Cleavable peptides and insecticidal and nematicidal proteins comprising same

Abstract

A peptide comprised of either a binary or a tertiary peptide, the peptide contains at least 4 amino acids and up to a maximum of 16 amino acids, comprised of 2 or 3 different regions, wherein the binary peptides have 2 different regions and the tertiary peptides have 3 different regions; wherein, the peptide can be cleaved by both an animal gut protease and an insect or nematode gut protease.

What is claimed is: 
     
         1 . A peptide comprised of either a binary or a tertiary peptide, that contains at least 4 amino acids and up to a maximum of 16 amino acids, comprised of 2 or 3 different regions, wherein the binary peptides have 2 different regions and the tertiary peptides have 3 different regions; wherein, the binary and tertiary peptide can be cleaved by both an animal gut protease and an insect or nematode gut protease. 
     
     
         2 . The peptide of  claim 1 , wherein the binary peptide contains at least 4 amino acids and up to a maximum of 8 amino acids; and the peptide comprises 2 different regions, wherein one of the regions can be cleaved by an animal gut protease and the other region can be cleaved by an insect or nematode gut protease. 
     
     
         3 . The peptide of  claim 1 , wherein the tertiary peptide contains at least 5 amino acids up to a maximum of 16 amino acids; and the tertiary peptide comprises a binary peptide and a spacer of 1 to 4 amino acids. 
     
     
         4 . The peptide of  claim 3 , wherein the spacer comprises the amino acid sequence GS, WN, WQ, MA, MV, AM, LH, MN, ES, WQ, or DT. 
     
     
         5 . The binary peptide of any one of  claims 1 - 4 , wherein the binary peptide comprises an amino acid sequence selected from the group consisting of: X 1 -X 2 -Y 1 -Y 2 , X 1 -X 2 -X 3 -Y 1 -Y 2 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 , X 1 -X 2 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 -Y 4 , Y 1 -Y 2 -X 1 -X 2 , Y 1 -Y 2 -X 1 -X 2 -X 3 , Y 1 -Y 2 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 -X 4 , wherein each X n  and each Y n  is an amino acid, and wherein each Xn and each Yn is an amino acid, and wherein X 1  is selected from the group consisting of: (all single letter amino acid format) A, D, E, G, I, K, L, N, P, Q, R, and V; X 2  is selected from the group consisting of A, F, G, I, K, L, N, P, Q, R, S, T, V, and Y; X 3  is selected from the group consisting of A, D, E, F, G, I, K, L, N, Q, R, S, T, V, and Y; X 4  is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. In some embodiments, Y 1  is selected from the group consisting of: (all single letter amino acid format) A, D, E, F, G, I, L, N, Q, S, T, V, and Y; Y 2  is selected from the group consisting of F, G, I, L, and Y; and Y 3  is selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, and Y; and Y 4  is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. 
     
     
         6 . The peptide of any one of  claims 1 - 5 , wherein the binary peptide has an amino acid sequence: AFVRLF, AKLFV, ALFALK, ALFLK, ALFLR, ALFR, ALFRLR, ALKALF, ALKFF, ALKFLV, ALKIFV, ALKLFV, FFADIK, FFALK, FFLK, FFLR, FFRLR, FGYRIK, FLRLF, FYARR, GGLRKK, IFVALK, IFVLK, IFVLR, IFVR, IFVRLR, ILFNIK, LFAAPF, FVALK, LFVLK, LFVLR, LFVR, LFVRLR, LFVRVFL, LGER, LKALF, LKFF, LKIFV, LKLFV, LRALF, LRFF, LRIFV, LRLFV, RALF, RIFV, RLFV, RLRALF, RLRFF, RLRIFV, RLRLFV, RRKAFV, RRKLIF, RRRFFA, VFGRKG, and YFVRK. 
     
     
         7 . The peptide of  claim 6 , wherein the binary peptide has an amino acid sequence: IFVALK, IFVRLR, ALKLFV, ALKFLV, RRKAFV, LFAAPF, LFVALK, LRLFV, LFVRLR, or LKLFV. 
     
     
         8 . The peptide of  claim 1 , wherein the tertiary peptide comprises an amino acid sequence of IFVALKGS, IFVRLRGS, ALKLFVGS, ALKFLVGS, RRKAFVGS, LFAAPFGS, LFVALKGS, LRLFVGS, LFVRLRGS, or LKLFVGS. 
     
     
         9 . The peptide of any one of  claims 1 - 7 , wherein the binary peptide has an amino acid sequence IFVRLR, ALKLFV, ALKFLV, RRKAFV, or LFAAPF. 
     
     
         10 . The peptide of  claim 1 - 9 , wherein the tertiary peptide comprises an amino acid sequence of: IFVRLRGS, ALKLFVGS, ALKFLVGS, RRKAFVGS, LFAAPFGS, GSIFVRLR, GSALKLFV, GSALKFLV, GSGSRRKAFV, or GSLFAAPF. 
     
     
         11 . The peptide of any one of  claims 1 - 10 , wherein the binary or tertiary peptide is linked to a toxic protein (TP). 
     
     
         12 . The peptide of  claim 11 , further comprising a second binary or tertiary peptide linked in frame to the C-terminus of the TP. 
     
     
         13 . The peptide of any one of  claims 1 - 12 , wherein the binary or tertiary peptide is linked to the C-terminus of an Endoplasmic Reticulum Signal Peptide (ERSP). 
     
     
         14 . An insecticidal and/or nematicidal protein having one or more cleavable linking peptides, the insecticidal and/or nematicidal protein comprising an ERSP fused in frame with the N-terminus of a binary or tertiary peptide, the binary or tertiary peptide is fused in frame with a construct comprising: (L-TP) n , (L-TP) n -L, (TP-L) n , or L-(TP-L) n , wherein TP is a toxic protein, L is a binary or tertiary peptide and n is an integer ranging from 1 to 200. 
     
     
         15 . The peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 14 , wherein the insect protease is a protease derived from a Lepidoptera or a Coleoptera insect species. 
     
     
         16 . The peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 15 , wherein the animal gut protease is a human gastrointestinal protease. 
     
     
         17 . The peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 16 , wherein the TP is a protein selected from the group consisting of a Pore Forming Insecticidal Protein (PFIP) and a Cysteine Rich Insecticidal Protein (CRIP). 
     
     
         18 . The peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 17 , wherein the TP is a peptide or protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity or sequence homology to a peptide or protein having an amino acid sequence as set forth in SEQ ID NOs: 5-1593 and 1761-1775. 
     
     
         19 . The peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 18 , wherein the TP is a peptide or protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity or sequence homology to a peptide or protein having an amino acid sequence as set forth in SEQ ID NOs: 5, 6 and 1761, 1762, 1767, 1771-1773, and variants thereof. 
     
     
         20 . The peptide or insecticidal and/or nematicidal protein of  claim 19 , wherein the TP is a peptide having an amino acid sequence of any one of SEQ ID NOs: 5, 6, 1761, 1762, 1767, 1771-1773, or variants thereof. 
     
     
         21 . A polynucleotide encoding a peptide or insecticidal and/or nematicidal protein of any one of  claims 1 - 20 . 
     
     
         22 . A vector comprising a polynucleotide of  claim 21 . 
     
     
         23 . A bacterial host cell, a yeast host cell, a plant host cell or an animal host cell comprising a vector of  claim 22 . 
     
     
         24 . A DNA construct comprising a polynucleotide linked to an operable promoter, wherein said polynucleotide encodes: a cleavable peptide comprising either a binary or a tertiary peptide, said cleavable peptide containing at least 4 amino acids and up to a maximum of 16 amino acids, and is comprised of 2 or 3 different regions, wherein the binary peptide has 2 different regions and the tertiary peptide has 3 different regions; wherein the cleavable peptide can be cleaved by both an animal gut protease and an insect or nematode gut protease, and the polynucleotide further encodes a toxic peptide fused in frame with said cleavable peptide. 
     
     
         25 . The DNA construct of  claim 24 , wherein the tertiary peptide contains at least 5 amino acids up to a maximum of 16 amino acids; and the tertiary peptide comprises a binary peptide and a spacer of 1 to 4 amino acids. 
     
     
         26 . The DNA construct of  claim 25 , wherein the spacer comprises the amino acid sequence GS, WN, WQ, MA, MV, AM, LH, MN, ES, WQ, or DT. 
     
     
         27 . The DNA construct of any one of  claims 24 - 26 , wherein the binary peptide comprises an amino acid sequence of: X 1 -X 2 -Y 1 -Y 2 , X 1 -X 2 -X 3 -Y 1 -Y 2 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 , X 1 -X 2 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 -Y 4 , Y 1 -Y 2 -X 1 -X 2 , Y 1 -Y 2 -X 1 -X 2 -X 3 , Y 1 -Y 2 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 -X 4 , wherein each Xn and each Yn is an amino acid, and wherein each Xn and each Yn is an amino acid, and wherein X1 is selected from the group consisting of: (all single letter amino acid format) A, D, E, G, I, K, L, N, P, Q, R, and V; X2 is selected from the group consisting of A, F, G, I, K, L, N, P, Q, R, S, T, V, and Y; X3 is selected from the group consisting of A, D, E, F, G, I, K, L, N, Q, R, S, T, V, and Y; X4 is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. In some embodiments, Y1 is selected from the group consisting of: (all single letter amino acid format) A, D, E, F, G, I, L, N, Q, S, T, V, and Y; Y2 is selected from the group consisting of F, G, I, L, and Y; and Y3 is selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, and Y; and Y4 is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. 
     
     
         28 . The DNA construct of  claim 27 , wherein the binary peptide comprises the amino acid sequence: AFVRLF, AKLFV, ALFALK, ALFLK, ALFLR, ALFR, ALFRLR, ALKALF, ALKFF, ALKFLV, ALKIFV, ALKLFV, FFADIK, FFALK, FFLK, FFLR, FFRLR, FGYRIK, FLRLF, FYARR, GGLRKK, IFVALK, IFVLK, IFVLR, IFVR, IFVRLR, ILFNIK, LFAAPF, FVALK, LFVLK, LFVLR, LFVR, LFVRLR, LFVRVFL, LGER, LKALF, LKFF, LKIFV, LKLFV, LRALF, LRFF, LRIFV, LRLFV, RALF, RIFV, RLFV, RLRALF, RLRFF, RLRIFV, RLRLFV, RRKAFV, RRKLIF, RRRFFA, VFGRKG, and YFVRK. 
     
     
         29 . The DNA construct of  claim 28 , wherein the binary peptide comprises an amino acid sequence: IFVRLR, ALKLFV, ALKFLV, RRKAFV, or LFAAPF. 
     
     
         30 . The DNA construct of  claim 24 , wherein said polynucleotide encodes a tertiary peptide comprising the amino acid sequence: AFVRLFGS, AKLFVGS, ALFALKGS, ALFLKGS, ALFLRGS, ALFRGS, ALFRLRGS, ALKALFGS, ALKFFGS, ALKFLVGS, ALKIFVGS, ALKLFVGS, FFADIKGS, FFALKGS, FFLKGS, FFLRGS, FFRLRGS, FGYRIKGS, FLRLFGS, FYARRGS, GGLRKKGS, IFVALKGS, IFVLKGS, IFVLRGS, IFVRGS, IFVRLRGS, ILFNIKGS, LFAAPFGS, LFVALKGS, LFVLKGS, LFVLRGS, LFVRGS, LFVRLRGS, LFVRVFLGS, LGERGS, LKALFGS, LKFFGS, LKIFVGS, LKLFVGS, LRALFGS, LRFFGS, LRIFVGS, LRLFVGS, RALFGS, RIFVGS, RLFVGS, RLRALFGS, RLRFFGS, RLRIFVGS, RLRLFVGS, RRKAFVGS, RRKLIFGS, RRRFFAGS, VFGRKGGS, YFVRKGS, GSAFVRLF, GSAKLFV, GSALFALK, GSALFLK, GSALFLR, GSALFR, GSALFRLR, GSALKALF, GSALKFF, GSALKFLV, GSALKIFV, GSALKLFV, GSFFADIK, GSFFALK, GSFFLK, GSFFLR, GSFFRLR, GSFGYRIK, GSFLRLF, GSFYARR, GSGGLRKK, GSIFVALK, GSIFVLK, GSIFVLR, GSIFVR, GSIFVRLR, GSILFNIK, GSLFAAPF, GSLFVALK, GSLFVLK, GSLFVLR, GSLFVR, GSLFVRLR, GSLFVRVFL, GSLGER, GSLKALF, GSLKFF, GSLKIFV, GSLKLFV, GSLRALF, GSLRFF, GSLRIFV, GSLRLFV, GSRALF, GSRIFV, GSRLFV, GSRLRALF, GSRLRFF, GSRLRIFV, GSRLRLFV, GSRRKAFV, GSRRKLIF, GSRRRFFA, GSVFGRKG, and GSYFVRK. 
     
     
         31 . The DNA construct of  claim 24 , wherein said insect protease is a protease derived from a Lepidoptera or a Coleoptera insect species. 
     
     
         32 . The DNA construct of  claim 24 , wherein said animal gut protease is a human gastrointestinal protease. 
     
     
         33 . The DNA construct of any one of  claims 24 - 32 , wherein the TP is a peptide or protein selected from the group consisting of a Pore Forming Insecticidal Protein (PFIP) and a Cysteine Rich Insecticidal Protein (CRIP). 
     
     
         34 . The DNA construct of  claim 33 , wherein the TP is a peptide or protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity or sequence homology to a peptide or protein having an amino acid sequence as set forth in SEQ ID NOs: 5-1593 and 1761-1775. 
     
     
         35 . The DNA construct of  claim 34 , wherein the TP is a peptide or protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity or sequence homology to a peptide or protein having an amino acid sequence as set forth in SEQ ID NOs: 5, 6 and 1761, 1762, 1767, 1771-1773, and variants thereof. 
     
     
         36 . The DNA construct of  claim 35 , wherein the TP is a peptide or protein having an amino acid sequence of any one of SEQ ID NOs: 5, 6, 1761, 1762, 1767, 1771-1773, or variants thereof. 
     
     
         37 . A host cell comprising a DNA construct of any one of  claims 24 - 36 . 
     
     
         38 . The host cell of  claim 37 , comprising a DNA construct of  claim 35 . 
     
     
         39 . The host cell of  claim 37 , wherein said host cell is a bacterial cell, a yeast cell, a plant cell, or an animal cell. 
     
     
         40 . The host cell of  claim 39 , wherein said bacterial cell is selected from the group consisting of an  Agrobacterium , a  Bacillus , an  Escherichia , a  Salmonella , a  Pseudomonas , and a  Rhizobium  cell. 
     
     
         41 . The host cell of  claim 39 , wherein said plant cell is selected from the group consisting of: alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant,  eucalyptus , flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,  Radiata  pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant cell. 
     
     
         42 . A plant, or part thereof comprising a polynucleotide linked to an operable promoter, wherein said polynucleotide encodes: a cleavable peptide comprising either a binary or a tertiary peptide, said cleavable peptide containing at least 4 amino acids and up to a maximum of 16 amino acids, and is comprised of 2 or 3 different regions, wherein the binary peptide has 2 different regions and the tertiary peptide has 3 different regions; wherein the cleavable peptide can be cleaved by both an animal gut protease and an insect or nematode gut protease, and wherein the polynucleotide further encodes a toxic peptide fused in frame with said cleavable peptide. 
     
     
         43 . The plant, or part thereof, of  claim 42 , wherein said plant is elected from the group consisting of a alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant,  eucalyptus , flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,  Radiata  pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant, and wherein said part is selected from the group consisting of a leaf, a stem a flower, a sepal, a fruit, a root, or a seed. 
     
     
         44 . A method of controlling a pest infection of a plant, said method comprising providing in a diet of the pest, a plant, or part thereof, the plant or part thereof expressing an insecticidal and/or nematicidal protein, or coated at least on a portion of the plant or part thereof, a composition comprising an insecticidal and/or nematicidal protein, wherein the insecticidal and/or nematicidal protein comprises one or more cleavable binary or tertiary peptides, and one or more TPs, wherein the binary and tertiary peptide can be cleaved by both an animal gut protease and an insect or nematode gut protease. 
     
     
         45 . The method of controlling a pest infection of a plant of  claim 44 , wherein said cleavable binary peptide comprises an amino acid sequence: X 1 -X 2 -Y 1 -Y 2 , X 1 -X 2 -X 3 -Y 1 -Y 2 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 , X 1 -X 2 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 , X 1 -X 2 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -Y 1 -Y 2 -Y 3 -Y 4 , X 1 -X 2 -X 3 -X 4 -Y 1 -Y 2 -Y 3 -Y 4 , Y 1 -Y 2 -X 1 -X 2 , Y 1 -Y 2 -X 1 -X 2 -X 3 , Y 1 -Y 2 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -X 1 -X 2 -X 3 -X 4 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 , Y 1 -Y 2 -Y 3 -Y 4 -X 1 -X 2 -X 3 -X 4 , wherein each Xn and each Yn is an amino acid, and wherein each Xn and each Yn is an amino acid, and wherein X1 is selected from the group consisting of: (all single letter amino acid format) A, D, E, G, I, K, L, N, P, Q, R, and V; X2 is selected from the group consisting of A, F, G, I, K, L, N, P, Q, R, S, T, V, and Y; X3 is selected from the group consisting of A, D, E, F, G, I, K, L, N, Q, R, S, T, V, and Y; X4 is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. In some embodiments, Y1 is selected from the group consisting of: (all single letter amino acid format) A, D, E, F, G, I, L, N, Q, S, T, V, and Y; Y2 is selected from the group consisting of F, G, I, L, and Y; and Y3 is selected from the group consisting of A, D, E, F, G, I, K, L, R, S, T, V, and Y; and Y4 is selected from the group consisting of: R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, W, Y, and V. 
     
     
         46 . The method of controlling a pest infection of a plant of  claim 44 , wherein the binary peptide comprises an amino acid sequence of: AFVRLF, AKLFV, ALFALK, ALFLK, ALFLR, ALFR, ALFRLR, ALKALF, ALKFF, ALKFLV, ALKIFV, ALKLFV, FFADIK, FFALK, FFLK, FFLR, FFRLR, FGYRIK, FLRLF, FYARR, GGLRKK, IFVALK, IFVLK, IFVLR, IFVR, IFVRLR, ILFNIK, LFAAPF, FVALK, LFVLK, LFVLR, LFVR, LFVRLR, LFVRVFL, LGER, LKALF, LKFF, LKIFV, LKLFV, LRALF, LRFF, LRIFV, LRLFV, RALF, RIFV, RLFV, RLRALF, RLRFF, RLRIFV, RLRLFV, RRKAFV, RRKLIF, RRRFFA, VFGRKG, and YFVRK. 
     
     
         47 . The method of controlling a pest infection of a plant of  claim 46 , wherein the binary peptide comprises an amino sequence IFVRLR, ALKLFV, ALKFLV, RRKAFV, or LFAAPF. 
     
     
         48 . The method of controlling a pest infection of a plant of  claim 44 , wherein the cleavable tertiary peptide at least 5 amino acids up to a maximum of 16 amino acids; and the tertiary peptide comprises a binary peptide and a spacer of 1 to 4 amino acids. 
     
     
         49 . The method of controlling a pest infection of a plant of  claim 48 , wherein the tertiary peptide comprises a binary peptide fused at its C-terminus to a di-peptide comprising the amino acid sequence GS, WN, WQ, MA, MV, AM, LH, MN, ES, WQ, or DT. 
     
     
         50 . The method of controlling a pest infection of a plant of  claim 44 , wherein the insect protease is a protease derived from a Lepidoptera or a Coleoptera insect species. 
     
     
         51 . The method of controlling a pest infection of a plant of  claim 44 , wherein the animal gut protease is a human gastrointestinal protease. 
     
     
         52 . The method of controlling a pest infection of a plant of any one of  claims 44 - 51 , wherein the TP is a protein selected from the group consisting of a Pore Forming Insecticidal Protein (PFIP) and a Cysteine Rich Insecticidal Protein (CRIP). 
     
     
         53 . The method of controlling a pest infection of a plant of  claim 44 , wherein said pest is an insect or a nematode. 
     
     
         54 . The method of controlling a pest infection of a plant of  claim 53 , wherein said pest is an insect, from the insect order selected from the group consisting of Coleoptera and Lepidoptera. 
     
     
         55 . The method of controlling a pest infection of a plant of 44, wherein said TP is a peptide or protein selected from the group consisting of: a Pore Forming Insecticidal Protein (PFIP) and a Cysteine Rich Insecticidal Peptide (CRIP). 
     
     
         56 . The method of controlling a pest infection of a plant of 55, wherein the TP is an Inhibitor Cysteine Knot (ICK) motif protein or a non-ICK protein. 
     
     
         57 . The method of controlling a pest infection of a plant of  claim 56 , wherein the TP is a peptide or protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity or sequence homology to a peptide or protein having an amino acid sequence as set forth in SEQ ID NOs: 5, 6 and 1761, 1762, 1767, 1771-1773, or variants thereof. 
     
     
         58 . The method of controlling a pest infection of a plant of  claim 57 , wherein the TP is a peptide or protein having an amino acid sequence of any one of SEQ ID NOs: 5, 6, 1761, 1762, 1767, 1771-1773, or variants thereof. 
     
     
         59 . The method of controlling a pest infection of a plant of  claim 44 , wherein a pesticidally effective amount of said TP is provided by the plant, which is a recombinant plant, a part of the plant, or a product of the plant or the plant part. 
     
     
         60 . The method of controlling a pest infection of a plant of  claim 44 , wherein a pesticidally effective amount of said insecticidal and/or nematicidal protein is provided in one or more formulations topically applied on the plant or a part of the plant, said one or more formulations comprising bacterial cells, bacterial spores, fungal cells, fungal spores, or parasporal crystals that comprise said insecticidal and/or nematicidal protein.