Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Marcia Saraiva, Irene de Bruijn, Laura Grenville-Briggs, Debbie McLaggan, Ariane Willems, Vincent Bulone, Pieter van West

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)
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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. Agene 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.

Original languageEnglish
Pages (from-to)621-629
Number of pages9
JournalFungal Biology
Volume118
Issue number7
DOIs
Publication statusPublished - Jul 2014
Externally publishedYes

Keywords

  • Cell wall
  • Gene expression
  • Melanin
  • Oomycete
  • Transient transformation

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