Antifungal plant proteins, peptides, and methods of use

Abstract

Provided are transgenic plants expressing MtDef5 antifungal proteins and peptides exhibiting high levels of resistance to susceptible fungi. Such transgenic plants contain a recombinant DNA construct comprising a natural or heterologous signal peptide sequence operably linked to a nucleic acid sequence encoding these molecules. Also provided are methods of producing such plants, methods of protecting plants against susceptible fungal infection and damage, as well as compositions that can be applied to the locus of plants, comprising microorganisms expressing these molecules, or these molecules themselves, as well as pharmaceutical compositions containing these molecules. Human and veterinary therapeutic use of MtDef5 antifungal proteins and peptides to treat susceptible fungal infections are also encompassed by the invention.

1 .- 20 . (canceled) 
     
     
         21 . A peptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 58, wherein the peptide comprises a fragment of a mature MtDef5 protein, wherein one or more amino acids are deleted from the N-terminal end, C-terminal end, the middle of the mature MtDef5 protein, or combinations thereof, and possesses antifungal activity. 
     
     
         22 . The peptide of  claim 21 , wherein the peptide comprises the gamma-core motif set forth as SEQ ID NO:81. 
     
     
         23 . The peptide of  claim 21 , wherein the peptide contains at least one conservative amino acid substitutions wherein one or more amino acids in the SEQ ID NO: 58 sequence are substituted with another amino acid having a charge and polarity which is similar to that of the amino acid of SEQ ID NO: 58. 
     
     
         24 . The peptide of  claim 23 , wherein one or more basic amino acids of SEQ ID NO: 58 are substituted with another basic amino acid. 
     
     
         25 . The peptide of  claim 23 , wherein one or more neutral nonpolar amino acids of SEQ ID NO: 58 are substituted with another neutral nonpolar amino acid. 
     
     
         26 . The peptide of  claim 21 , wherein the peptide comprises the gamma-core motif set forth as SEQ ID NO:81 and wherein any amino acid substitutions SEQ ID NO: 58 are conservative amino acid substitutions wherein one or more amino acids in the SEQ ID NO: 58 sequence are substituted with another amino acid having a charge and polarity which is similar to that of the native amino acid of SEQ ID NO: 58 which is substituted. 
     
     
         27 . The peptide of  claim 21 , wherein the fragment is a synthetic mutant of the mature MtDef5 protein. 
     
     
         28 . A composition comprising the peptide of  claim 21  and an agriculturally, pharmaceutically, or veterinarily acceptable carrier, diluent, or excipient. 
     
     
         29 . The composition of  claim 28 , wherein the composition further comprises a surfactant and/or a fungicide, wherein the fungicide is optionally a polyoxine, nikkomycine, carboxyamide, aromatic carbohydrates, carboxine, morpholine, an inhibitors of sterol biosynthesis, or an organophosphorus compound. 
     
     
         30 . The composition of  claim 27 , wherein the composition further comprises an antifungal compound, wherein the antifungal compound is a polyene, imidazole, triazole, thiazole, allylamine, or echinocandin. 
     
     
         31 . A DNA construct encoding the peptide of  claim 21 , wherein the nucleic acid is operably linked to a heterologous promoter. 
     
     
         32 . A cell comprising the DNA construct of  claim 31 . 
     
     
         33 . The cell of  claim 32 , wherein the cell is a plant, bacterial, or yeast cell. 
     
     
         34 . A method of treating a susceptible fungal infection in a patient or other subject in need thereof, comprising administering to said patient an antifungal effective amount of the peptide of  claim 21 . 
     
     
         35 . The method of  claim 34 , wherein said administering is performed topically. 
     
     
         36 . A method of preventing, treating, controlling, combating, reducing, or inhibiting damage to a plant susceptible to damage from a species of fungus selected from the group consisting of an  Alternaria  sp., an  Ascochyta  sp., an  Aspergillus  sp., a  Botrytis  sp.; a  Cercospora  sp., a  Colletotrichum  sp., a  Diplodia  sp., an  Erysiphe  sp., a  Fusarium  sp.,  Gaeumanomyces  sp.,  Helminthosporium  sp.,  Macrophomina  sp., a  Nectria  sp., a  Peronospora  sp., a  Phakopsora  sp., a  Phoma  sp., a  Phymatotrichum  sp., a  Phytophthora  sp., a  Plasmopara  sp., a  Puccinia  sp., a  Podosphaera  sp., a  Pyrenophora  sp., a  Pyricularia  sp., a  Pythium  sp., a  Rhizoctonia  sp., a  Scerotium  sp., a  Sclerotinia  sp., a  Septoria  sp., a  Thielaviopsis  sp., an  Uncinula  sp., a  Venturia  sp., and a  Verticillium  sp., comprising providing to the locus of said susceptible plant an antifungal effective amount of the peptide of claim  1 .