Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants
Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep).
Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect.
These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors.
KeywordsAfrican cassava mosaic virus (ACMV) DNA replication Replication initiator protein (Rep) Retinoblastoma-related protein (RBR) Proliferating cell nuclear antigen (PCNA) Virus-induced RNA silencing (VIGS)
African cassava mosaic virus (ACMV) is a single-stranded (ss) DNA virus in the genus Begomovirus, family Geminiviridae. ACMV possesses two circular DNAs, designated DNA-A and DNA-B of approximately 2.7 kb , both are required for systemic infection of plants . DNA-A and DNA-B share an almost identical common region that contains cis-acting elements required for replication and transcriptional modulation of viral gene expression [3, 4]. The bipartite ACMV genome encodes eight proteins that are responsible for the viral life cycle in and among host plants. The multifunctional replication initiator protein (Rep) is essential for the initiation of rolling circle replication (RCR) of both DNA A and DNA B [5, 6]. Rep also acts as a transcription repressor  and can trigger hypersensitive response and viral resistance in plants [5, 6, 8, 9]. ACMV infection can induce antiviral RNA silencing defence , affect siRNA production , disturb microRNA biosynthesis and cause abnormal developmental phenotypes in plants [12, 13].
Results and discussion
In each of three separate experiments, 3-4 young transgenic N. benthamiana line pOri-2 plants (25 days old) were inoculated with RNA transcripts for PVX/GFP, PVX/NbRBR1-GFP or PVX/NbPCNA-GFP as previously described . The transgenic pOri2 line contains a transgene consisting of a direct repeat of the ACMV replication origin (Ori) flanking a non-viral DNA region as previously described [5, 6]. In contrast to mock-treated controls [plants were inoculated with RNase-free water] (Figure 1b), plants inoculated with recombinant PVX RNAs developed local chlorotic lesions at 4-6 days post-inoculation (dpi). Subsequently, mosaic and chlorosis appeared in the systemic young leaves at approximately 10 dpi, which were maintained in plants infected with PVX/GFP (Figure 1c). By contrast, plants systemically infected with PVX/NbRBR1-GFP or PVX/NbPCNA-GFP started to show recovery from viral infection at approximately 14 dpi and typical PVX symptoms disappeared almost completely at 25 dpi. In contrast, VIGS of NbRBR1 resulted in growth retardation, abnormal leaf development, and newly emerged leaves were irregularly shaped and had a definite curl downwards running the whole circumference of the leaf (Figure 1d). VIGS of NbPCNA also caused stunted growth and distorted leaves. Young leaves growing at the apical meristem were heavily crinkled, curled upwards and rosette in shape (Figure 1e). These phenotypic changes are similar to that previously described for suppression of NbRBR1 or NbPCNA in N. benthamiana using various VIGS systems [14, 15].
Geminiviral Rep is not a DNA polymerase. However, during viral DNA replication the oligomeric Rep protein cleaves the viral replication origin TAATATT↓AC and acts as an ATP-dependent ligase to re-circularise progeny ssDNA [3, 16, 17]. Efficient viral DNA replication is also dependent on functional interplays between Rep and other viral proteins including the replication-enhancing protein and coat protein [18, 19], as well as with host factors such as replication factor C [20, 21]. Moreover, geminiviruses infect terminally differentiated tissues/cells in which host DNA polymerases are not functional. Therefore, to establish infection, geminiviruses need to re-programme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of the cell-cycle repressor RBR protein through direct Rep-RBR interactions and by Rep-triggered induction of host DNA synthesis PCNA expression [22, 23, 24, 25]. It is demonstrated that with the begomovirus Tomato golden mosaic virus, Rep-RBR interaction and PCNA accumulation are important for virus replication and infectivity in N. benthamiana[22, 24, 26]. On the other hand, in mastreviruses, such as Maize streak virus (MSV), an intact Rep RBR-interaction motif is not required for virus replication in culture cells or infectivity in maize, although it is possibly required for wild-type symptom development [27, 28, 29]. Indeed, wild-type MSV invades both vasculature and mesophyll cells of mature maize leaves. In contrast, MSV with a dysfunctional Rep RBR-interaction motif was restricted to the vasculature, in which dividing cells possess the active machinery for DNA synthesis. It is suggested that mature leaves contain high levels of RBR and the MSV Rep-RBR interaction is essential only in tissues with high levels of active RBR . However, prior to Rep-mediated deregulation of cell-cycle control to provide an environment that is able to accommodate replication, the Rep gene must be transcribed from double-stranded (ds) DNA intermediates to express mRNA from which the Rep protein can then be translated. It remains an open question how a geminivirus generates dsDNA from its ssDNA genomes in cells where the replication machinery is inactive.
ACMV Rep was unable to initiate RCR in cells of mature leaves of older pOri2 plants although it was effective in triggering RCR in cells of growing leaves of young plants. It is possible that young growing leaves possess many S-phase cells with an active DNA replication machinery, which ceases to function in cells of older mature leaves. One factor affecting the cellular DNA replication capacity can be different expression levels of genes that encode cell cycle modulators such as RBR1. For instance, there may be low expression of NbRBR1 in S-phase cells of growing leaves of young plants whereas in older leaves high NbRBR1 expression could effectively maintain cells in G1 phase with minimal replication activity. Indeed, in older tissues NbRBR1 mRNA was readily detectable (Figure 2). The ACMV Rep protein can hijack a functional replication apparatus in S-phase cells of young leaves to instigate and trans-replicate the episome (Figure 3a). However, Rep alone, or together with other ACMV CSG products AC2, AC3 and AC4, cannot re-programme a "ceased" replication machinery through its interaction with RBR. On the other hand, it is not surprising that silencing of NbPCNA, an auxiliary protein of DNA polymerase, could not restore the deficiency of DNA replication (Figure 3d). However, reduction of NbRBR1 expression by VIGS may allow G1 cells of older leaves progressing into the S phase. Consequently, DNA synthesis functionality is re-activated and then exploited by ACMV Rep (Figure 3c-d). Thus, our findings suggest that some uncharacterised viral and/or plant factors may participate in reactivating the host cellular machinery for geminiviral DNA replication in terminally differentiated cells, and this process is a more complex one than previously proposed [22, 23, 24, 25, 30]. This idea is supported by the facts that the curtovirus C4 protein can induce plant cell cycle regulator gene expression  and that in fission yeast the ACMV Rep can affect cell division cycle despite no RBR homologue has been identified to date .
We thank D. C. Baulcombe for providing the original PVX vector. This project was supported in part by BBSRC grants (BBS/E/H/00032637; BBS/E/H/00YH221 to Y.H.). G. B. was supported by a BBSRC Studentship (BBS/S/K/2003/10157A to Y.H.). We are grateful to referees for critical comments and reading of the manuscript.
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