PowerPoint Presentation : AGROBACTERIUM MEDIATED GENE TRANSFER IN PLANTS Part-I Agrobacterium – a natural genetic engineer Part-II Harnessing Agrobacterium in transferring foreign gene into plant By Dr Ichha Purak http://www.dripurak.com http://www.drichhapurak.webnode.com 1/4/2012 1 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : The introduction of desired/foreign /recombinant DNA into plants has enormous potential for agriculture, producing more nutritious and higher-yielding crops that are resistant to environmental stresses (cold and drought), insect pests and diseases. It is well known that plants can propagate vegetatively and some plant cells are totipotent having the potentiality of regeneration and differention to give complete plant. (Animal cells donot have this capacity) Fertile plants of at least some species may be generated from a single transformed cell ( cell in which foreign gene has been introduced) . Thus a gene introduced into a plant cell may ultimately be transmitted to progeny through seed in successive generations 1/4/2012 2 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : For introduction of desired gene into a plant cell there is no naturally occurring plasmid so it becomes a challenging task Fortunately scientist came to know about the soil bacteria Agrobacterium which is suitable for this tasnsformation. This bacterium invades plants at the site of a wound, transforming plant cells near the wound and inducing them to form a tumor called crown gall. This transformation takes place by transfer of T-DNA of Ti plasmid which integrates into plant genome and expresses itself 1/4/2012 3 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : This is a rare example of DNA transfer from a prokaryote to a eukaryote, it represents a natural genetic engineering process The integration of the T-DNA into a plant chromosome provides the vehicle necessary to introduce new genes into plants This ability of Agrobacterium to transfer T-DNA into plant genome can be harnessed in transferring desired gene into plant for transformation to produce transgenic plants . For this desired (new) genes can be inserted into T-DNA region of Ti plasmid ,such that when Agrobacterium infects plant cells these genes will automatically be transferred and become integrated into their genome. 1/4/2012 4 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 5 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 6 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Agrobacterium is a rod shaped plant pathogenic soil bacteria having two strains A . tumifaciens cause crown gall (tumor) and A rhizogenes hairy root disease in dicot plants by infecting through wounds on roots or stem at the soil surface. The bacterium contains Ti (Tumor inducing ) and Ri (Root inducing) plasmids . Both these plasmids can transfer part of their DNA (T-DNA) into plant cell chromosome by which Plant cells become transformed by expression of T-DNA gene which induce disease 1/4/2012 7 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Description of Agrobacterium as Natural Genetic Engineer Agrobacterium is a soil plant pathogenic bacteria. This bacterial cell possess a Ti plasmid in addition to bacterial chromosome. The bacteria can infect plants specifically dicots through wounds near soil surface. During infection a segment (T-DNA) of Ti plasmid gets transferred and become integrated in the plant cell genome. This T-DNA carries genes for expression of Disease 1/4/2012 8 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : COMPONENTS OF Ti / Ri PLASMID T- (Transferable) DNA region Vir (Virulence ) region Host Specificity Region Ori (origin of Replication) region The Ti plasmids are classified into different ( about 14) types depending upon the specific opine being synthesized. ( octapine/nopaline/Agropine ). To understand how T-DNA is transferred and Genes are expressed one must know the features/components of Ti plasmid 1/4/2012 9 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Generally all Ti plasmids share four conserved regions The most important is T DNA region which carries genes encoding plant hormones and opines. The Vir region contains the virulence genes which helps in transfer of T DNA Crown gall disease results from transformation of the plant genome with this part of the plasmid in a process analogous to bacterial conjugation Third region of plasmid contains conjugation genes concerned with whole plasmid transfer between bacteria. Fourth region encodes genes concerned with opine utilization . 1/4/2012 10 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Strucure of Ti Plasmid Components of Ti Plasmid 1/4/2012 11 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : It is ~200 kb megaplasmid T-DNA ( 15-40 kb) region contains genes for synthesis of Auxins,Cytokinins and Opines. Auxins and cytokinin genes are expressed in plant tissue inducing disease. Opines (unusual amino acids ) produced by infected cells are used as nutrients by Agrobacterium T-DNA region is bordered on both sides by 25bp repeat which helps in its transfer to plant genome. Virulence Region contains about 8 operons having about 24- 25 genes These genes help in transfer of T-DNA Host specificity region has gene for conjugative transfer and opine catabolism Ti plasmid also has origin of replication 1/4/2012 12 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : ORGANISATION OF T-DNA T-DNA is ~ 23kb ( 15-40kb) segment bordered on both sides by 25bp direct repeat sequences. T-DNA contains genes for tumor induction ( iaaM , iaaH & ipt ) by forming auxins , cytokinins and Opines. All the genes in T-DNA region contain eukaryotic regulatory sequences, so are expressed only in plant cells 1/4/2012 13 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : ORGANISATION OF Vir REGION Vir region contains 8 operons (VirA,B,C,D,E,F ,G &H) which together have 25 genes. Vir region mediates transfer of T-DNA into plant genome. It is itself not transferred. VirA and Vir G are constitutive operons encoding Vir A and VirG Proteins. Other Vir operons encode various proteins involved in T-DNA transfer 1/4/2012 14 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : MECHANISM OF TRANSFER OF T-DNA Transfer of T-DNA is a step wise process Vir region of Ti plasmid becomes activated by the phenolic signal molecules Acetosyringone and α-hydroxyacetosyringone released by wounded tissue of dicot plants which constitute wound response as follows 1/4/2012 15 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Acetosyringone and α-hydroxyacetosyringone bind with Vir A protein (located in the inner membrane) and activates it. It start functioning as autokinase to phosphorylate itself by ATP. Phosphorylated Vir A protein then phosphorylates Vir G protein which then dimerises. Phosphorylated Vir G protein has DNA binding function. It induces expression of rest of Vir operons Vir D1 protein has topoisomerase and endonuclease activity. It binds to right border sequence of T-DNA and facilitate the action of Vir D2 protein which is also endonuclease and nicks at the right border and remains bound to 5’end so generated. 1/4/2012 16 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : TRANSFER OF T- DNA by Vir genes 1/4/2012 17 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : The 3’end produced at the site of nick serves as a primer for DNA synthesis in 5’----3’ direction as a result of which one strand of T-DNA is displaced from the DNA duplex. The T-DNA strand is again nicked at the left border to generate a single strand copy of T-DNA. To this single strand copy Vir E 2 protein (single strand DNA binding proteins ) bind for its protection against exonucleases Vir B operon consisting of 11 genes encode membrane bound Vir B proteins. These along with Vir D4 proteins participate in conjugal tube formation between bacterial and plant cells for transfer of T-DNA Vir D2 which remains bound to 5’end of T DNA has a signal sequence which drives it into the nucleus of plant cell. 1/4/2012 18 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : INTEGRATION OF T-DNA INTO PLANT GENOME T-DNA enters plant cell as a single stranded structure which is immediately converted into double stranded form. Vir E2 also has nuclear localization sequence and is responsible for transfer of T DNA into plant cell nucleus Double stranded T-DNA integrate at random sites in the host plant genome. For integration 23-79 base pair deletion takes place at the integration or target site After integration of T-DNA into plant genome ,the genes for auxins, cytokinins and opines express themselves which result in uncontrolled growth in the form of tumor 1/4/2012 19 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 20 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : HARNESSING AGROBACTERIUM IN TRANSFERRING FOREIGN GENE INTO PLANT Part I of this class has made it clear that Agrobacterium has the potentiality to transfer prokaryotic DNA to eukaryotic Genome as a Natural Genetic Engineer In part two of this class discussion will be made on strategies involved for harnessing Agrobacterium for introducing new (desired) gene into plant cells 1/4/2012 21 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Agrobacterium’s ability to introduce its DNA into plant genome with efficiency makes Ti plasmid an attractive vector for gene transfer into plants After knowing about how T-DNA is transferred and what modifications are needed , Genetic engineers employed different strategies and before actual transfer of desired gene Genetically engineered Agrobacterium were produced with disarmed T-DNA region in which oncogenes and opine biosynthesis genes were replaced by desired (Foreign ) gene and a selectable marker gene . Along with this some other sequences as unique restriction sites were added. 1/4/2012 22 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : The natural Ti plasmids are unsuitable to be used directly as vectors for invitro manipulation due to following reasons Large size Tumor induction (Oncogenic ) property Absence of unique restriction enzyme site Now it is also well known that disarmed TDNA ,left and right borders along with genes of Vir region are essential elements for designing of transformation vectors Now let us see the strategies involved for harnessing Agrobacterium for introduction of new/desired/foreign genes into plants 1/4/2012 23 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Only useful attributes of Mega Ti plasmid (~200kb) have been exploited in designing plant transformation vector. For using Agrobacterium for in vitro genetic engineering purpose following manipulation are to be made Ti plasmids are disarmed .Genes from T DNA responsible for growth hormones (Oncogenes) and Opine formation are deleted so that no disease will be produced and as opines will not be synthesized further growth of Agrobacterium inside the host tissue stops The space between lB and RB is used to insert desired gene which is to be integrated and expressed in host tissue for transformation. The foreign gene is inserted by RDT A selectable marker gene providing resistance against antibiotic like kanamycin is also inserted in the T-DNA region The oncogenic problem can be solved by use of disarmed T-DNA from which these gene are removed 1/4/2012 24 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : The large size problem of Ti plasmid can be tackled by any of following two ways using manipulated Agrobacterium vectors The Co- integrated vector For this purpose a suitably modified E coli plasmid or vector is integrated into disarmed Ti plasmid ( pTi ),this gives rise to co-integrate vector. During disarming oncogenes of T-DNA are replaced by gene insert and other sequences of E coli plasmid. For this both plasmids are introduced into same Agrobacterium cell .Because of homology through recombination pBR plasmid is integrated into the T-DNA region. The gene to be cloned is therefore inserted into unique restriction site on the small pBR plasmid ,introduced into A tumifaciens cell carrying a Ti plasmid and natural recombination process left to integrate the new gene into T-DNA. 1/4/2012 25 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 26 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : ii) Binary (Two) Vector strategy (Agrobacterium containing two different recombinant plasmids) First is Agrobacterium E coli shuttle vector containing disarmed T DNA with 25 bp repeats flanking the gene insert to be introduced and a selective marker (often a Neo gene which provide cells resistence to antibiotic Kanamycin ) This Mini plasmid (pBIN19) is designed to replicate in both E coli and Agrobacterium and is capable of conjugal transfer between two bacterial species also has a copy of lacZ gene containing multiple cloning sites Second is helper Ti plasmid ( p AL4404) from which T DNA segment has been removed. It has a functional Vir region. The Vir genes of the helper Ti induce the transfer of T DNA present on first plasmid in the same bacterial cell. As a result a gene insert within T-region is also transferred into plant cell. 1/4/2012 27 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Ti helper pAL4404 pBIN19 Kan R LB RB Foreign gene 1/4/2012 28 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 29 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : 1/4/2012 30 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Many binary vectors have been developed which differ in size and source of 25bp repeat sequence, plant selection marker and cloning sites. In both cases (co-integrate or binary vector) ,the manipulated T-DNA is transferred to the plant genome efficiently TRANSFORMATION TECHNIQUE USING AGROBACTERIUM Some prerequisities for integration of foreign gene and production of transgenic plants are as follows The plant explants must produce acetosyringone to induce Vir genes for virulence Induced Agrobacterium should have access to cells that are competent for transformation or to take up DNA 1/4/2012 31 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Cells must be meristematic ,often transformed tissue or explants do not regenerate and are not totipotent, therefore transformation and regeneration competent cells should be taken as explants. The transformed explants cells should allow expression of transgene Disease causing genes should not be transferred and expressed,which is helped by disarmed T DNA Agrobacterium gene transfer can be achieved in the following two ways Co culture with tissue explants In planta transfusion 1/4/2012 32 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Co- culture with tissue explants For transformation explants like protoplast, callus , tissue slices, leaf disc, stem or floral tissue etc. can be co-cultured with genetically engineered Agrobacterium with recombinant vectors for about 2 days . During co-culture acetosyringone(phenolic signal molecule ) released from wounds (cut leaf disc) induce the Vir genes which bring about the transfer of recombinant T-DNA into many of the plant cells. The explants can then be transferred to shoot inducing (regeneration medium ) containing kanamycin and carbenicillin. Kanamycin allows only transformed plant cells to divide and regenerate shoots in about 3-4 weeks, while carbenicillin kills Agrobacterium cells . The shoots are separated and transferred to root inducing medium and finally after few weeks are transferred to soil. 1/4/2012 33 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Co-culture of cut explant with genetically engineered Agrobaterium 1/4/2012 34 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Agrobacterium with two plasmids co-cultured with explant 1/4/2012 35 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : By this co-culture method only dicot plants can be transformed. Agrobacterium mediated transformation of monocot plant ( cereals ) cells can be achieved by adding acetosyringone in the medium during co-culture In planta transformation Transformation can also be achieved by imbibition of seeds in fresh culturs of Agrobacterium. T-DNA becomes integrated into plant genome. It appears that Agrobacterium cells enter the seedling during germination, are retained within the plants, when flowers develop, the zygote or cells become transformed. 1/4/2012 36 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : Transgenes can be introduced into plants by various direct and indirect methods Agrobacterium mediated gene transfer is most frequenly used indirect method, it has many advantages Advantages of Agrobacterium mediated gene transfer Simple and comparatively less expensive High transformation efficiency Transgenic crops obtained have better fertility percentage Protocols for both dicotyledons and monocotyledon are available Relatively large length DNA segment can be transferred Disadvantages of Agrobacterium mediated gene transfer Time consuming Not all kind of cells can be treated by this method Sometimes leads to false positive result 1/4/2012 37 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : SUMMARY OF THE CLASS Some plant cells are totipotent ,can regenerate and develop complete plant vegetatively A single transformed cell may give rise to fertile transgenic plant For introduction of desired gene into a plant cell there is no naturally occurring plasmid Soil bacterium Agrobacterium which infects dicot plants near wounds is suitable for this purpose This transformation takes place by transfer of T-DNA of Ti plasmid which integrates into plant genome and expresses itself 1/4/2012 38 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : This is a rare example of DNA transfer from a prokaryote to a eukaryote, it represents a natural genetic engineering process . The bacterial system that transfers T-DNA into plant genome can be harnessed to transfer foreign (desired) /recombinant gene instead Agrobacterium’s ability to introduce its DNA into plant genome with efficiency makes Ti plasmid an attractive vector for gene transfer into plants The natural Ti plasmids are unsuitable to be used directly as vectors for invitro manipulation due to following reasons Large size Tumor induction (Oncogenic ) property Absence of unique restriction enzyme site 1/4/2012 39 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : For using Agrobacterium for in vitro genetic engineering purpose following manipulation are to be made T-DNA of Ti plasmid are disarmed,oncogenes and opine synthesis genes are removed Gene of interest and a selectable marker is inserted between LB and RB To tackle large size of Ti plasmid either co-integrate or binary vector strategy can be employed In both cases T-DNA containing gene of interest is transferred which induces transformation 1/4/2012 40 Agrobacterium mediated Gene Transfer in plant
PowerPoint Presentation : For transformation explant can be co cultured with genetically engineered Agrobacterium on a tissue culture medium with controlled levels of phytohormones along with antibiotics kanamycin and carbenicillin . Ultimately transformed plant lets are transferred to soil and are established as a transgenic plant. THANK YOU THANK YOU 1/4/2012 41 Agrobacterium mediated Gene Transfer in plant