Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens

Abstract

The invention is directed to nucleic acid sequences derived from Fusarium fungal genes which encode the cell wall-degrading enzymes glucanase, endochitinase, and exochitinase; isolated polypeptides having glucanase, endochitinase or exochitinase activity; recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule encoding a polypeptide having glucanase activity, selected from the group consisting of: 
 (a) a nucleic acid sequence having at least 70% nucleotide sequence identity with SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923;    (b) a nucleic acid sequence encoding a polypeptide having glucanase activity, said polypeptide having an amino acid sequence which has at least 80% identity with amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17.    (c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and    (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has glucanase activity.    
     
     
         2 . The nucleic acid molecule of  claim 1  as shown in SEQ ID NO:1, 3 or 16.  
     
     
         3 . The nucleic acid molecule of  claim 1  which is contained in plasmid Glu2, FvGluS or FvGluAS.  
     
     
         4 . A nucleic acid construct comprising a nucleic acid molecule of  claim 1  operably linked to one or more control sequences which direct the production of a polypeptide having glucanase activity in an expression host.  
     
     
         5 . A cell transformed with the isolated nucleic acid molecule of  claim 1 .  
     
     
         6 . A plant transformed with the isolated nucleic acid molecule of  claim 1 .  
     
     
         7 . A seed of the plant according to  claim 6 .  
     
     
         8 . The plant of  claim 6  wherein the plant is a monocot.  
     
     
         9 . The plant of  claim 8  wherein said monocotyledonous plant is wheat.  
     
     
         10 . Sexually or asexually derived progeny of the plant of  claim 6 .  
     
     
         11 . A glucanase polypeptide encoded by the nucleic acid molecule of  claim 1 .  
     
     
         12 . An isolated polypeptide having glucanase activity, selected from the group consisting of: 
 (a) a polypeptide having an amino acid sequence which has at least 80% identity with amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17;    (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii); and    (c) a fragment of (a), or (b) that has glucanase activity.    
     
     
         13 . An isolated nucleic acid molecule encoding a polypeptide having endochitinase activity, selected from the group consisting of: 
 (a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217;    (b) a nucleic acid sequence encoding a polypeptide having endochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 399 of SEQ ID NO:11 or 19;    (c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and    (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.    
     
     
         14 . The nucleic acid molecule of  claim 13  as shown in SEQ ID NO:10 or 18.  
     
     
         15 . The nucleic acid molecule of  claim 13  which is contained in plasmid Endo176, FvEndoS or FvEndoAS.  
     
     
         16 . A nucleic acid construct comprising a nucleic acid molecule of  claim 13  operably linked to one or more control sequences which direct the production of a polypeptide having endochitinase activity in an expression host.  
     
     
         17 . A cell transformed with the isolated nucleic acid molecule of  claim 13 .  
     
     
         18 . A plant transformed with the isolated nucleic acid molecule of  claim 13 .  
     
     
         19 . A seed of the plant according to  claim 18 .  
     
     
         20 . The plant of  claim 18  wherein the plant is a monocot.  
     
     
         21 . The plant of  claim 20  wherein said monocotyledonous plant is wheat.  
     
     
         22 . Sexually or asexually derived progeny of the plant of  claim 18 .  
     
     
         23 . An endochitinase polypeptide encoded by the nucleic acid molecule of  claim 13 .  
     
     
         24 . An isolated polypeptide having endochitinase activity, selected from the group consisting of: 
 (a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 399 of SEQ ID NO:11 or 19;    (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and    (c) a fragment of (a), or (b) that has endochitinase activity.    
     
     
         25 . An isolated nucleic acid molecule encoding a polypeptide having exochitinase activity, selected from the group consisting of: 
 (a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763;    (b) a nucleic acid sequence encoding a polypeptide having exochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 581 of SEQ ID NO:13 or 21;    (c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and    (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has exochitinase activity.    
     
     
         26 . The nucleic acid molecule of  claim 25  as shown in SEQ ID NO:8, 12 or 20.  
     
     
         27 . The nucleic acid molecule of  claim 25  which is contained in plasmid Exo9, FvExoS or FvExoAS.  
     
     
         28 . A nucleic acid construct comprising a nucleic acid molecule of  claim 25  operably linked to one or more control sequences which direct the production of a polypeptide having exochitinase activity in an expression host.  
     
     
         29 . A cell transformed with the isolated nucleic acid molecule of  claim 25 .  
     
     
         30 . A plant transformed with the isolated nucleic acid molecule of  claim 25 .  
     
     
         31 . A seed of the plant according to  claim 30 .  
     
     
         32 . The plant of  claim 30  wherein the plant is a monocot.  
     
     
         33 . The plant of  claim 30  wherein said monocotyledonous plant is wheat.  
     
     
         34 . Sexually or asexually derived progeny of  claim 30 .  
     
     
         35 . An exochitinase polypeptide encoded by the nucleic acid molecule of  claim 25 .  
     
     
         36 . An isolated polypeptide having exochitinase activity, selected from the group consisting of: 
 (a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 581 of SEQ ID NO:13 or 21;    (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and    (c) a fragment of (a), or (b) that has exochitinase activity.    
     
     
         37 . A plant transformed with one or more isolated nucleic acid molecules of claims  1 ,  13 , or  25 .  
     
     
         38 . A method of conferring or enhancing a plant's resistance to a fungal pathogen, which comprises transforming said plant with one or more isolated nucleic acid molecules which encode a polypeptide having glucanase activity, a polypeptide having endochitinase activity or a polypeptide having exochitinase activity.  
     
     
         39 . A method for producing a polypeptide having glucanase, endochitinase or exochitinase activity, which comprises cultivating a recombinant host cell comprising a transformed cell having a nucleic acid molecule which encodes a polypeptide having glucanase, endochitinase or exochitinase activity under conditions suitable for production of the polypeptide; and recovering the polypeptide.