Laboratoire de Biologie Cellulaire, INRA, Versailles 78026, France
*Author for correspondence (e-mail: herve.vaucheret{at}versailles.inra.fr)
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SUMMARY |
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Key words: Post-transcriptional gene silencing, RNA interference, Transgene, Virus, Mutants
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Introduction |
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PTGS results in RNA degradation after transcription |
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Initiation, propagation and maintenance of PTGS |
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Initiation
Because spontaneous initiation of PTGS in transgenic plants is localized and stochastic, it is particularly difficult to study. Most data concerning the control of initiation are indirect and result from the analysis of parameters that increase or decrease the efficiency of spontaneous triggering of PTGS. Such studies have revealed that two types of transgene loci efficiently trigger PTGS. The first type corresponds to highly transcribed single transgene copies. Several arguments suggest that the efficiency of triggering could depend on the probability that the transgene produces a particular form of RNA above a threshold level. Indeed, PTGS is triggered mostly when plants are homozygous for the transgene locus (de Carvalho et al., 1992). In addition, PTGS is triggered more efficiently when strong promoters are used (Que et al., 1997). Finally, PTGS is inhibited when transgene transcription is blocked (Vaucheret et al., 1997). The second type of transgene loci that efficiently triggers PTGS is those carrying two transgene copies arranged as an inverted repeat (IR). These IRs are usually transcribed at very low levels, which argues against the threshold model (van Blockland et al., 1994). To explain their ability to efficiently trigger PTGS, investigators have proposed that these IRs produce dsRNA by read-through transcription and that dsRNA efficiently triggers PTGS, even when produced at a low level. Indeed, introduction of single transgene copies that have a panhandle structure (i.e. carry the same sequence cloned in sense and antisense orientations downstream of the promoter) leads to efficient silencing of homologous (trans)genes, which suggests that such dsRNAs are efficient initiators of PTGS (Hamilton et al., 1998; Waterhouse et al., 1998).
The above results coincided with the discovery of RNAi in animals, a process that results in specific RNA degradation induced by injection of homologous dsRNA (Fire et al., 1998) or expression of panhandle transgenes (Tavernarakis et al., 2000). These similarities suggest that PTGS in plants and RNAi in animals could derive from an ancestral mechanism allowing degradation of RNAs that are homologous to dsRNAs abnormally present in a cell. However, the PTGS mechanisms triggered by highly transcribed single transgene loci and transgene IRs in plants are (at least in part) different. Indeed, mutants in which PTGS triggered by highly transcribed single transgene copies is impaired exhibit efficient PTGS triggered by transgene IRs (H.V. and P. Waterhouse, unpublished). This suggests that highly transcribed single transgene loci do not directly produce dsRNA and that the mutants that have been isolated are impaired in the steps leading to the formation of dsRNA (see below).
Systemic propagation
The transmission of PTGS of nitrate reductase, nitrite reductase or SAM-synthase (trans)genes from localized interveinal spots or vein-localized to the upper leaves of plants suggested that a PTGS propagation signal exists. The existence of such a signal was clearly established by grafting (Fig. 1). Silencing was transmitted with 100% efficiency from silenced stocks to target scions expressing the corresponding transgene but not to scions expressing a non-homologous transgene, which indicates that the signal is sequence-specific (Palauqui et al., 1997). Silencing of nitrate-reductase genes was also transmitted to a non-transgenic mutant scion overexpressing the endogenous Nia2 gene owing to metabolic derepression but not to a wild-type scion, which indicates that overaccumulation of Nia mRNA above the level of that in wild-type plants, rather than the presence of a transgene in the scion, is required for triggering of RNA degradation during PTGS (Palauqui and Vaucheret, 1998). The transmission of PTGS also occurred when silenced stocks and non-silenced target scions were physically separated by up to 30 cm of stem of a non-target wild-type plant, indicating long-distance propagation (Palauqui et al., 1997). Voinnet and co-workers drew similar conclusions when PTGS of a GFP transgene was systemically triggered after they inoculated one leaf of a non-silenced GFP transgenic N. benthamiana plant with an Agrobacterium strain carrying the GFP transgene (Voinnet and Baulcombe, 1997) or biolistically introduced the GFP transgene (Voinnet et al., 1998). Because it is sequence specific and mobile, this signal could be made (at least in part) of RNA. Whether it corresponds to dsRNA or siRNA remains to be determined.
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The experiments described above clearly show that PTGS is a dynamic process that can be separated into initiation, propagation and maintenance. However, a number of points remain mysterious. In particular, the nature of the systemic silencing signal remains to be determined.
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Genetic dissection of PTGS |
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Genes that inhibit PTGS in plants
Despite their identification in 1994, the EGS1 and EGS2 genes (which are presumed to encode proteins that negatively regulate PTGS) have not been cloned yet. Nevertheless, a tobacco gene that negatively regulates PTGS has been recently identified. This gene, named rgs-CaM (for regulator of gene silencing), encodes a calmodulin-related protein (Anandalakshmi et al., 2000), and its overexpression in tobacco inhibits PTGS of a 35S-GUS transgene (see below). However, its role in wild-type plants is still not known.
Genes that stimulate PTGS in plants
Our group has shown that the Arabidopsis sgs mutants are deficient for both PTGS of an exogenous 35S-GUS transgene and PTGS of a homologous 35S-Nia2 transgene and endogenous Nia genes, but not PTGS induced by transgenes producing dsRNA (Elmayan et al., 1998; Mourrain et al., 2000; H.V. and P. Waterhouse, unpublished). Dalmay et al. showed that the Arabidopsis sde1 and sde3 mutants are deficient for PTGS of a 35S-GFP transgene induced by a PVX-35S-GFP amplicon but not for virus-induced gene silencing (VIGS; see below) (Dalmay et al., 2000; Dalmay et al., 2001). This indicates that SGS and SDE genes positively control PTGS induced by highly transcribed transgenes but not PTGS induced by IR transgenes or viruses. Deficiency of PTGS in sgs and sde mutants correlates with a strong decrease in methylation of the transcribed region of the transgene, which confirms the correlation between PTGS and methylation. The sgs mutations have no effect on transgenes silenced at the transcriptional level, which indicates that they are specific for PTGS (Elmayan et al., 1998; Mourrain et al., 2000).
The Arabidopsis SGS2 gene (and the SDE1 gene, which is identical to SGS2) encodes a protein that has strong similarity to a tomato RdRP (Mourrain et al., 2000; Dalmay et al., 2000). The existence of an RdRP activity in plants was known for 30 years but its role was not. SGS2/SDE1 is similar to QDE-1, which is required for quelling in Neurospora (Cogoni and Macino, 1999), and EGO-1, which is required for RNAi of some genes in the germline of C. elegans (Smardon et al., 2000). The Arabidopsis SGS3 gene encodes a protein that has no significant similarity to other known proteins in plants or other kingdoms (Mourrain et al., 2000). Its function cannot be deduced, because it does not contain any known protein motif other than a coiled-coil domain present in the C-terminus of the protein, which suggests possible interactions with other proteins. The absence of similar proteins in C. elegans and Drosophila (two organisms that exhibit RNAi and whose genomes are entirely sequenced) and the absence of the corresponding mutant in Neurospora suggest that the function of the SGS3 protein is specific to plant PTGS.
A third gene that positively controls PTGS in Arabidopsis corresponds to a previously identified gene controlling development, AGO1 (Fagard et al., 2000). ago1 mutants display strong developmental alterations that affect plant architecture and fertility. The AGO1 protein shares similarity with a number of proteins containing Piwi and PAZ (Piwi/Argonaute/Zwille) domains (Cerutti et al., 2000): QDE-2, required for quelling in Neurospora (Catalanotto et al., 2000); RDE-1, required for RNAi in C. elegans (Tabara et al., 1999); eIF2C, presumed to play a role in the control of translation initiation in rabbit (Zou et al., 1998); STING, required for silencing of the repetitive Stellate locus in Drosophila (Schmidt et al., 1999); and PIWI, required for germline maintenance in Drosophila (Cox et al., 1998). Recently, the SDE3 gene that positively controls PTGS in Arabidopsis was isolated. It encodes an RNA helicase that shares similarity with MUT-6, which is required for PTGS in Chlamydomonas (Wu-Scharf et al., 2000), and SMG-2, which is required for RNAi in C. elegans (Domeier et al., 2000). Therefore, despite the absence of orthologs of SGS3, the identification of different sets of related proteins (SGS2/QDE-1/EGO-1, AGO1/QDE-2/RDE-1, SDE3/SMG-2) indicates that PTGS, quelling and RNAi probably derive from the same ancestral mechanism.
The influence of chromatin and methylation genes on plant PTGS
The Arabidopsis mutants ddm1 and met1 were isolated from a screen for mutations that result in a general reduction (70%) in methylation of the genome (Vongs et al., 1993). MET1 encodes the major DNA methyltransferase (Finnegan et al., 1996). DDM1 encodes a protein related to SNF2/SWI2 chromatin-remodelling proteins (Jeddeloh et al., 1999), which suggests that structural changes in chromatin can reduce the accessibility of DNA to the methylation machinery. Both ddm1 and met1 mutants exhibit impaired TGS (Steimer et al., 2000; Morel et al., 2000). Furthermore, they also exhibit impaired PTGS, which correlates with a decrease in transgene methylation (Morel et al., 2000). However, unlike sgs and ago1 mutants, ddm1 and met1 mutants do not show impaired PTGS in all plants. In addition, the impairments of PTGS in ddm1 and met1 mutants are different: in ddm1 mutants PTGS is inhibited in the whole plant throughout its life, whereas in met1 mutants, PTGS is progressively inhibited during the course of plant development. This suggests that MET1 and DDM1 are involved in the maintenance and initiation steps of PTGS, respectively. Together, these results confirm the existence of a nuclear step in PTGS and reveal a genetic link between PTGS and TGS.
A branched model for PTGS in plants
Several cellular components involved in the control of PTGS in plants have been identified. By extrapolation of genetic and biochemical results obtained in Neurospora, C. elegans and Drosophila to plants, we propose a branched model for PTGS in plants (see Fig. 2):
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2. The RdRP protein encoded by the SGS2/SDE1 gene could use such abRNAs as templates to synthesize dsRNA. SGS2 could also play a role in the production of the systemic silencing signal and/or its amplification.
3. The SGS3, AGO1 and SDE3 proteins, like SGS2/SDE1, are not required for PTGS induced by IR transgenes or viruses. They could facilitate the RdRP activity of SGS2/SDE1, by impeding translation of abRNAs, allowing them to be used as templates to synthesize dsRNA.
4. Unidentified plant RNases, similar to DICER, which is involved in RNAi in Drosophila (Bernstein et al., 2001), could participate in the degradation of dsRNA and in the formation of siRNAs. Arabidopsis mutants impaired in a gene sharing strong similarities with the Drosophila DICER gene caf (also known as sin1 or sus1) (Jacobsen et al., 1999) (A. Ray, personnal communication) are currently being analyzed to determine whether it plays a role similar to that of DICER in plants. siRNAs could subsequently direct the RNA-degradation complex (named RISC in Drosophila) to homologous mRNAs, allowing completion of their degradation.
5. The MET1 protein could be necessary for the maintenance of PTGS during plant development, methylating transgene sequences that are homologous to dsRNAs, and thus maintaining the chromatin state that is responsible for the synthesis of abRNA. Indeed, Wassenegger et al. have shown that dsRNAs direct DNA methylation of homologous sequences in the nucleus (Wassenegger et al., 1994).
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PTGS and plant resistance to viruses |
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Viruses as targets of PTGS
Introduction of transgenes constitutively expressing part of the genome of a virus can lead to resistance of the plant to infection by this virus (reviewed by Marathe et al., 2000). Plants can either resist infection (resistance is then referred to as immunity) or undergo a preliminary phase of infection from which they recover (resistance is then referred to as recovery) (Lindbo et al., 1993; Smith et al., 1994). Plants in which the transgene undergoes PTGS prior to infection are immune, whereas plants in which the transgene undergoes PTGS after infection show recovery. This suggests that homologous virus and transgene RNA are degraded by a PTGS-like mechanism. Both resistant plants and plants that exhibit recovery are immune to secondary infection by the same virus or by another recombinant virus carrying part of the genome of the first virus, which indicates that plants have a memory of the first virus. It is therefore tempting to hypothesize that this memory is based on the presence of a silencing signal similar to that revealed by grafting experiments.
Interestingly, recovery does not occur only in transgenic plants expressing part of the genome of a virus. In some cases, wild-type plants can recover from virus infection by specifically degrading virus RNA (Al-Kaff et al., 1998; Covey et al., 1997; Ratcliff et al., 1997; Ratcliff et al., 1999). Similarly to recovered transgenic plants, these wild-type recovered plants are immune to secondary infection by the same virus or by another recombinant virus carrying part of the genome of the first virus, showing that the memory signal can be maintained despite the absence of homologous transgenes (Ratcliff et al., 1997; Ratcliff et al., 1999). These results suggested that PTGS participates in a mechanism for plant resistance to viruses, a hypothesis that was confirmed by the discovery that sgs2/sde1, sgs3, sde3 and ago1 mutants are hypersusceptible to infection by a cucumovirus, CMV (Fig. 3) (Mourrain et al., 2000; Dalmay et al., 2001) (J.-B. Morel and H.V., unpublished). Therefore, there might be similarities between particular virus RNAs and transgene RNAs that make them targets for the PTGS machinery.
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The identification of a plant defence strategy based on the degradation of virus RNA is an important discovery of the 1990s. However, plant-virus interactions that are regulated at the RNA level are complex, because viruses can be targets, inducers or inhibitors of PTGS. Considering that PTGS and VIGS can be inhibited by viruses, we must now determine precisely how PTGS can degrade virus RNA and how viruses can induce VIGS. These apparent contradictions could be explained by the dynamics of infection. In particular, the differential spread speed and location of both the virus and the systemic silencing signal (Voinnet et al., 2000) could determine whether the virus or the plant wins the battle.
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Concluding remarks |
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