MicroRNA-Induced Gene Silencing (MIGS): A Tool for Multi-Gene Silencing and Targeting Viruses in Plants.

BackgroundMaize dwarf mosaic virus (MDMV), a member of the genus Potyvirus, infects maize and is non-persistently transmitted by aphids. Several plant viruses have been developed as tools for gene expression and gene silencing in plants. The capacity of MDMV for both gene expression and gene silencing were examined.ResultsInfectious clones of an Ohio isolate of MDMV, MDMV OH5, were obtained, and engineered for gene expression only, and for simultaneous marker gene expression and virus-induced gene silencing (VIGS) of three endogenous maize target genes. Single gene expression in single insertion constructs and simultaneous expression of green fluorescent protein (GFP) and silencing of three maize genes in a double insertion construct was demonstrated. Constructs with GFP inserted in the N-terminus of HCPro were more stable than those with insertion at the N-terminus of CP in our study. Unexpectedly, the construct with two insertion sites also retained insertions at a higher rate than single-insertion constructs. Engineered MDMV expression and VIGS constructs were transmissible by aphids (Rhopalosiphum padi).ConclusionsThese results demonstrate that MDMV-based vector can be used as a tool for simultaneous gene expression and multi-gene silencing in maize.

RNA Interference: Big Applause for Silencing in Stockholm

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is one of the widely used functional genomics method for suppressing the gene expression in plants. Initially, gene silencing by RNAi mechanism was believed to be specific requiring sequence homology between siRNA and target mRNA. However, several recent reports have showed that non-specific effects often referred as off-target gene silencing can occur during RNAi. This unintended gene silencing can lead to false conclusions in RNAi experiments that are aimed to study the functional role of a particular target gene in plants. This especially is a major problem in large-scale RNAi-based screens aiming for gene discovery. Hence, understanding the off-target effects is crucial for minimizing such effects to better conclude gene function analyzed by RNAi. We discuss here potential problems of off-target gene silencing and focus on possibilities that favor this effect during post-transcriptional gene silencing. Suggestions to overcome the off-target effects during RNAi studies are also presented. We believe that information available in present-day plant science literature about specificity of siRNA actions is inadequate. In-depth systematic studies to understand their molecular basis are necessary to enable improved design of more specific RNAi vectors. In the meantime, gene function and phenotype results from present-day RNAi studies need to be interpreted with caution.

Virus-mediated reprogramming of gene expression in plants

Combinatorial RNAi: A Winning Strategy for the Race Against Evolving Targets?

RNA interference (RNAi) is an important regulatory approach in functional genomics that has major impact on improvement of crop which allows down-regulation in gene expression. It regulates gene expression by either suppressing transcription or by activating a sequence specific RNA degradation process called post transcriptional gene silencing. RNAi mechanism helps in identification and functional evaluation of thousand of genes within any genome that is responsible for crop improvement. This prognostic approach also reduces the expression of any particular gene through short interfering RNA molecules in any target cell and moreover to appraise the changes that occur in signalling pathways. Recently, RNAi has become a powerful and reliable technique to inhibit the expression of targeted genes and also helps to determine gene loss of function phenotype which leads to gene functional analysis. Pathogens can cause huge damage to crop yield that can have a negative impact on economy and also they are threat to wipe out the entire plant species. This approach has opened new prospects in the development of eco-friendly techniques for plant improvement as specific genes are suppressed which cause stress and expression of novel genes for disease resistance. With the revolution in genetic engineering, biotechnologists were enthusiastic to employ this technology for improved crop quality and its nutritional status. RNAi being a novel approach has great potential to modify the gene expression in plants for better quality traits and nutritional improvement in different crops.

Small RNA-Mediated Epigenetic Myostatin Silencing

RNA gene silencing is a mechanism for gene regulation, which limits transcription level by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a process of degradation of specific RNA sequence (post-transcriptional gene silencing, PTGS), known also as RNA interference (RNAi). RNA interference was observed for the first time by chance during 1990, when in an attempt for over-expression of the chalcone synthase gene in petunias by insertion of its chimeric duplicate, the result was just the opposite – blocking of anthocyanin biosynthesis. After 16 years of experiments, the significance of this phenomenon has grown so much, that a Nobel Prize was awarded to Fire and Mello for its discovery. This chapter presents a retrospection of RNA gene silencing, its mechanism of action, corresponding participants, role in plants, and possible applications with a focus on the perspectives for utilizing this mechanism as a tool for control of viruses in plants.

RNA interference, transcriptional gene silencing, virus induced gene silencing, and micro RNAs comprise a series of mechanisms capable of suppressing gene expression in plants. These mechanisms reveal similar biochemical pathways and appear to be related in several levels. The ability to manipulate gene silencing has produced transgenic plants able to switch off endogenous genes and invading nucleic acids. This powerful biotechnological tool has provided plant breeders and researchers with great opportunity to accelerate breeding programs and developmental studies in woody plants. This research work reports on gene silencing in woody plants, and discuss applications and future perspectives.

Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

Epigenetics in Male Germ Cells

Recently, many small non-coding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become a powerful gene silencing tool in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described first the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural sRNAs in Gram-negative bacteria, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. The atsRNAs led to gene silencing and RNase E dependent degradation of target mRNA. We also designed a series of atsRNAs which targeted the toxic genes in Staphyloccocus aureus, but found no significant interfering effect. We established an effective method for specific gene silencing in Gram-negative bacteria.

RNA interference (RNAi)-based biotechnology has been previously implemented in decapod crustaceans. Unlike traditional RNAi methodologies that investigate single gene silencing, we employed a multigene silencing approach in decapods based on chimeric double-stranded RNA (dsRNA) molecules coined 'gene blocks'. Two dsRNA constructs, each targeting three genes of the crustacean hyperglycaemic hormone (CHH) superfamily of neuropeptides, were produced: Type II construct targeting Cq-Molt-inhibiting hormone 1 (MIH1), Cq-MIH-like 1 (MIHL1), and Cq-MIHL2 isoforms and Type I construct targeting Cq-ion transport peptide (Cq-ITP; a putative hybrid of CHH and MIH) and Cq-CHH and Cq-CHH-like (CHHL) isoforms. Both constructs were injected into juvenile redclaw crayfish, Cherax quadricarinatus, to determine the effects of multigene knockdown on molting and developmental processes. A 20-Hydroxyecdysone (20E) enzyme-linked immunosorbent assay (ELISA) and glucose assay were used to determine the effects of RNAi on molting and hemolymph glycemic activities, respectively. Multigene silencing reduced the intermolt interval by 23%. Statistically significant elevated 20E was recorded in treated intermolt individuals, consistent with the reduced intermolt interval as well as unique and abnormal phenotypes related to the molting process, which indicates a shift in 20E-induced cascade. There was no effect of RNAi treatment on hemolymph glucose level or molt increment. Through multigene silencing and subsequent annotation of gene networks, gene blocks may provide a tailored approach to investigate complex polygenic traits with RNAi in a more efficient and scalable manner.

RNA interference inhibitors were initially discovered in plant viruses, representing a unique mechanism employed by these viruses to counteract host RNA interference. This mechanism has found extensive applications in plant disease resistance breeding and other fields; however, the impact of such interference inhibitors on insect cell RNA interference remains largely unknown. In this study, we screened three distinct interference inhibitors from plant and mammal viruses that act through different mechanisms and systematically investigated their effects on the insect cell cycle and baculovirus infection period at various time intervals. Our findings demonstrated that the viral suppressors of RNA silencing (VSRs) derived from plant and mammal viruses significantly attenuated the RNA interference effect in insect cells, as evidenced by reduced apoptosis rates, altered gene regulation patterns in cells, enhanced expression of exogenous proteins, and improved production efficiency of recombinant virus progeny. Further investigations revealed that the early expression of VSRs yielded superior results compared with late expression during RNA interference processes. Additionally, our results indicated that dsRNA-binding inhibition exhibited more pronounced effects than other modes of action employed by these interference inhibitors. The outcomes presented herein provide novel insights into enhancing defense mechanisms within insect cells using plant and mammal single-stranded RNA virus-derived interference inhibitors and have potential implications for expanding the scope of transformation within insect cell expression systems.