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 33PO4.
Highlights
Phytopathogenic microorganisms are omnipresent in nature and pose a constant threat to plants
To further evaluate the applicability of this method, we investigated the role of bacterial titer on the outcome of hypersensitive response (HR)-Programmed cell death (PCD)
We have previously reported that Phosphatidic acid (PA) is formed quickly in tissue after elicitation with AvrRpm1 and that PA by itself can induce HR-like cell death in Arabidopsis (Andersson et al, 2006)
Summary
Phytopathogenic microorganisms are omnipresent in nature and pose a constant threat to plants. Plants rarely become infected and develop disease; a multi-layered innate immune system protects them from most pathogens. Plants can perceive microbes through pattern recognition receptors that recognizes conserved microbe-associated molecular patterns (MAMP), leading to activation of defense mechanisms and MAMPtriggered immunity (MTI) (Dodds & Rathjen, 2010). Pathogens have evolved several strategies which enable them to break plant defense and proliferate. The pathogens may for example secrete effector molecules that suppress MTI (Arnold & Jackson, 2011) or promote an outflow of nutrients from the host (Chen et al, 2010).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
