Molecular characterization of geminivirus-derived small RNAs in different plant species

Akbergenov, Rashid; Si‐Ammour, Azeddine; Blevins, Todd; Imran, Amin; Kutter, Claudia; Vanderschuren, Hervé; Zhang, Peng; Gruissem, Wilhelm; Meins, Frederick; Höhn, Thomas; Pooggin, Mikhail M.

doi:10.1093/nar/gkj447

Cited by 246 publications

(197 citation statements)

References 54 publications

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“…The profile of ⌬CP-specific siRNAs is generally consistent with previous reports (6,9,17). Their size was predominantly 21 nt in all but the dcl4 plants, where the DCL2-generated 22-nt siRNA predominates (Fig.…”

Section: Resultssupporting

confidence: 90%

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

Morris³

2008

Proc. Natl. Acad. Sci. U.S.A.

386

441

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Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.interpathway regulation ͉ plant antiviral defense R NA silencing is a cellular mechanism that uses small RNA molecules (21-30 nt in length) as sequence-specific mediators to regulate the expression of a diverse array of genes at the transcriptional, posttranscriptional, or translational levels (1). In plants, these very small RNA species are termed small interfering RNAs (siRNAs) or micro RNAs (miRNAs) depending on the source of their precursors. They are generated by a family of double-stranded RNA (dsRNA)-specific RNases called Dicerlike ribonucleases (DCLs) (2). Once produced, the siRNAs and miRNAs are recruited by Argonaute proteins (AGOs) into RNA-induced silencing complexes (RISCs) to direct the cleavage or translational repression of homologous mRNAs or to remodel the homologous chromosomal DNA to achieve transcriptional silencing (3). Another family of dsRNA-binding proteins (DRBs) has been found to modulate the function of DCLs (4). Plants also encode RNA-dependent RNA polymerases (RDRs) to produce some of the dsRNA precursors that serve as templates for DCLs (2). In Arabidopsis, 4 DCLs, 10 AGOs, 5 DRBs, and up to 6 RDRs have been identified. They participate in at least four different endogenous RNA silencing pathways to achieve spatial and temporal regulation of gene expression throughout the plant life cycle and to condition the plant response to biotic and abiotic stresses (5).Although the plant RNA silencing mechanism was first revealed through studies aimed to unravel the complexity of plant antiviral defense strategies, the details of plant antiviral RNA silencing pathway(s) are far...

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Section: Resultssupporting

confidence: 90%

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

Morris³

2008

Proc. Natl. Acad. Sci. U.S.A.

386

441

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“…In addition to the endogenous siRNAs, viruses and transgenes also give rise to siRNAs (reviewed in Brodersen and Voinnet, 2006;Vaucheret, 2006). We and others have shown that all types of siRNAs (except nat-siRNAs that have not been tested) are methylated in vivo in a HEN1-dependent manner (Akbergenov et al, 2006;Ebhardt et al, 2005;Li et al, 2005). Our biochemical studies with recombinant HEN1 protein also demonstrated that HEN1 is a siRNA methyltransferase (Yang et al, 2006).…”

Section: Introductionsupporting

confidence: 54%

Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo

Yang¹,

Vilkaitis²,

Yu³

et al. 2007

Methods in Enzymology

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The biogenesis of microRNAs (miRNAs) in plants is similar to that in animals, however, the processing of plant miRNAs consists of an additional step, the methylation of the miRNAs on the 3′ terminal nucleotides. The enzyme that methylates Arabidopsis miRNAs is encoded by a gene named HEN1, which has been shown genetically to be required for miRNA biogenesis in vivo. Small interfering RNAs (siRNAs) are also methylated in vivo in a HEN1-dependent manner. Our biochemical studies demonstrated that HEN1 is a methyltransferase acting on both miRNAs and siRNAs in vitro. HEN1 recognizes 21 to 24 nt small RNA duplexes, which are the products of Dicer-like enzymes, and transfers a methyl group from S-adenosylmethionine (SAM) to the 2′ OH of the last nucleotides of the small RNA duplexes. Here we describe methods to characterize the biochemical activities of the HEN1 protein both in vitro and in vivo, and methods to analyze the methylation status of small RNAs in vivo.

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“…Recent studies show that infections by ssDNA geminiviruses trigger accumulation of different vsRNA classes (14). This likely reflects the action of distinct DCLs that possibly use dsRNA resulting from annealing of converging sense͞antisense transcripts as substrate (15).…”

mentioning

confidence: 99%

RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins

Moissiard

Voinnet

2006

Proc. Natl. Acad. Sci. U.S.A.

215

172

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RNA silencing is an ancient mechanism of gene regulation with important antiviral roles in plants and insects. Although induction of RNA silencing by RNA viruses has been well documented in plants, the interactions between DNA viruses and the host silencing machinery remain poorly understood. We investigate this question with cauliflower mosaic virus (CaMV), a dsDNA virus that expresses its genome through the polycistronic 35S RNA, which carries an unusually extensive secondary structure known as translational leader. We show that CaMV-derived siRNAs accumulate in turnip- and Arabidopsis -infected plants and that the leader is a major, albeit not exclusive, source for those molecules. Biogenesis of leader-derived siRNA requires the coordinated and hierarchical action of the four Arabidopsis Dicer-like (DCL) proteins. Our study also uncovers a “facilitating” role exerted by the microRNA biosynthetic enzyme DCL1 on accumulation of DCL2-, DCL3-, and DCL4-dependent siRNAs derived from the 35S leader. This feature of DCL1 defines a small RNA biosynthetic pathway that might have relevance for endogenous gene regulation. Several leader-derived siRNAs were found to bear near-perfect sequence complementarity to Arabidopsis transcripts, and, using a sensor transgene, we provide direct evidence that at least one of those molecules acts as a bona fide siRNA in infected turnip. Extensive bioinformatics searches identified >100 transcripts potentially targeted by CaMV-derived siRNAs, several of which are effectively down-regulated during infection. The implications of virus-directed silencing of host gene expression are discussed.

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Molecular characterization of geminivirus-derived small RNAs in different plant species

Cited by 246 publications

References 54 publications

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo

RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins

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