This method can help to answer key questions in the fields of plant-microbe interaction. You can, for instance, identify important genes from bacterial strains that were isolated from the endosphere or rhizosphere during the interaction of the bacteria with the plant. It's a versatile method that can also be used for studies on other bacteria with plants.
The main advantage of this technique is that in a relatively short period a large number of bacteria genes that are important during plant-microbe interactions can be identified. To begin this procedure, rinse the potato surface with sterile water. Bathe the potato in 70%ethanol for five minutes and then in 3%sodium hypochlorite for another five minutes.
Afterward, rinse it again with sterile water to remove any remaining sodium hypochlorite. Next, prepare the materials needed for germinating and growing potato tubers. Sterilize the plastic pots, engraftment baskets, vermiculite, and water by autoclaving them at 121 degrees Celsius for 20 minutes.
Subsequently, put the surface-sterilized potato into the engraftment basket and place it in an autoclaved pot containing wet vermiculite. Use a large glass fiber box to cover the pot to avoid microbial contaminations. Keep the pot in a climate chamber at 24 degrees Celsius for three weeks, and set it on cycles of 16 hours of light and 8 hours of darkness.
To collect the potato root exudates, when the shoots sprout, transfer the basket with the whole potato into a sterilized beaker filled with 150 milliliters of autoclaved deionized water with the potato tubers placed just above the water surface and the roots submerged in the water. Keep the seedling in the climate chamber, and use the same settings as before. From the second day on, collect the water containing the exudates, and refill the beaker with sterile, deionized water.
Then store each sample separately at four degrees Celsius. For each sample, spread 100 microliters on a Luria-Bertani agar plate to check for microbial contaminations. Discard the contaminated samples.
Combine the collected samples of one seedling. Then transfer the combined root exudates into 50 milliliter tubes. Freeze the tubes containing root exudates at minus 80 degrees Celsius.
Concentrate them by freeze drying them at minus 40 degrees Celsius to a final volume of 150 milliliters. To grow the bacteria, streak a B.mycoides strain from a minus 80 degree Celsius glycerol stock onto an LB agar plate, and incubate the plate at 30 degrees Celsius overnight. Then inoculate the LB liquid medium with a single colony from the plate, and grow the culture in a shaking incubator at 200 rpm overnight at 30 degrees Celsius.
To treat and sample the bacteria, dilute an overnight B.mycoides culture with 90 milliliters of pre-warmed LB medium to the initial OD600 of zero point zero five in a 300 milliliter flask. Subsequently, add 10 milliliters of root exudates to the culture, and use a 10 milliliter sterile, deionized water treatment as a control. Then incubate them at 30 degrees Celsius for one hour.
After an hour, transfer the culture into 50 milliliter centrifuge tubes. Collect the cells from the culture by centrifugation at 9, 000 times G for two minutes at four degrees Celsius. Afterward, discard the supernatent, and immediately freeze the pellet in liquid nitrogen.
Then store it at minus 80 degrees Celsius until use. To isolate RNA, thaw the cell pellets on ice, and suspend each of them in 400 microliters of TE buffer. Then transfer the suspensions into the two milliliter screw cap tubes.
Add zero point five grams of glass beads into the tube. Then close the caps firmly and place the tubes in a bead mill homogenizer. Perform a 45-second pulse homogenization three times, with a one-minute interval on ice.
Afterward, centrifuge the samples for 10 minutes at 11, 000 times G, and transfer the upper phase of each sample to a new tube. Add 500 microliters of chloroform:isoamyl alcohol to the upper phase, and centrifuge the mixture for five minutes at 11, 000 times G.Then transfer 500 microliters of the upper phase to a fresh tube. Add one milliliter of the lysis binding buffer, and mix it by pipetting up and down.
Next, prepare one point five milliliter microcentrifuge tubes with 100 microliters of DNase buffer, 10 microliters of DNase I, and five microliters of an RNase inhibitor. Add the prepared solution mix on the filter of the filter tubes, and incubate them for 20 to 30 minutes at 15 to 25 degrees Celsius. Subsequently, perform the washing steps according to the manufacturer's instruction, and add 50 microliters of an elusion buffer to the tube.
After centrifugation, elute the RNA. Transfer a few microliters of the sample to a one point five milliliter microcentrifuge tube to perform a quality check. Finally, cover the eluded RNA with a plastic paraffin film and keep it at minus 80 degrees Celsius.
After collection, the root exudates were added to the B.mycoides culture at a 10%ratio, and the bacterial total RNA was isolated. The RNA was then subjected to a quality check by an automated electrophoresis instrument. These graphs represent the RNA isolated from B.mycoides treated with the root exudates, and these graphs represent the RNA isolated from the control group.
All samples scored a RIN value above nine, with two clear bands corresponding to the 16S and 23S RNA subunits, demonstrating that high-quality RNA was obtained by this protocol. After library preparation, pair end reads were obtained with a high-throughput sequencing platform. The raw RNA-seq reads were trimmed from the adaptor sequences, and mapped against the reference genome sequence.
The transcript home analysis was performed with the T-REx pipeline, and the ratio intensity plot of all the differentially expressed genes is shown here. After the potentially important gene's been identified, other in vivo methods like promoter GFP fusion, cue-pews-air, and fluorescent in situ hybridization can be performed in order to answer additional questions like time in reserved expression of certain genes during the interaction with host plant.
