Name: high-throughput identification of resistance to pseudomonas syringae pv. tomato in tomato using seedling flood assay
Uploaded: 2020-03-10T14:00:00
Description: Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

我们感谢杰米·卡尔玛测试媒体量对疾病或耐药性结果的影响。我们感谢刘易斯实验室的Maöl Baudin博士和卡尔·谢伯博士对手稿提出建设性意见和建议。Lewis实验室植物免疫研究得到了美国农业部ARS 2030-21000-046-00D和2030-21000-050-00D（JDL）和NSF生物科学局IOS-1557661（JDL）的支持。

1. 生物安全柜的制备和使用
<ol><li>用70%乙醇擦拭生物安全柜。</li>
	<li>关闭窗框，打开生物安全柜中的紫外线 15 分钟。</li>
	<li>15分钟后，关闭生物安全柜中的紫外线。提起窗框并打开鼓风机 15 分钟。</li>
	<li>在将物品放入消毒柜之前，用 70% 乙醇擦拭生物安全柜中使用的所有物品。</li>
	<li>在生物安全柜中工作之前，用 70% 乙醇清洁手套或徒手。</li>
	<li>在生物安全柜的中心工作，远离鼓风机。</...

<ol>
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介绍了一种使用PstDC3000或PstT1优化的洪水接种方案，以检测番茄幼苗中对这些细菌菌株的抗药性。在幼苗阻力测定中，有几个关键参数，包括细菌浓度和表面活性剂浓度，这是经验确定22。对于PstDC3000，光学密度进行了优化，在含有Pto/Prf簇的耐药品种上实现完全存活，并在缺乏Pto/Prf群集22的易感品种上完全死亡。对于PstT1...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60805">High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay</a>. The Introduction, Protocol, Representative Results and Discussion&#160;sections were&#160;updated.

Erratum: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

伪莫纳斯西林加是一种葛兰阴性致病菌，可感染多种植物宿主。细菌通过气孔或物理伤口进入宿主植物，并在凋亡1中增殖。植物已经进化出一种双层免疫反应，以防止细菌病原体感染。第一级发生在植物细胞表面，植物细胞膜上的模式识别受体在称为PAMP触发免疫（PTI）2的过程中感知高度保守的病原体相关分子模式（PAMPs）。在这个过程中，宿主植物加强防御反应途径，包括将钙质沉积到细胞壁上，关闭气孔，生产活性氧物种，以及诱导发病机制相关基因。
细菌可以通过利用III型分泌系统将蛋白质（称为效应器）直接输送到植物细胞3中，从而克服PTI。效应蛋白通常针对PTI的成分和促进病原体毒性4。第二层植物免疫在识别效应蛋白后发生在植物细胞内。这种识别依赖于抗药性基因，这些基因编码核苷酸结合位点富含白氨酸的重复含有受体（NLRs）。NLR 能够直接识别效应者或识别其在毒力目标或诱饵5上的活动。然后，它们触发一种称为效应器触发免疫（ETI）的过程的继发免疫反应，该反应通常与超敏反应（HR）相关，这是感染6地点的局部细胞死亡形式。与ETI相关的基因对基因电阻不同，植物可以表现出定量的局部阻力，这依赖于多个基因7的贡献。
P. 西林格加pv.番茄是番茄上细菌斑点的因果剂，是一个长期的农业问题.场上的主要菌株通常是Pst比赛0菌株，表示或两种III型效应器AvrPto和AvrPtoB。DC3000（PstDC3000） 是一种具有代表性的种族 0 菌株和一种模型病原体，可引起番茄中的细菌斑点。为了对抗细菌斑点病，育种者将Pto [P. syringae pv. 番茄]/Prf =Pto电阻和芬西翁敏感性] 基因簇从野生番茄物种索兰皮皮内利菌进入现代品种8，9.托基因编码的丝氨酸-threonine蛋白激酶，与Prf NLR一起，通过识别效应器AvrPto和AvrPtoB10，11，12，13，14，赋予对PstDC3000的电阻。然而，这种抵抗对新出现的比赛1株是无效的，允许他们快速和侵略性蔓延，近年来15，16。比赛1菌株逃避Pto/Prf集群的识别，因为AvrPto在这些菌株中丢失或突变，而AvrPtoB似乎累积最少15，17，18。
野生番茄种群是Pst抗药性自然变化的重要储藏库，以前曾用于识别潜在的抗性位点19、20、21。然而，目前用于病原体耐药性的屏幕使用4-5周大的成年植物20，21。因此，它们受生长时间、生长室空间和相对较小的样品尺寸的限制。为了解决传统方法的局限性，我们利用10天大的番茄苗22开发了高通量番茄P.syringae抗性测定。与使用成人工厂（即缩短生长时间、减少空间要求和提高吞吐量）比较，此方法具有几个优点。此外，我们已经证明，这种方法忠实地概括了在成年植物22中观察到的抗病现象。
在该协议中描述的幼苗洪水测定中，番茄幼苗生长在无菌的Murashige和Skoog（MS）介质的培养皿上，为期10天，然后被含有感兴趣的细菌和表面活性剂的细菌淹没。洪水过后，可以通过细菌生长测定定量评估幼苗的抗病能力。此外，幼苗的生存或死亡可以作为离散的抵抗或疾病表型7-14天后洪水。这种方法提供了一个高通量的替代方案，用于筛选大量的野生番茄加入，以抵抗Pst种族1菌株，如Pst菌株T1（PstT1），并可以很容易地适应其他细菌菌株的兴趣。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>3M Tape Micropore 1/2&#34; x 10 YD CS 240 (1.25 cm x 9.1 m)</td>
			<td>VWR International</td>
			<td>56222-182</td>
			<td></td>
		</tr>
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			<td>3mm borosilicate glass beads</td>
			<td>Friedrich &#38; Dimmock</td>
			<td>GB3000B</td>
			<td></td>
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			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
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			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
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			<td>Biophotometer Plus</td>
			<td>Eppendorf</td>
			<td>E952000006</td>
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			<td>Biosafety cabinet, class II type A2</td>
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			<td>BRAND Disposable Plastic Cuvettes, Polystyrene</td>
			<td>VWR International</td>
			<td>47744-642</td>
			<td></td>
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			<td>Chenille Kraft Flat Wood Toothpicks</td>
			<td>VWR International</td>
			<td>500029-808</td>
			<td></td>
		</tr>
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			<td>cycloheximide</td>
			<td>Research Products International</td>
			<td>C81040-5.0</td>
			<td></td>
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			<td>Dibasic potassium phosphate anhydrous, ACS grade</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
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			<td>Dimethylformamide</td>
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			<td>Dissecting microscope (Magnification of at least 10x)</td>
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			<td>Ethanol - 190 Proof</td>
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			<td>Falcon polystyrene 96 well microplates, flat-bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-3</td>
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			<td>Glass Alcohol Burner Wick</td>
			<td>Fisher Scientific</td>
			<td>S41898A / No. W-125</td>
			<td></td>
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			<td>Glass Alcohol Burners</td>
			<td>Fisher Scientific</td>
			<td>S41898 / No. BO125</td>
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			<td>Glycerol ACS reagent</td>
			<td>VWR International</td>
			<td>EMGX0185-5</td>
			<td></td>
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			<td>Kimberly-Clark&#8482; Kimtech Science&#8482; Kimwipes&#8482; Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
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			<td>Magnesium chloride, ACS grade</td>
			<td>VWR International</td>
			<td>97061-356</td>
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			<td>Magnesium sulfate heptahydrate, ACS grade</td>
			<td>VWR International</td>
			<td>97062-130</td>
			<td></td>
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			<td>Microcentrifuge tubes, 1.5 mL</td>
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			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2.2 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Beadbeater-96, 115 volt</td>
			<td>Bio Spec Products Inc.</td>
			<td>1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog, Basal Salts</td>
			<td>Caisson Laboratories, Inc.</td>
			<td>MSP01-50LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite XLS LTS 8-CH Pipet 20-200uL</td>
			<td>Rainin</td>
			<td>L8-200XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite XLS LTS 8-CH Pipet 2-20uL</td>
			<td>Rainin</td>
			<td>L8-20XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene 100mm x 25mm sterile petri dish</td>
			<td>VWR International</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene 150mm x 15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB08-757-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene 150x15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-757-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach)</td>
			<td>Staples</td>
			<td>1013131</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Gold Biotechnology</td>
			<td>R-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant)</td>
			<td>Fisher Scientific</td>
			<td>NCO138454</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 20 &#956;L 960/10 GPS-L10</td>
			<td>Rainin</td>
			<td>17005091</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 250 &#956;L 960/10 GPS-L250</td>
			<td>Rainin</td>
			<td>17005093</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR dissecting forceps fine tip, 4.5&#34;</td>
			<td>VWR International</td>
			<td>82027-386</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput identification of resistance to pseudomonas syringae pv. tomato in tomato using seedling flood assay

番茄是一种农学上重要的作物，可以感染伪莫纳斯西林加，一种革兰氏阴性细菌，导致细菌斑点病。番茄-P. 西林格pv。番茄病系广泛用于解剖植物先天反应和抗病的遗传基础。几十年来，通过将从索拉伦皮皮内利菌引入培养番茄的Pto/Prf基因簇成功地管理疾病，而P.syringae的种族1菌株已经进化，以克服托/普夫基因簇赋予的阻力，并在全世界发生。
野生番茄品种是病原体识别中自然多样性的重要蓄水池，因为它们在不同环境、不同病原体压力下进化。在野生番茄抗病的典型屏幕中，使用成年植物，这可以限制植物的数量，这些植物可以筛选，因为它们的生长时间延长，生长空间要求更大。我们开发了一种筛选10天大番茄幼苗的抗性方法，将植物生长时间和生长室空间降至最低，使植物快速周转，并允许测试大样本尺寸。生存或死亡的幼苗结果可视为离散表型或受洪水后存活幼苗新生长量定义的阻力尺度。该方法经过优化，筛选了10天大的番茄幼苗，以抵抗两个P.syringae菌株，并可轻松适应其他P.syringae菌株。

利用幼苗电阻测定检测培养物和等源系中的PtoR-介导免疫 图 5显示了货币制造商-PtoR和货币制造商 -PtoS品种在PstDC3000 泛滥 7-10 天后的代表性结果。在感染之前，10天大的幼苗完全出现并扩大，并出现第一个真正的叶子。幼苗在光学密度为0.005~ 0.015%的表面活性剂中，以负控制（未显示数据）和PstDC3000被淹没。幼苗在洪水?...

Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

幼苗洪水测定有助于快速筛选野生番茄加入，以抵抗伪莫纳斯西林加细菌。这种测定与幼苗细菌生长测定结合使用，有助于进一步描述细菌的潜在抗药性，并可用于筛选种群图以确定抗药性的遗传基础。

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

这种高通量协议允许快速筛选番茄幼苗从野生加入细菌病原体伪多多纳丝。幼苗洪水测定最大限度地减少了植物生长时间和生长室需求，允许植物的快速周转，并允许测试大样本量。这种测定是一个强大的工具，可以用来筛选在野生加入和其他线与复杂的遗传背景的耐药性。该协议为两个伪多莫纳斯西林加菌株的洪水接种提供了具体方向。然而，它是多功能的，可以修改，以检测宿主耐药性的其他细菌病原体。演示这个程序的将是亚娜哈桑，一个研究专家在我的实验室。首先种植番茄苗。将番茄种子放在2.2毫升微离心管中，加入两毫升50%漂白剂溶液。摇动管 25 分钟，然后用移液器取出漂白剂溶液。加入两毫升的超纯水，通过倒置管子五次来清洗种子。从管子中吸出液体，再重复洗涤四次。最后洗涤后，加入两毫升的超纯水，将种子倒入无菌的培养皿中。用乙醇燃烧钳子，然后用 0.5 XMS 加 0.8% 的加糖介质将 5 到 7 个种子转移到 100 x 25 毫升的盘子上， 对钳子进行灭菌。用手术胶带密封板的边缘。确保板堆积平坦，朝上。在黑暗中将灭菌的种子分层，至少三天，以同步发芽。三天后，垂直定向板，使根沿板的表面生长，种子线水平方向。将种子转移到一个生长室，温度为22摄氏度，有16小时的光，8小时的黑暗周期。在室内种植幼苗十天，此时它们通常会显示完全出现和扩大的长生，并出现第一个真正的叶子。用扁平的无菌牙签将新鲜细菌带到适当的 KB 阿加上。对于 PstT1，在洪水实验中使用细菌之前，在 28 摄氏度下孵育板 48 小时。为了准备注射液，无菌地将无菌的细菌重新灌装，将十毫摩尔氯化镁重新灌装到光学密度为0.1。然后进行两次连续稀释，以获得 OD600 0075 的工作浓度。还要在十毫摩尔氯化镁中准备一至十种稀释非离子有机体体表面活性剂共量体。漩涡它，并添加到细菌，旋转的管子混合。将带十天幼苗的盘子从生长室转移到生物安全柜，取出手术胶带。然后将六毫升的 inoculum 转移到每个盘子中。用移液器尖端轻轻将幼苗推入幼苗中，然后启动一个计时器三分钟。在每只手拿着一个盘子，向下倾斜盘子的前部，将幼苗的长子和叶子淹没。将幼崽侧向侧五到七次，然后将盘子倒回，用黄种素覆盖根部。再次向下倾斜板，并重复循环共三分钟。将浇注从盘子上倒下来，放在平坦的表面上，然后第二次倒掉任何残留的浇注。重新包装板，并放回生长室。赚钱者-PtoR和赚钱者-PtoS品种被洪水淹没的PSTDC3000和表型7至10天后洪水。赚钱者-PtoR幼苗携带PtoPRF基因簇，对PSTDC3000具有抗药性。虽然接近同源的造金人-PtoS幼苗，无法识别PSTDC3000效应器VRPto或VRPto-B在洪水后七天内迅速死亡。十天大的幼苗被PstT1淹没，在洪水发生至少十天后，表型被。易感的加入是死的，有棕色的脂肪，缺乏新的增长。相比之下，抗性幼苗表现出高水平的绿色生长，并幸存下来感染PstT1。进行细菌生长测定，以定量确认PstT1耐药性和索拉努姆NeoRici LA1329。耐药和易感LA1329和耐药LA1329和 Moneymaker-PtoS之间有1.7个日志的生长差异，这与表型结果相关。执行此检测时，要记住的最重要的事情是在整个协议中密切关注无菌技术，并确保板上的所有幼苗都彻底被淹没。一定要按照有关规定对受感染的植物组织和细菌进行核用处理，并处理材料。

Research

JoVE Journal

Bioengineering

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

使用Tomoauto：一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

中国仓鼠卵巢细胞培养中高通量自动化微生物反应器工艺优化

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...