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 modified viral satellite DNA that suppresses gene expression in plants

DNAbeta is a type of single-stranded (ss) circular satellite DNA found in association with monopartite-genome begomoviruses, such as Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10). Y10 DNAbeta is required for symptom expression in plants but depends on TYLCCNV-Y10 genomic DNA (DNA-A) for replication and encapsidation. When we converted DNAbeta into a gene-silencing vector (modified DNAbeta (DNAmbeta)) by replacing its C1 open-reading frame (ORF) with a multiple cloning site (MCS), it was replicated but no longer induced symptoms in association with TYLCCNV-Y10 DNA-A, so allowing the effects of gene inserts to be recognized easily. Insertion into DNAmbeta of sequences from any of the three host genes (proliferating cell nuclear antigen (PCNA), phytoene desaturase (PDS), and sulfur (Su)), or from a transgene (green fluorescent protein (GFP)), resulted in silencing of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than a month and was associated with decreased levels of mRNA of the gene targeted. Although DNAmbeta probably does not enter meristematic tissue, the PCNA gene could be silenced there. DNAmbeta was an effective silencing vector in tested N. glutinosa, N. tabacum Samsun (NN or nn), and Lycopersicon esculentum plants, and was able to silence two genes simultaneously. This satellite DNA vector-based form of virus-induced gene silencing (VIGS) promises to be applicable to other begomovirus/DNAbeta systems, which are recently reported to occur in several dicotyledonous crop species, thereby providing a powerful approach to gene discovery and the analysis of gene function in these crops.     

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus

The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.     

Jellyfish green fluorescent protein as a reporter for virus infections

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.     

Temporal and spatial Bean pod mottle virus‐induced gene silencing in soybean

Virus‐induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)‐based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in‐depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3′ sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near‐complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross‐sections of stems and leaf petioles, near‐complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post‐virus inoculation in leaves to 7 weeks post‐virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.     

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.     

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.     

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.     

Virus-induced Gene Silencing in Eggplant(Solanum melongena)

Eggplant(Solanum melongena)is an economically important vegetable requiring investigation into its various genomic functions.The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays.Virus-induced gene silencing(VIGS)has played a critical role in the functional genetic analyses.In this paper,TRV-mediated VIGS was successfully elicited in eggplant.We first cloned the CDS sequence of PDS(PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene.Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation,indicating that VIGS can be used to silence genes in eggplant.To further illustrate the reliability of VIGS in eggplant,we selected Chl H,Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method.Suppression of Chl H and Su led to yellow leaves,while the depletion of CLA1 resulted in albino.In conclusion,four genes,PDS,Chl H,Su(Sulfur),CLA1,were down-regulated significantly by VIGS,indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.     

Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.