A Co-Cultivation System to Study the Molecular Responses of Plant Hosts and Interacting Bacteria

Abstract

Source: Nathoo, N., et al. A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling. J. Vis. Exp. (2017).

This video demonstrates a hydroponic co-cultivation system for studying plant–bacteria interactions under sterile conditions. Plant seedlings are transferred into a sterile, acidic growth medium, incubated, and inspected for contamination before inoculation with Agrobacterium. The activated bacteria attach to plant roots, transfer T-DNA into plant cells, and promote molecular changes that enhance bacterial colonization.

Protocol

1. Hydroponic Co-cultivation System

1. Prepare liquid Murashige and Skoog (MS) medium: 2.165 g/L MS basal salts, 10 g/L sucrose, 0.25 g/L 2-(N-morpholino)ethanesulfonic acid (MES), and 59 mL/L B5 vitamin mix, pH 5.75.
2. Autoclave the medium. Pour 18 mL of it into each sterile cylindrical glass or clear plastic tank (crystallizing dishes, 100 x 80 mm2) with sterile lids.
   NOTE: Presterilize the tanks and lids by wrapping them in aluminum foil and autoclaving.
3. In a sterilized flow hood, use sterile forceps to gently transfer each mesh square with 10-14 day-old seedlings from the semi-solid medium to a hydroponic cylindrical tank (Figure 1d). Seal the lids onto the tanks using porous surgical tape.
4. Cultivate the seedlings on the mesh in the tank for 72 h at 22-24 °C, with a 16 h photoperiod and shaking at 50 rpm for aeration (Figure 1e).
   NOTE: This allows plants to adapt to the hydroponic environment prior to inoculation (cocultivation) with microorganisms. This waiting period will also allow accidental microbial contamination to be apparent prior to the inoculation.
   1. Begin the next step the day prior to the end of the incubation period.
5. Grow Agrobacterium tumefaciens in agrobacterium (AB) medium (0.5% w/v yeast extract, 0.5% w/v tryptone, 0.5% w/v sucrose, 50 mM MgSO₄, pH 7.0) at 28 °C with shaking overnight until the culture reaches a final optical density of 1.0 at 600 nm (OD₆₀₀), as measured using a spectrophotometer, with uninoculated AB medium as a blank.
   NOTE: An OD₆₀₀ of 1.0 is equivalent to approximately 10⁹ cells/mL.
6. Wash the A. tumefaciens three times in an equal volume of 0.85% NaCl. Resuspend in an equal volume of sterile double-distilled water.
7. Prior to the inoculation, examine the tank with seedlings carefully to ensure that there is no contamination; the medium in the uncontaminated tanks should be clear and transparent.
   1. Discard any tank with cloudy liquid (contamination) and number the rest of the tanks. Sample a small amount (20 µL) of hydroponic medium from each numbered tank and spot it on a petri dish filled with Luria broth (LB). Incubate the dish at 28 °C.
8. Immediately, add 50 µL of the A. tumefaciens suspension (approximately 5 x 10⁷ cells, estimated from the OD₆₀₀) into each numbered hydroponic tank. Cocultivate the Arabidopsis thaliana seedlings and A. tumefaciens at 22-24 °C with a 16 h photoperiod and shaking at 50 rpm (Figure 3f).
   1. Examine the spotted LB plate from step 1.7.1. after 12 and 28 h incubation to verify the sterility of the tanks. If there is any contamination, do not use the corresponding numbered tank (inoculated with bacteria) for further downstream assays.
9. If desired, monitor the growth of A. tumefaciens at regular intervals by removing a sample of medium from the tank and measuring the OD₆₀₀ using a spectrophotometer with medium from an un-inoculated control as a blank (Figure 2).
   NOTE: Plant disease symptoms should be apparent after 7 d (Figure 3).

Results

Figure 1: Hydroponic Plant-Microbe Cocultivation System. The left panel represents a flow chart o...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plant seeds (Arabidopsis thaliana Col-0)	Arabidopsis Biological Resource Centre	CS7000	https://abrc.osu.edu/order-stocks
Bacteria (Agrobacterium tumefaciens C58)	University of Washington	N/A
Labeled bacteria	in-house		optional, depends on downstream analyses
Double distilled water	(various)
Micropipette	(various)
Murashige and Skoog (MS) basal salts	Sigma-Aldrich	M5524
Sucrose	(various)
MES	(various)
B5 vitamin mix	Sigma-Aldrich	G1019
Phytoagar	(various)
Deep Petri dishes	(various)
Stainless steel mesh	Ferrier Wire Goods Company Ltd	N/A	grade: 304; mesh count: 40 × 40; wire DIA: 0.01
Micropore tape, 1"	3M	1530-1
Diurnal growth chamber	(various)
Cylindrical glass tanks, 100 × 80 mm	Pyrex	3250	other sizes can be used, in which case liquid content may need adjustment
Flow hood	(various)
Forceps	(various)
Yeast extract	(various)
Tryptone	(various)
MgSO₄	(various)
Shaking incubator	(various)