Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectin-degrading enzymes are thought to be important for pathogenesis. Antibodies prepared against a pectin-inducible pectate lyase (pectate lyase A [PLA]) produced by a phytopathogenic fungus, Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection. The gene (pelA) and its cDNA were cloned and sequenced. Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F. solani f. sp. pisi with the pelA cDNA as the probe. A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced. The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F. solani pisi culture grown in the presence of glucose as the sole carbon source. The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F. solani f. sp. pisi but no significant homology with other pectinolytic enzymes. The first 16 amino acid residues at the N terminus appeared to be a signal peptide. The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa. PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography. Purified PLB showed optimal lyase activity at pH 10.0. A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion. Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other. The 5' flanking regions of both pelA and pelB were translationally fused to the beta-glucuronidase gene and introduced into F. solani f. sp. pisi, and beta-glucuronidase activities of the transformants were measured. Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression. Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F. solani f. sp. pisi infects pea epicotyl.
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PMID:Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. 852 11

Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.
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PMID:Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity. 1046 62