second slide to obtain an even surface within the chamber.
    4. After 30 min, remove the upper cover slide by carefully moving it sideways and inoculate the agar surface with 300 conidia (15 µl of a 20 conidia µl-1 suspension) of mutants expressing HyPer or SypHer.
    5. Incubate in a sterile Petri dish (add 500 µl sterile ddH2O and use pipette tips as elevation) for 24 h at 28 °C in permanent darkness.
    6. On the next day, remove the cover foil of the second gene frame that covers the frame and the window and the remaining cover foil from the first gene frame. Take the second gene frame and attach it to the first frame on the slide.
    7. Cut a 1 mm and a 5 mm piece out of both gene frames to facilitate injection and drainage of a test substance, respectively (see Figure 3).


      Figure 3. Assembly of a customized fluidic chamber for the simultaneous image acquisition and injection of test substances. Detailed description in the main text (see also Video 1).

      Video 1. Assembly of a fluidic chamber

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    8. Remove the remaining cover foil from the gene frame.
    9. Place a cover slip on the second frame with an overhang to the drainage port. It serves as a small reservoir for excess volume.
    10. Rinse the fluidic chamber with ddH2O using a syringe with an endo needle for root canal rinsing attached.
    11. Prefill a Heidelberger extension with a test substance (e.g., 50 mM H2O2) using a syringe and attach it to another endo needle.
    12. Mount the fluidic chamber to the stage of the CSLM.
    13. Plug the endo needle into the injection port and secure the position of the Heidelberger extension and endo needle with adhesive tape.
    14. Program a sequential acquisition of xyt-frames (lateral resolution 512 x 512, color depth: 8 bit) using excitation at 405 and 488 nm over at least 40 min with the shortest possible acquisition time and repetition rate. Detect the fluorescence signal for both channels in a range from 493 to 598 nm. Use a 40x/1.2 W objective for image acquisition.
    15. Start the scanning. Adjust laser intensity and gain of both channels so that the fluorescence signal intensity is approximately levelled in both channels (405 and 488 nm excitation). Figure 4 represents exemplarily the HyPer fluorescence in a hypha of F. graminearum.


      Figure 4. Fluorescence emission and bright field image of a F. graminearum hypha expressing HyPer

    16. Before starting the injection monitor steady-state fluorescence over 10 min.
    17. Carefully inject the test substance while continue scanning. In order to prevent damage of the microscope always observe the microscope slide for spillage. Inject as much volume as it takes to completely wash out the initially added ddH2O.
    18. Aquire xyt frames over at least 30 min.
    19. [Optional] Wash out the first test substance, wash the chamber once with ddH2O and repeat the injection using a new, prefilled Heidelberger extension. Take up excess volume at the drainage port using absorbent paper (e.g., Whatman paper).
    20. For data analysis, open xyt stacks with ImageJ.
    21. To obtain the FEVs of each channel over time mark and measure fluorescence intensities in one or more custom-shaped regions of interest using the ‘ROI manager’ tool and ‘measure stacks’ plugin. Calculate the 488 nm/405 nm ratio (as similar as equation 1) and plot it on the y-axis against the acquisition time on the x-axis.
    22. For visual representation of the 488 nm/405 nm ratio follow the instructions (link) of Kardash et al. (2011). Figure 5 shows exemplarily a 488 nm/405 nm false color ratio image of a hypha.


      Figure 5. False color image of a 488 nm/405 nm ratio

Data analysis

The use of 96-well plates for the HyPer assays allows for high numbers of replicates. Each fungal colony growing in a well represents a biological replicate that is growing and monitored independently from the others. High numbers of replicates, in turn, allow for discrimination of colonies growing weakly due to an inappropriate distribution of media in the well or a low number of conidia due to pipetting mistakes. Results obtained from those wells can be excluded from the analysis.
For further details on data handling and statistics see Mentges and Bormann (2015).

Notes

HyPer measurements provide highly reproducible and reliable data on H2O2 fluctuations in hyphae of F. graminearum. The setup of a 96-well assay is easy and allows for a high number of replicates. This guarantees statistical accuracy. The CLSM analysis, although technically more sophisticated, enables monitoring intracellular H2O2 allocations, e.g., during nuclear division or directed growth (Mentges and Bormann, 2015). Critical for the CLSM assay is the plane agar surface provided by the two object slides stacked on top of each other separated by a gene frame.

Recipes

  1. Solution A
    100 g/L Ca(NO3)2·4H2O
    Sterilized by filtration
  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 g/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
    Sterilized by addition of 0.1% (v/v) chloroform
  4. Minimal medium (MM, 1 L)
    10 ml solution A
    10 ml solution B
    10 g sucrose
    1 ml suspension D
    16 g agar

Acknowledgments

The authors thank Dr. V.V. Belousov for providing the HyPer and SypHer plasmids and B. Hadeler and C. Kröger for technical assistance.

References

  1. Belousov, V. V., Fradkov, A. F., Lukyanov, K. A., Staroverov, D. B., Shakhbazov, K. S., Terskikh, A. V. and Lukyanov, S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4): 281-286.
  2. Guo, H., Aleyasin, H., Dickinson, B. C., Haskew-Layton, R. E. and Ratan, R. R. (2014). Recent advances in hydrogen peroxide imaging for biological applications. Cell Biosci 4: 64.
  3. Kardash, E., Bandemer, J. and Raz, E. (2011). Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat Protoc 6(12): 1835-1846.
  4. Maier, F. J., Malz, S., Lösch, A. P., Lacour, T. and Schäfer, W. (2005). Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker. FEMS Yeast Res 5(6-7): 653-662.
  5. Mentges, M. and Bormann, J. (2015). Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum. Sci Rep 5: 14980.

简介

活性氧(ROS)是有氧代谢的副产物。尽管作为信号分子的有利方面,ROS主要被认为是作用于核酸,蛋白质和脂质的有害物质。反应性氧物质,特别是过氧化氢(H 2 O 2 O 2),作为防御分子跨越诸如,通过植物为了击败入侵病原体如真菌。营养不良的植物致病真菌本身分泌H 2 O 2 O 2以诱导宿主细胞死亡并促进感染。过氧化氢在一定程度上可以通过膜自由扩散。为了能够监测真菌中的细胞内过氧化氢动力学,我们最近在丝状植物病原体禾谷镰刀菌(Mentges and Bormann,2015)中建立了多功能的HyPer成像技术。 HyPer由插入到原核H 2 O 2 O 2 - 感觉蛋白OxyR的调节结构域(RD)中的循环置换的黄色荧光蛋白(cpYFP)组成。 OxyR域使传感器高度特异于H 2 O 2 O 2。 HyPer的氧化增加了在488nm激发的cpYFP的荧光,并降低了在405nm激发的荧光,从而促进了比例式读数(Belousov等人,2006)。 HyPer原来是对pH敏感。 HyPer的H 2 O 2 O 2 - 感官域中的单个氨基酸突变使传感器对H 2 O 2不敏感。该记者称为SypHer,作为pH变化的对照。
   通过使用HyPer成像技术,我们可以证明:i)HyPer成像能够特异和准确地检测细胞内H 2 O 2 O 2平衡的快速变化,ii)。 gr um um um with with with sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub sub > 2 水平。
 该协议的目的是引导用户在基本上任何真菌中进行体外 HyPer成像实验的基本设置。它将提供用于荧光成像的具体参数以及用于体外应用的定制流动室的构建。

背景 HyPer是一种遗传编码的高度特异性的H 2 O 2 O 2传感器蛋白,使得能够实时检测细胞内H 2 O 2的波动 2 等级,例如,以响应外部刺激。遗传编码的传感器优于传统的染色方法,如2',7'-二氯二氢荧光素二乙酸酯(H2DCFDA),3,3'-二氨基联苯胺(DAB)或基于硼酸盐的H 2 O 2 探针,因为它们缺乏后者的主要缺点,例如技术上复杂的应用,细胞的化学固定的需要,摄取不足,细胞内分布的污渍不足,以及偶尔的不可逆性(Guo et al。 。,2014)。广泛用于体内检测细胞中氧化还原状态的遗传编码传感器是氧化还原敏感性GFP(roGFP)系统。然而,roGFP系统不是特定于某种氧化剂亚型,即,H 2 O 2 O 2,或(间接)监测器细胞的氧化还原状态。

关键字:过氧化氢, 禾谷镰刀菌, 比率计量, 活性氧, 菌丝体, 菌丝, 丝状真菌

材料和试剂

  1. 微孔板,96孔,黑色,F底(Greiner Bio One,目录号:655076)
  2. 盖板(24 x 40 mm)(Carl Roth,目录号:1870.2)
  3. 标准显微镜幻灯片(Carl Roth,目录号:0656.1)
  4. 10 ml一次性注射器(Carl Roth,目录号:0058.1)
  5. 0.35 x 25毫米内根针根管冲洗(Vedefar,Dilbeek,目录号:99010)
  6. 92毫米培养皿(SARSTEDT,目录号:82.1473)
  7. Whatman纸(Sigma-Aldrich,目录号:WHA10347511)(可选)
  8. 塑料处理袋(Carl Roth,目录号:E706.1)
  9. 双面胶框(Gene Frame)(Thermo Fisher Scientific,Thermo Fisher Scientific TM,目录号AB0576)
  10. 移液头(1000μl,200μl,10μl)
  11. PCR管
  12. 分生孢子。表示HyPer-2或SypHer的禾谷镰孢表达( ,优选新鲜,未冷冻)
  13. pC1-HyPer-2(Addgene,目录号:42211)
  14. pC1-HyPer-C199S(SypHer)(Addgene,目录号:42213)
  15. PCR引物用于HyPer和SypHer扩增:正向引物5'-ATG GAG ATG GCA AGC CCA GCA GGG CGA GAC GAT GT-3';反向引物5'-GCT TTT AAA CCG CCT GTT-3'
  16. 浸入油,Immersol W 2010(Pulch + Lorenz,目录号:444969-0000-000)
  17. 过氧化氢30%(Carl Roth,目录号:9681.4)
  18. 1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:000000010197777001)
  19. 硝酸钙四水合物,Ca(NO 3 3)2·4H 2 O(Carl Roth,目录号:X886.1)
  20. 磷酸二氢钾,KH 2 PO 4(Carl Roth,目录号:P018.1)
  21. 硫酸镁七水合物,MgSO 4·7H 2 O(Sigma-Aldrich,目录号:230391-500G)
  22. 氯化钠,NaCl(Carl Roth,目录号:9265.1)
  23. 硼酸,H 3 BO 3(Carl Roth,目录号:6943.1)
  24. 硫酸铜(II)五水合物,CuSO 4·5H 2 O(Sigma-Aldrich,目录号:209198-250G)
  25. 碘化钾,KI(Carl Roth,目录号:6750.1)
  26. 硫酸锰(II)一水合物,MnSO 4 H 2 O(Carl Roth,目录号:7347.2)
  27. 钼酸铵四水合物,(NH 4)6族Mo 7 O 24·4H 2/sub > O(Sigma-Aldrich,目录号:09880-100G)
  28. 硫酸锌七水合物,ZnSO 4·7H 2 O(Carl Roth,目录号:7316.1)
  29. 氯化铁(III)六水合物,FeCl 3·6H 2 O(Carl Roth,目录号:7119.1)
  30. 氯仿
  31. 蔗糖(Carl Roth,目录号:9097.2)
  32. 颗粒状琼脂(BD,目录号:214530)
  33. 解答A(见配方)
  34. 解决方案B(见配方)
  35. 悬挂D(见配方)
  36. 最小介质(见配方)

设备

  1. 微板多模式阅读器(例如,Berthold Technologies Multimode Microplate Reader Mithras ² LB 943,BERTHOLD TECHNOLOGIES,型号:Mithras2 LB 943),配备荧光激发滤光片(380×10nm和485×14nm,例如,BERTHOLD TECHNOLOGIES,目录号:40087-01和40271-01),荧光发射滤光片(520×10nm,例如,BERTHOLD TECHNOLOGIES ,目录号:38836-01)和注射器(例如,,BERTHOLD TECHNOLOGIES,型号:54116)
  2. Neubauer计数室改善(Carl Roth,目录号:T729.1)
  3. 共焦激光扫描显微镜(例如,安装在具有电动载物台的卡尔蔡司Axio Imager.Z2显微镜上的LSM 780)
  4. 40x物镜(例如,Carl Zeiss C-apochromat Carl Zeiss 40x/1.20W Korr M27)
  5. 28℃培养箱(Thermo Fisher Scientific,型号:Heraeus B20/UB20)
  6. 固体激光器405nm,50mW
  7. 氩离子激光458,488和514nm,30mW
  8. 多通道移液器(Eppendorf,目录号:3122000035)
  9. 移液器:10-100μl(Eppendorf,目录号:4920000059),100-1,000μl(Eppendorf,目录号:4920000083)
  10. 300 mm海德堡扩展(Dezember,Fresenius Kabi Deutschland,目录号:2873112)

软件

  1. 平板阅读器软件(例如,,Berthold MikroWin Lite软件[Berthold Technologies])
  2. 电子表格软件程序(例如,,Excel [Microsoft])
  3. ImageJ(版本1.46r, http://imagej.net/

程序

  1. 生成HyPer和SypHer报道突变体
    1. 使用正向引物5'-ATG GAG ATG GCA AGC CCA GCA GGG CGA GAC GAT GT-3'和反向引物5'-GCT TTT从pC1-HyPer-2和pC1-HyPer-C199S(SypHer)扩增HyPer和SypHer的序列AAA CCG CCT GTT-3'(可以在引物的5'末端添加限制酶识别位点,以便于在所需的二元载体中克隆)。
    2. 根据标准方案转化真菌。对于 F。 graminearum ,作者参考Maier等人。 (2005) 注意:建议用荧光显微镜或RT-PCR检查HyPer和SypHer的表达。

  2. 96孔板分析
    以下段落描述了使用96孔板和多模式阅读器分析"f"F的反应的分析。禾谷镰刀菌菌丝体至H 2 O 2 O 2,DTT和测试物质A和B,其可以是待测定的给定浓度下的任何选择物质因为其可能导致F中的H 2> 2 2 突发。 graminearum 。
    1. 用100μl基本培养基填充黑色96孔板的每个孔,等待30分钟直到固化。
    2. 根据图1所示的移液方案,将200个分生孢子(即,分别为10μl的20只分生孢子悬浮液)接种在96孔板中。 >

      图1.在荧光板读数器中装载和接种待分析的96孔测定板的方案每孔用100μl基本培养基填充,并接种野生型分生孢子(柱3和4),SypHer(第5列和第6列)和HyPer(第7-12列)。第1列和第2列保持未接受控制。

    3. 盖上96孔板,将其放入用无菌ddH 2 O加湿的塑料袋中,以防止脱水,并在28℃下在永久黑暗中孵育2天。
    4. 在第3天,应进行HyPer测定。长时间的孵化导致了扰动测量的气生菌丝的积累。
    5. 启动多模式阅读器,并允许内部室达到28°C。
    6. 按照制造商的说明注入喷油器。作为示例,注射器1可以用150mM H 2 O 2 O 2溶液,具有150mM DTT溶液的注射器2,具有测试物质A的注射器3和注射器4的测试物质B的浓度为孔中的最终浓度的三倍。
    7. 在读卡器软件中编程测量循环:
      1. 选择激发滤波器"380 x 10"和发射滤波器"520 x 10",用于测量HyPer在其降低状态下发出的荧光,激发滤波器'485 x 14'(发射滤波器'520 x 10'用于氧化状态)。
      2. 配置注射器。例如,注射器1将在循环31注入50μl的测试物质,注射器2将在循环61处注入50μl另外的物质。如果可能的话,在这两种情况下都可以选择缓慢到中等的注射速度以防止溢出。 > 注意:用户应避免长时间的测量循环,因为HyPer响应通常非常快。如果在荧光测量恢复之前将物质注入所有孔,则立即反应可能已经结束。然而,作者建议在注射后立即测量一口井。 
    8. 使用多通道移液管向所有孔中加入100μlddH 2 O,并在28℃放置30分钟。
    9. 将板插入读板器并开始测量循环。
    10. 运行后,在电子表格软件程序中导出原始数据进行进一步分析。
    11. 对于比率计算,将在该A1井中用485nm激发后的荧光发射值(FEV)除以在该特定孔中用380nm激发后的FEV(等式1)。

      >注意:等式1分别表示用485nm和380nm激发后测量的荧光强度比的计算。
    12. 将其应用于所有井。平均每个时间点生物重复的比例(即,即,介质对照的A1至H2的孔),并计算标准偏差。绘制y坐标上的平均比值与x轴上的时间。根据上述程序对H 2 O 2和DTT的真菌反应的测量通常将产生如图2所示的曲线。


      图2.对外部H 2 O 2和二硫苏糖醇(DTT)的真菌反应的比例时间过程测定。 表达HyPer,SypHer的菌丝的H 2 O 2(50mM)和DTT(50mM)诱导比[485/380nm]变化的时间变化进行比较到野生型(无荧光团)和培养基控制(MM)。如文中所述将菌丝体置于96孔板中,并用荧光计分析。误差条表示标准差。

  3. 共聚焦激光扫描显微镜分析
    本段描述了用于原位显微镜对外部H 2 O 2 O 2或任何测试物质的真菌反应的流体室的结构和用途。流体腔室的组件如图3和视频1所示
    1. 在乙醇或紫外光中表面灭菌显微镜载玻片,例如,
    2. 取下覆盖基因框架和窗口的盖子,并将其粘贴到幻灯片上。
    3. 将40μlMM填入框架中,并立即用第二张幻灯片盖住,以获得室内均匀的表面
    4. 30分钟后,通过小心地移动上盖板,通过侧面小心移动,并用300个分生孢子(15μl的20个分生孢子微悬浮液)分泌表达HyPer或SypHer的突变体接种琼脂表面。 />
    5. 在永久黑暗中,在28℃下孵育不育培养皿(加入500μl无菌ddH 2 O,并使用移液器吸头作为仰角)24小时。
    6. 第二天,从第一个基因框架中移除覆盖框架的第二个基因框架的盖子和窗口以及剩余的覆盖箔片。取第二个基因框架,并将其附加到幻灯片上的第一帧。
    7. 从两个基因框架中切出1毫米和5毫米片,以便分别注射和排出测试物质(见图3)。


      图3.用于同时进行图像采集和注入测试物质的定制流体室的组装。主要文本中的详细说明(另见视频1)。

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      视频1.装配流体腔
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      Get Adobe Flash Player

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    8. 从基因框架中移除剩余的覆盖箔。
    9. 在第二个框架上放置一个盖子滑块到伸出到排水口。它作为一个小容量的容积。
    10. 用带有内针针头的注射器冲洗带有ddH 2 O的流体腔,附着在根管上。
    11. 使用注射器使用测试物质(例如,50mM H 2 O 2 O 2)预先填充海德堡延长部分,并将其附着到另一个内针上。
    12. 将流体室安装到CSLM的阶段。
    13. 将内针插入注射口,用胶带固定海德堡延长杆和内针的位置。
    14. 以最短的采集时间和重复率在40分钟内使用405和488 nm的激发程序对xyt帧(横向分辨率512 x 512,颜色深度:8位)进行顺序采集。在493到598 nm的范围内检测两个通道的荧光信号。使用40x/1.2W的图像采集目标。
    15. 开始扫描调整两个通道的激光强度和增益,使得荧光信号强度在两个通道(405和488nm激发)中近似水平。图4示例性地表示了F的菌丝中的HyPer荧光。 graminearum 。


      图4. F的荧光发射和亮场图像。 graminearum 表达HyPer的菌丝
    16. 在10分钟内开始注入监测器稳态荧光之前。
    17. 继续扫描时小心注射测试物质。为了防止显微镜损坏,请始终观察显微镜载玻片的溢出。注入完全清除初始添加的ddH 2 O所需的体积。
    18. 至少30分钟以上的Aquire xyt框架。
    19. [可选]清洗第一个测试物质,用ddH 2 O 2清洗室一次,并使用新的预填充的海德堡延长重复注射。使用吸收纸(例如,Whatman论文)在排水口处收取多余的量。
    20. 对于数据分析,使用ImageJ打开xyt堆栈。
    21. 通过"ROI管理器"工具和"测量堆栈"插件,获取每个通道随时间的FEV值并测量一个或多个定制区域中的荧光强度。计算488 nm/405 nm比例(与等式1相似),并在y轴上绘制x轴上的采集时间。
    22. 对于488nm/405nm比例的视觉表示,遵循说明书( link )。图5示例性地示出了菌丝的488nm/405nm假色比图像。


      图5. 488nm/405nm比率的假彩色图像

数据分析

HyPer测量提供了高度可重现和可靠的H 2菌丝的H 2 O 2 O 2波动的数据。 graminearum 。 96孔测定的设置是容易的并允许大量的重复。这保证统计准确性。 CLSM分析尽管在技术上更复杂,但是在核分裂或定向生长期间能够监测细胞内H 2 O 2 O 2分配(例如,)(Mentges和Bormann,2015)。对于CLSM测定的关键是由由基因框架分开的彼此顶部堆叠的两个对象载玻片提供的平面琼脂表面。

食谱

  1. 解决方案A
    100g/L Ca(NO 3 3)2·4H 2 O
    通过过滤灭菌
  2. 解决方案B
    20g/L KH PO 4
    25g/L MgSO 4·7H 2 O
    10g/L NaCl
    通过过滤灭菌
  3. 暂停D
    60g/L H 3/3 3/3< 3>
    390g/L CuSO 4·5H 2 O
    13 mg/L KI
    60mg/L MnSO 4·H 2 O
    51mg/L(NH 4)6 O 24 2 O
    5.48g/L ZnSO 4·7H 2 O
    932mg/L FeCl 3·6H 2 O
    通过加入0.1%(v/v)氯仿消毒
  4. 最小介质(MM,1 L)
    10ml溶液A
    10ml溶液B
    10g蔗糖
    1ml悬浮液D
    16克琼脂

致谢

作者感谢V.V.V博士。 Belousov提供HyPer和SypHer质粒,B.Hadeler和C.Kröger提供技术援助。

参考文献

  1. Belousov,V.V.,Fradkov,A.F.,Lukyanov,K.A.,Staroverov,D.B.,Shakhbazov,K.S.Terskikh,A.V.和Lukyanov,S。(2006)。 细胞内过氧化氢的遗传编码荧光指示剂。 ; Nat方法 3(4):281-286。
  2. Guo,H.,Aleyasin,H.,Dickinson,B.C.,Haskew-Layton,R.E。和Ratan,R.R。(2014)。 用于生物应用的过氧化氢成像的最新进展。细胞生物学 4:64.
  3. Kardash,E.,Bandemer,J.和Raz,E。(2011)。 使用荧光共振能量转移生物传感器在活胚中成像蛋白质活性。/a>  Nat Protoc 6(12):1835-1846。
  4. Maier,F.J.,Malz,S.,Lösch,A.P.,Lacour,T。和Schäfer,W。(2005)。 开发禾谷镰刀菌的高效基因靶向系统使用聚酮化合物合酶基因的破坏作为可见标记。  FEMS酵母研究组织5(6-7):653-662。
  5. Mentges,M。和Bormann,J。(2015)。 营养和致病菌丝中过氧化氢动力学的实时成像镰刀菌镰刀菌。    5:14980.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mentges, M. and Bormann, J. (2016). Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi. Bio-protocol 6(24): e2080. DOI: 10.21769/BioProtoc.2080.
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