Inducible RNA Interference of brlAbeta in Aspergillus nidulans

Abstract

An inducible RNA interference (RNAi) construct composed of inverted repeating alcA promoters flanking the developmental regulatory gene brlAbeta was tested in Aspergillus nidulans. On inducing medium, the RNAi strains failed to sporulate and lacked brlAalpha and brlAbeta expression. RNAi was specific for brlAbeta, but not brlAalpha, silencing, indicating brlAalpha regulation by brlAbeta.

FIG. 1.

brlA locus and RNAi construct. (A) The brlA locus consists of two overlapping transcriptional units, brlAα and brlAβ. The top line represents the brlA genomic DNA (gDNA). The portion of the locus flanked by BamHI sites (−2902 to −404) used in the RNAi construct included only brlAβ. The line of the transcripts (mRNA) represents untranslated RNA, and the box represents translated mRNA. brlAβ has one intron (−99 to +293) where brlAα transcription begins. (B) The RNAi construct consists of inverted repeats of inducible alcA promoters [alcA_(p)] flanking brlAβ in a unique BamHI site. The RNAi construct also contains an argB marker for the nutritional selection of transformants.

FIG. 2.

Phenotypes of RNAi strains. (A) Plates that contained either glucose (G) or threonine (T) as the sole carbon source were scanned with a Microtek ScanMaker 4700. (B) Plates were photographed with a Canon Powershot A620 under a Nikon SMZ-U stereomicroscope at ×60 magnification. (C) Tape mounts were taken from the plates and photographed under a Nikon TMS microscope at ×1,000 magnification. These images show the wild type (WT) under both conditions and the RNAi strains (JB1 and JB3) on glucose with mature conidiophore development and viable conidia. The RNAi mutants on threonine show brlA phenotypes.

FIG. 3.

brlA expression and siRNA analysis. (A) Primers used in real-time RT-PCR were specific for brlAα, brlAβ, and the overlapping portion of brlAα and brlAβ (brlAαβ). The wild type (WT) had normal expression of brlA on both glucose (G) and threonine (T). JB1 had normal brlA expression on glucose but almost undetectable expression on threonine. JB3 had high levels of brlA expression on glucose but reduced expression on threonine. (B) siRNAs specific for brlAβ in the RNAi strains on threonine were detected. No siRNAs were detected in the wild-type or RNAi strains on glucose. Untreated probes (+) or probes treated with RNaseA/T1 (−) were used as controls. (C) No siRNAs were detected using a probe specific for the overlapping portion of brlAα and brlAβ. This indicates that the RNAi mechanism is specifically targeting for brlAβ, not brlAα. Low-molecular-weight (MW) RNA is shown to verify its integrity.