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Imunologia e Infecção

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Published: May 21, 2019
doi:
10.3791/59437
, , ,
1Department of Biology,Queen’s University

Summary

本文介绍了两种在暴露于免疫诱导剂后量化阿拉伯拟南芥的防御反应的方法:瞬时氧化性爆裂和对幼苗生长的抑制。

Abstract

植物已经进化出一个强大的免疫系统来感知病原体和防止疾病。本文介绍了两种检测,可用于测量在利用诱导分子治疗后,在阿拉伯拟南芥中免疫活化的强度。首先提出一种捕获快速诱导和动态氧化爆发的方法,可以使用基于发光醇的测定进行监测。第二种方法是描述如何测量免疫诱导的对幼苗生长的抑制。这些协议快速可靠,不需要专门的培训或设备,并广泛用于了解植物免疫的遗传基础。

Introduction

为了感知和防御病原体,植物已经进化出膜结合模式识别受体(PRRs),用于检测细胞外的保存微生物分子,称为微生物相关分子模式(MAMPs)1。MAMP与其共性PR的结合启动蛋白激酶介导的免疫信号,导致广谱抗病2。PRR激活后最早的反应之一是整体血浆膜的磷化和活化,这种蛋白质可催化细胞外活性氧物种(ROS)3的产生。,4.ROS在建立抗病性方面起着重要作用,既充当二次信使传播免疫信号,又直接传播抗菌剂5。首次观察到免疫诱导氧化性爆裂被描述使用马铃薯块茎cv.Rishiri后,植物磷酸菌接种6。ROS生产已评估在几个植物物种使用叶盘7,细胞悬浮培养8,和前顶器6。这里描述的是一个简单的方法,用于在阿拉伯拉多普西斯(阿拉伯)的叶盘中对模式触发的ROS进行化法。

作为对MAMP感知的一种反应,活性RBOH蛋白催化超氧化物基(O2-),羟基基(+OH)和单氧(1 O 2)的产生,这些基液被转化为过氧化氢(H2O)2)在细胞外空间9。H2O2可在氧化剂马萝卜过氧化物酶 (HRP)10的情况下通过基于发光的化学致发光进行量化。HRP氧化H2 O2产生氢氧化电(OH+)和氧气(O2),与发光醇发生反应,产生不稳定的中间体,释放出光10的光子。光子发射可以使用能够检测发光的微板读取器或成像器量化为相对光单位 (RLU),这些发光已成为大多数分子实验室的标准设备。通过测量在40-60分钟间隔内产生的光,在引出器处理后2-5分钟可以检测到瞬时氧化爆裂,在10-20分钟达到峰值,在+60分钟11后恢复到基础水平。这段时间内产生的累积光可用作与RBOH蛋白12激活对应的免疫强度的测量。方便的是,这种检测不需要专门的设备或繁琐的样品制备。

在MAMP检测后不久达到峰值,氧化性爆裂被认为是早期免疫反应,以及MAPK激活和乙烯生产5。后来的免疫反应包括转录重编程、口腔闭合和钙素沉积2,5。长期接触MAMP持续激活高能量昂贵的免疫信号,导致植物生长的抑制,表明在发育和免疫13之间的权衡。模式触发的幼苗生长抑制(SGI)被广泛用于评估阿拉伯的免疫输出,并且对于识别免疫信号的几个关键成分,包括PRRs 14,15 ,16.因此,本文还提出了在阿拉伯兰多普西的模式触发SGI的测定,即幼苗生长在含有标准介质或介质的多孔板中,并辅以免疫诱导器8-12天,然后称重使用分析量表。

为了演示如何使用ROS和SGI检测来监测PRR介导的信号,选择了三种基因型,表示不同的免疫输出:(1)野生型阿拉伯拟南芥加入哥伦比亚(Col-0),(2)占主导地位的负bak1-5 突变体,其中多功能PRR共受体BRASINSININININININININ1-ASSOCIATED KINASE 1(BAK1)在免疫信号17、18和(3)隐性cpk28-1突变体中不起作用,缺乏调节蛋白钙-依赖蛋白KINASE 28 (CPK28) 和显示增强的免疫触发反应19,20。ROS和SGI检测是针对细菌伸长因子Tu(EF-Tu)的合成生产的精灵18肽表位提出的,该表在阿拉伯兰多普西被PRR EF-Tu RECEPTOR(EFR)15所识别。这些协议可用于其他免疫诱导剂,如细菌活性蛋白鞭孢蛋白14或内源植物诱导蛋白(AtPeps)16,然而,应该注意,植物反应能力因引出者21。ROS 和 SGI 测定可用于快速定量评估早期和晚期 PRR 介导反应。

Protocol

1. 免疫诱导后在阿拉伯拉多普西叶盘中检测ROS爆裂 工厂生长和维护。 为了同步发芽,分层阿拉伯种子,将大约50个种子悬浮在1mL无菌0.1%琼脂[w/v]中,并在4°C(无光)下储存3-4天。注: 分层野生类型背景控制(例如,Col-0)和具有高和低免疫输出的基因型(例如,cpk28-1和bak1-5)作为内部控制。 在土壤中播种,在标准短日条件下(22 °C、10 小时光、150 mE/m2/s ?…

Representative Results

突变cpk28-119,25和bak1-517,18种植物分别用于证明在氧化性爆裂和SGI中具有高和低免疫反应的基因型的预期结果相对于野生型背景控制 (Col-0) 的测定。为了评估剂量依赖效应,使用了10倍肽稀释系列(1-1,1000 nM)的elf18。正如预期的那样,cpk28-1功能损耗线具有更高的累积(图<str…

Discussion

本文介绍了在阿拉伯拟南芥中检测模式触发免疫反应的两种方法,提供了定量方法,无需使用专用设备即可评估免疫输出。结合使用模式触发的ROS和SGI分别评估对微生物感知的早期和晚期反应。

氧化性爆裂测定的主要局限性是可变性。由于不完全理解的原因,绝对 RDO 在实验之间通常相差一个数量级。由于实验之间的可变性很高,因此建议除了野生型控制(例如Col-0)外,…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们实验室的工作由加拿大自然资源和工程研究委员会(NSERC)发现计划、加拿大创新基金会约翰·埃文斯领袖基金和皇后大学资助。KS 和 IS 由安大略省研究生奖学金和 NSERC 加拿大研究生奖学金(CGS-M)共同支持。

Materials

20-20-20 Fertilizer Plant Prod 10529 Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.
4 mm Biopsy Punch Medical Mart 232-33-34-P A cork borer set with a 0.125 cm^2 surface area can also be used.
48-Well Sterile Plates with Lid Sigma-Aldrich CLS3548
Analytical Scale with Draft Sheid VWR VWR-225AC Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.
BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL) Sartorius 725240 Any multichannel pipette can be used, as can a single pipetter if necessary.
elf18 (Ac-SKEKFERTKPHVNVGTIG) EZ Biolab cp7211 Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.
Forceps Fisher Scientific 22-327379
Horseradish Peroxidase Sigma-Aldrich P6782 Dissolve in pure water. Store at -20C and away from light.
Luminol Sigma-Aldrich A8511 Dissolve in DMSO. Store at -20C and away from light.
Murisage and Skoog Basal Salts Cedarlane Labs MSP09-100LT Store at 4C.
Soil SunGrow Horticulture Sunshine Mix #1 Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.
SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module Molecular Devices Must request a quote Any plate reader capable of detecting luminescence can be used for these assays.
Sucrose Sigma-Aldrich S0389-1KG Store at room temperature.
White Polystyrene 96-Well Plates Fisher Scientific 07-200-589
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Referências

  1. Couto, D. E., Zipfel, C. Regulation of pattern recognition receptor signalling in plants. Nature Reviews Immunology. 16, 537-552 (2016).
  2. Boller, T., Felix, G. A Renaissance of Elicitors: Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. Annual Review of Plant Biology. 60, 379-406 (2009).
  3. Marino, D., Dunand, C., Puppo, A., Pauly, N. A burst of plant NADPH oxidases. Trends in Plant Science. 56 (8), 1472-1480 (2012).
  4. Kadota, Y., Shirasu, K., Zipfel, C. Regulation of the NADPH Oxidase RBOHD during Plant Immunity. Plant and Cell Physiology. 56 (8), 1472-1480 (2015).
  5. Yu, X., Feng, B., He, P., Shan, L. From chaos to harmony: responses and signaling upon microbial pattern recognition. Annual Review of Phytopathology. 55, 109-137 (2017).
  6. Doke, N. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiological Plant Pathology. 23 (3), 345-357 (1983).
  7. Bindschedler, L. V., et al. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. The Plant Journal. 47 (6), 851-863 (2006).
  8. Keppler, L. D. Active Oxygen Production During a Bacteria-Induced Hypersensitive Reaction in Tobacco Suspension Cells. Phytopathology. 110 (3), 759-763 (1989).
  9. Wrzaczek, M., Brosché, M., Kangasjärvi, J. ROS signaling loops – production, perception, regulation. Current Opinion in Plant Biology. 16 (5), 575-582 (2013).
  10. Warm, E., Laties, G. G. Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol. Phytochemistry. 21 (4), 827-831 (1982).
  11. Trujillo, M. Analysis of the lmmunity-Related Oxidative Bursts by a Luminol-Based Assay. Methods in Molecular Biology. 1398, 323-329 (2016).
  12. Nühse, T. S., Bottrill, A. R., Jones, A. M. E., Peck, S. C. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. The Plant Journal. 51 (5), 931-940 (2007).
  13. Belkhadir, Y., Yang, L., Hetzel, J., Dangl, J. L., Chory, J. The growth-defense pivot: Crisis management in plants mediated by LRR-RK surface receptors. Trends in Biochemical Sciences. 39 (10), 447-456 (2014).
  14. Gómez-Gómez, L., Felix, G., Boller, T. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. The Plant Journal. 18 (3), 277-284 (1999).
  15. Zipfel, C., et al. Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation. Cell. 125 (4), 749-760 (2006).
  16. Krol, E., et al. Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2. Journal of Biological Chemistry. 285 (18), 13471-13479 (2010).
  17. Schwessinger, B., et al. Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genetics. 7 (4), e1002046 (2011).
  18. Roux, M., et al. The Arabidopsis Leucine-Rich Repeat Receptor-Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens. The Plant Cell. 23 (6), 2440-2455 (2011).
  19. Monaghan, J., et al. The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover. Cell Host and Microbe. 16 (5), 605-615 (2014).
  20. Wang, J., et al. A Regulatory Module Controlling Homeostasis of a Plant Immune Kinase. Molecular Cell. 69 (3), 493-504 (2018).
  21. Mott, G. A., et al. Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation. Genome Biology. 17, 98 (2016).
  22. Sang, Y., Macho, A. P. Analysis of PAMP-Triggered ROS Burst in Plant Immunity. Methods in Molecular Biology. 1578, 143-153 (2017).
  23. Smith, J. M., Heese, A. Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae. Plant Methods. 10 (1), 6 (2014).
  24. Lindsey, B. E., Rivero, L., Calhoun, C. S., Grotewold, E., Brkljacic, J. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds. Journal of Visualized Experiments. 128, (2017).
  25. Matschi, S., Werner, S., Schulze, W. X., Legen, J., Hilger, H. H., Romeis, T. Function of calcium-dependent protein kinase CPK28 of Arabidopsis thaliana in plant stem elongation and vascular development. The Plant Journal. 73 (6), 883-896 (2013).
  26. Felix, G., Duran, J. D., Volko, S., Boller, T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. The Plant Journal. 18 (3), 265-276 (2002).
  27. Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., Felix, G. The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants. The Plant Cell. 16 (12), 3496-3507 (2004).
  28. Zipfel, C., et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature. 428 (6984), 764-767 (2004).
  29. Mur, L. A. J., Kenton, P., Draper, J. In planta measurements of oxidative bursts elicited by avirulent and virulent bacterial pathogens suggests that H2O2 is insufficient to elicit cell death in tobacco. Plant, Cell and Environment. 28 (4), 548-561 (2005).
  30. Kobayashi, M., et al. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. The Plant Cell. 19 (3), 1065-1080 (2007).
  31. Yoshioka, H., et al. Induction of Plant gp91 phox Homolog by Fungal Cell Wall, Arachidonic Acid, and Salicylic Acid in Potato. Molecular Plant-Microbe Interactions. 14 (6), 725-736 (2001).
  32. Klauser, D., Flury, P., Boller, T., Bartels, S. Several MAMPs, including chitin fragments, enhance AtPep-triggered oxidative burst independently of wounding. Plant Signaling and Behavior. 8 (9), e25346 (2013).
  33. El Gueddari, N. E., Rauchhaus, U., Moerschbacher, B. M., Deising, H. B. Developmentally regulated conversion of surface-exposed chitin to chitosan in cell walls of plant pathogenic fungi. New Phytologist. 156 (1), 103-112 (2002).
  34. Daiber, A., et al. Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012. Free Radical Research. 38 (3), 259-269 (2004).
  35. Bauer, Z., Gómez-Gómez, L., Boller, T., Felix, G. Sensitivity of Different Ecotypes and Mutants of Arabidopsis thaliana toward the Bacterial Elicitor Flagellin Correlates with the Presence of Receptor-binding Sites. Journal of Biological Chemistry. 276 (49), 45669-45676 (2001).
  36. Vetter, M. M., et al. Flagellin perception varies quantitatively in arabidopsis thaliana and its relatives. Molecular Biology and Evolution. 29 (6), 1655-1667 (2012).
  37. Chinchilla, D. The Arabidopsis Receptor Kinase FLS2 Binds flg22 and Determines the Specificity of Flagellin Perception. The Plant Cell. 18 (2), 465-476 (2006).
  38. Lloyd, S. R., Schoonbeek, H., Trick, M., Zipfel, C., Ridout, C. J. Methods to Study PAMP-Triggered Immunity in Brassica Species. Molecular Plant-Microbe Interactions. 27 (3), 286-295 (2014).
  39. Clarke, C., Vinatzer, B. Characterizing the Immune-Eliciting Activity of Putative Microbe-Associated Molecular Patterns in Tomato. Methods in Molecular Biology. 1578, 249-261 (2017).
  40. Gimenez-Ibanez, S., Hann, D. R., Chang, J. H., Segonzac, C., Boller, T., Rathjen, J. P. Differential Suppression of Nicotiana benthamiana Innate Immune Responses by Transiently Expressed Pseudomonas syringae Type III Effectors. Frontiers in Plant Science. 9, 688 (2018).
  41. Wei, Y., et al. The Ralstonia solanacearum csp22 peptide, but not flagellin-derived peptides, is perceived by plants from the Solanaceae family. Plant Biotechnology Journal. 16 (7), 1349-1362 (2018).
  42. Melcher, R. L. J., Moerschbacher, B. M. An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures. Plant Methods. 12, 5 (2016).
  43. Perraki, A., et al. Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling. Nature. 561 (7722), 248-252 (2018).
  44. Yamaguchi, K., Kawasaki, T. Chitin-Triggered MAPK Activation and ROS Generation in Rice Suspension-Cultured Cells. Methods in Molecular Biology. 1578, 309-316 (2017).
  45. Ortmann, I., Conrath, U., Moerschbacher, B. M. Exopolysaccharides of Pantoea agglomerans have different priming and eliciting activities in suspension-cultured cells of monocots and dicots. FEBS Letters. 580 (18), 4491-4494 (2006).
  46. Ortmann, I., Sumowski, G., Bauknecht, H., Moerschbacher, B. M. Establishment of a reliable protocol for the quantification of an oxidative burst in suspension-cultured wheat cells upon elicitation. Physiological and Molecular Plant Pathology. 64 (5), 227-232 (2004).
  47. Dos Santos, A. L. W., El Gueddari, N. E., Trombotto, S., Moerschbacher, B. M. Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia. Biomacromolecules. 9 (12), 3411-3415 (2008).
  48. Bressendorff, S., Rasmussen, M., Petersen, M., Mundy, J. Chitin-Induced Responses in the Moss Physcomitrella patens. Methods in Molecular Biology. , 317-324 (2017).
  49. Lloyd, S. R., Ridout, C. J., Schoonbeek, H. Methods to Quantify PAMP-Triggered Oxidative Burst, MAP Kinase Phosphorylation, Gene Expression, and Lignification in Brassicas. Methods in Molecular Biology. 1578, 325-335 (2017).
  50. Gómez-Gómez, L., Boller, T. FLS2: An LRR Receptor-like Kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Molecular Cell. 5 (6), 1003-1011 (2000).
  51. Li, J., et al. Specific ER quality control components required for biogenesis of the plant innate immune receptor EFR. Proceedings of the National Academy of Sciences of the United States of America. 106 (37), 15973-15978 (2009).
  52. Lu, X., et al. Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele. Proceedings of the National Academy of Sciences of the United States of America. 106 (52), 22522-22527 (2009).
  53. Nekrasov, V., et al. Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity. EMBO Journal. 28 (21), 3428-3438 (2009).
  54. Boutrot, F., et al. Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1. Proceedings of the National Academy of Sciences of the United States of America. 107 (32), 14502-14507 (2010).
  55. Kadota, Y., et al. Direct Regulation of the NADPH Oxidase RBOHD by the PRR-Associated Kinase BIK1 during Plant Immunity. Molecular Cell. 54 (1), 43-55 (2014).

Tags

Keywords: Arabidopsis ThalianaPattern-triggeredOxidative BurstSeedling Growth InhibitionImmune OutputsPlant GenotypesLaboratory EquipmentLeaf DiscsElicitorsReactive Oxygen SpeciesMicroplate ReaderMS MediumSeedling GrowthGrowth Inhibition
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Bredow, M., Sementchoukova, I., Siegel, K., Monaghan, J. Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (147), e59437, doi:10.3791/59437 (2019).
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