New construct approaches for efficient gene silencing in plants

Abstract

An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts.

Figure 1.

Silencing constructs. A blue arrow indicates the position and orientation of the gus gene fragment used as trigger for gene silencing; a green box between arrows indicates a spacer. 35S, CMV 35S promoter; FMV, FMV35S promoter; FMV^T, FMV35S promoter lacking TATA box; Ubi, promoter of the potato ubiquitin-7 gene; T, nos gene terminator; mT, modified nos terminator lacking a NUE element; pA, synthetic poly(A) tail. A pink arrow depicts a fragment of the tobacco Ppo gene; an orange box indicates the intron of the gus gene. The expression cassette for the hpt selectable marker gene was oriented in such a way that the terminator of this cassette would not function in 3′-end processing of transcripts produced by the silencing constructs.

Figure 2.

RNA analysis of gus plants containing a terminator-free silencing construct. A, Diagrams of expression cassettes for the gus gene and the silencing constructs of pSIM717 and 715. The position of introns is indicated with horizontal striped bars. Primers (pr1–pr4) used for RT-PCR are shown as black arrows. Probes, depicted as delineated gray lines, are derived from: the gus gene (G), P_35S (1), intron of the potato Gbss gene (I; Rohde et al., 1990), and P_FMV (2). For further explanations, see the legend of Figure 1. B, RT-PCR analysis of silenced (underlined) and nonsilenced pSIM717 and pSIM715 plants. Amplified products are shown after separation on agarose gel and ethidium bromide staining. DNA fragments amplified with primers pr1 and pr2 can be derived from RNAs transcribed from P_35S, P_FMV, or both. Because pSIM717 contains a Gbss-derived intron that is shorter than that of pSIM715, products generated with the pr1 to pr3 primers are shorter for pSIM717 plants than for pSIM715 plants. C, RNA gel-blot analysis of three pSIM717 plants together with a wild-type (WT) and original gus (GUS) plant (16-h exposure). The white arrow shows the position of the gus gene transcript. Transcripts produced by the silencing construct of plants 717-12 and 717-36 vary in size from approximately 0.2 to approximately 1.0 kb. Silenced line numbers are underlined. CV, Transcripts specific for convergent transcription. D, RNA gel-blot hybridized with the gus gene-derived probe G (24-h exposure). WT, Untransformed; GUS, original gus expressing plant. E, Hybridization with probe I derived from the Gbss intron (15-h exposure). F, Hybridization with the P_35S probe 1 (5-d exposure). G, Hybridization with P_FMV (5-d exposure) probe 2.

Figure 3.

Effect of convergent-transcription constructs on expression of the neighboring hptII gene. A, Transformation frequencies for pSIM374 (gray) and 717 (white) were determined by infecting tobacco explants with the corresponding Agrobacterium strains, and, after 2 weeks, counting the number of calli/explant. Results are means ± se of at least 12 explants per experiment. B, Real-time PCR was performed to determine relative transcript levels of pSIM374 (gray) and pSIM717 (white). Data are the mean ± se of three independent measurements.

Figure 4.

Amplification of transcripts from plants transformed with pSIM717. The amplified DNAs are shown after separation on agarose gel and ethidium bromide staining. Silenced line numbers are underlined. The position of a cDNA representing the 3′ end of the gus transcript is indicated with a white arrowhead. The weaker band marked with a gray band relates to a tobacco PR52 cDNA, which contains an inadvertent pr1 binding site at 0.5 kb upstream from the cleavage site.

Figure 5.

pSIM789 plants. Histochemically stained leaf punches of plants containing both the gus gene and the silencing construct of pSIM789. Boxed leaf samples depict stained gus positive (top) and wild-type (bottom) plants.

Figure 6.

Silencing of the potato tuber-expressed genes. A, Diagram of expression cassettes pSIM217, pSIM764, and pSIM765, designed to target the Ppo gene. PG, Promoter of the Gbss gene; T, terminator of the potato ubiquitin-3 gene. An orange arrow indicates a Ppo gene fragment and a green box depicts the intron of the potato ubiquitin-7 gene (B) Ppo enzyme activity. Data are shown as percentages of wild type (OD410/g = 0.15 ± 0.01) and represent the mean of three tuber measurements per plant. C, Tubers stained for Ppo activity by pipetting 0.5 mL of catechol (50 mm) on cut surfaces. D, Expression cassettes pSIM216 and pSIM847 aimed to reduce Glc accumulation through silencing of the PhL gene. AG, ADP-Glc pyrophosphorylase promoter. E, Glc concentrations (milligrams per gram) in cold-stored tubers. CNTRL, Transgenic control. Data are the mean ± se of three independent measurements. F, PhL gene expression as determined by quantitative real-time PCR analyses of tuber RNA. Results are means ± se of three experiments.

Figure 7.

Promoter-targeted gene silencing. Representative histochemically stained leaf punches are shown for pSIM773 (A), pSIM1120 (B), pSIM788 (C), and pSIM1101 (D). Partially silenced leaf punches are shown in a green box.