Mobilization of viral genomes from T-DNA using site-specific recombination systems
The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium -mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention.
1. A method for providing to a plant cell a plurality of copies of a DNA sequence of interest flanked by nonidentical target sites comprising:
a) providing an Agrobacterium replicon having a T-DNA, wherein said T-DNA comprises in a 5′ to 3′ or 3′ to 5′ orientation, a first target site for a first recombinase, a viral replicon, a second target site for a second recombinase, said DNA sequence of interest, and a third target site for said first recombinase, wherein said first and said third target sites are directly repeated and identical with respect to each other, and said second target site is nonidentical to said first and said third target sites; and
b) infecting the plant cell with an Agrobacterium carrying said Agrobacterium replicon under conditions that allow transfer of said T-DNA and expression of said first recombinase in said cell;
wherein a nucleotide sequence encoding said first recombinase or an active fragment or an active variant thereof is introduced into the plant cell by sexual crossing and wherein said active fragment or said active variant retains the ability to catalyze a recombination event and said nucleotide sequence is operably linked to a promoter that drives expression in said cell.
2. The method of claim 1 , wherein said plant cell further comprises in its genome at least two target sites which correspond to the non-identical second and third target sites flanking the DNA sequence of interest.
3. The method of claim 1 , wherein said Agrobacterium replicon is a Ti plasmid.
4. The method of claim 1 , wherein said Agrobacterium replicon is a Ri plasmid.
5. The method of claim 1 , wherein said plant cell is from a monocot.
6. The method of claim 5 , wherein said monocot is a member of the family Graminae.
7. The method of claim 5 , wherein said monocot is maize , wheat, rice, barley, sorghum or rye.
8. The method of claim 1 , wherein said viral replicon is a MSV, a WDV, a TGMV or a TMV replicon.
9. The method of claim 1 , wherein said plant cell is from a dicot.
10. The method of claim 9 , wherein said dicot is soybean, canola, Brassica , sunflower, alfalfa or safflower.
11. The method of claim 1 , wherein said first and said second site specific recombinase are identical.
12. The method of claim 1 , wherein said first or said second recombinase is FLP, Cre, Int, SSVI, integrase R or an active fragment or an active variant thereof wherein said active fragment or said active variant retains the ability to catalyze a recombination event.
13. The method of claim 12 , wherein said first or said second recombinase is FLP or an active fragment or an active variant thereof wherein said active fragment or said active variant retains the ability to catalyze a recombination event.
14. The method of claim 13 , wherein said first, said second, or said third target site is selected from the group consisting of a FRT site, a mutant FRT site, a Lox site and a mutant Lox site.
15. The method of claim 14 , wherein said mutant FRT site is FRT5 (SEQ ID NO:4), FRT6 (SEQ ID NO:5) or FRT7 (SEQ ID NO:6).
16. The method of claim 13 , wherein said FLP recombinase is encoded by moFLP.
17. The method of claim 12 , wherein said first or said second recombinase is Cre or an active fragment or an active variant thereof wherein said active fragment or said active variant retains the ability to catalyze a recombination event.
18. The method of claim 17 , wherein said first, said second, or said third target site is selected from the group consisting of a Lox site, a mutant lox site, a FRT site and a mutant FRT site.
19. The method of claim 17 , wherein said Cre recombinase is encoded by moCre.