We thank all the members of the research project, &#39;Co-creation of plant adaptive traits via assembly of plant-microbe holobiont&#39;, for fruitful discussions. This work was supported by Grant-in-Aid for Transformative Research Areas (21H05151 and 21H05149 to A.M. and 21H05152 to Y.T.) and Grant-in-Aid for Challenging Exploratory Research (22K19178 to A. M.).

1. Growing plants
<ol>
	<li>To break dormancy, resuspend A. thaliana (Col-0) seeds in deionized water and incubate them at 4 &#176;C for 4 days in the dark.</li>
	<li>Sow the seeds on the soil and grow in a chamber equipped with white fluorescent light. Maintain the following growth conditions: temperature of 22 &#176;C, light intensity of 6,000 lux (ca. 100 &#181;mol/m2/s) for 10 h, and relative humidity of 60%.</li>
	<li>When needed, water the plants with a liquid fertilizer. Refrain from watering from 1 week to 2 days prior to inoculation and water well 1 day prior to inoculation.</li>
</ol>
2. Preparing bacterial inoculum
<ol>
	<li>Streak Pto from glycerol stock on solidified King&#39;s B (KB) medium (20 g of Tryptone, 1.5 g of K2HPO4, and 15 g of Glycerol for 1 L, 1.5% Agar) with 50 &#181;g/mL rifampicin and incubate at 28 &#176;C for 2 days.</li>
	<li>Inoculate a single colony to 5 mL of KB liquid medium with 50 &#181;g/mL rifampicin and incubate at 28 &#176;C with shaking at 200 rpm until late logarithmic growth phase.</li>
	<li>Centrifuge the culture at 6,000 x g for 2 min, discard the supernatant, and resuspend the pellet in 1 mL of sterile water. Repeat this step once again.</li>
	<li>Remove the supernatant, resuspend the pellet in 1 mL of stomata opening buffer (25 mM MES-KOH pH 6.15, 10 mM KCl), and measure the OD600.</li>
	<li>Dilute the suspension to OD600 0.2 with stomata opening buffer containing 0.04% silicone surfactant.</li>
</ol>
3. Spray&#160;inoculation of bacteria
<ol>
	<li>From 1 day before inoculation till the end of the experiment, expose plants to a light intensity of about 10,000 lux (ca. 170 &#181;mol/m2/s).</li>
	<li>To ensure that most stomata are open, keep plants on a tray covered with a transparent lid under the light for at least 3 h before spray inoculation.</li>
	<li>Remove the lid and use an airbrush to spray the abaxial side of leaves with 2.5 mL of bacterial suspension per three plants on a single pot.</li>
	<li>Incubate the inoculated plants on a tray covered with a transparent lid to maintain a relative humidity of around 85%.</li>
	<li>Acquire images of stomata at 1 h and 3 h after spray inoculation using the method described in section 4.</li>
</ol>
4. Direct observation of stomata using the portable imaging device
NOTE: The portable stomatal imaging device is equipped with an LED light and a camera module and can acquire 2,592 &#215; 1,944 (height &#215; width; pixels) images with a resolution of about 0.5 &#181;m/pixel.
<ol>
	<li>Connect the portable stomatal imaging device to a personal computer (PC) equipped with image acquisition software.</li>
	<li>Gently but completely remove water droplets from the inoculated leaves with a piece of paper.</li>
	<li>Open the top cover of the device, place the leaf on the stage, and close the top cover (Figure 1).</li>
	<li>Adjust the focus of the image by manipulating the adjuster screw, then click the Save Image button on the PC screen. The image will be acquired instantly. Typically, a focused image contains approximately 10 analyzable stomata. To obtain robust results, acquire stomatal images from six leaves of three different plants (two leaves per plant).</li>
</ol>
5. Manual measurement of stomatal aperture
NOTE: The ImageJ software can be downloaded at&#160;https://imagej.nih.gov/ij/download.html
<ol>
	<li>Open an image file in the ImageJ.</li>
	<li>Open the ROI manager by selecting Analyze &#62; Tools &#62; ROI Manager.</li>
	<li>Use the Straight-Line selection tool to draw a line corresponding to the width of a stoma (Figure 2) and register the ROI by clicking Add in the ROI Manager.</li>
	<li>Draw a line corresponding to the length of the same stoma (Figure 2A) and register the ROI as described in step 5.3.</li>
	<li>Click Measure in the ROI Manager to measure the width and length.</li>
	<li>Divide the width by the length to obtain the stomatal aperture (ratio). For robust quantification, use 60 or more stomata for each treatment and time point. Do not choose premature or obscure stomata for the analysis (Figure 2B, C).</li>
</ol>
6. Automated measurement of stomatal aperture
NOTE: The image analysis pipeline runs in Google Colaboratory, a cloud Python programming language executable environment. Users must have a valid Google account with a working Google Drive, Google Chrome browser, and a stable internet connection as a prerequisite.
<ol>
	<li>Download the Google Colaboratory notebook from Zenodo (https://doi.org/10.5281/zenodo.8062528), and open the notebook.</li>
	<li>Make a local copy of the notebook to Google Drive by selecting File &#62; Save a copy in Drive. After a new tab appears, safely close the tab of the original notebook.</li>
	<li>Press the Execute button once below the Environmental Setup section in the notebook without unfolding the cell blocks to import the required libraries.</li>
	<li>Execute the Directory Settings section to create three folders used for analysis (e.g., example_result, inference_results, and model) in Google Drive. 
	NOTE: In this case, the folders named example_result, inference_results, and model are used as the parental directory, storing inference results and trained models, respectively. This notebook shows an example of directory construction as a representative procedure. To change the name, rewrite the pardir, infdir, or modeldir path.</li>
	<li>According to the Preparation of the Images section, move the acquired images to example_result, grouped in image titles by treatment or sample (e.g., mock_1h_XXXXXX.jpg) for the final graph generation. Sample images are available from Zenodo (https://doi.org/10.5281/zenodo.8062528).</li>
	<li>Perform the Download Trained Models part to download the ONNX files of the trained models from Zenodo (https://doi.org/10.5281/zenodo.8062528) and place them under the&#160;model directory.</li>
	<li>Run the Inference and Measurement of Stomatal Aperture part to quantify the stomatal aperture from individual images. The result images with overlaid inference and the csv file named example_result.csv will be exported to the inference_results directory.</li>
	<li>Execute the Graph Generation section to create a graph about the stomatal aperture ratio, exported to the inference_results directory.</li>
</ol>

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

<ol>
	<li>Shimono, M., Higaki, T., Kaku, H., Shibuya, N., Hasezawa, S., Day, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+stomatal+cytoskeletal+patterns+during+the+activation+of+immune+signaling+in+Arabidopsis+thaliana.">Quantitative evaluation of stomatal cytoskeletal patterns during the activation of immune signaling in Arabidopsis thaliana.</a> PLoS One. 11, e0159291 (2016).</li><li>Bourdais, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+quantitative+imaging+to+investigate+regulators+of+membrane+trafficking+in+Arabidopsis+stomatal+closure.">The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure.</a> Traffic. 20 (2), 168-180 (2019).</li><li>Higaki, T., Kutsuna, N., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CARTA-based+semi-automatic+detection+of+stomatal+regions+on+an+Arabidopsis+cotyledon+surface.">CARTA-based semi-automatic detection of stomatal regions on an Arabidopsis cotyledon surface.</a> Plant Morphology. 26 (1), 9-12 (2014).</li><li>Eisele, J. F., F&#228;&#223;ler, F., B&#252;rgel, F., Chaban, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simple+method+for+microscopy-based+stomata+analyses.">A rapid and simple method for microscopy-based stomata analyses.</a> PLoS One. 11, e0164576 (2016).</li><li>Chitraker, R., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+stomatal+response+to+live+bacterial+cells+using+whole+leaf+imaging.">Assessing stomatal response to live bacterial cells using whole leaf imaging.</a> Journal of Visualized Experiments. 44, 2185 (2010).</li><li>Sai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=StomaAI:+an+efficient+and+user-friendly+tool+for+measurement+of+stomatal+pores+and+density+using+deep+computer+vision.">StomaAI: an efficient and user-friendly tool for measurement of stomatal pores and density using deep computer vision.</a> New Phytologist. 238 (2), 904-915 (2023).</li><li>Takagi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+quantification+of+Arabidopsis+thaliana+stomatal+aperture+from+leaf+images.">Image-based quantification of Arabidopsis thaliana stomatal aperture from leaf images.</a> Plant and Cell Physiology. pcad018, (2023).</li><li>Melotto, M., Zhang, L., Oblessuc, P. R., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+defense+a+decade+later.">Stomatal defense a decade later.</a> Plant Physiology. 174 (2), 561-571 (2017).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronatine+promotes+Pseudomonas+syringae+virulence+in+plants+by+activating+a+signaling+cascade+that+inhibits+salicylic+acid+accumulation.">Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation.</a> Cell Host and Microbe. 11 (6), 587-596 (2012).</li><li>Raffeiner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xanthomonas+type-III+effector+XopS+stabilizes+CaWRKY40a+to+regulate+defense+responses+and+stomatal+immunity+in+pepper+(Capsicum+annuum).">The Xanthomonas type-III effector XopS stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum).</a> The Plant Cell. 34 (5), 1684-1708 (2022).</li><li>Munemasa, S., Hauser, F., Park, J., Waadt, R., Brandt, B., Schroeder, J. 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The authors have no conflicts of interest to declare.

Previous studies used epidermal peels, leaf discs, or detached leaves to investigate stomatal responses to bacterial invasions9,11,12. In contrast, the method proposed in this study leverages the portable stomatal imaging device to directly observe stomata on a leaf attached to the plant after spray inoculation of Pto, mimicking natural conditions of bacterial invasion. In addition, because this method does not involve ...

Stomata are microscopic pores surrounded by a pair of guard cells on the surface of leaves and other aerial parts of plants. Under ever-changing environments, regulation of the stomatal aperture is central for plants to control the carbon dioxide uptake required for photosynthesis at the expense of water loss via transpiration. Thus, quantification of the stomatal aperture has been instrumental to understanding plant environmental adaptation. However, quantifying the stomatal aperture is inherently time-consuming and cumbersome as it requires human labor to spot and measure stomatal pores in a leaf image captured by a microscope. To circumvent these limitations, various methods have been developed to facilitate the quantification of stomatal aperture in Arabidopsis thaliana, a model plant extensively used to study stomatal biology1,2,3,4,5,6. For instance, a porometer can be used to measure transpiration rate as a metric of stomatal conductance. However, this method does not provide direct information on the stomatal number and aperture that determine stomatal conductance. Some studies have used confocal microscopy techniques highlighting stomatal pores using a fluorescent actin marker, a fluorescent dye, or cell wall autofluorescence1,2,3,4,5. While these approaches facilitate the detection of stomata, the cost of both operating a confocal microscopy facility and preparing microscopy samples can be an obstacle to routine application. In a ground-breaking work by Sai et al., a deep neural network model was developed to automatically measure stomatal aperture from bright-field microscopic images of A. thaliana epidermal peels6. Yet, this innovation does not exempt researchers from the task of preparing an epidermal peel for microscopic observation. Recently, this obstacle was overcome by developing a portable imaging device that can observe stomata by pinching a leaf of A. thaliana, together with a deep learning-based image analysis pipeline that automatically measures stomatal aperture from leaf images captured by the device7.
Stomata contribute to plant innate immunity against bacterial pathogens. The key to this immune response is stomatal closure that restricts bacterial entry through the microscopic pore into the leaf interior, where bacterial pathogens proliferate and cause diseases8. Stomatal closure is induced upon recognition of microbe-associated molecular patterns (MAMPs), immunogenic molecules that are often common to a class of microbes, by plasma membrane-localized pattern recognition receptors (PRRs)9. A 22 amino acid epitope of bacterial flagellin known as flg22 is a typical MAMP that induces stomatal closure through its recognition by the PRR FLS210. As a countermeasure, bacterial pathogens such as Pseudomonas syringae pv. tomato DC3000 (Pto) and Xanthomonas campestris pv. vesicatoria have evolved virulence mechanisms to reopen stomata9,11,12. These stomatal responses to bacterial pathogens have been conventionally analyzed in assays in which either leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion that occur in a leaf attached to the plant.
Here, a simple method is presented to investigate stomatal closure and reopening during Pto invasion under the condition that closely mimics the natural plant-bacteria interaction. This method leverages the portable imaging device for direct observation of A. thaliana stomata on a leaf attached to the plant inoculated with Pto, together with the image analysis pipeline for automated measurement of stomatal aperture.

<table><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 &amp;amp; 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>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Following spray inoculation of Pto, stomata on leaves attached to the inoculated plants were directly observed by the portable stomatal imaging device. Using manual and automated measurements, the same leaf images were used to calculate stomatal aperture by taking ratios of width to length of approximately 60 stomata. Manual and automated measurements consistently indicated a decrease in the stomatal aperture in Pto-inoculated plants compared with mock-inoculated plants at 1 hour post inoculation (hpi) ...

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

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

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

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

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Immunology and Infection

1.5K Views.  Kyoto University. Plantale huidmondjes spelen een centrale rol bij de aanpassing aan stressomstandigheden, dus we moeten de huidmondjesopening meten om te begrijpen hoe planten reageren op biotische en abiotische stress. Het meten van de huidmondjesopening is echter tijdrovend en omslachtig. Om de opening van huidmondjes te meten, pellen we de opperhuid en observeren we huidmondjes onder een microscoop.Vervolgens meten we handmatig de opening van huidmondjes, wat zelfs voor zeer ervaren onderzoekers lan...

Video: Auteur in de schijnwerpers: Stomataal onderzoek bevorderen met geautomatiseerde diafragmameting

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.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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).

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.

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

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.

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

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

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

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

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

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

Bioengineering

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.

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 ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Biology

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

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

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