**4. Applications**

#### **4.1. Virus-Induced Gene Silencing (VIGS)**

Virus-induced gene silencing (VIGS) is a technique derived from the knowledge of RNA silencing. It uses recombinant viruses to specifically reduce or knock-down endogenous gene activity; it is based on post-transcriptional gene silencing (PTGS) [132]. When used to infect plants, recombinant viral vectors carrying segments of host genes produce siRNAs that are specific to host mRNA. The RISC complex mediates the degradation of target host mRNAs, leading to downregulation of gene expression. Thus, the infected plant has a phenotype similar to a loss-of-function mutant of the gene of interest [133].

VIGS is used as a tool for turning down host gene expression, especially in plants. In principle, a plant gene of interest can be silenced by infecting the plant with a viral vector that has been modified to express a nucleic acid sequence homologous to the host gene. As a proof of concept, several endogenous genes have been silenced using VIGS. The plant gene *phytoene desaturase* (PDS), a regulator of carotenoid biosynthesis, was silenced in *N. benthamiana* plants by the use of a recombinant TMV vector. As a result, degradation of the host PDS mRNA and resultant alterations in the pigment synthesis pathway were observed [133].

There are four main reasons for the popularity of VIGS. First, the methodology is simple, often involving agroinfiltration or biolistic inoculation of plants. Second, the results are obtained rapidly, typically within two to three weeks of inoculation. Third, the technology bypasses transformation steps and hence is applicable to a number of plant species that are recalcitrant to transformation. Fourth, the method has the potential to silence multi-copy genes [134]. Efficient silencing depends mainly on the choice of VIGS vector. There are many factors to be considered when choosing the virus to be used for VIGS. Among the factors to be considered are (1) the virus must produce few or no symptoms during infection, thereby facilitating easy visualisation and interpretation of the mutant phenotype; (2) it must induce persistent silencing, thus viruses with strong silencing suppressors are to be avoided because they can interfere with the establishment of silencing; (3) it is advantageous to have infectious cDNA clones of the virus for cloning purposes; and (4) the virus must retain infectivity after insertion of foreign DNA. The virus should also show uniform spread, infect most cell types including the meristem, and preferably show a broad host range [133].

The induction of biotic or abiotic stress activates other defence responses that can divert the host from antiviral silencing [129]. Therefore, RAV2 is a critical control factor for carmovirus

270 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Other viruses make use of alternative mechanisms for suppression of silencing. The TrAP geminiviral protein AC2 upregulates a gene coding for the cold- and abscisic acid-inducible protein KIN1 as well as five additional known or putative cold-regulated genes [50]. As already mentioned, the efficiency of RNA silencing is dependent on temperature; at low temperatures, inhibition of silencing occurs and the plant becomes susceptible to viral infection [76]. The inhibition of silencing at low temperature is a pathway used by AC2 to accomplish the suppression of silencing [50]. Another strategy exploited by geminivirus is up-regulation of an endogenous RNA silencing suppressor, Werner exonuclease-like 1 (WEL1), which is mediated by AC2. Interestingly, the related proteins MUT-7 (mutate 7) and Werner syndromelike exonuclease (WEX) have been identified as positive regulators of RNA silencing in *C. elegans* and *A. thaliana*, respectively [130, 131]. Thus, AC2 up-regulation of Wel-1 results in interference with, or competition for, factors that are required for normal WEX function. Transient expression of a WEL-1 transcription unit is sufficient to suppress RNA silencing in

Virus-induced gene silencing (VIGS) is a technique derived from the knowledge of RNA silencing. It uses recombinant viruses to specifically reduce or knock-down endogenous gene activity; it is based on post-transcriptional gene silencing (PTGS) [132]. When used to infect plants, recombinant viral vectors carrying segments of host genes produce siRNAs that are specific to host mRNA. The RISC complex mediates the degradation of target host mRNAs, leading to downregulation of gene expression. Thus, the infected plant has a phenotype similar

VIGS is used as a tool for turning down host gene expression, especially in plants. In principle, a plant gene of interest can be silenced by infecting the plant with a viral vector that has been modified to express a nucleic acid sequence homologous to the host gene. As a proof of concept, several endogenous genes have been silenced using VIGS. The plant gene *phytoene desaturase* (PDS), a regulator of carotenoid biosynthesis, was silenced in *N. benthamiana* plants by the use of a recombinant TMV vector. As a result, degradation of the host PDS mRNA and resultant

There are four main reasons for the popularity of VIGS. First, the methodology is simple, often involving agroinfiltration or biolistic inoculation of plants. Second, the results are obtained rapidly, typically within two to three weeks of inoculation. Third, the technology bypasses transformation steps and hence is applicable to a number of plant species that are recalcitrant to transformation. Fourth, the method has the potential to silence multi-copy genes [134].

and potyvirus suppressors [128].

*N. benthamiana* [50].

**4. Applications**

**4.1. Virus-Induced Gene Silencing (VIGS)**

to a loss-of-function mutant of the gene of interest [133].

alterations in the pigment synthesis pathway were observed [133].

Several RNA and DNA viruses have been modified to create VIGS vectors. The gene to be silenced is cloned in an infectious derivative of a viral DNA (DNA virus-based vectors) or cDNA (RNA virus-based vectors) derived from viral RNA. Plant inoculation with viral vectors is most commonly achieved via *A. tumefaciens* infection, but can also be achieved by mechanical inoculation of *in vitro* synthesized transcripts, or for DNA-based vectors, by biolistic delivery methods. During the course of viral infection, either double-stranded RNA or RNA with a high degree of secondary structure is often produced; both of these are efficient initiators of RNA silencing directed against the infecting viral RNA. Other factors that play an important role in gene silencing in VIGS are the orientation of the insert (inverted repeats are more efficient than antisense orientation, which, in turn is more efficient than same sense orientation) and systemic spread of the silencing effect (the silencing signal is believed to spread independently of the VIGS vector to other parts of the plant) [134]. More than 30 VIGS vectors have been developed, and these vectors have been widely used to study the functions of genes involved in basic cellular functions, metabolic pathways, development, plant-microbe interaction, and abiotic mechanisms [132].

The first viral vector used for VIGS was TMV. Shortly thereafter, another vector was pro‐ duced based on another RNA virus, PVX carrying a cDNA fragment derived from the *PDS* gene [135]. However, although these first vectors were effective, they have intrinsic disadvantages. First, the VIGS phenotype is superimposed and sometimes complicated by chlorosis, leaf distortion and necrotic symptoms of virus infection. A second disadvant‐ age of these viral vectors is their inability to invade every cell, such that cells in which the target gene is not silenced may obscure VIGS phenotypes [136]. A novel VIGS vector based on TRV was then established. TRV was shown to induce more efficient silencing of transgenes and endogenous genes. It could spread more vigorously throughout the entire plant, including meristem tissue, and the symptoms induced by TRV are much milder than those produced by other viruses [136].

A primary limitation of VIGS technology is that a viral vector can be used only in plants that are hosts of the virus used. The first VIGS vectors (e.g., PVX) do not infect the model plant *A. thaliana*. Therefore, new vectors such as the TRV-based vector [136] were developed to overcome this difficulty. TRV is also one of the few viruses that have been modified into a highly efficient cloning and expression system for use in large-scale functional genomics screening. TRV vectors can induce VIGS in a number of solanaceous hosts like *N*. *benthami‐ ana*, tomato, potato, pepper, petunia, poppy (Eudicot species), and the model system *A.* *thaliana* (family Brassicaceae) [133, 137]. VIGS vectors have been applied not only in dicoty‐ ledonous plants but also in monocotyledonous plants. For this, a modified VIGS vector based on *Brome mosaic virus* (BMV) was developed and validated in barley, rice and maize [138].

would enhance expression of the genes of interest [101]. Studies with the P19 protein of TBSV were among those that provided the best results. Expression of a range of proteins was enhanced 50-fold or more in the presence of this suppressor, and experiments with GFP indicated that the co-infiltrated tissues accumulated the protein up to 7% of total soluble protein [142]. Due to its simplicity and rapidity, the P19-enhanced expression system is currently used in industrial production as well as used as a research tool for the isolation and biochemical characterisation of a broad range of proteins without the need for the time-

Viral Counter Defense X Antiviral Immunity in Plants: Mechanisms for Survival

http://dx.doi.org/10.5772/56253

273

The molecular basis of the silencing suppression of VSR proteins is quite complex and is currently incompletely understood. By the way, the discovery of the mode of action of different viral suppressors has demonstrated the existence of a complex interaction between VSR and plant silencing-regulated networks. For example, in addition to sequestering siRNA duplexes, the P19 protein of tombusviruses specifically controls antiviral AGO1 expression through enhanced miR168 expression, which arrests AGO1 translation [23]. It is likely that many other VSRs interact in diverse ways with RNA-silencing pathways. Many of these interactions remain to be discovered, and there are several gaps in our knowledge regarding the effectors of plant silencing machinery. Until very recently, the mechanisms of plant si/miRNA RISC assembly or the components of the plant RISC, which may also be potential targets of VSRs, were little known. The recently developed system of plant in vitro RISC [143] will likely accelerate the exploration of plant RISC assembly and RNA-targeting mechanisms mediated by this effector. This system will enable exploration of the mechanisms by which VSRs interact

More information about the replication, subcellular localisation and regulation of the expres‐ sion of viral genes, including VSRs, is required so that we may better understand the molecular mechanisms of VSR-mediated silencing suppression for the many plant viruses for which they are still not known. Because many VSRs have multiple functions in the virus life cycle, separate analysis of their silencing suppressor activities can lead to misinterpretations; thus, it is

Although common mechanisms of silencing suppression exist, there is also great variation in suppression mechanisms, likely driven by evolution and fitness, this variation has yielded viral strains with different properties. It is likely that additional differences will be found when plant viruses and their suppressors are tested in several plant species. This will provide us with a greater understanding of the parameters associated with the natural host range of a

Some of the already well described VSRs can be used as powerful tools for better understand‐ ing silencing pathways because they target specific steps of silencing machinery. Indeed, the P19 protein was recently used to demonstrate that siRNA duplexes function as mobile

consuming regeneration of stably transformed plants [141].

with one or more of the RISC components and prevent its assembly.

essential that VSRs be studied in their natural virus backgrounds [23].

virus and may possibly lead to new strategies for crop protection [52].

**5. Perspectives**

The VIGS system can be helpful in assessing gene function, especially for genes that cause zygotic/embryonic lethality when mutated and in species that are recalcitrant to genetic transformation. As aforementioned, it can be designed to silence multiple members of a gene family, thereby circumventing the problem of functional redundancy of genes [133].

#### **4.2. Use of viral suppressors**

The discovery of RNA silencing, and its derived technology (RNA interference; RNAi), has increased our knowledge of gene regulation and function. RNAi opened up novel avenues in biology, making it possible to develop fascinating strategies for application in genetic analysis, plant protection, and many other areas related to crop improvement [139]. In this context, a large number of silencing suppressor proteins have been described, and the discovery of the molecular basis of silencing suppression has inspired new concepts about the molecular basis of symptoms caused by viruses in plants [37].

Many biotechnological applications involving plants require high levels of protein expression. Generally, stably transformed plants are the preferred platform for large-scale production. To try to increase expression levels, transgenic lines that encode a replicating RNA virus vector carrying a gene of interest, a technology coined 'amplicon', have been exploited. The rationale of this method involves increasing the accumulation of the product of interest through transcription of an amplicon transgene that initiates viral RNA replication and gene expres‐ sion. However, the strategy failed because the transformants consistently exhibited RNA silencing of the amplicon transgene [140]. The viral dsRNA replication intermediates produced in every cell of the transgenic plants were recognised as potent triggers of the silencing-based defence mechanism that is normally elicited in the course of natural infections. Based on those findings, it was subsequently reasoned that co-expression of viral suppressors might prevent this adverse response and permit the high levels of gene expression initially envisioned with the use of amplicons [141].

To test this idea, in reference [94] crossed transgenic tobacco plants expressing TEV HC-Pro with amplicon lines designed to express a GUS reporter gene from the PVX genome. Pairing the suppressor and the amplicon locus resulted in a dramatic increase in virus accumulation and gene expression such that the leaves of mature plants accumulated the GUS protein up to 3% of total soluble protein. Remarkably, in spite of high virus accumulation, the plants did not suffer from viral disease and remained symptomless.

As opposed to stable, transgenic expression, transient expression is of interest for achieving expression of useful proteins. In plants, recombinant strains of *A. tumefaciens* can be used for transient gene expression. In principle, this system could allow high levels of gene expression; however, its utility has thus far been limited because ectopic protein expression usually ceases after 2– 3 days [141]. RNA silencing is, in fact, a major cause of this lack of efficiency. It was therefore anticipated that co-delivery of *A. tumefaciens* cultures with silencing suppressors would enhance expression of the genes of interest [101]. Studies with the P19 protein of TBSV were among those that provided the best results. Expression of a range of proteins was enhanced 50-fold or more in the presence of this suppressor, and experiments with GFP indicated that the co-infiltrated tissues accumulated the protein up to 7% of total soluble protein [142]. Due to its simplicity and rapidity, the P19-enhanced expression system is currently used in industrial production as well as used as a research tool for the isolation and biochemical characterisation of a broad range of proteins without the need for the timeconsuming regeneration of stably transformed plants [141].
