Novel Antifungal Proteins and Methods of Use

Abstract

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for novel variants of antipathogenic polypeptides generated through DNA shuffling that exhibit improved antipathogenic activity. Polynucleotides that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the polynucleotides disclosed herein is further provided. Compositions comprising an antipathogenic polypeptide or a microorganism comprising an antipathogenic polynucleotide of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Plants, plant cells, seeds, and microorganisms comprising an antipathogenic polynucleotide or polypeptide of the invention are also disclosed.

That which is claimed: 
     
         1 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence set forth in SEQ ID NO: 6 or 8;   (b) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8, wherein the polypeptide has antifungal activity;   (c) an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 6, wherein the polypeptide has antifungal activity;   (d) an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6, wherein the amino acid sequence comprises at least one of the amino acid residues selected from the group consisting of:
 (i) the arginine (Arg) residue at the position corresponding to residue 8 of SEQ ID NO: 6; and 
 (ii) the lysine (Lys) residue at the position corresponding to residue 42 of SEQ ID NO: 6, wherein the polypeptide has antifungal activity; and 
   (e) an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8, wherein the amino acid sequence comprises at least one of the amino acid residues selected from the group consisting of:
 (i) the arginine (Arg) residue at the position corresponding to residue 8 of SEQ ID NO: 8; 
 (ii) the glutamine (Glu) residue at the position corresponding to residue 41 of SEQ ID NO: 8; and 
 (iii) the lysine (Lys) residue at the position corresponding to residue 42 of SEQ ID NO: 8, wherein the polypeptide has antifungal activity. 
   
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the polypeptide has antifungal activity against at least one of  Colletotrichum graminocola, Diplodia maydis, Fusarium graminearum , and  Fusarium verticillioides.    
     
     
         3 . An expression cassette comprising a polynucleotide selected from the group consisting of:
 (a) a polynucleotide comprising SEQ ID NO: 5 or 7;   (b) polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 4, 6, or 8;   (c) polynucleotide having at least 85% sequence identity to SEQ ID NO: 5, or 7, wherein the polynucleotide encodes a polypeptide having antifungal activity;   (d) polynucleotide encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8, wherein the polynucleotide encodes a polypeptide having antifungal activity;   (e) polynucleotide encoding an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 6, wherein the polynucleotide encodes a polypeptide having antifungal activity;   (f) polynucleotide encoding an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6, wherein the amino acid sequence comprises at least one of the amino acid residues selected from the group consisting of:
 (i) the arginine (Arg) residue at the position corresponding to residue 8 of SEQ ID NO: 6; and 
 (ii) the lysine (Lys) residue at the position corresponding to residue 42 of SEQ ID NO: 6, wherein the polynucleotide encodes a polypeptide having antifungal activity; and 
   (g) polynucleotide encoding an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8, wherein the amino acid sequence comprises at least one of the amino acid residues selected from the group consisting of:
 (i) the arginine (Arg) residue at the position corresponding to residue 8 of SEQ ID NO: 8; 
 (ii) the glutamine (Glu) residue at the position corresponding to residue 41 of SEQ ID NO: 8; and 
 (iii) the lysine (Lys) residue at the position corresponding to residue 42 of SEQ ID NO: 8, wherein the polynucleotide encodes a polypeptide having antifungal activity; and 
   a heterologous promoter operably linked to the polynucleotide.   
     
     
         4 . The expression cassette of  claim 3 , wherein the encoded polypeptide has antifungal activity against at least one of  Colletotrichum graminocola, Diplodia maydis, Fusarium graminearum , or  Fusarium verticillioides.    
     
     
         5 . (canceled) 
     
     
         6 . A transformed plant comprising the expression cassette of  claim 3 , wherein the plant has improved fungal resistance over a non-transformed plant. 
     
     
         7 . A method of enhancing fungal resistance in a plant, the method comprising introducing into the plant the expression cassette of  claim 3 . 
     
     
         8 . A microorganism comprising the expression cassette of  claim 3 . 
     
     
         9 . An antifungal composition comprising the microorganism of  claim 8  and a suitable carrier. 
     
     
         10 . A method for protecting a plant from a fungal pathogen, the method comprising applying a composition according to  claim 9  to the environment of a plant fungal pathogen. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . A method of enhancing fungal pathogen resistance in a plant, the method comprising introducing into the plant the expression cassette of  claim 3 . 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled)