Detection of siRNA movement in Arabidopsis thaliana using P19 under root cell layer specific expression

We aimed at characterizing the identity of the silencing signal i.e. siRNA or longer dsRNA precursor thereof. We used transgenic dsRNA, endogenous dsRNA or virus infection as sources of siRNAs combined to sophisticated immunoprecipitation-based procedures coupled to deep-sequencing. We found that although they are diluted as they move away from their sites of production, siRNAs population are highly similar between incipient and recipient cells in the three systems. Additionally, a significant depletion of 5’U and 5’A content in the recipient cells suggested Argonaute-loading dependent depletion of these siRNAs during their movement over multiple cell layers. Using the PAZ-domain mutants ago1-18 and ago1-42 confirmed that the observed depletion of mobile siRNAs is the result of RISC loading. The results advocate movement of individual siRNA duplexes as opposed to their precursors and that loading into Argonaut proteins renders those small RNAs cell-autonomous. 8 sample examined: small RNA from input and Immunoprecipitation of Flag-HA-P19 protein expressed under stele (pSHR) or epidermis (pWER) promoter in Arabidopsis thaliana Col0 plants expressing SucSUL inverted repeat under SUC2 promoter (samples 1-4) or infected by TuYV (samples 5-8)