An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

Abstract

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.

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

“…Plasmid pREDi was previously described (Janus et al, 2007) and served for construction of pREDi_xyl. For this purpose, a xylP promoter fragment was generated by PCR using primers Pxyl_s and Pxyl_a with genomic DNA from P. chrysogenum as template.…”

“…Quantitative real-time PCR (qRT-PCR) was performed as described previously (Nowrousian et al, 2005), with the modifications described by Janus et al (2007).…”

“…Plasmid pBRLAi_xyl was designed by first introducing an annealed pair of complementary oligonucleotides (Nde_1 and Nde_2) into the ApaI-BglII restriction sites of plasmid pS-NAT1 (Janus et al, 2007) to insert a single NdeI restriction site. The resulting plasmid pS-NAT1-NdeI was then digested using EcoRI and ClaI for insertion of the above-mentioned xylP promoter to generate pS-NAT_xyl.…”

“…Recently, it was shown that RNAi can be used effectively to manipulate a number of biotechnologically relevant ascomycetes (Janus et al, 2007;Oliveira et al, 2008;Takeno et al, 2005;Ullán et al, 2008;Yamada et al, 2007;Zhang et al, 2007). For example, RNAi was used to silence regulators of biosynthetic pathways as well as to downregulate the expression of genes involved in the synthesis of secondary metabolites such as fatty acids or b-lactam antibiotics.…”

“…This system is based on the previously described vector pREDi that carries the DsRed gene as a reporter for direct monitoring of gene silencing on solid media (Janus et al, 2007). The vector was modified by using the xylP promoter from P. chrysogenum, which is induced when xylose is the sole carbon source in the medium (Zadra et al, 2000).…”