RNA silencing movement in plants ?™›-460/sil...RNA silencing movement in plants Review...

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  • Biol. Cell (2008) 100, 1326 (Printed in Great Britain) doi:10.1042/BC20070079 Review

    RNA silencing movement in plantsKriton Kalantidis*1, Heiko Tobias Schumacher*, Tasos Alexiadis* and Jutta Maria Helm**Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, P.O. Box 1527, GR-71110

    Heraklion/Crete, Greece, Department of Genetics, Kassel University, Heinrich-Plett-Str. 40, DE-34109 Kassel, Germany, Department

    of Biology, University of Crete, Vassilika Vouton, GR-71409 Heraklion/Crete, Greece, and Institute of Applied Genetics and Cell Biology,

    University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.

    Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation which is known as RNAsilencing. In plants and some animals, similar to the nematode Caenorhabditis elegans, RNA silencing is a non-cell-autonomous event. Hence, silencing initiation in one or a few cells leads progressively to the sequence-specificsuppression of homologous sequences in neighbouring cells in an RNA-mediated fashion. Spreading of silencingin plants occurs through plasmodesmata and results from a cell-to-cell movement of a short-range silenc-ing signal, most probably 21-nt siRNAs (short interfering RNAs) that are produced by one of the plant Dicerenzymes. In addition, silencing spreads systemically through the phloem system of the plants, which also translo-cates metabolites from source to sink tissues. Unlike the short-range silencing signal, there is little known about themediators of systemic silencing. Recent studies have revealed various and sometimes surprising genetic elementsof the short-range silencing spread pathway, elucidating several aspects of the processes involved. In this reviewwe attempt to clarify commonalities and differences between the individual silencing pathways of RNA silencingspread in plants.

    IntroductionHigher eukaryotes have developed a mechanism ofsequence-specific RNA degradation called RNA si-lencing, an idiom that combines the terms PTGS(post-transcriptional gene silencing) and RNAi(RNA interference). The central part of the RNA de-gradation pathway is the generation of siRNAs (shortinterfering RNAs) from dsRNA (double-strandedRNA) by an RNase III-type nuclease, Dicer. ThesiRNAs are incorporated into the RISC (RNA-induced silencing complex), and, after strand separ-ation, the remaining single-stranded RNA guides thesequence-specific cleavage of a target RNA. Despitecommon features of RNA silencing, there are dif-ferences between the animal and plant kingdoms andalso between species (reviewed in Meister and Tuschl,2004; Mello and Conte, 2004; Baulcombe, 2006).1To whom correspondence should be addressed (email kriton@imbb.forth.gr).Key words: green fluorescent protein (GFP) transgene, local silencing, plant,RNA silencing, short-range silencing, systemic silencing.Abbreviations used: AGO, Argonaute; AS-silencing, antisense-inducedsilencing; CLSY1, CLASSY1; DCL, Dicer-like; dsRNA, double-stranded RNA;DRB, dedicated dsRNA-binding protein; GFP, green fluorescent protein; GUS,-glucuronidase; HEN1, HUA enhancer 1; miRNA, microRNA; nat-siRNA,natural antisense transcript-derived siRNA; NRPD1A, DNA-dependent RNApolymerase IV large subunit; POLIV, DNA-dependent RNA-polymerase IV;RDR, RNA-directed RNA polymerase; RISC, RNA-induced silencing complex;RNAi, RNA interference; SGS, suppressor of gene silencing; siRNA, short inter-fering RNA; S-silencing, sense-induced silencing; SDE3, suppressor ofdefective silencing 3; tasiRNA, trans-acting siRNA; VIGS, virus-induced genesilencing.

    In addition to the RNAi pathway described above,which characterizes the response of cells to exogen-ous sequences (viruses, transgenes etc.), multiple si-lencing pathways that result from the processing ofendogenous RNAs operate in plants. miRNAs (mi-croRNAs) are probably the best characterized cat-egory of these small RNAs. They are processed fromlonger primary transcripts, with extensive fold-backstructures that are transcribed from specific endogen-ous non-protein-coding RNA genes. These miRNAgenes are under the control of regular polymerase IIpromoters (Xie et al., 2005a; Megraw et al., 2006)and are expressed in a tissue- and time-specific man-ner (Valoczi et al., 2006). In plants, miRNAs areconsidered to function by providing sequence spe-cificity to a protein complex, which slices mRNAscomplementary to the miRNAs in a manner sim-ilar to siRNAs. Moreover, they have been shown tohave important roles in plant growth, stress responseand development. miRNA biogenesis and functionboth in plants and animals have been extensivelyreviewed elsewhere (Bartel, 2004; He and Hannon,2004; Murchison and Hannon, 2004; Chen, 2005).

    RNase III enzymes: These specifically bind to and cleave dsRNA. RNAi- andmiRNA-related enzymes that contain an RNAse III domain include theDrosha and all Dicer proteins.

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    In addition to the well-characterized categor-ies of siRNAs and miRNAs, new kinds of smallRNAs have more recently been revealed in plants.These small RNAs, tasiRNAs (trans-acting siRNAs)and nat-siRNAs (natural antisense transcript-derived siRNAs), have biosynthetic pathways dis-tinct from siRNAs and miRNAs. tasiRNAs are de-rived from non-protein-coding transcripts that aretargeted by miRNAs, and one of the two cleav-age products is converted into a double-strand byRDR6 (RNA-directed RNA polymerase 6) (Vazquezet al., 2004; Allen et al., 2005). SGS3 (suppressorof gene silencing 3) is required in this step, as itseems to protect the miRNA cleavage products fromdegradation (Yoshikawa et al., 2005). The double-stranded intermediate is then processed by DCL4(Dicer-like 4), in a complex with DRB4 (dedicateddsRNA-binding protein 4), to phased 21-nt tas-iRNAs that are incorporated into AGO (Argonaute)-containing complexes to target complementary se-quences (Vazquez et al., 2004; Allen et al., 2005;Gasciolli et al., 2005; Xie et al., 2005b; Yoshikawaet al., 2005; Adenot et al., 2006). The miRNA cleav-age determines the phase and is critical for the pro-duction of specific tasiRNAs (Allen et al., 2005). InArabidopsis thaliana there are at least five tasiRNA-producing loci, TAS1aTAS1c, TAS2 and TAS3. Incontrast with TAS1 and TAS2 which do not seemto be found in other organisms, TAS3 is conservedin land plants. nat-siRNAs are derived from over-lapping transcripts. In the one example (Borsaniet al., 2005) salt-stress-induced SRO5 (similar toRCD-one 5) overlaps with P5CDH (1-pyroline-5-carboxylate dehydrogenase), which is implicated inproline catabolism. Under salt-stress conditions theoverlapping region gives rise to a 24-nt nat-siRNA,whose production depends on DCL2, RDR6, SGS3and NRPD1A (DNA-dependent RNA polymeraseIV large subunit). Since a considerable percentage ofgenes in eukaryotes come in overlapping pairs, theabove mechanism could be mediating various cellu-lar responses.

    It is widely accepted that the main biological func-tions of RNA silencing are to assist in the resistanceto viruses and other exogenous RNAs (for example,viroids or RNA from agrobacterial T-DNA-encodedgenes), secure the stability of the genome through thesuppression of transposon activity and provide an ad-ditional mechanism for the regulation of endogenes(Baulcombe, 2004).

    In some organisms, including plants and the nem-atode Caenorhabditis elegans, silencing is a non-cell-autonomous event. In these systems, silencing initi-ation in one cell eventually leads to the silencing ofthe same sequences in a whole group of cells or evenin the whole organism. Although the mechanisms ofsilencing spread have commonalities between the twosystems, such as the involvement of RDRs in silenc-ing spread, the details of silencing movement seemto be largely divergent (Voinnet, 2005). In a recentreview, Hunter et al. (2006) summarize the silencingspread in C. elegans, whereas the present review willconcentrate on silencing spread only in plants. Wewill attempt to clarify the distinctions between thethree different types of silencing spread, short-rangesilencing spread, extensive local silencing spread andsystemic silencing. The genetic and molecular factorsinvolved in systemic silencing are largely unknown.However, three recent studies have advanced our un-derstanding of short and extensive local spread, usingArabidopsis as a model system (Dunoyer et al., 2005,2007; Smith et al., 2007).

    Exogenous silencing activationManifestations of silencing spreadSystemic spread of silencing was first convincinglyshown in Nicotiana tabacum and Nicotiana benthamiana(Palauqui et al., 1997; Voinnet and Baulcombe,1997; Palauqui and Vaucheret, 1998; and reviewedin Fagard and Vaucheret, 2000). It was shown tomanifest as the sequence-specific suppression of anectopically overexpressed gene whether the sequencewas of endogenous origin (Palauqui et al., 1997;Fagard and Vaucheret, 2000) or of exogenous origin

    Argonaute (AGO) proteins: These are the catalytic components of the RISC, which is responsible for the post-transcriptional gene silencing phenomena. AGOproteins bind siRNAs and have endonuclease activity, which is known as slicer activity, directed against mRNA strands that are complementary to their boundsiRNA fragment.

    T-DNA: This is a DNA fragment of the Agrobacterium tumefaciens Ti plasmid which can be transferred by non-homologous recombination into the genomic DNAof the host cell.

    14 C The Authors Journal compilation C 2008 Portland Press Ltd

  • RNA silencing movement in plants Review

    (Voinnet et al., 1998). Grafting experiments have alsodemonstrated silencing spread in tomato (Shaharud-din et al., 2006)