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Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development

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Abstract

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360° without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3–4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

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Abbreviations

SU:

Sulfur gene

mTGMV:

Mutant TGMV

References

  • Ach RA, Durfee T, Miller AB, Taranto P, Hanley-Bowdoin L, Zambryski PC, Gruissem W (1997) RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein. Mol Cell Biol 17:5077–5086

    PubMed  CAS  Google Scholar 

  • Arguello-Astorga G, Lopez-Ochoa L, Kong LJ, Orozco BM, Settlage SB, Hanley-Bowdoin L (2004) A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein. J Virol 78:4817–4826

    Article  PubMed  CAS  Google Scholar 

  • Bass HW, Nagar S, Hanley-Bowdoin L, Robertson D (2000) Chromosome condensation induced by geminivirus infection of mature plant cells. J Cell Sci 113:1149–1160

    PubMed  CAS  Google Scholar 

  • Bouarab K, Melton R, Peart J, Baulcombe D, Osbourn A (2002) A saponin-detoxifying enzyme mediates suppression of plant defences. Nature 418:889–892

    Article  PubMed  CAS  Google Scholar 

  • Claudio PP, Tonini T, Giordano A (2002) The retinoblastoma family: twins or distant cousins? Genome Biol 3:reviews3012

  • Desvoyes B, Ramirez-Parra E, Xie Q, Chua NH, Gutierrez C (2006) Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development. Plant Physiol 140:67–80

    Article  PubMed  CAS  Google Scholar 

  • Ebel C, Mariconti L, Gruissem W (2004) Plant retinoblastoma homologues control nuclear proliferation in the female gametophyte. Nature 429:776–780

    Article  PubMed  CAS  Google Scholar 

  • Egelkrout EM, Robertson D, Hanley-Bowdoin L (2001) Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves. Plant Cell 13:1437–1452

    Article  PubMed  CAS  Google Scholar 

  • Gillet G, Brun G (1996) Viral inhibition of apoptosis. Trends Microbiol 4:312–317

    Article  PubMed  CAS  Google Scholar 

  • Grafi G, Burnett RJ, Helentjaris T, Larkins BA, DeCaprio JA, Sellers WR, Kaelin WG Jr (1996) A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication. Proc Natl Acad Sci USA 93:8962–8967

    Article  PubMed  CAS  Google Scholar 

  • Guivarc’h A, Rembur J, Goetz M, Roitsch T, Noin M, Schmulling T, Chriqui D (2002) Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation. J Exp Bot 53:621–629

    Article  PubMed  CAS  Google Scholar 

  • Hanley-Bowdoin L, Settlage S, Robertson D (2004) Reprogramming plant gene expression—a prerequisite to geminivirus DNA replication. Mol Plant Path 5:149–156

    Article  CAS  Google Scholar 

  • Hao L, Wang H, Sunter G, Bisaro DM (2003) Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15:1034–1048

    Article  PubMed  CAS  Google Scholar 

  • Huntley R, Healy S, Freeman D, Lavender P, de Jager S, Greenwood J, Makker J, Walker E, Jackman M, Xie Q, Bannister AJ, Kouzarides T, Gutierrez C, Doonan JH, Murray JA (1998) The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf development and displays conserved interactions with G1/S regulators and plant cyclin D (CycD) proteins. Plant Mol Biol 37:155–169

    Article  PubMed  CAS  Google Scholar 

  • Johnson SA, McCormick S (2001) Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant. Plant Physiol 126:685–695

    Article  PubMed  CAS  Google Scholar 

  • Kjemtrup S, Sampson KS, Peele CG, Nguyen LV, Conkling MA, Thompson WF, Robertson D (1998) Gene silencing from plant DNA carried by a Geminivirus. Plant J 14:91–100

    Article  PubMed  CAS  Google Scholar 

  • Kong LJ, Orozco BM, Roe JL, Nagar S, Ou S, Feiler HS, Durfee T, Miller AB, Gruissem W, Robertson D, Hanley-Bowdoin L (2000) A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants. Embo J 19:3485–3495

    Article  PubMed  CAS  Google Scholar 

  • Lipinski MM, Macleod KF, Williams BO, Mullaney TL, Crowley D, Jacks T (2001) Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system. Embo J 20:3402–3413

    Article  PubMed  CAS  Google Scholar 

  • Liu L, Saunders K, Thomas CL, Davies JW, Stanley J (1999) Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif. Virology 256:270–279

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Lucy AP, Boulton MI, Davies JW, Maule AJ (1996) Tissue specificity of Zea mays infection by maize streak virus. Mol Plant Microbe Interact 9:22–31

    CAS  Google Scholar 

  • McGivern DR, Findlay KC, Montague NP, Boulton MI (2005) An intact RBR-binding motif is not required for infectivity of Maize streak virus in cereals, but is required for invasion of mesophyll cells. J Gen Virol 86:797–801

    Article  PubMed  CAS  Google Scholar 

  • Menges M, Samland AK, Planchais S, Murray JA (2006) The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis. Plant Cell 18:893–906

    Article  PubMed  CAS  Google Scholar 

  • Muangsan N, Beclin C, Vaucheret H, Robertson D (2004) Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants. Plant J 38:1004–1014

    Article  PubMed  CAS  Google Scholar 

  • Nagar S, Pedersen TJ, Carrick KM, Hanley-Bowdoin L, Robertson D (1995) A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. Plant Cell 7:705–719

    Article  PubMed  CAS  Google Scholar 

  • Nagar S, Hanley-Bowdoin L, Robertson D (2002) Host DNA replication is induced by geminivirus infection of differentiated plant cells. Plant Cell 14:2995–3007

    Article  PubMed  CAS  Google Scholar 

  • Nahle Z, Polakoff J, Davuluri RV, McCurrach ME, Jacobson MD, Narita M, Zhang MQ, Lazebnik Y, Bar-Sagi D, Lowe SW (2002) Direct coupling of the cell cycle and cell death machinery by E2F. Nat Cell Biol 4:859–864

    Article  PubMed  CAS  Google Scholar 

  • Nakagami H, Sekine M, Murakami H, Shinmyo A (1999) Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro. Plant J 18:243–252

    Article  PubMed  CAS  Google Scholar 

  • Nevins JR (1992) E2F-a link between the Rb tumor suppressor protein and viral onco proteins. Science 258:424–429

    Article  PubMed  CAS  Google Scholar 

  • Orozco BM, Kong LJ, Batts LA, Elledge S, Hanley-Bowdoin L (2000) The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties. J Biol Chem 275:6114–6122

    Article  PubMed  CAS  Google Scholar 

  • Park JA, Ahn JW, Kim YK, Kim SJ, Kim JK, Kim WT, Pai HS (2005) Retinoblastoma protein regulates cell proliferation, differentiation, and endoreduplication in plants. Plant J 42:153–163

    Article  PubMed  CAS  Google Scholar 

  • Peele C, Jordan CV, Muangsan N, Turnage M, Egelkrout E, Eagle P, Hanley-Bowdoin L, Robertson D (2001) Silencing of a meristematic gene using geminivirus-derived vectors. Plant J 27:357–366

    Article  PubMed  CAS  Google Scholar 

  • Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245

    Article  PubMed  CAS  Google Scholar 

  • Robertson D (2004) VIGS VECTORS FOR GENE SILENCING: many targets, many tools. Annu Rev Plant Physiol Plant Mol Biol 55:495–519

    CAS  Google Scholar 

  • Sabelli PA, Larkins BA (2006) Grasses like mammals? Redundancy and compensatory regulation within the retinoblastoma protein family. Cell Cycle 5:352–355

    PubMed  CAS  Google Scholar 

  • Schaeffer GW (1977) Culture and morphoogenetic response of a lethal, chlorophyll-deficient mutant of tobacco to hormones, amino acids and sucrose. In Vitro 13:31–35

    Article  PubMed  CAS  Google Scholar 

  • Schaffer RL, Miller CG, Petty ITD (1995) Virus and host specific adaptations in the BL1 and BR1 genes of bipartite geminiviruses Virology 214:330–338

    Article  PubMed  CAS  Google Scholar 

  • Settlage SB, Miller AB, Gruissem W, Hanley-Bowdoin L (2001) Dual interaction of a geminivirus replication accessory factor with a viral replication protein, a plant cell cycle regulator. Virology 279:570–576

    Article  PubMed  CAS  Google Scholar 

  • Shan B, Durfee T, Lee WH (1996) Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis. Proc Natl Acad Sci USA 93:679–684

    Article  PubMed  CAS  Google Scholar 

  • Shen W, Hanley-Bowdoin L (2006) Geminivirus infection up-regulates the expression of two Arabidopsis protein kinases related to yeast SNF1- and mammalian AMPK-activating kinases. Plant Physiol 142:1642–1655

    Article  PubMed  CAS  Google Scholar 

  • Soni R, Carmichael JP, Shah ZH, Murray JAH (1995) A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. Plant Cell 7:85–103

    Article  PubMed  CAS  Google Scholar 

  • Tournier B, Tabler M, Kalantidis K (2006) Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J 47:383–394

    Article  PubMed  CAS  Google Scholar 

  • Trimarchi JM, Lees JA (2002) Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 3:11–20

    Article  PubMed  CAS  Google Scholar 

  • Wildwater M, Campilho A, Perez-Perez JM, Heidstra R, Blilou I, Korthout H, Chatterjee J, Mariconti L, Gruissem W, Scheres B (2005) The retinoblastoma-related gene regulates stem cell maintenance in Arabidopsis roots. Cell 123:1337–1349

    Article  PubMed  CAS  Google Scholar 

  • Wyrzykowska J, Schorderet M, Pien S, Gruissem W, Fleming AJ (2006) Induction of differentiation in the shoot apical meristem by transient overexpression of a retinoblastoma-related protein. Plant Physiol 141:1338–1348

    Article  PubMed  CAS  Google Scholar 

  • Yee AS, Shih HH, Tevosian SG (1998) New perspectives on retinoblastoma family functions in differentiation. Front Biosci 3:D532–D547

    PubMed  CAS  Google Scholar 

  • Zarkowska T, Mittnacht S (1997) Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272:12738–12746

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann P, Hennig L, Gruissem W (2005) Gene-expression analysis and network discovery using Genevestigator. Trends Plant Sci 10:407–409

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Petra Epple of the Jeff Dangl/Sarah Grant Lab in the Biology Department at the University of North Carolina at Chapel Hill for assistance with trypan blue staining. This work was supported by the North Carolina Agriculture Research Service (DR) and by NSF IBN-0235251 (LHB).

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Authors and Affiliations

  1. Department of Plant Biology, North Carolina State University, Box 7612, Raleigh, NC, 27695-7612, USA

    Chad V. Jordan & Dominique (Niki) Robertson

  2. Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA

    Wei Shen & Linda K. Hanley-Bowdoin

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  1. Chad V. Jordan
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  2. Wei Shen
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  3. Linda K. Hanley-Bowdoin
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  4. Dominique (Niki) Robertson
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Correspondence to Dominique (Niki) Robertson.

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Jordan, C.V., Shen, W., Hanley-Bowdoin, L.K. et al. Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development. Plant Mol Biol 65, 163–175 (2007). https://doi.org/10.1007/s11103-007-9206-3

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