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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A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...