High lysine derivatives of α-hordothionin

Abstract

Derivatives of α-hordothionin made by position-specific substitution with lysine residues provide lysine enrichment while retaining the antifungal activity of the parent compound.

1. A protein having the sequence of SEQUENCE  1  I .D. No. 1 wherein the amino acid residues at one or more of positions  5 ,  10 ,   11 ,  12 ,   17 ,  19 ,  22 ,  30  and  41  are lysine, and the remainder of the residues at those positions are the residues at the corresponding positions in SEQUENCE I.D. No. 1. 
     
     
       2. A protein according to  claim 1 , wherein one or more of the amino acid residues at positions  5 ,  11 ,  12 ,   17 ,  19 ,  22  and  41  are lysine. 
     
     
       3. A protein according to  claim 2 , wherein all of the amino acid residues at positions 5, 11, 12, 17, 19, 22 and 41 are lysine. 
     
     
       4. A nucleotide sequence which codes for a  the protein according to  claim 1 . 
     
     
       5. An RNA sequence according to  claim 4   that encodes the protein of  claim 1 . 
     
     
       6. A DNA sequence according to  claim 4   that encodes the protein of  claim 1 . 
     
     
       7. An expression cassette containing the DNA sequence of  claim 6  operably linked to plant regulatory sequences which cause the expression of the DNA sequence in plant cells. 
     
     
       8. A bacterial transformation vector comprising an  the expression cassette according to  claim 7 ,  operably linked to bacterial expression regulatory sequences which cause replication of the expression cassette in bacterial cells. 
     
     
       9. Bacterial cells containing as a foreign plasmid at least one copy of a  the bacterial transformation vector according to  claim 8 . 
     
     
       10. Transformed plant cells containing at least one copy of the expression cassette of  claim 7 . 
     
     
       11. Transformed  The transformed cells according to  claim 10 , further characterized in being cells of a monocotyledonous species. 
     
     
       12. Transformed  The transformed cells according to  claim 11 , further characterized in being maize, sorghum, wheat or rice cells. 
     
     
       13. Transformed  The transformed cells according to  claim 10 , further characterized in being cells of a dicotyledonous species. 
     
     
       14. Transformed  The transformed cells according to  claim 13 , further characterized in being soybean, alfalfa, rapeseed, sunflower, tobacco or tomato cells. 
     
     
       15. A maize cell or tissue culture comprising  The transformed cells according to  claim 12   of  claim 11  wherein the cells are maize cells. 
     
     
       16. A transformed plant comprising transformed cells according to  claim 10 . 
     
     
       17. A method for killing and inhibiting plant pathogenic microorganisms which are susceptible to a α-Hordothionin comprising , wherein the method comprises introducing into the environment of the pathogenic microorganisms an antimicrobial amount of a  the protein according to  claim 1 . 
     
     
       18. A method for killing and inhibiting plant pathogens selected from Fusarium graminearum, Fusarium moniliforme, Diplodia maydis, Colletototrichum graminicola, Verticillum alboatrum, Phytophthora megaspermae f.sp. glycinea, Macrophomina phaseolina, Diaporthe phaseolorum caulivora, Sclerotinia sclerotiorum, Sclerotinia trifoliorum, and Aspergillus flavus, comprising  wherein the method comprises introducing into the environment of the pathogenic microorganisms an antimicrobial amount of a  the protein according to  claim 1 . 
     
     
       19. A method according to  claim 17 , wherein the environment of the pathogen is the tissues of a living plant. 
     
     
       20. A method for enhancing the lysine content of a plant cell or seed comprising the step of causing a , wherein the method comprises transforming a plant cell by insertion of the expression cassette of  claim 7  such that the protein according to  claim 1  to be  is expressed in the cell or seed. 
     
     
       21. A method for enhancing the lysine content of a plant comprising the step of causing a , wherein the method comprises transforming a plant cell by insertion of the expression cassette of  claim 7  such that the protein according to  claim 1  to be  is expressed in tissues of the plant.