Permeabilization of fungal membranes by plant defensins inhibits fungal growth

Abstract

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.

FIG. 1

Membrane permeabilization induced by plant defensins and α-PT in N. crassa and S. cerevisiae in water. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence of N. crassa hyphae (A) and S. cerevisiae wild-type cells (B) are shown. Fungal cells were suspended in distilled water buffered with 100 μM HEPES (pH 6.5) and treated with α-PT (●), Rs-AFP2 (■), Hs-AFP1 (X), or Dm-AMP1 (▴) for 30 min, whereupon fluorescence was measured. The dotted line indicates the upper limit of fluorescence detection. Values correspond to one representative experiment of three.

FIG. 2

Membrane permeabilization induced by Dm-AMP1 in S. cerevisiae wild-type cells and S. cerevisiae DM1 cells in water. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence of S. cerevisiae wild-type cells (□) and S. cerevisiae DM1 cells (■) are shown. Fungal cells were suspended in distilled water buffered with 100 μM HEPES (pH 6.5) and treated with Dm-AMP1 for 30 min, whereupon fluorescence was measured. The dotted line indicates the upper limit of fluorescence detection. Values are averages with standard errors of triplicate measurements and correspond to one representative experiment of three.

FIG. 3

Membrane permeabilization and growth inhibition induced by plant defensins and α-PT in N. crassa in low- and high-ionic-strength growth media. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence and growth inhibition of N. crassa suspended in SMF1 (A), SMF1 plus 50 mM KCl (B), and SMF1 plus 5 mM MgCl₂ (C) are shown. Fungal hyphae were treated with α-PT, Rs-AFP2, Hs-AFP1, or Dm-AMP1, and fluorescence was measured at different time points. Time points (in minutes) are indicated at the left of the α-PT permeabilization curves. Values correspond to one representative experiment of three.

FIG. 4

Fluorescence microscopy of fungal cells in the presence of SYTOX Green and Dm-AMP1. N. crassa cells were suspended in SMF1 supplemented with 5 mM MgCl₂ and 0.2 μM SYTOX Green and treated for 360 min in the absence (A) or presence (B) of 20 μM Dm-AMP1. Upper panels are light microscopic images; lower panels are fluorescence microscopic images. Bar, 25 μm.

FIG. 5

Effect of CCCP on membrane permeabilization induced by Rs-AFP2 in N. crassa. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence of N. crassa suspended in SMF1 in the absence (●) or presence (○) of 2 μM CCCP are shown. Fluorescence was measured 4 h after the addition of SYTOX Green, CCCP, and Rs-AFP2. Values correspond to one representative experiment of two.

FIG. 6

Membrane permeabilization and growth inhibition induced by Rs-AFP2 and Rs-AFP2(Y38G) in N. crassa in low- and high-ionic-strength growth media. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence and growth inhibition of N. crassa suspended in SMF1 (A) and SMF1 plus 5 mM MgCl₂ (B) are shown. Fungal hyphae were incubated with Rs-AFP2 (■) and Rs-AFP2(Y38G) (□) for 4 h, whereupon fluorescence was measured. The dotted line indicates the upper limit of fluorescence detection. Values correspond to one representative experiment of two.

FIG. 7

Membrane permeabilization induced by plant defensins and α-PT in S. cerevisiae wild-type cells and S. cerevisiae DM1 cells in low- and high-ionic-strength growth media. Dose-response curves of membrane permeabilization measured by SYTOX Green fluorescence and growth inhibition of S. cerevisiae wild-type cells and S. cerevisiae DM1 cells suspended in YMM (A) and YMM plus 5 mM MgCl₂ (B) are shown. Fungal cells were incubated with Rs-AFP2, Hs-AFP1, or Dm-AMP1 for 12 h, whereupon fluorescence was measured. Values correspond to one representative experiment of two.