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

Medição Automatizada da Resposta Estomática à Invasão Bacteriana em 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.

Autor em destaque: Avançando na pesquisa estomática com medição automatizada de abertura

Observação Direta e Mensuração Automatizada das Respostas Estomáticas a Pseudomonas syringae pv. tomate DC3000 em Arabidopsis thaliana

Indução de Pseudomonas syringae pv. tomate Infecção por DC3000 em Arabidopsis thaliana para Medição Automatizada da Resposta Estomática

Para começar, plante sementes de Arabidopsis thaliana no solo. Cresça-os em uma câmara com luz fluorescente branca. Em seguida, inocular uma única colônia de Pseudomonas syringae pv.tomate DC3000 em 5 mililitros de meio líquido King's B. Incubar a cultura a 28 graus Celsius, com agitação a 200 rotações por minuto, até que a cultura atinja a fase logarítmica tardia de crescimento. Centrifugar a cultura a 6.000G por dois minutos.Em seguida, ressuspenda o pellet bacteriano em um mililitro de água estéril. Em seguida, pipetar o sobrenadante. E ressuspenda o pellet em um mililitro de tampão de abertura dos estômatos.Meça a densidade óptica da suspensão a 600 nanômetros. Diluir a suspensão bacteriana até uma densidade óptica de 0,2, com tampão de abertura dos estômatos contendo tensoativo de silicone a 0,4%. Para inocular a suspensão bacteriana, primeiro, coloque as plantas de Arabidopsis thaliana em uma bandeja coberta com uma tampa transparente sob luz por pelo menos três horas.Retire a tampa. Em seguida, use um aerógrafo para pulverizar a parte inferior das folhas com a suspensão bacteriana. Após a inoculação, incubar as plantas em uma bandeja coberta com tampa transparente para manter uma umidade relativa em torno de 85%

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

112 visualizações. Para começar, plante sementes de Arabidopsis thaliana no solo. Cresça-os em uma câmara com luz fluorescente branca. Em seguida, inocular uma única colônia de Pseudomonas syringae pv.tomate DC3000 em 5 mililitros de meio líquido King's B. Incubar a cultura a 28 graus Celsius, com agitação a 200 rotações por minuto, até que a cultura atinja a fase logarítmica tardia de crescimento. Centrifugar a cultura a 6.000G por dois minutos.Em seguida, ressuspenda o pellet bacteri...

Vídeo: Indução de Pseudomonas syringae pv. tomate Infecção por DC3000 em Arabidopsis thaliana para Medição Automatizada da Resposta Estomática

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 ...

Imunologia e Infecção

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

Medida Automatizada da Resposta Estomática em Arabidopsis thaliana em Resposta a Pseudomonas syringae pv. tomate DC3000 infecção