AGROBACTERIUM TUMEFACIENS MEDIATED GENE TRANSFER IN PLANTS Agrobacterium

AGROBACTERIUM TUMEFACIENS MEDIATED GENE TRANSFER IN PLANTS

Agrobacterium - mediated Gene Transfer Most common method of engineering dicots, but also used for monocots Pioneered by J. Schell (Max-Planck Institute, Cologne) Agrobacterium- Soil borne, gram negative, rod shaped, motile found in rhizosphere Causative agents of “Crown gall” disease of dicoltyledones Have ability transfer bacterial genes to plant genome Attracted to wound site via chemotaxis in response to chemicals (sugar and Phenolic molecules: acetosyringone) released from damaged plant cells Contains Ti plasmid which can transfer its T-DNA region into genome of host plants

Agrobacterium tumefaciens the species of choice for engineering dicot plants; monocots are generally resistant. some dicots more resistant than others (a genetic basis for this). complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes.

Infection and tumorigenesis Infection occurs at wound sites. Involves recognition and chemotaxis of the bacterium toward wounded cells. galls are “real tumors”, can be removed and will grow indefinitely without hormones. genetic cells. information must be transferred to plant

Tumor characteristics 1. Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate 2. Opine depends on the strain of A. tumefaciens. 3. Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.

Elucidation of the TIP (tumor-inducing principle) It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra-chromosomal element. Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.

Ti-plasmid features Two strains of Ti-plasmid: -Octopine strains- contains two T-DNA region: TL (14 kb) and TR ( 7 kb) -Nopaline strains- contain one T-DNA region(20 kb) Size is about 200 kb Has a central role in Crown-gall formation Contains one or more T-DNA region that is integrated into the genome of host plants Contain a vir region ~ 40 kb at least 8~11 vir genes Has origin of replication Contains a region enabling conjugative transfer Has genes for the catabolism of opines

Ti Plasmid (7 bp repeat) (14 bp repeat)

Ti plasmids and the bacterial chromosome act in concert to transform the plant Agrobacterium tumefaciens chromosomal genes: chv. A, chv. B, psc. A required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. Virulence region (vir) carried on p. Ti, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer. T-DNA encodes genes for opine synthesis and for tumor production. occ (opine catabolism) genes carried on the p. Ti allow the bacterium to utilize opines as nutrient.

Generation of the T-strand Left Border Right Border T-DNA overdrive 5’ vir. D/vir. C Vir. D nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

Generation of the T-strand Left border T-DNA Right border gap filled in T-strand vir. E vir. D/vir. C D 1. Helicases unwind the T-strand which is then coated by the vir. E protein. 2. ~one T-strand produced per cell.

Left border T-DNA Right border D T-strand coated with vir. E vir. D nicks at Left Border sequence 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome

Overview of the Infection Process

Important points: Monocots don't produce AS in response to wounding. Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell. Engineering plants with Agrobacterium: Two problems had to be overcome: Ti plasmids large, difficult to manipulate couldn't regenerate plants from tumors

Binary vector system: Strategy: Move T-DNA onto a separate, small plasmid. Remove aux and cyt genes. Insert selectable marker (kanamycin resistance) gene in T-DNA. Vir genes are retained on a separate plasmid. Put foreign gene between T-DNA borders. Co-transform Agrobacterium with both plasmids. Infect plant with the transformed bacteria.

Binary vector system:

Practical application of Agrobacterium-mediated plant transformation: Agrobacterium mediated transformation methods are thought to induce less rearrangement of the transgene. Lower transgene copy number that direct DNA delivery methods. Successful production of transgenic plants depends on the suitable transformation protocols.

Recent research

Conclusion: Agrobacteria are biological vector for introduction of genes into plants. Agrobacterium-mediated transformation is not restricted to eukaryotes as Agrobacterium is also able to act on the gram positive bacterium Streptomyces lividans. Agrobacterium can transfer not only DNA but also proteins to the host organisms through its type four secretion system.

References Bevan, M. (1984) “Binary Agrobacterium vectors for plant transformation”. Nucleic Acids Res 12: 8711 -8721. Deblaere R. , Bytebier B. , De Greve H. , Deboeck F. , Schell J. , Van Montagu M. and Leemans J. (1985) “Efficient octopine Ti plasmid-derived vectors for Agrobacteriummediated gene transfer to plants”. Nucleic Acid Research 13: 4777 -4788. Chilton, M. D. (1983) “A vector for introducing new genes into plants”. Scientific American, 248, 50 -9. Nadolska-Orczyk, A. , Orczyk, W. and Przetakiewicz, A. (2000) “Agrobacteriummediated transformation of cereals – from technique development to its application”. Acta Physiologiae Plantarum, 22, 77 -8. Kakkar, A. and Verma, V. K. , (2011) Agrobacterium mediated biotransformation. Journal of Applied Pharmaceutical Science 01 (07); 2011: 29 -35.