Diversity of Cationic Antimicrobial Peptides in Black Cumin (Nigella sativa L.) Seeds.

Black cumin (Nigella sativa L.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search in N. sativa seeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lacking N. sativa genome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in "next-generation" biopesticides development for agricultural use.

Oak bark (Quercus sp. cortex) protects plants through the inhibition of quorum sensing mediated virulence of Pectobacterium carotovorum.

Bacterial intercellular communication mediated by small diffusible molecules, known as quorum sensing (QS), is a common mechanism for regulating bacterial colonisation strategies and survival. Influence on QS by plant-derived molecules is proposed as a strategy for combating phytopathogens by modulating their virulence. This work builds upon other studies that have revealed plant-derived QS inhibitors extracted from oak bark (Quercus sp.). It was found that co-incubation of Pectobacterium carotovorum VKM-B-1247 with oak bark extract (OBE) reduced the production of acyl-HSL. This was accompanied by a dose-dependent decrease in the bacterial cellulolytic and protease activity. At the transcriptomic level, the OBE treatment suppressed the main QS-related genes expR/expI. Potato tubers pre-treated with OBE showed resistance to a manifestation of soft-rot symptoms. Analysis of the component composition of the OBE identified several biologically active molecules, such as n-hexadecanoic acid, 2,6-di-tert-butyl-4-methylphenol, butylated hydroxytoluene (BHT), gamma-sitosterol, lupeol, and others. Molecular docking of the binding energy between identified molecules and homology models of LuxR-LuxI type proteins allow to identify potential inhibitors. Collectively, obtained results figure out great potential of widely distributed oak-derived plant material for bacterial control during storage of potato.

Peptide Extracts from Seven Medicinal Plants Discovered to Inhibit Oomycete Phytophthora infestans, a Causative Agent of Potato Late Blight Disease.

We report the inhibitory effect of peptide extracts obtained from seven medicinal plants against a causative agent of late blight disease Phytophthora infestans. We find that all the extracts possess inhibitory activity toward the zoospores output, zoosporangium germination, and the development of P. infestans on potato disc tubers at different quantitative levels. Based on the biological effects detected, an extract of common horsetail (Equisetum arvense) biomass is recognized as the most effective and is selected for further structural analysis. We perform a combination of amino acid analysis and MALDI-TOF mass spectrometry, which reveal the presence of Asn/Asp- and Gln/Glu-rich short peptides with molecular masses in the range of 500-900 Da and not exceeding 1500 Da as the maximum. Analytical anion-exchange HPLC is successfully applied for separation of the peptide extract from common horsetail (E. arvense). We collect nine dominant components that are combined in two groups with differences in retention times. The N-terminal amino acid sequence of the prevalent compounds after analytical ion-exchange HPLC allows us to identify them as peptide fragments of functionally active proteins associated with photosynthesis, aquatic transport, and chitin binding. The anti-oomycete effects may be associated with the conversion of ribulose-1,5-bisphosphate carboxylase/oxygenase to produce a number of biologically active anionic peptides with possible regulatory functions. These data inform our knowledge regarding biologically active peptide fragments; they are the components of programmed or induced proteolysis of plant proteins and can realize secondary antimicrobial functions.

Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.

Antibacterial and quorum sensing regulatory activities of some traditional Eastern-European medicinal plants.

The objective of this study was to screen extracts of twenty Eastern European medicinal plants, using wild-type and reporter Chromobacterium violaceum bioassays, for novel components that target bacterial cells and their quorum sensing (QS) communication systems. Three types of activity and their combinations were revealed: (i) direct antimicrobial growth-inhibitory activity, (ii) non-specific and specific pro-QS activities, (iii) anti-QS activity. Among seven plant extracts showing direct growth-inhibitory activity, the strongest effect was shown by Arctostaphylos uva-ursi (bearberry) leaves. Many plants stimulated violacein production by wild-type C. violaceum ATCC 31532 in a non-specific manner, and only the herb Bidens tripartita (three-lobe beggarticks) contained compounds that mimic acyl-homoserine lactone and operated as a QS agonist. Anti-QS activity was found in eleven plants including Quercus robur (oak) cortex, Betula verrucosa (birch) buds and Eucalyptus viminalis (Manna Gum) leaves. Subsequent statistical analysis showed differences between antimicrobial and anti-QS activities, whereas both activities were defined by phylogenetic position of medical resource plant. Finally, extract from Quercus robur cortex revealed at least two fractions, showing different anti-QS mechanisms. These data confirm that multicomponent anti-infectious mechanisms are used by plants, which may be useful for drug development.

A novel hairpin-like antimicrobial peptide from barnyard grass (Echinochloa crusgalli L.) seeds: Structure-functional and molecular-genetics characterization.

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.

A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers.

A novel peptide named ToAMP4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, and RP-HPLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids, and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 does not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4, it was successfully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4-mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and functional diversity of AMPs in plants.