A Simplified Method for Agrobacterium-mediated Transformation of Phytophthora palmivora

This protocol offers a simple, effective, and reproducible system to transform phytophthora palmivora and phytophthora capsici. To begin, take a new LB auger plate supplemented with antibiotics. Add 20 microliters of LB liquid media to the center of the plate and scoop agrobacterium cells from a previously grown plate with a sterile 1.5 milliliter microcentrifuge tube.

Using the bottom and flat base of the tube, spread the cells evenly across the plate. Incubate the plate overnight at 28 degrees Celsius for approximately 16 to 18 hours. Next, scoop the overnight grown agrobacterium cells using a one milliliter pipette tip.

Resuspend the cells in one milliliter of agrobacterium induction medium by pipetting up and down. Now, transfer the resuspended cells into a sterile 50 milliliter tube covered with foil and add agrobacterium induction medium. Measure the optical density of the sample at 600 nanometers and dilute it with agrobacterium induction medium to achieve an optical density of 0.4.

Then loosen the lid of the foil covered tube containing the bacterial suspension. Place it on a platform shaker with gentle agitation at approximately 70 revolutions per minute. Flood a seven to eight day old phytophthora palmivora plate with 10 milliliters of water pre-cooled to four degrees Celsius.

Use a pipette tip to gently tap the surface and remove any air bubbles on the media. Incubate the plate at four degrees Celsius for 15 minutes. Then transfer it to room temperature under light for another 15 minutes.

Using a pipette, gently transfer approximately seven to eight milliliters of released zoospores into a sterile 15 milliliter centrifuge tube without disturbing the mycelia and sporangia. Then transfer 20 microliters of the zoospore suspension to a 1.5 milliliter microcentrifuge tube, and add 180 microliters of water to dilute the suspension tenfold. Vortex the tube for one minute to insist the zoospores and use a hemocytometer under a microscope to count the zoospores.

Add five milliliters of the prepared agrobacterium suspension to five milliliters of the zoospore suspension in a sterile 50 milliliter foil covered tube. Gently mix the contents, loosen the lid of the tube, and let it sit at room temperature for two hours. During the incubation, prepare the AZCA plates.

Once the medium solidifies, place one piece of sterilized Hybond-N+membrane onto the plate and keep the plate in the dark. After two hours of co-incubation, use a pipette tip to spread 300 microliters of the mixture onto the Hybond-N+membrane, ensuring the margins are free from the liquid. Leave the Petri dishes uncovered in the hood for about 10 minutes to allow the mixture to air dry.

Once the liquid stops moving on the membrane, cover the plates. Incubate the covered plates at 25 degrees Celsius in the dark for two days. After 48 hours of incubation, use sterilized forceps to transfer the Hybond-N+membrane upside down onto the Plich auger plate without dragging it across the auger surface.

Use the flat base of a 1.5 milliliter microcentrifuge tube to gently press and slide across the membrane surface to ensure full contact with the medium. Check the underside of the plate for air bubbles and gently press it out using the same flat base. Incubate the plate at 25 degrees Celsius under a 12-hour light and 12-hour dark cycle for two to three days.

Finally, after two to three days of incubation, remove the membrane using sterile forceps and continue incubating the plate under the same light and temperature conditions. Co-incubation of phytophthora palmivora zoospores with agrobacterium tumefaciens EHA 105 expressing pCB301TOR-GFP yielded G418-resistant colonies on Plich auger supplemented with 30 micrograms per milliliter G418, whereas no colonies appeared on control plates without agrobacterium infection. The number of G418-resistant transformants per 10 to the power seven zoospores was 55 in experiment one and 50 in experiment two.

Fluorescence microscopy confirmed a clear green fluorescent protein signal in phytophthora palmivora transformants. Additionally, this protocol was applied to phytophthora capsici to successfully generate transformants expressing GFP. Our protocol allows researchers to study gene functions, determine protein cellular localization, and track pathogen infection dynamics.

These protocol may generate a low percentage of false positives and hence requires additional approaches to confirm the true transformants. Application of this transformation system can be extended to other oomycetes and fungi. We will also try to further simplify this protocol in the future.