RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas.

1. RNA interference
Technology in crop
Improvement
Presented by:
J. KANMANI BHARATHI
II M.Sc., (Plant molecular Biology and Biotechnology

2. OUTLINE
INTRODUCTION
WHAT? RNAi PATHWAY
RNAi MEACHANISUMS
PROCESS OF RNAi
INTRODUCE
HOW ?
RNAi IN CROP
IMPROVEMENT
CHALLENGES
WHY RNAi IS
NEEDED
WHY

3. RNA Interference -- One of the gene silencing technology
 Plants - Co suppresser or Post transcriptional gene silencing (PTGS)
 Fungi - Quelling
 Animals - RNA Interference
Introduction
Several terms to describe

4. Definition
RNAi can be defined as the ability of exogenous or endogenous
double stranded RNA to suppress the expression of the gene
which corresponds to the sequence of double stranded RNA.
RNA interference (RNAi) is a method of blocking gene
function by inserting short sequences of ribonucleic acid
(RNA) that match part of the target gene’s sequence, thus no
proteins are produced.
RNA interference (RNAi) is a biological process in which RNA
molecules inhibit gene expression or translation, by
neutralizing targeted mRNA molecules.

5. 1990
Co-Suppressing
in Petunias
1992
Quelling
In Neurospora crassa
1998
RNAi in
Caenorhabditis elegans
2001
siRNA in
Mammalian cells
2002
Science named
RNAi Technology
RNAi T I M E L I N E
RNAi in Drosophila
2003
RNAi Mechanism
2005
Nobel Prize
Fire & Mello
2006 Artificial miRNA
2020
Now I presenting RNAi

6. Noble prize winners in C.elegans field
Craig Mello
John Sulston
Andrew Fire
Sydney Brenner
Robert Horvitz

7. RNAi Pathways
shRNA
siRNA
microRNA
small non-coding RNA
molecule functions in RNA
silencing and post-
transcriptional regulation.
Small (short) interfering RNA
is a class of double-stranded
non-coding RNA It interferes
with the expression of specific
genes with complementary
nucleotide sequences by
degrading mRNA after
transcription, preventing
translation.
Short (small) hairpin RNA
is an artificial RNA molecule with a
tight hairpin turn that can be used to
silence target gene expression.
Expression of shRNA in cells is
accomplished by plasmids / viral /
bacterial vectors.
20 - 22 bp
20 – 27 bp
22 – 25 bp
miRNA

8. E.g. For Some RNAi

9. RNAi machinery
03
01
04
02
DROSHA AGO
DICER RdRP
RICS

10. DROSHA
This gene encodes dsRNA specific ribonuclease & subunit of the microprocessor
protein complex, which catalyzes the initial processing of miRNA synthesis. The
encoded protein cleaves the stem loop structure from the pri-miRNA in the nucleus,
yielding the pre-miRNA
Drosha is a Class 2 ribonuclease III
enzyme
RNA-specific endoribonucleases participate in diverse
RNA maturation and decay pathways

11. DICER
 Belongs under RNase III endonuclease family
 Dicer cleaves dsRNA and pre-miRNA into
ssRNA
 ATP independent cutting mechanism
 Initiate RNAi
 cleavage – 2 nt overhangs at each 3′ end
 Dicer facilitates the activation of the RNA-induced silencing
complex (RISC)
Functional domains in dicer
 Helicase
 PAZ domain
 Tandem RNase III
domains
 dsRNA binding domain (dRBD)

12. ARGONAUTE
Once the Argonaute is associated with the small RNA, the enzymatic activity conferred by
the PIWI domain cleaves only the passenger strand of the small interfering RNA.
This protein family plays a central role in RNA silencing processes. Homology seeking
Essential components (active part) of the RNA-induced silencing complex (RISC)
Argonaute proteins bind different classes of small non-coding RNAs, including miRNA, siRNA,
shRNA
Small RNAs guide Argonaute proteins to their specific targets through sequence
complementarity, which then leads to mRNA cleavage or translation inhibition

13. RNA-dependent RNA polymerase
RdRP is an enzyme that catalyzes the replication of
RNA from an RNA template.
Specifically, it catalyses synthesis of the RNA strand
complementary to a given RNA template.
RNA replication process is a two-step
mechanism
1. First, the initiation step of RNA synthesis begins at or near the 3' end of the RNA template
by means of a primer-independent (de novo), or a primer-dependent mechanism
2. Elongation

14. RNA Induced Silencing Complex (RISC)
 RISC consists of both Protein and RNA
 Endonuclease and exonuclease “Slicer”
 Helicase
 Activities associated with RISC
 RNAi effector complex – Critical for mRNA degradation / Translation inhibition
 Targets and destroys endogenous mRNA complementary to interfering
RNA
 Homology seeking – RNA binding

15. PROCESS

16. RNA pol II
ssRNA
sequence
complementary
pre-miRNA
NUCLEUS
Dicing
miRNA-miRNA* duplex
2ATP 2ADP
EXP 5
SLICER Passenger
strand
mRNA
mRNA
Cleavege
mRNA
Degradation
A A A AA
CYTOPLASM

17. ssRNA
Sequence
Complimentary
Primary-miRNA (Pri-mRNA) transcript in nucleus (Hairpin)
Processing of pri-miRNA by
Dorsha
Transport of pre-miRNA into cytoplasm
Cleavage of pre-mRNA by
DICER
miRNA-miRNA*duplex
Unwinding of duplex Binding of mature
miRNA to Argonaute Degradation/ejection of
passenger strand
Interaction of RNA complex and target RNA
Formation of miRNA complex
RNAi effect
miRNA has only partial complementarity 3' un-translated
region of target mRNA, with therefore no slicing process
by Ago protein
Translation repression by : 1.Repression of mRNA
translation 2.Removal of mRNA poly (A)
(deadenylation)

18. RNA pol II
ssRNA
sequence
complementary
NUCLEUS
Dicing
shRNA-shRNA* duplex
2ATP 2ADP
EXP 5
SLICER Passenger
strand
mRNA
mRNA
Cleavage
mRNA
Degradation
A A A AA
CYTOPLASM

19. ssRNA
Sequence
Complimentary
ds shRNA transcript in nucleus (Hairpin)
Processing of shRNA by Dorsha
Transport of shRNA into cytoplasm
Cleavage of shRNA by DICER
shRNA-shRNA*duplex
Unwinding of duplex Binding of mature
shRNA to Argonaute Degradation/ejection of
passenger strand
Interaction of RNA complex and target RNA
Formation of shRNA complex
RNAi effect
miRNA has only partial complementarity 3' un-translated
region of target mRNA, with therefore no slicing process
by Ago protein
Translation repression by : 1.Repression of mRNA
translation 2.Removal of mRNA poly (A)
(deadenylation)

20. Plasmid
Virul Vector
siRNA (SYNTHETIC)
EXZOGENUS
SLICER
Passenger
strand
mRNA
mRNA
Cleavage
mRNA
Degradation
A A A AA
CYTOPLASM

21. ssRNA
Sequence
Complimentary
ds siRNA (Hairpin)
Processing of siRNA by Dorsha
Introduce of siRNA into cytoplasm
Cleavage of siRNA by DICER
siRNA-siRNA*duplex
Unwinding of duplex Binding of mature
siRNA to Argonaute Degradation/ejection of
passenger strand
Interaction of RNA complex and target RNA
Formation of siRNA complex
RNAi effect
miRNA has only partial complementarity 3' un-translated
region of target mRNA, with therefore no slicing process
by Ago protein
Translation repression by : 1.Repression of mRNA
translation 2.Removal of mRNA poly (A)
(deadenylation)

22. Roadway to perform RNA interference
STEP I
• Identification of target
gene & pathway
• Genome sequencing
• Applying bioinformatic
tools
• Analysis of
transcriptome, proteome
& metabolome
STEP II
• Vector development -
RNAi constructs &
screening for RNAi
constructs
• Selection of suitable
vector & promoter
• Screening by selectable
markers
STEP III
• Transforming and
screening transgenic
plant
• Delivery of RNAi
• Tissue culture of
transgenic line(s)
• Screening and selection
of transformed plants
4:00 – 4:30 PM
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STEP IV
• Evaluation of transgenic
lines for improved
quality
• Morphological
evaluation
• Transcriptome
evaluation
• Biochemical evaluation

23. 3
Insects, Parasitic weeds,
Pathogen
Biotic stress
4
Vitamin, carotenoids, Zinc,
Iron
Nutritional improvement
RNAi in crop
improvement
1
Hight, Branching, Leaf
morphology
Alteration of plant
Architecture 2
Drought, Flood, Temperature,
Salinity
Abiotic stress tolerance
5
Toxic substance
Deletion of Allergens
6
Caffeine, Gossypol,
Removal of Toxin
components
7
Tomato
Prolongation of self life
8
Morphine, Artemisinin
Secondary metabolites
9
Tomato, Grapes, Watermelon
Seedless Fruits
10
……………….
Male sterile plants
11
Flower color, Sent
Improve ethical value

24. Downregulation – two key FA
desaturase gene (ghSAD-1, ghFAD-
1).
Increasing – Steric acid 40%, Oleic
acid 77%
High Level of UFA /
Cottonseed
Enhancement in Nutritional Value and
Reduction in Antinutrient
Encoded by multigenic family (Glu A,
Glu B)
Stable for 20 generation .
Low glutelin content / Rice
Encoded by ∂ candinene gene.
First reported Tissue specific RNAi
based approach.
Gossypol Reduction /
Cottonseed
Over expression – biosynthetic
enzymes – improve C&F individually
(not combination)
RNAi suppression – endogenous.
photomorphogenesis regulatory gene
(DETI)
(Fruit specific promotor).
Simulation two independent
Increasing Carotenoid &
Flavonoid / Tomatoes

25. Starch ( Amylopectin & Amylose
polysaccharide) – Synthesis by two
competitive pathways.
Suppressing of starch hvanching
enzyme (SBE II, a,b) – more 70%
Amylose
High Amylase / Wheat
Enhancement in Nutritional Value and
Reduction in Antinutrient
Secondary Sulfur metabolites
(chopping)
Alliinase cleaves – Sulfonic acid &
volatile sulfur components – this
group includes - lachrymatory factor
– suppression – prevent sulfonic acid
conversion .
Tearless Onion
Essential ingredient in Beverages
Caffeine biosynthesis – Involves three distinct N-
methyltransferase.
In coffee – Caffeine synth. Mediated by theobromine synth. ---
-- RNAi.
100% decaffeination – Embryogenic tissue.
70% in plant.
Low Caffeine content In Coffee

26. RNAi Mediated
Crop
Improvement

27. Ms45 – male fertility
gene.
Lack of this gene –
Sterile.
Maize
Development Of Male Sterile Line
Silencing – Tapetum development Zinc Finger protein
(TAZI).
Degeneration.
Associate with flavanol accumulation – defect in pollen
wall formation & poor germination.
Petunia
MS1 – male
sterile
Suppress – TAZI
Arabidopsis

28. Bioremediation of Heavy Soil
ACR2 gene silencing – Arsenic
reductase.
10 to 16 fold more arsenic in shoots.
Less accumulation in roots.
High level of Arsenic

29. Improving Ethical Value
Downregulation of Chalcone Synthase (CHS) gene, Chalcone isomerase (CHI)
Involved in Anthocyanin biosynthesis – Manipulation of flower color - Flavonoid synthesis
Flower Color

30. PGTS – Suppression of foreign
genetic elements
Virus Resistance
Resistance Development
Encoded by multigenic family (Glu A,
Glu B)
Stable for 20 generation .
Bacterial Resistance
Encoded by ∂ candinene gene.
First reported Tissue specific RNAi
based approach.
Fungal Resistance Over expression – biosynthetic
enzymes – improve C&F individually
(not combination)
RNAi suppression – endogenous.
photomorphogenesis regulatory gene
(DETI)
(Fruit specific promotor).
Simulation two independent
Nematode Resistance

31. RNAi
Mediated
Resistance

32. Self Life Enhancement
Delay ripening (45 days)
Introduction of 1-amino cyclopro-pone-carboxylate (ACC) Oxidase .
dsRNA suppress the expression of Ethylene gene
Tomato

33. RNAi Mediated Metabolic Expression

34. RNAi In Rice

35. Very crucial – small RNA – delivered in sufficient amount in right time
CHALLENGES
Significant alteration in the expression of targeted gene – may result complex
downstream modification in the cell physiology
Careful selection of the target gene is important and critical step
Mutation is very common & frequent in miRNA based crop – loss of trait
stability

36. Off – target effect
RNAi is largely determine by the sequence
similarity of the involved small RNA with
the target mRNA or gene sequence.
therefore, the degree of identity between
small RNA and the target sequence is
crucial for the efficacy of the RNAi.
Candidate small RNAs may
also silence non-target genes,
with partial sequence
similarity.
Most of the off-target effect by
translation inhibition in
animals resulting in partial
homology of siRNA to the 3′
untranslated regions of non-
target genes
No such effects have been
reported in plants.
As in plants, silencing is
mainly due to the homology of
miRNA with coding sequences
resulting in mRNA cleavage.

37. Stability of transgene
The dsRNA-mediated transgene silencing
is systemic in nature, as it spreads from
one cell to another cell, transported long
distances via the vascular system in
plants.
Transgenes can also silence
systemically through grafting
The required factors for
transmission through grafting
are also involved in RNA-
directed DNA methylation
(RdDM) indicating the possible
role of chromatin modification
in the perception and
perpetuation of long-distance
silencing signals

38. Persistence of dsRNA
Food web is the next potential source of
exposure with dsRNA. Theoretically, the
ingested dsRNA by an organism serves as
prey for predators or host for parasitoids,
wherein dsRNAs may be potentially
transferred and amplified to different
successive trophic levels.
Transgene-encoded dsRNA or
naked dsRNA might become
transferred from plant system
to insect body through feeding,
resulting in targeted gene
silencing
The host plant has to preserve
dsRNAs without processing it
into matured small RNAs or
amplify sufficient quantities of
dsRNA to be exposed at the
level of secondary or tertiary
consumers (natural enemies)
for eliciting a persistent
response
Transferred to high tropic
level in food chain case
harmness to related species

39. Food Risk assesment
One of the major challenges of small
RNA-based technology is to identify the
potential adverse effects on health that
may arise due to changed transcriptome
and proteome in GM plants.
There is no report of any
heterologous proteins that
may be produced in RNAi-
based GM crops
RNA is considered safe as a
component of the human diet.
RNAi based GM crop Assessed
(i) the phenotypic and
agronomic characteristics, (ii)
the nutritional and
compositional characteristics,
and (iii) the toxicity potential
of the new protein(s) and their
metabolites.

40. Bio safety evaluation
Environmental Risk assessment
The wide-scale adoption of GM crops for
the long term has brought some
important ecological issues.
GM plants expressing dsRNA
to control insect pests -
Assessment of any adverse
effects on non-target
arthropods is necessary
Non-Targeted Arthropods
(NTAs)
Bt cotton. For example, in
north China, the population
size of mirid bugs
progressively increased

41. Defence
Protection against virus, Bacteria, Fungi
Silence fault codes
Translation suppression of unfavorable
mutant genes
Improvement of trait
Increase Nutrient quality by Suppression
of some antinutrients