Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.     

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.     

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.     

Use of geminiviral vectors for functional genomics

Virus-induced gene silencing (VIGS) can be used to study the function of a gene by downregulating its expression and analyzing the resulting phenotype. VIGS is a handy tool that is less time consuming and labor intensive than other methods for generating mutants. Geminiviruses are particularly convenient and valuable choices as VIGS vectors in functional genomics. The small size of their DNA genome, the simplicity of the methods for inoculation, their wide host range and their conserved genome organization are just a few of the advantageous characteristics that this group of viruses has to offer. Geminivirus-based vectors have proved to be very efficient in VIGS systems, and further development of these systems will most probably permit their application in studies of the functional genomics of important crops that are recalcitrant to other forms of analysis.     

A guide to the contained use of plant virus infectious clones

Plant virus infectious clones are important tools with wide‐ranging applications in different areas of biology and medicine. Their uses in plant pathology include the study of plant–virus interactions, and screening of germplasm as part of prebreeding programmes for virus resistance. They can also be modified to induce transient plant gene silencing (Virus Induced Gene Silencing – VIGS) and as expression vectors for plant or exogenous proteins, with applications in both plant pathology and more generally for the study of plant gene function. Plant viruses are also increasingly being investigated as expression vectors for in planta production of pharmaceutical products, known as molecular farming. However, plant virus infectious clones may pose a risk to the environment due to their ability to reconstitute fully functional, transmissible viruses. These risks arise from both their inherent pathogenicity and the effect of any introduced genetic modifications. Effective containment measures are therefore required. There has been no single comprehensive review of the biosafety considerations for the contained use of genetically modified plant viruses, despite their increasing importance across many biological fields. This review therefore explores the biosafety considerations for working with genetically modified plant viruses in contained environments, with focus on plant growth facilities. It includes regulatory frameworks, risk assessment, assignment of biosafety levels, facility features and working practices. The review is based on international guidance together with information provided by plant virus researchers.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

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.     

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.     

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.     

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.

     

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.     

Virus-induced gene silencing and its application in plant functional genomics

Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and reverse genetics to identify plant functional genes. Many viruses have been developed into virus-induced gene silencing vectors and gene functions involved in development, biotic and abiotic stresses, metabolism, and cellular signaling have been reported. In this review, we discuss the development and application of virus-induced gene silencing in plant functional genomics.     

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．     

烟草坏死病毒A诱导的基因沉默及其条件优化

以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.     

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.     

Gene silencing approaches through virus-based vectors: speeding up functional genomics in monocots

Plant Molecular Biology - The design and use of existing VIGS vectors for revealing monocot gene functions are described and potential new vectors are discussed, which may expand their repertoire....