Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress

LAURA DE LA CANAL; FERNANDA GOMES TRINDADE; GABRIEL BONAN TAVEIRA; EDÉSIO JOSÉ TENÓRIO DE MELO; VALDIRENE MOREIRA GOMES; MARIANA REGENTE; ANDRÉ DE OLIVEIRA CARVALHO; LIDIA DA SILVA PEREIRA; MARCELA PINEDO; MAURA DA CUNHA; SAMY PIMENTA; KÁTIA VALEVSKI SALES FERNANDES; JÚLIA RIBEIRO SOARES

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2017 vol. 1861 p. 3429 - 3443

0304-4165

BackgroundPlant defensins were discovered at beginning of the 90s´; however, their precise mechanism of action is still unknown.Herein, we studied ApDef1-Saccharomyces cerevisiae interaction.MethodsApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assaywas repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization,ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPIand FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructuralanalysis was done by electron microscopy.ResultsApDef1 caused S. cerevisiae cell death and MIC was 7.8 μM. Whole cell population died after 18 h of ApDef1 interaction.After 3 h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatincondensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization.Z-VAD-FMK prevented yeast cell death.ConclusionsApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preservedmembrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led toplasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependentapoptosis via.General significanceWe show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membranepotential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would bean indirect event of the ApDef1-S. cerevisiae interaction