Sulfonylurea-responsive repressor proteins

Abstract

Compositions and methods relating to the use of sulfonylurea-responsive repressors are provided. Compositions include polypeptides that specifically bind to an operator, wherein the specific binding is regulated by a sulfonylurea compound. Compositions also include polynucleotides encoding the polypeptides as well as constructs, vectors, prokaryotic and eukaryotic cells, and eukaryotic organisms including plants and seeds comprising the polynucleotide, and/or produced by the methods. Also provided are methods to provide a sulfonylurea-responsive repressor to a cell or organism, and to regulate expression of a polynucleotide of interest in a cell or organism, including a plant or plant cell.

1 . An isolated polypeptide comprising a sulfonylurea-responsive repressor that specifically binds to a polynucleotide comprising an operator sequence, wherein the binding is regulated by a sulfonylurea compound. 
     
     
         2 . An isolated polypeptide comprising an amino acid substitution to a wild type tetracycline repressor ligand binding domain, wherein the polypeptide or multimer thereof specifically binds to a polynucleotide comprising an operator sequence, wherein the specific binding is regulated by a sulfonylurea compound. 
     
     
         3 . The isolated polypeptide of  claim 2 , wherein the amino acid substitution is selected from the group consisting of an amino acid substitution at amino acid residue position 55, 60, 64, 67, 82, 86, 100, 104, 105, 108, 113, 116, 134, 135, 138, 139, 147, 151, 170, 173, 174 or 177, wherein the amino acid residue position corresponds to the equivalent position using the amino acid numbering of a wild type TetR(B) of SEQ ID NO: 1. 
     
     
         4 . (canceled) 
     
     
         5 . The isolated polypeptide of  claim 3 , wherein the amino acid substitution is selected from the group consisting of:
 (a) M at amino acid residue position 55;   (b) A, L or M at amino acid residue position 60;   (c) A, N, Q, L or H at amino acid residue position 64;   (d) Y at amino acid residue position 67;   (e) N, S or T at amino acid residue position 82;   (f) F, M, W or Y at amino acid residue position 86;   (g) C, V, L, M, F, W or Y at amino acid residue position 100;   (h) R, A or G at amino acid residue position 104   (i) A, I, V, F or W at amino acid residue position 105;   (j) Q at amino acid residue position 108;   (k) A, M, H, K, T, P or V at amino acid residue position 113;   (l) I, L, M, V, R, S, N, P or Q at amino acid residue position 116;   (m) I, L, V, M, R, S or W at amino acid residue position 134;   (n) R, S, N, Q, K, or A at amino acid residue position 135;   (o) A, C, G, H, I, V, R or T at amino acid residue position 138;   (p) A, G, I, V, M, W, N, R or T at amino acid residue position 139;   (q) I, L, V, F, W, T, S or R at amino acid residue position 147;   (r) M, L, W, Y, K, R or S at amino acid residue position 151;   (s) I, L, V or A at amino acid residue position 170;   (t) A, G or V at amino acid residue position 173;   (u) L, V, W, Y, H, R, K or S at amino acid residue position 174; and,   (v) A, G, I, L, Y, K, Q or Sat amino acid residue position 177,   wherein the amino acid residue position corresponds to the equivalent position using the amino acid numbering of a wild type TetR(B) of SEQ ID NO: 1.   
     
     
         6 . The isolated polypeptide of  claim 1 , wherein the polypeptide has at least 80% sequence identity to the ligand binding domain of SEQ ID NO: 1. 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The isolated polypeptide of  claim 1 , wherein the polypeptide is selected from the group consisting of SEQ ID NO: 3-401, 1206-1213, 1228-1233, and 1240-1243. 
     
     
         11 . (canceled) 
     
     
         12 . The isolated polypeptide of  claim 1 , wherein the polypeptide specifically releases from the polynucleotide comprising an operator sequence in the presence of the sulfonylurea compound. 
     
     
         13 . The isolated polypeptide of  claim 1 , wherein the polypeptide specifically binds to the polynucleotide comprising an operator sequence in the presence of the sulfonylurea compound. 
     
     
         14 . The isolated polypeptide of  claim 1 , wherein the sulfonylurea compound is a pyrimidinylsulfonylurea compound, a triazinylsulfonylurea compound or a thiadiazolylurea compound. 
     
     
         15 . The isolated polypeptide of  claim 1 , wherein the sulfonylurea compound is selected from the group consisting of a chlorsulfuron, a metsulfuron, an ethametsulfuron, a thifensulfuron, a sulfometuron, a tribenuron, a chlorimuron, a nicosulfuron and a rimsulfuron. 
     
     
         16 . The isolated polypeptide of  claim 1 , wherein the sulfonylurea compound has herbicidal activity. 
     
     
         17 . The isolated polypeptide of  claim 1 , wherein the sulfonylurea compound is a chlorsulfuron. 
     
     
         18 . The isolated polypeptide of  claim 17 , wherein the polypeptide is selected from the group consisting of SEQ ID NO: 14-204. 
     
     
         19 . The isolated polypeptide of  claim 1 , wherein the sulfonylurea compound is an ethametsulfuron. 
     
     
         20 . The isolated polypeptide of  claim 19 , wherein the polypeptide is selected from the group consisting of SEQ ID NO: 205-401, 1206-1213, 1228-1233, and 1240-1243. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . An isolated polynucleotide encoding the polypeptide of  claim 1 . 
     
     
         24 . The isolated polynucleotide of  claim 23 , wherein the polynucleotide is selected from the group consisting of SEQ ID NO: 434-832, 1214-1221, 1222-1227, 1234-1239, and 1244-1247. 
     
     
         25 . A non-human host cell comprising the isolated polynucleotide of  claim 24  stably incorporated into its genome. 
     
     
         26 - 29 . (canceled) 
     
     
         30 . The host cell of  claim 25 , wherein the host cell is a eukaryotic cell. 
     
     
         31 . The host cell of  claim 30 , wherein the eukaryotic cell is a plant cell. 
     
     
         32 . (canceled) 
     
     
         33 . The host cell of  claim 25 , wherein the host cell is in a plant. 
     
     
         34 . A method of regulating transcription of a polynucleotide of interest in a host cell comprising:
 (a) providing a host cell comprising the polynucleotide of interest, wherein the polynucleotide of interest is operably linked to a promoter comprising at least one operator sequence;   (b) providing the polypeptide of  claim 1 , wherein the polypeptide specifically binds to the operator sequence; and,   (c) providing a sulfonylurea compound, wherein the sulfonylurea binds to the polypeptide to form a complex that modifies the binding properties of the polypeptide to the operator.   
     
     
         35 - 55 . (canceled) 
     
     
         56 . The polypeptide of  claim 1 , wherein the polypeptide has at least 88% sequence identity to the ligand binding domain of SEQ ID NO: 1. 
     
     
         57 . The polypeptide of  claim 1 , wherein the polypeptide has at least 90% sequence identity to the ligand binding domain of SEQ ID NO: 1. 
     
     
         58 . wherein the polypeptide has at least 95% sequence identity to the ligand binding domain of SEQ ID NO: 1