JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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면역학 및 감염병학

细菌性叶渗透试验使用的植物防御反应精细表征<em>拟南芥，丁香假单胞菌</em>病害系统

Published: October 01, 2015
doi:
10.3791/53364
, , , , ,
1Department of Biology,University of Alabama at Birmingham

Summary

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Abstract

在没有专门的手机免疫细胞，植物利用其本地化的程序性细胞死亡和系统获得抗性反对病原体攻击自卫。具体拟南芥基因的整体植物免疫应答的贡献可以是具体地和定量通过测定内感染的组织的病原体生长评估。在过去的三十年里，hemibiotrophic细菌野火病菌 。 假单胞菌斑ES4326（PSM ES4326）已被广泛作为模型病原体研究拟南芥免疫反应的分子机制的应用。为了提供病原体进入叶组织，多个接种方法已经建立， 如注射器浸润，浸接种，喷涂，真空渗透，洪水接种。以下方案描述了优化注射器渗入法以提供强毒PSM ES4326到成年的叶土壤中生长的拟南芥植株，准确地筛选增强疾病易感性（EDS）对这种病菌。此外，该协议可以与多个预处理来补充不同层植物防御，包括水杨酸（SA），-Triggered抗扰度（STI）和MAMP触发抗扰度（MTI）内，以进一步解剖特异性免疫缺陷。

Introduction

由于其无柄性质，植物不断受到过多的病原体展示不同的生活方式和营养策略1的威胁。要第一个近似值，植物病毒保持自己的主机活着获取营养物质，而死体营养病原体积极秘密毒素和酶杀死宿主组织和食死细胞1。另一组的病原体，被称为hemibiotrophs，一旦到达病原体积累2的一定的阈值开始他们的感染过程的活体营养阶段和转移到死体营养阶段。为了对抗这些微生物有效地保护自己，植物进化配备了多个监测机制，以发现病原体的攻击，引发复杂的先天免疫系统的本地化程序性细胞死亡3以及系统获得抗性（SAR）4。目前的研究主要集中在定性的基本SIG南陵成分和植物免疫系统 5内跨会谈。

中提出的“之字形”的模式5中，植物天然免疫反应的第一层需要质膜本地化模式识别受体（的PRR）的存在来检测入侵微生物的。蓝耳病是能够识别微生物相关分子模式（毫安），并建立MAMP触发免疫（MTI）6。除了 ​​诱导基因编码的抗菌PR蛋白7的一个转录上调，MTI导致各种事件的逮捕病原体的生长，包括活性氧的产生（ROS）和活性氮粒子（RNS），胼胝质沉积于细胞壁以及多个激酶通路的激活通路8。

到现在为止，几个毫安已经确定触发MTI拟南芥，包括细菌flg22 9 </sup>（从鞭毛蛋白衍生的22氨基酸的片段），elf18 10和结构细胞壁成分（18个从细菌翻译延伸因子Tu氨基酸）肽聚糖11。要建立一个成功的感染，一些专业的病原体已经进化的能力，秘密毒性效应蛋白进入细胞内或细胞间隙，从而抑制MTI和触发效应触发易感性（ETS）12,13。例如，毒力效应可以灭活MTI的丝裂原活化蛋白激酶（MAPK）的磷酸化级联以诱导内感染组织14-16所述疾病的发展。在主机和病原体之间的动态协同进化，植物也制定了反击策略，以识别效应蛋白，减轻病原毒力分子17。这直接或间接的执行识别由疾病抗性（R）的蛋白18介导的。大多数的t下摆是NB-LRR成员（核苷酸结合和富含亮氨酸重 ​​复）系列19。一种无毒效应的由R蛋白的看法引发一个更强大和更广泛的免疫反应定性为效应触发免疫（ETI）20。除了 ​​诱导防御基因21的表达和生产防御代谢物22的 ，ETI往往导致被称为过敏反应（HR）快速本地化程序性细胞死亡，以散布到邻近组织3限制病原体。

除了 ​​局部程序性细胞死亡23，植物是能够引发长期和全系统的免疫应答称为系统获得性抗性（SAR）4。当与活体营养病原体的挑战，植物细胞触发内源性植物激素水杨酸（SA）和PR蛋白在局部和全身组织24的生物合成和积累。至T他的过程中，准备产生了高度的未感染叶子，允许通过的病原体24广谱在随后的感染上升较快的防御反应的实现。 SA和其合成的类似物，例如苯并（1,2,3） -噻二唑-7- 硫代羧酸S-甲酯（BTH）和2,6-二氯异烟酸（INA）能够化学诱导水杨酸（SA）的-Triggered抗扰度（STI）在外部应用程序24。的发病机制相关的基因1（NPR1）Nonexpressor建议成为该SA受体和功能期间在局部和全身组织21,25,26的SA介导的防卫反应的主要转录调节中的一个。已经决定性地证明了NPR1需要特区的建立和NPR1的缺失导致剧烈的敏感性对丁香假单胞菌 25。

到广泛表征植物的分子贡献在植物-病原体相互作用的组件，多个生物测定法已经被开发用于测量特异性防御事件，包括ROS的突发27，胼胝质沉积及其蛋白质产物21的28，防卫基因表达和积累。尽管这些个体测定可以提供深入了解植物的免疫反应的一种具体形式，其中没有，但是，都能够代表在整株水平完全防御反应。相反，在感染后量化病原体生长提供在有机体水平免疫应答的总体估计。因此，精确而高度标准化的病原体接种试验的开发和优化是非常重要的燃料上的拟南芥属的免疫反应的研究和发现。

丁香假单胞菌 ，一种革兰氏阴性细菌，被鉴定为能够引起疾病的范围内的植物宿主，包括Arabidops的植物病原菌29。作为模式植物-病原体系统，拟南芥- P.丁香互动已被广泛应用到了解的分子机制基础植物的防御反应29。到现在为止，超过50 P.基于它们感染不同的植物物种30能力丁香 pathovars已经确定 。P.斑点病菌 。 番茄DC3000（PST DC3000）31 和 P. 斑点病菌 。 假单胞菌斑ES4326（PSM ES4326）32是两种最广泛使用和广泛表征毒株。除了 ​​由工厂被识别并触发MTI响应的Pst DC3000 和 PSM ES4326能分泌致命效应蛋白来抑制MTI，引发ETS有利于病原体生长31,33。

从功能解剖拟南芥和P之间的相互作用丁香，多0;根据病原体交付方法病原体感染的方法被开发出来。对于土壤中生长的植物，病原体可以通过注射器渗透，真空渗透，浸接种，接种喷雾29,34传递。近日，苗汛接种法的开发是为了在组织培养进行大规模的屏板生长的年轻拟南芥植物35。注射器渗入，作为最常用的方法之一，手动开出病原体进入质外体通过被称为气孔29的天然叶开口。通过这种做法，等于P.量丁香可以渗透到受感染的叶和植物免疫反应的强度负相关病原体的增长水平。因此，病原体生长的定量用作在整株水平来评价的免疫功能的最佳方法。此外，注射器浸润可以区分局部和全身组织，其可以bE采用的表征基本特区36的分子机制。

在下面的协议中，我们描述了PSM ES4326优化的注射器渗透法来筛选拟南芥突变体，增强疾病易感性（EDS）。该协议将使用两只拟南芥基因型：野生型生态型哥伦比亚-0（Col-0中）植物（对照）和功能丧失的突变体npr1-1 （hypersusceptible） 将被感染致命的细菌菌株PSM ES4326 37在npr1-1突变体携带NPR1基因分子，它改变了高度保守的组氨酸酪氨酸，并呈现蛋白无功能25的锚蛋白重复共有序列中的点突变。此外，一些注射器浸润测定的变型描述了允许在免疫反应，包括MTI和性病的特定层中的缺陷定量。

Protocol

下面的文字描述了一个协议，逐步在拟南芥进行优化的PSM ES4326注射渗透法。该测定的主要程序中表示在一个简化流程图（ 图1）。 1.植物生长条件播种制备2盆（4-直径，竖直3.75）由从底部直径：浸泡松散装满泥土和水的盆/ N之前排出多余的水。 播下50-100拟南芥种子，野生型Col-0中或npr1-1突变体，到每个盆用折叠70毫米称重片或其…

Representative Results

我们在这里描述的协议代表了一个优化的P.丁香注射器浸润试验，以定量地评价在拟南芥植物的免疫应答。 如图1，PSM ES4326的注射器浸润之后是通过连续稀释和菌落计数病原体提取和定量。 在协议文本中步骤3所述，增强疾病易感性（EDS）对PSM ES4326可以通过感染有OD = 0.0002的接种物进行评估。 如图3所示，高度易感npr1-1</em…

Discussion

通过减少现有耕地和人口的不断增加，世界各地的研究人员正在与作物改良迫切的需求的挑战。产率可以通过各种生物和非生物胁迫可以极大影响。其中，病原体感染是其中作物产量减少的主要原因，负责在美国约12％的损失就45。要解决此问题，大量的研究已经在模型拟南芥进行- P.丁香病害系统全面表征组件和植物免疫反应的调节机制。在这里，我们提出了一个优化的P.丁香</e…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

Materials

MetroMix 360  Grosouth SNGMM360
Large pots Grosouth TEKUVCC10TC
12×6 Inserts Grosouth LM1206
11×21 Flats with no holes Grosouth LM1020
11×21 Flats with holes Grosouth LM1020H
Vinyl propagation domes Grosouth CW-221
Proteose Peptone Fisher Scientific DF0122-17-4
Potassium Phosphate Dibasic Trihydrate  MP Biomedicals 151946
Agar  Fisher Scientific A360-500
Streptomycin sulfate Bio Basic Inc SB0494
100x15mm Petri dishes Fisher Scientific FB0875713
150x15mm Petri dishes Fisher Scientific R80150
Rectangular plate Fisher Scientific 12-565-450 
MgCl2 Hexahydrate Bio Basic Inc MB0328
Glycerol Bio Basic Inc GB0232
MgSO4  Bio Basic Inc MN1988
1 mL syringe Fisher Scientific NC9992493 
Kimwipe Fisher Scientific 06-666-A
Grinding tubes  Denville Scientific B1257
Caps for grinding tubes Denville Scientific B1254
Stainless steel grinding ball Fisher Scientific 2150
96-well plate  Fisher Scientific 12-556-008
Sodium Salicylate Sigma Aldrich s3007-1kg
flg22 (QRLSTGSRINSAKDDAAGLQIA) Genescript Made to order
elf18 (Ac-SKEKFERTKPHVNVGTIG) Genescript Made to order
Hole puncher Staples 146308
Biophotometer plus Eppendorf 952000006
PowerGen High-Throughput Homogenizer Fisher Scientific 02-215-503
Accu spin micro centrifuge Fisher Scientific 13-100-675
Multichannel pipette (10-100 µl) Eppendorf 3122 000.043
Multichannel pipette (30-300µl) Eppendorf 3122 000.060
Pipette (20µl) Eppendorf 3120 000.038
Pipette tips Fisher Scientific 3552-HR
Sharpie permanent marker Staples 507130
1.5 mL tube Eppendorf 22363204
Forceps Fisher Scientific 08-890
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Tags

Bacterial Leaf InfiltrationArabidopsis ThalianaPseudomonas SyringaePathogen Growth AssaySystemic Acquired ResistanceProgrammed Cell DeathSyringe InfiltrationEnhanced Disease SusceptibilitySalicylic Acid-triggered ImmunityMAMP-triggered Immunity
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Liu, X., Sun, Y., Kørner, C. J., Du, X., Vollmer, M. E., Pajerowska-Mukhtar, K. M. Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem. J. Vis. Exp. (104), e53364, doi:10.3791/53364 (2015).
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