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Volumn 286, Issue 5441, 1999, Pages 950-952

A species of small antisense RNA in posttranscriptional gene silencing in plants

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY RNA;

EID: 0033615491     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5441.950     Document Type: Article
Times cited : (2425)

References (32)
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    • 33P-phosphorylated DNA oligonucleotides run on the same gels but imaged separately. Additionally, samples from different types of PTGS, including those shown, were frequently run on the same gel. Alignment of the filters after hybridization with different specific probes confirmed that the PTGS-specific signals were identical in size. The probes used are in each case sequence specific. We have observed no cross-hybridization between 25-nt signals in different PTGS systems using either filter hybridization or RNAase protection (www.sciencemag.org/feature/data/1042575.shl). We do not have an exact measurement of the amount of 25-nt RNA per cell, but given the short exposure times routinely used to detect these molecules and taking into account their size, they are likely to be abundant in cells exhibiting PTGS.
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    • note
    • The 25-nt ACO antisense signal was completely abolished by pretreatment with either RNAaseONE (Pro-mega) or NaOH.
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    • note
    • 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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    • note
    • 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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    • note
    • 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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