Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to Tomato Spotted Wilt Virus in transgenic tobacco

  • 124 Accesses

  • 19 Citations

Abstract

Matrix attachment regions (MARs) are DNA elements that can increase and stabilize transgene expression. We investigated the effect of the RB7 MAR on transgenic virus resistance. Constructs for resistance to tomato spotted wilt virus (TSWV) with and without flanking RB7 MARs were used to transform tobacco and produce homozygous lines. The population with the MAR construct had a significantly higher percentage of TSWV resistant plants in the R1 generation than the nonMAR population. Each resistant line was advanced to the R4 generation, and significantly fewer MAR lines lost resistance over generations compared to the nonMAR population. Lines with TSWV resistance in growth chamber tests were also resistant in field trials. Two lines that were resistant in the R1 generation and susceptible in the R4 were examined in more detail in order to determine if transcriptional silencing of the transgene was occurring in the later generation. Short interfering 21– 25 nt RNAs from the transgene that are characteristic of post-transcriptional gene silencing (PTGS) were present in the resistant R1 plants, but not the susceptible R4 plants, indicating that virus resistance was associated with PTGS of the transgene. Loss of resistance was accompanied by an increase in promoter methylation in both lines. In line M41, the transgene was fully silenced at the transcriptional level in the R4 as shown by nuclear run-on assays. In line NM13, transgene transcription and RNA accumulation was still present in the R4 generation, but the level of transcription was not sufficient to trigger PTGS, suggesting that this line may have partial transcriptional silencing. These results are consistent with the concept that MARs may prevent transcriptional silencing.

This is a preview of subscription content, log in to check access.

References

  1. GC Allen G Hall SuffixJr. S Michalowski W Newman S Spiker AK Weissinger WF Thompson (1996) ArticleTitleHigh-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco Plant Cell 8 899–913 Occurrence Handle10.1105/tpc.8.5.899 Occurrence Handle1:CAS:528:DyaK28XjtlOktr0%3D Occurrence Handle8672887

  2. GC Allen S Spiker WF Thompson (2000) ArticleTitleUse of matrix attachment regions (MARs) to minimize transgene silencing Plant Mol Biol 43 361–376 Occurrence Handle10.1023/A:1006424621037 Occurrence Handle1:CAS:528:DC%2BD3cXmsF2jsrY%3D Occurrence Handle10999416

  3. R Ascenzi B Ülker JJ Todd DA Sowinski CR Schimeneck GC Allen AK Weissinger WF Thompson (2003) ArticleTitle.Analysis of trans -silencing interactions using transcriptional silencers of varying strength and targets with and without flanking nuclear matrix attachment regions Transgenic Res 12 305–318 Occurrence Handle10.1023/A:1023310118231 Occurrence Handle1:CAS:528:DC%2BD3sXivFynurc%3D Occurrence Handle12779119

  4. FM Ausubel R Brent RE Kingston DD Moore JG Seidman JA Smith K Struhl () (Eds) (1995) Current Protocols in Molecular Biology John Wiley and Sons, Inc. New York

  5. D Becker E Kemper J Schell R Masterson (1992) ArticleTitleNew plant binary vectors with selectable markers located proximal to the left T-DNA border Plant Mol Biol 20 1195–1197 Occurrence Handle10.1007/BF00028908 Occurrence Handle1:CAS:528:DyaK3sXltlaqug%3D%3D Occurrence Handle1463855

  6. C Brouwer W Bruce S Maddock Z Avramova B Bowen (2002) ArticleTitleSuppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5′ ADH1matrix attachment region Plant Cell 14 2251–2264 Occurrence Handle10.1105/tpc.004028 Occurrence Handle1:CAS:528:DC%2BD38XnsFemurg%3D Occurrence Handle12215518

  7. LG Burk DU Gerstel EA Wernsman (1979) ArticleTitleMaternal haploids of Nicotiana tabacumL. from seed Science 206 585

  8. KG Campbell EA Wernsman WP Fitzmaurice JA Burns (1994) ArticleTitleConstruction of a designer chromosome in tobacco Theor Appl Genet 87 837–842 Occurrence Handle10.1007/BF00221136

  9. KH Cox RB Goldberg (1988) Analysis of plant gene expression C.H. Shaw (Eds) Plant Molecular Biology: A Practical Approach. Information Printing Ltd. Oxford 1–35

  10. AS Csinos HR Pappu RM McPherson MG Stephenson (2001) ArticleTitleManagement of Tomato spotted wilt virus in flue-cured tobacco with acibenzolar-S-methyl and imidacloprid Plant Dis 85 292–296 Occurrence Handle1:CAS:528:DC%2BD3MXitV2lsbk%3D

  11. T Dalmay AJ Hamilton S Rudd S Angell DC Baulcombe (2000) ArticleTitle.An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus Cell 101 543–553 Occurrence Handle10.1016/S0092-8674(00)80864-8 Occurrence Handle1:CAS:528:DC%2BD3cXjvFWntLY%3D Occurrence Handle10850496

  12. T Elmayan H Vaucheret (1996) ArticleTitleExpression of single copies of a strongly expressed 35S transgene can be silenced post-transcriptionally Plant J 9 787–797 Occurrence Handle10.1046/j.1365-313X.1996.9060787.x Occurrence Handle1:CAS:528:DyaK28XksFyqsr8%3D

  13. JJ English DC Baulcombe (1997) ArticleTitleThe influence of small changes in transgene transcription on homology-dependent virus resistance and gene silencing Plant J 12 1311–1318 Occurrence Handle10.1046/j.1365-313x.1997.12061311.x Occurrence Handle1:CAS:528:DyaK1cXlvVWisg%3D%3D

  14. EJ Finnegan M Wang P Waterhouse (2001) ArticleTitleGene silencing: fleshing out the bones Curr Biol 11 R99–R102 Occurrence Handle1:CAS:528:DC%2BD3MXhtlGlurk%3D Occurrence Handle11231168

  15. TL German DE Ullman JW Moyer (1992) ArticleTitleTospoviruses: diagnosis, molecular biology, phylogeny, and vector relationships Annu Rev Phytopathol 30 315–348 Occurrence Handle1:CAS:528:DyaK3sXns1KnsQ%3D%3D

  16. R Goldbach E Bucher M Prins (2003) ArticleTitleResistance mechanisms to plant viruses: an overview Virus Res 92 207–212 Occurrence Handle1:CAS:528:DC%2BD3sXis12rtLo%3D Occurrence Handle12686431

  17. C Gonsalves B Xue SZ Pang R Provvidenti JL Slightom D Gonsalves (1996) ArticleTitleBreeding transgenic tomatoes for resistance to tomato spotted wilt virus and cucumber mosaic virus Acta Horticult 431 442–44

  18. J Goodwin K Chapman S Swaney TD Parks Wernsman EA WG Dougherty (1996) ArticleTitleGenetic and biochemical dissection of transgenic RNA-mediated virus resistance Plant Cell 8 95–105 Occurrence Handle1:CAS:528:DyaK28Xnslylsw%3D%3D Occurrence Handle8597662

  19. R Grumet (1994) ArticleTitleDevelopment of virus resistant plants via genetic engineering Plant Breeding Rev 12 47–79

  20. A Gubba C Gonsalves MR Stevens DM Tricoli D Gonsalves (2002) ArticleTitleCombining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentummill) Mol Breeding 9 13–23 Occurrence Handle1:CAS:528:DC%2BD38XkvFCgt7c%3D

  21. GE Hall SuffixJr. GC Allen DS Loer WF Thompson S Spiker (1991) ArticleTitleNuclear scaffolds and scaffold-attachment regions in higher plants Proc Natl Acad Sci USA 88 9320–9324 Occurrence Handle1:CAS:528:DyaK3MXmsFait74%3D Occurrence Handle11607225

  22. AJ Hamilton O Voinnet L Chapell DC Baulcombe (2002) ArticleTitleTwo classes of short interfering RNA in RNA silencing EMBO J 21 4671–4679 Occurrence Handle1:CAS:528:DC%2BD38Xms1KmsrY%3D Occurrence Handle12198169

  23. S Herrero A Culbreath A Csinos H Pappu RC Rufty ME Daub (2000) ArticleTitleNucleocapsid gene-mediated transgenic resistance provides protection against Tomato spotted wilt virusepidemics in the field Phytopathology 90 139–147

  24. R Holmes-Davis L Comai (1998) ArticleTitleNuclear matrix attachment regions and plant gene expression Trends Plant Sci 3 91–96

  25. JJMR Jacobs K Litière V Van Dijk GJ Van Eldik M Van Montagu M Cornelissen (1997) ArticleTitlePost-transcriptional ß-1,3-glucanase gene silencing involves increased transcript turnover that is translation-independent Plant J 12 885–893 Occurrence Handle1:CAS:528:DyaK2sXns1yitrc%3D

  26. MJ Kasperbauer GB Collins (1972) ArticleTitleReconstitution of diploids from leaf tissue of anther-derived haploid in tobacco Crop Sci 12 98–101

  27. NJ Kilby HMO Leyser IJ Furner (1992) ArticleTitlePromoter methylation and progressive transgene inactivation in Arabidopsis Plant Mol Biol 20 103–112 Occurrence Handle1:CAS:528:DyaK38XlvVGrsrs%3D Occurrence Handle1325204

  28. JW Kim SSM Sun TL German (1994) ArticleTitleDisease resistance in tobacco and tomato plants transformed with the tomato spotted wilt virus nucleocapsid gene Plant Dis 78 615–621 Occurrence Handle1:CAS:528:DyaK2MXisVCnsQ%3D%3D

  29. SP Kumpatla W Teng WG Buchholz TC Hall (1997) ArticleTitleEpigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice Plant Physiol 115 361–373 Occurrence Handle1:CAS:528:DyaK2sXntFSmur0%3D Occurrence Handle9342860

  30. SP Kumpatla TC Hall (1998) ArticleTitle.Recurrent onset of epigenetic silencing in rice harboring a multi-copy transgene Plant J 14 129–135 Occurrence Handle1:CAS:528:DyaK1cXjtV2isLs%3D Occurrence Handle15494058

  31. JA Lindbo L Silva-Rosales WM Proebsting WG Dougherty (1993) ArticleTitleInduction of a highly specific antiviral state in transgenic plants: Implications for regulation of gene expression and virus resistance Plant Cell 5 1749–1759 Occurrence Handle1:CAS:528:DyaK2cXisVSgurg%3D Occurrence Handle12271055

  32. YS Liu JS Levin JS Murray EA Wernsman AK Weissinger (2003) ArticleTitleA multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines Mol Breeding 12 145–156 Occurrence Handle1:CAS:528:DC%2BD3sXnvVajt7w%3D

  33. ZV Magbanua HD Wilde JK Roberts K Chowdhury J Abad JW Moyer HY Wetzstein WA Parrot (2000) ArticleTitleField resistance to Tomato spotted wilt virus in transgenic peanut (Arachis hypogeaL.) expressing an antisense nucleocapsid gene sequence Mol Breeding 6 227–236 Occurrence Handle1:CAS:528:DC%2BD3cXjt1Crsrw%3D

  34. SL Mankin (2000) Chromatin elements and transgene expression in tobacco cells North Carolina State University, Raleigh NC, USA

  35. SL Mankin GC Allen T Phelan S Spiker WF Thompson (2003) ArticleTitleElevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3′ MAR Transgenic Res 12 3–12 Occurrence Handle1:CAS:528:DC%2BD3sXnsFanuw%3D%3D Occurrence Handle12650520

  36. R Marathe R Anandalakshmi TH Smith GJ Pruss VB Vance (2000) ArticleTitleRNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing Plant Mol Biol 43 295–306 Occurrence Handle1:CAS:528:DC%2BD3cXmsF2jsr4%3D Occurrence Handle10999412

  37. AJM Matzke MA Matzke (1998) ArticleTitlePosition effects and epigenetic silencing of plant transgenes Curr Opin Plant Biol 1 142–148 Occurrence Handle1:CAS:528:DyaK1cXivVynsLc%3D Occurrence Handle10066569

  38. MF Mette J Vander Winden Matzke MA AJM Matzke (1999) ArticleTitleProduction of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans EMBO J 18 241–248 Occurrence Handle1:CAS:528:DyaK1MXntV2msA%3D%3D Occurrence Handle9878066

  39. MF Mette W Aufsatz J Vander Winden Matzke MA AJM Matzke (2000) ArticleTitle.Transcriptional silencing and promoter methylation triggered by double-stranded RNA EMBO J 19 5194–5201 Occurrence Handle1:CAS:528:DC%2BD3cXnvF2qtLk%3D Occurrence Handle11013221

  40. KK Mishra AK Handa (1998) ArticleTitlePost-transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre-mRNA processing Plant J 14 583–592 Occurrence Handle1:CAS:528:DyaK1cXksVyhtb8%3D

  41. L Mlynárová RC Jansen AJ Conner Stiekema WJ J-P Nap (1995) ArticleTitleThe MAR-mediated reduction in position effect can be uncoupled from copy number-dependent expression in transgenic plants Plant Cell 7 599–609 Occurrence Handle12242378

  42. L Mlynárová A Hricová A Loonen J-P Nap (2003) ArticleTitlepresence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing Plant Cell 15 2203–2217 Occurrence Handle12953121

  43. E Mueller J Gilbert G Davenport G Brigneti DC Baulcombe (1995) ArticleTitleHomology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing Plant J 7 1001–1013 Occurrence Handle1:CAS:528:DyaK2MXnsF2lt7s%3D

  44. F Neuhuber Y-D Park AJM Matzke MA Matke (1994) ArticleTitleSusceptibility of transgene loci to homology-dependent gene silencing Mol Gen Genet 244 230–241 Occurrence Handle1:CAS:528:DyaK2cXmsVOqsLo%3D Occurrence Handle8058034

  45. S-Z Pang P Nagpala M Wang JL Slightom D Gonsalves (1992) ArticleTitleResistance to heterologous isolates of tomato spotted wilt virus in transgenic tobacco expressing its nucleocapsid protein gene Phytopathology 82 1223–1229 Occurrence Handle1:CAS:528:DyaK3sXhvVCmtrw%3D

  46. S-Z Pang F-J Jan K Carney J Stout DM Tricoli HD Quemada D Gonsalves (1996) ArticleTitlePost-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant development Plant J 9 899–909 Occurrence Handle1:CAS:528:DyaK28XksFyqs74%3D

  47. S-Z Pang F-J Jan D. Gonsalves (1997) ArticleTitleNontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants Proc Natl Acad Sci USA 94 8261–8266 Occurrence Handle1:CAS:528:DyaK2sXkvVSht70%3D Occurrence Handle9223349

  48. J Paszkowski SA Whitham (2001) ArticleTitleGene silencing and DNA methylation processes Curr Opin Plant Biol 4 123–129 Occurrence Handle1:CAS:528:DC%2BD3MXislCktLY%3D Occurrence Handle11228434

  49. Pickford AS C Cogoni (2003) ArticleTitleRNA-mediated gene silencing Cell Mol Life Sci 60 871–882 Occurrence Handle12827277

  50. Q Que H-Y Wang JJ English RA Jorgensen (1997) ArticleTitleThe 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 9 1357–1368 Occurrence Handle1:CAS:528:DyaK2sXlvVKgsL0%3D Occurrence Handle12237385

  51. JM Sherman JW Moyer ME Daub (1998) ArticleTitleTomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene Plant Dis 82 407–414

  52. P Stoeva M Yankulova V Nikolaeva R Bachvarova L Ivanova E Maiss G Adam V Vulkov S Guelemerov A Atanassov (1998) ArticleTitleLong-term resistance to tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests Mol Breeding 4 155–164 Occurrence Handle1:CAS:528:DyaK1cXjtFeiurc%3D

  53. P Susi M Hohkuri T Wahlroos NJ Kilby (2004) ArticleTitleCharacteristics of RNA silencing in plants: similarities and differences across kingdoms Plant Mol Biol 54 157–174 Occurrence Handle1:CAS:528:DC%2BD2cXkt1Squr8%3D Occurrence Handle15159620

  54. MM Tanzer WF Thompson MD Law EA Wernsman S Uknes (1997) ArticleTitleCharacterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco Plant Cell 9 1411–1423 Occurrence Handle1:CAS:528:DyaK2sXlvVKgsLk%3D Occurrence Handle12237389

  55. F Tenllado JR Díaz-Ruíz (1999) ArticleTitleComplete resistance to pepper mild mottle tobamovirus mediated by viral replicase sequences partially depends on transgene homozygosity and is based on a gene silencing mechanism Transgenic Res 8 83–93 Occurrence Handle1:CAS:528:DyaK1MXlt1egsLs%3D

  56. WF Thompson GC Allen G Hall SuffixJr. S Spiker (1996) attachment regions and transgene expression JP Gustafson RB Flavell (Eds) Genomes of Plants and Animals 21st Stadler Genetics Symposium. Plenum Press New York 243–269

  57. B Ülker GC Allen WF Thompson S Spiker AK Weissinger (1999) ArticleTitleA tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants Plant J 18 253–263

  58. P Vain B Worland A Kohli JW Snape P Christou Allen GC EM Thompson (1999) ArticleTitleMatrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny Plant J 18 233–242 Occurrence Handle1:CAS:528:DyaK1MXktVaku7Y%3D

  59. T Vanden Boogaart F Wen JW Davies GP Lomonossoff (2001) ArticleTitleReplicase-derived resistance against Pea early browning virus in Nicotiana benthamianais an unstable resistance based upon posttranscriptional gene silencing Mol Plant-Microbe Interact 14 196–203 Occurrence Handle1:CAS:528:DC%2BD3MXmsVGnsw%3D%3D Occurrence Handle11204783

  60. H Vaucheret T Elmayan D Thierry A Geest Particlevan der T Hall AJ Conner Mlynárová L J-P Nap (1998) ArticleTitleFlank matrix attachment regions (MARs) from chicken, bean, yeast, or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants Mol Gen Genet 259 388–392 Occurrence Handle1:CAS:528:DyaK1cXmvFCqtrw%3D Occurrence Handle9790594

  61. H Vaucheret M Fagard (2001) ArticleTitleTranscriptional gene silencing in plants: targets, inducers, and regulators Trends Genet 17 29–35 Occurrence Handle1:CAS:528:DC%2BD3MXktl2ksA%3D%3D Occurrence Handle11163919

  62. H Vaucheret C Béclin M Fagard (2001) ArticleTitlePost-transcriptional gene silencing in plants J Cell Science 114 3083–3091 Occurrence Handle1:CAS:528:DC%2BD3MXnsFOhtbo%3D Occurrence Handle11590235

  63. M Wassenegger (2002) ArticleTitlesilencing-based disease resistance Transgenic Res 11 639–653 Occurrence Handle1:CAS:528:DC%2BD38XovVGjuro%3D Occurrence Handle12509140

  64. JC Watson LS Kaufman WF Thompson (1987) ArticleTitleDevelopmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum J Mol Biol 193 15–26 Occurrence Handle1:CAS:528:DyaL2sXhtlaktbY%3D Occurrence Handle3035192

  65. H Yu PP Kumar (2003) ArticleTitlePost transcriptional gene silencing in plants by RNA Plant Cell Reports 22 167–174 Occurrence Handle1:CAS:528:DC%2BD3sXnvFaltrs%3D Occurrence Handle12937944

  66. PD Zamore T Tuschi PA Sharp DP Bartel (2000) ArticleTitleRNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21-23 nucleotide intervals Cell 101 25–33 Occurrence Handle1:CAS:528:DC%2BD3cXisVaitbo%3D Occurrence Handle10778853

Download references

Author information

Correspondence to Jennifer S. Levin.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Levin, J.S., Thompson, W.F., Csinos, A.S. et al. Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to Tomato Spotted Wilt Virus in transgenic tobacco. Transgenic Res 14, 193–206 (2005). https://doi.org/10.1007/s11248-004-5413-8

Download citation

Keywords

  • gene silencing
  • matrix attachment regions
  • RNA-mediated virus resistance
  • Tomato Spotted Wilt Virus