title: An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi.
abstract: BACKGROUND
  The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses.
  
  
  METHODS
  In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement.
  
  
  RESULTS
  The purified peptide with a molecular weight of 7kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP-fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum.
  
  
  CONCLUSIONS
  In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.
authors.affiliation: Universidade Vila Velha, Vila Velha, ES, Brazil.
authors.forename: Umberto
authors.initials: U
authors.lastname: Zottich
authors.forename: Maura
authors.initials: M
authors.lastname: Da Cunha
authors.forename: André O
authors.initials: AO
authors.lastname: Carvalho
authors.forename: Germana B
authors.initials: GB
authors.lastname: Dias
authors.forename: Nádia
authors.initials: N
authors.lastname: Casarin
authors.forename: Ilka M
authors.initials: IM
authors.lastname: Vasconcelos
authors.forename: Valdirene M
authors.initials: VM
authors.lastname: Gomes
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D040503:Coffea / Q000737:chemistry*; D005670:Fusarium / Q000254:growth & development*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D000935:Antifungal Agents; D010455:Peptides; D010940:Plant Proteins