Laboratory of Biotechnology, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Russia.
Int J Mol Sci. 2022 May 10;23(10):5325. doi: 10.3390/ijms23105325.
Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific -dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water, -dsRNA, or dsRNA. After plant foliar treatments, we detected the emergence of a large number of - and -encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous -dsRNAs were processed into siRNAs and induced RNAi-mediated gene silencing. The analysis showed that gene-specific sRNAs mapped to the and genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies.
最近的研究表明，通过植物叶面处理裸露的双链 RNA（dsRNA）有可能人工诱导 RNA 干扰（RNAi），从而沉默植物真菌病原体中的必需基因或靶向病毒 RNA。此外，几项研究已经记录了通过将裸露的基因特异性 dsRNA 和 siRNA 施加到植物表面来下调植物内源基因的表达。然而，关于外部 dsRNA 应用后 dsRNA 处理和基因沉默机制的研究有限。这些研究将有助于开发用于作物改良和植物功能研究的创新工具。在这项研究中，我们使用外源性基因特异性 dsRNA 下调拟南芥中类黄酮/花青素生物合成途径中的关键酶查尔酮合酶（CHS）的基因。使用编码非相关新霉素磷酸转移酶 II 细菌基因的非特异性 -dsRNA 处理植物，以验证任何观察到的效果和 mRNA 的处理都是序列特异性的。使用高通量小 RNA（sRNA）测序，我们获得了用水、-dsRNA 或 dsRNA 处理的植物的六个 sRNA-seq 文库。在植物叶面处理后，我们检测到大量的 - 和 -编码 sRNAs 的出现，而对照水治疗后则没有这些 sRNAs。因此，外源 -dsRNA 被加工成 siRNA，并诱导 RNAi 介导的基因沉默。分析表明，基因特异性 sRNA 不均匀地映射到基因的和基因上，在特定位置具有峰值读数，主要涉及正义链，并记录了从 17-nt 到 30-nt sRNA 的读数逐渐减少。本研究的结果突出表明，外源 dsRNA 具有很大的潜力，可作为一种有前途的策略，用于诱导基于 RNAi 的植物靶基因下调，以进行植物管理和基因功能研究。
