Agrobacterium tumefaciens transgenic plants

 Agrobacterium-mediated gene transfer

Agrobacterium tumefaciens could be a soil gram-negative infectious agent that naturally infects plants at their wound sites and causes unwellness} disease by transfer of (T)-DNA from microorganism cells into plant cells. Therefore, the bacteria genus has been extensively studied as an infectious agent and a very important biotechnological tool. The infection method involves the transfer of T-DNA and virulence proteins into the plant cell. At that point, the organic phenomenon patterns of host plants disagree betting on the bacteria genus strain, plant species, and cell-type used. 



It is the preeminent practical utilized vector to plant transformation. This particular method of activity has moreover made the microorganisms a significant device in plant breeding. once attachment of bacteria genus to plant cells and expression of multiple virulence (vir) genes, many effector proteins, in conjunction with T-DNA, area unit transported into the plant cell by a type-IV-secretion system.

History

Marc Van Montagu and Jozef Schell at the University of urban focus (Belgium) found the grouping move component between microscopic organisms variety and plants, that brought about the improvement of approaches to change microorganisms sort into Associate in the Nursing practical conveyance framework for arrangement designing in plants. A group of specialists diode by Dr. Mary-Dell Chilton was essential to show that the destructiveness qualities can be expelled while not unfavorably impactful the adaptability of microscopic organisms sort to embed its own deoxyribonucleic corrosive into the plant order (1983).

Types of bacteria genus species

1. Agrobacterium tumefaciens that form unwellness} disease/ crown gall disease.







2. Agrobacterium rhizogenes, Bacteria genus rhizogenes form hairy root disease. bacteria genus rhizogenes (recently revised as Rhizobium rhizogenes; Young et al., 2001) could be a soil-borne gram-negative bacteria that initiate textured roots after injuring and contamination of monocotyledon and eudicot plants. Its root-inducing inclusion, containing transfer deoxyribonucleic acid cryptography root locus (rol) sequence loci (rolA, rolB, and rolC), is answerable for the stable introduction of genetic material into host cells. This will trigger the lush production of extremely branched furry roots at the positioning of infection, typically the hypocotyl or seed leaf. 







3. Bacteria genus radiobacter is an Associate in Nursing avirulent strain. It contains 7.2 million base pairs with a G+C composition of 59.9%.  The hereditary material is composed inside one round body containing 4,005,130 base sets, in addition to four plasmids. A. radiobacter is distinguished by being the sole member of the genus that doesn't have morbific tendencies towards plants. The life form utilizes dead stuff inside the rhizosphere, unequivocal environs impacted by the essential frameworks of plants, as every its carbon and vitality gracefully.

Ti Plasmid 

The microscopic organisms contain Ti (Tumor causing/Inducing ) and Ri (Root initiating) plasmids. Every one of these plasmids will move a piece of their deoxyribonucleic corrosive (T-DNA) into plant cell chromosomes by that Plant cells become modified by the articulation of the T-DNA succession that actuates disease. 


1. A Ti consideration/plasmid is roundabout deoxyribonucleic corrosive found in most agrobacteria. 

2. There territory unit 3 principle areas of Ti plasmids 


• T-(Transferable) deoxyribonucleic corrosive locale between the left and right border sequence, Oncogenes, opine 


• Vir (Virulence ) locale 


• Host Specificity Region 


• Ori (origin of Replication) Region 


The Ti plasmids territory unit grouped into entirely unexpected ( with respect to 14) assortments depending upon the exact opine being incorporated. 


( octapine/nopaline/Agropine )

Depending upon the trans and cis offer of vir sequence merchandise 2 vector systems are developed i.e. cointegrate vector system and binary vector system

Cointegrate vector system

Co-integrated vectors were among the primary forms of changed and designed plasmids devised for Agrobacterium-mediated transformation, however aren't widely used nowadays. 



These vectors area unit created by the homologous recombination of an E. coli plasmid with the-DNA region of Associate in Nursing endogenous inclusion/Plasmid in bacteria genus. 



The integration of the 2 plasmids needs a part of similarity present in each. 

Co-integrated inclusion assembled by in vitro manipulation unremarkably contains: 




 the vir genes 



 the left and right T-DNA borders 



 an exogenous DNA sequence between the two T-DNA borders



  plant and microorganism selectable markers

Binary vector system

Binary Plant Transformation Vector within the binary vector system, the 2 totally different plasmids utilized area unit A wide-host-range tiny replicon, known as mini Ti plasmid that has Associate in Nursing origin of replication (ori) that allows the upkeep of the inclusion during a big selection of bacterium as well as E. coli and bacteria genus. 





This plasmid generally contains: 





 foreign deoxyribonucleic acid in situ of T-DNA 



 the left and right T-DNA borders (or a minimum of the proper T- border) 



 markers for choice and maintenance in each E. coli and A. tumefaciens 



 a selectable marker for plants 



 The inclusion is "disarmed" since its tumor-inducing genes situated within the T-DNA are removed

Transfer of bacteria genus into the Plant order

The bacterium will infect plants specifically dicots through a wound close to the soil surface. throughout infection T-DNA of Ti-plasmid gets transferred within the plant cells and becomes integrated into the order. T-DNA carries genes for the expression of illness. The T-DNA region contains genes for the synthesis of auxins, cytokinins, and opines. Auxins and cytokinins genes causing illness and opines area unit used as a nutrient by bacteria genus. Virulence region contains eight operons having 24-25 genes. All the genes within the T-DNA region contain eukaryotic regulative sequences, therefore the area unit expressed solely within the plant cells.

Function T-DNA

Auxin biosynthesis; encodes protein tryptophan-2-mono-oxygenase, that converts tryptophan into indole-3-acetamide (IAM).





Auxin biosynthesis; encodes protein indole-3-acetamide hydrolase, that converts IAM into IAA (indole-3-acetic acid).





Cytokinin biosynthesis; encodes protein isopentenyl enzyme, that catalyzes the formation of isopentenyl A. 

Nopaline biosynthesis; encodes the protein nopaline synthase, which produces nopaline from essential amino acid and acid.  

Vir region ( virulence region)

Wikipedia

Agrobacterium-mediated horizontal sequence transfer: Mechanism ...








Vir region contains eight operons ( VirA, B, C, D, E, F, G and H). Vir regions mediate the transfer of T-DNA into the plant order.




Vir region of Ti-plasmid becomes activated by the phenoplast signals ( acetosyringone ) that area unit discharged by the wounded tissue of exogen plants. phenoplast compounds bind with VirA supermolecule and activate it. 



Then  VirA supermolecules phosphorylate VirG protein that then dimerizes. It induces expression of the remainder of all operons. 



Then VirD1 supermolecule has nuclease activity, it binds to the proper border sequence of T-DNA and facilitates the action of VirD2 supermolecule that is additionally nuclease. The T-DNA strand is nicked at the left border to get a single-strand copy of T-DNA. to the present single-strand copy, VirE2 supermolecule binds for its protection against exonucleases.




VirB and VirD4 proteins participate in connubial tube formation between microorganisms and plant cells for the transfer of T-DNA. VirD2 remains bind to 5' finish of T-DNA that drives into the nucleus of the plant cell.




T-DNA enters into the plant cell as a fiber structure that is regenerate into ds structure. ds T-DNA integrates arbitrarily web sites within the host plant order for integration 23-79 bp deletion takes place at the target site. once the mixing of T-DNA into the plant order, the sequence for auxins, cytokinins, and opines categorical themselves which ends in uncontrolled growth of a tumor that is termed unwellness} disease in plants.

Vir region operate

vir A Encodes a receptor for acetosyringone that functions as Associate in Nursing autokinase; additionally phosphorylates VirG protein; constituent expression. 



virB Membrane proteins; probably kind a channel for T-DNA transport (conjugal tube formation); VirB eleven has ATPase activity.



virC Helicase; binds to the overdrive region simply outside the proper border; concerned in moving of T-DNA. 


VirDl has topoisomerase activity; it binds to the proper border of T-DNA; VirD2 is Associate in Nursing endonuclease; it nicks the proper border. 



virE Single-strand binding proteins (SSBP); bind to T-DNA throughout its transfer.



virF, virG deoxyribonucleic acid-binding protein; in all probability forms chemical compound once phosphorylation by VirA, and induces the expression of all vir operons; constituent expression.



virH Not standard.

TRANSFORMATION TECHNIQUE mistreatment bacteria genus 

Some prerequisites for the integration of foreign sequence and production of transgenic plants area unit as follows 



•The plant explants should turn out acetosyringone to induce Vir genes for virulence. 



•Induced bacteria genus ought to have access to cells that area unit competent for transformation or to require up deoxyribonucleic acid.


 • Cells should be meristematic, often reworked tissue or explants don't regenerate and aren't ability, thus transformation and regeneration competent cells ought to be taken as explants. 


• The reworked explants cells ought to enable expression of the transgene. 


• illness inflicting genes shouldn't be transferred and expressed, which is helped by disarmed T deoxyribonucleic acid.




Agrobacterium sequence transfer is achieved within the following 2 ways that



• Co-culture with tissue explants


• In planta transfusion




Co-culture with tissue explants For transformation explants like a body part, callus, tissue slices, leaf disc, stem or floral tissue, etc. is co-cultured with genetically designed bacteria genus with recombinant vectors for regarding two days. throughout co-culture acetosyringone(phenolic signal molecule ) discharged from wounds (cut-leaf disc) induce the Vir genes that cause the transfer of recombinant T-DNA into several of the plant cells. 



The explants will then be transferred to shoot causing (regeneration medium ) containing antibiotics and carbenicillin. antibiotic permits solely reworked plant cells to divide and regenerate shoots in regarding 3-4 weeks, whereas carbenicillin kills bacteria genus cells. The shoots area unit separated and transferred to root causing medium and eventually once a few weeks area unit transferred to soil. 



By this co-culture methodology solely exogen plants are reworked. Agrobacterium-mediated transformation of monocotyledon plant ( cereals ) cells is achieved by adding acetosyringone within the medium throughout co-culture In planta transformation.

field 

In planta transformation 



Transformation may also be achieved by the imbibition of seeds in the recent culture of bacteria genus. T-DNA becomes integrated into the plant order. It seems that bacteria genus cells enter the spermatophyte throughout germination, area unit maintained inside the plants, once flowers develop, the fertilized ovum or cells become reworked. 

Advantages of Agrobacterium-mediated sequence transfer 

• Easy and relatively less costly 


• High transformation potency 


• Transgenic crops obtained have higher fertility proportion 


• Protocols for each dicotyledon and monocot area unit offered 


• comparatively giant length deoxyribonucleic acid phase is transferred 

Disadvantages of Agrobacterium-mediated sequence transfer 

•Time overwhelming 

•Not all reasonably cells are treated by this methodology 


•Sometimes ends up in false-positive results.

References

Akiyoshi, D. E., Morris, R., Hinz, R., Mischke, B., Kosuge, T., Garfinkel, D., et al. (1983). Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc. Natl. Acad. Sci. U.S.A. 80, 407–411. DOI: 10.1073/pnas.80.2.407





Akiyoshi, D. E., Regier, D. A., and Gordon, M. P. (1987). Cytokinin production by Agrobacterium and Pseudomonas spp. J. Bacteriol. 169, 4242–4248.




Akiyoshi, D. E., Regier, D. A., Jen, G., and Gordon, M. P. (1985). Cloning and nucleotide sequence of the gene from Agrobacterium tumefaciens strain T37. Nucleic Acids Res. 13, 2773–2788. DOI: 10.1093/nar/13.8.2773





Aloni, R., Pradel, K. S., and Ullrich, C. I. (1995). The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L. Planta 196, 597–605. DOI: 10.1007/BF00203661






Aloni, R., Wolf, A., Feigenbaum, P., Avni, A., and Klee, H. J. (1998). The never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Plant Physiol. 117, 841–849. DOI: 10.1104/pp.117.3.841