Take a suspension of a pathogenic bacterium isolated from an infected plant.
The bacterial genome contains a transcriptional fusion, in which the promoter of a virulence-regulatory gene also drives expression of a downstream, promoterless fluorescent protein gene.
During plant infection, the promoter activation triggers the production of the regulatory and fluorescent proteins.
The regulatory protein activates virulence genes, while the fluorescence provides a measurable readout of virulence activation.
Centrifuge the cells, discard the supernatant, then resuspend to obtain a concentrated suspension.
Place a drop on a coverslip and overlay with an agarose pad to spread and immobilize the cells.
Capture a brightfield image to identify all bacterial cells, then a fluorescence image to detect single-cell fluorescence.
Merge both to visualize fluorescence intensity differences, which reveal subpopulations with high, low, or no virulence gene expression.
This phenotypic variation reflects an adaptive strategy that enhances bacterial survival during plant colonization.
For a single-cell analysis, prepare a 1.5% agarose solution in distilled water. Once melted, add enough volume to fill the space between two microscopy slides set side by side and place another slide on top. Let them drive for 15 minutes and remove the slide placed on the top carefully.
Using a blade, cut the agarose pad into 5 millimeter by 5 millimeter pieces before use. Parallely, centrifuge 1 milliliter of the apoplast-extracted bacteria. Carefully remove the supernatant using a pipette and resuspend the pellet into 20 microliters of water to concentrate the cells.
Place a 2 microliter drop of the concentrated cells onto a 0.17 millimeter cover slip and cover the drop with a piece of agarose pad. Visualize the bacterial preparation under the confocal microscope to identify green fluorescent bacteria using specific lasers. Identify all the bacteria using the bright-field and merge both fields.
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