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

애기장대(Arabidopsis)의 박테리아 침입에 대한 자동 기공 반응 측정

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.

저자 스포트라이트: 자동 조리개 측정을 통한 기공 연구 발전

Pseudomonas syringae pv에 대한 기공 반응의 직접 관찰 및 자동 측정. 토마토 애기장대(Arabidopsis thaliana)의 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 pv의 유도. 토마토 자동화된 기공 반응 측정을 위한 애기장대 탈리아나의 DC3000 감염

시작하려면 애기장대(Arabidopsis thaliana) 씨앗을 토양에 심습니다. 백색 형광등이 있는 방에서 키운다. 다음으로, Pseudomonas syringae pv의 단일 콜로니를 접종합니다.토마토 DC3000 5 밀리리터의 King's B 액체 배지. 배양액이 후기 로그 성장 단계에 도달할 때까지 분당 200회전으로 흔들면서 섭씨 28도에서 배양물을 배양합니다. 6, 2 분 동안 000G에 문화를 분리하십시오.그런 다음 박테리아 펠릿을 멸균수 1m리터에 재현탁시킵니다. 다음으로, 상층액을 피펫팅합니다. 그리고 펠릿을 기공 개방 완충액 1밀리리터에 재현탁시킵니다.현탁액의 광학 밀도를 600나노미터로 측정합니다. 박테리아 현탁액을 0.2의 광학 밀도로 희석하고 0.4% 실리콘 계면활성제가 포함된 기공 개방 완충액을 사용합니다. 박테리아 현탁액을 접종하려면 먼저 애기장대 탈리아나 식물을 투명한 뚜껑으로 덮인 트레이에 최소 3시간 동안 빛 아래에서 놓습니다.뚜껑을 제거합니다. 그런 다음 에어 브러시를 사용하여 잎 아래쪽에 박테리아 현탁액을 뿌립니다. 접종 후 상대 습도를 약 85%로 유지하기 위해 투명한 뚜껑으로 덮인 트레이에 식물을 배양합니다

induction-pseudomonas-syringae-pv-tomato-dc3000-infection-arabidopsis

Research

JoVE Journal

Immunology and Infection

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

97 조회수.  Kyoto University. 시작하려면 애기장대(Arabidopsis thaliana) 씨앗을 토양에 심습니다. 백색 형광등이 있는 방에서 키운다. 다음으로, Pseudomonas syringae pv의 단일 콜로니를 접종합니다.토마토 DC3000 5 밀리리터의 King's B 액체 배지. 배양액이 후기 로그 성장 단계에 도달할 때까지 분당 200회전으로 흔들면서 섭씨 28도에서 배양물을 배양합니다. 6, 2 분 동안 000G에 문화를 분리하십시오.그런 다?...

비디오: Pseudomonas syringae pv의 유도. 토마토 자동화된 기공 반응 측정을 위한 애기장대 탈리아나의 DC3000 감염

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.

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.

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

연구

면역학 및 감염병학

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