Evaluation of programmed cell death in Olea europaea var. galega vulgar suspension cell cultures elicited with Pseudomonas savastanoi

Abstract

To study the events that underlie the resistance mechanisms of certain Olea europaea L. varieties to the olive knot, a disease caused by the bacteria Pseudomonas savastanoi, we had previously developed an in vitro elicitation system using the bacteria and suspension cell cultures of the olive variety Galega Vulgar, which is known to be resistant to the disease. When characterizing the interactions between plant and pathogen in this system, we have observed typical events of a Hypersensitive Response (HR), corroborating that this variety of O. europaea is in fact resistant to the infection. Further studies have been conducted to clarify the resistance phenomena. Reactive Oxygen Species (ROS) accumulation in the elicited cells was qualitatively evaluated using a fluorescent probe, allowing the observation of a significant increase in ROS production distributed evenly across the cytoplasm. The activation of programmed cell death was accessed by TUNEL assay and determination of cell viability during the time course of elicitation. Challenged cells started to show nuclear staining typical of positive TUNEL assay at around 24 hours, with more than 90% of the cells presenting nuclear staining 24 hours later. Consistently, cell viability started to decrease rapidly at around 60 hours, with more than 95% of non-viable cells 24 hours later, indicating that the HR indeed triggered a program that ultimately leads to the death of the elicited cells. To give insights on the signal transduction pathways that lead to the activation of this program and other related defence mechanisms, we studied the effects of inhibitors of some key cellular events on the production of ROS during the Oxidative Burst of the HR. The results demonstrated that olive resistance to P. savastanoi attack depends on de novo protein synthesis and Ca2+ influx. In addition, NADPH oxidase and MAPK seem to be implicated in the defence signalling pathways.