Name: an assay for detecting reactive oxygen species production in rice tissues upon immune elicitation
Uploaded: 2024-01-31T13:36:01.000+00:00
Duration: 1 min 26 s
Description: NOTE: The protocol is applicable to different plant tissues. Rice sheath and leaf discs were used in this protocol for ROS detection upon PAMP ...

an assay for reactive oxygen species production in rice tissues upon immune elicitation

An Assay for Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

Cut the sheath from 10-day-old rice seedlings into 3-millimeter segments with a sharp razor blade or surgical blade for pretreatment one day before the ROS assay. Place 5 sheath segments in an individual well of a 96-well microtiter plate containing 100 microliters of double-distilled water for 10 to 12 hours.
Cut the leaf disks from four to six-week-old rice plants using a biopsy punch with a plunger. Always cut the leaf disks from the middle third of the second leaf of the main tiller to reduce data variation. Next place one leaf disk in an individual well of a 96-well microtiter plate containing 100 microliters of double-distilled water for 10 to 12 hours for pretreatment. Keep all the leaf disks floating and facing up in the wells of a microtiter plate for water pretreatment to avoid leaf-side associated variation.
Prepare elicitation solution by combining 9.4ml of 50 micromolar tris-HCl, 400 microliters of L-012 solution, 100 microliters of horseradish peroxidase, and 100 microliters of flg22. Add 200 microliters of elicitation solution to the wells.
Start the software, and click on the Experiments button to create a new protocol or use an existing protocol. Click Procedure in the pop-up to set up the plate and select the wells from the plate to be monitored. Click Start Kinetic, and set the runtime to 30 minutes or longer, depending on the experimental requirements. Select minimum interval to obtain the readings as frequently as possible, and for integration time, choose 1 second or longer, depending on the signal intensity.
Click on Validate followed by OK to confirm the settings. Then click on Detect a New Plate in the pop-up and wait for the software to prompt the load plate dialog box. Carefully remove the double-distilled water from the wells containing the pretreated tissues, avoiding any tissue damage or desiccation. Use a multichannel pipette to add 200 microliters of the elicitation solution to the wells containing the tissues. Place the plate to be tested on the carrier, and begin detection.

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An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

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NOTE: The protocol is applicable to different plant tissues. Rice sheath and leaf discs were used in this protocol for ROS detection upon PAMP elicitation. As differences mainly arise due to the method of sampling, only the common procedures are described below, with specific steps being mentioned wherever necessary.
1. Tissue preparation and pretreatment
<ol>
	<li>Rice sheath
	<ol>
		<li>Cut the sheath from 10-day-old rice seedlings into 3 mm segments with a sharp razor blade or surgical blade for pretreatment 1 day before the ROS assay (Figure 1B).</li>
		<li>Place five sheath segments in an individual well of a 96-well microtiter plate containing 100 &#181;L of double distilled water (ddH2O) for 10-12 h, in the dark at 25 &#176;C, which allows wound injury-related ion leakage and defense responses to abate (Figure 2). 
		NOTE: Taking care to keep the cuts vertical to ensure a consistent cutting surface area exposed to the elicitation solution is an important step to obtaining highly reproducible results. Move the segments gently. Do not make extra cuts or wounds on the segments, which could be a source of data variation. As a principle, each test must contain at least five replicates since the variation of the ROS value is large. The more replicates set, the more reliable the data are.</li>
	</ol>
	</li>
	<li>Leaf disc
	<ol>
		<li>Cut the leaf discs (4 mm in diameter) from 4-6-week-old rice plants using a biopsy punch with a plunger. Always cut leaf discs from the middle third of the second leaf (numbered from the top) of the main tiller to reduce data variation (Figure 1C).</li>
		<li>Place one leaf disc in an individual well of a 96-well microtiter plate containing 100 &#181;L of ddH2O for 10-12 h for pretreatment, which allows wounding-related responses to abate as these could interfere with the induction of ROS by PAMPs (Figure 2). 
		NOTE: Operate the leaf discs gently. Do not make extra cuts or wounds on the discs in the experiment, which could result in data variation. The induction of ROS mostly occurs from the cells of the cut edge, since the surfaces of rice tissues (leaves or sheaths) are covered with hydrophobic layers. Only the cells of the cut edges are in contact with the elicitation solution (refer to the discussion section).</li>
		<li>Keep all the leaf disks floating, with the abaxial surface facing up, in the wells of a microtiter plate for water pretreatment to avoid leaf side-associated variation.</li>
	</ol>
	</li>
</ol>
2. Preparing the elicitation solution
<ol>
	<li>Dissolve L-012 powder into 20 mM (6.23 mg/mL) aqueous solution with ddH2O to make the stock solution. Then, dilute the stock solution with 50 mM Tris HCl buffer (pH 7.5) to make the working solution at the final concentration of 500 &#181;M L-012. Keep the stock solution frozen and dilute to the working solution before use.</li>
	<li>Prepare the elicitation solution containing PAMP, L-012, and horseradish peroxidase (HRP; 10 mg/mL in ddH2O). For a 10 mL elicitation solution, mix 9.4 mL of 50 mM Tris HCl (pH 7.5) solution, 400 &#181;L of the L-012 solution, 100 &#181;L of HRP, and 100 &#181;L of flg22 (PAMP; 10 mM in ddH2O). For the negative control, add 100 &#181;L of ddH2O instead of PAMP. 
	NOTE: Keep the prepared elicitation solutions at room temperature to avoid cold stress on rice tissues. Other PAMPs can also be used for treatment as required, such as chitin (20 ng/mL in final concentration). Since L-012 is light-sensitive, cover all the tubes containing L-012 solution with aluminum foil.</li>
</ol>
3. Starting the software and setting up the protocol with the referenced microplate reader (see Table of Materials)
NOTE: It takes some time to set up the parameters of the microplate reader software. It is recommended to get the machine and protocol ready (one click to proceed) before adding the elicitation solution.
<ol>
	<li>Start the software. Click the Experiments button to create a new protocol or use an existing protocol.</li>
	<li>Click Procedure in the pop-up to set up the plate. Select the wells from the plate to be monitored.</li>
	<li>Click Start Kinetic to set up the total run time and reading interval. Set the Run time to 35 min or longer, depending on the experimental requirements. To obtain readings as frequently as possible, select Minimum Interval. For integration time, choose 1 s or longer, depending on the signal intensity. 
	NOTE: The reading interval depends on the number of samples and signal integration duration.</li>
	<li>Click Validate | OK to confirm the settings.</li>
	<li>Click on Detect the new plate in the pop-up and wait for the software to prompt the load plate dialog box. Place the plate to be tested on the carrier.</li>
	<li>Stop here to wait for the elicitation system to be established (in the next section). As soon as the elicitation system is ready, click on Run to start the reading.</li>
</ol>
4. Establishing the elicitation system and measuring real-time ROS production
<ol>
	<li>Carefully remove the ddH2O from the wells containing the pretreated tissues, avoiding any tissue damage or desiccation.</li>
	<li>Use a multichannel pipette to add 200 &#181;L of the elicitation solution to the wells containing the tissues.</li>
	<li>Gently shake to mix. Click Run to begin detection. 
	NOTE: With PAMP treatment, plant tissues respond and produce ROS very quickly. Therefore, it is suggested that the negative control without PAMP be treated first to reduce the operation time when there are multiple treatments. Operate as quickly as possible to reduce the elicitation delay between treatments. The shorter the time between the addition of the elicitation solution and the start of detection, the better the capture of important experimental data will be.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well microtiter plate</td>
			<td>WHB</td>
			<td>WHB-96-01</td>
			<td></td>
		</tr>
		<tr>
			<td>flg22</td>
			<td>Sangon Biotech</td>
			<td>p20973</td>
			<td>PAMP</td>
		</tr>
		<tr>
			<td>Gen5</td>
			<td>BioTek</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>L-012</td>
			<td>FUJIFILM</td>
			<td>120-04891</td>
			<td>8-amino-5-chloro-7-phenyl-2,3-dihydropyrido [3,4-d] pyridazine-1,4-dione, CAS #:143556-24-5</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase from horseradish (HRP)</td>
			<td>Sigma</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sampler</td>
			<td>Miltex</td>
			<td>15110-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sangon Biotech</td>
			<td>A610195</td>
			<td></td>
		</tr>
	</tbody>
</table>

an assay for detecting reactive oxygen species production in rice tissues upon immune elicitation

Source: Wang, Y., et al. <a href="https://app.jove.com/v/64776">Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay.</a> J. Vis. Exp. (2022).
This video demonstrates an assay for real-time detection of reactive oxygen species (ROS) production in rice tissues upon pathogen-associated molecular pattern (PAMP) elicitation. Cut sheath segments and leaf discs are exposed to a bacterial flagella-derived peptide functioning as a PAMP. Upon recognition of the peptide via pattern recognition receptors (PRRs) on plant cells, a rapid and transient production of ROS occurs, which is detected via a chemiluminescence assay.

<img alt="Figure 1" src="/files/ftp_upload/21911/21911_Figure1.jpg" /> 
Figure 1: The growth condition and stages of rice seedlings for sheath sampling and parts of the rice sheath and rice leaves used in the assay. (A) Rice seedlings grown on 1/2 MS medium under sterile conditions for 10 days can be sampled for ROS assay. Sterilized rice seeds were cultured on 1/2 MS medium and grown in a 12 h light/12 h dark photoperiod in A clear glass vial, 8.5 c...

NOTE: The protocol is applicable to different plant tissues. Rice sheath and leaf discs were used in this protocol for ROS detection upon PAMP ...

Take a microplate containing uniform sheath segments cut from rice seedlings and leaf discs obtained from rice plant leaves.
Add sterile water and incubate overnight to allow recovery from wound stress.
Add an immunogenic peptide derived from bacterial flagella that acts as a pathogen-associated molecular pattern or PAMP.
Plant cells at the cut edges bind to the PAMPs via pattern recognition receptors.
The binding triggers downstream signaling, activating a plasma membrane-localized enzyme termed respiratory burst oxidase homolog or RBOH.
Activated RBOH produces reactive oxygen species or ROS in the apoplast &#8212; the intercellular space. The produced ROS gets converted into hydrogen peroxide.
Add a peroxidase enzyme and a chemiluminescent substrate.
Peroxidase utilizes hydrogen peroxide to convert the substrate into an excited state intermediate, which releases energy as light and returns to the ground state.
Measure luminescence in both tissue types to detect a sharp increase immediately following PAMP treatment, indicating an ROS burst.

an-assay-for-detecting-reactive-oxygen-species-production-rice

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An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

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An Assay for Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation