Plant Molecular Biology

, Volume 65, Issue 1–2, pp 163–175 | Cite as

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

  • Chad V. Jordan
  • Wei Shen
  • Linda K. Hanley-Bowdoin
  • Dominique (Niki) Robertson
Article

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.

Keywords

DNA VIGS Retinoblastoma-related protein Cell death Geminivirus Hyperplasia Cell differentiation 

Abbreviations

SU

Sulfur gene

mTGMV

Mutant TGMV

Notes

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).

References

  1. 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 17:5077–5086PubMedGoogle Scholar
  2. 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 78:4817–4826PubMedCrossRefGoogle Scholar
  3. Bass HW, Nagar S, Hanley-Bowdoin L, Robertson D (2000) Chromosome condensation induced by geminivirus infection of mature plant cells. J Cell Sci 113:1149–1160PubMedGoogle Scholar
  4. Bouarab K, Melton R, Peart J, Baulcombe D, Osbourn A (2002) A saponin-detoxifying enzyme mediates suppression of plant defences. Nature 418:889–892PubMedCrossRefGoogle Scholar
  5. Claudio PP, Tonini T, Giordano A (2002) The retinoblastoma family: twins or distant cousins? Genome Biol 3:reviews3012Google Scholar
  6. 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 140:67–80PubMedCrossRefGoogle Scholar
  7. Ebel C, Mariconti L, Gruissem W (2004) Plant retinoblastoma homologues control nuclear proliferation in the female gametophyte. Nature 429:776–780PubMedCrossRefGoogle Scholar
  8. 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 13:1437–1452PubMedCrossRefGoogle Scholar
  9. Gillet G, Brun G (1996) Viral inhibition of apoptosis. Trends Microbiol 4:312–317PubMedCrossRefGoogle Scholar
  10. 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 93:8962–8967PubMedCrossRefGoogle Scholar
  11. 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 53:621–629PubMedCrossRefGoogle Scholar
  12. Hanley-Bowdoin L, Settlage S, Robertson D (2004) Reprogramming plant gene expression—a prerequisite to geminivirus DNA replication. Mol Plant Path 5:149–156CrossRefGoogle Scholar
  13. Hao L, Wang H, Sunter G, Bisaro DM (2003) Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15:1034–1048PubMedCrossRefGoogle Scholar
  14. 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 37:155–169PubMedCrossRefGoogle Scholar
  15. Johnson SA, McCormick S (2001) Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant. Plant Physiol 126:685–695PubMedCrossRefGoogle Scholar
  16. 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 14:91–100PubMedCrossRefGoogle Scholar
  17. 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 19:3485–3495PubMedCrossRefGoogle Scholar
  18. 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 20:3402–3413PubMedCrossRefGoogle Scholar
  19. 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 256:270–279PubMedCrossRefGoogle Scholar
  20. 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 25:402–408PubMedCrossRefGoogle Scholar
  21. Lucy AP, Boulton MI, Davies JW, Maule AJ (1996) Tissue specificity of Zea mays infection by maize streak virus. Mol Plant Microbe Interact 9:22–31Google Scholar
  22. 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 86:797–801PubMedCrossRefGoogle Scholar
  23. 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 18:893–906PubMedCrossRefGoogle Scholar
  24. 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 38:1004–1014PubMedCrossRefGoogle Scholar
  25. 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 7:705–719PubMedCrossRefGoogle Scholar
  26. Nagar S, Hanley-Bowdoin L, Robertson D (2002) Host DNA replication is induced by geminivirus infection of differentiated plant cells. Plant Cell 14:2995–3007PubMedCrossRefGoogle Scholar
  27. 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 4:859–864PubMedCrossRefGoogle Scholar
  28. 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 18:243–252PubMedCrossRefGoogle Scholar
  29. Nevins JR (1992) E2F-a link between the Rb tumor suppressor protein and viral onco proteins. Science 258:424–429PubMedCrossRefGoogle Scholar
  30. 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 275:6114–6122PubMedCrossRefGoogle Scholar
  31. 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 42:153–163PubMedCrossRefGoogle Scholar
  32. 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 27:357–366PubMedCrossRefGoogle Scholar
  33. 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 25:237–245PubMedCrossRefGoogle Scholar
  34. Robertson D (2004) VIGS VECTORS FOR GENE SILENCING: many targets, many tools. Annu Rev Plant Physiol Plant Mol Biol 55:495–519Google Scholar
  35. Sabelli PA, Larkins BA (2006) Grasses like mammals? Redundancy and compensatory regulation within the retinoblastoma protein family. Cell Cycle 5:352–355PubMedGoogle Scholar
  36. Schaeffer GW (1977) Culture and morphoogenetic response of a lethal, chlorophyll-deficient mutant of tobacco to hormones, amino acids and sucrose. In Vitro 13:31–35PubMedCrossRefGoogle Scholar
  37. Schaffer RL, Miller CG, Petty ITD (1995) Virus and host specific adaptations in the BL1 and BR1 genes of bipartite geminiviruses Virology 214:330–338PubMedCrossRefGoogle Scholar
  38. 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 279:570–576PubMedCrossRefGoogle Scholar
  39. 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 93:679–684PubMedCrossRefGoogle Scholar
  40. 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 142:1642–1655PubMedCrossRefGoogle Scholar
  41. 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 7:85–103PubMedCrossRefGoogle Scholar
  42. Tournier B, Tabler M, Kalantidis K (2006) Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J 47:383–394PubMedCrossRefGoogle Scholar
  43. Trimarchi JM, Lees JA (2002) Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 3:11–20PubMedCrossRefGoogle Scholar
  44. 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 123:1337–1349PubMedCrossRefGoogle Scholar
  45. 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 141:1338–1348PubMedCrossRefGoogle Scholar
  46. Yee AS, Shih HH, Tevosian SG (1998) New perspectives on retinoblastoma family functions in differentiation. Front Biosci 3:D532–D547PubMedGoogle Scholar
  47. Zarkowska T, Mittnacht S (1997) Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272:12738–12746PubMedCrossRefGoogle Scholar
  48. Zimmermann P, Hennig L, Gruissem W (2005) Gene-expression analysis and network discovery using Genevestigator. Trends Plant Sci 10:407–409PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Chad V. Jordan
    • 1
  • Wei Shen
    • 2
  • Linda K. Hanley-Bowdoin
    • 2
  • Dominique (Niki) Robertson
    • 1
  1. 1.Department of Plant BiologyNorth Carolina State UniversityRaleighUSA
  2. 2.Department of Molecular and Structural BiochemistryNorth Carolina State UniversityRaleighUSA

Personalised recommendations