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

Summary

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.

Abstract

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.

Introduction

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 (•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⁻) 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 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 deposition^2,5. 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 PRRs^14,15,16. Therefore, this paper additionally presents an assay for 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 (BAK1) is non-functional in immune signaling^17,18, and (3) the recessive cpk28-1 mutant, which lacks the regulatory protein CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) and displays heightened immune-triggered responses^19,20. 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 of early and late PRR-mediated responses.

Protocol

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

1. Plant growth and maintenance.
   1. 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 °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.
   2. Sow seeds on soil and germinate under standard short-day conditions (22 °C, 10 h light, 150 μE/m²/s light intensity, and 65-70% relative humidity).
   3. 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).
2. Collect leaf discs in 96-well plates for overnight recovery.
   1. 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 μL of ddH₂O with the adaxial side facing upwards to prevent desiccation²² (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 solution²³.
   2. 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.
3. Perform the elicitor treatment and measure ROS production.
   1. 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.
   2. 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.
   3. Prepare a reaction solution containing 100 μM luminol, 10 μg/mL 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 °C. When ready to use, dilute stocks in sterile H₂O to generate a 100 μM working stock and store at -20 °C.
   4. Use a multi-channel pipette to dispense 100 μL of the reaction mixture to each well, adding the solution to all leaf discs of the same treatment at the same time²². 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°C unless in use.
   5. 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 sensitivity¹¹, while ensuring that all samples in a 96-well plate can be measured within one interval.
4. Data interpretation.
   1. For each genotype, use a spreadsheet application to calculate the average photon count and standard error at each time point, and display ROS production as a function of time using a scatter plot.
      
      Where t = each time point
   2. Alternatively, sum the photon counts for each leaf disc and present the average of these values for each genotype using a bar graph with standard error bars or a box and whisker plot.
      

2. Seedling Growth Inhibition Assay

1. Sterilize seeds and sow on Murashige and Skoog (MS) plates.
   1. Prepare sterile half-strength (0.5x) MS medium (2.16 g/L) containing 0.8% agar [w/v]. Pour media into plates (90 x 15 mm) in a laminar flow hood.
   2. Sterilize approximately 100 seeds in a microcentrifuge tube by washing with 1 mL of 70% ethanol for 2 min and remove by aspiration. Add 1 mL of 40% bleach and gently rock for 17 mins at room temperature. Remove bleach by aspiration and wash three times in 1 mL of sterile water for 5 min. Resuspend in 1 mL of sterile 0.1% agar.
      NOTE: Alternatively, use a chlorine gas seed sterilization protocol²⁴.
   3. Sow approximately 100 seeds per genotype on MS agar plates using a pipette and seal with micropore tape in a laminar flow hood.
      NOTE: Avoid placing seeds in close proximity as this will make transplanting seedlings difficult later.
   4. Stratify seeds by placing plates at 4 °C (no light) for 3-4 days to synchronize germination (Figure 2A).
2. Transplant seedlings into 48-well plates containing immune elicitor peptides.
   1. After stratification, move plates to light under short day conditions (22 °C, 10 h light, 150 μE/m²/s light intensity, and 65-70% relative humidity) for 3-4 days to allow germination.
   2. In a laminar flow hood, prepare elicitor peptide dilutions (0 nM, 1 nM, 10 nM, 100 nM, and 1,000 nM) in sterile 0.5x MS liquid media containing 1% sucrose [w/v], using 25 mL of MS for each 48-well plate. Prepare plates by pipetting 500 µL of MS liquid medium or MS medium containing peptides per well. If available, use a repeat pipettor for plate preparation to expedite the process.
      NOTE: For each genotype, grow seedlings in MS without elicitor to account for any inherent differences in seedling growth.
   3. Using sterile forceps carefully transplant one seedling to each well of the same size and age, ensuring that there is no damage to the seedling or breakage to the root and that the root is submerged in media. For each genotype, transplant a minimum of 6-8 seedlings into each peptide dilution (Figure 2B).
      NOTE: Transplant seedlings at 4 days post-germination, as short roots are easier to manipulate, and older seedlings may yield less optimal results.
   4. Seal plates with micropore tape and move back to light under standard short-day conditions (22 °C, 10 h light, 150 μE/m²/s light intensity, and 65-70% relative humidity). Allow seedlings to grow for 8-12 days.
3. Determine percent growth inhibition.
   1. Carefully remove seedlings from 48-well plates and dry by dabbing on paper towel. Weigh seedlings on an analytical scale and record values. If available, use an analytical scale equipped with USB output to record fresh weight values on a spreadsheet. Before weighing seedlings, take a photo to visually display growth inhibition (Figure 2C).
   2. Determine percent growth inhibition of elicitor-treated seedlings compared to seedlings grown in MS only (Figure 2D) as follows:
      
   3. Plot data using a bar graph with standard error bars or using a box and whisker plot to better display inter-experimental variance.

Results

Mutant cpk28-1^19,25 and bak1-5^17,18 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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20-20-20 Fertilizer	Plant Prod	10529	Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.
4 mm Biopsy Punch	Medical Mart	232-33-34-P	A cork borer set with a 0.125 cm^2 surface area can also be used.
48-Well Sterile Plates with Lid	Sigma-Aldrich	CLS3548
Analytical Scale with Draft Sheid	VWR	VWR-225AC	Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.
BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL)	Sartorius	725240	Any multichannel pipette can be used, as can a single pipetter if necessary.
elf18 (Ac-SKEKFERTKPHVNVGTIG)	EZ Biolab	cp7211	Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.
Forceps	Fisher Scientific	22-327379
Horseradish Peroxidase	Sigma-Aldrich	P6782	Dissolve in pure water. Store at -20C and away from light.
Luminol	Sigma-Aldrich	A8511	Dissolve in DMSO. Store at -20C and away from light.
Murisage and Skoog Basal Salts	Cedarlane Labs	MSP09-100LT	Store at 4C.
Soil	SunGrow Horticulture	Sunshine Mix #1	Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.
SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module	Molecular Devices	Must request a quote	Any plate reader capable of detecting luminescence can be used for these assays.
Sucrose	Sigma-Aldrich	S0389-1KG	Store at room temperature.
White Polystyrene 96-Well Plates	Fisher Scientific	07-200-589