**1. Introduction**

[19] Bacon TH, Levin MJ, Leary JJ, Sarisky RT, & Sutton D (2003) Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clinical

microbiology reviews 16[1]:114-128.

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

The history of plant virology dates to the late 19th century, when Iwanowski and Beijerinck, who were investigating the cause of a mysterious disease of tobacco, independently described an unusual agent that caused tobacco mosaic disease. This agent was later named *Tobacco mosaic virus* (TMV) [1]. During this period, viruses including *Potato virus X* (PVX), *Potato virus Y* (PVY) and *Lettuce mosaic virus* (LMV) were described. These viruses could be distinguished based on their transmission and method of disease induction. In addition, numerous techni‐ ques for the study of viruses were developed.

Viruses are among the most agriculturally important groups of plant pathogens, causing serious economic losses in many major crops by reducing yield and quality. A virus can be defined as a set of one or more nucleic acid template molecules, often encased in a protective coat of protein or lipoprotein, which is able to organise its own replication only within suitable host cells [1]. Because the genetic information encoded by viral genomes is limited, viruses depend entirely on host cells to replicate their genome and produce infectious progeny. Both plant and animal viruses can be classified according to the type of nucleic acid that makes up their genome. In plants, the vast majority of viruses have positive-sense (+) RNA genomes (i.e., the RNA genome has the same polarity as cellular mRNA), although negative-sense (−) RNA and double-stranded RNA viral genomes also exist. Other plant viruses have DNA genomes; the DNA can be double-stranded (caulimoviruses) or single-stranded (geminiviruses) [1, 2]. Cell-to-cell movement of plant viruses occurs through cytoplasmic "bridges" between cells

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called plasmodesmata, and viruses are able to move systemically throughout plants via the phloem [1].

alternative pathway of host defence that occurs in parallel to RNA silencing and reinforces

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Over time, the strategies used by the virus to overcome these elaborate host defences can lead to a number of fundamental changes in the plant's physiology. Such changes, including structural modifications in the host cell, may give rise to intranuclear inclusions of various types and may affect the nucleolus or the size and shape of the nucleus. Within the mitochon‐ dria, abnormal membrane systems may develop [16, 17]. In plants infected with *Turnip yellow mosaic virus* (TYMV - Tymovirus), abnormalities such as clumping of chloroplasts and abnormal size and number of starch grains in leaf cells may occur, and small vesicles near the periphery and chloroplasts may become greatly enlarged and filled with starch grains [18]. These abnormalities were also observed in squash infected by the *Zucchini yellow mosaic potyvirus* (ZYMV - Potyvirus) [17]. In plant cell walls, three types of abnormality have been observed: abnormal thickening due to the deposition of callose near the edges of virus-induced lesions; cell wall protrusions involving the plasmodesmata (these protrusions may have one or more canals and may be quite short or of considerable length); paramural bodies, which are depositions of electron-dense material between the cell wall and the plasma membrane, may appear and extend over substantial areas of the cell wall, or be limited in extent occurring in association with plasmodesmata. Moreover, in the cytoplasm of an infected cell, virus particles may accumulate in sufficient numbers to form three-dimensional crystalline arrays. The ability to form crystals within the host cell cytoplasm depends on properties of the virus itself, and is not related to the overall concentration reached in the tissue or to the ability of the purified

Intriguingly, in carrot plants (*Daucus carota* L.) infected by *Cucumber mosaic virus* (CMV) some cytological and physiological changes were observed due to alterations in various host metabolites. With respect to cytological changes, scattered metaphase was observed in the diseased plant cells. The mitotic index of the diseased cells was decreased, while the nucleus / cytoplasm ratio was increased. Chromatin bridges were also observed at anaphase I and II. Physiological changes resulting in decreased carbon, nitrogen and protein content and

increased phosphorous content of the virally infected plants have been observed [19].

Other viral counterattack mechanisms involve changes in the plant cell cycle. In plants, as in all eukaryotes, the four phases of the mitotic cell cycle (G1, S, G2 and M) are conserved. During development, plant cells leave the cell division cycle, and in mature plants, DNA replication and the corresponding enzymes are confined to meristematic tissues [20]. Geminiviruses are good models for the study of the relationship between the cell cycle and viral DNA replication because they replicate in differentiated cells, such as mature cells of the leaves, stems and roots, in which most of the cellular factors required for viral DNA replication are normally absent. These cells have left the cell division cycle and no longer contain detectable levels of plant DNA replication enzymes necessary for geminivirus replication [21]. Due to the requirement for cellular factors, geminiviral DNA replication must be coupled to a special state of the infected cell, suggesting that the virus may have evolved mechanisms that affect the expression of cellular genes involved in S-phase progression and G1/S transition [22]. One such a mechanism involved in regulating changes of the host cell cycle appears to be the inactivation

cellular antiviral defence in plants [14].

virus to form crystals [1].

Viruses depend on other organisms (vectors) to transmit them from diseased to healthy plants. These vectors are often sap-sucking insects such as aphids, thrips, whiteflies, leaf-feeding beetles, plant-feeding mites, soil-inhabiting nematodes or fungal pathogens. Some viruses can be mechanically transmitted, on pruning knives or gardeners hands; or by grafting material, and a relatively small number of species can pass through infected seed [3]. Viruses use a variety of strategies that frequently induce disease in the plants they infect. Different viruses induce distinct diseases, and this can be true even for different strains of the same virus. Virus infection can profoundly alter the physiology of the host due to the interaction with cellular components. In plants, the severity of viral diseases varies considerably depending on the host genotype, the stage of infection and the environmental conditions. Diseases caused by viruses can vary broadly in intensity, from very mild symptoms observed in tolerant plants up to very severe symptoms and plant death [1, 4, 5].

Each plant virus encodes an average of 4-10 proteins necessary to coordinate the complex biochemical and intermolecular interactions required for viral infection cycles. The cycle of infection includes viral genome replication, cell-to-cell and systemic movement and transmis‐ sion [6]. For efficient viral infection to occur, viral proteins must be able to interact with factors in the host cell, thereby manipulating metabolic pathways and coordinating biochemical interactions that promote infection. Thus, during co-evolution between viruses and their hosts, a variety of complex interactions have developed that involve several distinct mechanisms of plant defence and virus attack.

During evolution, plants have developed diverse defence mechanisms that are activated during viral infection. One of these is the hypersensitive response (HR), which activates initial defence responses that prevent the infection from spreading further and then kills the cells within the infected zone. The onset of a second mechanism, systemic acquired resistance (SAR), then protects the plant together with HR against new attacks by the same pathogen. SAR is induced by a variety of agents after initial infection and can provide resistance to a wide range of pathogens for days [7-10]. The HR and SAR responses are accompanied by changes in gene expression that include the production of pathogenesis-related (PR) proteins and of several proteins involved in cell signalling [11].

Plants also possess other antiviral defence mechanisms such as RNA silencing, a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA [12], and the more recently described ubiquitin/26S proteasome system (UPS), which plays a central role in the degradation of proteins. The latter system is involved in almost all phases of the defence mechanisms of plants, regardless of the type of pathogen [13]. In addition to its proteolytic activity against ubiquitinated pathogen proteins, which directs their degradation by the 20S proteasome, the degradation of viral RNA can also occur via the ribonuclease activity of the 20S proteasome [14, 15]. While the proteasome as a structure, and RNA silencing as a mechanism, are two conserved features among eukaryotes, several lines of evidence suggest that the proteasome-linked RNase activity is most likely not directly related to RNA silencing. The selective degradation of viral RNAs by the 20S complex can represent an alternative pathway of host defence that occurs in parallel to RNA silencing and reinforces cellular antiviral defence in plants [14].

called plasmodesmata, and viruses are able to move systemically throughout plants via the

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

Viruses depend on other organisms (vectors) to transmit them from diseased to healthy plants. These vectors are often sap-sucking insects such as aphids, thrips, whiteflies, leaf-feeding beetles, plant-feeding mites, soil-inhabiting nematodes or fungal pathogens. Some viruses can be mechanically transmitted, on pruning knives or gardeners hands; or by grafting material, and a relatively small number of species can pass through infected seed [3]. Viruses use a variety of strategies that frequently induce disease in the plants they infect. Different viruses induce distinct diseases, and this can be true even for different strains of the same virus. Virus infection can profoundly alter the physiology of the host due to the interaction with cellular components. In plants, the severity of viral diseases varies considerably depending on the host genotype, the stage of infection and the environmental conditions. Diseases caused by viruses can vary broadly in intensity, from very mild symptoms observed in tolerant plants up to very

Each plant virus encodes an average of 4-10 proteins necessary to coordinate the complex biochemical and intermolecular interactions required for viral infection cycles. The cycle of infection includes viral genome replication, cell-to-cell and systemic movement and transmis‐ sion [6]. For efficient viral infection to occur, viral proteins must be able to interact with factors in the host cell, thereby manipulating metabolic pathways and coordinating biochemical interactions that promote infection. Thus, during co-evolution between viruses and their hosts, a variety of complex interactions have developed that involve several distinct mechanisms of

During evolution, plants have developed diverse defence mechanisms that are activated during viral infection. One of these is the hypersensitive response (HR), which activates initial defence responses that prevent the infection from spreading further and then kills the cells within the infected zone. The onset of a second mechanism, systemic acquired resistance (SAR), then protects the plant together with HR against new attacks by the same pathogen. SAR is induced by a variety of agents after initial infection and can provide resistance to a wide range of pathogens for days [7-10]. The HR and SAR responses are accompanied by changes in gene expression that include the production of pathogenesis-related (PR) proteins and of several

Plants also possess other antiviral defence mechanisms such as RNA silencing, a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA [12], and the more recently described ubiquitin/26S proteasome system (UPS), which plays a central role in the degradation of proteins. The latter system is involved in almost all phases of the defence mechanisms of plants, regardless of the type of pathogen [13]. In addition to its proteolytic activity against ubiquitinated pathogen proteins, which directs their degradation by the 20S proteasome, the degradation of viral RNA can also occur via the ribonuclease activity of the 20S proteasome [14, 15]. While the proteasome as a structure, and RNA silencing as a mechanism, are two conserved features among eukaryotes, several lines of evidence suggest that the proteasome-linked RNase activity is most likely not directly related to RNA silencing. The selective degradation of viral RNAs by the 20S complex can represent an

phloem [1].

severe symptoms and plant death [1, 4, 5].

plant defence and virus attack.

proteins involved in cell signalling [11].

Over time, the strategies used by the virus to overcome these elaborate host defences can lead to a number of fundamental changes in the plant's physiology. Such changes, including structural modifications in the host cell, may give rise to intranuclear inclusions of various types and may affect the nucleolus or the size and shape of the nucleus. Within the mitochon‐ dria, abnormal membrane systems may develop [16, 17]. In plants infected with *Turnip yellow mosaic virus* (TYMV - Tymovirus), abnormalities such as clumping of chloroplasts and abnormal size and number of starch grains in leaf cells may occur, and small vesicles near the periphery and chloroplasts may become greatly enlarged and filled with starch grains [18]. These abnormalities were also observed in squash infected by the *Zucchini yellow mosaic potyvirus* (ZYMV - Potyvirus) [17]. In plant cell walls, three types of abnormality have been observed: abnormal thickening due to the deposition of callose near the edges of virus-induced lesions; cell wall protrusions involving the plasmodesmata (these protrusions may have one or more canals and may be quite short or of considerable length); paramural bodies, which are depositions of electron-dense material between the cell wall and the plasma membrane, may appear and extend over substantial areas of the cell wall, or be limited in extent occurring in association with plasmodesmata. Moreover, in the cytoplasm of an infected cell, virus particles may accumulate in sufficient numbers to form three-dimensional crystalline arrays. The ability to form crystals within the host cell cytoplasm depends on properties of the virus itself, and is not related to the overall concentration reached in the tissue or to the ability of the purified virus to form crystals [1].

Intriguingly, in carrot plants (*Daucus carota* L.) infected by *Cucumber mosaic virus* (CMV) some cytological and physiological changes were observed due to alterations in various host metabolites. With respect to cytological changes, scattered metaphase was observed in the diseased plant cells. The mitotic index of the diseased cells was decreased, while the nucleus / cytoplasm ratio was increased. Chromatin bridges were also observed at anaphase I and II. Physiological changes resulting in decreased carbon, nitrogen and protein content and increased phosphorous content of the virally infected plants have been observed [19].

Other viral counterattack mechanisms involve changes in the plant cell cycle. In plants, as in all eukaryotes, the four phases of the mitotic cell cycle (G1, S, G2 and M) are conserved. During development, plant cells leave the cell division cycle, and in mature plants, DNA replication and the corresponding enzymes are confined to meristematic tissues [20]. Geminiviruses are good models for the study of the relationship between the cell cycle and viral DNA replication because they replicate in differentiated cells, such as mature cells of the leaves, stems and roots, in which most of the cellular factors required for viral DNA replication are normally absent. These cells have left the cell division cycle and no longer contain detectable levels of plant DNA replication enzymes necessary for geminivirus replication [21]. Due to the requirement for cellular factors, geminiviral DNA replication must be coupled to a special state of the infected cell, suggesting that the virus may have evolved mechanisms that affect the expression of cellular genes involved in S-phase progression and G1/S transition [22]. One such a mechanism involved in regulating changes of the host cell cycle appears to be the inactivation 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 replication [21, 22].

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

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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‐

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

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

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

in the response. In some situations, tissue- or cell-specific silencing is desirable.

nology of several agriculturally important cultures.

cell divisions or plant generations [33].

and crop improvement.

**2.1. Post-transcriptional gene silencing**

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 suppressors are known.

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.
