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

Abstract

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.

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Acknowledgments

We thank Dr. Sergey I. Kovalchuk for peptide synthesis, Kseniya S. Kudryashova for CD measurements and Natalia V. Khadeeva for antibacterial assay. Support from the Russian Foundation for Basic Research [Grants 12-04-00117a and 11-04-00190a], the Biodiversity Program and the Program of Molecular and Cell Biology of the Russian Academy of Sciences is acknowledged. AAS and AAV are recipients of the stipend of the President of Russian Federation.

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Correspondence to Anna A. Slavokhotova.

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Tsezi A. Egorov: Deceased 4 January 2012.

Database submissions The Sm-AMP-X sequence data is reported in the UniProt Knowledgebase under the accession number C0HJD6. The sm-amp-x gene sequence data is reported in the European Nucleotide Archive under the accession number HG423454.

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Slavokhotova, A.A., Rogozhin, E.A., Musolyamov, A.K. et al. Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution. Plant Mol Biol 84, 189–202 (2014). https://doi.org/10.1007/s11103-013-0127-z

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  • DOI: https://doi.org/10.1007/s11103-013-0127-z

Keywords

  • Weeds
  • cDNA cloning
  • Gene structure
  • Plant defense
  • Protein biosynthesis
  • α-hairpinin