This work was supported by Project Grant RTI2018-095069-B-I00 funded by MCIN/AEI/10.13039/501100011033/ and by &#34;ERDP A way of making Europe&#34;. J.S.R. was funded by Plan Andaluz de Investigaci&#243;n, Desarrollo e Innovaci&#243;n (PAIDI 2020). N.L.P. was funded by Project Grant P18-RT-2398 from Plan Andaluz de Investigaci&#243;n, Desarrollo e Innovaci&#243;n.

1. Plant preparation
<ol>
	<li>Prepare Arabidopsis Col-0 plants following the steps below.
	<ol>
		<li>Fill a 10 cm diameter pot with a 1:3 vermiculite-plant substrate mix (see Table of Materials), previously watered, and cover the pot with a 15 cm x 15 cm metal mesh with 1.6 mm x 1.6 mm holes. Adjust the metal mesh to the wet soil using a rubber band (Figure 1A).</li>
		<li>With a wet toothpick, sow Arabidopsis seeds into the holes of the metal mesh. Place three to four seeds in distant positions within the pot (Figure 1B, C).</li>
		<li>Cover the pots with a plastic dome to maintain high relative humidity and incubate them for 72 h at 4 &#176;C for stratification. 
		NOTE: Stratification (incubation at high humidity and low temperature, as described) improves the germination rate and synchrony of the seeds.</li>
		<li>Transfer the pots to a plant growth chamber under short-day conditions (8 h light/16 h dark at 21 &#176;C, light intensity: 100 &#181;mol&#183;m&#8722;2&#183;s&#8722;1, relative humidity: 70%).</li>
		<li>After seed germination (8-10 days), use the tweezers to remove most of the seedlings, keeping one seedling in each of the positions of the pot (six seedlings/pot) (Figure 1D). Remove the plastic dome to uncover the pots. 
		NOTE: The plants will be ready to use 4-5 weeks after germination.</li>
	</ol>
	</li>
	<li>Prepare Phaseolus vulgaris bean (cultivar Canadian Wonder) plants.
	<ol>
		<li>Cover the bottom of a Petri dish with a wet piece of towel paper, and place the bean seeds on top of it. Seal the Petri dish with surgical tape and incubate at 28 &#176;C for 3-4 days (Figure 2A).</li>
		<li>Transfer the germinated seeds into a 10 cm diameter pot filled with wet 1:3 vermiculite-plant substrate mix.</li>
		<li>Incubate in a plant growth chamber under long-day settings (16 h light/8 h dark at 23 &#176;C, light intensity: 100 &#181;mol&#183;m&#8722;2&#183;s&#8722;1, relative humidity: 70%). 
		NOTE: The plants will be ready to use 10 days after germination (Figure 2B).</li>
	</ol>
	</li>
</ol>
2. Inoculation of Arabidopsis and bean plants
NOTE: In this study, the strains P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A were used.
<ol>
	<li>Prepare the P. syringae inoculum.
	<ol>
		<li>Streak out the P. syringae strain of interest from a &#8722;80 &#176;C glycerol stock onto an LB plate (10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast extract, and 16 g/L bacteriological agar, see Table of Materials) supplemented with the appropriate antibiotics. Incubate at 28 &#176;C for 40-48 h. 
		NOTE: The use of antibiotics is recommended if the strain of interest carries a plasmid or a genomic resistance gene. The recommended antibiotic concentrations for P. syringae are as follows: kanamycin (15 &#181;g/mL), gentamycin (10 &#181;g/mL), ampicillin (300 &#181;g/mL) (see Table of Materials).</li>
		<li>Scrape out the bacterial biomass and resuspend in 5 mL of 10 mM MgCl2. Measure the OD600&#160;and adjust to 0.1 by adding 10 mM MgCl2. 
		NOTE: An OD600&#160;of 0.1 of a P. syringae culture corresponds to 5 x 107 CFU&#183;mL&#8722;1.</li>
		<li>Perform serial dilutions into 10 mM MgCl2&#160;to reach a final inoculum concentration of 5 x 105 CFU&#183;mL&#8722;1. Prepare 200 mL of inoculum for the Arabidopsis plants and 50 mL for the bean plants.</li>
		<li>Right before inoculation, add the surfactant Silwett L-77 (see Table of Materials) to a final concentration of 0.02% for bean inoculation and 0.01% for Arabidopsis. Note that Silwett is somewhat detrimental to the Arabidopsis tissue.</li>
	</ol>
	</li>
	<li>Perform vacuum infiltration.
	<ol>
		<li>For Arabidopsis infiltration, place two wood sticks forming an X over the pot (Figure 1E), and place the pot facing down over a 14 cm diameter Petri dish containing the 200 mL inoculum (Figure 1F).</li>
		<li>For bean leaves inoculation, introduce the leaf into a 50 mL conical centrifuge tube containing the inoculum (Figure 2C).</li>
		<li>Insert the plants immersed in the inoculum solution into a vacuum chamber (Figure 1G and Figure 2D) and give a pulse of 500 mbar for 30 s to infiltrate the leaves. Repeat the vacuum pulse 2-3 times until the leaf is completely infiltrated (Figure 1H and Figure 2E).</li>
		<li>Drain the excess inoculum solution with a piece of paper and return the plants to their corresponding growth chamber.</li>
	</ol>
	</li>
</ol>
3.&#160;Extraction of bacteria from the apoplast
<ol>
	<li>Four days after inoculation, cut either the aerial part of the Arabidopsis plant or the inoculated leaf from the bean plant and place it into a 20 mL syringe without a needle (Figure 2G). For bean leaves, roll the leaf on itself, leaving the abaxial face outward, as displayed in Figure 2F.</li>
	<li>Add enough volume of distilled water to cover the tissue (usually 10-15 mL).</li>
	<li>Insert the plunger, and with the syringe in the vertical position with the tip pointing up, remove the excess air and air bubbles inside the syringe by gently tapping the barrel until all the air is located near the tip. Slide then the plunger to take the air out. Once there is as little air as possible inside the syringe, cover the tip of the syringe barrel with paraffin film.</li>
	<li>Carefully press the plunger to generate positive pressure until the tissue turns darker (Figure 1I and Figure 2H). Then, pull the plunger to generate negative pressure (Figure 1J and Figure 2I). Repeat this step 3-5 times.</li>
	<li>Remove the paraffin film and the plunger and collect the fluid containing the apoplast-extracted bacteria, as in Figure 2J.</li>
</ol>
4. Single-cell analysis of the apoplast-extracted bacteria
<ol>
	<li>Visualize by confocal microscopy following the steps below.
	<ol>
		<li>Prepare a 1.5% agarose solution in distilled water. Once melted, add enough volume to fill the space between two microscopy slides set side by side and place another slide on top on it (Figure 3). Let them dry for 15 min and carefully remove the slide placed on the top. Using a blade, cut the agarose pad into 5 mm x 5 mm pieces right before use.</li>
		<li>Parallelly, centrifuge 1 mL of the apoplast-extracted bacteria at 12,000 x g for 1 min at room temperature, carefully remove the supernatant using a pipette, and resuspend the pellet into 20 &#181;L of water to concentrate the cells. Place a 2 &#181;L drop of the concentrated cells onto a 0.17 mm coverslip and cover the drop with a 5 mm x 5 mm piece of the agarose pad previously obtained in step 1, as is represented in Figure 3.</li>
		<li>Visualize the bacterial preparation under the confocal microscope (see Table of Materials). To identify green-fluorescent bacteria, use the excitation laser at 488 nm and an emission filter ranging from 500 nm to 550 nm. To identify all the bacteria, use the bright field and merge both fields.</li>
		<li>Process the confocal images using Fiji (see Table of Materials). To do this, use the MicrobeJ plugin to identify the contour of the bacterial cell and measure the fluorescence intensity within. 
		NOTE: Image acquisition from isolated bacteria (not clustered) is recommended for this analysis.</li>
	</ol>
	</li>
	<li>Perform analysis by flow cytometry.
	<ol>
		<li>Take an aliquot of the apoplast-extracted bacteria suspensions to analyze using the flow-cytometer. Acquire 100,000 events.</li>
		<li>To discriminate between bacteria and plant debris, analyze the apoplast extracted from a non-inoculated plant and compare the dot plot showing its forward scatter (FSC) cell size versus side scatter (SSC) cell size with that of the apoplast-extracted bacterial suspension. To identify non-fluorescent bacteria, use the apoplasts extracted from the plants inoculated with non-fluorescent isogenic bacteria and compare their fluorescent emissions.</li>
	</ol>
	</li>
</ol>

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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=Generating+chromosome-located+transcriptional+fusions+to+fluorescent+proteins+for+single-cell+gene+expression+analysis+in+Pseudomonas+syringae.">Generating chromosome-located transcriptional fusions to fluorescent proteins for single-cell gene expression analysis in Pseudomonas syringae.</a> Methods in Molecular Biology. 1734, 183-199 (2018).</li><li>Rufian, J. 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=Pseudomonas+syringae+differentiates+into+phenotypically+distinct+subpopulations+during+colonization+of+a+plant+host.">Pseudomonas syringae differentiates into phenotypically distinct subpopulations during colonization of a plant host.</a> Environmental Microbiology. 18 (10), 3593-3605 (2016).</li><li>Katagiri, F., Thilmony, R., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> Arabidopsis Book. 1, 0039 (2002).</li><li>Fouts, D. E., 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=Genomewide+identification+of+Pseudomonas+syringae+pv.+tomato+DC3000+promoters+controlled+by+the+HrpL+alternative+sigma+factor.">Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (4), 2275-2280 (2002).</li><li>Huber, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Robust Statistics. , (1981).</li><li>Freed, N. E., 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+simple+screen+to+identify+promoters+conferring+high+levels+of+phenotypic+noise.">A simple screen to identify promoters conferring high levels of phenotypic noise.</a> PLoS Genetics. 4 (12), 1000307 (2008).</li><li>Hirano, S. S., Upper, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+in+the+leaf+ecosystem+with+emphasis+on+Pseudomonas+syringae-a+pathogen,+ice+nucleus,+and+epiphyte.">Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae-a pathogen, ice nucleus, and epiphyte.</a> Microbiology and Molecular Biology Reviews. 64 (3), 624-653 (2000).</li><li>Lindeberg, M., Myers, C. R., Collmer, A., Schneider, D. 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(104), e53364 (2015).</li><li>O'Leary, B. M., Rico, A., McCraw, S., Fones, H. N., Preston, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infiltration-centrifugation+technique+for+extraction+of+apoplastic+fluid+from+plant+leaves+using+Phaseolus+vulgaris+as+an+example.">The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example.</a> Journal of Visualized Experiments. (94), e52113 (2014).</li><li>Tornero, P., Dangl, J. 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The authors have nothing to disclose.

The method presented here describes a non-invasive procedure that allows the infiltration of bacteria into the plant foliar tissue, allowing the rapid inoculation of large volumes while minimizing tissue disruption. One of the characteristics of the P. syringae species complex is the ability to survive and proliferate inside the plant apoplast and on the plant surface as epiphyte14. Thus, the possibility that the bacteria extracted using the present protocol come only from the plant apopl...

Pathogenic bacteria display differences in diverse phenotypes, giving rise to the formation of subpopulations within genetically identical populations. This phenomenon is known as phenotypic heterogeneity and has been proposed as an adaptation strategy during bacterial-host interactions1. Recent advances in the optical resolution of confocal microscopes, flow cytometry, and microfluidics, combined with fluorescent proteins, have fostered single-cell analyses of bacterial populations2.
The Gram-negative Pseudomonas syringae is an archetypal plant pathogenic bacteria due to both its academic and economic importance3. The life cycle of P. syringae is linked to the water cycle4. P. syringae enters the intercellular spaces between the mesophyll cells, the plant leaf apoplast, through natural apertures such as stomata or wounds5. Once within the apoplast, P. syringae relies on the type III secretion system (T3SS) and the type III-translocated effectors (T3E) to suppress plant immunity and manipulate plant cellular functions for the benefit of the pathogen6. The expression of T3SS and T3E depends on the master regulator HrpL, an alternative sigma factor that binds to the hrp-box motifs in the promoter region of the target genes7. 
By generating chromosome-located transcriptional fusions to fluorescent protein genes downstream of the gene of interest, one can monitor gene expression based on the fluorescence levels emitted at the single-cell level8. Using this method, it has been established that the expression of hrpL is heterogeneous both within bacterial cultures grown in the laboratory and within bacterial populations recovered from the plant apoplast8,9. Although gene expression analysis at the single-cell level is typically performed in bacterial cultures grown in laboratory media, such analyses can also be carried out on bacterial populations growing within the plant, thus providing valuable information on the formation of subpopulations in the natural context. A potential limitation for the analysis of bacterial populations extracted from the plant is that classic inoculation methods by syringe-pressure infiltration into the apoplast, followed by bacterial extraction by maceration of the leaf tissue, typically generate a large amount of cellular plant debris that interferes with downstream analysis10. Most cellular debris consists of autofluorescent fragments of chloroplasts that overlap with GFP fluorescence, resulting in misleading results. 
The present protocol describes the process of analyzing single-cell gene expression heterogeneity in two model pathosystems: the one formed by the P. syringae pv. tomato strain DC3000 and Arabidopsis thaliana (Col-0), and the other by the P. syringae pv. phaseolicola strain 1448A and bean plants (Phaseolus vulgaris cultivar Canadian Wonder). An inoculation method is proposed based on vacuum infiltration using a vacuum chamber and a pump, resulting in a fast and damage-free method to infiltrate whole leaves. Furthermore, as an improvement on conventional protocols, a gentler method is used to extract the bacterial population from the apoplast that significantly reduces tissue disruption, based on the extraction of apoplastic fluid by applying cycles of positive and negative pressure using a small amount of volume within a syringe.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.17 mm coverslip</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>1.6 x 1.6 mm metal mesh</td>
			<td>Buzifu</td>
			<td></td>
			<td>Fiberglass screen mesh</td>
		</tr>
		<tr>
			<td>10 cm diameter pots</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>140 mm Petri dishes</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium</td>
			<td>GoldBio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Roko</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope Stellaris</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSVerse cell analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Duchefa</td>
			<td>G-0124</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin monosulfate</td>
			<td>Phytotechnology</td>
			<td>K378</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Pechiney Plastic Packaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plant substrate</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Cromton Europe Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Vacuum chamber 25 cm diameter</td>
			<td>Kartell</td>
			<td>554</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>GAST</td>
			<td>DOA-P504-BN</td>
			<td></td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell analysis of the expression of pseudomonas syringae genes within the plant tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

The expression of the type III secretion system is essential for bacterial growth within the plant. The timely expression of T3SS genes is achieved through intricate regulation, at the center of which is the extracytoplasmic function (ECF) sigma factor HrpL, the key activator of the expression of T3SS-related genes11. An analysis of the expression of hrpL was previously carried out using a chromosome-located transcriptional fusion to a downstream promoterless gfp gene and by foll...

Watch this Scientific Journal Video about Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue at JoVE.com

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

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1.6K Views.  Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC). The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

Video: Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...