Most experiments study plant-microbe interactions by focusing on colonization, but our protocol is designed to look both at colonization and maintenance of the bacteria on the root. We can change the experiment to fit different bacteria and plants, and we can even test multiple bacteria in each 24-well plate. To begin, use a standard hole puncher to create disks of plastic mesh.
Collect the disks in a glass container, and cover loosely. Sterilize using an autoclave set to a 20-minute dry cycle. Use flame-sterilized tweezers to distribute approximately 40 sterilized mesh disks in a single layer across the surface of previously prepared plant growth medium agar plate.
Place two previously sterilized seeds at the center of each mesh. Seal the plate with surgical tape, and incubate for two to six days at four degrees Celsius in darkness to vernalize the seeds. Then, place the plate agar-side down in a plant growth chamber for eight to 10 days under short-day settings of nine hours of light at 21 degrees Celsius and 15 hours of dark at 18 degrees Celsius to germinate and grow seedlings.
Add one milliliter of bacterial growth medium to each well of a sterile 24-well plate. To transfer the germinated seedlings embedded in mesh, use flame-sterilized forceps to gently peel the mesh containing two germinated, equally sized, and undamaged seedlings up and off the agar plate. Transfer one float with seedlings to each well of bacterial growth liquid, root-side down.
Next, resuspend bacteria grown overnight on agar plates in liquid bacterial growth medium. Add 10 microliters of bacterial suspension to each well, except for media-only control wells, for a final concentration of one million colony-forming units of bacteria per well. To seal the plate for sterile growth, carefully press gas-permeable film across the plate without touching the sticky side.
Apply pressure around each of the rings made by the wells to ensure that each well has been individually sealed. Then, close the plate with its plastic lid snuggly over the gas-permeable film. Place the plate in a plant growth chamber on an orbital plate shaker set to 220 rpm, and incubate for 18 hours under the same conditions as the seedlings were originally germinated.
To rinse all floats with plants, add one milliliter of sterile water to each well of a new 24-well plate. Remove gas-permeable film from the plate with plants, and use sterile forceps to transfer floats to wells with water, and incubate for 10 minutes at room temperature without agitation. Then, fill each well of a new 24-well plate with one milliliter of plant growth medium.
Transfer one mesh to each well, and cover with a gas-permeable seal. Incubate for 72 hours on the orbital plate shaker at 220 rpm in plant growth chamber. After the incubation, rinse the plants as previously done.
After the rinsing, remove the seedlings from the mesh by gently placing flame-sterilized forceps below the leaves on the leaf side of the mesh. Lightly pinch the stem, and wiggle the seedlings up and away from the mesh to dislodge the root without breaking it. To remove bacteria from plant roots, transfer seedlings from each mesh into individual wells of a 24-well sonication plate containing one milliliter of double-distilled water.
Sonicate all samples within the plate at once using a multipronged sonicator as described in the manuscript. To quantify the bacteria, perform serial 10-fold dilutions of the sonicated samples in bacterial growth medium. Spread using sterile glass beads, and incubate at the optimal temperature for bacteria until individual colonies are countable.
To collect intact plant root, use forceps to remove the seedlings from the mesh. Then, transfer each plant to a microscope slide by placing the tip of the root on the slide and dragging it away from the tip to set the shoot down flush with the slide, ensuring a straightened root for best imaging. Add a drop of water or sterile plant growth medium to each sample to hydrate interfaces between the coverslips and slides.
Place a glass coverslip just above the root crown and below the shoot leaves to avoid slanting of the coverslip, and press down gently. Then, image the bacteria using appropriate excitation/emission filters to differentiate bacteria from each other and the plant root. Pseudomonas simiae, naturally fluorescent bacterium, colonized Arabidopsis thaliana roots and was maintained on the root following transfer to plant growth medium.
Root crown, mid-length, and tip show fluorescence due to colonization of Pseudomonas simiae. The no-bacteria negative control showed no colonization. When Pseudomonas simiae on Arabidopsis thaliana roots were quantified after 18 hours of colonization or 72 hours of maintenance, total number of colony-forming units per seedling at either time point showed good reproducibility across biological replicates performed on different days.
There was an increase in the number of colony-forming units per seedling after 72 hours in maintenance medium, as compared to the numbers observed at the post-colonization 18-hour time point, indicating that active growth of the colonized bacteria on the plant root occurred during the maintenance stage. Three species of bacteria isolated from Arabidopsis thaliana grown in natural soil under laboratory conditions were used to monitor the association of multiple species on plant roots. They were clearly differentiated on media containing X-gal due to differences in colony morphology and color, which allowed counting the colony-forming units per seedling of each species without antibiotic selection, even in multi-species coculture.
These three species of bacteria were all colonized and maintained on the root, whether alone or in bacterial coculture. Each species showed trends that were similar across different biological and technical replicates, showing that this protocol can be used to measure both relative or total colony-forming units per root of each species. When grown alone, no individual species showed substantial increase in abundance during the maintenance stage, but the overall colony-forming units per root of the combined community increased in cocultures, indicating that these bacteria do not prohibit the colonization of the other strains.
The most difficult steps are those that require the removal of intact plants from the mesh disks. Being patient and gentle will make this possible. If the bacterial cells are fluorescent, the samples could be sorted by flow cytometry to determine the relative numbers of cells.
