Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management

RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle-mediated RNAi, summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management, and finally address the prospects of nanoparticle-based RNA pesticides.     

病虫害RNAi技术中的外源RNA递送策略

病虫害严重威胁着作物安全生产。近年来,在RNA干扰(RNA interference,RNAi)基础上开发病虫害防控策略的研究得到越来越多的关注。RNAi是真核生物体内的一种基因调控过程,如何将外源RNA有效地递送到靶标生物体内,是病虫害RNAi技术能否成功的关键之一。国内外学者进行了大量研究和实践,探究影响病虫害吸收和传递外源双链RNA(double-stranded RNA,dsRNA)的因素,探索提高dsRNA递送效率的方法,取得了重要的进展。本文对相关研究进行了梳理,简述了影响病虫害对dsRNA吸收和递送的因素,对外源RNA的递送策略进行了综述,讨论了纳米颗粒复合物在dsRNA递送中的应用前景,以期为相关研究提供参考。     

A plasmid-based reverse genetics system for animal double-stranded RNA viruses

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.     

红细胞伪装纳米颗粒:一个具有潜力的药物及疫苗递送系统

红细胞伪装纳米颗粒是一种以红细胞或红细胞膜纳米囊泡为载体在体内递送药物、酶、多肽和抗原等物质的系统,具有生物相容性好、循环周期长、靶向性强等优势。本文从红细胞载体的种类、发展历程、递送策略应用以及其局限性和未来的挑战等方面进行了详细阐述,并展望了其未来的发展方向。     

A transgene-based,embryo-specific lethality system for insect pest management

Biological approaches to insect pest management offer alternatives to pesticidal control. In area-wide control programs that cover entire regions, the sterile insect technique (SIT) can be used to successfully suppress economically important pest species by the mass release of sterilized pest organisms. However, conventional sterilization by ionizing radiation reduces insect fitness, which can result in reduced competitiveness of the sterilized insects. Here we report a transgene-based, dominant embryonic lethality system that allows for generation of large quantities of competitive but sterile insects without the need of irradiation. The system involves the ectopic expression of a hyperactive pro-apoptotic gene that causes embryo-specific lethality when driven by the tetracycline-controlled transactivator (tTA) under the regulation of a cellularization gene enhancer-promoter. We have successfully tested this system in Drosophila melanogaster. The embryonic lethality can be suppressed maternally, which will allow it to be combined with transgenic female-specific lethality systems to raise only vigorous but sterile males.     

Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production

ABSTRACT: Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provide significant health benefits and this has led to an increased consumption as dietary supplements. Omega-3 fatty acids EPA and DHA are found in animals, transgenic plants, fungi and many microorganisms but are typically extracted from fatty fish, putting additional pressures on global fish stocks. As primary producers, many marine microalgae are rich in EPA (C20:5) and DHA (C22:6) and present a promising source of omega-3 fatty acids. Several heterotrophic microalgae have been used as biofactories for omega-3 fatty acids commercially, but a strong interest in autotrophic microalgae has emerged in recent years as microalgae are being developed as biofuel crops. This paper provides an overview of microalgal biotechnology and production platforms for the development of omega-3 fatty acids EPA and DHA. It refers to implications in current biotechnological uses of microalgae as aquaculture feed and future biofuel crops and explores potential applications of metabolic engineering and selective breeding to accumulate large amounts of omega-3 fatty acids in autotrophic microalgae.     

A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA.

X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it). X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA. X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance. X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle. This finding confirms our "head-full replication" model originally proposed for M1 and M2. Like M1 or M2, X lowers the copy number of L-A, especially in a ski host. Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.     

A promising biotechnical approach to pest management of Diabrotica virgifera virgifera in Illinois maize fields under kairomonal shielding with the new MSD technique

Environmentally compatible and sustainable plant protection requires novel approaches to pest management characterized by minimal emphasis on toxicants. Classical toxicants traditionally dominated economic entomology for half a century. But worldwide problems with environmental pollution and with increasing resistance levels in all major pesticide classes and in many key insect species including Diabrotica virgifera virgifera (D.v.v.) strongly advocate a rethinking and a change in management paradigms used. Soft, minimally invasive, biological, biotechnical and cultural approaches should replace hard pesticides which are in favor up to now. Fortunately, pheromones, kairomones, plant attractants, better traps, new plant varieties and cultural methods like crop rotation, in short more sophisticated methods are now available as pressure for finding and exploring novel strategies increases. Facing this situation, a new biotechnical approach of population reduction of D.v.v., called "MSD" technique, is introduced. MSD is characterized as an approach combining mass trapping, shielding and deflecting of adult insects along an invisible odor barrier of synthetic kairomone which diminishes the flux of insects across a high capacity trap line baited with kairomone, thus reducing both the population fluctuation and number and its reproductive success within the shielded area. In the case of D.v.v. in Zea mays fields, effects realized by the MSD technique have been measured simultaneously by a number of independent criteria during the summers of 2003 and 2004 at 2 different locations in Illinois maize fields of up to one half hectare size. Results observed are statistically significant and cannot be explained by mass trapping alone. There is also an additional shielding and deflection, in short "diversion" effect whose basic sensory and behavioral mechanisms call for future exploration.     

天敌昆虫控害机制与可持续利用

天敌昆虫是自然生态系统内抑制害虫种群的重要因子,利用天敌昆虫控制农业害虫是安全有效的害虫控制策略,也是未来害虫管理发展的方向.本文在系统总结国内外研究进展的基础上,提出害虫治理要从“被动应急控制”转变为内部助增的“主动促进自然调控”的新理念,创新多种天敌昆虫协同控制多种害虫的“网式协同调控”新途径,建立一个自我维持并可有效降低害虫种群水平的农业生态系统.未来的研究应针对“天敌昆虫调控害虫的内在机制”与“天敌昆虫在农业生态系统中持续发挥作用的生态学基础”等关键科学问题,从基因、个体、种群、群落和生态系统不同层次,重点开展:1)天敌昆虫寄生和捕食害虫的行为与适应机制;2)天敌昆虫大量繁育的营养与生殖生理基础;3)寄生性天敌昆虫与寄主互作的免疫机制;4)天敌昆虫协同控害的生态学机制;5)天敌昆虫可持续利用的生物防治新模式等方面的研究.     

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.     

A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M₁ and M₂ with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.     

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: A promising approach for the treatment of prostate cancer

Background

Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

Methods

Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

Results

Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery.

Conclusion

Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.     

A linear double-stranded RNA in Trichomonas vaginalis

A "double-stranded" RNA was identified in the anaerobic, parasitic protozoan Trichomonas vaginalis. Electron microscopic evidence indicated linear double-stranded structure 1.5 micron in length, with no apparent hairpins or loops. Boiling in 30% dimethyl sulfoxide denatured it into single strands of 1.5 micron and shorter fragments. It consists of 23.4% G, 23.4% C, 23.0% A, and 30.3% U and melts at a transition temperature of 81.7 degrees C in 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 7-15% hyperchromicity. The 32P-labeled double-stranded RNA hybridized specifically with T. vaginalis DNA fragments in a single DNA band from EcoRI digest and two DNA bands from HindIII digest. Of the 33 different strains or isolates of T. vaginalis examined, all contained this double-stranded RNA. However, the only two metronidazole-resistant T. vaginalis strains examined thus far (IR78 and 85) contained no detectable double-stranded RNA, although the corresponding DNA sequence was present. DNA fragments of Escherichia coli and Giardia lamblia did not hybridize with the double-stranded RNA. But DNA fragments of a metronidazole-sensitive Tritrichomonas foetus hybridized specifically with the double-stranded RNA, even though this organism does not contain the double-stranded RNA itself.     

A stepwise model for double-stranded RNA processing by ribonuclease III

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family represented by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the family. Previously, we have shown that RNase III uses two catalytic sites to create the 2-nucleotide (nt) 3' overhangs in its products. Here, we present three crystal structures of RNase III in complex with double-stranded RNA, demonstrating how Mg(2+) is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg(2+) ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modelled a protein-substrate complex and a protein-reaction intermediate (transition state) complex on the basis of the crystal structures. Together, the crystal structures and the models suggest a stepwise mechanism for RNase III to execute the phosphoryl transfer reaction.     

A phagocytic route for uptake of double-stranded RNA in RNAi

RNA interference (RNAi) has a range of physiological functions including as a defence mechanism against viruses. To protect uninfected cells in a multicellular organism, not only a cell-autonomous RNAi response is required but also a systemic one. However, the route of RNA spread in systemic RNAi remains unclear. Here we show that phagocytosis can be a route for double-stranded RNA uptake. Double-stranded RNA expressed in Escherichia coli induces robust RNAi in Drosophila S2 cells, with effectiveness comparable to that of naked dsRNA. We could separate this phagocytic uptake route from that for RNAi induced by naked dsRNA. Therefore, phagocytic uptake of dsRNA offers a potential route for systemic spread of RNAi.     

Environmentally sustainable pest control options for Drosophila suzukii

The spotted‐wing drosophila or cherry vinegar fly (Drosophila suzukii) is native to Asia but has invaded other continents since 2008 and has spread throughout Europe. The females have a serrated ovipositor allowing them to penetrate the skins of intact ripening fruits to deposit their eggs, and the developing larvae rapidly destroy the fruits close to harvest. Drosophila suzukii has a rapid life cycle and the larvae develop well beneath the fruit surface. This means that the use of pesticides is problematic and often not effective, first due to their restricted use close to harvest to protect consumers, and second because the larvae are deep enough inside the fruit to avoid contact. There are currently no effective and environmentally sustainable pest control methods for this species, resulting in extensive damage to fruit crops. Here, we review the current status of D. suzukii as a fruit crop pest and discuss the feasibility of current pesticide‐free control methods. We also consider the potential of new technologies as a basis for the urgently needed specific and long‐term control of this species.