title: Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption.
abstract: Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.
authors.affiliation: School of Biological Sciences, University of Bristol, Woodland Road, BS8 1UG Bristol, UK.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Amey
authors.forename: Peter R
authors.initials: PR
authors.lastname: Mills
authors.forename: Andy
authors.initials: A
authors.lastname: Bailey
authors.forename: Gary D
authors.initials: GD
authors.lastname: Foster
mesh_terms: D000364:Agaricus; D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry; D018628:Gene Dosage; D018390:Gene Targeting; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D017385:Sequence Homology; D014170:Transformation, Genetic; D020172:Verticillium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*
chemical_list: D004271:DNA, Fungal; D002686:Chitin; D006026:Glycoside Hydrolases; C020105:endo-1,6-beta-glucanase