RNA interference in insect pest management (RNAi in ipm)

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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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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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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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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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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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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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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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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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Slide 9
Transport of RNAi information
Two types of RNAi response can be defined (Whangbo and
Hunter, 2008)

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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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Mode of dsRNA delivery methods
Soaking or incubation
Spraying
Injection
Transgenic plants
 Coating leaf disc
 Floating leaf disc

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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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Overview of studies on the use of plant-RNAi against different insect

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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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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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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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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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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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 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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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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