The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.

1. Chemicals
<ol>
	<li>Prepare a solution of 500 mM CEPA (144.49 g/mol), and a solution consisting of both 500 mM NaCl (58.44 g/mol) and 500 mM NaH2PO4&#183;H2O (137.99 g/mol) in acidified (pH 2.5) ultra-pure water or 0.1 N HCl. Mix using a vortex until the solutions are clear.</li>
	<li>Serially dilute (10x) the 500 mM solutions in the same solvent to obtain 5 mM and 50 mM stocks.</li>
	<li>Prepare a 10 mM solution of 1-aminocyclopropane carboxylic acid (ACC; 101.1 g/mol) in ultra-pure water.</li>
	<li>Filter-sterilize stock solutions and store aliquots at -20 &#176;C.</li>
	<li>Filter-sterilize cellulase. Store aliquots at 4 &#176;C.</li>
</ol>
2. Verifying Ethylene Production from 2-Chloroethylphosphonic Acid Decomposition: Triple Response Assay
<ol>
	<li>Surface-sterilize Arabidopsis thaliana Ecotype Columbia Seeds Using the Vapor-phase Method: 
	CAUTION: The following step produces toxic chlorine gas. Conduct seed sterilization in a fume hood.
	<ol>
		<li>Obtain a sealable container deep enough to accommodate a 250 ml glass beaker for seed sterilization and place it in a fume hood.</li>
		<li>Add A. thaliana seeds to a microcentrifuge tube and place the tube in a rack. Place the rack containing the open tube into the sealable container. 
		Note: Do not fill individual tubes over half full to allow chlorine gas to penetrate the lower seeds.</li>
		<li>Place a 250 ml beaker containing 100 ml of commercial bleach in the sealable container. Carefully add 3 ml of concentrated HCl to the bleach and immediately seal the container with its lid. 
		CAUTION: Bleach and HCl react to produce toxic chlorine gas which acts to surface-sterilize the seeds.</li>
		<li>Incubate the seeds in the presence of chlorine gas for 4 hr in the fumehood. 
		Note: Longer sterilization times will reduce germination efficiency.</li>
		<li>After sterilization, allow the chlorine gas to vent in the fume hood for at least 1 hr then seal the microcentrifuge tube. Leave the bleach and HCl mixture in the fumehood for at least 24 hr before discarding. 
		Note: Sterilized seeds may be stored at room temperature for immediate use.</li>
		<li>Keep seeds at 4 &#176;C for long-term storage. Bring seeds to room temperature before opening the microcentrifuge tube to prevent condensation. Store seeds in the dark.</li>
	</ol>
	</li>
	<li>Prepare Agar Plates in 4-sector Petri Dishes (90 x 15 mm):
	<ol>
		<li>Prepare 110 ml of growth medium for A. thaliana seedlings: 1x Murashige and Skoog (MS) basal medium15 (4.33 g/L) containing 1% (w/v) sucrose and 0.8% (w/v) agar. Adjust the MS medium to pH 6 with NaOH.</li>
		<li>Prepare 100 ml of bacterial growth medium for CEPA decomposition: Schramm and Hestrin (SH) medium16 containing 1.5% (w/v) agar. Adjust the SH medium to pH 7 with NaOH.</li>
		<li>Sterilize media by autoclaving and temper in a 55 &#176;C water bath.</li>
		<li>Once the MS agar has tempered, add 40 ml to a sterile flask. To prepare the positive control growth medium for A. thaliana seedlings, supplement the 40 ml aliquot of MS agar with 40 &#181;l of 10 mM ACC to obtain a final concentration of 10 &#181;M ACC.</li>
		<li>Add 5 ml of medium to the appropriate quadrants of the sectored Petri dishes (Figure 1) and allow the agar to solidify. Prepare all plates in triplicate. 
		Note: CEPA is not added to the growth medium until later in the protocol.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54682/54682fig1.jpg" /> 
Figure 1: Setup of agar plates used for the triple response assay with CEPA. A schematic illustrates the quadrants specific for the negative control (A), positive control (B), and experimental plates (C). This figure has been modified from Augimeri and Strap9. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54682/54682fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Stratify A. thaliana Seeds to Ensure Synchronous Germination:
	<ol>
		<li>Add approximately fifty A. thaliana seeds onto each quadrant containing MS or MS+ACC agar. Ensure seeds are evenly distributed to facilitate seedling removal and analysis.</li>
		<li>Incubate plates containing seeds in the dark at 4 &#176;C for 3-4 days.</li>
	</ol>
	</li>
	<li>Decompose CEPA on Bacterial Growth Medium (SH) and Perform Triple Response Assay:
	<ol>
		<li>After stratification, expose the seeds to fluorescent light for 2 hr.</li>
		<li>Spread 10 &#181;l of the 500 mM CEPA stock solution onto the quadrants of the experimental plates containing SH agar (pH 7; Figure 1) to obtain a final CEPA concentration of 1 mM.</li>
		<li>Seal the plates with laboratory film and cover with foil to create a dark environment for the seeds.</li>
		<li>Germinate seeds by incubating plates in the dark at 23 &#176;C for 3 days with the agar side down. 
		Note: Plates can be stacked, but negative controls should be placed on the bottom since ethylene is lighter than air.</li>
	</ol>
	</li>
	<li>Analyze Triple Response Assay Data:
	<ol>
		<li>With flame-sterilized forceps, remove single seedlings from a plate corresponding to each treatment and view under a dissecting or digital USB microscope. Ensure the bottom of the seedlings are aligned and photograph.</li>
		<li>Remove 30 seedlings from each quadrant (60 seedlings per biological replicate and 180 seedlings per treatment). Align on a surface with a black background and photograph with a ruler.</li>
		<li>Measure the hypocotyl length (mm) of replicate seedlings using ImageJ software17. Set the scale by clicking and dragging the *Straight* tool to select a length of 10 mm. Select &#34;Set Scale&#34; under the &#34;Analyze&#34; tab and set the known distance to 10. Using the *Segmented* tool, click and drag to select the length of the hypocotyl and press M to measure distance. Compare the means of biological replicates using a one-way ANOVA with a Tukey&#39;s multiple comparison test. Differences are considered significant if p &#60; 0.05.</li>
	</ol>
	</li>
</ol>
3. Analysis of pH throughout Bacterial Growth
<ol>
	<li>Grow and Quantify Komagataeibacter xylinus Starter Cultures in Triplicate:
	<ol>
		<li>Inoculate a single colony of K. xylinus into 5 ml of SH medium (pH 5) supplemented with 0.2% (v/v) filter-sterilized cellulase. Incubate cultures at 30 &#176;C with agitation at 150 rpm until an OD600 of 0.3 to 0.4 is reached (about 72 hr).</li>
		<li>Harvest starter cultures by centrifugation (2,000 x g; 4 &#176;C; 10 min). With 5 ml sterile 0.85% (w/v) NaCl solution, wash the cells twice and resuspend the cell pellet. Keep cells on ice.</li>
		<li>Quantify cells using a Petroff-Hausser counting chamber.</li>
	</ol>
	</li>
	<li>Inoculate Cultures for Analysis of pH:
	<ol>
		<li>Add 150 ml of SH broth (pH 7) supplemented with 0.2% (v/v) filter-sterilized cellulase to twelve 500 ml flasks with foil lids. Inoculate flasks with K. xylinus starter cultures at a concentration of 105 cells/ml. Using the three starter cultures, prepare three biological replicates per treatment.</li>
		<li>Supplement cultures with 300 &#181;l of the 5, 50, or 500 mM CEPA stock solutions to obtain final CEPA concentrations of 0.01, 0.1, and 1.0 mM, respectively. Supplement the untreated control cultures with 300 &#181;l of the solvent used to dissolve CEPA.</li>
		<li>Seal the flasks by tightly taping the foil lids to the flasks and incubate cultures for 14 days at 30 &#176;C with agitation at 150 rpm.</li>
	</ol>
	</li>
	<li>Each day, aseptically remove 5 ml samples from each flask and pellet cells by centrifugation (2,000 x g; 4 &#176;C; 10 min). Transfer supernatants to a clean tube and measure the pH of each biological replicate using a pH meter.</li>
	<li>Analyze the time-course data by graphing the mean pH of the biological replicates. 
	Note: It is important that the culture pH does not drop below 3.5; a culture pH of 5 significantly reduces ethylene release from CEPA, and a pH below 3.5 completely inhibits ethylene release.</li>
	<li>To control for chlorine and phosphate levels, perform an identical experiment using 0.01, 0.1, and 1.0 mM of the NaCl-NaH2PO4 solution using the 5, 50, and 500 mM stocks, respectively.</li>
</ol>
4. Colony Morphology
<ol>
	<li>Grow K. xylinus Starter Cultures in Triplicate:
	<ol>
		<li>Inoculate a single colony of K. xylinus into 5 ml of SH medium (pH 5) supplemented with 0.2% (v/v) filter-sterilized cellulase. Incubate cultures at 30 &#176;C with agitation at 150 rpm until an OD600 of 0.3 to 0.4 is reached (about 72 hr).</li>
		<li>Harvest starter cultures by centrifugation (2,000 x g; 4 &#176;C; 10 min). With 5 ml of sterile saline, wash the cells and then resuspend the cell pellet. Keep cells on ice.</li>
	</ol>
	</li>
	<li>Prepare 24 agar plates containing 25 ml of SH (pH 7) medium with 1.5% (w/v) agar.</li>
	<li>Once the agar has solidified, spread 50 &#181;l of the 5, 50, and 500 mM CEPA stock solutions on the agar to obtain final CEPA concentrations of 0.01, 0.1, and 1.0 mM, respectively. Set up untreated and solvent control plates, which consist of no amendment and spreading of 50 &#181;l of the solvent used to dissolve the test compounds.</li>
	<li>Streak plates for isolated colonies with a loop-full (~5 &#956;l) of K. xylinus starter culture and seal them with paraffin film. Inoculate all plates in triplicate. Incubate plates for five days at 30 &#176;C.</li>
	<li>Photograph colonies at 20X magnification using a digital USB microscope and qualitatively assess colonial morphology and cellulose production on solid medium. 
	Note: Cellulose appears as a hazy substance along the margin of the colony.</li>
	<li>To control for chlorine and phosphate levels, perform an identical experiment using 0.01, 0.1, and 1.0 mM of the NaCl-NaH2PO4 solution using the 5, 50, and 500 mM stocks, respectively.</li>
</ol>
5. Pellicle Assays
<ol>
	<li>Grow and Quantify K. xylinus Starter Cultures in Triplicate:
	<ol>
		<li>In triplicate, inoculate a single colony of K. xylinus into 5 ml of SH medium (pH 5) supplemented with 0.2% (v/v) filter-sterilized cellulase. Incubate cultures at 30 &#176;C with agitation at 150 rpm until an OD600 of 0.3 to 0.4 is reached (about 72 hr).</li>
		<li>Harvest starter cultures by centrifugation (2,000 x g; 4 &#176;C; 10 min). With 5 ml of sterile saline, wash the cells twice and resuspend the cell pellet. Keep cells on ice.</li>
		<li>Quantify cells using a Petroff-Hausser counting chamber.</li>
	</ol>
	</li>
	<li>Prepare Master Mixes to Inoculate 24-well Plates:
	<ol>
		<li>Supplement 60 ml of SH medium (pH 7) with 120 &#181;l of the 5, 50, and 500 mM CEPA stocks to obtain final CEPA concentrations of 0.01, 0.1, and 1.0 mM, respectively. Supplement another 60 ml of SH medium (pH 7) with 120 &#181;l of the solvent used to dissolve CEPA. Vortex to mix.</li>
		<li>Divide each 60 ml of CEPA-containing medium into four 14 ml aliquots. Inoculate three of the 14 ml aliquots with biological replicates of starter culture at a concentration of 105 cells/ml. Keep tubes with cells on ice to prevent cellulose production. 
		Note: The remaining 14 ml aliquot will be used for sterile control wells.</li>
	</ol>
	</li>
	<li>Inoculate 24-well Plates:
	<ol>
		<li>Test each treatment in its own plate with three rows of biological replicates and a row of sterile controls (Figure 2).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54682/54682fig2.jpg" /> 
Figure 2: Flow-chart illustrating the protocol used for pellicle assay and analysis. Stock CEPA-supplemented pH 7 SH medium (60 ml) is aliquoted for three separate biological replicate inoculations and a sterile control (14 ml each). These cultures are then aliquoted into six technical replicates (2 ml) into a 24 well plate and then sealed with paraffin film. After incubation for 7 days at 30 &#176;C, pellicles are harvested and characterized by determining wet weight, thickness, dry weight, and crystallinity by FT-IR. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54682/54682fig2large.jpg" target="_blank">Please click here to view a larger version of this figure</a>
<ol start="2" style="margin-left: 40px;">
	<li>Using the 14 ml master mix, add 2 ml into each of six wells of a sterile 24-well plate. Complete for the three biological replicates and sterile control (Figure 2). Repeat for each treatment.</li>
	<li>Seal plates with paraffin film and incubate statically for 7 days at 30 &#176;C.</li>
</ol>
<ol start="4">
	<li>Harvest and Measure Pellicle Wet Weight, Thickness, dry Weight (Cellulose Yield) and Crystallinity (Figure 2):
	<ol>
		<li>Depress one side of the pellicle to elevate the opposing pellicle edge and remove individual pellicles with forceps. While retaining grip, place them on fresh paper towel for 3 sec to remove excess medium before weighing to determine their wet weights.</li>
		<li>Align pellicles adjacent to a ruler and photograph from the side using a high resolution digital camera.</li>
		<li>Using ImageJ software17, measure pellicle thickness on the left shoulder, right shoulder and the center of each pellicle. Average all technical replicates for each biological replicate.</li>
		<li>Individually transfer the pellicles into the wells of a 6-well plate. Treat pellicles with 12 ml of 0.1 N NaOH at 80 &#176;C for 20 min to lyse cells.</li>
		<li>Remove the NaOH and neutralize pellicles by washing with ultra-pure water for 24 hr with agitation. Change water every 6 hr. 
		Note: Pellicles should be white upon completion of the washing step.</li>
		<li>Place pellicles on silicon mats and dry at 50 &#176;C for 48 hr to constant weight. Once dry, remove from mats and measure pellicle weights on an analytical scale to determine bacterial cellulose yield.</li>
		<li>Analyze pellicle crystallinity using Fourier-transform infrared spectroscopy (FT-IR) using 32 scans and a resolution of 4 cm-1 in the range of 4,000 to 650 cm-1. Calculate the crystallinity index, CI(IR), using A1437/A895; the absorbance ratio of the &#34;crystalline band&#34; and &#34;amorphous band&#34; as previously described18.</li>
	</ol>
	</li>
	<li>To control for chlorine and phosphate levels, perform an identical experiment using 0.01, 0.1, and 1.0 mM of the NaCl-NaH2PO4 solution using the 5, 50, and 500 mM stocks, respectively.</li>
	<li>Analyze Pellicle Data:
	<ol>
		<li>Calculate pellicle hydration by determining the difference between pellicle wet weight and dry weight.</li>
		<li>Average the values of all technical replicates to obtain a single value for each biological replicate for statistical analysis. Compare treatments using a one-way ANOVA with Tukey&#39;s multiple comparison test. Differences are significant if p &#60;0.05.</li>
		<li>Normalize data as the percent of untreated controls and plot the means of biological replicates.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The methods described here outline the in situ production of ethylene from CEPA for the study of bacterial ethylene response using the model organism, K. xylinus. This method is very useful as ethylene can be produced by supplementing any aqueous medium that has a pH greater than 3.510,11 with CEPA negating the need for pure ethylene gas or specialized laboratory equipment. This method is not limited to studying the effects of CEPA-derived ethylene on bacteria but can be also be adapted to st...

The olefin ethylene (C2H4) was first discovered as a plant hormone in 1901 when it was observed that pea seedlings, grown in a laboratory that used coal gas lamps, exhibited an abnormal morphology in which stems (hypocotyls) were shorter, thicker and bent sideways compared to normal pea seedlings; a phenotype later termed the triple response1,2. Subsequent studies demonstrated that ethylene is a vital phytohormone that regulates numerous developmental processes such as growth, stress response, fruit ripening and senescence3. Arabidopsis thaliana, a model organism for plant biology research, has been well studied in regards to its response to ethylene. Several ethylene response mutants have been isolated by exploiting the triple response phenotype observed in dark-grown A. thaliana seedlings in the presence of ethylene1,4,5. The biosynthetic precursor for ethylene production in plants is 1-aminocyclopropane carboxylic acid (ACC)6 and is commonly used during the triple response assay to increase endogenous ethylene production that leads to the triple response phenotype1,4,5.
Although the ethylene response is widely studied in plants, the effect of exogenous ethylene on bacteria is vastly understudied despite the close association of bacteria with plants. One study reported that certain Pseudomonas strains can survive using ethylene as a sole source of carbon and energy7. However, only two studies have demonstrated that bacteria respond to ethylene. The first study showed that strains of Pseudomonas aeruginosa, P. fluorescens, P. putida, and P. syringae were chemotactic toward ethylene using an agarose plug assay in which molten agarose was mixed with a chemotaxis buffer equilibrated with pure ethylene gas8. However, to our knowledge, there have been no further reports using pure ethylene gas to characterize bacterial ethylene response, likely due to the difficultly of handling gases in the laboratory without specialized equipment. The second report of bacterial ethylene response demonstrated that ethylene increased bacterial cellulose production and influenced gene expression in the fruit-associated bacterium, Komagataeibacter (formerly Gluconacetobacter) xylinus9. In this case, the ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA) was used to produce ethylene in situ within the bacterial growth medium, bypassing the need for pure ethylene gas or specialized equipment.
CEPA produces ethylene at a 1:1 molar ratio above pH 3.510,11 through a base-catalyzed, first-order reaction12-14. The degradation of CEPA is positively correlated with pH and temperature13,14 and results in the production of ethylene, chloride and phosphate. CEPA provides researchers interested in studying bacterial responses to ethylene with a convenient alternative to gaseous ethylene.
The overall goal of the following protocols is to provide a simple and efficient method to study bacterial ethylene response and includes validation of physiologically relevant levels of ethylene production from CEPA decomposition in bacterial growth medium, analysis of culture pH to ensure CEPA decomposition is not impaired during bacterial growth, and assessment of the effect of ethylene on bacterial morphology and phenotype. We demonstrate these protocols using K. xylinus, however, these protocols can be adapted to study ethylene response in other bacteria by using the appropriate growth medium and phenotype analyses.

<table><tbody><tr><td>1-aminocyclopropane carboxylic acid (ACC)</td><td>Sigma</td><td>A3903</td><td>Biosynthetic precursor of ethylene in plants</td></tr><tr><td>4-sector Petri dish</td><td>Phoenix Biomedical</td><td>CA73370-022</td><td>For testing triple response</td></tr><tr><td>Agar</td><td>BioShop</td><td>AGR001.1</td><td>To solidify medium</td></tr><tr><td>Canon Rebel T1i DLSR camera</td><td>Canon</td><td>3818B004</td><td>For pictures of pellicles</td></tr><tr><td>Cellulase from Trichoderma reesei ATCC 26921&amp;nbsp;</td><td>Sigma</td><td>C2730</td><td>Aqueous solution</td></tr><tr><td>Citric acid</td><td>BioShop</td><td>CIT002.500</td><td>For SH medium</td></tr><tr><td>Commercial bleach</td><td>Life Brand</td><td>57800861874</td><td>Bleach for seed sterilization</td></tr><tr><td>Concentrated HCl</td><td>BioShop</td><td>HCL666.500</td><td>Hydrochloric acid for pH adjustment</td></tr><tr><td>Digital USB microscope</td><td>Plugable</td><td>N/A</td><td>For pictures of colonies</td></tr><tr><td>Ethephon (&amp;ge;96%; 2-chloroethylphosphonic acid)</td><td>Sigma</td><td>C0143</td><td>Ethylene-releasing compound</td></tr><tr><td>Glucose</td><td>BioBasic</td><td>GB0219</td><td>For SH medium</td></tr><tr><td>Komagataeibacter xylinus ATCC 53582</td><td>ATCC</td><td>53582</td><td>Bacterial cellulose-producing alphaproteobacterium</td></tr><tr><td>Microcentrifuge tube</td><td>LifeGene</td><td>LMCT1.7B</td><td>1.7 ml microcentrifuge tube</td></tr><tr><td>Murashige and Skoog (MS) basal medium&amp;nbsp;</td><td>Sigma</td><td>M5519</td><td>Arabidopsis thaliana growth medium</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>BioShop</td><td>SPD579.500</td><td>Sodium phosphate, dibasic heptahydrate for SH medium</td></tr><tr><td>NaCl</td><td>BioBasic</td><td>SOD001.1</td><td>Sodium chloride for saline and control solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>BioShop</td><td>SPM306.500</td><td>Sodium phosphate, monobasic monohydrate for control solution</td></tr><tr><td>NaOH</td><td>BioShop</td><td>SHY700.500</td><td>Sodium hydroxide for pH adjustment</td></tr><tr><td>Paraffin film</td><td>Parafilm</td><td>PM996</td><td>For sealing plates and flasks</td></tr><tr><td>Peptone (bacteriological)</td><td>BioShop</td><td>PEP403.1</td><td>For SH medium</td></tr><tr><td>Petroff-Hausser counting chamber</td><td>Hausser scientific</td><td>3900</td><td>Bacterial cell counting chamber</td></tr><tr><td>Polyethersulfone sterilization filter 0.2 &amp;micro;m</td><td>VWR</td><td>28145-501</td><td>For sterilizing cellulase</td></tr><tr><td>Sucrose</td><td>BioShop</td><td>SUC600.1</td><td>Sucrose for MS medium</td></tr><tr><td>Yeast extract</td><td>BioBasic</td><td>G0961</td><td>For SH medium</td></tr></tbody></table>

utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

A schematic plate setup for verification of ethylene liberation from CEPA in SH medium (pH 7) by the triple response assay is shown in Figure 1A-C. A flow-chart illustrating the pellicle protocol is shown in Figure 2. Dark-grown A. thaliana seedlings exhibit the triple response phenotype (shorter and thicker hypocotyl with an exaggerated apical hook) in the presence of ACC and in the presence of ethylene produced through the dec...

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Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

utilizing-ethylene-releasing-compound-2-chloroethylphosphonic-acid-as

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.8K Views.  University of Ontario Institute of Technology. The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas. 

Video: Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

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

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Biology

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

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 ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Summary

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Chapters in this video

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

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

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

Detection of Histone Modifications in Plant Leaves

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

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

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae