Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.     

A branched pathway for transgene-induced RNA silencing in plants

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.     

The role of PACT in the RNA silencing pathway

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.     

Gene silencing by double-stranded RNA

Thanks to the Nobel Foundation for permission to publish this Lecture. We report here the Nobel Lecture delivered by Professor Andrew Z Fire. Together with the accompanying lecture by Professor Mello this lecture describes the exciting years leading to the discovery of RNA interference (RNAi) and some of the underlying molecular mechanisms. Professor Fire nicely points out his own contribution and the contribution of other research groups to the development of this field. He also presents an interesting discussion on the role of RNAi in immunity and challenges us with a number of open questions. The lecture ends presenting the great potential of exploiting RNAi for therapeutical purposes.     

Gene silencing by double-stranded RNA

Eukaryotes silence gene expression in the presence of double-stranded RNA homologous to the silenced gene. Silencing occurs by the targeted degradation of mRNA. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21--23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21--23 nucleotides apart. This mechanism, termed RNAi, has functional links to viral defense and silencing phenomena, such as cosuppression. It also functions to repress the hopping of transposable elements.     

Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.     

Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus

As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs^RI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N^RI/RB protein, or when NSs^RB was expressed. Although NSs^RI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs^RB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.     

RNA silencing

Gene silencing through the increased degradation of mRNA appears to represent a novel cellular pathway that is functional in a broad range of organisms. Recent work has established a role for RNA silencing in host antiviral defense and transposon silencing, suggesting a potential application in plant functional genomics.     

Post-transcriptional gene silencing by double-stranded RNA

Imagine being able to knock out your favourite gene with only a day's work. Not just in one model system, but in virtually any organism: plants, flies, mice or cultured cells. This sort of experimental dream might one day become reality as we learn to harness the power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes. How this phenomenon works is slowly becoming clear, and might help us to develop an effortless tool to probe gene function in cells and animals.     

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.