Viral suppression of RNA silencing

Gene silencing (RNA silencing) plays a fundamental role in antiviral defense in plants, fungi and invertebrates. Viruses encode proteins that suppress gene silencing to counter host defense. Viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera and some viruses of insects and mammals. Recent studies have revealed that VSRs counter host defense and interfere with host gene regulation by interacting with RNA or important components of the RNA silencing pathway. Here, we review the current understanding of the complex mechanisms of VSRs that have been revealed by recent studies.

RNA silencing:From discovery and elucidation to application and perspectives

RNA silencing(or RNA interference,RNAi)is a conserved mechanism for regulating gene expression in eukaryotes.The discovery of natural trans-kingdom RNAi indicated that small RNAs act as signaling molecules and enable communication between organisms in different kingdoms.The phenomenon and potential mechanisms of trans-kingdom RNAi are among the most exciting research topics.To better understand trans-kingdom RNAi,we review the history of the discovery and elucidation of RNAi mechanisms.Based on canonical RNAi mechanisms,we summarize the major points of divergence around RNAi pathways in the main eukaryotes’kingdoms,including plants,animals,and fungi.We review the representative incidents associated with the mechanisms and applications of trans-kingdom RNAi in crop protection,and discuss the critical factors that should be considered to develop successful trans-kingdom RNAi-based crop protection strategies.

The role of virus-derived small interfering RNAs in RNA silencing in plants

Antiviral defense is one of the important roles of RNA silencing in plants. Virus-derived small interfering RNAs (vsiRNAs) are found in the infected host cells, indicating that the host RNA silencing machinery can target viral RNAs for destruction. With the development of high-throughput sequencing of vsiRNAs, recent genetic studies have shed light on the origin and composition of vsiRNAs and their potential functions in the regulation of gene expression. Here, we briefly describe the origin and biogenesis of vsiRNAs, and review the recent discoveries regarding vsiRNA-mediated RNA silencing of viral genomes and host transcripts. This will better our understanding of virus pathogenicity and RNA silencing-related host-pathogen inter- actions in plants.

RNA silencing movement in plants

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.

RNA silencing in fungi

RNA silencing is an evolutionarily conserved mechanism in eukaryotic organisms induced by double-stranded RNA(dsRNA)and plays an essential role in regulating gene expression and maintaining genome stability.RNA silencing occurs at both posttranscriptional levels through sequence-specific RNA degradation or translational repression and at transcriptional levels through RNA-directed DNA methylation and/or hetero-chromatin formation.RNA silencing pathways have been relatively well characterized in plants and animals,and are now also being widely investigated in diverse fungi,some of which are important plant pathogens.This review focuses primarily on the current understanding of the dsRNA-mediated posttranscriptional gene silencing pro-cesses in fungi,but also discusses briefly the known gene silencing pathways that appear to be independent of the RNA silencing machineries.We review RNA silencing studies for a variety of fungi and highlight some of the mechanistic differences observed in different fungal organisms.As RNA silencing is being exploited as a technology in gene function studies in fungi as well as in engineering anti-fungal resistance in plants and animals,we also discuss the recent progress towards understanding dsRNA uptake in fungi.

A non-structural protein encoded by Rice Dwarf Virus targets to the nucleus and chloroplast and inhibits local RNA silencing

RNA silencing is a potent antiviral mechanism in plants and animals. As a counter-defense, many viruses studied to date encode one or more viral suppressors of RNA silencing(VSR). In the latter case, how different VSRs encoded by a virus function in silencing remains to be fully understood. We previously showed that the nonstructural protein Pns10 of a Phytoreovirus, Rice dwarf virus(RDV), functions as a VSR. Here we present evidence that another nonstructural protein, Pns11, also functions as a VSR. While Pns10 was localized in the cytoplasm, Pns11 was localized both in the nucleus and chloroplasts. Pns11 has two bipartite nuclear localization signals(NLSs), which were required for nuclear as well as chloroplastic localization. The NLSs were also required for the silencing activities of Pns11. This is the first report that multiple VSRs encoded by a virus are localized in different subcellular compartments, and that a viral protein can be targeted to both the nucleus and chloroplast. These findings may have broad significance in studying the subcellular targeting of VSRs and other viral proteins in viral-host interactions.

RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content,Alters Transcripts of Phytic Acid Biosynthetic Genes,and Modulates Mineral Distribution in Rice Seeds

Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.

High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide （nt） small interfering RNA （siRNA） and microRNA （miRNA）. Infection of plants with viral pathogens or transformation of plants with RNA interference （RNAi） constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA （satRNA） infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus （CMV） Y-satellite RNA （Y-Sat）. We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA （hpRNA）-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）

Small Interfering RNA Targeting TNF-α Gene Significantly Attenuates Renal Ischemia-Reperfusion Injury in Mice

Tumor necrosis factor-alpha（TNF-α） has been found to be centrally involved in the development of ischemia-reperfusion injury（IRI）-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interfering RNA（si RNA） may protect renal IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. TNF-α si RNA was administered intravenously to silence the expression of TNF-α. The therapeutic effects of si RNA were evaluated in terms of renal function, histological examination, and overall survival following lethal IRI. A single systemic injection of TNF-α si RNA resulted in significant knockdown of TNF-α expression in ischemia-reperfusion injured kidney. In comparison with control mice, levels of BUN and serum creatinine were significantly reduced in mice treated with si RNA. Pathological examination demonstrated that tissue damage caused by IRI was markedly reduced as a result of TNF-α si RNA treatment. Furthermore, survival experiments showed that nearly 90% of control mice died from lethal IRI, whereas more than 50% of si RNApretreated mice survived until the end of the eight-day observation period. We have demonstrated for the first time that silencing TNF-α by specific si RNA can significantly reduce renal IRI and protect mice against lethal kidney ischemia, highlighting the potential for si RNA-based clinical therapy.

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 Arabi- dopsis 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 bio- informatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of ef- fective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.

Recent advances and emerging trends in antiviral defense networking in rice

Plants and viruses coexist in the natural ecosystem for extended periods of time,interacting with each other and even coevolving,maintaining a dynamic balance between plant disease resistance and virus pathogenicity.During virus–host interactions,plants often exhibit abnormal growth and development.However,plants do not passively withstand virus attacks but have evolved sophisticated and effective defense mechanisms to resist,limit,or undermine virus infections.It is widely believed that the initial stage of infection features the most intense interactions between the virus and the host and the greatest variety of activated signal transduction pathways.This review describes the most recent findings in rice antiviral research and discusses a variety of rice antiviral molecular mechanisms,including those based on R genes and recessive resistance,RNA silencing,phytohormone signaling,autophagy and WUSmediated antiviral immunity.Finally,we discuss the challenges and future prospects of breeding rice for enhanced virus resistance.

A novel strategy to enhance resistance to Cucumber mosaic virus in tomato by grafting to transgenic rootstocks

Cucumber mosaic virus（CMV） can infect a wide range of host species. For the lacking of CMV resistant varieties of tomato, RNA interference（RNAi） can be used as a fast and effective method for the generation of transgenic resistant varieties. In this current study, five intron-spliced hairpin RNA（ihp RNA） plant expression vectors aimed at five genes of CMV have been constructed. Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with expression vectors. Highly resistant generations of transgenic plants were employed as rootstocks and grafted onto non-transgenic tomatoes that resulted in the successful transfer of resistance to the scions. Using a novel method of plant cuttings for rootstock propagation, we obtained large quantities of disease-resistant material. Further, this method produces scions that can remain undetectable for transgenic resistance marker genes that may provide novel approaches to evade collective concerns about genetically-modified organism（GMO） biosafety.

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotic RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved functional amino acids between archaeal and eukaryotic Piwi domains suggested their fimctional similarity. Our results provide new clues to the evolution of RNA silencing pathways.

Characterization of Argonaute family members in the silkworm, Bombyx mori

The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgol and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5＇ end of the small RNA guide strand and of the conserved （aspartate, aspartate and histidine [DDH]） motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.