title: A quick and robust method for quantification of the hypersensitive response in plants.
abstract: One of the most studied defense reactions of plants against microbial pathogens is the hypersensitive response (HR). The HR is a complex multicellular process that involves programmed cell death at the site of infection. A standard method to quantify plant defense and the HR is to measure the release of cellular electrolytes into water after infiltration with pathogenic bacteria. In this type of experiment, the bacteria are typically delivered into the plant tissue through syringe infiltration. Here we report the development of a vacuum infiltration protocol that allows multiple plant lines to be infiltrated simultaneously and assayed for defense responses. Vacuum infiltration did not induce more wounding response in Arabidopsis leaf tissue than syringe inoculation, whereas throughput and reproducibility were improved. The method was used to study HR-induced electrolyte loss after treatment with the bacterium Pseudomonas syringae pv. tomato DC3000 harboring the effector AvrRpm1, AvrRpt2 or AvrRps4. Specifically, the influence of bacterial titer on AvrRpm1-induced HR was investigated. Not only the amplitude, but also the timing of the maximum rate of the HR reaction was found to be dose-dependent. Finally, using vacuum infiltration, we were able quantify induction of phospholipase D activity after AvrRpm1 recognition in leaves labeled with (33)PO4.
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Oskar N
authors.initials: ON
authors.lastname: Johansson
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Anders K
authors.initials: AK
authors.lastname: Nilsson
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Mikael B
authors.initials: MB
authors.lastname: Gustavsson
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Thomas
authors.initials: T
authors.lastname: Backhaus
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Mats X
authors.initials: MX
authors.lastname: Andersson
authors.affiliation: Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Ellerström
keywords: AvrRpm1; Effector-triggered-immunity (ETI); Electrolyte leakage; Hypersensitive response (HR); Programmed cell death (PCD); Pseudomonas syringae