Complementation between Two Tospoviruses Facilitates the Systemic Movement of a Plant Virus Silencing Suppressor in an Otherwise Restrictive Host

Background

New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature.

Methodology/Principal Findings

Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs.

Conclusion/Significance

Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.     

RNA沉默机制及其抗病毒应用

RNA沉默是发生在植物 (转录后基因沉默或共抑制 )、动物 (RNA干扰 )和真菌 (消除作用 )等真核生物细胞中的一种对外源遗传因子 (转座子、转基因或病毒 )的特异性和高效率的降解机制。随着对植物病毒分子遗传学认识的加深和对寄主防御系统研究的深入 ,发现了许多控制植物病毒病的方法 ,不过迄今为止最为成功的是通过RNA沉默机制获取抗病毒工程植株。在陈述了RNA沉默机制的研究最新进展基础上 ,提出了如何充分利用该机制进行植物抗病毒转基因研究。     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.     

转录后基因沉默--植物抵御外来病毒入侵的一种机制

基因沉默是近几年来在转基因植物中发现的一种后生遗传现象。基因沉默大体可以分为两类：位置效应引起的基因沉默和同源依赖的基因沉默。其中,同源依赖的基因沉默又可以分为转录水平的基因沉默和转录后水平的基因沉默。基因沉默的发现使得人们对植物和病毒的相互关系有了一个新的认识。基因沉默研究中所发现的转录后基因沉默现象是植物抵御病毒入侵、保持自身基因组完整性的一种防御机制,是植物与病毒共进化的结果。对于沉默产生的机理,尤其是转录后基因沉默,已经提出不少模型,但是都未能较全面地解释基因沉默中出现的各种实验现象。该文现就实验所取得的相关结果、转录后基因沉默机制和植物对病毒防御机制的相互关系,以及其研究进展进行综述。     

假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子

为了研究中国胜红蓟黄脉病毒(Ageratum yellow vein Chin virus,AYVCNV)和假马鞭曲叶病毒(Stachytarpheta leafcurl virus,StaLCV)C4蛋白的功能,利用烟草脆裂病毒(Tobacco rattle virus,TRV)载体在本氏烟(Nicotianabenthamiana)中分别表达了这两种病毒的C4蛋白,结果发现它们均能在本氏烟中引起类似于病毒侵染的症状,推测AYVCNV和StaLCV的C4蛋白是病毒的致病因子;在RNA沉默的抑制试验中,AYVCNV和StaLCV的C4蛋白均能够在表达gfp基因的转基因本氏烟(16c)上抑制由gfp基因正义链引起的基因沉默的建立,证明它们都是RNA沉默的抑制子。     

Crystal structure of p19--a universal suppressor of RNA silencing

RNA silencing in plants has an antiviral role and, consequently, plant viruses encode counter-defensive suppressor proteins that block this process. The recently reported crystal structure of two Tombusvirus suppressor proteins reveals a novel RNA-binding structure and illustrates precisely how the silencing mechanism is blocked. These suppressor protein structures, combined with molecular analyses of their effects in animal and plant cells, are informative about RNA silencing mechanisms. They also suggest various ways that Tombusvirus suppressors can be used to investigate RNA silencing in plants and animals.     

Escape of a plant virus from amplicon-mediated RNA silencing is associated with biotic or abiotic stress

Strong RNA silencing was induced in plants transformed with an amplicon consisting of full-length cDNA of potato leafroll virus (PLRV) expressing green fluorescent protein (GFP), as shown by low levels of PLRV-GFP accumulation, lack of symptoms and accumulation of amplicon-specific short interfering RNAs (siRNAs). Inoculation of these plants with various viruses known to encode silencing suppressor proteins induced a striking synergistic effect leading to the enhanced accumulation of PLRV-GFP, suggesting that it had escaped from silencing. However, PLRV-GFP escape also occurred following inoculation with viruses that do not encode known silencing suppressors and treatment of silenced plants with biotic or abiotic stress agents. We propose that viruses can evade host RNA-silencing defences by a previously unrecognized mechanism that may be associated with a host response to some types of abiotic stress such as heat shock.     

Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems

RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Correspondingly, plant viruses encode suppressors of silencing. In the positive-strand RNA virus Tobacco rattle virus (TRV), the suppressor of silencing is a 16-kDa (16K) protein encoded by RNA1. The suppressor action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by tomato bushy stunt virus. Mutant TRV that does not produce its suppressor, unlike other suppressor-defective viruses, is competent to accumulate and spread systemically in the infected plant. However, this mutant virus does not exhibit the transient invasion of the meristem that is characteristic of the wild-type virus. Based on this analysis, we propose that the 16K suppressor of silencing allows TRV to transiently invade the meristem. Our data are consistent with a mechanism of long-term meristem virus exclusion that is dependent on a transient invasion of the meristem early in the infection cycle. This novel mechanism of meristem exclusion may be associated with the phenomenon of recovery in virus-infected plants in which upper leaves have little or no virus and are immune to secondary infection by the same virus.     

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 .     

RNA沉默与植物病毒

植物中RNA沉默（RNAsilencing)亦称为转录后基因沉默（PTGS）或共抑制,是植物抵抗外来核酸（转座子、转基因或病毒）入侵,并保护自身基因组完整性的一种防御机制。RNA沉默是近十年来发现的植物界中普遍存在的现象,已成为植物分子生物学领域的一个新的研究方向。对RNA沉默特点和机制的研究表明,植物病毒与（转基因）植物内发生的RNA沉默有着密切的联系,作者从病毒对RNA沉默的诱导、抑制、防御等方面,简述了RNA沉默与病毒的关系。并对病毒载体所诱导的RNA沉默在植物发育和基因组功能分析等方面的应用价值进行了讨论。     

RNA沉默在植物生物逆境反应中的作用

RNA沉默是真核生物共有的基因表达调节机制和防御机制。在植物RNA沉默中, 一些小RNAs, 如微小 RNAs和小干扰RNAs, 在植物防御病毒、细菌或食草动物的反应中具有重要作用。为了抑制宿主的RNA沉默系统, 植物病毒或细菌进化出了在RNA沉默不同阶段起作用的病毒沉默抑制子或细菌沉默抑制子, 来克服寄主的RNA沉默反应。文章就植物RNA沉默、病毒沉默抑制子、细菌沉默抑制子及其相关防御反应的一些新进展做一概述。     

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.     

Induction and suppression of gene silencing in plants by nonviral microbes

Gene silencing is a conserved mechanism in eukaryotes that dynamically regulates gene expression. In plants, gene silencing is critical for development and for maintenance of genome integrity. Additionally, it is a critical component of antiviral defence in plants, nematodes, insects, and fungi. To overcome gene silencing, viruses encode effectors that suppress gene silencing. A growing body of evidence shows that gene silencing and suppression of silencing are also used by plants during their interaction with nonviral pathogens such as fungi, oomycetes, and bacteria. Plant–pathogen interactions involve trans-kingdom movement of small RNAs into the pathogens to alter the function of genes required for their development and virulence. In turn, plant-associated pathogenic and nonpathogenic microbes also produce small RNAs that move trans-kingdom into host plants to disrupt pathogen defence through silencing of plant genes. The mechanisms by which these small RNAs move from the microbe to the plant remain poorly understood. In this review, we examine the roles of trans-kingdom small RNAs and silencing suppressors produced by nonviral microbes in inducing and suppressing gene silencing in plants. The emerging model is that gene silencing and suppression of silencing play critical roles in the interactions between plants and their associated nonviral microbes.     

Role of siRNAs and miRNAs in the processes of RNA-mediated gene silencing during viral infections

Phenomenon of RNA-induced gene silencing is a highly conservative mechanism among eukaryotic organisms. Several classes of small RNAs (siRNAs and miRNAs) 21–25 nt in length, which play a significant role in the processes of development of an organism, occurred important components of antiviral defence in animals and plants. This review shortly describes the main stages of gene silencing mechanism, features of antiviral RNA silencing in plants, invertebrates, mammals, ways of suppression of RNA-interference by viruses, as well as possible approaches of utilization of abovementioned phenomenon for struggling against viral infections. The article is published in the original.