This document provides an overview of RNA interference (RNAi) and its applications in crop improvement. It discusses the history and discovery of RNAi, the mechanism of RNAi involving initiation by Dicer and effector function of RISC complexes, and methods of transforming plants with RNAi constructs. Applications of RNAi described in the document include modifying traits in rice, maize, barley, cotton, jute, tomato, lathyris, and coffee to improve nutritional quality, increase yields, confer virus resistance, and remove toxic compounds.

1. WELCOME

2. RNA INTERFERENCE
AND
ITS APPLICATIONS IN
CROP IMPROVEMENT
PRESENTED BY S.NAGALAKSHMI
RAD/10-28
DEPARTMENT OF AGRICULTURAL
BIOTECHNOLOGY

3. 1.RNA Interference(RNAi)
-Introduction
-History
-Mechanism
2.Transforming methods
-Direct method
-Indirect method
3.Significance
4.Applications in crop improvement
5.Conclusion
6.References
3

4. What is RNAiWhat is RNAi
• RNA interference (RNAi) is a process by
which double-stranded RNA (dsRNA)
homologous to the target locus can
specifically inactivate gene function in
plants, invertebrates, and mammalian
systems (Sharp 1999;Hammond et al.
2001).

5. RNAi was first discovered in 1998 byRNAi was first discovered in 1998 by
Andrew Fire and Craig Mello in theAndrew Fire and Craig Mello in the
nematode wormnematode worm Caenorhabditis elegans.Caenorhabditis elegans.
This technique has proven effective in
Drosophila, plants, and recently, in
mammalian cell culture.

6. History
• Post-transcriptional gene silencing (PTGS)
was observed first in Petunia.
• RNA interference was first observed in
petunias, when Napoli et al. (1990)
discovered that introduction of a pigment-
producing gene under control of a powerful
promoter suppressed expression of both the
introduced gene and the homologous
endogenous gene.
• The phenomenon they called cosuppression.”

7. • HISTORY: Late 1980s & Early
1990s Botanists;
PETUNIA
GENES wereGENES were SILENCEDSILENCED

8. Final Discovery
Andrew Fire , Craig Mello 1998 announced RNAi
discovery.
The crucial 1998 discovery by Fire et al., that injection
of dsRNA—a mixture of both sense and antisense
strands of the target mRNA, rather than either strand
alone—into gonads of the nematode Caenorhabditis
elegans resulted in extremely potent silencing
unequivocably identified dsRNA as the inducer of
RNA interference
Awarded Nobel Prize 2006 for this Path-breaking
concept.

9. In 2006, Andrew Z. Fire and Craig C. Mello shared the Nobel
Prize in Physiology or Medicine for their work on RNA
interference in the nematode worm Caenorhabditis elegans.
Andrew Z. Fire Craig C.Mello
w
Craig Mello at the 2006 Nobel Prize
lecture.

10. What are the
components
of RNAi machinery?

11. Components of RNA InterferenceComponents of RNA Interference
• DicerDicer
• RNA-Induced Silencing Complex (RISC)RNA-Induced Silencing Complex (RISC)
• DroshaDrosha
• siRNAsiRNA
• miRNAmiRNA

12. DicerDicer
Enzyme involved in the initiation of RNAi.
Rnase-III-like dsRNA-specific ribonuclease (200
KDa)
ATP-dependent nucleases.
Dicer homologs exist in many organisms including
C.elegans, Drosphila, yeast and humans.

13. Dicer’s domains
 Helicase domain at the amino-terminal end
 PAZ domain (110-130 amino-acid domain)
 Dual Rnase III motifs in the carboxy-terminal end
 dsRNA binding domain

14.  Cleaves dsRNA or pre-miRNA,Cleaves dsRNA or pre-miRNA, leaves 3’leaves 3’
overhangs and 5’ phosphate groupsoverhangs and 5’ phosphate groups

15. miRNA
• In genetics micro RNA are single standedRNA
mlecules of about 21-23 nucleotide in
length,which regulate gene expression.
• MicroRNA are encoded by genes that are
transcribed from DNA but not translated in to
protein,instead they are processed from primary
transcripts known as pri-miRNA to stem-
loopstructures called pre miRNA and finally to
functional miRNA.

16. miRNA ProcessingmiRNA Processing
• Transcribed from endogenous gene as pri-miRNATranscribed from endogenous gene as pri-miRNA
– Primary miRNA: long with multiple hairpinsPrimary miRNA: long with multiple hairpins
• Cleaved by Drosha into pre-miRNACleaved by Drosha into pre-miRNA
– Precursor miRNA: ~70nt imperfect hairpinsPrecursor miRNA: ~70nt imperfect hairpins
– Exported from nucleusExported from nucleus
• Cleaved by Dicer into mature miRNACleaved by Dicer into mature miRNA
– 21-25nt21-25nt

17. miRNA processingmiRNA processingmiRNA processingmiRNA processingmiRNA processingmiRNA processing
miRNA processingmiRNA processing
Pri-miRNA
DroshaDrosha
pre-miRNA
DicerDicer
mature
miRNA

18. Argonaute family protein.Argonaute family protein.
2 RNA binding proteins2 RNA binding proteins
RNA/DNA HelicaseRNA/DNA Helicase
RNA-Dependent RNARNA-Dependent RNA
PolymerasePolymerase
(RdRP)(RdRP)
18

19. RISC effector complexRISC effector complex
• siRNAs or miRNAs are incorporated intosiRNAs or miRNAs are incorporated into
RNA-induced silencing complex (RISC).RNA-induced silencing complex (RISC).
• siRNAs or miRNAs guides the RISC &siRNAs or miRNAs guides the RISC &
degrade mRNAs / inhibits translationdegrade mRNAs / inhibits translation
• Assembling the RISC complex requires ATP,Assembling the RISC complex requires ATP,
while RNA cleavage does not.while RNA cleavage does not.
– Approx 500 kDa nuclease complex
– sequence specific nuclease activity resulting in
ablation of target mRNA.

20. Structure of si-RNA
• si-RNAs have a well defined structure
• Short (usually 21 nucleotide) double-strand of RNA
(di-RNA)
• Each strand has a 5' phosphate group and a 3'
hydroxyl (-OH) group.
• This structure is the result of processing by Dicer,
an enzyme that converts either long dsRNAs or
hairpin RNAs into siRNAs.

21. Mechanism of RNAi

22. RNAi: Two Phase ProcessRNAi: Two Phase Process
• Initiation stepInitiation step
– Generation of mature siRNA or miRNAGeneration of mature siRNA or miRNA
• Effecter stepEffecter step
– Silencing of target geneSilencing of target gene
– Degradation or inhibition of translationDegradation or inhibition of translation

23. Initiation StepInitiation Step
ATP
ATP
ADP + ppi
ADP + ppi
DICER
KINASE
RdRP

24. Effector StepEffector Step
• siRNA bindingsiRNA binding
• siRNA unwindingsiRNA unwinding
• RISC activationRISC activation

25.  Initiation stepInitiation step
dsRNAs get processed intodsRNAs get processed into
siRNAs or miRNAs by DicersiRNAs or miRNAs by Dicer
They are assembled intoThey are assembled into
RISCsRISCs
Effecter stepEffecter step
They guide the RISCs toThey guide the RISCs to
complementary mRNA,complementary mRNA,
where they cleave andwhere they cleave and
destroy the cognate RNAdestroy the cognate RNA
Mechanism of RNAiMechanism of RNAi

26. The Mechanism of RNA Interference (RNAi)
• First, the dsRNAs get
processed into 20-25
nucleotide (nt) small
interfering RNAs (siRNAs) by
an RNase III-like enzyme
called Dicer (initiation step).
• Then, the siRNAs assemble
into endoribonuclease-
containing complexes known
as RNA-induced silencing
complexes (RISCs),
unwinding in the process.
• The siRNA strands
subsequently guide the
RISCs to complementary
RNA molecules, where they
cleave and destroy the
cognate RNA (effecter step).
Cleavage of cognate RNA
takes place near the middle
of the region bound by the
siRNA strand.

27. Transforming methods

28. Agrobacterium-mediated
transformation
• DNA-directed RNAi makes use of dsRNA-
expressing vectors introduced into plants via
Agrobacterium-mediated transformation .
• This approach has been shown to be effective in
reducing the amount of a specific gene product.
• One of the first studies testing DNA-directed
RNAi in plants was done to compare the ability
of sense, antisense,or dsRNA at generating
RNA-mediated virus resistance via PTGS in
tobacco and silencing of an endogenous GUS
reporter gene in rice (Waterhouse et al. 1998).
• In bothcases it was shown that duplex RNA was
more effective than either sense or antisense
RNA at silencing the target gene.

31. Particle Gun
• some applications of RNAi in plants have relied
on non-Agrobacterium-mediated methods of
introduction of dsRNA into the cells.
• For example, particle bombardment was used
to show that RNAi worked at the single cell level
in cereals (Schweizer et al. 2000).
• while Klink & Wolniak (2000) and Stout et al.
(2003) demonstrated that there is a direct
uptake of dsRNA by fern spores during
imbibition.

32. Particle Gun

33. Scheme of particle gun

34. • Most of these methods utilize an RNAi vector to
produce stable or transient dsRNA in vivo.
• Unnamalai et al. (2004), have recently devised a
way of introducing dsRNA without transforming
the plant with an RNAi vector.
• They used cationic oligopeptides for delivering
dsRNA into plant cells.
• Specifically, the scientists introduced dsRNA
into tobacco cells using cationic oligopeptide 12-
mer.
• Their results showed efficient silencing of the
target genes.

35. Vectors expressing siRNAsVectors expressing siRNAs
• Plasmid-based vectorsPlasmid-based vectors
– Low and variable transfection efficiencyLow and variable transfection efficiency
• Viral-based vectorsViral-based vectors
– Highly efficientHighly efficient
– Better than plasmid-basedBetter than plasmid-based
• Pol II promoter-based plasmid vectorsPol II promoter-based plasmid vectors
– Tissue specificTissue specific

36. Significance of RNA interferenceSignificance of RNA interference
 Protects against viral infections.
. Secures genome stability by keeping
mobile elements silent.
• Important role during development in
plants,C. elegans and mammals.

37. • Used to determine the function of a specific
gene by silencing its expression.
• Silencing of over-expressed or aberrantly
expressed disease causing genes.

38. Applications in crop improvement

39. RICE
• Kusaba and his team (Kusaba et al., 2003) have
made significant contribution by applying RNAi
to improve rice plants.
• They were able to reduce the level of glutenin
and produced a rice variety called LGC-1 (low
glutenin content 1).
• The low glutenin content was a relief to the
kidney patients unable to digest glutenin. The
trait was stable and was transmitted for a
number of generations.
• They showed that the procedure may apply to
both monogenic and polygenic agronomic
characters (Williams et al., 2004).

40. MAIZE
• In another study, RNAi has been
successfully used to generate a dominant
high-lysine maize variant by knocking out
the expression of the 22-kD maize zein
storage protein,a protein that is poor in
Lysine content (Segal et al., 2003).
• RNAi generates quality and normal maize
seeds with high levels of lysine-rich
proteins (Tang et al., 2004).

41. BARLEY
• Bayer Crop Science has acquired an exclusive
worldwide license to develop, market, and sell
selected crop plant varieties in which the RNAi
technology has been successfully applied.
• Using this technique this group has developed
varieties of barley that are resistant to BYDV (barley
yellow dwarf virus) (Wang et al., 2000).
• Their results showed that the barley plants
developed through RNAi technology are resistant to
viral infection while the control plants became
infected with the yellow dwarf virus.

42. COTTON
• Another nutritionally important crop is cotton.
• Cotton is mainly used for fiber production, and is an
important crop not only in developed ountries, but
also in many developing countries where
malnutrition and starvation are widespread
(Sunilkumar et al., 2006).
• In these developing areas, the cottonseeds that
remain after fiber extraction could be extensively
used as sources of protein and calories.
• But they are largely underutilized because they
contain a toxic gossypol terpenoid.

43. • Indeed, transgenic cotton plants expressing a
RNAi construct of the d-cadinene synthase
gene of gossypol synthesis fused to a seed-
specific promoter caused seed specific
reduction of this metabolite.
• These cotton plants are thus expected to
have similar insect and pathogen resistance
to that of wild type cotton, but to produce
seeds with higher nutritional value (Tang et
al., 2007).

44. JUTE
• A possible application of RNAi involves
the down regulation of a key enzyme in
the biosynthetic pathway of lignin in the
two economically important Corchorus
species, namely, C. capsularis and C.
olitorius.
• The enzyme 4-coumarate: CoA ligase (4-
Cl) is one of the key enzymes in the early
stages of lignin biosynthesis.

45. • With the availability of the sequence of the 4-Cl
gene, it would be possible to create a transgenic
jute variety expressing the RNAi construct to
down regulate the quantity of 4-Cl mRNA
thereby reducing the lignin production.
• Thus RNAi technology may prove to be a
powerful molecular tool by generating jute
varieties with low lignin content and production
of various economically important commodities
such as high quality paper and cloth (Williams et
al., 2004).

46. TOMATO
• Among the vegetables, tomato fruits are
relatively rich in a number of vitamins including
the strong antioxidant carotenoid, or lycopene,
which provides the tomato fruit with its typical
red color.
• Carotenoids are synthesized by the same
biosynthetic pathway that synthesizes
chlorophyll, and it has been shown that genes
controlling the light-mediated regulation of the
photosynthetic machinery also influence tomato
fruit quality by altering the levels of carotenoids
and flavonoids (Adams-Phillips et al., 2004).

47. • The tomato high pigment (hp-2) phenotype,
which accumulates elevated levels of
carotenoids and flavonoids, is due to mutations
in the regulatory gene DE-ETHIOLATED1
(DET1)
• which represses several light-dependent
signaling pathways (Levin et al., 2003; Mustilli et
al., 1999).
• Yet, RNAi-mediated suppression of DET1
expression under fruit-specific promoters has
shown to improve carotenoid and flavonoid
levels in tomato fruits with minimal effects on
plant growth and other fruit quality parameters
(Davuluri et al., 2005) .

48. Latyrus sativus
• In Ethiopia, Bangladesh and India, the
people in the lower socioeconomic class
use a leafy vegetable known as Lathyrus
sativus.
• It is a leguminous crop and contains a
neurotoxin called β-oxalylaminoalanine-L-
alanine (BOAA) (Spencer et al., 1986).
• People consuming this vegetable suffer
from a paralytic disease called, lathyrism.

49. • Paralysis in the limbs is a known symptom of
BOAA, yet people still consume this vegetable in
times of famine.
• This is an instance where RNAi technology can be
used to silence the gene(s) responsible for
production of BOAA.
• There may be one difficulty; in that the BOAA
genes may be linked to genes, which confer
immunity to this unique crop or impart drought and
flood tolerance.
• Bringing down the levels of BOAA to a safe
concentration, rather than totally silencing the
concerned genes, may overcome this obstacle
(Williams et al., 2004).

50. COFFEE
• 10% of the coffee on the world market is shared
by decaffeinate coffee (DECAF). Decaf is
wanted by buyers which are sensitive to
caffeine.
• Decaf is obtained from natural coffee by several
ways: by using water or solvent extraction.
• RNAi technology has enabled the creation of
varieties of Coffee that produces natural coffee
with low or very low caffeine content, thus by-
pass the need of extraction (Van Uyen, 2006).

51. BANANA
• Another instance where RNAi may be fruitfully
applied is in the production of banana varieties
resistant to the Banana Bract Mosaic Virus
(BBrMV), currently devastating the banana
population in Southeast Asia and India (Rodoni et
al., 1999).
• The BBrMV infects banana plants destroying the
fruit producing bract region, rendering them
useless to farmers. By designing an RNAi vector
aimed at silencing the Coat Protein (CP) region of
the virus.

52. APPLE
• Fruit quality has also been addressed by
silencing experiments.
• Several characteristics are involved in fruit
quality.
• Transgenic apple fruits silencing key
enzymes involved in autocatalytic ethylene
production were significantly firmer and
displayed an increased shelf-life
(Dandekar et al., 2004).

53. CITRUS
• In Citrus, the down regulation of putative
thermostable pectin methylesterase genes
is projected as a possible solution for the
undesirable separation of juice into clear
serum and particulate phase (Guo et al.,
2005).
• In this case, RNAi could be used to
achieve this goal (Amancio et al., 2007).

54. Healthier oil
• An application of RNAi in plants that is much
closer to agricultural use is the silencing of
genes involved with seedoil production.
• The best oil for heat stability and with no
negative effects on cholesterol levels is one
which is high in oleic acid.
• Used RNAi to silence the gene in cotton which
codes for the enzyme that converts oleic acid
into a different fatty acid. This has altered the
seed-oil from being around 10% to an
impressive 75% oleic acid.

55. Pest control
• dsRNA designed to suppress specific genes in
some pests,can be provided in the diet to
suppress or kill those pests.
• The sequence specificity of RNAi presents the
opportunity to selectively target some pest
species while sparing desirable species.
• All transgenic-plant-mediated insect control
technology is based on the plants producing
proteins derived from a specific type of bacteria,
Bacillus thuringiensis or Bt.

56. Flower color modification of plants by
RNAi-mediated gene silencing

57. Blue Rose
• The cyanidin gene is responsible for a synthethic
pathway that leads to formation of red pigment
and a correspondent Delphinidin gene is the key
gene for formation of blue color.
• Scientists at Florigene (Australia) and Suntory
(Japan) have been successful in knock -downing
the cyanidin genes in rose and carnation by
RNAi technology and introduce delphinidin
genes,

58. How did they produce it?How did they produce it?

59. The First Blue RoseThe First Blue Rose

60. RNAi and Flowering time
• One of the important aspects of crop
production is flowering time .
• So, being able to control flowering time in
plants could be a very useful tool in
horticulture and agriculture.

61. • In Arabidopsis, there is a gene called FLC
which represses flowering and used RNAi
to switch it off and bring on flowering.
• This clearly shows that the technology has
the potential to regulate flowering time in
crops (Waterhouse et al., 1998).

62. RNAi Assay
• RT-PCR
• Western blot
• Histological, physiological, structural,
biochemical assay of transgenic cells or
animals.

63. Conclusion
• Current agricultural technology needs
more and more molecular tools to
reduce current crop loss and feed extra
mouths,which according to a recent
estimate by the FAO will increase by
two billion over the next 30 years.
• An important gene regulation pathway.An important gene regulation pathway.
• A small world with mighty biologicalA small world with mighty biological
functions.functions.

64. • The technology is well developed and
can be applied directly to evolve a crop
resistant to stresses caused by
virus,bacteria,fungi,insects or natural
disasters.
• Since this technology offers great
potential in understanding gene
functions and utilize them to improve
crop quality and production.
• It is a matter of time before we see
the products of this RNAi research in
the farmer’s fields around the world.

65. References
• Wang M, Abbott D, Waterhouse P M (2000) A single copy of
a virus derived transgene encoding hairpin RNA gives
immunity to barley yellow dwarf virus. Molecular Plant
Pathology 1: 401-410.
• Kusaba M, Miyahara K, Lida S, Fukuoka H, Takario T, Sassa
H, Nishimura M, Nishio T (2003) Low glutenin content 1 : a
dominant mutation that suppresses the glutenin multigene
family via RNA silencing in rice. Plant Cell 15: 1455-1467.
• Williams M, Clark G, Sathasivan K, Islam AS (2004) RNA
Interference and its Application in Crop Improvement. Plant
tissue culture and Biotechnology 1-18.
• Tang G, Galili G (2004) Using RNAi to improve plant
nutritional value: from mechanism to application. TRENDS in
Biotechnology 22(9) 463-469.

66. The web
sites containing such RNAi related software applications
are listed here.
.
Silencing RNA Target Finder from Ambion
• http://www.ambion.com/techlib/misc/siRNA_finder.html
RNAi Design Tool from Integrated DNA Technologies
• http://biotools.idtdna.com/rnai/
Gene Specific siRNA Selector
• http://hydra1.wistar.upenn.edu/Projects/siRNA/siRNAindex.htm
RNAi Designer from Invitrogen
• https://rnaidesigner.invitrogen.com/sirna/
siRNA Selection Program, Whitehead Institute for Biomedical
Research.
• http://jura.wi.mit.edu/siRNAext/register.php
Summary of siRNA Design Rules and Tools
• http://www.protocol-online.org/prot/Molecular_Biology/
• RNA/RNA_Interference/siRNA_Design_Rules/
General Resources on RNAi in Ambion
• http://www.ambion.com/techlib/resources/RNAi/

67. THANK YOU