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Figure 3 | Virology Journal

Figure 3

From: 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

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.

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