Gaykar Amruta B.

PTGS in plants:PTGS in plants:

Discovery of Jorgensen inDiscovery of Jorgensen in 1990 1990

AIM: upregulate activity of chsA

Expected:more pigments

Observed:white sectors

Loss of mRNAs of both

endo-and transgene

(Etienne et al., 2006)

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

Mechanism of RNAiMechanism of RNAi

Two major pathways involvedmiRNA

Control of endogenous gene expression

siRNA

Defense against viruses and transposons

(Lindbo et al., 2005)

Geminivirus induced gene silencing in plantsGeminivirus induced gene silencing in plants

(Ramachandran et. al., 2005)

Transgene directed gene silencing in plansTransgene directed gene silencing in plans

Sense

Antisense

Sense- Antisense

(Waterhouse and Helliwell ,2003)

IntroductionIntroduction

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

Constructs used in gene silencingConstructs used in gene silencing

ResultsResults

Suppression of RNA silencing by viral proteinsSuppression of RNA silencing by viral proteins

(Miguel, 2007)

SGS3-mediated generation of siRNAsSGS3-mediated generation of siRNAs

Genome organization of RCNMVGenome organization of RCNMV

RNA1 isRNA1 is essential for suppressing RNAi essential for suppressing RNAi

IntroductionIntroduction

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

Materials and MethodsMaterials 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

Transcription cassettes for AgroinfiltrationTranscription cassettes for Agroinfiltration

BBTV B3 , B4 and B5 enhance the pathogenicity of chimeric PVX

N. benthamiana leaves infected with chimeric PVX at 14 days post infiltration (dpi)

Western blot analysisWestern blot analysis

Accumulation of PVX CP in N. benthamiana leaves

B3 and B4 reverse established RNA silencing of GFPB3 and B4 reverse established RNA silencing of GFP

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

Results:14 dpiResults:14 dpi

BBTV B4, but not B3 protein, suppresses systemic gene silencing in newly emerged leaves