Post Transcriptional Gene Silencing In Plant Biology Essay

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Post transcriptional gene silencing (PTGS) in plant is an RNA- degradation mechanism that shows similarities to RNA interference (RNAi) in animals. (Vaucheret et al. 2001) PTGS results in the specific degradation of a population of homologous RNAs. The basic requirements for RNA degradation in both animals and plants were found to have a great similarity as in the presence of double-stranded RNA (dsRNA), spreading within the organism from a localized area, the association with the accumulation of small interfering RNA (siRNA) and the presence of RNA-dependent RNA polymerases, RNA helicases besides, some proteins of undetermined functions, containing PAZ and Piwi domains. Concerning the mechanism of RNA-degradation in both animals and plants was also found to have common basics, so we could say the unifying feature of RNA silencing is, the cleavage of long double-stranded dsRNA into short-interfering (21-26 nt) RNAs by a Ribonuclease III-like enzyme termed DICER. In plants, four Dicer-like (DCL) enzymes have been identified in Arabidopsis thaliana; however, distinct functions are defined for DCL1, which is involved in miRNA biogenesis; DCL3 is required for retroelement and transposon siRNA production and chromatin silencing, and DCL2 has been implicated in viral siRNA production. The function of DCL4 is not known. MicroRNAs are endogenous, non-coding RNAs (18-25 nt) existing both in plants and in animals. These small-RNA fragments serve as the specificity determinant by being incorporated into the RNA-induced silencing complex (RISC) endonuclease, which degrades mRNAs in a sequence-specific manner or inhibits protein translation. However, some differences between both plants and animals are still quite clear. First, PTGS in plants requires at least two genes which are - SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA methyltransferase) - that were found to be absent in C. elegans thus consequently they are not required for RNAi. Second, it was found that all Arabidopsis mutants that reveal impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Although many viruses have developed strategies to counteract PTGS and could successfully infect the plants. On the cellular level, PTGS reduces mRNA accumulation in plant cytoplasm but does not affect transcription (de Carvalho et al., 1992; van Blockland et al., 1994). Detailed analyses of RNA content in plants carrying out PTGS has revealed the presence of several independent RNA degradation intermediates.

Initiation, Propagation and Maintenance

Studying how PTGS is started has showed the presence of basically three steps: initiation, propagation and maintenance. Indeed, spontaneous initiation of PTGS by nitrate reductase, nitrite reductase or SAM-synthase (trans) genes (which leads to particular chlorotic or necrotic phenotypes that correlate with the disappearance of the corresponding RNA) starts with interveinal or vein-localized spots on one leaf and then propagates to the upper leaves, in which it is subsequently maintained (Boerjan et al., 1994; Palauqui et al., 1996). These non clonal patterns were found reproducibly in all transgenic lines in which a given gene was silenced, suggesting that the analysis of spontaneous PTGS into localized initiation, systemic propagation and active maintenance is a general rule.


Spontaneous initiation of PTGS in transgenic plants is localized, so it is considered to be particularly difficult to study. Most data concerning the controlling the 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. It is suggested that the efficiency of triggering could depend on the probability that the transgene produces a particular form of RNA above a threshold level. Actually, 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 trigger 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 so 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 that 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.

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. 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 over expressing the endogenous Nia2 gene owing to metabolic derepression but not to a wild-type scion, which indicates that over accumulation 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). Other scientists were able to draw a 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.


Grafting experiments using nitrate-reductase-silenced tobacco stocks and a set of different transgenic and non-transgenic scions revealed similar requirements for spontaneous initiation and maintenance. Indeed, when grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild type plants, silencing was not maintained in lines that cannot trigger PTGS spontaneously (Fig. 1, class I and III plants). These lines seem to be able to 'sense' the systemic PTGS signal that induces the degradation of the mRNA, but cannot produce the signal. On the other hand, silencing was maintained in transgenic lines that are able to trigger PTGS spontaneously (Fig. 1, class II plants), which indicates that only the transgene loci that are able to initiate PTGS can maintain a silent state.

The ability of a transgenic line to produce the systemic silencing signal could depend on the genomic location and/or the structure of the transgene locus, which would thus involve a nuclear step in PTGS. Chemical modifications (e.g. DNA methylation or histone acetylation) or structural modification (i.e. chromatin remodeling) could correspond to an epigenetic imprint induced by the systemic silencing signal, allowing PTGS to be actively maintained during development. This could be maintained in newly developing tissues in a conservative manner during replication or could be imposed de novo in each new cell in response to the systemic signal. Recently, it was showed that a mutant in which the major maintenance DNA-methyltranferase was impaired exhibited impaired maintenance of PTGS (Morel et al., 2000), which supports this hypothesis. 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.

Fig. 1. Evidence for a systemic silencing signal and for a maintenance step in PTGS.

(A) Wild-type plants, (wt) do not undergo PTGS after grafting onto silenced transgenic plants. (B) Transgenic plants that do not spontaneously undergo PTGS (class I) undergo PTGS after grafting onto silenced transgenic plants but do not maintain silencing after elimination of the silenced rootstock. (C) Nonsilenced transgenic plants derived from lines that can spontaneously undergo PTGS (class II) undergo PTGS after grafting onto silenced transgenic plants and maintain silencing after elimination of the silenced rootstock. (D) Nontransgenic plants that express an endogenous gene at high level owing to metabolic derepression (class III) undergo PTGS after grafting onto silenced transgenic plants but do not maintain silencing after elimination of the silenced rootstock.