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.
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Acknowledgments
Research at CRAG was funded with grants PID2019-108595RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and AGL2016-78002-R funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” (NSC), fellowship PID2019-108595RB-I00 funded by Spanish MCIN/AEI/ 10.13039/501100011033 (NSC) and fellowships BES-2017-080210 funded by MCIN/AEI/ 10.13039/501100011033 and by “ESF Investing in your future” (JS-L); and through the “Severo Ochoa Programme for Centres of Excellence in R&D” (SEV-2015-0533 and CEX2019-000902-S funded by MCIN/AEI/ 10.13039/501100011033) and by the CERCA Programme /Generalitat de Catalunya.
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Salguero-Linares, J., Lema-Asqui, S., Salas-Gómez, M., Froilán-Soares, A., Coll, N.S. (2022). Detection and Quantification of the Hypersensitive Response Cell Death in Arabidopsis thaliana . In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_16
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DOI: https://doi.org/10.1007/978-1-0716-2079-3_16
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