Pseudomonas savastanoi triggers the hypersensitive response of Olea europaea var. Galega Vulgar suspension cell cultures

Abstract

The bacteria Pseudomonas savastanoi is a natural pathogen of Olea europaea L., and is responsible for the olive knot, a disease that seriously affects productivity of Portuguese olive orchards. In previous work, we established an in vitro elicitation system using the bacteria and suspension cell cultures of O. europaea var. Galega Vulgar, which is known to be resistant to this pathogen. This system is now being used to study the interaction between plant and pathogen and evaluate the Hypersensitive Response triggered by the infection. Evaluation of Reactive Oxygen Species (ROS) levels was performed by spectrofluorimetric quantification of the fluorescent probe H2DCFDA, and showed a significant increase in production by the elicitated cells, with a pattern typical of the Oxidative Burst (OB) of incompatible interactions. The levels of lipid peroxidation followed a similar pattern, indicating ROS production in the OB as an origin of this cellular damage. Phenylalanine ammonia lyase (PAL) activity was measured to determine changes in the phenylpropanoid pathway and, therefore, in secondary metabolism. Determination of total lignin content of cell walls allowed us to assess these changes. Challenged cells showed a significant increase in PAL activity during the time course of elicitation, as well as an increase in the lignin content. These results point to changes in the production of secondary metabolites, which are induced by the elicitation with an avirulent pathogen and lead to the reinforcement of the cell wall. The TUNEL assay was performed to evaluate if programmed cell death is triggered in challenged cells.