A quick and robust method for quantification of the hypersensitive response in plants.
Oskar N Johansson1, Anders K Nilsson1, Mikael B Gustavsson1
1Department of Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
Peerj
|January 7, 2016
Summary
A new vacuum infiltration method improves plant defense studies. This technique enhances throughput and reproducibility for analyzing the hypersensitive response (HR) and electrolyte leakage in plants challenged by pathogens.
Area of Science:
- Plant pathology
- Molecular plant-microbe interactions
- Plant immunology
Background:
- The hypersensitive response (HR) is a key plant defense against microbial pathogens, involving programmed cell death.
- Quantifying plant defense often relies on measuring electrolyte leakage following bacterial infiltration.
- Traditional syringe infiltration limits throughput and reproducibility.
Purpose of the Study:
- To develop and validate a vacuum infiltration protocol for simultaneous assaying of plant defense responses.
- To compare vacuum infiltration with traditional syringe infiltration in terms of wounding response, throughput, and reproducibility.
- To investigate the dose-dependent nature of the hypersensitive response and phospholipase D activity.
Main Methods:
- Development of a vacuum infiltration technique for plant tissue.
- Comparison of vacuum infiltration with syringe infiltration in Arabidopsis using electrolyte leakage assays.
- Infiltration with Pseudomonas syringae pv. tomato DC3000 strains expressing different effectors (AvrRpm1, AvrRpt2, AvrRps4).
- Investigation of bacterial titer effects on HR.
- Measurement of phospholipase D activity using (33)PO4 labeling.
Main Results:
- Vacuum infiltration offers improved throughput and reproducibility compared to syringe infiltration.
- Vacuum infiltration did not induce significantly more wounding response in Arabidopsis leaf tissue.
- HR-induced electrolyte loss and the timing of maximum HR rate were found to be dose-dependent on bacterial titer.
- Phospholipase D activity induction was quantified after AvrRpm1 recognition using vacuum infiltration.
Conclusions:
- Vacuum infiltration is a robust and efficient method for studying plant defense responses like the HR.
- The developed protocol facilitates high-throughput analysis of plant-pathogen interactions.
- Bacterial effector recognition and subsequent defense responses, including phospholipase D activity, are influenced by pathogen dose.
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