Permeabilization of fungal membranes by plant defensins inhibits fungal growth.
We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.
Thevissen
K
K
F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, B-3001 Heverlee-Leuven, Belgium.
Terras
F R
FR
Broekaert
W F
WF
D000935:Antifungal Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D003470:Culture Media; D023082:Defensins; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D009154:Mutation; D009492:Neurospora crassa / Q000187:drug effects / Q000254:growth & development*; D009930:Organic Chemicals; D010640:Phenothiazines / Q000494:pharmacology; D010940:Plant Proteins / Q000493:pharmacokinetics / Q000494:pharmacology*; D011506:Proteins / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*
D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D003470:Culture Media; D023082:Defensins; C099969:Dm-AMP1 protein, Dahlia merckii; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010640:Phenothiazines; D010940:Plant Proteins; D011506:Proteins; C402795:SYTOX Green; C009469:thionine