**5. Perspectives**

*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].

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

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

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

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

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

family, thereby circumventing the problem of functional redundancy of genes [133].

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

**4.2. Use of viral suppressors**

the use of amplicons [141].

of symptoms caused by viruses in plants [37].

suffer from viral disease and remained symptomless.

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

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

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

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 silencing signals between plant cells, in addition that P1, P38 and P0 proteins may prove to be powerful tools for studying the still unknown components of RISCs [118].

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