In this procedure who Leaf imaging is used to assess guard cell movement in response to live bacterial cells. First, the bacteria accultured from a glycerol stock and an inoculum is prepared. Then young healthy leaves are collected and stained with propidium iodide.
The stained leaves are cut and the leaf pieces are incubated on the top of a bacterial suspension. Finally, the leaf surface is observed under the microscope and stomatal apertures can be measured. The results obtained using fluorescence microscopy show changes in the stomatal aperture in response to bacteria.
The main advantage of this method over existing methods like use of epidermal peels, is that the leaf in contact with bacteria is intact, mimicking the natural conditions for bacterial penetration into the leaves To begin this procedure. So Arabidopsis seeds on a one-to-one, to one V to V to V mixture of growing medium. Find vermiculite amite light grow the plants in a growth chamber and water as needed.
The plants are ready to use in four to six weeks when they have young, fully expanded leaves prior to bolting. Two days before starting the experiment, prepare the bacterium culture streak. Pseudomonas ringe from glycerol stock on modified LB medium, and incubate for 24 hours at 28 degrees Celsius.
Use fresh culture to prepare the inoculum and always start culture plates from glycerol stocks. As bacteria become less virulent after subculture, use bacteria that grew on the plate to start a 10 milliliter liquid culture of pseudomona singe. In a 50 milliliter erlenmeyer flask incubate the culture overnight at 28 degrees Celsius with vigorous shaking until it reaches optical density or od at 600 nanometers between 0.8 and one.
Measure the OD at 600 and harvest the cells by centrifugation at 1, 360 times G for 10 minutes. Resus suspend the cells in distilled water so that the final OD is 0.2. This od corresponds to 10 to the eight colony forming units per milliliter.
With the bacteria ready, proceed to prepare the leaves and perform the assay in order to stain the leaves. First, prepare 20 micromolar propidium, iodide, or PI solution in water. 10 milliliters of solution is sufficient to stain three small leaves at a time.
Retrieve the plants from the growth chamber and with forceps, detach three young fully expanded leaves. Immerse the whole leaves in the PI solution for five minutes. Then remove the leaves and rinse them briefly with distilled water.
Next, place a leaf with the lower surface facing down on a microscope slide. Cut the leaf with a sharp razor blade into four pieces, excluding the mid vein so that the leaf lays flat on the slide to incubate the bacteria with the leaves. Add 300 microliters of bacterial suspension under the leaf pieces on the slide, make sure that the lower surface of the leaf is in contact with the inoculum.
Incubate the samples under the same environmental conditions that were used to grow the plants. When it is time to observe leaves under the microscope, transfer the leaf pieces to a new slide with the lower surface facing up. Note that a cover slip is not used when mounting the slides.
The same leaf sample can be imaged multiple times during incubation, and a time course may be set up according to the research objectives. Here, a laser scanning confocal microscope is used to examine the leaves, observe the lower surface of the leaves to detect the fluorescence of PI using the same leaf samples, capture images of stoma over time. Save the images for measuring the width of the stomatal aperture.
Measure stomatal aperture width of at least 60 stoma for each treatment at each time point. Using the LSM image browser, calculate the average and standard error for the stomatal aperture width. Statistical significance of the results can be calculated using two-tailed paired wise students.
T-test here is a typical microscopic image of an arabidopsis leaf surface under a laser scanning confocal microscope. In brightfield view, propidium iodide stains the cell wall of viable cells increasing the visibility of guard cells in addition to allowing for the identification of undamaged cells. A range of stomatal PO openings can be seen in these micrographs from completely closed stoma to wide open stoma.
Completely closed stoma are identified by the shape of the guard cells. The aperture width is considered to be zero micrometer for open stoma. The aperture width shown is measured by using a straight line drawn across the widest area of the stoma pore.
These are typical experimental results of the live leaf inoculation assay. Arabidopsis leaves were incubated in the dark with three different strains of pseudomonas Cy Ringe, only the bacterium P-S-T-D-C 3000 was able to open dark clo stoma as measured by the aperture width of 60 randomly selected stoma per bacterial strain. This technique has paved a way for researchers in the field of planned bacterium interactions to explore the mechanisms of early response to pathogens in plant species.
