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Plant viruses and gene silencing<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Post transcriptional gene silencing

In higher plants RNA silencing serves as an adaptive, antiviral defense system induced in response to a virus challenge. PTGS is similar to quelling in Neurospora crassa and RNA interference (RNAi) in animals. The three distinct phases in RNA silencing includes initiation, maintenance and systemic silencing. The three basic silencing pathways are:

1. siRNA mediated degeneration of aberrant mRNAs including viral mRNAs and RNA genomes.
2. miRNA mediated silencing involved in translational inhibition or degradation of cellular mRNAs and RNA genomes.
3. siRNA directed denovo methylation of DNA and histone proteins leading to Transcriptional Gene Silencing (TGS). The third pathway supports methylation of DNA virus genomes, inhibiting virus replication or transcription. That is, RNA directed DNA methylation (RdDM) which induces epigenetic modification of homologous sequences is a novel form of defense against viruses.

Both TGS and PTGS are nucleotide sequence homology dependent; however genes silenced transcriptionally are homologous in promoter regions, whereas genes targeted for PTGS share homology in transcribed region. TGS silences genes at the level of transcription in nucleus. In contrast PTGS has no apparent effect on transcription of target gene but promotes a rapid and specific turnover of RNA transcripts in cytoplasm.

It is observed that transcriptionally silenced transgenes acquire metastable epigenetic states that are characterized by altered methylation patterns and chromatin structure. These transgenes are often methylated in cytosine residues that are not located within CG or CNG sequences, the classical targets for DNA methylation. Also methylation patterns act as signal for chromatin condensation in induction of transgene silencing. Interaction between chromatin remodeling and methylation is responsible for maintenance of transcriptional transgene silencing. (Kooter et al., 1999).

Components of PTGS

Virus derived small RNAs mediate RNA silencing pathways in plants. These RNAs target viral RNAs that accumulate during infections. dsRNA from replication intermediates or viral RNA with extensive foldback structure are precursors for virus- derived small RNA (vsRNA). The small RNA species that are 21-28 nucleotides in length can be subdivided into at least two major classes. The first one are small interfering RNAs (siRNAs) are produced as populations from long dsRNAs that result from read through or bidirectional transcription of DNA repeats or transposon loci, and from the action of host encoded RNA-dependent RNA polymerases (RdRP) that synthesize complementary strands from cellular RNAs. Another class of small RNAs constitutes microRNAs (miRNAs). miRNAs arise from large precursors transcribed from miRNA genes that do not encode proteins. They also appear to arise from introns present in transcripts of encoding proteins. Perfectly base-paired dsRNA are the precursors of siRNA populations whereas primary miRNA transcripts contain imperfect intramolecular stemloops.

Both classes of RNAs from dsRNAs are processed by Dicer, a class III RNase III enzyme which processively chops its substrate into 21-28 nucleotide dsRNA. Dicer contains a distinct helicase domain and dual RNase III motif and also contains a region of homology to RDE/QDE/ARGO family of proteins. The RNase III domain forms an intramolecular processing center establishing two active sites in near proximity. Each catalytically active site cleaves one phosphodiester bond on the opposite strands of precursor, which give rise to the characteristic 21-28 nt long dsRNA with 2 nucleotide 3' overhangs. The model plant Arabidopsis has four specialized Dicer-like (DCL) proteins. DCL-1 catalyzes the cleavage of intergenic or intronic fold precursors to release miRNAs. These molecules incorporate into RNA induced silencing complex (RISC) to promote cleavage or translational repression of cellular transcripts carrying miRNA complementary sites. DCL3 produces 24-nt siRNAs guiding heterochromatin formation and transcriptional repression of transposon and DNA repeats. DCL4 has roles in RNA interference and processes noncoding RNA precursors into 21-nt trans-acting siRNAs that control developmental timing and leaf polarity .DCL2 synthesizes a 24-nt natural antisense transcript siRNA that regulates stress responses and, in plants with compromised DCL4 activity, it alternately processes <?xml:namespace prefix = v ns = "urn:schemas-microsoft-com:vml" />22-nt siRNAs from trans-acting siRNA precursors.

RNA-induced silencing complexes also called as effector complexes are assembled upon loading of one selected small RNA strand called as guide strand into one member of Argonaute (Ago) protein family. Argonauts have two conserved protein domains, the PAZ domain which is proposed to bind ssRNA and PIWI motif. Ago proteins cleave target ssRNAs at the duplex formed with the guide strand. The strand selection for RISC depends on the relative thermodynamic characteristics of two 5' termini of a siRNA and miRNA. The comparative sequence analysis have shown that 5' end of guiding strands of competent siRNAs are less stable than 5' end of passenger strand. The outcome of this event is Post transcriptional gene silencing.

fig 1: The core reaction in Post transcriptional gene silencing. dsRNA acts as the precursor and is cleaved by dicer into siRNAs. One of the strands of siRNA enters RNA induced silencing complex (RISC) and activates. Finally endogenous RNA homologous to siRNA is degraded.

Geminiviruses and RNA silencing

Geminiviruses are single stranded circular DNA viruses that cause economically significant diseases. They are characterized by small geminate particles (18x20nm) containing either one or two single stranded circular DNA molecules of around 2.7 kb. The family Geminiviridae is divided into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus. (Fauquet et. al., 2003). The majority of begomoviruses have two components, referred to as DNA-A and DNA-B, both of which are essential for infectivity. These types of viruses are called Bipartite. DNA-A has six genes: ac1 encodes a replication-associated protein (Rep) essential for viral DNA replication in association with host DNA polymerase ; ac2 encodes a transcription activator protein (TrAP); AC3 encodes a replication enhancer protein (REn); av1 and av2 encode coat protein and pre-coat protein, respectively; but no function has been attributed to ac4-encoded protein in relation to virus multiplication. DNA-B has bv1 and bc1 genes that encode a nuclear-shuttle protein (NSP) and movement protein (MP), respectively . The nucleotide sequence of DNA-A and DNA-B are different except for a region of approximately 200nt which shares 90% homology defined as common region (CR). CR carries regulatory sequences essential for viral replication and transcription.

Srilankan cassava mosaic virus (SLCMV) is a bipartitevirus and triggers post transcriptional gene silencing .How these viruses trigger PTGS has been a mystery since they are nuclear replicating single stranded DNA molecules with no known dsRNA form present in their replication cycle. However transcription in Geminiviruses is bidirectional with production of polycistronic mRNAs occurring from common region. The bidirectional transcription of these viruses with transcripts occurring from opposite polarity could overlap resulting in formation of dsRNA.

Approaches used to study PTGS

Amplicon system

Amplicon are transgenes comprising a promoter and terminator directing transcription of a modified viral vector mRNA in plant cell. PTGS could be targeted against endogenous RNA by Potato virus X (PVX ) amplicons with fragments of endogenous genes and a silencing phenotype was observed in Arabidopsis. The Arabidopsis lines carrying a single copy of 35S-GFP transgene were transformed with vectors carrying 35S-PVX:GFP constructs. None of the transformed exhibited GFP fluorescence indicating PTGS (Dalmay et al., 2000).

16c lines of Nicotiana benthamiana

16c are lines of N. benthamiana carrying a gfp transgene (Voinnet and Baulcombe, 1997). These plants accumulate a high level of GFP mRNA and appear uniformly green fluorescent under UV illumination, whereas non-transformed plants appear red due to chlorophyll. (Ruiz et al., 1998). 16c lines are super transformed with a construct in which gfp has been cloned under a constitutive promoter in sense as well as antisense orientation. This construct gives rise to dsGFP RNA which triggers PTGS which in turn knocks down the gfp transcripts also thus leading to loss of gfp expression. If suppressors of PTGS are introduced in 16ci lines, they interfere with PTGS pathway leading to recovery of GFP expression. Hence these transgenic lines have proven to be quite useful in studies of gene silencing.

Silencing mutants and genes involved in silencing

The production of mutants impaired in various types of transgene induced silencing is proving to be a powerful approach for identifying the proteins involved in different silencing mechanisms. Mutants defective in PTGS have been isolated from Neurospora (qde) and Arabidopsis (sgs). The qde-1 gene from Neurospora encodes a protein with homology to RdRP. SGS3 is the only one essential gene for RNAi in plants which show no similarity to any known protein (Mourrain et al., 2000). HYL1 (Arabidopsis hyponastic leaves) functions primarily in miRNA related pathways. It was shown to be required for the accumulation of several miRNAs and trans-acting smRNAs, (Han et al. 2004, Vazquez et al. 2004). The Arabidopsis Hua Enhancer (HEN1) gene is involved in miRNA biogenesis and natural virus resistance. It encodes a methyl transferase that methylates the last nucleotide of miRNAs at 2'-O and 3'-O position (Yu et al., 2005) with 2'-OH claimed to be major target of modification and hence protects them from 3' end uridylation activity (Li et al., 2005).

Viral supressors of PTGS

Many plant viruses encode proteins that suppress gene silencing reflecting the natural role of PTGS as an antiviral defense. One of the suppressors is helper component proteinase (HcPro) of potyviruses. It promotes maintenance of genome amplification (i.e., replication and accumulation) over extended periods and stimulates vasculature dependent, long-distance transport of virus (Klein et al., 1994; Cronin et al., 1995; Kasschau et al., 1997). HcPro recruits a calcium dependent regulatory pathway (rgs-CaM) that negatively controls silencing (Anandalakshmi et al., 2000). HCPro suppresses gene silencing at a step upstream of the accumulation of small RNAs but downstream of silencing signal. Another candidate suppressor for PTGS is the 2-b protein encoded in cucumber mosaic virus (CMV) (Ding et al., 1995). The HcPo acts by blocking the maintenance of PTGS in tissues where silencing had been already set, whereas the 2b protein prevents initiation of gene silencing at the growing points of plants (Brigneti et al., 1998). Another suppressor is p25 cell to cell movement protein of potato virus X (PVX). P25 prevents the accumulation or transport of mobile silencing signal, probably by interfering with the cellular RdRP branch of silencing pathway (Voinnet et al., 2000). Protein P6 from Cauliflower mosaic virus has also been found as suppressor of RNA silencing. A transgenic Arabidopsis line having silenced GFP expression was infected by CaMV. The infected leaves showed strong GFP florescence indicating that silencing of GFP had been suppressed during infection (Love et al., 2007).

Systemic silencing in plants

The potential to transmit a signal out of initially infected cell and activate viral RNA degradation in non infected cells located beyond the front of infection is systemic silencing. Silencing may spread from silenced rootstocks to scions or from locally silenced regions of plants to upper parts of same plant. In grafting experiments systemic silencing was transmitted across a graft junction from spontaneously silenced transgenic tobacco rootstocks to isogenic scions that had not silenced spontaneously (Palauqui et al., 1997).

The patterns of systemic silencing suggests that signal moves cell to cell through the phloem, mimicking patterns of viral movement through the plant. In 35S GFP plants, stomatal guard cells that have lost plasmodesmatal connections to other cells before induction of systemic silencing do not silence, providing evidence that signal moves cell to cell through plasmodesmata (Voinnet et al., 1998).

Candidate RNAs for mobile silencing signal

siRNAs

These RNA are long enough to convey sequence specificity yet small enough to move through plasmodesmata, it is possible that they are components of systemic signal and specificity determinants of PTGS. But two lines of evidence contradict this idea. First, HC-Pro suppression of silencing interferes with accumulation of the small RNAs but does not eliminate either the production or movement of the silencing signal (Mallory et al., 2001). Second, the PVX p25 protein interferes with the mobile silencing signal, but does not affect the accumulation of small RNAs (Voinnet et al., 2000)

dsRNA

long dsRNAs which act as precursors of siRNAs can be possible candidates for mobile signal but evidence for this is lacking. However viroid RNAs are able to enter a series of transport pathways starting with exit from the nucleus into cytoplasm; cell to cell movement through plasmodesmata; and systemic movement which requires special assistance by special plant proteins in phloem (Gomez and Pallas, 2001; Owens et al., 2001; Zhu et al., 2001).

Aberrant RNAs

RNA transcript from a silenced locus which is in some way aberrant triggers RNA silencing upon arrival in new cell. There are also several examples of endogenous mRNAs, such as the maize KNOTTED1 (Lucas et al., 1995) and SUT1 in tobacco and potato (Kuhn et al., 1997), which moves through plasmodesmata presumably using endogenous mechanisms for RNA trafficking.

Fig 2: RNA based model of silencing showing two modes of silencing: transcriptional gene silencing (TGS) which causes methylation of promoter sequences by mechanism known as RNA dependent DNA methylation (RdRM) and post transcriptional gene silencing (PTGS) which in a sequence specific manner can target both cellular and viral mRNA for degradation. Silencing signal is amplified by RNA dependent DNA polymerase (RdDP). Helper component proteins (Hypo), 2-b protein encoded in cucumber mosaic virus (CMV2b), p25 from PVX are suppressors of silencing, acting at different steps in PTGS pathway

Virus induced gene silencing (VIGS)

VIGS is the technology in which gene expression in plants can be suppressed in sequence specific manner by infection with virus vectors carrying fragments of host genes to be silenced. Another technique is transgenic VIGS that produces a stable and heritable silenced phenotype and is based on a transgene comprising a cDNA of replicating RNA to which a fragment of host gene is inserted. Transgenic VIGS is more difficult than conventional VIGS as it is more difficult to be adapted to a large scale genome wide screen with which one can establish the relationship from gene to phenotype within a week (Baulcombe 1999)

Advantages of VIGS

1. VIGS is rapid and can identify a loss of function phenotype for a specific gene within a single generation.
2. The gene responsible for an interesting phenotype can be quickly sequenced from a VIGS vector and identified.
3. It avoids plant transformation.
4. It overcomes functional redundancy.
5. Allows rapid comparison of gene function between species.
6. Works in different genetic backgrounds as well.

Disadvantages of VIGS

1. VIGS seldom results in the complete suppression of expression of a target gene. Because it is possible that a decreased transcript level will be sufficient to produce enough functional protein, a phenotype might not be observed in silenced plant.
2. It often does not result in uniform silencing of genes.

Virus control by PTGS

The use of viral sequences to produce virus resistant plants is now almost a standard technique. The two mechanisms operating for this purpose are-

1. Requiring expression of viral protein(s).
2. Other one is dependent only on presence of transgene derived RNA.

Pathogen Derived Resistance (PDR)

For PDR, a part, or a complete viral gene is introduced into the plant which subsequently interferes with one or more essential steps in the life cycle of the virus. PDR has been obtained by expressing various forms of CP genes, viral replicase genes and other genes.

First instance of PDR was reported by Powell et al., (1996) in which transgenic tobacco plants were produced having chimeric gene containing a cDNA copy of Coat protein (CP) of Tobacco mosaic virus (TMV). On inoculation with TMV the plant were either found to lack symptoms or exhibited a delay in symptom development as compared to inoculated non transgenic control plants. This type of protection was termed CP-mediated resistance (CPMR). Beachy et.al, 1997 described several mechanisms of CPMR that include prevention of uncoating of incoming virus, interference with viral translation, replication and interference with long distance and cell to cell movement. Nejidat and Beachy (1990) reported that the protection conferred by TMV CP against range of other tobamoviruses decreases as CP amino acid sequence homology decreased from 85% to 45%. Hence level of protection was found to be greatly dependent upon the extent of homology between the viruses. It was initially thought that PDR would operate through expression of protein (Powell et al., 1986, Lapidot et al., 1993 and Brederode et al., 1995). But later it was shown that untranslatable constructs were also able to confer resistance (Dougherty et al., 1992). This type of resistance based upon presence of RNA is called RNA mediated resistance.

Replicase Mediated resistance

Transformation with a DNA sequence derived from a gene encoding a viral replicase can endow a plant with high level of resistance to corresponding virus. Longstaff et al, 1993 studied the importance of the conserved GDD motif sequence in replicas which imparted resistance by introducing mutations in it. 54kDa replicas protein is the predicted protein involved and contain Gly-Asp-Asp (GDD) motif that is conserved among many positive strand viruses and was shown to be essential for replicas activity.

Protection by movement protein

Movement protein (MP) is essential for cell to cell movement of plant viruses. These proteins have been shown to modify the gating function of plasmodesmata, thereby allowing virus particles or their nucleoprotein derivatives to spread to adjacent cells. Transgenic expression of viral MP in such a way that dysfunctional MP protein is expressed provides resistance. This is MP-mediated protection (MPMP). Resistance conferred by transgenic expression of dysfunctionl TMV MP is likely due to competition for plasmodesmatal binding sites between mutant MP and wild type MP of inoculated virus (Lapidot et al., 1993).

RNA mediated resistance

Resistance responses determined by the transgene RNA rather than the expression of the transgene product are termed RNA-mediated resistance. This type of resistance is characterized by detection of low- steady state levels of the transgene transcript in the transgenic plants and was first reported in tobacco plants transformed with the CP gene of Tobacco etch virus (Lindbo and Dougherty, 1992)

Antisense RNA approach

Stable reduction of transcript in cells expressing high levels of anti-sense RNA have been attributed to the formation of duplex formation with the DNA template to block transcription, blocking intron splicing , failure of antisense RNA:mRNA duplex to be transported from nucleus, promoting rapid RNA degradation either in the nucleus or the cytoplasm, blocking initiation of translation.

Homology dependent gene silencing (HDGS)

HDGS is a phenomenon whereby the presence of (multiple) transgene(s) can lead to inactivation one or all of the transgenes (Dougherty and Parks 1995; Baulcombe and English 1996). When PTGS affects both the transgene and the endogeous gene the phenomenon is termed cosuppression. Napoli et al, 1990 over expressed chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia chs gene and noticed a dramatic fall in anthocyanin biosynthesis in plants. After doing RNase protection assay from RNAs isolated from white flowers it was seen that developmental timing of mRNA expression of endogenous chs gene is not altered but the level of mRNA produced was reduced 50 fold than wildtype.