**2. The importance of silencing pathways**

A major breakthrough in the history of biology was the discovery of an RNA-induced silencing response in the nematode *Caenorhabditis elegans*[25]. Prior to that discovery, this RNA-induced silencing had been interpreted as a defence mechanism against viruses and other invading nucleic acids; the discovery of endogenous small regulatory RNAs in many species led to the realisation that gene silencing is a fundamental genetic regulatory mechanism in eukaryotic organisms [26].

Several studies in genetics, biochemistry and the development of novel techniques in molec‐ ular biology have helped identify different components of the RNA silencing machinery and have confirmed that RNA interference (RNAi), co-suppression and virus-induced gene silencing share mechanistic similarities [27]. RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21–23 nucleotide (nt) RNA molecules [28]. Organisms utilise RNA silencing for three purposes: 1) creating and maintaining heterochromatin at repetitive DNA and transpo‐ sons; 2) regulating development, stress responses and other endogenous regulatory functions; 3) defending against viral and bacterial infections [29]. Silencing is utilised in developmental pathways and in cellular differentiation to repress genes whose products are not required at specific stages of development or in specific cell types; in plants silencing is also used to respond to internal and external stresses by changing the expression of specific genes involved in the response. In some situations, tissue- or cell-specific silencing is desirable.

Initially discovered in transgenic plants, especially in those created to acquire virus resistance, RNA silencing is now also believed to be responsible for various epigenetic effects and their maintenance; the silencing of transgenic loci in plants most likely results from the activation of defence mechanisms. A number of silencing studies with different plant systems have explored transgenes as indicators of silencing pathways; these works have received important attention in part because silencing reduces the reliability of transgenic approaches in biotech‐ nology of several agriculturally important cultures.

RNA silencing can be classified into two major categories: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). TGS is defined as inhibition of transcription, whereas PTGS involves the post-transcriptional degradation of RNA species but does not affect the transcription rate [30]. TGS occurs when double-stranded RNA molecules (dsRNA) containing promoter sequences are present, and PTGS occurs when dsRNA comprise open reading frames (ORF). Together, TGS and PTGS depend on small interfering RNAs (siRNA) or microRNAs (miRNA) that are produced from dsRNA precursors [31]. Because RNA silencing, mainly PTGS, also contributes to antiviral immunity in plants, fungi and inverte‐ brates, it is an important part of innate immunity [32]. The silencing may persist over many cell divisions or plant generations [33].

The basic steps in common to all RNA silencing pathways (Figure 1) include: (i) formation of a dsRNA; (ii) processing of the dsRNA by an RNase III–like enzyme named Dicer (DCL) to shorter (20-30 nucleotide) dsRNA duplexes, the so called siRNA (iii) binding of the small RNA duplexes to a protein from the Argonaute (AGO) family; and (iv) targeting of the RNA-induced complex to mRNA (or DNA) guided by strand complementary to the small dsRNA, which is called the guide [34]. At present, there is good evidence for the existence of at least four different types of RNA silencing pathways in plants. These pathways involve different types of small RNA molecules, specially siRNA and miRNA. Heterochromatin-associated siRNA (hcsiRNA), trans-acting siRNA (tasiRNA) and viral siRNA (primary and secondary) are also important in silencing [35]. A better understanding of silencing pathways is very important because of the potential usefulness of silencing as a powerful tool for gene function studies and crop improvement.

#### **2.1. Post-transcriptional gene silencing**

of the retinoblastoma protein (pRb), which negatively regulates the G1/S transition in cells. The Rep protein, which is encoded by all geminiviruses and is the only viral protein necessary for viral DNA replication, has been found to induce expression and also interacts with the host "proliferating cell nuclear antigen" (PCNA), an auxiliary protein of DNA polymerases required during replication and repair in non-dividing plant cells. This observation suggests that Rep protein can provide the necessary stimulus to induce the dedifferentiation process. Mechanisms other than sequestering plant pRb most likely contribute to the multiple effects of geminivirus proteins on cellular gene expression, cell growth control and cellular DNA

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

Ultimately, RNA silencing suppressor (RSS) proteins are able to block or attenuate plant host defence mechanisms, particularly post-transcriptional gene silencing and efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machi‐ nery, often mimicking their normal cellular functions [23]. Viral suppressors of RNA silencing have been identified from almost all plant virus genera; these VSRs are surprisingly diverse, exhibiting no obvious sequence similarities. Most identified VSRs are multifunctional; in addition to being RNA silencing suppressors, they often perform essential roles, functioning as coat proteins, replicases, movement proteins, helper components for viral transmission, proteases or transcriptional regulators. The first viral RNA silencing suppressor identified was the helper component-proteinase (HC-Pro) of potyviruses [24]; currently, many different

The aim of this chapter review is to discuss the current status of knowledge regarding various components of host silencing machinery and viral suppression. We also pretend to describe how the defence response in plants is directed against the virus and, in particular, how the virus can sidetrack the host's defence response. Relevant topics on the molecular bases of the induction and suppression of the RNA silencing mechanism, as well as the applications and perspectives of the use of silencing suppression in plant biotechnology, will be emphasised.

A major breakthrough in the history of biology was the discovery of an RNA-induced silencing response in the nematode *Caenorhabditis elegans*[25]. Prior to that discovery, this RNA-induced silencing had been interpreted as a defence mechanism against viruses and other invading nucleic acids; the discovery of endogenous small regulatory RNAs in many species led to the realisation that gene silencing is a fundamental genetic regulatory mechanism in eukaryotic

Several studies in genetics, biochemistry and the development of novel techniques in molec‐ ular biology have helped identify different components of the RNA silencing machinery and have confirmed that RNA interference (RNAi), co-suppression and virus-induced gene silencing share mechanistic similarities [27]. RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21–23 nucleotide (nt) RNA molecules [28]. Organisms utilise RNA silencing for

replication [21, 22].

suppressors are known.

organisms [26].

**2. The importance of silencing pathways**

As already mentioned, PTGS is essential to antiviral immunity in plants, thus our focus will be concentrated in this phenomenon. PTGS was first observed in 1990, and initially referred to as 'co- suppression', it was first discovered in transgenic petunia plants in which the introduction of the gene for chalcone synthase created a block in anthocyanin biosynthesis, resulting in variegated pigmentation [36].

*virus* (TEV) were initially susceptible to infection and showed symptoms. However, a few weeks after the transgenic plants recovered from the TEV infection, newly developed leaves were symptomless and virus-free. Remarkably, the recovered leaves were resistant against a

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

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

257

PTGS is a mechanism that has been preserved among eukaryote kingdoms. It is a genetic regulatory mechanism that is involved in several processes including defence of the genome against mobile DNA elements, establishment of heterochromatin, control of plant and animal development, and downregulation of gene expression by specific cleavage and translational repression of target mRNAs that contain complementary sites to miRNAs or siRNAs [40]. The involvement of short RNAs in PTGS was discovered when ~25 nt RNA molecules with sequence homology to a transgene were detected only in plants in which the corresponding transgene was silenced [41]. These molecules are also generated against replicating viruses. They are loaded in an active silencing complex called RISC (RNA-induced silencing complex). RISC shows slight variability in composition from one RNA silencing pathway to the next and from species to species; however, all RISCs contain a guide RNA strand bound to an AGO protein. AGO imparts endonuclease activity to the RISC (the so called slicer activity) being

During PTGS, RNA is degraded predominantly in the cytoplasm. The strongest evidence in support of this comes from study of PTGS-based degradation of RNA viruses that are expressed exclusively in the cytoplasm [42]. However, recent work has demonstrated that the enzymes involved in PTGS are localised in both the cytoplasm and the nucleus. Overall, the available data suggest that PTGS activity in plant cells occurs in both the cytoplasmic and nuclear compartments. Nuclear PTGS would allow regulation of a potentially larger set of

Some aspects of genomic silencing remain unknown. For a more complete understanding of genomic silencing, supplementary approaches are needed. This is especially important because gene silencing has the potential for use as a potent sequence-specific gene inactivation

RNA silencing is known to serve as a mechanism for plant defence against pathogens. To counteract this mechanism, viruses have evolved the ability to avoid or suppress RNA silencing. Using this strategy, viruses protect their genomes from degradation through the production of proteins that act as suppressors of RNA silencing. These viral proteins act through a variety of molecular mechanisms including, particularly, blockage of the intercel‐ lular and systemic spread of mobile small silencing RNA molecules. The ability of viruses to infect cells can have a profound impact on host endogenous RNA silencing regulatory pathways and can result in alterations in endogenous short RNA expression profile and gene expression [43]. A general overview of the RNA silencing pathway discriminating the different

endogenous targets that cannot be regulated through cytoplasmic PTGS [31].

tool in functional genomics and plant biotechnology.

steps targeted by different VSR is provided in Figure 2.

second TEV infection but were susceptible to infection by the heterologous PVY [39].

responsible for target RNA cleavage.

**3. RNA silencing suppression**

**Figure 1.** Overview of the transcriptional gene silencing (TGS), siRNA and miRNA pathways. The important steps of each pathway are depicted.

PTGS was also detected in transgenic plants engineered for virus resistance, and associated with the phenomenon of recovery of a host plant from viral infection. This recovery process was soon understood to be associated with the plant's inherent RNA silencing mechanism, which is an evolutionarily conserved antiviral system. The first report related to virus-induced gene silencing was published as long ago as 1929 by McKinney, who reported that tobacco plants infected with the "green" strain of TMV were protected against infection by a closely related second virus (TMV "yellow" strain). This phenomenon was later described as "crossprotection" [37]. However, a remarkable explanation of cross-protection was provided when it was shown that virus infection prevents infection by a second virus if the two viruses possess homologous sequences. Importantly, this viral-RNA-mediated cross-protection was function‐ ally equivalent to post-transcriptional gene silencing [38].

Viruses are not only targets of transgene induced RNA silencing but also elicit silencing themselves. Transgenic plants expressing a truncated version of the coat protein of *Tobacco etch* *virus* (TEV) were initially susceptible to infection and showed symptoms. However, a few weeks after the transgenic plants recovered from the TEV infection, newly developed leaves were symptomless and virus-free. Remarkably, the recovered leaves were resistant against a second TEV infection but were susceptible to infection by the heterologous PVY [39].

PTGS is a mechanism that has been preserved among eukaryote kingdoms. It is a genetic regulatory mechanism that is involved in several processes including defence of the genome against mobile DNA elements, establishment of heterochromatin, control of plant and animal development, and downregulation of gene expression by specific cleavage and translational repression of target mRNAs that contain complementary sites to miRNAs or siRNAs [40].

The involvement of short RNAs in PTGS was discovered when ~25 nt RNA molecules with sequence homology to a transgene were detected only in plants in which the corresponding transgene was silenced [41]. These molecules are also generated against replicating viruses. They are loaded in an active silencing complex called RISC (RNA-induced silencing complex). RISC shows slight variability in composition from one RNA silencing pathway to the next and from species to species; however, all RISCs contain a guide RNA strand bound to an AGO protein. AGO imparts endonuclease activity to the RISC (the so called slicer activity) being responsible for target RNA cleavage.

During PTGS, RNA is degraded predominantly in the cytoplasm. The strongest evidence in support of this comes from study of PTGS-based degradation of RNA viruses that are expressed exclusively in the cytoplasm [42]. However, recent work has demonstrated that the enzymes involved in PTGS are localised in both the cytoplasm and the nucleus. Overall, the available data suggest that PTGS activity in plant cells occurs in both the cytoplasmic and nuclear compartments. Nuclear PTGS would allow regulation of a potentially larger set of endogenous targets that cannot be regulated through cytoplasmic PTGS [31].

Some aspects of genomic silencing remain unknown. For a more complete understanding of genomic silencing, supplementary approaches are needed. This is especially important because gene silencing has the potential for use as a potent sequence-specific gene inactivation tool in functional genomics and plant biotechnology.
