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Analysis of RNA silencing transitivity in agroinfected leaves of rice (Oryza sativa L.)

Breitler J.C., Andrieu A., Sire C., Perin C., Meynard D., Guiderdoni E.. 2006. In : Plant Genomics European Meetings, Venice, 2006. Bologne : Ed. Avenue media, p. 129-129. Plant Genomics European Meetings. 5, 2006-10-11/2006-10-14, Venise (Italie).

RNA silencing is a nucleotide sequence-specific RNA degradation mechanism with the out-standing property of propagating throughout the organism. Here, transitivity and the cell-to-cell movement of RNA silencing in rice are investigated in agroinfected rice leaves and transgenic rice plants. Agrobacterium-mediated transient assays by agroinfiltration have been increasingly employed as a simple and rapid method for assaying the function of gene. However, in cereal species, leaf structure completely prevents infiltration of bacterial solution by pressure. We had developed an efficient method of indica and japonica rice leaves agroinfection based on soaking wounded tissues in bacterial solution at low temperature. Using this original method, we analysed several aspects of RNA silencing by transitory expression of DNA constructs encoding double-stranded RNAs derived from GUS transgene or endogenous PDS and SLR1 gene. The GUS and SLR1 siRNA concentration regularly increased between 2 to 20 days in the agroinfected tissues. Contrastingly, the PDS siRNAs concentration peaked at day 12 and highly decreased to reach a very low level at day 20. Whatever the hpRNA construct used, no detectable levels of siRNAs could be observed in the adjacent part of agroinfected leaf suggesting a very low or null siRNA movement in the rice leaf. Using probes located outside dsRNA trigger sequence, we demonstrated de novo synthesis of SLR1 and GUS secondary siRNAs produced by transitivity in agroinfected tissues, which belong exclusively to the 21-nt size class. Same results were obtained after analysis of transgenic rice expressing constitutively the GUS, PDS and SLR1 hpRNA constructs. These results are discussed in view of the current models of RNA silencing. (Texte intégral)
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