Quantification of the Humidity Effect on HR by Ion Leakage Assay
离子渗漏实验定量分析湿度对超敏反应的影响   

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Nature Communications
Oct 2018

 

Abstract

We describe a protocol to measure the contribution of humidity on cell death during the effector-triggered immunity (ETI), the plant immune response triggered by the recognition of pathogen effectors by plant resistance genes. This protocol quantifies tissue cell death by measuring ion leakage due to loss of membrane integrity during the hypersensitive response (HR), the ETI-associated cell death. The method is simple and short enough to handle many biological replicates, which improves the power of test of statistical significance. The protocol is easily applicable to other environmental cues, such as light and temperature, or treatment with chemicals.

Keywords: ETI (ETI), HR (HR), Cell death (细胞死亡), Humidity (湿度), Plant-pathogen interactions (植物-病原体相互作用), Ion leakage (离子渗漏)

Background

Environmental cues are important factors in determining the outcome of host-pathogen interactions. The disease triangle paradigm requires, among all, a favorable environment for disease to develop (Francl, 2001; Scholthof, 2007). High humidity, for example, represses HR development and negatively regulates ETI (Zhou et al., 2004; Xin et al., 2016; Mwimba et al., 2018). It is, thus, important to quantify the effect of environments on host-pathogen interactions.

Cell death by HR during ETI is often quantified using a time-course measurement of ion leakage from infected tissue to the aqueous solution surrounding the tissues (Hatsugai and Katagiri, 2018). However, when interested in assessing the contribution of the environment to the HR development, it is necessary for tissue to remain in the environment being studied until HR has developed.

In this protocol, we subject infected tissue to 50% RH or 90% RH for 36 h before conductivity is measured. Also, we have adjusted the dosage of the pathogen to OD600nm = 0.002 to delay the onset of HR and maximize the effect of the environment on HR development. The method adapted here was originally designed to measure cell death in senescing leaves (Woo et al., 2001). Leaves were immersed in 400 mM mannitol, and conductivity was expressed as percentage of the ratio of conductivity measurement before and after boiling. In this modified method, we use tissue of equal size (leaf disc), which make reporting percent ion leakage optional. This protocol was used in our study (Mwimba et al., 2018) and can be applied to other environmental cues or to chemical treatments.

Materials and Reagents

  1. 200 µl and 1,000 µl pipet tips
  2. 1.5 ml tube (Eppendorf, catalog number: 022363204)
  3. 15 ml sterilized tubes (VWR, catalog number: 89039-664)
  4. 50 ml sterilized tubes (VWR, catalog number: 89039-656)
  5. 1 ml needle-less syringes for bacterial inoculation (BD, catalog number: 309659)
  6. Kimwipes (Kimberly-Clarck, catalog number: 34120)
  7. Pseudomonas syringae pv. tomato DC3000 expressing the AvrRpt2 effector (Pst AvrRpt2)
  8. dH2O
  9. MgCl2 (Sigma, catalog number: M8266) or MgSO4 (Sigma, catalog number: M7506)
  10. Mannitol (Sigma, catalog number: M4125)
  11. Agar A (Bio Basic, catalog number: FB0010)
  12. Proteose peptone (BD Biosciences, catalog number: 211684)
  13. K2HPO4·3H2O (Fisher scientific, CAS: 16788-57-1)
  14. 1 M MgSO4 (VWR, catalog number: 0338-500G)
  15. 80% Glycerol (Sigma, catalog number: G5516)
  16. King’s B agar (see Recipes) plate with kanamycin (50 µg/ml) and rifampicin (25 µg/ml) antibiotic selection
  17. 400 mM mannitol solution (see Recipes)

Equipment

  1. 1 L beaker
  2. Shaker (Lab Companion, SI-600R)
  3. Water bath (Precision Scientific, Model 25)
  4. Growth chambers (Percival, AR36L3)
  5. Pipets (Eppendorf, 20-200 µl and 100-1,000 µl)
  6. Spectrophotometer (Amersham Biosciences, model: Ultraspec 2100 pro)
  7. Electro-conductivity meter (Thermo Scientific, model: Orion Star A322)
  8. Puncher and sampling tool (Electron Microscopy Sciences, model: EMS Rapid-core 6.0, catalog number: 69039-60)

Software

  1. Microsoft Excel
  2. Graph prism, or R

Procedure

  1. Grow 12 plants per genotype on soil for 3 weeks under 22 °C, 50% RH 12 h light/12 h dark condition.
  2. In a 15 ml tube, prepare Pst AvrRpt2 (OD600nm = 0.002) in 10 mM MgCl2 (or MgSO4).
  3. Using a 1 ml needle-less syringe, pressure-infiltrate 3 fully expanded leaves (leaves 4, 5 and 6)
    per plants with the Pst AvrRpt2.
    Note: It is much easier to infiltrate from the abaxial side of the leaf.
  4. Dry excess of Pst AvrRpt2 inoculum gently using Kimwipes. 
  5. Move plants to chambers set to conditions being compared. Keep 6 plants per genotype under 50% RH 22 °C 12 h light/12 h dark and the other 6 plants per genotype 90% RH 22 °C 12 h light/12 h dark conditions for 36 h.
    Note: We use Pst AvrRpt2 at OD600nm = 0.002 to capture the effect of the environment on the developing ETI. At this inoculant, the hypersensitive response becomes visible to the eye at around 24 hpi in plants under 50% RH.
  6. Collect dH2O in a 1 L beaker and confirm that the conductivity is below 1.5 µS/cm.
    Note: High dH2O conductivity generates high noise and can result in failure of the experiment.
  7. Using the EMS Rapid-core 6.0 puncher, collect 3 leaf discs per plant (1 disc per leaf) in a 50 ml tube (i.e., 6 tubes per genotype).
  8. Label each tube individually if interested to report data as a percentage (see Step 15)
  9. Add 30 ml dH2O to each tube and rinse discs by inverting tube 3 times.
  10. Using dH2O, prepare 100 ml of 400 mM mannitol (7.3 g mannitol in 100 ml dH2O) per genotype.
    Note: Confirm ion measurement of the 400 mM mannitol is not greater than 3 µS/cm.
  11. For each tube containing leaf discs, replace all dH2O with 6 ml 400 mM mannitol.
  12. Make a blank by adding 6 ml 400 mM mannitol to an empty 50 ml tube for measurement correction during data analysis
  13. Shake tubes at 100 rpm for 2 h at room temperature.
  14. Using the Thermo Scientific Orion Star A322 electro-conductivity meter, measure initial ion leakage (see Figure 1) and proceed to data analysis.
    Note: When measuring, be careful not to hurt leaf discs or move discs across tubes during measurement.
  15. (Optional Step) If presenting data as percent ion leakage.
    1. For 10 min, boil samples by moving tubes to a water bath set at 100 °C.
    2. Cool tubes to RT.
    3. Measure total ion leakage and proceed to data analysis.


    Figure 1. Picture describing ion leakage measurement using the Thermo Scientific Orion Star A322 Electro-conductivity meter

Data analysis

  1. Present data as corrected conductivity values or percent ratio of corrected initial to corrected total conductivity (see Figure 2).
    Corrected conductivity = Measured conductivity - Conductivity of blank.
    Percent ratio =100 x (Initial corrected conductivity/Total corrected conductivity)


    Figure 2. High humidity suppresses the hypersensitive response. A. Conductivity values measured 36 hpi and after boiling. Plants were moved to 50% RH or 90% RH after inoculation with Pst AvrRpt2 OD600nm = 0.002. B and C. Quantification of the hypersensitive response, described in (A), was graphed as membrane ion leakage measurement (B) and as percent membrane ion leakage (C). Data are mean ± S.D.

  2. Repeat the experiment at least three independent times for data reproducibility. 
  3. For statistical analysis, use two-way ANOVA. The three experimental replicates can be combined using linear mixed-effect models.

Recipes

  1. King’s B agar (1 L)
    15 g Agar A
    20 g Proteose peptone
    2 g K2HPO4·3H2O
    Autoclave for 20 min at 120 °C then add 6.1 ml of autoclaved 1 M MgSO4, 18.8 ml of autoclaved 80% Glycerol 
  2. 400 mM mannitol solution (1 L)
    Add 72.88 g mannitol to 500 ml dH2O
    Stir to dissolve mannitol
    Add dH2O to bring volume to 1 L

Acknowledgments

This work was supported by grants from the National Institutes of Health (NIH) (1R01-GM099839-01, 2R01-GM069594-09, and 5R35-GM118036) and by the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation (GBMF3032). We have adapted this protocol from Woo et al. (2001).

Competing interests

The authors have no conflicts of interest or competing interests.

References

  1. Francl, L. J. (2001). The disease triangel: a plant pathological paradigm revisited. The Plant Health Instructor. Doi: 10.1094/PHI-T-2001-0517-01.
  2. Hatsugai, N. and Katagiri, F. (2018). Quantification of plant cell death by electrolyte leakage assay. Bio-protocol 8(5): e2758. 
  3. Mwimba, M., Karapetyan, S., Liu, L., Marques, J., McGinnis, E. M., Buchler, N. E. and Dong, X. (2018). Daily humidity oscillation regulates the circadian clock to influence plant physiology. Nat Commun 9(1): 4290.
  4. Scholthof, K. B. (2007). The disease triangle: pathogens, the environment and society. Nat Rev Microbiol 5(2): 152-156.
  5. Woo, H. R., Chung, K. M., Park, J. H., Oh, S. A., Ahn, T., Hong, S. H., Jang, S. K. and Nam, H. G. (2001). ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. Plant Cell 13(8): 1779-1790.
  6. Xin, X. F., Nomura, K., Aung, K., Velasquez, A. C., Yao, J., Boutrot, F., Chang, J. H., Zipfel, C. and He, S. Y. (2016). Bacteria establish an aqueous living space in plants crucial for virulence. Nature 539(7630): 524-529.
  7. Zhou, F., Menke, F. L., Yoshioka, K., Moder, W., Shirano, Y. and Klessig, D. F. (2004). High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression. Plant J 39(6): 920-932.

简介

我们描述了一种方案来测量湿度对效应器触发免疫(ETI)过程中细胞死亡的影响,ETI是由植物抗性基因识别病原体效应子引发的植物免疫反应。 该协议通过测量在过敏反应(HR),即ETI相关细胞死亡期间膜完整性丧失引起的离子泄漏来量化组织细胞死亡。 该方法简单,短,足以处理许多生物学重复,提高了统计学意义的测试能力。 该协议很容易适用于其他环境因素,如光照和温度,或化学品处理。
【背景】环境因素是决定宿主 - 病原体相互作用结果的重要因素。疾病三角模式尤其需要一个有利于疾病发展的环境(Francl,2001; Scholthof,2007)。例如,高湿度会抑制人力资源开发并对ETI产生负面影响(Zhou et al。,2004; Xin et al。,2016; Mwimba et al。,2018)。因此,量化环境对宿主 - 病原体相互作用的影响是很重要的。

ETI期间HR的细胞死亡通常使用从受感染组织到组织周围水溶液的离子泄漏的时间过程测量来量化(Hatsugai和Katagiri,2018)。然而,当有兴趣评估环境对人力资源开发的贡献时,组织必须保留在正在研究的环境中,直到人力资源开发。

在该方案中,在测量电导率之前,我们将感染的组织置于50%RH或90%RH下36小时。此外,我们已将病原体的剂量调整至OD 600nm = 0.002,以延迟HR的发生并最大化环境对HR发展的影响。这里采用的方法最初设计用于测量衰老叶片中的细胞死亡(Woo et al。,2001)。将叶子浸入400mM甘露醇中,并将电导率表示为沸腾前后电导率测量值的百分比。在这种改进的方法中,我们使用相同尺寸的组织(叶盘),这使得报告离子泄漏百分比是可选的。该方案用于我们的研究(Mwimba et al。,2018),并可应用于其他环境线索或化学处理。

关键字:ETI, HR, 细胞死亡, 湿度, 植物-病原体相互作用, 离子渗漏

材料和试剂

  1. 200μl和1,000μl移液器吸头
  2. 1.5毫升管(Eppendorf,目录号:022363204)
  3. 15毫升灭菌管(VWR,目录号:89039-664)
  4. 50毫升灭菌管(VWR,目录号:89039-656)
  5. 用于细菌接种的1 ml无针注射器(BD,目录号:309659)
  6. Kimwipes(金佰利 - 克拉克,目录号:34120)
  7. Pseudomonas syringae pv。表达AvrRpt2效应子的番茄 DC3000( Pst AvrRpt2 )
  8. 卫生署<子> 2 0
  9. MgCl 2 (Sigma,目录号:M8266)或MgSO 4 (Sigma,目录号:M7506)
  10. 甘露醇(西格玛,目录号:M4125)
  11. Agar A(Bio Basic,目录号:FB0010)
  12. 蛋白胨(BD Biosciences,目录号:211684)
  13. K 2 HPO 4 ·3H 2 O(Fisher scientific,CAS:16788-57-1)
  14. 1 M MgSO 4 (VWR,目录号:0338-500G)
  15. 80%甘油(西格玛,目录号:G5516)
  16. King's B琼脂(参见食谱)板用卡那霉素(50μg/ ml)和利福平(25μg/ ml)抗生素选择
  17. 400毫升甘露醇溶液(见食谱)

设备

  1. 1升烧杯
  2. 振荡器(Lab Companion,SI-600R)
  3. 水浴(Precision Scientific,Model 25)
  4. 生长室(Percival,AR36L3)
  5. 移液管(Eppendorf,20-200μl和100-1,000μl)
  6. 分光光度计(Amersham Biosciences,型号:Ultraspec 2100 pro)
  7. 电导率仪(Thermo Scientific,型号:Orion Star A322)
  8. 打孔器和取样工具(Electron Microscopy Sciences,型号:EMS Rapid-core 6.0,目录号:69039-60)

软件

  1. Microsoft Excel
  2. 图形棱镜,或R

程序

  1. 在22℃,50%RH 12小时光照/ 12小时黑暗条件下,在土壤上每种基因型生长12株植物3周。
  2. 在15 ml管中,在10 mM MgCl 2 (或MgSO 4 中制备 Pst AvrRpt2 (OD 600nm = 0.002)子>)。
  3. 使用1毫升无针注射器,压力渗透3个完全展开的叶子(叶子4,5和6)
    每株植物含有 Pst AvrRpt2 。
    注意:从叶背面渗透更容易。
  4. 使用Kimwipes轻轻地干燥过量的 Pst AvrRpt2 接种物。&nbsp;
  5. 将植物移至设定为比较条件的室。在50%RH 22°C 12 h光照/ 12 h黑暗下保持每种基因型6株植物,其他6种植物每种基因型90%RH 22°C 12 h光照/ 12 h黑暗条件持续36 h。
    注意:我们在OD 600nm = 0.002时使用 Pst AvrRpt2 来捕捉环境对正在发展的ETI的影响。在这种接种物中,在50%RH的植物中,大约24 hpi的眼睛可以看到过敏反应。
  6. 在1L烧杯中收集dH 2 O并确认电导率低于1.5μS/ cm。
    注意:高dH 2 O电导率会产生很高的噪音,并可能导致实验失败。
  7. 使用EMS Rapid-core 6.0打孔机,在50ml管中收集每株植物3叶盘(每叶1个盘)(即,每个基因型6个管)。
  8. 如果有兴趣以百分比形式报告数据,请单独标记每个管(参见步骤15)
  9. 向每个管中加入30ml dH 2 O并通过倒置管3次冲洗圆盘。
  10. 使用dH 2 O,每种基因型制备100ml 400mM甘露醇(7.3g甘露醇,100ml dH 2 O)。
    注意:确认400 mM甘露醇的离子测量值不大于3μS/ cm。
  11. 对于每个含有叶盘的管,用6ml 400mM甘露醇替换所有dH 2 O.
  12. 通过在空的50ml管中加入6ml 400mM甘露醇进行空白,以便在数据分析期间进行测量校正
  13. 在室温下以100rpm摇动管2小时。
  14. 使用Thermo Scientific Orion Star A322电导率仪测量初始离子泄漏(见图1)并进行数据分析。
    注意:测量时,注意不要在测量过程中伤害叶片或将光盘移过管道。
  15. (可选步骤)如果以离子泄漏百分比的形式显示数据。
    1. 在10分钟内,通过将管移至设定在100℃的水浴中来煮沸样品。
    2. 冷却管到RT。
    3. 测量总离子泄漏并进行数据分析。


    图1.使用Thermo Scientific Orion Star A322电导率仪描述离子泄漏测量的图片

数据分析

  1. 将校正电导率值或校正初始电阻与校正总电导率的百分比表示为现有数据(见图2)。
    校正电导率=测量电导率 - 空白电导率。
    百分比= 100×(初始校正电导率/总校正电导率)


    图2.高湿度抑制过敏反应。 A.电导率值
    测量36 hpi和煮沸后。在用 Pst AvrRpt2 OD 600nm = 0.002接种后,将植物移至50%RH或90%RH。 B和C.将(A)中描述的过敏反应的定量作为膜离子泄漏测量(B)和膜离子泄漏百分比(C)绘制成图。数据是平均值±S.D.

  2. 至少重复三次实验,以获得数据重现性。&nbsp;
  3. 对于统计分析,使用双向ANOVA。可以使用线性混合效应模型组合三个实验重复。

食谱

  1. King's B琼脂(1升)
    15克琼脂A
    20克蛋白胨
    2g K 2 HPO 4 。 3H 2 O
    在120℃高压灭菌20分钟,然后加入6.1ml高压灭菌的1M MgSO 4 ,18.8ml高压灭菌的80%甘油&nbsp;
  2. 400mM甘露醇溶液(1L)
    将72.88g甘露醇加入500ml dH 2 O
    搅拌溶解甘露醇
    添加dH 2 O使体积达到1 L

致谢

这项工作得到了美国国立卫生研究院(NIH)(1R01-GM099839-01,2R01-GM069594-09和5R35-GM118036)以及霍华德休斯医学研究所和戈登和贝蒂摩尔基金会(GBMF3032)的资助。我们已经从Woo et al。(2001)改编了这个协议。

利益争夺

作者没有利益冲突或竞争利益。

参考

  1. Francl,L。J.(2001)。 疾病triangel:重新审视植物病理范例。 植物健康教练。 Doi:10.1094 / PHI-T-2001-0517-01。
  2. Hatsugai,N。和Katagiri,F。(2018)。 通过电解质渗漏检测定量植物细胞死亡。 生物协议 8(5 ):e2758。&nbsp;
  3. Mwimba,M.,Karapetyan,S.,Liu,L.,Marques,J.,McGinnis,E.M.,Buchler,N.E。和Dong,X。(2018)。 每日湿度振荡调节生物钟以影响植物生理学。 Nat Commun 9(1):4290。
  4. Scholthof,K。B.(2007)。 疾病三角:病原体,环境和社会。 Nat Rev Microbiol 5(2):152-156。
  5. Woo,H.R.,Chung,K.M.,Park,J.H.,Oh,S.A.,Ahn,T.,Hong,S.H.,Jang,S.K.and Nam,H.G。(2001)。 ORE9,一种调节拟南芥叶片衰老的F-box蛋白质。 植物细胞 13(8):1779-1790。
  6. Xin,X.F.,Nomura,K.,Aung,K.,Velasquez,A.C.,Yao,J.,Boutrot,F.,Chang,J.H.,Zipfel,C.and He,S.Y。(2016)。 细菌在对毒力至关重要的植物中建立水生生物空间。 自然 539(7630):524-529。
  7. Zhou,F.,Menke,F.L。,Yoshioka,K.,Moder,W.,Shirano,Y。和Klessig,D.F。(2004)。 高湿度抑制ssi4介导的细胞死亡和MAP激酶活化,H2O2产生和防御上游的疾病抵抗基因表达。 Plant J 39(6):920-932。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mwimba, M. and Dong, X. (2019). Quantification of the Humidity Effect on HR by Ion Leakage Assay. Bio-protocol 9(7): e3203. DOI: 10.21769/BioProtoc.3203.
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