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

In RNA interference (RNAi), small-interfering (si)RNAs processed from double-stranded RNA guide ARGONAUTE(AGO) proteins to silence sequence-complementary RNA/DNA. RNAi can propagate locally and systemically in plants, but despite recent mechanistic advances, basic questions remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata(PDs), 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 apparently recovered in RNAi-recipient tissues, yet only under some experimental settings. Shootward movement of endogenous RNAi in micro-grafted Arabidopsis is also yet-to-be achieved, while potential endogenous functions of mobile RNAi remain scarcely documented. Here, we show i) that temporal, localised PD occlusion in source leaves' companion-cells(CCs) suffice to abrogate all systemic manifestations of CC-activated mobile transgene silencing, including in sink leaves; ii) that the presence or absence of specific AGOs in incipient/traversed/recipient tissues, likely explains the apparent siRNA-length selectivity observed upon vascular movement; iii) that stress enhancement allows endo-siRNAs of a single inverted-repeat (IR) locus to translocate against the shoot-to-root phloem flow, and iv) that mobile endo-siRNAs generated from this 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 mobile endo-siRNA research.