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

シロイヌナズナにおける細菌の侵入に対する自動気孔応答測定

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.

著者スポットライト:自動開口測定による気孔研究の進歩

Pseudomonas syringae pvに対する気孔応答の直接観察と自動測定。トマトシロイヌナズナのDC3000

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.

Pseudomonas syringae pvに応答したシロイヌナズナの気孔応答の自動測定。トマトDC3000感染

まず、ポータブル気孔イメージングデバイスを画像取得ソフトウェアを搭載したコンピューターに接続します。一枚の紙を使用して、シロイヌナズナ植物のシュードモナス注射器を接種した葉から水滴を慎重に取り除きます。次に、デバイスのトップカバーを開き、リーフをステージに置き、カバーを閉じます。アジャスターネジを使用してフォーカスを調整し、コンピューターの[画像を保存]をクリックして画像をキャプチャします。気孔開口部の自動測定を実行するには、Google Colaboratory ノートブックを開きます。[ファイル]、[コピーをドライブに保存]の順にクリックして、Googleドライブにローカルコピーを作成します。新しいタブが開いたら、元のノートブックのタブを閉じます。ノートブックの [環境設定] セクションの下にある [実行] ボタンを押して、セル ブロックを展開せずに必要なライブラリをインポートします。[ディレクトリ設定] セクションで、分析用に Google ドライブに 3 つのフォルダを作成します。取得した画像をexample_resultフォルダに移動し、処理またはサンプルごとに分類して、最終的なグラフを生成します。[トレーニング済みモデルのダウンロード] をクリックして、トレーニング済みモデルの ONNX ファイルを Zenodo からダウンロードします。ダウンロードしたファイルをモデルディレクトリに配置します。ノートブックの気孔開口部分の推論と測定を実行して、個々の画像から気孔開口を定量化します。推論とexample_resultを重ね合わせた結果の画像。csv ファイルが inference_results ディレクトリにエクスポートされます。グラフ生成セクションを実行して、気孔開口率に関するグラフを作成します。このグラフを inference_results ディレクトリにエクスポートします。Pto接種植物では、模擬接種植物と比較して、接種後1時間で気孔開口部の減少が観察されました。感染後3時間では、Pto接種植物と模擬接種植物の両方で気孔開口部はほぼ同じでした。気孔開口の自動測定は、1枚の画像を処理するのに約5秒かかり、測定時間を95%短縮しました。

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 ...

免疫・感染学

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

Pseudomonas syringae pvの誘導。トマトシロイヌナズナにおけるDC3000感染による気孔反応の自動測定