An antifungal peptide from Fagopyrum tataricum seeds

A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14 kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys⁸ to Cys⁶⁵ and Cys⁴⁹ to Cys⁵⁸. The active site of the inhibitor was found to contain an Asp⁶⁶-Arg⁶⁷ bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838 kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6 nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.     

An antifungal peptide from passion fruit (Passiflora edulis) seeds with similarities to 2S albumin proteins

An actual worldwide problem consists of an expressive increase of economic losses and health problems caused by fungi. In order to solve this problem, several studies have been concentrating on the screening of novel plant defence peptides with antifungal activities. These peptides are commonly characterized by having low molecular masses and cationic charges. This present work reports on the purification and characterization of a novel plant peptide of 5.0 kDa, Pe-AFP1, purified from the seeds of passion fruit (Passiflora edulis). Purification was achieved using a Red-Sepharose Cl-6B affinity column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Pe-AFP1 was able of inhibiting the development of the filamentous fungi Trichoderma harzianum, Fusarium oxysporum, and Aspergillus fumigatus with IC50 values of 32, 34, and 40 microg ml(-1), respectively, but not of Rhyzoctonia solani, Paracoccidioides brasiliensis and Candida albicans. This protein was also subjected to automated N-terminal amino acid sequence, showing high degree of similarities to storage 2S albumins, adding a new member to this protein-defence family. The discovery of Pe-AFP1 could contribute, in a near future, to the development of biotechnological products as antifungal drugs and transgenic plants with enhanced resistance to pathogenic fungi.     

A sensitive and specific PCR-based discrimination of split red vetch and lentil seeds

A PCR-based marker technique was developed to discriminate between morphologically similar split seed of vetch (Vicia sativa) and lentil (Lens culinaris subsp.culinaris). Sequence tagged microsatellite site (STMS) markers were more discriminatory than markers produced from the nontranscribed spacer (NTS) region of the 5S ribosomal RNA gene. A sequence characterized amplified region (SCAR) marker, developed from the 5S rRNA NTS region, was sensitive when resolved on agarose. However, the fluorescent-labeled 5S rRNA SCAR marker was unable to discriminate between vetch and lentil, probably because of the low copy number of the marker, and was not visualized on agarose. An STMS primer-pair (PSMPSAD123), developed from field pea, was able to discriminate split red cotyledon vetch from split red cotyledon lentil because it produced specific markers at 563 bp for lentil and 353 and 474 bp for vetch. The vetch-specific STMS marker was conserved among all species of theVicia genus used in this study and was sensitive enough to discriminate both on agarose gels and on polyacrylamide gel-based fluorescent systems. The fluorescent-tagged STMS analysis revealed peaks for vetch and lentil at the expected sizes in admixtures of milled vetch and lentil seeds, and it was sensitive enough to detect one vetch seed in 1999 lentil seeds. The development of PCR-based tests for detecting the level of vetch seed contamination in lentil export seed may provide a method for quality assurance of export lentil seed.     

A novel defensin from the lentil Lens culinaris seeds

A novel 47-residue plant defensin was purified from germinated seeds of the lentil Lens culinaris by ammonium sulfate precipitation, gel filtration, chromatography, and RP-HPLC. The molecular mass (5440.41 Da) and complete amino acid sequence (KTCENLSDSFKGPCIPDGNCNKHCKEKEHLLSGRCRDDFRCWCTRNC)¹ of defensin, termed Lc-def, were determined. Lc-def has eight cysteines forming four disulfide bonds. The total RNA was isolated from lentil germinated seeds, RT-PCR and subsequent cloning were performed, and cDNA was sequenced. A 74-residue predefensin contains a putative signal peptide (27 amino acid) and a mature protein. Lc-def shows high sequence homology with legumes defensins, exhibits an activity against Aspergillus niger, but does not inhibit proteolytic enzymes.     

First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

An antifungal peptide from Phaseolus vulgaris cv. brown kidney bean

A 5.4-kDa antifungal peptide, with an N-terminal sequence highly homologous to defensins and inhibitory activity against Mycosphaerella arachidicola (IC(50)= 3 μM), Setospaeria turcica and Bipolaris maydis, was isolated from the seeds of Phaseolus vulgaris cv. brown kidney bean. The peptide was purified by employing a protocol that entailed adsorption on Affi-gel blue gel and Mono S and finally gel filtration on Superdex 75. The antifungal activity of the peptide against M. arachidicola was stable in the pH range 3-12 and in the temperature range 0°C to 80°C. There was a slight reduction of the antifungal activity at pH 2 and 13, and the activity was indiscernible at pH 0, 1, and 14. The activity at 90°C and 100°C was slightly diminished. Deposition of Congo red at the hyphal tips of M. arachidicola was induced by the peptide indicating inhibition of hyphal growth. The lack of antiproliferative activity of brown kidney bean antifungal peptide toward tumor cells, in contrast to the presence of such activity of other antifungal peptides, indicates that different domains are responsible for the antifungal and antiproliferative activities.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi

Background

The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses.

Methods

In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement.

Results

The purified peptide with a molecular weight of 7 kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP–fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum.

Conclusions and general significance

In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.     

Autolysis of protein bodies in germinating lentil seeds

Protein bodies isolated from lentil (Lens culinaris, Medik) cotyledons exhibit autolytic activity which increases during seed germination. Such autolytic capacity is active across a broad pH range and shows a maximum at pH 6.5. Excision of the embryonic axis reduces autolytic capacity and application during incubation of the seeds without axis of both 6-benzylaminopurine and kinetin is able to replace it. On the other hand, the proteolytic activity in the protein body membrane, is located towards the proteinaceous matrix and is obviously partially responsible for this autolytic activity.     

An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds

A 30-residue antimicrobial peptide Ar-AMP was isolated from the seeds of amaranth Amaranthus retroflexus L. essentially by a single step procedure using reversed-phase HPLC, and its in vitro biological activities were studied. The complete amino acid sequence of Ar-AMP was determined by Edman degradation in combination with mass spectrometric methods. In addition, the cDNA encoding Ar-AMP was obtained and sequenced. The cDNA encodes a precursor protein consisting of the N-terminal putative signal sequence of 25 amino acids, a mature peptide of 30 amino acids and a 34-residue long C-terminal region cleaved during post-translational processing. According to sequence similarity the Ar-AMP belongs to the hevein-like family of antimicrobial peptides with six cysteine residues. In spite of the fact that seeds were collected in 1967 and lost their germination capacity, Ar-AMP retained its biological activities. It effectively inhibited the growth of different fungi tested: Fusarium culmorium (Smith) Sacc., Helminthosporium sativum Pammel., King et Bakke, Alternaria consortiale Fr., and Botrytis cinerea Pers., caused morphological changes in Rhizoctonia solani Kühn at micromolar concentrations and protected barley seedlings from H. sativum infection.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

A novel antifungal analog peptide derived from protaetiamycine

Previously, the 9-mer analog peptides, 9Pbw2 and 9Pbw4, were designed based on a defensin-like peptide, protaetiamycine isolated from Protaetia brevitarsis. In this study, antifungal effects of the analog peptides were investigated. The antifungal susceptibility testing exhibited that 9Pbw4 contained more potent antifungal activities than 9Pbw2. A PI influx assay confirmed the effects of the analog peptides and demonstrated that the peptides exerted their activity by a membrane-active mechanism, in an energy-independent manner. As the noteworthy potency of 9Pbw4, the mechanism(s) of 9Pbw4 were further investigated. The membrane studies, using rhodamine-labeled giant unilamellar vesicle (GUV) and fluorescein isothiocyanate (FITC)-dextran loaded liposome, suggested that the membrane-active mechanism of 9Pbw4 could have originated from the poreforming action and the radii of pores was presumed to be anywhere from 1.8 nm to 3.3 nm. These results were confirmed by 3D-flow cytometric contour-plot analysis. The present study suggests a potential of 9Pbw4 as a novel antifungal peptide.