Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.15 (pectinase)
2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Posttranscriptional regulation mediated by the regulator of secondary metabolites (RSM) RsmA-rsmB pair is the most important factor in the expression of genes for extracellular enzymes and HarpinEcc in Erwinia carotovora subsp. carotovora. RsmA is a small RNA-binding protein, which acts by lowering the half-life of a mRNA species. rsmB specifies an untranslated regulatory RNA and neutralizes the RsmA effect. It has been speculated that GacA-GacS, members of a two-component system, may affect gene expression via RsmA. Because expA, a gacA homolog, and expS (or rpfA), a gacS homolog, have been identified in E. carotovora subsp. carotovora, we examined the effects of these gacA and gacS homologs on the expression of rsmA, rsmB, and an assortment of exoprotein genes. The gacA gene of E. carotovora subsp. carotovora strain 71 stimulated transcription of genes for several extracellular enzymes (pel-1, a pectate lyase gene; peh-1, a polygalacturonase gene; and celV, a cellulase gene), hrpNEcc (an E. carotovora subsp. carotovora gene specifying the elicitor of hypersensitive reaction), and rsmB in GacA+ and GacS+ E. carotovora subsp. carotovora strains. Similarly, the E. carotovora subsp. carotovora gacA gene stimulated csrB (rsmB) transcription in Escherichia coli. A GacS- mutant of E. carotovora subsp. carotovora strain AH2 and a GacA- mutant of E. carotovora subsp. carotovora strain Ecc71 compared with their parent strains produced very low levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts but produced similar levels of rsmA RNA and RsmA protein as well as transcripts of hyperproduction of extracellular enzymes (Hex) hexA, kdgR (repressor of genes for uronate and pectate catabolism), rsmC, and rpoS (gene for Sigma-S, an alternate Sigma factor). The levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts as well as production of pectate lyase, polygalacturonase, cellulase, protease, and HarpinEcc proteins were stimulated in GacS- and GacA- mutants by GacS+ or GacA+ plasmids, respectively. The GacA effect on exoenzyme genes and hrpNEcc was abrogated in E. carotovora subsp. carotovora mutants deficient in RsmA and RsmC or RsmA, RsmC, and rsmB RNA. The expression of lacZ transcriptional fusions of rsmB of Erwinia amylovora and Erwinia herbicola pv. gypsophilae was markedly reduced in a GacA- and a GacS- mutant of Pseudomonas syringae pv. syringae. Southern blot hybridization revealed the presence of gacA and gacS homologs in all tested strains of soft-rotting Erwinia spp. and several nonsoft-rotting Erwinia species such as E. amylovora, E. rhapontici, E. herbicola, E. stewartii, and E. herbicola pv. gypsophilae. These findings establish that the GacA-GacS system controls transcription of rsmB of E. carotovora subsp. carotovora, E. amylovora, and E. herbicola pv. gypsophilae and support the hypothesis that the effects of this two-component system on extracellular protein production in E. carotovora subsp. carotovora is mediated, at least in part, via the levels of rsmB transcripts.
Mol Plant Microbe Interact 2001 Apr
PMID:Effects of the two-component system comprising GacA and GacS of Erwinia carotovora subsp. carotovora on the production of global regulatory rsmB RNA, extracellular enzymes, and harpinEcc. 1131 Jul 39

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici, whose predicted amino acid sequence shows significant homology with subtilisin-like fungal proteinases. Prt1 is a single-copy gene, and its structure is highly conserved among different formae speciales of F. oxysporum. Prt1 is expressed constitutively at low levels during growth on different carbon and nitrogen sources and strongly induced in medium containing collagen and glucose. As shown by reverse transcription-polymerase chain reaction and fluorescence microscopy of F. oxysporum strains carrying a prt1-promoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F. oxysporum strains transformed with an expression vector containing the prt1 coding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extracellular subtilase activity that resolved into a single peak of pI 4.0 upon isoelectric focusing. The active fraction produced two clearing bands of 29 and 32 kDa in sodium dodecyl sulfate gels containing gelatin. Targeted inactivation of prt1 in F. oxysporum f. sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.
Mol Plant Microbe Interact 2001 May
PMID:Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum. 1133 29

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.
Mol Plant Microbe Interact 2001 Jun
PMID:Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata. 1138 70

The alkaloid-rich latex of the opium poppy, Papaver somniferum L., is valued as a source of pharmaceuticals including thebaine, codeine, and morphine, but is also harvested for heroin production. The poppy laticifer system develops through the gradual disappearance of the common walls between differentiating laticifer elements throughout the plant. Gene homologues for cell-wall-degrading enzymes were found during random sequencing of an opium poppy latex cDNA library. RNA gel blot analysis of cellulase, polygalacturonase beta-subunit, 1,3-beta-glucanase, and xyloglucan endotransglycosylase homologues showed their expression was not limited to laticifers. In contrast, poppy gene homologues to pectin methylesterase (PME), pectin acetylesterase (PAE) and pectate lyase (PL) where all highly expressed and latex-specific. Enzyme assays confirmed the presence of PME, PAE, and PL activities in latex serum. The abundance of transcripts encoding pectin-degrading enzymes in latex suggests that these enzymes may play an important role in laticifer development.
Plant Mol Biol 2001 Mar
PMID:Expression and activity of cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L. 1141 15

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.
Plant Mol Biol 2001 Jul
PMID:Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth. 1148 3

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.
Mol Plant Microbe Interact 2001 Aug
PMID:Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction. 1149 71

Polygalacturonases hydrolyze the alpha-(1-4) glycosidic bonds of de-esterified pectate in the smooth region of the plant cell wall. Crystal structures of polygalacturonase from Aspergillus aculeatus were determined at pH 4.5 and 8.5 both to 2.0 A resolution. A. aculeatus polygalacturonase is a glycoprotein with one N and ten O-glycosylation sites and folds into a right-handed parallel beta-helix. The structures of the three independent molecules are essentially the same, showing no dependency on pH or crystal packing, and are very similar to that of Aspergillus niger polygalacturonase. However, the structures of the long T1 loop containing a catalytic tyrosine residue are significantly different in the two proteins. A three-dimensional model showing the substrate binding mode for a family 28 hydrolase was obtained by a combined approach of flexible docking, molecular dynamics simulations, and energy minimization. The octagalacturonate substrate was modeled as an unbent irregular helix with the -1 ring in a half-chair ((4)H(3)) form that approaches the transition state conformation. A comparative modeling of the three polygalacturonases with known structure shows that six subsites ranging from -4 to +2 are clearly defined but subsites -5 and +3 may or may not be shaped depending on the nearby amino acid residues. Both distal subsites are mostly exposed to the solvent region and have weak binding affinity even if they exist. The complex model provides a clear explanation for the functions, either in catalysis or in substrate binding, of all conserved amino acid residues in the polygalacturonase family of proteins. Modeling suggests that the role of the conserved Asn157 and Tyr270, which had previously been unidentified, may be in transition state stabilization. In A. niger polygalacturonase, the long T1 loop may have to undergo conformational change upon binding of the substrate to bring the tyrosine residue close to subsite -1.
J Mol Biol 2001 Aug 24
PMID:The X-ray structure of Aspergillus aculeatus polygalacturonase and a modeled structure of the polygalacturonase-octagalacturonate complex. 1151 36

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of beta-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1-->4)beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.
Plant Mol Biol 2001 Sep
PMID:Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. 1155 79

Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.
Mol Microbiol 2002 Feb
PMID:A putative three-dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora. 1192 17

This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to localize gene expression in plant tissues. This RT-PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier, was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7 microm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50 microl of the RT-PCR reaction mixture supplemented with 0.5 microM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.
Cell Mol Biol Lett 2002
PMID:A useful protocol for in situ RT-PCR on plant tissues. 1194 46


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