Virus-induced gene silencing in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.     

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots

Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof-of-concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus-induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize–microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize–microbe interactions and more broadly in the context of the monocot functional genomics toolbox.     

A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.     

Graft‐accelerated virus‐induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS', where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR_1, RhA G, and RhNUDX_1 in rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.     

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

A pseudorecombinant-chimeric Cucumber mosaic virus-based virus-induced gene silencing system rapidly and efficiently triggers persistent gene silencing in maize.     

植物瞬间表达系统与功能基因组学研究

结构基因组学和功能基因组学的发展使特定植物基因组和转录组序列的获取更为方便和快捷。随之而来的是对各种基因和调控序列的功能注释,探索植物生长和发育的遗传机理。表达和调控表达是遗传物质的自身语言和动态属性,因此通过植物细胞内表达来分析目标基因和序列的表达和调控行为是功能分析的主要立足点。除创造转基因植株外,近几年来植物细胞瞬间表达系统得到了广泛的使用,与基因重排、病毒诱导基因沉默和RNA干扰等新兴技术的结合使其在植物功能基因组研究中扮演了越来越重要的角色。     

病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

     

Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato

Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the duration of endogenous gene silencing and developing a method for nonintegration-based persistent VIGS in progeny seedlings will widen the application of VIGS. We used three marker genes that provoke visible phenotypes in plants upon silencing to study persistence and transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato. We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltration greatly increased the effectiveness and duration of VIGS. Our results also showed transmittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate detailed study of target genes in plant development and stress tolerance. Further, the transmittance of VIGS to progeny will be useful in studying the effect of gene silencing in young seedlings. Our results provide a new dimension for the application of VIGS in plants.     

RETRACTED ARTICLE: Virus-induced gene silencing for functional analysis of selected genes

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics. Circumvention of plant transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is well established in Nicotiana benthamiana, and efforts are being made to improve VIGS in other species, including monocots. Here, we discuss the issues specific to the application of VIGS technology to determine gene function, which has revealed the roles of a variety of genes in disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis. M. R. Godge and A. Purkayastha made equal contributions and hence should be treated as joint first authors for this paper.     

Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy

Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.     

Efficient virus-induced gene silencing in Arabidopsis

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.     

Virus-induced gene silencing as a tool for analysis of gene functions in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antivirus defense mechanism in plants and has been shown to have great potential in plant reverse genetics. When the virus vector carries sequences of plant genes, virus infection triggers VIGS that results in the degradation of endogenous mRNAs homologous to the plant genes. The system is well established in Nicotiana benthamiana and several reliable VIGS vectors have been developed for other plant species including important agricultural crops. Here, we describe the use of VIGS technology to determine gene function and plant virus vectors for induction of VIGS in plants.     

植物病毒诱导的基因沉默在基因功能研究中的应用

传统的植物遗传转化方法周期长、工作量大、过程繁琐,不利于基因功能的快速高通量鉴定．近年来随着基因沉默机制研究的深入和不断发展,利用病毒诱导的基因沉默(Virus induced gene silencing,VIGS)进行植物功能基因组研究作为一种快速、高通量的反向遗传学工具已被广泛应用在烟草、马铃薯、番茄等植物中, 在大规模的植物基因组功能鉴定中展示了广阔的应用前景．综述了 VIGS 的作用机制、植物病毒栽体、转化方法以及在植物基因功能研究等方面的应用及前景．