RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties.     

DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next-generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.     

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.     

Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

RNAi suppressors encoded by pathogenic human viruses

RNA silencing or RNAi interference (RNAi) serves as an innate antiviral mechanism in plants, fungi and animals. Human viruses, like plant viruses, encode suppressor proteins or RNAs that block or modulate the RNAi pathway. This review summarizes the mechanisms by which pathogenic human viruses affect the RNAi pathway. Furthermore, some applications of the viral RNAi suppressor functions and the consequences for antiviral therapeutic strategies that are based on RNAi are discussed.     

miR398在植物逆境胁迫应答中的作用

MicroRNA (miRNA)是一类新型的调控基因表达的小分子RNA, 它作为基因表达的负调控因子, 在转录后水平调节靶基因的表达。miRNA参与调控植物的生长发育, 并在多种非生物与生物胁迫响应中发挥重要作用。miR398是第一个被报道的受氧化胁迫负调控的miRNA。它通过负调控其靶基因Cu/Zn过氧化物歧化酶(Cu/Zn-superoxide dismutase, CSD)的表达, 在多种逆境胁迫响应中扮演重要角色, 如调节铜代谢平衡, 应答重金属、蔗糖、臭氧等非生物胁迫, 以及参与应答生物胁迫等。文章综述了miR398在多种逆境胁迫响应中重要的调节作用及miR398自身的转录调控。     

RNA沉默在植物生物逆境反应中的作用

RNA沉默是真核生物共有的基因表达调节机制和防御机制。在植物RNA沉默中, 一些小RNAs, 如微小 RNAs和小干扰RNAs, 在植物防御病毒、细菌或食草动物的反应中具有重要作用。为了抑制宿主的RNA沉默系统, 植物病毒或细菌进化出了在RNA沉默不同阶段起作用的病毒沉默抑制子或细菌沉默抑制子, 来克服寄主的RNA沉默反应。文章就植物RNA沉默、病毒沉默抑制子、细菌沉默抑制子及其相关防御反应的一些新进展做一概述。     

植物RNA沉默机制的研究进展

RNA沉默是真核生物中普遍存在的抵抗病毒复制表达、抑制转座子转座及调控基因表达的监控机制。植物中的RNA沉默(转录水平的基因沉默PTGS)在RdRP的作用、transitive RNAi的双向延伸、沉默效应的系统性传播等方面与动物中有所不同,且植物中的内源性miRNA在数量和种类上也更具多样性。文中就以上方面及RNA沉默在植物中的应用进行了综述。     

The Ebola virus VP35 protein is a suppressor of RNA silencing

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.