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

Abstract

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 ΔPcdcl2 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.

Members of the Penicillium chrysogenum Velvet Complex Play Functionally Opposing Roles in the Regulation of Penicillin Biosynthesis and Conidiation

“…S2 in the supplemental material). Together with the previously described ⌬PclaeA mutant (26,33), five mutants lacking single genes for components of the velvet complex were available for our genetic study. Note that our genetic analysis was extended by the construction of four double-knockout strains, using single knockouts as a reference, and the corresponding genealogy of all strains is depicted in Fig.…”

“…The marker-free ⌬Pcku70 strain served as a recipient for the construction of knockout mutants (32). In addition to newly generated deletion strains, the recently described ⌬PclaeA and ⌬PclaeA ⌬PcvelA strains were used for functional characterization (33). All P. chrysogenum strains were grown either in liquid complex medium (CCM) or minimal medium (MM) at 27°C and 120 rpm or on solid medium as already described (33).…”

“…In addition to newly generated deletion strains, the recently described ⌬PclaeA and ⌬PclaeA ⌬PcvelA strains were used for functional characterization (33). All P. chrysogenum strains were grown either in liquid complex medium (CCM) or minimal medium (MM) at 27°C and 120 rpm or on solid medium as already described (33). For induction of the P. chrysogenum Pcflp-mediated site-specific recombination, appropriate strains were grown on solid MM supplemented with 2% xylose (32,36).…”

“…To inoculate shake flasks and solid medium, we used spores collected from 7-day-old cultures grown on medium M322. Transformation of individual P. chrysogenum strains was performed as described previously (33,34), and transformants were selected by growth on solid medium supplemented with 200 g ml Ϫ1 nourseothricin, 40 g ml Ϫ1 phleomycin, or 700 g ml Ϫ1 pyrithiamine. Construction of plasmids.…”

Special issue: Physiology and Systems Biology of the Fungal Cell

“…The special collection of research papers found in this issue spans a range of topics and organisms. Topics covered include primary metabolism (Heyland et al, 2009;Domin et al, 2009;Dawe et al, 2009) and its regulation (Teves et al, 2009;Vongsangnak et al, 2009;Wong et al, 2009), as well as the cellular and systems biology of key processes that underlie fungal growth (Bormann & Tudzynski, 2009;Hutchison et al, 2009;Etxebeste et al, 2009;Janus et al, 2009;Eisenman et al, 2009;Gale et al, 2009;Avanzo et al, 2009;Mukaiyama et al, 2009;Zara et al, 2009). Organisms include well-known model systems (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, Aspergillus nidulans), human pathogens (Candida albicans, Cryptococcus neoformans), plant pathogens (Claviceps purpurea, Cryphonectria parasitica, Fusarium solani) and industrial producers (Penicllium chrysogenum).…”