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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 3M Tape Micropore 1/2" x 10 YD CS 240 (1.25 cm x 9.1 m) | VWR International | 56222-182 | |
| 3mm borosilicate glass beads | Friedrich & Dimmock | GB3000B | |
| Bacto peptone | BD | 211677 | |
| Bacto agar | BD | 214010 | |
| Biophotometer Plus | Eppendorf | E952000006 | |
| Biosafety cabinet, class II type A2 | |||
| BRAND Disposable Plastic Cuvettes, Polystyrene | VWR International | 47744-642 | |
| Chenille Kraft Flat Wood Toothpicks | VWR International | 500029-808 | |
| cycloheximide | Research Products International | C81040-5.0 | |
| Dibasic potassium phosphate anhydrous, ACS grade | Fisher Scientific | P288-500 | |
| Dimethylformamide | |||
| Dissecting microscope (Magnification of at least 10x) | |||
| Ethanol – 190 Proof | |||
| Falcon polystyrene 96 well microplates, flat-bottom | Fisher Scientific | 08-772-3 | |
| Glass Alcohol Burner Wick | Fisher Scientific | S41898A / No. W-125 | |
| Glass Alcohol Burners | Fisher Scientific | S41898 / No. BO125 | |
| Glycerol ACS reagent | VWR International | EMGX0185-5 | |
| Kimberly-Clark™ Kimtech Science™ Kimwipes™ Delicate Task Wipers | Fisher Scientific | 06-666-A | |
| Magnesium chloride, ACS grade | VWR International | 97061-356 | |
| Magnesium sulfate heptahydrate, ACS grade | VWR International | 97062-130 | |
| Microcentrifuge tubes, 1.5 mL | |||
| Microcentrifuge tubes, 2.2 mL | |||
| Mini Beadbeater-96, 115 volt | Bio Spec Products Inc. | 1001 | |
| Murashige & Skoog, Basal Salts | Caisson Laboratories, Inc. | MSP01-50LT | |
| Pipet-Lite XLS LTS 8-CH Pipet 20-200uL | Rainin | L8-200XLS | |
| Pipet-Lite XLS LTS 8-CH Pipet 2-20uL | Rainin | L8-20XLS | |
| Polystyrene 100mm x 25mm sterile petri dish | VWR International | 89107-632 | |
| Polystyrene 150mm x 15mm sterile petri dish | Fisher Scientific | FB08-757-14 | |
| Polystyrene 150x15mm sterile petri dish | Fisher Scientific | 08-757-148 | |
| Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach) | Staples | 1013131 | |
| Rifampicin | Gold Biotechnology | R-120-25 | |
| Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant) | Fisher Scientific | NCO138454 | |
| Tips LTS 20 μL 960/10 GPS-L10 | Rainin | 17005091 | |
| Tips LTS 250 μL 960/10 GPS-L250 | Rainin | 17005093 | |
| VWR dissecting forceps fine tip, 4.5" | VWR International | 82027-386 |
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