INTRODUCTION

RNA silencing is a novel gene regulatory mechanism that limits the transcript level by
either suppressing transcription (transcriptional gene silencing [TGS]) or by activating a
sequence-specific RNA degradation process (post transcriptional gene silencing
[PTGS]/RNA interference [RNAi]). Three phenotypically different but mechanistically
similar forms of RNAi are PTGS in plants, quelling in fungi, and RNAi in the
nimal kingdom have been described.

RNA silencing might have arisen as an ancient RNA surveillance system that is
conserved among eukaryotes,and that acts as a natural defense mechanism against
invasive nucleic acids, including viruses, transposons and perhaps other highly
repetitive genomic sequences. RNA silencing also plays a n important role in plant and
animal development by providing a system of gene control that can occur through RNA
degradation, translational inhibition or chromatin modification.
There are two overlapping but distinct RNA silencing pathways in plants and animals,
the siRNA pathway and the micro-RNA (miRNA) pathway. The siRNA pathway is
induced by the presence of perfect dsRNAs, and is believed to play a defensive role
against viruses and transposons. miRNAs are small 22-nt RNAs that are generated by
Dicer cleavage of short hairpin structures in primary miRNA transcripts. Many of these
miRNAs control the expression of key regulatory genes in plants and animals by binding
to mRNA, either targeting its destruction by cleavage or preventing its translation into
protein.

.The natural functions of RNAi and its related processes seem to be protection of the
genome against invasion by mobile genetic elements such as viruses and transposons
as well as regulated functioning of the developmental programs of eukaryotic
organisms.

THE DISCOVERY OF RNAi

In a 1998 issue of Nature (19 February) the laboratories of Fire and Mello described the
unexpected effects of injecting dsRNA into the nematode Caenorhabditis elegans.
dsRNA induced gene silencing in a gene-specific manner and phenotypes
corresponding to null mutant were observed. (Fire et al. 1998). The silencing activity of
dsRNA spread from the cells at the site of injection to most of the cells in the worm and
could be passed on to the next generation. The term RNAi was then coined to indicate
genetic interference by dsRNA (Fire et al. 1998). it was further shown that for dsRNA to
be an effective trigger of RNAi its sequence needs be homologous to the mRNA coding
region of the gene of interest and should be a few hundred base pairs in length
(Montgomery et al. 1998). While this work was in progress RNAi was also discovered in
the parasitic protozoa Trypanosoma brucei electroporation of dsRNA homologous to
tubulin mRNA led to downregulation of tubulin synthesis (Ngo et al. 1998). Within a
short time thereafter several laboratories simultaneously reported RNAi in a variety of
other organisms, including Drosophila, Planaria and Hydra, and, subsequently, RNAi
was described in mouse embryos and zebrafish (Sharp 1999). At an early stage, it was
established that gene silencing by dsRNA is widespread throughout the eukaryotic
lineage and trypanosomatid protozoa that separated relatively early from the main
eukaryotic lineage, also has the ability to induce gene silencing. Thus, gene silencing
might represent an ancient function of eukaryotic cells.
In plants, and in the fungus Neurospora crassa, it has been known for quite some time
that there exist PTGS phenomena induced by transgenes in the form of inverted
repeats,viruses or viroids (Depicker &Montagu 1997). In these instances the term
cosuppression is commonly used to indicate PTGS of homologous genes induced by
the genetic elements. In N. crassa co-suppression is referred to as quelling (Cogoni &
Macino 1997) ,RNAi in C. elegans (Ketting et al. 1999; Tabara et al. 1999) and PTGS in
plants (Kooter et al. 1999; Dalmay et al. 2000; Fagard & Vaucheret 2000; Mourrain et
al. 2000; Plasterk & Ketting 2000). Genetic analysis of all these has identified a subset
of genes shared by these gene silencing phenomena, thus establishing a relationship
between RNAi, PTGS and quelling. Whereas in plants PTGS/RNAi functions both at the
transcriptional and post-transcriptional levels, in animals and protozoa only post-
transcriptional RNAi has so far been detected.

RNAi as ribonuclease engine

The famous short interfering RNA (siRNA) was first detected in plant systems, and its
linkage to RNAi in animals provided the connection between gene silencing pathways
across kingdoms. The third experimental component of RNAi began 20 years ago with
the study of developmental timing genes in C. elegans. Ruvkun andAmbros had
identified lin-4, a small untranslated RNA that regulated the expression of the mRNA for
lin-14. This was the first discovered microRNA. We now know that microRNAs are
naturally occurring triggers of the RNAi pathway and play an important role in gene
regulation in many organisms ranging from nematodes to plants to humans.

A simplified model for the RNAi pathway is based on two steps, each involving a
ribonuclease machine. In the first step, the RNA (either dsRNA or microRNA primary
transcript) is processed into an siRNA by the RNaseIII enzymes Dicer and Drosha.
dsRNA binding domain proteins (dsRBD) Pasha, Loquacious, and R2D2 are cofactors
for processing events. In the second step, siRNAs are loaded into the effector complex
RNA-induced silencing complex (RISC). The siRNA is unwound in a strand specific
manner during RISC assembly.
This single-stranded siRNA locates mRNA targets by Watson– Crick base pairing. Gene
silencing is a result of the nucleolytic degradation of the targeted mRNA by the RNaseH
enzyme Argonaute (Slicer). If the siRNA/mRNA duplex contains mismatches at the
important sites, often the case for microRNAs, the mRNA is not cleaved. And gene
silencing gene is a result of translational inhibition.
The outline of this biochemical pathway are derived from the earliest studies by Fire and
Mello . Recent work on RNAi also fill our our understanding of some of the details of the
RNAi pathway. For example, one strand of the siRNA is preferentially incorporated into
RISC. This was first observed with microRNAs, but also occurs with siRNAs and long
dsRNAs. One immediate impact of this discovery was the improvement in siRNA
design. Secondly, RISC assembly is a multistep process. Dicer and RISC do not
function independently, but act as part of a coordinated pathway.

Antiviral action of RNAi in Plants

The infection of plants by both RNA(replicate in cytoplasm) and DNA viruses(replicate
in nucleus) results in the accumulation of viral siRNAs. Viruses are therefore inducers of
RNA silencing that is directed against their own replication. The siRNA pathway of RNA
silencing is believed to be a natural antiviral defense mechanism in plants. The exact
pathway for the biogenesis of viral siRNAs is unclear. It is thought that dsRNA
replication intermediates are the source of viral siRNAs. However, direct processing by
Dicer of duplex structures formed within single-stranded viral RNAs could also
contribute to the siRNA pool. Furthermore, the involvement of RdRP in antiviral
defense and in DNA virus-induced gene silencing in plants suggests that RdRP-
mediated synthesis of secondary viral dsRNA might also play a role in viral siRNA
accumulation.WANG

The discovery that almost all plant viruses encode silencing suppressors provides
evidence for the involvement of RNA silencing in plant antiviral defense.
These suppressor proteins operate through a variety of mechanisms. For instance, the
P1/HC-Pro suppressor from the potyviruses inhibits silencing at a step downstream of
dsRNA processing, by preventing the unwinding of duplex siRNAs or the incorporation
of siRNA into RISC, or both . The tombusvirus p19 protein also functions downstream of
dsRNA processing, but it physically binds to duplex siRNAs and hence prevents their
incorporation into RISC. The cucumisvirus 2b protein and the p25 protein of
potexviruses, on the other hand, inhibit the systemic transmission of silencing signals .
Thus, plant viruses appear to have evolved diverse counter-defense strategies against
RNA silencing.

None of these silencing suppressors appear to block dsRNA processing, but tend to
operate by sequestering siRNAs, preventing siRNA unwinding, or blocking the
cell-to-cell movement of siRNAs. This might have significant implications for viral self-
defense strategies. It is possible that silencing suppressors only function in those cells
in which viruses are actively replicating, and might lose their suppressor activity once
the viruses have completed their life cycle and moved into neighboring cells. The
siRNA-charged suppressor proteins in the preinfected
cells would then release their siRNAs, making them available for silencing against
secondary infection by the same or a related virus. Thus, viruses might have evolved a
survival mechanism by protecting their hosts from secondary viral infection.
RNA silencing and viral pathogenicity
Overexpression of viral silencing suppressors can affect miRNA accumulation and
function, and can result in developmental abnormalities in plants . This has led to the
suggestion that viral pathogenicity is largely determined by the effect of viral silencing
suppressors on the host miRNA pathway. Evidence against the universality of this
pathogenicity model comes from the observation that not all viral suppressors appear to
affect the miRNA pathway in plants .
An alternative RNA-silencing-mediated pathogenicity model (illustrated in Figure) is
suggested by the finding that plant subviral RNAs appear to induce symptoms by
inducing silencing against host genes .
This model predicts three possible scenarios: first, viral siRNAs induce cleavage of host
mRNA because of their sequence identity; second, viral siRNAs are partially
complementary to the host mRNA and serve as primers to initiate RdRP-mediated
synthesis of secondary dsRNA against the host mRNA; or third, certain host mRNAs
contain sequence motifs that resemble viral origins of replication, and consequently,
viral-encoded RNA dependent RNA polymerase recognizes the sequences and initiates
the synthesis of antisense RNA against the host mRNA. Each of these scenarios would
result in the silencing of host genes, leading to disease symptoms.
.

As early as in the 1920s, it was known that plants could be protected from a severe
virus by prior infection with a mild strain of a closely related virus. Although the
mechanism of such cross protection in plants remained unknown for a long time, such
phenomena could be explained partly in terms of PTGS that could be induced by the
mild strain and targeted later against the virulent viral genome. It was also found that
transforming plants with virus-derived transgenes gave protection against the challenge
viruses even when no transgene protein was produced .Analyses of these virus-
resistant plants revealed that the transgenes were highly transcribed in the nucleus,
whereas the steady-state level of cytoplasmic mRNA was very low.
Further analysis suggested that some of the transgenic mRNA molecules assumed the
conformation of dsRNA, which triggered sequence-specific degradation of self and other
homologous or cRNA sequences in the cytoplasm. Thus, in the virus-resistant lines, not
only the transgene mRNAs but also the mRNA from the homologous endogenous gene
and the invading viral RNA (with homology to the transgene) were degraded.
Another form of virus-induced gene silencing is the phenomenon of viral recovery itself.
When Brassica napus was inoculated with cauliflower mosaic virus (a DNA virus),
lesions at the site of virus entry were visible 5 to 7 days postinoculation. Symptoms of
systemic infections were apparent by 10 to 14 days postinoculation. Symptoms were
most prominent at 30 to 40 days postinoculation and declined thereafter (i.e., the plants
recovered), with the newly emergent leaves remaining asymptomatic at 50 days
postinoculation .
 Figure diagrammatically illustrates the systemic spread of RNAi in plants. Such
recovery occurred by a PTGS-like mechanism because 19S and 35S RNAs encoded by
the cauliflower mosaic virus were degraded while cauliflower mosaic virus DNA was still
replicating in the nucleus. Induction of PTGS was visualized if the cauliflower mosaic
virus infection and subsequent recovery were followed up in a transgenic B. napus
expressing a p35S-GUS (glucuronidase) transgene. At the site of inoculation, GUS
silencing associated with local lesions
was first observed 7 days postinoculation. GUS silencing eventually spread
systemically, and the GUS activity of the entire plant was suppressed by 50 days
postinoculation. In this particular example, cauliflower mosaic virus acted as the inducer
of PTGS for the transgenes sharing homology with the virus within the transcribed
region. However, the virus itself was also the target of the induced PTGS, since 19S
and 35S RNAs were found degraded.

Fig. Schematic illustration of

systemic viral spread as well
as RNAi and subsequent
viral recovery in plants.
Virus lesion at
CaMV-GFP Green and red indicate the
GFP Transgenic 7 days post
Transgenic presence and loss of GFP
transgenic infection.
fluorescence, respectively,
New leaf
and orange denotes the
(without
infection presence of both colors. The
red dots on leaves show viral
lesions. The bold arrows
indicate the stages of plant
growth.

Systemic infection and

AT 50 days post infection
the emerging leaf is
GFP silencing at 30 to
asymptomatic (free of 40 days.
virus) and shows GFP
expression.

Based on the principles of virus-induced gene silencing, vectors designed with the
genome sequence of RNA viruses tobacco mosaic virus, potato virus X, and tobacco
rattle virus are being widely used to knock down the expression of host genes. The
characteristics of many plant genes were revealed by observing the loss-of-function-
related phenotypic changes when the recombinant vectors incorporating the concerned
host genes were introduced into plants. Of these vectors, the TRV-based are more
promising because these are capable of inducing-meristematic gene silencing, which
has not been possible to achieve with other RNA virus-based vectors. Meristematic
gene silencing employing TGMV vectors has also been reported. Thus, virus-induced
gene silencing-based techniques are extremely useful for studies related to functional
genomics in plants.