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

Abstract

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) levels in the cells. This Oxidative Burst has various consequences, in particular the induction of the synthesis of secondary metabolites like phythoalexins (compounds with anti-microbial activity) and lignin (to reinforce the cell wall), and the triggering of a programmed cell death. We used a previously established in vitro elicitation system to study the interaction between Olea europaea L. and its pathogen Pseudomonas savastanoi, responsible for the olive knot, a disease that drastically affects olive oil production in Portugal. Material and Methods: The in vitro elicitation system used in this work was composed of suspension cell cultures of the resistant Olea europaea variety Galega Vulgar and by an avirulent strain of the pathogen Pseudomonas savastanoi. ROS detection was performed using a modification of the method described by Parsons et al. (1999), lipid peroxidation was measured using the TBA test and phenylalanine ammonia lyase (PAL) activity was evaluated by the quantification of trans-cinnamic acid determined spectrophotometrically at 290 nm. Results and Conclusions: Evaluation of ROS levels show a significant increase in production by the elicitated O. europaea cells, with a pattern typical of incompatible interactions, indicating that the Galega Vulgar variety is in fact resistant to the pathogen. This OB leads to significant cellular damage, illustrated by increased levels of lipid peroxidation. 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 the total soluble phenolics allowed us to assess these changes. In order to evaluate the programmed cell death of the challenged cells, the T.U.N.E.L. assay was carried out during the time course of elicitation. References: 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.

Since the ROS produced in the OB function mainly as an intracellular signal, we studied the effect of Hydrogen peroxide as a signal molecule. Various concentrations of H2O2 were added to suspension cell cultures of O. europaea and PAL activity was measured. The increase in PAL activity when the added concentrations were low indicates that hydrogen peroxide may in fact function as a transduction signal during the HR.

Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1); 189-98.