RPS gene family, primers, probes, and detection methods

Abstract

Disclosed is substantially pure DNA encoding an Arabidopsis thaliana Rps2 polypeptide; substantially pure Rps2 polypeptide; and methods of using such DNA to express the Rps2 polypeptide in plant cells and whole plants to provide, in transgenic plants, disease resistance to pathogens. Also disclosed are conserved regions characteristic of the RPS family and primers and probes for the identification and isolation of additional RPS disease-resistance genes.

What is claimed is:  
     
         1 . Substantially pure DNA encoding an Rps polypeptide.  
     
     
         2 . The DNA of  claim 1 , wherein said DNA contains the RPS2 gene.  
     
     
         3 . The DNA of  claim 1 , wherein said DNA is genomic DNA.  
     
     
         4 . The DNA of  claim 1 , wherein said DNA is cDNA.  
     
     
         5 . The DNA of  claim 1 , wherein said DNA is of a plant of the genus Arabidopsis.  
     
     
         6 . Substantially pure DNA having the sequence of FIG. 2, or degenerate variants thereof, and encoding the amino acid sequence of open reading frame “a” of FIG. 2.  
     
     
         7 . Substantially pure DNA having about 50% or greater sequence identity to the DNA sequence of FIG. 2.  
     
     
         8 . The DNA of  claim 1  or  2 , wherein said DNA is operably linked to regulatory sequences for expression of said polypeptide; and 
 wherein said regulatory sequences comprise a promoter.  
 
     
     
         9 . The DNA of  claim 8 , wherein said promoter is a constitutive promoter.  
     
     
         10 . The DNA of  claim 8 , wherein said promoter is inducible by one or more external agents.  
     
     
         11 . The DNA of  claim 8 , wherein said promoter is cell-type specific.  
     
     
         12 . A cell which contains the DNA of  claim 1 .  
     
     
         13 . The cell of  claim 12 , said cell being a plant cell.  
     
     
         14 . The plant cell of  claim 13 , said plant cell being resistant to disease caused by a plant pathogen carrying an avirulence gene generating a signal recognized by an Rps polypeptide.  
     
     
         15 . The plant cell of  claim 14 , said plant pathogen carrying an avrRpt2 gene.  
     
     
         16 . The plant cell of  claim 14 , said plant cell being from the group of plants comprising Arabidopsis, tomato, soybean, bean, maize, wheat, and rice.  
     
     
         17 . The plant cell of  claim 14 , said plant pathogen being  Pseudomonas syringae.    
     
     
         18 . The plant cell of  claim 13 , wherein said plant cell further contains an avrRpt2 gene operably linked to regulatory sequences; and 
 wherein said regulatory sequences comprise a promoter.    
     
     
         19 . The plant cell of  claim 18 , wherein said promoter is a constitutive promoter.  
     
     
         20 . The plant cell of  claim 18 , wherein said promoter is inducible by one or more external agents.  
     
     
         21 . The plant cell of  claim 18 , wherein said promoter is cell-type specific.  
     
     
         22 . A transgenic plant which contains the DNA of  claim 1  integrated into the genome of said plant, wherein said DNA is expressed in said transgenic plant.  
     
     
         23 . A transgenic plant which contains the DNA of  claim 8  integrated into the genome of said plant, wherein said DNA is expressed in said transgenic plant.  
     
     
         24 . A transgenic plant generated from the plant cell of  claim 18  wherein said DNA and said avrRpt2 gene are expressed in said transgenic plant.  
     
     
         25 . A seed from a transgenic plant of  claim 22 .  
     
     
         26 . A seed from a transgenic plant of  claim 23 .  
     
     
         27 . A seed from a transgenic plant of  claim 24 .  
     
     
         28 . A cell from a transgenic plant of  claim 22 .  
     
     
         29 . A cell from a transgenic plant of  claim 23 .  
     
     
         30 . A method of providing resistance to a plant pathogen in a plant, said method comprising: 
 producing a transgenic plant cell comprising the DNA of  claim 1  integrated into the genome of said transgenic plant cell and positioned for expression in said plant cell; and    growing a transgenic plant from said plant cell wherein said DNA is expressed in said transgenic plant.    
     
     
         31 . A method of detecting a resistance gene in a plant cell, said method comprising: 
 contacting the DNA of  claim 1  or a portion thereof greater than about 18 nucleic acids in length with a preparation of genomic DNA from said plant cell under hybridization conditions providing detection of DNA sequences having about 50% or greater sequence identity to the sequence of FIG. 2.    
     
     
         32 . A method of producing an Rps2 polypeptide comprising: 
 providing a cell transformed with DNA encoding an Rps2 polypeptide positioned for expression in said cell; culturing said transformed cell under conditions for expressing said DNA; and    isolating said Rps2 polypeptide.    
     
     
         33 . A method of providing, in a transgenic plant, resistance to a plant pathogen, said method comprising: 
 producing a transgenic plant cell comprising the DNA of  claim 8  integrated into the genome of said transgenic plant cell and positioned for expression in said plant cell; and    growing said transgenic plant from said plant cell wherein said DNA is expressed in said transgenic plant.    
     
     
         34 . A method of providing, in a transgenic plant, resistance to a plant pathogen, said method comprising: 
 growing said transgenic plant from the plant cell of  claim 18  wherein said DNA and said avrRpt2 gene are expressed in said transgenic plant.    
     
     
         35 . A method of isolating a disease resistance gene or portion thereof in plants having sequence identity to RPS2, said method comprising: 
 amplifying by PCR said disease resistance gene or portion thereof using oligonucleotide primers wherein said primers 
 (a) are each greater than 13 nucleotides in length;  
 (b) each have regions of complementarily to opposite DNA strands in a region of the nucleotide sequence of FIG. 2; and  
 (c) optionally contain sequences capable of producing restriction enzyme cut sites in the amplified product; and  
   isolating said disease resistance gene or portion thereof.    
     
     
         36 . A substantially pure Rps2 polypeptide.  
     
     
         37 . The polypeptide of  claim 32 , comprising an amino acid sequence substantially identical to an amino acid sequence shown in FIG. 2.  
     
     
         38 . A vector comprising the DNA of  claim 1 , said vector being capable of directing expression of the peptide encoded by said DNA in a vector-containing cell.  
     
     
         39 . A vector comprising the DNA of the avrRpt2 gene operably linked to regulatory sequences wherein said regulatory sequences comprise a promoter.  
     
     
         40 . A vector comprising the DNA of  claim 1  and the DNA of the avrRpt2 gene operably linked to regulatory sequences wherein said regulatory sequences comprise a promoter.  
     
     
         41 . A substantially pure oligonucleotide comprising the sequence: 
 5′ GGNATGGGNGGNNTNGGNAARACNAC 3′, wherein N is A, T, G, or C; and R is A or G.    
     
     
         42 . A substantially pure oligonucleotide comprising the sequence: 
 5′ NARNGGNARNCC 3′, wherein N is A, T, G or C; and R is A or G.    
     
     
         43 . A substantially pure oligonucleotide comprising the sequence: 
 5′NCGNGWNGTNAKDAWNCGNGA 3′, 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.    
     
     
         44 . A substantially pure oligonucleotide comprising the sequence: 
 5′ GGWNTBGGWAARACHAC 3′, 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.    
     
     
         45 . A substantially pure oligonucleotide comprising the sequence: 
 5′ TYGAYGAYRTBKRBRA 3′, 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.    
     
     
         46 . A substantially pure oligonucleotide comprising the sequence: 
 5′ TYCCAVAYRTCRTCNA 3′, 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.    
     
     
         47 . A substantially pure oligonucleotide comprising the sequence: 
 5′ GGWYTBCCWYTBGCHYT 3′, wherein B is C, G, or T; H is A, C, or T; W is A or T; and Y is T or C.    
     
     
         48 . A substantially pure oligonucleotide comprising the sequence: 
 5′ ARDGCVARWGGVARNCC 3′, 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.    
     
     
         49 . A substantially pure oligonucleotide comprising the sequence: 
 5′ ARRTTRTCRTADSWRAWYTT 3′, 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.    
     
     
         50 . A recombinant plant gene comprising the DNA sequence: 
 5′ GGNATGGGNGGNNTNGGNAARACNAC 3′, wherein N is A, T, G or C; and R is A or G.    
     
     
         51 . The gene of  claim 50 , further comprising the sequence: 
 5′ NARNGGNARNCC 3′, wherein N is A, T, G or C; and R is A or G.    
     
     
         52 . The gene of  claim 51 , further comprising the sequence: 
 5′ NCGNGWNGTNAKDAWNCGNGA 3′, 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.    
     
     
         53 . A recombinant plant gene comprising a combination of any two or more sequences of claims  50 ,  51 , and  52 .  
     
     
         54 . A substantially pure plant polypeptide comprising the amino acid sequence: 
 Gly Xaa 1  Xaa 2  Gly Xaa 3  Gly Lys Thr Thr Xaa 4  Xaa 5 , wherein Xaa 1  is Met or Pro; Xaa 2  is Gly or Pro; Xaa 3  is Ile, Leu, or Val; Xaa 4  is Ile, Leu, or Thr; and Xaa 5  is Ala or Met.    
     
     
         55 . A substantially pure plant polypeptide comprising the amino acid sequence: 
 Xaa 1  Xaa 2  Xaa 3  Leu Xaa 4  Xaa 5  Xaa 6  Asp Asp Xaa 7  Xaa 8 , wherein Xaa 1  is Phe or Lys; Xaa 2  is Arg or Lys; Xaa 3  is Ile, Val, or Phe; Xaa 4  is Ile, Leu, or Val; Xaa 5  is Ile or Leu; Xaa 6  is Ile or Val; Xaa 7  is Ile, Leu, or Val; and Xaa 8  is Asp or Trp.    
     
     
         56 . A substantially pure plant polypeptide comprising the amino acid sequence: 
 Xaa 1  Xaa 2  Xaa 3  Xaa 4  Xaa 5  Thr Xaa 6  Arg, wherein Xaa 1  is Ser or Cys; Xaa 2  is Arg or Lys; Xaa 3  is Phe, Ile, or Val; Xaa 4  is Ile, or Met; Xaa 5  is Ile, Leu, or Phe; Xaa 6  is Ser, Cys, or Thr.    
     
     
         57 . A substantially pure plant polypeptide comprising the amino acid sequence: 
 Gly Leu Pro Leu Xaa 1  Xaa 2  Xaa 3  Xaa 4 , wherein Xaa 1  is Thr, Ala, or Ser; Xaa 2  is Leu or Val; Xaa 3  is Ile, Val, or Lys; and Xaa 4  is Val or Thr.    
     
     
         58 . A substantially pure plant polypeptide comprising the amino acid sequence: 
 Xaa 1  Xaa 2  Ser Tyr Xaa 3  Xaa 4  Leu, wherein Xaa 1  is Lys or Gly; Xaa 2  is Ile or Phe; Xaa 3  is Asp or Lys; and Xaa 4  is Ala, Gly, or Asn.    
     
     
         59 . A method of isolating a disease-resistance gene or fragment thereof from a plant cell, comprising: 
 (a) providing a sample of plant cell DNA;    (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an RPS disease-resistance gene;    (c) combining said pair of oligonucleotides with said plant cell DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and    (d) isolating said amplified disease-resistance gene or fragment thereof.    
     
     
         60 . The method of  claim 59 , wherein said amplification is carried out using a reverse-transcription polymerase chain reaction.  
     
     
         61 . The method of  claim 59 , wherein said reverse-transcription polymerase chain reaction is RACE.  
     
     
         62 . A method of identifying a plant disease-resistance gene in a plant cell, comprising: 
 (a) providing a preparation of plant cell DNA;    (b) providing a detectably-labelled DNA sequence having homology to a conserved region of an RPS gene;    (c) contacting said preparation of plant cell DNA with said detectablly-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and    (d) identifying a disease-resistance gene by its association with said detectable label.    
     
     
         63 . The method of  claim 62 , wherein said DNA sequence is produced according to the method of  claim 59 .  
     
     
         64 . The method of  claim 62 , wherein said preparation of plant cell DNA is isolated from a plant genome.  
     
     
         65 . A method of isolating a disease-resistance gene from a recombinant plant cell library, comprising: 
 (a) providing a recombinant plant cell library;    (b) contacting said recombinant plant cell library with a detectably-labelled gene fragment produced according to the method of  claim 59  under hybridization conditions providing detection of genes having 50% or greater sequence identity; and    (c) isolating a member of a disease-resistance gene by its association with said detectable label.    
     
     
         66 . A method of isolating a disease-resistance gene from a recombinant plant cell library, comprising: 
 (a) providing a recombinant plant cell library;    (b) contacting said recombinant plant cell library with a detectably-labelled oligonucleotide of any of claims  41 - 49  under hybridization conditions providing detection of genes having 50% or greater sequence identity; and    (c) isolating a disease-resistance gene by its association with said detectable label.    
     
     
         67 . A recombinant plant polypeptide capable of conferring disease-resistance wherein said plant polypeptide comprises a P-loop domain or nucleotide binding site domain.  
     
     
         68 . The recombinant plant polypeptide of  claim 67 , wherein said polypeptide further comprises a leucine-rich repeating domain.  
     
     
         69 . A recombinant plant polypeptide capable of conferring disease-resistance wherein said plant polypeptide contains a leucine-rich repeating domain.  
     
     
         70 . A plant disease-resistance gene isolated according to the method comprising: 
 (a) providing a sample of plant cell DNA;    (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an RPS disease-resistance gene;    (c) combining said pair of oligonucleotides with said plant cell DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and    (d) isolating said amplified disease-resistance gene or fragment thereof.    
     
     
         71 . A plant disease-resistance gene isolated according to the method comprising: 
 (a) providing a preparation of plant cell DNA;    (b) providing a detectably-labelled DNA sequence having homology to a conserved region of an RPS gene;    (c) contacting said preparation of plant cell DNA with said detectably-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and    (d) identifying a disease-resistance gene by its association with said detectable label.    
     
     
         72 . A plant disease-resistance gene according to the method comprising: 
 (a) providing a recombinant plant cell library;    (b) contacting said recombinant plant cell library with a detectably-labelled gene fragment produced according to the method of claims  41 - 49  under hybridization conditions providing detection of genes having 50% or greater sequence identity; and    (c) isolating a disease-resistance gene by its association with said detectable label.    
     
     
         73 . A method of identifying a plant disease-resistance gene comprising: 
 (a) providing a plant tissue sample;    (b) introducing by biolistic transformation into said plant tissue sample a candidate plant disease-resistance gene;    (c) expressing said candidate plant disease-resistance gene within said plant tissue sample; and    (d) determining whether said plant tissue sample exhibits a disease-resistance response, whereby a response identifies a plant disease-resistance gene.    
     
     
         74 . The method of  claim 73 , wherein said plant tissue sample comprises leaf, root, flower, fruit, or stem tissue.  
     
     
         75 . The method of  claim 73 , wherein said candidate plant disease-resistance gene is obtained from a cDNA expression library.  
     
     
         76 . The method of  claim 73 , wherein said disease-resistance response is the hypersensitive response.  
     
     
         77 . A plant disease-resistance gene isolated according to the method comprising: 
 (a) providing a plant tissue sample;    (b) introducing by biolistic transformation into said plant tissue sample a candidate plant disease-resistance gene;    (c) expressing said candidate plant disease-resistance gene within said plant tissue sample; and    (d) determining whether said plant tissue sample exhibits a disease-resistance response, whereby a response identifies a plant disease-resistance gene.    
     
     
         78 . A purified antibody which binds specifically to an rps family protein.  
     
     
         79 . A DNA sequence substantially identical to the DNA sequence shown in FIG. 12.  
     
     
         80 . A substantially pure polypeptide having a sequence substantially identical to a Prf amino acid sequence shown in FIG. 5(A or B).