Measuring Immune-Induced Inhibition of Seedling Growth

Transplant seedlings into wells containing desired concentrations of an immune elicitor peptide — a protein fragment derived from pathogen-associated molecules.

This peptide binds to the pattern recognition receptors, or PRRs, on the plant cells, initiating a signaling cascade.

This cascade includes phosphorylation of respiratory burst oxidase homolog, or RBOH proteins, that become enzymatically active and catalyze the production of reactive oxygen species or ROS.

ROS act as secondary messengers in immune signaling pathways, amplifying the defense response initiated by PRRs, and function as direct antimicrobial agents, inhibiting pathogen growth by damaging their cellular components.

During ROS production, the plant allocates energy and nutrients to support its immune response, diverting resources away from growth-related processes that result in growth inhibition.

Determine the percent growth inhibition in the elicitor-treated seedlings compared to untreated seedlings by correlating their fresh weights.

Increasing concentrations of peptide elicitors inhibit seedling growth due to heightened immune responses and associated biochemical alterations.

At four days post-termination, 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.