In planta dynamics, transport-biases and endogenous functions of mobile siRNAs in Arabidopsis

Abstract

ABSTRACTIn RNA interference (RNAi), small-interfering (si)RNAs processed from double-stranded RNA guide ARGONAUTE(AGO) proteins to silence sequence-complementary RNA/DNA. Plant RNAi can propagate locally and systemically, but despite recent mechanistic advances, basic questions/hurdles remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata, yet how its dynamics in planta compares with that of established symplastic-diffusion markers remains unknown. Also unknown is why select siRNA species, or size-classes thereof, are recovered in RNAi-recipient tissues, yet only under some experimental settings. Finally, RNAi shootward movement in micro-grafted Arabidopsis – necessary to study its presumptive transgenerational effects– has not been achieved thus far and endogenous functions of mobile RNAi remain scarcely documented.Focusing on non-amplified RNAi in Arabidopsis, we show here that (i) transgenic RNAi-movement, although symplasmic, only partially recapitulates the diffusion pattern of free GFP in planta, (ii) the presence/absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA-selectivity observed during vascular movement, (iii) stress application allows endo-siRNA translocation against the shoot-to-root phloem flow, and (iv) mobile endo-siRNAs generated from a single inverted-repeat(IR) locus, have the potential to regulate hundreds of transcripts.Our results close important knowledge-gaps, rationalize previously-noted inconsistencies between mobile RNAi settings, and provide a framework for functional endo-siRNA studies.