Vector-initiated transitive RNA interference in the filamentous fungus Aspergillus oryzae

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.

► Small gene fragments inserted upstream of a vector transcribed inverted repeat undergo silencing.
► Spore color screens demonstrate efficiency of transitive RNAi comparable to that of hairpin induced RNAi.
► Over expression of the endogenous RdRP increases the incidence of transitive RNAi nearly twofold.
► Vector initiated transitive RNAi purports an alternative means for cloning and silencing large mutant libraries.