RNA silencing and plant viral diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.     

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.     

植物抗病毒分子机制

在与植物病毒的长期斗争中,植物进化出多种抗病毒机制,其中RNA沉默和R基因介导的病毒抗性是最受人们关注的两种机制.一方面,RNA沉默是植物抵抗病毒侵染的重要手段.植物在病毒侵染过程中可形成病毒来源的双链RNA,经过DCL蛋白的切割、加工形成sRNA,与AGO蛋白结合形成RISC指导病毒RNA的沉默,用于清除病毒.相应地,病毒在与植物的竞争中进化出RNA沉默抑制子,抑制宿主RNA沉默系统以逃避宿主RNA沉默抗病毒反应,增强致病能力.另一方面,植物也进化出R基因介导植物对包括病毒在内的多类病原的抗性.R蛋白直接或间接识别病毒因子,通过一系列的信号转导途径激活植物防御反应,限制病毒的进一步侵染.对植物抗病毒的研究有助于人们对植物抗病分子基础的理解,有重要的科学意义和潜在应用价值.本文综述了植物抗病毒分子机制的重要进展.     

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.     

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.Following virus entry and replication, successful infection of a host requires viral spread to distal parts of the organism through the vascular tissue. In plants, virus movement involves mostly symplastic trafficking of different viral components through the connections of plasmodesmata (PD) (13). With this aim, plant viruses encode one or more movement proteins (MPs), which allow viral genomes to cross the host cell wall by altering the size exclusion limit (SEL) or the structure of PD (6, 11). Plant viruses have evolved distinct mechanisms to move their genomes within the host. These mechanisms can be grouped into two general strategies: one in which the genome is transported in the form of a nucleoprotein complex (NPC) and another in which nucleic acids are encapsidated and move as virus particles. In both cases, besides altering PD SEL, MPs are involved either in NPC assembly or in forming tubules traversing modified PD and helping transport of either NPC or virions to the neighboring cell. Within these two major strategies, there exists a wide range of variability in terms of the number and type of viral and host proteins helping MPs to mediate virus spread within the host (11).In spite of such variability, several different MPs have been classified into a 30K superfamily; these MPs, from 20 genera including both RNA and DNA genome viruses, are structurally related to the 30-kDa MP of Tobacco mosaic virus (TMV), independent of the movement strategy followed (14). Members of this family have a common core of predicted secondary structure elements (α-helices and β-elements) containing a nucleic acid binding domain. Distinct MPs belong to this family, including several tubule-forming MPs, although these are phylogenetically separated from the other members (14). Thus, 30K superfamily MPs are closely related, and some of them are functionally interchangeable in the viral context (2, 20). In particular, MPs from five distinct genera with an RNA genome can successfully replace the corresponding gene of Alfalfa mosaic virus (AMV) (19), indicating that one or more basic and fundamental movement properties might be associated with the common 30K structural core.Among all known plant viruses, only three viral families have evolved a DNA genome: Geminiviridae, Caulimoviridae, and Nanoviridae (6). One possible explanation for this restriction is that endogenous cell-to-cell transport via PD is specialized to use RNA as the communication and signaling molecule (12). To circumvent this restriction, and to allow the efficient exploitation of endogenous transport machineries, DNA genome viruses have evolved appropriate mechanisms involving their MPs. Interestingly, Begomovirus and Caulimovirus MPs also belong to the 30K superfamily discussed above (14). The MP encoded by Cauliflower mosaic virus (CaMV), the type member of Caulimoviridae, forms tubules that guide the movement of encapsidated virus via an indirect MP-virion interaction (16, 21), whereas geminivirus MPs selectively bind their genomes and transport them as NPCs (6, 9, 17). In this study, we investigated the evolutionary convergence of MPs encoded by DNA and RNA viruses by testing their exchangeability in the viral context.     

RNA silencing pathways of plants: silencing and its suppression by plant DNA viruses

RNA silencing refers to processes that depend on small (s)RNAs to regulate the expression of eukaryotic genomes. In plants, these processes play critical roles in development, in responses to a wide array of stresses, in maintaining genome integrity and in defense against viral and bacterial pathogens. We provide here an updated view on the array of endogenous sRNA pathways, including microRNAs (miRNAs), discovered in the model plant Arabidopsis, which are also the basis for antiviral silencing. We emphasize the current knowledge as well as the recent advances made on understanding the defense and counter-defense strategies evolved in the arms race between plants and DNA viruses on both the nuclear and the cytoplasmic front. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

RNA 沉默的病毒抑制子

RNA 沉默是一种在真核生物体内普遍保守的、通过核酸序列特异性的相互作用来抑制基因表达的调控机制 . RNA 沉默的一种重要生物学效应是防御病毒的侵染,而针对寄主的这种防御机制,许多植物病毒已演化通过编码 RNA 沉默的抑制子来克服这种防御反应 . 目前,已从植物、动物和人类病毒中鉴定了 20 多种 RNA 沉默的抑制子,围绕抑制子的鉴定和作用机理研究已成为病毒学研究的一个热点 . 对 RNA 沉默抑制子的发现、鉴定方法、作用机理及与病毒病症状形成的关系、动物病毒的沉默抑制子等方面的最新进展做了综述,并对沉默抑制子的应用和存在的问题进行了讨论 .     

Use of viral replicons for the expression of genes in plants

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.     

Micro RNA and viral infections in mammals

RNA silencing plays an important role in development through the action of micro (mi) RNAs that fine tune the expression of a large portion of the genome. But, in plants and insects, it is also a very important player in innate immune responses, especially in antiviral defense. It is now well established that the RNA silencing machinery targets plant as well as insect viruses. While the genetic basis underlying this defense mechanism in these organisms starts being elucidated, much less is known about the possible antiviral role of RNA silencing in mammals. In order to identify siRNAs coming from viruses in infected human cells, small RNAs from cells infected with RNA viruses, such as hepatitis C virus, yellow fever virus or HIV-1, were cloned and sequenced, but no virus-specific siRNAs could be detected. On the contrary, viral small RNAs were found in cells infected by the DNA virus Epstein-Barr. A closer look at these revealed that they were not siRNAs, but rather resembled miRNAs. This finding indicated that, rather than being targeted by RNA silencing, human DNA viruses seem to have evolved their own miRNAs to modulate the expression of host genes. This primary observation has been extended to other members of the herpesvirus family as well as other DNA viruses such as the polyomavirus SV40. Viral miRNAs have the potential to act both in cis to regulate expression of viral genes, or in trans on host genes. There are good indications for the cis mode of action, but the identification of cellular targets of these small viral regulators is only in its infancy.     

The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K-Akt-mTOR signalling pathway

The successful replication of mammalian DNA viruses requires that they gain control of key cellular signalling pathways that affect broad aspects of cellular macromolecular synthesis, metabolism, growth and survival. The phosphatidylinositol 3'-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway is one such pathway. Mammalian DNA viruses have evolved various mechanisms to activate this pathway to obtain the benefits of Akt activation, including the maintenance of translation through the activation of mTOR. In addition, viruses must overcome the inhibition of this pathway that results from the activation of cellular stress responses during viral infection. This Review will discuss the range of mechanisms that mammalian DNA viruses use to activate this pathway, as well as the multiple mechanisms these viruses have evolved to circumvent inhibitory stress signalling.     

Movement protein Pns6 of rice dwarf phytoreovirus has both ATPase and RNA binding activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5'- and 3'-terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions.     

Identification of an RNA silencing suppressor from a plant double-stranded RNA virus

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus (RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of beta-glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.     

CRISPR/Cas‐based tools for the targeted control of plant viruses

Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the “Genetic modification” issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas‐based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research.