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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 border="0" cellpadding="0" cellspacing="0" width="663">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">plant seeds (Arabidopsis thaliana Col-0)</td>
			<td style="width:163px;">Arabidopsis Biological Resource Centre</td>
			<td style="width:119px;">CS7000</td>
			<td style="width:167px;">https://abrc.osu.edu/order-stocks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">bacteria (Agrobacterium tumefaciens C58)</td>
			<td style="width:163px;">University of Washington</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">labeled bacteria</td>
			<td style="width:163px;">in-house</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">vortex</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">microcentrifuge tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">microcentrifuge</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">5% sodium hypochlorite</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">double distilled water</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">autoclave</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">micropipette&#160;</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70 % ethanol</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Murashige and Skoog (MS) basal salts</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sucrose</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MES</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">B5 vitamin mix</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td>G1019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">phytoagar</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">deep Petri dishes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">stainless steel mesh</td>
			<td style="width:163px;">Ferrier Wire Goods Company Ltd</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">grade: 304; mesh count: 40 &#215; 40; wire DIA: 0.01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">micropore tape, 1 inch</td>
			<td style="width:163px;">3M</td>
			<td style="width:119px;">1530-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">diurnal growth chamber</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">cylindrical glass tanks, 100 &#215; 80 mm&#160;</td>
			<td style="width:163px;">Pyrex</td>
			<td style="width:119px;">3250</td>
			<td style="width:167px;">other sizes can be used, in which case liquid content may need adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">flow hood</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">forcepts</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">yeast extract</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">tryptone</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">MgSO4</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">shaking incubator</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">spectrophotometer</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NaCl</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">shaker</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">scissors</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">fluorescence microscope</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">microscope slides and cover slips</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">nail polish</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bacterial RNA extraction kit</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">plant RNA extraction kit (RNeasy Plant Mini Kit)</td>
			<td style="width:163px;">Qiagen</td>
			<td>74903 or 74904</td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for RT-qPCR</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for microarray analysis</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">liquid nitrogen</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">mortar and pestle</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">0.2 &#181;m pore filter</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">50 mL conical tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">freeze dryer</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">sealable test tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ethyl acetate</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">nitrogen gas</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for HPLC</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for ESI-TOF-MS</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">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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13.9K 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

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 ...

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生物学

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层:牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...