Abstract：
Artificial small RNAs (art-sRNAs) are 21-nucleotide small RNAs (sRNAs) computationally designed to silence plant genes or pathogenic RNAs with high efficacy and specificity. They are typically produced in transgenic plants to induce silencing at the whole-organism level, although their expression in selected tissues for inactivating genes in distal tissues has not been reported. Here, art-sRNAs designed against the magnesium chelatase subunit CHLI-encoding SULFUR gene (NbSu) were agroinfiltrated in Nicotiana benthamiana leaves, and the induction of local and systemic silencing was analyzed phenotypically by monitoring the appearance of the characteristic bleached phenotype, as well as molecularly by analyzing art-sRNA processing, accumulation and targeting activity and efficacy. We found that the two classes of art-sRNAs, artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are able to induce systemic silencing of NbSu, which requires high art-sRNA expression in the vicinity of the leaf petiole but is independent on the production of secondary sRNAs from NbSu mRNAs. Moreover, we revealed that 21-nucleotide amiRNA and syn-tasiRNA duplexes, and not their precursors, are the molecules moving between cells and through the phloem to systemically silence NbSu in upper leaves. In sum, our results indicate that 21-nucleotide art-sRNAs can move throughout the plant to silence plant genes in tissues different from where they are produced. This highlights the biotechnological potential of art-sRNAs, which might be applied locally for triggering whole-plant and highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops. The present study demonstrates that artificial small RNAs, such as artificial microRNAs and synthetic trans-acting small interfering RNAs, can move long distances in plants as 21-nucleotide duplexes, specifically silencing endogenous genes in tissues different from where they are applied. This highlights the biotechnological potential of artificial small RNAs, which might be applied locally for triggering whole-plant, highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops.
摘要：
人工小RNA（art-sRNA）是计算设计的21个核苷酸的小RNA（sRNA），以高效和特异性沉默植物基因或致病性RNA。它们通常在转基因植物中产生，以在整个生物体水平上诱导沉默，尽管它们在所选组织中的表达使远端组织中的基因失活尚未报道。这里,针对镁螯合酶亚基CHLI编码SULFUR基因（NbSu）设计的art-sRNAs被农业浸润在烟草叶片中，通过监测特征性漂白表型的出现，对局部和系统沉默的诱导进行了表型分析，以及通过分析Art-sRNA加工的分子，积累和靶向活性和功效。我们发现两类art-sRNAs，人工microRNAs（amiRNAs）和合成反式作用小干扰RNAs（syn-tasiRNAs），能够诱导NbSu的系统性沉默，这需要在叶柄附近的高art-sRNA表达，但与NbSumRNA的次级sRNA产生无关。此外，我们揭示了21个核苷酸的amiRNA和syn-tasiRNA双链体，而不是它们的前体，是分子在细胞之间移动并通过韧皮部系统地沉默上部叶片中的NbSu。总之,我们的结果表明，21个核苷酸的art-sRNAs可以在整个植物中移动，以沉默与产生它们的地方不同的组织中的植物基因。这凸显了art-sRNAs的生物技术潜力，这可能是局部应用于触发整个植物和高度特异性沉默，以调节基因表达或诱导对下一代作物致病性RNA的抗性。本研究表明，人工小RNA，如人工microRNA和合成反式小干扰RNA，可以作为21个核苷酸的双链体在植物中长距离移动，在与应用它们的地方不同的组织中特异性沉默内源基因。这凸显了人造小RNA的生物技术潜力，这可能会在当地应用于触发整个工厂，在下一代作物中调节基因表达或诱导对致病性RNA的抗性。
