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

Abstract

Source: Wang, Y., et al. Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay. J. Vis. Exp. (2022).

This video demonstrates an assay for real-time detection of reactive oxygen species (ROS) production in rice tissues upon pathogen-associated molecular pattern (PAMP) elicitation. Cut sheath segments and leaf discs are exposed to a bacterial flagella-derived peptide functioning as a PAMP. Upon recognition of the peptide via pattern recognition receptors (PRRs) on plant cells, a rapid and transient production of ROS occurs, which is detected via a chemiluminescence assay.

Protocol

NOTE: The protocol is applicable to different plant tissues. Rice sheath and leaf discs were used in this protocol for ROS detection upon PAMP elicitation. As differences mainly arise due to the method of sampling, only the common procedures are described below, with specific steps being mentioned wherever necessary.

1. Tissue preparation and pretreatment

1. Rice sheath
   1. Cut the sheath from 10-day-old rice seedlings into 3 mm segments with a sharp razor blade or surgical blade for pretreatment 1 day before the ROS assay (Figure 1B).
   2. Place five sheath segments ....

Results

Figure 1: The growth condition and stages of rice seedlings for sheath sampling and parts of the rice sheath and rice leaves used in the assay. (A) Rice seedlings grown on 1/2 MS medium under sterile conditions for 10 days can be sampled for ROS as.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well microtiter plate	WHB	WHB-96-01
flg22	Sangon Biotech	p20973	PAMP
Gen5	BioTek		software
L-012	FUJIFILM	120-04891	8-amino-5-chloro-7-phenyl-2,3-dihydropyrido [3,4-d] pyridazine-1,4-dione, CAS #:143556-24-5
Microplate reader	BioTek	Synergy 2
Peroxidase from horseradish (HRP)	Sigma	P8375
Sampler	Miltex	15110-40
Tris	Sangon Biotech	A610195