An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation
Published: January 31, 2024
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 Rice sheath Cut the sheath from 10-day-old rice seedlings into 3 mm segments with …
Representative 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 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 c…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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An Assay for Detecting Reactive Oxygen Species Production in Rice Tissues upon Immune Elicitation. J. Vis. Exp. (Pending Publication), e21911, doi: (2024).