Gene silencing is a process that regulates gene expression through transcriptional or post-transcriptional mechanisms. Virus-induced gene silencing (VIGS) is a technique used to study gene function in plants using viral vectors. The Tobacco rattle virus (TRV) is a commonly used VIGS vector that can be modified to carry fragments of target plant genes, inducing post-transcriptional gene silencing through small interfering RNAs (siRNAs). VIGS provides an efficient and rapid method to study gene function without genetic transformation of plants.

1. Presented by:
Mesele Tilahun
(Integ.1st year)
Advisor: MoonJae Sun
GENE
SILENCING
Jun 18, 2020
KRIBB,UST, S.Korea

2. Contents
INTRODUCTION TO GENE SILENCING
MOLECULAR MECHANISM OF VIGS
APPLICATION
SUMMARY

3. GENE SILENCING
• It is a general term describing epigenetic process of gene regulation
• It is used to describe the “switching off” of a gene by a mechanism other than
genetic mutation
• Gene are regulated by transcriptional or post-transcriptional level
INTRODUCTION

4. GENE SILENCING (level)
 TRANSCRIPPTIONAL GENE SILENCING. The result of histone
modifications, creating an environment of heterochromatin around a gene that
makes it in accessible to transcriptional machinery (RNA polymerase
transcription factors etc.)
 DNA methylation and chromatin remodeling- blocking gene expression
 POST TRANSCRIPTIONAL GENE SILENCING/PTGS. It is the result of
mRNA of a particular gene being destroyed or blocked.

5. About PTGS
• PTGS was first described in transgenic petunia and was named as “co-
suppressor of gene expression” (the molecular mechanism remain unknown )
• Later plant virologists made similar observation and called it “virus induced
gene silencing” (VIGS)
• Also named transgene silencing
• PTGS is now known as RNAi
• A mechanism: evolved for protection of plant from virus infection

6. Early Findings Of GS In Plants
• First discovered in plants by R. Jorgensen in1990
• When Jorgensen introduced a re-engineered gene into petunia that had a lot
of homology with an endogenous petunia gene, both genes became
suppressed!
(Stam et al., 1997)
Aim: to increase pigment, Result: loss of pigmentation in segments

7. That cosuppression in Petunia
overexpression of pigment
gene (enzyme for pigment
synthesis) caused loss of
pigmentation in flower sectors
dsRNA
Aberrant transcripts from overexpression
- formation of dsRNA
- Degradation to siRNAs that silence both
transgene and internal gene

8. Virus type Virus vector Virus genus Silenced hosts Genes silenced Natural host Reference
RNA virus
Tobacco mosaic virus (TMV) Tobamovirus Nicotiana
benthamiana,
nicotiana tabacum
pds Tomato, squash, potato, tobacco (Tran et al., 2016)
Potato virus X (PVX) Potexvirus Nicotiana
Benthamiana,
Arabidopsis
pds Potato, oilseed, rape (Ryu et al., 2004)
Tobacco rattle virus (TRV) Tobravirus Nicotiana
benthamiana, tomato,
Arabidopsi, solanum species, chilli
pepper, opium poppy,
Aquilegia vulgaris
Rar1, EDS1, NPR1/NIM1 pds, rbcS, Spinach, beet, potato, tobacco (Kirigia et al., 2014)
Barley stripe mosaic virus (BSMV) Hordeivirus Barley pds, Lr21, Rar1, Sgt1, Hsp90 Barley, wheat (Bennypaul et al.,
2012;(Gunupuru et al., 2019)
Bean pod mottle virus (BPMV) Comovirus Glycine max pds Phaseolus vulgaris,glycine max (Pflieger et al., 2013)
Pea early browning virus (PEBV) Tobravirus Pisum sativu,
Medicago truncatula,
Medicago truncatula,
pspds, uni, kor, pds Pisum sativum
Phaseolus,
vulgaris
(Pflieger et al., 2013)
Poplar mosaic virus (PopMV) Carlavirus Poplar gfp Nicotiana
Brome mosaic virus (BMV) Bromovirus Barley, rice, maize pds, actin 1, rubisco activase Barley (Singh et al., 2018)
Tomato bushy shunt virus (TBSV) Tombusvirus Nicotiana
benthamiana,
gfp Lycopersicon esculentum (Scholthof et al., 2011)
DNA virus
Cabbage leaf curl virus (CaLCuV) Begomovirus Arabidopsis CH42, pds Cabbage,
broccoli,
cauliflower
(Trejo-Saavedra et al., 2009)
African cassava mosaic virus
(ACMV)
Begomovirus Nicotiana
benthamiana,
Manihot esculenta
pds, su, cyp79d2 Manihot esculenta (Beyene et al., 2017)
Tomato yellow leaf curl China Virus
(TYLCV)
DNAbeta satellite DNA Nicotiana benthamiana,
Lycopersicon esculentum
N.glutinosa, N.tabacum
pcna, pds, su, gfp Tomato (Scholthof et al., 2011)
Tobacco golden mosaic virus
(TGMV)
Begomovirus Nicotiana
benthamiana,
su Tomato (Sunter et al., 1993)
Satellite virus Satellite tobacco mosaic virus
(STMV)
RNA satellite virus Nicotiana tabacum pds, rbcS, rbcL and various genes Nicotiana glauca, pepper (Tiwari et al., 2014)
Viruses used for silencing of genes and their hosts with targeted genes are listed

9. The genes used as markers for silencing system identification
Gene Function Silence phenotype
PDS phytonene desaturase Carotenoid synthesis related enzymes Leaf blade bleaching
pcna Proliferating cell
Nuclear antigen
Factor of DNA polymerase δ Meristems stop growing
ChlH magnesium chelatase Enzymes involved in chlorophyll
synthesis pathways
Leaves bright yellow
ChsA chalcone synthasa Enzyme of anthocyanin synthesis
pathway
A part or whole of a design or color that turns
white
su Sulfur gene Magnesium ion chelase in chlorophyll
synthesis pathway
Superior leaf
rpIIa RNA polymerase II a RNA synthesis Leaves twisted and whitened
gfp green fiuorescent protein Fluorescence occurs under ultraviolet
excitatio
The leaves appear red under ultraviolet light
luc luciferase Fluorescence occurs under ultraviolet
excitation
There was no fluorescence under ultraviolet
light

10. Molecular mechanism
(Courdavault et al. 2020)

11. Example of gene silencing
Experiments to modify flower color in
petunia gave early evidence of RNA
silencing.
Aim: Designing RNAi strategies based
on host derived resistance.
How Virus infection causes systemic
siRNA accumulation?
(Senthil-Kumar & Mysore, 2014)

12. Tobacco Rattle Virus (TRV)
 widely used VIGS vector to down regulate the expression of target gene.
 1ST modified viruses of VIGS used for effective pds gene silencing
 Bipartite, positive-strand RNA virus with TRV1 and TRV2genomes
 To induce PTGS, the TRV2 genome is genetically modified to carry a
fragment of the target gene and delivered into the plant (along with the
TRV1 genome) by agro-inoculation

13. Overview of the VIGS protocol in Selection of GOI fragment N. benthamiana
(Senthil-Kumar & Mysore, 2014)
Methods of TRV inoculation
Large scale expt
Small scale expt

14. Silencing the NbPDS gene in N. benthamiana plants by TRV-VIGS.
(Senthil-Kumar & Mysore, 2014)
photo bleached phenotype x-cs of PDS silencing
TRV2: GFP used as a vector control. GFP sequence
does not have any homology to plant DNA, and not
cause silencing

15. Plant-herbivore interaction
Plant-pathogen interaction
Plant symbiotic interaction
Plant development
Plant lethality
Evolution
Abiotic stress
TRV-VIGS approach in functional genomics

17.  GABA accumulates in many plant species in response to environmental stress.
Authors studied the 3 metabolic pathway controlling steps of GABA
Through VIGS approach
- glutamate decarboxylases (SlGADs),
- GABA transaminases (SlGABA-Ts) and
- succinic semi aldehyde dehydrogenase (SlSSADH),
(Bao et al., 2015)

18. The silecing of
-SlGADs- GABA biosynthetic genes
-SlGABA-Ts- GABA catabolic genes
accumulate (ROS) than control plants
salt sensitivity.
-SlSSADH-silenced plants accumulated
higher level of ROS under normal conditions
- less sensitive to salt stress
 These results suggest that GABA shunt is involved in salt
tolerance of tomato, probably by affecting the homeostasis of
metabolites such as succinate and γ-hydroxy butyrate and
subsequent ROS accumulation under salt stress.

19. Viral-induced Gene silencing
• rapid and highly efficient
• Simple to performed
• applicable to mature plants
• useful for species hard to transform
(Independent of Genetic
transformation)
• Suitable for analysis of large-scale
gene function and lethal phenotype
gene analysis
• Low cost
• host range limitations
• restricted regions of silencing
• viral symptoms superimposed
on silencing phenotype
• limited inoculation methods between
different plants and
• Difficulty in observing characteristics of
some genes after deletion
Advantages Limitation

20. Application
Courdavault et al. 2020

21. Summary
 Generally, VIGS is one of the reverse genetics tools for analysis of gene
function that uses viral vectors carrying a target gene fragment to produce
dsRNA which trigger RNA-mediated gene silencing.
 siRNAs interfere with viral replication.
 siRNAs act systemically to aid in host plant recovery and resistance
 While several VIGS vectors have been developed, TRV provides the most
robust results in terms of
- efficiency,
-ease of application, and
-absence of disease symptoms