幼苗淹水法可快速筛选野生番茄种质资源对Pseudomonas syringae细菌的抗性。该方法与幼苗细菌增殖测定法联合使用，有助于进一步表征对这种细菌的抗性机制，并可用于筛选作图群体，以确定抗性的遗传基础。
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勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
番茄是一种具有重要农艺价值的作物，可被革兰氏阴性细菌Pseudomonas syringae侵染，导致细菌性斑点病。番茄与P. syringae pv. tomato的互作体系被广泛用于解析植物先天免疫反应和抗病性的遗传基础。尽管通过将Solanum pimpinellifolium中的Pto/Prf基因簇导入栽培番茄，已成功防控该病害数十年，但P. syringae的1号生理小种已进化出克服Pto/Prf基因簇所介导抗性的能力，并已在世界范围内出现。
野生番茄物种是病原体识别中天然多样性的重要资源库，因为它们在具有不同病原体压力的多样化环境中进化而来。在对野生番茄进行抗病性筛选时，通常使用成株植物，但由于成株生长期较长且需要更大的生长空间，限制了可筛选植株的数量。我们开发了一种针对10日龄番茄幼苗的抗性筛选方法，该方法可最大限度地缩短植株生长时间和生长室空间占用，实现植株的快速轮替，并支持大规模样本的测试。幼苗的存活或死亡结果可视为离散表型，也可根据淹水后存活幼苗的新组织生长量定义为一个抗性等级。该方法已优化用于10日龄番茄幼苗对两种P. syringae菌株的抗性筛选，并可轻松适用于其他P. syringae菌株。
引言
Pseudomonas syringae 是一种革兰氏阴性致病细菌，可感染多种植物宿主。细菌通过气孔或物理伤口进入寄主植物，并在质外体中增殖1。植物已进化出两层免疫反应以抵御细菌病原体的侵染。第一层免疫反应发生在植物细胞表面，植物细胞膜上的模式识别受体识别病原体中高度保守的病原体相关分子模式（PAMP），该过程称为PAMP触发的免疫（PTI）2。在此过程中，寄主植物会上调防御反应通路，包括在细胞壁沉积胼胝质、关闭气孔、产生活性氧物种，以及诱导病程相关基因的表达。
细菌可通过利用III型分泌系统将称为效应子的蛋白质直接递送至植物细胞内，从而克服PTI3。效应子蛋白通常靶向PTI的组分并促进病原菌的毒力4。植物免疫的第二道防线发生在植物细胞内，当细胞识别到效应子蛋白时被激活。这种识别依赖于抗性基因，这些基因编码含有核苷酸结合位点和富含亮氨酸重复序列的受体（NLRs）。NLRs能够直接识别效应子，或识别效应子对其毒力靶标或诱饵蛋白的作用5。随后，它们触发一种称为效应子触发免疫（ETI）的次级免疫反应，该反应常伴随超敏反应（HR），即在感染部位发生的局部细胞死亡6。与ETI相关的基因....
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方案
1. 生物安全柜的准备与使用
- 用 70% 乙醇擦拭生物安全柜。
- 关闭前窗，开启生物安全柜内的紫外灯照射 15 分钟。
- 15 分钟后，关闭生物安全柜内的紫外灯，升起前窗并开启风机运行 15 分钟。
- 将所有需放入生物安全柜的物品在放入前均用 70% 乙醇擦拭。
- 在进入生物安全柜操作前，用 70% 乙醇清洁手套或裸露的手部。
- 在生物安全柜中央区域操作，远离风机位置。
- 实验中使用未开封的高压灭菌无菌 10 mM MgCl2 溶液和超纯 H2O。将瓶装试剂放入生物安全柜后，仅在柜内开启，不得在台面上打开。
- 在灭菌的生物安全柜内操作时，使用专用的玻璃移液管和移液器吸头。确保这些耗材仅在生物安全柜内开启，绝不在台面上打开。
- 使用完毕后，所有废弃物（漂白剂废液除外）均需高压灭菌，并用 70% 乙醇擦拭柜内表面。
2. 植物培养基的制备
- 称取并溶解0.5x MS基础盐于超纯H2O中。称取0.8%的细菌培养用琼脂，加入已溶解的0.5x MS溶液中。
- 高压灭菌后，将培养基置于50 °C水浴中冷却1小时，再进行倾注或移液操作。
- 为防止平板过量填充，预先在聚苯乙烯一次性无菌100....
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结果
检测 PtoR利用苗期抗性测定法评估品种和近等基因系中的介导免疫反应
图5 展示了Moneymaker的代表性结果PtoR 和 Moneymaker-PtoS 淹水后7–10天的品种 PstDC3000。接种前，10日龄幼苗已长出完全展开的子叶和初生的第一对真叶。将幼苗用10 mM MgCl₂溶液浸润2 + 0.015% 表面活性剂作为阴性对照（数据未显示）以及 PstDC3000（OD600 = 0.005）并添加0.015%表面活性剂。幼苗在淹水处理后7–10天进行表型观察。图5）。来自基因型同质系（如 Moneymaker-PtoR 和 Moneymaker-PtoS 在幼苗淹水试验中表现出高度一致且呈二元的表型。当Moneymaker-PtoR，携带 Pto/Prf 基因簇（n = 5）经.......
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讨论
一种用于淹水接种的方案 PstDC3000 或 Pst19 种用于检测番茄幼苗对这些细菌菌株抗性的优化方法已被描述。幼苗抗性测定中获得最佳结果的关键参数包括细菌浓度和表面活性剂浓度，这些参数均通过实验确定。22。对于 PstDC3000，通过优化光密度以实现在含有抗性基因的抗性品种上完全存活 Pto/Prf 在缺乏该基因的感病品种上形成簇状并完全致死 Pto/Prf 簇22对于像这样的菌株 Pst19，由于尚无已知的抗性品种，因此将光密度优化至尽可能低，以确保植株一致且完全死亡22. Uppalapati 等24 设计了一种番茄幼苗检测方法以研究其致病机制 PstDC3000 及其冠菌素的毒力功能。在此毒力测定实验中，感染过程使用浓度调整至 OD 的细菌进行600 0.1的
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披露
作者无任何利益冲突需要披露。
致谢
感谢Jamie Calma对培养基体积对疾病或抗性结果影响的测试。感谢Lewis实验室的Maël Baudin博士和Karl J. Scheiber博士对本手稿提供的建设性意见和建议。Lewis实验室的植物免疫研究得到了美国农业部农业研究局（USDA ARS）项目2030-21000-046-00D和2030-21000-050-00D（JDL）以及美国国家科学基金会生物科学局（NSF Directorate for Biological Sciences）项目IOS-1557661（JDL）的支持。
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 3M 胶带 Micropore 1/2" x 10 码 CS 240（1.25 cm x 9.1 m） | VWR International | 56222-182 | |
| 3 mm 硼硅酸盐玻璃珠 | Friedrich & Dimmock | GB3000B | |
| 胰蛋白胨（Bacto Peptone） | BD | 211677 | |
| 琼脂粉（Bacto Agar） | BD | 214010 | |
| Biophotometer Plus 分光光度计 | Eppendorf | E952000006 | |
| II 级 A2 型生物安全柜 | |||
| BRAND 一次性塑料比色皿，聚苯乙烯材质 | VWR International | 47744-642 | |
| Chenille Kraft 平头木制牙签 | VWR International | 500029-808 | |
| 放线菌酮（cycloheximide） | Research Products International | C81040-5.0 | |
| 无水磷酸氢二钾，ACS 级 | Fisher Scientific | P288-500 | |
| 二甲基甲酰胺（Dimethylformamide） | |||
| 解剖显微镜（放大倍数至少 10 倍） | |||
| 乙醇 - 190 证明 | |||
| Falcon 聚苯乙烯 96 孔微孔板，平底 | Fisher Scientific | 08-772-3 | |
| 玻璃酒精灯灯芯 | Fisher Scientific | S41898A / No. W-125 | |
| 玻璃酒精灯 | Fisher Scientific | S41898 / No. BO125 | |
| 甘油，ACS 级试剂 | VWR International | EMGX0185-5 | |
| Kimberly-Clark™ Kimtech Science™ Kimwipes™ 精密擦拭纸 | Fisher Scientific | 06-666-A | |
| 氯化镁，ACS 级 | VWR International | 97061-356 | |
| 七水合硫酸镁，ACS 级 | VWR International | 97062-130 | |
| 微量离心管，1.5 mL | |||
| 微量离心管，2.2 mL | |||
| Mini Beadbeater-96，115 伏 | Bio Spec Products Inc. | 1001 | |
| Murashige & Skoog 基本盐混合物 | Caisson Laboratories, Inc. | MSP01-50LT | |
| Pipet-Lite XLS LTS 8 通道移液器，20–200 μL | Rainin | L8-200XLS | |
| Pipet-Lite XLS LTS 8 通道移液器，2–20 μL | Rainin | L8-20XLS | |
| 聚苯乙烯 100 mm x 25 mm 无菌培养皿 | VWR International | 89107-632 | |
| 聚苯乙烯 150 mm x 15 mm 无菌培养皿 | Fisher Scientific | FB08-757-14 | |
| 聚苯乙烯 150 mm x 15 mm 无菌培养皿 | Fisher Scientific | 08-757-148 | |
| Pure Bright 杀菌漂白剂，有效氯含量 5.7%（定义为 100% 漂白剂） | Staples | 1013131 | |
| 利福平（Rifampicin） | Gold Biotechnology | R-120-25 | |
| Silwet L-77（非离子型有机硅表面活性剂共聚物，C13H34O4Si3 表面活性剂） | Fisher Scientific | NCO138454 | |
| 移液器吸头 LTS 20 μL，960/10，GPS-L10 | Rainin | 17005091 | |
| 移液器吸头 LTS 250 μL，960/10，GPS-L250 | Rainin | 17005093 | |
| VWR 解剖镊，细尖头，4.5" | VWR International | 82027-386 |
参考文献
- Underwood, W., Melotto, M., He, S. Y. Role of plant stomata in bacterial invasion. Cell Microbiology. 9 (7), 1621-1629 (2007).
- Zipfel, C. Early molecular events in PAMP-triggered immunity. Current Opinion in Plant Biology. 12 (4), 414-420 (2009).
- Galan, J. E., ....
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重印与许可
勘误
Formal Correction: Erratum: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay
Posted by JoVE Editors on 10/18/2023. Citeable Link.
An erratum was issued for: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay. The Introduction, Protocol, Representative Results and Discussion sections were updated.
The last paragraph of the Introduction section was updated from:
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7–14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain T1 (PstT1), and can easily be adapted to other bacterial strains of interest.
to:
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7–14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain 19 (Pst19), and can easily be adapted to other bacterial strains of interest.
Step 4.8 of the Protocol section was updated from:
- After 1 h, transfer the bottle to the biosafety cabinet and under aseptic conditions, add 1,600 µL of sterile 1 M MgSO4, and appropriate antibiotics to the media.
NOTE: For rifampicin resistant strains PstDC3000 and PstT1, use rifampicin dissolved in dimethylformamide at a final concentration of 50 µg/mL. Use cycloheximide dissolved in ethanol at a final concentration of 50 µg/mL to prevent fungal growth on the plates.
to:
- After 1 h, transfer the bottle to the biosafety cabinet and under aseptic conditions, add 1,600 µL of sterile 1 M MgSO4, and appropriate antibiotics to the media.
NOTE: For rifampicin resistant strains PstDC3000 and Pst19, use rifampicin dissolved in dimethylformamide at a final concentration of 50 µg/mL. Use cycloheximide dissolved in ethanol at a final concentration of 50 µg/mL to prevent fungal growth on the plates.
Step 5.2 of the Protocol section was updated from:
- Patch bacteria (i.e., PstT1) from a glycerol stock onto KB agar with appropriate antibiotics (section 4).
to:
- Patch bacteria (i.e., Pst19) from a glycerol stock onto KB agar with appropriate antibiotics (section 4).
Step 5.6 of the Protocol section was updated from:
- For PstT1, incubate the KB plate at 28 ˚C for 48 h prior to using bacteria in the flood experiment.
to:
- For Pst19, incubate the KB plate at 28 ˚C for 48 h prior to using bacteria in the flood experiment.
Step 6 of the Protocol section was updated from:
6. Preparation of PstT1 inoculum
to
6. Preparation of Pst19 inoculum
Step 6.2 of the Protocol section was updated from:
- Perform serial dilutions using sterile 10 mM MgCl2 solution in the biosafety cabinet. For PstT1, use a spectrophotometer to make inoculum with a starting concentration of OD600 = 0.1.
to:
- Perform serial dilutions using sterile 10 mM MgCl2 solution in the biosafety cabinet. For Pst19, use a spectrophotometer to make inoculum with a starting concentration of OD600 = 0.1.
Step 6.3 of the Protocol section was updated from:
- For PstT1, make a 1/10 dilution from the initial resuspension at OD600 = 0.1 to obtain a serial dilution at a concentration of OD600 = 0.01.
to:
- For Pst19, make a 1/10 dilution from the initial resuspension at OD600 = 0.1 to obtain a serial dilution at a concentration of OD600 = 0.01.
Step 8.3 of the Protocol section was updated from:
- Set a timer for 3 min. Measure 6 mL of final inoculum (PstT1 OD600 = 0.0075 [section 6] or PstDC3000 OD600 = 0.005 [section 7]) and transfer 6 mL of inoculum to each plate with the 10-day-old seedlings.
to:
- Set a timer for 3 min. Measure 6 mL of final inoculum (Pst19 OD600 = 0.0075 [section 6] or PstDC3000 OD600 = 0.005 [section 7]) and transfer 6 mL of inoculum to each plate with the 10-day-old seedlings.
Step 8.11 of the Protocol section was updated from:
- Phenotype after 7–10 days for PstDC3000 or 10–14 days for PstT1 (section 11). If carrying out bacterial growth assays, collect leaf tissue after 4 days (sections 9 and 10) and then phenotype (section 11). Alternatively, perform phenotypic analysis and bacterial growth assays on separate sets of plants.
to:
- Phenotype after 7–10 days for PstDC3000 or 10–14 days for Pst19 (section 11). If carrying out bacterial growth assays, collect leaf tissue after 4 days (sections 9 and 10) and then phenotype (section 11). Alternatively, perform phenotypic analysis and bacterial growth assays on separate sets of plants.
Step 10.7 of the Protocol section was updated from:
- After obtaining colony counts (Figure 2B), normalize the counts to 0.01 g of tissue for seedlings and convert to log bacterial growth (Table 1).
NOTE: The average mass of one Moneymaker-PtoS cotyledon is 0.01 g and is empirically determined22.
| Genotype1 Column A | Tissue Weight (g) Column B | # of Colonies in a spot Column C | Dilution factor for spot2 Column D | Adjusted # of Colonies3 Column E | Dilution factor for serial dilution Column F | Total # of Colonies Column G (cfu/0.01 g)4 | Average # of Colonies (cfu/0.01 g) Column H | Average Log Growth (cfu/0.01 g (log10)) Column I |
| Sample 1 | 0.004 g | 10 | 200 | calculated as: (C2 x 0.01 g) / B2 = 25 | 1000 | calculated as: (D2 x E2 x F2) = 5000000 | average for sample 1 through last sample: (ie. average G1:G3) = 7000000 | log of average ie. log(H2) = 6.85 |
| Sample 2 | 0.003 g | 15 | 200 | 50 | 1000 | 10000000 | ||
| Sample 3 | 0.002 g | 6 | 200 | 30 | 1000 | 6000000 | ||
| 1Data shown for 3 samples | ||||||||
| 2Based on plating 5 µL x 200 for 1 mL | ||||||||
| 3Cotyledons are too small to core so colony counts were normalized to 0.01 g of tissue based on the average mass of one MoneyMaker-PtoS cotyledon (data not shown) | ||||||||
| 4Adjusted per mL based on volume plated | ||||||||
Table 1: Sample calculations for seedling bacterial growth assay. Sample calculations demonstrate how to normalize bacterial counts and determine log bacterial growth.
to:
- After obtaining colony counts (Figure 2B), normalize the counts to 0.1 g of tissue for seedlings and convert to log bacterial growth (Table 1).
NOTE: The average mass of one Moneymaker-PtoS cotyledon is 0.1 g and is empirically determined22.
| Genotype1 Column A | Tissue Weight (g) Column B | # of Colonies in a spot Column C | Dilution factor for spot2 Column D | Adjusted # of Colonies3 Column E | Dilution factor for serial dilution Column F | Total # of Colonies Column G (cfu/0.01 g)4 | Average # of Colonies (cfu/0.01 g) Column H | Average Log Growth (cfu/0.1 g (log10)) Column I |
| Sample 1 | 0.04 g | 10 | 200 | calculated as: (C2 x 0.1 g) / B2 = 25 | 1000 | calculated as: (D2 x E2 x F2) = 5000000 | average for sample 1 through last sample: (ie. average G1:G3) = 7000000 | log of average ie. log(H2) = 6.85 |
| Sample 2 | 0.03 g | 15 | 200 | 50 | 1000 | 10000000 | ||
| Sample 3 | 0.02 g | 6 | 200 | 30 | 1000 | 6000000 | ||
| 1Data shown for 3 samples | ||||||||
| 2Based on plating 5 µL x 200 for 1 mL | ||||||||
| 3Cotyledons are too small to core so colony counts were normalized to 0.1 g of tissue based on the average mass of one MoneyMaker-PtoS cotyledon (data not shown) | ||||||||
| 4Adjusted per mL based on volume plated | ||||||||
Table 1: Sample calculations for seedling bacterial growth assay. Sample calculations demonstrate how to normalize bacterial counts and determine log bacterial growth.
Step 11.3 of the Protocol section was updated from:
- Phenotype plants infected with PstT1 at 10–14 days after flood inoculation.
to:
- Phenotype plants infected with Pst19 at 10–14 days after flood inoculation.
Figure 4 in the Protocol section was updated from:
Figure 4: Schematic representation of expected phenotypes for seedling resistance and death in various genetic backgrounds. (A) Seedlings of Rio Grande-PtoR and the near-isogenic cultivar Rio Grande-PtoS are displayed 7 days after flooding with PstDC3000 (OD600 = 0.005) + 0.015% surfactant. Rio Grande-PtoR displays consistent resistance, and Rio Grande-PtoS displays consistent susceptibility to infection with PstDC3000. These lines give rise to discrete and binary phenotypes. (B) Seedlings of a wild accession, such as Solanum neorickii LA1329, are shown 10 days after flooding with PstT1 (OD600 = 0.0075) + 0.015% surfactant. Seedlings display phenotypic variability but were recorded as binary phenotypes. The amount of phenotypic variability and the method of phenotyping (binary resistance or resistance spectrum) will depend on the particular accession tested. (C) Mapping populations generated by outcrossing wild accessions to susceptible cultivars may display a wider spectrum of phenotypes in F2 segregating populations. In this case, it may be most appropriate to record seedling phenotypes on a spectrum. Highly susceptible seedlings from a mapping population may be phenotyped for death as early as day 7 when flooded with PstT1, and typically show a brown apical meristem, no to very little extension of the epicotyl, and no new, green vegetative growth. The apical meristem of susceptible seedlings may stay green or very light brown for more time, and there may be some extension of the epicotyl and very little vegetative growth, which turns brown and arrests by day 10. Individual seedlings can be phenotyped for resistance based on the amount of new and ongoing vegetative growth by day 14. Seedlings can then be grouped based on the phenotypes described above into different categories of resistance such as weak, medium, or strong resistance. Please click here to view a larger version of this figure.
to:
Figure 4: Schematic representation of expected phenotypes for seedling resistance and death in various genetic backgrounds. (A) Seedlings of Rio Grande-PtoR and the near-isogenic cultivar Rio Grande-PtoS are displayed 7 days after flooding with PstDC3000 (OD600 = 0.005) + 0.015% surfactant. Rio Grande-PtoR displays consistent resistance, and Rio Grande-PtoS displays consistent susceptibility to infection with PstDC3000. These lines give rise to discrete and binary phenotypes. (B) Seedlings of a wild accession, such as Solanum neorickii LA1329, are shown 10 days after flooding with Pst19 (OD600 = 0.0075) + 0.015% surfactant. Seedlings display phenotypic variability but were recorded as binary phenotypes. The amount of phenotypic variability and the method of phenotyping (binary resistance or resistance spectrum) will depend on the particular accession tested. (C) Mapping populations generated by outcrossing wild accessions to susceptible cultivars may display a wider spectrum of phenotypes in F2 segregating populations. In this case, it may be most appropriate to record seedling phenotypes on a spectrum. Highly susceptible seedlings from a mapping population may be phenotyped for death as early as day 7 when flooded with Pst19, and typically show a brown apical meristem, no to very little extension of the epicotyl, and no new, green vegetative growth. The apical meristem of susceptible seedlings may stay green or very light brown for more time, and there may be some extension of the epicotyl and very little vegetative growth, which turns brown and arrests by day 10. Individual seedlings can be phenotyped for resistance based on the amount of new and ongoing vegetative growth by day 14. Seedlings can then be grouped based on the phenotypes described above into different categories of resistance such as weak, medium, or strong resistance. Please click here to view a larger version of this figure.
The second paragraph of the Representative Results section was updated from:
Phenotypic screening of wild accessions using the seedling resistance assay
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10–14 days after flooding with PstT1. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and PstT1 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as PstT1-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10–14 days after inoculation with PstT1. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with PstT1 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5–7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with PstT1, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
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Phenotypic screening of wild accessions using the seedling resistance assay
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10–14 days after flooding with Pst19. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and Pst19 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as Pst19-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10–14 days after inoculation with Pst19. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with Pst19 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5–7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with Pst19, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
Figure 6 in the Representative Results section was updated from:
Figure 6: Phenotypic characterization of resistance or disease symptoms 10–14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with PstT1 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
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Figure 6: Phenotypic characterization of resistance or disease symptoms 10–14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with Pst19 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
The third paragraph of the Representative Results section was updated from:
Quantitative assessment of bacterial growth using the seedling flood assay
To confirm that the observed resistance in LA1329 to PstT1 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of PstT1 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to PstT1 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
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Quantitative assessment of bacterial growth using the seedling flood assay
To confirm that the observed resistance in LA1329 to Pst19 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of Pst19 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to Pst19 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
Figure 7 in the Representative Results section was updated from:
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Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with PstT1 and normalization was performed to 0.01 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p < 0.001) followed by a multiple comparison of means using Tukey's post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. Please click here to view a larger version of this figure.
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Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with Pst19 and normalization was performed to 0.1 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p < 0.001) followed by a multiple comparison of means using Tukey's post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. Please click here to view a larger version of this figure.
The first paragraph of the Discussion section was updated from:
A protocol for flood inoculation with PstDC3000 or PstT1 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as PstT1, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
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A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as Pst19, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
The third paragraph of the Discussion section was updated from:
Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7–10 days after inoculation with PstDC3000 or 10–14 days after inoculation with PstT1, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
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Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7–10 days after inoculation with PstDC3000 or 10–14 days after inoculation with Pst19, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
The fourth paragraph of the Discussion section was updated from:
Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to PstT1 in adult plants as previously described22.
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Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to Pst19 in adult plants as previously described22.
