Interaction between the plant ApDef1defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.
Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction.



ApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was 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, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy.



ApDef1caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef1interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death.



ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via.



We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Júlia Ribeiro
JR
Soares
Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Edésio
E
José Tenório de Melo
Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Maura
M
da Cunha
Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Kátia Valevski Sales
KVS
Fernandes
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Gabriel Bonan
GB
Taveira
Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Lidia
L
da Silva Pereira
Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Samy
S
Pimenta
Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Fernanda Gomes
FG
Trindade
Instituto de Investigaciones Biologicas, Universidad Nacional de Mar del Plata -CONICET, Mar del Plata, Argentina.
Mariana
M
Regente
Instituto de Investigaciones Biologicas, Universidad Nacional de Mar del Plata -CONICET, Mar del Plata, Argentina.
Marcela
M
Pinedo
Instituto de Investigaciones Biologicas, Universidad Nacional de Mar del Plata -CONICET, Mar del Plata, Argentina.
Laura
L
de la Canal
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
Valdirene Moreira
VM
Gomes
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil. Electronic address: andreocarvalhouenf@gmail.com.
André
A
de Oliveira Carvalho
D000935:Antifungal Agents / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects; D023082:Defensins / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D050296:Microbial Viability / Q000187:drug effects*; D008954:Models, Biological; D018384:Oxidative Stress / Q000187:drug effects*; D010940:Plant Proteins / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D000935:Antifungal Agents; D023082:Defensins; D010940:Plant Proteins; D006861:Hydrogen Peroxide; D020169:Caspases
Antimicrobial peptides; Caspase; Cell cycle; Membrane permeabilization; Microbicidal; ROS induction