A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins

Elmorjani, Khalil; Lurquin, Vanessa; Lelion, André; Rogniaux, Hélène; Marion, Didier

doi:10.1016/j.bbrc.2004.02.173

Cited by 26 publications

(20 citation statements)

References 28 publications

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“…Therefore, Ha-DEF1 antifungal activity regroups features from two distinct defensin types. These bioassays conWrm that the Ha-DEF1 recombinant peptide has a typical defensin activity, not altered by the dipeptide extension as already established for a wheat nsLTP1 by Elmorjani et al (2004).…”

Section: Discussionmentioning

confidence: 60%

“…Consequently, we produced a soluble and active mature Ha-DEF1 recombinant protein using a bacterial expression system developed by Elmorjani et al (2004). The recombinant Ha-DEF1 peptide exhibits an inhibitory activity on Saccharomyces cerevisiae growth.…”

Section: Discussionmentioning

confidence: 99%

“…An aliquot was used to inoculate the 4l TB medium (1/1,000) supplemented with the same antibiotics and growth was initiated at the physical and chemical parameters use for the expression of cysteine-rich protein Richard et al 2004). At appropriate cell densities (OD 600 : 2), IPTG was added (0.5 mM) to induce expression of the recombinant protein for 3 h. The production and puriWcation of the resulting TRX-Ha-DEF1 fusion protein were conducted according to Elmorjani et al (2004). Owing to the presence of the acid labile D-P dipeptide, the Ha-DEF1 peptide was released by incubating the puriWed fusion protein at pH 1.4, 24 h at 50°C and the resulting products were resolved by HPLC.…”

Section: Production Of Ha-def1mentioning

confidence: 99%

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Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

Zélicourt

Letousey

Thoiron

et al. 2007

Planta

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Plant defensins are small basic peptides of 5-10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Production Of Ha-def1mentioning

confidence: 99%

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Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

Zélicourt

Letousey

Thoiron

et al. 2007

Planta

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“…Recombinant GDSL1 was produced in Escherichia coli (Origami) as previously described (Elmorjani et al, 2004;de Zélicourt et al, 2007) after amplification using specific primers (GDSL39, 59-TAAAGCTTATGCATG-TGAAT-39; and GDSL59, 59-ATCCATGGGGCAAAGTGAAGCTAGGGC-ATTT-39) and insertion into expression vectors.…”

Section: Production Of Recombinant Gdsl1 For Generation Of Polyclonalmentioning

confidence: 99%

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

et al. 2012

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The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

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“…The purity of LTPs was checked by SDS-PAGE and electrospray mass ionization spectrometry analyses (ESI-MS), as detailed in a previous work (32).…”

Section: Methodsmentioning

confidence: 99%

Specific Adduction of Plant Lipid Transfer Protein by an Allene Oxide Generated by 9-Lipoxygenase and Allene Oxide Synthase

Bakan

Hámberg

Perrocheau

et al. 2006

Journal of Biological Chemistry

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Lipid transfer proteins (LTPs) are ubiquitous plant lipidbinding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the ␣-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development.

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A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins

Cited by 26 publications

References 28 publications

Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

Specific Adduction of Plant Lipid Transfer Protein by an Allene Oxide Generated by 9-Lipoxygenase and Allene Oxide Synthase

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