Name: a hydroponic co-cultivation system for simultaneous and systematic analysis of plant/microbe molecular interactions and signaling
Uploaded: 2017-07-22T14:00:00
Description: Watch this Scientific Journal Video about A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling at JoVE.com

We would like to thank Brian Weselowski and Alexander W. Eastman for their help and useful discussion. We would also like to thank Drs. Eugene W. Nester, Lingrui Zhang, Haitao Shen, Yuhai Cui, and Greg Thorn for their help, useful discussions, and critical reading of the manuscript. This research was funded by Agriculture and Agri-Food Canada, Growing Forward-AgriFlex (RBPI number 2555) and Growing Forward II project number 1670, conducted by the authors as a part of their duties. This study was also partially funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2015-06052 awarded to Z-C Yuan.

1. Experimental Planning
<ol>
	<li>Determine the specific goals of the experiment.</li>
	<li>Read through the entire protocol below. Determine which sections are relevant to the specific goals and whether any modifications need to be made. See below for examples.
	<ol>
		<li>Consider whether the experiments will use A. thaliana plants and A. tumefaciens bacterium, as below, or another plant-microbe combination. 
		NOTE: While various plant species can be used, the thickness of th...

Given the gradual nature of root secretion, the concentration of virulence-inducing chemicals produced in planta and their effects on dynamic plant-microbe interactions occur in spatial and temporal gradients. In this hydroponic co-cultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defenses is supplemented. In contrast, using conventional approaches, the addition of synthetic chemicals, such as acetosyringone, creates a sudden, artificial spike in concentrations. ...

Plant-associated microbes play important roles in biogeochemical cycling, bioremediation, mitigation of climate change, plant growth and health, and plant tolerance to biotic and abiotic stresses. Microorganisms interact with plants both directly through plant cell wall contact and indirectly via chemical secretion and signaling1,2,3. As sessile organisms, plants have developed direct and indirect mechanisms to resist infection by pathogens. Direct defenses include structural defenses and the expression of defense proteins, whereas indirect defenses include secondary plant metabolite production and the attraction of organisms antagonistic to invading pathogens4,5. Plant-derived root exudates, secretions, mucilages, mucigel, and lysates alter the physical-chemical properties of the rhizosphere to attract or repel microbes towards their hosts6. The chemical composition of root secretion is species-specific, thereby serving as a selective filter that allows certain microorganisms capable of recognizing such compounds to flourish in the rhizosphere6. Thus, compatible microbial species may be stimulated to activate and enhance their associations, either to the benefit or detriment of the plant host1.
Understanding plant-microbe interactions in the rhizosphere is key to enhancing plant productivity and ecosystem functioning, since a majority of the microbial and chemical exposure occurs at the root structure and soil-air interface2,6,7,8. However, the examination of subterranean plant-microbe interactions and reciprocal responses has been a challenge due to its intriguingly complex and dynamic nature and the lack of suitable experimental models with natural root structure and plant morphology under tightly controllable growth conditions. As one of the most heavily studied phytopathogens, Agrobacterium infects a wide range of plants with agricultural and horticultural importance, including cherry, apple, pear, grape, and rose9. Agrobacterium is an important model organism for understanding plant-pathogen interactions and is a powerful tool in plant transformation and plant engineering10,11,12,13,14.
Molecular plant-Agrobacterium interactions have been well studied for several decades, and the current understanding of Agrobacterium pathogenicity is extensive9,11,15,16. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of perceiving plant-derived signals, resulting in the fine modulation of its virulence program and cell-to-cell communication, so-called quorum sensing17. The Agrobacterium virulence program is regulated by several signals available in the rhizosphere and involves two sets of 2-component systems, the ChvG/I system and the VirA/G system. Acidic conditions in the rhizosphere activate the transcription of chvG/I, virA/G, and several other genes involved in Agrobacterium pathogenicity, including virE0, virE1, virH1, virH2, and genes of the type VI secretion system (T6SS)18. Plant-derived phenolic compounds, including acetosyringone (4&#39;-hydroxy-3&#39;,5&#39;-dimethoxyacetophenone), activate the VirA/G 2-component system through phosphorylation signaling mechanisms19. VirA/G then activates the entire vir regulon, resulting in the transfer and integration of a ~20 kb bacterial DNA fragment called transfer DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the plant nucleus16. T-DNA carries genes responsible for the synthesis of the plant hormones indole-3-acetic acid (IAA) (iaaM and iaaH) and cytokinin (ipt), and once expressed in plant cells, large amounts of these phytohormones are produced. This results in abnormal tissue proliferation and plant tumor development, known as crown gall disease, which is a chronic and resurgent problem for plants9,11,20. IAA also acts collectively with salicylic acid and gamma-amino butyric acid to repress Agrobacterium virulence or to reduce Agrobacterium quorum sensing (QS)17,21,22. To counter this repression, T-DNA also carries genes for opine biosynthesis, which activates Agrobacterium quorum sensing to promote Agrobacterium pathogenicity and also serves as a nutrient source for the pathogen22,23.
Despite an overall deep understanding of Agrobacterium-plant interactions and the resultant T-DNA transfer into the plant host, the complex signaling events at the initial stage of interaction are less well understood. This is partially due to the limitations of conventional approaches to investigate Agrobacterium-plant signaling. Plant cell suspension cultures and artificial site-specific wounding are commonly used to study molecular plant-microbe interactions24,26,27. However, cell suspensions lack typical plant morphology; in particular, plant suspension cells do not have root structures and root exudates, which are very important for activating microbial chemotaxis and virulence28,29. The maintenance of plant morphology and root structure has been addressed by artificially wounding plants, which facilitates site-specific infection, resulting in the detection of induced plant defense-related genes in directly infected plant tissue30,31. However, artificial wounding is significantly different from pathogen infection in nature, particularly as wounding leads to jasmonic acid (JA) accumulation, which systemically interferes with natural plant signaling and defense26. In addition, synthetic chemicals are typically used to artificially induce plant host responses or pathogen virulence. Although the supplementation of such chemical compounds reflective of concentrations in planta is possible, such supplementation does not account for the diffusion of root exudates gradually into the surrounding rhizosphere, which generates a chemotactic gradient sensed by microbes28,32. Given the limitations of conventional approaches to study plant-microbe interactions, the accuracy and depth of the data obtained might be impeded and restrictive, and the knowledge generated from the conventional approaches may not translate directly in planta. Many aspects of plant-Agrobacterium signaling are not yet fully understood, particularly at the early stage of interactions, when the disease symptoms have not yet developed.
To amend the limitations of conventional approaches, this work presents an inexpensive, tightly controllable, and flexible hydroponic cocultivation system that allows researchers to gain deeper insights into the complex signaling and response pathways at the initial stage of molecular plant-microbe interactions. Hydroponics has been widely used to study plant nutrients, root exudates, growth conditions, and the effects of metallic toxicity on plants33,34. There are several advantages of hydroponic models, including the small spatial requirements, the accessibility of various plant tissues, the tight control of nutrient/environmental conditions, and the pest/disease control. Hydroponic systems are also less limiting to plant growth in comparison to agar/phytoagar plating techniques, which typically restrict growth after 2-3 weeks. Importantly, the maintenance of whole-plant structures facilitates the natural root secretion necessary for microbial chemotaxis and virulence induction8,29. The system described here is simpler and less labor-intensive than the alternatives33,34. It uses fewer parts and does not require any tools other than standard scissors. It uses metal mesh (as opposed to nylon33) as a strong support for plant growth and a simple method of aeration under sterile conditions through shaking to support microbial growth. In addition, the system can use metal mesh of various sizes to support plant growth, which accommodates diverse plant species without restricting the width of their roots.
In the hydroponic cocultivation system presented here, plants are cultivated in a sterile hydroponic system where the plant roots secrete organic compounds supporting the growth of inoculated bacteria. In this cocultivation system, no artificial chemicals, such as plant hormones, defense elicitor, or virulence-inducing chemicals, are supplemented, which reflects the natural cell-signaling homeostasis during plant-microbe interactions. With this hydroponic cocultivation system, it was possible to simultaneously determine gene expression in Arabidopsis thaliana Col-0 root tissue upon infection by Agrobacterium, as well as the activation of Agrobacterium genes upon cocultivation with Arabidopsis. It was further demonstrated that this system is suitable to study Agrobacterium attachment to plant roots, as well as the plant root secretome profile, upon cocultivation (infection) with Agrobacterium (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55955/55955fig1.jpg" /> 
Figure 1: Overview of the Hydroponic Cocultivation System, with Sample Analyses. Plants are grown on top of the mesh (shoots above the mesh), with the roots immersed in hydroponic medium that is then inoculated with bacteria for coculture. Plant tissues and bacteria are then separated for simultaneous extractions and analyses. This figure has been modified from reference35.

<table border="0" cellpadding="0" cellspacing="0" width="663">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">plant seeds (Arabidopsis thaliana Col-0)</td>
			<td style="width:163px;">Arabidopsis Biological Resource Centre</td>
			<td style="width:119px;">CS7000</td>
			<td style="width:167px;">https://abrc.osu.edu/order-stocks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">bacteria (Agrobacterium tumefaciens C58)</td>
			<td style="width:163px;">University of Washington</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">labeled bacteria</td>
			<td style="width:163px;">in-house</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">vortex</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">microcentrifuge tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">microcentrifuge</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">5% sodium hypochlorite</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">double distilled water</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">autoclave</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">micropipette&#160;</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70 % ethanol</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Murashige and Skoog (MS) basal salts</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sucrose</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MES</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">B5 vitamin mix</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td>G1019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">phytoagar</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">deep Petri dishes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">stainless steel mesh</td>
			<td style="width:163px;">Ferrier Wire Goods Company Ltd</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">grade: 304; mesh count: 40 &#215; 40; wire DIA: 0.01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">micropore tape, 1 inch</td>
			<td style="width:163px;">3M</td>
			<td style="width:119px;">1530-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">diurnal growth chamber</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">cylindrical glass tanks, 100 &#215; 80 mm&#160;</td>
			<td style="width:163px;">Pyrex</td>
			<td style="width:119px;">3250</td>
			<td style="width:167px;">other sizes can be used, in which case liquid content may need adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">flow hood</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">forcepts</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">yeast extract</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">tryptone</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">MgSO4</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">shaking incubator</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">spectrophotometer</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NaCl</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">shaker</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">scissors</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">fluorescence microscope</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">microscope slides and cover slips</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">nail polish</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bacterial RNA extraction kit</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">plant RNA extraction kit (RNeasy Plant Mini Kit)</td>
			<td style="width:163px;">Qiagen</td>
			<td>74903 or 74904</td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for RT-qPCR</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for microarray analysis</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">liquid nitrogen</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">mortar and pestle</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">0.2 &#181;m pore filter</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">50 mL conical tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">freeze dryer</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">sealable test tubes</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ethyl acetate</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">nitrogen gas</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for HPLC</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">material and equipment for ESI-TOF-MS</td>
			<td style="width:163px;">(various)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">optional, depends on downstream analyses</td>
		</tr>
	</tbody>
</table>

a hydroponic co-cultivation system for simultaneous and systematic analysis of plant/microbe molecular interactions and signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

Growth in the hydroponic co-cultivation system
The growth curve of A. tumefaciens C58 demonstrated a significant lag phase in the initial 16 h of cocultivation, followed by a very stable growth when co-cultivated with A. thaliana Col-0, up to a maximum OD600 of about 0.9 starting at 48 h postinoculation. By contrast, essentially no bacterial growth was observed in the control culture without A. thal...

Watch this Scientific Journal Video about A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling at JoVE.com

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

The overall goal of this system is to study the reciprocal signaling interaction and responses between plant and micro-partners or plant responses to chemicals or abiotic factors in a simply experimental setup that closely mimics the natural environment. This video shows co-cultivation of plants with Agrobacterium tumefaciens bacterium. This method can help answer key questions in plants'microbial interactions, microbial or plants stress response, and in molecular signaling, including complex signaling even at an initial stage of a plant's microbial interactions.The main advantage of this technique is that it helps to understand the nature signaling and response of a plant's microbial interactions, in a simple set-up that includes the mimic of natural environment. Though this method can be used to provide insight into plant interactions with a single microbial species, it can also be used to study plant interactions with multiple microbial species or with abiotic factors. We had the idea for this method while searching for cell signaling homeostasis during plant microbe interactions that closely mimic natural situations.To begin the experiment, seeds must be sterilized. For Arabidopsis thaliana, combine approximately 200 seeds with 500 microliters of deionized water in a micro centrifuge tube, and vortex the tube at high speed. Centrifuge the tube at 9000 x g for 30 seconds in a tabletop micro centrifuge.Using a pipet, remove the water supernatant. Next, add 300 microliters of 2%sodium hypochlorite to the seeds and vortex them. Let the mixture settle at room temperature.After one minute, centrifuge the seeds. With the pipet, remove the sodium hypochlorite solution. Then add 500 microliters of sterile, deionized water to the seeds, and vortex the mixture.After centrifuging the tube, remove the water. Add 500 microliters of 70%ethanol to the seeds, and vortex the mixture. After allowing the seeds to settle for one minute, centrifuge the tube.Remove the 70%ethanol using a pipet. Wash the seeds five times with 500 microliters of sterile, deionized water. Then, re-suspend the sterilized seeds in 500 microliters of sterile, ultra pure water.Pour 25 milliliters of half strength Murashige and Skoog, or MS medium with 04%phyto agar into each sterile, deep Petri dish. For each Petri dish, cut out a 90 millimeter by 90 millimeter square of stainless steel mesh. Bend the corners of each square mesh just enough so that the mesh fits inside the Petri plate.Bend the corners at a 90 degree angle to the bulk of the mesh to allow the mesh to be propped up by the corners, leaving enough space below for root development. Put the cut mesh squares in a beaker and cover them with aluminum foil. Sterilize the mesh squares by autoclaving using a 30 minute dry cycle.Once the medium has solidified in the Petri dishes, and the mesh is sterile, place each mesh square on top of the solid medium with the bent corners facing down. Push the mesh so that the corners penetrate the medium and the bulk of the mesh touches the top surface of the medium. Next, use a sterile 200 microliter pipet to draw out the individual surface-sterilized A thaliana seeds and gently transfer each seed on top of the mesh.Place the seeds so that they are spaced per the plant species and experimental needs. Seal the Petri dishes with lids and place porous surgical tape around the edges of the plates. Wrap the seeds in aluminum foil.Stratify the seeds by placing the entire Petri dish. After two days, cultivate the seeds in the Petri dish at 22 to 24 degrees Celsius with a 16 hour photo period for 10 to 14 days. In a sterilized flow hood, pour 18 milliliters of autoclaved liquid MS medium into sterile cylindrical glass tanks with sterile lids.Next, use sterile forceps to gently transfer each mesh square with 10 to 14 day old seedlings from the Petri dish of semi solid medium to a hydroponical cylindrical tank. The seedlings shown here are alfalfa. Seal the lids onto the tanks using porous surgical tape.Then, cultivate the seedlings at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Prior to the inoculation, examine the tank with seedlings carefully for any potential contamination. The medium in the uncontaminated tanks should be clear and transparent.If tanks are cloudy with contamination, discard them and number the rest of the tanks. Sample 20 microliters of hydroponic medium from each numbered tank and spot it on a Petri dish with Luria broth or LB agar. Incubate the dish at 28 degrees Celsius for 28 hours.Immediately inoculate each numbered hydroponic tank with 50 microliters of the agrobacterium tumefacians suspension into each numbered hydroponic tank. Co-cultivate the seedlings and A tumefacians bacterium at 22 to 24 degrees Celsius with a 16 hour photo period and shaking at 50 rpm. Examine the spotted LB plate after 12 and 28 hours of incubation to verify the sterility of the hydroponic growth medium.If there is any contamination, do not use the corresponding numbered tank inoculated with bacteria for further downstream assays. Next, separate roots from the bacterial suspension by lifting the mesh plate. If using roots for subsequent analyses, cut the roots, quickly rinse them in double distilled water, and dry on sterilized filter paper.If using leaf or other tissue, cut off the tissue. For plant transcript analysis, the roots or shoots should be immediately deposited into liquid nitrogen. For bacterial transcript analysis, transfer 1.5 milliliters of the culture from the co-cultivation tank and pellet the cells by centrifugation.Following an appropriate co-cultivation, use sterile scissors to separate individual secondary roots, the side branches of the main root. Next, rinse the roots in double distilled water to remove loosely bound material. Submerge each root in 30 microliters of water on a microscope slide, and cover it with a glass cover slip.Seal the edges of the coverslip with nail polish to protect the roots from dehydration. Then visualize the attachment of A tumefacians to A thaliana roots by fluorescence microscopy. After an appropriate co-cultivation, separate the plants from the hydroponic medium.Sterilize the 18 milliliters of hydroponic medium by passing it through a 0.2 micron pore filter into 50 milliliter conical tubes. Freeze the samples at 80 degrees Celsius overnight. The following day, loosen the caps on the 50 milliliter conical tubes and place them in a freeze drying machine for 36 hours.Finally, proceed to store or process the samples for HPLC analysis and compound detection. Without artificially supplied nutrients, A tumefacians c58 grew in the co-cultivation system. By contrast, essentially no bacterial growth was observed in the control culture without A thaliana.The hydroponic co-cultivation systems can be used to study the attachment of bacteria to the root structure as seen in this microscopic visualization, where PCherry red flourescence-labeled bacteria were localized to the plant root structure with a typical rock-like or filamenta shape. Gene expression profiles of both plant posts and interacting microbes can be ascertained via QRTPCR. After eight hours of co-cultivation, eight tumefacians cells revealed up regulation of expression of virulence genes.At the same time, roots of A thaliana revealed differential gene expression in response to co-cultivation. After 72 hours of co-cultivation, 35 compounds were enhanced and 76 compounds were decreased in the secretome of inoculated plants compared to the control. This technique paves the way for researchers to study plant-microbe interactions and explore the reciprocal responses between plant and microbe or microbe biome partners, or plant responses to abiotic factors in a simple set-up that closely mimics the natural environment.After watching this with you, we hope you are able to set up a plant microbial co-cultivation system in hydroponics to study molecular plants, microbial interactions and the signaling responses in a more natural and manageable environment.

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