Júlia Ribeiro Soares1, Edésio José Tenório de Melo2, Maura da Cunha2, Kátia Valevski Sales Fernandes3, Gabriel Bonan Taveira1, Lidia da Silva Pereira4, Samy Pimenta4, Fernanda Gomes Trindade2, Mariana Regente5, Marcela Pinedo5, Laura de la Canal5, Valdirene Moreira Gomes1, André de Oliveira Carvalho6. 1. Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil. 2. Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil. 3. Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil. 4. Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense - Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil. 5. Instituto de Investigaciones Biologicas, Universidad Nacional de Mar del Plata -CONICET, Mar del Plata, Argentina. 6. 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.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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. Copyright Â
BACKGROUND: 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. METHODS: 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. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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. Copyright Â
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