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Evaluating Leaf Responses to Microbial Secondary Metabolites Using A High-Throughput Format.

Journal of visualized experiments : JoVE · 5 Dec 2025 · 10.3791/69026

Abstract

Microbes secrete structurally diverse secondary metabolites during plant infection, some of which are detected by plant cells, which trigger stress responses. In this method, the induction of ion leakage, peroxidase activity, and callose production is measured in the same leaf disk sample. First, Arabidopsis or barley leaf disks are vacuum infiltrated in a 96-well plate. After 4-6 hours, conductivity is measured, followed by peroxidase activity and callose deposition at 24 hours. The flg22 peptide induces all three responses and is an affordable positive control. Surfactin and gramillin cyclic lipopeptides induce peroxidase activity and ion leakage, respectively, while the phytotoxic T-2 trichothecene suppresses peroxidase activity. Overall, this approach enables multiple comparisons across either plant genotypes or metabolite treatments. This approach can be applied to chemical genetics or bioprotection to identify stress-modulating compounds for further study. In plant genetics, this approach can be used to compare responses across plant populations for genetic mapping and to improve our understanding of plant-microbe interactions.

Plant phenotyping relevance

植物葉のストレス応答(イオン漏出、ペルオキシダーゼ活性、カロース蓄積)を同一試料で高スループット測定する方法が研究の中心であり、植物表現型取得法に該当する。

abstractIn this method, the induction of ion leakage, peroxidase activity, and callose production is measured in the same leaf disk sample.
abstractFirst, Arabidopsis or barley leaf disks are vacuum infiltrated in a 96-well plate.
abstractOverall, this approach enables multiple comparisons across either plant genotypes or metabolite treatments.

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