Gene Silencing Vector Research Articles

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

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

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

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

Development of new potato virus X-based vectors for gene over-expression and gene silencing assay

Multiple plant viruses, including potato virus X (PVX), have been modified as vectors for expressing heterologous genes or silencing endogenous genes in plants. PVX-based vectors facilitate the functional analysis of genes in plant. However, they can only express one protein in a time. In this paper we report the construction of new vectors based on a 35S promoter-driven PVX infectious clone, pCaPVX100. Vector pCaPVX440 contains two additional subgenomic promoters and can be utilized to express two foreign genes at the same time. Plasmid pCaPVX760 is a CP minus vector and can be used to express foreign proteins through the gene substitution strategy. In addition, plasmid pCaPVX100 was engineered into a gene silencing vector (pCaPVX440-LIC) by introducing a ligation independent cloning (LIC) site into the vector. These results indicate that the newly developed PVX vectors are competent for multiple research purposes.

Maize Elongin C interacts with the viral genome-linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection.

The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified.ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts.Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virus-induced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression.Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation.

Chitosan hydrogel as siRNA vector for prolonged gene silencing.

BackgroundThe periodontitis is one of the most prevalent diseases with alveolar resorption in adult people and is the main cause of the tooth loss. To investigate the possibility for protecting the loss of alveolar bone in periodontal diseases, a RNAi-based therapeutic strategy is applied for silencing RANK signaling using thermosensitive chitosan hydrogel as siRNA reservoir and vector.ResultsThe thermosensitive chitosan hydrogel was formed from solution (PH = 7.2, at 4°C) at 37°C within 8 minutes. The degradation rates of hydrogel were ~50% and 5% (W remaining/W beginning) in the presence and absence of lysozyme, respectively, over a period of 20 days. The concurrent cumulative in vitro release of Cy3-labeled siRNA from the hydrogel was 50% and 17% over 14 days, with or without lysozyme digestion, respectively. High cell viability (>88%) was maintained for cells treated with hydrogel loaded with RANK specific siRNA and RANK knockdown was prolonged for up to 9 days when cells were incubated with siRNA/hydrogel complex. In vivo release of siRNA was investigated in a subcutaneous delivery setup in mice. The fluorescent signal from siRNA within hydrogel was remained for up to 14 days compared to less than one day for siRNA alone.ConclusionsChitosan hydrogel can potentially serve as a suitable reservoir and vector for local sustained delivery of siRNA in potential therapy.

New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.

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.

Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

Gossypium barbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G . barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G . barbadense . In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G . barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G . barbadense . The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G . barbadense , and help to contribute desirable traits for breeding of G . barbadense and G. hirsutum.

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

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

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

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

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P<0.01) change in expression levels were screened according to the screening standard (signal log ratio≥1 or ≤-1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335±0.0116 72 h after transfection. This value was significantly (P<0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P<0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.

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

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

A single-step mixing cloning method for assembly of lentiviral short hairpin RNA expression vectors for gene silencing

Lentiviral expression vectors encoding short hairpin RNA (shRNA) are widely used for RNAi-based gene silencing in mammalian cells. However, current methods for the construction of shRNA expression vectors require multiple steps, which are expensive, time-consuming, and error-prone. Here, we developed a single-step mixing cloning method for the generation of lentiviral shRNA expression vectors. With this method, a pair of short oligonucleotides (∼50nt) is required and a lentiviral shRNA vector can be constructed with only one step. This method has been used to construct 30 lentiviral shRNA expression vectors successfully.

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

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

Isolation and Characterization of Medicago truncatula U6 Promoters for the Construction of Small Hairpin RNA-Mediated Gene Silencing Vectors

RNA silencing using vector-based double-stranded RNA is an attractive strategy to suppress gene expression in plants. Especially, a short hairpin RNA (shRNA)-mediated approach is potent for specific target gene silencing. Here, shRNA expression vectors were constructed based on U6 small nuclear RNA (snRNA) gene promoters in Medicago truncatula. Ten U6 snRNA gene loci identified showed highly homologous sequences with those of other eukaryotes. Their RNA polymerase III-specific promoters had a conserved upstream sequence element and TATA box that were located approximately three helical DNA turns apart. The U6 promoters were also insensitive to α-amanitin, actively guiding transcription of fused GUS reporter gene fragments. When the U6 fusion constructs were introduced into M. truncatula via Agrobacterium rhizogenes-mediated transformation, GUS fragment transcripts were detected in nearly every transformed root albeit in varying amounts. Consistently, under the control of U6 promoters, constructs of 21-nucleotide-stem shRNAs directed silencing of GUS expression in transformed roots. Moreover, a phytoene desaturase gene (MtPDS3)-targeting 27-nucleotide-stem U6-shRNA constructs, when introduced into hairy roots, decreased the MtPDS3 transcript levels by approximately 80 % as a result of endogenous gene silencing, as verified by the presence of shPDS-derived siRNAs detected by stem-loop PCR. The U6 promoter-directed shRNA expression plasmids constructed herein can be a useful tool for functional gene analyses in this model legume.

Multi-armed cationic cyclodextrin:poly(ethylene glycol) polyrotaxanes as efficient gene silencing vectors.

A family of branched polyrotaxanes (bPRTx(+)), threaded with multiple cationic α-cyclodextrins (α-CDs) onto a multi-armed poly(ethylene glycol) (PEG) core, were synthesized and studied as gene silencing vectors. These bPRTx(+) formed stable, positively charged complexes with diameters of 150-250 nm at N/P ratios as low as 2.5. The bPRTx(+) materials were shown to have gene-silencing efficiencies comparable to those of Lipofectamine 2000 (L2k) and bPEI, while displaying similar toxicity profiles. The unique structure of these polyrotaxanes allows them to effectively condense and complex siRNA into nanoparticles at much lower N/P ratios than L2k or bPEI. These findings suggest that bPRTx(+) may be useful materials for gene therapy applications.

VEGF-C facilitates development of esophageal squamous cancer via regulating expression of CNTN-1

Objective To investigate the biological significance and mechanism of VEGF-C in e- sophageal tumor development, and correlation of CNTN-1 level with VEGF-C. Methods The expression of VEGF-C and its receptors in esophageal squamous cancer cell （ESCC） and in corresponding noncancerous esophageal tissue specimens were detected by real-time PCR. Esophageal squamous cancer cell line TE-1 was transinfected by VEGF-C overexpression and gene silencing vectors, respectively, and the relative amount of C/EBP bound to CNTN-1 promoter was determined by quantitative CHIP, to explore the possibility that VEGF-C was involved in development of esophageal cancer through mediating transcription of CNTN-1. Re- suits The mRNA levels of VEGF-C was significantly higher in ESCC than in normal esophageal tissues. VEGF-C expression was significantly increased in VEGF-C-overexpressing TE-1 cells compared to untrans- fected cells （mock）. Cells transfected with either of the VEGF-C targeting shRNA vectors, shRNA-1 and shRNA-2, showed reduced VEGF-C transcripts （P 〈 0. 01 ）. Expression levels of VEGF-C and CNTN-1 mR- NA correlated significantly with each other. The binding site of C/EBP in CNTN-1 was detected by CHIP, and the relative amount of C/EBP binding to CNTN-1 promoter was significantly increased in TE-1 after transfecting by VEGF-C overexpression vector （ P 〈 0. 05 ）. Conclusion VEGF-C and its receptor are highly expressed in esophageal cancer tissues, which may be associated with ESCC carcinogenesis and development. VEGF-C may influence on growth and migration in TE-1 cells through CNTN-1. Key words: Esophageal tumor; VEGF-C; CNTN-1

Begomovirus research in India: A critical appraisal and the way ahead

Begomoviruses are a large group of whitefly-transmitted plant viruses containing single-stranded circular DNA encapsidated in geminate particles. They are responsible for significant yield losses in a wide variety of crops in India. Research on begomoviruses has focussed on the molecular characterization of the viruses, their phylogenetic analyses, infectivities on host plants, DNA replication, transgenic resistance, promoter analysis and development of virus-based gene silencing vectors. There have been a number of reports of satellite molecules associated with begomoviruses. This article aims to summarize the major developments in begomoviral research in India in the last approximately 15 years and identifies future areas that need more attention.

RNAi-Mediated Silencing of the Flavanone 3-Hydroxylase Gene and Its Effect on Flavonoid Biosynthesis in Strawberry Fruit

Anthocyanin biosynthesis requires the activities of several enzymes in vivo. Flavanone 3-hydroxylase (F3H) converts flavanone into dihydroflavanol at an early step in the anthocyanin biosynthesis pathway. In this study we constructed an RNAi gene-silencing vector that encodes a hairpin F3H RNA. Agrobacterium strain GV3101 harboring the F3H RNAi vector was injected into strawberry fruits which were still attached to the plants 14 days after pollination. The phenotype was observed 10 days postinjection, and fruits were tested by RT-PCR and northern blot assays. The results showed that the F3H gene was downregulated by approximately 70 % in the agroinfiltrated fruits compared with the control. HPLC–MS analysis showed that anthocyanin content was greatly reduced, flavonol was also decreased, and the levels of p-coumaroyl glucoside and p-coumaroyl-1-acetate were markedly increased. We conclude that the precursors were shunted to the phenylpropanoid pathway, and that F3H is one of the key enzymes required for the biosynthesis of flavonoids in strawberry fruit. According to our results, reducing gene function via RNA interference is a rapid, simple, and effective way to identify gene function in strawberry fruit.

A High-Throughput Virus-Induced Gene-Silencing Vector for Screening Transcription Factors in Virus-Induced Plant Defense Response in Orchid

The large number of species and worldwide spread of species of Orchidaceae indicates their successful adaptation to environmental stresses. Thus, orchids provide rich resources to study how plants have evolved to cope with stresses. This report describes our improvement of our previously reported orchid virus-induced gene silencing vector, pCymMV-pro60, with a modified Gateway cloning system which requires only one recombination and can be inoculated by agroinfiltration. We cloned 1,700 DNA fragments, including 187 predicted transcription factors derived from an established expression sequence tag library of orchid, into pCymMV-Gateway. Phalaenopsis aphrodite was inoculated with these vectors that contained DNA fragments of the 187 predicted transcription factors. The viral vector initially triggered the expression of the salicylic acid (SA)-related plant defense responses and later induced silencing of the endogenous target transcription factor genes. By monitoring the expression of the SA-related plant defense marker PhaPR1 (homolog of PR1), we identified a gene, PhaTF15, involved in the expression of PhaPR1. Knockdown of PhaTF15 by virus-induced gene silencing and by transient delivery of double-stranded RNA (dsRNA) reduced expression of the orchid homolog of the conserved positive defense regulator NPR1, PhaNPR1. Cymbidium mosaic virus also accumulated to high levels with knockdown of PhaTF15 by transient delivery of dsRNA. We demonstrated efficient cloning and screening strategies for high-throughput analysis of orchid and identify a gene, PhaTF15, involved in regulation of SA-related plant defense.