Parallel, Continuous Monitoring and Quantification of Programmed Cell Death in Plant Tissue

Abstract

The accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease-resistant crop varieties. In this study, we report an accelerated phenotyping platform for the continuous-time, rapid and quantitative assessment of HR: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating the detection of microscopic levels of cell death. We validated PASTEL by transiently expressing the effector protein AVRblb2 in transgenic lines of the model plantNicotiana benthamiana(expressing the corresponding resistance protein Rpi-blb2) to reliably induce HR. We were able to detect cell death at microscopic intensities, where leaf tissue appeared healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously (sub-seconds to minutes). Using this data, we developed a supervised machine learning models for classification of HR. We were able to classify input data (inclusive of our entire tested concentration range) as HR-positive or negative with 84.1% mean accuracy (F1score = 0.75) at 1 hour and with 87.8% mean accuracy (F1score = 0.81) at 22 hours. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks.

Time-resolved chemical monitoring of whole plant roots with printed electrochemical sensors and machine learning

“…We also demonstrated uptake of ions by the roots via an electrolyte leakage assay (a widely used method in plant research) of the stems of the kale seedlings (fig. S6) ( 45 , 46 ).…”

“…Following on from salt uptake experiments, stem samples were tested using a simultaneous multiwell impedance measurement system developed for electrolyte leakage assays ( 45 ). The system consisted of an array of measurement wells, each with a two-electrode assembly interfaced with external electronics that facilitated the measurement of solution conductivity in each well.…”