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Sep 2018

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MAMP-triggered Medium Alkalinization of Plant Cell Cultures
MAMP诱导植物细胞培养基质碱化   

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Abstract

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

Keywords: Plant immunity (植物免疫), Host-microbe interactions (宿主微生物相互作用), Elicitor (诱导子), Effector Biology (效应生物学), Phytopathology (植物病理学)

Background

Throughout evolution, plants developed the ability to detect microbe-derived molecules and mount immune responses that seize detrimental interactions (Boutrot and Zipfel, 2017). The microbial-associated molecular patterns (MAMPs) that induce such immune responses are often broadly conserved structural components of microbes, such as chitin from fungi and flagellin from bacteria (Cook et al., 2015). Recognition of MAMPs by extracellular plant receptors leads to MAMP-triggered immunity (MTI), which contributes to halt microbial invaders and maintain plant health (Böhm et al., 2014).

At the molecular level, MAMP perception is followed by an orchestrated set of biochemical events. Firstly, within seconds to few minutes, influx of Ca2+ and H+ ions occurs, leading to membrane depolarization and extracellular alkalinization (Ranf et al., 2011). Subsequently, production of reactive oxygen species (ROS) is observed, which may locally block pathogen growth and mediate downstream signaling (Bigeard et al., 2015). Phosphorylation of MAP kinases occurs within minutes after MAMP perception and promotes transcriptional activation of defense-related genes, many of which encode antimicrobial proteins or enzymes involved in the synthesis of hormones and secondary metabolites (Bigeard et al., 2015). Lastly, accumulation of antimicrobial compounds and callose deposition are observed, further hampering microbial growth and spread.

Mechanistic understanding of the plant immune system and its activity in plant-microbe interactions has advanced through methods that monitor many of the biochemical events underlying MTI. The most widely used experimental approaches are based on detection of ROS burst, MAP kinase activation, induction of defense-related marker genes and medium alkalinization of cell suspensions (Flury et al., 2013; Bisceglia et al., 2015; Liu et al., 2018). ROS measurement is typically performed by means of luminol-based chemiluminesce assays. MAP kinase activation is detected by western blots using specific antibodies. Transcriptional reprogramming is usually accessed by monitoring the expression levels of one or a few marker genes using real-time PCR. Medium alkalinization assays are based on recording the pH changes in MAMP-treated plant cell suspensions over time using a pH sensor for small volumes. Compared to the other methods, medium alkalinization assays are less laborious and require low cost laboratory equipment. Both model plant species and crops have been studied using medium alkalinization assays (Nühse et al., 2000; Moroz et al., 2017).

Materials and Reagents

  1. 12-well cell culture plate (Greiner Bio-One, CELLSTAR®, catalog number: 665180 )
  2. Serological pipette without tip 10 ml (SARSTEDT, catalog number: 86.1688.010 )
  3. P200 pipette tips
  4. 20 ml syringe (VWR, catalog number: 613-2046 )
  5. 0.22 µm syringe filter (GE Healthcare, WhatmanTM Uniflo, catalog number: 9915-2502 )
  6. Hexa-N-acetylchitohexaose (two manufacturers are recommended: Santa Cruz Biotechnology, catalog number: sc-222018 ; Isosep, catalog number: 56/11-0010 ). Store at -20 °C
    Note: Chitoheptaose (NAG7) and chitooctaose (NAG8) or even chitin (Sigma, catalog number: C7170) may be used instead of chitohexaose.
  7. Flagellin 22 peptide (flg22: QRLSTGSRINSAKDDAAGLQIA) (Genscript, catalog number: RP19986 ). Store at -20 °C
  8. Gamborg’s vitamin Solution 1,000x (sterile) (Sigma-Aldrich, catalog number: G1019-50ML ). Store at 4 °C
    Note: Before using, inspect for the presence of precipitates. If present, warm the solution up to 42 °C and vortex until all precipitates dissolve.
  9. Murashige and Skoog Basal salt mixture (PhytoTechnology Laboratories, catalog number: M524 ). Store at 4 °C
  10. Sucrose (Sigma-Aldrich, catalog number: S5391 )
  11. Myo-inositol (Sigma-Aldrich, catalog number: I7508 )
  12. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 ). Store at 4 °C
  13. KH2PO4 (Sigma-Aldrich, catalog number: P5655 )
  14. Tryptone (Oxoid, catalog number: LP0042 )
  15. 2,4-Dichlorophenoxyacetic acid (Sigma-Aldrich, catalog number: D7299 )
  16. 2-(N-Morpholino)ethanosulfonic acid (MES) hydrate (Sigma-Aldrich, catalog number: M2933
  17. Kinetin (Sigma-Aldrich, catalog number: K0753 )
  18. Chitohexamers (NAG6) solution (see Recipes)
  19. Flg22 solution (see Recipes)
  20. Kinetin stock solution (1,000x) (see Recipes)
  21. 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution (1,000x) (see Recipes)
  22. KH2PO4 stock solution (see Recipes)
  23. Thiamine/myo-inositol stock solution (see Recipes)
  24. Tryptone stock solution (10%) (see Recipes)
  25. MES/Phosphate-buffered culture medium (see Recipes)
  26. Subculturing medium (see Recipes)

Equipment

  1. 125 ml Erlenmeyer flasks (PyrexTM, catalog number: 15685767 )
  2. 250 ml Erlenmeyer flasks (PyrexTM, catalog number: 11902619 )
  3. P200 adjustable pipette
  4. Flow hood (Thermo Scientific, model: 1300 Series Class II , catalog number: 1323TS )
  5. Autoclave (VWR, catalog number: 481-0666 )
  6. pH meter (Mettler Toledo, Seven Compact S220 Basic, catalog number: 30019028 ). Alternative models may be used as long as the pH resolution is at least 0.001 
  7. pH sensor InLab Micro for small volumes (Mettler Toledo, catalog number: 51343160 )
  8. Orbital shaker (LaboTech, model: RS150 )

Software

  1. Microsoft Excel
  2. EasyDirectTM pH Software (Mettler Toledo, 30323214)

Procedure

  1. Cell suspension preparation and maintenance
    The protocol described here uses Nicotiana tabacum cultivar Bright Yellow 2 (BY-2) cell suspensions, which are established from in vitro-induced calli (Figure 1A). The BY-2 cell line was produced from tobacco seedlings by Kato et al. in 1972 (Kato et al.,1972) and has since been propagated and shared among plant scientists (Nagata et al., 1992). BY-2 cells are currently used as models in a number of experimental systems and are available from various research groups.
    1. To generate a suspension of plant cells, transfer approximately 1 ml of calli to a sterile 125 ml Erlenmeyer flask containing 20 ml of MES/Phosphate-buffered culture medium (Recipe 8) using a sterile spatula or inoculation loop (in the flow hood). Be gentle to avoid unnecessary mechanical stress to the calli. In order to prevent contamination, work carefully and orderly at the flow hood. Incubate at 27 °C in the dark and under gentle agitation (100-120 rpm) in an orbital shaker. The calli will release small cell aggregates that will eventually result in a cell suspension.
    2. After 10-14 days, transfer 5 ml of the resulting cell suspension using a 10 ml tipless serological pipette to a sterile 250 ml Erlenmeyer flask containing 45 ml of subculturing medium (Recipe 9). Incubate at 27 °C in the dark and under gentle agitation (100-120 rpm) in an orbital shaker.
    3. After 7 days, repeat the subculturing as described in Step A2. At this point, the newly established cell suspension should be similar to the one shown in Figures 1B-1C. Inspect an aliquot of the suspension under the microscope to ensure that it is free from contaminants.
    4. For regular maintenance of BY-2 cell suspensions, subculture the cells weekly as described in Step A2. It is advisable to routinely keep two or three independent flasks of cell suspensions to prevent culture loss due to contamination.


      Figure 1. Nicotiana tabacum cv. Bright Yellow 2 (BY-2) cells growing as calli and cell suspensions. A. Calli growing on solid MES/Phosphate-buffered culture medium. B. Cell suspension just after subculturing. C. A 7-day-old cell suspension. Note the pale-yellow color indicating saturation.

  2. MAMP treatment and measurement of medium alkalinization
    Once established cell suspensions are available (as described in the previous section), MAMP-triggered medium alkalinization can be accessed. Aliquots of cell suspension are individually treated with MAMPs and probed for response (Figure 2).


    Figure 2. Experimental setup for medium alkalinization measurement. A 12-well microplate containing 2.5 ml of cell suspensions in each well is kept under gentle agitation on an orbital shaker. The pH sensor is inserted so that the probe is completely submerged and does not touch the walls.

    1. Carefully transfer a 3- to 5-day-old cell suspension to a 12-well plate (2.5 ml per well) using a 10 ml tipless serological pipette. The cell cultures to be used should be similar to the ones shown in Figure 1C, with a pale-yellow coloration. Dark yellow or brownish colored suspensions are typically stressed and should not be used.
      Note: One key factor that affects the medium alkalinization assay is the age of the cell suspension. In our experiments with BY-2 cells, we used 3- to 5-day-old suspensions. However, the optimum age may vary from lab to lab and therefore needs to be determined experimentally.
    2. Incubate the plate for at least 2 h and up to 4 h at room temperature and under gentle agitation in an orbital shaker (typically 100-200 rpm; the speed should be just enough to keep the cells in suspension). This step allows the cells to recover from stresses caused by the transference to the plate.
    3. Before starting the measurements, adjust the pH meter settings appropriately: measurement resolution of 0.001, endpoint format set as manual and timed interval readings of 3 s. Calibrate the sensor according to the manufacturer’s recommendations using calibration standard solutions.
    4. Open the EasyDirectTM pH Software on a computer connected to the pH meter and set the export option to Excel. An Excel sheet will automatically be opened.
    5. Keeping the plate under agitation on the orbital shaker (100-200 rpm), insert the sensor in one well, carefully adjusting the position to ensure that the sensor is sufficiently inserted (colored tip submerged) and is not touching the walls or the bottom of the well.
    6. Press the Start button on the pH meter. The instrument will start recording the pH values in the Excel sheet.
    7. Once the measurements become stable (same value for at least 12 s), add 25 μl of 1 µM NAG6 (Recipe 1) (final concentration: 10 nM) or 25 μl of 10 µM flg22 (Recipe 2) (final concentration: 100 nM) to the well. To avoid artefactual changes in pH, equilibrate the elicitors to room temperature for 10 min before adding to the cell suspension. Make sure that each plate includes at least one well treated with elicitor solvent (H2O) as non-elicited control.
    8. Record the pH values for 10 min for NAG6 and 60 min for flg22.
    9. After the measurement is complete, remove the sensor from the well, wash thoroughly with ultrapure water and dry using tissue paper.
    10. Repeat Steps B5-B9 until all samples included in the experiment have been tested.

Data analysis

The EasyDirectTM pH Software features an export tool that records pH measurements directly into Excel sheets. Therefore, once all the raw data from an experiment is collected, the variation in pH (ΔpH) over time for each tested condition can be calculated by subtracting the pH value measured at time 0 (i.e., the pH of the medium before MAMP treatment) from the pH at each subsequent time point (i.e., the pH of the medium after MAMP treatment). Line plots depicting the variation in pH over time (Figure 3A) or bar plots showing the maximum ΔpH value (Figure 3B) for each treatment may be plotted to visualize the results. Standard deviation should be presented to indicate variation among replicates. It is desirable to have at least 3 biological replicates for each condition tested.


Figure 3. NAG6-induced medium alkalinization of tobacco BY-2 cell suspensions. The pH of cell suspensions treated with 10 nM NAG6 or ultrapure water (mock) was recorded for 10 min after the treatment. A. Variation in pH (ΔpH) over time (in seconds) shown in a line plot. B. Maximum variation in pH plotted as a bar graph. Shades in the line graph and error bars in the bar plot indicate standard deviation of three biological replicates.

Notes

  1. As an alternative to the tipless serological pipette, it is possible to cut the end of a regular serological pipette or even from a P5000 tip and use it for handling of the cell suspensions.

Recipes

  1. Chitohexamers (NAG6) solution
    Dissolve in sterile ultrapure water to 1 µM (1.24 µg/ml)
    Store at -20 °C
  2. Flg22 solution
    Dissolve in sterile ultrapure water to 10 µM (22.7 µg/ml)
    Store at -20 °C
  3. Kinetin stock solution (1,000x)
    1. Dissolve 0.1 mg/ml kinetin in 1/10 volume (100 µl per ml) of 1 M NaOH aqueous solution
    2. Once the powder is dissolved, complete the final volume with ultrapure water
    3. Filter sterilize and store at -20 °C
  4. 2,4-Dichlorophenoxyacetic acid (2,4-D) stock solution (1,000x)
    Dissolve 1 mg/ml 2,4-D in absolute ethanol
    Store at -20 °C
  5. KH2PO4 stock solution
    1. Dissolve 60 mg/ml KH2PO4 in deionized water
    2. Prepare 10 ml aliquots and store at -20 °C
  6. Thiamine/myo-inositol stock solution
    1. Dissolve 0.1 mg/ml thiamine hydrochloride and 10 mg/ml myo-inositol in deionized water
    2. Prepare 3 ml aliquots and store at -20 °C
  7. Tryptone stock solution (10%)
    1. Dissolve 10 g/L tryptone in deionized water
    2. Sterilize by autoclavation or filtration through a 0.22 µm syringe filter
    3. Store at room temperature
  8. MES/Phosphate-buffered culture medium
    Composition:
    0.5 g/L 2-(N-Morpholino)ethanosulfonic acid (MES) hydrate
    30 g/L sucrose
    1x Murashige and Skoog basal salts
    100 mg/L myo-inositol
    1 mg/L Thiamine hydrochloride (vitamin B1)
    0.18 g/L KH2PO4
    0.22 mg/L (0.1 µM) 2,4-Dichlorophenoxyacetic acid
    1% tryptone
    pH 5.7

    Preparation (1 L):
    1. Dissolve 4.3 g of Murashige and Skoog basal salt mixture, 30 g sucrose and 0.5 g MES hydrate in 900 ml of deionized water
    2. Adjust the pH to 5.7 using a 1 M KOH aqueous solution
    3. Add 3 ml of KH2PO4 stock solution, 10 ml of Thiamine/myo-inositol stock solution and 220 µl of 2,4-D stock solution
    4. Complete the final volume to 1 L with deionized water
    5. Transfer 18 ml aliquots to 125 ml Erlenmeyer flasks
    6. Autoclave at 121 °C and 15 psi for 20 min
    7. After equilibration at room temperature, add 2 ml of tryptone stock solution. The medium is ready to be used
  1. Subculturing medium
    Composition:
    1x Murashige and Skoog basal salts
    30 g/L sucrose
    1x Gamborg’s vitamins
    1 mg/L (4.5 µM) 2,4-Dichlorophenoxyacetic acid
    0.1 mg/L (0.46 µM) kinetin
    pH 5.7

    Preparation (1 L):
    1. Dissolve 4.3 g of Murashige and Skoog basal salt mixture and 30 g sucrose in 900 ml of deionized water
    2. Adjust the pH to 5.7 using a 1 M KOH aqueous solution
    3. Complete the final volume with deionized water
    4. Transfer 45 ml aliquots to 250 ml Erlenmeyer flasks
    5. Autoclave at 121 °C and 15 psi for 20 min
    6. After equilibration at room temperature, transfer the autoclaved flasks to a flow hood and add 45 µl of 1,000x Gamborg’s vitamin solution (sterile), 45 µl of 2,4-Dichlorophenoxyacetic acid stock solution and 45 µl of kinetin stock solution. The medium is ready to be used

Acknowledgments

This protocol describes in detail the procedure to access MAMP-triggered medium alkalinization of cell suspensions used in the research report by Fiorin et al. (2018). This research was supported by the São Paulo Research Foundation (FAPESP) through fellowships 09/51018-1, 09/50119-9, 11/23315-1, 13/09878-9 and 14/06181-0.

Competing interests

The authors declare no conflict of interest.

References

  1. Bigeard, J., Colcombet, J. and Hirt, H. (2015). Signaling mechanisms in pattern-triggered immunity (PTI). Mol Plant 8(4): 521-539.
  2. Bisceglia, N. G., Gravino, M. and Savatin, D. V. (2015). Luminol-based assay for detection of immunity elicitor-induced hydrogen peroxide production in Arabidopsis thaliana leaves. Bio-protocol 5(24): e1685.
  3. Böhm, H., Albert, I., Fan, L., Reinhard, A. and Nürnberger, T. (2014). Immune receptor complexes at the plant cell surface. Curr Opin Plant Biol 20: 47-54.
  4. Boutrot, F. and Zipfel, C. (2017). Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance. Annu Rev Phytopathol 55(1): 257-286.
  5. Cook, D. E., Mesarich, C. H. and Thomma, B. P. H. J. (2015). Understanding plant immunity as a surveillance system to detect invasion. Annu Rev Phytopathol 53(1): 541-563.
  6. Fiorin, G. L., Sanchéz-Vallet, A., Thomazella, D. P. de T., do Prado, P. F. V., do Nascimento, L. C., Figueira, A. V. and de O. Teixeira, P. J. P. L. (2018). Suppression of plant immunity by fungal chitinase-like effectors. Current Biology 28(18): 3023-3030.
  7. Flury, P., Klauser, D., Boller, T. and Bartels, S. (2013). MAPK phosphorylation assay with leaf disks of Arabidopsis. Bio-protocol 3(19): e929.
  8. Kato, K., Matsumoto, T., Koiwai, A., Mizusaki, S., Nishida, K., Noguchi, M. and Tamaki, E. (1972). Liquid suspension culture of tobacco cells. In: Ferment Technology Today. Terui, G. (Ed.) Society of Fermentation Technology, Osaka, pp 689-695.
  9. Liu, F., Xu, Y., Wang, Y. and Wang, Y. (2018). Real-time PCR analysis of PAMP-induced marker gene expression in Nicotiana benthamiana. Bio-protocol 8(19): e3031.
  10. Moroz, N., Fritch, K. R., Marcec, M. J., Tripathi, D., Smertenko, A. and Tanaka, K. (2017). Extracellular alkalinization as a defense response in potato cells. Front Plant Sci 8:32.
  11. Nagata, T., Nemoto, Y. and Hasezawa, S. (1992). Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132: 1-30.
  12. Nühse, T. S., Peck, S. C., Hirt, H. and Boller, T. (2000). Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6. J Biol Chem 275(11): 7521-7526.
  13. Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J. and Scheel, D. (2011). Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant J 68(1): 100-113.

简介

[摘要 ] 植物识别多种微生物分子以检测和响应潜在的入侵者,通过细胞表面受体识别微生物相关的分子模式(MAMP)引发一系列生化反应,其中包括跨血浆的离子通量此类事件的后果是细胞外H + 离子浓度降低,这可以通过实验检测到植物细胞悬浮液中培养基pH值的变化,因此类似于活性氧(ROS)积累, MAP激酶的磷酸化和防御相关基因的诱导,MAMP诱导的培养基碱化可以用作激活植物免疫反应的代理。在这里,我们描述了测量烟草BY-2细胞培养基碱化的详细方案两种不同MAMP处理后的悬浮液:衍生自真菌细胞壁的壳六聚体(NAG6; N-乙酰氨基葡萄糖)和鞭毛蛋白表位flg22 ,在细菌鞭毛发现。氏S分析提供了可靠和快速的平台来访问毫安时触发免疫(在MTI)在烟草悬浮细胞,并且可以很容易地适应其他植物物种以及其他毫安。

[背景 ] 在整个进化过程中,植物都具有检测微生物衍生分子并建立起能够抓住有害相互作用的免疫反应的能力(Boutrot和Zipfel,2017年)。诱导此类免疫反应的微生物相关分子模式(MAMP)通常被广泛保存。微生物的结构成分,例如真菌的几丁质和细菌的鞭毛蛋白(Cook 等人,2015)。胞外植物受体对MAMP的识别会导致MAMP触发的免疫(MTI),从而阻止微生物入侵并维持植物健康。(Böhmet al。,2014)。

从分子水平在,甲基苯丙胺的看法是跟一个精心策划的集生化事件。首先,在几秒钟内要几分钟,在助焊剂钙2Tasu 和h Tasu 离子发生,导致细胞膜去极化和Extracell 的UI 氩碱化(Ranf 等。,2011) 。随后,生产活性氧(ROS)观察到,这可以在本地阻止病原体生长和介导下游信号(比雅尔等,2015)。磷酸化MAP激酶发生在几分钟之内MAMP感知后,促进转录防御相关基因的激活,其中许多编码与激素和次生代谢产物合成有关的抗菌蛋白或酶(Bigeard 等人,2015)。最后,观察到抗菌化合物的积累和accumulation 质沉积,进一步阻碍了微生物的生长和传播。

通过监测许多MTI潜在的生化事件的方法,对植物免疫系统及其在植物与微生物相互作用中的活性的机理有了更深入的了解,最广泛使用的实验方法是基于检测ROS爆发,MAP激酶激活,诱导防御标记基因和-相关介质碱化细胞悬浮液(Flury 等人,2013; Bisceglia 等人,2015;刘等人,2018)ROS测量通常执行借助于中。鲁米诺-Based 化学发光测定法MAP激酶的活化。转录重编程通常通过实时PCR监测一个或几个标记基因的表达水平来进行,中等碱化试验基于记录经MAMP处理的植物细胞悬液的pH变化。使用pH传感器处理小体积样品所需的时间与其他方法相比,中度碱化测定省时省力 成本实验室设备。已经使用中等碱化法研究了两种模型植物物种和农作物(Nühse 等,2000; Moroz 等,2017 )。

关键字:植物免疫, 宿主微生物相互作用, 诱导子, 效应生物学, 植物病理学

材料和试剂


 


-Well细胞培养12板(ģ 莱纳乙IO ø NE,蜂星® ,目录号的:665180)
不含吸头的血清移液管10 ml(SARSTEDT,目录号:86.1688.010)
P200移液器吸头
M 20 大号注射器(VWR,目录号:613-2046)
0.22 µm针头过滤器(GE Healthcare,Whatman TM Uniflo ,目录号:9915-2502)
六-N- 乙酰基乙酰己糖(推荐两家制造商:圣克鲁斯生物技术,目录号:sc-222018; 伊索塞普(Isosep),目录号:56 / 11-0010)。存储在-20 °C
注意:可以使用壳庚糖(NAG7)和壳八糖(NAG8)甚至几丁质(Sigma ,目录号:C7170)代替壳六糖。


鞭毛蛋白22肽(flg22:QRLSTGSRINSAKDDAAGLQIA)(Genscript,目录号:RP19986)。在-20°C下保存
作者:的Gamborg 维生素溶液1 ,000X(无菌)(Sigma-Aldrich公司,目录号:G1019-50 ML 。)储存在4 ℃下
注意:在使用前,请检查是否有沉淀物存在(如果存在),将溶液加热至42°C并涡旋直至所有沉淀物溶解。


Murashige 和Skoog基础盐混合物(PhytoTechnology 实验室,目录号:M524)。储存在4 °C
蔗糖(Sigma-Aldrich,目录号:S5391)
肌醇(Sigma-Aldrich,目录号:I7508)
盐酸硫胺素(Sigma-Aldrich,目录号:T1270)。储存在4 °C
KH 2 PO 4 (Sigma-Aldrich,目录号:P5655)
胰蛋白((Oxoid ,目录号:LP0042 )。
2,4-二氯苯氧基乙酸(Sigma-Aldrich,目录号:D7299)
2-(N- 吗啉代)乙磺酸(MES)水合物(Sigma-Aldrich,目录号:M2933)
Kinetin(Sigma-Aldrich,目录号:K0753)
六聚体(NAG6)解决方案(请参阅食谱)
Flg22解决方案(请参阅食谱)
Kinetin储备溶液(1,000x)(请参阅食谱)
2,4-二氯苯氧基乙酸(2,4-D)储备溶液(1,000x)(请参阅配方)
KH 2 PO 4 储备溶液(请参阅配方)
硫胺素/肌醇原液(参见食谱)
胰蛋白stock储备溶液(10%)(请参阅食谱)
MES /磷酸盐缓冲培养基(请参阅食谱)
传代培养基(请参见食谱)
 


配套设备


 


125 ml锥形瓶(Pyrex TM ,目录号:15685767)
250 ml锥形瓶(Pyrex TM ,目录号:11902619)
P200可调移液器
通风橱(Thermo Scientific,型号:1300系列II类,目录号:1323TS)
高压灭菌器(VWR,目录号:481-0666)
pH计(Mettler Toledo,Seven Compact S220 Basic ,目录号:30019028)。可以使用其他型号,只要pH分辨率至少为0.001
小体积pH传感器InLab Micro (Mettler Toledo,目录号:51343160)
轨道振动筛(LaboTech ,型号:RS150)
 


软体类


 


微软Excel
EasyDirect TM pH软件(Mettler Toledo,30323214)
 


程序


 


细胞悬液的制备和维护
这里描述的协议使用的是烟草(Nicot iana tabacum)品种亮黄色2(BY-2)细胞悬液,该细胞悬液是由体外诱导的愈伤组织建立的(图1A).BY-2细胞系由Kato 等人从烟草幼苗中产生。1972年(加藤等人,1972年)一钕,此后一直传播并共享其中植物科学家(永田等,1992)。BY-2 细胞是否目前作为模型在一些实验系统,并且可从不同研究小组。


以生成悬浮植物细胞的,输送约1 毫升的愈伤组织到无菌的125毫升锥形瓶中含有20ml的MES /磷酸盐缓冲培养基(配方8 )使用无菌稚贝ù Ia或INOC ù 特征研环路(在..流罩)温和地避免不必要的机械应力愈伤组织,以防止污染,工作Caref ü LLY 有序的流罩孵育27 ℃,在黑暗中缓慢的搅拌下(100 - 120转)甲在ñ 轨道振荡器。愈伤组织将释放小细胞聚集体,将最终的RE 的UI T IN的细胞悬浮液。
10-14天之后,将5 毫升的RES 的UI 汀细胞悬浮,用10个毫升无针尖血清移液管至无菌250 毫升Erlenmeyer烧瓶中含有45 毫升中传代培养培养基(配方9 )。孵育于27℃在黑暗中温和搅拌下(100 - 120转)在甲ñ 轨道摇床。
7天之后,重复SUBC 的UI 图灵如上所述在步骤阿2。此时,新建立的细胞悬浮昭的UI d类似于示出一个在图小号1B- 1 C.检查等分试样悬浮液下有关的显微镜确保没有污染物。
登记费的UI 氩维护BY-2细胞悬液,SUBC 的UI 真实姿态细胞周刊所描述的步骤一2.最好是按惯例保留两个或三个独立瓶悬浮细胞,防止的C 的UI 真实姿态造成的损失污染。
 


D:\重新格式化\ 2020-2-7 \ 1902926--1315 Paulo Teixeira 696431 \ Figs jpg \ Figure1.jpg


图1 。烟草栽培种生长为愈伤组织和细胞悬浮液嫩黄2(BY-2)细胞。阿。愈伤组织在固体MES生长/磷酸盐缓冲Ç UL TURE平台。乙。刚过细胞悬液SUBC UL 图灵。Ç 。A 7日龄细胞悬液。注意:浅黄色颜色指示饱和度。


 


MAMP处理和中等碱度的测量
一旦建立了可用的细胞悬浮液(如上一节所述),就可以使用MAMP触发的培养基碱化,将等分试样的细胞悬浮液分别用MAMPs处理并探查是否有应答(图2)。


 


D:\重新格式化\ 2020-2-7 \ 1902926--1315 Paulo Teixeira 696431 \ Figs jpg \ Figure2.jpg


图2 。实验设置对于中等碱化测量。12孔微孔板含2.5 毫升细胞悬液每孔保持缓慢的搅拌下在轨道振荡器上。pH传感器插入使探头完全被浸没,而不接触墙壁。


 


用10毫升无针血清移液器小心地将3至5天大的细胞悬液转移至12孔板(每孔2.5毫升)中。所用的细胞培养物应与图1C所示的相似,浅黄色或棕褐色的悬浮液通常会受到压力,因此不应使用。
注意:影响培养基碱化分析的一个关键因素是细胞悬浮液的年龄。在我们的BY-2细胞实验中,我们使用了3至5天的悬浮液,但是最佳年龄可能因实验室而异实验室,因此需要通过实验确定。


将培养板至少2 ħ 和最多4 ħ 在室温和在温和搅拌下在轨道摇床(通常为100 - 200转;速度昭的UI 。d恰好足以使细胞保持悬浮)该步骤允许细胞可以从转移到板引起的应力中恢复。
开始测量之前,请适当调整pH计设置:0.001的测量分辨率,终点格式设置为3 s的手动和定时间隔读数。根据制造商的建议,使用校准标准溶液校准传感器。
在连接到pH 计的计算机上打开EasyDirect TM pH软件,并将导出选项设置为Excel 。将自动打开Excel工作表。
维持板块在搅拌下对轨道摇床(100 - 200转),插入传感器于一体的,Caref 的UI Ly的调整位置,以确保传感器完全插入(吸头浸入式),而不是触摸墙壁或井底。
按下pH计上的开始按钮。仪器将开始在Excel表中记录pH值。
一旦测量变得稳定(相同的值的至少12 S),添加25 Myueru 1 MyuM NAG6(配方1)(终浓度:10 NM )或25 Myueru 中10 MYU 中号Flg22(拍摄蚁2 )(最终浓度:100 NM )到井,为了避免人工的变化在PH值,下平衡的诱抗剂至室温10 敏之前添加到细胞悬浮液。确保每个板包括至少一个处理好的随着诱发剂解决Nt个(H 2 O)作为未引起的控制。
记录NAG6 10分钟和flg22 60分钟的pH值。
测量完成后,从孔中取出传感器,用超纯水彻底清洗并用薄纸擦干。
重复步骤B5-B9,直到测试了实验中包括的所有样品。
 


资料分析


 


该EasyDirect TM PH软件功能导出工具,用于记录测量pH值直接插入Excel工作表。因此,一旦所有原始数据从实验中被收集,则变化在PH(DerutapH )一段时间内各个测试条件,可以计算为S Ubtracting测量的时间0(PH值即,为媒介的PH MAMP处理前)从pH在每个随后的时间点(即,为媒介的PH甲基苯丙胺治疗后)。线图描绘了变化pH值随时间(图3a)或酒吧图,显示的最大DerutapH 值(图3B),每次治疗可绘制可视化水库的UI TS,标准偏差翔的UI d提交指明变化在重复测试,最好是至少有3个生物针对每个测试条件进行复制。


 


D:\重新格式化\ 2020-2-7 \ 1902926--1315 Paulo Teixeira 696431 \ Figs jpg \ Figure3.jpg


图3 。NAG6诱导培养基碱化烟草BY-2细胞悬液。治疗用10细胞悬浮液的PH NM NAG6或者的UI Trapure水(模拟)被记录为10 闵后的治疗。甲。变化在PH(DerutapH )时间过(在秒所示)线图; B 。中号Aximum变化在PH绘制成的柱状图。灯罩在线路图和误差条柱状图表示标准偏差的3个生物重复中。


 


注意事项


 


替代为无针尖血清移液管,它可以切割终结的注册ü 大号氩血清移液管,甚至从P5000提示,并用它来处理的细胞悬液。
 


菜谱


 


六聚体(NAG6)解决方案
溶于无菌超纯水中至1 µM(1.24 µg / ml )。


储存在-20°C


Flg22 解决方案
溶于无菌超纯水中至10 µM(22.7 µg / ml )。


储存在-20°C


Kinetin储备溶液(1,000x)
将0.1 mg / ml激动素溶解在1/10体积(每毫升100 µl)的1 M NaOH水溶液中
粉末溶解后,用超纯水补足最终体积
过滤除菌并储存在-20°C
2,4-二氯苯氧基乙酸(2,4-D)储备溶液(1,000x)
将1 mg / ml 2,4-D溶于无水乙醇


储存在-20°C


KH 2 PO 4 储备溶液
将60 mg / ml KH 2 PO 4 溶解在去离子水中
准备10毫升等分试样并储存在-20°C
硫胺素/肌醇原液
将0.1 mg / ml盐酸硫胺素和10 mg / ml肌醇溶于去离子水中
准备3毫升等分试样并储存在-20°C
胰蛋白stock原液(10%)
在去离子水中溶解10 g / L胰蛋白water
通过高压灭菌或通过0.22 µm注射器式过滤器进行灭菌
室温保存
MES /磷酸盐缓冲培养基
组成:


0.5 g / L 2-(N- 吗啉代)乙磺酸(MES)水合物


30克/ 升蔗糖


1 x Murashige 和Skoog基础盐


100 mg / L 肌醇


1毫克/ 升盐酸硫胺素(维生素B1)


                                                                      0.18 g / L KH 2 PO 4


                                          0.22 mg / L (0.1 µM)2,4-二氯苯氧基乙酸


1%胰蛋白p


pH值5.7


 


准备(1 升):


将4.3克Murashige 和Skoog基础盐混合物,30克蔗糖和0.5克MES 水合物溶解在900毫升去离子水中
使用1 M KOH水溶液将pH调节至5.7
加入3 ml KH 2 PO 4 储备溶液,10 ml硫胺/肌醇储备溶液和220 µl 2,4-D储备溶液  
用去离子水将最终体积完成至1 L
18毫升分装转移至125ml Ë Rlenmeyer瓶
在121°C和15 psi下高压灭菌20分钟
平衡在室温后,加入2毫升蛋白胨储备液。介质是随时可以使用
传代培养基
组成:


1x Murashige 和Skoog基础盐


30克/ 升蔗糖


1x Gamborg的维生素


1 mg / L (4.5 µM)2,4-二氯苯氧基乙酸


0.1毫克/ 升(0.46 µM)激动素


pH值5.7


 


准备(1升):


将4.3克Murashige 和Skoog基础盐混合物和30克蔗糖溶解在900毫升去离子水中
使用1 M KOH水溶液将pH调节至5.7
用去离子水完成最终体积
将45毫升等分试样转移到25 0毫升锥形瓶中
在121°C和15 psi下高压灭菌20分钟
平衡在室温后,转移蒸压瓶给一个流罩添加45 Myueru 1 ,000X 的Gamborg 维生素溶液(无菌),45 Myueru中2,4-二氯苯氧乙酸原液和45 Myueru中激动素原液。中型企业准备使用
 


致谢


 


该协议详细描述了Fiorin 等人(2018)的研究报告中使用的MAMP触发的细胞悬浮液培养基碱化的程序。这项研究由圣保罗研究基金会(FAPESP)通过研究金09 / 51018- 1、09 / 50119-9、11 / 23315-1、13 / 09878-9和14 / 06181-0。






竞争利益


 


作者声明没有利益冲突。


 


参考文献


 


Bigeard ,J.,Colcombet ,J. and Hirt,H.(2015)。模式触发免疫(PTI)的信号传导机制。分子植物8(4):521-539。
Bisceglia ,NG,Gravino ,M。和Savatin ,DV(2015)。鲁米诺基于检测方法的免疫激发子诱导的过氧化氢生产中拟南芥叶片。生物协议5(24):E1685。
波姆,H.,阿尔伯特,I.,电风扇,L.,莱因哈德,A. 和纽伦堡,T。(2014)。免疫受体复合在植物细胞表面。 CURR OPIN 植物生物学20:47 - 54。
Boutrot ,F. 和Zipfel,C。(2017)。功能,发现和开发植物的模式识别受体为广谱抗病性。 Annu 启Phytopathol 55(1):257-286。
库克,DE,Mesarich ,CH 和托马,BPHJ(2015年)理解植物免疫作为监视系统来检测入侵。 Annu 版本Phytopathol 53(1):541-563。
Fiorin,GL,Sanchéz-Vallet,A.,Thomazella,DP de T.,do Prado,PFV,do Nascimento,LC,Figueira,AV and de O.Teixeira,PJPL(2018)。真菌几丁质酶抑制植物免疫力。效应一样。当代生物学28(18):302 3 - 3030 。
Flury ,P.,Klauser ,D.,Boller ,T. 和Bartels,S.(2013)。拟南芥叶盘的 MAPK 磷酸化测定。生物协议3(19):e929。
加藤,K.,松本,T.,小岩井,A.,水崎。,S.,西田,K.,野口,M。和玉置,E。(1972)液体悬浮Ç ù Lture烟草细胞。我Ñ :。发酵技术今日特锐,G ^ 。(编。)发酵技术,大阪,第689学会- 695。
Liu,F.,Xu,Y.,Wang,Y. and Wang,Y.(2018)。PAMP 诱导的本氏烟草标记基因表达的实时PCR分析。生物协议8(19):e3031。
Moroz ,N.,Fritch,KR,Marcec ,MJ,Tripathi,D.,Smertenko ,A. 和Tanaka,K.(2017)。细胞外碱化作为马铃薯细胞的防御反应。植物科学8:32。
Nagata,T.,Nemoto,Y.和Hasezawa,S.(1992)。烟草BY-2细胞系作为高等植物细胞生物学中的“ HeLa”细胞, Int Rev Cytol 132:1-30。
Nühse ,TS,Peck,SC,Hirt,H。和Boller ,T。(2000)。微生物引发剂诱导拟南芥MAPK 6的活化和双重磷酸化。JBiol Chem 275(11):7521-7526。
Ranf ,S.,Eschen-Lippold ,L.,Pecher ,P.,Lee,J. and Scheel,D.(2011)。在对微生物或与损伤相关的分子模式的防御反应中,钙信号与早期信号元素之间的相互作用。 。植物杂志68(1):100-113。
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引用:Fiorin, G. L., Sánchez-Vallet, A., Thomma, B. P., Pereira, G. A. and Teixeira, P. J. (2020). MAMP-triggered Medium Alkalinization of Plant Cell Cultures. Bio-protocol 10(8): e3588. DOI: 10.21769/BioProtoc.3588.
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