A Branched Pathway for Transgene-Induced RNA Silencing in Plants
2002; Elsevier BV; Volume: 12; Issue: 8 Linguagem: Inglês
10.1016/s0960-9822(02)00792-3
ISSN1879-0445
AutoresChristophe Béclin, Stéphanie Boutet, Peter M. Waterhouse, Hervé Vaucheret,
Tópico(s)CRISPR and Genetic Engineering
ResumoIn plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) [1Que Q. Wang H.-Y. English J.J. Jorgensen R.A. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence.Plant Cell. 1997; 9: 1357-1368Crossref PubMed Scopus (167) Google Scholar, 2Vaucheret H. Nussaume L. Palauqui J.-C. Quilléré I. Elmayan T. A transcriptionally active state is required for post-transcriptional silencing (cosupprssion) of nitrate reductase host genes and transgenes.Plant Cell. 1997; 9: 1495-1504Crossref PubMed Scopus (67) Google Scholar] or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS) [3Stam M. Viterbo A. Mol J.N. Kooter J.M. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.Mol. Cell. Biol. 1998; 18: 6165-6177Crossref PubMed Scopus (121) Google Scholar, 4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar, 5Chuang C.F. Meyerowitz E.M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2000; 97: 4985-4990Crossref PubMed Scopus (565) Google Scholar, 6Sijen T. Vijn I. Rebocho A. van Blokl R. Roelofs D. Mol J.N. Kooter J.M. Transcriptional and posttranscriptional gene silencing are mechanistically related.Curr. Biol. 2001; 11: 436-440Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar]. Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene [7Hamilton A. Baulcombe D. A species of small antisense RNA in posttranscriptional gene silencing in plants.Science. 1999; 286: 950-952Crossref PubMed Scopus (2318) Google Scholar] and with methylation of the coding sequence [3Stam M. Viterbo A. Mol J.N. Kooter J.M. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.Mol. Cell. Biol. 1998; 18: 6165-6177Crossref PubMed Scopus (121) Google Scholar, 4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar, 6Sijen T. Vijn I. Rebocho A. van Blokl R. Roelofs D. Mol J.N. Kooter J.M. Transcriptional and posttranscriptional gene silencing are mechanistically related.Curr. Biol. 2001; 11: 436-440Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar]. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ([9Béclin C. Berthome R. Palauqui J.-C. Tepfer M. Vaucheret H. Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes.Virology. 1998; 252: 313-317Crossref PubMed Scopus (141) Google Scholar, 10Brigneti G. Voinnet O. Li W.X. Ji L.H. Ding S.W. Baulcombe D.C. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.EMBO J. 1998; 17: 6739-6746Crossref PubMed Scopus (925) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar] this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 12Dalmay T. Hamilton A. Rudd S. Angell S. Baulcombe D.C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.Cell. 2000; 101: 543-553Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 13Fagard M. Boutet S. Morel J.-B. Bellini C. Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA. 2000; 97: 11650-11654Crossref PubMed Scopus (436) Google Scholar, 14Morel J.B. Godon C. Mourrain P. Béclin C. Boutet S. Fagard M. Feuerbach F. Proux F. Vaucheret H. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.Plant Cell. 2002; 14: 629-639Crossref PubMed Scopus (493) Google Scholar]. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1. In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) [1Que Q. Wang H.-Y. English J.J. Jorgensen R.A. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence.Plant Cell. 1997; 9: 1357-1368Crossref PubMed Scopus (167) Google Scholar, 2Vaucheret H. Nussaume L. Palauqui J.-C. Quilléré I. Elmayan T. A transcriptionally active state is required for post-transcriptional silencing (cosupprssion) of nitrate reductase host genes and transgenes.Plant Cell. 1997; 9: 1495-1504Crossref PubMed Scopus (67) Google Scholar] or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS) [3Stam M. Viterbo A. Mol J.N. Kooter J.M. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.Mol. Cell. Biol. 1998; 18: 6165-6177Crossref PubMed Scopus (121) Google Scholar, 4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar, 5Chuang C.F. Meyerowitz E.M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2000; 97: 4985-4990Crossref PubMed Scopus (565) Google Scholar, 6Sijen T. Vijn I. Rebocho A. van Blokl R. Roelofs D. Mol J.N. Kooter J.M. Transcriptional and posttranscriptional gene silencing are mechanistically related.Curr. Biol. 2001; 11: 436-440Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar]. Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene [7Hamilton A. Baulcombe D. A species of small antisense RNA in posttranscriptional gene silencing in plants.Science. 1999; 286: 950-952Crossref PubMed Scopus (2318) Google Scholar] and with methylation of the coding sequence [3Stam M. Viterbo A. Mol J.N. Kooter J.M. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.Mol. Cell. Biol. 1998; 18: 6165-6177Crossref PubMed Scopus (121) Google Scholar, 4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar, 6Sijen T. Vijn I. Rebocho A. van Blokl R. Roelofs D. Mol J.N. Kooter J.M. Transcriptional and posttranscriptional gene silencing are mechanistically related.Curr. Biol. 2001; 11: 436-440Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar]. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ([9Béclin C. Berthome R. Palauqui J.-C. Tepfer M. Vaucheret H. Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes.Virology. 1998; 252: 313-317Crossref PubMed Scopus (141) Google Scholar, 10Brigneti G. Voinnet O. Li W.X. Ji L.H. Ding S.W. Baulcombe D.C. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.EMBO J. 1998; 17: 6739-6746Crossref PubMed Scopus (925) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar] this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 12Dalmay T. Hamilton A. Rudd S. Angell S. Baulcombe D.C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.Cell. 2000; 101: 543-553Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 13Fagard M. Boutet S. Morel J.-B. Bellini C. Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA. 2000; 97: 11650-11654Crossref PubMed Scopus (436) Google Scholar, 14Morel J.B. Godon C. Mourrain P. Béclin C. Boutet S. Fagard M. Feuerbach F. Proux F. Vaucheret H. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.Plant Cell. 2002; 14: 629-639Crossref PubMed Scopus (493) Google Scholar]. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1. Mutations in the SGS2/SDE1, SGS3, and AGO1 genes controlling S-PTGS result in CMV hypersensitivity, indicating that PTGS is a mechanism of resistance against viruses [10Brigneti G. Voinnet O. Li W.X. Ji L.H. Ding S.W. Baulcombe D.C. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.EMBO J. 1998; 17: 6739-6746Crossref PubMed Scopus (925) Google Scholar]. However, numerous viruses, including potyviruses, cucumoviruses, and tobamoviruses, are able to counteract these defenses by inhibiting this type of PTGS [9Béclin C. Berthome R. Palauqui J.-C. Tepfer M. Vaucheret H. Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes.Virology. 1998; 252: 313-317Crossref PubMed Scopus (141) Google Scholar, 10Brigneti G. Voinnet O. Li W.X. Ji L.H. Ding S.W. Baulcombe D.C. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.EMBO J. 1998; 17: 6739-6746Crossref PubMed Scopus (925) Google Scholar, 15Voinnet O. Pinto Y.M. Baulcombe D. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants.Proc. Natl. Acad. Sci. USA. 1999; 23: 14147-14152Crossref Scopus (835) Google Scholar]. In addition, recently, it was shown that potyviruses also inhibit IR-PTGS in a transient system [4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar]. To further investigate the ability of viruses to counteract both S-PTGS and IR-PTGS, we infected hybrids between the Arabidopsis transgenic line 306-1 carrying a panhandle ΔGUS-SUG transgene shown to be a very efficient silencer of GUS [16Johansen L.K. Carrington J.C. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system.Plant Physiol. 2001; 126: 930-938Crossref PubMed Scopus (414) Google Scholar] and the transgenic lines 6b4 and 526-3 stably expressing a 35S-GUS sense GUS transgene (see Experimental Procedures). After infection with cucumber mosaic cucumovirus (CMV), turnip crinckle carmovirus (TCV), turnip mosaic potyvirus (TuMV), and turnip vein clearing tobamovirus (TVCV), GUS expression was recovered in all the plants infected with any of the four viruses, whereas mock-infected plants remained silenced (Table 1), indicating that these viruses are able to inhibit both S-PTGS and IR-PTGS, thus reinforcing the similarities between both mechanisms.Table 1Inhibition of GUS IR-PTGS by VirusesGUS Expressing LineX WTX 306-1MockMockTCVTVCVTuMVCMV6b419240113041832055526-384801050645325NDSilent hybrids obtained by crossing lines 6b4 and 526-3 stably expressing a 35S-GUS transgene to line 306-1 carrying a ΔGUS-SUG panhandle construct were infected with TCV, TVCV, TuMV, and CMV. GUS activity was determined at the same developmental stage in young leaves of the mock-inoculated plants and in symptomatic leaves of the infected plants. Open table in a new tab Silent hybrids obtained by crossing lines 6b4 and 526-3 stably expressing a 35S-GUS transgene to line 306-1 carrying a ΔGUS-SUG panhandle construct were infected with TCV, TVCV, TuMV, and CMV. GUS activity was determined at the same developmental stage in young leaves of the mock-inoculated plants and in symptomatic leaves of the infected plants. The sgs2, sgs3, and ago1 mutations were recovered after mutagenesis followed by a screen based on the reactivation of the PTG-Silenced sense 35S-GUS transgene at the L1 locus [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 13Fagard M. Boutet S. Morel J.-B. Bellini C. Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA. 2000; 97: 11650-11654Crossref PubMed Scopus (436) Google Scholar, 14Morel J.B. Godon C. Mourrain P. Béclin C. Boutet S. Fagard M. Feuerbach F. Proux F. Vaucheret H. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.Plant Cell. 2002; 14: 629-639Crossref PubMed Scopus (493) Google Scholar]. These three mutations not only suppress GUS S-PTGS but also also impair cosuppression of the nitrate reductase gene and lead to hypersusceptibility toward CMV infection [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 13Fagard M. Boutet S. Morel J.-B. Bellini C. Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA. 2000; 97: 11650-11654Crossref PubMed Scopus (436) Google Scholar], indicating that SGS2/SDE1, SGS3, and AGO1 genes control S-PTGS of (trans)genes and CMV tolerance. To investigate whether these genes also control IR-PTGS, the transgenic line 6b4 and the sgs2-1, sgs3-1, and ago1-27 mutants carrying the L1 locus were transformed with the ΔGUS-SUG panhandle construct [4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar]. Efficient silencing was observed in line 6b4 and in the three mutants (Figure 1). Indeed, the percentage of transformants showing more than 99% reduction of GUS activity ranged between 50% and 71% in the four genetic backgrounds, while most of the remaining population showed between 50% and 99% reduction. Less than 4% of transformants showed no sign of silencing, probably because of incomplete or nonfunctional T-DNA integration The analysis of the progeny of the silenced transformants revealed a mendelian segregation of [GUS−]/[GUS+] plants, indicating that silencing was fully reversible after elimination of the panhandle construct (data not shown). We further investigated whether SGS2/SDE1, SGS3, and AGO1 genes are required for methylation of the GUS coding sequence induced by S-PTGS and IR-PTGS [3Stam M. Viterbo A. Mol J.N. Kooter J.M. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.Mol. Cell. Biol. 1998; 18: 6165-6177Crossref PubMed Scopus (121) Google Scholar, 4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar, 6Sijen T. Vijn I. Rebocho A. van Blokl R. Roelofs D. Mol J.N. Kooter J.M. Transcriptional and posttranscriptional gene silencing are mechanistically related.Curr. Biol. 2001; 11: 436-440Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar]. Genomic DNA was digested with the methylation-sensitive enzyme HpaII and hybridized with a probe specific to the GUS target transgene and which does not share homology with the panhandle construct (Figure 2). GUS coding sequence contains seven HpaII sites, including one in the region covered by the probe. In the sgs2, sgs3, and ago1 mutants (lanes 2, 3, and 4), the 35S-GUS carried at the L1 locus is not silenced, and the probe reveals two bands of 70 and 230 bp which are indicative of the absence of methylation in the GUS coding sequence. In the silenced line L1 (lane 1) and in mutants retransformed with the ΔGUS-SUG panhandle construct (lanes 5, 6, 7, and 8), HpaII sites in or surrounding the region covered by the probe are methylated in all or part of the plant, and the probe reveals bands equal or larger than 300 bp (Figure 2 and data not shown). The methylation pattern was similar in the self-progeny of mutants retransformed with the ΔGUS-SUG panhandle construct and in isogenic progenies obtained by backcrossing to line L1 (thus annulating the effect of the recessive sgs2, sgs3, and ago1 mutations) (data not shown). This result confirms that IR-PTGS mediated by a panhandle transgene correlates with an increase in methylation and shows that the SGS2/SDE1, SGS3, and AGO1 genes are dispensable for such silencing-mediated methylation. To confirm that the mechanism of IR-PTGS is not controlled by SGS2/SDE1, SGS3, and AGO1 genes, we transformed wild-type plants and sgs2-1, sgs3-1, and ago1-27 mutants with panhandle constructs shown to be able to silence the endogenous AG, AP1, and CLV3 genes involved in flower development [5Chuang C.F. Meyerowitz E.M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2000; 97: 4985-4990Crossref PubMed Scopus (565) Google Scholar]. None of the three mutations was able to inhibit IR-PTGS. Indeed, similar homeotic alterations of flower organs were observed in the wild-type plants and in the three mutants, resulting in included sterile flowers (AG), flowers without petals (AP1), or enlarged meristems and extra carpels (CLV3). Silencing was observed with high frequency in each genetic background (see the Supplementary Material available with this article online). This result shows that neither IR-PTGS of GUS nor IR-PTGS of AG, AP1, or CLV3 genes require the SGS2/SDE1, SGS3, and AGO1 genes. Although we cannot exclude the hypothesis that the AG, AP1, and CLV3 genes are expressed in cellular regions where the SGS2/SDE1, SGS3, and AGO1 are not expressed, the fact that SGS2/SDE1, SGS3, and AGO1 genes are dispensable for IR-PTGS of transgenes and endogenous genes strongly reinforces the assumption that the genetic requirements for S-PTGS and IR-PTGS are different, a result which is reminiscent of the observation that VIGS also occur in the sde1 mutant [12Dalmay T. Hamilton A. Rudd S. Angell S. Baulcombe D.C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.Cell. 2000; 101: 543-553Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar]. IR-PTGS mediated by constructs designed to transcribe duplex of RNA is also sometimes referred to as RNAi, by analogy with the phenomenon leading to RNA silencing in animals by injection of dsRNA [17Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11739) Google Scholar]. However, SGS2/SDE1 and AGO1 are not required for IR-PTGS, although similar proteins (EGO1, RRF1, and RDE1) are required for RNAi in C. elegans[18Smardon A. Spoerke J.M. Stacey S.C. Klein M.E. Mackin N. Maine E.M. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.Curr. Biol. 2000; 10: 169-178Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 19Tabara H. Sarkissian M. Kelly W.G. Fleenor J. Grishok A. Timmons L. Fire A. Mello C.C. The rde-1 gene, RNA interference, and transposon silencing in C. elegans.Cell. 1999; 99: 123-132Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 20Sijen T. Fleenor J. Simmer F. Thijssen K.L. Parrish S. Timmons L. Plasterk R.H. Fire A. On the role of RNA amplification in dsRNA-triggered gene silencing.Cell. 2001; 107: 465-476Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar]. Similarly, RNAi in Drosophila involves an RdRP [21Lipardi C. Wei Q. Paterson B.M. RNAi as random degradative PCR siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell. 2001; 107: 297-307Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar]. This could indicate that these related proteins, indeed, part of multigene families, do not have the same function in plants and in worms or flies and that different members of these gene families are involved at different steps in PTGS and RNAi. Consistent with this hypothesis is the finding that the AGO2 protein from Drosophila is a component of the RNA degradation complex [22Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Argonaute2, a link between genetic and biochemical analyses of RNAi.Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1180) Google Scholar] involved in the final steps of RNAi, whereas the related proteins RDE1 and AGO1 are dispensable for the mRNA degradation process of RNAi and for IR-PTGS, respectively ([23Grishok A. Tabara H. Mello C.C. Genetic requirements for inheritance of RNAi in C. elegans.Science. 2000; 287: 2494-2497Crossref PubMed Scopus (362) Google Scholar] and this work). Alternatively, this could indicate differences in the mechanism of RNA silencing, depending on whether dsRNA is produced continuously by a transgene at a high level in each cell (IR-PTGS) or is injected locally and at a given time (RNAi). Considering all these results, we propose the hypothesis that plant S-PTGS, plant IR-PTGS, and animal RNAi are not equivalent mechanisms but are related and share common steps (Figure 3). The final steps of RNAi leading to the degradation of the target RNA have been dissected in an in vitro system in Drosophila[24Sharp P.A. Zamore P.D. Molecular biology. RNA interference.Science. 2000; 287: 2431-2432Crossref PubMed Scopus (105) Google Scholar]. The inducer of these steps are dsRNA molecules which are cleaved by the DICER enzyme into small RNAs (called siRNAs) [25Bernstein E. Caudy E.E. Hammond S.M. Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3776) Google Scholar]. These siRNAs then target the enzymatic degradation complex RISC to the mRNA to be degraded [26Hammond S.M. Bernstein E. Beach D. Hannon G.J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature. 2000; 404: 293-296Crossref PubMed Scopus (2400) Google Scholar]. Since RNA silencing in plants and animals leads to the accumulation of siRNAs, we suppose that the steps shared by all these phenomena and targeted by the viruses in plants are similar to the final steps of RNA degradation described in Drosophila. Moreover, considering its role in the induction of these final steps, it appears that the formation of dsRNA is probably involved also in S-PTGS mediated by a sense construct. In this case, dsRNA would be a branch point between S-PTGS and IR-PTGS pathways (Figure 3). This model of two alternative branches in the induction of PTGS, both leading to the formation of dsRNA, is consistent with the model previously developed distinguishing two branches: the "VIGS branch," where the dsRNA is produced by virus replication in an SDE1/SGS2- and SDE3-independent manner, and the "transgene branch," where dsRNA is produced by the transcript transgene replication by SDE1/SGS2 [27Voinnet O. Lederer C. Baulcombe D.C. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.Cell. 2000; 103: 157-167Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 28Dalmay T. Horsefield R. Braunstein T.H. Baulcombe D.C. SDE3 encodes an RNA helicase for post-transcriptional gene silencing in Arabidopsis.EMBO J. 2001; 20: 2069-2078Crossref PubMed Scopus (298) Google Scholar]. Our results indicate that, like SGS2/SDE1 and SDE3, SGS3 and AGO1 are also involved in the early steps of S-PTGS. Assuming that SGS2/SDE1 and AGO1 have functions similar to EGO1 and RRF1 putative RdRPs and to RDE1, respectively, one of the functions assumed by SGS2/SDE1 and AGO1 could be the transition from a localized induction to a continuous production of dsRNA in all the organism. In this case, the function of these molecules could be the "amplification" of dsRNA, probably by replicating complementary single-stranded RNA using siRNA as primers, as it has been shown to occur in C. elegans and Drosophila[20Sijen T. Fleenor J. Simmer F. Thijssen K.L. Parrish S. Timmons L. Plasterk R.H. Fire A. On the role of RNA amplification in dsRNA-triggered gene silencing.Cell. 2001; 107: 465-476Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar, 21Lipardi C. Wei Q. Paterson B.M. RNAi as random degradative PCR siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell. 2001; 107: 297-307Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar]. SGS3 has no clear signature or motif, except a coiled-coil domain in its C terminus that could suggest interactions with another protein involved in S-PTGS [11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar]. Nevertheless, since SGS3 is also dispensable for IR-PTGS induced by panhandle transgenes, this protein would be involved with SGS2/SDE1 and AGO1 in the formation and/or replication of dsRNA. We have shown previously that PTGS of a sense transgene is associated with the methylation of the transcribed region and that the inhibition of S-PTGS in the sgs2, sgs3, or ago1 mutant backgrounds was associated with a strong reduction of cytosine methylation [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar, 11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 13Fagard M. Boutet S. Morel J.-B. Bellini C. Vaucheret H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA. 2000; 97: 11650-11654Crossref PubMed Scopus (436) Google Scholar]. Here we show that IR-PTGS mediated by a panhandle construct in these mutant backgrounds is also associated with methylation in the transcribed sequence of the target transgene. These results demonstrate that methylation does not result from the direct action of SGS2/SDE1, SGS3, or AGO1 genes. Rather, methylation is probably induced by a component downstream of the branch point of S-PTGS and IR-PTGS pathways, i.e., dsRNA or siRNAs. However, it was shown that the inhibition of PTGS by the potyvirus protein HC-Pro suppresses the accumulation of siRNAs but does not affect methylation, suggesting that these siRNA are not required for methylation [29Mallory A.C. Ely L. Smith T.H. Marathe R. Analakshmi R. Fagard M. Vaucheret H. Pruss G. Bowman L. Vance V.B. HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.Plant Cell. 2001; 13: 571-583Crossref PubMed Scopus (262) Google Scholar]. Thus, methylation could be triggered by dsRNA, a hypothesis which is in accordance to the fact that nuclear dsRNA are able to trigger methylation to homologous sequences of the genomic DNA [30Wassenegger M. Heimes S. Riedel L. Sanger H.L. RNA-directed de novo methylation of genomic sequences in plants.Cell. 1994; 76: 567-576Abstract Full Text PDF PubMed Scopus (717) Google Scholar]. Lines L1, 6b4, and 526-3 carry a 35S-GUS transgene. Line L1 is silenced by PTGS, whereas lines 6b4 and 526-3 are not silenced. The panhandle constructs targeting genes AP1, CLV3, and AG were described in [5Chuang C.F. Meyerowitz E.M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2000; 97: 4985-4990Crossref PubMed Scopus (565) Google Scholar]. To investigate the effect of viruses on GUS IR-PTGS, lines 6b4 and 526-3 were crossed with line 306-1 carrying at a single locus the ΔGUS-SUG panhandle construct described in [4Waterhouse P.M. Graham M.W. Wang M.-B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl. Acad. Sci. USA. 1998; 95: 13959-13964Crossref PubMed Scopus (902) Google Scholar]. Hybrids were then infected with CMV, TVCV, and TuMV as described in [11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar] and with TCV using the same procedure as with TVCV and TuMV. For genetic transformation and multiplication, homozygous line 6b4 and homozygous sgs2-1[8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar], sgs3-1[11Mourrain P. Béclin C. Elmayan T. Feuerbach F. Godon C. Morel J.-B. Jouette D. Lacombe A.-M. Nikic S. Picault N. et al.Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell. 2000; 101: 533-542Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar], and ago1-27 mutants [14Morel J.B. Godon C. Mourrain P. Béclin C. Boutet S. Fagard M. Feuerbach F. Proux F. Vaucheret H. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.Plant Cell. 2002; 14: 629-639Crossref PubMed Scopus (493) Google Scholar] were used. GUS activity was determined as previously described [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar]. For measuring GUS activity in virus-infected plants, analysis was performed on young symptomatic leaves. DNA extraction and southern blotting were performed as in [8Elmayan T. Balzergue S. Béon F. Bourdon V. Daubremet J. Guénet Y. Mourrain P. Palauqui J.-C. Vernhettes S. Vialle T. et al.Arabidopsis mutants impaired in cosuppression.Plant Cell. 1998; 10: 1447-1457Crossref Scopus (193) Google Scholar]. To analyze methylation in the GUS coding sequence at the L1 locus in the presence of the ΔGUS-SUG panhandle construct, genomic DNA was digested with HpaII and was hybridized with the 231 bp EcoRV fragment of the GUS coding sequence, which is missing in the ΔGUS-SUG panhandle construct. Supplementary Material including additional Results and Discussion; Experimental Procedures; a figure showing that IR-PTGS of endogenous genes occurs in the sgs2-1, sgs3-1, ago1-27 mutants; and a table showing the proportion of transformants exhibiting IR-PTGS of AG, AP1, and CLV3 endogenous genes in wild-type and S-PTGS-deficient mutants background can be found online at http://images.cellpress.com/supmat/supmatin.htm. We thank E. Meyerowitz for kindly providing the panhandle constructs derived from the AG, AP1, and CLV3 genes. We thank J.-L. Macia and J.-M. Pollien for plant culture. This work was partly supported by the French Ministry of Research and Technology through the PRFMMIP program. Download .pdf (.07 MB) Help with pdf files Supplementary material
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