Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development
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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.
KeywordsDNA VIGS Retinoblastoma-related protein Cell death Geminivirus Hyperplasia Cell differentiation
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).
- Claudio PP, Tonini T, Giordano A (2002) The retinoblastoma family: twins or distant cousins? Genome Biol 3:reviews3012Google 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 53:621–629PubMedCrossRefGoogle 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 37:155–169PubMedCrossRefGoogle 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 19:3485–3495PubMedCrossRefGoogle Scholar
- Lucy AP, Boulton MI, Davies JW, Maule AJ (1996) Tissue specificity of Zea mays infection by maize streak virus. Mol Plant Microbe Interact 9:22–31Google Scholar
- Robertson D (2004) VIGS VECTORS FOR GENE SILENCING: many targets, many tools. Annu Rev Plant Physiol Plant Mol Biol 55:495–519Google Scholar