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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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相关的基因对基因抗性不同，植物还可表现出数量性部分抗性，这种抗性依赖于多个基因的共同作用7。
P. syringae pv. tomato（Pst）是番茄细菌性斑点病的致病菌，也是农业生产中长期存在的问题。田间主要流行菌株通常为Pst小种0（race 0）菌株，其表达一种或两种III型效应子AvrPto和AvrPtoB。DC3000（PstDC3000）是小种0的代表性菌株，也是一种可引起番茄细菌性斑点病的模式病原菌。为防治细菌性斑点病，育种人员已将来自野生番茄物种Solanum pimpinellifolium的Pto [P. syringae pv. tomato]/Prf [Pto抗性与芬硫磷敏感性]基因簇导入现代栽培品种中8,9。Pto基因编码一种丝氨酸-苏氨酸蛋白激酶，与Prf NLR蛋白共同作用，通过识别效应子AvrPto和AvrPtoB，赋予植物对PstDC3000的抗性10,11,12,13,14。然而，该抗性对近年来迅速且广泛传播的新兴小种1（race 1）菌株无效15,16。小种1菌株能够逃避Pto/Prf基因簇的识别，原因在于这些菌株中AvrPto基因缺失或发生突变，且AvrPtoB蛋白似乎积累量极低15,17,18。
野生番茄群体是丁香假单胞菌番茄致病变种（Pst）抗性自然变异的重要资源库，此前已被用于鉴定潜在的抗性位点19,20,21。然而，目前对病原菌抗性的筛选通常使用4至5周龄的成株植物20,21，因此受限于生长周期、生长室空间以及相对较小的样本量。为克服传统方法的局限性，我们开发了一种基于10日龄番茄幼苗的高通量番茄假单胞菌（P. syringae）抗性检测方法22。该方法相较于使用成株植物具有多项优势：生长周期更短、空间需求更小、通量更高。此外，我们已证实该方法能够真实再现成株植物中观察到的抗病表型22。
在本实验方案所述的幼苗淹水试验中，将番茄幼苗在无菌的Murashige和Skoog（MS）培养基平板上培养10天，随后用含有目标细菌及表面活性剂的接种液进行淹水处理。淹水处理后，可通过细菌增殖实验对幼苗的抗病性进行定量评估。此外，在淹水处理7至14天后，幼苗的存活或死亡可作为明确的抗性或感病表型指标。该方法为大量野生番茄种质资源对丁香假单胞菌番茄致病变种（Pst）小种1菌株（如Pst菌株19（Pst19））抗性的筛选提供了一种高通量的替代方案，并可轻松适用于其他感兴趣的细菌菌株。
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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 × 25 mm培养皿上标记40 mL的填充刻度。在无菌生物安全柜中将培养基倒入100 × 25 mm无菌培养皿中。
3. 植物材料的准备与培养条件
- 将番茄种子放入2.2 mL微量离心管中，加入2.0 mL 50%次氯酸钠溶液。
- 在摇床上轻轻摇动试管 25 分钟。
- 25分钟后，从摇床上取出种子，在超净生物安全柜中用移液器吸除次氯酸钠溶液，确保完全去除所有次氯酸钠。
- 加入 2 mL 无菌超纯 H2O 洗涤种子。将离心管颠倒 5 次。
- 用移液器从试管中移除液体。
- 重复步骤 3.3–3.5，再清洗种子 4 次。
- 加入 2 mL 无菌超纯 H2O，并将种子倒入一个无菌的空培养皿中。
- 将火焰灭菌后的镊子浸入乙醇中，待冷却后用于转移种子，并将其均匀放置于含有0.5×MS+0.8%琼脂培养基的100×25 mm培养皿中。
- 将5–7粒种子沿平板中央呈直线放置，并用手术胶带（1.25 cm × 9.1 m）密封平板边缘。
- 将灭菌后的种子在4 ˚C避光条件下进行分层处理至少3天，以同步萌发。确保培养皿平放且朝上叠放，防止种子在培养皿上发生位移。
- 将培养皿垂直放置，使种子排成水平线，转移至生长室后，根沿培养皿表面向下生长。
注意：将生长室温度设置为 22 ˚C，并提供光照强度约为 200–220 µE 米-2 s-1 和8小时黑暗。 - 在淹水处理前，将幼苗在生长培养箱中培养10天，此时幼苗通常已完全长出并展开子叶，并开始长出第一片真叶。图1).
图1：典型10天大番茄幼苗的发育阶段。 Rio Grande-PtoR 番茄种子经表面消毒后接种于培养基，并在4 ˚C黑暗条件下层积至少3天。幼苗在22 ˚C下于0.5x MS培养基中培养10天后进行淹水处理。通常在第10天时，子叶已完全展开，第一对真叶开始萌出。请点击此处查看该图的放大版本。
4. King's B23 (KB) 培养基的制备
- 在烧杯中加入 500 mL 超纯 H2O，并在搅拌器上搅拌。
- 将 20 g 细菌蛋白胨、1.5 g 无水 K2HPO4 和 12.5 mL 甘油完全溶解于盛有超纯 H2O 的烧杯中。
- 将溶解后的混合液倒入 1 L 量筒中，并用超纯 H2O 定容至 1 L。
- 将肉汤倒回烧杯中，搅拌至均匀。
- 称取 7.5 g 细菌琼脂，分装至两个 500 mL 玻璃瓶中，每瓶加入 500 mL 第 4.4 步制备的 KB 肉汤。高压灭菌 20 分钟。
- 从高压灭菌器中取出瓶子，轻轻旋转摇匀以分散琼脂。
- 将瓶子转移至 50 ˚C 水浴中，放置 1 小时。
- 1 小时后，将瓶子转移至生物安全柜中，在无菌条件下向培养基中加入 1,600 µL 无菌的 1 M MgSO4 以及适当的抗生素。
注意：对于利福平抗性菌株 PstDC3000 和 Pst19，使用溶解于二甲基甲酰胺中的利福平，终浓度为 50 µg/mL；使用溶解于乙醇中的放线菌酮，终浓度为 50 µg/mL，以防止平板上真菌生长。 - 轻轻旋转混合培养基，然后倾注平板，使培养基覆盖整个平板底部。
- 让平板静置至少 1 小时使其凝固，然后倒置存放于 4 ˚C。
5. 细菌菌株的保藏与培养条件
- 将单菌落细菌的甘油保存液保存在 -80 °C，其中含 1 mL 饱和细菌培养物和 333 µL 无菌 80% 甘油。
- 从甘油保存液中挑取细菌（例如 Pst19）至含适当抗生素的 KB 琼脂平板上（见第 4 节）。
- 在 28 °C 下培养 2 天，使细菌恢复生长，然后用无菌扁头牙签将新鲜细菌划线接种至选择性 KB 琼脂平板上。
- 使用无菌扁头牙签从甘油保存液中直接划线接种新鲜细菌至相应选择性 KB 琼脂平板上。
注意：确保所用甘油保存液不超过 2 周。 - 对于 PstDC3000，将 KB 平板在 28 °C 培养 24 小时，之后用于浸润实验。
- 对于 Pst19，将 KB 平板在 28 °C 培养 48 小时，之后用于浸润实验。
6. Pst19 接种体的制备
- 在无菌条件下，将细菌重悬于无菌的 10 mM MgCl2 溶液中，使其在 600 nm 处的光密度（OD600）为 0.1，或约 5 × 107 菌落形成单位（CFU）/mL。
- 在生物安全柜中使用无菌的 10 mM MgCl2 溶液进行系列稀释。对于 Pst19，使用分光光度计制备起始浓度为 OD600 = 0.1 的接种液。
- 对于 Pst19，将初始重悬液（OD600 = 0.1）进行 1/10 稀释，得到浓度为 OD600 = 0.01 的系列稀释液。
- 使用第 6.3 步中 OD600 = 0.01 的系列稀释液，再进行 3/4 稀释，获得最终 OD600 = 0.0075 的稀释液。
- 将非离子型有机硅表面活性剂共聚物 C13H34O4Si3（即表面活性剂）用 10 mM MgCl2 溶液进行 1/10 稀释，并涡旋振荡 15 秒。将该 1/10 倍浓度的表面活性剂母液加入到最后一次系列稀释液（OD600 = 0.0075）中，使其终浓度为 0.015%，充分混匀。
7. 准备PstDC3000 接种物
- 在无菌条件下，用无菌的 10 mM MgCl2 将细菌重悬，使其在 600 nm 处的光密度（OD600）为 0.1（约 5 × 107 CFU/mL）。
- 在生物安全柜中使用无菌的 10 mM MgCl2 溶液进行系列稀释。对于 PstDC3000，使用分光光度计制备起始浓度为 OD600 = 0.1 的接种液。
- 对于 PstDC3000，将 OD600 = 0.1 的初始重悬液进行 1/10 稀释，得到浓度为 OD600 = 0.01 的系列稀释液。
- 使用第 3 步中 OD600 = 0.01 的系列稀释液，再进行 1/2 稀释，获得最终 OD600 = 0.005 的稀释液。
- 将表面活性剂用 10 mM MgCl2 进行 1/10 稀释，涡旋振荡 15 秒。将该 1/10 的表面活性剂储备液加入到最后一步的稀释液（OD600 = 0.005）中，使表面活性剂终浓度为 0.015%，充分混匀。
8. 番茄幼苗淹水法
- 从生长室中取出含有10天龄幼苗的培养皿，放入生物安全柜中，准备进行淹水处理。
- 从两个培养皿上移除外科胶带。
- 设置计时器为3分钟。量取6 mL终浓度的接种液（Pst19 OD600 = 0.0075 [第6节] 或 PstDC3000 OD600 = 0.005 [第7节]），并将6 mL接种液分别转移至含有10天龄幼苗的每个培养皿中。
- 用无菌移液器吸头轻轻将幼苗压入接种液中。启动计时器。
- 双手各持一个培养皿，将培养皿前端倾斜向下，使接种液聚集并主要浸没幼苗的子叶和叶片。
- 左右轻轻晃动5–7次，然后将培养皿向后倾斜，使根部及整个培养皿表面均被接种液覆盖。
- 再次将培养皿倾斜向下，使子叶和叶片浸入接种液中，并重复此操作，持续共3分钟。
- 将接种液从培养皿中倒出，将培养皿平放在台面上，再次倾倒以去除残留的接种液。
- 重新用外科胶带包裹培养皿，并对剩余的所有培养皿重复步骤8.2–8.8。
- 所有培养皿完成淹水处理后，将其重新放回生长室中继续培养（参见步骤3.11的注释）。
- 接种PstDC3000后7–10天或接种Pst19后10–14天进行表型观察（第11节）。若进行细菌增殖测定，应在接种后第4天采集叶片组织（第9和第10节），随后进行表型分析（第11节）。或者，可分别在不同组植物上进行表型分析和细菌增殖测定。
9. 子叶表面灭菌以进行细菌生长测定
- 在淹水处理并重新将幼苗放回生长室培养四天后（第8节），从生长室中取出带有番茄幼苗的培养皿。
- 在培养皿底部外侧对应每株幼苗与皿壁连接处，为每个基因型的幼苗编号。
- 用对应幼苗编号标记无菌的1.5 mL微量离心管，并使用洁净镊子向每管中加入一颗3 mm无菌硼硅酸盐小珠，用于珠磨处理。（参见步骤10.1中的注释。）
- 向每管中加入200 µL的10 mM MgCl2溶液，并盖紧管盖。
- 配制70%乙醇，并将100 mL倒入洁净的烧杯中；另取一个洁净烧杯，倒入100 mL无菌超纯H2O。
- 用乙醇清洁带锯齿尖端的不锈钢细头直镊，略微打开培养皿，以便用洁净镊子无菌取出一片子叶。
- 用镊子夹住子叶基部的叶柄，取下一片叶片，放入盛有70%乙醇的烧杯中，表面灭菌10秒；随后将子叶在超纯H2O中漂洗10秒。
- 将子叶置于纸巾上，用精密科学擦拭纸吸干表面水分。
- 表面灭菌并吸干后，单独称量每片子叶的重量，并记录数据。
- 将子叶放入先前已准备好的1.5 mL微量离心管中（步骤9.3和9.4），管上已标记对应的基因型和个体编号。
- 用无菌胶带重新密封培养皿，并将幼苗放回生长室继续培养（参见第3.11步注释）。
10. 细菌生长实验
- 使用步骤 9.10 中的样品，在 10 mM MgCl2 中利用珠磨机对组织进行 1–2 分钟的匀浆处理。如果组织未充分破碎，需再次进行匀浆。
注意：许多厂商生产珠磨机匀浆器。珠子的数量和类型，以及匀浆时间与速度（若可编程）应根据每种类型的匀浆器进行优化。确保样品在匀浆过程中不过热。 - 向含有步骤 10.1 中破碎组织的每个试管中加入 800 µL 的 10 mM MgCl2，反复倒置数次以混匀。
- 使用多通道移液器，在 96 孔板中用 10 mM MgCl2 对每个样品进行系列稀释（100、10-1、10-2、10-3、10-4、10-5）（图 2A）。
- 使用多通道移液器从每个稀释系列中吸取 5 µL，点样于含环己酰亚胺及目标菌株所需选择性抗生素的 KB 琼脂平板（150 mm × 15 mm）上（参见步骤 4.8 的注意说明）。让平板完全干燥。
- 将平板倒置于 28 ˚C 培养 36 小时，然后使用解剖显微镜观察平板上的菌落（图 2B），判断菌落是否足够大以进行计数。
注意：如果菌落尚未足够大，可重新孵育平板，并每隔几小时重新检查菌落大小。通常情况下，菌落在培养约 36–48 小时后即可计数。
图 2：幼苗细菌生长测定的系列稀释。 （A）来自感病植株的研磨叶片组织在进行菌落计数前进行稀释。稀释在96孔板中进行（100为未稀释）。通常稀释梯度为10-1至10-5。（B）用于细菌菌落计数的稀释液涂布。将稀释系列中每一列共5 µL样品从最稀释到最浓缩依次涂布。待菌落完全干燥后，将平板在28 ˚C下培养36–48 h。在10x解剖显微镜下计数菌落。请点击此处查看该图的放大版本。
- 在菌落融合之前，使用解剖显微镜对菌落进行计数（图2B）。选择稀释系列平板中菌落数少于100的平板进行计数。
- 获得菌落计数后（图2B），将计数值标准化为每0.1 g组织（用于幼苗），并转换为细菌生长对数值（表1）。
注：Moneymaker-PtoS 子叶的平均质量为0.1 g，该数值为经验测定22。
| 基因型1 第A列 | 组织重量 (g) 第B列 | 单个点上的菌落数 第C列 | 点样稀释倍数2 第D列 | 校正后菌落数3 第E列 | 系列稀释倍数 第F列 | 总菌落数 第G列 (cfu/0.01 g)4 | 平均菌落数 (cfu/0.01 g) 第H列 | 平均对数生长量 (cfu/0.1 g (log10)) 第I列 |
| 样品 1 | 0.04 g | 10 | 200 | 计算方法: (C2 × 0.1 g) / B2 = 25 | 1000 | 计算方法: (D2 × E2 × F2) = 5000000 | 样品1至最后一个样品的平均值: (例如，G1:G3的平均值) = 7000000 | 平均值的对数，即 log(H2) = 6.85 |
| 样品 2 | 0.03 g | 15 | 200 | 50 | 1000 | 10000000 | ||
| 样品 3 | 0.02 g | 6 | 200 | 30 | 1000 | 6000000 | ||
| 1数据显示为3个样品 | ||||||||
| 2基于将5 µL × 200点样至1 mL | ||||||||
| 3子叶过小无法打孔取样，因此菌落计数根据一个MoneyMaker-PtoS子叶的平均质量归一化至0.1 g组织（数据未显示） | ||||||||
| 4根据点样体积调整为每毫升的数值 | ||||||||
表1：幼苗细菌生长测定的示例计算。 示例计算展示了如何对细菌计数进行归一化处理，并确定细菌生长的对数值。
- 对于野生种质及其他遗传背景复杂的品系，将单个幼苗中的细菌生长水平与其表型相关联，具体方法如第11节所述。
11. 抗性表型分析
- 从生长室中取出培养皿，在接种后7–14天对单株幼苗进行表型观察，判断其死亡（由病害导致）或存活（由抗性导致）。
- 对于接种了高毒力菌株（如PstDC3000）的植株，应在接种后7–10天较早进行表型观察。
- 对于接种了Pst19的植株，应在接种后10–14天进行表型观察。
- 根据观察到的抗性表型范围建立评分系统。对于表现出稳定、强至中等抗性表型的品种、近等基因系和野生种质材料，记录其二元表型（图4A, 4B）。
- 若幼苗在表型观察期间从顶端分生组织长出新叶，则记为存活；若幼苗顶端分生组织呈褐色，且无任何新的绿色营养生长，则记为死亡（图3）。
图 3：番茄幼苗的示意图。 图示番茄幼苗的不同部位，包括下胚轴、子叶、上胚轴、茎尖分生组织和真叶。 请点击此处查看此图的放大版本。
- 在具有广泛抗性表型（如 F2 作图群体）的群体中，记录疾病谱上的表型（图 4C）。
- 密切监测幼苗是否出现病害症状及死亡，以确定表型鉴定的合适时间窗口。
图4：不同遗传背景下幼苗抗性与死亡表型的示意图。（A）将Rio Grande-PtoR与近等基因系品种Rio Grande-PtoS的幼苗用PstDC3000（OD600 = 0.005）+ 0.015% 表面活性剂浸润处理后第7天的表型。Rio Grande-PtoR表现出稳定的抗性，而Rio Grande-PtoS对PstDC3000感染表现出稳定的感病性。这两个品系产生明确且呈二元分布的表型。（B）将野生种质（如Solanum neorickii LA1329）的幼苗用Pst19（OD600 = 0.0075）+ 0.015% 表面活性剂浸润处理后第10天的表型。幼苗表现出表型变异，但记录为二元表型。表型变异的程度以及表型记录方法（二元抗性或抗性谱）取决于所测试的具体种质。（C）通过将野生种质与感病品种杂交产生的作图群体，其F2分离群体可能表现出更广泛的表型谱。在此情况下，以连续谱形式记录幼苗表型可能更为合适。来自作图群体的高感病幼苗在用Pst19浸润处理后最早第7天即可观察到死亡表型，通常表现为顶端分生组织呈褐色、上胚轴无或极少伸长、无新的绿色营养生长。感病幼苗的顶端分生组织可能在较长时间内保持绿色或浅褐色，上胚轴可能有一定伸长，营养生长极少，至第10天转为褐色并停止生长。可根据第14天时新发生长和持续营养生长的程度对单株幼苗进行抗性表型鉴定。随后可根据上述描述的表型将幼苗划分为不同的抗性等级，例如弱抗性、中等抗性或强抗性。请点击此处查看此图的放大版本。
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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 |
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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.
to:
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.
to:
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.
to:
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:
x
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.
x
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.
to:
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.
to:
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.
to:
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.
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