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.     

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.     

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.     

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata

Defensins are a large family of small, cysteine‐rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C‐terminal pro‐peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt‐thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC₅₀) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5–10 times less effective at killing necrotrophs, but only two‐fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad‐spectrum resistance to pathogens.     

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.     

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.     

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.     

Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.     

Molecular cloning and characterization of Scotch pine defensin 2

A 252 bp cDNA fragment that corresponds to defensin 2 (PsDef2) was amplified from a cDNA library from seven-day plantlets of Pinus sylvestris L. This fragment encodes a protein that consists of 83 amino acid residues. The protein contains an N-terminal signal peptide, which includes 33 amino acid residues. A mature form of defensin 2 of Scotch pine contains a gamma-thionine domain and it is also characterized by specific conservative residues that are common to all plant defensins.     

Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.     

Production of an Arabidopsis halleri foliar defensin in Escherichia coli

Aims:  Production of the recombinant Arabidopsis halleri defensin AhPDF1.1 in a native-like form.
Methods and Results:  Mature AhPDF1.1 cDNA was cloned into pET-28-a(+) and expressed in Escherichia coli Rosetta. After a denaturing extraction, purification by metal affinity chromatography and CNBr cleavage of the His-tag, a protein without extra amino acids at the N-terminus was obtained. An oxidative folding step was then required to renature the protein that was then purified to homogeneity by a C18 HPLC separation. Mass spectroscopy and circular dichroism analyses showed that the recombinant AhPDF1.1 has the expected molecular mass and 3D-structure features of a folded defensin with four-disulfide bridges. The recombinant protein is active against the filamentous fungus Fusarium oxysporum with a minimal inhibitory concentration of 0·6 μmol l⁻¹.
Conclusion:  The proposed purification protocol produces a native-like defensin suitable for tests of new biological roles.
Significance and Impact of the Study:  Plant defensins are essentially known as anti-fungal proteins; however, some unexpected actions on plant cells have recently been discovered. AhPDF1.1, for example, has been shown to confer zinc tolerance. Efficient production of native-like defensins is required to explore the different targets and roles of plant defensins.     

Characterization of a defensin in bark and fruit tissues of peach and antimicrobial activity of a recombinant defensin in the yeast, Pichia pastoris

Herein the cloning and characterization of a defensin gene ( PpDfn1 ) from a cDNA library made from peach ( Prunus persica [L.] Batsch) winter bark tissues is described. A partial clone obtained from the library was extended to full length by 5' Rapid Amplification of cDNA Ends (RACE). The open reading frame of 237 bp codes for a 79 amino acid peptide related to the defensin family of proteins. Sequence comparison of the encoded protein using blast analysis revealed significant homology to defensins from other plant species. RNA gel blot analysis indicated that the gene is seasonally expressed in bark tissues of 1-year-old shoots, and is also expressed in early fruit development. Results of quantitative RT-PCR and protein blot analysis were similar to those of RNA gel blot analyses for the bark tissues. A recombinant version, rDFN1 was expressed in the yeast, Pichia pastoris . It was found that rDFN1 inhibited germination of the fungal pathogens Penicillium expansum and Botrytis cinerea , but not the Gram-negative bacterium Erwinia amylovora . The potential physiological role of PpDFN1 and its antimicrobial properties are discussed.     

Structural characterization of plant defensin protein superfamily

Plant defensins represent a major innate immune protein superfamily with strong inhibitory effects on infectious diseases of humans, antifungal/antibacterial activities, proteinase and insect amylase inhibitory activities. They are generally defined by their conserved cysteine scaffold with α-helix and triple strand anti parallel β-sheet connected to the scaffold. With the genome of more plant species being fully sequenced, significant information about newly sequenced defensin proteins has been revealed. In this paper, we identify members of defensin protein families across plant species and use protein-modeling-based structural reconstitution to reveal specific three dimensional hidden features of plant defensins mediating defense responses and other interesting biological activities in plants. Our data revealed that plant defensins are structurally similar to their insect counterparts despite the low amino acid sequence similarity between these two organisms. The molecular and structural relationship among plant defensins and defensins from other species is discussed.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

Expression of murine poly(A) binding protein II gene in testis

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three-dimensional structure highly similar to that of scorpion toxins. A full-length cDNA encoding an insect defensin-like peptide was isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor, the overall organization of which is similar to that of insect defensins, consists of 61 amino acid residues with a putative signal peptide of 15 residues, a propeptide of 7 residues, and a mature peptide of 39 residues (named BmTXKS2). The positions of six cysteines and a conserved glycine in mature BmTXKS2 are the same as those in LqDef, the first defensin found in scorpions, which suggests these peptides should present a similar cysteine-stabilized alphabetamotif. Phylogenetic analysis further shows that the structure of BmTXKS2 is closer to that of ancient defensins (e.g., LqDef and AaDef, two insect defensins present in the scorpion hemolymph) than to scorpion toxins.     

Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.     

Cloning and characterization of a cDNA encoding a cowpea seed defensin and analysis of its expression

We have previously isolated a cowpea seed defensin with potent antifungal activity. Here we report the cloning of its cDNA obtained from RT-PCR. The sequence of the deduced protein VUDEF exhibited homology to plant defensins. Northern analysis revealed that VUDEF mRNA is accumulated during cowpea seed development and its level increases in seedling tissues after exposure to fungal pathogen and cold.