Studying the intercellular movement of RNA silencing signals in Arabidopsis embryos

NICOLÁS CARLOTTO; KEN KOBAYASHI; NICOLÁS FURMAN

Berlin

Workshop; 2017 EMBO Workshop of Intercellular communication in development and disease; 2017

European Molecular Biology Organization

RNA silencing is a crucial mechanism during plant development and pathogen/stressresponses. It consists of a sequence-specific negative regulation of gene expressionand can be triggered by several types of RNA molecules (lncRNA, hairpin RNA,dsRNA; Ariel et al., 2015). RNA silencing signals are non-cell autonomous whichresults in a local and/or systemic propagation in the plant. Previously, RNA silencingspreading pattern was studied in Arabidopsis thaliana embryos (Kobayashi andZambryski, 2007), by expressing ectopically hairpin RNA sequences againstendogenous chlorin subunit of magnesium chelatase mRNA (AT4G18480), whichcould be visualized as a disappearance of chlorophyll in silenced regions. By using anauxin-induced DR5 promoter , this work demonstrated RNA silencing is functional inArabidopsis embryos and can propagate from cotyledon and radicle tips toneighboring cells. Intriguingly, silencing signals originated at the tip of the radicle canpropagate throughout the hypocotyl until a sharp border located just below the SAM(shoot apical meristem), suggesting a potential symplastic transport barrier in thatregion.We aim to characterize if this and other potential symplastic barriers to the propagationof silencing signals exist. If so, do they work in both directions of the movement (root toshoot and vice versa)? Do they depend on the distance of silencing triggering placewithin the embryo?To answer these questions, we engaged in inducing RNA silencing in differentregions of the embryo. For this, we developed a transgenic expression system basedon Jim Haseloff´s GAL4-VP16 transcriptional activator and yeast Upstream ActivatingSequence (UAS12X), to promote the expression of the CHLI hairpin in a tissuespecific manner. After confirming the functionality of our expression system inNicotiana benthamiana leaves and Arabidopsis transgenic lines, selected Haseloff´s?enhancer trap? lines(http://www.haseloff-lab.org/tools/gal4system/page138.html) were crossed to our UASCHLI:hairpin lines. A potential symplastic domains map for RNA silencing inArabidopsis embryo will be presented and compared to those reported for proteintransport, and their potential relevance during embryo development will be discussed.Ariel F., Romero-Barrios N., Jégu T., Benhamed M. and Crespi M. (2015) Battles andhijacks: noncoding transcription in plants. Trends in Plant Science, 20, 6: 362-371,ISSN 1360-1385, http://dx.doi.org/10.1016/j.tplants.2015.03.003.Kobayashi, K. and Zambryski, P. (2007), RNA silencing and its cell-to-cell spreadduring Arabidopsis embryogenesis. The Plant Journal, 50: 597?604.doi:10.1111/j.1365-313X.2007.03073.x