Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-26
2003-06-17
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C800S279000, C800S301000, C514S002600, C514S012200, C514S013800, C514S015800, C530S324000, C530S326000, C530S328000, C530S370000, C530S379000
Reexamination Certificate
active
06579677
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to plant disease resistance, in particular to plant genes conferring pathogen resistance.
Whether a plant is resistant or susceptible to attack by a given pathogen is frequently under the control of a single, dominant resistance gene (Flor,
Annu. Rev. Phytopathol
. 9:275-296, 1971). Resistance gene products are thought to recognize signal molecules produced by the pathogen and respond by initiating rapid changes in host cell physiology and metabolism that directly inhibit pathogen growth.
Classic genetic analyses of disease resistance in plants have demonstrated that resistance to pathogens is often highly specific, requiring single corresponding genetic loci in both the plant and the pathogen. Several plant genes corresponding to these resistance loci have recently been cloned and characterized. The Pto gene from tomato, which confers resistance to
Pseudomonas syringae
pv.
tomato
, encodes a serine-threonine protein kinase (Martin et al.,
Science
262:1432-1436, 1993). The RPS2, RPM1 and RPP5 genes from Arabidopsis (Bent et al.,
Science
265:1856-1860, 1994; Mindrinos et al.,
Cell
78:1089-1099, 1994; Grant et al.,
Science
269:843-846, 1995), the N gene from tobacco (Whitham et al.,
Cell
78:1101-1115, 1994), and the L6 gene from flax (Lawrence et al.,
Plant Cell
7:1195-1206, 1995) all have several motifs suggestive of signaling roles for these proteins. These motifs include nucleotide binding sites (NBSs) and leucine-rich repeats (LRRs); thus, these proteins are commonly referred to as the NBS/LRR class. LRR motifs have been implicated in protein-protein interactions (Kobe and Deisenhofer,
Trends Biochem. Sci
. 19:415-421, 1994), raising the possibility that the LRR portion of these resistance gene products is required for specific recognition of a protein elicitor, or for interaction with other protein components of a signal transduction pathway. RPP5, L6 and N also contain N-terminal domains with homology to the mammalian interleukin 1 receptor (IL-1R) and the Drosophila Toll gene. These genes encode transmembrane receptor tyrosine kinases that act upstream of the rel family transcription factors NF-&kgr;B and Dorsal. A kinase with high levels of homology to Pto—the Pelle gene product—functions downstream of Toll in Drosophila.
It is possible that molecular recognition of an avirulent pathogen requires proteins with all of these motifs. The identification of Prf, an NBS/LRR protein required for function of the Pto gene, and of Xa-21, a gene that confers resistance to bacterial blight of rice and has both an LRR and a kinase domain, supports this hypothesis. This striking similarity between genes that confer resistance to bacterial, viral and fungal pathogens suggests that mechanisms of resistance are conserved within and among different plant species. Therefore, the signal transduction pathways utilized by the different resistance gene products may converge at some common step. However, genes that integrate different upstream molecular recognition signals and activate a hypothetical common downstream resistance pathway have not been previously identified.
SUMMARY OF THE INVENTION
We have identified a gene in
Arabidopsis thaliana
that encodes a pathogen-induced signal integrator required for disease resistance, referred to herein as NDR1. The nucleotide sequence of a genomic clone of NDR1 and the corresponding amino acid sequence are provided. The NDR1 gene is required for resistance to the bacterial pathogen
Pseudomonas syringae
pv.
tomato
(
Pst
) and the fungal pathogen
Peronospora parasitica
, among others. Expression of NDR1 in transgenic plants confers resistance to a broad variety of plant pathogens, including, but not limited to, bacteria, viruses, fungi, nematodes, and other plant pathogens. According to one aspect of the present invention, isolated nucleic acids are provided that include a promoter that is expressible in a plant cell, and, operably linked to the promoter, (a) a polynucleotide comprising at least 100 consecutive nucleotides having at least 70% nucleotide sequence similarity with SEQ ID NO:1; or (b) a polynucleotide that encodes a polypeptide of at least 100 amino acid residues having at least 70% amino acid sequence similarity with SEQ ID NO:2. Preferably, the polynucleotide includes only silent nucleotide sequence changes to the NDR1 sequence of SEQ ID NO:1 or changes that result in conservative amino acid substitutions. Since NDR1 has two putative transmembrane domains, preferably the polynucleotide encodes two transmembrane domains. Transformation of a plant cell with the polynucleotide sequence increases resistance of the plant cell to the pathogen.
Therefore, according to another aspect of the invention, transgenic plants are provided that comprise nucleic acids as described above. Such transgenic plants can be produced by introducing such nucleic acids into the cell of a plant, thereby producing a transformed plant cell, and regenerating the transformed cell to produce a transgenic plant. As discussed in the Example, such transgenic plants display greater pathogen resistance than control plants.
Alleles and homologs of the NDR1 gene from Arabidopsis accession Col-0 can be obtained, for example, by contacting a plurality of polynucleotides of a plant other than Arabidopsis accession Col-0 with a probe or primer comprising at least 15 contiguous nucleotides of SEQ ID NO:1 under at least moderately stringent hybridization conditions, to permit the probe or primer to hybridize to an NDR1 polynucleotide of the plant, and isolating the NDR1 polynucleotide of the plant species to which the probe or primer hybridizes. For example, a plant cDNA or genomic library can be screened with a NDR1 probe that includes a detectable label. Alternately, an amplification reaction (e.g., the polymerase chain reaction, PCR) can be performed on the plant's mRNA, cDNA, or genomic DNA to produce an amplified NDR1 polynucleotide, which can be isolated.
The availability of the cloned NDR1 gene also makes it possible to produce isolated NDR1 polypeptides, e.g., by recombinant expression of NDR1 constructs in host cells. NDR1-specific antibodies can be raised against purified NDR1 and used, for example, for purifying NDR1 polypeptides, in immunoassays, and for expression cloning efforts.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.
SEQUENCE LISTING
The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
SEQ ID NO: 1 shows a nucleotide sequence encoding an NDR1 polypeptide.
SEQ ID NO: 2 shows the amino acid sequence of an NDR1 polypeptide.
REFERENCES:
patent: WO95 05731 (1995-03-01), None
patent: WO95 28423 (1995-10-01), None
Century et al. NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen. 1995. (Proc. Natl. Acad. Sci. vol. 92. pp. 6597-6601), pp. 6597 (abstract), 6599-6601.*
Century et al. NDR1, a Pathogen-Induced Component Required for Arabidopsis Disease Resistance. 1997. (Science vol. 278: 1963-1965.*
Century et al., “NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.” Proceedings of the national Academy of Sciences of the United States of America, pp. 6597-6601 (1995).
Linthorst et al. The Plant Cell. 1989. Mar. Issue. vol. 1: 285-291.
Newman et al., 3321 Lambda-PRL2 Arabidopsis Thaliana cDNA Clone 95G3T7, see whole document (1994).
Newman, T., et al., Genes Galore: A Summary of Methods for Accessing Results from Large-Scale Partial Sequencing of Anonymous Araidopsis cDNA Clones,Plant Physiol.(1994( 106:1241-1255.
Sequence Listing Accession No. T21313 from theEntrezdatab
Century Karen S.
Dahlbeck Douglas
Repetti Peter P.
Shapiro Allan
Staskawics Brian J.
Chism Billy D.
Klarquist & Sparkman, LLP
Low Christopher S. F.
The Regents of the University of California
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