Pathogen resistant citrus compositions, organisms, systems, and methods

Abstract

The present disclosure relates, according to some embodiments, to pathogen resistant citrus compositions, organisms, systems, and methods. For example, a composition may comprise a peptide (e.g., a defensin peptide) and/or a nucleic acid (e.g., a defensin nucleic acid). A pathogen resistant citrus plant may comprise, in some embodiments, a defensin peptide and/or an expressable nucleic acid encoding a defensin peptide.

What is claimed is: 
     
         1 . A nucleic acid comprising a nucleic acid sequence having about 98% identity to a sequence selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 5 and encoding a peptide having an amino acid sequence having at least 99% identity to SEQ ID NO: 1. 
     
     
         2 . A nucleic acid comprising a nucleic acid sequence having about 98% identity to a sequence selected from the group consisting of SEQ ID NO: 31 and SEQ ID NO: 6 and encoding a peptide having an amino acid sequence having at least 99% identity to SEQ ID NO: 2. 
     
     
         3 . A nucleic acid comprising a nucleic acid sequence having about 98% identity to a sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 11 and encoding a peptide having an amino acid sequence having at least 99% identity to SEQ ID NO: 7. 
     
     
         4 . A nucleic acid comprising a nucleic acid sequence having about 98% identity to a sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12 and encoding a peptide having an amino acid sequence having at least 99% identity to SEQ ID NO: 8. 
     
     
         5 . An expression vector comprising, in a 5′ to 3′ direction:
 an expression control sequence; 
 an exogenous nucleic acid operably linked to the expression control sequence; and 
 a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:30, and SEQ ID NO:31. 
 
     
     
         6 . An expression vector according to  claim 5 , wherein the expression vector is located in a bacterial cell. 
     
     
         7 . An expression vector according to  claim 5 , wherein the expression vector is located in a plant cell. 
     
     
         8 . An expression vector according to  claim 5 , wherein the nucleotide sequence of positions −4 to +4 is AACAATGG. 
     
     
         9 . An expression vector according to  claim 5  further comprising a linker 3′ of the expression control sequence, 5′ of the exogenous nucleic acid, and having a length of from about 1 to about 200 nucleotides. 
     
     
         10 . A bacterial cell comprising an expression vector, the expression vector comprising, in a 5′ to 3′ direction:
 an expression control sequence; 
 an exogenous nucleic acid operably linked to the expression control sequence; and 
 a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:30, and SEQ ID NO:31. 
 
     
     
         11 . A citrus plant cell comprising an expression vector, the expression vector comprising, in a 5′ to 3′ direction:
 an expression control sequence; 
 an exogenous nucleic acid operably linked to the expression control sequence; and 
 a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:30, and SEQ ID NO:31. 
 
     
     
         12 . A citrus plant cell according to  claim 11 , wherein the citrus plant cell is located in a citrus plant. 
     
     
         13 . A citrus plant cell according to  claim 11 , wherein the plant is selected from the group consisting of orange and grapefruit. 
     
     
         14 . A citrus plant cell according to  claim 11  further comprising a peptide having an amino acid sequence having at least about 99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         15 . A citrus plant comprising an expression vector, the expression vector comprising, in a 5′ to 3′ direction:
 an expression control sequence; 
 an exogenous nucleic acid operably linked to the expression control sequence; and 
 a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:30, and SEQ ID NO:31. 
 
     
     
         16 . A citrus plant according to  claim 15 , wherein the plant is selected from the group consisting of orange and grapefruit. 
     
     
         17 . A citrus plant according to  claim 15  further comprising a peptide having an amino acid sequence having at least about 99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         18 . A citrus plant cell comprising
 (a) a first expression vector, the first expression vector comprising, in a 5′ to 3′ direction:
 a first expression control sequence; 
 a first exogenous nucleic acid operably linked to the first expression control sequence; and 
 a first 3′ termination sequence operably linked to the first exogenous nucleic acid, wherein the first exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 30; and 
   (b) a second expression vector, the second expression vector comprising, in a 5′ to 3′ direction:
 a second expression control sequence; 
 a second exogenous nucleic acid operably linked to the second expression control sequence; and 
 a second 3′ termination sequence operably linked to the second exogenous nucleic acid, wherein the second exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 31. 
   
     
     
         19 . A citrus plant according to  claim 18 , wherein the plant is selected from the group consisting of orange and grapefruit. 
     
     
         20 . A citrus plant according to  claim 18  further comprising:
 a first peptide having an amino acid sequence having at least about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 7; and 
 a second peptide having an amino acid sequence having at least about 99% identity to SEQ ID NO: 2 or SEQ ID NO: 8. 
 
     
     
         21 . A method of expressing in a citrus plant an exogenous nucleic acid comprising a nucleic acid sequence encoding an expressed peptide, the method comprising:
 contacting an expression cassette comprising the exogenous nucleic acid or an expression vector comprising the exogenous nucleic acid with the cytosol of a cell of the citrus plant under conditions that permit expression of the exogenous nucleic acid and formation of the expressed peptide,   wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:30, and SEQ ID NO:31,   the expression cassette or expression vector comprises, in a 5′ to 3′ direction, an expression control sequence, the exogenous nucleic acid operably linked to the expression control sequence, and a 3′ termination sequence operably linked to the exogenous nucleic acid, and   the expressed peptide comprises an amino acid sequence having at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8.   
     
     
         22 . A method according to  claim 21 , wherein the contacting further comprises co-cultivating the cell with a  Agrobacterium  cell comprising the expression vector or expression cassette to form a co-cultivated cell. 
     
     
         23 . A method according to  claim 21  further comprising regenerating a plant from the co-cultivated cell.