WEL COME

Department of Plant Pathology

Rajasthan College of Agriculture,

Udaipur
Credit seminar

: M.Sc.(Plant Pathology)
Speaker
: Praveen Molekar
Registration No
: 14-01-02-13-56
Topic
: Virus Induced Gene Silencing
Post Graduate Programme

VIRUS INDUCED GENE

SILENCING

Content

Gene silencing
VIGS
Components of VIGS
Molecular mechanism of VIGS
Application of VIGS
Example of VIGS vector
Case studies
Limitations
Conclusion
4

GENE SILENCING:
Refers to the ability of a cell to prevent
the expression of a certain gene.
Gene silencing is often confused
withgene knockout.
Gene Silencing
Gene
Knockout

Gene expression is
Gene completely
reduced
erased from the
organism'sgenome
NCBI : National Center for Biotechnology Information
5

WHY GENE SILENCING..?

Pathogen-derived

resistance has also been

achieved through the expression of virus
sequences, the acquisition of resistance being
dependent on the transcribed RNA.

This

RNA mediated virus resistance can be

considered to be an example of posttranscriptional gene silencing (PTGS) in plants
(Prins et. al., 2008).

Napoli

et. al.,1990 firstly reported PTGS in

Petunia hybrida transgenically expressing the
chalcone synthase gene
7

 The silencing process involves the cleavage of a dsRNA

precursor into short (21-26 nucleotides) RNAs by an enzyme,
Dicer, that has RNase III domains.
These RNAs are known as short interfering RNAs (siRNA) and
microRNAs (miRNAs).
Both siRNA and miRNA are able to guide an RNA-induced
silencing complex (RISC) to destroy single-strand cognate RNA.
Longer siRNAs (24-26 nt) have been shown to result in
methylation of homologous DNA causing chromatin remodeling
and transcriptional gene silencing (TGS).
8

RNA silencing was first recognized as an

antiviral mechanism that protected
organisms against RNA viruses or the
random integration of transposable
elements.
However a general role for RNA silencing
in the regulation of gene expression only
became evident after it had been
demonstrated that specific short miRNAs
precursor molecules (foldback dsRNA)
were actively involved in RNA silencing in
plants and animals (Bartel, 2004; Naqvi
et. al., 2009).

TG
TGS
S

PTG
PTGS
S

10

 There are three RNA silencing pathways have

been described in plants (Baulcombe, 2004).

I.
II.
III.

Cytoplasmic siRNA silencing, important in virus

infected cells,
The silencing of endogenous mRNAs by miRNAs
DNA methylation and the suppression of
transcription

Sequence-specific DNA methylation (RNA-

directed DNA methylation RdDM) can be

induced by dsRNA molecules in various plant
systems and in response to various dsRNA
inducers.
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DNA methylation
Methylation of DNA and the
subsequent recruitment of binding
proteins that preferentially recognize
methylated DNA.
Histone deacetylase and chromatin
remodelling complexes to cause the
stabilization of condensed chromatin.
Eu chromatin
chromatin

Hetero
Hongmei Li-Byarlaya et.al,
12

Source: NCBI13

Agent
Most drugs

Mechanism

Remarks

Binds to many
protein

Protein inhibition

RNAase H
idepedent
ODNs
RNAase Hdependent
ODNs
Ribozymes and
DNA enzymes
SiRNA

Hybridize to target
mRNA

Inhibition of
translation of target
protein.

Hybridize to target
mRNA

Degradation of the
mRNA by RNAase H

Catalyse cleavage
of target mRNA

Degradation of the
mRNA

Hybridize to target
Degradation of the
m RNA by
mRNA
antisense strand
and guide it in endo
ribonuclease
enzyme complex
Source: NCBI14
(RISC)

Viral Induced Gene

Silencing
The term VIGS was coined by A. Van
Kammen to describe the resistance
event against viral infection.
VIGS

Post-Transcriptional Gene
Silencing
Mechanism.

15

VIGS
Virus-induced gene silencing
(VIGS) is a technology that
exploits an RNA-mediated
antiviral defence mechanism.
One important function of this
pathway in plants is in defence
against viruses, in which, viral
RNA acts as a trigger to induce

16

VIG
S
Based upon the phenomenon of
RNA-interference ( RNAi).
RNAi
interference in gene
expression, mediated by small RNA
in a sequence specific manner.

17

Cellular components needed for

VIGS
dsRNA, SiRNA, miRNA
VIGS Vector
RdRP-RNA dependent RNA polymerase
Sid-1 , Sid-2: Transfer Gene silencing
to next cells.
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 RNA-induced silencing complex

(RISC): Argonate protein (AGO) is a
core component and exhibits
structural similarity to RNase H.
dsRNA binding protein (DRB):
required for loading of small RNA into
RISC (Adenot et al, 2006; Nakazawa
et al, 2007).

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DICE
DICE
R:
R:

RNA double- strandedribonucleases

3 DICER proteins in
plants(DCLs)
DCL1- Production of miRNAs
DCL2-Cleaves dsRNAs from replicating
virus.
DCL3-Cleaves ds RNAs derived from
endogenous transcripts.

20

Argon
ate

21

22

Molecular mechanism of
VIGS
Viral vector having viral genome and
multiple cloning site To insert Target gene.
Plant inoculation with viral vector.
Production of ds RNA with the help of RdRp.
The ds RNA is cleaved into 20-25 nucleotide
siRNA .
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Formation of DICER Complex.

Unwinding of siRNA , sense strand degraded

and anti sense strand activated.

RISC complex with the help of antisense

strand recognises target mRNA.

Activation of DICER, cleaves the mRNA.

24

Length of mRNA
reduced.
No protein synthesis.
GENE IS SILENCED.

25

26

27

28

Successful application of
VIGS
In Nicotiana benthamiana silenced
the gene PHYTOENE DESATURASE (PDS).
PDS- governs carotenoid
biosynthesis pathway.
No carotenoids synthesis.
Reduced levels of photo protective
carotenoids lead to the rapid
destruction of chlorophyll by photo
oxidation.
29

Hybrid viral vector composed of

sequences from the tobacco
mosaic virus and the tomato
mosaic virus (TMV).
30

31

32

Turgay Unver and Hikmet Budak

33

The popularity of VIGS

The methodology is simple often involving
agro infiltration or biolistic inoculation of
plants.
The results are obtained rapidly typically
within two-three weeks of inoculation.
The technology by passes transformation
steps and
hence is applicable to number of plant
species
recalcitrant
to
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transformation.

Limitation of VIGS
Virus vector can be used only in plants
that are hosts of the virus used.
Ex: VIGS vectors (e.g.: PVX) do not
infect the model plant Arabidopsis
thaliana.
Single resistance genes
Highly virus isolate restricted (Specific)

35

1. Virus Induced Gene Silencing in

Tomato

Objectives:
Systemic transform of TRV vector
Silencing of the tomato PDS gene
using TRV-VIGS vector.
Silencing of CTR1 homology in
tomato.
Vector: TRV vector.
Gene silenced: PDS, CTR1 and CTR2
Yule Liu et.al,
36

 In Arabidopsis spp.

The CTR1 (constitutive triple response

1) gene encodes a mitogen-activated
protein kinase kinase kinase (MAPKKK)
that functions down stream of an
ethylene receptor and negatively
regulates the ethylene response
(Kieber et al., 1993).
In tomato 2 CTR 1 like proteins: t
CTR1 and
t CTR2
37

Infection of tomato plants with recombinant TRV alone (a)

or
TRV carrying the tomato PDS (TRV-tPDS) (b). Infection
with TRV-tPDS silences endogenous PDS in Micro-tom
tomato plants and causes inhibition of carotenoid
biosynthesis resulting in photo-bleaching phenotype (b).
38

2. Potential Application of VirusInduced Gene Silencing (VIGS) in

Flower Senescence Studies
Objectives:
To know the host range of TRV Vector
in ornamental plants.
To silence the CSH (chalcone
synthase) gene in Petunia.

Chenet.al
,
39

Materials and method

Vector: pTRV1 and pTRV2 (Tobacco
Rattle Virus) vector.
Plant species: Ornamental plants.
Petunia hybrida, Nicotiana
benthamiana.
Gene of interest: PDS, CHS.

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Mechanism
Silencing of anthocyanin production
in infected flower which is
responsible for purple color.
White (silenced) flowers or flower
sectors produced less ethylene and
senesced later than purple (nonsilenced) tissues.

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Hypothesis: sense RNA production

enhances pigmentation and antisense
RNA production blocks pigmentation
Endogenous gene
PRO

ORF

Transgene
Sense construct:
PRO

mRNA

Protein translated
mRNA
Extra protein translated

Sense RNA

ORF

mRNA
mRNA

Transgene
Antisense construct:
PRO

ORF

Antisense
RNA

Sense-antisense duplex forms

and prohibits translation

Surprisingly, both antisense and sense

gene constructs can inhibit pigment
production

Plants carrying CHS transgene

CaMV 35S pro : CHS

Sense

OR

CaMV 35S pro :

CHS

Antisense
Photo credit Richard Jorgensen

CONCLUSION

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