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. 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. 2 samples examined: shoot apex from Arabidopsis thaliana Col0 wild type and ir71 mutant

在RNA干扰（RNA interference, RNAi）过程中，由双链RNA加工生成的小干扰RNA（small-interfering RNAs, siRNAs）引导ARGONAUTE（AGO）蛋白沉默序列互补的RNA/DNA分子。植物RNAi可进行局部与系统性传播，尽管近年来在机制研究上取得了一定进展，但仍有诸多基础问题与技术障碍尚未得到解决。例如，虽推测RNAi通过胞间连丝进行扩散，但目前尚不明确其在植物体内的动态变化与已确立的共质体扩散标记物之间存在何种差异。同样尚未明晰的是，为何仅在部分实验条件下，特定的siRNA种类或其大小亚型会在RNAi受体组织中被检出。此外，为探究RNAi的推测性跨代效应，需在微嫁接拟南芥中研究其向地上部的移动能力，但该尝试此前尚未成功；同时可移动RNAi的内源功能也鲜有报道。本研究聚焦于拟南芥中的非扩增型RNAi，结果显示：（i）尽管转基因RNAi移动属于共质体运输过程，但其仅部分复刻了游离绿色荧光蛋白（free GFP）在植物体内的扩散模式；（ii）初始组织、途经组织以及受体组织中特定AGO蛋白的存在与否，或可解释维管运输过程中观察到的siRNA选择性分选特征；（iii）施加胁迫可使内源siRNA逆向突破茎向根的韧皮部流动进行转运；（iv）由单个反向重复（inverted-repeat, IR）位点产生的可移动内源siRNA，具备调控数百条转录本的潜力。本研究共检测2个样本：拟南芥Col0野生型与ir71突变体的茎尖。