This document summarizes a seminar on viral-induced gene silencing presented by Sushila Choudhary. The seminar covered the definition of gene silencing, a short history of post-transcriptional gene silencing discovered in the 1990s, the mechanism of RNA interference, applications of RNAi in modulating viral replication and development, and the use of virus-induced gene silencing (VIGS) in crop improvement including developing barley varieties resistant to barley yellow dwarf virus. The advantages and disadvantages of gene silencing techniques were also discussed.

1. Seminar Incharge:-
Dr.Pratibha Sharma
(Emeritus Scientist)
Speaker:-
Sushila Choudhary
Ph.D Scholar
(Plant Pathology)
S.K.N. College of Agriculture, Jobner
Course Seminar
On
VIRALINDUCED
GeneSilencing

2. Welcome
Course Seminar
on VIRAL INDUCED
Gene Silencing

3. Gene Silencing
• Gene silencing is the regulation of gene expression
in a cell to prevent the expression of a certain gene.
• Gene silencing is same as gene knock down but is
totally different from gene knock out.
• When genes are silenced, their expression is
reduced. In contrast, when genes are knocked out,
they are completely erased from the organism's
genome and, thus, have no expression.

4. Short history of post-transcriptional gene silencing
Definition: the ability of exogenous double-stranded RNA
(dsRNA) to suppress the expression of the gene which
corresponds to the dsRNA sequence.
Term VIGS first coined by A. Van Kammen to describe
resistance against viral infection (Kammen,1997).
1990 Jorgensen :
Introduction of transgenes homologous to endogenous genes
often resulted in plants with both genes suppressed!
Called Co-suppression
Resulted in degradation of the endogenous and the
transgene mRNA

5. 1995 Guo and Kemphues:
-injection of either antisense or sense RNAs in the
germline of C. elegans was equally effective at
silencing homologous target genes
1998 Mello and Fire:
-extension of above experiments, combination of
sense and antisense RNA (= dsRNA) was 10
times more effective than single strand RNA
Contd….

6. Types of Gene silencing
• Genes are regulated at either the transcriptional level or
post-transcriptional level, therefore silencing can be induced
either at transcriptional level or post transcriptional level.
• There are mainly two types of gene silencing:
1.Transcriptional gene silencing
2.Post transcriptional gene silencing

8. Other names of post-transcriptional gene
silencing (PTGS) :
– gene silencing
– RNA silencing
– RNA interference
– In certain fungi: quelling
RNAi can spread throughout certain organisms
(Caenorhabditis elegans, plants).

9. What is RNA interference /PTGS?
dsRNA needs to be directed against an exon, not an
intron in order to be effective
homology of the dsRNA and the target gene/mRNA is
required
targeted mRNA is lost (degraded) after RNAi
the effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA

10. Why is PTGS important?
Most widely held view is that RNAi evolved to
protect the genome from viruses (or other
invading DNAs or RNAs)
Recently, very small (micro) RNAs have been
discovered in several eukaryotes that regulate
developmentally other large RNAs
–May be a new use for the RNAi mechanism
besides defense

11. Recent applications of RNAi
Modulation of HIV-1 replication by RNA interference.
Hannon(2002).
Potent and specific inhibition of human immunodeficiency
virus type 1 replication by RNA interference.
An et al.(1999)
Selective silencing of viral gene expression in HPV-positive
human cervical carcinoma cells treated with siRNA, a primer
of RNA interference.
Jung et al. 2002.
Successful inactivation of endogenous Oct-3/4 and c-mos
genes in mouse pre implantation embryos and oocytes using
short interfering RNAs.
Le Bon et al.2002

12. Uses of VIGS
Some virus species were previously modified and used
for silencing the gene of interest. Tobacco mosaic virus
(TMV) is one of the modified viruses which was used for
effective pds gene silencing in Nicotiana benthamiana
plants [Kumagai et al., 1995].
TMV is the first modified virus for application of VIGS
methods to plants. The viral delivery leads
downregulation of transcript of target gene through its
homology dependent degradation so potential of VIGS
for analysis of gene function was easily recognized
[Boulcombe, 1999].

13. Advantages of Gene Silencing
Downregulation of gene expression simplifies
"knockdown" analysis.
Cost effective
 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.

14. Disadvantages of Gene Silencing
 High pressure injection” and electroporation can cause
significant injection damage to the integrity of the normal
tissues and organs.
Liposomes/cationic encapsulated Si RNA may also be toxic
to the host and may cause severe host immune responses.

15. Application of Gene Silencing in crop
improvement

16. Virus resistance :

17. Developed varieties of
Barley that resistance to BYDV
(Barley Yellow Dwarf Virus)
through the RNAi technology
using hpRNA as a construct
(Gould and Krammer, 2007).

18. Conclusion
• It is the epigenetic regulation of gene expression and
widely used in agriculture and biotechnology.
• Besides the all types of gene silencing the RNAi is
the important post transcriptional gene silencing.

19. References
• A. van Kammen, “Virus-induced gene silencing in infected and
transgenic plants,” Trends in Plant Science, vol. 2, no. 11, pp.
409–411, 1997.
• B. Gould and E. M. Kramer, “Virus-induced gene silencing as a
tool for functional analyses in the emerging model plant
Aquilegia (columbine, Ranunculaceae),” The Plant Methods,
vol. 3, no. 1, article 6, pp. 1–12, 2007.
• R. Lu, A. M. Malcuit, J. R. Mart ´ ın-Hern´andez, I. Malcuit, and
D. C. Baulcombe, “Virus-induced gene silencing in plants,”
Methods, vol. 30, no. 4, pp. 296–303, 2003.

20. Thank
You