RPS gene family, primers, probes, and detection methods

Details RPS gene family, primers, probes, and detection methods RPS gene family, primers, probes, and detection methods

1999-04-28

2001-07-17

LeGuyader, John L. (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024330

Reexamination Certificate

active

06262248

ABSTRACT:

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made in part with Government funding and the Government therefore has certain rights in the invention.
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of application Ser. No. 08/227,360, filed Apr. 13, 1994.
The invention relates to recombinant plant nucleic acids and polypeptides and uses thereof to confer disease resistance to pathogens in transgenic plants.
Plants employ a variety of defensive strategies to combat pathogens. One defense response, the so-called hypersensitive response (HR), involves rapid localized necrosis of infected tissue. In several host-pathogen interactions, genetic analysis has revealed a gene-for-gene correspondence between a particular avirulence (avr) gene in an avirulent pathogen that elicits an HR in a host possessing a particular resistance gene.
SUMMARY OF THE INVENTION
In general, the invention features substantially pure DNA (for example, genomic DNA, cDNA, or synthetic DNA) encoding an Rps polypeptide as defined below. In related aspects, the invention also features a vector, a cell (e.g., a plant cell), and a transgenic plant or seed thereof which includes such a substantially pure DNA encoding an Rps polypeptide.
In preferred embodiments, an RPS gene is the RPS2 gene of a plant of the genus Arabidopsis. In various preferred embodiments, the cell is a transformed plant cell derived from a cell of a transgenic plant. In related aspects, the invention features a transgenic plant containing a transgene which encodes an Rps polypeptide that is expressed in plant tissue susceptible to infection by pathogens expressing the avrRpt2 avirulence gene or pathogens expressing an avirulence signal similarly recognized by an Rps polypeptide.
In a second aspect, the invention features a substantially pure DNA which includes a promoter capable of expressing the RPS2 gene in plant tissue susceptible to infection by bacterial pathogens expressing the avrRpt2 avirulence gene.
In preferred embodiments, the promoter is the promoter native to an RPS gene. Additionally, transcriptional and translational regulatory regions are preferably native to an RPS gene.
The transgenic plants of the invention are preferably plants which are susceptible to infection by a pathogen expressing an avirulence gene, preferably the avrRpt2 avirulence gene. In preferred embodiments the transgenic plant is from the group of plants consisting of but not limited to Arabidopsis, tomato, soybean, bean, maize, wheat and rice.
In another aspect, the invention features a method of providing resistance in a plant to a pathogen which involves: (a) producing a transgenic plant cell having a transgene encoding an Rps2 polypeptide wherein the transgene is integrated into the genome of the transgenic plant and is positioned for expression in the plant cell; and (b) growing a transgenic plant from the transgenic plant cell wherein the RPS2 transgene is expressed in the transgenic plant.
In another aspect, the invention features a method of detecting a resistance gene in a plant cell involving: (a) contacting the RPS2 gene or a portion thereof greater than 9 nucleic acids, preferably greater than 18 nucleic acids in length with a preparation of genomic DNA from the plant cell under hybridization conditions providing detection of DNA sequences having about 50% or greater sequence identity to the DNA sequence of
FIGS. 2A-2H
encoding the Rps2 polypeptide.
In another aspect, the invention features a method of producing an Rps2 polypeptide which involves: (a) providing a cell transformed with DNA encoding an Rps2 polypeptide positioned for expression in the cell; (b) culturing the transformed cell under conditions for expressing the DNA; and (c) isolating the Rps2 polypeptide.
In another aspect, the invention features substantially pure Rps2 polypeptide. Preferably, the polypeptide includes a greater than 50 amino acid sequence substantially identical to a greater than 50 amino acid sequence shown in
FIGS. 2A-2H
, open reading frame “a”. Most preferably, the polypeptide is the
Arabidopsis thaliana
Rps2 polypeptide.
In another aspect, the invention features a method of providing resistance in a transgenic plant to infection by pathogens which do not carry the avrRpt2 avirulence gene wherein the method includes: (a) producing a transgenic plant cell having transgenes encoding an Rps2 polypeptide as well as a transgene encoding the avrRpt2 gene product wherein the transgenes are integrated into the genome of the transgenic plant; are positioned for expression in the plant cell; and the avrRpt2 transgene and, if desired, the RPS2 gene, are under the control of regulatory sequences suitable for controlled expression of the gene(s); and (b) growing a transgenic plant from the transgenic plant cell wherein the RPS2 and avrRpt2 transgenes are expressed in the transgenic plant.
In another aspect, the invention features a method of providing resistance in a transgenic plant to infection by pathogens in the absence of avirulence gene expression in the pathogen wherein the method involves: (a) producing a transgenic plant cell having integrated in the genome a transgene containing the RPS2 gene under the control of a promoter providing constitutive expression of the RPS2 gene; and (b) growing a transgenic plant from the transgenic plant cell wherein the RPS2 transgene is expressed constitutively in the transgenic plant.
In another aspect, the invention features a method of providing controllable resistance in a transgenic plant to infection by pathogens in the absence of avirulence gene expression in the pathogen wherein the method involves: (a) producing a transgenic plant cell having integrated in the genome a transgene containing the RPS2 gene under the control of a promoter providing controllable expression of the RPS2 gene; and (b) growing a transgenic plant from the transgenic plant cell wherein the RPS2 transgene is controllably expressed in the transgenic plant. In preferred embodiments, the RPS2 gene is expressed using a tissue-specific or cell type-specific promoter, or by a promoter that is activated by the introduction of an external signal or agent, such as a chemical signal or agent.
In other aspects, the invention features a substantially pure oligonucleotide including one or a combination of the sequences:
5′ GGNATGGGNGGNNTNGGNAARACNAC 3′ (SEQ ID NO:158), wherein N is A, T, G, or C; and R is A or G;
5′ NARNGGNARNCC 3′ (SEQ ID NO:169), wherein N is A, T, G or C; and R is A or G;
5′ NCGNGWNGTNAKDAWNCGNGA 3′ (SEQ ID NO:159), wherein N is A, T, G or C; W is A or T; D is A, G, or T; and K is G or T;
5′ GGWNTBGGWAARACHAC 3′ (SEQ ID NO:160), wherein N is A, T, G or C; R is G or A; B is C, G, or T; H is A, C, or T; and W is A or T;
5′ TYGAYGAYRTBKRBRA 3′ (SEQ ID NO:163), wherein R is G or A; B is C, G, or T; D is A, G, or T; Y is T or C; and K is G or T;
5′ TYCCAVAYRTCRTCNA 3′ (SEQ ID NO:164), wherein N is A, T, G or C; R is G or A; V is G or C or A; and Y is T or C;
5′ GGWYTBCCWYTBGCHYT 3′ (SEQ ID NO:170), wherein B is C, G, or T; H is A, C, or T; W is A or T; and Y is T or C;
5′ ARDGCVARWGGVARNCC 3′ (SEQ ID NO:171), wherein N is A, T, G or C; R is G or A; W is A or T; D is A, G, or T; and V is G, C, or A; and
5′ ARRTTRTCRTADSWRAWTT 3′ (SEQ ID NO:174), wherein R is G or A; W is A or T; D is A, G, or T; S is G or C; and Y is C or T.
In other aspects, the invention features a recombinant plant gene including one or a combination of the DNA sequences:
5′ GGNATGGGNGGNNTNGGNAARACNAC 3′ (SEQ ID NO:162), wherein N is A, T, G or C; and R is A or G;
5′ NARNGGNARNCC 3′ (SEQ ID NO:169), wherein N is A, T, G or C; and R is A or G;
5′ NCGNGWNGTNAKDAWNCGNGA 3′ (SEQ ID NO:167), wherein N is A, T, G or C; W is A or T; D is A, G or T; and K is G or T.
In another aspect, the invention feaures a substantially pure plant polypeptide including one or a combiantion of the amino acid sequences:
G

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