title: Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing.
abstract: Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK.
authors.forename: Marcia
authors.initials: M
authors.lastname: Saraiva
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK.
authors.forename: Irene
authors.initials: I
authors.lastname: de Bruijn
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK; Division of Glycoscience, School of Biotechnology, KTH - Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
authors.forename: Laura
authors.initials: L
authors.lastname: Grenville-Briggs
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK.
authors.forename: Debbie
authors.initials: D
authors.lastname: McLaggan
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK.
authors.forename: Ariane
authors.initials: A
authors.lastname: Willems
authors.affiliation: Division of Glycoscience, School of Biotechnology, KTH - Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
authors.forename: Vincent
authors.initials: V
authors.lastname: Bulone
authors.affiliation: Aberdeen Oomycete Laboratory, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, UK. Electronic address: p.vanwest@abdn.ac.uk.
authors.forename: Pieter
authors.initials: P
authors.lastname: van West
mesh_terms: D002473:Cell Wall / Q000648:ultrastructure; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D008543:Melanins / Q000378:metabolism; D008854:Microscopy, Electron; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044762:Saprolegnia / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D058432:Sporangia / Q000254:growth & development / Q000378:metabolism
chemical_list: D008543:Melanins; D034741:RNA, Small Interfering; D014442:Monophenol Monooxygenase
keywords: Cell wall; Gene expression; Melanin; Oomycete; Transient transformation
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