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>

<ol><li>Weigel, W. A., Dersch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+heterogeneity%3A+A+bacterial+virulence+strategy%5BTitle%5D)+AND+%22Microbes+and+Infection%22%5BJournal%5D)">Phenotypic heterogeneity: A bacterial virulence strategy.</a> Microbes and Infection. 20 (9-10), 570-577 (2018).</li>
<li>Hare, P. J., LaGree, T. J., Byrd, B. A., DeMarco, A. M., Mok, W. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+technologies+to+study+phenotypic+heterogeneity+and+bacterial+persisters%5BTitle%5D)+AND+%22Microorganisms%22%5BJournal%5D)">Single-cell technologies to study phenotypic heterogeneity and bacterial persisters.</a> Microorganisms. 9 (11), 2277(2021).</li>
<li>Mansfield, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Top+10+plant+pathogenic+bacteria+in+molecular+plant+pathology%5BTitle%5D)+AND+%22Molecular+Plant+Pathology%22%5BJournal%5D)">Top 10 plant pathogenic bacteria in molecular plant pathology.</a> Molecular Plant Pathology. 13 (6), 614-629 (2012).</li>
<li>Morris, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+life+history+of+the+plant+pathogen+Pseudomonas+syringae+is+linked+to+the+water+cycle%5BTitle%5D)+AND+%22The+ISME+Journal%22%5BJournal%5D)">The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.</a> The ISME Journal. 2 (3), 321-334 (2008).</li>
<li>Rufián, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confocal+microscopy+reveals+in+planta+dynamic+interactions+between+pathogenic%2C+avirulent+and+non-pathogenic+Pseudomonas+syringae+strains%5BTitle%5D)+AND+%22Molecular+Plant+Pathology%22%5BJournal%5D)">Confocal microscopy reveals in planta dynamic interactions between pathogenic, avirulent and non-pathogenic Pseudomonas syringae strains.</a> Molecular Plant Pathology. 19 (3), 537-551 (2018).</li>
<li>Macho, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subversion+of+plant+cellular+functions+by+bacterial+type-III+effectors%3A+Beyond+suppression+of+immunity%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">Subversion of plant cellular functions by bacterial type-III effectors: Beyond suppression of immunity.</a> New Phytologist. 210 (1), 51-57 (2016).</li>
<li>Xiao, Y., Hutcheson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+promoter+sequence+recognized+by+a+newly+identified+alternate+sigma+factor+directs+expression+of+pathogenicity+and+host+range+determinants+in+Pseudomonas+syringae%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae.</a> Journal of Bacteriology. 176 (10), 3089-3091 (1994).</li>
<li>Rufián, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generating+chromosome-located+transcriptional+fusions+to+fluorescent+proteins+for+single-cell+gene+expression+analysis+in+Pseudomonas+syringae%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Pseudomonas+syringae+differentiates+into+phenotypically+distinct+subpopulations+during+colonization+of+a+plant+host%5BTitle%5D)+AND+%22Environmental+Microbiology%22%5BJournal%5D)">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/pubmed?term=((The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction%5BTitle%5D)+AND+%22Arabidopsis+Book%22%5BJournal%5D)">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/pubmed?term=((Genomewide+identification+of+Pseudomonas+syringae+pv.+tomato+DC3000+promoters+controlled+by+the+HrpL+alternative+sigma+factor%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHuber%2C+PJ+%22Robust+Statistics%22">Robust Statistics</a>. , Wiley. New York, NY. (1981).</li>
<li>Freed, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+screen+to+identify+promoters+conferring+high+levels+of+phenotypic+noise%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">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/pubmed?term=((Bacteria+in+the+leaf+ecosystem+with+emphasis+on+Pseudomonas+syringae-a+pathogen%2C+ice+nucleus%2C+and+epiphyte%5BTitle%5D)+AND+%22Microbiology+and+Molecular+Biology+Reviews%22%5BJournal%5D)">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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roadmap+to+new+virulence+determinants+in+Pseudomonas+syringae%3A+Insights+from+comparative+genomics+and+genome+organization%5BTitle%5D)+AND+%22Molecular+Plant+Microbe+Interactions%22%5BJournal%5D)">Roadmap to new virulence determinants in Pseudomonas syringae: Insights from comparative genomics and genome organization.</a> Molecular Plant Microbe Interactions. 21 (6), 685-700 (2008).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+leaf+infiltration+assay+for+fine+characterization+of+plant+defense+responses+using+the+Arabidopsis+thaliana-Pseudomonas+syringae+pathosystem%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Bacterial leaf infiltration assay for fine characterization of plant defense responses using the Arabidopsis thaliana-Pseudomonas syringae pathosystem.</a> Journal of Visualized Experiments. (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/pubmed?term=((The+infiltration-centrifugation+technique+for+extraction+of+apoplastic+fluid+from+plant+leaves+using+Phaseolus+vulgaris+as+an+example%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+method+for+quantifying+growth+of+phytopathogenic+bacteria+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana.</a> The Plant Journal. 28 (4), 475-481 (2001).</li>
</ol>

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><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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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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1.7K Views.  Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC). Ons protocol biedt een zachte planteninentings- en extractiemethode waarmee bacteriepopulaties die in de bladapoplast zijn gekweekt, kunnen worden hersteld die geschikt zijn voor eencellige genexpressieanalyse zoals flowcytometrie. Deze methode maakt de extractie van een aanzienlijke hoeveelheid bacteriën uit de apoplast mogelijk zonder de monsters aanzienlijk te besmetten met plantencellulair afval. Deze geoptimaliseerde methode voor het herstel van apoplastische bacteriën kan direct of me...

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Immunology and Infection

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetics

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Bioengineering

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

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

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