title: Cloning, purification and characterisation of brassinin glucosyltransferase, a phytoalexin-detoxifying enzyme from the plant pathogen Sclerotinia sclerotiorum.
abstract: The plant-pathogenic fungus Sclerotinia sclerotiorum can detoxify cruciferous phytoalexins such as brassinin via glucosylation. Here we describe a multifaceted approach including genome mining, transcriptional induction, phytoalexin quantification, protein expression and enzyme purification that led to identification of a S. sclerotiorum glucosyltransferase that detoxifies brassinin. Transcription of this gene, denoted as brassinin glucosyltransferase 1 (SsBGT1), was induced significantly in response to the cruciferous phytoalexins camalexin, cyclobrassinin, brassilexin, brassinin and 3-phenylindole, a camalexin analogue. This gene was also up-regulated during infection of Brassica napus leaves. Levels of brassinin decreased significantly between 48 and 72h post-inoculation, with a concomitant increase in levels of 1-beta-d-glucopyranosylbrassinin, the product of the reaction catalysed by SsBGT1. These findings strongly implicate the involvement of this gene during infection of B. napus. This gene was cloned and expressed in Saccharomyces cerevisiae. The purified recombinant enzyme was able to glucosylate brassinin and two other phytoalexins, albeit much less effectively. This is the first report of a fungal gene involved in detoxification of plant defence molecules via glucosylation.
authors.affiliation: School of Botany, The University of Melbourne, Vic. 3010, Australia.
authors.forename: Adrienne C
authors.initials: AC
authors.lastname: Sexton
authors.forename: Zoran
authors.initials: Z
authors.lastname: Minic
authors.forename: Anton J
authors.initials: AJ
authors.lastname: Cozijnsen
authors.forename: M Soledade C
authors.initials: MS
authors.lastname: Pedras
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Howlett
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029688:Brassica napus / Q000378:metabolism / Q000382:microbiology; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000378:metabolism / Q000382:microbiology; D016415:Sequence Alignment; D012717:Sesquiterpenes; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D000094162:Phytoalexins
chemical_list: D005656:Fungal Proteins; D012717:Sesquiterpenes; D013729:Terpenes; D005964:Glucosyltransferases; D000094162:Phytoalexins