automated measurement of stomatal response in arabidopsis thaliana in response to pseudomonas syringae pv. tomato dc3000 infection

Mesure automatisée de la réponse stomatique à l’invasion bactérienne chez Arabidopsis

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.

Pleins feux sur l’auteur : Faire progresser la recherche sur les statistiques grâce à la mesure automatisée de l’ouverture

Observation directe et mesure automatisée des réponses stomatiques à Pseudomonas syringae pv. tomate DC3000 chez Arabidopsis thaliana

Plantal stomata play central roles in adaptation to stress conditions, so we need to measure stomata aperture to understand how plants respond to biotic and abiotic stresses. However, stomata aperture measurement is time-consuming and cumbersome. To measure stomata aperture, we peel the epidermis and observe stomata under a microscope.Then we manually measure stomata aperture, which takes a long time even for very experienced researchers. We challenged these barriers and developed a tool and a technique that can automatically measure stomata aperture in intact Arabidopsis leaves. So we strongly believe that stomata measuring device and deep-learning algorithm that we developed will facilitate functional analysis of stomata responses to various biotic and abiotic stresses because these technical advances significantly reduce time and human labor required for stomata aperture measurement.

Mesure automatisée de la réponse stomatique chez Arabidopsis thaliana en réponse à Pseudomonas syringae pv. tomate DC3000 Infection

Pour commencer, connectez un appareil d’imagerie stomatique portable à un ordinateur équipé d’un logiciel d’acquisition d’images. Utilisez un morceau de papier pour retirer soigneusement les gouttelettes d’eau des feuilles inoculées à Pseudomonas syringae de la plante Arabidopsis thaliana. Ouvrez maintenant le capot supérieur de l’appareil, placez la feuille sur la platine et fermez le couvercle.Ajustez la mise au point à l’aide de la vis de réglage et cliquez sur Enregistrer l’image sur l’ordinateur pour capturer l’image. Pour effectuer une mesure automatisée de l’ouverture des stomates, ouvrez le carnet Google Colaboratory. Cliquez sur Fichier puis sur Enregistrer une copie dans le lecteur pour faire une copie locale dans Google Drive.Lorsqu’un nouvel onglet s’ouvre, fermez l’onglet du bloc-notes d’origine. Appuyez sur le bouton Exécuter sous la section Configuration de l’environnement dans le bloc-notes pour importer les bibliothèques requises sans déplier les blocs de cellules. Dans la section Paramètres du répertoire, créez trois dossiers dans Google Drive pour analyse.Déplacez les images acquises dans le dossier example_result, en les classant par traitement ou par échantillon pour la génération finale du graphique. Cliquez sur Télécharger les modèles entraînés pour télécharger les fichiers ONNX des modèles entraînés depuis Zenodo. Placez les fichiers téléchargés dans le répertoire du modèle.Exécutez l’inférence et la mesure de la partie de l’ouverture stomatique du carnet pour quantifier l’ouverture stomatique à partir d’images individuelles. Les images résultantes avec inférence superposée et la example_result. CSV sera exporté vers le répertoire inference_results.Exécutez la section Génération de graphiques pour créer un graphique sur le rapport d’ouverture des stomates. Exportez ce graphique dans le répertoire inference_results. Une diminution de l’ouverture stomatique chez les plantes inoculées par PTO par rapport aux plantes inoculées a été observée une heure après l’inoculation.Trois heures après l’infection, l’ouverture stomatique chez les plantes auto-inoculées à la PTO et les plantes faussement inoculées était pratiquement la même. La mesure automatisée de l’ouverture des stomates a pris environ cinq secondes pour traiter une image, ce qui a entraîné une réduction de 95 % du temps de mesure.

Research

JoVE Journal

Immunology and Infection

Automated Measurement of Stomatal Response in Arabidopsis thaliana in Response to Pseudomonas syringae pv. tomato DC3000 Infection

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 ...

Recherche

Immunologie et infection

Induction of Pseudomonas syringae pv. tomato DC3000 Infection in Arabidopsis thaliana for Automated Stomatal Response Measurement

Induction de Pseudomonas syringae pv. tomate Infection à DC3000 chez Arabidopsis thaliana pour la mesure automatisée de la réponse stomatique