Serine Hydroxymethyltransferase as Target for Herbicides

Abstract

The present invention relates to serine hydroxymethyltransferase (E.C. 2.1.2.1) as novel target for herbicides, and to nucleic acid sequences encoding a polypeptide with the biological activity of a serine hydroxymethyltransferase, which, when not present, bring about growth retardation symptoms and chlorotic leaves, comprising the nucleic acid sequence SEQ ID No:3, and functional equivalents of the abovementioned nucleic acid sequence or the nucleic acid sequence SEQ ID NO:7 and functional equivalents of the abovementioned nucleic acid sequence. Moreover, the present invention relates to the use of the abovementioned nucleic acid sequences, of functional analogs of the SEQ ID NO:3 or SEQ ID NO:7 or of polypeptides encoded by one of the abovementioned nucleic acid sequences in a method for identifying herbicidally active compounds which inhibit serine hydroxymethyltransferases, and to the use of these compounds which have been identified by the method as herbicides.

1 . A method for detecting functional analogs of the SEQ ID NO: 3 or SEQ ID NO: 7, comprising
 a) preparing a probe and subsequently screening a genomic library or cDNA library of the species in question; or   b) conducting a computer search for analogous sequences in an electronic data base.   
     
     
         2 . A nonhuman transgenic organism comprising at least one nucleic acid molecule, expression cassette or vector selected from the group consisting of:
 a) a nucleic acid molecule selected from the group consisting of:   i) a nucleic acid sequence as shown in SEQ ID NO:3;   ii) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:4;   iii) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 7;   iv) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:8; and   v) functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 95% identity to SEQ ID NO:7,   b) an expression cassette comprising genetic control sequences in operable linkage with a nucleic acid sequence as in a), and   c) a vector comprising an expression cassette according to b),   wherein said organism is selected from the group consisting of bacteria, yeasts, fungi, animals and plants.   
     
     
         3 . A method for identifying herbicidally active substances comprising
 a) bringing a nucleic acid molecule encoding a polypeptide with the enzymatic activity of a serine hydroxymethyltransferase or a polypeptide with the enzymatic activity of a serine hydroxymethyltransferase into contact with one or more test compounds under conditions which permit the test compound(s) to bind to the nucleic acid molecule or the polypeptide; and   b) detecting whether the test compound binds to the polypeptide of a); reduces or blocks the activity of the polypeptide of a); or reduces or blocks the transcription, translation or expression of the nucleic acid of a), as an indication that the test compound is herbicidally active.   
     
     
         4 . The method according to  claim 3 , wherein said polypeptide with the enzymatic activity of a serine hydroxymethyltransferase has the biological activity of a serine hydroxymethyltransferase. 
     
     
         5 . A method of identifying herbicidally active substances as claimed in  claim 3 , wherein said nucleic acid molecule is a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence as shown in SEQ ID NO:3;   b) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:4;   c) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 7;   d) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:8;   e) functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 95% identity to SEQ ID NO:7, and   f) a nucleic acid molecule comprising a region with at least 60% identity to SEQ ID NO:3.   
     
     
         6 . The method according to  claim 5 , wherein said polypeptide is encoded by a nucleic acid molecule selected from the group consisting of
 a) a nucleic acid sequence as shown in SEQ ID NO:3;   b) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:4;   c) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 7;   d) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:8; and   e) functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 95% identity to SEQ ID NO:7, and   f) a nucleic acid molecule comprising a region with at least 60% identity to SEQ ID NO:3.   
     
     
         7 . A method for identifying herbicidally active substances, comprising
 a) expressing in a transgenic organism, a plant serine hydroxymethyltransferase which is encoded by a nucleic acid sequence selected from the group consisting of:   i) a nucleic acid sequence as shown in SEQ ID NO:3;   ii) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:4;   iii) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 7;   iv) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:8;   v) functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 95% identity to SEQ ID NO:7; and   vi) a nucleic acid sequence comprising a part-region with at least 60% identity to the SEQ ID NO:3,   or culturing an organism which naturally encodes a plant serine hydroxymethyltransferase which is encoded by a nucleic acid sequence according to i)-vi);   b) preparing a cell digest of the transgenic or nontransgenic organism and bringing the serine hydroxymethyltransferase in the cell digest, in partially or homogeneously purified form, into contact with a test compound; and   c) determining whether the test compound reduces or blocks the activity of the serine hydroxymethyltransferase, where the activity of the serine hydroxymethyltransferase incubated with the test compound is compared with the activity of a serine hydroxymethyltransferase which is not incubated with a test compound; as an indication that the test compound is herbicidally active.   
     
     
         8 . The method according to  claim 7 , wherein, in step c), the activity of the serine hydroxymethyltransferase is determined by the chromatographic isolation and quantification of tetrahydrofolate and Cl-tetrahydrofolate. 
     
     
         9 . A method for identifying herbicidally active substances, comprising
 a) generating a transgenic organism as claimed in  claim 2  or a transgenic organism with a nucleic acid sequence comprising a region with at least 60% identity to SEQ ID NO:3;   b) applying a test substance to the transgenic organism generated in a) and to a nontransgenic organism of the same type;   c) determining the growth or the viability of the transgenic and the nontransgenic organisms after application of the test substance; and   d) determining test substances which bring about a reduced growth or a reduced viability of the nontransgenic organism in comparison with the growth of the transgenic organism as an indication that the test compounds are herbicidally active.   
     
     
         10 . The method as claimed in  claim 9 , wherein said transgenic organism is a plant or a yeast. 
     
     
         11 . The method according to  claim 10 , wherein said reduced growth or a reduced viability of the nontransgenic organism in comparison with the growth of the transgenic organism is between 10-50%. 
     
     
         12 . A method for identifying substances with a growth-regulatory action, comprising:
 a) generating a transgenic plant with a nucleic acid sequence comprising a region with at least 60% identity with SEQ ID NO:3;   b) applying a test substance to the transgenic plant generated in a) and to a nontransgenic plant of the same type;   c) determining the growth or the viability of the transgenic and the nontransgenic plants after application of the test substance; and   d) selecting test substances which bring about altered growth of the nontransgenic plant in comparison with the growth of the transgenic plant as an indication that the test compounds are herbicidally active.   
     
     
         13 . The method as claimed in  claim 3 , wherein the substances are identified in a high-throughput screening. 
     
     
         14 . A method for generating modified gene products which are encoded by variants of the nucleic acid sequence of SEQ ID NO: 7, comprising
 a) expressing a protein encoded by a nucleic acid as shown in SEQ ID NO: 7 in a heterologous system or in a cell-free system;   b) modifying the protein by randomized or directed mutagenesis of the nucleic acid;   c) measuring the interaction of the modified gene product with the herbicide;   d) identifying derivatives of the protein which show less interaction;   e) assaying the biological activity of the protein after application of the herbicide; and   f) selecting the nucleic acid sequences which have a modified biological activity against the herbicide.   
     
     
         15 . A method as claimed in  claim 14 , further comprising introducing the sequences selected in accordance with  claim 14  f) into an organism. 
     
     
         16 . An organism generated by the method as claimed in  claim 15 . 
     
     
         17 . An organism generated by a method for generating transgenic plants which are resistant to herbicidally active substances which reduce or inhibit the biological activity of a serine hydroxymethyltransferase, comprising overexpressing in said transgenic plants, nucleic acid sequences selected from the group consisting of:
 i) a nucleic acid sequence as shown in SEQ ID NO:3;   ii) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:4;   iii) a nucleic acid sequence with the nucleic acid sequence shown in SEQ ID NO: 7;   iv) a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO:8; and   v) functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 95% identity to SEQ ID NO:7,   or functional equivalents of the nucleic acid sequence SEQ ID NO:7 with at least 88% identity to SEQ ID NO:7.