Development of T-DNA Essay

Question: Describe the development of T-DNA-based vector systems from the Ti plasmid and the mechanisms of their delivery into plant cells. Answer:   Tumor-inducing plasmids (Ti plasmids) are used extensively in the construction of vectors and transgenic plants (Binns and Thomashow, 1988).   Ti plasmids are ~200-kb in size, derived from Agrobacterium tumefaciens, Gram-negative phytopathogenic soil bacteria that deliver DNA and proteins to plant cells at wound sites, resulting in crown gall tumorigenesis (Chilton et al., 1977). The generation of tumors depends on the induction of a set of Ti plasmid-encoded virulence (vir) genes acting through a virA/virG regulatory system, which primarily responds to monosaccharide and phenolic levels released by wounded plants.   The transferred DNA (T-DNA) of Ti plasmids is randomly integrated into the plant nuclear genome through a process known as non-homologous recombination (NHR) (Offringa et al., 1990). T-DNA is a single-stranded DNA molecule produced by a virDl/D2-encoded site-specific endonuclease that nicks within two border sequences of 24-bp in length, flanking the T-DNA   (van Haaren et al., 1987).   After cleavage and excision, the T-DNA binds with the DNA-binding protein VirE2 and the resulting complex is transferred to the plant cell via type IV-type secretion (Zupan and Zambryski, 1995). For genetic engineering purposes, the T-DNA region is modified into a non-tumor generating DNA segment by removal of genes that encode enzymes controlling auxin and cytokinin synthesis.   Cloned genes may be inserted into the T-DNA of a Ti plasmid that will eventually be introduced into cultured plant cells, leaf discs or root slices by infection.    Genes for antibiotic resistance are also incorporated into the T-DNA to facilitate selection of transformed cells.   Transformed cells are cultured in media containing auxins and cytokinins for growth and a specific antibiotic to aid identification of transformed clones.   There are reports of successful introduction of foreign genes for disease resistance, herbicide resistance and salt tolerance into commercially important plants.   Another way of transforming plants is by immersion of whole plants in a solution containing engineered-Ti Agrobacterium (Bechtold et al. 1993). Transformation may also be performed by exposing whole plants to a solution containing Agrobacterium that is carrying engineered or wild-type Ti plasmids. The plants must be treated in such a way to allow the Agrobacterium to enter tissue, either by applying a vacuum or by treating with detergents. The Agrobacterium penetrates the floral tissue and transforms the developing ovules. Isolation of seeds from these Agrobacterium-exposed plants yields up to 2% of the seeds that are transformed with the T-DNA. This approach is very useful for molecular genetic studies, such as for characterizing DNA sequences involved in the control of gene expression, or constructing large libraries of insertional mutants. Question:   Explain why transformation of certain species has been problematical and to what extent this has been overcome. Answer:   Ti plasmids encounter compatibility problems wherein closely related plasmids exclude each other.   The repABC genes have been identified to play a major role in this incompatibility.   This problem has been overcome by a curing method (Uragi et al., 2002) which is based on three steps.   Firstly, a curing plasmid is introduced, followed by a screening for Ti-less clones by either opine utilization or hybridization by using a highly conserved region of the virulence cluster as probe, and lastly, detection and deletion of the curing plasmid. Question:   What improvements can be made to the expression systems to overcome some of the objectives of the GM technology? The transformation mechanism of Ti plasmids is so powerful that it becomes a concern on whether other crops might be accidentally modified and propagated.   Termed as â€Å"xenogenic† plants, these plants result from the insertion of laboratory-designed DNA for which no naturally evolved genetic counterpart can be found.   Such DNA segments may integrate into the plant genome causing rearrangements in the nuclear material which may later result in species differentiation.   A silencing mechanism should be constructed to the expression systems of Ti plasmids to overcome such freak accident in GM technology. References Bechtold, N., Ellis, J. and Pelletier, G. (1993):   Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci., 316: 1194–1199. Binns, A.N. and Thomashow, M.F.,   (1988):   Cell biology of Agrobacterium infection and transformation of plants.   Annu. Rev. Microbiol.,   42:575-606. Chilton, M.D., Drummond, M.H., Merio, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, M.P.   (1977):   Stable incorporation of plasmid DNA into higher plant cells: The molecular basis of crown gall tumorigenesis.   Cell,   11:263-271. Matzke, A. J. M. and Chilton, M-D. (1981) Site-specific insertion of gene into T-DNA of the Agrobacterium tumor-inducing plasmid: An approach to genetic engineering of higher plant cells. J. Mol. Appl. Genet. 1: 39–49. Offringa, R., De Groot, M.J.A., Haagsman, H.J., Does, M.P., van den Elzen, P.J.M. and Hooykaas, P.J.J.   (1990):   Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.   EMBO J., 9:3077-3084. Uragi, M., Suzuki, K. and Yoshida, K.   (2002):   A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.   Genes Genet. Syst.   77:1-9. van Haaren, M.J., Sedee, N.J., Schilperoort, R.A. and Hooykaas, P.J. (1987): Overdrive is a T-region transfer enhancer which stimulates T-strand production in Agrobacterium tumefaciens. Nucl. Acids Res., 15: 8983–8997. Zupan, J., Muth, T., Draper, O. and Zambryski, P. (2000). The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J.,   23: 11–28. Zupan, J.R. and Zambryski, P. (1995): Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol., 107: 1041–1047.