Ti plasmid ss

Ti plasmid

1. Ti plasmid
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. Nature’sgeneticengineer
1.Agrobacteriumisconsideredasthenature’sgeneticengineer.
2.Agrobacteriumtumefaciensisarodshaped,gramnegativebacteriafoundin
thesoilthatcausestumorousgrowthtermedascrowngalldiseaseindicot
plants.
3.TheinvolvementofbacteriainthisdiseasewasestablishedbySmithand
Townsend(1907).
4.AgrobacteriumcontainsatransferDNA(T-DNA)locatedinitstumor-inducing
(Ti)plasmidthatistransferredintothenucleusofaninfectedplantcell.
5.TheT-DNAgetsincorporatedintotheplantgenomeandissubsequently
transcribed.TheT-DNAintegratedintotheplantgenomecarriesnotonly
oncogenicgenesbutalsoopinesynthesizinggenes.
Agrobacterium“Species”AndHostRange
ThegenusAgrobacteriumhasbeendividedintoanumberofspeciesonthe
basisofsymptomsofdiseaseandhostrange.
A.radiobacterisan“avirulent”species,
A.tumefacienscausescrowngalldisease,
A.rubicausescanegalldisease,
A.rhizogenescauseshairyrootdiseaseand
A.vitiscausesgallsongrapeandafewotherplantspecies

3. MolecularbasisofAgrobacterium-mediatedtransformation
Ti-plasmid
ThevirulentstrainsofA.tumefaciensharborlargeplasmids(140–235kbp)knownas
tumor-inducing(Ti)plasmidinvolvingelementslike
A.T-DNA–LeftborderandRightborder,Auxinandcytokininandopinesynthesizing
genes
B.virregion-GenesinthevirulenceregionaregroupedintotheoperonsvirABCDEFG,
whichcodefortheenzymesresponsibleformediatingconjugativetransferofT-DNAto
plantcells.
C.originofreplication,
D.regionenablingconjugativetransferand
E.o-catregion(requiredforcatabolismofopines).
•Theplasmidhas196genesthatcodefor195proteins.
•Theplasmidis206,479nucleotideslong,theGCcontentis56%and81%ofthematerialis
codinggenes.
•Therearenopseudogenes.

4. •TheTiplasmidislostwhenAgrobacteriumisgrownabove28°C.
•Themodificationofthisplasmidisveryimportantinthecreationof
transgenicplants.
A.)T-DNA(TransferDNA)
1.Itisasmall,specificsegmentoftheplasmid,about24kbinsize
andfoundintegratedintheplantnuclearDNAatrandomsite.
ThisDNAsegmentisflankedbyrightandleftborders.
2.Thisregionhasthegenesforthebiosynthesisofauxin(aux),
cytokinin(cyt)andopine(ocs),andisflankedbyleftandright
borders.Itisclearlyestablishedthattherightborderismore
criticalforT-DNAtransfer.
3.TheT-DNAcontainstwogroupsofgenes,whichpossessthe
abilitytoexpressinplantsasfollows-
i.Oncogenesforsynthesisofauxinsandcytokinins
(phytohormones).Theoverproductionofphytohormonesleads
toproliferationofcallusortumourformationinplants.
ii.Opinesynthesizinggenesforthesynthesisofopines.Thus
opinesactassourceofnutrientforbacterialgrowth,e.g.
Octopine,Nopaline.

5. The functions of T-DNA genes are listed:

6. B)Virulencegenes(virgenes)
1.VirulencegenesaidinthetransferofT-DNAintothehostplantcell.
2.Tiplasmidcontains35virgenesarrangedin8operons.
3.Atleastninevir-geneoperonshavebeenidentified.TheseincludevirA,virG,virB1,vir
C1,virD1,D2,virD4andvirE1,E2.
4.TransfertheT-DNAtoplantcell
5.Acetosyringone(AS)(aflavonoid)releasedbywoundedplantcellsactivatesvirgenes.
6.virA,B,C,D,E,F,G(7complementationgroups,butsomehavemultipleORFs),spanabout
30kbofTiplasmid.
Functionofvirgenes
virA-transportsacetosyringone(AS)intobacterium,activatesvirGpost-translationally(by
phosphorylation)
virG-promotestranscriptionofothervirgenes
virD2-endonuclease/integrasethatcutsT-DNAatthebordersbutonlyononestrand.
virE2-canformchannelsinmembranes
virE1-chaperoneforvirE2
virD2&virE2alsohaveNLSs,getsT-DNAtothenucleusofplantcell
virB-operonof11proteins,getsT-DNAthroughbacterialmembranes

7. C)Opines:
DerivativesofaminoacidssynthesizedbyT-DNA.
Thisregioncodesforproteinsinvolvedintheuptakeandmetabolismsofopines.
Tiplasmidscanbeclassifiedaccordingtotheopinesproduced:
1.Nopalineplasmids-carrygeneforsynthesizingnopalineintheplantandfor
utilization(catabolism)inthebacteria.
2.Octopineplasmids-carrygenestosynthesizeoctopineintheplantandcatabolismin
thebacteria.
3.Agropineplasmids-carrygenesforagropinesynthesisandcatabolism.
Opinegenescanbeusedasmarkergenes-
Forscreeningoftransformantsor
recombinants

9. DNA transfer into the plant genome
Ti plasmid
Ti Plasmid

10. Tiplasmidbasedvectors:
1.DisarmedTi-plasmidderivativesasplantvectors
2.BinaryVectors
3.C0-integratevectors
1.DisarmedTivector
1.Tiplasmidisanaturalvectorforgeneticallyengineeringplantcellsduetoitsabilityto
transferT-DNAfromthebacteriumtotheplantgenome.
2.Butwild-typeTiplasmidsarenotsuitableasvectorsduetothepresenceofoncogenes
inT-DNAthatcausetumorgrowthintherecipientplantcells.
3.Forefficientplantregeneration,vectorswithdisarmedT-DNAareusedbymakingit
non-oncogenicbydeletingallofitsoncogenes.
4.TheforeignDNAisinsertedbetweentheRBandLBandthenintegratedintotheplant
genomewithoutcausingtumors.

11. Construction:StructureoftheTi-plasmidpGV3850withdisarmed
T-DNA
1.Zambryskietal.(1983)substitutedpBR322sequencesintheT-DNAofpTiC58,without
disturbingtheleftandrightborderregionsandthenosgene.
2.TheresultingconstructwascalledpGV3850.Notumourcellformationtakesplace
whenmodifiedT-DNAistransferredfromAgrobacteriumcarryingpGV3850plasmid.
3.Theevidenceoftransferisdonebyscreeningthecellsfornopalineproduction.
Drawbacks
Severaldrawbacksareassociatedwith
disarmedTi-vectorsystemsare:
•Necessitytocarryoutenzymaticassayson
allpotentialtransformants.
•Notconvenientasexperimentalgene
vectorsduetolargesize.
•Difficultyininvitromanipulationand
•Absenceofuniquerestrictionsitesinthe
T-DNA.

12. 2.Binaryvector
•BinaryvectorwasdevelopedbyHoekmaetal(1983)andBevanin(1984).
•Itutilizesthetrans-actingfunctionsofthevirgenesoftheTi-plasmidandcanactonanyT-
DNAsequencepresentinthesamecell.
•Binaryvectorcontainstransferapparatus-helperplasmid(containsthevirgenes)andthe
disarmedT-DNAcontainingthetransgeneonseparateplasmids.
Binaryvectorsystem
Binaryvectorconsistsofapairofplasmids:
1)AdisarmedTiplasmid:ThisplasmidhasT-DNAcontainingLBandRBwithgeneofinterest+
oriforbothE.coliandAgrobacterium.Alsocalledasmini-TiormicroTiplasmideg:Bin19.
2)HelperTiplasmidhasvirulenceregionthatmediatestransferofT-DNAinmicroTiplasmidto
theplant.
Plasmid with disarmed
T-DNA but without
virgene
Plasmid with virregion but
without G0I

13. A binary vector system
Plasmid with disarmed T-
DNA but without virgene
Plasmid with virregion but
without G0I

14. Binary vector Cloning Strategy:
1. Move T-DNA onto a separate, small
plasmid.
2. Remove auxand cytgenes.
3.Insert selectable marker (kanamycin
resistance) gene in T-DNA.
4. Virgenes are retained on a separate
plasmid.
5. Put foreign gene between T-DNA
borders.
6. Co-transform Agrobacteriumwith both
plasmids.
7. Infect plant with the transformed
bacteria.
Examples of Binary vector system
pBIN19-one of the first binary vectors developed in 1980s and was widely used.
pGreen-A newly developed vector with advanced features than pBIN19.

15. 3. Co-integrate vectors
Transferisachievedusinga‘triparentalmating’inwhichthreebacterialstrainsaremixed
together:
(i)AnE.colistraincarryingahelperplasmidabletomobilizetheintermediatevectorin
trans;
(ii)TheE.colistraincarryingtherecombinantintermediatevector;
(iii)A.tumefacienscarryingtheTiplasmid
1.InfirstE.colistrain:TheDNAtobeintroducedintotheplanttransformationvectoris
subclonedinaconventionalEscherichiacoliplasmidvectorforeasymanipulation,
producingaso-calledintermediatevector.Thesevectorsareincapableofreplication
inA.tumefaciensandalsolackconjugationfunctions.
2.---AnanotherE.colistraincarryingahelperplasmidabletomobilizethe
intermediatevector.
3.ConjugationbetweenthetwoE.colistrainstransfersthehelperplasmidtothe
carrieroftheintermediatevector,whichinturnismobilizedandtransferredtothe
recipientAgrobacterium.
4.HomologousrecombinationbetweentheT-DNAsequencesoftheTiplasmidand
intermediatevectorformsalargeco-integrateplasmidresultinginthetransferof
recombinantT-DNAtotheplantgenome.

16. Construction of a Co-integrate vector (foreign gene cloned into an appropriate plasmid is
integrated with a disarmed Ti-plasmid through homologous recombination).
1.At first; an intermediate vector is made using E. coliplasmid + origin of replication +
pBR322 sequences + some markers + gene of interest.
2.-Second vector is a disarmed pTivector = Left and right borders+ some markers +
pBR322 sequences + virregion.
3.-Both intermediate vector and disarmed pTihas some sequences in common (pBR322
sequences).
4.-Therefore by homologous recombination, co-integration of two plasmids will take
place within Agrobacterium.
5.Now we have a co-integrate vector that has both T-DNA with our gene of interest with
in the T-DNA borders and virregion. This complete vector is used for transformation in
plant cells. eg: pGV2260.
in
Agrobac
terium
tumefac
iens