A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced ...and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.
This study presents purification, activity characterization, and 1H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a ...disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.
A novel peptide named SmAMP3 was isolated from leaves of common chickweed (Stellaria media L.) by a combination of acidic extraction and a single-step reversed-phase HPLC and sequenced. The peptide ...is basic and cysteine-rich, consists of 35 amino acids, and contains three disulphide bridges. Homology search revealed that SmAMP3 belongs to the family of hevein-like antimicrobial peptides carrying a conserved chitin-binding site. Efficient binding of chitin by SmAMP3 was proved by in vitro assays. Molecular modeling confirmed conservation of the chitin-binding module in SmAMP3 locating the variable amino acid residues to the solvent-exposed loops of the molecule. The peptide exhibits potent antifungal activity against important plant pathogens in the micromolar range, although it is devoid of antibacterial activity at concentrations below 10 μM. As judged by chromatographic behavior and mass spectrometric data, the peptide is constitutively expressed in above-ground organs and seeds of S. media plants, thus representing an important player in the preformed branch of the plant immune system.
•A novel peptide named SmAMP3 was isolated from leaves of common chickweed (Stellaria media L.).•SmAMP3 belongs to the family of hevein-like AMPs carrying a conserved chitin-binding site.•The peptide exhibits potent antifungal activity against plant pathogens in the micromolar range.•This peptide is constitutively expressed in above-ground organs and seeds of S. media plants.
Two novel antifungal hevein-like peptides, SmAMP1.1a and SmAMP2.2a, were previously isolated from seeds of Stellaria media. It has been established that these peptides accumulate in this weed as a ...result of proteolysis of two propeptides, pro-SmAMP1 and pro-SmAMP2. The primary structure of these propeptides is unique; in addition to having a signal peptide and negatively charged C-terminus, each of these structures consists of two hevein-like peptides of different length separated by a space rather than a single peptide. In this work, we demonstrated that the expression of the pro-SmAMP1 and pro-SmAMP2 genes was tissue-specific and increased substantially under exposure to fungal infection. To elucidate whether S. media has any advantages in defending against phytopathogens due to its unusual structure of pro-SmAMP1 and pro-SmAMP2, on the basis of the pro-SmAMP1 gene, we created three genetic constructs. Arabidopsis and tobacco plants were subsequently transformed with these constructs. Transgenic plants bearing the full-length pro-SmAMP1 gene exhibited the best resistance to the phytopathogens Bipolaris sorokiniana and Thielaviopsis basicola. The resistance of S. media plants to phytopathogenic fungi was likely due to the fungal-inducible expression of pro-SmAMP1 and pro-SmAMP2 genes, and due to the specific features of the primary structure of the corresponding propeptides. As a result of the processing of these propeptides, two different antimicrobial peptides were released simultaneously. Based on our results, we conclude that the genes for antimicrobial peptides from S. media may be promising genetic tools for the improvement of plant resistance to fungal diseases.
A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk‐AMP‐X1 and Tk‐AMP‐X2, were completely sequenced and ...shown to belong to the α‐hairpinin structural family of plant peptides with a characteristic C1XXXC2‐X(n)‐C3XXXC4 motif. The peptides inhibit the spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk‐AMP‐X peptides. They code for precursor proteins of unusual multimodular structure, consisting of a signal peptide, several α‐hairpinin (4‐Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C‐terminal prodomain. Three types of precursor proteins, with five, six or seven 4‐Cys peptide modules, were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk‐AMP‐X precursors have no introns in the protein‐coding regions and are upregulated by fungal pathogens and abiotic stress, providing conclusive evidence for their role in stress response. A combined PCR‐based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically‐related Triticum and Aegilops species, including polyploid wheat genome donors. Association of the Tk‐AMP‐X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk‐AMP‐X‐related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize.
Database
Nucleotide sequence data have been deposited in the EMBL database under accession numbers: HF562347, HF562348, HF562349, HF562350, HF562351, HF562352, HF562353, HF562354, HF562355, HF562356, HF562357, HF562358, HF562359, HF562360, HF562361, HF562362, HF562363 and HF562364.
Genes encoded new wheat peptides code for precursor proteins of unusual multimodular structure consisting of a signal peptide, several α‐hairpinin (4‐Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C‐terminal prodomain. Three types of precursor proteins, with five, six or seven 4‐Cys peptide modules were found.
Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in ...pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C¹X₃C²XₙC³X₃C⁴), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C¹–C⁴ and C²–C³. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.
A novel plant hairpin-like defense polypeptide named EcAMP3 was isolated from latent barnyard grass (Echinochloa crusgalli L.) seeds. The native peptide and its recombinant analogue were ...characterized. EcAMP3 displays antifungal and antibacterial activity in vitro. The gene family encoding EcAMPs precursor protein was also characterized; the genes and pseudogenes of this family show 97–100% homology. Every member of EcAMPs precursor family contains seven identical cysteine motifs: C1XXXC2(11–13)C3XXXC4. One of those motifs corresponds to the isolated peptide. EcAMP3 is the first member of the plant hairpin-like peptide family that inhibits the growth of phytopathogenic bacteria. Obtained results can explain the nature of the complex resistance of barnyard grass to a variety of pathogenic microorganisms.
•The polypeptide EcAMP3 was isolated from latent barnyard grass seeds.•The recombinant EcAMP3 shown antifungal and antibacterial activities in vitro.•The full-length gene encoding AMPs precursor protein was established.
Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of ...the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC50≤1μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3′-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.
► Two highly homologous defensins from common chickweed were isolated and sequenced. ► Defensins exhibit strong antifungal activity against important plant pathogens. ► Three defensin-encoding genes were identified, two of which are expressed in seeds. ► Defensins precursors consist of a signal peptide and a mature peptide domains. ► The genomic DNA sequences contain an intron in the signal peptide region.
Diversity of wheat anti-microbial peptides Egorov, Tsezi A.; Odintsova, Tatyana I.; Pukhalsky, Vitaliy A. ...
Peptides (New York, N.Y. : 1980),
11/2005, Letnik:
26, Številka:
11
Journal Article
Recenzirano
From seeds of
Triticum kiharae Dorof. et Migusch., 24 novel anti-microbial peptides were isolated and characterized by a combination of three-step HPLC (affinity, size-exclusion and reversed-phase) ...with matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Edman degradation. Based on sequence similarity and cysteine motifs, partially sequenced peptides were assigned to 7 families: defensins, thionins, lipid-transfer proteins, hevein-like peptides, knottin-like peptides, glycine-rich peptides, and MBP-1 homologs. A novel subfamily of defensins consisting of 6 peptides and a new family of glycine-rich (8 peptides with different repeat motifs) were identified. Three 6-cysteine knottin-like peptides represented by N- and C-terminally truncated variants revealed no sequence homology to any known plant anti-microbial peptides. A new 8-cysteine hevein-like peptide and three 4-cysteine peptides homologous to MBP-1 from maize were isolated. This is the first communication on the occurrence of nearly all families of plant anti-microbial peptides in a single species.
From seeds of
Nigella sativa L. (Ranunculaceae), an endemic plant of Uzbekistan, two novel defensins named Ns-D1 and Ns-D2, were isolated and sequenced. The peptides differ by a single amino acid ...residue and show high sequence similarity to
Raphanus sativus L. defensins Rs-AFP1 and Rs-AFP2. The Ns-D1 and Ns-D2 defensins display strong although divergent antifungal activity towards a number of phytopathogenic fungi. High antifungal activity of
N. sativa defensins makes them promising candidates for engineering pathogen-resistant plants.
► In this study we isolated and characterized two novel antimicrobial peptides from seeds of
Nigella sativa (blackseed), an endemic plant of Uzbekistan. ► We showed these peptides differed by a single amino acid residue and show high sequence similarity to
Raphanus sativus L. defensins. ► The blackseed defensins display strong although divergent antifungal activity towards a number of phytopathogenic fungi and oomycete-
Phytophthora infestans. ► In this study we conclude high antifungal activity of
Nigella sativa defensins makes them promising candidates for engineering pathogen-resistant plants.
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