Insecticidal proteins derived from bacillus thuringiensis

Abstract

The present invention relates to the field of plant pest control, particularly insect control. Provided are nucleotide sequences from Bacillus thuringiensis encoding insecticidal proteins. Further provided are methods and means for using said nucleotide sequence for controlling plant insect pests.

1 .- 19 . (canceled) 
     
     
         20 . An isolated insecticidal protein comprising an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence of the smallest toxic fragment thereof, wherein said smallest toxic fragment is the smallest portion of the protein of SEQ ID NO: 4 retaining insecticidal activity which can be obtained by incubating insect gut-juice extracts with solutions comprising the protein of SEQ ID NO:4, said gut-juice extracts obtained from any one of the following insects:  Helicoverpa zea, Heliothis virescens, Ostrinia nubilalis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus  and  Anticarsia gemmatalis.    
     
     
         21 . The protein of  claim 20 , comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 55 to amino acid position 788, from amino acid position 55 to amino acid position 455, or from amino acid position 200 to amino acid position 455. 
     
     
         22 . The protein of  claim 21  comprising the amino acid sequence of SEQ ID NO: 4. 
     
     
         23 . The protein of any one of  claim 22 , wherein an Asp or Ala amino acid is inserted as a new second amino acid so that the protein starts with a Met-Asp or Met-Ala dipeptide. 
     
     
         24 . A hybrid or chimeric protein comprising a toxic fragment of the protein of SEQ ID NO: 4, wherein said toxic fragment comprises the amino acid sequence of SEQ ID NO: 4 from amino acid position 55 to amino acid position 788 or from amino acid position 200 to amino acid position 455, or wherein said toxic fragment is the smallest portion of the protein of SEQ ID NO: 4 retaining insecticidal activity which can be obtained by incubating insect gut-juice extracts with solutions comprising the protein of SEQ ID NO:4, said gut-juice extracts obtained from any one of the following insects:  Helicoverpa zea, Heliothis virescens, Ostrinia nubilalis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus  and  Anticarsia gemmatalis.    
     
     
         25 . A method of protecting a plant against lepidopteran insect damage comprising contacting said plant with the insecticidal protein of  claim 20 . 
     
     
         26 . The method of  claim 25 , wherein said protein is applied externally to said plant. 
     
     
         27 . The method of any one of  claim 25 , wherein said plant is a maize, cotton, soybean or rice plant. 
     
     
         28 . An insecticidal composition comprising the protein of any one of  claim 20 . 
     
     
         29 . The composition of  claim 28  which comprises suitable carriers, diluents, emulsifiers and/or dispersants, which composition, when applied externally to a plant increases resistance to lepidopteran insect damage compared to control plants to which no such composition is applied. 
     
     
         30 . A process to control a Lepidopteran cotton, maize, rice or soybean insect pest, comprising the step of applying a composition comprising the protein of  claim 20 . 
     
     
         31 . A process to control insects selected from the group consisting of:  Scirphophaga  spp.,  Cnaphalocrocis  spp.,  Sesamia  spp,  Chilo  spp.,  Anticarsia  spp.,  Pseudoplusia  spp.,  Epinotia  spp.,  Rachiplusia  spp,  Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus, Anticarsia gemmatalis, Pseudoplusia includens, Epinotia aporema, Rachiplusia nu, Spodoptera ornithogalli,  and  Plathypena scabra,  comprising using an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 or an insecticidal variant thereof against said insects, wherein said variant is an insecticidal protein selected from the group consisting of: a) an insecticidal protein with at least 95% sequence identity to the protein of SEQ ID NO: 4, b) an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 55 to amino acid position 788 or from amino acid position 200 to amino acid position 455, c) a variant of the protein of SEQ ID NO: 4 or the protein in b) wherein less than 5, or 5-10, amino acids are added, replaced or deleted without changing the insecticidal activity of the protein. 
     
     
         32 . The process of  claim 31 , wherein said insect is selected from the group consisting of:  Scirphophaga  spp.,  Cnaphalocrocis  spp.,  Sesamia  spp,  Chilo  spp.,  Anticarsia  spp.,  Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus,  and  Anticarsia gemmatalis.    
     
     
         33 . The process of  claim 31 , wherein said insect is selected from the group consisting of:  Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus,  and  Anticarsia gemmatalis.    
     
     
         34 . A process wherein two insecticidal proteins are used to control insects pests, wherein :
 a) the first protein is an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 or an insecticidal variant thereof, wherein said variant is an insecticidal protein selected from the group consisting of: a) an insecticidal protein with at least 95% sequence identity to the protein of SEQ ID NO: 4, b) an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 55 to amino acid position 788 or from amino acid position 200 to amino acid position 455, c) a variant of the protein of SEQ ID NO: 4 or the protein in b) wherein less than 5, or 5-10, amino acids were added, replaced or deleted without changing the insecticidal activity of the protein, and   b) the second insecticidal protein is a Cry1F protein, a hybrid derived from a Cry1F protein, a Cry1A-type protein, a Cry1Ac protein, a hybrid derived from a Cry1Ac protein, a Cry1Ab protein or an insecticidal fragment thereof, a Cry2Ae protein, a Cry2Af protein, a Cry2Ag protein, or a VIP3Aa protein.   
     
     
         35 . The process of  claim 34 , wherein said first and second protein are expressed in maize, rice, cotton or soybean plants, and wherein said second insecticidal protein is: a Cry1Ac protein, a Cry1Ab protein, a Cry2Ae protein, or a VIP3Aa protein. 
     
     
         36 . A method for controlling Lepidopteran soybean insect pests selected from the group consisting of: Velvet Bean Caterpillar ( Anticarsia gemmatalis ), Soybean Looper ( Pseudoplusia includens ), Yellowstriped Armyworm ( Spodoptera ornithogalli ), Pod Borer ( Epinotia aporema ) and  Rachiplusia nu,  which method comprises applying to an area or plant to be protected, an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 or an insecticidal variant thereof, wherein said variant is an insecticidal protein selected from the group consisting of: a) an insecticidal protein with at least 95% sequence identity to the protein of SEQ ID NO: 4, b) an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 55 to amino acid position 788 or from amino acid position 200 to amino acid position 455, c) a variant of the protein of SEQ ID NO: 4 or the protein in b) wherein less than 5, or 5-10, amino acids were added, replaced or deleted without changing the insecticidal activity of the protein. 
     
     
         37 . The method of  claim 36  wherein a soybean plant is contacted with said insecticidal protein. 
     
     
         38 . The method of  claim 36  which comprises planting a soybean plant transformed with a gene encoding said insecticidal protein, or spraying a composition containing said insecticidal protein.