Virus Induced Gene Silencing (VIGS)

8/19/2019 Virus Induced Gene Silencing (VIGS)

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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

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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&

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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

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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&

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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 &

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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;&

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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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Virus Induced Gene Silencing (VIGS)

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