an assay to detect oxidative burst in arabidopsis leaf discs upon immune elicitation

An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

At 4 to 5 weeks post-germination, use a sharp 4-millimeter biopsy punch to remove one leaf disk per Arabidopsis plant, avoiding the mid-vein and being careful to limit wounding. Collect the disks into individual wells of an unused 96-well luminometer plate containing 100 microliters of double-distilled water per well at axial side up to prevent desiccation.
If assessing multiple elicitors, remove a second leaf disk from the same leaf for each elicitor treatment, and cover the plate with the lid to allow the leaf disks to recover overnight at room temperature. The next morning, set the microplate reader parameters to a 1,000-millisecond integration time in 2-minute intervals over a 40 to 60-minute period to capture the dynamic oxidative burst.
Next, use a multichannel pipette to replace the water in each well with 100 microliters of freshly prepared reaction solution, including a control no-elicitor reaction for each genotype to assess the basal reaction oxygen species levels in the absence of elicitation. Then, immediately, measure the light emission for all of the wavelengths in the visible spectrum on the microplate reader.

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1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation
<ol>
	<li>Plant growth and maintenance.
	<ol>
		<li>To synchronize germination, stratify Arabidopsis seeds by suspending approximately 50 seeds in 1 mL of sterile 0.1% agar [w/v] and store at 4 &#176;C (no light) for 3-4 days. 
		NOTE: Stratify a wild-type background control (for example, Col-0) and genotypes with high and low immune outputs (for example, cpk28-1 and bak1-5, respectively) to serve as internal controls.</li>
		<li>Sow seeds on the soil and germinate under standard short-day conditions (22 &#176;C, 10 h light, 150 &#956;E/m2/s light intensity, and 65-70% relative humidity).</li>
		<li>After approximately 7 days, use forceps to transplant individual seedlings to new pots, separated by at least 4 cm to permit full rosette development. Transplant 6-12 seedlings per genotype and return to standard short-day conditions. Regularly water and fertilize plants (1 g/L of 20-20-20 fertilizer every 2 weeks).</li>
	</ol>
	</li>
	<li>Collect leaf discs in 96-well plates for overnight recovery.
	<ol>
		<li>At 4-5 weeks post-germination (Figure 1A), use a sharp 4 mm biopsy punch to remove one leaf disc per plant, avoiding the mid-vein and being careful to limit wounding (Figure 1B). Place leaf discs in an unused 96-well luminometer plate containing 100 &#956;L of double distilled water (ddH2O) with the adaxial side facing upwards to prevent desiccation (Figure 1C). If assessing multiple elicitors, remove 1 leaf disc from the same leaf for each elicitor treatment. 
		NOTE: Sample leaves that are fully expanded, third to fifth from the top of the rosette, and approximately the same size and age to limit variability. If possible, cut leaf discs in half using a razor blade prior to overnight recovery, as this increases the surface area exposed to elicitor solution.</li>
		<li>Recover overnight at room temperature to prevent the interference of ROS produced by wounding during leaf disc collection. Cover plates with a lid to prevent evaporation.</li>
	</ol>
	</li>
	<li>Perform the elicitor treatment and measure ROS production.
	<ol>
		<li>Program the plate reader prior to adding the reaction solution, as this will reduce the time between ROS burst initiation and the first measurements recorded. Use a commercial software that is appropriate for the plate reader. In our case (see Table of Contents), click Settings, and select the LUM96 Cartridge and the Kinetic Read Type. Click the Read Area category and drag to select a subset of wells or the entire plate to be read. Under the PMT and Optics tab, set the integration time to 1,000 ms. Under the Timing tab, set the Total Run Time to 40-60 min and the Interval to 2 min.</li>
		<li>Remove water from each well using a multi-channel pipette. 
		NOTE: Do not puncture leaf discs, as this may cause wounding stress and result in more variable ROS outputs.</li>
		<li>Prepare a reaction solution containing 100 &#956;M luminol, 10 &#956;g/mL horseradish peroxidase (HRP), and the desired concentration of elicitor (for example: elf18 at 1 nM, 10 nM, 100 nM, or 1,000 nM) in sterile ddH2O using 10 mL of solution for one 96-well plate. 
		NOTE: Dissolve lyophilized peptides in sterile H2O in low-binding tubes to make a 10 mM stock, flash-freeze in liquid N2, and store at -80 &#176;C. When ready to use, dilute stocks in sterile H2O to generate a 100 &#956;M working stock and store at -20 &#176;C.</li>
		<li>Use a multi-channel pipette to dispense 100 &#956;L of the reaction mixture to each well, adding the solution to all leaf discs of the same treatment at the same time. Include a control reaction (no elicitor) for each genotype to assess basal ROS levels in the absence of elicitation. Immediately measure light emission for all wavelengths in the visible spectrum using a microplate reader. 
		NOTE: Prepare and apply the reaction solution under low light, as luminol and HRP are light-sensitive reagents. Keep reagents on ice or at -20&#176;C unless in use.</li>
		<li>Measure light emission with a 1,000 ms integration time in 2 min intervals over a 40-60 min period in a microplate reader in order to capture the dynamic oxidative burst (Figure 1D). 
		NOTE: Use a longer integration time to improve assay sensitivity, while ensuring that all samples in a 96-well plate can be measured within one interval.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20-20-20 Fertilizer</td>
			<td>Plant Prod</td>
			<td>10529</td>
			<td>Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.</td>
		</tr>
		<tr>
			<td>4 mm Biopsy Punch</td>
			<td>Medical Mart</td>
			<td>232-33-34-P</td>
			<td>A cork borer set with a 0.125 cm^2 surface area can also be used.</td>
		</tr>
		<tr>
			<td>BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL)</td>
			<td>Sartorius</td>
			<td>725240</td>
			<td>Any multichannel pipette can be used, as can a single pipetter if necessary.</td>
		</tr>
		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>EZ Biolab</td>
			<td>cp7211</td>
			<td>Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase</td>
			<td>Sigma-Aldrich</td>
			<td>P6782</td>
			<td>Dissolve in pure water. Store at -20C and away from light.</td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Sigma-Aldrich</td>
			<td>A8511</td>
			<td>Dissolve in DMSO. Store at -20C and away from light.</td>
		</tr>
		<tr>
			<td>Murisage and Skoog Basal Salts</td>
			<td>Cedarlane Labs</td>
			<td>MSP09-100LT</td>
			<td>Store at 4C.</td>
		</tr>
		<tr>
			<td>Soil</td>
			<td>SunGrow Horticulture</td>
			<td>Sunshine Mix #1</td>
			<td>Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.</td>
		</tr>
		<tr>
			<td>SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Any plate reader capable of detecting luminescence can be used for these assays.</td>
		</tr>
		<tr>
			<td>White Polystyrene 96-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Bredow, M., et al. <a href="https://app.jove.com/v/59437">Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to measure oxidative burst in Arabidopsis leaves upon immune elicitation. Leaf discs, cut from plant leaves, are incubated with bacterial peptides functioning as microbe-associated molecular patterns or MAMPs. Upon detection of the MAMPs, plant cells induce the production of extracellular ROS, which is detected using a chemiluminescence assay.

<img alt="Figure 1" src="/files/ftp_upload/21909/21909_Figure1.jpg" /> 
Figure 1. Luminol-based oxidative burst assay following immune induction in Arabidopsis. (A) Grow plants on soil in short-day conditions for 4-5 weeks. (B) Use a 4 mm biopsy punch to collect leaf discs from each plant and recover overnight in ddH2O. (C) Add reaction solution (100 &#956;M luminol, 10 &#956;g/mL HRP, and the desired concentration of elicitor) and measure light emission ove...

1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation

	Plant growth and maintenance.
	
		To synchronize germination, stratify ...

Obtain Arabidopsis thaliana leaf discs. Immerse them in microplate wells containing double-distilled water to prevent desiccation.
Incubate the discs to allow recovery from wounding.
Post-incubation, remove the water. Add a reaction mixture containing peptides derived from bacteria, horseradish peroxidase enzyme, and luminol.
Using a microplate reader, measure the luminescence over time.
Pattern recognition receptors, or PRRs, on the plant cells, bind the bacterial peptides, which are microbe-associated molecular patterns.
Further, the PRR associates with its co-receptor, undergoing mutual phosphorylation, activating receptor-like protein kinases, and causing intracellular calcium influx.
This, in turn, activates the transmembrane respiratory burst oxidase homolog, or RBOH proteins.
Activated RBOH catalyzes the production of extracellular reactive oxygen species, or ROS, causing an oxidative burst. Further, ROS gets converted into hydrogen peroxide.
Horseradish peroxidase utilizes hydrogen peroxide to oxidize luminol into an excited state, emitting luminescence upon returning to the ground state.
Measure the luminescence to monitor the rapid oxidative burst following microbial pattern recognition.

68 Views. Test do wykrywania wybuchu oksydacyjnego w krążkach liści Arabidopsis po wywołaniu immunologicznym

Video: Test do wykrywania wybuchu oksydacyjnego w krążkach liści Arabidopsis po wywołaniu immunologicznym - Experiment

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Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

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An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

Transkrypcja

An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation