Inoculating a Bacterial Pathogen into Arabidopsis Leaves via Vacuum Infiltration

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 vacuum infiltration method to introduce a bacterial pathogen into Arabidopsis plant leaves. The approach uses a prepared bacterial suspension and vacuum pulses to enable uniform entry through stomata, resulting in controlled leaf infection.

Protocol

1. Inoculation of Arabidopsis plants

NOTE: In this study, the strains Pseudomonas syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A were used.

1. Prepare the P. syringae inoculum.
   1. Streak out the P. syringae strain of interest from a −80 °C glycerol stock onto an LB plate (10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast extract, and 16 g/L bacteriological agar) supplemented with the appropriate antibiotics. Incubate at 28 °C for 40-48 h.
      NOTE: The use of antibiotics is recommended if the strain of interest carries a plasmid or a genomic resistance gene. The recommended antibiotic concentrations for P. syringae are as follows: kanamycin (15 µg/mL), gentamycin (10 µg/mL), ampicillin (300 µg/mL).
   2. Scrape out the bacterial biomass and resuspend in 5 mL of 10 mM MgCl₂. Measure the OD₆₀₀ and adjust to 0.1 by adding 10 mM MgCl₂.
      NOTE: An OD₆₀₀ of 0.1 of a P. syringae culture corresponds to 5 x 10⁷ CFU·mL⁻¹.
   3. Perform serial dilutions into 10 mM MgCl₂ to reach a final inoculum concentration of 5 x 10⁵ CFU·mL⁻¹. Prepare 200 mL of inoculum for the Arabidopsis plants.
   4. Right before inoculation, add the surfactant Silwett L-77 to a final concentration of 0.01% for Arabidopsis. Note that Silwett is somewhat detrimental to the Arabidopsis tissue.
2. Perform vacuum infiltration.
   1. For Arabidopsis infiltration, place two wood sticks forming an X over the pot (Figure 1E), and place the pot facing down over a 14 cm diameter Petri dish containing the 200 mL inoculum (Figure 1F).
   2. Insert the plants immersed in the inoculum solution into a vacuum chamber (Figure 1G) and give a pulse of 500 mbar for 30 s to infiltrate the leaves. Repeat the vacuum pulse 2-3 times until the leaf is completely infiltrated (Figure 1H).
   3. Drain the excess inoculum solution with a piece of paper and return the plants to their corresponding growth chamber.

Results

Figure 1: Bacterial inoculation and apoplast extraction using Arabidopsis plants...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
140 mm Petri dishes			No special requirements
Bacteriological agar	Roko
Gentamycin sulfate	Duchefa	G-0124
Kanamycin monosulfate	Phytotechnology	K378
MgCl₂	Merk
NaCl	Merk
Silwet L-77	Cromton Europe Ltd
Tryptone	Merk
Vacuum chamber 25 cm diameter	Kartell	554
Vacuum pump	GAST	DOA-P504-BN
Yeast Extract	Merk
Ampicillin sodium	GoldBio