我们感谢研究项目“通过组装植物-微生物全息生物共同创造植物适应性状”的所有成员进行了富有成效的讨论。这项工作得到了变革性研究领域的补助金（21H05151 和 21H05149 到 A.M. 和 21H05152 到 Y.T.）和挑战性探索性研究的补助金（22K19178 到 A.M.）的支持。

1. 种植植物
<ol><li>为了打破休眠，将 拟南芥 （Col-0）种子重悬于去离子水中，并在4°C下在黑暗中孵育4天。</li>
	<li>将种子播种在土壤上，并在装有白色荧光灯的房间中生长。保持以下生长条件：温度为22°C，光强度为6,000勒克斯（约100μmol /m 2 / s）10小时，相对湿度为60%。</li>
	<li>需要时，用液体肥料给植物浇水。接种前 1 周至 2 天不要浇水，接种前 1 天浇水。</li>
</ol>2.制备细菌接种物
<ol><li>在凝固的King's B（KB）培养基（20g胰蛋白胨，1.5gK2HPO4和15g甘油的1L，1.5%琼脂）上从甘油储备中提取条纹Pto与50μg/ mL利福平，并在28°C下孵育2天。</li>
	<li>将单个菌落接种至含有50μg/ mL利福平的5mLKB液体培养基中，并在28°C下以200rpm振荡孵育至对数生长后期。</li>
	<li>以 6,000 x g 离心培养物 2 分钟，弃去上清液，并将沉淀重悬于 1 mL 无菌水中。再次重复此步骤。</li>
	<li>除去上清液，将沉淀重悬于1mL气孔开口缓冲液（25mM MES-KOH pH 6.15,10mM KCl）中，并测量OD600。</li>
	<li>用含有0.04%有机硅表面活性剂的气孔开口缓冲液将悬浮液稀释至OD600 0.2。</li>
</ol>3.喷雾接种细菌
<ol><li>从接种前1天到实验结束，将植物暴露在约10,000勒克斯（约170μmol/m2/s）的光照强度下。</li>
	<li>为确保大多数气孔是开放的，在喷雾接种之前，将植物放在覆盖有透明盖子的托盘上至少3小时。</li>
	<li>取下盖子，用喷枪在单个花盆上每三株植物喷洒 2.5 mL 细菌悬浮液的叶子背面。</li>
	<li>将接种的植物在覆盖有透明盖子的托盘上孵育，以保持约85%的相对湿度。</li>
	<li>使用第 4 节中描述的方法在喷雾接种后 1 小时和 3 小时获取气孔图像。</li>
</ol>4.使用便携式成像设备直接观察气孔
注意：便携式气孔成像设备配备 LED 灯和摄像头模块，可以采集 2,592 × 1,944（高×宽;像素）图像，分辨率约为 0.5 μm/像素。
<ol><li>将便携式气孔成像设备连接到配备图像采集软件的个人计算机 （PC）。</li>
	<li>用一张纸轻轻但完全去除接种叶子上的水滴。</li>
	<li>打开设备的顶盖，将叶子放在载物台上，然后关闭顶盖（图 1）。</li>
	<li>通过操作调节螺钉调整图像的焦距，然后单击 保存 图像 PC屏幕上的按钮。图像将立即被获取。通常，聚焦图像包含大约 10 个可分析的气孔。为了获得可靠的结果，从三种不同植物的六片叶子（每株植物两片叶子）获取气孔图像。</li>
</ol>5.气孔孔径的手动测量
注意：ImageJ 软件可在 https://imagej.nih.gov/ij/download.html 下载
<ol><li>在 ImageJ 中打开图像文件。</li>
	<li>通过选择 “分析>工具”>“ROI 管理器”打开 ROI 管理器。</li>
	<li>使用直线选择工具绘制一条与造口宽度相对应的线（图 2），并通过单击 ROI 管理器中的添加来注册 ROI。</li>
	<li>画一条对应于相同造口长度的线（图2A）并按照步骤5.3中的描述记录ROI。</li>
	<li>单击 ROI 管理器中的测量以测量宽度和长度。</li>
	<li>将宽度除以长度，得到气孔孔径（比率）。为了进行稳健的定量，每个治疗和时间点使用 60 个或更多气孔。不要选择过早或模糊的气孔进行分析（图2B，C）。</li>
</ol>6. 气孔孔径自动测量
注意：图像分析管道在 Google Colaboratory 中运行，这是一个云 Python 编程语言可执行环境。用户必须拥有有效的 Google 帐户，并且必须拥有有效的 Google Drive、Google Chrome 浏览器和稳定的互联网连接作为先决条件。
<ol><li>从 Zenodo （https://doi.org/10.5281/zenodo.8062528） 下载 Google Colaboratory 笔记本，然后打开笔记本。</li>
	<li>通过选择 “文件”>“在云端硬盘中保存副本”，将笔记本本地复制到 Google 云端硬盘。出现新选项卡后，安全地关闭原始笔记本的选项卡。</li>
	<li>按一下笔记本中“环境设置”部分下方的“执行”按钮，而无需展开单元块以导入所需的库。</li>
	<li>执行 “目录设置 ”部分，在 Google 云端硬盘中创建三个用于分析的文件夹（例如 example_result、inference_results 和 model）。 注意：在本例中，名为 example_result、inference_results 和 model 的文件夹用作父目录，分别存储推理结果和训练模型。此笔记本显示了作为代表过程的目录构造示例。若要更改名称，请重写 pardir、 infdir 或 modeldir 路径。</li>
	<li>根据 “图像的准备 ”部分，将采集的图像移动到 example_result，按处理或样本（例如，mock_1h_XXXXXX.jpg）分组在图像标题中，以生成最终的图形。示例图像可从 Zenodo （https://doi.org/10.5281/zenodo.8062528） 获得。</li>
	<li>执行下载 训练模型 部分，从 Zenodo （https://doi.org/10.5281/zenodo.8062528） 下载训练模型的 ONNX 文件，并将其放在模型 目录下。</li>
	<li>运行气 孔孔径的推断和测量 部分，从单个图像量化气孔径。具有叠加推理的结果图像和名为 example_result.csv 的 csv 文件将导出到 inference_results 目录。</li>
	<li>执行 “图形生成 ”部分以创建有关气孔孔径比的图形，并导出到 inference_results 目录。</li>
</ol>

拟南芥中细菌入侵的气孔反应自动测量

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作者没有利益冲突需要声明。

以前的研究使用表皮剥离、叶盘或分离的叶子来研究气孔对细菌入侵的反应 9,11,12。相比之下，本研究提出的方法利用便携式气孔成像设备直接观察喷洒接种Pto后附着在植物上的叶子上的气孔，模拟细菌入侵的自然条件。此外，由于该方法不涉及破坏性的样品制备过程，例如叶片脱落、叶盘切除和表皮剥离，因此可以避?...

气孔是植物叶子和其他地上部分表面被一对保卫细胞包围的微观孔隙。在不断变化的环境中，气孔的调节是植物控制光合作用所需的二氧化碳吸收的核心，而代价是通过蒸腾作用流失水分。因此，气孔孔径的量化有助于了解植物的环境适应性。然而，量化气孔本质上是耗时和繁琐的，因为它需要人工来发现和测量显微镜捕获的叶片图像中的气孔。为了规避这些限制，已经开发了各种方法来促进拟南芥气孔的量化，拟南芥是一种广泛用于研究气孔生物学的模式植物1,2,3,4,5,6。例如，孔径计可用于测量蒸腾速率，作为气孔导度的指标。然而，该方法不能提供关于确定气孔导率的气孔数和孔径的直接信息。一些研究使用共聚焦显微镜技术，使用荧光肌动蛋白标记物、荧光染料或细胞壁自发荧光1、2、3、4、5 突出气孔。虽然这些方法有助于气孔的检测，但操作共聚焦显微镜设施和制备显微镜样品的成本可能会成为常规应用的障碍。在Sai等人的一项开创性工作中，开发了一种深度神经网络模型，用于从拟南芥表皮剥离的明场显微图像中自动测量气孔孔径6。然而，这项创新并没有免除研究人员为显微镜观察准备表皮剥离的任务。最近，通过开发一种便携式成像设备克服了这一障碍，该设备可以通过捏住拟南芥的叶子来观察气孔，以及基于深度学习的图像分析管道，该管道可以从设备捕获的叶子图像中自动测量气孔孔径7。
气孔有助于植物对细菌病原体的先天免疫力。这种免疫反应的关键是气孔闭合，它限制细菌通过微观孔进入叶片内部，细菌病原体在那里增殖并引起疾病8.通过质膜定位模式识别受体 （PRR） 识别微生物相关分子模式 （MAMP） 时，诱导气孔闭合，MAMP 是一类微生物通常共有的免疫原性分子9。细菌鞭毛蛋白的 22 个氨基酸表位称为 flg22，是一种典型的 MAMP，它通过被 PRR FLS210 识别来诱导气孔闭合。作为对策，细菌病原体如丁香假单胞菌pv。番茄DC3000 （Pto） 和 Xanthomonas campestris pv.囊泡已经进化出毒力机制来重新打开气孔 9,11,12。这些对细菌病原体的气孔反应已在测定中进行了常规分析，其中叶表皮皮、叶盘或分离的叶子漂浮在细菌悬浮液上，然后在显微镜下观察气孔，然后手动测量气孔孔径。然而，这些测定很麻烦，可能无法反映附着在植物上的叶子中发生的对自然细菌入侵的气孔反应。
在这里，提出了一种简单的方法，用于在密切模拟自然植物-细菌相互作用的条件下研究 Pto 入侵期间的气孔关闭和重新开放。该方法利用便携式成像设备直接观察附着在接种Pto的植物上的叶片上的拟南芥气孔，以及用于自动测量气孔孔径的图像分析管道。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Nakarai tesque</td>
			<td>01028-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Airbrush kits</td>
			<td>ANEST IWATA</td>
			<td>MX2900</td>
			<td>Accessory kits for SPRINT JET</td>
		</tr>
		<tr>
			<td>Biotron</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>LPH-411S</td>
			<td>Plant Growth Chamber with white fluorescent light</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Wako</td>
			<td>072-00626</td>
			<td></td>
		</tr>
		<tr>
			<td>Half tray</td>
			<td>Sakata</td>
			<td>72000113</td>
			<td>A set of tray and lid</td>
		</tr>
		<tr>
			<td>Hyponex</td>
			<td>Hyponex</td>
			<td>No catalogue number available</td>
			<td>Dilute the solution of Hyponex at a ratio of 1:2000 in deionized water for watering plants</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>Natinal Institute of Health</td>
			<td>Download at https://imagej.nih.gov/ij/download.html</td>
			<td>Used for manual measurement of stomatal aperture</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>164-04295</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Wako</td>
			<td>168-21815</td>
			<td>For MES-KOH</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Wako</td>
			<td>343-01621</td>
			<td>For MES-KOH</td>
		</tr>
		<tr>
			<td>Portable stomatal imaging device</td>
			<td>Phytometrics</td>
			<td>Order at https://www.phytometrics.jp/</td>
			<td>Takagi et al.(2023) doi: 10.1093/pcp/pcad018.</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Wako</td>
			<td>185-01003</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr>
			<td>Silwet-L77</td>
			<td>Bio medical science</td>
			<td>BMS-SL7755</td>
			<td>silicone surfactant used in spray inoculation</td>
		</tr>
		<tr>
			<td>SPRINT JET</td>
			<td>ANEST IWATA</td>
			<td>IS-800</td>
			<td>Airbrush used for spray inoculation</td>
		</tr>
		<tr>
			<td>SuperMix A</td>
			<td>Sakata seed</td>
			<td>72000083</td>
			<td>Mix with Vermiculite G20 in equal proportions for preparing soil</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Nakarai tesque</td>
			<td>35640-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Vermiculite G20</td>
			<td>Nittai</td>
			<td>No catalogue number available</td>
			<td>Mix with Super Mix A in equal proportions for preparing soil</td>
		</tr>
		<tr>
			<td>White fluorescent light</td>
			<td>NEC</td>
			<td>FHF32EX-N-HX-S</td>
			<td>Used for Biotron</td>
		</tr>
	</tbody>
</table>

direct observation and automated measurement of stomatal responses to pseudomonas syringae pv. tomato dc3000 in arabidopsis thaliana

气孔是在植物叶片表皮中发现的微小孔隙。气孔的调节不仅对于平衡光合作用和蒸腾水分损失的二氧化碳吸收至关重要，而且对于限制细菌入侵也至关重要。当植物在识别微生物时关闭气孔，致病细菌，如 丁香假单胞菌 pv。 番茄 DC3000 （Pto），重新打开闭合的气孔以进入叶片内部。在评估气孔对细菌入侵反应的常规测定中，将叶表皮皮、叶盘或分离的叶漂浮在细菌悬浮液上，然后在显微镜下观察气孔，然后手动测量气孔孔径。然而，这些测定很麻烦，可能无法反映附着在植物上的叶子对自然细菌入侵的气孔反应。最近，开发了一种便携式成像设备，可以通过捏住叶子而不将其从植物上分离来观察气孔，以及基于深度学习的图像分析管道，旨在从设备捕获的叶子图像中自动测量气孔孔径。在这里，在这些技术进步的基础上，介绍了一种评估 拟南芥 细菌入侵的气孔反应的新方法。该方法包括三个简单的步骤：模拟自然感染过程的 Pto 喷雾接种，使用便携式成像设备直接观察 Pto接种植物叶子上的气孔，以及通过图像分析管道自动测量气孔径。该方法成功地用于在 Pto 侵袭期间在密切模拟天然植物-细菌相互作用的条件下气孔关闭和重新开放。

喷洒接种Pto后，通过便携式气孔成像设备直接观察附着在接种植物上的叶子上的气孔。使用手动和自动测量，使用相同的叶片图像通过获取大约 60 个气孔的宽度与长度之比来计算气孔孔径。手动和自动测量一致表明，在接种后 1 小时 （hpi） 时，与模拟接种的植物相比，Pto 接种植物的气孔孔径减小（图 3A，B），表明拟南芥植物在响应 Pto 入侵<stron...

Watch this Scientific Journal Video about Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement at JoVE.com

作者聚焦：通过自动孔径测量推进气孔研究

在这里，我们提出了一种简单的方法，用于直接观察和自动测量 拟南芥对细菌入侵的气孔反应。该方法利用便携式气孔成像设备，以及专为设备捕获的叶片图像而设计的图像分析管道。

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

植物气孔在适应胁迫条件方面起着核心作用，因此我们需要测量气孔孔径，以了解植物如何响应生物和非生物胁迫。然而，气孔孔径测量既费时又繁琐。为了测量气孔孔径，我们剥离表皮并在显微镜下观察气孔。然后我们手动测量气孔孔径，即使对于非常有经验的研究人员来说，这也需要很长时间。我们挑战了这些障碍，并开发了一种工具和技术，可以自动测量完整拟南芥叶片中的气孔孔径。因此，我们坚信，我们开发的气孔测量装置和深度学习算法将促进气孔对各种生物和非生物胁迫响应的功能分析，因为这些技术进步大大减少了气孔孔径测量所需的时间和人力。

direct-observation-automated-measurement-stomatal-responses-to

Research

JoVE Journal

Immunology and Infection

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

1.4K 次观看.  Kyoto University. 植物气孔在适应胁迫条件方面起着核心作用，因此我们需要测量气孔孔径，以了解植物如何响应生物和非生物胁迫。然而，气孔孔径测量既费时又繁琐。为了测量气孔孔径，我们剥离表皮并在显微镜下观察气孔。然后我们手动测量气孔孔径，即使对于非常有经验的研究人员来说，这也需要很长时间。我们挑战了这些障碍，并开发了一种工具和技术，可以自动测量完整拟?...

视频: 作者聚焦：通过自动孔径测量推进气孔研究

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

科研

免疫与感染

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

自动测量隐球菌种多糖胶囊和细胞体

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...