Post on 07-Jul-2018
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WEL COME
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Rajasthan College of Agriculture,
Udaipur
Credit seminar
• Post Graduate Programme : M.Sc.(Plant Pathology)• Speaker : Praveen Molekar
• Registration No : 14-01-02-13-!
• Topic : "ir#s $nd#ced %ene Silencing
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VIRUS INDUCED GENE
SILENCING
3
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Content
• Gene silencing
• VIGS
• Components of VIGS
• Molecular mechanism of VIGS
• Application of VIGS
• Eample of VIGS !ector
• Case studies• "imitations
• Conclusion
#
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GENE SILENCING:
• Refers to the a$ilit% of a cell to pre!entthe epression of a certain gene&
• Gene silencing is often confused
'ith gene knockout. Gene Silencing Gene
)noc*out
Gene epression is Gene completel%
reduced erased from the
organism+s genome -C.I / -ational Center for .iotechnolog% Informatio
http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/
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012 GE-E SI"E-CI-G&&4
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Pathogen-derived resistance has also beenachieved through the expression of virus
sequences, the acquisition of resistance beingdependent on the transcribed RNA.
This RNA mediated virus resistance can be
considered to be an example of post-transcriptional gene silencing (PT!" in plants(Prins et. al., #$$%".
Napoli et. al.,&''$ firstl reported PT! inPetunia hybrida transgenicall expressing thechalcone snthase gene
)
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• &he silencing 'rocess involves the cleavage o a ds*+
'rec#rsor into short (21-2! n#cleotides) *+s ,y an enzyme,
Dicer, that has RNase III domains.
• &hese *+s are knon as short interering *+s (si*+) and
micro*+s (mi*+s).
• oth si*+ and mi*+ are a,le to g#ide an *+-ind#ced
silencing com'le/ ($SC) to destroy single-strand cognate *+.
• onger si*+s (24-2! nt) have ,een shon to res#lt inmethylation o homologo#s *+ ca#sing chromatin remodeling
and transcri'tional gene silencing (&%S).8
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RNA silencing *as first recogni+ed as anantiviral mechanism that protected
organisms against RNA viruses or therandom integration of transposableelements.
o*ever a general role for RNA silencing
in the regulation of gene expression onlbecame evident after it had beendemonstrated that specific short miRNAsprecursor molecules (foldbac dsRNA"
*ere activel involved in RNA silencing inplants and animals (artel, #$$/0 Naqviet. al., #$$'".
'
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GS PGS
67
GS
PGS
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8here are three R-A silencing path'a%s ha!e$een descri$ed in plants 9.aulcom$e, :77#;&
I& C%toplasmic siR-A silencing, important in !irusinfected cells,
II& 8he silencing of endogenous mR-As $% miR-As
III& speci?c directed
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63Source/ -C.I
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!gent "echan#$m Remark$
"o$t %rug$ .inds to man%protein
Brotein inhi$ition
RN!a$e & '#%epe%ent(DN$
1%$ridie to targetmR-A
Inhi$ition oftranslation of targetprotein&
RN!a$e &)
%epen%ent(DN$
1%$ridie to target
mR-A
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V#ral In%uce% GeneS#lenc#ng
• 8he term VIGS 'as coined $% !. Van,ammen to descri$e the resistancee!ent against !iral infection&
• VIGS Bost>8ranscriptional Gene
SilencingMechanism&
-6
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VIGS
• Virus>induced gene silencing9VIGS; is a technolog% thateploits an R-A>mediatedanti!iral defence mechanism&
• ne important function of thispath'a% in plants is in defenceagainst !iruses, in 'hich, !iralR-A acts as a trigger to induce
65
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VIGS
• .ased upon the phenomenon ofR-A>interference 9 R-Ai;&
R-Ai interference in geneepression, mediated $% small R-Ain a se=uence speci?c manner&
6D
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Cellular components needed for
VIGS
• dsR-A, SiR-A, miR-A
• VIGS Vector
• RdRB>R-A dependent R-A pol%merase
• Sid>6 , Sid>:/ 8ransfer Gene silencingto net cells&
6
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• R-A>induced silencing comple9RISC;/ Argonate protein 9AG; is acore component and ehi$itsstructural similarit% to R-ase 1&
• dsR-A $inding protein 9
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DICER: R-A HHH dou$le> stranded>ri$onucleases
DICER prote#n$ #nplant$/DCL$0
Clea!es dsR-As from replicating!irus&
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:6
!rgonate
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::
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Molecular mechanism ofVIGS
• Viral !ector ha!ing !iral genome andmultiple cloning site 8o insert 8arget gene&
Blant inoculation 'ith !iral !ector&
Broduction of ds R-A 'ith the help of RdRp&
8he ds R-A is clea!ed into :7>: nucleotidesiR-A &
:3
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ormation of
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"ength of mR-Areduced&
-o protein s%nthesis&
GENE IS SILENCED.
:
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:5
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:D
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:
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Successful application ofVIGS
• In Nicotiana benthamiana silencedthe gene B128E-E
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1%$rid !iral !ector composed of
se=uences from the to$accomosaic !irus and the tomatomosaic !irus 98MV;&
37
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36
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3:
d i* d *
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33
8urga% Un!er and 1i*met .uda*
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he popular#ty of VIGS
8he methodolog% is simple often in!ol!ing agro in?ltration or $iolistic inoculation ofplants&
8he results are o$tained rapidl% t%picall%'ithin t'o>three 'ee*s of inoculation&
8he technolog% $% passes transformationsteps and
hence is applica$le to num$er of plantspecies recalcitrant to
transformation& 3#
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"imitation of VIGS
• Virus !ector can $e used onl% in plantsthat are hosts of the !irus used&
• E/ VIGS !ectors 9e&g&/ BVJ; do notinfect the model plant Ara$idopsisthaliana&
• Single resistance genes
• 1ighl% !irus isolate restricted 9Speci?c;3
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6& Virus Induced Gene Silencing in 8omato
• $jecti!es/
• S%stemic transform of 8RV !ector
• Silencing of the tomato BVIGS !ector&
• Silencing of C8R6 homolog% intomato&
• Vector/ 8RV !ector&
• Gene silenced/ B
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In Arabidopsis spp&
• 8he C8R6 9constituti!e triple response
6; gene encodes a mitogen>acti!atedprotein *inase *inase *inase 9MAB)));that functions do'n stream of aneth%lene receptor and negati!el%regulates the eth%lene response9)ie$er et al&, 63;&
• In tomato : C8R 6 li*e proteins/ tC8R6 and
t C8R:
3D
:
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• Infection of tomato plants 'ith recom$inant 8RV alone 9a;or
• 8RV carr%ing the tomato BtB
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:& Botential Application of Virus>Induced Gene Silencing 9VIGS; in
lo'er Senescence Studies
$jecti!es/
• 8o *no' the host range of 8RV Vectorin ornamental plants&
• 8o silence the CS1 9chalcones%nthase; gene in Petunia.
3
Chen et.al3
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Materials and method
• Vector/ p8RV6 and p8RV: 98o$accoRattle Virus; !ector&
• Blant species/ rnamental plants&Petunia hybrida, Nicotianabenthamiana.
• Gene of interest/ B
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Mechanism
• Silencing of anthoc%anin productionin infected Ko'er 'hich isresponsi$le for purple color&
• 0hite 9silenced; Ko'ers or Ko'ersectors produced less eth%lene andsenesced later than purple 9non>
silenced; tissues&
#6
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Sense RNA
Antisense
RNA
Sense construct:
PRO ORF
Endogenous genemRNA
Transgene
PRO ORF
mRNA
Protein translated
mRNA
mRNA
Extra protein translated
Antisense construct:
PRO ORF
Transgene Sense-antisense duplex forms
and prohibits translation
Hypothesis: $en$e RN! pro%uct#onenhance$ p#gmentat#on an% ant#$en$eRN! pro%uct#on *lock$ p#gmentat#on
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Surprisingly, both antisense and sense
gene constructs can inhibit pigment
production
Photo credit ichard orgensen
Plants carrying CHS transgene
Ca! "#S pro : CHS Ca! "#S pro : CHS
Sense Antisense
OR
http://ag.arizona.edu/pls/faculty/jorgensen.htmlhttp://ag.arizona.edu/pls/faculty/jorgensen.html
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C(NCLUSI(N
##
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