Suppression of Resistance in Insects to Bacillus Thuringiensis Cry Toxins, Using Toxins that do not Require the Cadherin Receptor

Abstract

The present invention provides a method to obtain DNA constructs that encode 3-domain Cry toxins (also called Cry toxins, Bt toxins, or β-endotoxins) lacking helix α-1. These DNA constructs have been modified to encode proteins that kill insects that are resistant to the corresponding unmodified Cry toxins. The DNA constructs encoding the Modified 3-Domain Cry Toxins and the encoded Modified 3-Domain Cry Toxins are provided together with the molecular vectors and the host cell comprising said constructs and the recombinant methods to produce the Modified 3-Domain Cry Toxins. Additionally, compositions comprising the Modified 3-Domain Cry Toxins are disclosed. The resistance of the insects to unmodified Cry toxins is due to reduced toxin binding to the insects' midgut receptors. The invention further provides methods to overcome resistance in crop pests.

1 . A Method for obtaining DNA constructions that encode Modified 3-Domains Cry Toxins lacking the α-1 helix, where the encoded Modified 3-Domain Cry Toxins are capable of killing the Eligible Resistant Insects, suppressing their resistance. This method consists of the following steps:
 a) Selection of a coding gene for a 3-Domain Cry Toxin as a target.   b) Identification of the promoter area, modifying areas of the protein fragment of the respective unmodified toxin from the α-2 helix to the β-23 sheet, the coding area of the α-1 helix and the C-Terminal area of the protoxin, if it is present in the selected 3-Domain Cry Toxin.   c) Amplification of the genetic construction excluding the coding area of the α-1 helix.   
     
     
         2 . The method of  claim 1 , characterized by the amplification step, is performed by successive amplifications that consist of the following steps:
 a) A first amplification step to amplify only the promoter area.   b) One or more amplification steps to amplify the coding area beginning from α-2 helix to β-23 sheet, and the carboxy terminal area of the protoxin, if present.   c) Linking of the PCR products obtained in steps a) and b).   
     
     
         3 . The method of  claim 1 , characterized in that the 3-Domain Cry Toxin is selected from the group consisting of all the 3-Domain Cry Toxins of the subfamilies: Cry1, Cry2, Cry3, Cry4, Cry5, Cry7 Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry16, Cry17, Cry18, Cry 19, Cry20, Cry21, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry47, Cry48 and Cry50. 
     
     
         4 . The method of  claim 3 , characterized in that the 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1. 
     
     
         5 . The method of  claim 4 , characterized in that the 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1A. 
     
     
         6 . The method of  claim 5 , characterized in that the 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1Ab y Cry1Ac. 
     
     
         7 . An isolated DNA fragment that includes a coding area for a Modified 3-Domains Cry Toxin, characterized in that the encoded Modified 3-Domain Cry Toxin lack the α-1 helix and consequently is capable of killing the Eligible Resistant Insects, suppressing their resistance to the Unmodified 3-Domain Cry Toxins. 
     
     
         8 . The isolated DNA fragment of  claim 7 , characterized in that the Modified 3-Domain Cry Toxin is selected from the group consisting of all the 3-Domain Cry Toxins of the subfamilies: Cry1, Cry2, Cry3, Cry4, Cry5, Cry7 Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry10, Cry17, Cry18, Cry19, Cry20, Cry21, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry47, Cry48 and Cry50, modified so that they lack the α-1 helix. 
     
     
         9 . The isolated DNA fragment of  claim 8 , characterized in that the Modified 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1, modified so that they lack the α-1 helix. 
     
     
         10 . The isolated DNA fragment of  claim 9 , characterized in that the Modified 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily CryIA, modified so that they lack the α-1 helix. 
     
     
         11 . The isolated DNA fragment of  claim 10 , characterized in that the Modified 3-Domain Cry Toxin is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamilies Cry1Ab and Cry1Ac, modified so that they lack the α-1 helix. 
     
     
         12 . A molecular vector characterized in that it includes the isolated DNA fragment, of  claim 7 . 
     
     
         13 . The molecular vector of  claim 12 , characterized in that the vector is a vector with a double origin replication that may be replicated in the cells of  Bacillus thuringiensis  and  Escherichia coli.    
     
     
         14 . The molecular vector of  claim 12 , characterized in that the vector is suitable for the transformation of and expression in vegetable cells. 
     
     
         15 . A genetically altered cell selected from the group consisting of bacterial cells or vegetable cells, characterized in that said host cell includes the isolated DNA fragment of  claim 7 . 
     
     
         16 . A Modified 3-Domains Cry Toxins lacking the α-1 helix, characterized in that it is capable of killing the Eligible Resistant Insects, suppressing their resistance to the Unmodified 3-Domain Cry Toxins. 
     
     
         17 . The Modified 3-Domain Cry Toxin of  claim 16 , characterized in that it is selected from the group consisting of all the 3-Domain Cry Toxins of the subfamilies: Cry1, Cry2, Cry3, Cry4, Cry5, Cry7 Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry 14, Cry10, Cry17, Cry18, Cry19, Cry20, Cry21, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry47, Cry48 and Cry50, modified so that they lack the α-1 helix. 
     
     
         18 . The Modified 3-Domain Cry Toxin of  claim 17 , characterized in that it is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1, modified so that they lack the α-1 helix. 
     
     
         19 . The Modified 3-Domain Cry Toxin of  claim 18 , characterized in that it is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamily Cry1A, modified so that they lack the α-1 helix. 
     
     
         20 . The Modified 3-Domain Cry Toxin of  claim 19 , characterized in that it is selected from a group consisting of all the 3-Domain Cry Toxins of the subfamilies Cry1Ab and Cry1Ac, modified so that they lack the α-1 helix. 
     
     
         21 . A method to obtain a Modified 3-Domain Cry Toxin that lacks an α-1 helix, characterized in that it includes the steps to:
 a) cultivate genetically altered host cells that include a DNA construction that encodes a Modified 3-domain Cry Toxin that lack the α-1 helix, under conditions suitable for allowing the expression of the Modified 3-Domain Cry Toxin, and optionally   b) isolate the Modified 3-Domain Cry Toxin, and optionally   c) purify the Modified 3-Domain Cry Toxin.   
     
     
         22 . A compound that kills the Eligible Resistant Insects in order to suppress their resistance, characterized in that it contains one or more Modified 3-Domain Cry Toxins in a quantity that is sufficient to control the target infestations and a mixture with an agronomically and ecologically acceptable excipient. 
     
     
         23 . The compound of  claim 22 , characterized in that the Modified 3-Domain Cry Toxin is present in a quantity of between 0.0001% and 95% by weight. 
     
     
         24 . A transgenic plant that includes the DNA fragment of  claim 7 . 
     
     
         25 . A method to suppress the resistance of the Eligible Resistant Insects, characterized in that it includes the step to feed said Eligible Resistant Insects with lethal doses of the Modified 3-Domain Cry Toxins. 
     
     
         26 . The method of  claim 25 , characterized in that said Modified 3-Domain Cry Toxins are fed as part of a compound. 
     
     
         27 . The method of  claim 25 , characterized in that said Modified 3-Domain Cry Toxins are present in a transgenic plant.