1. Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds
Patrícia D Games, et al. Peptides. 2008 Dec;29(12):2090-100. doi: 10.1016/j.peptides.2008.08.008. Epub 2008 Aug 22.
The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.
2. Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv
Tatyana I Odintsova, Eugene A Rogozhin, Yurij Baranov, Alexander Kh Musolyamov, Nasser Yalpani, Tsezi A Egorov, Eugene V Grishin Biochimie. 2008 Nov-Dec;90(11-12):1667-73. doi: 10.1016/j.biochi.2008.06.007. Epub 2008 Jun 26.
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.
3. Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds
Anna A Slavokhotova, Tatyana I Odintsova, Eugene A Rogozhin, Alexander K Musolyamov, Yaroslav A Andreev, Eugene V Grishin, Tsezi A Egorov Biochimie. 2011 Mar;93(3):450-6. doi: 10.1016/j.biochi.2010.10.019. Epub 2010 Nov 4.
Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC(50)≤1μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3'-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.
