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(12) Patent Application Publication (10) Pub. No.: US 2005/0289672 A1 Jefferson (43) Pub US 2005O289672A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0289672 A1 Jefferson (43) Pub. Date: Dec. 29, 2005 (54) BIOLOGICAL GENE TRANSFER SYSTEM Publication Classification FOR EUKARYOTC CELLS (75) Inventor: Richard A. Jefferson, Canberra (AU) (51) Int. Cl. ............................. A01H 1700; C12N 15/82 (52) U.S. Cl. .............................................................. 800,294 Correspondence Address: CAROL NOTTENBURG 81432ND AVE 5 SEATTLE, WA 98144 (US) (57) ABSTRACT (73) Assignee: CAMBIA Appl. No.: 10/954,147 This invention relates generally to technologies for the (21) transfer of nucleic acids molecules to eukaryotic cells. In Filed: Sep. 28, 2004 particular non-pathogenic Species of bacteria that interact (22) with plant cells are used to transfer nucleic acid Sequences. Related U.S. Application Data The bacteria for transforming plants usually contain binary vectors, Such as a plasmid with a Vir region of a Tiplasmid (60) Provisional application No. 60/583,426, filed on Jun. and a plasmid with a T region containing a DNA sequence 28, 2004. of interest. pEHA105 244981 bp M3REW M13Fw f1 origin accA W pEHA105::pWBE58 (Km, Ap) moaa. Patent Application Publication Dec. 29, 2005 Sheet 1 of 24 US 2005/0289672 A1 FIGURE 1A CLASS ALPHAPROTEOBACTERIA ORDER Rhizobiales family Rhizobiaceae bgenus Rhizobium (includes former Agrobacterium) bgenus Chelatobacter bgenus Sinorhizobium Dunclassified Rhizobiaceae family Bartonellaceae bgenus Bartonella Dunclassified Bartonellaceae family Brucellaceae bgenus Brucella genus Mycoplana D genus Ochrobactrum Dunclassified Brucellaceae family Phyllobacteriaceae Dgenus Phyllobacterium Pgenus Aminobacter genus Aquamicrobium >genus Defluvibacter Dgenus Mesorhizobium genus Pseudaminobacter Dunclassified Phyllobacteriaceae family Methylocystaceae bgenus Methylocystis D genus Albibacter genus Methylosinus >genus Terasakiella Dunclassified Methylocystaceae family Beijerinckiaceae Dgenus Beijerinckia Dunclassified Beijerinckiaceae family Bradyrhizobiaceae genus Bradyrhizobium genus Afipia >genus Blastobacter genus Bosea >genus Nitrobacter genus Rhodoblastus genus Rhodopseudomonas Dunclassified Bradyrhizobiaceae Patent Application Publication Dec. 29, 2005 Sheet 2 of 24 US 2005/0289672 A1 FIGURE 1B family Hyphomicrobiaceae genus Hyphomicrobium P genus Ancylobacter genus Azorhizobium genus Blastochloris genus Devosia genus Pedomicrobium genus Rhodomicrobium genus Rhodoplanes genus Starkeya genus Xanthobacter Punclassified Hyphomicrobiaceae family Methylobacteriaceae Pgenus Methylobacterium Dunclassified Methylobacteriaceae family Rhodobiaceae genus Rhodobium Punclassified Rhodobiaceae Punclassified Rhizobiales Patent Application Publication Dec. 29, 2005 Sheet 3 of 24 US 2005/0289672 A1 FIGURE 2 T-ONA region removed Wirulence region Origin of replication Promoter Gene of interest Pronuts Terminator Part selectable merkor T&TTiirator left T-DNA border Wido-host-range replicon Bacterial selectable marker Repbcation origin for E. coi Replication origin for Agrobacteriun Patent Application Publication Dec. 29, 2005 Sheet 10 of 24 US 2005/0289672 A1 FIGURE 4 Patent Application Publication Dec. 29, 2005 Sheet 11 of 24 US 2005/0289672 A1 FIGURE 5 LBA288 Transformant Transformant EHA101 1 2 a b c d e f a b c d e f a b c d e f M a b c d e f Chromosomal piti markers markers Patent Application Publication Dec. 29, 2005 Sheet 12 of 24 US 2005/0289672 A1 FIGURE 6 pEHA105 244981 bp M13REV f1 origin acCA W pEHA105::pWBE58 (Km, Ap) moaA Patent Application Publication Dec. 29, 2005 Sheet 13 of 24 US 2005/0289672 A1 FIGURE 7 pEHA105::pWBE58 EHA105::pWBE60 (2xvirC) = pTil (AccA) = pTi2 EHA105 EHA105 LBA pWBE58 EHA105 EHA105 LBA pWBE60 pTil wt 288 (virG)M pTi2 wt 288 (accA) s i i i VirG probe ACCA probe Patent Application Publication Dec. 29, 2005 Sheet 14 of 24 US 2005/0289672 A1 FIGURE 8 TBORDER(L) POLY ASE HYG(R) Sna I (1598) specistrepresistance gene ' CAMV35s Nsi I (10859) - g said: pBR322 or Pu II (2167) pBR322 bom si A pCAMBIA1105.1 12136bp 35S promoter ThGUSupstreamCAT No I (3456) Catalase intron GusPlusrev BGUS Ns Iss6 NOS polyA 1 T-BORDER(R) sh IGs) Patent Application Publication Dec. 29, 2005 Sheet 15 of 24 US 2005/0289672 A1 FIGURE 9 TBORDER (L) POLY A SITE HYG(R) Sima I (1598) CAMV35S Pu II (2467) pCambia 1105.1r 1227 bp 35S promoter TmgUSupstreamCAT Catalase intron GusPlusrev BGUS NOS polyA T-BORDER(R) Sph I(ooo) Patent Application Publication Dec. 29, 2005 Sheet 16 of 24 US 2005/0289672 A1 FIGURE 10 binary virs pTi1 rhizo Rhizobium spp. NGR234 Rhizobium spp. NGR234 pTi1 + pC1105.1r S. meliloti S. meliloti pTi1 + pC1105.1r PCR positive controls Patent Application Publication Dec. 29, 2005 Sheet 17 of 24 US 2005/0289672 A1 FIGURE 11 Patent Application Publication Dec. 29, 2005 Sheet 18 of 24 US 2005/0289672 A1 FIGURE 12 Agrobacterium tunefaciens Patent Application Publication Dec. 29, 2005 Sheet 19 of 24 US 2005/0289672 A1 FIGURE 13 Patent Application Publication Dec. 29, 2005 Sheet 20 of 24 US 2005/0289672 A1 FIGURE 14 Untransformed Leaf tips from three independent tobacco shoots leaf Patent Application Publication Dec. 29, 2005 Sheet 21 of 24 US 2005/0289672 A1 FIGURE 15 5 2, 3,O 5 bp a N A. 8 a 5 Hyg Multiple cloning Site Patent Application Publication Dec. 29, 2005 Sheet 22 of 24 US 2005/0289672 A1 FIGURE 16 Patent Application Publication Dec. 29, 2005 Sheet 23 of 24 US 2005/0289672 A1 FIGURE 17 Patent Application Publication Dec. 29, 2005 Sheet 24 of 24 US 2005/0289672 A1 Kb 2-2 3-2 6 13 + BV Kb 2-2 3-2 6 13 + BV FIGURE 18 US 2005/0289672 A1 Dec. 29, 2005 BIOLOGICAL GENE TRANSFER SYSTEM FOR Seven chromosomal virulence genes and Several other genes EUKARYOTC CELLS that affect virulence that are still present in commonly employed Agrobacterium Strains. CROSS-RELATED APPLICATION 0008. Despite this disadvantage, Agrobacterium-medi 0001) This application claims the benefit of U.S. Provi ated transformation of plants has been widely used for sional Application No. 60/583,426, filed 28 Jun. 2004, transformation of plant cells. Other shortcomings of using which is incorporated by reference in its entirety. Agrobacterium include a limited host range, and it can only REFERENCE TO SEQUENCE LISTING ON infect a limited number of cell types in that range. Of COMPACT DISK particular importance, whereas Agrobacterium can infect many dicots, monocotyledonous plants (monocots) are more 0002 The sequence listing of this application is provided resistant to infection. Monocotyledonous plants (monocots) Separately in a file named "414A Seq list.txt (on one (1) however, constitute most of the important food crops in the compact disc. The content of this file, which was created on world (e.g., rice, corn). Monocots are only able to be 28 Sep. 2004 and is 30,596 bytes, is incorporated in its transformed by Agrobacterium under Special conditions and entirety. using a special type of cell, the callus cells or other dedif ferentiated tissue (e.g., U.S. Pat. No. 5,591,616; No. 6,037, BACKGROUND OF THE INVENTION 552; No. 5,187,073; No. 6,074,877). Nonetheless, some 0003. This invention relates generally to technologies for monocots and Some dicots, e.g. Soybean and other legumi the transfer of nucleic acids molecules to eukaryotic cells nous plants, are still notoriously difficult to transform with and in particular technologies using non-pathogenic bacteria Agrobacterium. There also exist huge differences in trans to transfer nucleic acid Sequences to eukaryotic cells, e.g. to formation efficiency between varieties of a given plant plant cells. Species, with Some being completely recalcitrant to gene 0004. There are three essential processes for commercial transfer by Agrobacterium. use of transformation technology in crops: (i) introduction of 0009. Despite these drawbacks of Agrobacterium, other new DNA into appropriate plant cells/organs; (ii) growth or bacteria Systems have not been developed for transformation multiplication of Successfully transformed cells/plants, often of eukaryotic cells. Other bacteria genera were not believed involving Selection or discrimination methodologies, and to be suitable for transforming plants. Indeed, Agrobacte (iii) expression of transgene(s) in target cells/organs/stages. rium is widely known as the only bacterial genus that has the 0005 Each of these processes is represented by several capacity for trans-kingdom gene transfer. While Some alternative technologies of varying quality and efficiencies. reports allegedly demonstrated that the tumor-inducing abil The first Step, however, is the most critical, not only for ity of Agrobacterium could be transferred to other related plants but for transformation of any eukaryotic organism and genera, including rhizobia (Klein and Klein, Arch Microbiol. cell type. There are currently two classes of DNA introduc 52:325-344, 1953; Kern, Arch. Microbiol. 52:325-344, tion methods widely used to generate transgenic organisms, 1965), the results were not uniformly repeatable nor was physical methods and biological methods. there any physical proof of gene transfer. For example, Hooykaas, Schilperoort and their colleagues in the mid to 0006 Physical methods for introducing DNA include late 70's reported that some bacterial species, Rhizobium particle bombardment, electroporation and direct DNA trifolii and R. leguminoSarum in particular, were capable of uptake by or injection into protoplasts. These methods-in tumor formation on plants after introduction of a Tiplasmid their currently practiced forms-have
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