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Detection and Quantification of the Hypersensitive Response Cell Death in Arabidopsis thaliana

Abstract:

In plants, the hypersensitive response (HR) is a programmed cell death modality that occurs upon recognition of harmful non-self. It occurs at the site of pathogen infection, thus preventing pathogens to live off plant tissue and proliferate. Shedding light on the molecular constituents underlying this process requires robust and quantitative methods that can determine whether plants lacking functional genes are defective in HR execution compared to wild-type controls. In this chapter, we provide two quantitative protocols in which we measure cell death from Arabidopsis thaliana leaves infected with avirulent HR-causing bacterial strains. Firstly, we use trypan blue staining to quantify the stained area of leaves upon bacterial infection using a personalized macro in the Image J (Fiji) software. Alternately, we incorporate an electrolyte leakage protocol in order to measure HR caused by different avirulent bacterial strains at different bacterial titers. We encourage users to perform a combination of both methods when assessing HR in different plant genotypes.