Name: monitoring bacterial colonization and maintenance on arabidopsis thaliana roots in a floating hydroponic system
Uploaded: 2019-05-28T09:00:00
Description: Watch this Scientific Journal Video about Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System at JoVE.com

This work was supported by research funds provided by the Department of Energy Biological and Environmental Research (DOE-BER 0000217519 to E.A.S.), the National Science Foundation (INSPIRE IOS-1343020 to E.A.S). SLH was also supported by the National Science Foundation Graduate Research Fellowship Program. We thank Dr. Jeffery Dangl for providing bacterial strains and invaluable insight. We thank Dr. Andrew Klein and Matthew J. Powers for experimental suggestions. Finally, SLH would like to thank connections on social media for reminding us that disseminating science is a privilege and a responsibility, especially through creative and accessible means.

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
<ol>
	<li>Determine the morphology of bacteria on the growth medium agar plate. Resuspend cells at an approximate OD600 = 0.5...

<ol>
	<li>Strange, R. N., Scott, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+disease:+a+threat+to+global+food+security.">Plant disease: a threat to global food security.</a> Annual Review of Phytopathology. 43, 83-116 (2005).</li><li>Cook, A. M., Grossenbacher, H., H&#252;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+microbes+with+biodegradative+potential.">Isolation and cultivation of microbes with biodegradative potential.</a> Experientia. 39 (11), 1191-1198 (1983).</li><li>Vacheron, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+growth-promoting+rhizobacteria+and+root+system+functioning.">Plant growth-promoting rhizobacteria and root system functioning.</a> Fronteirs in Plant Science. 4, 356 (2013).</li><li>Backer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+Growth-Promoting+Rhizobacteria:+Context,+Mechanisms+of+Action,+and+Roadmap+to+Commercialization+of+Biostimulants+for+Sustainable+Agriculture.">Plant Growth-Promoting Rhizobacteria: Context, Mechanisms of Action, and Roadmap to Commercialization of Biostimulants for Sustainable Agriculture.</a> Fronteirs in Plant Science. 9, 1473 (2018).</li><li>Zamioudis, C., Pieterse, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+immunity+by+beneficial+microbes.">Modulation of host immunity by beneficial microbes.</a> Molecular Plant-Microbe Interactions. 25 (2), 139-150 (2012).</li><li>Kr&#246;ber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+strain+Bacillus+amyloliquefaciens+FZB42+on+the+microbial+community+in+the+rhizosphere+of+lettuce+under+field+conditions+analyzed+by+whole+metagenome+sequencing.">Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing.</a> Frontiers in Microbiology. 5, 252 (2014).</li><li>Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., Schulze-Lefert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+bacterial+microbiota+of+plants.">Structure and functions of the bacterial microbiota of plants.</a> Annual Review of Plant Biology. 64, 807-838 (2013).</li><li>Niu, B., Paulson, J. N., Zheng, X., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+representative+bacterial+community+of+maize+roots.">Simplified and representative bacterial community of maize roots.</a> Proceedings of the National Academy of Sciences USA. 114 (12), E2450-E2459 (2017).</li><li>Richter-Heitmann, T., Eickhorst, T., Knauth, S., Friedrich, M. W., Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Strategies+to+Separate+Root-Associated+Microbial+Communities:+A+Crucial+Choice+in+Rhizobiome+Research.">Evaluation of Strategies to Separate Root-Associated Microbial Communities: A Crucial Choice in Rhizobiome Research.</a> Frontiers in Microbiology. 7, 773 (2016).</li><li>Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+coculture+to+detect+chemically+mediated+interspecies+interactions.">Using coculture to detect chemically mediated interspecies interactions.</a> Journal of Visual Experiments. (80), e50863 (2013).</li><li>Woodward, A. W., Bartel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+in+Bloom:+A+Primer+on+the+Arabidopsis+thaliana+Model+System.">Biology in Bloom: A Primer on the Arabidopsis thaliana Model System.</a> Genetics. 208 (4), 1337-1349 (2018).</li><li>Alatorre-Cobos, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved,+low-cost,+hydroponic+system+for+growing+Arabidopsis+and+other+plant+species+under+aseptic+conditions.">An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions.</a> BMC Plant Biology. 14, 69 (2014).</li><li>Haney, C. H., Samuel, B. S., Bush, J., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+with+rhizosphere+bacteria+can+confer+an+adaptive+advantage+to+plants.">Associations with rhizosphere bacteria can confer an adaptive advantage to plants.</a> Nature Plants. 1 (6), (2015).</li><li>Massalha, H., Korenblum, E., Malitsky, S., Shapiro, O. H., Aharoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+root-bacteria+interactions+in+a+microfluidics+setup.">Live imaging of root-bacteria interactions in a microfluidics setup.</a> Proceedings of the National Academy of Sciences USA. 114 (17), 4549-4554 (2017).</li><li>Townsley, L., Yannarell, S. M., Huynh, T. N., Woodward, J. J., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-AMP+Acts+as+an+Extracellular+Signal+That+Impacts.">Cyclic di-AMP Acts as an Extracellular Signal That Impacts.</a> MBio. 9 (2), (2018).</li><li>Beauregard, P. B., Chai, Y., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+biofilm+induction+by+plant+polysaccharides.">Bacillus subtilis biofilm induction by plant polysaccharides.</a> Proceedings of the National Academy of Sciences U S A. 110 (17), E1621-E1630 (2013).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+of+Bacteria+to+Plant+Surfaces+Measured+in+the+Laboratory.">Adherence of Bacteria to Plant Surfaces Measured in the Laboratory.</a> Journal of Visual Experiments. 136 (136), (2018).</li><li>Garcia-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> Journal of Visual Experiments. (60), (2012).</li><li>Cole, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+bacterial+plant+colonization+genes.">Genome-wide identification of bacterial plant colonization genes.</a> PLoS Biology. 15 (9), e2002860 (2017).</li><li>Lundberg, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+core+Arabidopsis+thaliana+root+microbiome.">Defining the core Arabidopsis thaliana root microbiome.</a> Nature. 488 (7409), 86-90 (2012).</li><li>Grandchamp, G. M., Caro, L., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pirated+Siderophores+Promote+Sporulation+in+Bacillus+subtilis.">Pirated Siderophores Promote Sporulation in Bacillus subtilis.</a> Applied Environmental Microbiology. 83 (10), (2017).</li><li>Gange, A. C., Gadhave, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+growth-promoting+rhizobacteria+promote+plant+size+inequality.">Plant growth-promoting rhizobacteria promote plant size inequality.</a> Science Reports. 8 (1), 13828 (2018).</li><li>Levy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+features+of+bacterial+adaptation+to+plants.">Genomic features of bacterial adaptation to plants.</a> Nature Genetics. 50 (1), 138-150 (2018).</li><li>Mart&#237;nez-Hidalgo, P., Maymon, M., Pule-Meulenberg, F., Hirsch, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+root+microbiomes+for+healthier+crops+and+soils+using+beneficial,+environmentally+safe+bacteria.">Engineering root microbiomes for healthier crops and soils using beneficial, environmentally safe bacteria.</a> Canada Journal of Microbiology. , 1-14 (2018).</li><li>Niu, B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+Composition+Dynamics+of+a+Maize+Root-associated+Simplified+Bacterial+Community+and+Evaluation+of+Its+Biological+Control+Effect.">Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect.</a> Bio Protocol. 8 (12), (2018).</li></ol>

Plants in all environments interact with thousands to millions of different bacteria and fungi5,7. 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...

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 fields7,8. 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 metabolites7,9. 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 plants14,15. 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.&#160;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&#8217; interactions)7. This protocol is designed to be adaptable for use with various bacteria, plants, and environmental conditions.

<table>
	<tbody>
		<tr>
			<td>Required Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL eppendorf tubes</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>BD Falcon</td>
			<td>1801343</td>
			<td></td>
		</tr>
		<tr>
			<td>Aeraseal</td>
			<td>Excel Scientific</td>
			<td>BE255A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria of Interest</td>
			<td>any</td>
			<td>N/A</td>
			<td>Stored at -80&#730;C in 40% glycerol preferred</td>
		</tr>
		<tr>
			<td>BactoAgar</td>
			<td>BD</td>
			<td>2306428; REF 214010</td>
			<td></td>
		</tr>
		<tr>
			<td>bleach</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Conviron</td>
			<td>any</td>
			<td>N/A</td>
			<td>Short Day Light-Dark Cycles: 460-600 &#181;moles/m&#178;/s set at 9/15 hours light/dark at 18/21&#730;C, with inner power outlet</td>
		</tr>
		<tr>
			<td>Dessicator Jar: glass or heavy plastic</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Flame</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>any</td>
			<td>N/A</td>
			<td>At optimal temperature for growth of specified bacteria</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lennox LB Broth</td>
			<td>RPI</td>
			<td>L24066-1000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetters</td>
			<td>any</td>
			<td>N/A</td>
			<td>Volumes 5 &#181;L to 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Microscope (preferably fluorescence)</td>
			<td>any</td>
			<td>N/A</td>
			<td>Could be light if best definition not important</td>
		</tr>
		<tr>
			<td>MS Salts + MES</td>
			<td>RPI</td>
			<td>M70300-50.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Plate Shaker</td>
			<td>any</td>
			<td>N/A</td>
			<td>Capable of running at 220 rpm for at least 96 hours</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>any</td>
			<td>N/A</td>
			<td>50 mL total volume</td>
		</tr>
		<tr>
			<td>Reservoirs</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Hole Punch</td>
			<td>any</td>
			<td>N/A</td>
			<td>Approximately 7mm punch diameter</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tape</td>
			<td>3M</td>
			<td>MMM1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon Mesh</td>
			<td>McMaster-Carr</td>
			<td>1100t41</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>GoldBio</td>
			<td>x4281c</td>
			<td>other vendors available</td>
		</tr>
		<tr>
			<td>Suggested Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Prong Ultrasonicator attachment</td>
			<td>any</td>
			<td>N/A</td>
			<td>For sonicating multiple samples at once. Can be done individually</td>
		</tr>
		<tr>
			<td>Alumaseal II</td>
			<td>Excel Scientific</td>
			<td>FE124F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipetter/Repetter</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 96-well plates</td>
			<td>any</td>
			<td>N/A</td>
			<td>For serial dilutions. Can be replaced by eppendorf tubes</td>
		</tr>
		<tr>
			<td>Biological Materials Used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arabidopsis thaliana seeds</td>
			<td>any</td>
			<td>N/A</td>
			<td>We recommend Arabidopsis Biological Resource Center for seed stocks</td>
		</tr>
		<tr>
			<td>Arthrobacter nicotinovorans</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Curtobacterium oceanosedimentum</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Microbacterium oleivorans</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Pseudomonas simiae WCS417r</td>
			<td></td>
			<td></td>
			<td>Published in a similar system in Haney, et al. 2015. Strain used developed in Cole, et al. 2017</td>
		</tr>
	</tbody>
</table>

monitoring bacterial colonization and maintenance on arabidopsis thaliana roots in a floating hydroponic system

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&#8217;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.

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&#8217; (4&#8211;6 mm length) roots, doing so for many plants would take a prohibitive amount of time....

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

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.

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

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Most experiments study plant-microbe interactions by focusing on colonization, but our protocol is designed to look both at colonization and maintenance of the bacteria on the root. We can change the experiment to fit different bacteria and plants, and we can even test multiple bacteria in each 24-well plate. To begin, use a standard hole puncher to create disks of plastic mesh.Collect the disks in a glass container, and cover loosely. Sterilize using an autoclave set to a 20-minute dry cycle. Use flame-sterilized tweezers to distribute approximately 40 sterilized mesh disks in a single layer across the surface of previously prepared plant growth medium agar plate.Place two previously sterilized seeds at the center of each mesh. Seal the plate with surgical tape, and incubate for two to six days at four degrees Celsius in darkness to vernalize the seeds. Then, place the plate agar-side down in a plant growth chamber for eight to 10 days under short-day settings of nine hours of light at 21 degrees Celsius and 15 hours of dark at 18 degrees Celsius to germinate and grow seedlings.Add one milliliter of bacterial growth medium to each well of a sterile 24-well plate. To transfer the germinated seedlings embedded in mesh, use flame-sterilized forceps to gently peel the mesh containing two germinated, equally sized, and undamaged seedlings up and off the agar plate. Transfer one float with seedlings to each well of bacterial growth liquid, root-side down.Next, resuspend bacteria grown overnight on agar plates in liquid bacterial growth medium. Add 10 microliters of bacterial suspension to each well, except for media-only control wells, for a final concentration of one million colony-forming units of bacteria per well. To seal the plate for sterile growth, carefully press gas-permeable film across the plate without touching the sticky side.Apply pressure around each of the rings made by the wells to ensure that each well has been individually sealed. Then, close the plate with its plastic lid snuggly over the gas-permeable film. Place the plate in a plant growth chamber on an orbital plate shaker set to 220 rpm, and incubate for 18 hours under the same conditions as the seedlings were originally germinated.To rinse all floats with plants, add one milliliter of sterile water to each well of a new 24-well plate. Remove gas-permeable film from the plate with plants, and use sterile forceps to transfer floats to wells with water, and incubate for 10 minutes at room temperature without agitation. Then, fill each well of a new 24-well plate with one milliliter of plant growth medium.Transfer one mesh to each well, and cover with a gas-permeable seal. Incubate for 72 hours on the orbital plate shaker at 220 rpm in plant growth chamber. After the incubation, rinse the plants as previously done.After the rinsing, remove the seedlings from the mesh by gently placing flame-sterilized forceps below the leaves on the leaf side of the mesh. Lightly pinch the stem, and wiggle the seedlings up and away from the mesh to dislodge the root without breaking it. To remove bacteria from plant roots, transfer seedlings from each mesh into individual wells of a 24-well sonication plate containing one milliliter of double-distilled water.Sonicate all samples within the plate at once using a multipronged sonicator as described in the manuscript. To quantify the bacteria, perform serial 10-fold dilutions of the sonicated samples in bacterial growth medium. Spread using sterile glass beads, and incubate at the optimal temperature for bacteria until individual colonies are countable.To collect intact plant root, use forceps to remove the seedlings from the mesh. Then, transfer each plant to a microscope slide by placing the tip of the root on the slide and dragging it 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 each sample to hydrate interfaces between the coverslips and slides.Place a glass coverslip just above the root crown and below the shoot leaves to avoid slanting of the coverslip, and press down gently. Then, image the bacteria using appropriate excitation/emission filters to differentiate bacteria from each other and the plant root. Pseudomonas simiae, naturally fluorescent bacterium, colonized Arabidopsis thaliana roots and was maintained on the root following transfer to plant growth medium.Root crown, mid-length, and tip show fluorescence due to colonization of Pseudomonas simiae. The no-bacteria negative control showed no colonization. When Pseudomonas simiae on Arabidopsis thaliana roots were quantified after 18 hours of colonization or 72 hours of maintenance, total number of colony-forming units per seedling at either time point showed good reproducibility across biological replicates performed on different days.There was an increase in the number of colony-forming units per seedling after 72 hours in maintenance medium, as compared to the numbers observed at the post-colonization 18-hour time point, indicating that active growth of the colonized bacteria on the plant root occurred during the maintenance stage. Three species of bacteria isolated from Arabidopsis thaliana grown in natural soil under laboratory conditions were used to monitor the association of multiple species on plant roots. They were clearly differentiated on media containing X-gal due to differences in colony morphology and color, which allowed counting the colony-forming units per seedling of each species without antibiotic selection, even in multi-species coculture.These three species of bacteria were all colonized and maintained on the root, whether alone or in bacterial coculture. Each species showed trends that were similar across different biological and technical replicates, showing that this protocol can be used to measure both relative or total colony-forming units per root of each species. When grown alone, no individual species showed substantial increase in abundance during the maintenance stage, but the overall colony-forming units per root of the combined community increased in cocultures, indicating that these bacteria do not prohibit the colonization of the other strains.The most difficult steps are those that require the removal of intact plants from the mesh disks. Being patient and gentle will make this possible. If the bacterial cells are fluorescent, the samples could be sorted by flow cytometry to determine the relative numbers of cells.

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