Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-11-04
2004-07-06
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S279000, C800S260000, C800S298000, C536S023100, C435S468000
Reexamination Certificate
active
06759574
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed generally to compositions and methods for producing plants resistant to gall disease. The methods involve transforming plants with nucleic acid constructs encoding sense-strand untranslatable RNA molecules and/or double-stranded RNA molecules, RNA molecules that are similar to (but not identical to) certain Agrobacterium tumor-inducing genes.
BACKGROUND OF THE INVENTION
Several different recombinant DNA technologies have been employed to interfere with gene expression in plants including technologies based on ribozyme molecules, antisense RNA molecules, and co-suppression constructs. For a general discussion of ribozyme technology, antisense technology and co-suppression technology, see Castanotto,
Crit. Rev. Eukaryot Gen. Expr.
2:331-357,1997; Simons,
Gene,
72:35-44, 1988; and Jorgensen,
Biotechnol.
8:340-344, 1990, respectively.
In addition to standard co-suppression techniques, researchers have studied the suppression of a target gene caused by the introduction of an untranslatable sense-strand transgene. (Dougherty and Parks,
Curr. Opin. Cell Biol.
7:399-405, 1995; Jorgensen et al.,
Science,
279:1486-1487, 1998). This method has been used to silence a number of endogenous plant genes, as well as viral genes. (Marano and Baulcombe,
Plant J.
13:537-546, 1998; Conner et al.,
Plant J.
11:809-823, 1997, Dougherty et al.,
Mol. Plant-Microbe Interact.
7:544-552, 1994; and Smith et al.,
Plant Cell
6:1441-1453, 1994).
The bacterial genus Agrobacterium contains several species that cause disease in economically important plants. For example,
Agrobacterium tumefaciens
causes gall tumors when it infects the wounded tissue of certain dicotyledonous plants such as apple trees and grape vines (DeCleene and DeLey,
Bot. Rev.
42:389-466, 1976). Pathogenic strains of Agrobacterium harbor a tumor-inducing (Ti) plasmid that carries genes essential for gall tumorigenesis (Watson et al., J.
Bacteriol.
123:255-264, 1975; Van Larebeke et al.,
Nature
252:169-170, 1974). The transferred DNA (“T-DNA”;
FIG. 1
) portion of the Ti plasmid enters the cells of susceptible plants and integrates into nuclear DNA (Chilton et al.,
Cell
11:263-271, 1977; Willmitzer et al.,
Nature
287:359-361, 1980; Chilton et al.,
Proc. Natl. Acad Sci. USA
77:4060-4064, 1980). Following T-DNA integration in cellular DNA, expression of three T-DNA genes, iaaM, iaaH, and ipt, leads to overproduction of the plant growth hormones auxin and cytokinin, resulting in tumorous growths referred to as “galls.” (Ream,
Annual Rev. Phytopathol.
27:583-618, 1989; Winans,
Micro. Revs.
56:12-31, 1992; and Zambryski,
Annu. Rev. Plant Physiol. Plant Mol. Bio.
43:465-490, 1992) (FIG.
2
). The iaaM, iaaH, and ipt genes have been well characterized, and the sequences of these genes are available for example in the Genbank database under accession numbers X56185, M25805, and X17428, respectively.
iaaM and iaaH are required for auxin production: the enzyme encoded by iaaM converts tryptophan into indole acetamide, which the enzyme encoded by iaaH converts into indole acetic acid, an auxin (Schroeder et al.,
Eur. J. Biochem.
138:387-391, 1984; Thomashow et al.,
Proc. Natl. Acad. Sci.
81:5071-5075, 1985; Thomashow et al.,
Science
231:616-618, 1986; and Inze et al.,
Mol. Gen. Genet.
194:265-274, 1984). Loss of either enzyme prevents auxin production. ipt is required for cytokinin production; the enzyme encoded by this gene converts adenosine monophosphate into isopentenyl adenosine monophosphate, a cytokinin (Akiyoshi et al.
Proc. Nall. Acad Sci. USA
81:5994-5998, 1984; Barry et al.,
Proc. Natl. Acad. Sci. USA
81:4776-4780, 1984; and Buchmann et al.,
EMBO J.
4:853-859, 1985). Inactivation of ipt and either one of the two auxin-biosynthesis genes on the Ti plasmid will reduce gall formation (Ream et al.,
Proc. Natl. Acad. Sci. USA
80:1660-1664, 1983).
Additionally, galls can be formed in cases in which just the cytokinin pathway, or only the auxin biosynthesis pathway, is functional. In these cases the galls are visually distinguishable based on their different morphological characteristics. For example, plants infected with an Agrobacterium strain that carries only the gene necessary for the production of auxin develop necrotic galls. In contrast, plants infected with strains of the bacteria that induce only the production of cytokinin produce shooty galls.
To date, adequate means do not exist to control gall disease on grape vines, fruit trees, nut trees, chrysanthemums, roses, cane berries, ornamental shrubs, and other nursery crops. Inoculation of plants with
Agrobacterium radiobacter
strain K84 affords some protection against specific strains of
A. tumefaciens
(Moore,
MicrobioL
Sci 5:92-95, 1988); however, gall disease remains a multimillion dollar worldwide problem.
Arabidopsis thaliana
plants resistant to gall disease have been produced by a traditional genetic approach (Nam et al.,
Plant Cell
9:317-333, 1997), but this strategy currently is not applicable to plants in which gall is a problem.
This invention is directed towards a new, more effective method of producing plants that are substantially resistant to gall disease caused by bacteria.
SUMMARY OF THE INVENTION
The present invention provides compositions that can be used to prevent tumor (gall) formation in plants infected with bacterial pathogens such as
Agrobacterium tumefaciens
and other species of Agrobacterium. These compositions comprise nucleic acid molecules that, when introduced into plants, can produce transgenic plants having enhanced resistance to tumors caused by such pathogens. The invention also encompasses transgenic plants that contain these nucleic acid molecules.
In general terms, the invention involves the use of nucleic acid constructs that encode untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules that share specified high levels of sequence identity with target genes in the bacterial pathogen. For example, the constructs may encode one or more types of untranslatable RNA molecules sharing high levels of sequence identity with one or more of the iaaM, iaah, or ipt tumor genes of Agrobacterium. The invention also provides nucleic acid constructs that encode multiple different untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules, including both sense and antisense RNA molecules. For example, one construct provided by the invention encodes untranslatable RNA forms of both the ipt and iaaM genes of
Agrobacterium tumefaciens
; this construct can suppress both shooty and necrotic gall formation in plants caused by a pathogenic strain of
A. tumefaciens
infecting the iplant.
The untranslatable RNA molecules appear to inhibit gall formation by triggering sequence-specific destruction of RNA molecules encoded by certain “target genes” of the infecting pathogen in the plant. However, the mechanism by which this occurs is not fully understood. For optimal efficacy, the untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules should be of a sufficient length and share a minimum degree of sequence identity with the target bacterial gene such that disease resistance is conferred to the host cell. The level of disease resistance conferred to the host cell by the RNA molecule can be determined using the experiments described below. Thus, the RNA molecules encoded by the constructs provided herein are typically at least 25 nucleotides in length and share at least 60% sequence identity with the target gene of the pathogen. However, enhanced tumor suppression may be obtained by increasing the length of the untranslatable single-stranded RNA, double-stranded RNA, and/or untranslatable double-stranded RNA molecules (e.g., to at least 100, or at least 200 nucleotides) and/or by enhancing the level of sequence identity with the target gene (e.g., to at least 70%, 75%, 80%, or 90% sequence identity).
The inventi
Lee Hyewon
Mok Machteld
Ream, Jr. Lloyd Walter
Baum Stuart F.
Klarquist & Sparkman, LLP
McElwain Elizabeth F.
The State of Oregon Acting by and through the State Board of Hig
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