Antifungal Plant Defensins Research Articles

The antifungal plant defensin AhPDF1.1b is a beneficial factor involved in adaptive response to zinc overload when it is expressed in yeast cells.

Antimicrobial peptides represent an expanding family of peptides involved in innate immunity of many living organisms. They show an amazing diversity in their sequence, structure, and mechanism of action. Among them, plant defensins are renowned for their antifungal activity but various side activities have also been described. Usually, a new biological role is reported along with the discovery of a new defensin and it is thus not clear if this multifunctionality exists at the family level or at the peptide level. We previously showed that the plant defensin AhPDF1.1b exhibits an unexpected role by conferring zinc tolerance to yeast and plant cells. In this paper, we further explored this activity using different yeast genetic backgrounds: especially the zrc1 mutant and an UPRE-GFP reporter yeast strain. We showed that AhPDF1.1b interferes with adaptive cell response in the endoplasmic reticulum to confer cellular zinc tolerance. We thus highlighted that, depending on its cellular localization, AhPDF1.1b exerts quite separate activities: when it is applied exogenously, it is a toxin against fungal and also root cells, but when it is expressed in yeast cells, it is a peptide that modulates the cellular adaptive response to zinc overload.

Antifungal plant defensins: mechanisms of action and production.

Plant defensins are small, cysteine-rich peptides that possess biological activity towards a broad range of organisms. Their activity is primarily directed against fungi, but bactericidal and insecticidal actions have also been reported. The mode of action of various antifungal plant defensins has been studied extensively during the last decades and several of their fungal targets have been identified to date. This review summarizes the mechanism of action of well-characterized antifungal plant defensins, including RsAFP2, MsDef1, MtDef4, NaD1 and Psd1, and points out the variety by which antifungal plant defensins affect microbial cell viability. Furthermore, this review summarizes production routes for plant defensins, either via heterologous expression or chemical synthesis. As plant defensins are generally considered non-toxic for plant and mammalian cells, they are regarded as attractive candidates for further development into novel antimicrobial agents.

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

Structure-Activity Determinants in Antifungal Plant Defensins MsDef1 and MtDef4 with Different Modes of Action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX3–9C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.

The Antifungal Plant Defensin HsAFP1 from Heuchera Sanguinea Induces Apoptosis in Candida Albicans

Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin.

The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.

Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins inFusarium graminearum

The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The DeltaFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the DeltaFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No DeltaFgmt1DeltaFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The DeltaFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the DeltaFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The DeltaFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the DeltaFgmt2 mutant was less virulent on different host plants tested than the previously characterized DeltaFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the DeltaFgmt1 and DeltaFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani

Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice.

The Plant Defensin, NaD1, Enters the Cytoplasm of Fusarium Oxysporum Hyphae

The plant defensin, NaD1, from the flowers of Nicotiana alata displays potent antifungal activity against a variety of agronomically important filamentous fungi including Fusarium oxysporum f. sp. vasinfectum (Fov). To understand the mechanism of this antifungal activity, the effect of NaD1 on Fov fungal membranes and the location of NaD1 in treated hyphae was examined using various fluorescence techniques. NaD1 permeabilized fungal plasma membranes via the formation of an aperture with an internal diameter of between 14 and 22A. NaD1 bound to the cell walls of all treated hyphae and entered several hyphae, resulting in granulation of the cytoplasm and cell death. These results suggest that the activity of antifungal plant defensins may not be restricted to the hyphal membrane and that they enter cells and affect intracellular targets.

Two mitogen-activated protein kinase signalling cascades mediate basal resistance to antifungal plant defensins in Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of Fusarium graminearum, which causes head blight disease in cereals. In order to determine the signalling cascades that are modulated by these defensins, we have isolated several insertional mutants of F. graminearum that exhibit hypersensitivity to MsDef1, but not to MtDef4. The molecular characterization of two of these mutants, designated enhanced sensitivity to defensin (esd), has revealed that the Mgv1 and Gpmk1 MAP kinase signalling cascades play a major role in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4. The Hog1 MAP kinase signalling cascade, which is responsible for adaptation of this fungus to hyperosmotic stress, does not participate in the fungal response to these defensins. Significantly, the esd mutants also exhibit hypersensitivity to other tested defensins and are highly compromised in their pathogenesis on wheat heads and tomato fruits. The studies reported here for the first time implicate two MAP kinase signalling cascades in a plant defensin-mediated alteration of fungal growth. Based on our findings, we propose that specific MAP kinase signalling cascades are essential for protection of a fungal pathogen from the antimicrobial proteins of its host plant.

Level of M(IP) 2C sphingolipid affects plant defensin sensitivity, oxidative stress resistance and chronological life-span in yeast

The antifungal plant defensin DmAMP1 interacts with fungal sphingolipids of mannosyldiinositolphosphorylceramide (M(IP) 2C) class. We screened a Saccharomyces cerevisiae transposon (Tn) mutant library against DmAMP1 and identified one DmAMP1-resistant mutant with the Tn inserted in the M(IP) 2C biosynthesis gene IPT1 (DmTn11) and one DmAMP1-hypersensitive mutant with the Tn inserted in rDNA (HsTnII). However, tetrad analysis pointed to HsTnII as a spontaneous mutant. Apparently, membranes of DmTn11 lack M(IP) 2C, whereas membranes of HsTnII have increased M(IP) 2C levels. In addition, DmTn11 and HsTnII are characterized by increased and reduced oxidative stress resistance/chronological life-span (CL), respectively. A putative involvement of M(IP) 2C in oxidative stress and CL in yeast is discussed.

Detection of Fungal Infections Using Radiolabeled Antifungal Agents

The outcome of antifungal therapy depends on the progression of the infection at the start of therapy. Unfortunately, most patients are diagnosed once the fungal infection has progressed considerably as a result of the non-specific clinical signs of fungal infections in immunocompromised patients and the poor sensitivity of current mycological diagnostic tests. This review will highlight current fungal diagnostic techniques and will focus on scintigraphic methods for the specific detection of fungal infections in mice. For this purpose, antifungal components (e.g. fluconazole and antifungal peptides) are radiolabeled e.g. with technetium-99m ((99m)Tc) and their in vivo distribution is monitored in infected mice. It has been demonstrated that (99m)Tc-fluconazole is an excellent tracer to detect Candida albicans infections in mice as it distinguishes these infections from bacterial infections and sterile inflammations. However, this radiopharmaceutical only poorly detects infections with Aspergillus fumigatus in mice. (99m)Tc-peptides derived from antifungal peptides/proteins, such as human ubiquicidin and lactoferrin, can distinguish C. albicans and A. fumigatus infections from sterile inflammations, but not from bacterial infections, in mice. Furthermore, the efficacy of fluconazole in C. albicans-infected mice could be successfully monitored using (99m)Tc-ubiquicidin. In conclusion, neither (99m)Tc-fluconazole nor the (99m)Tc-peptides tested are optimal tracers for fungal infections. Nonetheless, since early initiation of antifungal therapy for candidemia reduces its high mortality rate, a positive result with (99m)Tc-fluconazole scintigraphy is of clinical relevance. Finally, the possibility that other (radiolabeled) antifungal agents, e.g. voriconazole, caspofungin, antifungal plant or insect defensins, can be useful for detection of fungal infections should be considered.

Neutral glycolipids of the filamentous fungus Neurospora crassa:altered expression in plant defensin-resistant mutants

To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705-1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as beta-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta3-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-beta-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.

SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis

The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP) 2C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP) 2C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in β-1,6-glucan biosynthesis. We demonstrate that a Δ skn1 mutant lacks M(IP) 2C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP) 2C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae.

Interactions of antifungal plant defensins with fungal membrane components

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.

DmAMP1, an antifungal plant defensin from dahlia ( Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae

DmAMP1, an antifungal plant defensin from Dahlia merckii, was shown previously to require the presence of sphingolipids for fungicidal action against Saccharomyces cerevisiae. Sphingolipids may stabilize glycosylphosphatidylinositol (GPI)-anchored proteins, which interact with DmAMP1, or they may directly serve as DmAMP1 binding sites. In the present study, we demonstrate that S. cerevisiae disruptants in GPI-anchored proteins showed small or no increased resistance towards DmAMP1 indicating no involvement of these proteins in DmAMP1 action. Further, studies using an enzyme-linked immunosorbent assay (ELISA)-based binding assay revealed that DmAMP1 interacts directly with sphingolipids isolated from S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol. Therefore, DmAMP1 antifungal action involving membrane interaction with sphingolipids and ergosterol is proposed.

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.

Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities.

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.