Methods and compositions for making and using compatible insecticidal proteins

Abstract

Pesticidal proteins (FFPP's) are used to produce derivatives (DP's) that are ineffective and disabled relative to conferring toxic properties upon a target pest, yet the ability of the DP to bind to the receptor to which said FFPP binds is unaffected. Such DP's are useful in inhibiting the FFPP from which it was derived when both are fed to a target pest and for comparing receptor binding capability and efficiency relative to different FFPP's from which the DP has been derived, providing for an assessment of different FFPP's relative to each other, and providing uniformity and certainty in combinations of such FFPP's for compositions, including transgenic plants, that can be used to control pest populations susceptible to both FFPP's, creating more durable transgenic plant products, inhibiting the development of resistance to such FFPP's when used in plants commercially, and in providing a durable and viable resistance management strategy for crops using such FFPP combinations. Polynucleotide sequences intended for use in expression of the DP's and FFPP's are also provided. Particular embodiments provide methods of designing and preparing DP's, as well as compositions and methods of using DP's and the FFPP's from which the DP's have been derived in more effective pesticidal compositions and products.

What is claimed is: 
     
         1 . A method for selecting two pesticidal agents compatible for use together in a composition for controlling a target pest, said method comprising:
 a) selecting a first and a second toxic agent, each agent being different from the other and each agent causing toxic properties when provided individually in the diet of a target pest;   b) producing a DT from said first toxic agent that, upon ingestion by said target pest, blocks the toxic properties conferred by said first toxic agent but does not itself confer toxic properties;   c) producing a plurality of different mixtures containing a fixed but pesticidally effective amount of a second toxic agent, and increasing amounts of said DT;   d) providing in the diet of said target pest, a pesticidally effective amount of said second toxic agent;   e) providing a dose of each mixture of step c) separately to each of at least three different individuals of said target pest;   
       wherein observing toxic properties in any individual in step e) is determinative that said first and second toxic agents are compatible for use together to control said target pest. 
     
     
         2 . A recombinant plant or seed expressing two or more pesticidal agents, wherein said agents are selected for use together as compatible agents according to the method of  claim 1 . 
     
     
         3 . Selecting a first FFPP and a second FFPP to be combined together in a composition or for use on or in a plant to control insect infestation of said plant by a target insect pest species, wherein said combination of said FFPP's optionally provide:
 (a) for decreasing the likelihood of the development of resistance by said target insect pest to any of the FFPP's in said composition;   (b) for aiding in or improving resistance management practices for controlling said target insect pest; or   (c) for delaying the onset of resistance to any of the FFPP's in said composition;
 Wherein
 (i) said two or more FFPP's are selected for use together by the steps of  claim 1 ; 
 (ii) said DP, when used in a plurality of molar ratios with a constant amount of said first FFPP, is effective in titering the toxic effect of the first FFPP upon said target pest; and 
 (iii) said DP, when used in a plurality of molar ratios with a constant amount of said second FFPP, does not titer the toxic effect of the second FFPP upon said target pest; and 
 
 Wherein said first and said second FFPP are compatible for use together in said composition on or in said plant. 
   
     
     
         4 . A method of assessing the mode of action of a first FFPP for compatibility with a second FFPP to be used in a common pesticidal composition, said method comprising the steps of:
 (a) preparing a DP from a first FFPP that is toxic to a target pest;   (b) confirming that said DP when used alone in a bioassay with said target pest has diminished toxicity against the target pest when compared to the toxicity of the first FFPP;   (c) comparing said DP to a second FFPP different from said first FFPP alone and in a plurality of molar ratios in which the DP is present in a greater concentration than said second FFPP in the diet of the target pest;   
       wherein the inability to titer the toxic properties of said second FFPP with said DP is determinative of the binding of said first FFPP and said second FFPP to different receptors in said target pest. 
     
     
         5 . The method of any of  claim 3  or  4 , wherein a composition comprising said first FFPP and said second FFPP is effective in controlling an insect pest infestation wherein said insects are selected from the group consisting of Arachnida, Coleoptera,  Ctenocephalides , Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera and Thysanoptera insects. 
     
     
         6 . The method of  claim 1 , wherein a composition comprising said first toxic agent and said second toxic agent is effective in controlling an insect pest infestation wherein said insects are selected from the group consisting of Arachnida, Coleoptera,  Ctenocephalides , Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera and Thysanoptera insects. 
     
     
         7 . A plant or a seed from a plant comprising a first recombinant nucleic acid molecule comprising a first heterologous promoter operably linked to a first polynucleotide segment encoding a first FFPP and a second recombinant nucleic acid molecule comprising a second heterologous promoter operably linked to a second polynucleotide segment encoding a second FFPP different from said first FFPP, wherein said first FFPP and said second FFPP are selected for use together from the steps as set forth in any of  claim 3  or  4 , wherein, optionally:
 a) said plant or said seed are produced from the breeding together by the hand of man of two different plants of the same or substantially similar species, a first plant comprising said first recombinant nucleic acid molecule expressing said first FFPP and a second plant comprising said second recombinant nucleic acid molecule expressing said second FFPP; 
 b) said plant or seed are produced from the regeneration of a plant from the transformation of a first plant cell by said first recombinant nucleic acid molecule and the transformation of a second plant cell by said second recombinant nucleic acid molecule; 
 c) said plant or seed are produced from the transformation of a single plant cell by said first and said second recombinant nucleic acid molecule; and 
 d) said plant or seed are grown from a plant or seed of any of a), b), or c), wherein said plant or seed comprise said first and said second recombinant nucleic acid molecule. 
 
     
     
         8 . A composition comprising a first polypeptide and a second polypeptide that is different from the first polypeptide, wherein said first and second polypeptides are each toxic to a target pest and do not bind to the same target receptor in said pest, and wherein said first and second polypeptide are selected for use together by the steps of:
 a) producing a DT from said first polypeptide that, upon ingestion by said target pest, blocks the toxic properties conferred by said first polypeptide but does not itself confer toxic properties;   b) producing a plurality of different mixtures containing a fixed but pesticidally effective amount of said second polypeptide, and increasing amounts of said DT;   c) providing in the diet of said target pest, a pesticidally effective amount of said second polypeptide;   d) providing a dose of each mixture of step c) separately to each of at least three different individuals of said target pest;
 wherein observing toxic properties in any individual in step 
   e) is determinative that said first and second polypeptides are compatible for use together to control said target pest.