Cysmotif Searcher Pipeline for Antimicrobial Peptide Identification in Plant Transcriptomes.

In this paper, we present the new Cysmotif searcher pipeline for identification of various antimicrobial peptides (AMPs), the most important components of innate immunity, in plant transcriptomes. Cysmotif searcher reveals and classifies short cysteine-rich amino acid sequences containing an open reading frame and a signal peptide cleavage site. Due to the combination of various search methods, Cysmotif searcher allows to obtain the most complete repertoire of AMPs for one or more transcriptomes in a short amount of time. The pipeline performance is estimated on the model plant Arabidopsis thaliana and nine other plants, including cultivated and wild species. The obtained results are compared to the existing annotation (A. thaliana) and results of conventional homology search (other plants). The comparison is carried out for known families of plant AMPs and newly discovered peptides that could not be assigned to existing families. The applicability of Cysmotif searcher in detecting new AMPs is discussed, and some practical recommendations on the pipeline usage for end users are given. The Cysmotif searcher pipeline is free for academic use and can be downloaded from Github (http://github.com/fallandar/cysmotifsearcher).

Hevein-Like Antimicrobial Peptides of Plants.

Plant antimicrobial peptides represent one of the evolutionarily oldest innate immunity components providing the first line of host defense to pathogen attacks. This review is dedicated to a small, currently actively studied family of hevein-like peptides that can be found in various monocot and dicot plants. The review thoroughly describes all known peptides belonging to this family including data on their structures, functions, and antimicrobial activity. The main features allowing to assign these peptides to a separate family are given, and the specific characteristics of each peptide are described. Further, the mode of action for hevein-like peptides, their role in plant immune system, and the applications of these molecules in biotechnology and medicine are considered.

[Genes encoding hevein-like antimicrobial peptides WAMPs: Expression in response to phytohormones and environmental factors].

We investigated the role of genes of hevein-like antimicrobial peptides of the WAMP family in the protection of wheat plants against biotic and abiotic stress. The semiquantitative RT-PCR method was used to examine the expression of wamp genes in wheat seedlings in response to infection by pathogens and exposure to phytohormones and ions of a heavy metal ion­cadmium. We discovered that wheat germ contamination by harmful fungi significantly increases expression of genes of the wamp family, and the primary transcript is wamp-2. We determined that salicylic acid, rather than methyl jasmonate, induces expression of genes of the wamp family. We showed that abiotic stress induced by cadmium ions inhibits expression of wamp genes in the roots with no effect on their expression in shoots. The results support the protective role of wamp genes in the response of wheat plants to infections by pathogens. In turn, the resistance to abiotic stress induced by cadmium ions does not appear to be associated with expression of genes of the wamp family.

Antimutagenic activity of wheat polypeptides in human cells exposed to cadmium chloride.

Antimutagenic effects of polypeptides isolated from Triticum kiharae wheat plantule extracts have been studied on human cells exposed to cadmium chloride. The most effective polypeptide Tk-AMP-BP ß -purothionin exhibited higher antimutagenic activity than wheat water extract and another peptide isolated from the same wheat species, Tk-AMP-γ 2 defensin; it also produced a pronounced antioxidant effect. This polypeptide can be used as a preventive agent for reducing the mutagenic potential of some environmental pollutants and for correction of human diseases associated with the defense system defects.

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.

[Defense peptides of plant immune system].

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms to combat pathogens. They are important effector molecules of the immune system both in animals and plants. AMPs are diverse in structure and mode of action. Based on homology of amino acid sequences and 3D structures several AMP families have been distinguished. They are defensins, thionins, lipid transfer proteins, hevein- and knottin-like peptides, and cyclotides. AMPs display broad-spectrum antimicrobial activity and thus show promise for the development of disease- resistant crops by genetic engineering and for the production of new-generation drugs. In this paper, the properties of the main AMP families (defensins and hevein-like peptides) and of a new 4-Cys plant AMP family are reviewed.

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.

[Antimutagenic activity of wheat beta-purothionin Tk-AMP-BP].

Antimutagenic activity on human RD cells was studied for beta-purothionin Tk-AMP-BP isolated from seeds of wheat Triticum kiharae, which has a higher stress resistance. Cadmium chloride at 5 x 10(-6) M was used as a mutagen. The numbers of DNA breaks in mutagen-treated and intact cells were inferred from the single-stranded to double-stranded DNA ratio and expressed as protection coefficients. The protective effect was simultaneously assayed for aqueous plant extracts known to possess antioxidant properties. Wheat thionin was the most active among all of the antimutagens examined; its protection coefficient reached 85-88% at micromolar peptide concentrations (8-32 microg/ml). Thus, wheat beta-purothionin was for the first time demonstrated to be highly efficient in protecting human cell DNA from the damaging effect of cadmium chloride.

[Heterologous expression of a synthetic gene encoding a novel hevein-type antimicrobial peptide of Leymus arenarius in Escherichia coli cells].

A novel antifungal peptide, LAMP-Ia, was isolated from sand-elymus (Leymus arenarius) seeds. Expression of a synthetic gene encoding this peptide in Escherichia coli cells was obtained. The target peptide was expressed as a fusion with thioredoxin. Identity of the recombinant peptide to native LAMP-Ia was confirmed by chromatography, mass spectrometry, and amino acid sequencing. LAMP-Ia displayed a high inhibitory activity in respect of a number of phytopathogenic fungi in in vitro assays, which opens up possibilities for the gene encoding it to be used for genetic transformation of plants and for engineering pathogen-resistant crops.

Antifungal activity of storage 2S albumins from seeds of the invasive weed dandelion Taraxacum officinale Wigg.

In this work, we isolated and characterized novel antifungal proteins from seeds of dandelion (Taraxacum officinale Wigg.). We showed that they are represented by five isoforms, each consisting of two disulphide-bonded large and small subunits. One of them, To-A1 was studied in detail, including N-terminal amino acid sequencing of both subunits, and shown to display sequence homology with the sunflower 2S albumin. Using different assays we demonstrated that dandelion 2S albumins possess inhibitory activity against phytopathogenic fungi and the oomycete Phytophtora infestans at micromolar concentrations with various isoforms differing in their antifungal activity. Thus, 2S albumins of dandelion seeds represent a novel example of storage proteins with defense functions.

[The purification and characterization of a novel lipid transfer protein from caryopsis of barnyard grass (Echinochloa crusgalli)].

A novel lipid transfer protein called Ec-LTP was isolated from resting caryopsis of weed barnyard grass Echinochloa crusgalli (L.) Beauv.; its molecular weight, amino acid content and N-terminal amino acid sequence were determined. Ec-LTP was a 9150 Da protein, containing eight cysteine residues, which formed four disulfide bonds. The isolated protein could significantly inhibit the development of pathogenic fungi Phytophthora infestans and Helminthosporium sativum, causing the late blight of potato and tomato and the root rot of herbs, respectively.

[Isolation of the lipid-transporting protein Ns-LTP1 from seeds of the garden fennel flower (Nigella sativa)].

A novel lipid-transporting protein (Ns-LTP1) has been isolated from seeds of the garden fennel flower Nigella sativa. The molecular mass, N-terminal amino acid sequence, and amino acid composition of the protein have been determined. Ns-LTP1 has a molecular mass of 9602 Da and contains eight cysteine residues which form four disulfide bridges. The protein is capable of suppressing the development of some phytopathogenic fungi and oomycetes.

[Specifics of the coat protein gene in Russian strains of the cucumber green mottle mosaic virus].

The primary structure of the coat protein (CP) gene was examined for pathogenic strain MS-1 and vaccine strain VIROG-43M of the cucumber green mottle mosaic virus (CGMMV). In CP amino acid composition, strains MS-1 and VIROG-43M are typical representatives of CGMMV: their CPs have 98-100% homology to CPs of other tobamoviruses of the group. The CP gene has the same nucleotide composition in pathogenic MS-1 and vaccine VIROG-43M, indicating that strain attenuation is not determined by this gene. The CP amino acid sequences of the two Russian strains are fully identical to the CP sequences of two Greek strains, GR-3 and GR-5. However, the nucleotide sequences of their genes differ in 13 bp, testifying to the difference between the Russian and Greek strains.

[Leaf proteins in transgenic tobacco plants, expressing dual-stranded RNA in viral infection]. / Issledovanie belkov list'ev v transgennykh rasteniiakh tabaka, ékspressiruiushchikh dvunitevuiu RNK, pri virusnoi infektsii.

Generation of transgenic tobacco plants, producing double-stranded RNA with no homology to tobacco genome sequences is reported. The RNA synthesis is mediated by a construct containing an inverted repeat of the pBR322 tetracycline-resistance gene fragment under control of the 35S CaMV promoter. Analysis of the resistance of transgenic plants to the tobacco mosaic virus revealed the changes in the protein spectra of the infected plants. The 25- and 30-kDa proteins found were not detected in the extracts of normal plants. Amino acid sequencing of the 30-kDa peptide with subsequent computer database search revealed the homology of this protein to the hydrolases belonging to the group of plant beta-glucanases. The role of the novel polypeptides in an increase of the resistance of transgenic plants to TMV, and also the possibility of the regulation of their expression by nonhomologous dsRNA are discussed.

[Isolation of endo-1,4-beta-xylanase from Geotrichum candidum 3C with various ability of sorption on an insoluble substrate]. / Razdelenie éndo-1,4-beta-ksilanaz Geotrichum candidum 3C s razlichnoi sposobnost'iu k sorbtsii na nerastvorimom substrate.

Culture liquid from Geotrichum candidum 3C was shown to contain three endoxylanase types: endoxylanase I that binds to cellulose, endoxylanase II that sorbs to insoluble xylan, and endoxylanase III that cannot sorb to dissoluble substrate. The catalytic and substrate-binding domains of endoxylanase II were isolated.

Structural analysis of the coat protein of cucumber green mottle mosaic virus.

Using reversed-phase high-performance liquid chromatography, two components of the coat protein of isolate No. 3 of the cucumber green mottle mosaic virus (CGMMV, cucumber strain), Cp1 (minor) and Cp2 (major), were isolated and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In the Cp2 mass spectrum, two polypeptides with Mr of 16,727.0 and 16,813.5 were detected. By Edman degradation in combination with mass spectrometry, the primary structure of the tryptic peptides of Cp2 comprising in total 150 amino acid residues was determined. Two amino acid substitutions, Val-56-->Ala-56 and Asp-64-->Ser-64, were revealed in Cp2, as compared to the watermelon strain of the virus. Cp1 was shown to consist of three polypeptides with Mr of 10,014.2, 10,224.9, and 10,355.9 corresponding to the N-terminal regions of Cp2 (positions 1-92, 1-94, and 1-95). The observed heterogeneity of the coat protein of CGMMV, cucumber strain, may be due to proteolysis during protein isolation.

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.

Determination of disulfide bonds in gamma-46 gliadin.

The disulfide bonds in gamma-46 gliadin were identified: Cys173--Cys192, Cys212--Cys291, Cys165--Cys199 (or Cys200), Cys283--Cys200 (or Cys199). The disulfide-containing peptides were obtained by limited hydrolysis of the intact protein with chymotrypsin at an enzyme/substrate ratio of 1:1000 at 20 degrees C for 22 h with subsequent digestion of disulfide-containing fragments with trypsin and chymotrypsin. The locations of disulfide bonds were determined by sequencing disulfide-containing fractions and constituent peptides and comparison of the obtained sequences with the partial amino acid sequence of gamma-46 gliadin determined earlier.