Tatyana I. Odintsova

Diversity of wheat anti-microbial peptides.

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.

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing.

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.

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.

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.

Solution structure of a defense peptide from wheat with a 10-cysteine motif.

Hevein, a well-studied lectin from the rubber tree Hevea brasiliensis, is the title representative of a broad family of chitin-binding polypeptides. WAMP-1a, a peptide isolated from the wheat Triticum kiharae, shares considerable similarity with hevein. The peptide possesses antifungal, antibacterial activity and is thought to play an important role in the defense system of wheat. Importantly, it features a substitution of the conserved serine residue to glycine reducing its carbohydrate-binding capacity. We used NMR spectroscopy to derive the spatial structure of WAMP-1a in aqueous solution. Notably, the mutation was found to strengthen amphiphilicity of the molecule, associated with its mode of action, an indication of the hevein domain multi-functionality. Both primary and tertiary structure of WAMP-1a suggest its evolutionary origin from the hevein domain of plant chitinases.

DISULPHIDE STRUCTURE OF A SUNFLOWER SEED ALBUMIN : CONSERVED AND VARIANT DISULPHIDE BONDS IN THE CEREAL PROLAMIN SUPERFAMILY

Disulphide mapping of a methionine‐rich 2S albumin from sunflower seeds showed four intra‐chain disulphide bonds which are homologous with those in a related heterodimeric albumin from lupin seeds (conglutin δ). Similar conserved disulphide bonds are also present in α‐gliadin and γ‐gliadin storage proteins of wheat, but a lower level of conservation is present in a further related group of proteins, the cereal inhibitors of α‐amylase and trypsin. These differences may relate to the different functions of the proteins.

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.