Eugene A. Rogozhin

Novel antifungal defensins from Nigella sativa L. seeds.

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.

A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif.

Two forms of a novel antimicrobial peptide (AMP), named WAMP‐1a and WAMP‐1b, that differ by a single C‐terminal amino acid residue and belong to a new structural type of plant AMP were purified from seeds of Triticum kiharae Dorof. et Migusch. Although WAMP‐1a and WAMP‐1b share similarity with hevein‐type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described previously for plant AMPs, but is characteristic of the chitin‐binding domains of cereal class I chitinases. An unusual substitution of a serine for a glycine residue in the chitin‐binding domain was detected for the first time in hevein‐like polypeptides. Recombinant WAMP‐1a was successfully produced in Escherichia coli. This is the first case of high‐yield production of a cysteine‐rich plant AMP from a synthetic gene. Assays of recombinant WAMP‐1a activity showed that the peptide possessed high broad‐spectrum inhibitory activity against diverse chitin‐containing and chitin‐free pathogens, with IC50 values in the micromolar range. The discovery of a new type of AMP active against structurally dissimilar microorganisms implies divergent modes of action and discloses the complexity of plant–microbe interactions.

Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli).

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.

Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv.

From the annual weed barnyard grass Echinochloa crusgalli (L.) Beauv., two novel defensins Ec-AMP-D1 and Ec-AMP-D2 that differ by a single amino acid substitution were isolated by a combination of different chromatographic procedures. Both defensins were active against several phytopathogenic fungi and the oomycete Phytophthora infestans at micromolar concentrations. The Ec-AMP-D1 showed higher activity against the oomycete than Ec-AMP-D2. The comparison of the amino acid sequences of the antifungal E. crusgalli defensins with those of earlier characterized T. kiharae defensins [T.I. Odintsova, Ts.A. Egorov, A.Kh. Musolyamov, M.S. Odintsova, V.A. Pukhalsky, E.V. Grishin, Seed defensins from T. kiharae and related species: genome localization of defensin-encoding genes, Biochimie, 89 (2007) 605-612.] that were devoid of substantial antifungal activity point to the C-terminal region of the molecule as the main determinant of the antifungal activity of E. crusgalli defensins.

Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases.

The multilayered plant immune system relies on rapid recognition of pathogen‐associated molecular patterns followed by activation of defense‐related genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn‐metalloproteinase from Fusarium verticillioides, named fungalysin Fv‐cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein‐like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP‐1b or WAMP‐2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.

Molecular characterisation and diagnostics of some Longidorus species (Nematoda: Longidoridae) from Russia and other countries using rRNA genes

The needle nematodes of the genus Longidorus can cause diseases of various crops and trees, and are comprised of more than 150 valid species. Eleven valid and six unidentified species of the genus Longidorus collected in different regions of Russia, two states of USA, Germany, New Zealand and Ukraine were molecularly characterized using analysis of the partial 18S rRNA and the D2–D3 expansion segments of the 28S rRNA gene sequences. Fifty-four partial 28S rRNA and fifteen partial 18S rRNA gene sequences were obtained for the present study. Using molecular criteria, we confirmed the morphological identification and distinguished between the following species: L. aetnaeus, L. africanus, L. andalusicus, L. artemisiae, L. caespiticola, L. distinctus, L. elongatus, L. euonymus, L. intermedius, L. leptocephalus and L. lignosus. Two longidorid populations from Russia and four from California were not identified to a species level. We obtained the full length D2–D3 of 28S rRNA gene sequence from several freshly-collected L. artemisiae samples. We confirmed the identity of the D2 region of 28S rRNA gene sequence with a short D2 of 28S rRNA gene fragment sequence previously obtained from formalin-fixed nematodes embedded in the L. artemisiae paratype slides. Longidorus lignosus was molecularly characterized and L. aetnaeus was reported from Russia for the first time. PCR-D2-D3-RFLP diagnostic profiles generated by five restriction enzymes: AluI, HinfI, Bsp143I, Tru1I and RsaI are presented for sixteen Longidorus species.

Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence

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.

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.

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution

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.

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

Abstract 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