Figure 7.

35S:DCL1 Does Not Rescue P1/HC-Pro Suppression of either Sense Transgene–Induced or Virus-Induced RNA Silencing.

(A) Sense transgene–induced RNA silencing was monitored by RNA gel blot analysis of high molecular weight RNA to detect GUS mRNA (top blot) and of low molecular weight RNA to detect GUS siRNAs (bottom blot). The RNAs in lanes 1 to 5 are derived from plants hemizygous for the L1 GUS-silenced locus. The presence or absence of the P1/HC-Pro and 35S:DCL1 transgenes is indicated above the blot. The RNA in lane 2 is from a single plant hemizygous for both 35S:DCL1 and L1 transgenes and is representative of four separate individuals that were assayed. The RNAs in lanes 3 and 4 are from two different individual plants hemizygous for all three transgenes. Lanes 5 and 6 show RNA from control plants lacking both P1/HC-Pro and 35S:DCL1 transgenes and either hemizygous (lane 5) or homozygous (lane 6) for the L1 locus.

(B) VIGS was assayed by RNA gel blot analysis of high molecular weight RNA to detect CH42 mRNA (top blot) and of low molecular weight RNA to detect CH42 siRNA (bottom blot). RNA was isolated from tissue pooled from 10 plants at 21 d after bombardment with the CH42 silencing vector. The presence or absence of the P1/HC-Pro and 35S:DCL1 transgenes is indicated above the blot. Ethidium bromide (EtBr) staining of 25S rRNA and the predominant RNA species in the low molecular weight fraction are shown as loading controls in (A) and (B). The molecular weight of the indicated small RNA species in (A) and (B) was inferred from the migration of DNA oligonucleotides that were subsequently standardized to RNA markers.