Virus-induced Gene Silencing Vector Research Articles

Virus-induced gene silencing (VIGS) for elucidation of pathogen defense role of serine/threonine protein kinase in the non-model plant Piper colubrinum Link.

Piper colubrinum Link., an exotic species, is a wild relative of cultivated black pepper (Piper nigrum L.), and shows high degree of resistance to the oomycete pathogen Phytophthora capsici, which causes the devastating ‘quick wilt’ disease in P. nigrum. Serine/threonine protein kinase (PcSTPK) is a prospective candidate gene identified from P. colubrinum for future genetic improvement of P. nigrum. RACE PCR amplified a full-length PcSTPK of 1.7 kbp which encodes 575 amino acid residues with a calculated molecular weight of 64.45 kDa. PcSTPK forms a part of a distinct clade in phylogeny analysis, sharing higher similarity to receptor-like protein kinase class of Ser/Thr protein kinases. Real-time qRT-PCR showed the enhanced expression of PcSTPK gene upon P. capsici inoculation and the maximum accumulation of STPK transcripts was observed at 12 h post inoculation. A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) was established using Phytoene desaturase endogenous gene as a reporter. TRV:PcSTPK VIGS vector was infiltrated into young leaves of P. colubrinum. A time course study revealed that STPK transcript levels were significantly downregulated. Knock down of PcSTPK by VIGS increased the susceptibility to P. capsici infection, as evidenced by the appearance of foliar necrotic lesions and increased proliferation and sporulation of P. capsici on the leaf surface. We report the successful implementation of VIGS as a systemic strategy for defense gene functional validation in P. colubrinum and our data provide direct evidence for the possible role of PcSTPK in modulating antifungal defense response in the plant.

The impacts of BSMV on vegetative growth and water status in hulless barley (Hordeum vulgare var. nudum) in VIGS study

Barley stripe mosaic virus (BSMV) is an established and extensively used virus-induced gene silencing (VIGS) vector for gene function analysis in monocots. However, the phenotypes generated by targeted gene silencing may be affected or masked by symptoms of BSMV infection. To better understand the potential effects of BSMV-VIGS in hulless barley (<em>Hordeum vulgare</em> var. <em>nudum</em>), the accumulation pattern of BSMV and its impacts on vegetative growth and water status were investigated. The results indicated that the vegetative growth of infected plants was significantly and continuously impacted by BSMV from 10 to 40 days post inoculation (dpi). When the accumulation of BSMV was extremely high (7 to 11 dpi), infected plants displayed twisted leaf tips with an increased water lose rate (WLR) and decreased water content (WC). Virus accumulation declined and stabilized after 25 dpi, at this stage, the WLR and WC were unaffected in the infected plants. The efficiency of VIGS was tested by the silencing of Phytoene desaturase (PDS). RT-qPCR indicated that BSMV-VIGS can be sustained with good efficiency for up to 40 dpi under an altered condition with lower temperature (22 ±1°C) and higher relative humidity (70 ±10%). It was concluded that 25 to 40 dpi was the appropriate time zone for drought-related gene analysis by BSMV-VIGS under such condition.

Virus-induced gene silencing of fiber-related genes in cotton.

Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.

Advances in plant gene silencing methods.

Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

Tobacco rattle virus mediated gene silencing in strawberry plants

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of plant gene function; however, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether the use of a VIGS vector based on the tobacco rattle virus (TRV) would result in phenotypic changes in strawberry (Fragaria × ananassa). In this study we successfully silenced the expression of a strawberry phytoene desaturase (PDS) gene (FaPDS) and showed a significant reduction in FaPDS mRNA levels correlated with a concomitant photobleached phenotype. The spread of the virus harboring the TRV vector containing the coding sequence of the green fluorescent protein (GFP) marker protein (TRV-GFP) virus was readily monitored using fluorescent microscopy and a hand-held UV lamp, such that the infiltrated plants could be screened by detecting the GFP fluorescence. We also demonstrated that post-agroinfiltration water culture is an effective means to enhance the viability of vacuum-infiltrated plants. Taken together, the results confirm that VIGS using the TRV/TRV-GFP vector represents a new potentially valuable tool for strawberry functional genomic studies.

Citrus tristeza virus-based RNA-interference (RNAi) vector and its potential in combating citrus Huanglongbing (HLB)

Citrus tristeza virus (CTV), a plus-sense ssRNA virus, is member of the genus Closterovirus, family Closteroviridae. RNA viruses are inducers as-well-as targets of gene silencing defense mechanism of host plants and this has been exploited as a tool in functional genomics. CTV was developed into virus-induced gene silencing (VIGS) or RNA-interference (RNAi) vector, which interferes with expression of endogenous genes in citrus or GFP-transgene in Nicotiana benthamiana (16c) in a sequence specific manner. Photobleaching phenotype indicative of silencing of endogenous gene, phytoene desaturase in citrus, and red color under UV indicative of silencing of transgene GFP in N. benthamiana (16c) was observed using CTV-RNAi vector. CTV-RNAi vector has great potentials in combating huanglongbing (HLB) disease through (1) enhancing basal defense of citrus by silencing of auxin signaling F-Box receptor genes while simultaneously overexpressing microRNAs; (2) down-regulation of overexpressed genes, callose synthase and phloem protein-2, responsible for phloem-plugging in citrus by HLB; (3) expressing dsRNA specific to essential genes of insect vector psyllid (Diaphorina citri) to disable transmission of 'Candidatus' Liberibacter asiaticus pathogen. Simultaneous silencing of multiple endogenous genes of a metabolic pathway is possible through tandem engineering of potential siRNA eliciting regions in CTV-RNAi vector.

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.

Development of a VIGS vector based on the β-satellite DNA associated with bhendi yellow vein mosaic virus

Bhendi yellow vein mosaic virus (BYMV) is a monopartite begomovirus with an associated β-satellite. βC1 ORF encoded by the β-satellite is the symptom determinant and a strong suppressor of post transcriptional gene silencing. To create a virus induced gene silencing vector based upon the β-satellite associated with BYVMV the βC1 ORF was replaced with multiple cloning sites. GFP transgene and plant endogenous genes Su, PDS, PCNA and AGO1 were cloned into β-satellite based VIGS vector. GFP expression was silenced in the GFP expressing transgenic 16c Nicotiana benthamiana plants infiltrated with VIGS vector carrying GFP gene inside. N. benthamiana plants infiltrated with the VIGS vector harboring the endogenous genes Su, PDS, PCNA and AGO1 produced the phenotypic symptoms yellowing of the veins, photobleaching of the veins, stunting of the plant and upward leaf curling, respectively. Real time PCR analyses revealed a reduction in the levels of the corresponding transgene or endogenous target mRNA. The β-satellite based VIGS vector was able to silence the target genes effectively. Hence, BYVMV β-satellite based VIGS vector can be used in functional genomics studies.

Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.

Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.

A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica.

BackgroundStriga hermonthica is a hemiparasitic weed that infects cereals in Sub Sahara Africa (SSA) resulting in up to 100% grain yield loss. This significant loss in grain yields is a major contributor to food insecurity and poverty in the region. Current strategies to control the parasite are costly, unavailable and remain unpracticed by small-scale farmers, underscoring the need for more economical and sustainable control strategies. Development of resistant germplasm is the most sustainable strategy in the control of S. hermonthica, but is constrained by paucity of resistance genes for introduction into crop germplasm. RNA interference (RNAi) has potential for developing host-derived resistance against S. hermonthica by transformation of host crops with RNAi sequences targeted at critical Striga genes. The application of RNAi in management of S. hermonthica is however constrained by lack of efficient high throughput screening protocols for the candidate genes for silencing, as well as sub optimal delivery of siRNAs into the parasite. In comparison to stable transformation, viral induced gene silencing (VIGS) is a rapid and powerful tool for plant functional genomics and provides an easy and effective strategy in screening for putative candidate genes to target through RNAi. In addition, VIGS allows for a secondary amplification of the RNAi signal increasing the siRNA threshold and facilitates siRNA transport through viral movement proteins. We tested the efficiency of the Tobacco rattle virus (TRV1 and TRV2) VIGS vectors in silencing S. hermonthica phytoene desaturase (PDS) gene through agrodrench and agro-infiltration.ResultsWe report the validation of VIGS in S. hermonthica using a silencing cassette generated from TRV with a PDS gene insert. Agro-infiltrated and agro-drenched S. hermonthica leaves showed photo-bleaching phenotypes typical for PDS silencing within 7 and 14 days post infection respectively. In both cases S. hermonthica plants recovered from photo-bleaching effects within 28 days post inoculation. The transformation efficiency of the VIGS protocol in S. hermonthica was (60 ± 2.9)%.ConclusionThese results demonstrate that the TRV-VIGS system work in S. hermonthica and can be used for candidate gene validation for their role in the parasite development and parasitism, with the ultimate goal of developing resistant transgenic maize.

A novel method for the evaluation of virus-induced gene silencing efficiency.

Virus-induced gene silencing (VIGS) is an important tool for studying gene function. However, a number of factors highly restrict the application of VIGS, such as unstable efficiency and tissue-specific silencing. We developed a novel evaluation method for improving the applicability of VIGS vectors. In this method, 4 indexes were defined and utilized to evaluate VIGS efficiency by silencing the endogenous phytoene desaturase (PDS) gene with a tobacco rattle virus-based VIGS vector. To illustrate the reliability of this evaluation method, we assessed the silencing efficiency of SpPDS and SpMPK1 in Solanum pimpinellifolium. The silencing results of SpPDS showed that an optical density at 600 nm of 2.0 was more suitable than 1.0 for VIGS in S. pimpinellifolium. This suggests that the proposed evaluation method is a valid technique for optimizing the VIGS system of plants. Moreover, the SpMPK1 gene was highly silenced in the 4th-9th leaves with a 50-95% reduction in transcription levels, further demonstrating that this method can be used to select highly silenced candidates for further experiments, particularly when the target gene shows no phenotypic change after being silenced.

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.

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.

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana. In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

A virus-induced gene silencing method to study soybean cyst nematode parasitism in Glycine max

BackgroundBean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed.FindingsHere we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described.ConclusionsThe VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations.

The SNARE Protein Syp71 Is Essential for Turnip Mosaic Virus Infection by Mediating Fusion of Virus-Induced Vesicles with Chloroplasts

All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection.

Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim

Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

The closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology

Important progress in understanding replication, interactions with host plants, and evolution of closteroviruses enabled engineering of several vectors for gene expression and virus-induced gene silencing. Due to the broad host range of closteroviruses, these vectors expanded vector applicability to include important woody plants such as citrus and grapevine. Furthermore, large closterovirus genomes offer genetic capacity and stability unrivaled by other plant viral vectors. These features provided immense opportunities for using closterovirus vectors for the functional genomics studies and pathogen control in economically valuable crops. This review briefly summarizes advances in closterovirus research during the last decade, explores the relationships between virus biology and vector design, and outlines the most promising directions for future application of closterovirus vectors.

Virus induced gene silencing in Lolium temulentum

Lolium temulentum L. is valuable as a model species for studying abiotic stress in closely related forage and turf grasses, many of which are polyploid outcrossing species. As with most monocot species, Agrobacterium-mediated transformation of L. temulentum is still challenging, time consuming and inefficient. The aim of this study was to use the Barley stripe mosaic virus (BSMV) vector to develop a virus induced gene silencing (VIGS) system in L. temulentum to facilitate functional gene analysis. Plants infected with the BSMV vector containing a small region of the L. temulentum phytoene desaturase (PDS) gene, which is commonly used as a phenotypic marker in VIGS studies, displayed the characteristic albino phenotype observed in PDS silenced plants. Decreased PDS gene expression in these albino leaves was also confirmed by quantitative reverse transcriptase polymerase chain reaction. To evaluate the utility of using the VIGS vector for gene interactions in L. temulentum, a region of the translation factor eIF4A gene was incorporated into the VIGS vector. Inoculation of young seedlings with this modified vector resulted in a partial reduction in the expression of eIF4A suggesting that this vector will be a useful tool to examine gene interactions in L. temulentum.