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Gene silencing in transgenic soybean plants transformed via particle bombardment

  • Genetics and Genomics
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Abstract

Transgenes are susceptible to silencing in plants especially when multiple copies of the gene of interest are introduced. Transgenic plants derived by particle bombardment, which is the common method for transforming soybean, have a tendency to have multiple integration events. Three independent transgenic soybean plants obtained via particle bombardment were analyzed for transgene silencing. A GUS transgenic soybean line had at least 100 copies of the GUS gene while there were approximately 60 copies of the transgene in the two soybean lines transformed with a 15-kDa zein storage protein gene from maize. Soybean plants transformed with the GUS gene showed variable GUS expression. The coding region and promoter of the GUS gene in the plants with low expression of GUS were heavily methylated. Variability in GUS expression was observed in the progeny of the high expressors in the T2 and T3 generations as well. Expression level of the 15-kDa zein gene in transgenic soybean plants showed correlation with the level of transgene methylation. The helper component-proteinase from potyviruses is known to suppress post-transcriptional gene silencing. Transgenic plants were inoculated with the soybean mosaic potyvirus (SMV) to test possible effects on transgene silencing in soybean. Infection with SMV did not suppress transgene silencing in these plants and suggests that the silencing in these plants may not be due to post-transcriptional gene silencing.

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References

  • Al-Kaff NS, Covey SN, Kreike MM, Page AM, Pinder R, Dale PJ (1998) Transcriptional and posttranscriptional plant gene silencing in response to a pathogen. Science 279:2113–2115

    Article  CAS  PubMed  Google Scholar 

  • Al-Kaff NS, Kreike MM, Covey SN, Pitcher R, Page AM, Dale PJ (2000) Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene. Nat Biotechnol 18:995–999

    CAS  PubMed  Google Scholar 

  • Anandalakshmi R, Pruss GJ, Ge X, Marathe R, Mallory AC, Smith TH, Vance VB (1998) A viral suppressor of gene silencing in plants. Proc Natl Acad Sci USA 95:13079–13084

    Article  CAS  PubMed  Google Scholar 

  • Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau C, Mallory A, Pruss G, Bowman L, Vance VB (2000) A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290:142–144

    Article  CAS  PubMed  Google Scholar 

  • Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218

    CAS  Google Scholar 

  • Bagga S, Adams H, Kemp JD, Sengupta-Gopalan C (1995) Accumulation of 15-kilodalton zein in novel protein bodies in transgenic tobacco. Plant Physiol 107:13–23

    CAS  PubMed  Google Scholar 

  • Blokland R van, ten Lohuis M, Meyer P (1997) Condensation of chromatin in transcriptional regions of an inactivated plant transgene:evidence for an active role of transcription in gene silencing. Mol Gen Genet 257:1–13

    PubMed  Google Scholar 

  • Burow MD, Sen P, Chlan CA, Murai N (1992) Developmental control of the β-phaseolin gene requires positive, negative and temporal seed specific transcriptional regulatory elements and a negative element for stem and root expression. Plant J 2:537–548

    CAS  Google Scholar 

  • Calvert LA, Ghabrial SA (1983) Enhancement by soybean mosaic virus of bean pod mottle virus titer in doubly infected soybean. Phytopathology 73:992–997

    Google Scholar 

  • Chandler VL, Vaucheret H (2001) Gene activation and gene silencing. Plant Physiol 125:145–148

    Google Scholar 

  • Cho EK, Goodman RM (1982) Field spread of soybean mosaic virus strains (transmitted by aphids, Myzus in Korea). Korean J Plant Prot 21:53–60

    Google Scholar 

  • Cogoni C, Macino G (1999) Homology dependent gene silencing in plants and fungi:a number of variations on the same theme. Curr Opin Microbiol 2:657–662

    CAS  PubMed  Google Scholar 

  • Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC (2000) An RNA-dependent RNA polymerase is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543–553

    CAS  PubMed  Google Scholar 

  • De Buck S, Depicker A (2001) Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated GUS transgenes in Arabidopsis. Mol Gen Genet 265:1060–1068

    Google Scholar 

  • De Buck S, Van Montagu M, Depicker A (2001) Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved. Plant Mol Biol 46:433–445

    PubMed  Google Scholar 

  • De Neve M, De Buck S, De Wilde C, Van Houdt H, Strobbe I, Jocobs A, Van Montagu M, Depicker A (1999) Gene silencing results in instability of antibody production in transgenic plants. Mol Gen Genet 260:582–592

    Article  PubMed  Google Scholar 

  • Domínguez A, Fagoaga C, Navarro L, Moreno P, Peña L (2002) Regenration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes. Mol Gen Genet 267:544–556

    Google Scholar 

  • Fu X, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low copy number transgenic plants with simple integration patterns. Transgen Res 9:11–19

    Article  CAS  Google Scholar 

  • Grant S (1999) Dissecting the mechanisms of posttranscriptional gene silencing:divide and conquer. Cell 96:303–306

    CAS  PubMed  Google Scholar 

  • Guo HS, Ding SW (2002) A Viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 21:398–407

    Article  CAS  PubMed  Google Scholar 

  • Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    CAS  PubMed  Google Scholar 

  • Han Y, Grierson D (2002) Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato. Plant J 29:509–519

    Google Scholar 

  • Jakowitsch J, Papp I, Moscone EA, van der Winden J, Matzke M, Matzke AJM (1999) Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters in trans. Plant J 17:131–140

    Article  CAS  PubMed  Google Scholar 

  • Jeddeloh JA, Stokes TL, Richards EJ (1999) Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat Genet 22:94–97

    CAS  PubMed  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants:the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Jeong B-R, Wu-Scharf D, Zhang C, Cerutti H (2002) Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements. Proc Natl Acad Sci USA 99:1076–1081

    Article  CAS  PubMed  Google Scholar 

  • Jones L, Thomas CL, Maule AJ (1998) De novo methylation and cosuppression induced by a cytoplasmically replicating plant RNA virus. EMBO J 17:6385–6393

    CAS  PubMed  Google Scholar 

  • Jones L, Ratcliff F, Baulcombe DC (2001) RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr Biol 11:747–757

    Article  CAS  PubMed  Google Scholar 

  • Kasschau KD, Carrington JC (1998) A counterdefensive strategy of plant viruses:suppression of posttranscriptional gene silencing. Cell 95:461–470

    CAS  PubMed  Google Scholar 

  • Kinney AJ, Jung R, Hermann EM (2001) Cosuppression of the alpha subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies. Plant Cell 13:1165–1178

    Article  CAS  PubMed  Google Scholar 

  • Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17:591–601

    CAS  PubMed  Google Scholar 

  • Kumpatla SP, Teng W, Buchholz WG, Hall TC (1997) Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol 115:361–373

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

    PubMed  Google Scholar 

  • Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA 97:13401–13406

    Article  CAS  PubMed  Google Scholar 

  • Luff B, Pawlowski L, Bender J (1999) An inverted repeat triggers cytosine methylation of identical sequences in Arabidopsis. Mol Cell 3:505–511

    CAS  PubMed  Google Scholar 

  • Marathe R, Smith TH, Anandalakshmi R, Bowman LH, Fagard M, Mourrain P, Vaucheret H, Vance VB (2000) Plant viral suppressors of post-transcriptional gene silencing do not suppress transcriptional silencing. Plant J 22:51–59

    Article  CAS  PubMed  Google Scholar 

  • Matzke AJ, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148

    Google Scholar 

  • Matzke MA, Mette MF, Matzke AJ (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415

    CAS  PubMed  Google Scholar 

  • Matzke MA, Matzke AJ, Kooter JM (2001) RNA: guiding gene silencing. Science 293:1080–1083

    CAS  PubMed  Google Scholar 

  • Mette MF, van der Winden J, Matzke MA, Matzke AJ (1999) Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans. EMBO J 18:241–248

    Article  CAS  PubMed  Google Scholar 

  • Mette MF, Aufastz W, van Der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201

    CAS  PubMed  Google Scholar 

  • Ng H-H, Bird A (1999) DNA methylation and chromatin modification. Curr Opin Genet Dev 9:158–163

    CAS  PubMed  Google Scholar 

  • Pacumbaba RP (1995) Seed transmission of soybean mosaic virus in mottled and nonmottled soybean seeds. Plant Dis 79:193–195

    Google Scholar 

  • Reddy MSS, Ghabrial SA, Redmond CT, Dinkins RD, Collins BG (2001) Resistance to Bean pod mottle virus in transgenic soybean lines expressing the capsid polyprotein. Phytopathology 91:831–838

    CAS  Google Scholar 

  • Sharp PA (2001) RNA interference — 2001. Genes Dev 15:485–490

    CAS  PubMed  Google Scholar 

  • Sijen T, Vijn I, Rebocho A, van Blokland R, Roelofs D, Mol JN, Kooter JM (2001) Transcriptional and posttranscriptional gene silencing are mechanistically related. Curr Biol 11:436–440

    Article  CAS  PubMed  Google Scholar 

  • Stam M, Viterbo A, Mol JN, Kooter JM (1998) 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 18:6165–6177

    CAS  PubMed  Google Scholar 

  • Steimer A, Amedeo P, Afsar K, Fransz P, Scheid OM, Paszkowski J (2000) Endogenous targets of transcriptional gene silencing in Arabidopsis. Plant Cell 12:1165–1178

    Article  CAS  PubMed  Google Scholar 

  • Takano M, Egawa H, Ikeda J-E, Wakasa K (1997) The structure of integration sites in transgenic rice. Plant J 11:353–361

    CAS  PubMed  Google Scholar 

  • Trick HN, Dinkins RD, Santarem ER, Di R, Samoylov V, Meurer CA, Walker DR, Parrott WA, Finer JJ, Collins GB (1997) Recent advances in soybean transformation. Plant Tiss Cult Biotechnol 3:9–26

    Google Scholar 

  • Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants:targets, inducers and regulators. Trends Genet 17:29–35

    Article  CAS  PubMed  Google Scholar 

  • Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17:449–459

    Article  CAS  PubMed  Google Scholar 

  • Wallrath LL (1998) Unfolding the mysteries of heterochromatin. Curr Opin Genet Dev 8:147–153

    CAS  PubMed  Google Scholar 

  • Waterhouse PM, Wang M, Laugh T (2001) Gene silencing as an adaptive defense against viruses. Nature 411:834–842

    Article  CAS  PubMed  Google Scholar 

  • Wu-Scharf D, Jeong B, Zhang C, Cerutti H (2000) Transgene and transposon silencing in Chlamydomonas reinhardtii by DEAH-box RNA helicase. Science 290:1159–1163

    Article  CAS  PubMed  Google Scholar 

  • Yan B, Reddy MSS, Collins GB, Dinkins RD (2000) Agrobacterium tumefacien-mediated transformation of soybean [Glycine max (L.) Merril.] using immature zygotic cotyledon explants. Plant Cell Rep 19:1090–1097

    CAS  Google Scholar 

  • Zamore PD (2001) RNA interference:listening to the sound of silence. Nat Struct Biol 8:746–750

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to thank Carl Redmond and Michael Hayes for valuable technical support, Dr Wayne Parrott and Dr John Finer for providing materials, Dr Said A. Ghabrial for the SMV strain and SMV specific antibodies, Curtis A. Meurer and Kay McAllister for critical evaluation of the manuscript. This research work was supported by funds from the United Soybean Board. This paper (00–06–204) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.

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  1. Department of Agronomy, N109 Agriculture Science Center Building North, University of Kentucky, Lexington, KY 40546–0091, USA

    M. S. Srinivasa Reddy, Randy D. Dinkins & Glenn B. Collins

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  1. M. S. Srinivasa Reddy
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  2. Randy D. Dinkins
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  3. Glenn B. Collins
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Correspondence to Glenn B. Collins.

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Communicated by: G.C. Phillips

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Reddy, M.S.S., Dinkins, R.D. & Collins, G.B. Gene silencing in transgenic soybean plants transformed via particle bombardment. Plant Cell Rep 21, 676–683 (2003). https://doi.org/10.1007/s00299-002-0567-4

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