Rps gene family, primers, probes and detection methods

Abstract

Disclosed is substantially pure DNA encoding an Arabidopsis thaliana Rps 2 polypeptide, substantially pure Rps 2 polyneptide; 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.

1 . 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.   
     
     
         2 . 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.   
     
     
         3 . The method of  claim 1 , wherein said amplification is carried out using a reverse-transcription polymerase chain reaction. 
     
     
         4 . The method of  claim 1 , wherein said reverse-transcription polymerase chain reaction is RACE. 
     
     
         5 . 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.   
     
     
         6 . The method of  claim 5 , wherein said DNA sequence is produced according to the method of  claim 1 . 
     
     
         7 . The method of  claim 5 , wherein said preparation of plant cell DNA is isolated from a plant genome. 
     
     
         8 . A method of isolating a disease-resistance gene or portion thereof from a plant cell, plant tissue sample, or recombinant plant cell library, comprising the steps of:
 (a) providing DNA sample from a plant cell, plant tissue, or recombinant plant cell library;   (b) providing a pair of oligonucleotides having sequence homology to any one of SEQ ID NOs: 110-113, 145-156, 158-162, 209-211, or 213;   (c) combining said pair of oligonucleotides with said DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and   (d) isolating said amplified disease-resistance gene or portion thereof.