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.
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
2. Preparing the elicitation solution
3. Starting the software and setting up the protocol with the referenced microplate reader (see Table of Materials)
NOTE: It takes some time to set up the parameters of the microplate reader software. It is recommended to get the machine and protocol ready (one click to proceed) before adding the elicitation solution.
4. Establishing the elicitation system and measuring real-time ROS production
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 assay. Sterilized rice seeds were cultured on 1/2 MS medium and grown in a 12 h light/12 h dark photoperiod in A clear glass vial, 8.5 cm in diameter and 15 cm in height. (B) Schematic diagram of the sampling parts of leaf sheaths. Leaf sheaths were cut from 10-day-old rice seedlings. The positions of leaf sheaths were above the roots and below the first leaf. (C) Schematic diagram of the sampling position of leaf discs. The leaf discs can be cut from the middle third of the second leaf (count from the top) of the main tiller of healthy rice plants at any growth stage. Abbreviations: ROS = reactive oxygen species; MS = Murashige and Skoog.
Figure 2: Schematic diagram of the plate setup for measuring ROS production with different lines of Oryza sativa. Pretreatment and test of rice tissues using a 96-well plate. Line 1, Line 2, and Line 3 (up to eight lines on one plate) can be any material of interest, different cultivars, mutants, or transgenic lines. The tissues were stimulated with elicitation solutions with PAMP (PAMP, white) or without PAMP (ddH2O, gray) to measure ROS response. It should be noted that the more the samples to be tested, the longer the time interval between readings. Abbreviations: ROS = reactive oxygen species; PAMP = pathogen-associated molecular pattern; ddH2O = double-distilled water.
The authors have nothing to disclose.
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 |