Herbicide tolerance achieved through plastid transformation

Abstract

The present invention provides novel DNA sequences coding for protoporphyrinogen oxidase (protox) enzymes from soybean, wheat, cotton, sugar beet, oilseed rape, rice, sorghum, and sugar cane. In addition, the present invention teaches modified forms of protox enzymes that are herbicide tolerant. Plants expressing herbicide tolerant protox enzymes taught herein are also provided. These plants may be engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or they may be transformed with a gene encoding an herbicide tolerant form of a plant protox enzyme. The present invention further provides shuffled DNA molecules encoding protox enzymes having enhanced tolerance to a herbicide that inhibits the protox activity encoded by a template DNA molecule from which the shuffled DNA molecule is derived.

1 - 27 . (canceled)  
     
     
         28 . A method for expressing a mature enzyme in a plant plastid comprising: 
 (a) introducing into the plastome of a plant a chimeric gene comprising: 
 (1) a modified DNA molecule that encodes a mature enzyme that is normally targeted to a plant plastid by a plastid transit peptide, wherein said DNA molecule is modified such that a coding sequence of the plastid transit peptide is absent from said modified DNA molecule; and  
 (2) a promoter capable of expressing said DNA molecule in a plastid, wherein said promoter is operatively linked to said DNA molecule,  
   (b) expressing said DNA molecule in a plastid of said plant, wherein said mature enzyme is produced in said plastid.    
     
     
         29 . The method according to  claim 28 , wherein said mature enzyme is normally inhibited by a herbicidal compound.  
     
     
         30 . The method according to  claim 29 , wherein said mature enzyme has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP synthase) activity, acetolactate synthase (ALS) activity, acetoxyhydroxyacid synthase (AHAS) activity, acetylcoenzyme A carboxylase (ACCase) activity, glutamine synthase (GS) activity or protoporphyrinogen oxidase (protox) activity.  
     
     
         31 . The method according to  claim 30 , wherein said mature enzyme has protoporphyrinogen oxidase (protox) activity.  
     
     
         32 . The method according to  claim 31 , wherein said mature enzyme has at least one amino acid substitution compared to a naturally occurring protox enzyme, wherein said at least one amino acid substitution confers resistance to an inhibitor of the naturally occurring protox enzyme.  
     
     
         33 . The method according to  claim 32 , wherein said at least one amino acid substitution comprises an amino acid substitution occurring at a position corresponding to position 240, 245, 246, 388, 390, 451, 455, 500, or 536 of the comparitive alignment shown in Table 1.  
     
     
         34 . The method according to  claim 29 , wherein said mature enzyme produced in said plastid confers upon said plant tolerance to the herbicidal compound in an amount that inhibits growth of an untransformed plant.  
     
     
         35 . A method for expressing a mature enzyme in a plant plastid comprising: 
 (a) introducing into the plastome of a plant a chimeric gene comprising: 
 (1) a modified DNA molecule that encodes a polypeptide comprising: 
 (i) a modified, non-functional plastid transit peptide, wherein said modified, non-functional transit peptide is not competent for import in a plastid, and  
 (ii) a mature enzyme that is normally targeted to a plant plastid by a functional plastid transit peptide; and  
 
 (2) a promoter capable of expressing said DNA molecule in a plastid, wherein said promoter is operatively linked to said DNA molecule,  
   (b) expressing said DNA molecule in a plastid of said plant,    wherein said polypeptide is produced in said plastid.    
     
     
         36 . The method according to  claim 35 , wherein said mature enzyme is normally inhibited by a herbicidal compound.  
     
     
         37 . The method according to  claim 36 , wherein said mature enzyme has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP synthase) activity, acetolactate synthase (ALS) activity, acetoxyhydroxyacid synthase (AHAS) activity, acetylcoenzyme A carboxylase (ACCase) activity, glutamine synthase (GS) activity or protoporphyrinogen oxidase (protox) activity.  
     
     
         38 . The method according to  claim 37 , wherein said mature enzyme has protoporphyrinogen oxidase (protox) activity.  
     
     
         39 . The method according to  claim 38 , wherein said mature enzyme has at least one amino acid substitution compared to a naturally occurring protox enzyme, wherein said at least one amino acid substitution confers resistance to an inhibitor of the naturally occurring protox enzyme.  
     
     
         40 . The method according to  claim 39 , wherein said at least one amino acid substitution comprises an amino acid substitution occurring at a position corresponding to position 240, 245, 246, 388, 390, 451, 455, 500, or 536 of the comparitive alignment shown in Table 1.  
     
     
         41 . The method according to  claim 36 , wherein said polypeptide confers upon said plant tolerance to the herbicidal compound in an amount that inhibits growth of an untransformed plant.