The overall goal of this system is to study the reciprocal signaling interaction and responses between plant and micro-partners or plant responses to chemicals or abiotic factors in a simply experimental setup that closely mimics the natural environment. This video shows co-cultivation of plants with Agrobacterium tumefaciens bacterium. This method can help answer key questions in plants'microbial interactions, microbial or plants stress response, and in molecular signaling, including complex signaling even at an initial stage of a plant's microbial interactions.
The main advantage of this technique is that it helps to understand the nature signaling and response of a plant's microbial interactions, in a simple set-up that includes the mimic of natural environment. Though this method can be used to provide insight into plant interactions with a single microbial species, it can also be used to study plant interactions with multiple microbial species or with abiotic factors. We had the idea for this method while searching for cell signaling homeostasis during plant microbe interactions that closely mimic natural situations.
To begin the experiment, seeds must be sterilized. For Arabidopsis thaliana, combine approximately 200 seeds with 500 microliters of deionized water in a micro centrifuge tube, and vortex the tube at high speed. Centrifuge the tube at 9000 x g for 30 seconds in a tabletop micro centrifuge.
Using a pipet, remove the water supernatant. Next, add 300 microliters of 2%sodium hypochlorite to the seeds and vortex them. Let the mixture settle at room temperature.
After one minute, centrifuge the seeds. With the pipet, remove the sodium hypochlorite solution. Then add 500 microliters of sterile, deionized water to the seeds, and vortex the mixture.
After centrifuging the tube, remove the water. Add 500 microliters of 70%ethanol to the seeds, and vortex the mixture. After allowing the seeds to settle for one minute, centrifuge the tube.
Remove the 70%ethanol using a pipet. Wash the seeds five times with 500 microliters of sterile, deionized water. Then, re-suspend the sterilized seeds in 500 microliters of sterile, ultra pure water.
Pour 25 milliliters of half strength Murashige and Skoog, or MS medium with 04%phyto agar into each sterile, deep Petri dish. For each Petri dish, cut out a 90 millimeter by 90 millimeter square of stainless steel mesh. Bend the corners of each square mesh just enough so that the mesh fits inside the Petri plate.
Bend the corners at a 90 degree angle to the bulk of the mesh to allow the mesh to be propped up by the corners, leaving enough space below for root development. Put the cut mesh squares in a beaker and cover them with aluminum foil. Sterilize the mesh squares by autoclaving using a 30 minute dry cycle.
Once the medium has solidified in the Petri dishes, and the mesh is sterile, place each mesh square on top of the solid medium with the bent corners facing down. Push the mesh so that the corners penetrate the medium and the bulk of the mesh touches the top surface of the medium. Next, use a sterile 200 microliter pipet to draw out the individual surface-sterilized A thaliana seeds and gently transfer each seed on top of the mesh.
Place the seeds so that they are spaced per the plant species and experimental needs. Seal the Petri dishes with lids and place porous surgical tape around the edges of the plates. Wrap the seeds in aluminum foil.
Stratify the seeds by placing the entire Petri dish. After two days, cultivate the seeds in the Petri dish at 22 to 24 degrees Celsius with a 16 hour photo period for 10 to 14 days. In a sterilized flow hood, pour 18 milliliters of autoclaved liquid MS medium into sterile cylindrical glass tanks with sterile lids.
Next, use sterile forceps to gently transfer each mesh square with 10 to 14 day old seedlings from the Petri dish of semi solid medium to a hydroponical cylindrical tank. The seedlings shown here are alfalfa. Seal the lids onto the tanks using porous surgical tape.
Then, cultivate the seedlings at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Prior to the inoculation, examine the tank with seedlings carefully for any potential contamination. The medium in the uncontaminated tanks should be clear and transparent.
If tanks are cloudy with contamination, discard them and number the rest of the tanks. Sample 20 microliters of hydroponic medium from each numbered tank and spot it on a Petri dish with Luria broth or LB agar. Incubate the dish at 28 degrees Celsius for 28 hours.
Immediately inoculate each numbered hydroponic tank with 50 microliters of the agrobacterium tumefacians suspension into each numbered hydroponic tank. Co-cultivate the seedlings and A tumefacians bacterium at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Examine the spotted LB plate after 12 and 28 hours of incubation to verify the sterility of the hydroponic growth medium.
If there is any contamination, do not use the corresponding numbered tank inoculated with bacteria for further downstream assays. Next, separate roots from the bacterial suspension by lifting the mesh plate. If using roots for subsequent analyses, cut the roots, quickly rinse them in double distilled water, and dry on sterilized filter paper.
If using leaf or other tissue, cut off the tissue. For plant transcript analysis, the roots or shoots should be immediately deposited into liquid nitrogen. For bacterial transcript analysis, transfer 1.5 milliliters of the culture from the co-cultivation tank and pellet the cells by centrifugation.
Following an appropriate co-cultivation, use sterile scissors to separate individual secondary roots, the side branches of the main root. Next, rinse the roots in double distilled water to remove loosely bound material. Submerge each root in 30 microliters of water on a microscope slide, and cover it with a glass cover slip.
Seal the edges of the coverslip with nail polish to protect the roots from dehydration. Then visualize the attachment of A tumefacians to A thaliana roots by fluorescence microscopy. After an appropriate co-cultivation, separate the plants from the hydroponic medium.
Sterilize the 18 milliliters of hydroponic medium by passing it through a 0.2 micron pore filter into 50 milliliter conical tubes. Freeze the samples at 80 degrees Celsius overnight. The following day, loosen the caps on the 50 milliliter conical tubes and place them in a freeze drying machine for 36 hours.
Finally, proceed to store or process the samples for HPLC analysis and compound detection. Without artificially supplied nutrients, A tumefacians c58 grew in the co-cultivation system. By contrast, essentially no bacterial growth was observed in the control culture without A thaliana.
The hydroponic co-cultivation systems can be used to study the attachment of bacteria to the root structure as seen in this microscopic visualization, where PCherry red flourescence-labeled bacteria were localized to the plant root structure with a typical rock-like or filamenta shape. Gene expression profiles of both plant posts and interacting microbes can be ascertained via QRTPCR. After eight hours of co-cultivation, eight tumefacians cells revealed up regulation of expression of virulence genes.
At the same time, roots of A thaliana revealed differential gene expression in response to co-cultivation. After 72 hours of co-cultivation, 35 compounds were enhanced and 76 compounds were decreased in the secretome of inoculated plants compared to the control. This technique paves the way for researchers to study plant-microbe interactions and explore the reciprocal responses between plant and microbe or microbe biome partners, or plant responses to abiotic factors in a simple set-up that closely mimics the natural environment.
After watching this with you, we hope you are able to set up a plant microbial co-cultivation system in hydroponics to study molecular plants, microbial interactions and the signaling responses in a more natural and manageable environment.
