RNAi – Mechanism and Its Application In Crop Improvement

1. Presented by:-
Jadhao Kundansingh R.
09ABT/11
Department of Agril-biotechnology,
Orissa University of Agriculture & Technology,
BBSR.

2. • Small RNA Family
• Mechanism of action of RNAi
• Application of RNAi in crop
improvement
• Case study I
• Conclusions

3. DNA
RNA
protein
AAAA
mRNA
rRNA
tRNA
other noncoding RNAs

4. scnRNA
Constituent RNAs
tRNA
Regulatory RNAs
rRNA
Non-coding RNAs
RNA
mRNA
Coding RNA
miRNA
(stRNA)
siRNA snoRNA

5. Name Organism Length
(nt)
Proteins Source of trigger Function
miRNA Plants,algae,animals,v
iruses, protists
20-25 Drosha (animals
only) + Dicer
Pol II transcription (pri-miRNAs Regulation of mRNA stability,
Translation
rasiRNA Plants 24 DCL3 Transposons, repeats Chromatin modification
tasiRNA Plants 21 DCL4 miRNA-cleaved TAS RNAs Post transcriptional regulation
Exo -siRNA Animals, fungi,
protists, plants
21-24 Dicer Transgenic, viral or other
exogenous dsRNA
Post transcriptional regulation,
antiviral defense
Endosi RNA Plants,algae,animals,f
ungi, protists
21 Dicer Structured loci, convergent and
bidirectional transcription, mRNAs
paired to antisense pseudogene
transcripts
Post transcriptional regulation of
transcripts and transposons
Transcriptional gene silencing
piRNA germ line Drosophila
melanogaster,
mammals, zebrafish
24-30 Dicer- independent Long, primary transcripts Transposon regulation, unknown
functions
piRNA like Drosophila
melanogaster
24-30 Dicer- independent In ago2 mutants in
Drosophila
Unknown
21U-RNA piRNAs Caenorhabditis
elegans
21 Dicer- independent Individual transcription of each
piRNA
Transposon regulation ,unknown
functions
26G RNA Caenorhabditis
elegans
26 RdRP Enriched in sperm Unknown

6. • RNA interference (RNAi) is an evolutionally
highly conserved process of post-transcriptional
gene silencing (PTGS) by which double stranded
RNA (dsRNA) causes sequence-specific
degradation of mRNA sequences.
• It was first discovered in 1998 by Andrew Fire
and Craig Mello in the nematode worm
Caenorhabditis elegans and later found in a wide
variety of organisms, including mammals.

7. •Plants
•Petunias
•Fungi
•Neurospora
•Animals
•Caenorhabditis elegans

8. • PTGS (Posttranscriptional Gene
Silencing)
• Cosuppression
• Quelling
• Virus-induced gene silencing

10. • An Unexpected Result…
• petunias
surprisingly
developed areas of
hypopigmentation
when transduced
with the gene
encoding an
enzyme required
for pigment
synthesis.
1990-
Petunias

11. • Napoli et al. defined an RNAi-like
phenomenon and called it
“cosupression.”
• chalcone synthase (CHS), a key
enzyme in flavonoid biosynthesis,
the rate-limiting enzyme in
anthocyanin biosynthesis,
responsible for the purple
coloration.

12. • Carlo Cogoni and Guiseppe Macino of the Università
di Roma La Sapienza in Italy introduced a gene
needed for carotenoid synthesis in the mold
Neurospora crassa:
• The introduced gene led to inactivation of
the mold's own gene in about 30% of the
transformed cells. They called this gene
inactivation "quelling."
A rosette of the asci

13. • The answer actually came in the year 1988 from
researchers working on C. elegans
Potent and genetic interference
by ds RNA in C. elegans
Fire.A,Xus,Montogomery ,Mk Kosta SA Driver SE
,Mello CG 1998 Feb 19.391:806-11.
Double stranded RNA Posses
Puzzle
Wagner RW, Sun.L Nature 1988 19,744-5

14. • Codes for a non essential myofilament
• It is present several thousand copies/cell

15. • 4-6 hours after injection, eggs
collected.
• Screened for phenotypic
changes
• twiching
Exon Size RNA Phenotype
Exon 21-22 742 Sense
Antisense
Sense+antisense
Wildtype
Wildtype
Twicher (100%)
Exon 27 1033 Sense
Antisense
Sense+antisense
Wildtype
Wildtype
Twicher (100%)

16. Timeline for RNAi Dicsoveries

17. Attempting to use antisense RNA to knock down gene
expression, they found synergistic effects on gene
silencing when antisense and sense RNA strands where
delivered together,
this phenomenon is later termed as

19. • Targetted disruption of gene
function in Drosophylla by RNA
interference : a role for nautilus in
embryonic somatic formation.
L.Misquitta,B.Paterson,proc.Nat.
Acad .Sci,USA,1999.96-1451-1453.
• Double stranded RNA induces m-
RNA degradation in Trypanosoma
bruci.
H.Nigo,C.Tschudi,K.Gull.Proc.Nati.
Acad.Sci.USA.1998,95,14687-14692

20. •Virus resistance and gene
silencing in plants can be
induced by simultaneous
expression of sense and
antisense RNA.
R.Water house,M.GrahamM.wang proc.Nat. Acad
.sci,USA,1998.95,13959-13964.

21. It was shown that plants contain
an enzyme RNA dependent RNA
polymerase .
It was responsible for the
synthesis of ds RNA in presence of
high level of m-RNA

24. Dicer
Double-stranded RNA triggers processed into siRNAs
by enzyme RNAseIII family, specifically the Dicer family
Processive enzyme - no larger intermediates.
Dicer family proteins are ATP-dependent nucleases.
These proteins contain an amino-terminal helicase
domain, dual RNAseIII domains in the carboxy-
terminal segment, and dsRNA-binding motifs.

25. Contd…..
They can also contain a PAZ domain, which is thought
to be important for protein-protein interaction.
Dicer homologs exist in many organisms including
C. elegans, Drosphila, yeast and humans
Loss of dicer: loss of silencing, processing in vitro
Developmental consequence in Drosophila and
C. elegans

26. RISC complex
RISC is a large (~500-kDa) RNA-multiprotein complex, which
triggers mRNA degradation in response to siRNA
some components have been defined by genetics, but function
is unknown, e.g.
– unwinding of double-stranded siRNA (Helicase !?)
– ribonuclease component cleaves mRNA (Nuclease !?)
– amplification of silencing signal (RNA-dependent RNA
polymerase !?)
cleaved mRNA is degraded by cellular exonucleases

29. • Two-step model to explain
RNAi.
• I. dsRNA is diced by an ATP-
dependent ribonuclease (Dicer)
into short interfering RNAs
(siRNAs).
• duplexes of 21 23 nucleotides
bearing two-nucleotide 3'
overhanging ends.
• II. siRNAs are transferred to a
second enzyme complex,
designated RISC for RNAi-
induced silencing complex.
The siRNA guides RISC to the
target mRNA, leading to its

31. AAAA
RNAi is mediated by small
(~21-25 nucleotide) noncoding RNAs
complementary to the targeted gene
cleavage of
targeted mRNA
(siRNA)
inhibitsprotein translation or
causes mRNA degradation
(miRNA)
mRNA:
dsRNA
intermediate

33.  Crop quality traits : Sunilkumar et al., 2006.
reduced the toxic terpenoid gossypol in cotton
seeds and cotton oil by engineering small RNAs
for the cadinene synthase gene in the gossypol
biosynthesis pathway.
 Virus resistance : the toxic terpenoid gossypol in
cotton seeds and cotton oil by engineering small
RNAs for the cadinene synthase gene in the
gossypol biosynthesis pathway.

34.  Protection from insect pests :
 Baum et al. 2007. showed that silencing of a vacuolar ATPase
gene (V-type ATPase A gene) in midgut cells of western corn
rootworm (WCR) led to larval mortality and stunted growth.
 Researchers identified a cytochrome P450 monooxygenase
(CYP6AE14) gene important for larval growth expressed in
midgut cells with a causal relationship to gossypol tolerance.
 Transgenic tobacco and Arabidopsis producing CYP6AE14
dsRNA were fed to larvae, successfully decreasing
endogenous CYP6AE14 mRNA in the insect, stunting larval
growth and increasing sensitivity to gossypol.

35.  Nematode resistance :
Yadav et al., 2006. showed transgenic
tobacco having dsRNA targetting two
Meloidogyne (root knot) nematode genes
had more than 95% resistance to
Meloidogyne incognita.
Huang et al., 2006. showed that Arabidopsis
plants expressing dsRNA for a gene
involved in plant–parasite interaction
(16D10) had suppressed formation of root
galls by Meloidogyne nematodes and
reduced egg production.

36. Bacterial and fungal risistance :
Little progress.
Escobar et al. 2001. showed that silencing
of two bacterial genes (iaaM and ipt) could
decrease the production of crown gall
tumors (Agrobacterium tumefaciens) to
nearly zero in Arabidopsis, suggesting that
resistance to crown gall disease could be
engineered in trees and woody ornamental
plants.

37. Application Case study authors
the level of lysine Reduction of lysine
catabolism and improving
seed
germination generating a
dominant high-lysine
maize
variant by knocking out
the expression of the 22-
kD
maize zein storage
protein
Zhu et al., Tang et al.,
Segal et al.
Barley and Rice Resistance of barley to
BYDV and producing a
rice
variety called LGC-1 (low
glutenin content 1) by
RNAi technology
Wang et al.,
Kusaba et al.
Williams et al.
Banana Production of banana
varieties resistant to the
Banana
Bract Mosaic Virus
(BBrMV) by RNAi
Rodoni et al.

38. Cotton 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 Gossypol
Sunilkumar et al.
Jute
.
Generating jute varieties with
low lignin content by
RNAi technology
Williams et al.
Lathyrus sativus RNAi construct designed to
silence the genes
encoding the two starch-
branching isozymes of
amylopectin synthesisRNAi
technology can be used to
silence the gene(s) responsible
for production of
BOAA
Regina et al

39. Tomato RNAi-mediated
suppression of DET1
expression
under fruit-specific
promoters has recently
shown to
improve carotenoid
and flavonoid levels in
tomato
fruits with minimal
effects on plant growth
Williams et al
Coffee RNAi technology has
enabled the creation of
varieties
of Coffee that produces
natural coffee with low or
very low caffeine content
Davuluri et al.

40. Trait
Target Gene Host Application
Enhanced
nutrient content
Lyc Tomato Increased concentration of lycopene
(carotenoid antioxidant)
DET1 Tomato Higher flavonoid and b-carotene
contents
SBEII Wheat, Sweet potato, Maize Increased levels of amylose for
glycemic management and digestive
health
FAD2 Canola, Peanut, Cotton Increased oleic acid content
SAD1 Cotton Increased stearic acid content
ZLKR/SDH Maize Lysine-fortified maize
Reduced alkaloid
production
CaMXMT1 Coffee Decaffeinated coffee
COR Opium poppy Production of non-narcotic alkaloid,
instead of morphine
CYP82E4 Tobacco Reduced levels of the carcinogen
nornicotine in cured leaves
Heavy metal
accumulation
ACR2 Arabidopsis Arsenic hyperaccumulation for
phytoremediation
Reduced polyphenol
production
s-cadinene synthase
gene
Cotton Lower gossypol levels in
cottonseeds, for safe consumption
Ethylene
sensitivity
LeETR4 Tomato Early ripening tomatoes
ACC oxidase gene Tomato Longer shelf life because of slow
ripening
Reduced
allergenicity
Arah2 Peanut Allergen-free peanuts
Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass

41. RNAi-mediated disruption of squalene synthase improves drought tolerance and yi
in rice
Lakshmi P. Manavalan et.al.2012