This document discusses post-transcriptional gene silencing (PTGS) in plants. It describes key discoveries such as Jorgensen discovering PTGS in 1990 by observing unexpected white color sectors in transgenic plants aimed at increasing pigment production. It also discusses mechanisms of RNA interference (RNAi) such as the two major pathways of microRNA and small interfering RNA (siRNA). Additionally, it examines gene silencing induced by geminiviruses and transgenes in plants. Various constructs and viral proteins that affect RNA silencing suppression are also reviewed.

1. Gaykar Amruta B.

2. PTGS in plants:
Discovery of Jorgensen in 1990
 AIM: upregulate activity of chsA
Expected:more pigments
Observed:white sectors
Loss of mRNAs of both
endo-and transgene
(Etienne et al., 2006)

3. RNA interference – gene silencing by double-stranded
RNA:The 2006 Nobel Prize for Physiology or Medicine
CURRENT SCIENCE, VOL. 91, NO. 11, 10 DECEMBER 2006

4. Mechanism of RNAi
 Two major pathways
involved
miRNA
Control of endogenous
gene expression
siRNA
Defense against viruses
and transposons
(Lindbo et al., 2005)

5. Geminivirus induced gene silencing in plants
(Ramachandran et. al., 2005)

6. Transgene directed gene silencing in plans
 Sense
 Antisense
 Sense- Antisense
(Waterhouse and Helliwell ,2003)

8. Introduction
Four major tomato-infecting tospoviruses
 TSWV
 Groundnut ringspot virus (GRSV)
 Tomato chlorotic spot virus (TCSV) and
 Watermelon silver mottle virus (WSMoV), in a single small
chimaeric hairpin (hp) RNA construct

9. Constructs used in gene silencing

10. Results

12. Suppression of RNA silencing by viral proteins

13. (Miguel, 2007)

15. SGS3-mediated generation of siRNAs

21. Genome organization of RCNMV

22. RNA1 is essential for suppressing RNAi

25. Introduction
 BBTV, a circular single-stranded DNA virus
 Genus- Babuvirus
 Family- Nanoviridae
 Multi-segmented genome
 B3 and B4,encoded by DNA3 and DNA4 of virus(BBTV),
exhibit RNA silencing suppression activity

26. Materials and Methods
 Plasmids and Agrobacterium
 The ORF fragments B1, B3, B4, B5 and B6 of BBTV
DNA1, DNA3, DNA4, DNA5, DNA6 were obtained by
PCR using primers
 The obtained fragments were inserted into pND108
 Agroinfiltration and GFP imaging
 Western blot analysis

27. Transcription cassettes for Agroinfiltration

28.  BBTV B3 , B4 and B5
enhance the pathogenicity
of chimeric PVX
N. benthamiana leaves infected with
chimeric PVX at 14 days post
infiltration (dpi)

29. Western blot analysis
Accumulation of PVX CP in N. benthamiana leaves

30. N. benthamiana leaves infected
with chimeric PVX under UV
light at 5 dpi GFP accumulation detected by western
blot analysis with anantibody specific for
GFP at 5 dpi
B3 and B4 reverse established RNA silencing of GFP

31. Results:14 dpi
 BBTV B4, but not B3 protein, suppresses
systemic gene silencing in newly emerged
leaves