Silencing Of Phytoene Desaturase Research Articles

Establishment of Transcriptional Gene Silencing Targeting the Promoter Regions of GFP, PDS, and PSY Genes in Cotton using Virus-Induced Gene Silencing.

Virus-induced gene silencing (VIGS) by deploying viral-based vectors such as tobacco rattle virus (TRV) is a homology-based gene silencing technique in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) to validate the function of particular genes. The study presented here showed the induction of DNA methylation in the promoter regions of three phenotypic marker genes in different cotton accessions, including two endogenous genes such as phytoene desaturase (PDS) and phytoene synthase (PSY), and an exogenous gene, such as green fluorescent protein (GFP). First, DNA methylation was established in transgenic GFP cotton where methylation persisted up to S3 generation. Afterward, the promoter of PSY was targeted following the same conditions. Significant silencing of PSY was observed and methylation of the promoter was found up to S2 generation in red leaf cotton as detected in GFP cotton. Silencing of PDS resulted in a photobleaching phenotype; interestingly, the strength of this phenotype was diverse within the plants and was not observed in the next generation. Bisulfite sequencing results showed methylation percentage of the cytosine residues was high at CG and CHG sites of the targeted promoter sequences in the silenced plants. The findings of this paper suggest that TRV-based vector system can be used to monitor DNA methylation for both exogenous and endogenous gene levels in cotton and offer a very useful tool for plant epigenetic modification.

Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics Screens and Analysis of Gene Function in Cannabis sativa L.

Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

BackgroundLupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV).ResultsWe identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)—a gene likely to be involved in toxic alkaloid biosynthesis—we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a ‘PDS co-silencing’ approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL.ConclusionsWe provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector

BackgroundVirus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes.ResultsIn this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants.ConclusionsThe CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum

BackgroundAntirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates.ResultsA. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter.ConclusionsWe demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.

A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

BackgroundCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiency. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits.ResultsHere, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69–300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged.ConclusionsThese results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function, controlling viral pathogens or even performing resistance breeding in cucurbits. Moreover, this study will possess considerable important reference value for developing different viral vectors.

Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

Silencing of the phytoene desaturase (PDS) gene affects the expression of fruit-ripening genes in tomatoes

BackgroundPast research has shown that virus-induced phytoene desaturase (PDS) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer.ResultsWe found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase (PDS), zeta-carotene (ZDS), prolycopene isomerase (CrtlSO), and beta-carotene hydroxylase (CrtR-b2), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 (TAGL1), RIPENING INHIBITOR (RIN), pectin esterase gene (PE), lipoxygenase (LOX), FRUITFULL1/FRUITFUL2 (FUL1/FUL2), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 (ACO1 and ACO3) and ethylene-responsive genes (E4 and E8).ConclusionThese results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

Abscisic acid deficiency caused by phytoene desaturase silencing is associated with dwarfing syndrome in citrus.

In citrus, abscisic acid-deficiency was associated with a dwarfing phenotype, slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation reversed the dwarfing phenotype and enhanced growth. Abscisic acid (ABA) is a ubiquitously distributed phytohormone, which is almost produced by all living kingdoms. In plants, ABA plays pleiotropic physiological roles in growth, development, and stress responses. We explored the hidden relationship between ABA deficiency, and citrus dwarfing. We used targeted-HPLC, targeted-GC-MS, molecular genetics, immunoassays, and gene expression techniques to investigate the effects of the silencing of phytoene desaturase (PDS) gene on the ABA-biosynthetic pathway, endogenous ABA content, and other phytohormones. Silencing of PDS directly suppressed the carotenoids compounds involved in ABA biosynthesis, altered phytohormonal profile, and caused phytoene accumulation and ABA deficiency. The reduction of ABA presumably due to the limited availability of its precursor, zeaxanthin. The ABA-deficient citrus cuttings displayed photobleaching, a dwarf phenotype with impaired growth characteristics that included slow growth, small leaves, decreased fresh weight, and faster water loss. ABA supplementation enhanced the growth and reversed the dwarfing phenotype of the ABA-deficient cuttings. Our data demonstrate that ABA-deficiency may lead to dwarfing phenotype and impaired growth in citrus cuttings. The negative influence of ABA-deficiency on growth rate is the result of altered water relations. Addition of ABA to the CTV-tPDS roots restored shoot growth and reversed the dwarfing phenotype.

Disruption of Chloroplast Function Through Downregulation of Phytoene Desaturase Enhances the Systemic Accumulation of an Aphid-Borne, Phloem-Restricted Virus.

Chloroplasts play a central role in pathogen defense in plants. However, most studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae includes RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins that is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll, and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.

Transient silencing of phytoene desaturase reveals critical roles on plant response to salinity stress

In this work, we investigated the importance of phytoene desaturase (PDS) during salt stress in plants. For this aim, we transiently suppressed the corresponding gene of Nicotiana benthamiana using a Tobacco Rattle Virus silencing system. After salinity stress, the silenced plants were assayed for different growth and physiological parameters. The silenced plants were found to be induced more stunted and feeble shoots and roots than the negative controls. The results indicated that the concurrent incidence of salinity stress and viral infection did not have an additive influences on the plant growth and physiological parameters. Importantly, our results showed a more detrimental influence of salinity on plant growth and physiology after PDS silencing. Compared to the control, salt-stressed plants contained more proline, and malondialdehyde, less amount of photosynthetic pigments, higher Na+/K+ ratios in shoots and roots and lower relative water content after PDS silencing. The activity of antioxidant enzymes were also decreased in the plants coped with salinity or Tobacco Rattle Virus. It was also represented that simultaneous silencing of the PDS gene and salinity treatment resulted in a significant decrease of the performance index (PIABS) and an increase of dissipation per active reaction center (DIo/RC) reflecting severe injuries in photosynthetic machinery under such a condition. Overall results suggested that PDS silencing could increase the sensitivity of plants to salinity.

PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing.

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.

Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit

BackgroundVirus-induced gene silencing (VIGS) has become a powerful tool for post-genomic technology in plant species. This is important, especially in select plants, such as the pepper plant, that are recalcitrant to Agrobacterium-mediated transformation. Although VIGS in plants has been widely employed as a powerful tool for functional genomics, scattering phenotypic effects by uneven gene silencing has been implemented in order to overcome challenges in experiments with fruit tissues.ResultsWe improved the VIGS system based on the tobacco rattle virus (TRV) containing the An2 MYB transcription factor, which is the genetic determinant of purple colored- or anthocyanin-rich pepper. Silencing of endogenous An2 in the anthocyanin-rich pepper with the modified TRV vector for ligation-independent cloning (LIC) lacked purple pigment in its leaves, flowers, and fruits. Infection with TRV–LIC containing a tandem construct of An2 and phytoene desaturase (PDS) resulted in a typical photobleaching event in leaves without the purple pigment, whereas silencing of PDS led to the presence of photobleached and purple-colored leaves. Cosilencing of endogenous An2 and capsaicin synthase in fruits resulted in decreased levels of capsaicin and dihydrocapsaicin as assessed by high performance liquid chromatography analysis coupled with the absence of the purple pigment in fruits.ConclusionsVIGS with tandem constructs harboring An2 as a visible reporter in anthocyanin-rich pepper plants can facilitate the application of functional genomics in the study of metabolic pathways and fruit biology.

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes.

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes.

Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS.

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.

Optimization of Virus-induced Gene Silencing to Facilitate Evo-devo Studies in the Emerging Model SpeciesMimulus guttatus(Phrymaceae)1

Abstract Mimulus guttatus DC. (yellow monkey-flower; Phrymaceae) is an important model species for ecological and evolutionary studies, being locally adapted to a wide range of elevation, moisture and temperature gradients, soil types, and pollinator availabilities. In order to advance this species as a model for evolutionary genetic studies, we have developed virus-induced gene silencing (VIGS) using the tobacco rattle virus (TRV) to assay gene function. We demonstrate the effectiveness of Agrobacterium-mediated VIGS in two divergent populations of M. guttatus, Iron Mountain 767 (IM767) and Point Reyes (PR). Plants infected with a fragment of the carotenoid biosynthesis pathway gene PHYTOENE DESATURASE (PDS) cloned into the TRV2 vector exhibited endogenous PDS silencing and photobleached phenotypes. We further assayed for VIGS-induced floral phenotypes by silencing paralogous genes putatively affecting floral symmetry, CYCLOIDEA1 (CYC1) and CYCLOIDEA2 (CYC2). Simultaneous silencing of CYC1 and CYC2 resul...

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 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.

A UNIVERSAL PRIMER FOR ISOLATION OF FRAGMENTS OF A GENE ENCODING PHYTOENE DESATURASE FOR USE IN VIRUS-INDUCED GENE SILENCING (VIGS) STUDIES

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.