Abstract
SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal α-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
M.S. Almeida K.S. Cabral L. Nevesde Medeiros A.P. Valente F.C.L. Almeida E. Kurtenbach (2001) ArticleTitlecDNA Cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris Arch Biochem Biophys 395 199–207
J.L. Bennetzen B.D. Hall (1981) ArticleTitleCodon selection in yeast J Biol Chem 257 3026–3031
C.Jr. Bloch M. Richardson (1991) ArticleTitleA new family of small (5 kDa) protein inhibitors of insect alpha-amylases from seeds or sorghum (Sorghum bicolar (L) Moench) have sequence homologies with wheat gamma-purothionins FEBS Lett 279 101–104
B.P. Cammue M.F. De Bolle F.R. Terras P. Proost J. Van Damme S.B. Rees J. Vanderleyden W.F. Broekaert (1992) ArticleTitleIsolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds J. Biol. Chem. 267 2228–2233
S. Cociancich A. Ghazio C. Hetru J.A. Hoffmann L. Letelliers (1993) ArticleTitleInsect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus J. Biol. Chem. 268 19239–19245
G.W. De Samblanx I.J. Goderis K. Thevissen R. Raemaekers F. Fant F. Borremans D.P. Acland R.W. Osborn S. Patel W.F. Broekaert (1997) ArticleTitleMutational analysis of a plant defensin from radish (Raphanus sativusL.) reveals two adjacent sites important for antifungal activity J. Biol. Chem. 272 1171–1179
F. Fant W. Vranken W. Broekaert F. Borremans (1998) ArticleTitleDetermination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR J. Mol. Biol. 279 257–270
C.P. Hill J. Yee M.E. Selsted D. Eisenberg (1991) ArticleTitleThe crystal structure of defensin HNP-3, an amphophilic simer, suggests mechanisms of membrane permeabilization Science 251 1481–1485
D.M. Hoover K.R. Rajashankar R. Blumenthal A. Puri J.J. Oppenheim O. Chertov J. Lubkowski (2000) ArticleTitleThe structure of human β-Defensin-2 shows evidence of higher order oligomerization J. Biol. Chem. 275 4232911–4232918
B.L. Kagan M.E. Selsted T. Ganz R.I. Lehre (1990) ArticleTitleAntimicrobial defensin peptides from voltage-dependent ion-permeable channels in planar lipid bilayer membranes Proc. Natl. Acad. Sci. U.S.A. 87 210–214
P. Kraulis (1991) ArticleTitleMolScript, a program to produce both detailed and schematic plots of protein structures J. Appl. Crystallog. 24 946–950
R.I. Lehrer T. Ganz D. Szklarek M.E. Selsted (1988) ArticleTitleModulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations J. Clin. Invest. 81 1829–1835
E. Mendez A. Moreno F.J. Colilla F. Pelaez G.G. Limas R. Mendez F. Soriano M. Salinas C. Haro Particlede (1990) ArticleTitlePrimary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-horodthionin, from barley endosperm Eur. J. Biochem. 194 533–539
E.A. Merritt D.J. Bacon (1997) ArticleTitleRaster3D-Photorealistic molecular graphics Methods Enzymol. 277 505–524
M. Moreno A. Segura F. Garcia-Olmedo (1994) ArticleTitlePseudothionin-St1, a potato peptide active against potato pathogens Eur. J. Biochem. 223 135–139
R.W. Osborn G.W. De Samblanx K. Thevissen I. Goderis S. Torrekens F. Van Leuven S. Attenborough S.B. Rees W.F. Broekaert (1995) ArticleTitleIsolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae FEBS Lett 368 257–262
M. Osusky G. Zhou L. Osuska R.E. Hancock W.W. Kay S. Misra (2000) ArticleTitleTransgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens Nat. Biotechnol. 18 1162–1166
Z.K. Punja (2001) ArticleTitleGenetic engineering of plants to enhance resistance to fungal pathogens – a review of progress and future prospects Can. J. Plant Pathol. 23 216–235
P.A. Raj A.R. Dentino (2002) ArticleTitleCurrent status of defensins and their role in innate and adaptive immunity FEMS Microbiol. Lett. 206 9–18
D.J. Schibli H.N. Hunter V. Aseyev T.D. Starner J.M. Wiencek P.B. McCray B.F. Tack H.J. Vogel (2002) ArticleTitleThe solution structures of the human ß-Defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus J. Biol. Chem. 277 8279–8289
A. Segura M. Moreno A. Molina F. Garcia-Olmedo (1998) ArticleTitleNovel defensin subfamily from spinach (Spinacia oleracea) FEBS Lett. 435 159–162
X. Song Z. Zhou J. Wang F. Wu W. Gong (2004) ArticleTitlePurification, characterization and preliminary crystallographic studies of a novel plant defensin from Pachyrrhizus erosus seeds Acta Cryst. D 60 1121–1124
F.R. Terras H.M. Schoofs M.F. De Bolle F. Van Leuven S.B. Rees J. Vanderleyden B.P. Cammue W.F. Broekaert (1992) ArticleTitleAnalysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds J. Biol. Chem. 267 15301–15309
F.R. Terras K. Eggermont V. Kovaleva N.V. Raikhel R.W. Osborn A. Kester S.B. Rees S. Torrekens F. Van Leuven J. Vanderleyden B.P. Cammue W.F. Broekaert (1995) ArticleTitleSmall cysteine-rich antifungal proteins from radish: their role in host defense Plant Cell 7 568–573
K. Thevissen R.W. Osborn D.P. Acland W.F. Broekaert (1997) ArticleTitleSpecific, high affinity binding sites for an antifungal plant defensin onNeurospora crassa hyphaeand microsomal membranes J. Biol. Chem. 272 32176–32181
K. Thevissen A. Ghazi G.W. De Samblanx C. Brownleei R.W. Osborn W.F. Broekaert (1996) ArticleTitleFungal Membrane Responses Induced by Plant Defensins and Thionins J Biol Chem 271 15018–15025
J.D. Thompson D.G. Higgins T.J. Gibson (1994) ArticleTitleCLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res. 22 4673–4680 Occurrence Handle1:CAS:528:DyaK2MXitlSgu74%3D Occurrence Handle7984417
W.C. Wimley M.E. Selsted S.H. White (1994) ArticleTitleInteractions between human defensins and lipid bilayers: evidence for formation of multimeric pores Protein Sci. 3 1362–1373
D. Yang A. Biragyn L.W. Kwak J.J. Oppenheim (2002) ArticleTitleMammalian defensins in immunity: more than just microbicidal Trends Immunol., 23 291–296
Author information
Authors and Affiliations
Corresponding author
Additional information
Nucleotide sequence data reported in this paper are available in the DDBJ/EMBL/GenBank database under accession number AY679170
Rights and permissions
About this article
Cite this article
Song, X., Wang, J., Wu, F. et al. cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds. Plant Mol Biol 57, 13–20 (2005). https://doi.org/10.1007/s11103-004-6637-y
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/s11103-004-6637-y