Host-Pathogen Interactions

Weinstein, Lawrence I.; Hahn, Michael G.; Albersheim, Peter

doi:10.1104/pp.68.2.358

Cited by 42 publications

(22 citation statements)

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“…The Gram-negative bacterium E. carotovora has been shown to elicit the accumulation of pterocarpan phytoalexins in wounded soybean cotyledons (33). Ninety-five per cent of this elicitor activity was abolished by killing the bacteria with heat treatments or antibiotics (13).…”

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Host-Pathogen Interactions

Davis

Lyon²,

Darvill³

et al. 1984

Plant Physiol.

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191

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ABSTRACrHeat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defed medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-05m gel filtration column. The heat-labile elicitor activity co-purified with two a- (14) and from citrus pectin (25).The Gram-negative bacterium E. carotovora has been shown to elicit the accumulation of pterocarpan phytoalexins in wounded soybean cotyledons (33). Ninety-five per cent of this elicitor activity was abolished by killing the bacteria with heat treatments or antibiotics (13). These results suggested that viable E. carotovora cells produce an elicitor when in contact with plant tissue. We report in this paper the purification and characterization of heat-labile elicitors produced by E. carotovora grown on a defined medium containing citrus pectin. These elicitors were found to be PGA2 lyases, pectin-degrading enzymes that have been shown to be secreted by many plant pathogens (3). We also present evidence that the release, by PGA lyase, of oligosaccharides from the pectic polymers of plant cell walls triggered the elicitation of phytoalexin accumulation. This evidence suggests that the release of endogenous elicitors (14, 25) from plant cell walls by pectin-degrading enzymes plays a role in general plant disease resistance to microorganisms. A preliminary report of these results has been published (10).MATERIALS AND METHODS Chemicals. Electrophoresis-grade acrylamide and N,N'-methylenebisacrylamide, low-mol-wt protein standards, silver-stain reagents, dye reagent concentrate for protein assay, and Bio-Gel A-0.5m agarose beads were from Bio-Rad. Sephadex G-15, CMSephadex (C-50), BSA (fraction V), streptomycin sulfate, and Tris were from Sigma. Ion-exchange resin PB 118 and Pharmalyte pH 8-10.5 ampholytes were from Pharmacia. Electrophoresis-grade SDS was from BDH Chemicals Ltd., D-galacturonic acid was from Aldrich, and D-glucose was from Fisher. All other chemicals and solvents were of reagent grade or better.Polysaccharides. Sodium polypectate (grade II), polygalacturonic acid (grade III), and citrus pectin were from Sigma. Cell walls from soybean stems were prepared by M. Woodward and M. Hahn, as described (14). Before use, the cell walls (1 g) were washed with 1 L 5 mm Tris-HCl, 1 mm CaCl2 at pH 8.5, and 1 L deionized H20. The walls were then dried in a vacuum oven. A crude glucan elicitor from Phytophthora megasperma var glycinea was a gift from J. K. Sharp of this laboratory.

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confidence: 99%

Host-Pathogen Interactions

Davis

Lyon²,

Darvill³

et al. 1984

Plant Physiol.

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191

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“…Furthermore, the maximum amount of phytoalexins induced by the decagalacturonide appeared to be approximately half of the maximum amount induced by the ,3-glucan elicitor and equivalent to that induced by PGA lyase (7). The amount of the phytoalexin, glycinol, induced by 5 ug/ cotyledon of the decagalacturonide was estimated to be approximately 100 Mg/ml (7), which is a concentration previously shown to significantly inhibit the growth of various microorganisms (36). Experiments in which a decagalacturonide-rich fraction was assayed in the presence of fractions rich in other specific sized oligogalacturonides demonstrated that these fractions modified the elicitor activity of the decagalacturonide (Table VII).…”

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confidence: 99%

“…This paper is based on part of a Ph.D. thesis presented to the University of Colorado toalexins (1, 1 1). Phytoalexins are a diverse group of compounds of low mol wt that have broad antibiotic activity against many prokaryotic and eukaryotic organisms (32,36). In some hostpathogen combinations, phytoalexins accumulate at the site of attempted infection rapidly enough and in sufficient concentration to prevent further growth of the invading microorganism (19,24).…”

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confidence: 99%

Host-Pathogen Interactions

Davis¹,

Darvill²,

Albersheim³

et al. 1986

Plant Physiol.

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161

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Recent studies have demonstrated that an apparently homogeneous preparation of an a-1,4-D-endopolyplacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max jL.1Merr. cv Wayne) and that this pectin-degrading enzyme released heatstable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of a-1,4-D-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-,6-L-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/ cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) plactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-a-1,4-D-galactosyluronic acid that contained 4,5-unsaturated gaactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants. Although (1, 1 1). Phytoalexins are a diverse group of compounds of low mol wt that have broad antibiotic activity against many prokaryotic and eukaryotic organisms (32, 36). In some hostpathogen combinations, phytoalexins accumulate at the site of attempted infection rapidly enough and in sufficient concentration to prevent further growth of the invading microorganism (19,24).Phytoalexin accumulation is also induced by molecules called elicitors. Various compounds isolated from microorganisms have been found to have elicitor activity (reviewed in 1, 2, 6, 37). Molecules derived from plant tissues also induce phytoalexin accumulation (14-16, 23, 28).Recent work has demonstrated that a heat-labile elicitor of phytoalexins produced by the phytopathogenic bacterium Erwinia carotovora var carotovora is a p...

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“…Among isoflavonoids, glyceollins have a characteristic tetracyclic structure and are known as pterocarpan. Glyceollins are synthesized via glycinol as the major phytoalexin in soybeans (Glycine max) and are known for their antibiotic and antitumor activities (Boué et al 2009;Salvo et al 2006;Weinstein et al 1981). Medicarpin is another pterocarpan-type phytoalexin that is distributed in various legume lineages, including Canavalia, Cicer, Glycyrrhiza, Medicago, Melilotus, and Trifolium.…”

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Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Uchida

Akashi

Aoki

2015

Plant Biotechnology

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Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l −1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.

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Host-Pathogen Interactions

Cited by 42 publications

References 19 publications

Host-Pathogen Interactions

Host-Pathogen Interactions

Host-Pathogen Interactions

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

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