Dimerization of plant defensin NaD1 enhances its antifungal activity

The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys⁴ as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys⁴ that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys⁴ mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.     

The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a “cationic grip” conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.     

Prokaryotic expression of a constitutively expressed <Emphasis Type="Italic">Tephrosia villosa</Emphasis> defensin and its potent antifungal activity

Plant defensins are small, highly stable, cysteine-rich antimicrobial peptides produced by the plants for inhibiting a broad-spectrum of microbial pathogens. Some of the well-characterized plant defensins exhibit potent antifungal activity on certain pathogenic fungal species only. We characterized a defensin, TvD1 from a weedy leguminous herb, Tephrosia villosa. The open reading frame of the cDNA was 228 bp, which codes for a peptide with 75 amino acids. Expression analyses indicated that this defensin is expressed constitutively in T. villosa with leaf, stem, root, and seed showing almost similar levels of high expression. The recombinant peptide (rTvD1), expressed in the Escherichia coli expression system, exhibited potent in vitro antifungal activity against several filamentous soil-borne fungal pathogens. The purified peptide also showed significant inhibition of root elongation in Arabidopsis seedlings, subsequently affecting the extension of growing root hairs indicating that it has the potential to disturb the plant growth and development.     

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.     

The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.     

Fungicidal and Cytotoxic Activity of a Capsicum chinense Defensin Expressed by Endothelial Cells

Plant defensins are antimicrobial peptides that exhibit mainly antifungal activity against a broad range of plant fungal pathogens. However, their actions against Candida albicans have not been extensively studied. The mRNA for γ-thionin, a defensin from Capsicum chinense, has been expressed in bovine endothelial cells. The conditioned medium of these cells showed antifungal activity on germ tube formation (60–70% of inhibition) and on the viability of C. albicans (70–80% of inhibition). Additionally, C. albicans was not able to penetrate transfected cells. Conditioned medium from these cells also inhibited the viability (80%) of the human tumor cell line, HeLa.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Characterization of a Heterobasidion irregulare endo‐rhamnogalacturonase that mediate growth on pectin

Heterobasidion irregulare is one of five Heterobasidion annosum sensu lato (s.l.) species, which are destructive pathogens in boreal and temperate forests of the northern hemisphere that causes root and butt rot in conifer. A gene encoding endo‐rhamnogalacturonase (HIRHG), which belongs to the glycoside hydrolase family 28 (GH28), was found in a quantitative trait loci (QTL) region for virulence in Heterobasidion. In this study, we showed that HIRHG is highly upregulated during necrotrophic infection of Norway spruce compared with growth in liquid culture and that the HIRHG encoded protein is produced during fungal growth on complex carbon sources. Phylogenetic analysis of endo‐rhamnogalacturonases revealed that rhamnogalacturonase genes have been lost in most of the biotrophic and hemibiotrophic plant pathogens investigated but were common in necrotrophic pathogens and saprophytic fungi. Heterologous expression of the HIRHG gene in the hemibiotrophic fungus Magnaporthe oryzae increased its capacity to grow on pectin; however, the transformed M. oryzae isolates showed significant less infection of rice leaves compared to the wild type.     

Incidence of Fungal Necrotrophic and Biotrophic Pathogens in Pioneer and Shade‐tolerant Tropical Rain Forest Trees

This study assessed the hypothesis that plant life history traits determine the incidence of fungal biotrophic and necrotrophic pathogens in pioneer vs. shade‐tolerant tropical plant species. Considering that pioneer species mainly invest in induced defenses, we expected a negative relationship between the incidence of biotrophic and necrotrophic pathogens; in contrast, as shade‐tolerant species invest heavily in constitutive defenses, we expected to find no correlation between the incidence of biotrophic and necrotrophic pathogens. These ideas were evaluated by assessing standing levels of fungal damage in a set of pioneer and shade‐tolerant species from the Lacandona tropical rain forest (Mexico). The results showed that among pioneer plant species, leaves with biotrophic lesions were between 34 and 44 percent more abundant than those with necrotic lesions. In contrast, among shade‐tolerant species, the proportions of leaves with necrotic lesions were 17–23 percent higher than those of leaves with injuries caused by biotrophic pathogens. Our study suggests that tropical tree species might present different defense strategies depending on the life‐style of the pathogens that attack them, and the life history strategy of the attacked host plant species. Thus, the host constitutive and induced defenses, as well as the mechanisms used by different types of pathogens to circumvent those defenses maybe responsible for the patterns of attack observed in perennial tropical plants. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

Specific domains of plant defensins differentially disrupt colony initiation,cell fusion and calcium homeostasis in Neurospora crassa

MsDef1 and MtDef4 from Medicago spp. are small cysteine‐rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ‐core motif (GXCX_3–9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ‐core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ‐core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca²⁺ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca²⁺ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca²⁺ were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ‐core motifs play important roles in their different modes of antifungal action.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.     

Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.     

Ha-DEF1, a sunflower defensin,induces cell death in <Emphasis Type="Italic">Orobanche</Emphasis> parasitic plants

Plant defensins are small basic peptides of 5–10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.     

Antifungal activity of the ribosome‐inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mould Penicillium digitatum

The ribosome‐inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter‐ and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N‐glycosylase activity on the sarcin–ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C‐terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α‐helix and a β‐hairpin, similar to the γ‐core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.     

Tryptophan‐derived secondary metabolites in Arabidopsis thaliana confer non‐host resistance to necrotrophic Plectosphaerella cucumerina fungi

A defence pathway contributing to non‐host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)‐derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate‐limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non‐adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp‐derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non‐adapted P. cucumerina isolates, and super‐susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp‐derived secondary metabolites in limiting the growth of both non‐adapted and adapted necrotrophic fungi. However, 4‐methoxy‐indol‐3‐ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non‐adapted necrotrophs. This contrasts with a major role of these two trp‐derived phytochemicals in limiting invasive growth of non‐adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp‐derived metabolites in resistance responses to non‐adapted necrotrophic P. cucumerina. Impaired defence to non‐adapted P. cucumerina, but not to the non‐adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non‐host resistance to necrotrophic and biotrophic pathogens.     

Insights into the role of jasmonic acid-mediated defenses against necrotrophic and biotrophic fungal pathogens

Jasmonic acid (JA) is a natural hormone regulator involved in development,responses against wounding and pathogen attack.Upon perception of pathogens,JA is synthesized and mediates a signaling cascade ...     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.