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A high-titer, synchronized PVX infection on leaves of untransformed N. benthamiana was initiated by infiltration of single leaves with A. tumefaciens containing a binary plasmid incorporating a 35S-PVX-GFP sequence. Once transcribed, the PVX RNA replicon is independent of the 35S-PVX-GFP DNA, replicates to high levels, and moves systemically through the plant. The A. tumefaciens does not spread beyond the infiltrated patch and is not present in systemic leaves (20). The GFP reporter in the virus was used to allow visual monitoring of infection progress. We have obtained similar signals with wild-type PVX inoculated as virions in sap taken from an infected plant.
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The other examples of PTGS tested were in N. benthamiana (spontaneous silencing of a 35S-GFP transgene), tomato (35S-ACO containing an internal direct and inverted repeat), petunia (cosuppression of chalcone synthase transgenes and endogenes), and Arabidopsis thaliana (PTGS of 35S-GFP by a 35S-PVX-GFP transgene).
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We thank D. Grierson, C. DeLong, H. Vaucheret, and R. Hellens for transgenic plants. We are also grateful to O. Voinnet, D. Bradley, A. Bendahmane, and F. Ratcliff for helpful comments and suggestions. This work was carried out under M.A.F.F. licence PHL 24A/2921. Funded by the Biotechnology and Biological Sciences Research Council and the Gatsby Charitable Foundation.
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