Agrobacterium-mediated Gene Transfer

An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell

A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.

The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.

1. Agrobacterium mediated Gene Transfer Dr. Divya Sharma Assistant Professor

2. Agrobacterium -mediated gene transfer Agrobacterium species Ti-Plasmid Organization of T-DNA Genes responsible for transfer of T-DNA Mode of action Gene of interest Gene cloning

3. Introduction Genes Agrobacterium is a rod shaped, Gram negative soil bacterium that naturally infect the dicot plants (commonly), but also used for monocots. Common species of Agrobacterium like A. tumefaciens , A.rhizogenes , A. rubi and A. vitis are naturally infect plants at wound sites and causing ‘ Crown gall ' and ‘ Hairy root ’ diseases. These bacteria are natural genetic engineer due to they insert their genes into the genome of higher plants.

4. Agrobacterium mediated transformation in plant

5. The bacterium contains a plasmid (called Ti-plasmid or Tumour-inducing plasmid), their part of T-DNA (Transfer DNA) integrates into the host plant chromosomes. The Ti-plasmid contains several genes including the vir genes which control the process of infection of the plant and transfer of the T-DNA to the chromosome. The T-DNA contains the auxin and cytokines genes and these gene expressed specific compounds, like opines, resulting in tumor and changes in plant metabolism. A. tumefaciens , now used as a tool for engineering desired genes into plants (called as Gene cloning ).

6. A. tumefaciens : Infects wounded plant tissue which is induced plant tumor i.e., ‘ Crown gall ’ disease. Crown gall occurs when bacterium releases Ti-plasmid (Tumour-inducing plasmid) into cytoplasm. Serious pathogen of walnut, grapevines, stone fruits, nut trees, sugar beets, horse radish and rhubarb. It grows optimally at 28 °C. The doubling time can range from 2.5-4h depending on the media, culture format, grow aerobically, without forming endospores. Agrobacterium species

7. Ti - Plasmid Ri - Plasmid

8. 2. A. rhizogenes : Responsible for inducing ‘ Hairy root ’ diseases. Bacterial genes transfer its T-DNA from its Ri -plasmid (Root-inducing plasmid) into plant through wound. Ri -plasmid is analogue to Ti-plasmid. 3. A. rubi : Cause ‘ Cane gall ’ disease in sugarcane plant 4. A. vitis : Cause ‘ Gall ’ in grapes plant

9. Tumour inducing plasmid

10. Large size plasmid of 200 kbp . The Ti-plasmid is damage when Agrobacterium is grown above 28 °C (curing of plasmid). The modification of this plasmid is very important in the creation of transgenic plants. Tumour inducing plasmid Ti-Plasmid have T-DNA, Right border (RB), Left border (LB), virulence ( vir gene) region, phytochrome region, origin of replication and opine catabolism region.

11. For T-DNA transfer Breakdown of opines For gene transfer T-DNA region or oncogenic region (for tumor induction): Plant hormone synthesis region Opine synthesis region

12. Difference between NOPALine and oCTOpine T-DNA NOPALINE OCTOPINE Has one 23kb region as T-DNA Two adjoining region one is L (13kb) and another is R (8kb) Has 13 ORFs Has 8 (L) and 6 (R) ORFs Nopaline and Octopine plasmids are similar; carry a variety of genes, including T-regions that have overlapping functions

13. Composition of Oncogenic Region

14. Organization of t- dna

15. The transfer DNA (T-DNA) is the transferred gene, part of Ti-plasmid of some species of Agrobacterium . Size of T-DNA is between 15-30 kbp . It has LB and RB, RB plays a important role in transfer and integrated of T-DNA. Absence of RB will terminate the T-DNA transfer. T-DNA carry genes for phytohormones ( Auxin and Cytokinin ) and opine that are expressed in plant cell. Organization of t- dna

16. Over production of these hormones at the sites of infection is responsible for the proliferation of wound cell into a gall/tumor. These tumor can harbor a plenty of bacteria. Opines synthesis is a unique characteristics cells. some opines are showing high plant species specificity. Opines are low molecular weight compounds found in plant’s crown gall tumors or hairy root diseases produced by parasitic bacteria of the genes Agrobacterium. Different types of opines like: Nopaline , Octapine , Agropine and Succinamopine types . These opines are important source of nitrogen, carbon and energy. These opines are condensation product of : a). An amino acid and a keto acid; and b). An amino acid and a sugar. Naturally occuring T-DNA codes for: ( i ) Opine synthesis ; and (ii) Phytohormones to induce tumor genesis .

17. Structure of various opines

18. Functions of T-DNA genes in Ti-plasmid Genes Ti-plasmid Functions vir All DNA transfer into plant shi All Shoot induction roi All Root induction ocs Octapine Octopine synthesis occ Octapine Octopine catabolism nos Nopaline Nopaline synthesis noc Nopaline Nopaline catabolism tra All Bacterial transfer genes Inc All Incompatibility genes oriV All Origin of replication tms1 Trytophane-2-mono-oxygenase Auxin synthesis tms2 Indoleacetamide hydrolase Auxin synthesis tmr Isopentyl transferase Cytokinin synthesis frs Fructopine synthase Opine synthesis mas Mannopine synthase Opine synthesis ags Agropine synthase Opine synthesis

19. Mode of action

20. genes responsible for transfer T-DNA Vir ulence Gene Function in Agrobacterium Function in plant Essential genes vir A Kinase protein in bacterial membrane; Receptor of phenolic compounds, sensor of a 2 component regulatory system - vir A and G Phenolic response regulator of a 2 component regulatory system ( acetosyringone ) - vir G Activates other vir genes vir D1 Endonuclease Required for T-DNA processing in-vivo and for ds T-DNA border nicking in-vitro - vir B/D4 Type IV secretion system ( T4SS - binding system apparatus ) Synthesis and assembly of the T- pilus ( vir B2 encodes a prepropilin) Formation of pore channel to transfer T-DNA from bacterium to plant cell - vir C1 Stimulate transfer Putative “overdrive” binding protein; Enhancement of T-DNA transfer - vir D2 T-DNA border specific endonuclease; Nuclear targeting of the T-stranded;

21. Vir ulence Gene Function in Agrobacterium Function in plant vir D2 Prevent attack of exonuclease at 5’ end of T-DNA; Cutting phosphodiester bond Putative ‘pilot protein’ that leads the T-strand through the transfer apparatus and into the plant Protection of the T-strand from 5’ exonucleolytic degradation; T-strand integration into the plant genome vir E/E2 Act as single stranded binding protein Protect T-DNA against nuclease and target T-DNA to plant cell vir E1 Required for vir E2 except from Agrobacterium; Chaperone for vir E2 - vir E2 Formation of a putative “T-complex” in Agrobacterium Have nuclear localization signal (NLS) Formation of a putative “T-complex” in plant; Protection of the T-strand from nucleolytic degradation; Nuclear targeting of the T-strand; Passage of the T-strand through the nuclear pore complex Non Essentials Genes vir F - Host range factor; Possible interaction with skip proteins to regulate plant cell division cycle vir H Putative cytochrome P450 enzyme - vir J Putative T-strand binding proteins T-strand export from Agrobacterium -

22. Attachment of A. tumefaciens to the plant cells Sensing plant signals by A. tumefaciens and regulation of virulence genes in bacteria following transduction of the sensed signals Generation and transport of T-DNA complex and virulence proteins from the bacterial cells into plant cells Nuclear import of T-DNA and effecter proteins in the plant cells T-DNA integration and expression in the plant genome Steps of transformation process Plant stress conditions Production of phenolics compounds form plant cell

23. Role as genetic tool T-DNA region get replaced by any gene of interest and then targeted to Plant cell for transformation. Note: Monocot are not good host for Agrobacterium. There is a hypothetical believe that monocot are resistant to Agrobacterium because they do not produce phenolics that can induce Virulence genes.

24. Advantages and disadvantages of gene transfer S.No . Requirements Advantages Disadvantages 1. The explants of plant must produce acetosyringone or other related phenolic compounds Natural means of transfer hence plant friendly Limited host range. Can not infect cereal plants. 2. The induce bacteria should have access to cell that are competent for transformation It is capable of infecting intact plant cells Sometimes cells in a tissue that are able to regenerate are difficult to transform 3. Transformation competent cells and tissue should be able to regenerate into whole plants Capable of transferring large fragmented of DNA very efficiently without substantial rearrangements. The stability of gene transferred is excellent.

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