Bacterial resistance in grapevine

Details Bacterial resistance in grapevine Bacterial resistance in grapevine

1998-10-15

2001-01-09

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C435S419000, C435S468000, C800S288000, C800S301000

Reexamination Certificate

active

06172280

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to disease resistance in plants.
Grapes are the most widely grown fruit crop in the world and are difficult to breed by conventional methods (Mullins et al.,
BioTechnology
8:1041-1045, 1990). There has been considerable effort targeted toward the production of transgenic grape cultivars. Recently, transformation of grape cultivars has been successful using embryogenic calli or suspension cultures for transformation. Transgenic plants have been recovered for rootstock cultivars Rupestris St. George (
Vitis rupestris
, Mullins et al.,
BioTechnology
8:1041-1045, 1990), Richter 110(
V. rupestris x V. berlandieri
, LeGall et al.,
Plant Science
102:161-170, 1994; Krastanova et al.,
Plant Cell Reports
14:550-554, 1995), 41B (
V. berlandieri x V. rupestris
, Mauro et al.,
Plant Science
112:97-106, 1995), as well as scion cultivars Chancellor (
Vitis spp
., Kikkert et al.,
Plant Cell Reports
15:311-316, 1996), Thompson Seedless (
V. vinifera
, Scorza et al.,
J. Amer. Soc. Hort. Sci
. 121:616-619, 1996), and Superior seedless (
V. vinifera
, Perl et al.,
Plant Science
104:193-200, 1996). Many of the genes introduced into such grape cultivars have been coat protein genes of grapevine viruses, including grapevine fanleaf virus (Mauro et al., 1995; Krastanova et al.,
Plant Cell Reports
14:550-554, 1995), grapevine chrome mosaic virus (LeGall et al.,
Plant Science
102:161-170, 1994), and tomato ringspot virus (Scorza et al.,
J. Amer. Soc. Hort. Sci
. 121:616-619, 1996). Additionally, transformation with a gene encoding a lytic peptide, Shiva-1, has been reported (Scorza et al.,
J. Amer. Soc. Hort. Sci
. 121:616-619, 1995). Use of these techniques and others provides the basis for engineering disease resistance in grapes; e.g., developing transgenic grape plants that are resistant to infestation by pests (e.g., insects and nematodes) and to attack by pathogenic microorganisms (e.g., fungi, bacteria, and viruses).
One microbial induced disease found in grapes is crown gall disease. It is caused by Agrobacterium spp., a soil-inhabiting, gram-negative bacterium. Persisting for long-periods of time in plant debris found in the soil, this bacterium has one of the widest host ranges of any plant pathogen. These bacteria induce galls or plant tumors on the roots, crowns, trunks, and canes of plants.
Agrobacterium causes crown gall by copying and transferring a segment of its tumor-inducing (Ti) plasmid, referred to as the transfer DNA (T-DNA), to the plant cell where it is integrated into the plant cell genome. Transfer of the T-DNA into the plant cell nucleus is dependent on the expression of various virulence (vir) genes that are also located on the Ti plasmid. One virulence protein, VirD2, is a site-specific endonuclease which, when assisted by the VirD1 protein, recognizes and nicks the ‘bottom strand’ of the left and right T-DNA border sequences (Stachel et al.,
EMBO J
. 6:857-863, 1987). The VirD2-protein becomes covalently attached to the 5′ end of the nicked strand (Ward and Barnes,
Science
242:927-930, 1988). The single-stranded T-DNA (ssT-DNA) is then exported to the plant cell (Tinland et al.,
Proc. Natl. Acad. Sci
. 91:8000-8004, 1994, Yusibov et al.,
Proc. Natl. Acad. Sci
. 91:2994-2998, 1994).
A current hypothesis for the transport of T-DNA to the plant cell nucleus is that the ssT-DNA is coated by molecules of the VirE2 protein, which has been shown to bind ssT-DNA. Coated T-DNA, referred to as the T-complex, is thought to be exported to the plant cell via a pore formed by VirB proteins. T-complexes may be imported into the nucleus using a plant-protein pathway; involving the transport of proteins having nuclear localization signals to the nucleus. Both VirD2 and VirE2 are required for optimal transfer of T-DNA to the plant cell nucleus. The T-complex therefore travels from Agrobacterium into the plant cell where the T-DNA is integrated into the plant genome.
Agrobacteriurn's T-DNA encodes a number of enzymes involved in auxin and cytokinin biosynthetic pathways. Infected cells overproduce the plant hormones auxin and cytokinin, leading to rapid and uncontrolled cell division, and the formation of galls or plant tumors. Gall formation interferes with water and nutrient flow in the plant. Infected plants typically become unproductive and are more susceptible to adverse environmental conditions. The disease is especially destructive on fruit crops such as grapes.
Crown gall of grape is caused almost exclusively by
A. vitis
and to a lesser extent by
A. tumefaciens
. The bacterium infects the grapevine at wound sites in the trunk, and leads to the formation of galls. Bacteria survive in grapevine xylem, and are disseminated by infected propagating materials. Agrobacterium infections are particularly damaging to young vines during vineyard establishment; rapidly growing galls are capable of girdling young vines in a single season (Agrios,
Plant Pathology
, 3rd edition, Academic Press, 1998). Infected vines have reduced yield and productivity.
SUMMARY OF THE INVENTION
In general, the invention features a method for providing resistance to a bacterial pathogen in a plant (e.g., a grapevine such as Vitis). The method generally involves the steps of: (a) transforming plant cells with a virulence (vir) gene or an anti-pathogenic fragment thereof; (b) regenerating the plant cells to provide a differentiated plant; and (c) selecting a transformed plant which expresses the vir gene or the anti-pathogenic fragment thereof, wherein expression of the vir gene or the anti-pathogenic fragment thereof provides resistance to the plant bacterial pathogen. In general, the processing and transfer of T-DNA from bacterium to plant are mediated by gene products encoded by the vir region residing on the tumor-inducing (Ti) or root-inducing (Ri) plasmids of Agrobacteriurn. Exemplary vir genes useful in the invention include, without limitation, virE2 and virD2.
In preferred embodiments of the invention, the vir gene or anti-pathogenic fragment thereof is integrated into the genome of the plant. Preferably, the vir gene is virE2, virD2, or both; or is mutated (e.g., is an anti-pathogenic vir gene fragment such as one encoding a deletion of a Vir protein such as the virE2 deletion B, virE2 deletion C, or virE2 deletion E). vir sequences that mediate an increased resistance to crown gall disease are considered useful in the invention. As used herein, the term “fragment,” as applied to sequences of a nucleic acid molecule, means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and most preferably at least 40 to 80 or more contiguous nucleotides. Natural or synthetic fragments of a vir gene (e.g., virE2 or virD2) can be produced and, subsequently, integrated into any standard plant expression vector (e.g., those described herein) according to methods known to those skilled in the art. The ability of such vir gene fragments (e.g., a vir gene encoding deletion B, deletion C, deletion D, or deletion E of the virE2 gene) to confer resistance, when expressed in a plant, to a bacterial plant pathogen can be tested according to standard methods (e.g., those described herein). Fragments conferring resistance to a plant bacterial pathogen are referred to as “anti-pathogenic fragments.”
In preferred embodiments, the plant is a grapevine or a grapevine component (e.g., a somatic embryo, scion, or a rootstock); the bacterial pathogen is
Agrobacterium vitis
or
Agrobacterium tumefaciens
; and resistance to
Agrobacterium vitis
or
Agrobacterium tumefaciens
reduces crown gall formation or growth of such bacteria on the infected plant. In yet other preferred embodiments, the vir gene (or an anti-pathogenic fragment thereof) is from a tumor-inducing (Ti) or root-inducing (Ri) plasmid of Agrobacterium (e.g.,
Agrobacterium vitis, Agrobacterium tumefaciens, Agrobacterium rhizogenes
). Examples of such Ti plasmids, without limitation, include nopalin

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