Modified Cry3A toxins and nucleic acid sequences coding therefor

A transgenic maize plant comprising a nucleotide sequence that encodes a modified Cry3A toxin comprising a cathepsin G recognition site, wherein said cathepsin G recognition site modifies a Cry3A toxin and is located at a position selected from the group consisting of:

• a) a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4;
• b) a position between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4;
• and
• c) a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4, and between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4,
• wherein said cathepsin G recognition site is recognizable by a gut protease of western corn rootworm, and wherein said transgenic plant expresses said modified Cry3A toxin in root tissue at a level that causes mortality to western corn rootworm.

A modified Cry3A toxin comprising a cathepsin G recognition site, wherein said cathepsin G recognition site modifies a Cry3A toxin and is located at a position selected from the group consisting of:

• a) a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4;
• b) a position between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4;
• and
• c) a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4, and between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4,
• wherein said cathepsin G recognition site is recognizable by a gut protease of western corn rootworm, and wherein said modified Cry3A toxin causes higher mortality to western corn rootworm than the mortality caused by said Cry3A toxin to western corn rootworm in an artificial diet bioassay.

A method of making a modified Cry3A toxin, comprising:

• (a) obtaining a cry3A gene which encodes a Cry3A toxin;
• (b) identifying a gut protease of western corn rootworm;
• (c) obtaining a nucleotide sequence which encodes a recognition site for said gut protease;
• (d) inserting said nucleotide sequence into said cry3A gene, such that said recognition site is located in said Cry3A toxin at a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4, or at a position between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4, or at a position between amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4 and between amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4, thus creating a modified cry3A gene;
• (e) inserting said modified cry3A gene into an expression cassette;
• and
• (f) transforming said expression cassette into a non-human host cell, wherein said host cell produces a modified Cry3A toxin.

A modified cry3A gene comprising a nucleotide sequence that encodes a modified Cry3A toxin comprising a cathepsin G recognition site, wherein said modified cry3A gene comprises a coding sequence encoding said cathepsin G recognition site, wherein said coding sequence modifies a cry3A gene and is inserted at a position selected from the group consisting of:

• a) a position between the codons that code for amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4;
• b) a position between the codons that code for amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4;
• and
• c) a position between the codons that code for amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4, and between codons that code for amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4,
• wherein said cathepsin G recognition site is recognizable by a gut protease of western corn rootworm, and wherein said modified Cry3A toxin causes higher mortality to western corn rootworm than the mortality caused by said Cry3A toxin to western corn rootworm in an artificial diet bioassay.

A transgenic maize plant comprising a modified cry3A gene comprising a nucleotide sequence that encodes a modified Cry3A toxin comprising a cathepsin G recognition site, wherein said modified cry3A gene comprises a coding sequence encoding said cathepsin G recognition site, wherein said coding sequence modifies a cry3A gene and is inserted at a position selected from the group consisting of:

• a) a position between the codons that code for amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4;
• b) a position between the codons that code for amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4;
• and
• c) a position between the codons that code for amino acids corresponding to amino acid numbers 107 and 115 of SEQ ID NO:4, and between codons that code for amino acids corresponding to amino acid numbers 536 and 542 of SEQ ID NO:4,
• wherein said cathepsin G recognition site is recognizable by a gut protease of western corn rootworm, and wherein said transgenic plant expresses said modified Cry3A toxin in root tissue at a level that causes mortality to western corn rootworm.