This article details the methodology for studying plant-microbe interactions using Bacillus mycoides, a plant growth-promoting rhizobacterium. The process involves culturing the bacteria, treating them with potato root exudates, and analyzing the resulting changes in gene expression.
Take a stock culture of Bacillus mycoides, a plant growth-promoting rhizobacterium, and streak an inoculum onto a culture plate.
Incubate the plate to promote bacterial proliferation into colonies.
Inoculate a single colony into a liquid medium and incubate with shaking to promote bacterial growth.
Dilute the culture using the liquid medium to achieve the optimal density for the assay.
Add potato root exudates to the culture to emulate plant-microbe interactions.
Incubate the culture with agitation, facilitating the interaction of the signaling molecules with bacterial receptors.
This interaction induces signaling pathways that activate regulatory proteins, which bind to DNA and alter gene expression.
Transfer the culture into a tube and spin down the bacterial cells into a pellet.
Discard the supernatant and dip the tube into liquid nitrogen to flash-freeze the cells.
Store the cells to analyze plant root exudate-induced changes in bacterial gene expression.
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 supernatant, and immediately freeze the pellet in liquid nitrogen.
Then store it at minus 80 degrees Celsius until use.
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