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Figure 3. At 4 dpi, a large fraction of eGFP+ transgenic leaves have regenerated. For each time point, two plant samples were taken and analysed by non-targeted RNAseq to determine the relative accumulation of endogenous viral RNA to eGFP+ transgenic RNA. IT, Ion Torrent; NG, Next Generation. Errors are standard deviations from the mean.
By 4 dpi, the population of cells at 16 hpi had undergone extensive growth, and this coincided with the cessation of accumulation of eGFP-specific RNA in comparison to eGFP+ transgenic tissue. The amount eGFP-specific RNA accumulated was significantly lower than that of eGFP+ transgenic RNA. There was also a significant reduction in the level of Accumulation Factor, which was significantly higher for eGFP-specific accumulation than for the accumulation of eGFP+ RNA at 8 and 16 dpi (Figure 3, Table 1). At 4 dpi, the effect of the virus on host genotype was small compared to that present earlier in infection, but by 12 dpi, the magnitude of the difference was similar to its effect on time point 16 (Table 1). By 16 dpi, transgene repression had been reduced to the point that eGFP+ and all eGFP-specific transcripts are present at very similar levels (Figure 4). This might be expected if repression was mediated by the presence of the virus replicase, or the simultaneous accumulation of more vRNA in a single cell.
The difference of accumulation of eGFP-specific RNA and eGFP+ transgenic RNA from 8-16 dpi is a striking example of the timing of a transient repressed transgene accumulation that takes place following transient expression from a single biolistic shot. The spatio-temporal profile of this repression is evident in the accumulating eGFP-specific RNA (Figure 3). d2c66b5586
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