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

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 degrees 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.

ArabidopsisRETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

“…The construction of an inducible RBRi system allowed us to reduce RBR levels rapidly and transiently, in the absence of confounding effects that may be associated with downregulation of RBR expression by VIGS (Park et al, 2005;Jordan et al, 2007) or expression of plant virus proteins that interact with RBR (Desvoyes et al, 2006;Lageix et al, 2007). Arabidopsis can recover from transient downregulation of RBR by reconstituting meristems and resuming growth (Figure 1), although certain cell populations that developed during the loss of RBR function may have been irreversibly changed in their differentiation status, causing transient alterations in morphology and anatomy (Figures 4 and 5).…”

“…The inactivation of RBR function by viral proteins or increased E2F/DP expression results in strongly increased DNA endoreduplication (Desvoyes et al, 2006;Jordan et al, 2007), while RBR downregulation by VIGS in tobacco leaves (Park et al, 2005), RNAi in Arabidopsis cell cultures (Hirano et al, 2008), and RNAi in Arabidopsis plants resulted in a similar increase in cells with a 4C DNA content. It is therefore possible that binding of plant viral proteins interferes with a function of RBR in the development-dependent regulation of DNA endoreduplication.…”

“…Previously, virus-induced gene silencing (VIGS) using Tobacco rattle virus (Park et al, 2005) or Tomato golden mosaic virus (Jordan et al, 2007) in Nicotiana benthamiana has been used to downregulate RBR in leaves. Similarly, conditional expression of viral proteins that bind RBR to interfere with RBR function has been reported (Desvoyes et al, 2006;Lageix et al, 2007).…”

“…Similar results were obtained with 5-d-old whole seedlings germinated on plates containing b-estradiol ( Figure 1C). The inducible, rapid, and effective downregulation of RBR transcript and protein using our RBRi system therefore provides a spatially and developmentally more tightly controlled conditional mechanism to specifically interfere with RBR function than RBR Control of Postembryonic Development 1793 virus-induced RBR silencing systems (Park et al, 2005;Jordan et al, 2007). …”

A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

“…These and other studies have shed light on the complicated transcriptional network that is governed by pocket proteins and that regulates various classes of target genes including cell-cycle genes, DNA damage-response genes, differentiation-associated genes, and others (Lavia and Jansen-Durr 1999;Stevaux and Dyson 2002;Cam and Dynlacht 2003;RamirezParra et al 2003;Blais and Dynlacht 2004;Bracken et al 2004;Chaussepied and Ginsberg 2005;Dimova and Dyson 2005;Vandepoele et al 2005). In higher plants the RB-E2F pathway is also critical for cell-cycle progression and development, but much less is known about plant pocket protein complexes and their regulation (Ebel et al 2004;Park et al 2005;Wildwater et al 2005;Wyrzykowska et al 2006;Jordan et al 2007). Thus, despite recent advances many questions about the RB pathway remain open, including the identification of key targets that are important for cell-cycle progression and tumorigenesis (Knudsen and Knudsen 2006).…”