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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.

SUBMITTER: Johansson ON

PROVIDER: S-EPMC4699783 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A quick and robust method for quantification of the hypersensitive response in plants.

Johansson Oskar N ON Nilsson Anders K AK Gustavsson Mikael B MB Backhaus Thomas T Andersson Mats X MX Ellerström Mats M

PeerJ 20151201

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. ...[more]

PMID: 26734506

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Project description:BackgroundQuantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.ResultsThe presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency.ConclusionThe fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.

Project description:BackgroundIn cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community.ResultsIn this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (-?10?dB) without apparent signal distortion.ConclusionsWe demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at https://doi.org/10.6084/m9.figshare.11344046.v1 , and its code and datasets are available in GitHub at https://github.com/zoccoler/metroid .

Dataset Information

A quick and robust method for quantification of the hypersensitive response in plants.

Publications

A quick and robust method for quantification of the hypersensitive response in plants.

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