PAMP-Triggered ROS Assay in N. benthamiana

Pathogen-associated molecular pattern (PAMP)-triggered ROS was performed according to the previous reports (Wang et al., 2020 (link)). Briefly, the indicated GmMRLKs were expressed in N. benthamiana leaves for 2 days by Agrobacterium-mediated transient transformation. Leaf disks were taken and incubated overnight with 200 μL of sterile water in a 96-well plate. Leaf disks were treated with luminescence detection mixture containing 1 μM of flg22 (Sangon, Shanghai, China) or 200 μg/mL of chitin (Sigma-Aldrich, St. Louis, MO, United States), 20 mM of luminol (Sigma-Aldrich, St. Louis, MO, United States) and 10 mg/ML of horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, United States), and relative luminescence unit (RLU) was recorded by a luminometer (TECAN, Männedorf, Switzerland).

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Zhang Q., Chen S., Bao Y., Wang D., Wang W., Chen R., Li Y., Xu G., Feng X., Liang X, & Dou D. (2022). Functional Diversification Analysis of Soybean Malectin/Malectin-Like Domain-Containing Receptor-Like Kinases in Immunity by Transient Expression Assays. Frontiers in Plant Science, 13, 938876.

Publication 2022

flg22 chitin luminol horseradish peroxidase

Agrobacterium Chitin Horseradish peroxidase Leaf Luminescence Luminol Pamp Pathogen associated molecular pattern Sterile Transient

Corresponding Organization :

Other organizations : China Agricultural University, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, South China Agricultural University, Nanjing Agricultural University

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Variable analysis

independent variables

Expression of indicated GmMRLKs in N. benthamiana leaves for 2 days by Agrobacterium-mediated transient transformation

dependent variables

Relative luminescence unit (RLU) measured by a luminometer

control variables

Leaf disks incubated overnight with 200 μL of sterile water in a 96-well plate
Luminescence detection mixture containing 20 mM of luminol and 10 mg/ML of horseradish peroxidase

controls

Positive control: Leaf disks treated with 1 μM of flg22
Positive control: Leaf disks treated with 200 μg/mL of chitin

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