This section introduces a new type of vector for Agrobacterium-mediated transformation. Some scientific information and patenting aspects are discussed.
Conjugation is a bacterial mechanism through which a plasmid genome or a host chromosome is transferred from one bacterial cell to another. Conjugation requires a whole complex of sequences and gene products. Some bacterial plasmids are conjugative plasmids that have the ability of transfer themselves into another host.
Mobilisable plasmids are not able to promote their own transfer unless an appropriate conjugation system is provided by a helper plasmid. Mobilisable vectors contain a site for transfer initiation called origin of transfer , oriT, and have sequences encoding proteins (Mob) involved in the mobilization of the DNA during the conjugative process. Mob proteins alone are not sufficient to achieve the transfer of the genome. Additional proteins for transfer (Tra ) are involved in the formation of a pore or pilus through which the genome passes to the recipient bacteria. Mobilisable plasmids do not encode Tra proteins and for this reason they require a helper plasmid providing the tra genes. In general, the process involves the following steps:
- double-stranded plasmid DNA is nicked at a specific site in oriT
- a single-strand DNA is released to the recipient through a pore or pilus structure
The enzyme that cleaves the double-stranded circular DNA at oriT and binds to an exposed 5′ end is called relaxase, and the intermediate structure formed is called a relaxosome. A complex of auxiliary proteins assemble at oriT and assist in the nicking process to form this intermediate in the DNA transfer.
Agrobacterium T-DNA transfer
The transfer of the Agrobacterium T-DNA to a host cell is comparable to a conjugation process. The virulence (vir) genes are involved in the mobilization and transfer of the T-DNA to the host plant cell. The virD operon contains genes that encode:
- proteins for DNA cleavage at T-DNA borders: virD1 and virD2;
- a protein that remains covalently bound to the 5′ end of the T-strand and contains a nuclear localization site: virD2; and
- a coupling protein, which binds to the T-DNA complex (T-strand plus vir proteins attached to it) and mediates its transfer through the mating bridge: virD4.
The T-DNA transfer apparatus is encoded by the virB operon. The proteins of the virB genes are located in the inner and outer membrane of the bacterium and are involved in the production of the pilus/pore structure. They also play an essential role in tumorigenesis.
Leiden University, in The Netherlands, have national phase entries of a PCT application related to the use of mobilisable plasmids for genetic transformation of eukaryotic cells. The invention disclosed by the applicant combines mobilisable plasmids with Agrobacterium-mediated transformation. A mobilisable plasmid contains at least an oriT and some mob genes, and Agrobacterium provides the transfer genes, basically a virB operon.
Unlike the binary and co-integrate vector systems described previously, there is no Agrobacterium T-DNA or nucleotide sequences surrounded by T-borders transferred in the mobilisable vector system. The mobilisable plasmid is engineered into an Agrobacterium and the genetic material contained in the mobilisable plasmid is transferred to the eukaryotic host using the transfer machinery of Agrobacterium. VirD4, the coupling factor, and mobilization functions can either be part of the mobilisable plasmid or can be provided byAgrobacterium.
A mobilisable plasmid is defined in the patent as “a plasmid that has the capability of forming a relaxosome in a suitable surrounding such as Agrobacterium and being capable of being transferred by an Agrobacterium vir-like system into eukaryotic cells.”
According to the applicant, the advantages of mobilisable plasmids such as the plasmid CloDF13 include:
- small size;
- easy to manipulate;
- can be maintained at high copy number in enterobacteria (e.g., E. coli);
- can be transferred using the Agrobacterium virulence system;
- their transfer to fungi and plants is very efficient compared to other plasmids;
- can be used for nuclear and organelle transformation as well as homologous and site-specific recombination.