RNA silencing in plants by the expression of siRNA duplexes

Abstract

In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.

Figure 1

Preparation of human H1 RNA gene promoter-based siRNA expression constructs. The 19 nt GUS gene specific sequence (GT1 or GT2) separated by a 9 nt spacer from the reverse complement of the same sequence followed by a termination signal of five thymidines (H1-GT1 or H1-GT2) was cloned into pSUPER (34) downstream of the H1 promoter (H1-P). The H1-P::GT expression construct harboring H1-GT1 or H1-GT2 was then excised and cloned into the binary vector pGPTV-HPT (41). The resulting vector, pGPH1-HPT-GT1 or pGPH1-HPT-GT2, which contained a hygromycin phosphotransferase (hpt) selectable marker gene under the control of a nopaline synthase promoter (Pnos)-transcription terminator (pAg7, agropine synthase polyadenylation signal sequence) pair, was then mobilized into A.tumefaciens C58 for transforming tobacco. The predicted secondary siRNA structures of H1-GT1 and H1-GT2 are depicted.

Figure 2

Preparation of plant 7SL RNA gene promoter-based siRNA expression constructs. A promoter fragment (7SL-P, 289 bp) containing USE and TATA elements (47) and a 3′-UT region (267 bp) of Arabidopsis At7SL4 (AY525344) gene were cloned and ligated in pUC19, from which the 7SL-P::UT construct was excised and cloned into the pGPTV-HPT vector (41) to replace the pAnos-uidA fragment. The resulting vector, pGPSL, contained an hpt selectable marker gene under the control of a nopaline synthase promoter (Pnos)-transcription terminator (pAg7, agropine synthase polyadenylation signal sequence) pair. GUS gene-specific 7SL-GT1, 7SL-GT2 or 7SL-GT3 sequence module, which contained a termination signal of seven thymidines, for the generation of the corresponding hairpin, siRNA was inserted into pGPSL between 7SL-P and 3′-UT. The resulting binary vectors were named pGPSL-HPT-GT1, pGPSL-HPT-GT2 and pGPSL-HPT-GT3, respectively. The binary vector was then mobilized into A.tumefaciens C58 for transforming tobacco. The predicted secondary siRNA structures of 7SL-GT1, 7SL-GT2 and 7SL-GT3 are depicted.

Figure 3

Histological staining of GUS protein activity in tobacco plants harboring human H1 RNA gene promoter-based siRNA expression vectors. GUS staining of stem cross-section, leaf and root from 1-month-old siRNA-transgenic (pGPH1-HPT-GT1 and pGPH1-HPT-GT2) and GUS-expressing control (C) tobacco plants.

Figure 4

Analysis of human H1 promoter-mediated siRNA silencing of GUS gene expression in transgenic tobacco. (A) GUS protein activity in leaves of the control plants (C) and 10 pGPH1-HPT-GT2 transgenic lines. Mean values were calculated from three independent measurements per line. (B) Loading control for gel blot analysis showing 25S rRNA transcript levels. (C) The same gel blot as in (B) was used to characterize the GUS mRNA level with a GUS cDNA probe. (D) Gel blot detection of small RNAs of ∼21 nt, as indicated, using a GUS cDNA probe. RNA was isolated from a portion of the leaves used for GUS protein activity assay in (A).

Figure 5

Analysis of plant 7SL promoter-mediated siRNA silencing of GUS gene expression in transgenic tobacco. (A) GUS protein activity in leaves of the control plants (C) and 11 pGPSL-HPT-GT2 transgenic lines. Mean values were calculated from three independent measurements per line. (B) RNA loading control. (C) Same gel blot used in (B) above was used to characterize the GUS mRNA level with a GUS cDNA probe. (D) Gel blot detection of small RNAs of ∼21 nt, as indicated, using a GUS cDNA probe. RNA was isolated from a portion of the leaves used for GUS protein activity assay in (A).