Viruses of gene to access their function. The

Viruses induced gene silencing as a tool for gene functional analysisin crop plantsIntroduction                       Viruses induced genesilencing is the rapid tool of gene to access their function. Thefunctionality  of VIGS can be used for atleast four reasons.

First, is that the usage is simple often involvingagroinfiltration or biotic inoculation of plants. Second, one is the theresults are gotten rapidly typically with the time duration of two-three weeks  of inoculation. Third, is the technologybypasses  transformation points and hencecan be used to number of crop plant  recalcitrant to transformation. Fourth, is the method has the strengthto silence multiple copy genes.

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VIGS is based on the phenomenon of RNA-interference.VIGS is based on the methodology of RNA-interference (RNAi), which refers todisturbance in gene phenotype, provided by small RNA in a sequence in aspecific manner. Manifestations of this pathway are differently termed aspost-transcriptional gene silencing (PTGS) in crop plants, quelling in fungiand RNAi in animals. One important usage of this pathway in plants is indefense mechanism against viruses, in which, viral RNA acts as a initiator toinduce RNA caused gene silencing which, in return, is directed towards theviral genes. In VIGS, this viral RNA-induced defense strategy against viruseshas been developed as a tool for reversion of genetics and observation of genefunctions in plants, known as VIGS. In comparison to other PTGS-based methodsrequiring genetic transformation points, a ”functional knock-down” for aspecific plant gene can be developed using VIGS within a duration of weekswithout going to transform.

Besides being rapid and simple, VIGS in practice isuseful in observing gene functions in specific crop species stable fortransformation and genes that cause embryo death in knock-outs. Anotherpotential of the VIGS method is that it can be managed to silence multiplecomponents of a gene family, thereby incorporating the problem of functionalretardnace of genes. Several RNA and DNA viruses have been developed toestablish VIGS vectors. The gene to be silenced is cloned in an infectious modeof a viral DNA (DNA virus-based vectors) or cDNA (RNA virus-based vectors)obtained from viral RNA. The VIG vectors are inoculated introduction of invitro into plants by mechanical transcripts, Agrobacterium-mediated agroinfiltration or, for DNA-based vectors through the biolistic delivery methods.During the method of viral infection, either double-stranded RNA or RNA withhigh level of secondary structure is mostly obtained, both of which are goodinitiators of RNAi directed towards the infecting viral RNA.Establishment and development of VIGS                                                          In1997, van Kammen first described the term “VIGS” to describe the method ofrecovery from virus infection.

After that, the term “VIGS” has been usedimportantly for the method of using newly modified viruses to knockdownexpression of endogenous genes. In the initial stages, most of the VIGS systemswere other examples are also shown in the table as given below. based upon RNAviruses. In 1995, Kumagai et al. inoculated a fragment of phytoene desaturase(PDS), a basal enzyme of the carotenoid biosynthetic pathway, into the Tobaccomosaic virus (TMV) . When this new developed virus was inserted into Nicotianabenthamiana, a blench expression in the leaves was shown and this phenomenonwas developed by reduction in endogenous PDS mRNA                                         In1998, similar results were obtained using another RNA virus, Potato X virus(PVX), carrying a fragment of the PDS cDNA . Thus, VIGS is taken as to be auseful method for masking endogenous gene expression and opening plant genefunctions. In 2001, a novel VIGS vector was developed based on Tobacco rattlevirus (TRV).

TRV was provided to develop more rapid silencing of transgenes anddisaster genes. TRV could be released more vigorously throughout the allmorphology plant, including its meristem tissue, and the visible phenotypicexpression caused by TRV are much lower as compared with other viruses. The TRVvector has been extensively used in gene function studies of tomato, tobacco,Petunia hybrida, chili pepper, Arabidopsis, and cotton plants.

Similar examplesare also shown in the tables on the next page table no 1.ar his mturase (PDS), a keypid tool theirfunction.ysis in crop plantsDifferent methods of developing  VIGSIn a VIGS system, to reduceexpression of an disasteros plant gene, a part of the gene to be silencedshould be cloned and inoculate into the VIGS vector and then introduced intoplants.

The VIGS phenotype can be subsequently shown. Generally, to enhance theefficiency of silencing, the VIGS system should be maximized. First, the sizerange of the inserted segment of target endogenous gene may affect theperformance of VIGS. Most VIGS vectors have the ability to carry a fragment oflength upto 150 and 800 bp. VIGS vectors may fail to cause gene silencing if afragment of more than 1500 bp is introduced. Although some studies give risethat a 23 bp inoculation was able to cause VIGS, fragments of 200-350 bp in length is usuallytaken for VIGS to get higher silencing efficiency. Furthermore, some studiesgiven that the orientation of the inoculated gene fragment was also animportant cause that could affect the efficiency of VIGS, with higher silencingefficiency being caused by a reverse oriented insertion in comparison with thatof a forward oriented insertion. However, it is not possible  to all vectors.

                                For example,the ability of the TYLCCNV DNA? vector is the identical whatever theorientation of the inoculated fragment. In addition, the silencing efficiencycould be markly enhanced if the target segments were constructed as a hairpintype structure. Selection of the target gene is useful for VIGS.  Evidence has given that an improper genefragment might caused off-target silencing, developing an inaccurate phenotype.

Many appearing fragments can be chosen for silencing of a specific targetedgene. However, if the target gene refers to a gene family, some sequences mayhave conserved domains between various genes in the gene family, and thesegments 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 ofnon-target genes. That’s why, a more pointed fragment requires to be chosen.Generally, a fragment from UTR region is a good one to choose. At the otherhand, the restricted domains should be taken to avoid functional complementationby genes from the identical family; in this thing all the genes in the familyare silenced.                         The progress of genesilencing may be manipulated by different inoculation methods.

The commonmethods in use for inoculation are agro-infiltration, rub-inoculation with RNAtranscripts, and particle bombardment as given in the above table. For someviruses, effecting plants will be inoculated firstly for multiplication of thevirus, and then the sap or the virus RNA extract will be used to inoculatechosen plants. High silencing efficiency was obtained using agro drench, amethod of watering the plant roots with agro-inoculation directly. Liu et ALsuccessfully caused the TRV vector into tomato by spraying a TRV agro-cultureby using an airbrush. Ding et al.

reported that enhanced gene silencing couldbe obtained by vacuum agro-infiltration in crop plants that are difficult toinoculate by conventional methods.                      In the fruits, directinjection with an agro-culture produces a more desirable silencing expressionthan inoculation of cotyledons or seedlings. Some studies given that effectivesilencing could be caused by incorporating plucked tomato, strawberry andbilberry fruits with an agro-culture along with the VIGS vector, which isuseful for analyzing gene functions during the postharvest stage.Co-incorporating of viral suppressors with VIGS vectors may also enhanced thesilencing efficiency. When plants were inserted by a mixture of VIGS vector anda gene-silencing suppressor, higher accumulation of virus in native inoculatedcells induced a higher enhancement of silencing in systemic leaves. With theestablishment of more and more new virus inoculation techniques, VIGS will beapplicable to more plant spices. As following diagram represents the methodology.

                    At the end, environmental processes of plantgrowth will cause the efficiency of gene silencing. At higher temperaturesareas, the getting of virus are markedly reduced, which cause the progress ofvirus induced silencing. At the other hand, lower temperatures areas lead tohigher virus concentration and silencing progress. For TRV vectors, tomatoplants should be taken at less than 21°C. Lowertemperature and humidity will enhance silencing progress. However, for someother vectors, temperature is not so essential; for example, both DNAb and DNA 1 vectors can inducehighly effective silencing from the temperature of 22 to 32°C. Validity of functionality of genes via VIGS                         The high effectivenessof VIGS has caused to its enhanced use in unwinding the functions of hundredsof crop plant genes involved in defense mechanism pathways, plant growth, andmetabolism.

Recent studies in gene function identification by VIGS are detailedbelow.Gene involved in pathogen stresses and insects and abiotic stresses                                           Plantsgrowth in an environment surrounded by a variety of microbes and abiotic hazards.A highly efficient defense system has been introduced to resist effectiveattack by biotic and abiotic stresses. Past studies have developed thefunctions of different plant genes involved in virus-, bacteria-, fungi-, andinsect-resistance and stress to give response. In the study of plant resistanceto virus inoculation, the most effective examples of using VIGS to open genefunctions in defense mechanism pathway was the N gene against TMV and Rx geneagainst PVX. Up to recent times, a lot of genes have been observed, such asNRG1, NbCA1, NbCAM1, NbrbohB, RAR1, EDS1, NPR1/NIM1, MEK1, MAPKK, NTF6, MAPK, andWRKY/MYB of the transcription factors, COI1 and CTR1 genes. The power of VIGSas a way in reverse genetics is more manifested by the following studies of theroles of BECLIN-1 and NRIP1 in N-gene and RanGAP2 in Rx-gene caused programmedcell death (PCD). Silencing of BECLIN-1 by TRV in N.

benthamiana plantsconsisting the N gene showed an unconfined PCD response on TMV 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 Ngene-mediated almost resistance 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 Rxsignaling complex. In addition, a number of host genes used in virusreplication and apart movement in plants have been observed by VIGS. VIGS hasalso been used to study plant defense system against fungi. A series of hostgenes used in Cladosporium folium-tomato resistance have been characterized.                       VIGS was used to observegenes that reduce stress. Later on embryogenic abundant 4 (lea4) was observedto be involved in used moisture stress.

SlGRX1 was observed to for regulationof the abiotic defense against oxidative, drought, and salt stresses. Inpepper, CaOXR1 was observed to play roles in defending to high salinity andosmotic stress. In tobacco plants, NbPHB1 and NbPHB2, two subunits of prohibitions,were observed to have a critical role in mitochondrial biogenesis and defenseagainst stress and senescence in plants. NaHD20 has a target in responses todehydration. In more explanation, VIGS has been used to manipulate waterdeficit-induced genes in peanut.Plant growth genes                            VIGS is an effectiveassay for reducing gene expression; therefore, VIGS facilitates the observationof 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 thatPapsAG-1 has a role in stamen and carpel identification; however, the samegene, PapsAG-2, while showing redundancy in these functions, has a major rolein the development of the setae, ovules, and stigmas.

                                             Intobacco and Petunia hybrid plants, many flower development associating genes,such as flowering duration determine genes (FCA and FY), floral organ identitygenes (AP3 and DEFICIENS) and flower development genes (NbMADS4-1, NbMADS4-2, PhPHB1and PhPHB2) have been uncovered by VIGS. In a study of leaf and shoot growth,Kang et al. showed that the silencing of the NbBPS1 gene obtained in growthreduction, abnormal leaf development, and cell ultimate death. This phenotypeis diverse from the case of the Arabidopsis bps mutant.

Boozier et al used VIGSto reduce the expression of SAMT1 in N. benthamiana. The disastrous growthreduction phenotype in silenced plants reveals that this methylation-relatedprotein has an important role in plant development.

The plant vascular developmentgene (RPN9), Retinoblastoma-concerning gene (RBR), a plant root developmentgene and some genes in meristem, as Dt1 and ML1 have been characterized byVIGS. These results give rise that VIGS is one of the most powerful method forthe observation of genes whose loss-of-function mutants induce embryonic andseedling death.Cellular metabolism and function                          VIGS has been impliedto study plant cellular functions and metabolic paths, such as biotin, enzymebiosynthesis, and organic manifesto. Burton et al. and Held et al. used PVX andBSMV vectors, alternatively, to study the usage of Cellulose synthase (Cesar)142,143. VIGS was also implied to show the genes used in the biosynthesis ofcapsaicinoids (AT3, Comet, pat, and KS), D-appose (UDP-D-appose/UDP-D-xylosesynthase, and AXS1), flaming 146, histone, and major proteins in the RNAsilencing pathway, such as Argonaute1- and Argonaut 4-like genes.

In moreanalysis, genes involved in the regulating the functions of PCD have been nowidentified using VIGS.                         For example, themitochondrial-related hexokinase Hxk1 gene, 20S proteasome, the 19S regulatoryunit of the 26S proteasome and a regulatory gene of PCD (CDC5). VIGS has alsobeen developed to characterized cellular functions of genes used inchloroplasts and mitochondria bio genes, plastid biogenetics, peroxisomebiogenesis, alkaloid biosynthesis, isoprenoid biosynthesis, ascorbic acid biosynthesis,sterol biosynthesis, and membrane biogenesis.

Merits and demerits of VIGS                      As comparing with othergenomic methods, VIGS has many advantages:(i)                VIGS is much faster. An important characteristicof VIGS is that it can cause loss-of-function expression of a specific gene ina short period of time. Therefore, the gene functionality can be accessedquickly, obviating the tedious method of plant regeneration. (ii)              (ii) Plant transformation is excluded, whichmeans that studies of gene functionality in plants that are more difficult totransfer (e.

g., cotton and soybean) would be more productive once the VIGSsystem is developed.(iii)             (iii) VIGSallows the study of genes that are necessary for plant viability. VIGS can beinduced at the seedling or early development stages, and has been developed asa powerful tool in the observation of genes whose mutations induce embryonicand seedling-death. VIGS is the only method that allows the analysis of suchplant genes that are used in plant development.(iv)              (iv) Thephenotype of multiple genes with functional reduction can be silenced togetherthrough VIGS using conserved domains.

On the other hand, a specific site can beused for VIGS if only one gene in a gene family is projected to be silenced. (v)               (v) It allows rapid comparison of the functionsof same genes between different plant species simultaneously, developing moreidentical gene function identification. VIGSalso has some disadvantages or limitations. For example(i)                In most cases, the phenotypes of gene cannot becompletely reduced through VIGS. As the expression of the target gene isretarded, the remaining phenotype of the target gene can be same for itsfunction. Therefore, for those genes, the loss-of-function expression cannot beobserved by VIGS. (ii)              (ii) VIGS requires before knowledge of targetgene sequence information. The effectiveness of silencing may be compared byretarded genes, unless the full genome or sufficient EST sequences are present.

(iii)            (iii) Genes give expression during germination orthe early seedling stage cannot be observed by VIGS, because VIGS is usuallyshown on adult plants and most of the VIGS phenotype is not transfer to nextgeneration. (iv)             (iv) The efficiency may differ and the expressionof VIGS is not very stable one. Results may not be same among differentexperiments or different plants. To get solution of this problem, it is commonto use a marker gene that gives a visible silencing phenotype as a positivecontrol.

  Conclusionand future of this VIGS           Over the previous 15 years, VIGS hasbeen successfully implied to give discovery and confirm gene functions in manycrop plants, including both dicotyledonous and monocotyledonous crop plants.Furthermore understanding of the method of gene silencing and growth of vectorsfor VIGS will led to most plant species already studied by newly made VIGSsystems, especially those that are difficult to observe by conventionalmethods. Now, more plant genomes have been sequenced, and new molecular biologymethods have been established for VIGS.                                     For example,artificial miRNA silencing vectors have been implemented in VIGS, and a VIGScDNA library was made using the gateway system.

With more technicalenhancements, VIGS will continuously to be frequently used in plant functionalgenomes.                                               References 1 Matthew L. RNAi for plantfunctional genomics. Comp Funct Genomics, 2004, 5: 240–2 Baulcombe D C. Fast forwardgenetics based on virus-induced gene silencing. Curr Opin Plant Biol, 1999, 2:109–113 Burch-Smith T M, Anderson J C,Martin G B, et al. Applications and advantages of virus-induced gene silencingfor gene function studies in plants. Plant J, 2004, 39: 734–746 4 Robertson D.

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