viruses fight back against rn ai technology
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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
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