Name: a hydroponic co-cultivation system for simultaneous and systematic analysis of plant/microbe molecular interactions and signaling
Uploaded: 2017-07-22T14:00:00.000+00:00
Duration: 11 min 16 s
Description: Watch this Scientific Journal Video about A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling at JoVE.com

我们要感谢Brian Weselowski和Alexander W. Eastman的帮助和有益的讨论。我们也要感谢Drs。尤金·韦斯特，张敏瑞，沉海涛，崔翠翠，格雷格·索恩为他们的帮助，有益的讨论和对稿件的批判性阅读。该研究由作者作为其职责的一部分由加拿大农业和农业食品加拿大农业和农业发展组织（RBPI号码2555）和成长前进II项目1670号资助。本研究还部分由加拿大自然科学与工程研究理事会（NSERC）发现奖励RGPIN-2015-06052授予中国元。

实验规划<ol><li>确定实验的具体目标。 </li><li> 请阅读下面的整个协议。确定哪些部分与具体目标相关，以及是否需要进行任何修改。见下面的例子。 <ol><li>考虑实验是否将使用拟南芥植物和根癌土壤杆菌细菌，如下所述，或另一种植物 - 微生物组合。 注意:虽然可以使用各种植物种类，植物根部的厚度可能需要不同尺寸的网格（步骤3.3），生长参数（步骤3.10-3.11和4.4）以及水培池尺寸和中等体积（步骤4.2）也可能需要调整（ 图2 ）。微生物可以容易地作为纯培养物，混合物种（财团）或环境样品（微生物）接种，但任何变化都可能影响方案，特别是第4.5步。 </li><li>考虑一下将用于分析样品的实验类型。 注意:荧光显微镜（步骤5）需要用荧光报告基因构建体标记的生物体。 </li></ol></li><li>设计适当的控制。包括没有接种有对照细菌的幼苗的水栽池和没有接种细菌的对照幼苗。 </li><li>用适当数量的种子和水培池计划实验。考虑对照，生物重复（至少3个）和所有分析所需的组织量。 </li><li>确定实验目标的合适的共培养长度（步骤4.8）。 </li><li>根据需要修改以下任何步骤。 </li></ol><img alt="图2" class="xfigimg" src="/files/ftp_upload/55955/55955fig2.jpg" /> 图2.在水培系统中可以栽培的其他植物的实例，由P支持<html>金属网拉丝。一套用于各种植物种子和栽培的金属网的兼容性。 （ A ）不锈钢304型焊缝3×3目×.047" 蚕豆蚕丝（ B ）不锈钢304型焊缝4×4目×.035" 玉米线。 （C）不锈钢型304 weldmesh 4×4网格×0.032&quot;直径导线对大豆 （大豆）。（D）不锈钢型304 weldmesh 6×6×网状0.047&quot;直径导线对萝卜 （冬萝卜）。 （ E ）不锈钢304型焊缝6×6目×.047"用于小麦的直径线（ F ）不锈钢304型焊缝6×6目×.035" 黄瓜丝。这个数字已经从参考文献35中修改过。955fig2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 2.植物种子表面灭菌<ol><li>在微量离心管中涡旋（高速）约200种拟南芥的种子和500μL的去离子水。对于具有较大种子的植物，使用大管来促进表面灭菌。 </li><li>在台式微量离心机中以约9,000 xg离心30秒钟。用移液器去除水（上清液）。 </li><li>向种子中加入300μL的2％次氯酸钠并涡旋。在室温下放置1分钟。对于具有较大种子的植物物种，使用较大体积来覆盖种子。 </li><li>在台式微量离心机中以约9,000 xg离心30秒。使用移液管去除次氯酸钠溶液。 </li><li>向种子中加入500μL无菌去离子水并涡旋。离心机约为9,000 xg30秒，并使用无菌移液器取出水。 </li><li>重复步骤2.5另外4次。 </li><li>向种子中加入500μL的70％乙醇，并涡旋。在室温下放置1分钟。 </li><li>在台式微量离心机中以约9,000 xg离心30秒。使用移液器去除70％乙醇。 </li><li>重复步骤2.5另外5次。 </li><li>将灭菌的种子重新悬浮于500μL无菌超纯水中。 </li></ol> 3.植物种子萌发和半固体栽培<ol><li>制备半固体Murashige和Skoog（MS）培养基:2.165 g / L MS基础盐; 10g / L蔗糖; 0.25 g / L MES;和59mL / L B5维生素混合物，pH 5.75，具有4g / L植物药。 </li><li>高压灭菌MS培养基。倒入25毫升无菌深培养皿（100 x 25毫米2 ）。 </li><li>对于每个培养皿，切出90×90平方毫米不锈钢网。 注意: 拟南芥的推荐网格<html> /标准等级为304（标准级），网格数（每线性英寸的孔数）为40×40，线径为0.01"（0.0254cm），较大的水平槽需要较大的网格或更高的平台。 </li><li>弯曲每个正方形网格的角落就足够了，使网格适合Petri板。将角部以90°的角度弯曲到网格的大部分，以允许网格由角落支撑，为根部开发留下足够的空间（ 图3a ）。 </li><li>将切割的网格方格放入烧杯中，用铝箔盖住，并用30分钟的干燥循环通过高压灭菌灭菌。 </li><li>一旦培养基在培养皿中固化并且网状物是无菌的，则将每个消毒的网格方形放置在半固体培养基的顶部，使弯曲的角落朝下。 </li><li>推动网格，使角落穿透介质，大部分网状物接触介质的顶部表面（&gt;图3b）。 </li><li>使用200μL转移移液器来绘制单个表面灭菌的拟南芥种子（种子将由于真空力而停留在移液管的尖端），并轻轻地将每个种子转移到网格顶部。放置种子，使其适当地适应实验需要（ 例如， 4 6个种子/板）。 </li><li>用盖子密封培养皿，将多孔手术胶带放在边缘周围。 </li><li>通过将整个培养皿放置在4℃，在黑暗中2 d（ 例如，包裹在锡箔中以模拟黑暗）来分层种子。 </li><li>在22-24℃培养培养皿中的种子16小时光周期10-14天（ 图3c ）。 </li></ol><img alt="图3" class="xfigimg" src="/files/ftp_upload/55955/55955fig3.jpg" /> 图3:水培植物 - 微生物共培养系统。左边的nel是一个流程图，概述了组合和操作水培共培养系统的六个关键步骤。正确的小组演示了当研究拟南芥 - 农杆菌相互作用时水培共培养系统的实际实验材料，设备和操作程序。现在显示植物或细菌取样的接种步骤和最后步骤。这个数字已经从参考文献35中修改过。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55955/55955fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 水培共培养系统<ol><li>制备液体Murashige和Skoog（MS）培养基:2.165g / L MS基础盐，10g / L蔗糖，0.25g / L MES和59mL / L B5维生素混合物，pH 5.75。 </li><li>高压灭菌介质。倒入18毫升的无菌圆柱形玻璃或透明塑料罐（结晶盘，100 x 80毫米2）用无菌盖。 注意:通过将其包裹在铝箔中并进行高压灭菌来预防罐和盖。 </li><li>在灭菌的流动罩中，使用无菌钳子将每个网格平方从10-14天的幼苗从半固体培养基转移到水培圆柱形罐（ 图3d ）。使用多孔手术胶带将盖子密封在罐体上。 </li><li> 在22-24℃下在槽中的网上培养72小时的幼苗，并在16小时光周期下振荡50rpm进行曝气（ 图3e ）。 注意:这允许植物在与微生物接种（共培养）之前适应水培环境。这个等待期也将允许在接种之前意外的微生物污染是明显的。 <ol><li>在潜伏期结束前一天开始下一步。 </li></ol></li><li>在AB培养基中培养A.根癌土壤杆菌（0.5％w / v酵母提取物，0.5％w / v胰蛋白胨，0.5％w / v蔗糖，50mM MgSO 4 ，pH7.0）在28℃振荡过夜直到培养物在600℃达到1.0的最终光密度nm（OD 600 ），使用分光光度计测量，未接种的AB培养基为空白。 注意:1.0的OD 600相当于约10 9个细胞/ mL。 </li><li>在等体积的0.85％NaCl中洗涤根癌土壤杆菌三次。重悬于等体积的无菌双蒸水中。 </li><li> 在接种之前，仔细检查带有幼苗的罐，以确保没有污染;未受污染的坦克中的介质应清晰透明。 <ol><li>丢弃任何有混浊液体（污染物）的坦克，并对其余的坦克进行编号。从每个编号的罐中取少量（20μL）的水培培养基，并将其放在装有Luria肉汤（LB）的培养皿上。孵化菜在28°C。 </li></ol></li><li> 立即将50μL的根癌土壤杆菌悬浮液（大约5×10 7个细胞，从OD 600估计）加入到每个编号的水培槽中。在22-24℃下用16小时光周期将培养拟南芥幼苗和根瘤农杆菌 ，并以50rpm摇动 （ 图3f ）。 <ol><li>从步骤4.7.1检查点样的LB板。 12和28小时孵化后验证坦克的不育性。如果有任何污染，请勿使用相应编号的罐（接种细菌）进一步下游测定。 </li></ol></li><li>如果需要，通过从罐中移除培养基样品并用分光光度计测量OD 600 ，从未接种对照的培养基作为空白，以规则的间隔监测根癌农杆菌的生长（ 图4 ）。 没有TE:7 d后，植物病症状明显（ 图5 ）。 </li><li>通过提起网板从细菌悬浮液中分离拟南芥根;此步骤的时间取决于下游流程。详见步骤5-8。 </li><li>如果使用根进行后续分析，请立即用双蒸水冲洗根部。如果使用叶子或其他组织，切断组织。对于RNA分析，请立即执行步骤7.1。 </li></ol><img alt="图4" class="xfigimg" src="/files/ftp_upload/55955/55955fig4.jpg" /> 图4:水培共培养系统中的农杆菌生长。每4小时监测植物宿主（ 拟南芥 ）存在或不存在的农杆菌生长。 农杆菌细胞在AB培养基O / N中生长，用0.85％NaCl洗涤3次，并接种到水培系统中，用或w没有拟南芥共培养，从初始OD 600约0.1。 OD 600值是三个生物重复的平均值，标准偏差（ OD 600为1.0 = 1×10 9个细胞/ mL）。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55955/55955fig5.jpg" /> 图5:共培养期间的代表性植物表型和可观察到的疾病症状。在接种后4天内，与非接种植物（ B ）相比，没有可见的疾病症状（ A ）。在7天的共培养（感染）后，感染植物（ C ）中观察到疾病症状，而非接种的植物保持健康（ D ）。 荧光显微镜<ol><li>使用携带自体荧光蛋白的报道构建物的根瘤土壤杆菌在步骤4.5中进行协同设置。 注意:在此使用表达pCherry的修饰的pJP2质粒，dsRed 36的衍生物。 </li><li>在步骤4.8中进行适当的共培养（ 例如 48小时）后，使用无菌剪刀分离个体次生根（主根的侧枝）。 </li><li>用双蒸水冲洗根以去除松散结合的物质。将每根根在30微升的水中浸在显微镜载玻片上，盖上玻璃盖玻片。 </li><li>用指甲油密封盖玻片的边缘以防止根部脱水。 </li><li>通过荧光显微镜观察根癌农杆菌对拟南芥根的附着。 注意:这里，使用来自氦氖（He-Ne）543/594nm激光器的激发的共聚焦显微镜在590-630nm下在倒数63X水透镜物镜上显现pCherry红色荧光，数值孔径为1.4（ </ol><img alt="图6" class="xfigimg" src="/files/ftp_upload/55955/55955fig6.jpg" /> 图6:通过共聚焦显微镜测定的农杆菌根附着物。通过来自氦氖（He-Ne）543/594nm激光的激发，在590-630nm处观察pCherry红色荧光标记的农杆菌 。可视化在数值孔径为1.4的倒置63X水透镜物镜下进行。比例尺=11μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55955/55955fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 细菌成绩单分析<ol><li>在步骤4.8中进行适当的共培养（ 例如 8小时）后，如步骤4.9那样将根与水培培养基分开。将1.5mL的水培培养基转移至1.5mL的微量离心管和离心机以12,000xg离心2分钟以沉淀细胞。 </li><li>去除上清液。将另外1.5 mL的水培培养基转移到相同的1.5 mL微量离心管中，并以12,000 g离心2分钟以沉淀细胞。 </li><li>去除上清液。 注意:通过将样品冷冻至-80°C直至使用，此协议可暂停。 </li><li>使用标准方法37或具有DNA酶处理的合适的商业RNA分离和纯化试剂盒从根癌农杆菌细胞中提取RNA以避免DNA污染。 注意:通过在-80°C下将RNA的等分部分冻结直到使用，可以暂停该协议。 </li><li> 使用分离的RNA进行各种分析，使用标准方法，包括以下内容。 <ol><li>根据制造商的方案使用商业微阵列来检测在共培养对照中差异表达的基因组文化。 </li><li>使用定量实时聚合酶链反应（qRT-PCR）检测特异性基因的表达或验证微阵列数据38 。 </li></ol></li></ol> 7.植物成绩单分析<ol><li>在步骤4.8中进行合适的共培养（ 例如 8小时）后，如步骤4.9中那样从水培培养基中分离根，或根据需要分开其它组织。立即将大约150毫克的根或其他植物组织置于液氮中。 注意:通过将样品冷冻至-80°C直至使用，此协议可暂停。 </li><li>使用研钵和研杵将冷冻样品研磨成液氮粉末。 </li><li>使用标准方法39或使用DNase处理的合适的商业RNA分离和纯化试剂盒从粉末中提取RNA，以避免DNA污染。 注意:通过冻结RNA的等分试样，可以暂停该协议-80°C直到使用。 </li><li> 使用分离的RNA进行各种分析，使用标准方法，包括以下内容。 <ol><li>根据制造商的方案使用商业微阵列检测在共培养对照植物中差异表达的基因组。 </li><li>使用qRT-PCR检测特定基因的差异表达或验证微阵列数据38 。 </li></ol></li></ol> 8. Secretome分析<ol><li>在步骤4.8中进行适当的共培养（ 例如， 72小时）后，如步骤4.9那样将根与水培培养基分开。将18 mL的水培培养基通过0.2μm孔过滤器进行灭菌，使其成为50mL锥形管。 </li><li>在-80°CO / N下冷冻样品。 </li><li>松开50mL锥形管上的盖子并将其放在冷冻干燥机中36小时。继续存储或处理样品（如des用于HPLC分析和化合物检测。 注意:协议可以在这里暂停。 </li><li>在可密封的试管中将每个样品重悬在5 mL双蒸水中。 </li><li>加入5mL乙酸乙酯，并将反应管混合数次。 </li><li>允许相在室温下分离5分钟。 </li><li>通过移液将顶部（有机相）转移到新的容器。如果需要，汇集多个有机部分。 </li><li>在温和的氮气流（约45分钟）下干燥。 </li><li>将样品重新悬浮在合适的HPLC溶液中（ 如 100％甲醇）。 </li><li>按照标准方法进行HPLC 40 。 </li><li>通过适当的方式检测化合物。 注意:这里使用电喷雾离子化飞行时间质谱（ESI-TOF-MS） 40 。 </li></ol>

作者没有什么可以披露的。

鉴于根分泌的逐渐性质， 在植物中产生的毒力诱导化学物质的浓度及其对动植物 - 微生物相互作用的影响发生在空间和时间梯度上。在这种水培共培养系统中，不补充诱导微生物毒力或植物防御的合成植物激素或化学物质。相比之下，使用常规方法，添加合成化学品，如乙酰丁香酮，会产生突然的，人为的浓度峰值。因此，这种水培共培养系统更加模仿自然环境，微生物和宿主植物在空间和时间上识别和响应。 在这里，可以证明农杆菌毒力基因在与拟南芥共同培养时被激活。这表明天然植物来源的化学物质会诱导土壤杆菌毒力。未来e需要确定这些化学物质，并验证其中是否引起毒力诱导化合物乙酰丁香酮。此外，在与农杆菌共培养的同时观察到拟南芥根系基因表达和分泌物谱的显着变化，证明了水培共培养系统在植物 - 微生物相互作用期间研究植物宿主反应和分泌谱的潜力。事实上，使用水培共培养技术，可以在导致微生物相互作用之前和之后的任何阶段研究植物宿主的基因表达和分泌物谱，这将有助于阐明这些转录或分泌物变化如何促进或阻止随后的植物 - 微生物相互作用。 除了研究拟南芥 - 农杆菌信号，水培共培养系统可以适应于研究其他重要的植物 - 微生物系统。它可以用来阐明植物对各种致病或有益微生物的作用，单独或共同作用，以及在相对"自然"的条件下研究微生物对不同植物宿主的附着和反应。典型的幼苗形态的维持对于澄清分子植物 - 微生物相互作用是重要的。在其他细菌，真菌或食草病原体的情况下，可以补充额外的营养物以在共培养期间支持微生物生长。 然而，有一些参数要考虑与不同的植物 - 微生物系统共培养。根据种子大小和根部形态，可能需要较大的种子和幼苗，具有较大的孔和较大的生长容器的金属网，以确保根系结构在网表面下充分发育，并且枝条组织在上面增殖。该设置也可能需要更改以包括更高的平台。这可以实现by切割更大的网格方形，并从拐角进一步弯曲，以使网格表面从容器的底部更高，或者通过切割希腊十字形状的网格，并在凹陷处弯曲每个折翼。当然，每一个系统都有局限性，而这里提出的一个对于长时间生长的大型植物（ 如成熟的玉米）来说并不有用。此外，一些生物可能需要比在使用该系统时通过摇动提供的更多的曝气。 诚然，在实验室设计一个完全完美地复制自然界中发生的动植物 - 微生物相互作用的系统是非常困难甚至不可能的。虽然水培共培养更密切地代表根际的自然条件，至少在概念上，完整的根结构和植物形态，系统中没有土壤或土壤微生物。但是，该系统可以适应以满足不同的需求，例如在可控环境中研究微生物，并了解植物或微生物如何看待和应对不同的生物或非生物刺激。这种水培共培养系统还可以促进对水生植物或生态系统中微生物及其功能的理解。这对于水培温室工业的疾病管理和植物健康或植​​物和微生物的水生生态非常重要。 此外，这种水培共培养系统可以适应于研究生物修复和阐明微生物或植物中的相关代谢过程和调控途径。此外，该共培养水培系统便于上的预先存在的（化感物质）的影响的调查和/或施加的（合成的）的营养物和化学信号/上的微生物或植物化合物主机44。间接影响植物组织的可获得性（ 即0;叶子，芽）在水培共培养系统中提供了探索其他植物防御过程（如植物灌注）以及这些信号如何转导的机会。 这种共同培育体系最关键的部分是防止污染。特别是，必须非常小心地将植物从半固体培养基转移到罐中，以防止水培料的污染。因此，必须在生物安全柜的无菌条件下进行该实验步骤，实验者必须等待2-3天后才能接种任何微生物进行实验。 总而言之，水培共培养系统通过在整个实验过程中保持典型的植物形态和植物根结构的完整性来改善常规方法的各种限制。此外，系统避免补充合成化学品，如植物激素或防御/毒力诱导化学品。因此，它更接近模仿自然环境，微生物和宿主植物在空间和时间上识别和响应。这种共培养系统可以适应于在更"自然"和可管理的条件下研究不同的植物 - 微生物系统，包括内生或附生微生物。 在植物中的相互作用，如发病机理，共生，或植物生长促进的各个方面，可以进行检查。水培共培养系统可以为植物和微生物的生理，生物化学和分子反应提供新的见解。在任何给定的时间点/间隔或生长阶段，可以分别对生长在水培共培养系统中的细菌或植物进行各种"omics"分析，包括植物和微生物转录组学，植物和微生物代谢组学，植物和微生物分泌物以及生物介导上。此外，这种水培共培养系统可以适应于在可控环境中研究植物宿主和微生物群之间的相互作用。总而言之，水培共培养的优点体现了揭示分子植物 - 微生物相互作用和信号传导的优越系统。

植物相关微生物在生物地球化学循环，生物修复，减缓气候变化，植物生长和健康以及植物对生物和非生物胁迫的耐受方面起重要作用。微生物与两个直接通过植物细胞壁接触，并通过化学分泌间接和信令1，2，3株植物相互作用。作为无生物，植物已经开发出直接和间接的机制来抵抗病原体的感染。直接防御包括结构防御和防御蛋白的表达，而间接防御包括次级代谢产物的植物的生产和生物拮抗对侵入病原体4,5的吸引力。植物来源的根系分泌物，分泌物，粘液，粘液和裂解物改变根际的物理化学性质，以吸引或排斥微生物往其主人6 。根分泌的化学成分是物种特异性的，从而作为选择性过滤器，其允许某些能够识别这些化合物的微生物在根际6中繁殖。因此，相容的微生物物种可以被刺激以激活和增强它们的相互关系，这是为了植物宿主1的益处或损害。 在根际理解植物-微生物相互作用的关键是增强植物生产力和生态系统功能，因为大多数微生物和化学暴露的发生在根部结构和土壤-空气界面2，6，7，8。然而，考虑地下植物 - 微生物的相互作用和互惠的反应是一个挑战，因为它有趣的<html>复杂和动态的性质，以及在紧密可控的生长条件下缺乏具有天然根结构和植物形态的合适的实验模型。作为研究最多的植物病原体之一， 农杆菌感染了广泛的农业和园艺重要植物，包括樱桃，苹果，梨，葡萄和玫瑰9 。 农杆菌是了解植物病原体相互作用的一个重要的模式生物，是在植物转化和植物工程10，11，12，13，14的有力工具。 分子植物 - 农杆菌相互作用已经被很好地研究了几十年，目前对农杆菌致病性的理解是广泛的9 ，F"&gt; 11，15，16。 土壤杆菌的致病性在很大程度上归因于感知植物衍生的信号，从而导致在其毒力方案和细胞-细胞通信的精细调制的其演进能力，所谓群体感应17. 土壤杆菌毒力程序由几种根际信号调节，涉及两组双组分系统，即ChvG / I系统和VirA / G系统，根际酸性条件激活了chvG / I ， virA / G的转录，以及涉及农杆菌致病性的其他几个基因，包括virE0 ， virE1 ， virH1 ， virH2和VI型分泌系统（T6SS）的基因18。植物衍生的酚类化合物，包括乙酰丁香酮（4&#39;-羟基-3&#39;，5 &#39;二甲氧基苯乙酮），激活VirA / G 2成分系统通过磷酸化信号传导机制19 。 VirA / G然后激活整个vir调节子，导致将来自其肿瘤诱导（Ti）质粒的称为转移DNA（T-DNA）的〜20kb细菌DNA片段转移并整合到植物核16中 。 T-DNA携带负责合成植物激素吲哚-3-乙酸（IAA）（ iaaM和iaaH ）和细胞分裂素（ ipt ）的基因 ，一旦在植物细胞中表达，就会产生大量的这些植物激素。这导致异常组织增殖和植物肿瘤的发展，被称为冠瘿病，这是植物9，11，20中的慢性和复苏问题。 IAA还与水杨酸和γ-氨基丁酸共同作用以抑制农杆菌毒力或减少农杆菌<html>米群体感应（QS）17，21，22。为了对抗这种压制，T-DNA还携带基因冠瘿碱的合成，其激活农杆菌群体感应来促进土壤杆菌的致病性，并且还用作对于该病原体22,23的营养源。 尽管对农杆菌 -植物相互作用的总体了解以及由此产生的T-DNA转移到植物宿主中，但在相互作用的初始阶段的复合信号传导事件了解甚少。这部分是由于传统方法调查农杆菌植物信号传导的局限性。植物细胞悬浮培养和人工位点特异性伤害通常用于研究分子植物 - 微生物相互作用24 ，。EF"&gt; 26,27然而，细胞悬浮液缺乏典型植物形态学，特别是，植物悬浮细胞不具有根结构和根渗出物，其是用于激活微生物趋化性和毒力28,29非常重要的植物形态学的维持。和根部结构已经通过人为伤害植物，这有利于特定于站点的感染，从而导致在检测诱导的植物防御相关基因的直接感染植物组织30，31寻址的。然而，人工伤人是从病原体感染性质显著不同，特别是作为伤人导致茉莉酸（JA）的积累，其全身地与天然植物信令和防御26干涉。另外，合成的化学物质通常用于人工诱导植物宿主反应或病原体毒力。虽然反射在植物中的浓度这样的化学化合物的补充是可行的，例如补充不占根分泌物扩散逐渐进入周围根际，其产生由微生物28,32感测的趋化梯度。鉴于常规方法研究植物 - 微生物相互作用的局限性，所获得的数据的准确性和深度可能受到阻碍和限制，而常规方法产生的知识可能不会直接在植物中转化。植物 - 农杆菌信号传导的许多方面尚未完全了解，特别是在相互作用的早期阶段，当疾病症状尚未发展时。 为了修正常规方法的局限性，本文提出了一种廉价，可控的，灵活的水培c摄像系统，允许研究人员在分子植物 - 微生物相互作用的初始阶段更深入地了解复杂的信号和反应途径。水耕已被广泛用来研究植物营养素，根渗出物，生长条件，和金属毒性的植物33，34的影响。水培模型有几个优点，包括小空间要求，各种植物组织的可及性，营养/环境条件的严格控制以及病虫害防治。与琼脂/植物电镀技术相比，水培系统对植物生长的限制也较少，其通常限制2-3周后的生长。重要的是，全植物结构的维护便于必要微生物趋化性和毒力感应8,29自然根分泌。系统描述这里的床比其它的33，34简单，劳动密集程度较低。它使用的部件较少，不需要标准剪刀以外的任何工具。它使用金属网（与尼龙33相反）作为植物生长的强支撑和通过摇动以支持微生物生长的无菌条件下通气的简单方法。此外，该系统可以使用各种尺寸的金属网来支持植物生长，其适应不同植物物种而不限制其根部的宽度。 在这里介绍的水培共培养系统中，植物在无菌水培系统中培养，其中植物根分泌支持接种细菌生长的有机化合物。在这种共培养系统中，补充了人造化学物质，如植物激素，防御诱发剂或毒力诱导化学品，反映了天然细胞在植物 - 微生物相互作用期间进行稳态。利用这种水培共培养系统，可以同时测定农杆菌感染后的拟南芥 Col-0根组织中的基因表达，以及用拟南芥共培养的农杆菌基因的活化。进一步表明，该系统适用于研究农杆菌与植物根系的连接以及植物根系分泌物分布，与土壤杆菌共培养（感染）（ 图1 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55955/55955fig1.jpg" /> 图1:具有样品分析的水培共培养系统概述。将植物生长在网格顶部（在网格上方的芽上），将根浸入水培培养基中，然后用细菌f或共培养。然后分离植物组织和细菌用于同时提取和分析。这个数字已经从参考文献35中修改过。

<table><tbody><tr><td>plant seeds (Arabidopsis thaliana Col-0)</td><td>Arabidopsis Biological Resource Centre</td><td>CS7000</td><td>https://abrc.osu.edu/order-stocks</td></tr><tr><td>bacteria (Agrobacterium tumefaciens C58)</td><td>University of Washington</td><td>N/A</td><td></td></tr><tr><td>labeled bacteria</td><td>in-house</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>vortex</td><td>(various)</td><td></td><td></td></tr><tr><td>microcentrifuge tubes</td><td>(various)</td><td></td><td></td></tr><tr><td>microcentrifuge</td><td>(various)</td><td></td><td></td></tr><tr><td>5% sodium hypochlorite</td><td>(various)</td><td></td><td></td></tr><tr><td>double distilled water</td><td>(various)</td><td></td><td></td></tr><tr><td>autoclave</td><td>(various)</td><td></td><td></td></tr><tr><td>micropipette&amp;nbsp;</td><td>(various)</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>(various)</td><td></td><td></td></tr><tr><td>Murashige and Skoog (MS) basal salts</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>sucrose</td><td>(various)</td><td></td><td></td></tr><tr><td>MES</td><td>(various)</td><td></td><td></td></tr><tr><td>B5 vitamin mix</td><td>Sigma-Aldrich</td><td>G1019</td><td></td></tr><tr><td>phytoagar</td><td>(various)</td><td></td><td></td></tr><tr><td>Deep Petri dishes</td><td>(various)</td><td></td><td></td></tr><tr><td>stainless steel mesh</td><td>Ferrier Wire Goods Company Ltd</td><td>N/A</td><td>grade: 304; mesh count: 40 &amp;times; 40; wire DIA: 0.01</td></tr><tr><td>micropore tape, 1&quot;</td><td>3M</td><td>1530-1</td><td></td></tr><tr><td>diurnal growth chamber</td><td>(various)</td><td></td><td></td></tr><tr><td>cylindrical glass tanks, 100 &amp;times; 80 mm&amp;nbsp;</td><td>Pyrex</td><td>3250</td><td>other sizes can be used, in which case liquid content may need adjustment</td></tr><tr><td>flow hood</td><td>(various)</td><td></td><td></td></tr><tr><td>forcepts</td><td>(various)</td><td></td><td></td></tr><tr><td>yeast extract</td><td>(various)</td><td></td><td></td></tr><tr><td>tryptone</td><td>(various)</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>(various)</td><td></td><td></td></tr><tr><td>shaking incubator</td><td>(various)</td><td></td><td></td></tr><tr><td>spectrophotometer</td><td>(various)</td><td></td><td></td></tr><tr><td>NaCl</td><td>(various)</td><td></td><td></td></tr><tr><td>shaker</td><td>(various)</td><td></td><td></td></tr><tr><td>scissors</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>fluorescence microscope</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>microscope slides and cover slips</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>nail polish</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>Bacterial RNA extraction kit</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>plant RNA extraction kit (RNeasy Plant Mini Kit)</td><td>Qiagen</td><td>74903 or 74904</td><td>optional, depends on downstream analyses</td></tr><tr><td>material and equipment for qRT-PCR</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>material and equipment for microarray analysis</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>liquid nitrogen</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>mortar and pestle</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>0.2 &amp;micro;m pore filter</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>50 mL conical tubes</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>freeze dryer</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>sealable test tubes</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>ethyl acetate</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>nitrogen gas</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>material and equipment for HPLC</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr><tr><td>material and equipment for ESI-TOF-MS</td><td>(various)</td><td></td><td>optional, depends on downstream analyses</td></tr></tbody></table>

a hydroponic co-cultivation system for simultaneous and systematic analysis of plant/microbe molecular interactions and signaling

模拟天然植物 - 微生物相互作用的实验设计对于描绘复杂的植物 - 微生物信号传导过程非常重要。 拟南芥 - 根癌土壤杆菌 提供了一个优秀的模型系统来研究细菌发病和植物相互作用。以前对植物 - 土壤杆菌相互作用的研究在很大程度上依赖于植物细胞悬浮培养物，植物的人造伤害，或通过合成化学品人工诱导微生物毒力因子或植物防御。然而，这些方法与植物中的自然信号不同，其中植物和微生物以空间和时间的方式识别和响应。这项工作提出了一个水培共培养系统，其中完整的植物由金属筛网支持并与农杆菌共培养。在这种共培养系统中，没有合成的植物激素或诱导微生物的化学物质补充抗性毒性或植物防御。水培共培养系统非常类似于天然植物-微生物相互作用和在植物信号动态平衡。植物根部可以从含有农杆菌的培养基中分离出来，同时有效地研究植物宿主和相互作用的微生物的信号传导和反应。在任何给定的时间点/间隔，可以分别收集植物组织或细菌用于各种"omics"分析，证明该系统的功效和功效。水培共培养系统可以容易地适应于研究:1）不同植物 - 微生物系统的相互信号转导，2）植物宿主与多种微生物物种（ 即微生物菌群或微生物群落）之间的信号传导，3）营养物质和化学物质如何牵连在植物微生物信号传导中，以及4）微生物如何与植物宿主相互作用，并有助于植物对生物的耐受性非生物压力。

水培栽培系统的生长 根癌土壤杆菌 C58的生长曲线在共培养的初始16小时中显示出显着的滞后期，随后在与拟南芥 Col-0共培养时达到非常稳定的生长，直到约0.9的最大OD 600开始于接种后48小时相比之下，在没有拟南芥的对照培养物中基本上没有观察到细菌生长（ 图4 ）。这些结果表明，没有人工提供的其他营养物质，植物根系分泌的化合物能够在水培共培养系统中支持农杆菌的生长。在共培养的初始16小时期间的显着滞后期可能归因于与病原体共培养相当复杂的宿主感知和逐渐的根分泌。/ P&gt; 相对于在不存在根癌土壤杆菌的情况下生长的对照植物，在开始3天期间， 拟南芥植物显示出没有疾病症状（ 图5a ， 5b ）。然而，在共培养5天后，差异逐渐变得明显，模拟处理的对照植物更环保，更健康。在与土壤杆菌共培养7天后，植物组织显示萎黄病和坏死，以及早开花（ 图 5c， 5d ）。早期开花在拟南芥中是由一系列的系统发生学上不同的病原体41,42感染的一般结果。 农杆菌附着到植物根的显微镜观察根癌土壤杆菌以典型的棒状或丝状形状局限于植物根结构，大约宽度为0.5μm，长度为3-5μm（ 图6 ）。大多数细菌细胞垂直连接到根细胞壁的表面，这是根据以前的发现， 农杆菌以极性的方式结合细胞壁32 。 这些结果表明，水培共培养系统可用于实时研究植物的细菌动力学，如根结构和感染过程。事实上，在没有其他微生物的情况下，这种共培养系统为在植物中直接观察细菌植物附着提供了一个无噪声的平台。 </p&gt; 农杆菌 毒力的转录 激活 在共培养8小时后，收获根癌土壤杆菌细胞用于RNA分离，并通过qRT-PCR评价代表性毒力基因的转录水平（ 图7 ）。与没有拟南芥的对照相比，在与植物宿主共培养期间，毒力基因ropB，virA，virD1，virH1，virE0和chvG的表达显着上调。具体地，mRNA的丰度增加了100％为chvG，70％，持续virD1，94％为的virA，由40％virE0，由330％为ROPB，和48％为virH1。 这些结果表明，没有补充具有合成的毒力诱导化学物质，在水培共培养系统中分泌化学物质的植物能够激活农杆菌毒力程序。 土壤杆菌毒力基因的诱导表明在水培共培养系统中农杆菌对植物来源的信号的反应。这是非常有趣的是， 农杆菌ROPB和chvG都在共培养与拟南芥诱导，如先前的研究已经表明，这些基因在酸性条件引起的18，但不是由乙酰丁香酮，一个知名的毒力诱导化合物19。该结果表明，来自拟南芥根系的未鉴定化合物能够诱导ropB和chvG表达。然而，这种毒力诱导化学品必须进一步鉴定。 <img alt="图7" class ="xfigimg"src ="/ files / ftp_upload / 55955 / 55955fig7.jpg"/&gt; 图7. 农杆菌毒力在水培共培养系统中的活化。通过qRT-PCR定量测定6种农杆菌毒力因子的mRNA水平。毒力因子转录物的丰度与16S rRNA转录物的丰度成正比。数据表示3个生物重复的平均值±标准偏差（误差条）， P值通过学生t检验获得。 植物转录物的表达对以前在拟南芥中差异表达的5个基因进行了qRT-PCR分析（未发表的数据）。如预期的那样，qRT-PCR证实， AGP 30（AT2G33790）和NAS 1（AT5G04950）被下调（分别为97％和89％），而HSP21（AT4G27670）， PGIP 1（AT5G06860）和ELIP 1（AT3G22840）在3次重复相对于对照（ 图8 ）中分别上调（分别为3,800,540和510％）。 <img alt="图8" class="xfigimg" src="/files/ftp_upload/55955/55955fig8.jpg" /> 图8: 拟南芥根目录的qRT-PCR。每个mRNA转录物的丰度被标准化为At ACTIN II转录物的丰度。数据是3个生物重复的平均值±标准偏差（误差条）， P值是通过学生t检验获得的。 改变宿主分泌谱根衍生的化合物已经显示出植物物种6 8之间，在很大程度上是不同的。罗OT渗出物包括在植物-微生物相互作用1，43引起的各种信令响应的分子信号。然而，微生物相互作用后随着时间的推移，宿主分泌谱的渐进差异远远不能理解，除了根分泌的化合物对有益细菌的吸引是至关重要的29 。在使用根癌土壤杆菌 C58共培养72小时后，使用正离子模式LC-MS分析和比较未接种的拟南芥 Col-0（ 即在不存在农杆菌的情况下，建立了拟南芥 Col-0分泌物谱的差异）。观察到分泌物的明显转变，其中新化合物作为农杆菌接种的结果出现（ 图9 ）。在农杆菌接种方案的分泌物中，共有35种化合物相对增强ts，而在非接种植物的分泌物中增强了76个（ 图9 ）。后者可能代表根癌土壤杆菌 C58在感染前会遇到的化合物。虽然这些化合物没有被鉴定，并且在本研究的上下文中没有确定特异性根源化合物的不同分泌，但是分泌物分析使用该水培共培养系统证实了宿主分泌谱的可检测差异。 <img alt="图9" class="xfigimg" src="/files/ftp_upload/55955/55955fig9.jpg" /> 图9: 拟南芥 Col-0 Root Secretome分析。 拟南芥 Col-0分泌物谱的分析一式三份完成，用于模拟接种和接种的样品。对每个治疗组分泌物谱中发现的138个独特质量信号（化合物）的峰面积进行平均，使用平均值计算每种化合物（模拟接种的减去接种）的丰度差异，以便可视化可检测化合物的差异。在接种植物的分泌物中具有较大的负差异值的化合物更丰富，而具有较大阳性差异的化合物在模拟接种的植物的分泌物中更丰富。

Watch this Scientific Journal Video about A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling at JoVE.com

用于同时系统分析植物/微生物分子相互作用和信号传导的水培共培养系统|生物学 |视频

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

所描述的水培共培养系统支持具有金属筛网的完整植物，并将其与细菌共培养。然后可以单独收集植物组织，细菌和分泌的分子用于下游分析，同时允许研究植物宿主和相互作用的微生物或微生物群的分子响应。

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

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Nanyang Technological University

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14.0K 次观看.  Agriculture & Agri-Food Canada. 所描述的水培共培养系统支持具有金属筛网的完整植物，并将其与细菌共培养。然后可以单独收集植物组织，细菌和分泌的分子用于下游分析，同时允许研究植物宿主和相互作用的微生物或微生物群的分子响应。 

视频: A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

科研

环境科学

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

旋转芯在常见菌根网络中植物相互作用的研究

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

免疫与感染

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培:一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

半水培玻璃罐系统，用于多种植物物种的渗出物分析

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

作者聚焦:在新型生长系统中探索植物与微生物通过根系分泌物的相互作用 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

用于半水培根系渗出物分析的多功能玻璃罐系统

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...