Single-Cell Analysis of Gene Expression from Plant-Extracted Pathogens

Abstract

Source: Rufián, J. S., et al. Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue. J. Vis. Exp. (2022).

This video demonstrates a method for analyzing gene expression in plant-extracted pathogenic bacteria at the single-cell level using a fluorescently tagged transcription factor. Confocal imaging reveals heterogeneity in virulence gene expression, highlighting functional diversity within a clonal bacterial population.

Protocol

1. Single-cell analysis of the apoplast-extracted bacteria

1. 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 on it (Figure 1). Let them dry for 15 min and carefully remove the slide placed on the top. Using a blade, cut the agarose pad into 5 mm x 5 mm pieces right before use.
2. Parallelly, centrifuge 1 mL of the apoplast-extracted bacteria at 12,000 x g for 1 min at room temperature, carefully remove the supernatant using a pipette, and resuspend the pellet into 20 µL of water to concentrate the cells. Place a 2 µL drop of the concentrated cells onto a 0.17 mm coverslip and cover the drop with a 5 mm x 5 mm piece of the agarose pad previously obtained in step 1, as is represented in Figure 1.
3. Visualize the bacterial preparation under the confocal microscope (see Table of Materials). To identify green-fluorescent bacteria, use the excitation laser at 488 nm and an emission filter ranging from 500 nm to 550 nm. To identify all the bacteria, use the bright field and merge both fields.
4. Process the confocal images using Fiji (see Table of Materials). To do this, use the MicrobeJ plugin to identify the contour of the bacterial cell and measure the fluorescence intensity within.
   NOTE: Image acquisition from isolated bacteria (not clustered) is recommended for this analysis.

Results

Figure 1: Schematic representation of the setting and preparation of the agar pad.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.17 mm coverslip			No special requirements
1.6 x 1.6 mm metal mesh	Buzifu		Fiberglass screen mesh
50 mL conical tubes	Sarstedt
Agarose	Merk
Confocal Microscope Stellaris	Leica Microsystems
Fiji software