The hypersensitive response of Olea europaea L. elicitated by Pseudomonas savastanoi

Abstract

Introduction: In plants, the Hypersensitive Response (HR) is an early defense mechanism that is elicited by the recognition of an incompatible pathogen, with the objective of restricting its spread. One of the earliest events in the HR is the rapid and significant increase in Reactive Oxygen Species (ROS) cellular levels. This Oxidative Burst (OB) has various consequences, in particular the induction of synthesis of secondary metabolites like phythoalexins (compounds with anti-microbial activity) and lignin (cell wall reinforcement), and the triggering of programmed cell death. We used a previously established in vitro elicitation system to study the interaction between Olea europaea L. and Pseudomonas savastanoi, causal agent of olive-knot, a disease that drastically affects olive oil production in Portugal. Material and Methods: Suspension cell cultures of Olea europaea variety Galega Vulgar were inoculated with a middle-exponential grown phase suspension of Pseudomonas savastanoi. ROS detection was performed according to a modification of Parsons et al. (1999), lipid peroxidation was measured using TBA (Heath and Packer, 1968), and phenylalanine ammonia-lyase (PAL) activity was evaluated by spectrophotometrical quantification of trans-cinnamic acid at 290 nm. T.U.N.E.L. assay was performed using the “In situ cell death detection kit, POD” (Roche). Results and Conclusions: Evaluation of ROS levels show a significant increase in elicitated O. europaea cells, with two oxidative bursts characteristic of incompatible interactions, suggesting that the Galega Vulgar variety exhibits resistance to the pathogen. The increase in ROS and lipid peroxidation suggest significant cellular damage. PAL activity also increased in elicitated cells, pointing toward changes in the phenylpropanoid pathway and, therefore, in the production of secondary metabolites. Quantification of lignin and of total soluble phenolics allowed us to assess these changes. In order to evaluate the programmed cell death of the challenged cells, T.U.N.E.L. assay was carried out during the time course of elicitation. References: Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1); 189-98. Parsons HL, Yip YHJ, Vanlerberghe GC. 1999. Increased Respiratory Restriction during Phosphate-Limited Growth in Transgenic Tobacco Cells Lacking Alternative Oxidase. Plant Physiol 121; 1309-1320.