SIGS vs HIGS: a study on the efficacy of two dsRNA delivery strategies to silence Fusarium FgCYP51 genes in infected host and non-host plants

CYP3RNA, a double-stranded (ds)RNA designed to concomitantly target the two sterol 14α-demethylase genes FgCYP51A and FgCYP51B and the fungal virulence factor FgCYP51C, inhibits the growth of the ascomycete fungus Fusarium graminearum (Fg) in vitro and in planta. Here we compare two different methods (setups) of dsRNA delivery, viz. transgene expression (host-induced gene silencing, HIGS) and spray application (spray-induced gene silencing, SIGS), to assess the activity of CYP3RNA and novel dsRNA species designed to target one or two FgCYP51 genes. Using Arabidopsis and barley, we found that dsRNA designed to target two FgCYP51 genes inhibited fungal growth more efficiently than dsRNA targeting a single gene, although both dsRNA species reduced fungal infection. Either dsRNA delivery method reduced fungal growth stronger than anticipated from previous mutational knock-out (KO) strategies, where single gene KO had no significant effect on fungal viability. Consistent with the strong inhibitory effects of the dsRNAs on fungal development in both setups, we detected to a large extent dsRNA-mediated co-silencing of respective non-target FgCYP51 genes. Together, our data further support the valuation that dsRNA applications have an interesting potential for pesticide target validation and gene function studies, apart from their potential for crop protection.     

RNA干涉及其应用前景

RNA干涉是指由特定双链RNA(dsRNA)引起的转录后基因沉默现象。研究表明,Dicer断裂dsRNA产生的小干涉RNA可以抑制哺乳动物体细胞和胚胎中的基因的表达。RdRP在扩增RNAi中起着关键性的作用,RdRP活性复制较长的触发性dsRNA或以一种非引物的方式复制短的siRNA,即以siRNA为引物的RdRP反应使靶mRNA转变为dsRNA,同时复制触发性dsRNA。所有的产物又可作为Dicer的底物,起始RdRP级联反应。本文综述了RNAi可能的作用机制,并对RNAi在分析功能基因组、药物治疗等方面的应用前景进行了展望。     

Analysis of the 3-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3^′-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed “degradative PCR” and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3^′-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

Reduction of Tubulin Expression in Angomonas deanei by RNAi Modifies the Ultrastructure of the Trypanosomatid Protozoan and Impairs Division of Its Endosymbiotic Bacterium

In the last two decades, RNA interference pathways have been employed as a useful tool for reverse genetics in trypanosomatids. Angomonas deanei is a nonpathogenic trypanosomatid that maintains an obligatory endosymbiosis with a bacterium related to the Alcaligenaceae family. Studies of this symbiosis can help us to understand the origin of eukaryotic organelles. The recent elucidation of both the A. deanei and the bacterium symbiont genomes revealed that the host protozoan codes for the enzymes necessary for RNAi activity in trypanosomatids. Here, we tested the functionality of the RNAi machinery by transfecting cells with dsRNA to a reporter gene (green fluorescent protein), which had been previously expressed in the parasite and to α‐tubulin, an endogenous gene. In both cases, protein expression was reduced by the presence of specific dsRNA, inducing, respectively, a decreased GFP fluorescence and the formation of enlarged cells with modified arrangement of subpellicular microtubules. Furthermore, symbiont division was impaired. These results indicate that the RNAi system is active in A. deanei and can be used to further explore gene function in symbiont‐containing trypanosomatids and to clarify important aspects of symbiosis and cell evolution.     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Effects of injecting cadherin gene dsRNA on growth and development in diamondback moth Plutella xylostella (Lep.: Plutellidae)

Two laboratory diamondback moth (DBM) strains were used to study the effects of injecting cadherin gene double‐stranded RNA (dsRNA) on the growth and development of Plutella xylostella (L.). Specifically, the susceptible strain named DBM.1Ac‐S and the low resistant strain DBM.1Ac‐R selected with Cry1Ac toxin were studied. The third larvae of the two strains were injected dsRNA of cadherin gene and their corresponding controls, DBM.1Ac‐RH and DBM.1Ac‐SH, were both injected diethypyrocarbonate (DEPC)‐treated water respectively. The basic biological properties such as death rate, hatching ratio, fecundity, weight of pupa and eclosion rate of the strains mentioned above were likewise studied. Meanwhile, the length and width of the egg and pupa were also measured. The results showed that the cadherin gene dsRNA injection resulted in a significant increase of the death rate and sex ratio. On the other hand, hatching ratio, fecundity, weight of pupa, eclosion rate and adult longevity for male and female of treatments decreased compared to their corresponding controls. As such, there was no significant difference on the length of egg and pupa in between treatments and the corresponding controls. However, their width increased inversely with their corresponding controls. Hence, the results suggest that cadherin gene dsRNA injection retarded the larval growth and development of P. xylostella. Also, these results can help reveal the function of cadherin gene through the RNA interference technique.     

Control of specific gene expression in mammalian cells by co-expression of long complementary RNAs

The use of long double-stranded RNA (dsRNA) for gene silencing in mammalian cells has generally been restricted to embryonic cell types and proposed to induce non-specific effects on gene expression in differentiated cells. In this study, we report that foreign and endogenous gene expression can be regulated in immortalised human cell lines by co-expression of long complementary RNAs with the potential to form dsRNA. The observed gene silencing effect was transferable to recipient control cells, occurred independently of cytoplasmic Dicer and produced an epi-allelic series of clones suitable for gene function studies. This complementary RNA co-expression approach permits the use of long complementary RNAs for regulating specific gene expression in mammalian cells.     

RNA interference‐mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre‐adult life stages

In recent years, RNA interference (RNAi) has been validated as a viable approach for functional genetic studies in non‐model organisms. In this report we demonstrate the efficacy of RNAi in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). A L. lineolaris inhibitor of apoptosis gene (LlIAP) has been identified and cloned. The translated sequence encodes a 381 amino acid protein similar to other insect IAPs and contains two conserved baculovirus inhibitor of apoptosis protein repeat (BIR) domains. Microinjection of double stranded RNA (dsRNA) corresponding to two disparate portions of the gene resulted in decreased LlIAP mRNA quantities relative to controls. Both nymphs and adult specimens injected with IAP dsRNA exhibited significantly reduced lifespan compared with those injected with non‐insect dsRNA (eGFP). Thus, RNAi‐mediated knockdown of LlIAP expression has been correlated with a lethal phenotype in adults and nymphs.