Measuring Immune-Induced Inhibition of Seedling Growth

1. 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 1A).
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 1B).
      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.

2. Determine percent growth inhibition.

1. Carefully remove seedlings from 48-well plates and dry them by dabbing them on a 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 1C).
2. Determine percent growth inhibition of elicitor-treated seedlings compared to seedlings grown in MS only 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.