An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation

Take a microplate containing uniform sheath segments cut from rice seedlings and leaf discs obtained from rice plant leaves.

Add sterile water and incubate overnight to allow recovery from wound stress.

Add an immunogenic peptide derived from bacterial flagella that acts as a pathogen-associated molecular pattern or PAMP.

Plant cells at the cut edges bind to the PAMPs via pattern recognition receptors.

The binding triggers downstream signaling, activating a plasma membrane-localized enzyme termed respiratory burst oxidase homolog or RBOH.

Activated RBOH produces reactive oxygen species or ROS in the apoplast — the intercellular space. The produced ROS gets converted into hydrogen peroxide.

Add a peroxidase enzyme and a chemiluminescent substrate.

Peroxidase utilizes hydrogen peroxide to convert the substrate into an excited state intermediate, which releases energy as light and returns to the ground state.

Measure luminescence in both tissue types to detect a sharp increase immediately following PAMP treatment, indicating an ROS burst.

Cut the sheath from 10-day-old rice seedlings into 3-millimeter segments with a sharp razor blade or surgical blade for pretreatment one day before