Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Virus or bacteriophage
Reexamination Certificate
1998-12-11
2001-03-13
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Virus or bacteriophage
C424S204100, C424S115000, C043S132100, C047S05810R
Reexamination Certificate
active
06200561
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of controlling insects, including particularly weevils, cotton boll weevils etc. by use of a viral protein extract which may be applied directly to the boll weevil or larva or cotton plants. In another aspect, the invention relates to an isolated gene from the viral toxin which can be transferred to plant crops such as cotton so that toxin will be produced in such engineered plants.
BACKGROUND OF THE INVENTION
The use of natural products, including proteins, is a well-known method of controlling many insect pests. Endotoxins of
Bacillus thruringiensis
(
B.t
.) are used to control both lepidopteran and coleopteran insect pests. Genes producing the
B.t
. toxin have been introduced and expressed in several plants, including cotton, tomato, and tobacco, and have also been expressed by various microorganisms. However, there are several economically important insect pests that are not susceptible to
B.t
.endotoxins, and this group includes the cotton boll weevil. Researchers at the Monsanto Co. have identified a bacterial enzyme (cholesterol oxidase) that induces mortality and stunting in boll weevil larvae and in several lepidopteran species. These workers have isolated the gene for this enzyme and expressed it in plant-colonizing bacteria and in cotton tissue culture. Experience with
B.t
. toxins suggests that development of resistance will be a problem with use of protein toxins for insect control, and a number of approaches have been recommended to minimize this. These include the use of refugia, dosage control, and use of multiple toxins, etc. An important strategy against the development of resistance will be the identification of alternate toxins that have a different mode of action. This approach will allow use of lower doses for all toxins and will minimize the probability of mutations that result in resistance to two or more toxins.
There are, however, several economically important insect pests that are not susceptible to
B.t
. endotoxins. One such important pest is the cotton boll weevil. There is also a need for additional proteins which control insects for which
B.t
. or other toxins provides control in order to manage any development of resistance in the population.
Interest in the biological control of insect pests has arisen as a result of disadvantages of conventional chemical pesticides. Chemical pesticides generally affect beneficial as well as nonbeneficial species. Insect pests tend to acquire resistance to such chemicals so that new insect pest populations can rapidly develop that are resistant to these pesticides. Furthermore, chemical residues pose environmental hazards and possible health concerns. Biological control presents an alternative means of pest control which can reduce dependence on chemical pesticides.
The primary strategies for biological control include the deployment of naturally-occurring organisms which are pathogenic to insects (entomopathogens) and the development of crops that are more resistant to insect pests. Approaches include the identification and characterization of insect genes or gene products which may serve as suitable targets for insect control agents, the identification and exploitation of previously unused microorganisms (including the modification of naturally-occurring nonpathogenic microorganisms to render them pathogenic to insects), the modification and refinement of currently used entomopathogens, and the development of genetically engineered crops which display greater resistance to insect pests.
In 1972 McLaughlin et al. published their work on the effect of CIV on the cotton boll weevil. They showed that infection with whole virus arrested metamorphosis and death. Researchers in France showed that soluble extracts from CIV inhibited host protein synthesis and gene expression in cell cultures from mosquitoes and some caterpillar species (Cerutti and Devauchelle, 1980). However, no group has previously shown that a protein fraction from CIV kills boll weevil larvae, nor has any group shown whole virus or viral protein preparations causing inhibition of protein synthesis in boll weevil cell lines or induction of programmed cell death (apoptosis) in any cell line.
The cotton boll weevil will have an economic impact exceeding $500 million per year in Texas alone. More than 9,200 jobs will be lost and at least 60 cotton gins will close if no new technology is developed. Chemical control of the weevil is not working well because of resistance problems and adverse effects on beneficial insects. In addition, there are difficulties in discovering new chemistry and problems with insecticide contamination of ground reserves. Therefore, the development of alternative, biological (especially microbial) control systems is critical. Because larvae develop inside the cotton boll and cannot be sprayed externally, the best control strategy will be to engineer transgenic cotton that produces insecticidal proteins.
Our laboratory has shown that Chilo iridescent virus (CIV) induces metamorphic deformity in boll weevil larvae, and kills them. We have shown that CIV replicates efficiently in this host. A protein extract from the virus induces mortality in neonate larvae. The Extract also inhibits host protein synthesis and minduces programmed cell death or apoptosis as evidenced by cell blebbing and DNA fragmentation. Heating at 60 degrees C. for 30 minute or treatment with protease destroys these activities.
Prior art has shown that Chilo iridescent virus (CIV) induces mortality and metamorphic deformity in the cotton boll weevil. Prior art also shows that CIV protein extracts inhibited host gene expression in lepidopteran and dipteran cells. However, the use of CIV protein extracts in the control of in sect infestation have not been demonstrated nor has it been demonstrated that CIV protein extract induces programmed cell death or apoptosis in any cell line or organism.
What is needed is a biological pesticide which reduces the adverse effects of chemical pesticide. A biological pesticide is preferred because it creates less of an environmental hazard than a chemical pesticide. A pesticide that causes insect death more rapidly is additionally needed. What is also needed to the identification and isolation of a gene that codes for a protein which will control insect development. Such a gene or its protein product could then be incorporated into various organisms for the improved biological control of insect pests.
SUMMARY OF THE INVENTION
It has been discovered that a protein extract from purified Chilo iridescent virus (CIV) particles will control infestations by boll weevils, and whole virus particles will control aphid populations.
It has been discovered that proteins or extracts of proteins that are insecticidal proteins provide composition and methods for using certain viral inhibitors to protect plants otherwise susceptible to insect infestation by one or more of Mexican bean beetle, red flower beetle, confused flower beetle, boll weevil, Colorado potato beetle, 3-line potato beetle, rice weevil, maze weevil, granularly weevil, Egyptian alfalfa weevil, bean weevil, yellow mill worm weevil, asparagus beetle and a variety of other insects including other beetles and weevils.
We have shown that a protein fraction from purified Chilo iridescent virus particles causes mortality in freshly hatched larvae of the cotton boll weevil. This toxin preparation also inhibits wholesale protein synthesis, and induces programmed cell death (apoptosis) in cell cultures of the cotton boll weevil,
Anthonomus grandis
. Knowledge of the viral toxin will allow isolation of the gene responsible for the toxin. The gene would then be transferred to crop plants (such as cotton) so that toxin will be produced in such engineered plants. Pest insects will be arrested in their development or die upon contact with toxin producing plant tissues. The toxin is the only way viral protein component is known to kill boll weevil larvae. It is also the only viral toxin that inhibits host protein synthesis
Croskell Henry
Mosher Mary E.
LandOfFree
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