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

Christian Doppler Laboratory for ‘Fungal Biotechnology’, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780 Bochum, Germany

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific ³²P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the Pcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

Correspondence
Ulrich Kück
ulrich.kueck{at}rub.de

Supplementary tables of oligonucleotides and plasmids, and supplementary figures showing amino acid sequence alignments of Pcdcl1 and Pcdcl2 with homologous proteins of other filamentous fungi are available with the online version of this paper.

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