Tobacco Rattle Virus Vector Research Articles

Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments

ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

Virus‐induced gene silencing for Asteraceae—a reverse genetics approach for functional genomics inGerbera hybrida

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

BackgroundPlant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.ResultsReplicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass.ConclusionsFor the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

BackgroundRNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.Methodology/Principal FindingsStable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.ConclusionsBoth stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.

Co-silencing of the Mirabilis antiviral protein (MAP) permits virus-induced gene silencing (VIGS) of other genes in Four O’Clock plants (Mirabilis jalapa)

SummaryVirus-induced gene silencing (VIGS) is a powerful and rapid technique for the analysis of gene function during plant growth and development. We attempted to use VIGS for the functional analysis of genes associated with floral senescence and abscission in Four O’Clock plants (Mirabilis jalapa). Plants infected with a Tobacco Rattle Virus (TRV) vector containing a fragment of the phytoene desaturase (PDS) gene did not show the expected photo-bleaching phenotype. We hypothesised that the failure of VIGS in this species might be due to the presence of the Mirabilis antiviral protein (MAP) which is known to have potent activity in preventing viral infection. Plants infected with a TRV vector containing fragments of both the PDS and MAP genes showed the typical photo-bleaching phenotype in photosynthetic tissues, and this was accompanied by reductions in the abundance of PDS and MAP transcripts. We also demonstrated reduced transcript abundance (and the expected phenotypes) when VIGS with a TRV-MAP vector was used to silence the chlorata42 (Ch42) and the proliferating cell nuclear antigen (PCNA) genes. Silencing of the 3,4-dihydroxy-L-phenylalanine 5-glucosyl transferase (DOPA5GT) gene reduced pigmentation in stems, leaves, and flowers. This is the first direct demonstration of the role of an enzyme that has been proposed to be a key step in the synthesis of betalain pigments. Co-silencing of endogenous anti-viral proteins may increase the range of taxa that are amenable to the use of VIGS for functional gene analysis.

The Feasibility Analysis of PVX and TRV Vectors as the VIGS Tool for Studying the Gene Function

Abstract A method of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and potato X virus vector as materials in this study. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR, and protein levels of rbcS by the antibodies of rbcS. For feasibility assessment of the PVX and TRV vectors on the VIGS for studying gene function, the statistical analysis was first employed in which it contained the two-factor variance analysis model and F-test based upon a large amount of data of the response of photosynthetic rates, the photosynthetic active radiation intensity and CO2 concentrations generated from the rbcS silenced and control plants. Our findings suggested that TRV vector was superior to PVX vector.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of "sectoring" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Petunia. Evolutionary, developmental and physiological genetics

Petunia. Evolutionary, developmental and physiological genetics

A VIRAL INDUCED GENE SILENCING APPROACH TO STUDY GALACTOSE LOSS IN CELL WALLS DURING FLOWER DEVELOPMENT AND SENESCENCE

ISHS International Symposium Postharvest Pacifica 2009 - Pathways to Quality: V International Symposium on Managing Quality in Chains + Australasian Postharvest Horticultural Conference A VIRAL INDUCED GENE SILENCING APPROACH TO STUDY GALACTOSE LOSS IN CELL WALLS DURING FLOWER DEVELOPMENT AND SENESCENCE

First Report of the Stubby Root Nematode, Paratrichodorus minor, in Mainland China.

The stubby root nematode, Paratrichodorus minor (Colbran, 1956) Siddiqi, 1974, is an economically important species. It not only causes direct damage to the meristemic tissues of plants, but also can be a vector of Tobacco rattle virus and Pepper ringspot virus (2). It has been documented in Afghanistan, Argentina, Australia, Brazil, Canary Islands, Cuba, Egypt, Fiji, India, Israel, Ivory Coast, Japan, Java, Mauritania, New Zealand, Nicaragua, Philippines, Puerto Rico, Senegal, South Africa, Taiwan, Upper Volta, the United States, the former USSR, and Venezuela (1). During a recent investigation on Trichodoridae occurring in China, eight stubby root nematode populations were detected in Hainan, Yunnan, and Fujian provinces in the rhizospheres of eggplant (Solanum melongena), Chinaberry (Melia azedarach), lettuce (Lactuca sativa), longan (Dimocarpus longan), peach (Prunus persica), pear (Pyrus sp.), and walnut (Juglans regia) in soils ranging from clay to sand. The eight populations had similar morphological and molecular characters. The females were cigar shaped with rod-shaped vaginal sclerotization parallel to the longitudinal body axis with a quadrangular vagina in side view having a short vulval slit. The key morphometrics of females were consistent with those of P. minor: body length, 631 to 757 μm; body diameter, 27 to 43 μm; onchiostyle, 35 to 36 μm; a, 18 to 24; b, 4.9 to 5.5; v%, 53 to 55; length of vagina, 10.8 to 11.1 μm; and size of vaginal pieces, 1.7 to 1.9 μm. Males in P. minor are rare and were not found in any of the eight populations. Partial sequences of the 18s RNA gene of each population were amplified with forward primer 5'-AAA GAT TAA GCC ATG CAT G-3' (2), and reverse primer 5'-AGT CAA ATT AAG CCG CAG-3' (3), which yielded a PCR fragment of 1,153 bp. PCR products from all eight populations were sequenced and submitted to GenBank and assigned Accession Nos. GQ995703, GQ995704, GQ995705, GQ995706, GQ995707, GQ995708, GQ995709, and GQ995710. The sequences exhibited 99.8 to 100% similarity with those of P. minor isolates DQ345526, AM269897, AJ438052, AJ438053, and AJ438058 from GenBank. Morphological and molecular identification confirmed that all populations were P. minor. To our knowledge, this is the first report of P. minor in mainland China.

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

SummaryCompared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.

Plastocyanin Transit Peptide Interacts withPotato virus XCoat Protein, While Silencing of Plastocyanin Reduces Coat Protein Accumulation in Chloroplasts and Symptom Severity in Host Plants

Potato virus X coat protein (PVXCP) is, through communication with host proteins, involved in processes such as virus movement and symptom development. Here, we report that PVXCP also interacts with the precursor of plastocyanin, a protein involved in photosynthesis, both in vitro and in vivo. Yeast two-hybrid analysis indicated that PVXCP interacted with only the plastocyanin transit peptide. In subsequent bimolecular fluorescence complementation assays, both proteins were collocated within chloroplasts. Western blot analyses of chloroplast fractions showed that PVXCP could be detected in the envelope, stroma, and lumen fractions. Transmission electron microscopy demonstrated that grana were dilated in PVX-infected Nicotiana benthamiana. Furthermore, virus-induced gene silencing of plastocyanin by prior infection of N. benthamiana using a Tobacco rattle virus vector reduced the severity of symptoms that developed following subsequent PVX infection as well as the accumulation of PVXCP in isolated chloroplasts. However, PVXCP could not be detected in pea chloroplasts in an in vitro re-uptake assay using the plastocyanin precursor protein. Taken together, these data suggest that PVXCP interacts with the plastocyanin precursor protein and that silencing the expression of this protein leads to reduced PVXCP accumulation in chloroplasts and ameliorates symptom severity in host plants.

Rapid analysis of Jatropha curcas gene functions by virus‐induced gene silencing

Jatropha curcas L. is a small, woody tree of the Euphorbiaceae family. This plant can grow on marginal land in the tropical and subtropical regions and produces seeds containing up to 30% oil. Several Asian countries have selected Jatropha for large scale planting as a biodiesel feedstock. Nevertheless, Jatropha also possesses several undesirable traits that may limit its wide adoption. An improved understanding of plant development and the regulation of fatty acid (FA) and triacylglyceride biosynthesis in Jatropha is particularly facilitative for the development of elite crops. Here, we show that a tobacco rattle virus (TRV) vector can trigger virus-induced gene silencing (VIGS) in Jatropha. Our optimized method produced robust and reliable gene silencing in plants agroinoculated with recombinant TRV harbouring Jatropha gene sequences. We used VIGS to investigate possible functions of 13 Jatropha genes of several functional categories, including FA biosynthesis, developmental regulation and toxin biosynthesis, etc. Based on the effects of VIGS on the FA composition of newly emerged leaves, we determined the function of several genes implicated in FA biosynthesis. Moreover, VIGS was able to discriminate independent functions of related gene family members. Our results show that VIGS can be used for high-throughput screening of Jatropha genes whose functions can be assayed in leaves.

Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants: Figure 1.

INTRODUCTIONThe inherent RNA silencing mechanism in plants has been effectively manipulated as a tool for the targeted down-regulation of genes. Numerous methods have been employed to initiate this homology-based RNA degradation process, but all rely on the activity of double-stranded RNAs (dsRNAs) corresponding to the gene of interest. Virus-induced gene silencing (VIGS) has gained acceptance as the tool of choice for transient induction of silencing. It involves creation of engineered viruses carrying sequences corresponding to the host gene to be silenced. Infection leads to synthesis of viral dsRNA, an intermediate step in viral replication. This activates the anti-viral RNA silencing pathway, resulting in down-regulation of the host gene transcript. While several VIGS vectors have been developed, the Tobacco Rattle Virus (TRV) provides the most robust results in terms of efficiency, ease of application, and absence of disease symptoms. Engineered TRV vectors carrying host-derived segments are transformed into Agrobacterium tumefaciens, which is then introduced into the plant. This protocol outlines a simple procedure for introducing the TRV-based binary vectors pTRV1 and pTRV2 into solanaceous plants such as Nicotiana benthamiana and Solanum lycopersicon (tomato), as well as Arabidopsis.

Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls.

Virus-induced gene silencing (VIGS) enables high-throughput analysis of gene function in plants but is not universally applicable and requires optimization for each species. Here a VIGS system is described for Solanum nigrum, a wild relative of tomato and potato and a valuable model species for ecogenomics. The efficiency of the two most widely used Tobacco rattle virus (TRV) vectors to silence phytoene desaturase (PDS) in S. nigrum was tested. Additionally, the infiltration method and growth temperatures for gene silencing were optimized and the suitability of different control vectors evaluated. Using leucine aminopeptidase (LAP), a herbivore-induced protein, silencing efficiency and the applicability of silenced plants for herbivore feeding assays were assessed. Vacuum infiltration of seedlings with Agrobacterium carrying the vector, pYL156, proved the most efficient means of silencing genes. Empty-vector controls decreased plant growth but control vectors carrying a piece of noncoding sequence did not. Silencing LAP significantly increased the larval mass of Manduca sexta that fed on silenced plants. This VIGS protocol proved highly successful for S. nigrum, which should include control vectors carrying noncoding sequence as control treatments. Silencing LAP provided the first experimental evidence that LAP has a defensive function against herbivores.

Role of a xyloglucan‐specific endo‐β‐1,4‐glucanase inhibitor in the interactions ofNicotiana benthamianawithColletotrichum destructivum,C. orbiculareorPseudomonas syringaepv.tabaci

A xyloglucan-specific endo-beta-1,4-glucanase inhibitor cDNA, NbXEGIP1, was amplified from diseased leaves of Nicotiana benthamiana. The sequence was similar to the tomato xyloglucan-specific endo-beta-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-beta-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare. Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto. A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana. Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci, silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1-silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2. NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan-cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.

Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus

BackgroundVirus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV).ResultsNbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT), with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.ConclusionBased on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.

A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development.

Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.

Delivery of macromolecules to plant parasitic nematodes using a tobacco rattle virus vector

Plant parasitic nematodes cause significant damage to crops on a worldwide scale. These nematodes are often soil dwelling but rely on plants for food and to sustain them during reproduction. Complex interactions occur between plants and nematodes during the nematode life cycle with plant roots developing specialized feeding structures through which nematodes withdraw nutrients. Here we describe a novel method for delivering macromolecules to feeding nematodes using a virus-based vector [tobacco rattle virus (TRV)]. We show that the parasitic nematode Heterodera schachtii will ingest fluorescent proteins transiently expressed in plant roots infected with a TRV construct carrying the appropriate protein sequence. A prerequisite for this delivery is the presence of replicating virus in root tips prior to the formation of nematode-induced syncytia. We show also that TRV vectors expressing nematode gene sequences can be used to induce RNAi in the feeding nematodes.

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.