Virus Induced Gene Silencing (VIGS)
Virus Induced Gene Silencing (VIGS)
Virus Induced Gene Silencing (VIGS)
: M.Sc.(Plant Pathology)
Speaker
: Praveen Molekar
Registration No
: 14-01-02-13-56
Topic
: Virus Induced Gene Silencing
Post Graduate Programme
Content
Gene silencing
VIGS
Components of VIGS
Molecular mechanism of VIGS
Application of VIGS
Example of VIGS vector
Case studies
Limitations
Conclusion
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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
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Pathogen-derived
This
Napoli
TG
TGS
S
PTG
PTGS
S
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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,
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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)
Post-Transcriptional Gene
Silencing
Mechanism.
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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
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VIG
S
Based upon the phenomenon of
RNA-interference ( RNAi).
RNAi
interference in gene
expression, mediated by small RNA
in a sequence specific manner.
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DICE
DICE
R:
R:
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Argon
ate
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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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Length of mRNA
reduced.
No protein synthesis.
GENE IS SILENCED.
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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.
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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)
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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,
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In Arabidopsis spp.
Chenet.al
,
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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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ORF
Transgene
Sense construct:
PRO
mRNA
Protein translated
mRNA
Extra protein translated
Sense RNA
ORF
mRNA
mRNA
Transgene
Antisense construct:
PRO
ORF
Antisense
RNA
OR
CHS
Antisense
Photo credit Richard Jorgensen
CONCLUSION
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