Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum.
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 beta-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 (32)P-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 DeltaPcdcl2 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.
Christian Doppler Laboratory for 'Fungal Biotechnology', Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780 Bochum, Germany.
Danielle
D
Janus
Christian Doppler Laboratory for 'Fungal Biotechnology', Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780 Bochum, Germany.
Birgit
B
Hoff
Christian Doppler Laboratory for 'Fungal Biotechnology', Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Universitätsstr. 150, 44780 Bochum, Germany.
Ulrich
U
Kück
D005800:Genes, Fungal; D017930:Genes, Reporter; D007218:Industrial Microbiology; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D011995:Recombination, Genetic; D043244:Ribonuclease III / Q000378:metabolism*
D008164:Luminescent Proteins; D010406:Penicillins; C413663:fluorescent protein 583; D043244:Ribonuclease III