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RNA INTERFERENCE IN INSECT PEST MANAGEMENT
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RNAi Insect Pest Control
1.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 1 RNA INTERFERENCE IN INSECT PEST MANAGEMENT Term-Paper Presentation on ENT-606, INSECT BIOCHEMISTRY Presented by Course Leader Devendra K. Meena Dr. Vinay Kalia Division of Entomology Indian Agricultural Research Institute New Delhi- 110012
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Default! A Free sample background from www.awesomebackgrounds.com Slide 2 Contents RNAI (RNA INTERFERENCE) Discovery of RNAi Small RNA molecules are central to RNA interference siRNA Pathways miRNA Pathways Transport of RNAi information Mode of dsRNA delivery methods Challenges for RNAi for successful control of insect control strategy Overview of studies on the use of plant-RNAi against different insect Applications in pest management Conclusion References
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Default! A Free sample background from www.awesomebackgrounds.com Slide 3 RNAi is a conserved mechanism for post transcriptional sequence specific gene silencing and it is initiated by the introduction of double stranded RNA (dsRNA) (Zotti et al., 2018). Plays a natural role in suppression of transposon activity, resistance to viral infection, post transcriptional and post translational regulation of gene expression and epigenetic regulation of chromatin structure. It was also known as co-suppression, post-transcriptional gene silencing (PTGS), and quelling. RNAI (RNA INTERFERENCE)
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Default! A Free sample background from www.awesomebackgrounds.com Slide 4 Discovery of RNA Interference Chalcone synthase (sense strand) Phenomenon of co-suppression for Chalcone Synthase- A (CHS-A) gene in Petunia (Rich Jorgensen, 1990) Quelling in Neurospora crassa (Cogoni & Macino, 1992) par-1 Caenorhabditis elegans (Guo et al.,1995) Twitching in C. elegans due to defective muscle gene (Fire and Mello, 1998)
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Default! A Free sample background from www.awesomebackgrounds.com Slide 5 The advent of RNAi Andrew Z. Fire and Craig C. Mello were awarded by the Nobel Prize in Physiology or Medicine in 2006 Working on the nematode Caenorhabditis elegans, Fire and Mello showed that exogenous double- stranded RNA triggered gene silencing, thus, they discovered RNAi.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 6 Small RNA molecules are central to RNA interference RNAi can be sorted into three pathways depending on the type of small RNA biogenesis and the Argonaute protein involved: micro RNA (miRNA), small interfering RNA (siRNA) and piwi-interacting RNA pathways. Among them, the siRNA pathway has shown the most potential in insect pest control. siRNAs – 21-25 nt in length, formed by cleavage of long dsRNAs (Dicer) miRNAs – 21-23 nt in length encoded by specific genes & repress mRNA translation/degradation. Processed from long ssRNA sequences that fold into hairpin structures (Drosha & Dicer) piRNAs – 20-30 nt generated from long ssRNA precursors, associate PIWI proteins, important for germ cell development
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Default! A Free sample background from www.awesomebackgrounds.com Slide 7 I. dsRNA enter into RNAi pathway II. Dicer proteins convert these dsRNA into small RNA molecules (21-25nt) known as siRNAs small interfering RNA’s III. These siRNA then assemble into a complex called RNA induced silencing complex known as (RISC), It starts the unwinding IV. The siRNA strands then recognize the complementary sequences on a particular mRNA and bind to them V. Cleavage of mRNA at the points where siRNA binds. siRNA Pathways
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Default! A Free sample background from www.awesomebackgrounds.com Slide 8 miRNA pathway pri-miRNAs are transcribed from the genome. Drosha endonuclease cleaves pri- miRNAs into approximately 70 nt precursor miRNAs which are then transported from the nucleus to the cytoplasm by Exportin-5 (XPO5) via the nuclear pore complex. further cleaved by Dicer into mature 21–23 nt miRNA fragments. Once the strands separate, the guide strand is loaded into the RISC complex (AGO and different cofactors) to scan the transcriptome for partial complementary target transcripts.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 9 Transport of RNAi information Two types of RNAi response can be defined (Whangbo and Hunter, 2008)
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Default! A Free sample background from www.awesomebackgrounds.com Slide 10 1. Cell autonomous: the silencing effect is encompassed within the cells where dsRNA is constitutively expressed or exogenously introduced. 2. Non- cell autonomous: the interfering take place in tissue/cells different from the site of replication or production of the dsRNA. Environmental: silencing signal is directly picked up by cells from the immediate environment, such as gut or hemocoel. Systemic: the silencing signal spreads to neighboring cells from an epicenter of cells.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 11 Mode of dsRNA delivery methods Soaking or incubation Spraying Injection Transgenic plants Coating leaf disc Floating leaf disc
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Default! A Free sample background from www.awesomebackgrounds.com Slide 12 Challenges for RNAi for successful control of insect control strategy 1. Insect Nucleases 2. Gut pH 3. dsRNA length 4. Delivery method 5.Off target effects 6.RNA concentration 7.dsRNA availability 8.Target genes 9.dsRNA resistance 10.Sequence Polymorphism 11.Variation in RNAi effects 12.Life stage of target insect
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Default! A Free sample background from www.awesomebackgrounds.com Slide 13 Overview of studies on the use of plant-RNAi against different insect
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Default! A Free sample background from www.awesomebackgrounds.com Slide 14 Soon after RNAi methods were developed, two groups employed this technology to express dsRNA targeting insect genes in plants and showed that the expression of target genes in insects feeding on transgenic plants containing dsRNA was reduced and affected survival of target insect pests on transgenic plants. One reason could be that there is not much interest in developing GM crops expressing dsRNA to control insect pests because of public hesitance in adoption of transgenic food. In addition, methods for making transgenic plants are available for only a few commercial crops; therefore, alternative methods of production and delivery of dsRNA are being explored Application of RNAi in Pest Management 1. Controlling insect pests
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Default! A Free sample background from www.awesomebackgrounds.com Slide 15 Recent studies in Colorado potato beetle, tephritid fruit fly and beet armyworm showed that the efficient induction of RNAi when bacteria expressed dsRNA was fed to these insects suggesting that using bacteria to deliver dsRNA is a possible method that could be used for the management of pests. The cost per application is much lower for bacterially expressed dsRNA when compared with in vitro synthesized dsRNA.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 16 2. Controlling pathogens that affect beneficial insects Feeding or injection of dsRNA targeting specific genes in the Israeli acute paralysis virus that infects honey bees caused an efficient knockdown in the expression of target genes, and resulted in a reduction in the virus disease. Recently, a dsRNA product Remebee -1 from Beeologics Inc.,Florida, USA was successfully tested in field trails to control Israeli acute paralysis virus infection of honey bees. Bombyx mori nucleo polyhedrovirus (BmNPV) causes devastating viral disease in the silkworm. Transgenic silkworms expressing dsRNA targeting lef-1 and ie1 genes of BmNPV were produced.
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Default! A Free sample background from www.awesomebackgrounds.com Slide 17 3. Blocking disease transmission by insect vectors RNAi methods are also being used to understand host parasite interactions in insects that transmit pathogens of deadly diseases of humans with an ultimate goal of using this information to block transmission of pathogens by insects. Injection of dsRNA into Anopheles gambiae adults caused an efficient and reproducible silencing of defensin gene and showed that defensin plays an important role in mosquito antimicrobial defence against Gram-positive bacteria. 4. Resistance Management When the diamondback moth, Plutella xylostella, was fed synthetic dsRNA against CYP6BG1, expression of the P450 gene was suppressed, resulting in reduced larval resistance to the insecticide permethrin
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Default! A Free sample background from www.awesomebackgrounds.com Slide 18 Advantages of RNAi High molecular potency High level of safety to the non-target organisms Ecofriendly and no residues Cost effective Compatible with other insect pest management approaches (e.g. Bt) Varied modes of delivery viz. spray & transgenic Futuristic total solution for insect pests, virus, bacteria, fungi & nematode
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Default! A Free sample background from www.awesomebackgrounds.com Slide 19 RNAi is innovative tool in genomics and has wide scope in pest control. It is target specific tool. Significant point is that this technology is useful for resistance management by gene silencing. Conclusion
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Default! A Free sample background from www.awesomebackgrounds.com Slide 20 References Niu, J., Taning, C.N., Christiaens, O., Smagghe, G. and Wang, J.J. 2018. Rethink RNAi in Insect Pest Control: Challenges and Perspectives. Advances in Insect Physiology ,Elsevier Ltd ISSN 0065-2806. Rodrigues, T.B. and Figueira, A. 2016. Management of Insect Pest by RNAi — A New Tool for Crop Protection, Intech. Vogel, E., Santos, D., Mingels, L., Verdonckt, T.W. and Broeck, J.B. 2019. RNA Interference in Insects: Protecting Beneficials and Controlling Pests. Frontiers in physiology, 2018.01912.
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