Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-05-12
2004-10-05
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C536S023400, C536S023100, C435S424000, C435S425000, C435S430000, C424S234100
Reexamination Certificate
active
06800791
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to delivering proteins to plant cells. More specifically, the invention provides methods and compositions to increase the efficiency of plant cell transformation and to engineer desirable plant traits through bacterial-mediated delivery of specific proteins into plants.
BACKGROUND OF THE INVENTION
Gene transfer has offered great promise in the genetic manipulation of organisms. The movement of genes within plant species has played an important role in crop improvement for many decades. The recombinant DNA methods which have been developed have greatly extended the sources from which genetic information can be obtained for crop improvement. A variety of methods have been developed for the transformation of plants and plant cells with DNA. Indeed, many of the recent advances in plant science have resulted from the power of recombinant DNA technology coupled with plant transformation. These approaches facilitate studies of the effects of specific gene alterations and additions on plant development and physiology. They also make possible the direct manipulation of genes to bio-engineer improved plant varieties.
While strides have been made in the genetic transformation of plants, it is by no means a routine matter. In fact, low transformation efficiencies preclude many genetic studies and commercial applications.
There is evidence to suggest that cells must be dividing for transformation to occur. It has been observed that dividing transformed cells represent only a fraction of cells that transiently express a transgene. Furthermore, the presence of damaged DNA in non-plant systems (similar to DNA introduced by particle gun or other physical means) has been well documented to rapidly induce cell cycle arrest (Siede,
Mutation Res
. 337(2):73-84). Therefore it would be desirable to provide a method for increasing the number of cells undergoing division.
Cell division in higher eukaryotes is controlled by two main checkpoints in the cell cycle that prevent the cell from entering either M- or S-phase of the cycle prematurely. Evidence from yeast and mammalian systems has repeatedly shown that over-expression of key cell cycle genes can either trigger cell division in non-dividing cells, or stimulate division in previously dividing cells (i.e. the duration of the cell cycle is decreased and cell size is reduced). Examples of genes whose over-expression has been shown to stimulate cell division include cyclins (see, e.g. Doemer et al.(1996)
Nature
380:520-423; Wang et al., (1994)
Nature
369:669-671; Quelle et al (1993)
Genes Dev
. 7:1559-1571, E2F transcription factors (see, e.g. Johnson et al. (1993)
Nature
365:349-352; Lukas et al. (1996)
Mol Cell. Biol
. 16:1047-1057), cdc25 (see, e.g. Bell et al. (1993)
Plant Mol Biol
. 23:445-451; Draetta et al. (1996)
BBA
1332:53-63), mdm2 (see, e.g. Teoh et al. (1997)
Blood
90: 1982-1992.
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly growing callus cells or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into literally thousands of cells, yet transformants are recovered at frequencies of 10
−5
relative to transiently expressing cells. Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest, and accumulating evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Bowen et al.,
Tucson International Mot. Biol. Meetings
).
In addition to low transformation frequencies, in some cases the stable incorporation of DNA into recipient cells can be problematic. For example, the continuous expression of transformation enhancing genes can have negative consequences and transformed DNA may be incorporated into the host genome in a location that results in interference with other cellular functions.
The direct transfer of proteins to plant cells could be used to address both the issue of low transformation efficiencies and the undesirable effects of stable incorporation of transformed DNA. For example, a protein possessing transformation enhancing activity, that is delivered directly to a plant cell, would increase transformation efficiency and would only need to be present transiently.
Certain species of micro-organisms are known to transfer T-DNA into recipient cells by a mechanism similar to bacterial conjugation. The T-DNA traverses the bacterial membrane, the host cell wall and cell membranes, and the host nuclear membrane before integrating into the host genome through illegitimate recombination. Numerous bacterial proteins are included in these processes and have been characterized. Among these proteins are at least three from Agrobacterium: VirD2, VirE2, and VirF, whose genes are transcribed from the virulence region of the Ti plasmid, following which the proteins are transferred directly into plant cells.
VirD2 is a multifunctional protein which participates in the endonucleolytic cleavage of the T-DNA border sequences, the ligation of the left border nick for replacement strand synthesis, nuclear import of the T-complex, and precise integration of the 5′ end of T-DNA into the host genome. VirD2 establishes a covalent association with the T-DNA between a specific right-border (RB) nucleotide and TYR-29 of the protein.
VirE2 encodes a multifunctional protein that has single-stranded DNA binding (SSB) activity and coats the T-strand. VirE2 is also likely to be involved both in nuclear import and with the integration of full-length T-DNA into the host genome. VirE2 is the most abundant of the virulence proteins with 350 to 700 copies thought to be required to coat a 20 kb T-strand.
The coding sequence of the virF gene is present in octopine strains but not in nopaline strains. The transfer of VirF protein to plant cells has not been directly shown, but has been inferred from the observation that transgenic
N. glauca
plants expressing bacterial virulence gene virF are converted into hosts for nopaline stains of
A. tumefaciens
(Regensburg-Tuink and Hooykaas (1993)
Nature
363:69-71).
Agrobacterium-mediated transfer of proteins to plant cells in the form of virulence protein fusion constructs could be used to deliver proteins to plant cells to increase transformation efficiencies and to engineer specific desirable plant traits.
SUMMARY OF THE INVENTION
Methods and compositions are provided for the direct delivery of proteins into plant cells. Methods are provided for bacterial-mediated delivery of proteins to plant cells using Agrobacterium cells comprising nucleotide sequences encoding fusion proteins comprising a polypeptide of interest operably linked to the Agrobacterium virulence protein VirF. Agrobacterium cells are provided that are modified to comprise at least one fusion protein, such a fusion protein is expressed from a replicon or DNA construct comprising a virF coding sequence, or variant thereof, operably linked to a promoter that drives expression in a bacterium, wherein the polynucleotide encoding a VirF protein is operably linked to a polynucleotide encoding a polypeptide of interest.
The polynucleotide encoding the Agrobacterium VirF fusion is constructed on a replicon outside the T-DNA borders, so that the polynucleotide is not incorporated into the plant host genome upon infection of the plant cell with the modified bacterium. Thus, the problems associated with stable transformation of gene products into plant cells are avoided.
Agrobacterium fusion proteins are constructed to retain the functional properties of the VirF protein so that a selected protein as well as a transgene can be delivered to the same cell simultaneously.
This invention finds use in facilitating plant transformation and particularly in the bio-engineering of desirable plant traits into crop plants. Any protein or polypeptide may be utilized in the practice of the inven
Bailey Matthew A.
Bhattacharjee Saikat
Gelvin Stanton B.
Gordon-Kamm William J.
Lowe Keith S.
Alston & Bird LLP
Bui Phuong T.
Helmer Georgia
Pioneer Hi-Bred International , Inc.
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