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

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 the 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 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.
   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 Tabla de Contenidos), 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 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 °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 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°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 sensitivity, while ensuring that all samples in a 96-well plate can be measured within one interval.