Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1998-09-28
2003-12-23
Mehta, Ashwin (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
C435S320100, C435S419000, C435S468000, C536S023720, C800S286000, C800S301000
Reexamination Certificate
active
06667426
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to disease resistance in plants.
Grapevine fanleaf virus (GFLV) is a grape nepovirus, which is transmitted from plant to plant by the dagger nematode,
Xiphinema index
. GFLV is the agent responsible for grapevine fanleaf disease, which occurs worldwide. The disease is named for the fan-leaf shaped appearance of GFLV-infected leaves. It is one of the most damaging and widespread diseases of grapevine. Symptoms of GFLV infection include abnormal shoot morphology and discolorations of the leaves, yielding a fan-like appearance (Agrios,
Plant Pathology
, 3
rd
Edition, Academic Press, 1988, pp. 687-688). In addition, fruit production of infected vines is low, with grapevines producing small bunches having abnormal fruit set and ripening. Ultimately, infected grapevines degenerate and die.
Long range spread of GFLV is believed to be by use of infected planting material. While the natural host range is thought to be restricted to grape, GFLV is also transmissible to a wide range of herbaceous species by sap-rubbing inoculation.
Chenopodium quinoa
is a useful diagnostic species for the virus. In general, GFLV isolates are antigenically uniform and diagnosis by ELISA is a standard procedure.
Current strategies for controlling grapevine fanleaf disease and other nepovirus-induced diseases in vineyards include nematode control (for example, soil fumigation and use of other pesticides), breeding rootstocks for resistance to nematode feeding, breeding grapevines for resistance to GFLV, and planting certified disease-free grapevines.
SUMMARY OF THE INVENTION
In general, the invention features a method for producing and selecting a transgenic grapevine or grapevine component having increased resistance to a fanleaf disease. The method generally involves: (a) transforming a grape plant cell with a grape nepovirus coat protein nucleic acid molecule or fragment thereof (for example, a grape nepovirus coat protein nucleic acid molecule or fragment thereof having about 50% or greater sequence identity to SEQ ID NO: 1) which is capable of being expressed in a plant cell; (b) regenerating a transgenic grapevine or grapevine component from the plant cell; and (c) selecting a transgenic grapevine or grapevine component which expresses, at a low level, the nucleic acid molecule or fragment thereof, wherein the low level expression increases the resistance of the transgenic grapevine or grapevine component to fanleaf disease as compared to plants expressing the nucleic acid molecule at a high level. Low level expression of the grape nepovirus mRNA or of the expressed coat protein itself in the transgenic plant is measured according to standard methods including, without limitation, Northern blot analysis, ELISA, and inoculation of transgenic plants with virus and selection of resistant vines. In preferred embodiments, the nucleic acid molecule or fragment thereof is encoded by a transgene found in the transgenic grapevine. In other preferred embodiments, the nucleic acid molecule or fragment thereof is expressed as in a sense or antisense orientation. In yet other preferred embodiments, such grape nepovirus coat protein nucleic acid molecules or fragments thereof (a non-limiting example being a sense nontranslatable grape nepovirus viral coat protein mRNA having an out-of-reading frame initiation ATG initiation codon with the remainder of the mRNA being out of frame) is expressed in the transgenic grapevine or grapevine component.
As is discussed above, the invention also includes fragments of a grape nepovirus coat protein nucleic acid molecule that facilitate, when expressed at low levels, an increased resistance of a transgenic grapevine or grapevine component thereof, to a fanleaf disease. Thus, grape nepovirus coat protein nucleic acid sequences described herein or portions thereof may be expressed in a plant to facilitate disease resistance. Sequences that mediate an increased resistance to a fanleaf 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. Fragments of a grape nepovirus nucleic acid molecule can be produced and, subsequently, integrated into any standard expression vector (for example, those described herein) according to methods known to those skilled in the art.
Preferably, the grapevine useful in the invention is a member of the genus Vitis; and the grapevine component is a somatic embryo, a scion, a rootstock, or a mother block. In still other preferred embodiments, the fanleaf disease is grapevine fanleaf disease caused by a grape nepovirus. In yet other preferred embodiments, the grape nepovirus is a grapevine fanleaf virus or an arabis mosaic virus.
In another aspect, the invention features a vineyard including three or more transgenic grapevines or grapevine components each of which express, at a low level, a grape nepovirus coat protein nucleic acid molecule or fragment thereof, wherein the low level expression of the nucleic acid molecule or fragment thereof increases resistance of the transgenic grapevines or grapevine components in the vineyard to fanleaf disease.
In still another aspect, the invention features a substantially pure protein (for example, a recombinant protein) including an amino acid sequence having at least 97% amino acid identity to the amino acid sequence of the ‘Geneva’ isolate grape nepovirus coat protein shown in
FIGS. 1A-1C
(SEQ ID NO: 2). In preferred embodiments, the protein includes the amino acid sequence of the grape nepovirus coat protein shown in
FIGS. 1A-1C
(SEQ ID NO: 2). In yet other preferred embodiments, the protein has the amino acid sequence of the grape nepovirus coat protein shown in
FIGS. 1A-1C
(SEQ ID NO: 2) or fragments thereof.
In yet another aspect, the invention features an isolated nucleic acid molecule encoding a protein (for example, a recombinant protein) including an amino acid sequence having at least 97% amino acid identity to the amino acid sequence of the ‘Geneva’ isolate grape nepovirus coat protein shown in
FIGS. 1A-1C
(SEQ ID NO: 2). In preferred embodiments, the protein encoded by the nucleic acid molecule includes the amino acid sequence of SEQ ID NO: 2. In yet other preferred embodiments, the protein encoded by the nucleic acid molecule has the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.
In another aspect, the invention features an isolated nucleic acid molecule (for example, a DNA molecule) that encodes a grape nepovirus coat protein that specifically hybridizes to a nucleic acid molecule that includes the nucleic acid sequence of
FIGS. 1A-1C
(SEQ ID NO: 1). Preferably, the specifically hybridizing nucleic acid molecule encodes a grape nepovirus sequence that mediates resistance when expressed at low levels in a grape plant cell to a fanleaf disease (for example, grapevine fanleaf disease). The invention also features an RNA transcript having a sequence complementary to any of the isolated nucleic acid molecules described above.
In related aspects, the invention further features a cell (for example, a prokaryotic cell or a eukaryotic cell such a mammalian cell or yeast cell) which includes an isolated nucleic acid molecule of the invention. In preferred embodiments, the cell is a bacterium (for example,
E. coli
or
Agrobacterium tumefaciens
) or is a plant cell (for example, a grape plant cell from any of the cultivars listed herein). Such a plant cell has resistance against a fanleaf disease (for example, grapevine fanleaf disease).
In still other related aspects, the invention further features a vector (for example, a plant expression vector) which includes an isolated nucleic acid molecule of the invention. In a preferred embodiment, the isolated nucleic acid molecule is operably linked to an expression control region that mediates
Fuchs Marc
Gonsalves Dennis
Krastanova Tania
Ling Kai-Shu
Xue Baodi
Clark & Elbing LLP
Cornell Research Foundation Inc.
Mehta Ashwin
LandOfFree
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