The work was supported by Pomona College Start-up Funds and Hirsch Research Initiation Grants Fund (to FJ) as well as the Pomona College Molecular Biology Program through the Stellar Summer Research Assistant Program (to KG).

1. Growing the plants
<ol>
	<li>Add a 4 cm-thick layer of fine vermiculite to standard 10 in. x 20 in. (254 mm x 501 mm) plant trays with no holes.</li>
	<li>Place the seed holders (see Table of Materials) 2 cm apart in the plant trays.</li>
	<li>Fill seed holders with vermiculite.</li>
	<li>Place a sunflower seed with its pointed end down in each seed holder, pushing down so half of the seed remains exposed. 
	NOTE: A sunflower seed is asymmetrical and the pointed end from where the radicle will emerge should point downwards. Proper seed placement is important as the reorientation of root and stem are not possible within the seed holders. The rounded end of the seed should extend past the top of the seed holder.</li>
	<li>Once the seeds are in place, cover them with an additional 2 cm-thick layer of fine vermiculite. Water by misting from above. The surface should remain wet after an hour. If that is the case, cover the trays with lids.</li>
	<li>Grow plants in a growth chamber or a greenhouse. Recommended conditions are a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1 and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 5 days. 
	NOTE: Watering should not be necessary unless the surface becomes visibly dry.</li>
</ol>
2. Set up of the hydroponic system
<ol>
	<li>Find an appropriately sized container suitable for growing plants hydroponically. The size of the container should be adapted to the space available in the growth chamber or greenhouse. A minimal depth of 15 cm is recommended.</li>
	<li>Fill the container with distilled water and add general hydroponics fertilizer as indicated by the manufacturer. The resulting hydroponic solution should be aerated and in constant movement, which can be achieved by using air and water pumps.</li>
	<li>Prepare the hydroponics floaters.
	<ol>
		<li>Cut a sheet of 2 cm-thick expanded polystyrene foam (see Table of Materials) to the dimensions of the container. The sheet should cover most of the surface of the container in order to limit the growth of algae. A wood burning tool is effective for cutting polystyrene foam and is versatile enough for this protocol. 
		CAUTION: Fumes or vapor released during hot cutting of polystyrene foam are serious health hazards. Use proper respiratory protection. Users can also satisfy ventilation requirements by cutting the foam under a fume hood.</li>
		<li>Make holes (1-2 cm in diameter) in the polystyrene foamsheet using a wood burning tool. The distance between the centers of two holes can be adjusted to the needs of the experiment. However, a minimal distance of 2.5 cm is recommended.</li>
	</ol>
	</li>
</ol>
3. Transfer of seedlings to hydroponics and plant growth
<ol>
	<li>Gently pull out 5-day old seedlings from the vermiculite and transfer immediately to a container filled with water for 30 min. This step will remove excess vermiculite and soften the remaining pericarps. The emerging primary root should be visible.</li>
	<li>Remove the pericarp walls by hand if needed in order to optimize the future expansion of the cotyledons.</li>
	<li>Transfer the seedlings within the seed holders to the polystyrene foam floater. Grow the plants with a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1, a relative humidity of 65% and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 2 days.</li>
</ol>
4. Preparation prior to treatment
NOTE: This procedure is for testing 20 chemicals from a small compound library in triplicate, with 100 &#181;M ABA in 10 mM MES-KOH (pH = 6.2) and 10 mM MES-KOH (pH = 6.2) containing 1% (v/v) dimethyl sulfoxide (DMSO) as positive and negative controls, respectively.
<ol>
	<li>Ensure that there are enough plants ready for treatment. Plants ready for treatment should be mature enough to have fully unfolded cotyledons, but young enough that lateral root proliferation is minimal. To do a standard screening, 69 such plants are needed.</li>
	<li>Remove the 96-well plate containing the small compounds from the -80 &#176;C freezer. Thaw at room temperature.</li>
	<li>Prepare 80 mL of 10 mM MES-KOH buffer adjusted to a pH of 6.2 with 1 M KOH.</li>
	<li>Label cap-less 2 mL microtubes. Prepare at least six tubes for the negative control treatment (10 mM MES-KOH (pH = 6.2) containing 1% (v/v) DMSO). Use three tubes for ABA treatment (100 &#181;M ABA in 10 mM MES-KOH pH = 6.2), positive control). Use the remaining 60 tubes to analyze the effect of the 20 chemicals in triplicate.</li>
	<li>Transfer 10 &#181;L of each chemical (10 mM in DMSO) into each of the three appropriately labeled tubes. Pipet 10 &#181;L of 10 mM ABA dissolved in DMSO into three tubes and 10 &#181;L of DMSO into the six control tubes. 
	CAUTION: By nature, some compounds may cause serious health effects and users must take appropriate protective measures.</li>
	<li>Add 990 &#181;L of 10 mM MES-KOH (pH = 6.2) to each of the 69 tubes. Dispense the MES buffer with enough force to mix the chemical with the MES buffer but be very careful not to use so much force that the chemicals and MES buffer squirt out of the tube. Alternatively, vortex at low speed.</li>
</ol>
5. Set up the thermal imaging camera
<ol>
	<li>Mount the thermal imaging camera on a copy stand. Connect all cables to a laptop. 
	NOTE: The recording is done under conditions of temperature (20 &#176;C to 25 &#176;C), humidity (50% to 70%) and light quality (110 to 140 &#181;mol photons&#183;m-2&#183;s-1) similar to those used to grow the plants.</li>
	<li>Turn on the camera then the laptop and open the thermal imaging analysis software. 
	NOTE: The subsequent instructions for recording apply to a specific software used (see Table of Materials).</li>
	<li>Adjust the recording settings.
	<ol>
		<li>Mouse over the record red button at the top of the central window. A dropdown menu will appear. Click on the wrench icon Record Settings.</li>
		<li>Select the appropriate record mode and options. The record periodically option, with one frame captured per minute and a manual stop could be used. Note the file destination where the software will save the video. Close the Record Settings window.</li>
	</ol>
	</li>
</ol>
6. Plant preparation and treatment
<ol>
	<li>Place the cap-less tubes containing the chemicals in tube racks. Alternatively, a polystyrene foam sheet can be cut and poked with a wood burning tool as described in step 2.3 to make a custom tube rack. The diameter of each hole should be very close to the external diameter of the capless tubes in order to hold them firmly. 
	NOTE: The field of view of the camera is a limiting factor that should be taken into consideration when deciding on how the cap-less tubes will be held in place.</li>
	<li>Evenly distribute the positive and negative control tubes as well as the experimental tubes in the racks to account for position-related bias17.</li>
	<li>Prepare the following materials next to the plants grown hydroponically: microdissection scissors, a shallow dish with water, delicate task wipes, the 69 cap-less tubes containing the different chemicals.</li>
	<li>Repeat the following steps for each plant to be treated. The sunflower sprout will always remain in the seed holder.
	<ol>
		<li>Carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.</li>
		<li>Cut the primary root underwater to prevent cavitation. The cut should occur 0.8-1 cm underneath the most basal end of the seed holder.</li>
		<li>Inset the freshly cut plant into one of the tubes containing the chemicals.</li>
		<li>If there are any drops of water on the cotyledons, gently dab them dry with a delicate task wipe. 
		NOTE: These four steps must be done as quickly as possible (10 min or less) to prevent inconsistencies in the kinetics study.</li>
	</ol>
	</li>
	<li>Move the plants under the thermal imaging camera and ensure that all plants are within the field of view of the camera. Adjust camera height and racks position as necessary.</li>
</ol>
7. Recording
<ol>
	<li>Focus the camera to the surface of the cotyledons by pressing Ctrl + Alt + A.</li>
	<li>Mouse over the red button and click on the Record a Movie option. A new window confirming the recording should open.</li>
	<li>Stop the recording 1-2 hours later. 
	NOTE: The protocol can be paused here.</li>
</ol>
8. Data collection
<ol>
	<li>Go to File | Open | Browse for the correct .SEQ file and open it.</li>
	<li>Stop playing the movie.</li>
	<li>On the left side of the main window, click on add a measurement cursor ROI (3x3 pixels) icon. ROI stands for Region of Interest.</li>
	<li>Mouse over the center of a cotyledon of the first plant and left-click once. The cursor 1 is now in place. Label the second cotyledon of the first plant by repeating the procedure. The order of the labels should be noted.</li>
	<li>Repeat the procedure. All plants should be labeled with two cursors.</li>
	<li>Click on the Edit ROIs icon. In the main window, left click and hold on the top left corner and scroll to the bottom right corner to select all the ROIs.</li>
	<li>Mouse over the Statistic Viewer icon and select Temporal Plot. A new window will open.</li>
	<li>Run the movie. A graph will fill with the data.</li>
	<li>In this window, click on the double arrow in the top right corner to open a new menu.</li>
	<li>Click on the Save icon. Save as X and Y values in the plot (.csv). Close the software once data has been exported.</li>
</ol>
9. Data analysis
<ol>
	<li>Open the .csv file using a data analysis software (e.g., Microsoft Excel). Note that the three first columns (A to C) provide information about the frame number, the absolute time and the relative time. The remaining columns give the temperature of each ROI over time.</li>
	<li>Decide on the nature of the statistical tool to be used; this decision depends on different factors including the experimental design. 
	NOTE: In our example, a standard score, or z-score, is calculated for each sample based on population mean and population standard deviation. For each sample, a p-value is then calculated from the z-score. This method allows the confirmation of the positive and negative controls as well as the identification of new compounds to be tested further.</li>
</ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetics+reveals+negative+regulation+of+abscisic+acid+signaling+by+a+plant+immune+response+pathway.">Chemical genetics reveals negative regulation of abscisic acid signaling by a plant immune response pathway.</a> Current Biology. 21 (11), 990-997 (2011).</li><li>Ito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Abscisic+Acid+Antagonists+Identified+with+Chemical+Array+Screening.">Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.</a> ChemBioChem. 16 (17), 2471-2478 (2015).</li><li>Ye, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Chemical+Inhibitor+of+ABA+Signaling+Targets+All+ABA+Receptors.">A Novel Chemical Inhibitor of ABA Signaling Targets All ABA Receptors.</a> Plant Physiology. 173 (4), 2356-2369 (2017).</li><li>Takeuchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+abscisic+acid+analogs+as+antagonists+of+PYL-PP2C+receptor+interactions.">Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.</a> Nature Chemical Biology. 10 (6), 477-482 (2014).</li><li>Rauf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+modification+of+sunflower+oil+to+expand+its+industrial+value.">Progress in modification of sunflower oil to expand its industrial value.</a> Journal of the Science of Food and Agriculture. 97 (7), 1997-2006 (2017).</li><li>Caraus, I., Alsuwailem, A. 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The authors have nothing to disclose.

The number of compounds that can be tested on a given day mostly depends on (i) the environmentally controlled space available to grow the plants and to perform the screen, as well as (ii) the number of individuals who can be involved in step 6 of the protocol. We recommend the use of three experimental replicates to consolidate the interpretation of the results after statistical treatment. In a typical day, one to two individuals can screen 60 compounds in triplicates without difficulty by testing for example [60 chemic...

Stress tolerance in plants is a polygenic trait influenced by a variety of molecular, cellular, developmental and physiological features and mechanisms1. Plants in a fluctuating environment need to continuously modulate their stomatal movements to balance the photosynthetic demand for carbon while maintaining sufficient water and preventing pathogen invasion2; however, the mechanisms by which these trade-off &#34;decisions&#34; are made are poorly understood3. Introducing bioactive molecules into plants can modulate their physiology and help in probing new mechanisms of regulation.
The large-scale screening of small molecules is an effective strategy used in anti-cancer drug discovery and pharmacological assays to test the physiological effects of hundreds to thousands of molecules in a short period of time4,5. In plant biology, high-throughput screening has showed its effectiveness for example in the identification of the synthetic molecule pyrabactin6, as well as the discovery of the long-sought receptor of abscisic acid (ABA)7,8. Since then, agonists and antagonists of ABA receptors, and small molecules able to modulate the expression of ABA-inducible reporter genes have been identified9,10,11,12,13,14,15. High-throughput screening approaches currently available to identify small compounds that can modulate stomatal aperture have some drawbacks: (i) protocols revolving around the ABA signaling pathway may prevent the identification of novel ABA-independent mechanisms, and (ii) in vivo strategies used for the identification of bioactive small molecules rely primarily on their physiological effects on seed germination or seedling growth, and not on the regulation of plant transpiration per se.
Additionally, while there are many ways to treat plants with bioactive molecules, most of them are not well suited for a large-scale study of stomatal movement. Briefly, the three most common techniques are foliar application by spraying or dipping, treatment of the root system, and root irrigation. Foliar application is not compatible with the most common and rapid methodologies to measure stomatal aperture since the presence of droplets at the leaf surface interfere with large-scale data collection. The major limitations of root irrigation are the large sample volume requirements, the potential retention of the compounds by elements in the rhizosphere, and the reliance on active root uptake.
Here, we present a large-scale method to identify new compounds regulating plant transpiration that does not necessarily involve ABA- or known drought-responsive mechanisms and allows for efficient and reliable treatment of plants. In this system, Helianthus annuus plants are treated using a root feeding approach that consists of cutting the primary root of seedlings grown hydroponically and dipping the cut site into the sample solution. Once treated, the effect of each compound on the transpiration of plants is measured using an infrared thermal imaging camera. Since a major determinant of leaf surface temperature is the rate of evaporation from the leaf, thermal imaging data can be directly correlated to stomatal conductance. The relative change in foliar temperature following chemical treatment thus provides a direct means to quantify the plant transpiration.
H. annuus is one of the five largest oilseed crops in the world16 and discoveries made directly on this plant may facilitate future transfers of technology. In addition, H. annuus seedlings have large and flat cotyledons, as well a thick primary root, which was ideal for the development of this protocol. However, this method can be readily adapted to other plants and a variety of compounds.
This protocol can be used to effectively identify molecules able to trigger stomatal closure or promote stomatal opening, which has major implications for understanding the signals that regulate stomatal conductance and plant adaptation to environmental stresses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1020 plastic growing trays without drain holes</td>
			<td></td>
			<td></td>
			<td>Standard 10 x 20 inch trays</td>
		</tr>
		<tr>
			<td>2.0 mL microtubes, capless</td>
			<td>Genesee Scientific</td>
			<td>22-283NC</td>
			<td></td>
		</tr>
		<tr>
			<td>Abscisic acid (ABA)</td>
			<td>Sigma-Aldrich</td>
			<td>A1049</td>
			<td></td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Active Aqua</td>
			<td>AAPA7.8L</td>
			<td>2 Outlets, 3W, 7.8 L/min</td>
		</tr>
		<tr>
			<td>Airstones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical compound library</td>
			<td>MicroSource Discovery</td>
			<td></td>
			<td>Natural Product Collection</td>
		</tr>
		<tr>
			<td>Creative Versa-Tool (wood burning tool)</td>
			<td>Nasco</td>
			<td>9724549</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO), plant cell culture tested</td>
			<td>Sigma-Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Dwarf Sunspot Sunflower seeds</td>
			<td>Outsidepride.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrosin B</td>
			<td>Sigma-Aldrich</td>
			<td>200964</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroponics fertilizer set (FloraBloom, FloraGrow, FloraMicro)</td>
			<td>General Hydroponics</td>
			<td>GL51GH1421.31.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Dell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissection scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr>
			<td>ResearchIR Software</td>
			<td>FLIR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R-Tech Rigid Polystyrene Foam Board</td>
			<td>Insulfoam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seedholders</td>
			<td>Araponics</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Tub (plastic utility tub)</td>
			<td>Maccourt</td>
			<td>ST3608</td>
			<td>36 x 24 x 8 inch tub used for hydroponics</td>
		</tr>
		<tr>
			<td>T450sc LWIR (Long-Wave Infrared) Handheld Thermal Imaging Camera</td>
			<td>FLIR</td>
			<td>FLIR-T62101</td>
			<td>Comes with required charging cable and USB cable needed to connect to laptop</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water filter</td>
			<td>SunSun</td>
			<td>HW-304B Pro Canister Filter</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of novel regulators of plant transpiration by large-scale thermal imaging screening in helianthus annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

An experiment using the red dye Erythrosine B (0.8 kDa) demonstrates the ability of chemicals to be visibly absorbed through a cut root into the cotyledons of a sunflower seedling within 10 minutes (Figure 1).
When plants are treated with ABA, an increase in leaf temperature is detected in sunflower cotyledons within minutes. This increase in leaf temperature is associated with a decrease in stomata...

Watch this Scientific Journal Video about Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus at JoVE.com

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus | Biology | Video

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

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5.9K Views.  Pomona College. We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Video: Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...