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Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method

Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2009, Article ID 198680, 8 pages doi:10.1155/2009/198680 Review Article Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method 1, 2 1 Turgay Unver and Hikmet Budak Biological Sciences & Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla, Turkey Kocaeli University, Arslanbey MYO, Izmit, Turkey Correspondence should be addressed to Hikmet Budak, budak@sabanciuniv.edu Received 1 December 2008; Accepted 30 March 2009 Recommended by Chunji Liu Virus-induced gene silencing (VIGS) is one of the reverse genetics tools for analysis of gene function that uses viral vectors carrying a target gene fragment to produce dsRNA which trigger RNA-mediated gene silencing. There are a number of viruses which have been modified to silence the gene of interest effectively with a sequence-specific manner. Therefore, different types of methodologies have been advanced and modified for VIGS approach. Virus-derived inoculations are performed on host plants using different methods such as agro-infiltration and in vitro transcriptions. VIGS has many advantages compared to other loss- of-gene function approaches. The approach provides the generation of rapid phenotype and no need for plant transformation. The cost of VIGS experiment is relatively low, and large-scale analysis of screening studies can be achieved by the VIGS. However, there are still limitations of VIGS to be overcome. Nowadays, many virus-derived vectors are optimized to silence more than one host plant such as TRV-derived viral vectors which are used for Arabidopsis and Nicothiana benthamiana. By development of viral silencing systems monocot plants can also be targeted as silencing host in addition to dicotyledonous plants. For instance, Barley stripe mosaic virus (BSMV)-mediated VIGS allows silencing of barley and wheat genes. Here we summarize current protocols and recent modified viral systems to lead silencing of genes in different host species. Copyright © 2009 T. Unver and H. Budak. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction genetic approach VIGS provides silencing of target gene in sequence specific manner. RNA-induced gene silencing Gene silencing at posttranscriptional level, posttranscrip- mechanism is also acting on VIGS in which 21–25 nucleotide tional gene silencing (PTGS), is an RNA-mediated systemic sequence of small interfering RNAs (siRNAs) guides specific silencing mechanism which was described as quelling in cleavage or suppression of target mRNAs at posttranscrip- fungi [1] and RNA interference in animals [2]. To specifically tional level [2, 7]. siRNAs which are processed from long silence or knock down the expression of targeted gene in double-stranded RNAs (dsRNA) by DICER, an RNAse-like plants several approaches of PTGS have been developed. enzyme, are then incorporated into RNA-induced silencing Virus-Induced Gene Silencing (VIGS) is one of these tools complex (RISC). This complex with siRNA targets specific to suppress expression level of the gene of interest in plants mRNA transcripts having sequence complementarity with [3, 4]. The term VIGS was first coined by A. van Kammen to the specific siRNA. In other words the antisense strand describe the resistance event against viral infection [5]. Plants of the siRNA associates with the RNAi silencing complex infected by many viruses induce RNA-mediated defense (RISC) to target homologous RNA for degradation [8]. which targets viral RNAs and any transgene RNA products dsRNAs may be originated in infected plant during cytoplas- inserted into it [6]. As a gene silencing method VIGS has mic replication of positive-sense single-stranded (ss)RNA several advantageous such as fast, transient suppression of viruses and in the case of replicative form and replicative gene expression, and it involves cloning of short sequence intermediates may represent the pool of dsRNAs [6]. For fragments of targeted gene to be silenced. As a reverse transgenes dsRNA may be generated by host RNA dependent 2 International Journal of Plant Genomics RNA polymerases (RdRp) [9]. To be a PTGS inducers a study Tomato yellow leaf curl China virus being helper transgenes also designed and constructed to produce dsRNA and a modified satellite DNA ware used to silence gene in [10]. N. benthamiana [38]. There are other viruses modified for silencing of dicotyledonous plants such as African cassava mosaic virus in cassava [37], Pea early browning virus in pea [29], and Bean pod mottle virus in soybean [28]. 2. Development of VIGS Methodology Previously barley stripe mosaic virus (BSMV) was devel- Some virus species were previously modified and used for oped for efficient silencing of pds gene in barley [26]. This system was then used for silencing of wheat genes [27]. silencing the gene of interest (Table 1). Tobacco mosaic virus (TMV) is one of the modified viruses which was used for BSMV is a positive sense RNA virus containing a tripartite effective pds gene silencing in Nicotiana benthamiana plants (α, β, γ) genome. The modified γ of BSMV genome replaced [11]. TMV is the first modified virus for application of VIGS by DNA vector was used for plant gene cloning. β genome methods to plants. The viral delivery leads downregulation has been deleted for viral coat protein production defect. of transcript of target gene through its homology dependent Each of the modified DNAs is used to synthesize RNAs by in degradation so potential of VIGS for analysis of gene vitro transcription. Recently, Brome mosaic virus strain has function was easily recognized [3]. Thomas et al. detected been modified for VIGS of pds, actin, and rubisco activase. These genes were also silenced in important model plants the minimum length of RNA for PTGS. A minimum of 23 nucleotide possessing 100% homology to the target gene was such as rice [33]. observed to be required but not enough for efficient PTGS, and longer identical sequence is needed to initiate silencing 3. Methods Used in VIGS [12, 13]. Tobacco rattle virus (TRV) was also modified to be a tool for gene silencing in plants. VIGS has been effectively 3.1. PVX (Potato Virus X)-Derived VIGS for Potato Silencing. applied in N. benthamiana [14] and in tomato [15]by PVX is RNA virus and infects broad range of solanaceous using TRV vectors. The significant advantage of TRV-based plants. A PVX derivative vector, an agroinfection vector, VIGS in Solanaceous species is the ease of introduction of pGR106, has been previously constructed for gene silencing the VIGS vector into plants. The VIGS vector is placed [19]. The vector was also used for the PVX-mediated VIGS between Rigth Border (RB) and Left Border (LB) sites of T- in leaves andtubersofpotatoplants[21]. DNA and inserted into Agrobacterium tumefaciens [15, 16]. Another property of TRV is the more vigorous spreading 3.1.1. Construction of PVX-Derived Vectors. PVX.GFP and all over the entire plant including meristem, and infection PVX.PDS can be constructed via PCR-based cloning using AS symptoms of TRV are mild [15]. Modified TRV vectors such specific oligonucleotide primers incorporating AscIand NotI as pYL156 and pYL279 have strong duplicate 35S promoter restrictions sites, respectively, at the 5 -and 3 -termini into and a ribozyme at C-terminus for more efficient and faster pGR106, a PVX derivative vector (Sainsbury Laboratory, spreading. These vectors are also able to infect other plant Norwich, UK). species [13, 14]. TRV-based vector has been used by Liu et al. for gene silencing in tomato [14]. Dalmay et al. have also used TRV-based VIGS to silence gene in A. thaliana [9]. Burch- 3.1.2. Agrobacterium Tumefaciens Transformation. Transfor- Smith et al. [17] have developed an efficient TRV-based mation procedure can be followed as outlined previously VIGS method to silence the A. thaliana genes with minimal [40]. A. tumefaciens strains (such as LB4404 and GV3101) modification of widely used TRV-based VIGS technique. should be prepared, and 500 mL of SOB medium (2% Bacto Very recently, Pflieger et al. [18] have shown that a viral tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM vector derived from Turnip yellow mosaic virus [TYMV) has KCl) in a flask should be inoculated with 1.0 mL of an the ability to induce VIGS in Arabidopsis thaliana.VIGSof N. overnight culture of bacteria for 6 hours at 28 C with shaking till OD550 reaches 0.7. The culture then chilled on ice for benthamiana using Potato virus X (PVX) was also achieved [19]. PVX-based vectors have more limited host range (only 30 minutes. The cells should be harvested at 6000 rpm for three families of plants are susceptible to PVX) than TMV- 10 minutes at 4 C. The pellet will be washed four times based vectors (nine plant families show susceptibility for with 200 mL 10% glycerol (90% sterile water). The final re- TMV) but PVX-based vectors are more stabile compared to suspension can be made with 0.5 mL in ice cold 10% glycerol. TMV [20]. The prepared competent cells can be used immediately Geminivirus-derived vectors can be used for VIGS stud- or stored at −80 C in small aliquots. Transformation of ies especially to study function of genes involved in meristem electrocompetent A. tumefaciens cells is performed by an function. Tomato golden mosaic virus (TGMV) was used to electroporator. A prechilled electroporation cuvette is filled silence a meristematic gene, proliferating cell nuclear antigen with 20–30 μL electrocompetent cells and up to 5 μL ligation (PCNA) in N. benthamiana [34]. The TGMV-based silencing products and should be treated with recommended 330 μF vector had been used for also silencing of nonmeristematic capacitance, 4000 Ω resistance, and 380 V voltage. Cells gene silencing [39]. Satellite-virus-based vectors are also are then put into 0.5 mL of SOC medium and incubated used for efficient gene silencing in plants only with the for 1 hour with shaking (100 rpm). The transformed cells help of other helper viruses. This two-component system are selected via antibiotic selection on spread plates with is called Satellite-virus-induced silencing system, SVISS. In supplemented selection [40]. International Journal of Plant Genomics 3 Table 1: Viruses used for silencing of genes and their hosts with targeted genes are listed. Natural host Viruse/viruse type Silencing host species Group Genes silenced Reference species Nicotiana Tobacco mosaic virus Tomato, squash, Tobamovirus pds benthamiana, [11] (TMV)/RNA virus potato, tobacco nicotiana tabacum Nicotiana Potato virus X Potato, oilseed, Potexvirus pds benthamiana, [21, 22] (PVX)/RNA virus rape Arabidopsis Nicotiana benthamiana,tomato, Rar1, EDS1, Tobacco rattle virus Spinach, beet, Tobravirus NPR1/NIM1 Arabidopsi,solanum species, [14, 16, 23–25] (TRV)/RNA virus potato, tobacco pds, rbcS, chilli pepper, opium poppy, Aquilegia vulgaris Barly stripe mosaic virus pds, Lr21, Rar1, Barley [26, 27] Hordeivirus Barley, wheat (BSMV) RNA virus Sgt1, Hsp90 Phaseolus Bean pod mottle virus Glycine max [28] Comovirus pds vulgaris,glycine (BPMV)/RNA virus max Pisum sativu, Pisum sativum, Pea early browning virus pspds, uni, kor, Tobravirus Medicago truncatula, [29, 30] Phaseolus (PEBV)/RNA viruse pds Lathyrus odorata vulgaris Satellite tobaccomosaic pds, rbcS, rbcL RNA satellite Nicotiana virus (STMV)/Satellite Nicotiana tabacum and various [31] virus glauca,pepper virus genes Poplar mosaic virus Nicotiana Poplar Carlavirus gfp (PopMV)/RNA virus [32] benthamiana Brome mosaic virus pds, actin 1, Barley, rice, maize [33] Bromovirus Barley (BMV)/RNA virus rubisco activase Tobacco golden mosaic Nicotiana Begomovirus su Tomato virus (TGMV)/DNA [34] benthamiana, virus Nicotiana Tomato bushy shunt Lycopersicon Tombusvirus gfp [35] virus (TBSV)/RNA virus esculentum benthamiana, Cabbage, Cabbage leaf curl virus Arabidopsis Begomovirus CH42, pds [36] broccoli, (CaLCuV)/DNA cauliflower Nicotiana African cassava mosaic Manihot virus (ACMV)/DNA Begomovirus pds, su, cyp79d2 benthamiana, [37] esculenta virus Manihot esculenta Nicotiana benthamiana, Tomato yellow leaf curl Lycopersicon esculentum, DNAbeta pcna, pds, su, [38] China Virus Tomato satellite DNA gfp (TYLCV)/DNA virus N. glutinosa, N. tabacum 4 International Journal of Plant Genomics 3.1.3. Agrobacterium Infection of Plants. Agrobacterium tumefaciens strain possessing helper plasmid pSoup is RdRp generally transformed with PVX.GFP or PVX.GOI using LB RB pTRV1 procedure described above. Agroinfiltration of N. benthami- ana and Solanum species should be performed as follows. PVX.GOI construct containing A. tumefaciens culture will be grown overnight at 28 C, harvested at 3000 rpm for 20 GOI minutes, and resuspended in the same volume of 10 mM A. tumefaciens GV3101 LB RB MgCl , with 100 μM acetosyringone and 1 mM Mes, pH 5.6. 2 pTRV2 The culture should be infiltrated into leaves by a syringe at cDNA pool lower face [40]. cDNA of GOI 3.2. TRV-Derived VIGS for Arabidopsis Silencing. The most widely used viral delivery vectors are Tobacco rattle viruses (a) (TRV, 16] because introduction of virus into plant including Syringe is easy in meristematic tissue [16]. TRV-mediated gene Toothpick silencing was applied to many plants from diverse genera such as Nicotiana benthamiana [14, 16], tomato [15], pepper (Capsicum annuum; 32), potato (Solanum tuberosum; 33), and petunia (Petunia hybrida; 34) from Solanaceae family, opium poppy (Papaver somniferum)fromPapaveraceae[23], and Arabidopsis thaliana being a model organism [17]. The TRV silencing in plants is usually mediated by Agrobacterium tumefaciens. TRV vectors pTRV1 and pTRV2 are placed between LB and RB sites separately. One of these vectors pTRV1, is constructed with GOI for targeted gene silencing Vacuum (Figure 1). 3.2.1. Construction of TRV Vectors and Agrobacterium- Mediated Infiltration. The TRV vectors pTRY1 (pYL192) and pTRY2 (pYL156) have been described earlier [14], and 2 to 3 weeks for the procedure can be followed described by Birch-Smith phenotype observation et al. [17]. XbaI-EcoRI-cut pTRV2 vector is ligated with XbaI-EcoRI-engineered PCR fragment of GOI and then (b) transformed into A. tumefaciens GV3101 strain which is made electrocompotent (described in Section 3.1.2). The Figure 1: TRV-mediated VIGS in N. benthamiana.TRV-based virus induced gene silencing assay covers many steps; the gene with Agrobacterium culture transformed with both pTRV1 and known sequence is first selected and then genetically engineered pTRV2-GOI (grown in 50 mg/L gentamycin and 50 mg/L for cloning into pTRV2. pTRV1 consists of a TRV1-based cassette kanamycin overnight culture) and infiltrated into Arabidopsis (RNA-dependent RNA polymerase gene, movement protein, etc.), leaves by pressing a syringe (described in Section 3.1.3, LB and RB site for plant transformation. The plasmids are Figure 1). transformed into A. tumefaciens, and then agro-inoculation is applied. Agrobacterium can be inoculated on plant into seedling by 3.3. “One-Step” TYMV-Derived Arabidopsis Silencing. a toothpick, a syringe and a vacuum infiltration as shown in the Turnip yellow mosaic virus is a positive strand of RNA virus picture. from the genus Tymovirus and infects many Brassicaseae including Arabidopsis [41]. Recently, Pflieger et al. [18] have developed a TYMV-derived vector to induce VIGS cDNA clone under the control of the duplicated CaMV in Arabidopsis. The TYMV-derived vector for efficient 35S promoter and terminator. This vector can be used for silencing includes inverted repeats of target gene fragments. efficient gene silencing by cloning the gene(s) of interest The system has ability to silence the gene even expressed into the vector. For example, pTY-PDS52-IR can be obtained in meristem and contains only a single vector. The other by cloning the self-hybridized palindromic oligonucleotides advantage of the TYMV mediated VIGS system that allows PDS52 into the SnaBI site of pTY-S. direct delivery of plasmid DNA to plant cells using rub- inoculation is the precluding of in vitro transcription, biolistic, and agroinfiltration steps [18]. 3.3.2. Preparation and Transfection of Protoplasts. Protoplasts of A. thaliana can be prepared from cell suspension culture 3.3.1. Cloning of Plasmid DNAs. The plasmid pTY has been using the procedure described by [42]. A total of 106 generated by Pflieger et al. [18] using full-length TYMV protoplasts are transfected DNA plasmids (prepared as International Journal of Plant Genomics 5 in Section 3.1), using the quantities indicated. Transfected combined with 50 μLFES.50 mLFES requires GP solution protoplasts are incubated at 24 Cinthe dark for48hours (10X GP: (18.77 g glycine, 26.13 g K2HPO4, ddH 0upto (18). 500 mL, sterilized by 20 minute autoclaving) which is then combined with 2.5 g sodium pyrophosphate, 2.5 g bentonite, 2.5 g celite with ddH 0upto250mL andre-autoclaved 3.4. Barley Stripe Mosaic Virus (BSMV)-Mediated Silencing. [44], and directly applied to the second leaf (when it is 5– The pγ.bpds4As can be used to make construction as 7 cm long) from the bottom of leaf to the tip. After 7–10 pγ.(gene of interest, GOI)As by replacing pds4 insert with days post inoculation (dpi), appearance of mosaic symptoms short GOI fragment applying restriction digestion. The same on leaves should be observed showing systemic spread of procedures can be followed for pγ.(gene of interest, GOI)S the virus. Leaves from inoculated plants are collected after silencing using pγ.bpds4S as template [26](Figure 2). approximately 14-15 day postinoculation (dpi) in order to check pds gene silencing level by qRT-PCR [26, 43]. 3.4.1. Barley and Wheat Pds Gene Silencing and Measurement of Silencing Levels 4. Improvements of Virus-Induced Linearization of Plasmids. For linearization, pα,pβΔβa, pγ, Gene Silencing pγ.bpds4S, and pγ.bpds4As plasmids should be digested with following restriction enzymes. pα plasmid DNA is digested Gene specific silencing via VIGS system is now used with MluI enzyme. To perform digestion, 10 μgpurified pα for diverse monocot and dicot plant species. Therefore, plasmid DNA, 1X RE buffer, 10 U MluI enzyme, and PCR a number of viral-derived vectors have been developed grade water are combined in a sterile eppendorf tube to a (Table 1), and many procedures have been optimized by final volume of 50 μL. Mixture is incubated at 37 Cfor 2 the researchers. TRV system was efficiently optimized for hours. BcuIenzymecan be used forpβΔβa plasmid DNA efficient silencing of Solanaceous plants [14, 15], and the digestion. For digestion, 10 μgpurified pβΔβa plasmid DNA, system was also applied for tomato to study role of fruit 1X, 10 U BcuI are combined in a sterile tube to reach a final ripening genes [45]. TRV-mediated VIGS has been modified volume of 50 μL PCR water is used. Mixture is incubated for robust and effective gene silencing in a model organism, at 37 C for approximately 2 hours. pγ plasmid can be Arabidopsis by [17]. The emerging model plant columbine digested with BssHII enzyme. To generate linearization of Aquilegia vulgaris has been efficiently silenced via TRV- pγ vectors 10 μgpγ plasmid DNA, 1X enzyme buffer, 10 U mediated VIGS [24]. Many economically important plants BssHII enzyme, and PCR grade water are combined in a were studied to optimize TRV-derived VIGS silencing such tube to handle a final volume of 50 μL. Mixture is generally as opium poppy [23]. Efficiency of the TRV-derived viral incubated at 50 C for 2-3 hours. After the incubation vector used VIGS system on tomato fruit via agro-injection samples should be observed on 1% agarose gel. Linearized has been improved up to 90% silencing compared to agro- plasmids should then be excised and purified [26, 27, 43]. infiltration of cotyledons and first leaves of plants (66%) [46]. Lacomme et al. [47] have described a method to In Vitro Transcription. In vitro transcription is performed enhance the robustness of the VIGS phenotype by increasing for the silencing of selected target gene. It requires at least the level of dsRNA by incorporation of 40–60 base direct three separate in vitro transcription reactions which are the inverted-repeats into a plant viral vector. Cheapness and transcription of α, βΔβa, and γ linearized genomes. Accord- easiness of Arabidopsis silencing have been improved via ing to manufacturer’s procedure mMessage mMachine T7 in “one-step”TYMV -derived VIGS [18]. Monocot plants are vitro transcription kit (cat no: 1344, Ambion, Austin, TX) also subjected to be silenced via VIGS. For this propose, transcriptions are performed. Components are mixture in Holzberg et al. [26] developed a BSMV-mediated VIGS a sterile tube: separately for each linarized plasmids (MluI system for barley, and Scofield et al. [27] have applied the digested pα-BcuI digested pβΔβa, BssHII digested pγ-or system to wheat. BMV has also been used to silence genes in BssHII digested pγ.bpds4S and BssHII digested pγ.bpds4As) monocot plants. Ding et al. [33]efficiently silenced the genes 80 ng template is used per one silencing reaction (linearized in barley, rice and maize. plasmid DNA), 1X Buffer (Ambion), 1X nucleotide mix with NTP Cap (Ambion), 0.3 μL of T7 RNA polymerase mix (Ambion), and sterile distilled water are combined up to 5. Comparison of VIGS with Other Gene 3 μL. Mixture is incubated at 37 Cfor 2hours andstored Silencing Methods at −80 C until use [26, 27, 43]. VIGS has many advantages and disadvantages compared BSMV Transcript Inoculations on Plants. Barley and wheat to other techniques used for functional analysis of plant plants can be used for BSMV-mediated PTGS. The second genes. Generally, the method is chosen for its reliability, leaves (approximately 7–10 days upon germination) should low cost, easiness, and rapidness. Several tools have been be inoculated with BSMV for silencing. All BSMV transcripts used for identification of loss-of-function of gene(s) such as, which are α, βΔβa, and γ will be mixedina1 : 1 : 1ratio TILLING, chemical and physical mutagenesis, T-DNA, and (1.0–1.5 μg of each transcript concentration is observed on transposon insertion techniques. However, VIGS presents spectrophotometer, Figure 2, Table 2). Transcription mix is an intended potential for the researchers working with 6 International Journal of Plant Genomics β β b d γ γ γ.GOI a b GOI In vitro transcripts are mixed (1:1:1) γ γ γ.GOIAs b GOI Modified tripartite BSMV genome Silenced leaves Transcripts are rubbed with FES 2 to 3 weeks for phenotype observation Figure 2: BSMV-mediated VIGS in barley. Barley stripe mosaic virus has a tripartite genome, and it has been modified to specific VIGS in barley plants [26, 27, 43]. Table 2: Construction of transcripts for the BSMV inoculation [26, 27, 43]. pα pβΔβ a pγ pγ.GOIS pγ.GOIAs FES Inoculation for silencing transcript transcript transcript transcript transcript solution BSMV:00 (viral control) 1.0–1.5 μg 1.0–1.5 μg 1.0–1.5 μg — — 50–55 μL BSMV:GOIS (sense version) 1.0–1.5 μg 1.0–1.5 μg — 1.0–1.5 μg — 50–55 μL BSMV:GOIAs (anti-sense version) 1.0–1.5 μg 1.0–1.5 μg — — 1.0–1.5 μg 50–55 μL FES (non silencing control) ————— 50–55 μL functional genomics due to the fact that it avoids many application using most conserved region of the gene family of limitations of the above approaches. Its main advantage [26, 27]. Despite the valuable advantages of VIGS approach, is the generation of rapid phenotype and that there is no there are also limitations. One of the most important need for plant transformation. The cost of VIGS experiment limitation is that complete loss-of-function by VIGS might is relatively low; Agrobacterium or in vitro transcription not be achieved. Generally 75–90% downregulation in the mediated VIGS assays do not cost effectively. VIGS method expression level of the targeted gene is accomplished [18, also provides a large-scale screening of genes for functional 43, 46]. Unfortunately the low level of gene expression can analysis. Moreover, there is no need to screen large pop- be enough to produce functional protein and phenotype in ulations to detect the function of a specific gene; only a silenced plant. Some of viral infections can cause symptoms single plant is enough to follow phenotype with targeted on plant that might mask the phenotype caused by the silencing. Therefore, repeating the experiment is easy and phenotype. This problem might be minimized as TRV-VIGS time effective. Host range wideness of viral vectors is the system because of mild symptoms [14, 16]. VIGS aims to other versatility of the approach. For instance TRV can infect silence the specific gene, which can only be achieved by spinach, beet, potato, and tobacco naturally. Hence TRV- sequence specific manner so the system relays on sequence based VIGS is applied to Nicotiana benthamiana,tomato, information. The approach also depends on pathogen-host Arabidopsis, chilli pepper, opium poppy, and Aquilegia vul- interaction, so the disadvantage is that pathogen infection garis(Table 1). Since it does not require plant transformation, may manipulate host function and alter development and VIGS is particularly useful on plants which are difficult morphology. There should be positive control in all VIGS or impossible to transform. Therefore, VIGS system can assays to mark the effect of viral inoculation on silenced be applied to the genes associated with embryonic devel- plant. Lastly, VIGS might suppress nontargeted gene in opment or essential housekeeping functions in plants [33, silenced plant cell or tissue [17]. This response should be 38]. Functional redundancy problem is overcome by VIGS addressed before the next genomic era. International Journal of Plant Genomics 7 6. Concluding Remarks [11] M. H. Kumagai, J. Donson, G. Della-Cioppa, D. Harvey, K. Hanley, and L. K. Grill, “Cytoplasmic inhibition of carotenoid VIGS as a reverse genetics tool for functional genomics biosynthesis with virus-derived RNA,” Proceedings of the studies presenting many advantages promises rapid gen- National Academy of Sciences of the United States of America, vol. 92, no. 5, pp. 1679–1683, 1995. eration of functional genomics even proteomics. By the [12] C. L. Thomas,L.Jones,D.C.Baulcombe,and A. 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Marathe, and S. P. Dinesh-Kumar, great contribution of VIGS usage. With the whole genome “Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required sequence availability, Brachypodium distachyon (L.) Beauv., for N -mediated resistance to tobacco mosaic virus,” The Plant Journal, vol. 30, no. 4, pp. 415–429, 2002. a model temperate grass species, should also be used in [15] Y. Liu, M. Schiff, and S. P. Dinesh-Kumar, “Virus-induced gene application of VIGS system for generation of genomics infor- silencing in tomato,” The Plant Journal, vol. 31, no. 6, pp. 777– mation to improve temperate crops. Large-scale screening 786, 2002. via VIGS-based method to detect important and fascinating [16] F. Ratcliff,A.M.Mart´ ın-Hernandez, ´ and D. C. Baulcombe, phenotypes should be performed. “Tobacco rattle virus as a vector for analysis of gene function by silencing,” The Plant Journal, vol. 25, no. 2, pp. 237–245, [17] T. M. Burch-Smith, M. Schiff, Y. Liu, and S. P. 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Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method

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Copyright © 2009 Turgay Unver and Hikmet Budak. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2009/198680
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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2009, Article ID 198680, 8 pages doi:10.1155/2009/198680 Review Article Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method 1, 2 1 Turgay Unver and Hikmet Budak Biological Sciences & Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla, Turkey Kocaeli University, Arslanbey MYO, Izmit, Turkey Correspondence should be addressed to Hikmet Budak, budak@sabanciuniv.edu Received 1 December 2008; Accepted 30 March 2009 Recommended by Chunji Liu Virus-induced gene silencing (VIGS) is one of the reverse genetics tools for analysis of gene function that uses viral vectors carrying a target gene fragment to produce dsRNA which trigger RNA-mediated gene silencing. There are a number of viruses which have been modified to silence the gene of interest effectively with a sequence-specific manner. Therefore, different types of methodologies have been advanced and modified for VIGS approach. Virus-derived inoculations are performed on host plants using different methods such as agro-infiltration and in vitro transcriptions. VIGS has many advantages compared to other loss- of-gene function approaches. The approach provides the generation of rapid phenotype and no need for plant transformation. The cost of VIGS experiment is relatively low, and large-scale analysis of screening studies can be achieved by the VIGS. However, there are still limitations of VIGS to be overcome. Nowadays, many virus-derived vectors are optimized to silence more than one host plant such as TRV-derived viral vectors which are used for Arabidopsis and Nicothiana benthamiana. By development of viral silencing systems monocot plants can also be targeted as silencing host in addition to dicotyledonous plants. For instance, Barley stripe mosaic virus (BSMV)-mediated VIGS allows silencing of barley and wheat genes. Here we summarize current protocols and recent modified viral systems to lead silencing of genes in different host species. Copyright © 2009 T. Unver and H. Budak. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction genetic approach VIGS provides silencing of target gene in sequence specific manner. RNA-induced gene silencing Gene silencing at posttranscriptional level, posttranscrip- mechanism is also acting on VIGS in which 21–25 nucleotide tional gene silencing (PTGS), is an RNA-mediated systemic sequence of small interfering RNAs (siRNAs) guides specific silencing mechanism which was described as quelling in cleavage or suppression of target mRNAs at posttranscrip- fungi [1] and RNA interference in animals [2]. To specifically tional level [2, 7]. siRNAs which are processed from long silence or knock down the expression of targeted gene in double-stranded RNAs (dsRNA) by DICER, an RNAse-like plants several approaches of PTGS have been developed. enzyme, are then incorporated into RNA-induced silencing Virus-Induced Gene Silencing (VIGS) is one of these tools complex (RISC). This complex with siRNA targets specific to suppress expression level of the gene of interest in plants mRNA transcripts having sequence complementarity with [3, 4]. The term VIGS was first coined by A. van Kammen to the specific siRNA. In other words the antisense strand describe the resistance event against viral infection [5]. Plants of the siRNA associates with the RNAi silencing complex infected by many viruses induce RNA-mediated defense (RISC) to target homologous RNA for degradation [8]. which targets viral RNAs and any transgene RNA products dsRNAs may be originated in infected plant during cytoplas- inserted into it [6]. As a gene silencing method VIGS has mic replication of positive-sense single-stranded (ss)RNA several advantageous such as fast, transient suppression of viruses and in the case of replicative form and replicative gene expression, and it involves cloning of short sequence intermediates may represent the pool of dsRNAs [6]. For fragments of targeted gene to be silenced. As a reverse transgenes dsRNA may be generated by host RNA dependent 2 International Journal of Plant Genomics RNA polymerases (RdRp) [9]. To be a PTGS inducers a study Tomato yellow leaf curl China virus being helper transgenes also designed and constructed to produce dsRNA and a modified satellite DNA ware used to silence gene in [10]. N. benthamiana [38]. There are other viruses modified for silencing of dicotyledonous plants such as African cassava mosaic virus in cassava [37], Pea early browning virus in pea [29], and Bean pod mottle virus in soybean [28]. 2. Development of VIGS Methodology Previously barley stripe mosaic virus (BSMV) was devel- Some virus species were previously modified and used for oped for efficient silencing of pds gene in barley [26]. This system was then used for silencing of wheat genes [27]. silencing the gene of interest (Table 1). Tobacco mosaic virus (TMV) is one of the modified viruses which was used for BSMV is a positive sense RNA virus containing a tripartite effective pds gene silencing in Nicotiana benthamiana plants (α, β, γ) genome. The modified γ of BSMV genome replaced [11]. TMV is the first modified virus for application of VIGS by DNA vector was used for plant gene cloning. β genome methods to plants. The viral delivery leads downregulation has been deleted for viral coat protein production defect. of transcript of target gene through its homology dependent Each of the modified DNAs is used to synthesize RNAs by in degradation so potential of VIGS for analysis of gene vitro transcription. Recently, Brome mosaic virus strain has function was easily recognized [3]. Thomas et al. detected been modified for VIGS of pds, actin, and rubisco activase. These genes were also silenced in important model plants the minimum length of RNA for PTGS. A minimum of 23 nucleotide possessing 100% homology to the target gene was such as rice [33]. observed to be required but not enough for efficient PTGS, and longer identical sequence is needed to initiate silencing 3. Methods Used in VIGS [12, 13]. Tobacco rattle virus (TRV) was also modified to be a tool for gene silencing in plants. VIGS has been effectively 3.1. PVX (Potato Virus X)-Derived VIGS for Potato Silencing. applied in N. benthamiana [14] and in tomato [15]by PVX is RNA virus and infects broad range of solanaceous using TRV vectors. The significant advantage of TRV-based plants. A PVX derivative vector, an agroinfection vector, VIGS in Solanaceous species is the ease of introduction of pGR106, has been previously constructed for gene silencing the VIGS vector into plants. The VIGS vector is placed [19]. The vector was also used for the PVX-mediated VIGS between Rigth Border (RB) and Left Border (LB) sites of T- in leaves andtubersofpotatoplants[21]. DNA and inserted into Agrobacterium tumefaciens [15, 16]. Another property of TRV is the more vigorous spreading 3.1.1. Construction of PVX-Derived Vectors. PVX.GFP and all over the entire plant including meristem, and infection PVX.PDS can be constructed via PCR-based cloning using AS symptoms of TRV are mild [15]. Modified TRV vectors such specific oligonucleotide primers incorporating AscIand NotI as pYL156 and pYL279 have strong duplicate 35S promoter restrictions sites, respectively, at the 5 -and 3 -termini into and a ribozyme at C-terminus for more efficient and faster pGR106, a PVX derivative vector (Sainsbury Laboratory, spreading. These vectors are also able to infect other plant Norwich, UK). species [13, 14]. TRV-based vector has been used by Liu et al. for gene silencing in tomato [14]. Dalmay et al. have also used TRV-based VIGS to silence gene in A. thaliana [9]. Burch- 3.1.2. Agrobacterium Tumefaciens Transformation. Transfor- Smith et al. [17] have developed an efficient TRV-based mation procedure can be followed as outlined previously VIGS method to silence the A. thaliana genes with minimal [40]. A. tumefaciens strains (such as LB4404 and GV3101) modification of widely used TRV-based VIGS technique. should be prepared, and 500 mL of SOB medium (2% Bacto Very recently, Pflieger et al. [18] have shown that a viral tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM vector derived from Turnip yellow mosaic virus [TYMV) has KCl) in a flask should be inoculated with 1.0 mL of an the ability to induce VIGS in Arabidopsis thaliana.VIGSof N. overnight culture of bacteria for 6 hours at 28 C with shaking till OD550 reaches 0.7. The culture then chilled on ice for benthamiana using Potato virus X (PVX) was also achieved [19]. PVX-based vectors have more limited host range (only 30 minutes. The cells should be harvested at 6000 rpm for three families of plants are susceptible to PVX) than TMV- 10 minutes at 4 C. The pellet will be washed four times based vectors (nine plant families show susceptibility for with 200 mL 10% glycerol (90% sterile water). The final re- TMV) but PVX-based vectors are more stabile compared to suspension can be made with 0.5 mL in ice cold 10% glycerol. TMV [20]. The prepared competent cells can be used immediately Geminivirus-derived vectors can be used for VIGS stud- or stored at −80 C in small aliquots. Transformation of ies especially to study function of genes involved in meristem electrocompetent A. tumefaciens cells is performed by an function. Tomato golden mosaic virus (TGMV) was used to electroporator. A prechilled electroporation cuvette is filled silence a meristematic gene, proliferating cell nuclear antigen with 20–30 μL electrocompetent cells and up to 5 μL ligation (PCNA) in N. benthamiana [34]. The TGMV-based silencing products and should be treated with recommended 330 μF vector had been used for also silencing of nonmeristematic capacitance, 4000 Ω resistance, and 380 V voltage. Cells gene silencing [39]. Satellite-virus-based vectors are also are then put into 0.5 mL of SOC medium and incubated used for efficient gene silencing in plants only with the for 1 hour with shaking (100 rpm). The transformed cells help of other helper viruses. This two-component system are selected via antibiotic selection on spread plates with is called Satellite-virus-induced silencing system, SVISS. In supplemented selection [40]. International Journal of Plant Genomics 3 Table 1: Viruses used for silencing of genes and their hosts with targeted genes are listed. Natural host Viruse/viruse type Silencing host species Group Genes silenced Reference species Nicotiana Tobacco mosaic virus Tomato, squash, Tobamovirus pds benthamiana, [11] (TMV)/RNA virus potato, tobacco nicotiana tabacum Nicotiana Potato virus X Potato, oilseed, Potexvirus pds benthamiana, [21, 22] (PVX)/RNA virus rape Arabidopsis Nicotiana benthamiana,tomato, Rar1, EDS1, Tobacco rattle virus Spinach, beet, Tobravirus NPR1/NIM1 Arabidopsi,solanum species, [14, 16, 23–25] (TRV)/RNA virus potato, tobacco pds, rbcS, chilli pepper, opium poppy, Aquilegia vulgaris Barly stripe mosaic virus pds, Lr21, Rar1, Barley [26, 27] Hordeivirus Barley, wheat (BSMV) RNA virus Sgt1, Hsp90 Phaseolus Bean pod mottle virus Glycine max [28] Comovirus pds vulgaris,glycine (BPMV)/RNA virus max Pisum sativu, Pisum sativum, Pea early browning virus pspds, uni, kor, Tobravirus Medicago truncatula, [29, 30] Phaseolus (PEBV)/RNA viruse pds Lathyrus odorata vulgaris Satellite tobaccomosaic pds, rbcS, rbcL RNA satellite Nicotiana virus (STMV)/Satellite Nicotiana tabacum and various [31] virus glauca,pepper virus genes Poplar mosaic virus Nicotiana Poplar Carlavirus gfp (PopMV)/RNA virus [32] benthamiana Brome mosaic virus pds, actin 1, Barley, rice, maize [33] Bromovirus Barley (BMV)/RNA virus rubisco activase Tobacco golden mosaic Nicotiana Begomovirus su Tomato virus (TGMV)/DNA [34] benthamiana, virus Nicotiana Tomato bushy shunt Lycopersicon Tombusvirus gfp [35] virus (TBSV)/RNA virus esculentum benthamiana, Cabbage, Cabbage leaf curl virus Arabidopsis Begomovirus CH42, pds [36] broccoli, (CaLCuV)/DNA cauliflower Nicotiana African cassava mosaic Manihot virus (ACMV)/DNA Begomovirus pds, su, cyp79d2 benthamiana, [37] esculenta virus Manihot esculenta Nicotiana benthamiana, Tomato yellow leaf curl Lycopersicon esculentum, DNAbeta pcna, pds, su, [38] China Virus Tomato satellite DNA gfp (TYLCV)/DNA virus N. glutinosa, N. tabacum 4 International Journal of Plant Genomics 3.1.3. Agrobacterium Infection of Plants. Agrobacterium tumefaciens strain possessing helper plasmid pSoup is RdRp generally transformed with PVX.GFP or PVX.GOI using LB RB pTRV1 procedure described above. Agroinfiltration of N. benthami- ana and Solanum species should be performed as follows. PVX.GOI construct containing A. tumefaciens culture will be grown overnight at 28 C, harvested at 3000 rpm for 20 GOI minutes, and resuspended in the same volume of 10 mM A. tumefaciens GV3101 LB RB MgCl , with 100 μM acetosyringone and 1 mM Mes, pH 5.6. 2 pTRV2 The culture should be infiltrated into leaves by a syringe at cDNA pool lower face [40]. cDNA of GOI 3.2. TRV-Derived VIGS for Arabidopsis Silencing. The most widely used viral delivery vectors are Tobacco rattle viruses (a) (TRV, 16] because introduction of virus into plant including Syringe is easy in meristematic tissue [16]. TRV-mediated gene Toothpick silencing was applied to many plants from diverse genera such as Nicotiana benthamiana [14, 16], tomato [15], pepper (Capsicum annuum; 32), potato (Solanum tuberosum; 33), and petunia (Petunia hybrida; 34) from Solanaceae family, opium poppy (Papaver somniferum)fromPapaveraceae[23], and Arabidopsis thaliana being a model organism [17]. The TRV silencing in plants is usually mediated by Agrobacterium tumefaciens. TRV vectors pTRV1 and pTRV2 are placed between LB and RB sites separately. One of these vectors pTRV1, is constructed with GOI for targeted gene silencing Vacuum (Figure 1). 3.2.1. Construction of TRV Vectors and Agrobacterium- Mediated Infiltration. The TRV vectors pTRY1 (pYL192) and pTRY2 (pYL156) have been described earlier [14], and 2 to 3 weeks for the procedure can be followed described by Birch-Smith phenotype observation et al. [17]. XbaI-EcoRI-cut pTRV2 vector is ligated with XbaI-EcoRI-engineered PCR fragment of GOI and then (b) transformed into A. tumefaciens GV3101 strain which is made electrocompotent (described in Section 3.1.2). The Figure 1: TRV-mediated VIGS in N. benthamiana.TRV-based virus induced gene silencing assay covers many steps; the gene with Agrobacterium culture transformed with both pTRV1 and known sequence is first selected and then genetically engineered pTRV2-GOI (grown in 50 mg/L gentamycin and 50 mg/L for cloning into pTRV2. pTRV1 consists of a TRV1-based cassette kanamycin overnight culture) and infiltrated into Arabidopsis (RNA-dependent RNA polymerase gene, movement protein, etc.), leaves by pressing a syringe (described in Section 3.1.3, LB and RB site for plant transformation. The plasmids are Figure 1). transformed into A. tumefaciens, and then agro-inoculation is applied. Agrobacterium can be inoculated on plant into seedling by 3.3. “One-Step” TYMV-Derived Arabidopsis Silencing. a toothpick, a syringe and a vacuum infiltration as shown in the Turnip yellow mosaic virus is a positive strand of RNA virus picture. from the genus Tymovirus and infects many Brassicaseae including Arabidopsis [41]. Recently, Pflieger et al. [18] have developed a TYMV-derived vector to induce VIGS cDNA clone under the control of the duplicated CaMV in Arabidopsis. The TYMV-derived vector for efficient 35S promoter and terminator. This vector can be used for silencing includes inverted repeats of target gene fragments. efficient gene silencing by cloning the gene(s) of interest The system has ability to silence the gene even expressed into the vector. For example, pTY-PDS52-IR can be obtained in meristem and contains only a single vector. The other by cloning the self-hybridized palindromic oligonucleotides advantage of the TYMV mediated VIGS system that allows PDS52 into the SnaBI site of pTY-S. direct delivery of plasmid DNA to plant cells using rub- inoculation is the precluding of in vitro transcription, biolistic, and agroinfiltration steps [18]. 3.3.2. Preparation and Transfection of Protoplasts. Protoplasts of A. thaliana can be prepared from cell suspension culture 3.3.1. Cloning of Plasmid DNAs. The plasmid pTY has been using the procedure described by [42]. A total of 106 generated by Pflieger et al. [18] using full-length TYMV protoplasts are transfected DNA plasmids (prepared as International Journal of Plant Genomics 5 in Section 3.1), using the quantities indicated. Transfected combined with 50 μLFES.50 mLFES requires GP solution protoplasts are incubated at 24 Cinthe dark for48hours (10X GP: (18.77 g glycine, 26.13 g K2HPO4, ddH 0upto (18). 500 mL, sterilized by 20 minute autoclaving) which is then combined with 2.5 g sodium pyrophosphate, 2.5 g bentonite, 2.5 g celite with ddH 0upto250mL andre-autoclaved 3.4. Barley Stripe Mosaic Virus (BSMV)-Mediated Silencing. [44], and directly applied to the second leaf (when it is 5– The pγ.bpds4As can be used to make construction as 7 cm long) from the bottom of leaf to the tip. After 7–10 pγ.(gene of interest, GOI)As by replacing pds4 insert with days post inoculation (dpi), appearance of mosaic symptoms short GOI fragment applying restriction digestion. The same on leaves should be observed showing systemic spread of procedures can be followed for pγ.(gene of interest, GOI)S the virus. Leaves from inoculated plants are collected after silencing using pγ.bpds4S as template [26](Figure 2). approximately 14-15 day postinoculation (dpi) in order to check pds gene silencing level by qRT-PCR [26, 43]. 3.4.1. Barley and Wheat Pds Gene Silencing and Measurement of Silencing Levels 4. Improvements of Virus-Induced Linearization of Plasmids. For linearization, pα,pβΔβa, pγ, Gene Silencing pγ.bpds4S, and pγ.bpds4As plasmids should be digested with following restriction enzymes. pα plasmid DNA is digested Gene specific silencing via VIGS system is now used with MluI enzyme. To perform digestion, 10 μgpurified pα for diverse monocot and dicot plant species. Therefore, plasmid DNA, 1X RE buffer, 10 U MluI enzyme, and PCR a number of viral-derived vectors have been developed grade water are combined in a sterile eppendorf tube to a (Table 1), and many procedures have been optimized by final volume of 50 μL. Mixture is incubated at 37 Cfor 2 the researchers. TRV system was efficiently optimized for hours. BcuIenzymecan be used forpβΔβa plasmid DNA efficient silencing of Solanaceous plants [14, 15], and the digestion. For digestion, 10 μgpurified pβΔβa plasmid DNA, system was also applied for tomato to study role of fruit 1X, 10 U BcuI are combined in a sterile tube to reach a final ripening genes [45]. TRV-mediated VIGS has been modified volume of 50 μL PCR water is used. Mixture is incubated for robust and effective gene silencing in a model organism, at 37 C for approximately 2 hours. pγ plasmid can be Arabidopsis by [17]. The emerging model plant columbine digested with BssHII enzyme. To generate linearization of Aquilegia vulgaris has been efficiently silenced via TRV- pγ vectors 10 μgpγ plasmid DNA, 1X enzyme buffer, 10 U mediated VIGS [24]. Many economically important plants BssHII enzyme, and PCR grade water are combined in a were studied to optimize TRV-derived VIGS silencing such tube to handle a final volume of 50 μL. Mixture is generally as opium poppy [23]. Efficiency of the TRV-derived viral incubated at 50 C for 2-3 hours. After the incubation vector used VIGS system on tomato fruit via agro-injection samples should be observed on 1% agarose gel. Linearized has been improved up to 90% silencing compared to agro- plasmids should then be excised and purified [26, 27, 43]. infiltration of cotyledons and first leaves of plants (66%) [46]. Lacomme et al. [47] have described a method to In Vitro Transcription. In vitro transcription is performed enhance the robustness of the VIGS phenotype by increasing for the silencing of selected target gene. It requires at least the level of dsRNA by incorporation of 40–60 base direct three separate in vitro transcription reactions which are the inverted-repeats into a plant viral vector. Cheapness and transcription of α, βΔβa, and γ linearized genomes. Accord- easiness of Arabidopsis silencing have been improved via ing to manufacturer’s procedure mMessage mMachine T7 in “one-step”TYMV -derived VIGS [18]. Monocot plants are vitro transcription kit (cat no: 1344, Ambion, Austin, TX) also subjected to be silenced via VIGS. For this propose, transcriptions are performed. Components are mixture in Holzberg et al. [26] developed a BSMV-mediated VIGS a sterile tube: separately for each linarized plasmids (MluI system for barley, and Scofield et al. [27] have applied the digested pα-BcuI digested pβΔβa, BssHII digested pγ-or system to wheat. BMV has also been used to silence genes in BssHII digested pγ.bpds4S and BssHII digested pγ.bpds4As) monocot plants. Ding et al. [33]efficiently silenced the genes 80 ng template is used per one silencing reaction (linearized in barley, rice and maize. plasmid DNA), 1X Buffer (Ambion), 1X nucleotide mix with NTP Cap (Ambion), 0.3 μL of T7 RNA polymerase mix (Ambion), and sterile distilled water are combined up to 5. Comparison of VIGS with Other Gene 3 μL. Mixture is incubated at 37 Cfor 2hours andstored Silencing Methods at −80 C until use [26, 27, 43]. VIGS has many advantages and disadvantages compared BSMV Transcript Inoculations on Plants. Barley and wheat to other techniques used for functional analysis of plant plants can be used for BSMV-mediated PTGS. The second genes. Generally, the method is chosen for its reliability, leaves (approximately 7–10 days upon germination) should low cost, easiness, and rapidness. Several tools have been be inoculated with BSMV for silencing. All BSMV transcripts used for identification of loss-of-function of gene(s) such as, which are α, βΔβa, and γ will be mixedina1 : 1 : 1ratio TILLING, chemical and physical mutagenesis, T-DNA, and (1.0–1.5 μg of each transcript concentration is observed on transposon insertion techniques. However, VIGS presents spectrophotometer, Figure 2, Table 2). Transcription mix is an intended potential for the researchers working with 6 International Journal of Plant Genomics β β b d γ γ γ.GOI a b GOI In vitro transcripts are mixed (1:1:1) γ γ γ.GOIAs b GOI Modified tripartite BSMV genome Silenced leaves Transcripts are rubbed with FES 2 to 3 weeks for phenotype observation Figure 2: BSMV-mediated VIGS in barley. Barley stripe mosaic virus has a tripartite genome, and it has been modified to specific VIGS in barley plants [26, 27, 43]. Table 2: Construction of transcripts for the BSMV inoculation [26, 27, 43]. pα pβΔβ a pγ pγ.GOIS pγ.GOIAs FES Inoculation for silencing transcript transcript transcript transcript transcript solution BSMV:00 (viral control) 1.0–1.5 μg 1.0–1.5 μg 1.0–1.5 μg — — 50–55 μL BSMV:GOIS (sense version) 1.0–1.5 μg 1.0–1.5 μg — 1.0–1.5 μg — 50–55 μL BSMV:GOIAs (anti-sense version) 1.0–1.5 μg 1.0–1.5 μg — — 1.0–1.5 μg 50–55 μL FES (non silencing control) ————— 50–55 μL functional genomics due to the fact that it avoids many application using most conserved region of the gene family of limitations of the above approaches. Its main advantage [26, 27]. Despite the valuable advantages of VIGS approach, is the generation of rapid phenotype and that there is no there are also limitations. One of the most important need for plant transformation. The cost of VIGS experiment limitation is that complete loss-of-function by VIGS might is relatively low; Agrobacterium or in vitro transcription not be achieved. Generally 75–90% downregulation in the mediated VIGS assays do not cost effectively. VIGS method expression level of the targeted gene is accomplished [18, also provides a large-scale screening of genes for functional 43, 46]. Unfortunately the low level of gene expression can analysis. Moreover, there is no need to screen large pop- be enough to produce functional protein and phenotype in ulations to detect the function of a specific gene; only a silenced plant. Some of viral infections can cause symptoms single plant is enough to follow phenotype with targeted on plant that might mask the phenotype caused by the silencing. Therefore, repeating the experiment is easy and phenotype. This problem might be minimized as TRV-VIGS time effective. Host range wideness of viral vectors is the system because of mild symptoms [14, 16]. VIGS aims to other versatility of the approach. For instance TRV can infect silence the specific gene, which can only be achieved by spinach, beet, potato, and tobacco naturally. Hence TRV- sequence specific manner so the system relays on sequence based VIGS is applied to Nicotiana benthamiana,tomato, information. The approach also depends on pathogen-host Arabidopsis, chilli pepper, opium poppy, and Aquilegia vul- interaction, so the disadvantage is that pathogen infection garis(Table 1). Since it does not require plant transformation, may manipulate host function and alter development and VIGS is particularly useful on plants which are difficult morphology. There should be positive control in all VIGS or impossible to transform. Therefore, VIGS system can assays to mark the effect of viral inoculation on silenced be applied to the genes associated with embryonic devel- plant. Lastly, VIGS might suppress nontargeted gene in opment or essential housekeeping functions in plants [33, silenced plant cell or tissue [17]. This response should be 38]. Functional redundancy problem is overcome by VIGS addressed before the next genomic era. International Journal of Plant Genomics 7 6. Concluding Remarks [11] M. H. Kumagai, J. Donson, G. Della-Cioppa, D. Harvey, K. Hanley, and L. K. Grill, “Cytoplasmic inhibition of carotenoid VIGS as a reverse genetics tool for functional genomics biosynthesis with virus-derived RNA,” Proceedings of the studies presenting many advantages promises rapid gen- National Academy of Sciences of the United States of America, vol. 92, no. 5, pp. 1679–1683, 1995. eration of functional genomics even proteomics. By the [12] C. L. Thomas,L.Jones,D.C.Baulcombe,and A. 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International Journal of Plant GenomicsHindawi Publishing Corporation

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