An efficient fungal RNA-silencing system using the DsRed reporter gene.

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.