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.     

Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross‐linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host‐induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae. When aphids were fed on transgenic barley expressing shp double‐stranded RNA (shp‐dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild‐type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2 weeks, respectively, on wild‐type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild‐type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S. avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

Transgenic cassava resistance to <Emphasis Type="Italic">African cassava mosaic virus</Emphasis> is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21–24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.     

Structural insights into the arms race between host and virus along RNA silencing pathways in Arabidopsis thaliana

RNA silencing refers to a conserved sequence‐specific gene‐regulation mechanism mediated by small RNA molecules. In plants, microRNA (miRNA) and small interfering RNA (siRNA) represent two major types of small RNA molecules which play pivotal roles in plant developmental control and antiviral defences. To escape these plant defences, plant viruses have encoded a vast array of viral suppressors of RNA silencing (VSRs) to attack the host antiviral silencing pathway by interfering with small RNA processing, RNA‐induced silencing complex (RISC) assembly, viral mRNA cleavage etc. Transgenic plants expressing distinct VSRs often show developmental aberrations that resemble the phenotype of miRNA‐deficient mutants, implying a potential intrinsic link between VSRs and the miRNA pathway (at least in Arabidopsis thaliana) even though their pathogenic mechanisms remain largely unknown. In this review, we summarise our current structural understandings of the arms race between the host and virus along the RNA silencing pathway in A. thaliana by focusing on several important ribonucleoprotein (RNP) structures involved in RNA silencing and unique structural features adopted by VSRs.     

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.     

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.