Our protocol provides a gentle plant inoculation and extraction method that allows recovering bacterial populations grown in the leaf apoplast suitable for single-cell gene expression analysis such as flow cytometry. This method allows the extraction of a considerable amount of bacteria from the apoplast without significantly contaminating the samples with plant cellular debris. This optimized method for apoplastic bacteria recovery can be applied directly or with minor modifications to a number of plant bacterial systems comprising either plant pathogenic bacteria or beneficial endocytic bacteria.
Demonstrating the procedure will be Nieves Lopez-Pagan, a Ph.D student from our laboratory. To begin, fill a 10-centimeter-diameter pot with previously watered 1:3 vermiculite plant substrate mix. Cover the pot with a perforated metal mesh and adjust the metal mesh to the wet soil using a rubber band.
Next, sow Arabidopsis seeds into the holes of the metal mesh with a wet toothpick. Place three to four seeds in distant positions within the pot. Cover the pot with a plastic dome to maintain high relative humidity and incubate for stratification at four degrees Celsius for 72 hours.
Transfer the pot to a plant growth chamber under short-day conditions. To uncover the pot, remove the plastic dome. Once the seeds are germinated, use the tweezers to remove most of the seedlings, keeping one seedling in each of the positions of the pot.
Then prepare Phaseolus vulgaris bean plants by covering the bottom of a Petri dish with a wet piece of paper towel and placing the bean seeds on top of it. Seal the Petri dish with surgical tape and incubate at 28 degrees Celsius for three to four days. Transfer the germinated seeds into a 10-centimeter-diameter pot filled with wet 1:3 vermiculite plant substrate mix and incubate in a plant growth chamber under long-day settings.
Streak out the glycerol stock of Pseudomonas syringae strain of interest from minus 80 degrees Celsius into an LB plate supplemented with the appropriate antibiotics. Incubate the plate at 28 degrees Celsius for 40 to 48 hours. Scrape the bacterial biomass and resuspend it in five milliliters of 10-millimolar magnesium chloride.
Measure the optical density at 600 nanometers and adjust it to 0.1 by adding 10-millimolar magnesium chloride. Perform serial dilutions in 10-millimolar magnesium chloride to get a final inoculum concentration of five times 10 to the fifth CFU per milliliter. Then, prepare 200 milliliters of inoculum for the Arabidopsis plants and 50 milliliters for the bean plants.
Before inoculation, add the surfactant Silwet L-77 to a final concentration of 0.02%for bean inoculation and 0.01%for Arabidopsis. For Arabidopsis infiltration, place two wood sticks forming an X over the pot and place the pot facing down over a 14-centimeter diameter Petri dish containing 200 milliliters of inoculum. For bean leaves inoculation, insert the plants and the inoculum solution into a vacuum chamber and introduce the leaf into a 50-milliliter conical centrifuge tube containing the inoculum.
Give a pulse of 500 millibars for 30 seconds to infiltrate the leaves. Drain the excess inoculum solution with a piece of paper and return the plans to their corresponding growth chamber. Four days after inoculation, cut either the aerial part of the Arabidopsis plant or the inoculated leaf from the bean plant and place it into a 20-milliliter syringe without a needle.
For bean leaves, roll the leaf on itself, leaving the abaxial face outward. Add enough volume of distilled water to cover the tissue. Then, insert the plunger holding the syringe in an upright position and remove the excess air and air bubbles inside the syringe by gently tapping the barrel until all the air is located near the tip, and, after removing the air, cover the tip of the syringe barrel with paraffin film.
Now carefully press the plunger to generate positive pressure until the tissue turns darker. Then, pull the plunger to generate negative pressure. Remove the paraffin film and the plunger and collect the fluid containing the apoplast-extracted bacteria.
For a single-cell analysis, prepare a 1.5%agarose solution in distilled water. Once melted, add enough volume to fill the space between two microscopy slides set side by side and place another slide on top. Let them drive for 15 minutes and remove the slide placed on the top carefully.
Using a blade, cut the agarose pad into five millimeter by five millimeter pieces before use. Parallely, centrifuge one millimeter of the apoplast-extracted bacteria. Carefully remove the supernatant using a pipette and resuspend the pellet into 20 microliters of water to concentrate the cells.
Place a two-microliter drop of the concentrated cells onto a 0.17-millimeter cover slip and cover the drop with a piece of agarose pad. Visualize the bacterial preparation under the confocal microscope to identify green fluorescent bacteria using specific lasers. Identify all the bacteria using the bright-field and merge both fields.
To perform analysis by flow cytometry, take an aliquot of the apoplast-extracted bacteria suspensions. Analyze using the flow cytometer and acquire 100, 000 events. Expression of hrpL GFP is monitored by GFP fluorescence.
The dot plot graphs show the fluorescence intensity distribution of GFP versus cell size in the population, while the histograms show the fluorescence intensity of GFP versus cell counts. The hrpL ON percentages were higher than the corresponding percentages of hrpL OFF. Fluorescence microscopy images show heterogeneous levels of GFP associated with the expression of hrpL.
The bacteria displaying low or no GFP fluorescence are observed. For optimal results, be gentle during the bacteria extraction steps to avoid damaging the plant tissue and thus limit contamination from plant cellular content. The bacteria samples obtained can be used for other purposes where reducing plant contamination is an advantage, such as nucleic acid extractions for subsequent bacterial genomic or transcriptomic analysis.
