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Mar 2018
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High-throughput Screening for Defense Priming-inducing Compounds in Parsley Cell Cultures
欧芹细胞防御引物诱导化合物的高通量筛选   

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Abstract

Defense priming describes the enhanced potency of cells to activate defense responses. Priming accompanies local and systemic immune responses and can be triggered by microbial infection or upon treatment with certain chemicals. Thus, chemically activating defense priming is promising for biomedicine and agriculture. However, test systems for spotting priming-inducing chemicals are rare. Here, we describe a high-throughput screen for compounds that prime microbial pattern-spurred secretion of antimicrobial furanocoumarins in parsley culture cells. For the best possible throughput, we perform the assay with 1-ml aliquots of cell culture in 24-well microtiter plates. The advantages of the non-invasive test over competitive assays are its simplicity, remarkable reliability, and high sensitivity, which is based on furanocoumarin fluorescence in UV light.

Keywords: Agrochemicals (农药), Cell culture (细胞培养), Coumarins (香豆素), Crop protection (作物保护), Defense priming (防御启动), High-throughput screen (高通量筛选), Induced resistance (感生阻力), Parsley (欧芹), Plant immunity (植物免疫), Priming (启动效应)

Background

The pressing global issue of food insecurity due to population growth, diminishing arable land, and climate change can only be addressed in agriculture by effective crop protection (UN Food and Agriculture Organization, 2009). Synthetic agrochemicals can effectively protect crops from disease but also raise ecological and health concerns (Lamberth et al., 2013; Mascarelli, 2013). Therefore, safe and eco-friendly disease control products are needed (Lamberth et al., 2013).


Chemicals that prime the plant immune system for enhanced defense are promising for sustainable crop protection (Beckers and Conrath, 2007; Conrath et al., 2015). When primed, plants respond to very low pathogen stimuli with more robust activation of defense than unprimed plants. This frequently reduces disease susceptibility of the primed plant and lowers the risk of pathogen adaptation (Conrath et al., 2002, 2006, and 2015; Beckers and Conrath, 2007; Frost et al., 2008). Thus, inducing priming by harmless chemicals represents a promising means for sustainable crop disease control. Previous work with several chemical compounds provided proof of this concept (summarized in Conrath et al., 2015). However, the economic success of these compounds was limited, mainly because farmers preferred the strong curative performance of standard pesticides.


Today, the commercial success of crop protectants often relies on their ability to combine antimicrobial activity with defense priming in the target crop (Beckers and Conrath, 2007; Conrath et al., 2015). However, straightforward test systems for identifying priming-inducing chemistry are rare. Here, we describe a high-throughput screen for identifying chemical compounds that prime microbial pattern-induced furanocoumarin (phytoalexin) secretion in suspension-cultured parsley (Petroselinum crispum) cells. The test measures the enhancement by priming-activating chemicals of furanocoumarin secretion provoked in parsley culture cells by a moderate concentration of Pep13 (Kauss et al., 1992), a molecular pattern peptide in the plant pathogen Phytophthora sojae (Brunner et al., 2002). Over the past 25 years, the test helped to identify novel priming-inducing chemistry (Katz et al., 1998; Siegrist et al., 1998; Schillheim et al., 2018). For higher throughput, we now perform the test with 1-mL aliquots of cell culture in 24-well microtiter plates (Schillheim et al., 2018). Other advantages of the test over competitive assays are the extraordinary reliability, simplicity, high sensitivity of fluorescence detection, and the need for only two simple treatments (activation of priming and Pep13 challenge) before final analysis. In principle, the assay should be feasible also with cell cultures of other plant species that synthesize furanocoumarins (e.g., giant hogweed, common hogweed, and garden angelica), provided they respond to treatment with a given molecular pattern with furanocoumarin secretion to the culture medium. If that is not the case, other test readouts need to be chosen [e.g., the production of reactive oxygen species, as described byKauss et al. (1994)]. A recently introduced competitive assay to the screen introduced here evaluates the enhancement of Pseudomonas syringae pv. tomato avrRpm1-induced cell death in an Arabidopsis cell culture by priming agents (Noutoshi et al., 2012). The identification of several immune-priming compounds verified the power of the assay for discovering priming-inducing chemistry. However, because of the requirement of bacterial challenge, cytochemical staining, washing, dye extraction, and absorbance measurement, the screen is highly elaborate. The same holds true for a more recent respiratory activity-monitoring system for discovering immune-priming chemistry (Schilling et al., 2015), which is highly innovative but suffers from low throughput.

Materials and Reagents

  1. Sealing film (Roth, catalog number: H666.1)

  2. Sealing foil for cell cultures (Roth, catalog number: EN85.1)

  3. Serological pipettes 10 ml (Sarstedt, catalog number: 86.1254.025)

  4. Serological pipettes 10 ml without tip (Sarstedt, catalog number: 86.1688.010)

  5. Suction bottle 1,000 ml (Roth, catalog number: NY68.1)

  6. Syringe filters, 0.22 µm (Roth, catalog number: KH54.1)

  7. Aluminum foil (Roth, catalog number: 2596.1)

  8. Büchner funnel, 70 ml (Roth, catalog number: XX45.1)

  9. Cell culture 24-multiwell plates (Greiner, catalog number: 662160)

  10. Cellulose plugs “Zellstoppers” (Diagonal, catalog number: ZE153)

  11. Circular filter papers, Type 113A, 55 mm (Roth, catalog number: AP74.1)

  12. Disposable syringe 20 ml with Luer-Lock fitting (Roth, catalog number: T550.1)

  13. Flasks, 250 ml with a baffle (Diagonal, catalog number: 2121636S1)

  14. Flasks, 500 ml with a baffle (Diagonal, catalog number: 2121644S1)

  15. Sealing gaskets (Roth, catalog numbers: 9757.1 [33 mm]; 9758.1 [41 mm])

  16. NuncTM MicroWellTM 96-well, Nunclon Delta-Treated, flat-bottom microplate (ThermoFisher Scientific, catalog number: 137101)

  17. Pasteur pipettes, 2 ml (Roth, catalog number: 4522.1)

  18. Pep13 peptide (Thermo Fisher Scientific, amino acid sequence: VWNQPVRGFKVYE)

  19. Petri dishes, 94/16 mm (Greiner, catalog number: 633180)

  20. Pipette tips Gilson (Greiner, catalog numbers: 740290 [1,000 µl]; 739290 [200 µl])

  21. Pipette tips Starlab (TipOne®, catalog number: 1110-3000 [10/20 µl])

  22. Reaction tubes 1.5 ml (Sarstedt, catalog number: 72706)

  23. Reaction tubes 50 ml (Greiner, catalog number: 227261)

  24. Petroselinum crispum PC794 callus culture (DSMZ - Leibniz Institute, catalog number: PC794)

  25. Plant agar (Duchefa, catalog number: P1001.1000)

  26. Potassium hydroxide, 1 M KOH (Roth, catalog number: 9522.1)

  27. Salicylic acid, C7H6O3 (TCI, catalog number: H1342)

  28. Test compounds

  29. Gamborg’s B5 medium including vitamins (Duchefa, catalog number: G0210.0050)

  30. D-Sucrose, C12H22O11 (Roth, catalog number: 4621.2)

  31. Hand sanitizer (Roth, catalog number: EH72.2)

  32. Magnesium sulfate heptahydrate, MgSO4·7H2O (Roth, catalog number: P027.1)

  33. 2,4-Dichlorophenoxyacetic acid, C8H6Cl2O3 (Merck, catalog number: 8204510005)

  34. Dimethyl sulfoxide, C2H6OS (Roth, catalog number: A994.1)

  35. Ethanol, C2H5OH, ≥99.5 %, extra pure (Roth, catalog number: 5054.4)

  36. 70% ethanol (see Recipes)

  37. 2,4-Dichlorophenoxyacetic acid, 0.2 mg/ml stock solution (see Recipes)

  38. Magnesium sulfate, 0.05 mg/ml stock solution (see Recipes)

  39. Modified Gamborg’s B5 Medium (see Recipes)

  40. Salicylic acid in water (SA in water), 10 mM stock solution (see Recipes, store aliquots at -20°C)

  41. Salicylic acid in DMSO (SA in DMSO), 80 mM stock solution (see Recipes, store aliquots at -20°C)

  42. Pep13, 0.005 µM stock solution (see Recipes, store aliquots at -20°C)

  43. Callus medium (see Recipes)

Equipment

  1. 10-µl, 100-µl and 1,000-µl micropipettes (ErgoOne, Starlab)

  2. Magnetic bars (Roth, catalog number: 2153.2)

  3. Scalpel (Roth, catalog number: X003.1)

  4. Scissors (Roth, catalog number: 5489.1)

  5. Measuring cylinder, 1,000 ml (Roth, catalog number: P153.1)

  6. Measuring cylinder, 100 ml (Roth, catalog number: P150.1)

  7. Analytical balance (Mettler Toledo, model: MS204)

  8. Autoclave (Systec, model: DX65)

  9. Darkroom, temperature controlled (25°C), with two shaker incubators and clean bench with gas burner (see below)

  10. Clean bench (IBS Tecomara, model: NU-543-4000EC)

  11. Large shaker incubator, 90 rpm, 25°C (Infors HT, model: Multitron Standard)

  12. Safety laboratory gas burner (Roth, catalog number: AN82.1) with propane gas bottle (Westphalengas; 11 kg)

  13. Small shaker incubator, 130 rpm, 25°C (Infors HT, model: Ecotron)

  14. Laboratory vacuum system (Fisher Scientific, model: Welch IlmvacTM LVS 210T, catalog number: 11882253)

  15. Magnetic stirrer (Roth, catalog number: AAN2.1)

  16. Microplate Reader (BMG Labtech, model: CLARIOstar Plus)

  17. pH meter (Mettler Toledo, model: SevenMultiTM)

  18. Pipette bulb (Roth, catalog number: YX53.1)

  19. Pipette controller (BRAND, model: accu-jet® pro)

  20. Ultrapure water system (Satorius, model: arium® pro VF)

  21. UV lamp (Thermo Fisher Scientific, catalog number: 12813029)

Software

  1. CLARIOstar Reader Control Software (obtained with microplate reader)

    Plate: NUNC96

    Excitation: 335 nm (Bandwidth: 20)

    Dichroic filter: 365.2

    Emission: 398 nm (Bandwidth: 20)

  2. MARS Data Analysis Software (obtained with microplate reader)

  3. Statistical software (e.g., GraphPad Prism)

Procedure

  1. Growth and maintenance of parsley cell culture

    1. Setting up the parsley cell culture

      1. Work aseptically. At the clean bench and in the darkroom, use a scalpel to transfer 2-3 cm of callus culture (obtained from DSMZ) to 50 ml of freshly prepared modified Gamborg’s B5 medium in a 250-ml flask. In addition, transfer a 1 × 1-cm piece of callus to a Petri dish with freshly prepared modified Gamborg’s B5 agar. The continuously kept cell callus serves as a backup for future cell culture restarts.

      2. Incubate the 250-ml flask with cell culture on a shaker incubator (25°C, 90 rpm) in the darkroom for 10 days.

      3. Work aseptically. At the clean bench, add 25 ml of modified Gamborg’s B5 medium to the culture, and repeat this step after 7 and 14 days.

      4. After another 7 days, use a 50-ml plastic tube to transfer 35 ml of cell culture to 120 ml sterile (autoclaved) modified Gamborg’s B5 medium in a 500-ml flask.

      5. Incubate the cell culture on a shaker incubator (25°C, 90 rpm) in the darkroom for 7 days.

      6. Determine the cell fresh weight by filtrating 10 ml of cell culture as follows:

        1. Weigh an empty Petri dish containing a circular filter paper.

        2. Assemble a suction bottle, sealing gasket, and funnel and connect to a vacuum pump.

        3. Slightly hand-shake the flask containing the cell culture to achieve an as even as possible dispersal of the cells in their culture medium.

        4. Use the pipette controller and a 10-ml serological pipette without tip to slowly transfer 10 ml of cell culture onto the circular filter paper in the funnel. Apply vacuum (150-200 pascal) and filtrate for 1 min.

        5. Place the filter paper with the cells onto a Petri dish, weigh, and determine the cell fresh weight by subtracting the tare weight (empty Petri dish with a circular filter paper).

          If the cells multiplied to 0.2 g fresh weight/ml within a week, initiate weekly propagation of the cell culture (Step A2).

          Note: If weight differs much, go back to Step A1d and transfer more (if cell fresh weight is too low) or less (if cell fresh weight is too high) than 35 ml of cell culture.

    2. Weekly propagation of the cell culture

      1. Work aseptically. Using a 50-ml reaction tube, transfer 35 ml of a 7-day-grown parsley cell culture to a 500-ml flask equipped with baffle and filled with 120 ml sterile modified Gamborg’s B5 medium prewarmed to 25°C.

        Note: Do not leave the cell culture unshaken for more than 5 min.

      2. Optional: Repeat Step A2a with more flasks.

        Note: In our lab, we use 4 flasks a week: one for propagation and three for performing the high-throughput screening.

      3. Incubate the cell culture on the large shaker incubator (25°C, 90 rpm) in the darkroom. Use 3-day-grown cultures for the screening experiments and 7-day-old cultures for weekly propagation.

    3. Propagation of callus for cell culture restarts

      Work aseptically. Every four weeks, transfer a 1 × 1-cm piece of grown callus to the center of a fresh Petri dish containing Gamborg’s B5 agar using a scalpel. Seal the Petri dish with sealing film and incubate at 25°C in a darkroom.


  2. High-throughput screening for defense priming-inducing compounds using parsley cell cultures

    1. Compound treatment of cells

      1. Powders or crystals of chemical compounds are to be dissolved in DMSO or sterile double-distilled water (depending on the compound‘s solubility in these solvents). If the substance is already in solution, continue with the next step.

      2. Work aseptically. Prepare a dilution series with the desired stock concentrations.

        Note: For substances dissolved in DMSO, a maximum of 2.5 µl should eventually be added per ml of cell culture. For substances dissolved in water, the volume to be added to 1 ml of cell culture should not exceed 20 µl.

      3. Work aseptically. Using a 1,000 µl micropipette and a truncated pipette tip transfer 1-ml aliquots of a 3-day-old parsley cell culture from Step A2c (cell fresh weight ~0.1 g/ml) to a 24-multiwell plate (Figure 1). Seal the plate with foil and put it into the small shaker incubator (25°C, 130 rpm) in the darkroom until further treatment.

        Note: Cut off ~1.3 cm of the tip with scissors to widen the opening and autoclave them. This ensures the easy and non-invasive transfer of cells.

      4. Work aseptically. Supply the cell culture with either DMSO or water as an appropriate negative control. The maximal volume of the adequate solvents should be the same as for the compound treatment samples. Use either 20 µl of a 10 mM solution of salicylic acid (SA) in water (see Recipes) or 2.5 µl of 80 mM SA in DMSO (see Recipes, final concentration: 200 µM) as a positive control for defense priming. For the candidate compounds, treat aliquots of cell culture with the desired concentration of compound (Figure 2). Seal the multiwell plate and place it back in the shaker incubator.



      Figure 1. Aliquots of parsley cell culture in a 24-multiwell plate. Three-day-old parsley cell culture 1-ml aliquots were transferred to a 24-multiwell plate using a truncated pipette tip.


    2. Treatment with Pep13

      1. Work aseptically. After 24 h of incubation in the presence of test compound or solvent, add 10 µl of 0.005 µM Pep13 (final concentration: 50 pM) or water (as a control for the Pep13 treatment) to the adequate cell culture (Figure 2).

        Optional: To further improve the identification of priming-specific chemicals, include a sample in which test compound and solvent are added together after the 24-h preincubation period. This may enable discrimination of priming responses from synergistic effects.

      2. Seal the plate and put it back in the shaker incubator.

    3. Furanocoumarin fluorescence measurement

      1. From this point on, working in the darkroom is no longer required. After another 24 h of cultivation, place the plate on a work bench and let cells settle down (approx. 30 s). Transfer 100 µl cell-free supernatant of each sample to a 96-well microtiter plate and determine the relative fluorescence of secreted furanocoumarins in the Microplate Reader at 335 nm excitation and 398 nm emission (Figure 2).

        Optional: For visualization of the fluorescence, take a picture of the 24-multiwell plate in UV light [e.g., when placed under a UV lamp (Figure 3)].



    Figure 2. Workflow of high-throughput screening for defense priming compounds using parsley cell culture. Transfer 1-ml aliquots of a 3-day-old parsley cell culture to 24-multiwell plates. Treat the cell cultures with the test compounds dissolved or diluted in DMSO or sterile double-distilled water at different concentrations (A, B, C). Use DMSO or sterile double-distilled water as a negative control (NC) and SA in DMSO or SA in sterile double-distilled water as positive (priming) control (PC). After 24 h of incubation in a shaker incubator (25°C, 130 rpm) in a darkroom, add 10 µl of 0.005 µM Pep13 to the respective aliquots of cell culture. After shaking for another 24 h, transfer the supernatants to a 96-well microtiter plate and determine the relative furanocoumarin fluorescence in the Microplate Reader. SA, salicylic acid. Figure modified from Schillheim et al. (2018) .



    Figure 3. Fluorescence of secreted furanocoumarins in aliquots of parsley cell culture in a 24-multiwell plate and in UV light. Parsley cell culture 1-ml aliquots in a 24-multiwell plate were left untreated (-) or were treated with SA (+; final concentration: 200 µM). After 24 h, cell culture aliquots in the absence or presence of SA were left untreated (-) or supplemented with Pep13 (+; final concentration: 50 pM) to activate furanocoumarin synthesis and secretion. After another 24 h, the multiwell plate was exposed to UV light and a photo taken. SA, salicylic acid.

Data analysis

For each experiment, determine the relative furanocoumarin fluorescence of four technical replicates for each treatment and repeat the experiment three times. For each treatment, calculate the mean values and standard deviation for all the experiments done. Statistically analyze the data by performing one-way analysis of variance (one-way ANOVA) followed by posthoc Student’s t-test using appropriate statistical software (e.g., GraphPad Prism).

Notes

It is very important to work aseptically wherever indicated. Before each step, disinfect your hands and clean and sterilize all materials with 70% ethanol before you work at the clean bench. For all cultivation and propagation steps, sterilize flasks, cellulose plugs, and scalpels using the flame of the gas burner. Avoid physical contact of reaction tubes and flasks while transferring the cell culture.

Recipes

  1. 70% ethanol

    Mix 350 ml ethanol (≥99.5 %, extra pure) and double distilled water up to 500 ml.

  2. 2,4-Dichlorophenoxyacetic acid (0.2 mg/ml stock solution)

    Dissolve 40 mg 2,4-dichlorophenoxyacetic acid in 1 ml ethanol (≥99.5 %, extra pure).

    Add double distilled water up to 200 ml.

  3. Magnesium sulfate (0.05 mg/ml stock solution)

    Dissolve 10 g magnesium sulfate heptahydrate in 200 ml double distilled water.

  4. Modified Gamborg’s B5 medium

    3.16 g Gamborg’s B5 medium including vitamins

    20 g D-sucrose

    10 ml of 0.2 mg/ml 2,4-dichlorophenoxyacetic acid stock solution

    5 ml of 0.05 mg/ml magnesium sulfate stock solution

    Add double distilled water up to 1 L.

    Adjust pH to 5.5 with 1 M KOH (approx. 3 droplets).

    Autoclave at 121°C for 15 min (Note: End temperature set to 80°C).

    For solid medium, add 10 g plant agar before autoclaving.

  5. SA in water (10 mM stock solution)

    345.25 mg SA

    Add double distilled water up to 200 ml.

    Adjust pH to 5.5 with 1 M KOH.

    Fill up to 250 ml with double distilled water.

    Sterile-filter and aliquot in 1.5 ml reaction tubes.

    Store at -20°C until use.

  6. SA in DMSO (80 mM stock solution)

    276.2 mg SA

    Add DMSO up to 25 ml.

    Aliquot in 1.5-ml reaction tubes.

    Store at -20°C until use.

  7. Pep13 (0.005 µM stock solution)

    4.055 mg Pep13

    Add double distilled water up to 50 ml.

    Dilute 1:1,000 with double distilled water.

    Filter sterilize and distribute to 1.5-ml reaction tubes.

    Store at -20°C until use.

Acknowledgments

We would like to thank our collaborators, whether public or private, with whom we used the parsley cell culture to spot priming-inducing chemistry over the past 25 years and who helped us optimize the screen continuously. This protocol is based on our previous publication (Schillheim et al., 2018; doi: 10.1104/pp.17.00124).

Competing interests

We declare we have no financial or non-financial competing interests.

References

  1. Beckers, G. J. and Conrath, U. (2007). Priming for stress resistance: from the lab to the field. Curr Opin Plant Biol 10(4): 425-431.
  2. Brunner, F., Rosahl, S., Lee, J., Rudd, J. J., Geiler, C., Kauppinen, S., Rasmussen, G., Scheel, D. and Nurnberger, T. (2002). Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases. EMBO J 21(24): 6681-6688.
  3. Conrath, U., Pieterse, C. M. and Mauch-Mani, B. (2002). Priming in plant-pathogen interactions. Trends Plant Sci 7(5): 210-216.
  4. Conrath, U., Beckers, G. J., Flors, V., García-Agustín, P., Jakab, G., Mauch, F., Newman, M. A., Pieterse, C. M., Poinssot, B., Pozo, M. J., et al. (2006). Priming: getting ready for battle. Mol Plant Microbe Interact 19(10): 1062-1071.
  5. Conrath, U., Beckers, G. J., Langenbach, C. J. and Jaskiewicz, M. R. (2015). Priming for enhanced defense. Annu Rev Phytopathol 53: 97-119.
  6. Frost, C. J., Mescher, M. C., Carlson, J. E. and De Moraes, C. M. (2008). Plant defense priming against herbivores: getting ready for a different battle. Plant Physiol 146(3): 818-824.
  7. Katz, V. A., Thulke, O. U. and Conrath, U. (1998). A benzothiadiazole primes parsley cells for augmented elicitation of defense responses. Plant Physiol 117(4): 1333-1339.
  8. Kauss, H., Theisinger-Hinkel, E., Mindermann, R., Conrath, U. (1992). Dichloroisonicotinic and salicylic acid, inducers of systemic acquired resistance, enhance fungal elicitor responses in parsley cells. Plant J 2: 655-660.
  9. Kauss, H., Jeblick, W., Ziegler, J. and Grabler, W. (1994). Pretreatment of parsley (Petroselinum crispum L.) suspension cultures with methyl jasmonates enhances elicitation of activated oxygen species. Plant Physiol 105(1): 89-94.
  10. Lamberth, C., Jeanmart, S., Luksch, T. and Plant, A. (2013). Current challenges and trends in the discovery of agrochemicals. Science 341(6147): 742-746.
  11. Mascarelli, A. (2013). Growing up with pesticides. Science 341(6147): 740-741.
  12. Noutoshi, Y., Okazaki, M., Kida, T., Nishina, Y., Morishita, Y., Ogawa, T., Suzuki, H., Shibata, D., Jikumaru, Y., Hanada, A., et al. (2012). Novel plant immune-priming compounds identified via high-throughput chemical screening target salicylic acid glucosyltransferases in Arabidopsis. Plant Cell 24(9): 3795-3804.
  13. Schillheim, B., Jansen, I., Baum, S., Beesley, A., Bolm, C. and Conrath, U. (2018). Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense. Plant Physiol 176(3): 2395-2405.
  14. Schilling, J. V., Schillheim, B., Mahr, S., Reufer, Y., Sanjoyo, S., Conrath, U. and Büchs, J. (2015). Oxygen transfer rate identifies priming compounds in parsley cells. BMC Plant Biol 15: 282.
  15. Siegrist, J., Mühlenbeck, S., Buchenauer, H. (1998). Cultured parsley cells, a model system for the rapid testing of abiotic and natural substances as inducers of systemic acquired resistance. Physiol Mol Plant Pathol 53:223-238.
  16. UN Food and Agriculture Organization (2009). Global agriculture towards 2050. Proceedings of the High-Level Expert Forum, Rome, October 12-13, 2009.

简介

[抽象的] 防御启动描述了细胞激活防御反应的增强效力。引发伴随局部和全身免疫反应,可由微生物感染或在用某些化学物质处理时触发。因此,化学激活防御启动对于生物医学和农业是有希望的。然而,用于检测引发引发化学物质的测试系统很少见。在这里,我们描述了一种化合物的高通量筛选,这些化合物在欧芹培养细胞中引发微生物模式刺激抗菌呋喃香豆素的分泌。为获得最佳通量,我们在 24 孔微量滴定板中使用 1 毫升细胞培养物进行检测。与竞争性检测相比,非侵入性检测的优势在于其简单性、卓越的可靠性和高灵敏度,这是基于紫外光下的呋喃香豆素荧光。


[背景] 由于人口增长、耕地减少和气候变化导致的紧迫的全球粮食不安全问题只能通过有效的作物保护在农业中得到解决(联合国粮食及农业组织,2009 年)。合成农用化学品可以有效保护作物免受病害,但也会引起生态和健康问题(Lamberth等,2013;Mascarelli,2013)。因此,需要安全环保的疾病控制产品(Lamberth等,2013)。
启动植物免疫系统以增强防御能力的化学品有望实现可持续的作物保护(Beckers 和 Conrath,2007 年;Conrath等人,2015 年)。当被激发时,植物对非常低的病原体刺激做出反应,比未激发的植物更强大的防御激活。这通常会降低已引发植物的疾病易感性并降低病原体适应的风险(Conrath等,2002、2006 和 2015;Beckers 和 Conrath,2007;Frost等,2008)。因此,通过无害的化学品诱导引发是可持续作物病害控制的一种有前途的手段。之前对几种化合物的研究证明了这一概念(Conrath等人,2015 年总结)。然而,这些化合物的经济成功是有限的,主要是因为农民更喜欢标准杀虫剂的强大疗效。
如今,作物保护剂的商业成功通常依赖于它们将抗菌活性与目标作物的防御启动相结合的能力(Beckers 和 Conrath,2007 年;Conrath等人,2015 年)。然而,用于识别引发诱导化学的直接测试系统很少见。在这里,我们描述了一种高通量筛选,用于识别在悬浮培养的欧芹 ( Petroselinumcrispum ) 细胞中引发微生物模式诱导的呋喃香豆素 (植物抗毒素) 分泌的化合物。该测试测量了中等浓度的 Pep13(Kauss等人,1992 年),一种植物病原体大豆疫霉中的分子模式肽(Brunner等人,2002 )。在过去的 25 年中,该测试有助于识别新的引发诱导化学(Katz等人,1998 年;Siegrist等人,1998 年;Schillheim等人,2018 年)。为了获得更高的通量,我们现在在 24 孔微量滴定板中使用 1 mL 细胞培养物进行测试(Schillheim等人,2018 年)。与竞争性测定相比,该测试的其他优势是荧光检测的非凡可靠性、简单性和高灵敏度,并且在最终分析之前只需要两个简单的处理(启动和 Pep13 挑战)。原则上,该测定也应该适用于合成呋喃香豆素的其他植物物种(例如,巨型猪草、普通猪草和花园当归)的细胞培养物,前提是它们对用特定分子模式的处理产生反应,并分泌到培养物中的呋喃香豆素中等的。如果不是这种情况,则需要选择其他测试读数 [例如,活性氧的产生,如 Kauss等人所述。(1994)]。最近引入的对此处介绍的屏幕的竞争性测定评估了丁香假单胞菌pv的增强。番茄avrRpm1通过引发剂诱导拟南芥细胞培养物中的细胞死亡(Noutoshi等,2012)。几种免疫引发化合物的鉴定证实了该测定法在发现引发诱导化学方面的能力。然而,由于细菌挑战、细胞化学染色、洗涤、染料提取和吸光度测量的要求,筛选非常复杂。对于最近用于发现免疫启动化学的呼吸活动监测系统也是如此(Schilling等,2015),该系统具有高度创新性,但吞吐量低。

关键字:农药, 细胞培养, 香豆素, 作物保护, 防御启动, 高通量筛选, 感生阻力, 欧芹, 植物免疫, 启动效应



材料和试剂
 
密封膜(Roth,目录号:H666.1)
用于细胞培养的密封箔(Roth,目录号:EN85.1)
血清移液管 10 ml(Sarstedt,目录号:86.1254.025)
血清移液管 10 ml 无尖端(Sarstedt,目录号:86.1688.010)
吸瓶1,000毫升(Roth,目录号:NY68.1)
注射器过滤器,0.22 µm(Roth,目录号:KH54.1)
铝箔(Roth,目录号:2596.1)
Büchner漏斗,70 ml(Roth,目录号:XX45.1)
细胞培养 24 孔板(Greiner,目录号:662160)
纤维素塞“Zellstoppers”(对角线,目录号:ZE153)
圆形滤纸,113A 型,55 毫米(Roth,目录号:AP74.1)
带 Luer-Lock 接头的一次性注射器 20 ml(Roth,目录号:T550.1)
烧瓶,250 毫升,带挡板(对角线,目录号:2121636S1)
带挡板的 500 毫升烧瓶(对角线,目录号:2121644S1)
密封垫圈(Roth,目录号:9757.1 [33 mm];9758.1 [41 mm])
Nunc TM MicroWell TM 96 孔,Nunclon Delta 处理,平底微孔板(ThermoFisher Scientific,目录号:137101)
巴斯德移液管,2 ml(Roth,目录号:4522.1)
Pep13 肽(Thermo Fisher Scientific,氨基酸序列:VWNQPVRGFKVYE)
培养皿,94/16 mm(Greiner,目录号:633180)
移液器吸头 Gilson(Greiner,目录号:740290 [1,000 µl];739290 [200 µl])
移液器吸头 Starlab(TipOne ® ,目录号:1110-3000 [10/20 µl])
反应管 1.5 ml(Sarstedt,目录号:72706)
反应管 50 ml(Greiner,目录号:227261)
Petroselinum creamum PC794愈伤组织培养物(DSMZ - Leibniz Institute,目录号:PC794)
植物琼脂(Duchefa,目录号:P1001.1000)
氢氧化钾,1 M KOH(Roth,目录号:9522.1)
水杨酸,C 7 H 6 O 3 (TCI,目录号:H1342)
测试化合物
包含维生素的 Gamborg B5 培养基(Duchefa,目录号:G0210.0050)
D-蔗糖,C 12 H 22 O 11 (Roth,目录号:4621.2)
洗手液(Roth,目录号:EH72.2)
七水硫酸镁,MgSO 4 ·7H 2 O(Roth,目录号:P027.1)
2,4-二氯苯氧基乙酸,C 8 H 6 Cl 2 O 3 (默克,目录号:8204510005)
二甲基亚砜,C 2 H 6 OS(Roth,目录号:A994.1)
乙醇,C 2 H 5 OH,≥99.5%,超纯(Roth,目录号:5054.4)
70% 乙醇(见配方)
2,4-二氯苯氧乙酸,0.2 mg/ml 原液(见配方)
硫酸镁,0.05 mg/ml 原液(见配方)
改良 Gamborg 的 B5 培养基(见食谱)
水中的水杨酸(水中的 SA),10 mM 储备溶液(参见配方,将等分试样储存在 -20°C 下)
DMSO 中的水杨酸(DMSO 中的 SA),80 mM 储备液(参见食谱,将等分试样储存在 -20°C 下)
Pep13,0.005 µM 储备溶液(参见配方,将等分试样储存在 -20°C)
愈伤组织培养基(见食谱)
 
设备
 
10-µl、100-µl 和 1,000-µl 微量移液器(ErgoOne、Starlab)
磁棒(Roth,目录号:2153.2)
手术刀(Roth,目录号:X003.1)
剪刀(Roth,目录号:5489.1)
量筒,1,000 ml(Roth,目录号:P153.1)
量筒,100 ml(Roth,目录号:P150.1)
分析天平(梅特勒托利多,型号:MS204)
高压釜(Systec,型号:DX65)
暗室,温度控制 (25°C),带有两个摇床培养箱和带燃气燃烧器的洁净工作台(见下文)
超净工作台(IBS Tecomara,型号:NU-543-4000EC)
大型摇床培养箱,90 rpm,25°C(Infors HT,型号:Multitron Standard)
带丙烷气瓶(Westphalengas;11 kg)的安全实验室燃气燃烧器(Roth,目录号:AN82.1)
小型摇床培养箱,130 rpm,25°C(Infors HT,型号:Ecotron)
实验室真空系统(Fisher Scientific,型号:Welch Ilmvac TM LVS 210T,目录号:11882253)
磁力搅拌器(Roth,目录号:AAN2.1)
酶标仪(BMG Labtech,型号:CLARIOstar Plus)
pH计(梅特勒托利多,型号:SevenMulti TM )
移液管灯泡(Roth,目录号:YX53.1)
移液器控制器(品牌,型号:accu-jet ® pro)
超纯水系统(Satorius,型号:arium ® pro VF)
紫外灯(Thermo Fisher Scientific,目录号:12813029)
 
软件
 
CLARIOstar Reader Control Software(通过酶标仪获得)
板:NUNC96
激发:335 nm(带宽:20)
二向色滤光片:365.2
发射:398 nm(带宽:20)
MARS 数据分析软件(通过酶标仪获得)
统计软件(例如,GraphPad Prism)
 
程序
 
欧芹细胞培养的生长和维持
设置欧芹细胞培养
无菌工作。在洁净工作台和暗室中,使用手术刀将 2-3 厘米的愈伤组织培养物(从 DSMZ 获得)转移到 250 毫升烧瓶中的 50 毫升新鲜制备的改良 Gamborg B5 培养基中。此外,将一块 1 × 1 厘米的愈伤组织转移到带有新鲜制备的改良甘博格 B5 琼脂的培养皿中。持续保存的细胞愈伤组织作为未来细胞培养重启的备份。
在暗室中的摇床培养箱(25°C,90 rpm)上孵育 250 毫升烧瓶和细胞培养物 10 天。
无菌工作。在超净工作台上,向培养物中加入 25 ml 改良 Gamborg's B5 培养基,并在 7 天和 14 天后重复此步骤。
再过 7 天后,使用 50 毫升塑料管将 35 毫升细胞培养物转移到 500 毫升烧瓶中的 120 毫升无菌(高压灭菌)改良 Gamborg B5 培养基中。
在暗室中的摇床培养箱(25°C,90 rpm)上孵育细胞培养物 7 天。
通过过滤 10 ml 细胞培养物来确定细胞鲜重,如下所示:
称量一个含有圆形滤纸的空培养皿。
组装吸瓶、密封垫圈和漏斗并连接到真空泵。
轻轻摇晃装有细胞培养物的烧瓶,使细胞尽可能均匀地分散在培养基中。
使用移液器控制器和 10 毫升无吸头血清移液器将 10 毫升细胞培养物缓慢转移到漏斗中的圆形滤纸上。施加真空 (150-200 帕斯卡) 并过滤 1 分钟。
将带有细胞的滤纸放在培养皿上,称重,并通过减去皮重(带有圆形滤纸的空培养皿)来确定细胞的鲜重。
如果细胞在一周内繁殖到 0.2 g 鲜重/ml,则开始每周培养细胞培养(步骤 A2)。
注意:如果重量差异很大,返回步骤 A1d 并转移更多(如果细胞鲜重太低)或少于(如果细胞鲜重太高)35 ml 细胞培养物。
细胞培养的每周繁殖
无菌工作。使用 50 毫升反应管,将 35 毫升培养 7 天的欧芹细胞培养物转移到装有挡板的 500 毫升烧瓶中,并装有 120 毫升已预热至 25°C 的无菌改良 Gamborg's B5 培养基。
注意:不要让细胞培养物保持不动超过 5 分钟。
可选:使用更多烧瓶重复步骤 A2a。
注意:在我们的实验室中,我们每周使用 4 个烧瓶:一个用于繁殖,三个用于执行高通量筛选。
在暗室的大型摇床培养箱(25°C,90 rpm)上培养细胞培养物。使用 3 天培养的培养物进行筛选实验,使用 7 天培养的培养物进行每周繁殖。
用于细胞培养的愈伤组织繁殖重新开始
无菌工作。每四个星期,使用手术刀将一块 1 × 1 厘米长的愈伤组织转移到含有甘博格 B5 琼脂的新鲜培养皿的中心。用密封膜密封培养皿,并在 25°C 的暗室中孵育。
 
使用欧芹细胞培养物高通量筛选防御引发诱导化合物
细胞复合处理
将化合物的粉末或晶体溶解在 DMSO 或无菌双蒸水中(取决于化合物在这些溶剂中的溶解度)。如果该物质已在溶液中,则继续下一步。
无菌工作。准备具有所需库存浓度的稀释系列。
注意:对于溶解在 DMSO 中的物质,每毫升细胞培养物最终应最多添加 2.5 µl。对于溶于水的物质,加入 1 ml 细胞培养物的体积不应超过 20 µl。
无菌工作。使用 1,000 µl 微量移液器和截短的移液器吸头将 3 天大的欧芹细胞培养物的 1 毫升等分试样从步骤 A2c(细胞鲜重约 0.1 克/毫升)转移到 24 孔板(图 1)。用箔纸密封板,然后将其放入暗室中的小型摇床培养箱(25°C,130 rpm)中,直至进一步处理。
注意:用剪刀剪掉约 1.3 厘米的尖端以扩大开口并对其进行高压灭菌。这确保了细胞的轻松和非侵入性转移。
无菌工作。用 DMSO 或水为细胞培养物提供适当的阴性对照。适当溶剂的最大体积应与化合物处理样品的最大体积相同。使用 20 µl 10 mM 水杨酸 (SA) 水溶液(参见配方)或 2.5 µl 80 mM SA DMSO(参见配方,最终浓度:200 µM)作为防御启动的阳性对照。对于候选化合物,用所需浓度的化合物处理细胞培养物的等分试样(图 2)。密封多孔板并将其放回摇床培养箱中。
 
 
图 1. 24 孔板中欧芹细胞培养物的等分试样。使用截短的移液管吸头将 1 毫升的 1 毫升欧芹细胞培养物转移到 24 孔板中。
 
用 Pep13 治疗
无菌工作。在测试化合物或溶剂存在下孵育 24 小时后,将 10 µl 0.005 µM Pep13(最终浓度:50 pM)或水(作为 Pep13 处理的对照)添加到足够的细胞培养物中(图 2)。
可选:为了进一步提高对启动特定化学品的识别,包括一个样品,其中测试化合物和溶剂在 24 小时预孵育期后一起添加。这可以从协同效应中区分引发反应。
密封板并将其放回摇床培养箱中。
呋喃香豆素荧光测定
从此,不再需要在暗房工作。再培养 24 小时后,将板放在工作台上,让细胞安定下来(约 30 秒)。将每个样品的 100 µl 无细胞上清液转移到 96 孔微量滴定板,并在酶标仪中测定分泌的呋喃香豆素在 335 nm 激发和 398 nm 发射下的相对荧光(图 2)。
可选:对于荧光的可视化,在紫外线下拍摄 24 孔板的照片 [例如,当放置在紫外线灯下时(图 3)]。
 
 
图 2. 使用欧芹细胞培养物对防御引发化合物进行高通量筛选的工作流程。将 3 天大的欧芹细胞培养物的 1 毫升等分试样转移到 24 孔板中。用溶解或稀释在 DMSO 或无菌双蒸水中的不同浓度(A、B、C)的测试化合物处理细胞培养物。使用 DMSO 或无菌双蒸水作为阴性对照 (NC),使用 DMSO 中的 SA 或无菌双蒸水中的 SA 作为阳性(启动)控制(PC)。在暗室中的摇床培养箱(25°C,130 rpm)中孵育 24 小时后,将 10 µl 0.005 µM Pep13 添加到相应的细胞培养物等分试样中。再摇晃 24 小时后,将上清液转移到 96 孔微量滴定板,并在微孔板读数器中测定相对呋喃香豆素荧光。SA,水杨酸。图修改自 Schillheim等人。(2018)。
 
 
图 3. 在 24 孔板和紫外光下,欧芹细胞培养物等分试样中分泌的呋喃香豆素的荧光。24 孔板中的欧芹细胞培养物 1 毫升等分试样未经处理 (-) 或用 SA 处理 (+;最终浓度:200 µM)。24 小时后,不存在或存在 SA 的细胞培养等分试样未经处理 (-) 或补充 Pep13 (+;最终浓度:50 pM) 以激活呋喃香豆素合成和分泌。再过 24 小时后,将多孔板暴露在紫外线下并拍照。SA,水杨酸。
 
数据分析
 
对于每个实验,确定每个处理四个技术重复的相对呋喃香豆素荧光,并重复实验 3 次。对于每个处理,计算所有实验的平均值和标准偏差。通过执行单向方差分析(单向方差分析),然后使用适当的统计软件(例如,GraphPad Prism)进行事后学生t检验,对数据进行统计分析。
 
笔记
 
在任何指示的地方进行无菌操作是非常重要的。在每一步之前,在超净工作台上工作之前,先对双手进行消毒,并用 70% 乙醇对所有材料进行清洁和消毒。对于所有培养和繁殖步骤,使用燃气燃烧器的火焰对烧瓶、纤维素塞和手术刀进行消毒。转移细胞培养物时避免反应管和烧瓶的物理接触。
 
食谱
 
70%乙醇
将 350 ml 乙醇(≥99.5%,超纯)和双蒸水混合至 500 ml。
2,4-二氯苯氧乙酸(0.2 毫克/毫升储备溶液)
将 40 mg 2,4-二氯苯氧乙酸溶解在 1 ml 乙醇中(≥99.5%,超纯)。
添加双蒸水至 200 毫升。
硫酸镁(0.05 mg/ml 原液)
将 10 克七水硫酸镁溶解在 200 毫升双蒸水中。
改良 Gamborg 的 B5 培养基
3.16 克 Gamborg's B5 培养基,包括维生素
20 克 D-蔗糖
10 ml 0.2 mg/ml 2,4-二氯苯氧乙酸储备溶液
5 毫升 0.05 毫克/毫升硫酸镁原液
添加双蒸水至 1 L。
使用 1 M KOH(约 3 滴)将 pH 值调节至 5.5。
在 121°C 下高压灭菌 15 分钟(注意:最终温度设置为 80°C )。
对于固体培养基,在高压灭菌前加入 10 g 植物琼脂。
SA 水溶液(10 mM 原液)
345.25 毫克 SA
添加双蒸水至 200 毫升。
用 1 M KOH 将 pH 值调节到 5.5。
用双蒸水填充至 250 毫升。
无菌过滤器并在 1.5 ml 反应管中分装。
储存在 -20°C 直至使用。
DMSO 中的 SA(80 mM 储备溶液)
276.2 毫克 SA
添加 DMSO 至 25 毫升。
分装在 1.5 毫升反应管中。
储存在 -20°C 直至使用。
Pep13(0.005 µM 原液)
4.055 毫克 Pep13
添加双蒸水至 50 毫升。
用双蒸水按 1:1,000 稀释。
过滤灭菌并分配到 1.5 毫升反应管中。
储存在 -20°C 直至使用。
 
致谢
 
我们要感谢我们的合作者,无论是公共的还是私人的,在过去的 25 年里,我们与他们一起使用欧芹细胞培养物发现了引发引发的化学反应,并帮助我们不断优化屏幕。该协议基于我们之前的出版物(Schillheim等人,2018 年;doi:10.1104/pp.17.00124)。
 
 
利益争夺
 
我们声明我们没有财务或非财务竞争利益。
 
参考
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schmitz, K., Werner, L. and Conrath, U. (2021). High-throughput Screening for Defense Priming-inducing Compounds in Parsley Cell Cultures. Bio-protocol 11(20): e4200. DOI: 10.21769/BioProtoc.4200.
  2. Schillheim, B., Jansen, I., Baum, S., Beesley, A., Bolm, C. and Conrath, U. (2018). Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense. Plant Physiol 176(3): 2395-2405.
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