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.

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.