Materials

• Distilled water
• Plastic bacterial inoculation loops
• Scalpel
• Petri dishes
• 96 well plates (Corning 96-Well Solid White Polystyrene Microplates)
• Luminometer (TECAN infinite M200)
• 500x L-012 stock solution (10μM): 3.1mg L-012 (a new luminol derivative with a higher activity which reacts with various types of reactive oxygen species) was completely dissolved in 1 ml of DMSO and used at a final concentration of 20μM. Because L-012 is light-sensitive, all solutions containing L-012 must be protected from light by wrapping tubes in aluminum foil. The 500x L-012 stock solution is stored at -20°C, thawn aliquots are thrown away after use.
• 10,000x Horseradish Peroxidase (HRP) stock solution: dissolve 10 mg/ml in sterile MilliQ. Store 10-30μl aliquots in -20°C (use at a final concentration of 1μg/ml).
• flg22
• 500mM MOPS pH7: Store at 4°C, make fresh every 1-2 weeks (once it turns yellowish)

When agro-infilitrating

• 24 hour prior to experiment start: Grow Agrobacteria strains from glycerol in appropriate antibiotics. Grow in 28°C over night.
• Measure OD₆₀₀ of Agrobacterial strains
• Calculate required volume of bacterial culture for a final OD₆₀₀ = 0.25/construct in the final volume
• Mix strains, spin down at 2000x g, RT
• Decant supernatant and resuspend bacteria in 1ml infiltration buffer (10mM MgCl₂, 10mM MES-KOH pH5.6, 200μM Acetosyringone)
• Fill up to final volume and leave shaking at 28°C for 2 hours prior to infiltration
• Infiltrate 4-5 week old N. benthamiana plants, minimize amount of pushes to fuly infiltrate leave, mark un-infiltrated zones

Leaf-discs

• Make leaf discs using a cork borer number 2 (6.25mm) by punching them out against a piece of Styrofoam. If possible, avoid the midvein and edges of the N. benthamiana leaf. When using Arabidopsis take the center of the leaf. Put the discs into a 50 ml plastic centrifuge tube. Approximately 40-50 discs per construct are needed. If using agro-infiltrated leaves determine how long post agro-infiltration is optimal for the given experiment.
• Place each leafdisc adaxial side up into a well containing 150μl distilled water (dH₂O)of a 96-well titer plate for in between 8-18 hours (no shorter or longer) in continuous light 22°C to reduce wounding effect

Elicitation mixture and elicitation

• Either use Pseudomonas syringae pv. tomato DC3000 ΔHopQ for N. benthamiana, or flg22 peptide or other elicitor. Keep solutions at room temperature.
  • For bacteria: take bacteria from plate after growing for 2 days and wash twice in sterile dH₂O. Adjust to OD₆₀₀ = 0.1 and add 20mu;M L-012, 1μg/ml HRP, and 10mM MOPS pH7.0
  • For flg22: prepare solution of 100nM flg22 and add 20μM L-012, 1μg/ml HRP, and 10mM MOPS pH7.0
• Immediately prior to elicitation carefully remove all dH₂O (150μl) from each well avoiding any tissue damage or desiccation
• Using a multichannel pipetman, quickly add 100μl of the elicitation solution to each leaf disc containing well
• Place plate directly in TECAN infinite M200

Note: some protocols mention addition of HRP and luminol solution first (substrate solution), waiting approximately 10min for the signal to stabilize, and then adding elicitor. This is because there is an initial peak when the substrate solution is added to the leaf discs. If a very fast response is expected this is vital. Alternatively, when adding the elicitor together with the substrate solution discard the first few minutes for the statistical analysis, as this is background luminescence and may differ in samples based on the well position and measurement time.

Plate-reader settings

• Measure ROS production between 0-80 minutes for Pto samples, and between 0-60 minutes for flg22 samples
• Measure for 500ms/well integration time, measure every minute (if less than the entire plate is taken the integration time can be increased)

Statistics

• Take at least 16 technical replicates per treatment
• Repeat each experiment independently at least 3 times (biological replicates = 3)
• Statistical analysis is peformed on the sum of luminescence over a defined time-period. Discard first minutes (see note above).
• Perform Wicoxon rank test for 2-sample comparison, Kruskall-Wallis for multiple samples if non-normally distributed. Alternatively if data is normally distributes use Welch two-sample t-test for 2-sample comparison, or one-way ANOVA for multiple samples.

Data and script

• In the the first tab of the excel sheet an example is given of what the data should look like to work with the script. The second tab has the raw output from the device. In the first sheet the first column is the well number, the second column the construct/condition used, and the remainder columns are the raw data over time (in seconds).
• Use the R script to make graphs and do the statistics!