Viruses viruses, in which, viral RNA acts as a

Viruses induced gene silencing as a tool for gene functional analysis
in crop plants


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                       Viruses 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 simple often involving
agroinfiltration or biotic inoculation of plants. Second, one is the the
results are gotten rapidly typically with the time duration of two-three weeks  of inoculation. Third, is the technology
bypasses  transformation points and hence
can be used to number of crop plant  
recalcitrant to transformation. Fourth, is the method has the strength
to silence multiple copy genes. VIGS is based on the phenomenon of RNA-interference.
VIGS is based on the methodology 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 are differently termed as
post-transcriptional gene silencing (PTGS) in crop plants, quelling in fungi
and RNAi in animals. One important usage of this pathway in plants is in
defense mechanism against viruses, in which, viral RNA acts as a initiator to
induce RNA caused gene silencing which, in return, is directed towards the
viral genes. In VIGS, this viral RNA-induced defense strategy against viruses
has been developed as a tool for reversion of genetics and observation of gene
functions in plants, known as VIGS. In comparison to other PTGS-based methods
requiring genetic transformation points, a ”functional knock-down” for a
specific plant gene can be developed using VIGS within a duration of weeks
without going to transform. Besides being rapid and simple, VIGS in practice is
useful in observing gene functions in specific crop species stable for
transformation and genes that cause embryo death in knock-outs. Another
potential of the VIGS method is that it can be managed to silence multiple
components of a gene family, thereby incorporating the problem of functional
retardnace of genes. Several RNA and DNA viruses have been developed to
establish VIGS vectors. The gene to be silenced is cloned 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
vitro into 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 VIGS                                   

1997, van Kammen first described the term “VIGS” to describe the method of
recovery from virus infection. After that, the term “VIGS” has been used
importantly for the method of using newly modified viruses to knockdown
expression of endogenous genes. In the initial stages, most of the VIGS systems
were other examples are also shown in the table as given below. based upon RNA
viruses. In 1995, Kumagai et al. inoculated a fragment of phytoene desaturase
(PDS), a basal enzyme of the carotenoid biosynthetic pathway, into the Tobacco
mosaic virus (TMV) . When this new developed virus was inserted into Nicotiana
benthamiana, a blench expression in the leaves was shown and this phenomenon
was developed by reduction in endogenous PDS mRNA

1998, 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 a
useful method for masking endogenous gene expression and opening plant gene
functions. In 2001, a novel VIGS vector was developed based on Tobacco rattle
virus (TRV). TRV was provided to develop more rapid silencing of transgenes and
disaster genes. TRV could be released more vigorously throughout the all
morphology plant, including its meristem tissue, and the visible phenotypic
expression caused by TRV are much lower as compared with other viruses. The TRV
vector has been extensively used in gene function studies of tomato, tobacco,
Petunia hybrida, chili pepper, Arabidopsis, and cotton plants. Similar examples
are also shown in the tables on the next page table no his mturase (PDS), a keypid tool their
function.ysis in crop plants

Different methods of developing  VIGS

In a VIGS system, to reduce
expression of an disasteros plant gene, a part of the gene to be silenced
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 of silencing, the VIGS system should be maximized. First, the size
range of the inserted segment of target endogenous gene may affect the
performance of VIGS. Most VIGS vectors have the ability to carry a fragment of
length upto 150 and 800 bp. VIGS vectors may fail to cause gene silencing if a
fragment of more than 1500 bp is introduced. Although some studies give rise
that a 23 bp inoculation was able to cause VIGS, fragments of 200-350 bp in length is usually
taken for VIGS to get higher silencing efficiency. Furthermore, some studies
given that the orientation of the inoculated gene fragment was also an
important cause that could affect the efficiency of VIGS, with higher silencing
efficiency being caused by a reverse oriented insertion in comparison 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 whatever the
orientation of the inoculated fragment. In addition, the silencing efficiency
could be markly enhanced if the target segments were constructed as a hairpin
type structure. Selection of the target gene is useful for VIGS.  Evidence has given that an improper gene
fragment might caused off-target silencing, developing an inaccurate phenotype.
Many appearing fragments can be chosen for silencing of a specific targeted
gene. However, if the target gene refers to a gene family, some sequences may
have conserved domains between various genes in the gene family, and the
segments of the target gene may have more than 23 bp that is same but not
identical 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 UTR region is a good one to choose. At the other
hand, the restricted domains should be taken to avoid functional complementation
by genes from the identical family; in this thing all the genes in the family
are silenced.

                        The progress of gene
silencing may be manipulated by different inoculation methods. The common
methods in use for inoculation are agro-infiltration, rub-inoculation with 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 the virus RNA extract will be used to inoculate
chosen plants. High silencing efficiency was obtained using agro drench, a
method of watering the plant roots with agro-inoculation directly. Liu et AL
successfully caused the TRV vector into tomato by spraying a TRV agro-culture
by using an airbrush. 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 desirable silencing expression
than inoculation of cotyledons or seedlings. Some studies given that effective
silencing could be caused by incorporating plucked tomato, strawberry and
bilberry fruits with an agro-culture along with the VIGS vector, which is
useful for analyzing gene functions during the postharvest stage.
Co-incorporating of viral suppressors with VIGS vectors may also enhanced the
silencing efficiency. When plants were inserted by a mixture of VIGS vector and
a gene-silencing suppressor, higher accumulation of virus in native inoculated
cells induced a higher enhancement of silencing in systemic leaves. With the
establishment of more and more new virus inoculation techniques, VIGS will be
applicable to more plant spices. As following diagram represents the methodology.

                    At the end, environmental processes of plant
growth will cause the efficiency of gene silencing. At higher temperatures
areas, the getting of virus are markedly reduced, which cause the progress of
virus induced silencing. At the other hand, lower temperatures areas lead to
higher virus concentration and silencing progress. For TRV vectors, tomato
plants should be taken 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 DNAb and DNA 1 vectors can induce
highly effective silencing from the temperature of 22 to 32°C.

Validity of functionality of genes via VIGS

                         The high effectiveness
of VIGS has caused to its enhanced use in unwinding the functions of hundreds
of crop plant genes involved in defense mechanism pathways, plant growth, and
metabolism. Recent studies in gene function identification by VIGS are detailed

Gene involved in pathogen stresses and insects and abiotic stresses

growth in an environment surrounded by a variety of microbes and abiotic hazards.
A highly efficient defense system has been introduced to resist effective
attack by biotic and abiotic stresses. Past studies have developed the
functions of different plant genes involved in virus-, bacteria-, fungi-, and
insect-resistance and stress to give response. In the study of plant resistance
to virus inoculation, the most effective examples of using VIGS to open gene
functions in defense mechanism pathway was the N gene against TMV and 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, and
WRKY/MYB of the transcription factors, COI1 and CTR1 genes. The power of VIGS
as a way in reverse genetics is more manifested by the following studies of the
roles of BECLIN-1 and NRIP1 in N-gene and RanGAP2 in Rx-gene caused programmed
cell death (PCD). Silencing of BECLIN-1 by TRV in N. benthamiana plants
consisting 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 N
gene-mediated almost resistance to TMV.

The association of Rx and RanGAP2 in N. benthamiana or potato is
required for severe resistance to PVX, where RanGAP2 is portion of the Rx
signaling complex. In addition, a number of host genes used in virus
replication and apart movement in plants have been observed by VIGS. VIGS has
also been used to study plant defense system against fungi. A series of host
genes used in Cladosporium folium-tomato resistance have been characterized.

                      VIGS was used to observe
genes that reduce stress. Later on embryogenic abundant 4 (lea4) was observed
to be involved in used moisture stress. SlGRX1 was observed to for 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 tobacco plants, NbPHB1 and NbPHB2, two subunits of prohibitions,
were observed to have a critical role in mitochondrial biogenesis and defense
against stress and senescence in plants. NaHD20 has a target in responses to
dehydration. In more explanation, VIGS has been used to manipulate water
deficit-induced genes in peanut.

Plant growth genes    

                       VIGS is an effective
assay for reducing gene expression; therefore, VIGS facilitates the observation
of genes whose loss of functionality could be able to die the plants. Up to
recent 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 in stamen and carpel identification; however, the same
gene, PapsAG-2, while showing redundancy in these functions, has a major role
in the development of the setae, ovules, and stigmas.

tobacco and Petunia hybrid plants, many flower development associating genes,
such as flowering duration determine genes (FCA and FY), floral organ identity
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,
Kang et al. showed that the silencing of the NbBPS1 gene obtained in growth
reduction, abnormal leaf development, and cell ultimate death. This phenotype
is diverse from the case of the Arabidopsis 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 this methylation-related
protein has an important role in plant development. The plant vascular development
gene (RPN9), Retinoblastoma-concerning gene (RBR), a plant root development
gene and some genes in meristem, as Dt1 and ML1 have been characterized by
VIGS. These results give rise that VIGS is one of the most powerful method for
the observation of genes whose loss-of-function mutants induce embryonic and
seedling death.

Cellular metabolism and function

                          VIGS has been implied
to study plant cellular functions and metabolic paths, such as biotin, enzyme
biosynthesis, and organic manifesto. Burton et al. and Held et al. used PVX and
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 been now
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 has also
been developed to characterized cellular functions of genes used in
chloroplasts and mitochondria bio genes, plastid biogenetics, peroxisome
biogenesis, alkaloid biosynthesis, isoprenoid biosynthesis, ascorbic acid biosynthesis,
sterol biosynthesis, and membrane biogenesis.

Merits and demerits of VIGS

                      As comparing with other
genomic methods, VIGS has many advantages:

VIGS is much faster. An important characteristic
of VIGS is that it can cause loss-of-function expression of a specific gene in
a short period of time. Therefore, the gene functionality can be accessed
quickly, obviating the tedious method of plant regeneration.

(ii) Plant transformation is excluded, which
means that studies of gene functionality in plants that are more difficult to
transfer (e.g., cotton and soybean) would be more productive once the VIGS
system is developed.

 (iii) VIGS
allows the study of genes that are necessary for plant viability. VIGS can be
induced at the seedling or early development stages, and has been developed as
a powerful tool in the observation of genes whose mutations induce embryonic
and seedling-death. VIGS is the only method that allows the analysis of such
plant genes that are used in plant development.

 (iv) The
phenotype of multiple genes with functional reduction can be silenced together
through VIGS using conserved domains. On the other hand, a specific site can be
used for VIGS if only one gene in a gene family is projected to be silenced.

(v) It allows rapid comparison of the functions
of same genes between different plant species simultaneously, developing more
identical gene function identification.

also has some disadvantages or limitations. For example

In most cases, the phenotypes of gene cannot be
completely reduced through VIGS. As the expression of the target gene is
retarded, the remaining phenotype of the target gene can be same for its
function. Therefore, for those genes, the loss-of-function expression cannot be
observed by VIGS.

(ii) VIGS requires before knowledge of target
gene sequence information. The effectiveness of silencing may be compared by
retarded genes, unless the full genome or sufficient EST sequences are present.

(iii) Genes give expression during germination or
the early seedling stage cannot be observed by VIGS, because VIGS is usually
shown on adult plants and most of the VIGS phenotype is not transfer to next

(iv) The efficiency may differ and the expression
of VIGS is not very stable one. Results may not be same among different
experiments or different plants. To get solution of this problem, it is common
to use a marker gene that gives a visible silencing phenotype as a positive


and future of this VIGS

           Over the previous 15 years, VIGS has
been successfully implied to give discovery and confirm gene functions in many
crop plants, including both dicotyledonous and monocotyledonous crop plants.
Furthermore understanding of the method of gene silencing and growth of vectors
for VIGS will led to most plant species already studied by newly made VIGS
systems, especially those that are difficult to observe by conventional
methods. Now, more plant genomes have been sequenced, and new molecular biology
methods have been established for VIGS.

                                     For example,
artificial miRNA silencing vectors have been implemented in VIGS, and a VIGS
cDNA library was made using the gateway system. With more technical
enhancements, VIGS will continuously to be frequently used in plant functional


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