Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Summary

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Abstract

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system’s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Introduction

Crop destruction by bacterial and fungal diseases results in lowered food production and can severely disrupt global stability¹. Based on the discovery that microbes in suppressive soils are responsible for increasing plant health², scientists have asked whether the plant microbiome can be leveraged to support plant growth by modifying the presence and abundance of particular bacterial species³. Bacteria found to aid in plant growth or development are collectively termed plant growth-promoting bacteria (PGPB). More recently, studies have shifted from simply identifying potential PGPB to understanding how interkingdom interactions in the soil, around roots, or in the rhizosphere (the area directly surrounding and including the root surface) may be impacting PGPB activity⁴.

Rhizosphere colonization by PGPB can increase the health or the development of host plants in response to diverse stressors relative to uncolonized plants⁵. However, results are often more variable in native soil conditions compared to those observed in the closely controlled greenhouse and laboratory settings⁶. One hypothesis for this difference is that the growth or behavior of PGPB may be inhibited by native soil bacteria or fungi in the fields^7,8. Beneficial effects by rhizosphere bacteria generally depend on the ability of the bacteria to 1) locate and move towards the root, 2) colonize the root through biofilm formation, and 3) interact with the host plant or pathogens via production of small molecule metabolites^7,9. Any of these colonization behaviors may be affected by the presence and activity of neighboring microbes¹⁰.

We designed a system to quantify and visualize these distinct bacterial colonization stages of the rhizosphere (Figure 1). This approach will facilitate studies investigating why long-term PGPB maintenance is sometimes not observed following transfer of plants into new environments, such as during the planting of pre-inoculated seedlings. Arabidopsis thaliana as were chosen as a plant model due to its extensive use in laboratory studies as well as the ample data available about its microbial interactions¹¹. There are three stages in the system: 1) A. thaliana growth, 2) bacterial colonization, and 3) bacterial maintenance (see Figure 1). Because A. thaliana is a terrestrial plant, it was important to ensure that it was not suffering undue water stress in the hydroponic system¹². Inspired by the methods used by Haney et al.¹³, the seedlings are grown on plastic mesh to separate the shoot from the liquid growth medium. This system does not appear to compromise the health and development of the plant host, and it improves A. thaliana growth in liquid¹¹. As the plant shoot floats above the surface, the roots are fully exposed to colonization by bacteria inoculated into the liquid bacterial growth medium. This permits bacteria of interest to be examined for colonization in nutrients that are most conducive to growth, while then shifting conditions to allow the plant to continue growing in a nutrient medium designed to support its growth. Both stages include steady shaking to prevent anoxia of the root¹³. Bacteria can be visualized or quantified from the plant roots following transfer from either the colonization medium or the maintenance medium. This hydroponic system is very flexible, allowing experimental conditions and applied stresses to be easily altered depending on interests of the researchers.

This described method is important in the context of the larger body of literature about plant-microbe interactions because it provides a robust system for studying these interactions at the root surface while also being customizable to the growth preferences of different bacteria. Plant biology labs often perform plant-microbe colonization experiments on solid agar, allowing for only planar movement (if that) of bacteria while also requiring the potentially destructive manipulation of plants during subsequent transfer. In contrast, microbiology labs have frequently prioritized the health of the bacteria within their experiments, to the detriment of the plants^14,15. These different priorities of plant- and microbiology-focused labs have historically made it difficult to compare results between these groups, since each typically optimizes experimental conditions to optimize their organism of interest¹⁵. The floating-mesh-plant-growth system described here prevents full plant submersion, a notable advantage to previous microbiology-oriented studies, while also temporarily optimizing the growth and survival of bacteria to facilitate colonization. Thus, the assay we present here may address concerns from both plant biologists (about over-hydration and tactile manipulation of the plant) while satisfying the criteria of microbiologists (allowing for different bacterial growth conditions and multiple species’ interactions)⁷. This protocol is designed to be adaptable for use with various bacteria, plants, and environmental conditions.

Protocol

NOTE: The experimental setup is described for clarity and used to generate the representative results included in this report, but conditions can be modified as desired. All steps should be performed using PPE and following institutional and federal reccomendations for safety, according to the BSL status of the bacteria used.

1. Characterization of bacteria

1. Determine the morphology of bacteria on the growth medium agar plate. Resuspend cells at an approximate OD₆₀₀ = 0.5 and plate a 1 µL volume onto agar medium of choice. Add X-gal to agar plates to a final concentration of 20 mg/mL to better differentiate individual members of the specific bacterial community. Grow at 24 °C or 30 °C until colonies form, then take pictures of and notes on colony morphology.
2. Define the correlation between each bacterial strain’s optical density and the number of CFU (colony forming units) per mL¹⁶. Resuspend bacteria in 1 mL of water in a 24-well plate to an approximate OD₆₀₀ = 5, perform two-fold serial dilutions, monitor OD₆₀₀ of all dilutions, and plate each to determine the viable CFU/mL in each sample at multiple optical densities.
3. Determine the maximum sonication tolerance for each bacterial strain. To do this, aliquot cells into a 24-well plate containing liquid medium, reserving some cells as an unsonicated control sample. Using an ultrasonicator with a 24-tip horn attachment, apply three rounds of 12 s of sonication at 40 amp with 2 s pulses.
   NOTE: The use of a 24-well ultrasonicator is advised to facilitate the downstream multiplexing of processing plant samples, but if one is not available, use an ultrasonicator fitted with a microtip and perform each sample sonication independently. Always wear earmuffs rated to at least 25 NRR protection.
4. Perform 10-fold serial dilutions of the sonicated and unsonicated samples and spot onto agar plates. Determine whether there is a reduction in viable cells after sonication. If so, use a fresh sample and repeat sonication step using a reduced total sonication time or amplitude until the treatment has no effect on final CFU/mL¹⁷.

2. Preparation of Arabidopsis thaliana seedlings on a plastic mesh

1. Create disks of the plastic mesh using a standard hole puncher.
   1. Collect the disks in a glass container with a loose cover of aluminum foil, and sterilize using an autoclave set to a 20 min dry cycle¹³.
   2. Using flame-sterilized tweezers, distribute approximately 40 sterilized mesh disks in a single layer across the surface of a plant-growth-medium agar plate. Use 0.5x Murashige and Skoog (MS) salts, containing 500 mg/L of MES buffer [2-(N-morpholino)ethanesulfonic acid] and 1.5% Bacto agar, as plant growth medium, with 50 µg/mL benomyl added to the limit fungal contamination of the seedlings.
2. Prepare axenic seeds of A. thaliana as previously described¹⁷.
   1. Place approximately 100–300 seeds each into individual centrifuge tubes in a rack and place into a resealable glass or heavy plastic container (“jar”) in a fume hood.
   2. Using caution, place a beaker of 100 mL of bleach into the jar, add 3 mL of concentrated HCl to the bleach, and immediately seal the jar and allow fumes to sterilize seeds for at least 4 h.
   3. Carefully remove the tubes of sterilized seeds from underneath the jar and seal.
3. Place two seeds at the center of each mesh. Seal plates with surgical tape and incubate for 2–6 days at 4 °C in darkness to vernalize seeds.
4. To germinate and grow seedlings, place the plate agar side down in a plant growth chamber for 8–10 days under short day settings: 9 h of light at 21 °C and 15 h of dark at 18 °C (Figure 1, step 2).

3. Colonization of plants in liquid bacterial growth medium

1. Add 1 mL of bacterial growth medium to each well of a sterile 24-well plate, except for media-only control wells. Use Lennox Luria Broth (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl) as the bacterial growth medium.
2. Transfer the germinated seedlings embedded in mesh from agar plates to the liquid (Figure 1, step 3a).
   1. Gently peel the mesh containing two germinated seedlings up and off the agar plate using flame-sterilized forceps. Choose mesh with equally sized and undamaged seedlings.
   2. If removal from the agar is not smooth, discard that mesh and plant. Transfer one float to each well of bacterial growth liquid, root side down.
3. Inoculate bacteria into wells containing floating seedlings.
   1. Resuspend bacteria grown overnight on agar plates to an OD₆₀₀ equivalent to 10⁸ CFU/mL in the bacterial growth medium liquid. Add 10 µL of bacterial suspension to each well for a final concentration of 10⁶ CFU bacteria per well.
   2. If preparing a mix of bacteria, resuspend each to the OD₆₀₀ equivalent to 10⁸ CFU/mL, mix in equal proportions, and add 10 µL of the final mix per well of liquid.
4. Seal the plate for sterile growth. Without touching the sticky side, carefully press the gas-permeable film across the plate. Ensure that each well has been individually sealed by applying pressure around each of the rings made by the wells. Replace the plate’s plastic lid snuggly over the plate and gas-permeable film (Figure 1, step 3b).
5. Incubate the plates for 18 h in a plant growth chamber, under the same conditions as the seedlings were originally germinated, except on an orbital plate shaker set to 220 rpm.

4. Maintenance of bacterial colonization

1. To rinse all floats (plants on mesh), add 1 mL of sterile water to wells of a new 24-well plate. Remove gas-permeable film. Using sterile forceps, transfer floats to wells with water (Figure 1, step 4a). Rinse by resting for 10 min at room temperature (RT) without agitation.
   NOTE: To determine bacterial colonization efficiency of roots rather than their ability to maintain colonization over time, plants can be sacrificed at this step by taking them directly to step 5.1.
2. Fill the wells of a new 24-well plate with 1 mL of plant growth medium. Transfer one mesh to each well. Cover with a gas-permeable seal and incubate for 72 h on the orbital plate shaker at 220 rpm in plant growth chamber (Figure 1, step 4b).
3. Repeat the rinsing as performed in step 4.1 with floats after the 72 h incubation period.

5. Collection of bacteria for viable cell counts

NOTE: The number of bacteria per seedling root can be determined at any incubation timepoint. Colonization can be monitored between 0 h and 18 h, while maintenance can be monitored from 18 h onwards. Plants destined for imaging can proceed directly to section 6.

1. Remove the seedlings from the mesh (Figure 1, step 5). Gently place flame-sterilized forceps below the leaves (but on the leaf side of the mesh), and lightly pinch the stem. Wiggle the seedlings up and away from the mesh to dislodge the root without breaking it. If the root breaks, gently scrape the mesh bottom to collect the full length.
2. Remove bacteria from plant roots. Transfer the bacteria to wells of a 24-well plate containing 1 mL of ddH₂0. Sonicate the samples as described in step 1.3.
   NOTE: Using a microscope, look for any remaining bacteria on the root surface on a sonicated sample. If bacteria remain, increase the total sonication time or intensity until no bacteria remain bound, up to the highest level of sonication that does not affect viable cell counts as determined in section 1.
3. Quantify the bacteria on roots.
   1. Perform serial 10-fold dilutions of the sonicated samples up to a 10^-6 dilution in bacterial growth medium. Add 50 µL of each dilution to individual agar plates and spread with sterile glass beads (or bacterial spreader). Incubate plates at the optimal temperature for bacteria until individual colonies are countable.
   2. Once distinguishable, count the number of each colony morphology (as determined in section 1), and calculate CFU of each bacterial species per seedling. Discard any samples showing contamination, as contamination during colonization or maintenance may affect bacterial presence.

6. Collection of intact plant roots for microscopy

1. Using forceps, remove the seedlings from mesh as in section 5.
2. Transfer each plant to microscope slides.
   1. Place the tip of the root on the slide and drag away from the tip to set the shoot down flush with the slide, ensuring a straightened root for best imaging. Add a drop of water or sterile plant growth medium to the samples to hydrate interfaces between the coverslips and slides.
   2. Place a glass coverslip just above the root crown (uppermost boxed region in Figure 1) and below the shoot leaves to avoid slanting of the coverslip (to allow for root crown imaging), and press down gently¹⁷.
3. If using fluorescent bacteria, image using appropriate excitation/emission filters to differentiate bacteria from each other and the plant root¹⁸.

Results

The well-characterized PGPB P. simiae WCS417r is known to colonize the roots of A. thaliana in hydroponic culture. This naturally fluorescent bacterium can easily be visualized using microscopy on the roots of seedlings following colonization (Figure 2). Although it is possible to image the full length of these A. thaliana seedlings’ (4–6 mm length) roots, doing so for many plants would take a prohibitive amount of time....

Discussion

Plants in all environments interact with thousands to millions of different bacteria and fungi^5,7. These interactions can either negatively and positively impact plant health, with potential effects on crop yield and food production. Recent work also suggests that variable colonization of crops by PGPBs may account for unpredictable plant size and crop yield in field trials²². Understanding the mechanisms behind these interactions might al...

Materials

Name	Company	Catalog Number	Comments
Required Materials
1.5 mL eppendorf tubes	any	N/A
24-well plates	BD Falcon	1801343
Aeraseal	Excel Scientific	BE255A2
Autoclave	any	N/A
Bacteria of Interest	any	N/A	Stored at -80˚C in 40% glycerol preferred
BactoAgar	BD	2306428; REF 214010
bleach	any	N/A
Conviron	any	N/A	Short Day Light-Dark Cycles: 460-600 µmoles/m²/s set at 9/15 hours light/dark at 18/21˚C, with inner power outlet
Dessicator Jar: glass or heavy plastic	any	N/A
Ethanol	any	N/A
Flame	any	N/A
Forceps	any	N/A
Incubator	any	N/A	At optimal temperature for growth of specified bacteria
Hydrochloric Acid	any	N/A
Lennox LB Broth	RPI	L24066-1000.0
Microcentrifuge	any	N/A
Micropipetters	any	N/A	Volumes 5 µL to 1000 µL
Microscope (preferably fluorescence)	any	N/A	Could be light if best definition not important
MS Salts + MES	RPI	M70300-50.0
Orbital Plate Shaker	any	N/A	Capable of running at 220 rpm for at least 96 hours
Petri Dishes	any	N/A	50 mL total volume
Reservoirs	any	N/A
Spectrophotometer	any	N/A
Standard Hole Punch	any	N/A	Approximately 7mm punch diameter
Sterile water	any	N/A
Surgical Tape	3M	MMM1538-1
Teflon Mesh	McMaster-Carr	1100t41
Ultrasonicator	any	N/A
Vortex Mixer	any	N/A
X-gal	GoldBio	x4281c	other vendors available
Suggested Materials
24 Prong Ultrasonicator attachment	any	N/A	For sonicating multiple samples at once. Can be done individually
Alumaseal II	Excel Scientific	FE124F
Glass beads	any	N/A
Multipetter/Repetter	any	N/A
Sterile 96-well plates	any	N/A	For serial dilutions. Can be replaced by eppendorf tubes
Biological Materials Used
Arabidopsis thaliana seeds	any	N/A	We recommend Arabidopsis Biological Resource Center for seed stocks
Arthrobacter nicotinovorans			Levy, et al. 2018
Curtobacterium oceanosedimentum			Levy, et al. 2018
Microbacterium oleivorans			Levy, et al. 2018
Pseudomonas simiae WCS417r			Published in a similar system in Haney, et al. 2015. Strain used developed in Cole, et al. 2017