Viruses term “VIGS” has been used importantly for

Viruses induced gene silencing as a tool for gene functional analysis incrop plantsIntroductionViruses induced gene silencing is the rapid tool of gene to access their function.The functionality of VIGS can be used for at least four reasons. First, is that the usage is simpleoften involving agroinfiltration or biotic inoculation of plants. Second, one is the the results aregotten rapidly typically with the time duration of two-three weeks of inoculation. Third, is thetechnology bypasses transformation points and hence can be used to number of crop plantrecalcitrant to transformation. Fourth, is the method has the strength to silence multiple copygenes. VIGS is based on the phenomenon of RNA-interference. VIGS is based on themethodology of RNA-interference (RNAi), which refers to disturbance in gene phenotype,provided by small RNA in a sequence in a specific manner. Manifestations of this pathway aredifferently termed as post-transcriptional gene silencing (PTGS) in crop plants, quelling in fungiand RNAi in animals. One important usage of this pathway in plants is in defense mechanismagainst viruses, in which, viral RNA acts as a initiator to induce RNA caused gene silencingwhich, in return, is directed towards the viral genes. In VIGS, this viral RNA-induced defensestrategy against viruses has been developed as a tool for reversion of genetics and observationof gene functions in plants, known as VIGS. In comparison to other PTGS-based methodsrequiring genetic transformation points, a ”functional knock-down” for a specific plant genecan be developed using VIGS within a duration of weeks without going to transform. Besidesbeing rapid and simple, VIGS in practice is useful in observing gene functions in specific cropspecies stable for transformation and genes that cause embryo death in knock-outs. Anotherpotential of the VIGS method is that it can be managed to silence multiple components of agene family, thereby incorporating the problem of functional retardnace of genes. Several RNAand DNA viruses have been developed to establish VIGS vectors. The gene to be silenced iscloned in an infectious mode of a viral DNA (DNA virus-based vectors) or cDNA (RNA virus-based vectors) obtained from viral RNA. The VIG vectors are inoculated introduction of in vitrointo plants by mechanical transcripts, Agrobacterium-mediated agro infiltration or, for DNA-based vectors through the biolistic delivery methods. During the method of viral infection,either double-stranded RNA or RNA with high level of secondary structure is mostly obtained,both of which are good initiators of RNAi directed towards the infecting viral RNA.Establishment and development of VIGSIn 1997, van Kammen first described the term “VIGS” to describe the method ofrecovery from virus infection. After that, the term “VIGS” has been used importantly for themethod of using newly modified viruses to knockdown expression of endogenous genes. In theinitial stages, most of the VIGS systems were other examples are also shown in the table asgiven below. based upon RNA viruses. In 1995, Kumagai et al. inoculated a fragment ofphytoene desaturase (PDS), a basal enzyme of the carotenoid biosynthetic pathway, into theTobacco mosaic virus (TMV) . When this new developed virus was inserted into Nicotianabenthamiana, a blench expression in the leaves was shown and this phenomenon wasdeveloped by reduction in endogenous PDS mRNAIn 1998, similar results were obtained using another RNA virus, Potato Xvirus (PVX), carrying a fragment of the PDS cDNA . Thus, VIGS is taken as to be a useful methodfor masking endogenous gene expression and opening plant gene functions. In 2001, a novelVIGS vector was developed based on Tobacco rattle virus (TRV). TRV was provided to developmore rapid silencing of transgenes and disaster genes. TRV could be released more vigorouslythroughout the all morphology plant, including its meristem tissue, and the visible phenotypicexpression caused by TRV are much lower as compared with other viruses. The TRV vector hasbeen extensively used in gene function studies of tomato, tobacco, Petunia hybrida, chilipepper, Arabidopsis, and cotton plants. Similar examples are also shown in the tables on thenext page table no 1.Different methods of developing VIGSIn a VIGS system, to reduce expression of an disasteros plant gene, a part of the gene to besilenced should be cloned and inoculate into the VIGS vector and then introduced into plants.The VIGS phenotype can be subsequently shown. Generally, to enhance the efficiency ofsilencing, the VIGS system should be maximized. First, the size range of the inserted segment oftarget endogenous gene may affect the performance of VIGS. Most VIGS vectors have theability to carry a fragment of length upto 150 and 800 bp. VIGS vectors may fail to cause genesilencing if a fragment of more than 1500 bp is introduced. Although some studies give rise thata 23 bp inoculation was able to cause VIGS, fragments of 200?350 bp in length is usually takenfor VIGS to get higher silencing efficiency. Furthermore, some studies given that the orientationof the inoculated gene fragment was also an important cause that could affect the efficiency ofVIGS, with higher silencing efficiency being caused by a reverse oriented insertion incomparison with that of a forward oriented insertion. However, it is not possible to all vectors.For example, the ability of the TYLCCNV DNA? vector is the identical whateverthe orientation of the inoculated fragment. In addition, the silencing efficiency could be marklyenhanced if the target segments were constructed as a hairpin type structure. Selection of thetarget gene is useful for VIGS. Evidence has given that an improper gene fragment mightcaused off-target silencing, developing an inaccurate phenotype. Many appearing fragmentscan be chosen for silencing of a specific targeted gene. However, if the target gene refers to agene family, some sequences may have conserved domains between various genes in the genefamily, and the segments of the target gene may have more than 23 bp that is same but notidentical to other genes in the gene family obtained in the degradation of non-target genes.That’s why, a more pointed fragment requires to be chosen. Generally, a fragment from UTRregion is a good one to choose. At the other hand, the restricted domains should be taken toavoid functional complementation by genes from the identical family; in this thing all the genesin the family are silenced.The progress of gene silencing may be manipulated by different inoculationmethods. The common methods in use for inoculation are agro-infiltration, rub-inoculationwith RNA transcripts, and particle bombardment as given in the above table. For some viruses,effecting plants will be inoculated firstly for multiplication of the virus, and then the sap or thevirus RNA extract will be used to inoculate chosen plants. High silencing efficiency was obtainedusing agro drench, a method of watering the plant roots with agro-inoculation directly. Liu et ALsuccessfully caused the TRV vector into tomato by spraying a TRV agro-culture by using anairbrush. Ding et al. reported that enhanced gene silencing could be obtained by vacuum agro-infiltration in crop plants that are difficult to inoculate by conventional methods.In the fruits, direct injection with an agro-culture produces a more desirablesilencing expression than inoculation of cotyledons or seedlings. Some studies given thateffective silencing could be caused by incorporating plucked tomato, strawberry and bilberryfruits with an agro-culture along with the VIGS vector, which is useful for analyzing genefunctions during the postharvest stage. Co-incorporating of viral suppressors with VIGS vectorsmay also enhanced the silencing efficiency. When plants were inserted by a mixture of VIGSvector and a gene-silencing suppressor, higher accumulation of virus in native inoculated cellsinduced a higher enhancement of silencing in systemic leaves. With the establishment of moreand more new virus inoculation techniques, VIGS will be applicable to more plant spices. Asfollowing diagram represents the methodology.At the end, environmental processes of plant growth will cause the efficiency ofgene silencing. At higher temperatures areas, the getting of virus are markedly reduced, whichcause the progress of virus induced silencing. At the other hand, lower temperatures areas leadto higher virus concentration and silencing progress. For TRV vectors, tomato plants should betaken at less than 21?C. Lower temperature and humidity will enhance silencing progress.However, for some other vectors, temperature is not so essential; for example, both DNA? andDNA 1 vectors can induce highly effective silencing from the temperature of 22 to 32°C.Validity of functionality of genes via VIGSThe high effectiveness of VIGS has caused to its enhanced use in unwinding thefunctions of hundreds of crop plant genes involved in defense mechanism pathways, plantgrowth, and metabolism. Recent studies in gene function identification by VIGS are detailedbelow.Gene involved in pathogen stresses and insects and abiotic stressesPlants growth in an environment surrounded by a variety of microbesand abiotic hazards. A highly efficient defense system has been introduced to resist effectiveattack by biotic and abiotic stresses. Past studies have developed the functions of differentplant genes involved in virus-, bacteria-, fungi-, and insect-resistance and stress to giveresponse. In the study of plant resistance to virus inoculation, the most effective examples ofusing VIGS to open gene functions in defense mechanism pathway was the N gene against TMVand Rx gene against PVX. Up to recent times, a lot of genes have been observed, such as NRG1,NbCA1, NbCAM1, NbrbohB, RAR1, EDS1, NPR1/NIM1, MEK1, MAPKK, NTF6, MAPK, andWRKY/MYB of the transcription factors, COI1 and CTR1 genes. The power of VIGS as a way inreverse genetics is more manifested by the following studies of the roles of BECLIN-1 and NRIP1in N-gene and RanGAP2 in Rx-gene caused programmed cell death (PCD). Silencing of BECLIN-1by TRV in N. benthamiana plants consisting the N gene showed an unconfined PCD response onTMV infection. NRIP1, which can directly attach with both the N gene and the 50 kid helicase(p50) of TMV, is used in pathogen recognition, and is required for N gene-mediated almostresistance to TMV.The association of Rx and RanGAP2 in N. benthamiana or potato isrequired for severe resistance to PVX, where RanGAP2 is portion of the Rx signaling complex. Inaddition, a number of host genes used in virus replication and apart movement in plants havebeen observed by VIGS. VIGS has also been used to study plant defense system against fungi. Aseries of host genes used in Cladosporium folium-tomato resistance have been characterized.VIGS was used to observe genes that reduce stress. Later on embryogenicabundant 4 (lea4) was observed to be involved in used moisture stress. SlGRX1 was observed tofor regulation of the abiotic defense against oxidative, drought, and salt stresses. In pepper,CaOXR1 was observed to play roles in defending to high salinity and osmotic stress. In tobaccoplants, NbPHB1 and NbPHB2, two subunits of prohibitions, were observed to have a critical rolein mitochondrial biogenesis and defense against stress and senescence in plants. NaHD20 has atarget in responses to dehydration. In more explanation, VIGS has been used to manipulatewater deficit-induced genes in peanut.Plant growth genesVIGS is an effective assay for reducing gene expression; therefore, VIGS facilitatesthe observation of genes whose loss of functionality could be able to die the plants. Up torecent advances, many development-concerning genes have been developed by VIGS. Recently,a study on the flowering of the opium poppy using VIGS shown that PapsAG-1 has a role instamen and carpel identification; however, the same gene, PapsAG-2, while showingredundancy in these functions, has a major role in the development of the setae, ovules, andstigmas.In tobacco and Petunia hybrid plants, many flower developmentassociating genes, such as flowering duration determine genes (FCA and FY), floral organidentity genes (AP3 and DEFICIENS) and flower development genes (NbMADS4-1, NbMADS4-2,PhPHB1 and PhPHB2) have been uncovered by VIGS. In a study of leaf and shoot growth, Kanget al. showed that the silencing of the NbBPS1 gene obtained in growth reduction, abnormalleaf development, and cell ultimate death. This phenotype is diverse from the case of theArabidopsis bps mutant. Boozier et al used VIGS to reduce the expression of SAMT1 in N.benthamiana. The disastrous growth reduction phenotype in silenced plants reveals that thismethylation-related protein has an important role in plant development. The plant vasculardevelopment gene (RPN9), Retinoblastoma-concerning gene (RBR), a plant root developmentgene and some genes in meristem, as Dt1 and ML1 have been characterized by VIGS. Theseresults give rise that VIGS is one of the most powerful method for the observation of geneswhose loss-of- function mutants induce embryonic and seedling death.Cellular metabolism and functionVIGS has been implied to study plant cellular functions and metabolic paths, suchas biotin, enzyme biosynthesis, and organic manifesto. Burton et al. and Held et al. used PVXand BSMV vectors, alternatively, to study the usage of Cellulose synthase (Cesar) 142,143.VIGS was also implied to show the genes used in the biosynthesis of capsaicinoids (AT3, Comet,pat, and KS), D-appose (UDP-D- appose/UDP-D- xylose synthase, and AXS1), flaming 146,histone, and major proteins in the RNA silencing pathway, such as Argonaute1- and Argonaut 4-like genes. In more analysis, genes involved in the regulating the functions of PCD have beennow identified using VIGS.For example, the mitochondrial-related hexokinase Hxk1 gene, 20S proteasome,the 19S regulatory unit of the 26S proteasome and a regulatory gene of PCD (CDC5). VIGS hasalso been developed to characterized cellular functions of genes used in chloroplasts andmitochondria bio genes, plastid biogenetics, peroxisome biogenesis, alkaloid biosynthesis,isoprenoid biosynthesis, ascorbic acid biosynthesis, sterol biosynthesis, and membranebiogenesis.Merits and demerits of VIGSAs comparing with other genomic methods, VIGS has many advantages:(i) VIGS is much faster. An important characteristic of VIGS is that it cancause loss-of- function expression of a specific gene in a short period oftime. Therefore, the gene functionality can be accessed quickly,obviating the tedious method of plant regeneration.(ii) (ii) Plant transformation is excluded, which means that studies of genefunctionality in plants that are more difficult to transfer (e.g., cottonand soybean) would be more productive once the VIGS system isdeveloped.(iii) (iii) VIGS allows the study of genes that are necessary for plant viability.VIGS can be induced at the seedling or early development stages, andhas been developed as a powerful tool in the observation of geneswhose mutations induce embryonic and seedling-death. VIGS is the onlymethod that allows the analysis of such plant genes that are used inplant development.(iv) (iv) The phenotype of multiple genes with functional reduction can besilenced together through VIGS using conserved domains. On the otherhand, a specific site can be used for VIGS if only one gene in a genefamily is projected to be silenced.(v) (v) It allows rapid comparison of the functions of same genes betweendifferent plant species simultaneously, developing more identical genefunction identification.VIGS also has some disadvantages or limitations. For example(i) In most cases, the phenotypes of gene cannot be completelyreduced through VIGS. As the expression of the target gene isretarded, the remaining phenotype of the target gene can besame for its function. Therefore, for those genes, the loss-of-function expression cannot be observed by VIGS.(ii) (ii) VIGS requires before knowledge of target gene sequenceinformation. The effectiveness of silencing may be compared byretarded genes, unless the full genome or sufficient ESTsequences are present.(iii) (iii) Genes give expression during germination or the earlyseedling stage cannot be observed by VIGS, because VIGS isusually shown on adult plants and most of the VIGS phenotypeis not transfer to next generation.(iv) (iv) The efficiency may differ and the expression of VIGS is notvery stable one. Results may not be same among differentexperiments or different plants. To get solution of this problem,it is common to use a marker gene that gives a visible silencingphenotype as a positive control.Conclusion and future of this VIGSOver the previous 15 years, VIGS has been successfully implied to give discovery andconfirm gene functions in many crop plants, including both dicotyledonous andmonocotyledonous crop plants. Furthermore understanding of the method of gene silencingand growth of vectors for VIGS will led to most plant species already studied by newly madeVIGS systems, especially those that are difficult to observe by conventional methods. Now,more plant genomes have been sequenced, and new molecular biology methods have beenestablished for VIGS.For example, artificial miRNA silencing vectors have been implemented inVIGS, and a VIGS cDNA library was made using the gateway system. With more technicalenhancements, VIGS will continuously to be frequently used in plant functional genomes.References1 Matthew L. RNAi for plant functional genomics. Comp Funct Genomics, 2004, 5: 240–2 Baulcombe D C. Fast forward genetics based on virus-induced gene silencing. Curr Opin PlantBiol, 1999, 2: 109–113 Burch-Smith T M, Anderson J C, Martin G B, et al. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J, 2004, 39: 734–7464 Robertson D. VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol,2004, 55: 495–5195 Becker A, Lange M. VIGS––genomics goes functional. Trends Plant Sci, 2010, 15: 1–46 Purkayastha A, Dasgupta I. Virus-induced gene silencing: a versatile tool for discovery of genefunctions in plants. Plant Physiol Biochem, 2009, 47: 967–9767 van Kammen A. Virus-induced gene silencing in infected and transgenic plants. Trends PlantSci, 1997, 2: 409–4118 Ruiz M T, Voinnet O, Baulcombe D C. Initiation and maintenance of virus-induced genesilencing. Plant Cell, 1998, 10: 937–9469 Kumagai M H, Donson J, della-Cioppa G, et al. Cytoplasmic inhibition of carotenoidbiosynthesis with virus-derived RNA. Proc Natl Acad Sci USA, 1995, 92: 1679–168310 Ratcliff F, Martin-Hernandez A M, Baulcombe D C. Tobacco rattle virus as a vector foranalysis of gene function by silencing. Plant J, 2001, 25: 237–24511 Kjemtrup S, Sampson K S, Peele C G, et al. Gene silencing from plant DNA carried by ageminivirus. Plant J, 1998, 14: 91–10012 Tuttle J R, Idris A M, Brown J K, et al. Geminivirus-mediated gene silencing from cotton leafcrumple virus is enhanced by low temperature in cotton. Plant Physiol, 2008, 148: 41–513 Fofana I B, Sangare A, Collier R, et al. A geminivirus-induced gene silencing system for genefunction validation in cassava. Plant Mol Biol, 2004, 56: 613–62414 Gossele V, Fache I, Meulewaeter F, et al. SVISS––a novel transient gene silencing system forgene function discovery and validation in tobacco plants. Plant J, 2002, 32: 859–86615 Tao X, Zhou X. A modified viral satellite DNA that suppresses gene expression in plants. PlantJ, 2004, 38: 850–860 16 Huang C, Xie Y, Zhou X. Efficient virus-induced gene silencing in plantsusing a modified geminivirus DNA1 component. Plant Biotechnol J, 2009, 7: 254–265 Huang C J,et al. Sci China Life Sci February (2012) Vol.55 No.2 105

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