Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-07-12
2002-08-06
Nashed, Nashaat T. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S410000, C435S252300, C435S252310, C435S252330, C514S012200, C530S350000, C536S023710
Reexamination Certificate
active
06429360
ABSTRACT:
The present invention relates to a novel class of proteins for the control of plant pests. Plant pests are a major factor in the loss of the world's commercially important agricultural crops resulting both in economic hardship to farmers and nutritional deprivation for local populations in many parts of the world. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. There is, however, substantial interest in developing effective alternative pesticides.
Control of various pests through the use of biological molecules has been possible in only a limited number of cases. The best known examples of biological molecules with pesticidal uses are the &dgr;-endotoxins from
Bacillus thuringiensis
(Bt), which is a gram-positive spore forming microorganism. Varieties of Bt are known that produce more than 25 different but related &dgr;-endotoxins. Bt strains produce &dgr;-endotoxins during sporulation the use of which is limited because they are active against only a very few of the many insect pests.
The limited specificity of the Bt endotoxins is dependent, at least in part, on both the activation of the toxin in the insect gut (Haider, M. Z et al., 1986, Eur. J. Biochem. 156:531-540) and its ability to bind to specific receptors present on the insects midgut epithelial cells (Hofmann, C. P. et al., 1988, PNAS 85.7844-7848). Therefore, the ability to control a specific insect pest using &dgr;-endotoxins at present depends on the ability to find an appropriate &dgr;-endotoxin with the desired range of activity. In many cases, no such &dgr;-endotoxin is known, and It is not certain that one even exists.
Plants also routinely become infected by fungi and bacteria, and many microbial species have evolved to utilize the different niches provided by the growing plant. In addition to infection by fungi and bacteria, many plant diseases are caused by nematodes which are soil-borne and infect roots, typically causing serious damage when the same crop species is cultivated for successive years on the same area of ground.
The severity of the destructive process of disease depends on the aggressiveness of the phytopathogen and the response of the host, and one aim of most plant breeding programs is to increase the resistance of host plants to disease. Novel gene sources and combinations developed for resistance to disease have typically only had a limited period of successful use in many crop-pathogen systems due to the rapid evolution of phytopathogens to overcome resistance genes.
It is apparent, therefore, that scientists must constantly be in search of new methods with which to protect crops against plant pests. It has been found in the present invention a novel class of proteins which can be used to control plant pests.
Programmed cell death is a process whereby developmental or environmental stimuli activate a genetic program that culminate in the death of the cell (Jacobson, M. D. et al., 1997, Cell 8B: 347-354). This genetic potential exists in most, if not all, multicellular organisms. In the case of invertebrates, programmed cell death appears to play a dual role by being an integral part of both the insect development process and a response mechanism to infections particularly of viral nature (Clem, R. J. et al., 1991, Science 254: 1388-1390). Programmed cell death appears to be executed in several different manners leading to either apoptosis, atrophy or differentiation. Apoptosis is one of the best characterized types of programmed cell death encompassing cytological changes including membrane-bound apoptotic bodies and cytoplasmic blebbing as well as molecular changes such as endonucleolysis typified by the generation of oligosomal length fragments (Vaux, D. L and Strasser, A., 1996, PNAS 93:2239-2244). Although the overall apoptotic phenomenology is rather conserved among the different organisms, It is interesting to point out that, for many insect cells, cytoplasmic vacuolization and swelling rather than condensation seem to be the cytological features associated with apoptotic processes (Bowen, I. D., et al., 1996, Micros. Res. Techniq.34:202-217). The novel class of proteins disclosed within the present invention are shown to induce programmed cell death and exert a pesticidal effect.
The present invention is drawn to VIP3A(c) proteins including homologues thereof. Also provided by the invention are domains of proteins of the VIP3 class, including the toxic domain and the stabilizing domain. A preferred embodiment of the invention is the toxic domain of the VIP3A(a) protein and homologues thereof. Another preferred embodiment are antibodies to proteins of the VIP3 class, but preferably to the VIP3A(c) protein.
The invention also provides hybrid toxins comprising a toxic domain of a protein of the VIP3 class. In a preferred embodiment, the hybrid toxin is a chimeric proteins having a toxic core domain operably linked to a heterologous stabilizing domain. In another preferred embodiment, the hybrid toxin comprises an antibody, or immunologically-active fragment thereof, which immunologically recognizes the VIP3 receptor operably linked to a toxic domain from other proteins, wherein the toxin domain is obtained from a number of cytotoxic proteins including but not limited to Bacilus toxins, including endotoxins and vegetative insecticidal proteins.
Also encompassed by the invention are plants comprising a DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein. Preferred embodiments include plants selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, cole crops, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape. In a particularly preferred embodiment, the plant is a maize plant.
The invention also provides microorganisms comprising a heterologous DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein. In a preferred embodiment, the microorganism is selected from the group consisting of bacteria, baculovirus, algae and fungi. In another preferred embodiment, the microorganism is selected from the group consisting of Bacillus, Pseudomonas, Clavibacter, and Rhizobium. Further encompassed by the invention are entomocidal compositions comprising microorganisms comprising a heterologous DNA sequence which encodes a protein of the VIP3 class, but preferably a VIP3A(c) protein.
The invention further relates to plants and microorganisms further comprising a second DNA sequence which encodes a second insecticidal protein. Particularly preferred second DNA sequences are those which encode a &dgr;-endotoxin, those which encode another protein of the VIP3 class, or those which encode a protein of the VIP1 or VIP2 classes. In a more preferred embodiment, the &dgr;-endotoxin is active against an insect selected from the group consisting of Lepidoptera and Coleoptera. In a more particularly preferred embodiment the &dgr;-endotoxin is active against Ostrinia, or Diabrotica. In another particularly preferred is a second DNA sequence which encodes a &dgr;-endotoxin protein selected from the group consisting of Cry1, Cry3, Cry5 and Cry9. In a more particularly preferred embodiment, the &dgr;-endotoxin is selected from the group consisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9C and Cry9B. Most particularly preferred are Sendotoxins selected from the group consisting of Cry1Ab, Cry1Ba and Cry9C proteins.
The invention further provides a method of controlling insects by contacting the insects with an insecticidal amount of a protein of the VIP3 class, but preferably a VIP3A(c) protein, or an insecticidal amount of a chemical ligand to a receptor of the VIP3 class of proteins. In one preferred embodiment, the insects are contacted with a transgenic plant comprising a DNA sequence which expresses a protein of the VIP3 class, but preferably a VIP3A(c) protein in another preferred embodiment, the insects are contacted with a an entomocidal composition comprising a prote
Desai Nalini Manoj
Estruch Juan Jose
Koziel Michael Gene
Nye Gordon James
Warren Gregory Wayne
Meigs J. Timothy
Nashed Nashaat T.
Pace Gary
Syngenta Investment Corporation
Warren Gregory
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