Work in our lab is funded through the Natural Resources and Engineering Research Council of Canada (NSERC) Discovery Program, the Canadian Foundation for Innovation John R. Evans Leader&#39;s Fund, and Queen&#39;s University. KS and IS are supported by tandem Ontario Graduate Scholarships and NSERC Canada Graduate Scholarships for master&#39;s students (CGS-M).

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:&#160;Stratify a wild type background control (for example, Col-0) and genotypes with high and low immune outputs (for example,&#160;cpk28-1&#160;and&#160;bak1-5,&#160;</em...

This paper describes two methods for assaying pattern-triggered immune responses in Arabidopsis, offering quantitative approaches to evaluating immune output without the use of specialized equipment. In combination, pattern-triggered ROS and SGI can be used to assess early and late responses to microbe perception, respectively.
The major limitation of the oxidative burst assay is variability. For reasons that are not completely understood, absolute RLUs often differ by an order of mag...

To perceive and defend against pathogens, plants have evolved membrane-bound pattern recognition receptors (PRRs) that detect conserved microbial molecules outside the cell known as microbe-associated molecular patterns (MAMPs)1. The binding of MAMPs to their cognate PRRs initiates protein kinase-mediated immune signaling resulting in broad-spectrum disease resistance2. One of the earliest responses following PRR activation is the phosphorylation and activation of integral plasma membrane RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins that catalyze the production of extracellular reactive oxygen species (ROS)3,4. ROS play an important role in establishing disease resistance, acting both as secondary messengers to propagate immune signaling as well as direct antimicrobial agents5. The first observation of an immune-elicited oxidative burst was described using potato tubers of cv. Rishiri following Phytophthora infestans inoculation6. ROS production has been evaluated in several plant species using leaf discs7, cell suspension cultures8, and protoplasts6. Described here is a simple method for assaying pattern-triggered ROS production in leaf discs of Arabidopsis thaliana (Arabidopsis).
As a response to MAMP perception, activated RBOH proteins catalyze the production of superoxide radicals (O2-), hydroxyl radicals (&#8226;OH), and singlet oxygen (1O2) that are converted into hydrogen peroxide (H2O2) in the extracellular space9. H2O2 can be quantified by luminol-based chemiluminescence in the presence of the oxidizing agent horseradish peroxidase (HRP)10. HRP oxidizes H2O2 generating a hydroxide ion (OH&#8722;) and oxygen gas (O2) which react with luminol to produce an unstable intermediate that releases a photon of light10. Photon emission can be quantified as relative light units (RLUs) using a microplate reader or imager capable of detecting luminescence, which have&#160;become standard pieces of equipment in most molecular laboratories. By measuring the light produced over a 40-60-minute interval, a transient oxidative burst can be detected as early as 2-5 minutes after the elicitor treatment, peaking at 10-20 minutes, and returning to basal levels after ~60 minutes11. The cumulative light produced over this time course can be used as a measure of immune strength corresponding to the activation of RBOH proteins12. Conveniently, this assay does not require specialized equipment or cumbersome sample preparation.
Peaking shortly after MAMP detection, the oxidative burst is considered an early immune response, along with MAPK activation and ethylene production5. Later immune responses include transcriptional reprogramming, stomatal closure, and callose deposition2,5. Prolonged exposure to MAMPs continually activates energetically-costly immune signaling resulting in the inhibition of plant growth, indicative of a trade-off between development and immunity13. Pattern-triggered seedling growth inhibition (SGI) is widely used to assess immune output in Arabidopsis and has been integral to the identification of several key components of immune signaling including PRRs14,15,16. Therefore, this paper additionally presents an assay for&#160;pattern-triggered SGI in Arabidopsis, whereby seedlings are grown in multi-well plates containing standard media or media supplemented with an immune elicitor for 8-12 days and then weighed using an analytical scale.
To demonstrate how ROS and SGI assays can be used to monitor PRR-mediated signaling, three genotypes that represent varying immune outputs were chosen: (1) the wild type Arabidopsis accession Columbia (Col-0), (2) the dominant-negative bak1-5 mutant in which the multi-functional PRR co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1&#160;(BAK1) is non-functional in immune signaling17,18, and (3) the recessive cpk28-1 mutant, which lacks the regulatory protein CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) and displays heightened immune-triggered responses19,20. ROS and SGI assays are presented in response to a synthetically-produced elf18 peptide epitope of bacterial Elongation Factor Tu (EF-Tu), recognized in Arabidopsis by the PRR EF-Tu RECEPTOR (EFR)15. These protocols can be used with other immune elicitors such as the bacterial motility protein flagellin14 or endogenous Plant Elicitor Proteins (AtPeps)16, however, it should be noted that plant responsiveness differs depending on the elicitor21. Together, ROS and SGI assays can be used for the quick and quantitative assessment&#160;of early and late PRR-mediated responses.

<table>
	<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>48-Well Sterile Plates with Lid</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Scale with Draft Sheid</td>
			<td>VWR</td>
			<td>VWR-225AC</td>
			<td>Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.</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>Must request a quote</td>
			<td>Any plate reader capable of detecting luminescence can be used for these assays.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-1KG</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>White Polystyrene 96-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
	</tbody>
</table>

pattern-triggered oxidative burst and seedling growth inhibition assays in arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Mutant cpk28-119,25 and bak1-517,18 plants were used to demonstrate expected outcomes for genotypes with high and low immune responses, respectively, in oxidative burst and SGI assays relative to a wild-type background control (Col-0). To assess dose-dependent effects, a 10-fold peptide dilution series (1-1,000 nM) of elf18 was used. As expected, cpk2...

Watch this Scientific Journal Video about Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana at JoVE.com

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

These assays can be used to assess and compare immune outputs of different plant genotypes. The main advantage of these techniques is that they are quick, reliable, and use common laboratory equipment. For the growth inhibition assay, seedlings of the same size and age should be transplanted and fully blotted dry before weighing to limit variation.At four to five weeks post-germination, use a sharp four-millimeter biopsy punch to remove one leaf disc per Arabidopsis plant, avoiding the midvein and being careful to limit wounding. Collect the discs into individual wells of an unused 96-well luminometer plate containing 100 microliters of double-distilled water per well, adaxial side up, to prevent desiccation. If assessing multiple elicitors, remove a second leaf disc from the same leaf for each elicitor treatment, and cover the plate with the lid to allow the leaf discs to recover overnight at room temperature.The next morning, set the microplate reader parameters to a 1, 000-millisecond integration time in two-minute intervals over a 40-to 60-minute period to capture the dynamic oxidative burst. Next, use a multi-channel 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.At four days post-germination, load each well of a 48-well plate with 500 microliters of MS medium supplemented with a dilution of elicitor peptide. Next, use sterile forceps to carefully transplant six to eight seedlings of the same size, age, and genotype to each peptide dilution, taking care that there is no damage to the seedling or breakage to the root and that the root is submerged in medium. When all of the seedlings have been plated, seal the plates with Micropore tape, and place the plants under standard short-day conditions for eight to 12 days.To determine the percent growth inhibition, take a photo to visually record the growth inhibition before carefully removing the seedlings from each well. Then, dab the roots on a paper towel to dry, and weigh each seedling on an analytical scale. In this representative experiment, mutant plants with hyperactive immune signaling demonstrated a higher cumulative and average oxidative burst compared to wild-type Arabidopsis plants, whereas mutant plants with impaired immune signaling displayed a reduced oxidative burst at concentrations between 10 and 1, 000 nanomolar.Expected differences in seedling growth inhibition could also be discerned between all of the genotypes grown in 100 and 1, 000 nanomolar elicitor peptide concentrations. When grown in the 1, 000 nanomolar peptide dilution, the mutants with hyperactive immune signaling were markedly smaller than wild-type plants grown under the same conditions, while the mutants with impaired immune signaling displayed a weak inhibition of growth relative to wild-type plants. These methods provide evidence for early and late immune responses.Several other techniques, including MAP kinase assays, pathogen-induced gene expression, and infection assays, can be used to delineate these pathways. These techniques have been modified for use in large-scale screens, which have successfully identified pathogen receptor proteins and several other signaling components.

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JoVE Journal

Immunology and Infection

12.9K Views.  Queen's University. These assays can be used to assess and compare immune outputs of different plant genotypes. The main advantage of these techniques is that they are quick, reliable, and use common laboratory equipment. For the growth inhibition assay, seedlings of the same size and age should be transplanted and fully blotted dry before weighing to limit variation.At four to five weeks post-germination, use a sharp four-millimeter biopsy punch to remove one leaf disc per Arabidopsis plant, avoiding the...