RNA Silencing In Plants Research Articles

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.

DICER-LIKE2 Plays a Primary Role in Transitive Silencing of Transgenes in Arabidopsis

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2–but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

Small RNA mediated silencing to target Tomato leaf curl virus

Plant RNA silencing systems are organized into different networks with overlapping functions. Primarily, micro RNAs (miRNAs) help in regulating development and small interfering RNAs (siRNAs) regulate transgene induced or virus induced silencing functions at the post transcriptional level. Small RNA silencing, using potent RNAi silencing target sequences from viral genome of leaf curl virus, was designed via artificial micro RNAs (amiRNAs) and transgene derived siRNAs to generate leaf curl resistance in plants. The comparative analysis of both the RNAi silencing systems is discussed.

A T7-driven silencing system in transgenic plants expressing T7 RNA polymerase is a nuclear process

We previously demonstrated a case of silencing in transgenic plants expressing T7 RNA polymerase in which expression of a reporter gene placed under the control of the T7 promoter was silenced. Here we demonstrate that endogenous genes can be silenced by the same system. The T7-driven silencing system does not conform to several aspects characteristic of post-transcriptional RNA silencing in plants, and this prompted an investigation into the mechanisms underlying this type of silencing. The present paper demonstrates that T7-driven silencing is a post-transcriptional process that is restricted to the nucleus. Nuclear run-on assays indicated the presence of silenced gene transcripts in both orientations. SiRNA corresponding to the silenced gene could not be traced in the cytoplasm but was found in nuclei. The silenced gene was hypermethylated. We present evidence that a tobacco RNA-dependent RNA polymerase (RdRP) is not involved in T7-mediated silencing, but indicate the involvement of a nuclear RdRP in this type of silencing.

The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways

One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.

Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.

Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19

The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19 kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni 2+–NTA coated 96-well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent p K a values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.

A CLASSY RNA Silencing Signaling Mutant in Arabidopsis

RNA silencing in plants is associated with movement of a silencing signal from cell to cell and through the phloem. It is likely that the mobile silencing signals are RNA molecules: either short interfering RNAs generated by Dicer-liker (DCL) RNaseIII enzymes or longer double stranded RNAs. Smith et

A practical approach to the understanding and teaching of RNA silencing in plants

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein ( gfp ) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool.

Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting

RNA silencing is often associated with methylation of the target gene. The DNA methylation level of transgenes was investigated in post-transcriptionally silenced or non-silenced Nicotiana benthamiana carrying either the 5′ region (200 or 400 bp) or the entire region of the coat protein gene ( CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Higher levels of transgene cytosine methylation were observed in both symmetrical (CpG, CpNpG) and non-symmetrical (CpHpH) contexts (CpG > CpNpG > CpHpH) in silenced lines, but there was very lower levels or no transgene methylation in non-silenced lines. RNA silencing was induced in non-silenced scions from silenced rootstocks and spread to the 3′ region of the transgene mRNA (Haque et al., Plant Mol. Biol. 2007; 63: 35–47). In this system, transgene methylation levels were analyzed in scions at different time intervals after being grafted onto silenced or non-silenced rootstocks to investigate if transgene methylation was associated with induction or transitivity of RNA silencing. We observed that, there was no change of transgene methylation level in the initial target or in extended regions in scions. These results showed that transgene methylation was associated with RNA silencing in individual transformants, but it was not associated with systemic RNA silencing and/or transitive RNA silencing through grafting.

Tomato Chlorotic Mottle Virus Is a Target of RNA Silencing but the Presence of Specific Short Interfering RNAs Does Not Guarantee Resistance in Transgenic Plants

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.

Viruses, microRNAs and cancer

Viruses represent one of the main factors that cause normal cells to proliferate and to become malignant: up to 15% of all human cancers are associated with single or multiple virus infections, and several viruses have been recognized as causal agents of specific types of cancer. Viruses have evolved many strategies to prevent infected cells from becoming apoptotic and to evade the innate and adaptive immune responses of their hosts. The recent discovery that Epstein-Barr virus and other herpesviruses produce their own sets of micro (mi)RNAs brings an additional layer of complexity in this ongoing host-virus arms race and changes our initial views of the antiviral roles of RNA silencing in plants and insects. It seems that, rather than being inhibited by this process, many mammalian viruses can usurp or divert the host RNA silencing machinery to their advantage. Viral-encoded miRNAs can act both in cis, to ensure accurate expression of viral genomes, and in trans, to modify the expression of host transcripts. Here, we review the current knowledge on viral miRNAs and discuss how mammalian viruses can also perturb host miRNA expression. Those recent findings provide new insights into the role of viruses and miRNAs in cancer development.

Characterization of the Recombinant Forms Arising from aPotato virus XChimeric Virus Infection under RNA Silencing Pressure

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.

Efficient virus-induced gene silencing in Arabidopsis.

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.

Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.

Regulation of microRNA on plant development and viral infection

MicroRNAs (miRNAs) are a class of ~22 nt noncoding regulatory RNAs which are universal in eukaryotes. They act as negative regulator of gene expression by targeting mRNAs for cleavage or translational repression. miRNAs widely participate in regulation of gene expression associated with plant developmental processes, such as organs morpho- genesis and signal transduction pathway in plants. Similar to antiviral RNA silencing in plants, miRNA pathway is also interfered by silencing suppressors encoded by viruses. In this review we give a brief summary of the differences between miRNA-mediated regulatory pathway and siRNA-mediated RNA silenc- ing. In particular, we review the recent studies on miRNA regulatory roles in plant developmental processes, as well as the interference of viral sup- pressors in miRNA pathway, aiming to get an insight into the complexity of gene expression regulation in eukaryotic organisms.

Using Satellite Tobacco Mosaic Virus Vectors for Gene Silencing

This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants.

BackgroundWe describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.ResultsWe give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs.ConclusionIn developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

RNA SILENCING SYSTEMS AND THEIR RELEVANCE TO PLANT DEVELOPMENT

RNA silencing refers to a broad range of phenomena sharing the common feature that large, double-stranded RNAs or stem-loop precursors are processed to ca. 21-26 nucleotide small RNAs, which then guide the cleavage of cognate RNAs, block productive translation of these RNAs, or induce methylation of specific target DNAs. Although the core mechanisms are evolutionarily conserved, epigenetic maintenance of silencing by amplification of small RNAs and the elaboration of mobile, RNA-based silencing signals occur predominantly in plants. Plant RNA silencing systems are organized into a network with shared components and overlapping functions. MicroRNAs, and probably trans-acting small RNAs, help regulate development at the posttranscriptional level. Small interfering RNAs associated with transgene- and virus-induced silencing function primarily in defending against foreign nucleic acids. Another system, which is concerned with RNA-directed methylation of DNA repeats, seems to have roles in epigenetic silencing of certain transposable elements and genes under their control.