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)

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 intensit....

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