FIG 2.

Generation of reporter transgene array ty48. (A) Coinjection of the plasmid constructs shown in Fig. 1A restored antiviral RNAi in rde-1;rde-4 double mutants that contain an FR1gfp transgene. Shown here is the visualization of green fluorescence in the rde-1;rde-4 double mutants that contain extrachromosomal arrays formed by the injected plasmids 48 h after heat induction. The image was produced by merging images recorded under red and green fluorescence using the same exposure. (B) Accumulation of FR1gfp transcripts in single and double mutants corresponding to rde-1 and rde-4 as indicated. An asterisk indicates worms that carry the ty48 transgene array. The FR1gfp transcripts were detected by Northern blotting, for which the probe was prepared using GFP coding sequence; RNA1, FR1gfp genomic RNA; RNA3, FR1gfp subgenomic RNA. Methylene blue-stained rRNA serves as an equal loading control. (C) Northern blot detection of Orsay virus RNA1, ovRNA1, in single and double mutants corresponding to rde-1 and rde-4 as indicated. An asterisk indicates worms that carry the ty48 transgene array. Orsay virus RNA1 cDNA was used to prepare probe for the detection of Orsay virus RNA1 through Northern blotting. (D) Accumulation of Orsay virus RNA1 in response to rde-1 or rde-4 dsRNA feeding in rde-1;rde-4 double mutants with or without the ty48 transgene array. An asterisk indicates worms that were fed with E. coli food expressing rde-1 dsRNA. A double asterisk indicates worms that were fed with E. coli food expressing rde-4 dsRNA. (E) The FR1gfp transgene in the transgene array ty48 is functional. Shown here is the visualization of green fluorescence in the wild-type N2 worms carrying the ty48 transgene array 48 h after heat induction. The worms have been fed using E. coli food expressing rde-4 dsRNA.