Inoculating a Bacterial Pathogen into Arabidopsis Leaves via Vacuum Infiltration

Take a bacterial culture capable of infecting leaf tissue.

Scrape the colonies and suspend in a salt solution.

Measure the optical density to assess bacterial concentration, then dilute to the desired OD.

Perform serial dilutions to reach the final inoculum concentration.

Add a surfactant to reduce surface tension and ensure even bacterial spreading on the leaf.

Transfer the suspension to a Petri dish.

Next, place two wooden sticks across the top of an Arabidopsis pot.

Invert the plant over the dish, immersing the leaves in the suspension.

Place the setup in a vacuum chamber and apply a vacuum pulse

The applied vacuum removes air from the leaf tissue through natural openings.

Releasing the vacuum restores atmospheric pressure, forcing bacteria into the leaves.

Repeat the pulses to achieve maximum infiltration.

Remove the pot. Then blot the excess inoculum.

The bacteria-infected Arabidopsis leaves are ready for downstream analysis.

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 5 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 plants to their corresponding growth chamber.