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.

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<ol><li>播种<ol><li>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。 </li><li>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毫米称重片或其它的纸张。 </li><li>覆盖用与水喷透明圆顶的锅，以增加相对湿度80-90％（图2A）。 </li></ol></li><li>孵育罐，在4℃条件下72小时，以便完成分层和同步发芽。 </li><li>转移锅的标准生长条件（12小时光照/ 12小时黑暗，21℃，光照强度100微摩尔/米2 /秒，相对湿度40％），以允许种子发芽。破解圆顶在转移到生长室，这将有助于避免凝结水后，立即打开生成一个2-3。 </li><li>当第一片真叶达到2-3毫米长的大小，移植从器皿中的幼苗到72井平板充满了土壤。 <ol><li>使用手指或1毫升枪头在在平坦的土壤表面的中间建立一个1英寸深的抑郁症。 </li><li>轻轻分开单独苗少根损害的可能。小心地拿起具有完整的根用钳子苗。 </li><li>将一个单一的分隔苗连同附着土壤丛移植冲击减少到凹陷，轻轻拍打下来周围的土壤，填补了抑郁症。丢弃多余的苗土成生物危险废物的容器和处置依据与当地的生物危险废物处置的指导方针。 </li><li>水转移从顶部幼苗和完全覆盖平板用与水喷洒透明圆顶以保持80-90％的湿度。保持圆顶上3天，然后裂纹圆顶在4天的早上和完全由该天结束时将其删除。 注意：播种种子可以通过分层种子含有1ml 0.1％琼脂48小时，在4℃1.5毫升离心管中，接着通过转移2-3种子用玻璃吸管上一个72井平板填满土可选地进行的。单位需覆盖有透明的圆顶可在发芽后一周被除去。额外的苗可以去掉，留下每孔只有一个苗子。 </li></ol></li><li>水草每两天通过浸泡单位以1在水中20分钟，然后沥干多余的水。 注：浇水的时间间隔取决于生长室内湿度和需要确定基地D对连续观察用户的植物生长设施。检查工厂每天以确保他们充分浇水。如果只有少数斑点干燥，水从用喷射瓶的顶部的那些植物。 </li><li>关于植物是处于或接近发育阶段＃3.50（当莲座尺寸为50％的最终尺寸），其对应取决于生长条件下（光周期，温度），以3-5周龄执行病原体感染测定（步骤3-5）。不感染花序出现（阶段＃5）由于发病年龄相关的电阻38,39后的植物。 </li></ol> 2.准备文化传媒和盘子<ol><li>让1升国王B（KB）液体介质。轻轻搅动20个克蛋白胨和2g磷酸氢二钾三水合物使用1000毫升去离子H 2 O的，直到没有可见的沉淀磁力搅拌棒。分装百毫升到容量为150ml的玻璃瓶和高压锅在20分钟的液体循环。 </li><li>备培养板KB固体培养基。 <ol><li>轻轻搅动20个克蛋白胨，2克磷酸氢二钾三水和15g琼脂用1,000ml去离子H 2 O高压灭菌器。 </li><li>冷静下来的磁力搅拌板中，直到它很酷的触摸，以减少未来的凝结，并避免抗生素降解。加入18毫升无菌80％甘油和5毫升无菌的1M 硫酸镁向培养基。鉴于PSM ES4326运载反对链霉素抗性，添加链霉素入冷却的介质的50微克ml的终浓度- 1。 </li><li>倒入平板上。准备两个不同尺寸的KB介质板：100毫米×15毫米和150毫米×15毫米的培养皿中。较小的Petri培养皿含有介质作为主要的细菌培养板，和较大的陪替氏培养皿用作细菌计数板。 注意：对于细菌计数板，矩形板可被用于降低耗材成本，因为两倍的治疗可以每盘相比传统的圆板绘制。 <ol><li>倒板前的感染实验。商店的KB培养基平板在4℃下，因为硫酸链霉素长达2-3个月逐渐失去抗生素活性40。 </li></ol></li></ol></li></ol> 3.增强疾病易感性（EDS） <ol><li>第1天 - 在平板Streak的细菌<ol><li> EDS的检测前两天，连胜PSM ES4326从-80℃甘油在KB-链球菌细菌培养板和孵化在28℃，24〜48小时。 </li></ol></li><li>第2天 - 发起液体培养和水草<ol><li>启动液体培养在无菌试管中含50微克毫升4毫升液体KB培养基- ​​1链霉素和摇晃在250转，28°CO / N。 注意：对于EDS感染测定，使用6植物每基因型推荐编辑。对于STI和MTI测定，每基因型6厂每处理建议。为细菌接种物，它建议用新鲜液体培养用的光密度在600nm的 λ在0.3和0.6之间。 </li><li>标记的叶数5和6的钝端防水标记以易于识别感染组织中的采样的叶柄（ 图1）。为了提高气孔开放和促进病原体的解决方案进入叶，浸泡从底部平20分钟植物浇水好，然后沥干多余的水分。 注：另外，盖平，透明的穹顶和浸泡在水中2-4小时，以增加气孔开放。 </li><li>第3天 - 病原稀释和注射渗透 注：病原感染期间早晨是最适合的易感基因型，最明显的疾病症状病原体增殖和发育。尽一切努力保持侵染离子定时一致，以消除昼夜节律和昼夜基因调控41,42的作用，这有助于减少实验重复中的变化。 <ol><li>沉淀细菌培养在微量1.5毫升管在9600×g离心在RT 2分钟，并弃去上清液。重悬细菌沉淀用1毫升无菌的10mM的MgCl 2。 注意：镁阳离子可以提高P ​​的运动和粘附丁香43。或者，使用MgSO 4干燥以相同的浓度。无菌水是一种可接受的替代物中的Mg盐溶液。 </li><li>稀释的细菌悬浮液用9ml的10mM的MgCl 2在50毫升离心管中，并测量细菌的光密度（OD）用分光光度计在λ= 600纳米。稀释细菌用10mM的MgCl 2的最终的OD 600nm的 = 0.0002 EDS感染测定。 </li><li>渗透叶用1毫升钝端针注射器（俗称胰岛素注射器），包含稀释细菌溶液。 </li><li>不填注射器至其满负荷生产。与0.5-0.6毫升加满，以便在渗透过程中更好地控制。 </li><li>暴露于食指顶部的叶的下表面，然后轻轻地调整与拇指的帮助的叶位置。垂直地定位在注射器靠在叶面上，以保证压力均匀分布。尽量避免脉地区的渗透，以减少危害叶片。 </li><li>慢慢推柱塞渗透到细菌的解决方案;由较暗叶色所示液体进入叶肉将被可视化。试图渗透到叶的整个表面。如果这不是在一个单一的企图实现的，选择另一浸润点，并重复上述步骤，直到整个叶表面被覆盖所述的操作。</li><li>完成后，轻轻地涂抹用吸水组织叶删除多余的病原体的解决方案。目视检查感染叶组织的损伤。 注：圆形注射器的印象就不会再看见后，渗透完成。 </li><li>离开渗入植物它们返回到原来的生长条件之前​​，干燥1-2小时。接下来，喷透明球罩与水并覆盖感染的植物2小时，然后破解圆顶产生开2-3，让它在整个感染剩余的实验。 注：由于P.丁香不是空中病原体，存在传播感染因子在同一设施生长其它植物的危险。然而，作为一个附加的预防措施，避免在附近感染的植物和其他实验工厂之间的身体接触。覆盖平面呈透明圆顶会增加病原体毒力，加速病情恶化。该需要在此步骤中的覆盖物期间的和最佳持续时间需要根据用户的植物生长设施的湿度条件被确定。 </li></ol></li><li>第5天 - 预干燥介质板<ol><li>就拿150毫米×15毫米的KB片了冷藏单元和干任何预先存在的冷凝水盘上。干板通过保持其在室温约24小时。为了加快这一进程，将它们放置在层流罩与盖破获30-60分钟。 注意：此步骤是为圆形液滴的板的表面上形成在接下来的步骤是至关重​​要的。 </li></ol></li><li>第6天 - 量化的病原体生长 注意：以下病原体量化过程可在更早的时间点进行感染后，确认细菌被递送到叶，特别是当植物具有改变的叶形态等量。 <ol><li>过程中，出苗后感染的组织萎黄的指示通过在易感基因型的感染组织的变黄，但坏死性病变的发展（图2B）之前。 注：建议的采样时间为病原体接种后三天。然而，由于病原体生长取决于多种环境因素，孵化的长度可以为2½-3天半内变化，且需要获得在用户的植物生长设施仔细的观察感染进展来确定。 <ol><li>制备6磨削管每种基因型（图1）。将其中不锈钢磨球，加入500微升无菌的10毫氯化镁到每个管。 </li><li>分离从植物中的感染叶和冲头叶盘用1孔纸冲头（ 图1）。 注意：为了尽量减少抽样误差，尽量在同一位置每次打孔取样的叶子。从顶部叶盘的采样叶的建议。 </li><li>随机放置2叶盘（从两个不同的植物）到使用镊子每个研磨管。 </li><li>密封该管并均化组织在最大速度高吞吐量均化器（每分钟1600冲程）10分钟。如果需要的话，直到组织是良好均质化并且溶液变成绿色，由于从感染叶叶绿素释放重复此过程。 </li></ol></li><li>同时等待均质化，使用多道移液器，并移液贮存器填充第6行到96孔培养板的180微升的10mM的MgCl 2。 </li><li>粉碎后，转移20微升基本组织悬浮进96孔板的第一行和反复吹打液体上下混合。如果组织的小碎片堵塞的前端，将它夹2-3毫米，以帮助获得溶液的正确体积。 <ol><li>为了提供在顶O足够的空间液滴f显示板，空间从不同基因型中替换行的组织（ 图1）。要准备十倍连续稀释，转移20微升液体到第二行并重复上述步骤，直到第六次稀释。 </li></ol></li><li>转移20微升从96孔板的溶液到使用划分枪头（图1）150毫米×15毫米的KB板。从最稀的悬浮最集中的工作。 注：从最稀释至最集中出发，就没有必要改变稀释液的吸头。如果该板是公预干燥后，所传输的液滴应该保持不变在介质的顶部，直到被吸收（通常15-30分钟）。干燥时间随RT和湿度。 </li><li>干燥板在室温盖破裂。一次没有更多的液体可以在板表面上可以观察到，盖上盖子，倒置，并培育该板在RT或28℃培养箱中。 </li><li>板块孵育40-60小时，直到殖民地变得可见。确认在板上生长反映形成单位（cfu）的菌落的预测的10倍下降（图2C）。算上细菌，他们长满和殖民地保险丝之前。确定细菌在最低稀释不具有重叠的菌落数。通常情况下，首选的稀释要算将包含10-50殖民地之间。 注：变异之间可能会技术重复出现;因此，最低的稀释每个重复应分别确定。 </li><li>要计算的细菌的增殖水平，记录行（R），以及每个技术复制（T），以书面内的细菌的数量的数量。确定集落形成单位的数量-通过公式CFU /叶盘：CFU /叶盘=（T×10 R / 20）×500/2 </li><li>在下面的电子表格模板输入数据到机生产线CE的曲线图（图1）（对于电子表格的数据分析的模板文件，见表2）。每个数据点被表示为对数刻度6技术重复的平均值。误差棒代表平均值的95％置信区间（N = 6）。 注：95％置信区间的计算需要基于技术复制的数目进行调整。 </li></ol></li></ol></li></ol> 4. SA-触发免疫（STI） <ol><li>第1天：如步骤3.1中所述按照相同的步骤。 </li><li>第2天：除了植物浇水并起始液体细菌培养（步骤3.2），水杨酸衍生物水杨酸钠21的外部应用诱导阻力。 注：纯SA具有水溶性较差，需要事先pH值调节，因此不建议这个实验。 <ol><li>诱导针对P. SA介导的防御丁香和总理对未来21感染的植物，24，喷1毫米水杨酸钠细雾或H 2 O（如模拟）植物病原体感染前16-24小时。 </li></ol></li><li>第3天：准备病原体稀释OD 600nm的为0.001和推入细菌通过注射器浸润（步骤3.3）整个叶面。 </li><li>第6天：病原体量化和计票过程，如针对EDS分析（步骤3.4和3.5），按照该过程。板块和算上H 2 O和水杨酸钠-预处理样品分别。对于野生型拟南芥，在病原体生长1-2数减少，预计在水杨酸钠 - 预处理工厂。 </li></ol> 5. MAMP触发免疫（MTI） 注意：在该试验中，我们使用的拟南芥野生型Col-0中和突变FLS2和EFR植物。 FLS2和EFR是细胞膜本地化模式识别受体（蓝耳病），可以识别flg22和elf18，respe沉着应对9,10。损失的各PRR导致不敏感的特定类型MAMP的，这是由下列外部MAMP预处理和注射器浸润PSM ES4326未改变的病原体生长的突变体表示。 <ol><li>天1和2：如步骤3.1和3.2中所述按照相同的步骤。 </li><li>第3天：甲基苯丙胺前处理和病原体感染<ol><li>要建立MAMP触发免疫，准备鞭毛抗原flg22或EF-Tu的表位elf18和1微米的解决方案注射器渗透入病原体感染（步骤3.3.3-3.3.6）之前，整个叶面上4小时。这将允许诱导对铜绿甲基苯丙胺介导的防御丁香和总理对未来感染6,37植物。 注：公开提供芯片数据揭示了甲基苯丙胺反应基因的转录最大重编程后flg22或elf18治疗37,44发生4小时。因此，4小时是推荐持续时间MAMP预处理导致在实现之间的甲基苯丙胺治疗COL-0和FLS2和EFR突变的病原体生长有着明显的区别的。 </li><li>取出用吸水组织额外的甲基苯丙胺的解决方案，并留下破获加快叶片内的液体吸收过程中的植物。以考虑从注射器压力渗透伤人效果，渗透H 2 O的进对照植物的叶子上。 </li><li>准备病原体稀释至OD 600nm的 = 0.001和推入细菌MAMP / H 2 O的预处理叶由注射器浸润（步骤3.3）的整个叶表面。 </li></ol></li><li>第6天：为了病原体量化及数量的过程中，如在步骤3.4和3.5中所述遵循的过程。板块和单独算H 2 O和甲基苯丙胺预处理的样本。在野生型拟南芥中病原体生长1-2数减少，预计在MAMP预处理厂，有点强ER通过降低介flg22相比elf18。 </li></ol>

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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...

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&gt;（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。

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			<td>Grosouth</td>
			<td>SNGMM360</td>
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			<td>11x21 Flats with no holes</td>
			<td>Grosouth</td>
			<td>LM1020</td>
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			<td>11x21 Flats with holes</td>
			<td>Grosouth</td>
			<td>LM1020H</td>
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			<td>Grosouth</td>
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			<td>Fisher Scientific</td>
			<td>DF0122-17-4</td>
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			<td>Potassium Phosphate Dibasic Trihydrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>151946</td>
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			<td>Fisher Scientific</td>
			<td>A360-500</td>
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			<td>Bio Basic Inc</td>
			<td>SB0494</td>
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			<td>100x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
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			<td>150x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80150</td>
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			<td>Rectangular plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-450&#160;</td>
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			<td>MgCl2 Hexahydrate</td>
			<td>Bio Basic Inc</td>
			<td>MB0328</td>
			<td></td>
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			<td>Glycerol</td>
			<td>Bio Basic Inc</td>
			<td>GB0232</td>
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			<td>MgSO4&#160;</td>
			<td>Bio Basic Inc</td>
			<td>MN1988</td>
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			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>NC9992493&#160;</td>
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			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
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			<td>Grinding tubes&#160;</td>
			<td>Denville Scientific</td>
			<td>B1257</td>
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			<td>Caps for grinding tubes</td>
			<td>Denville Scientific</td>
			<td>B1254</td>
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			<td>Stainless steel grinding ball</td>
			<td>Fisher Scientific</td>
			<td>2150</td>
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			<td>96-well plate&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-556-008</td>
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			<td>Sodium Salicylate</td>
			<td>Sigma Aldrich</td>
			<td>s3007-1kg</td>
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			<td>flg22 (QRLSTGSRINSAKDDAAGLQIA)</td>
			<td>Genescript</td>
			<td>Made to order</td>
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		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
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		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td>146308</td>
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			<td>Biophotometer plus</td>
			<td>Eppendorf</td>
			<td>952000006</td>
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			<td>PowerGen High-Throughput Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>02-215-503</td>
			<td></td>
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		<tr>
			<td>Accu spin micro centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (10-100 &#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.043</td>
			<td></td>
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			<td>Multichannel pipette (30-300&#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.060</td>
			<td></td>
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			<td>Pipette (20&#181;l)</td>
			<td>Eppendorf</td>
			<td>3120 000.038</td>
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			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>3552-HR</td>
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			<td>Sharpie permanent marker</td>
			<td>Staples</td>
			<td>507130</td>
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			<td>1.5 mL tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
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			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>08-890</td>
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bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

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。

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...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

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.

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

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Plant Biology

Plant Responses to the Environment

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21.5K 次观看.  University of Alabama at Birmingham. 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.

视频: Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

含量为病原体相关分子模式（PAMP）触发免疫功能的植物（公共交通交汇处）

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

科研

免疫与感染

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...