Effective Gene Silencing in Plants by Synthetic Trans-Acting siRNAs Derived From Minimal Precursors

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

In this issue of Circulation , Zheng and coworkers1 demonstrate the efficacy of specific small interfering RNAs (siRNAs) added to University of Wisconsin solution in protecting donor hearts against cold ischemic injury during storage and subsequent reperfusion. siRNAs targeted tumor necrosis factor-α, C3, and Fas. The study design was well rationalized and performed systematically to confirm the efficacy of these siRNA constructs in silencing their respective targets, first in cell culture and subsequently in whole mouse hearts. Quantitative polymerase chain reaction was used to demonstrate knockdown of each of these proteins after ischemia and reperfusion in contrast to their strong upregulation in control hearts. Beneficial effects were confirmed by enhanced function, decreased apoptosis, diminished neutrophil activation and infiltration, and less histological evidence of tissue damage in the treated hearts. Article see p 1099 First discovered as a method for gene silencing in plants, siRNA effectively silences or reduces posttranscriptional efficacy of …

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

Conservation of transgene-induced post-transcriptional gene silencing in plants and fungi

Posttranscriptional gene silencing (PTGS) also known as RNA silencing or RNA interference is an evolutionarily conserved innate immunity in eukaryotes that targets the complementary RNA sequences to slice/degrade the target RNA or repress the translation of mRNA. In the past two decades, RNA silencing as an important antiviral mechanism has been studied extensively in plants. Intriguingly, almost every virus encodes at least a viral suppressor of RNA silencing (VSR) to counterattack RNA silencing with many strategies to interfere with different steps of RNA silencing. Therefore, the molecular identification of VSRs and elucidation of their functional mechanisms contribute to a better understanding of host resistance and viral pathogenicity. Here, we describe a protocol for the transient expression-induced gene silencing in 16c GFP transgenic and wild type Nicotiana benthamiana plants, and the suppression of single-stranded GFP and double-stranded GFP induced RNA silencing with a VSR in N. benthamiana plants. This protocol is simple and can serve as a standard for the identification and functional analysis of a VSR.

RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3'-5' direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3' end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a beta-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5' fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3'-untranslated region. Conversely, silencing induced by a 3' fragment spread only for a limited distance in the 3'-5' direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3' end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.

Successful application of posttranscriptional gene silencing (PTGS) for gene function study in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5'-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Genetic engineering of virus resistance in plants may be conferred by transgenes based on sequences from the viral genome. In many instances the underlying mechanism involves the transgenically expressed proteins. However there are other examples in which the mechanism is based on RNA. It appears that this mechanism is related to post transcriptional gene silencing in transgenic plants. This gene silencing is likely to involve antisense RNA produced by the action of a host-encoded RNA dependent RNA polymerase. The natural role of this mechanism is as a genetic immune system conferring protection against viruses. There may also be a genomic role of the process reflected in RNA directed methylation of transgenes. Further understanding of this mechanism has obvious implications for virus resistance in plants. In addition the gene silencing can be used as a component of a new technology with application in functional genomics.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species, and are nearly impossible to control with conventional methods. During the past few decades, RNAi has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented, indicating potential for the development of methods to protect them via the delivery of the sRNAs to parasites, a method called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to increase the efficiency of HIGS in parasitic plants. We also investigate the important biological processes during the life cycle of the parasites, with a focus on broomrape species, providing several appropriate target genes that can be used, in particular, in multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for control of parasitic plants. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

Inverted repeat (IR) RNA silencing vectors containing homologous fragments of target endogenous plant genes, or pathogen genes, are the most widely used vectors to either study the function of genes involved in biotic stress or silence pathogens to induce plant resistance, respectively. RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of IR constructs during cloning and plant transformation. We have therefore developed a robust method for the production of long IR vector constructs by introducing base pair mismatches in the form of cytosine to thymine mutations on the sense arm by exposure to sodium bisulfite prior to assembly of the IR.

We developed a novel RNA virus vector based on the Cucumber mosaic virus (CMV), which is able to efficiently induce gene silencing in plants. We manipulated the RNA 2 of the CMV Y strain, whose genome consists of tripartite components, and introduced restriction sites for cloning a foreign sequence into the vector. To evaluate the vector (designated CMV2-A1) in terms of the ability to induce gene silencing, we cloned portions of the green fluorescent protein (GFP) cDNA or Cauliflower mosaic virus (CaMV) 35S promoter sequences into the vector and inoculated the infectious transcripts into Nicotiana benthamiana plants that express the GFP gene under the control of the CaMV 35S promoter. In both cases, a loss of GFP fluorescence accompanying a reduction in the level of GFP mRNA was induced. The short interfering RNAs (siRNAs) harboring the sequences inserted in the CMV2-A1 vector were detected in the silenced plants. When plants were infected with the virus containing the CaMV 35S promoter sequence, the CaMV 35S promoter sequence in the genomic DNA was heavily methylated. A reduction in the mRNA level of the GFP gene and loss of GFP fluorescence were induced as early as 6 and 12 days post-inoculation, respectively, earlier than the 20 days previously achieved with a Potato virus X vector. These results suggest that the CMV2-A1 vector is suitable for the rapid induction of both transcriptional and post-transcriptional gene silencing.

Plant transformation is a versatile method to introduce or alter a trait-of-interest through expression of a transgene or through transgene-induced mutation and/or expression changes of endogenes. Transgenes in plants, however, are subject to gene-silencing effects that are an obstacle to stable and heritable expression of transgene-encoded traits. In an exciting twist, natural gene-silencing processes have been harnessed by researchers to achieve highly specific and targeted down-regulation of endogenous or pathogen-derived genes for functional studies, crop protection and crop improvement. Part 1 of this chapter attempts to give an overview of the mechanistic pillars of gene silencing in plants. Part 2 summarizes the factors that may cause unintended silencing of transgene expression, with practical advice on how to minimize the risk of transgene silencing. Part 3 addresses the intentional use of gene silencing for biotechnological applications in transgenic plants, with particular emphasis on RNA interference approaches.

The discovery that double-stranded RNA is responsible for posttranscriptional gene silencing (PTGS) in plants has led to the development of a new generation of vectors for this purpose. These usually enable the cloning of an inverted repeat of the gene to be silenced, separated by an excisable loop, often of intron origin. Here we present the construction and analysis of a novel vector for the induction of PTGS. This vector, named pJM007, allows a single pair of primers to be used in the directional cloning of sense and antisense cDNA strands. To avoid cloning problems associated with the presence of recognition sites for the usual restriction enzymes, 2 rare 8-bp cutters have been included, 1 at each side of the intron loop.Arabidopsis thaliana lines ectopically expressing enhanced green fluorescent protein (EGFP), were transformed with pJM007 containing a silencing cassette for this gene and subsequently characterised. The results obtained demonstrate that pJM007 can induce PTGS with an efficiency of up to 95%. Furthermore, silencing is shown to remain stable up to the F2 generation of transformed plants.