Arabidopsis thaliana small RNA from epidermis and vasculature tissues of Col-0 and ago1 mutants leaves

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. Overall design: We examined small RNA from vasculature and epidermis tissus from Arabidopsis thaliana Col0 WT leaves as well as ago1-18 and ago1-42 mutants (3 replicates for each)