Figure 1.
Highly conserved structural motifs and homology-based three-dimensional structures of plant defensins MsDef1 and MtDef4.
(A) Amino acid sequences of MsDef1 and MtDef4 showing highly conserved α-core and γ-core motifs. The consensus α-core (pink color) and γ-core (orange color) sequences were created using 16 homologs of MsDef1 and 28 homologs of MtDef4. For both sequences, conserved amino acids of the core regions are listed underneath the respective motifs with highly conserved amino acids on the top followed by less conserved ones. α-helix and β-strands are underlined. (B) Homology-based models of MsDef1 and MtDef4. The α-core motif is indicated in pink color; the γ-core motif is indicated in orange color and the four disulfide bridges are indicated in yellow color. Models were developed using the I-TASSER website for protein structure and function predictions as described in Materials and Methods.
Figure 2.
MsDef1 γ-core substitution variant, MsDef1-γ4, exhibits antifungal activity similar to that of MtDef4 against the wild-type F. graminearum PH-1 and the MsDef1-resistant mutant ΔFggcs1 lacking glucosylceramide.
(A) Images showing the inhibition of conidial germination and hyphal growth at different concentrations of MsDef1, MtDef4 and the variant MsDef1-γ4. Images were taken after 16 hours of incubation of PH-1 conidia with defensins. Bar = 50 µm. Hyperbranching of hyphae in the presence of MsDef1 is indicated with black arrow. (B) Quantitative assessment of the antifungal activity of MsDef1, MtDef4 and the variant MsDef1-γ4 against PH-1 strain. (C) Images showing the inhibition of conidial germination and hyphal growth of MsDef1-resistant mutant ΔFggcs1 lacking glucosylceramaide at different concentrations of MsDef1 and the variant MsDef1-γ4. Images were taken after 16 hours of incubation of ΔFggcs1 conidia with defensins. Bar = 50 µm. (D) Quantitative measurement of the antifungal activity of MsDef1 and its γ-core substitution variant MsDef1-γ4 against ΔFggcs1 strain. Values are means of three replications. Error bars indicate standard deviations.
Table 1.
List of amino acid sequences, net charge and growth inhibitory concentrations against F. graminearum PH-1.
Figure 3.
MsDef1 induces significant changes in the growing hyphal tip morphology of F. graminearum, but MtDef4 and MsDef1-γ4 do not.
Images showing the hyphal tips of untreated fungal hyphae and those of fungal hyphae treated with IC100 concentrations of MsDef1 (12 µM), MtDef4 (1.5 µM) and the variant MsDef1-γ4 (3 µM). Hyphal tips of fungal hyphae treated with MtDef4 and MsDef1-γ4 appear similar to those of untreated fungal hyphae, whereas hyphal tips of fungal hyphae treated with MsDef1 appear swollen. Black arrows point to the hyphal tips. The division of hyphal tips in the presence of only MsDef1 is indicated with gray arrows. Bar = 20 µm. All images were taken between 7–8 hours after incubation of PH-1 germlings with defensins.
Figure 4.
MsDef1, MtDef4 and the MsDef1 variants, MsDef1-γ4 and MsDef1-R38Q, permeabilize the plasma membrane of F. graminearum, but with different intensities.
(A) Quantitative measurement of Sytox Green (SG) uptake by F. graminearum hyphae treated with MsDef1, MsDef1-γ4, MtDef4, and MsDef1-R38Q (488 excitation; 540 emission (530 cut-off)) for 5 hours, (B) Fluorescence image of SG binding to the nuclei of F. graminearum following treatment with MtDef4. Bar = 20 µm.
Figure 5.
GMA1-C and GMA4-C peptides containing the γ-core sequences plus the carboxy-terminal 6 amino acids of MsDef1 and MtDef4 exhibit antifungal activity and induce plasma membrane permeabilization in F. graminearum.
(A) Quantitative measurement of fungal growth inhibition caused by treatment with GMA1-C and GMA4-C. Values are means of at least three replications. Error bars indicate standard deviations, (B) Quantitative measurement of SG uptake by F. graminearum PH-1 hyphae treated with GMA1-C or GMA4-C.
Figure 6.
GMA4 peptide containing only the γ-core sequence of MtDef4 exhibits antifungal activity, but the GMA1 peptide containing only the γ-core sequence of MsDef1 and the α-core peptides, ALP1 & ALP4, of both defensins do not.
(A) GMA4 shows potent antifungal activity at concentration as low as 6 µM, while GMA1 as well as ALP1 and ALP4 show no antifungal activity even at 48 µM. (B) Quantitative measurement of fungal growth inhibition caused by treatment with GMA1, GMA4, ALP1 and ALP4. Values are means of at least three replications. Error bars indicate standard deviations.
Figure 7.
The hexapeptide RGFRRR of the MtDef4 γ-core motif alone is capable of inhibiting fungal growth, but its activity depends on the presence of F37 and R38 residues.
(A) Images showing the inhibition of conidial germination and hyphal growth at different concentrations of GMA1-L with a sequence RDDFR and GMA4-L with a sequence RGFRRR. Images were taken after 16 hours of incubation of PH-1 conidia with peptides. Bar = 50 µm. Note the potent in vitro antifungal activity of GMA4-L and complete lack of antifungal activity of GMA1-L, (B) Morphology of inhibition of conidial germination and hyphal growth inhibition at different concentrations of GMA4-L1 and GMA4-L2. Values are means of at least three replications. Error bars indicate standard deviations. (C) Quantitative measurement of the inhibition of fungal growth and (D) SG uptake by the peptides GMA1-L, GMA4-L and its variants GMA4-L1 and GMA4-L2.
Figure 8.
MsDef1, MtDef4 and peptides GMA1-C and GMA4-C inhibit the growth of another ascomycetous fungal pathogen F. verticillioides M3125 in vitro.
(A) Images showing the inhibition of F. verticillioides conidial germination and hyphal growth and (B) Quantitative measurement of the inhibition of fungal growth at different concentrations of MsDef1, MtDef4 and the peptides, GMA1-C and GMA4-C. Images were taken after 16 hours of incubation of M3125 microconidia with proteins/peptides. Bar = 50 µm. Values are means of at least three replications. Error bars indicate standard deviations.
Figure 9.
Antifungal activity of MsDef1, MtDef4 and derived peptides against an ascomycetous pathogen Aspergillus flavus.
(A) Images showing the inhibition of A. flavus conidial germination and hyphal growth and (B) Quantitative measurement of the inhibition of fungal growth at different concentrations of MsDef1, MtDef4 and the peptides, GMA1-C and GMA4-C. Images were taken after 16 hours of incubation of A. flavus conidiospores with proteins/peptides. Bar = 50 µm. Values are means of at least three replications. Error bars indicate standard deviations.