Gene silencing is describing as epigenetic processes of gene regulation. Gene silencing is a technique used to turn down or switch off the activity of genes by a mechanism other than genetic modification. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell. Gene silencing (GS) is defined as a molecular process involved in the down regulation of specific genes, the mechanisms of Gene silencing that suppress gene activity in plants has extended that control of gene expression. Currently, there are several routes of GS identified in plants, such as: transcriptional gene silencing and post transcriptional (PTGS or RNAi) gene silencing (Fire et al. 1998), microRNA silencing and virus induced gene silencing. All these pathways play an important role at the cellular level, affecting gene regulation and protection against viruses and transposons. The post-transcriptional gene silencing involves breakdown of the mRNA itself by various techniques like Ribozymes, antisense RNA, DNAzymes and RNA interference (RNAi). Among all these techniques RNA interference has emerged as most potent tool to effect targeted gene silencing and is being used to determine the function of genes which are expressed in a constitutive or cell-fate dependent manner.

1. Gene silencing and
its application in
crop improvement
Speaker:- Vinod Kumar
(A-2014-40-014)
GP-691
DOCTORAL SEMINAR

2. • introduced additional copies of a gene
encoding chalcone synthase (flower
pigmentation)
• Unexpectedly the flowers produced were less
pigmented, fully or partially white.
• both the transgene and endogenous gene
were down regulated in white flowers.
• Phenomenon was called co-suppression of
gene expression. Jorgensen (1990 )
SHORT HISTORY OF
GENE SILENCING
2

3. SHORT HISTORY OF
GENE SILENCING
• Quelling in Neurospora crassa
• Introduced extra copies of
carotenoid pigment genes (aL1)
• Expected :-orange pigment
• Observed:- albino
Macino (1992)
3

4. SHORT HISTORY OF
GENE SILENCING
• Discovery of the first miRNA: lin-4
• lin-4 required for larval development
Victor Ambros 1993
Gary Ruvkun 1993
4

5. • used antisense RNA to down regulate
par1 gene in Caenorhabditis elegans
• antisense RNA disrupted expression
of par-1
• they coined the term antisense
mediated silencing
Kempheus 1995
SHORT HISTORY OF
GENE SILENCING
Sub Guo 1995
5

6. C. elegans
Injected dsRNA
corresponding
to unc22 gene
ssRNA was effective only when sense strand was
injected followed by the antisense or vice versa
Craig C.Mello and Andrew Fire (1998)
SHORT HISTORY OF
GENE SILENCING
Discovery of RNAi 6
Noble Prize in 2006

7. • discovered that RNAi could
be trigger through the use of
shorter pieces of RNA known as
small interfering RNAs (siRNAs).
• demonstrated that siRNAs could
be designed to silence specific
genes
Thomas Tuschl 2001
identified, described, and named
• "Dicer" enzyme,
•RNA-induced silencing complex
(RISC) Gregory Hannon 2001
SHORT HISTORY OF
GENE SILENCING
7

8. Introduction
• Epigenetic process
• Gene silencing:- “switching off” of a gene by a
mechanism other than genetic modification
• same as gene knock down but different from gene
knock out.
• Gene silencing can take place at the post-
transcriptional level (PTGS) or at the transcriptional
level (TGS)
8

9. Before PTGS:- Antisense RNA
• Antisense RNA is a single-
stranded RNA that is
complimentry to a messenger
RNA (target mRNA)
• Also known as micRNA (mRNA-
interfering complementary RNA
• In this technique Short
segments of single stranded
DNA (antisense gene) are
introduced.
• tool used for inhibition of gene
expression 9

10. Antisense RNA
• Disadvantages :-
– some genes could not be silenced at all
– the degree of silencing varied considerably
between cells and tissues within an experiment
– silenced cells tended to revert and lose silencing
after prolonged growth
10

11. Post transcriptional gene
silencing (PTGS)
• different names in different organism
– Post-transcriptional gene silencing (PTGS) - Plants
– Quelling - Fungi
– RNA interference (RNAi) – Animals
• commonly known as RNA interference (RNAi)
11

12. Post transcriptional gene
silencing (PTGS)
Post transcriptional gene silencing
(PTGS)
RNA interference
(RNAi)
Virus induced gene
silencing (VIGS)
Host induced gene
silencing (HIGS)
DNA interference
(DNAi)
12
RNAi Based techniques

13. RNA interference
(RNAi)
• highly conserved process of PTGS by which dsRNA causes
sequence-specific degradation of target mRNA
• selective gene knock-down phenomenon
Major component of RNAi
1. Small RNA
• siRNA
• miRNA
2. Dicer
3. RISC
13

14. siRNA
• These are dsRNA from
exogenous sources like viruses,
transgenes or transposons
• Dicer enzyme cut the larger
fragment of dsRNA into 20-25
nucleotides length
• have Symmetric 2nt 3’overhangs,
5’ phosphate groups
• Incorporated into the RISC
guiding it to mRNA
• Complementary to a specific
sequence of target mRNA for
degradation.
14

15. miRNA
• about 22 nucleotides (nt) in length, and are processed
by Dicer out of the hairpin (pre-miRNAs) of an
endogenous RNA
• miRNA regulates post-transcriptional gene expression
and is often not 100% complementary to the target
• they down-regulate/silent genes by
– mRNA degradation
– translational repression
15

16. miRNA biosynthesis
miRNAs are processed
through two steps:
1. generation of the ~70
nucleotide precursor
microRNAs from the
longer transcripts
(termed pri-miRNAs);
2. processing of pre-miRNAs
into mature miRNAs.
16

17. Differences between
miRNAs and siRNAs
Property miRNAs siRNAs
Definition Regulators of endogenous genes Defenders of genome integrity in response to
foreign or invasive nucleic acids
Length 20-22 nt 21-24 nt
Precursors Hairpin shaped ssRNAs Long dsRNAs
Nature of precursors Endogenous precursor gene of host’s
genome
Transposons, transgenes, repeat elements or
viruses, i.e., exogenous precursor
Argonaute required AGO1, AGO10 AGO1, AGO4, AGO6, AGO7
Mode of action mRNA degradation, translational
repression
DNA methylation, histone modification and
mRNA degradation
Mechanism of gene
regulation
Post-transcriptional only Transcriptional as well as post-transcriptional
Complementarity with target
sequences
Partially or fully complementary Fully complementary
Functions •Cell development
•cell differentiation,
•development processes,
•biotic and abiotic stress responses
Defense against transposons and viruses,
17

18. Dicer
• Dicer is an endo-ribonuclease in the RNase III family.
• It cleaves double-stranded RNA (dsRNA) into short
double-stranded RNA fragments called small
interfering RNA(siRNA) about 20-25 nucleotides long,
usually with a two-base overhang on the 3' end.
• Dicer catalyzes the first step in the RNA
interference pathway and initiates formation of
the RNA- induced silencing complex (RISC),
18

19. DICER’s domain
• 4 distinct domains:
1. Amino-terminal
helicase domain
2. Dual RNAse III motifs in
the carboxyl terminal
segment
3. dsRNA binding domain
4. PAZ domain (110-130
amino-acid domain
present in protein like
Argo, Piwi.);it is thought
to be important for
protein-protein
interaction 19

20. RNA-Induced Silencing Complex
(RISC)
• large multi-protein complex (~500-kDa) specifically
a ribonucleoprotein, which incorporates one strand
of a ssRNA fragment, such as miRNA, and double-
stranded siRNA
• Unwinding of double- stranded siRNA by ATP
independent helicase.
• The active components of an RISC are
endonucleases called argonaute proteins which
cleave the target mRNA strand.
20

21. Mechanism of RNAi
1. Initiation step:
– dsRNA molecule is cleaved to
form 21-23 bp double stranded
fragments called siRNA
2. Effector step:
– siRNA is unbound by helicase
activity associated with a
multiprotein complex known as
RNA-induced silencing
complex(RISC).
– The antisense RNA complexed
with RISC binds to its
corresponding mRNA which is
cleaved by the enzyme Slicer
rendering it inactive.
21

22. Virus induced gene
silencing (VIGS)
• defined as the silencing of endogenous plant genes
initiated by recombinant viral vectors
• It is one of the plant defense mechanisms against
invading viruses.
• VIGS utilizes the RNAi pathway in plants to induce
transient gene knock-down
• introduction of modified virus that also contains
fragment of endogenous gene sequence.
• Once expressed in vivo, dsRNAs are generated as the
virus replicates and spread through the plant
• dsRNAs using RNAi pathway silence the target gene
22

23. Method and molecular
mechanism of VIGS
Becker and Lange 200923

24. VIGS
Virus/virus type Silencing host species Genes
silenced
Natural host
species
Tobacco mosaic virus
(TMV)/RNA virus
Nicotiana benthamiana,
nicotiana tabacum
pds Tomato, squash, potato,
tobacco
Potato virus X
(PVX)/RNA virus
Arabidopsis, Nicotiana
benthamiana
pds Potato, oilseed, rape
Tobacco rattle virus
(TRV)/RNA virus
Nicotiana benthamiana,
tomato,
Arabidopsi,solanum species,
chilli pepper, opium poppy,
Aquilegia vulgaris
Rar1, EDS1,
NPR1/NIM1
pds, rbcS,
Spinach, beet,
potato, tobacco
Barly stripe mosaic virus
(BSMV) RNA virus
Barley pds, Lr21,
Rar1,
Sgt1, Hsp90
Barley, wheat
Unver et al. 2009
24

25. Host-induced gene silencing (HIGS)
• gene silencing strategy that involves
silencing the highly conserved genes of
pathogens by the host plants
• For HIGS sense-intron-antisense
hairpin construct is transformed in to
the host
• This lead to dsRNA production of
pathogen specific sequence
• Pathogens uptake siRNAs from host
(nutrient transport or vesicle
trafficking mechanism)
25

26. Host-induced gene silencing
• When the pathogen attacks the host expressing a HIGS
construct, the gene of interest can be down regulated in
the pathogen
• A. thaliana expressing a dsRNA targeting the rootknot
gene 16D10 led to effective disease resistance against
four major nematode species
Huang et al 2006
• expression of siRNAs targeted against two fungal genes in
transgenic banana to achieve effective resistance against
Fusarium oxysporum f. sp. Cubense
Ghag et al 2014
26

27. DNA interference (DNAi)
• Promoter-less dsDNA molecules can induce
sequence-specific gene silencing in plants
– amplification of the DNA by PCR
– cloning in suitable cloning vector.
– digested DNA fragments are then introduced into
plants by particle bombardment.
– transformed plants are then analyzed for silencing
of the target genes
27

28. Comparison of various
PTGS method
Methods Mechanism of
action
Size of
trigger
Method of
delivery in plants
Off targets Specific
advantages
Short comings
RNAi PTGS 250-350 bp
Vector-mediated /
plant
transformation
More Efficient
Off targets,
dependence on
plant
transformation
VIGS PTGS TGS 200-500 bp Virus -mediated
Vacuum
infiltration
More Quick
Off targets,
Transient, virus
interference
with plant
metabolism
HIGS PTGS -
Infiltration/plant
transformation
No off-targets
in host Specificity
Tedious, Silencing
efficiency
DNAi TGS -500 bp Particle
bombardment
- -
Pandey et al 2015
28

29. Transcriptional gene
silencing (TGS)
• TGS is the result of DNA methylation and histone
modifications
• Which create an environment of heterochromatin
around a gene that makes it inaccessible to
transcriptional machinery (RNA polymerase,
transcription factors, etc.)
Transcriptional gene silencing
(TGS)
DNA
methylation
Histone
modification
Genomic
imprinting
Paramutation Position Effect
Transposone
Silencing
RdDM
29

30. DNA METHYLATION
• process by which methyl groups are added to DNA
• cytosine and adenine, can be methylated but adenine
methylation is restricted to prokaryotes
• Occurs largely in the CG dinucleotide ( CpG islands when
found in high conc upstream of a gene)
• DNA methylation of the promoter region interferes with
binding of transcription factors thus suppressing gene
expression.
cytosine 5-methylcytosine
Methyltransferase
30

31. Histone modification/
chromatin remodeling
Histone
modification
Acetylation Methylation Phosphorylation Ubiquitination
31

32. Histone Acetylation
32
HAT- Histone acetyltransferases
HDAC- Histone deacetylaces

33. METHYLATED
DNA
DEACETYLATED
HISTONES
INHIBITION OF
TRANSCRIPTION
DEMETHYLATED
DNA
HISTONES
REMAIN
ACETYLATED
PROMOTION
OF
TRANSCRIPTION
33

34. Genomic imprinting
• Genomic imprinting refers to differential expression
of maternally versus paternally inherited alleles
Or
• genetic phenomenon by which certain genes are
expressed in a parent-of-origin-specific manner
• independent of the classical Mendelian inheritance
• found in insects, mammals and flowering plants
34

35. Genomic imprinting
• Example :- Igf2 (insulin growth factor 2)
35

36. Paramutation
• allelic interaction in which one allele
(paramutagenic) causes a heritable change in the
expression of a homologous (paramutable) allele
• Crossing a plant carrying a paramutable allele with
a plant carrying a paramutagenic allele results in
reduced gene expression of the susceptible allele.
36

37. Position effect
• Effect on the expression of
a gene when its location in
a chromosome is changed,
often by translocation/
inversion
• This has been well
described in Drosophila
with respect to eye colour
and is known as position
effect variegation (PEV).
37

38. Transposon silencing
• It is a form of transcriptional gene silencing targeting transposons.
• Transposon silencing is achieved through
– DNA methylation
 Molecular analyses of the Activator (Ac), Suppressor-mutator (Spm) and
Mutator (Mu) elements revealed that inactivation of these elements
was correlated with the methylation of their DNA.
Banks et al 1988
– Histone modification
 Histone deacetylase mutants in Arabidopsis such as hda6, has revealed
that several classes of TEs are transcriptionally activated
Lippman et al 2003
– small RNA-based pathways
 The endo-siRNA pathways have been shown to target heterochromatin
formation and transcriptional silencing of TEs in Drosophila
Fagegaltier et al 2009
38

39. RNA directed DNA
Methylation (RdDM)
RdDM
– dsRNA induced both TGS and PTGS (RNAi)
– de novo DNA methylation in plants
– confined to transposons and repeats suggests a
role for RNAi as a targeting mechanism for specific
sequence chromatin remodeling or TGS
– RdDM signal transmitted from the cytoplasm to
the nucleus is most likely siRNA
39

40. Souza et al 2007
Link between PTGS and TGS
40

41. Application in crop
improvement
Gene silencing
Biotic stress
Virus resistance
Fungus
resistance
Bacterial
resistance
Insect and
nematode
resistance
Abiotic stress
Drought and
salinity tolerance
UV radiation
stress tolerance
Cold and heat
stress tolerance
Quality
improvement
Enhanced nutrient
Allergen and
anti-nutrient elimination
Seedless fruit
development
(parthenocarpy
Enhanced shelf
life
Functional
genomics
Male Sterility
41

42. Biotic resistance
Traits improvement Targeted gene Plant Reference
Virus resistance
Bean Golden Mosaic Virus (BGMV) AC1 Bean Bonfim et al. (2007)
Barley Yellow Dwarf Virus (BYDV) BYDV-PAV Barley Wang et al. (2000)
Rice Dwarf Virus (RDV) PNS12 Rice Shimizu et al. (2009)
Turnip Mosaic Virus (TuMV) HC-Pro Tobacco Niu et al. (2006)
Insect resistance
Helicoverpa armigera CYPAE14 Cotton Mao et al. (2007)
Corn rootworm V-ATPase A Maize Baum et al. (2007)
Nematode resistance
Meloidogyne incognita
Splicing factor
and integrase Tobacco Yadav et al. (2006)
Meloidogyne incognita 16D10 Arabidopsis Huang et al. (2006)
Bacterial resistance
Xanthomonas citri subsp. citri (Xcc) PDS and CalS1 Lemon Enrique et al. (2011)
Agrobacterium tumefaciens iaaM and ipt Arabidopsis
Escobar et al. (2001),
Dunoyer
et al. (2006)
Fungal resistance
Magnaporthe grisea Xanthomonas oryzae OsSSI2 Rice Jiang et al. (2009)
Magnaporthe grisea OsFAD7 and OsFAD8 Rice Yara et al. (2007)
Phytophthora infestans SYR1 Potato Eschen-Lippold et al.
(2012)
Blumeria graminis f. sp. tritici MLO Wheat Riechen (2007)42

43. Abiotic resistance
Trait improved Target Plant Reference
Drought tolerance NFYA5 Arabidopsis Li et al. 2008
GmNFYA3
Soybean and
Arabidopsis
Ni et al. 2013
OsDSG1 Rice Park et al. 2010
Cold tolerance PCF5/PCF8 Rice Yang et al. 2013
Heat stress tolerance CSD1 CSD2 CCS Arabidopsis
Guan et al.
2013
43
Kamthan et al. 2015

44. Quality improvement
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
ZLKR/SD H Maize Lysine-fortified maize
Reduced alkaloid
production
CaMXMT1 Coffee Decaffeinated coffee
COR Opium poppy
Production of nonnarcotic alkaloid, instead of
morphine
CYP82E4 Tobacco
Reduced levels of the carcinogen nornicotine in
cured leaves
Reduced Polyphenol
production
a-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 44

45. Advantage of GS
• Down regulation of gene expression
• Easier than use of antisense oligo-nucleotides.
• siRNA more effective and sensitive at lower concentration.
• High Specificity
• blocking expression of unwanted genes and undesirable
substances.
• Inducing viral resistance
• Powerful tool for analysing unknown genes in sequenced
genomes.
• Useful approach in future gene therapy.
• Oligonucleotides can be manufactured quickly, some within one
week; the sequence of the mRNA is all that is needed
45

46. Disadvantages
• Delivery system:- getting those exquisitely specific
siRNAs to the appropriate sites in the appropriate
amounts to ensure appropriate uptake and the
intended silencing remains a considerable challenge
• Off target effect:- when siRNA can affect
unintended genes in the organism which may be
vital
46

47. Case Studies
RNAi-mediated disruption of a rice squalene synthase (SQS) by maize squalene synthase improves
drought tolerance at both the vegetative and reproductive stages.
47

48. Improved drought resistance of SQS RNAi rice at vegetative stage.
A. Transgenic plants showing less drying at 15 days after drought.
B. Recovery after 32 days drought and 2 days after re-watering
C. Reduced water loss of transgenic plants depicted by weightof soil+plant
D. Survival rate and number of green leaves retained by transgenics in comparison with WT
and negative controls.
48

49. Case study
• wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing
antisense sequences against target genes of the Fusarium head blight (FHB) fungus F.
culmorum caused a reduction of corresponding transcript levels in the pathogen and
reduced disease symptoms
• Stable transgenic wheat plants carrying an RNAi hairpin construct against the β-1, 3-
glucan synthase gene FcGls1 of F. culmorum showed enhanced FHB resistance in leaf
and spike inoculation assays under greenhouse and near-field conditions
49

50. Case studies
50
Avr3 gene is largely responsible for virulence of oomycete plant pathogen
Phytophthora infestans.
Avr3 gene of P. infestans is silenced through RNAi technology.
Transgenic plants induced moderate silencing of Avr3 resulting in moderate
resistance

51. conclusion
• it is the epigenetic regulation of gene expression and
widely used in agriculture and in biotechnology.
• Besides the all types of gene silencing the RNAi is
the important post transcriptional gene silencing.
• RNAi-mediated gene suppression approaches
provide direct way of crop improvement by
knocking-out the specific genes, resulting
insect/pest/pathogen resistance and enhanced
nutritional status
• VIGS is commonly used in some plant species for
quick and transient gene silencing
51

52. 52