Method of producing exogenic allelism in a plant

Abstract

A non-human eukaryotic organism having a genome which includes a first exogene being in a first chromosome of a chromosome pair of the genome and a second exogene being in a second chromosome of the chromosome pair, the first and the second exogenes being in allelic relationship, such that the first and the second exogenes obligatorily segregate to different gametes.

1 . A method of generating exogenic allelism in a plant, the method comprising the steps of: 
 (a) providing a first plant and a second plant each comprising an expression cassette in the same chromosomal location, said expression cassette comprising: 
 (i) a first segment comprising a first transcribable polynucleotide sequence, said first transcribable polynucleotide sequence being operatively linked to a first promoter sequence, wherein the first segment is flanked by first site-specific recombination sequences; and  
 (ii) a second segment, being linked to the first segment, the second segment comprising a second transcribable polynucleotide sequence that is operatively linked to a second promoter sequence, wherein the second segment is flanked by second site-specific recombination sequences;  
   (b) introducing, by transformation or crossing, a first polynucleotide sequence encoding a first recombinase into the first plant, wherein the first recombinase recognizes the first site-specific recombination sequences so as to excise said first segment and produce a first plant comprising the first recombinase, selfing said first plant comprising the first recombinase, and selecting a progeny devoid of the first polynucleotide sequence encoding the first recombinase, wherein the progeny comprises the second segment of the expression cassette, but not the first segment;    (c) introducing, by transformation or crossing, a second polynucleotide sequence encoding a second recombinase into said second plant, wherein the second recombinase recognizes the second site-specific recombination sequences so as to excise the second segment and produce a second plant comprising the second recombinase, selfing the second plant comprising the second recombinase, and selecting a progeny devoid of the second polynucleotide sequence encoding said second recombinase, wherein the progeny comprises the first segment of the expression cassette, but not the second segment; and    (d) crossing the progeny resulting from step (b) with the progeny resulting from step (c), so as to generate an offspring plant characterized by exogenic allelism.    
     
     
         2 . The method of  claim 1 , wherein the second transcribable polynucleotide sequence encodes an expression product that transactivates the expression of the first transcribable polynucleotide sequence.  
     
     
         3 . The method of  claim 2 , wherein the first transcribable polynucleotide sequence encodes a cytotoxic polypeptide or a cytostatic polypeptide.  
     
     
         4 . The method of  claim 3 , wherein the polypeptide is pectate lyase, 1-3 β-glucanase, avidin, streptavidin, diphtheria toxin A-chain, URF13, indole acetic acid-lysine synthetase, CytA toxin, RNase-TI or Barnase.  
     
     
         5 . The method of  claim 2 , wherein the first transcribable polynucleotide sequence encodes an antisense RNA molecule.  
     
     
         6 . The method of  claim 2 , wherein the expression product of the second transcribable polynucleotide is a bacterial RNA polymerase or a bacteriophage RNA polymerase.  
     
     
         7 . The method of  claim 2 , wherein the second promoter sequence is selected from a group consisting of constitutive promoters and induced promoters.  
     
     
         8 . The method of  claim 2 , wherein the second promoter sequence is a tissue specific promoter.  
     
     
         9 . The method of  claim 1 , wherein the first and the second transcribable polynucleotide sequences encode expression products that assemble into a hetero-oligomeric protein.  
     
     
         10 . The method according to  claim 10 , wherein the hetero-oligomeric protein is cytotoxic or cytostatic protein.  
     
     
         11 . A plant or plant seed produced according to the method of  claim 1 , wherein the plant or the plant seed is characterized by exogenic allelism, and by a genome that lacks a polynucleotide sequence encoding an exogenic recombinase.  
     
     
         12 . A plant characterized by exogenic allelism comprising a chromosome pair, wherein the chromosome pair comprises: 
 (a) a first chromosome comprising a first exogenic segment comprising a first transcribable polynucleotide sequence that is operatively linked to a first promoter sequence, wherein the first segment is flanked by first site-specific recombination sequences; and    (b) a second chromosome comprising a second exogenic segment comprising a second transcribable polynucleotide sequence that is operatively linked to a second promoter sequence, wherein the second segment is flanked by second site-specific recombination sequences;    wherein the first exogenic segment and the second exogenic segment are in an allelic relationship, such that the first and second transcribable polynucleotide sequences obligatorily segregate to different gametes.    
     
     
         13 . The plant of  claim 12 , wherein either: (i) the first transcribable polynucleotide sequence encodes a polypeptide or an RNA molecule that regulates the expression of a product of the second transcribable polynucelotide sequence; or (ii) the second transcribable polynucleotide sequence encodes a polypeptide or an RNA molecule that regulates the expression of a product of the first transcribable polynucelotide sequence.  
     
     
         14 . A method of generating a male sterile plant, the method comprising the steps of: 
 (a) providing a first plant and a second plant each comprising an expression cassette in the same chromosomal location, said expression cassette comprising: 
 (i) a first segment comprising a first promoter sequence;  
 (ii) a second segment comprising a first transcribable polynucleotide sequence; and  
 (iii) a third segment comprising a second transcribable polynucleotide sequence, wherein the second transcribable polynucleotide sequence being operatively linked to a second promoter sequence, wherein the third segment is flanked by the first and the second segments, and wherein site-specific recombination sequences are disposed between said first segment and said third segment and between said third segment and said second segment, such that the first promoter sequence is operatively linked with the first transcribable polynucleotide sequence only following excision of said third segment from the expression cassette by site specific recombination via the site-specific recombination sequences;  
   (b) introducing, by transformation or crossing, a polynucleotide sequence encoding a recombinase into the first plant, wherein the recombinase recognizes said site-specific recombination sequences so as to excise said third segment and produce a first plant comprising the recombinase, selfing said first plant comprising the recombinase and selecting a progeny devoid of the polynucleotide sequence encoding said recombinase, wherein the progeny comprises said first segment of the expression cassette operatively linked to said second segment so as to express said first transcribable polynucleotide sequence, but does not comprise said third segment; and    (c) crossing the progeny resulting from step (b) with the plant of step (a) so as to generate an offspring plant characterized by exogenic allelism wherein expression of said first and said second transcribable polynucleotide sequences results in male sterility of the plant.    
     
     
         15 . The method of  claim 14 , wherein either: (i) the expression product of the first transcribable polynucleotide sequence encodes an expression product that transactivates the expression of the second transcribable polynucleotide sequence or (ii) the expression product of the second transcribable polynucleotide sequence encodes an expression product that transactivates the expression of the first transcribable polynucleotide sequence.  
     
     
         16 . The method of  claim 15 , wherein the transactivator is non-operable with plant promoters.  
     
     
         17 . The method of  claim 16 , wherein the transactivator is a polynucleotide sequence encoding bacterial RNA polymerase or bacteriophage RNA polymerase.  
     
     
         18 . The method of  claim 15 , wherein either the first or second transcribable polynucleotide sequence encodes a cytotoxic polypeptide or a cytostatic polypeptide.  
     
     
         19 . The method of  claim 18 , wherein the cytotoxic or cytostatic polypeptide is selected from the group consisting of pectate lyase, 1-3 β-glucanase, avidin, streptavidin, diphtheria toxin A-chain, URF13, indole acetic acid-lysine synthetase, CytA toxin, RNase-TI, Barnase, protease, DNAse, and salicylate hydroxylase.  
     
     
         20 . The method of  claim 15 , wherein either the first or second transcribable polynucleotide sequence encodes an RNA molecule selected from the group consisting of an antisense RNA molecule and a ribozyme RNA molecule.  
     
     
         21 . The method of  claim 15 , wherein either the first or second promoter sequence is selected from the group consisting of constitutive promoters and induced promoters.  
     
     
         22 . The method of  claim 21 , wherein the promoter sequence is a tissue specific promoter.  
     
     
         23 . A plant or a plant seed produced according to the method of  claim 14 , wherein the plant or the plant seed is characterized by exogenic allelism, and by a genome that lacks a polynucleotide sequence encoding an exogenic recombinase.  
     
     
         24 . A male sterile plant comprising a chromosome pair, wherein the chromosome pair comprises: 
 (a) a first chromosome comprising a first expression cassette comprising a first exogenic segment, the first segment comprising a first promoter operatively linked to a second exogenic segment comprising a first polynucleotide sequence; and    (b) a second chromosome comprising a second expression cassette comprising a third exogenic segment that is flanked by the first segment and the second segment, the third segment comprising a second transcribable polynucleotide sequence that is operatively linked to a second promoter sequence, wherein the third segment is also flanked by a pair of site-specific recombination sequences, one recombination sequence between the first segment and the third segment and other recombination sequence between the second segment and the third segment,    wherein the first expression cassette and the second expression cassette are in an allelic relationship, such that the first and second transcribable polynucleotide sequences obligatorily segregate to different gametes, and the expression of the first transcribable polynucleotide and said the second transcribable polynucleotide sequences results in male sterility in the plant.    
     
     
         25 . The male sterile plant of  claim 24 , wherein either: (i) the first transcribable polynucleotide sequence encodes a polypeptide or an RNA molecule that regulates the expression of a product of the second transcribable polynucelotide sequence; or (ii) the second transcribable polynucleotide sequence encodes a polypeptide or an RNA molecule that regulates the expression of a product of the first transcribable polynucelotide sequence.