Virus-derived Small RNAs Research Articles

RNA Silencing against Geminivirus: Complementary Action of Posttranscriptional Gene Silencing and Transcriptional Gene Silencing in Host Recovery

RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.

Mechanism of Induction and Suppression of Antiviral Immunity Directed by Virus-Derived Small RNAs in Drosophila

The small RNA-directed viral immunity pathway in plants and invertebrates begins with the production by Dicer nuclease of virus-derived siRNAs (viRNAs), which guide specific antiviral silencing by Argonaute protein in an RNA-induced silencing complex (RISC). Molecular identity of the viral RNA precursor of viRNAs remains a matter of debate. Using Flock house virus (FHV) infection of Drosophila as a model, we show that replication of FHV positive-strand RNA genome produces an approximately 400 bp dsRNA from its 5' terminus that serves as the major Dicer-2 substrate. ViRNAs thus generated are loaded in Argonaute-2 and methylated at their 3' ends. Notably, FHV-encoded RNAi suppressor B2 protein interacts with both viral dsRNA and RNA replicase and inhibits production of the 5'-terminal viRNAs. Our findings, therefore, provide a model in which small RNA-directed viral immunity is induced during the initiation of viral progeny (+)RNA synthesis and suppressed by B2 inside the viral RNA replication complex.

Endogenous small RNAs and antibacterial immunity in plants

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins

Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway.

The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased ( approximately 35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.

Hierarchical Action and Inhibition of Plant Dicer-Like Proteins in Antiviral Defense

The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by Arabidopsis Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non-cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.

Isolation and Cloning of Small RNAs from Virus‐Infected Plants

RNA silencing is an evolutionarily conserved, RNA-mediated, regulatory system of eukaryotic organisms that also acts as an antiviral system in plants and animals. A defining feature of RNA silencing is the presence of 21- to 26-nucleotide small RNAs corresponding to the silencing target sequence, which in virus-infected plants are derived from the viral genome. The virus-derived small RNAs have a nonrandom distribution along the viral genome, suggesting hotspots for viral small-RNA generation. The isolated small RNAs can be used either as probes for hybridization studies or for directional cloning in order to get detailed information about their sizes, origins, and functions. This unit describes an isotope-free small-RNA cloning procedure that utilizes unmodified small RNAs and is routinely used to characterize small RNAs from various plant tissues.

The complex interplay between plant viruses and host RNA-silencing pathways

RNA silencing was originally identified as an immune system targeted against transposons and viruses, but is now also recognized as a major regulatory process that affects all layers of host gene expression through the activities of various small RNA species. Recent work in plants and animals indicates that viruses not only suppress, but can also exploit, endogenous RNA silencing pathways to redirect host gene expression. There are also indications that cellular, as opposed to virus-derived small RNAs, might well constitute an unsuspected defense layer against foreign nucleic acids. This complex interplay has implications in the context of disease resistance and evolution of viral genomes.