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.

What is claimed is: 
     
         1 . An isolated, purified protein or peptide, comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64. 
     
     
         2 . The isolated, purified protein or peptide of  claim 1 , comprising a plant apoplast, vacuolar, or endoplasmic reticulum targeting amino acid sequence at its N-terminus. 
     
     
         3 . The isolated, purified protein or peptide of  claim 2 , wherein said targeting sequence comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences shown in shown in SEQ ID NOs:30-35 and 42-48. 
     
     
         4 . An isolated nucleotide sequence encoding said isolated, purified protein or peptide of any one of  claims 1 - 3 . 
     
     
         5 . The isolated nucleotide sequence of  claim 4 , codon-optimized for expression in a plant of interest. 
     
     
         6 . The isolated nucleotide sequence of  claim 4  or  5 , wherein said plant of interest is a food crop plant. 
     
     
         7 . The isolated nucleotide sequence of  claim 6 , wherein said food crop plant is selected from the group consisting of soybean, wheat, maize, sugarcane, rice, and potato. 
     
     
         8 . A transgenic plant, cells of which contain a protein or peptide comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64. 
     
     
         9 . The transgenic plant of  claim 8 , wherein said protein or peptide further comprises a plant apoplast, vacuolar, or endoplasmic reticulum targeting amino acid sequence at its N-terminus. 
     
     
         10 . The transgenic plant of  claim 8  or  9 , the genome of which further comprises:
 DNA encoding a plant defensin selected from the group consisting of MsDef1, MtDef2, MtDef4, NaD1, Rs-AFP1, Rs-AFP2, KP4, and KP6, wherein said DNA is expressed and produces an anti-fungal effective amount of said defensin, and/or 
 DNA encoding a  Bacillus thuringiensis  endotoxin, wherein said DNA is expressed and produces an anti-insect effective amount of said  Bacillus thuringiensis  endotoxin, and/or 
 DNA encoding a protein that confers herbicide resistance to said plant, wherein said DNA is expressed and produces an anti-herbicide effective amount of said protein that confers herbicide resistance. 
 
     
     
         11 . A composition, comprising a protein or peptide comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and any combination thereof. 
     
     
         12 . The composition of  claim 11 , further comprising an agriculturally or pharmaceutically acceptable carrier, diluent, or excipient. 
     
     
         13 . The composition of  claim 11  or  12 , formulated in a mixture of one or more other active agents selected from the group consisting of a pesticidally active substance, a fertilizer, an insecticide, an attractant, a sterilizing agent, an acaricide, a nematocide, a herbicide, and a growth regulator. 
     
     
         14 . 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 a antifungal protein or peptide comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and any combination thereof. 
     
     
         15 . The method of  claim 14 , wherein said antifungal protein or peptide is provided to said susceptible plant locus by expressing DNA encoding said antifungal protein or peptide within cells of said susceptible plant. 
     
     
         16 . The method of  claim 14 , wherein said antifungal protein or peptide is provided to said susceptible plant locus by applying a composition comprising said antifungal protein or peptide to said plant. 
     
     
         17 . The method of  claim 14 , wherein said antifungal protein or peptide is provided to said susceptible plant locus by plant colonizing microorganisms that produce said antifungal protein or peptide. 
     
     
         18 . The method of  claim 14 , wherein said antifungal protein or peptide is provided to said susceptible plant locus by applying a composition comprising plant colonizing microorganisms that produce said antifungal protein or peptide, or by applying said antifungal protein or peptide itself thereto. 
     
     
         19 . The method of  claim 18 , wherein said composition comprises an agriculturally acceptable diluent, excipient, or carrier. 
     
     
         20 . The method of any one of  claims 14 - 19 , wherein said susceptible plant is selected from the group consisting of maize, soybean, wheat, sugarcane, rice, and potato.