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.     

Virus-induced gene silencing: A versatile tool for discovery of gene functions in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an antiviral defense mechanism in plants as a tool for plant reverse genetics. VIGS circumvents the need for plant transformation, is methodologically simple and yields rapid results. Various VIGS vectors have been developed and have helped to unravel the functions of genes involved in processes such as disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.     

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.     

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.     

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．     

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.     

Development of a virus induced gene silencing vector from a legumes infecting tobamovirus

Medicago truncatula, the model plant of legumes, is well characterized, but there is only a little knowledge about it as a viral host. Viral vectors can be used for expressing foreign genes or for virus-induced gene silencing (VIGS), what is a fast and powerful tool to determine gene functions in plants. Viral vectors effective on Nicotiana benthamiana have been constructed from a number of viruses, however, only few of them were effective in other plants. A Tobamovirus, Sunnhemp mosaic virus (SHMV) systemically infects Medicago truncatula without causing severe symptoms. To set up a viral vector for Medicago truncatula, we prepared an infectious cDNA clone of SHMV. We constructed two VIGS vectors differing in the promoter element to drive foreign gene expression. The vectors were effective both in the expression and in the silencing of a transgene Green Fluorescent Protein (GFP) and in silencing of an endogenous gene Phytoene desaturase (PDS) on N. benthamiana. Still only one of the vectors was able to successfully silence the endogenous Chlorata 42 gene in M. truncatula.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species

Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. Key message SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.     

Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy)

Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are resistant to conventional genetic approaches. However, VIGS has been shown to be effective in only a few, closely related plant species. Tobacco rattle virus (TRV), a bipartite RNA virus, has a wide host range and so in principle could serve as an efficient vector for VIGS in a diverse array of plant species. Here we show that a vector based on TRV sequences is effective at silencing the endogenous phytoene desaturase (PapsPDS) gene in Papaver somniferum (opium poppy). We show that this vector does not compromise the growth or reproduction of poppy and the plants did not display viral symptoms. The silencing of PapsPDS resulted in a significant reduction in PapsPDS mRNA and a concomitant photobleached phenotype. The ability to rapidly assay gene function in P. somniferum will be valuable in manipulation of the opiate pathway in this pharmaceutically important species. We suggest that our vacuum infiltration method used to deliver TRV-based vectors into poppy is a promising approach for expanding VIGS to diverse angiosperm species in which traditional delivery methods fail to induce VIGS. Furthermore, these studies demonstrate the utility of TRV-VIGS for probing gene function in a basal eudicot species that is phylogenetically distant from model plant species.     

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.     

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.