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

SUMMARY In RNA interference (RNAi), small interfering RNAs (siRNAs) produced 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 advances in our understanding of the underlying mechanisms, 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 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, localized PD occlusion in source leaves' companion cells (CCs) suffices 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. Significance Statement Despite some major advances, many questions remain regarding how siRNA‐mediated RNAi moves in plants. Here, we show (i) that temporal plasmodesmata closure within companion cells (CCs) suffices to abrogate CC‐triggered systemic RNAi in whole transgenic Arabidopsis, (ii) that ARGONAUTE‐mediated siRNA consumption underlies size selectivity in long‐distance RNAi vascular movement, and (iii) that stress application can overcome biases and previously noted inconsistencies between experimental settings, thereby enabling discovery of mobile endo‐siRNA functions.