Lipid binding proteins from the endosperms of wheat and Oats

Ponz, F; Hernández-Lucas, C.; Carbonero, Pilar; Garcı́a-Olmedo, Francisco

doi:10.1016/s0031-9422(00)80515-4

Cited by 13 publications

(13 citation statements)

References 19 publications

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“…Thionins were purified as described by Ponz et al (44). One kilogram of wheat flour was extracted with petroleum-ether (boiling point, 40 to 60°C), concentrated in a rotoevaporator, mixed with 3 volumes of 0.5 M HCl in ethanol, and centrifuged at 5,000 ϫ g for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco

Titarenko¹,

López‐Solanilla²,

Garcı́a-Olmedo³

et al. 1997

J Bacteriol

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Ralstonia solanacearum K60 was mutagenized with the transposon Tn5, and two mutants, M2 and M88, were isolated. Both mutants were selected based on their increased sensitivity to thionins, and they had the Tn5 insertion in the same gene, 34 bp apart. Sequence analysis of the interrupted gene showed clear homology with the rfaF gene from Escherichia coli and Salmonella typhimurium (66% similarity), which encodes a heptosyltransferase involved in the synthesis of the lipopolysaccharide (LPS) core. Mutants M2 and M88 had an altered LPS electrophoretic pattern, consistent with synthesis of incomplete LPS cores. For these reasons, the R. solanacearum gene was designated rfaF. The mutants were also sensitive to purified lipid transfer proteins (LTPs) and to an LTP-enriched, cell wall extract from tobacco leaves. Mutants M2 and M88 died rapidly in planta and failed to produce necrosis when infiltrated in tobacco leaves or to cause wilting when injected in tobacco stems. Complemented strains M2* and M88* were respectively obtained from mutants M2 and M88 by transformation with a DNA fragment harboring gene rfaF. They had a different degree of wild-type reconstituted phenotype. Both strains retained the rough phenotype of the mutants, and their LPS electrophoretic patterns were intermediate between those of the wild type and those of the mutants.

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Section: Methodsmentioning

confidence: 99%

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco

Titarenko¹,

López‐Solanilla²,

Garcı́a-Olmedo³

et al. 1997

J Bacteriol

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“…Candeal, by petroleumether extraction and HCl/ethanol precipitation, as previously described (Garcia- Olmedo et al, 1968). A mixture of genetic variants of wheat thionins was obtained from the crude extract by preparative electrophoresis on 10% polyacrylamide colums (1.5 X 10 cm) with 0.1 M acetic acid buffer, pH 2.9, at 20 V/cm, essentially as reported (Ponz et al, 1984). The yield obtained was 50 mg/kg of dry endosperm.…”

Section: Purification Of the Proteinmentioning

confidence: 99%

Interaction of wheat α‐thionin with large unilamellar vesicles

Caaveiro

Molina

Rodrı́guez-Palenzuela

et al. 1998

Protein Science

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The interaction of the wheat antibacterial peptide a-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat a-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethy1ene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-arninonaphthalene-l,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.

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“…Betzes, by petroleum-ether extraction and HCl/ethanol precipitation, as previously described [12]. Mixtures of genetic variants of type I thionins were obtained from these preparations by preparative electrophoresis on 10% polyacrylamide columns (1.5 X 10 cm) with 0.1 M acetic acid buffer, pH 2.9, at 20 V/cm, essentially as reported [13]. Resolution of type I genetic variants [16]; AP, apricot médium [17]; PA, pea agar [16]; CB, carrot broth [16]; V-8, v-8 médium [18]; SMA, Sabouraud maltose agar (Difco); CDV-8, cdv-8 médium [19]; RPA, rice polish agar [20].…”

Section: Purification Of Thioninsmentioning

confidence: 99%

“…A crude preparation of type I thionins was obtained by precipitation with ethanol-HCl from the petroleum-ether extracts of mature endosperms from wheat and barley, followed by preparative electrophoresis, as previously described [12,13]. This procedure yields an essentially puré mixture of type I thionins both in wheat and in barley [12,13].…”

Section: Purification Of Thioninsmentioning

confidence: 99%

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Inhibition of bacterial and fungal plant pathogens by thionins of types I and II

Molina¹,

Goy

Fraile³

et al. 1993

Plant Science

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Thionins are cysteine-rich, 5 kDa polypeptides which are active against plant pathogens. Thionins of type I, from the endosperms of wheat (Wal, Wa2, W/3) and barley (Ba, B/3), and of type II, from barley leaves (BLa, BLb, BLc), have been purified to apparent homogeneity. For a given pathogen, the effective concentration giving 50% inhibition (EC-50) determined for the different thionins varied over a less than fivefold range. The ranking of the variants according to their activity differed among different pathogens, but certain variants, such as Wcd, W/3 or B/3, tended to be more active than the others. Strains of some bacterial species, such as Clavibacter michiganensis subsp. sepedonicus or Pseudomonas solanacearum were sensitive in the 2-3 X 10 -7 M concentration range, whereas the most sensitive fungal pathogens, such as Rosellinia necatrix, Colletotrichum lagenarium and Fusarium solani, had EC-50 valúes in the 1-4 X 10~6 M range. Thionins, which were not particularly effective in liquid médium against Phytophthora infestans (EC-50 = 3.9 x 10" 5 M) were more effective than the fungicide Ridomil on a molar basis in a drop application assay on leaf discs from potato.

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Lipid binding proteins from the endosperms of wheat and Oats

Cited by 13 publications

References 19 publications

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco

Interaction of wheat α‐thionin with large unilamellar vesicles

Inhibition of bacterial and fungal plant pathogens by thionins of types I and II

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