Cyclopaldic Acid, the Main Phytotoxic Metabolite of Diplodia cupressi, Induces Programmed Cell Death and Autophagy in Arabidopsis thaliana

Cyclopaldic acid is one of the main phytotoxic metabolites produced by fungal pathogens ofthe genus Seiridium, causal agents, among others, of the canker disease of plants of the Cupressaceaefamily. Previous studies showed that the metabolite can partially reproduce the symptoms of theinfection and that it is toxic to different plant species, thereby proving to be a non-specific phytotoxin.Despite the remarkable biological effects of the compound, which revealed also insecticidal, fungicidaland herbicidal properties, information about its mode of action is still lacking. In this study, weinvestigated the effects of cyclopaldic acid in Arabidopsis thaliana plants and protoplasts, in order to getinformation about subcellular targets and mechanism of action. Results of biochemical assays showedthat cyclopaldic acid induced leaf chlorosis, ion leakage, membrane-lipid peroxidation, hydrogenperoxide production, inhibited root proton extrusion in vivo and plasma membrane H+-ATPaseactivity in vitro. qRT-PCR experiments demonstrated that the toxin elicited the transcription ofkey regulators of the immune response to necrotrophic fungi, of hormone biosynthesis, as wellas of genes involved in senescence and programmed cell death. Confocal microscopy analysisof protoplasts allowed to address the question of subcellular targets of the toxin. Cyclopaldicacid targeted the plasma membrane H+-ATPase, inducing depolarization of the transmembranepotential, mitochondria, disrupting the mitochondrial network and eliciting overproduction ofreactive oxygen species, and vacuole, determining tonoplast disgregation and induction of vacuole-mediated programmed cell death and autophagy