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Analysis of RNA silencing systemic signal in agroinfected leaves of cocoa (Theobroma cacao L.)

Andrieu A., Lanaud C., Dedieu F., Breitler J.C.. 2006. In : Plant Genomics European Meetings, Venice, 2006. Bologne : Ed. Avenue media, p. 124-124. Plant Genomics European Meetings. 5, 2006-10-11/2006-10-14, Venise (Italie).

RNA interference or post-transcriptional gene silencing is now used as a means to easily manipulate gene expression experimentally and to probe gene function on a whole-genome scale. RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some case of RNA silencing can spread systemically. Because they provide a rapid, versatile and convenient way for achieving a very high level of gene expression in a distinct and defined zone of leaf, Agrobacterium-mediated transient expression systems have been useful for inducing silencing processes and for dissecting systemic silencing signal. The overall objective of this study is to evaluate the feasibility of an agrobinfiltration approach of generation of knock outs mediated by expression of hairpin RNA in cacao leaves after RNA silencing systemic signal production. This approach could be further used as a tool for large-scale discovery and validation of gene function. Based on the Nicotiana benthamiana agroinfiltration method, we developed an efficient and reproducible technique for transient expression of T -DNA vectors in cocoa leaves. Using gene specific tag (GST) from phytoen desaturase gene (pds) and efflux carrier pin-formed gene (pin1), we demonstrated the feasibility of an agroinfiltration method for inducing gene-silencing processes in a defined zone of leaf. SiRNA amplification was demonstrated for the two genes in cocoa-tree, indicating that the RNAi mechanism was initiated in planta. However, the RNA interference spreading throughout the whole plant in cocoa-tree still remains to be demonstrated. (Texte intégral)
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