This method can help answer the spatiotemporal regulation of a gene of interest during dynamic biological event such as immunity in intact arabidopsis leaves. The main advantage of this technique is that it allows us to capture the spatiotemporal dynamics of the promoter activity in soil grown intact plant leaves over few days. To begin this procedure, fill a plastic-cell plug tray with autoclaved soil.
Sow one transgenic arabidopsis seed into each cell. Transfer the tray into a growth room that is maintained at 23 degrees celsius and grow the plants under continuous white-light conditions for two to three weeks. Two days prior to pathogen inoculation, streak the pathogen carrying avrRpt2 from a glycerol stock onto NYG medium that contains rifampicin at 100 milligrams per liter and kanamycin at 50 milligrams per liter.
Incubate at 28 degree Celsius for 48 hours. Using plastic tips, harvest the bacterial cells that appear on the surface of the medium. Transfer the bacteria to a plastic tube containing 10 milliMolar magnesium chloride and resuspend them.
Then measure the optical density of the solution at 600 nanometers. Adjust the final concentration of bacterial cells to 100 million colony forming units per milliliter which normally corresponds to a OD600 of 0.2. First, carefully cut out a cell plug containing a two to three week old plant making sure not to damage the plant.
Set the cell in an empty cell plug tray to maintain a good balance. Select a visibly healthy leaf for inoculation. Noting that generally, the third, fourth, and fifth leaves from the bottom of the plant are easy to handle.
Water the soil holding the plant before inoculation for long-term time-lapse imaging. Optionally, when analyzing stress-responsive promoters examine the leaves under a fluorescence stereomicroscope prior to pathogen inoculation to verify the absence of YFP signal. Next, put on disposable latex gloves to avoid direct contact with the pathogen during infiltration.
Using a one milliliter needleless plastic syringe, carefully infiltrate the abaxial side of the leaf with the bacterial suspension. Inoculation of a small portion on one-half of the leaf enables a good visualization of the PR1 promoter activity. Use a soft paper towel to absorb any excess bacterial suspension from the area surrounding the infiltrated area on the infiltrated leaf.
Immediately after inoculation, use surgical tape to fix a glass slide to the plastic tray such that the infiltrated leaf is located at the center of the glass slide. Ensure that the inoculated leaf blade is completely fitted within the glass slide. Cut a piece of double-layered plastic tape into two pieces to fit the spaces along the petiole of the infiltrated leaf, and cut a corner off each piece.
Using a pair of fine tweezers, stick these pieces of tape on either side of the petiole such that the cut corners of each piece align with the base of the leaf blade, making sure that the tape pieces do not touch the petiole or the leaf blade. Next prepare an additional piece of double-layered plastic tape as outlined in figure two of the text protocol. Stick this piece of tape on top of the previously adhered pieces to form a bridge over the petiole, being careful to not catch the petiole or leaf blade directly between the tape pieces.
Then gently stick a small piece of surgical tape onto the glass slide above the tip of the leaf blade so that it is fixed very softly onto slide. Press down firmly only on the portion of the tape that is directly touching the glass slide. Gently place another small piece of surgical tape onto the border of the petiole and plastic tape pieces so that the petiole is very softly fixed onto both the glass slide and plastic tape pieces, making sure to only firmly attach the portion of the surgical tape that is directly touching the glass slide or the plastic tape pieces.
Insert 200 microliter pipette tips gently into the soil to gently hold the neighboring leaves away from the infiltrated leaf. First turn on the fluorescent stereomicroscope. Set the plant into the space under the objective lens of the stereomicroscope for imaging.
Set up the parameters for time-lapse imaging. Use the conventional YFP filter to visualize the YFP signal. Use the Texas Red filter to visualize chlorophyll autofluorescence so that the PCD domain is visible as a dark area surrounded by YFP-positive cell layers.
Next use the conventional epi-bright field setup for additional light exposure steps, making sure to program steps for light exposure during the interval period of the time-lapse imaging as light has a major impact on plant immunity. Run the time-lapse imaging program. For long-term observation over several days, consider watering the plant appropriately.
After image acquisition, omit extra channels used for light exposure in the intervals from the data set. Using image analysis software, analyze the data with various methods, such as region-of-interest analysis. In this study, a versatile method is demonstrated for the mounting of arabidopsis leaves for intravital time-lapse imaging.
A representative example of time-lapse image data using avrRpt2 induced ETI is obtained as both a series of images and as a time-lapse movie. In successful experiments using pPR1-YPF-NLS during avrRpt2 induced ETI, transient activation of the PR1 promoter is observed, as evident from YFP expressing foci and several layers of cells surrounding the PCD domain. The activation of the PR1 promoter in cells surrounding the PCD domain usually starts at approximately five hours post inoculation, peaks at approximately 12 hours post inoculation and lasts up to 40 hours post inoculation.
When attempting this procedure, time-lapse conditions need to be established carefully as described in the text. Following this procedure, the promoter activity can be analyzed quantitatively and qualitatively, using analysis software. These analysis help us to explore the spatiotemporal dynamics of gene expression in any biological event occurring in living leaves of arabidopsis plants.
