Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-11
2001-06-12
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S419000, C435S011000, C435S430000, C435S418000, C435S423000, C800S279000, C536S023100, C536S023600, C536S024100
Reexamination Certificate
active
06245510
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.
A well-studied model for interactions of plant pathogens with their hosts is that between tomato (
Lycopersicon esculentum
) and
Pseudomonas syringae
pv.
tomato
(Pst; Carland and Staskawicz,
Mol. Gen. Genet.
239:17-27, 1993; Martin et al.,
Mol. Plant
-
Microbe Interact.
6:26-34, 1993). Two genes required for the tomato signaling pathway that leads to resistance to Pst strains that express the avirulence gene avrPto (Ronald et al.,
J. Bacterol.
174:1604-1611, 1992; Salmeron and Staskawicz,
Mol. Gen. Genet.
239:6-16, 1993) have been identified through analyses of naturally-occurring resistant and susceptible tomato lines (Pitblado and MacNeill,
Canad. J. Plant Pathol.
5:251-255, 1983) and by mutational studies (Salmeron et al.,
Plant Cell
6:511-520, 1994).
The Pto gene (Pitblado and MacNeill,
Canad. J. Plant Pathol.
5:251-255, 1983) encodes a serine/threonine protein kinase with a potential amino-terminal myristoylation site (Martin et al.,
Science
262:1432-1436, 1993) that lacks additional motifs such as a leucine-rich repeat. Pto is a member of a tightly clustered family of five genes located on the short arm of chromosome five. It encodes a protein highly similar to the cytoplasmic domain of the Brassica self-incompatability gene SRK and the mammalian signaling factor Raf (Martin et al.,
Science
262:1432-1436, 1993).
The identification of Pto as a protein kinase suggests that intracellular phosphorylation events are important in the response of tomato to pathogen strains expressing avrPto. The tomato Pti1 protein is a substrate for Pto (Zhou et al.,
Cell
83:925-935, 1995) and Pti1 itself is predicted to be a serine/threonine protein kinase (Zhou et al.,
Cell
83:925-935, 1995). Therefore, the pathway for defense against Pst may incorporate a protein kinase cascade similar to those employed in numerous other eukaryotic signaling pathways (Hunter,
Cell
80:225-236, 1995).
The second gene required for resistance of tomato to Pst, designated Prf, was identified through a mutational approach and shown to be tightly linked to Pto (Salmeron et al.,
Plant Cell
6:511-520, 1994). Analysis of prf mutant alleles suggested that in addition to its role in disease resistance, the Prf protein also functions in the response of tomato to the organophosphate insecticide Fenthion (Salmeron et al.,
Plant Cell
6:511-520, 1994), a trait that co-segregates with Pto in genetic crosses (Carland and Staskawicz,
Mol. Gen. Genet.
239:17-27, 1993). In sensitive tomato lines, Fenthion induces rapid necrosis that mimics the hypersensitive response observed after inoculation with Pst strains expressing avrPto (Laterrot and Philouze,
Tomato Genet. Research Coop. Newsletter
35:6, 1985). This observation suggests that Fenthion mimics an elicitor produced under control of the avrPto gene in Pst.
Necrosis in response to Fenthion does not appear to require the Pto kinase (Martin et al.,
Science
262:1432-1436, 1993; Salmeron et al.,
Plant Cell
6:511-520, 1994), but rather is conferred by another member of the Pto gene cluster, designated Fen. Fen encodes a protein kinase 80% identical in amino acid sequence to Pto (Martin et al.,
Plant Cell
6:1543-1552, 1994; Rommens et al.,
Plant Cell
7:249-257, 1995). Thus, Prf is involved with two similar but distinct kinases, Pto and Fen, to induce hypersensitive-like necrosis in response to pathogen elicitor and Fenthion signals, respectively.
SUMMARY OF THE INVENTION
The tomato Prf genomic and cDNA sequences have been cloned and the corresponding DNA and amino acid sequences are provided herein. Expression of the Prf gene in transgenic plants confers resistance to Pst and, surprisingly, to a broad variety of unrelated pathogens. Also encompassed by the present invention are such transgenic plants. The tomato Prf gene hybridizes to homologous sequences from a variety of other plant species under moderately stringent hybridization conditions, and probes and primers based on the tomato Prf sequence can be used to isolate such Prf homologs. Based on these discoveries, the present invention provides compositions and methods related to the isolated tomato Prf gene.
For example, the present invention provides nucleic acid sequences that hybridize specifically to a native Prf sequence under at least moderately stringent conditions, preferably including at least 15 contiguous nucleotides of a native tomato Prf nucleic acid sequence. Such sequences are useful, for example, as probes and primers for isolating Prf homologs from other plant species. When expressed in transgenic plants (or plant cells or tissues), longer portions of the native Prf nucleic acid sequence, including all or a significant portion of the Prf coding region, confer pathogen resistance and/or Fenthion sensitivity.
The present invention also provides, for example, the native tomato Prf promoter sequence, which is useful, for example, for expressing a Prf gene or a heterologous gene in plant cells.
Also provided are sequences corresponding to various functional domains of the tomato Prf polypeptide, including, for example: (1) three motifs comprising the predicted ATP/GTP binding site, the “P-loop” domain occurring at residues 1120-1132, followed by the companion kinase domains 2 and 3a at 1195-1205 and 1224-1231, respectively; (2) sequences resembling leucine-rich repeat domains with approximately fourteen to eighteen imperfect copies of the leucine-rich repeat motif with a consensus sequence of LXXLXXLXXLXLXXN/CXXLXXIPSX, beginning at residue 1398; (3) a leucine zipper spanning residues 959-994; (4) a block of residues from 716-858 that includes two copies of a direct repeat, with 49% amino acid identity between the two copies; and (5) a string of seven amino acids (1058-1064) that corresponds precisely to one half of the binding site for interleukin-8 in the mammalian interleukin-8 receptor.
Armed with the disclosed tomato Prf nucleotide and amino acid sequences and taking advantage of the degeneracy of the genetic code, it is possible to design nucleic acids that are similar to the tomato Prf gene and that encode functional Prf polypeptides. Preferably, such nucleic acids include only silent or conservative changes to the native tomato Prf gene sequence. The present invention therefore also provides isolated nucleic acids that include a sequence that encodes a polypeptide that is at least about 70% homologous to a native tomato Prf polypeptide and that has Prf activity. That is, when expressed in a transgenic plant (or plant cell or tissue), the nucleic acid encodes a polypeptide that confers pathogen resistance or Fenthion sensitivity.
The present invention also encompasses cells, tissues, or transgenic plants comprising such nucleic acids.
The present invention also provides isolated Prf polypeptides comprising the at least 10 consecutive amino acids of a native tomato Prf polypeptide. Such polypeptides are useful for a number of purposes, including raising Prf-specific antibodies that can be used to purify Prf polypeptides, in immunoassays, or for expression cloning efforts.
Also provided by the present invention are related methods of producing Prf polypeptides, methods of producing pathogen-resistant transgenic plants, and methods of obtaining Prf genes from various plant species (and Prf homologs obtained thereby).
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 shows the nucleotide and ami
Oldroyd Giles Edward
Rommens Caius
Salmeron John M.
Staskawicz Brian S.
Jones W. Gary
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
The Regents of the University of California
Wilder Cynthia B.
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