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

Abstract

Background: 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).

Findings: 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.

Conclusions: 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.

Figure 1

Virus induced RNA silencing (VIGS) (a) Construction of PVX/NbRBR1-GFP and PVX/NbPCNA-GFP in PVX/GFP. The PVX genome organisation is shown to encode the RNA-dependent RNA polymerase (RDRP), the triple-gene block (25 K, 12 K and 8 K) and the capsid protein (CP) as well as an insertion of gene of interest (GOI). (b-e) VIGS phenotypes of transgenic Nicotiana benthamiana line pOri2 plants. Plants (25 days old) were mock-inoculated (b) or inoculated with PVX/GFP (c), PVX/NbRBR1-GFP (d) or PVX/NbPCNA-GFP (e). PVX/GFP infected plant showed mosaic and chlorosis at 10 days post-inoculation (dpi) whilst mock inoculated plant remained healthy. Plants challenged with either PVX/NbRBR1-GFP or PVX/NbPCNA-GFP showed local and systemic infection at early stage (7-14 dpi) of viral invasion, but later on (approximately 21 dpi onwards) newly growing leaves showed no viral symptoms. VIGS of NbRBR1 or NbPCNA caused abnormal leaf development and growth retardation. Plants (50 days old) were photographed at 25 dpi. The boxed portions of plants are enlarged to show VIGS-related phenotypes. Leaves of plants with mock inoculation (b) or infected with PVX/GFP (c) at the same developmental stage as these boxed leaves are indicated with an arrow. There leaves were infiltrated with agrobacterium and subsequently used for sqRT-PCR and PCR (see Figures 2 and 3).

Figure 2

VIGS reduction of NbRBR1 and NbPCNA expression (a) Semi-quantitative reverse transcription PCR (sqRT-PCR) assays of NbRBR1 mRNA levels in transgenic Nicotinana benthamiana line pOri-2 plants inoculated with PVX/GFP (GFP) or PVX/NbRBR1-GFP (NbRBR1-GFP, P1-P4 for four individual plants). All plants were at the age of 56 days. (b) SqRT-PCR assays of NbPCNA mRNA levels in healthy pOri2 plant (mock), or plants inoculated with PVX/GFP (GFP), PVX/NbRRB1-GFP (NbRBR1-GFP), or PVX/NbPCNA-GFP (NbPCNA-GFP). SqRT-PCR of 18S rRNA indicates similar amount of total RNAs used in each sample. The positions and sizes of 1-kb DNA marker (Ladder) and positions of RT-PCR products specific for NbRBR1 and NbPCNA mRNA as well as 18S rRNA are indicated.

Figure 3

VIGS of NbRBR1 and NbPCNA affects episiomal DNA replication (a) Outline of the trans-DNA replication system in the transgenic pOri2 line [5,6] . The replication origin (Ori) of the African cassava mosaic virus (ACMV) and the P1/P2 primers for PCR detection of the circular episomal replicon in the presence of ACMV Rep are indicated. (b) Constructs for pGreen0029/AC1234, pGreen0029/AC1m2m3m4 and pGreen0029/ACm1m2m3m4 expressing the ACMV complementary-sense genes (CSG, AC1 - AC4], AC1 alone or no gene under the control of the CaMV 35S promoter and polyA signal. Mutated genes are indicated by asterisks. (c) PCR assays for circular episomal replica in young N. benthamiana line pOri-2 plants at the age of 31 days. In three separate experiments, young growing leaves were infiltrated with Agrobacteria carrying pGreen0029/AC1234 (AC1234), pGreen0029/AC1m2m3m4 (AC1m2m3m4), or pGreen0029/ACm1m2m3m4 (ACm1m2m3m4). (d) Impact of VIGS-mediated reduction of NbRBR1 or NbPCNA expression on viral replication. In each of three experiments, line pOri2 plants (25 days old) were mock-inoculated (Mock), or inoculated with PVX/GFP (GFP, two plants P1-P2), PVX/NbRBR1-GFP (NbRBR1, four plants P1-P4) or PVX/NbPCNA-GFP (NbPCNA, two plants P1-P2). At 25 days post-inoculation, leaves of these plants (50 days old) as indicated in Figure 1 were subsequently infiltrated with Agrabacteria carrying pGreen0029/AC1234 and analysed by sqRT-PCR (see Figure 2) and PCR 6 days later. The positions and sizes of 1-kb DNA marker (Ladder) are indicated.