Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 59 rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

RNA silencing in plants: Yesterday, today, and tomorrow

RNA silencing has become a major focus of molecular biology and biomedical research around the world. This is highlighted by a simple PubMed search for “RNA silencing,” which retrieves almost 9,000 articles. Interest in gene silencing-related mechanisms stemmed from the early 1990s, when this phenomenon was first noted as a surprise observation by plant scientists during the course of plant transformation experiments, in which the introduction of a transgene into the genome led to the silencing of both the transgene and homologous endogenes. From these initial studies, plant biologists have continued to generate a wealth of information into not only gene silencing mechanisms but also the complexity of these biological pathways as well as revealing their multilevel interactions with one another. The plant biology community has also made significant advancements in exploiting RNA silencing as a powerful tool for gene function studies and crop improvements. In this article, we (1) review the rich history of gene silencing research and the knowledge it has generated into our understanding of this fundamental mechanism of gene regulation in plants; (2) describe examples of the current applications of RNA silencing in crop plants; and (3) discuss improvements in RNA silencing technology and its potential application in plant science.

Gene silencing as an adaptive defence against viruses

Gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants. It now seems that it is the consequence of accidentally triggering the plant's adaptive defence mechanism against viruses and transposable elements. This recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals.

Production of transgenic rice with rice ragged stunt virus synthetic resistance genes

Rice ragged stunt virus (RRSV) is an important pathogen of rice affecting its cultivation in South and South East Asia. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV genome segments 7 and 10 (non-structural genes), and 5, 8 and 9 (structural genes) were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, Ubil, Actl and RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-mediated delivery of one or more of these PDR gene constructs. A large number of transgenic lines were produced from calli derived from mature or immature embryos, co-bombarded with the marker gene hph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Mendelian segregations were observed in transgenic progeny, especially with transgenic lines produced by biolistics. Preliminary tests conducted in China on selected transgenic lines indicate that plants with RRSV segment 5 antisense PDR gene confer RRSV resistance.

Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

Mobile silencing in plants: what is the signal and what defines the target

RNA-mediated silencing in plants can spread from cell to cell and over a long distance, and such mobile silencing has been extensively studied in the past decade. However, major questions remain as to what is the exact nature of the mobile silencing signals, how the components of the RNA-directed DNA methylation pathway are involved, and why systemic spread of silencing has only been observed for transgenes but not endogenous genes. In this review, we provide an overview of the current knowledge on mobile gene silencing in plants and present a model where systemic silencing involves long nuclear RNA transcripts that serve as a template to amplify primary siRNA signals.

Application of gene silencing in plants

Recent studies of gene silencing in plants have revealed two RNA-mediated epigenetic processes, RNA-directed RNA degradation and RNA-directed DNA methylation. These natural processes have provided new avenues for developing high-efficiency, high-throughput technology for gene suppression in plants.

Gene silencing: Fleshing out the bones

Genetic studies are revealing the pathway for RNA-mediated gene silencing. Short RNA molecules are the key, giving sequence specificity for RNA degradation and mediating communication within and between cells; these short RNAs are common to transcriptional and post-transcriptional silencing pathways. The expression of transgenes in plants varies between independent transformants and there are many examples where the transgenic trait is not expressed, or disappears in subsequent generations, despite the presence of the transgene. This loss of a trait, but not of the transgene, has become known as gene silencing and can take two forms, transcriptional or post-transcriptional. As their names imply, transcriptional gene silencing occurs when a transgene is not transcribed, whereas in post-transcriptional gene silencing, the transgene mRNA is produced but degraded before it is translated (reviewed in [1]). Both forms of silencing seem to be the result of inherent mechanisms for protecting plants against mobile or invading DNA — for example, transposable elements or the T-DNA of Agrobacterium — or RNA viruses. Plants are not alone in their capacity for transgene silencing; both forms of silencing occur in flies and fungi, where it is known as RIP or quelling, while nematodes exhibit post-transcriptional silencing, generally referred to as RNA interference (RNAi). A clearer picture of the mechanisms and relationships of the different types of transgene silencing is beginning to emerge from a number of recent studies [2], [3], [4], [5], [6], [7] and [8]. Some of these studies [2], [3], [4] and [5] have enhanced our understanding of the steps within the post-transcriptional silencing pathway, and others [6], [7] and [8] have demonstrated that the two forms of silencing may be mechanistically linked.

C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis. © 2012 Springer Basel.

Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana

We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants. © The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer

RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract. © CSIRO 2008.

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus. © 2007 Federation of European Biochemical Societies.