Basement membrane degrading proteases as insect toxins and methods of use for same

Abstract

Novel insect toxins are disclosed for use in pesticidal compositions and methods. According to the invention basement membrane degrading proteases are identified which are capable of acting as insecticidal agents. Polynucleotides are provided which include expression constructs for the expression of the recombinant insecticidal proteases of the invention as recombinant insect pathogens, as well as transgenic plants with a substantial degree of insect resistance.

What is claimed is:  
     
         1 . A method of imparting substantial insect resistance to a plant comprising: introducing to said plant a polynucleotide comprising: a nucleotide sequence which encodes upon expression a basement membrane degrading protease insect toxin, said nucleotide sequence operably linked to regulatory elements capable of directing expression thereof in a host cell.  
     
     
         2 . The method of  claim 1  wherein said host cell is a plant cell.  
     
     
         3 . The method of  claim 1  wherein said polynucleotide is a vector.  
     
     
         4 . The method of  claim 3  wherein said vector is a plasmid vector.  
     
     
         5 . The method of  claim 3  wherein said vector is a viral vector.  
     
     
         6 . The method of  claim 5  wherein said viral vector is capable of transforming a plant cell.  
     
     
         7 . The method of  claim 6  wherein said vector is a Agrobacterium based vector.  
     
     
         8 . The method of  claim 3  wherein said vector is capable of transforming an insect cell.  
     
     
         9 . The method of  claim 8  wherein said vector is an infectious baculovirus based vector.  
     
     
         10 . The method of  claim 1  wherein said step of introducing comprises: topically administering said vector to plants.  
     
     
         11 . The method of  claim 1  wherein said step of introducing further comprises: transforming a plant cell with said vector.  
     
     
         12 . The method of  claim 1  wherein said protease is selected from the group consisting of: a metalloproteinase, a collagenase, a gelatinase, a stromelysin, a cysteine protease, and basement membrane degrading proteases from snake venom, invertebrates, fungi, and bacteria.  
     
     
         13 . The method of  claim 12  wherein said protease is selected from the group consisting of: interstitial collagenase, fibroblast collagenase, Collagenase-3 Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa type IV collagenase), Gelatinase B (92 kDa type IV collagenase), Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin, Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N,  Crotalus atrox , hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e),  Hypoderma lineatum  (fly) collagenase,  Uca pugilator  (crab) collagenolytic endopeptidase,  Entomophthora collagenase, Clostridium histolyticum collagenase, Streptomyces collagenase , and  Serratia marcescens  cysteine endopeptidase.  
     
     
         14 . The method of  claim 12  wherein said protease is a cathepsin L protease.  
     
     
         15 . The method of  claim 14  wherein said cathepsin L protease is from the facefly  Sarcophaga peregrina.    
     
     
         16 . An insecticidal composition comprising: a recombinant basement membrane degrading protease protein and a carrier.  
     
     
         17 . The composition of  claim 1  wherein said protease is selected from the group consisting of: a metalloprotease, a collagenase, a gelatinase, a stromelysin, a cysteine protease, and basement membrane degrading proteases from snake venom, invertebrates, fungi, and bacteria.  
     
     
         18 . The composition of  claim 2  wherein said protease is selected from the group consisting of: interstitial collagenase, fibroblast collagenase, Collagenase-3 Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa type IV collagenase), Gelatinase B (92 kDa type IV collagenase), Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin, Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N,  Crotalus atrox , hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e),  Hypoderma lineatum  (fly) collagenase,  Uca pugilator  (crab) collagenolytic endopeptidase,  Entomophthora collagenase, Clostridium histolyticum collagenase, Streptomyces collagenase , and  Serratia marcescens  cysteine endopeptidase.  
     
     
         19 . The composition of  claim 17  wherein said protease is a cathepsin L protease.  
     
     
         20 . The composition of  claim 19  wherein said cathepsin L protease is from the facefly  Sarcophaga peregrina.    
     
     
         21 . An expression construct comprising: a gene which encodes a basement membrane degrading protease operably linked to a promoter, said promoter capable of directing expression in an insect cell.  
     
     
         22 . The expression construct of  claim 21  wherein said promoter is selected from the group consisting of: p6.9 and ie-1.  
     
     
         23 . The expression construct of  claim 21  wherein said protease is selected from the group consisting of: a metalloprotease, a collagenase, a gelatinase, a stromelysin, a cysteine protease, and basement membrane degrading proteases from snake venom, invertebrates, fungi, and bacteria.  
     
     
         24 . The expression construct of  claim 23  wherein said protease is selected from the group consisting of: interstitial collagenase, fibroblast collagenase, Collagenase-3 Neutrophil collagenase, PMN-type collagenase, Gelatinase A, (72 kDa type IV collagenase), Gelatinase B (92 kDa type IV collagenase), Stromelysin-1, Stromelysin-2, Stromelysin-3, Matrilysin, Metalloelastase, Cathepsin B, Cathepsin L, Cathepsin N,  Crotalus atrox , hemorrhagic metalloproteinases: (Ht-a, Ht-c, Ht-d, and Ht-e),  Hypoderma lineatum  (fly) collagenase,  Uca pugilator  (crab) collagenolytic endopeptidase,  Entomophthora collagenase, Clostridium histolyticum collagenase, Streptomyces collagenase , and  Serratia marcescens  cysteine endopeptidase.  
     
     
         25 . The expression construct of  claim 24  wherein said protease is a cathepsin L protease.  
     
     
         26 . The expression construct of  claim 25  wherein said cathepsin L protease is from the facefly  Sarcophaga peregrina.    
     
     
         27 . A vector comprising the expression construct of  claim 21 .  
     
     
         28 . The vector of  claim 27  wherein said vector is a viral vector.  
     
     
         29 . The vector of  claim 28  wherein said vector is capable of infecting an insect cell.  
     
     
         30 . The vector of  claim 27  wherein said vector is selected form the group consisting of: bacteria (e.g.,  Bacillus thuringiensis  and  Bacillus popiliae ), viruses (e.g., nucleopolyhedroviruses) and protozoa (e.g., the microsporidian  Nosema locustae ).  
     
     
         31 . The vector of  claim 30  wherein said vector is AcIE1TV3.STR1, AcMLF9.STR1, AcIE1TV3.GEL, AcMLF9.GEL, AcIE1TV3.ScathL, and AcMLF9.ScathL.  
     
     
         32 . A host cell comprising the vector of  claim 27 .  
     
     
         33 . The host cell of  claim 32  wherein said host cell is a plant cell.  
     
     
         34 . A transgenic plant which is substantially resistant to insect infestation comprising the vector of  claim 27 .  
     
     
         35 . Seed of the plant of  claim 34.