EurekaMag
+ Translate
+ Most Popular
Gaucher's disease;thirty-two years experience at Siriraj Hospital
A study of Macrobathra Meyrick from China (Lepidoptera, Cosmopterigidae)
First occurrence in ores of tetragonal chalcocite
Effects of trace element nutrition on sleep patterns in adult women
N.Z. range management guidelines. 2. Design of grazing management systems for tussock country
A case of lipoma of the esophagus
A revision of world Acanthosomatidae (Heteroptera: Pentatomidae): keys to and descriptions of subfamilies, tribes and genera, with designation of types
Life history of the coronate scyphozoan Linuche unguiculata (Swartz, 1788)
Perceptual restoration of obliterated sounds
Mutagenicity studies on two chromium(III) coordination compounds
The formation of the skeleton. I. Growth of a long bone. 1st appearance of a center of calcification
Leucopenia and abnormal liver function in travellers on malaria chemoprophylaxis
The joint commission: four key root causes loom large in sentinel event data
Treatment of vitiligo with topical 15% lactic acid solution in combination with ultra violet-A
Behaviour of dairy cows within three hours after feed supply: I. Influence of housing type and time elapsing after feed supply
Observations of the propagation velocity and formation mechanism of burst fractures caused by gunshot
Management and control of patients with type 2 diabetes mellitus in Lebanon: results from the International Diabetes Management Practices Study (IDMPS)
The diet composition and nutritional knowledge of patients with anorexia nervosa
Physoporella croatica Herak, 1958 of the Slovak karst Anisian (Slovakia, the West Carpathians Mts.)
Bright lights, big noise. How effective are vehicle warning systems?
Ein Plesiosaurier-Rest mit Magensteinen aus mittlerem Lias von Quedlinburg
Incidence of Chlamydia trachomatis in patients with sterility
Monster soup: the microscope and Victorian fantasy
Preliminary tests with residual sprays against poultry lice
Duration of the life of plants in phylogeny

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


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



Journal of Bacteriology 177(24): 7070-7077



ISSN/ISBN: 0021-9193

PMID: 8522511

DOI: 10.1128/jb.177.24.7070-7077.1995

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.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 008334561

Download citation: RISBibTeXText

Related references

Cloning of a new pectate lyase gene pelC from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. Archives of Biochemistry and Biophysics 323(2): 352-360, 1995

Identification of a novel pelD gene expressed uniquely in planta by Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of its protein product as an endo-pectate lyase. Archives of Biochemistry and Biophysics 332(2): 305-312, 1996

Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI). Journal of Bacteriology 174(20): 6343-6349, 1992

Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi) that is expressed in planta. Molecular Genetics and Genomics: Mgg 265(2): 215-224, 2001

Regulation of constitutively expressed and induced cutinase genes by different zinc finger transcription factors in Fusarium solani f. sp. pisi (Nectria haematococca). Journal of Biological Chemistry 277(10): 7905-7912, 2002

A pectate lyase gene family from Fusarium solani f sp pisi. Phytopathology 86(11 Suppl): S20, 1996

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. Journal of Bacteriology 169(9): 4379-4383, 1987

Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta. International Journal of Biological Macromolecules 84: 94-100, 2016

Cloning of porcine pancreatic alpha-amylase gene and characterization of the enzyme over-expressed in Pichia pastoris. Journal of Dairy Science 93: 662-662, 2010

Pectate lyase from Fusarium solani f. sp. pisi: purification, characterization, in vitro translation of the mRNA, and involvement in pathogenicity. Archives of Biochemistry and Biophysics 258(1): 196-205, 1987

Identification of regulatory elements in the cutinase promoter from Fusarium solani f. sp. pisi (Nectria haematococca). Journal of Biological Chemistry 269(12): 9195-9204, 1994

The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber. Bmc Biotechnology 13: 26, 2013

Cutinase of Nectria haematococca (Fusarium solani f.sp. pisi) is not required for fungal virulence or organ specificity on pea. Molecular Plant Microbe Interactions 7(6): 713-725, 1994

Molecular cloning of an exo pectate lyase gene from erwinia chrysanthemi and characterization of the gene product. Phytopathology 79(10): 1211, 1989

Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines. Journal of Nematology 34(1): 9-11, 2002