Antifungal proteins

Abstract

Anfifungal proteins which are analogues of the Rs-AFP2 protein and contain particular mutations in their amino acid sequence are disclosed. The mutated proteins possess enhanced salt-tolerant antifungal activity. The proteins are useful for combating fungal diseases in agricultural, pharmaceutical or preservative applications.

1 . A transgenic plant comprising a DNA sequence encoding an antifungal protein having an amino acid sequence which is at least 80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid sequence of said antifungal protein contains at least one mutation selected from the group consisting of a basic residue at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9).  
     
     
         2 . The transgenic plant according to  claim 1 , wherein said plant is selected from the group consisting of canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, and onion.  
     
     
         3 . The transgenic plant of  claim 1  having improved resistance to a fungal pathogen.  
     
     
         4 . The transgenic plant according to  claim 3 , wherein said fungal pathogen is selected from the group consisting of  Alternaria brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum, Nectria haematococca, Phoma betae , and  Verticillium dahlias.    
     
     
         5 . A transgenic plant comprising a DNA sequence encoding an antifungal protein having an amino acid sequence selected from the group consisting of Rs-AFP2 (SEQ ID NO: 9), Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10) and Rs-AFP4 (SEQ ID NO: 11), wherein said amino acid sequence of said antifungal protein contains at least one mutation selected from the group consisting of a basic residue at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9).  
     
     
         6 . The transgenic plant according to  claim 5 , wherein said plant is selected from the group consisting of canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, and onion.  
     
     
         7 . The transgenic plant of  claim 5  having improved resistance to a fungal pathogen.  
     
     
         8 . The transgenic plant according to  claim 7 , wherein said fungal pathogen is selected from the group consisting of  Alternaria brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum, Nectria haematococca, Phoma betae , and  Verticillium dahliae.    
     
     
         9 . A transgenic plant comprising a DNA sequence encoding an antifungal peptide having at least six amino acid residues identical to a run of amino acid residues in an antifungal protein which is at least 80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid sequence of said antifungal protein contains at least one mutation selected from the group consisting of a basic residue at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9), said run of residues including at least one of the mutated residues.  
     
     
         10 . The transgenic plant according to  claim 9 , wherein said plant is selected from the group consisting of canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, and onion.  
     
     
         11 . The transgenic plant of  claim 9  having improved resistance to a fungal pathogen.  
     
     
         12 . The transgenic plant according to  claim 11 , wherein said fungal pathogen is selected from the group consisting of  Alternaria brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum, Nectria haematococca, Phoma betae , and  Verticillium dahliae.    
     
     
         13 . A method of producing a transgenic plant having improved resistance to a fungal pathogen comprising: 
 a) introducing into a plant cell a DNA sequence encoding an antifungal protein having an amino acid sequence which is at least 80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid sequence of said antifungal protein contains at least one mutation selected from the group consisting of a basic residue at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9); and    b) regenerating a transformed plant from said plant cell, wherein said transformed plant has improved resistance to a fungal pathogen as compared to an untransformed plant.    
     
     
         14 . The method according to  claim 13 , wherein said plant is selected from the group consisting of canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, and onion.  
     
     
         15 . The method according to  claim 13 , wherein said fungal pathogen is selected from the group consisting of  Alternaria brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum, Nectria haematococca, Phoma betae , and  Verticillium dahliae.    
     
     
         16 . A method of combating a fungal pathogen comprising contacting said fungal pathogen with the transgenic plant of any one of claims  1 ,  5 , or  9 .  
     
     
         17 . A method of making a transgenic plant capable of producing an antifungal peptide, comprising: 
 a) stably integrating into the genome of a plant cell a DNA sequence encoding an antifungal protein having an amino acid sequence which is at least 80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid sequence of said antifungal protein contains at least one mutation selected from the group consisting of a basic residue at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9); and    b) regenerating stably transformed plants from said transformed plant cell, wherein said stably transformed plants produce at least one antifungal peptide.