title: Vector-initiated transitive RNA interference in the filamentous fungus Aspergillus oryzae.
abstract: RNA interference (RNAi), modulates gene expression via cleavage of double-stranded RNA (dsRNA) by Dicer, producing 21-25 nucleotide silence-inducing RNAs (siRNAs). In association with Argonaute containing complexes, these siRNAs target sequence-specific degradation of the homologous single-stranded messenger RNA. In the majority of eukaryotes, degradation occurs within the boundaries of the dsRNA target. In Arabidopsis thaliana and Caenorhabditis elegans, gene silencing can also take place transitively, impacting transcripts from coding sequences that are adjacent to the intended target gene. Here we demonstrate effective transitive RNAi in the ascomycete Aspergillus oryzae. Fragments of 174 bp and 499 bp derived from the A. oryzae wA gene involved in spore color development, were inserted immediately upstream of an inverted repeat derived from the Escherichia coli gene encoding for Hygromycin Phosphotransferase B (hph), which provided a double-stranded hph RNAi trigger. Introduction of this construct into A. oryzae host cells produced transformants with spores that were lighter in color than those of wild type. Real-time RT-qPCR analysis demonstrated a direct correspondence of steady-state wA mRNA level to spore color. An A. oryzae strain deficient in RNA-dependent RNA Polymerase (RdRP) produced exclusively wild type colored spores when transformed with a wA transitive RNAi construct. Conversely, increased expression of RdRP enhanced the incidence of wA gene silencing via transitive RNAi.
authors.affiliation: Novozymes, Inc., Davis, CA 95618, United States.
authors.forename: Evee Q
authors.initials: EQ
authors.lastname: Fernandez
authors.forename: Donna L
authors.initials: DL
authors.lastname: Moyer
authors.forename: Suchindra
authors.initials: S
authors.lastname: Maiyuran
authors.forename: Anna
authors.initials: A
authors.lastname: Labaro
authors.forename: Howard
authors.initials: H
authors.lastname: Brody
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D020868:Gene Silencing; D005822:Genetic Vectors; D055029:Inverted Repeat Sequences; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D048630:Polyketide Synthases / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D013172:Spores, Fungal
chemical_list: D005656:Fungal Proteins; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D012333:RNA, Messenger; D048630:Polyketide Synthases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D012324:RNA-Dependent RNA Polymerase