Compositions and Methods for Protecting Plants Against Bacterial Infections

Abstract

A method of creating a genetically altered plant and parts thereof with a chimeric protein comprising a first domain, a second domain, and a third domain, wherein said first domain comprises either i) a recognition element comprising a Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence, said second domain comprises either i) a recognition element comprising a sequence selected from Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence wherein the second domain is an element that is different from the element of the first domain and said third domain comprises a linker; wherein said linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity.

1 . A chimeric protein comprising a first domain, a second domain, and a third domain, wherein said first domain comprises either i) a recognition element comprising a Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence, said second domain comprises either i) a recognition element comprising a sequence selected from Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence wherein the second domain is an element that is different from the element of the first domain and said third domain comprises a linker; wherein said linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity. 
     
     
         2 . The chimeric protein of  claim 1 , wherein said first domain is located at the amino terminus of said chimeric protein and has an amino acid sequence selected from the group consisting of SEQ ID NOs:1-3, 8-10, 12; and wherein said second domain is located at the carboxyl terminus of said chimeric protein and has an amino acid sequence selected from the group consisting of SEQ ID NOs 14-16. 
     
     
         3 . The chimeric protein of  claim 2  wherein said chimeric protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 17-23 or a variant thereof. 
     
     
         4 . The chimeric protein of  claim 3  is encoded for by a polynucleotide comprising a nucleic acid sequence. 
     
     
         5 . The polynucleotide of  claim 4  operably linked to a promoter to produce an expression vector. 
     
     
         6 . A genetically altered plant or part thereof and its progeny comprising the polynucleotide of  claim 4  operably linked to a promoter, wherein said plant or parts thereof and its progeny produce said chimeric protein. 
     
     
         7 . The genetically altered plant or part thereof of  claim 6  wherein its progeny is a seed from the genetically altered plant. 
     
     
         8 . The genetically altered plant or part thereof of  claim 6  wherein its parts is pollen from the genetically altered plant. 
     
     
         9 . The genetically altered plant or part thereof of  claim 6  wherein its parts are plant cells from the genetically altered plant cell comprising the polynucleotide of  claim 4  operably linked to a promoter, wherein said plant cell produces said chimeric protein. 
     
     
         10 . The plant cell of  claim 9  wherein the plant cell form a tissue culture. 
     
     
         11 . The chimeric protein of  claim 2 , wherein said first domain is located at the amino terminus of said chimeric protein and wherein said second domain is located at the carboxyl terminus of said chimeric protein. 
     
     
         12 . The chimeric protein of  claim 11 , wherein said chimeric protein has the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-7 or a homologue thereof. 
     
     
         13 . The chimeric protein of  claim 12  wherein a polynucleotide comprising a nucleic acid sequence encodes said chimeric protein of  claim 12 . 
     
     
         14 . The polynucleotide of  claim 13  operably linked to a promoter to form an expression vector. 
     
     
         15 . A method for constructing a genetically altered plant or part thereof having increased resistance to bacterial infections compared to a non-genetically altered plant or part thereof, the method comprising the steps of:
 a. introducing a polynucleotide encoding a chimeric protein into a plant or part thereof to provide a genetically altered plant or part thereof, wherein said chimeric protein comprising a first domain, a second domain, and a third domain, wherein said first domain comprises either i) a recognition element comprising a Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence, said second domain comprises either i) a recognition element comprising a sequence selected from Proteinase K sequence, or BPI/LBP sequence, or ii) a lysis element comprising a thionin sequence wherein an element of the second domain is a different one than the element of the first domain and said third domain comprises a linker; wherein said linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity, and said linker ranges in length between three amino acids and approximately forty-four amino acids; and   b. selecting a genetically altered plant or part thereof that expresses said chimeric protein, wherein said expressed chimeric protein has anti-bacterial activity; and wherein said genetically altered plant or part thereof has increased resistance to bacterial infections compared to the resistance to bacterial infections of said non-genetically altered plant or part thereof.   
     
     
         16 . The method of  claim 15 , wherein said introducing said polynucleotide encoding said chimeric protein occurs via introgression or transforming said plant with an expression vector comprising said polynucleotide operably linked to a promoter. 
     
     
         17 . The method of  claim 15 , wherein said first domain is located at the amino terminus of said chimeric protein and has an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NOs:1-3, 8-10, or 12 and wherein said second domain is located at the carboxyl terminus of said chimeric protein and has the amino acid sequence set forth in SEQ ID NO: 14-16. 
     
     
         18 . A method of enhancing a wild-type plant's resistance to bacterial diseases comprising transforming a cell from said wild-type plant with a polynucleotide encoding a chimeric protein to generate a transformed plant cell; and growing said transformed plant cell to generate a genetically altered plant wherein said chimeric protein comprises a first domain, a second domain, and a third domain; wherein said first domain comprises a thionin or pro-thionin, said second domain comprises Proteinase K or pro-Proteinase K, and said third domain comprises a peptide linker; wherein said peptide linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity; wherein said peptide linker ranges in length between three amino acids and approximately forty-four amino acids; wherein said genetically altered plant or part thereof produces said chimeric protein; and wherein said chimeric protein kills bacteria that cause said bacterial diseases; and wherein said genetically altered plant's resistance to said bacterial diseases is greater than said wild-type plant's resistance to said bacterial diseases. 
     
     
         19 . The method of  claim 18 , wherein said first domain is located at the amino terminus of said chimeric protein and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 8-10, or 12 and wherein said second domain is located at the carboxyl terminus of said chimeric protein and has the amino acid sequence set forth in SEQ ID NOs: 14-16. 
     
     
         20 . A genetically altered plant or part thereof produced by the method of  claim 17 . 
     
     
         21 . A genetically altered plant or part thereof produced by the method of  claim 19 . 
     
     
         22 . The genetically altered plant or part thereof of  claim 18  wherein the plan is a grape. 
     
     
         23 . An expression vector for  agrobacterium -mediated transformation using the polynucleotide in  claim 4 . 
     
     
         24 . A composition comprising the chimera protein of  claim 1  for use as a topical treatment of plants at risk for infection with xf. 
     
     
         25 . A method of treating plants at risk for infection with xf comprising the steps of: applying the composition of  claim 24  to the plants at risk of infection with xf.