Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context.

BACKGROUND: In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. METHODS: In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts. RESULTS: DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci. CONCLUSIONS: We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.

Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA.

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.

RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants.

Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and 'G-U wobbles' on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.

Endogenous activated small interfering RNAs in virus-infected Brassicaceae crops show a common host gene-silencing pattern affecting photosynthesis and stress response.

Viral infections are accompanied by a massive production of small interfering RNAs (siRNAs) of plant origin, such as virus-activated (va)siRNAs, which drive the widespread silencing of host gene expression, and whose effects in plant pathogen interactions remain unknown. By combining phenotyping and molecular analyses, we characterized vasiRNAs that are associated with typical mosaic symptoms of cauliflower mosaic virus infection in two crops, turnip (Brassica rapa) and oilseed rape (Brassica napus), and the reference plant Arabidopsis thaliana. We identified 15 loci in the three infected plant species, whose transcripts originate vasiRNAs. These loci appear to be generally affected by virus infections in Brassicaceae and encode factors that are centrally involved in photosynthesis and stress response, such as Rubisco activase (RCA), senescence-associated protein, heat shock protein HSP70, light harvesting complex, and membrane-related protein CP5. During infection, the expression of these factors is significantly downregulated, suggesting that their silencing is a central component of the plant's response to virus infections. Further findings indicate an important role for 22 nt long vasiRNAs in the plant's endogenous RNA silencing response. Our study considerably enhances knowledge about the new class of vasiRNAs that are triggered in virus-infected plants and will help to advance strategies for the engineering of gene clusters involved in the development of crop diseases.

Regulation of plant antiviral defense genes via host RNA-silencing mechanisms.

BACKGROUND: Plants in nature or crops in the field interact with a multitude of beneficial or parasitic organisms, including bacteria, fungi and viruses. Viruses are highly specialized to infect a limited range of host plants, leading in extreme cases to the full invasion of the host and a diseased phenotype. Resistance to viruses can be mediated by various passive or active mechanisms, including the RNA-silencing machinery and the innate immune system. MAIN TEXT: RNA-silencing mechanisms may inhibit viral replication, while viral components can elicit the innate immune system. Viruses that successfully enter the plant cell can elicit pattern-triggered immunity (PTI), albeit by yet unknown mechanisms. As a counter defense, viruses suppress PTI. Furthermore, viral Avirulence proteins (Avr) may be detected by intracellular immune receptors (Resistance proteins) to elicit effector-triggered immunity (ETI). ETI often culminates in a localized programmed cell death reaction, the hypersensitive response (HR), and is accompanied by a potent systemic defense response. In a dichotomous view, RNA silencing and innate immunity are seen as two separate mechanisms of resistance. Here, we review the intricate connections and similarities between these two regulatory systems, which are collectively required to ensure plant fitness and resilience. CONCLUSIONS: The detailed understanding of immune regulation at the transcriptional level provides novel opportunities for enhancing plant resistance to viruses by RNA-based technologies. However, extensive use of RNA technologies requires a thorough understanding of the molecular mechanisms of RNA gene regulation. We describe the main examples of host RNA-mediated regulation of virus resistance.

High throughput sequencing from Angolan citrus accessions discloses the presence of emerging CTV strains.

BACKGROUND: Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use. METHODS: Double-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3). RESULTS: From the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs. CONCLUSION: Phylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).

Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro.

In response to a viral infection, the plant's RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control.

The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm.

A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their full-length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.

Distribution of Small RNAs Along Transposable Elements in Vitis vinifera During Somatic Embryogenesis.

Metaviridae is a family of reverse-transcribing viruses, closely related to retroviruses; they exist within their host's DNA as transposable elements. Transposable element study requires the use of specialized tools, in part because of their repetitive nature. By combining data from transcript RNA-Seq, small RNA-Seq, and parallel analysis of RNA ends-Seq from grapevine somatic embryos, we set up a bioinformatics flowchart that could be able to assemble and identify transposable elements.

A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

Viral induction and suppression of RNA silencing in plants.

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Structural and functional analysis of viral siRNAs.

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

Diversity of viral RNA silencing suppressors and their involvement in virus-specific symptoms.

RNA silencing is an evolutionarily conserved and homology-dependent gene inactivation system that regulates most biological processes at either the transcriptional or post-transcriptional level. In plants, insects and certain mammalian systems, RNA silencing constitutes the basis of the antiviral defense mechanism. To counteract RNA silencing-based antiviral responses viruses adopt strategies of replication and host invasion that include mechanisms of RNA silencing suppression. Indeed, viruses can express proteins known as RNA silencing suppressors (RSSs). Over the last two decades, silencing studies in plant virology have been largely devoted to the discovery and description of RSSs. The result has been exciting and these studies have revealed (i) an incredible diversity of proteins and mechanisms of RSSs belonging to various viral taxonomic groups, (ii) the multifunctionality of RSSs: they can fulfill several functions during viral infection and target one or more key points in the RNA silencing machinery. Some RSSs of model viral systems have been the subject of exceptional in-depth studies; they have proven to be real molecular tools for studying plant physiology, plant biology and virus-plant interactions, even in some cases extending the knowledge of the response of plants to other biotic and abiotic stressors. RSS diversity in phylogenesis, in mechanism of action and the frequent presence of more than one RSS in a single viral genome all suggest that they are extremely plastic in evolving to overcome host defenses. In this chapter, we present and discuss the most recent findings related to the well-studied RSSs of four viral taxonomic groups: geminiviruses, potyviruses, tombusviruses and cucumoviruses.

Identification of Known and Novel Arundo donax L. MicroRNAs and Their Targets Using High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are a class of non-coding molecules involved in the regulation of a variety of biological processes. They have been identified and characterized in several plant species, but only limited data are available for Arundo donax L., one of the most promising bioenergy crops. Here we identified, for the first time, A. donax conserved and novel miRNAs together with their targets, through a combined analysis of high-throughput sequencing of small RNAs, transcriptome and degradome data. A total of 134 conserved miRNAs, belonging to 45 families, and 27 novel miRNA candidates were identified, along with the corresponding primary and precursor miRNA sequences. A total of 96 targets, 69 for known miRNAs and 27 for novel miRNA candidates, were also identified by degradome analysis and selected slice sites were validated by 5'-RACE. The identified set of conserved and novel candidate miRNAs, together with their targets, extends our knowledge about miRNAs in monocots and pave the way to further investigations on miRNAs-mediated regulatory processes in A. donax, Poaceae and other bioenergy crops.

Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Plant RNA silencing in viral defence.

RNA silencing is described in plants and insects as a defence mechanism against foreign nucleic acids, such as invading viruses. The RNA silencing-based antiviral defence involves the production of virus-derived small interfering RNAs and their association to effector proteins, which together drive the sequence specific inactivation of viruses. The entire process of antiviral defence 'borrows' several plant factors involved in other specialized RNA silencing endogenous pathways. Different viruses use variable strategies to infect different host plants, which render the antiviral RNA silencing a complex phenomenon far to be completely clarified. This chapter reports current advances in understanding the main steps of the plant's RNA-silencing response to viral invasion and discusses some of the key questions still to be answered.

Composted Municipal Green Waste Infused with Biocontrol Agents to Control Plant Parasitic Nematodes-A Review.

The last few years have witnessed the emergence of alternative measures to control plant parasitic nematodes (PPNs). We briefly reviewed the potential of compost and the direct or indirect roles of soil-dwelling organisms against PPNs. We compiled and assessed the most intensively researched factors of suppressivity. Municipal green waste (MGW) was identified and profiled. We found that compost, with or without beneficial microorganisms as biocontrol agents (BCAs) against PPNs, were shown to have mechanisms for the control of plant parasitic nematodes. Compost supports a diverse microbiome, introduces and enhances populations of antagonistic microorganisms, releases nematicidal compounds, increases the tolerance and resistance of plants, and encourages the establishment of a "soil environment" that is unsuitable for PPNs. Our compilation of recent papers reveals that while the scope of research on compost and BCAs is extensive, the role of MGW-based compost (MGWC) in the control of PPNs has been given less attention. We conclude that the most environmentally friendly and long-term, sustainable form of PPN control is to encourage and enhance the soil microbiome. MGW is a valuable resource material produced in significant amounts worldwide. More studies are suggested on the use of MGWC, because it has a considerable potential to create and maintain soil suppressivity against PPNs. To expand knowledge, future research directions shall include trials investigating MGWC, inoculated with BCAs.

Viral and subviral derived small RNAs as pathogenic determinants in plants and insects.

The phenotypic manifestations of disease induced by viruses and subviral infectious entities are the result of complex molecular interactions between host and viral factors. The viral determinants of the diseased phenotype have traditionally been sought at the level of structural or non-structural proteins. However, the discovery of RNA silencing mechanisms has led to speculations that determinants of the diseased phenotype are caused by viral nucleic acid sequences in addition to proteins. RNA silencing is a gene regulation mechanism conserved within eukaryotic kingdoms (with the exception of some yeast species), and in plants and insects it also functions as an antiviral mechanism. Non-coding RNAs of viral origin, ranging in size from 21 to 24 nucleotides (viral small interfering RNAs, vsiRNAs) accumulate in virus-infected tissues and organs, in some cases to comparable levels as the entire complement of host-encoded small interfering RNAs. Upon incorporation into RNA-induced silencing complexes, vsiRNAs can mediate cleavage or induce translational inhibition of nucleic acid targets in a sequence-specific manner. This review focuses on recent findings that suggest an increased complexity of small RNA-based interactions between virus and host. We mainly address plant viruses, but where applicable discuss insect viruses as well. Prominence is given to studies that have indisputably demonstrated that vsiRNAs determine diseased phenotype by either carrying sequence determinants or, indirectly, by altering host-gene regulatory pathways. Results from these studies suggest biotechnological applications, which are also discussed.