Abstract
Understanding the influence of secondary metabolites from fungi on the mitochondria of the host plant during infection is of great importance for the knowledge of fungus-plant interactions in general; it could help generate resistant plants in the future and in the development of specifically acting plant protection products. For this purpose, it must first be possible to record the mitochondrial parameters in the host plant. As of the date of this protocol, no measurements of mitochondrial respiration parameters have been performed in wheat paleae. The protocol shown here describes the measurements using the XF24 analyzer, which measures the rate of oxygen consumption in the sample by changes in the fluorescence of solid-state fluorophores. This procedure covers the preparation of samples for the XF24 analyzer and the measurement of mitochondrial parameters by adding specific mitochondrial inhibitors. It also shows the necessary approach and steps to be followed to obtain reliable, reproducible results. This is a robust protocol that allows the analysis of mitochondrial respiration directly in the wheat paleae. It demonstrates an important add-on method to existing screenings and also offers the possibility to test the effects of early infection of plants by harmful fungi (e.g., Fusarium graminearum) on mitochondrial respiration parameters. Key features This protocol offers the possibility of testing the effects of early infection of plants by pathogens on mitochondrial respiration parameters. This protocol requires a Seahorse XF24 Flux Analyzer with Islet Capture Microplates and the Seahorse Capture Screen Insert Tool. Graphical overview.