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.     

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.     

Geminivirus-based vectors for gene silencing in Arabidopsis

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.     

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.     

Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component

Virus‐induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite‐like and single‐stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication‐associated protein open reading frame and the A‐rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co‐agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co‐agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene‐silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.     

Virus-induced gene silencing in tomato

We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs.     

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.     

Silencing of a meristematic gene using geminivirus-derived vectors

Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.     

A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.     

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.