Year: 1998
Type: review
Abstract: The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker & Van Montagu 1997;Stam et al. 1997b ). Epigenetics is commonly defined as 'the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence' ( Russo et al. 1996 ). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals ( Dorer 1997;Foss & Selker 1991;Rossignol & Faugeron 1994;Ye 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals ( Lacroute 1971;Prusiner 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes ( Cogoni et al. 1996 ;Fire et al. 1998 ;Napoli et al. 1990 ). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and the position of the integration site cannot be predicted, regardless of whether Agrobacterium-mediated transfer or direct gene transfer are used. Therefore, one or multiple intact or rearranged copies can integrate at one or multiple unlinked loci. Cis-inactivation will be used to refer to silencing events that affect the expression of transgenes integrated at a single locus, irrespective of the copy number, while trans-inactivation will be used to define the silencing effect of one locus on another. As defined by Stam et al. (1997b) , transcriptional gene silencing will be referred to as TGS, and post-transcriptional gene silencing as PTGS. Transgenes can undergo TGS in cis when one or multiple copies integrate at a locus located in or next to silent hypermethylated genomic sequences. This case closely resembles position effect variegation (PEV) in Drosophila which occurs when a euchromatic gene is moved next to heterochromatin by chromosomal rearrangement. Heterochromatin can then spread into the gene and affect its expression in a stochastic, cell-autonomous and clonal manner, thus leading to variegation (for review see Karpen 1994). Methylation in plants may spread like heterochromatin in Drosophila from the adjacent sequences into the transgene, thus leading to silencing ( Pröls & Meyer 1992). Transgenes can also undergo TGS in cis when multiple copies become methylated although they integrate at a hypomethylated locus. This case resembles local heterochromatin formation and silencing in Drosophila induced by the extension of transgene repeats (for review see Dorer 1997). Such repeat-induced gene silencing in plants, defined as RIGS by Assaad et al. (1993) , correlates with increased methylation and increased resistance to both DNase I and microccocal nuclease in transgenic Arabidopsis plants, indicating that RIGS correlates with changes in chromatin configuration ( Ye & Signer 1996). Occasionally, transgenes inserted as single copies at a hypomethylated locus can undergo TGS ( Meyer & Heidmann 1994;Meyer et al. 1992 ). Silencing was observed when a transgene derived from a monocotyledonous plant was introduced into a dicotyledonous plant, whereas it was not observed with the corresponding dicot gene ( Elomaa et al. 1995 ), suggesting that a strong discrepancy between the DNA composition of the transgene and that of the surrounding genomic sequences can be recognized by the cellular machinery, leading to the specific methylation and silencing of foreign sequences. In this case also, TGS correlated with increased methylation and increased resistance to both DNase I and microccocal nuclease ( van Blokland et al. 1997 ), thus indicating that hypermethylation and chromatin condensation are general characteristics associated with transcriptional silencing. TGS can result from the uni-directional effect of one transgene on another. An active copy of a transgene can become silent and methylated when brought by crossing into the presence of a silenced homologous copy, and can acquire the capacity to inactivate another copies in subsequent crosses ( Meyer et al. 1993 ). This phenomenon resembles 'paramutation', a natural epigenetic phenomenon affecting host genes in the absence of transgenes. Paramutator (inactive) alleles inhibit the expression of paramutable (active) alleles which themselves become paramutators ( Brink 1956). Paramutation indicates that homologous chromosomes can exchange information in somatic cells. The mechanism invoked for paramutation involves DNA–DNA pairing and transmission of the chromatin structure from the silent copy to the active copy, as shown in Drosophila where PEV can be transmitted in a dominant manner from the rearranged silent chromosome to the wild-type active chromosome (for review see Karpen 1994). Active transgenes can also become silent and methylated when brought into the presence of an unlinked silenced homologous transgene ( Matzke et al. 1989 ;Vaucheret 1993). This phenomenon can be defined as 'ectopic trans-inactivation'. It differs from paramutation because the target-silenced transgenes do not acquire the capacity to inactivate in trans other unlinked transgenes ( Park et al. 1996 ). It can affect any transgene that is expressed under the control of the same promoter, irrespective of the coding sequence being expressed ( Matzke et al. 1994 ;Vaucheret 1993). This specificity indicates that the promoter of the transgene is the target for this form of transcriptional silencing ( Thierry & Vaucheret 1996). Deletion analysis indicated that 90 bp of homology between a silencing locus and the promoter of a target transgene is sufficient for directing silencing and methylation ( Vaucheret 1993). Two silencing loci showing transcriptional silencing of unlinked promoter–homologous transgenes irrespective of their positions within the genome have been identified ( Matzke et al. 1994 ;Vaucheret 1993). These two loci consist of multiple and rearranged copies of the transgene and are each located on or near a telomere ( Park et al. 1996 ). These data suggest that the ability to trigger ectopic trans-inactivation may depend on the ability of the silencing loci to interact with any other position of the genome by direct DNA–DNA pairing. Alternatively, it could involve the production of diffusible RNA by the silencing locus that leads to heritable silencing and methylation of homologous target loci via an RNA–DNA interaction ( Park et al. 1996 ;Wassenegger & Pélissier 1998). However, since one of these two silencing loci was shown to be unable to silence the expression of extra-chromosomal copies of a target transgene ( Vaucheret 1994), the transmission of silencing is more likely to occur through DNA–DNA pairing between stably integrated homologous copies. When the silenced target transgenes are separated from a silencing locus by segregation, they reactivate more or less quickly, i.e. during a period that ranges between immediately after segregation and two or three generations following the segregation. The fact that target transgenes which remain silent in the absence of the silencing locus are still methylated ( Park et al. 1996 ;Vaucheret 1994) indicates that methylation is probably involved in the maintenance of the silent state. Target transgenes driven by a 35S promoter devoid of CG and CNG methylation acceptor sites were as susceptible to silencing in trans as those driven by a wild-type promoter, indicating that methylation at CG and CNG sites is not a prerequisite for silencing. However, using this promoter, silencing was immediately relieved in the absence of the trans-silencer, thus further supporting the hypothesis that CG/CNG methylation is essential for the maintenance of silencing ( Diéguez et al. 1998 ). Transgene silencing is defined as occurring at the post-transcriptional level when RNA does not accumulate even though transcription occurs. As opposed to TGS, which is meiotically heritable ( Assaad et al. 1993 ;Matzke et al. 1989 ;Mittelsten Scheid et al. 1998 ;Park et al. 1996 ;Vaucheret 1993), PTGS is reset (i.e. affected genes are reactivated) after meiosis ( Balandin & Castresana 1997;Dehio & Schell 1994;Dorlhac de Borne et al. 1994 ;Hart et al. 1992 ;Vaucheret et al. 1995 ). However, in affected lines, PTGS recurs every generation at some time during plant development. Up until now, post-transcriptional cis-inactivation has been observed when foreign (bacterial) transgenes (uidA, npt, rolB) were introduced under the control of the strong viral 35S promoter ( Dehio & Schell 1994;Elmayan & Vaucheret 1996;English et al. 1996 ;Ingelbrecht et al. 1994 ). In all cases, PTGS occurred more efficiently (or exclusively) in haploids and homozygous plants as compared with hemizygous plants, suggesting a transgene dose effect. PTGS was observed in a larger proportion of transformants using a 35S promoter with a double enhancer as compared to the classical 35S promoter ( Elmayan & Vaucheret 1996;English et al. 1996 ). These results initially suggested that PTGS was due to the over-production of transgene RNA above a putative threshold level that triggers the irreversible degradation of RNA ( Dehio & Schell 1994). However, the level of transgene transcription was not always found to be significantly higher in silenced lines as compared to non-silenced lines ( English et al. 1996 ), thus suggesting that some other parameters may also play a role in the triggering of PTGS. The presence of repeats at the transgene locus of the silenced lines was proposed to play such a role ( English et al. 1996 ). Multiple models of PTGS have been proposed, considering mainly the roles of RNA thresholds and DNA repeats (reviewed by Baulcombe 1996). These models may not be exclusive if we consider that only a particular subpopulation of transgene RNA is important for the triggering of PTGS. Transgene RNA could be specifically degraded if tagged by specific molecules. To account for sequence-specific RNA degradation, these tag molecules are hypothesized to be small complementary RNA (cRNA). They could be synthesized by a plant RNA dependent RNA polymerase (RdRp) using transgene RNA as template ( Dougherty & Parks 1995). Alternatively, they could be internal fragments of transgene RNA produced by pairing-cleavage cycles between aberrant poly (A)– RNA and normal transgene RNA as a result of internal sequence complementarity ( Metzlaff et al. 1997 ). These cRNA could interact with mRNA, thus forming duplexes that behave as targets for cellular enzymes like double-strand RNA-specific RNase. Not all transgenic lines undergo PTGS. Therefore, the transgene loci that trigger PTGS may have some specific characteristics. First, the presence of repeats could allow DNA–DNA interactions and subsequent changes in methylation and/or chromatin structure that impedes correct transcription and leads to the production of malformed (aberrant) RNA that are a better template for RdRp than mRNA. Second, the utilisation of a very strong promoter to drive the transgene may contribute to elevate the amount of malformed (aberrant) RNA produced spontaneously by RNA polymerase errors up to a threshold that triggers PTGS. These aberrant RNA and/or the high amount of mRNA accumulated in the cytoplasm due to the use of a strong promoter may also trigger methylation of the coding sequence of the corresponding transgene by a feedback mechanism ( Wassenegger et al. 1994 ). Feedback methylated transgenes may therefore produce aberrant RNA as might do methylated transgene repeats. Therefore, either the use of a strong promoter or the presence of transgene repeats may lead to post-transcriptional silencing as a consequence of the production of aberrant RNA and subsequently of cRNA. PTGS was originally discovered as the reciprocal and co-ordinated silencing of transgenes and homologous host genes, and is usually referred to as 'co-suppression' ( Napoli et al. 1990 ). Since that time, a number of transgenes encoding part or the entire transcribed sequence of a host gene have been shown to trigger co-suppression of homologous host genes (for recent reviews see Depicker & Van Montagu 1997;Stam et al. 1997b ). Co-suppression occurs more efficiently (or exclusively) in haploids and homozygous plants as compared with hemizygous plants, suggesting a transgene dose effect ( de Carvalho et al. 1992 ;Dorlhac de Borne et al. 1994 ;Hart et al. 1992 ). As shown by a large scale analysis of petunia plants transformed with a chalcone synthase transgene, the efficiency of co-suppression correlates with the strength of the promoter driving the transgene, suggesting a transgene product dose effect rather than a transgene dose effect ( Que et al. 1997 ). Co-suppression of nitrate reductase is inhibited when the transgenes homologous to the host genes are themselves silenced at the transcriptional level, thus indicating that transgene transcription is required ( Vaucheret et al. 1997 ). In addition, the efficiency of co-suppression is reduced or delayed when host genes are not expressed ( Dorlhac de Borne et al. 1994 ;Smith et al. 1990 ), or when transgenes are introduced into mutants lacking a functional host gene ( Vaucheret et al. 1997 ). These results suggest that co-suppression cannot be considered as the unidirectional silencing effect of transgenes onto host genes, but rather as a reciprocal and synergistic phenomenon where host genes and transgenes can co-operate to produce aberrant RNA and/or cRNA above the threshold level that activates the RNA degradation machinery. Although most of the co-suppression events result from the introduction of strongly expressed transgenes, striking and intriguing data concerning PTGS of host genes by weakly transcribed or promoterless (untranscribed) transgenes have been reported ( van Blokland et al. 1994 ). These data can be reconciled with the aberrant RNA/cRNA hypothesis described above if we consider that these transgene loci always consist of transgene repeats ( Stam et al. 1997a ). Therefore, as proposed previously, DNA–DNA pairing could play a role in the initial step of production of aberrant RNA. Either cis-interactions between transgene repeats or trans-interactions between the transgene repeats and the homologous host genes could therefore trigger changes in methylation or chromatin structure, leading to the production of aberrant RNA either by a transcribed transgene or by the host genes only when the transgene does not contain a promoter. When the transgene which undergoes PTGS encodes part of the genome of a plant RNA virus, silenced transgenic plants become resistant against virus infection (reviewed by Baulcombe 1996) In some cases, virus resistance can be reached after a phase of sensitivity, a phenomenon called 'recovery' ( Lindbo et al. 1993 ). In this case, the silent state is not pre-established in the transgenic line, and only the infection by the virus triggers transgene silencing and virus resistance. It is assumed, therefore, that transgene transcription and virus replication both contribute to reach the level of accumulation of RNA that triggers PTGS. Viruses carrying a transgene inserted within their genome fail to infect transgenic plants that display spontaneous PTGS of this transgene ( English et al. 1996 ). These results are consistent with a model in which viral RNA and transgene RNA carrying the same sequence are tagged by the same cRNA molecules, and are subsequently degraded by the RNA degradation machinery. Conversely, viruses carrying a transgene inserted within their genome succeed in infecting transgenic plants that do not display spontaneous PTGS of this transgene. However, the transgene may become silenced after infection, a phenomenon called 'virus-induced gene silencing' ( Ruiz et al. 1998 ). PTGS behaves as a non-clonal event ( Boerjan et al. 1994 ;Hart et al. 1992 ;Palauqui et al. 1996 ). Some striking and reproducible features in the evolution of the silencing patterns during plant development suggest the propagation of a silencing message through the plant. Co-suppression of nitrate reductase, nitrite reductase and SAM-synthase host genes and transgenes in tobacco lead to particular chlorotic or necrotic phenotypes (which correlate with the disappearance of the corresponding RNA) that can be followed throughout the plant's life. Co-suppression appears as interveinal spots or vein-localized areas on one leaf and then propagates to the upper leaves with increasing silencing efficiency ( Boerjan et al. 1994 ;Palauqui et al. 1996 ). Since these types of non-clonal patterns are found reproducibly in all transgenic lines silenced for a given gene, this suggests that a sequence-specific message involved in the control of PTGS diffuses through the plant in a specific manner. Using grafting procedures, the transmission of a PTGS signal was investigated in tobacco. Silencing was transmitted with 100% efficiency from silenced stocks to target scions expressing the corresponding transgene ( Palauqui et al. 1997 ), indicating the propagation of a transgene-specific signal. The transmission of co-suppression 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. These results therefore reinforce the hypothesis that a transgene-specific, diffusable effector mediates the propagation of de novo post-transcriptional silencing over long distances within the plant, a phenomenon called systemic acquired silencing or SAS ( Palauqui et al. 1997 ). Similar conclusions were drawn when PTGS was systemically triggered after infection of one leaf of a transgenic N. benthamiana plant carrying a non-silenced transgene by an Agrobacterium strain carrying the same transgene ( Voinnet & Baulcombe 1997). Grafting experiments performed using nitrate reductase (Nia) silenced tobacco stocks and a set of different transgenic and non-transgenic scions revealed distinct requirements for the different steps of co-suppression: initiation, RNA degradation, and maintenance. Nia RNA degradation occurred in both grafted transgenic scions over-accumulating Nia mRNA due to the presence of a 35S-driven transgene and grafted non-transgenic scions over-accumulating host Nia mRNA due to metabolic derepression. However, RNA degradation did not occur in grafted wild-type scions, thus indicating that mRNA over-accumulation above the level of wild-type plants rather than the presence of a transgene in the scion is required for the RNA degradation step of co-suppression ( Palauqui & Vaucheret 1998). When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in non-transgenic plants and in transgenic lines that are not able to trigger co-suppression spontaneously. Conversely, silencing was maintained in transgenic lines that are able to trigger co-suppression spontaneously, thus indicating that only the transgene loci that are able to initiate co-suppression can also maintain a silent state ( Fig. 1). Systemic acquired silencing – systemic transient silencing. Particular transgene loci are competent to produce a signal that initiates sequence-specific RNA degradation in the cell. This signal moves systemically to neighbouring cells through plasmodesmata and at long distances through the phloem, then triggering degradation of homologous RNA. Because this systemic silencing signal is sequence-specific, it is likely that it derives from the transgene. Whether it is a direct product of the transgene (for example, an aberrant RNA) or a secondary product (for example, a cRNA), and whether such RNA circulates through the plant alone or complexed with a host factor remains to be determined. The absence of maintenance of silencing in non-transgenic plants that undergo grafting-induced RNA degradation suggests that the systemic silencing signal is not made of RNA degradation products only. It also indicates that the transgene locus as a to maintain co-suppression in each that the signal. In the absence of a transgene locus, a production of the systemic silencing signal by the silenced stocks is required to RNA degradation in the grafted This phenomenon could therefore be described as systemic transient silencing because it is not maintained when the silenced stocks are Conversely, in scions carrying a transgene locus, the systemic signal could an epigenetic of the transgene locus that the of the production of both the RNA degradation signal and the systemic silencing thus leading to systemic acquired silencing in the plant. Whether a systemic silencing signal is involved in all PTGS events reported remains to be determined. In phenotypes were produced by transgenes that control As proposed by co-suppression patterns may features of plant to of This hypothesis is in with the fact that wild-type scions grafted onto transgenic silenced stocks do not undergo whereas non-transgenic scions which accumulate the host mRNA above the level of the wild-type do undergo silencing ( Palauqui & Vaucheret 1998). Therefore, both a sequence-specific silencing signal and a high amount of target mRNA (or a high of to be required to a silent state. Dougherty et al. did not the transmission of virus resistance in grafting experiments plants expressing a viral thus suggesting that and/or systemic is not a general However, this result be in the of recent studies showing that virus infection systemic PTGS of et al. reported that infection PTGS of nitrate reductase (Nia) or transgenes in leaves of tobacco and Arabidopsis plants, suggesting that the systemic spread of (which has sequence to the Nia or may inhibit the systemic spread of the PTGS signal. genes are likely to be involved in both TGS and PTGS. In Drosophila, of genes PEV has the of more than loci (for review see Karpen 1994). These loci to two that PEV and that Some of the corresponding genes have been of the are involved in the formation of heterochromatin while genes of the transcription in which PTGS is have been in These called (for define three genetic loci ( Cogoni & 1997). in the control of TGS have been in Arabidopsis and are called mutants (for These mutants which are able to reactivate a silent transgene ( Scheid et al. 1998 ). These mutants also a in the methylation of sequences of the as do methylation mutants called in DNA ( et al. 1993 ). is to The function by the gene is It is that it encodes a because the plants display a normal plants lacking were using an transgene against the gene TGS was not in such plants ( Scheid et al. 1998 ). Therefore, these results that remains in the between TGS and affected in PTGS have also been in Arabidopsis These mutants to two The to mutants called (for enhancer of gene in which PTGS of a transgene is These are and define two genetic loci ( Dehio & Schell 1994). The to mutants called (for of gene which PTGS of a transgene as as post-transcriptional co-suppression of nitrate reductase host genes and transgenes. These are and define two genetic loci ( Elmayan et al. 1998 ), which probably to loci in and are not able to reactivate a silent transgene, and do not trigger a in the methylation of sequences of the genome. Therefore, genes are to specifically control PTGS. The of and genes will the of the of TGS and PTGS. gene silencing in plants can occur at the transcriptional or post-transcriptional TGS occurs mainly when multiple repeats of a transgene are inserted in the genome of transgenic plants. It correlates with condensation of chromatin and with The transfer of methylation and silencing from one locus to another indicates that of the genome and exchange This transfer may occur through direct DNA–DNA pairing. Alternatively, it could involve the production of diffusible RNA by one locus, leading to of homologous targets via an RNA–DNA interaction ( Park et al. 1996 ;Wassenegger & Pélissier 1998). Epigenetic of sequences may be an important because it might Either or between transgene repeats may be for the cell. between induced methylated and DNA molecules may not be thus This hypothesis may not only to the genome of transformants carrying transgene but also to wild-type plants in which an in the number of can the of the genome if they can with each Such a to in other In the fungi and DNA that the are very silenced and In addition, in N. methylation of is by a very high of in a phenomenon called repeat-induced or ( et al. 1989 ). Such methylation and are proposed to also occur in to the homology between repeats and to efficiently ( et al. 1992 ). In plants, for such a has been observed ( Scheid et al. 1994 ), suggesting that alone may be sufficient to the genome against transgene-induced PTGS occurs mainly when transgene RNA is produced at high under the control of the 35S promoter of the it has been shown that non-transgenic and initially systemic when by from which they by of This 'recovery' phenomenon correlates with the absence of accumulation of and 35S RNA although their of transcription remain ( et al. 1998 et al. 1997 ). non-transgenic plants spontaneously from infection by the ( et al. 1997 ). These results suggest that plants can virus infection in a post-transcriptional manner. Therefore, most of the of PTGS of 35S-driven transgenes may result from the of high of RNA transcription and from the subsequent degradation of this RNA by the cellular involved in post-transcriptional However, since promoterless or weakly transcribed transgenes can lead to post-transcriptional silencing of homologous host genes, some molecules (for example, the aberrant RNA and/or cRNA defined involved in the of events leading to RNA degradation might be produced in an manner, for by and subsequently methylated Grafting experiments that diffusible can throughout the plant to trigger silencing in the other ( Palauqui et al. 1997 ), as virus propagates Whether aberrant RNA and/or cRNA as diffusible of silencing and whether they can or complexed with remains to be The similarities between transgene-induced PTGS and spontaneous virus in non-transgenic plants ( et al. 1997 ), and the between the systemic spread of viruses and the systemic spread of PTGS ( et al. 1998 ), reinforce the hypothesis that transgene-induced PTGS derives from a natural post-transcriptional TGS and PTGS phenomena may natural of plant at the DNA or RNA level against or like TGS may cellular against DNA that into the while PTGS may cellular against DNA that in the or RNA that in the Because large of transgene and/or or can be introduced in plants to their silencing and as plant mutants affected in the control of silencing become it is to further in the analysis of the and the natural roles of epigenetic control in plants. from the on Silencing for and and for of the
Source: The Plant Journal
Language: English
Subfield: Plant Science
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W1661448904