Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Oxidoreductases
Reexamination Certificate
1998-04-21
2002-04-16
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Oxidoreductases
C424S094200, C424S094500
Reexamination Certificate
active
06372211
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to compositions and methods for controlling coleopteran insects by use of two proteins in combination which may be applied directly to the plant or produced thereon by microorganisms or by genetically modifying the plant to produce the proteins, to genes encoding these proteins, to methods for identifying such genes and proteins, and to recombinant microorganisms and plants capable of expressing these genes for use in controlling plant infestation by the target coleopteran insects.
BACKGROUND OF THE INVENTION
The control of insect pests by naturally occurring proteins is a well established practice. The most commonly used insect control proteins are the endotoxins derived from
Bacillus thuringiensis
(B.t.) that are used to control both lepidopteran and coleopteran insect pests. Expression of these proteins in transgenic plants also confers protection against certain insect pests (Barton et al., 1987; Fischhoff et al., 1987 Perlak et al., 1990; Vaeck et al., 1987).
A variety of insect pests that cause significant economic losses were not previously known to be controlled by B.t. endotoxins. Boll weevil (BWV),
Anthonomus grandis,
corn rootworm (CRW), Diabrotica spp., and wireworm (WW), Melanotus spp. are examples of coleopteran insect pests that inflict significant crop damage yet, until recently were not known to be controlled by known B.t. endotoxins. Thus, it would be useful to identify new insecticidal proteins which, alone or in combination, are able to control these coleopteran insects. Furthermore, it would be useful to identify new insecticidal proteins with different modes of action to delay the development of B.t. endotoxin resistance in coleopteran pests such as the Colorado potato beetle (CPB),
Leptinotarsa decemlineata
(Say), that are currently controlled by certain B.t. endotoxins (Krieg et al., 1983).
Preparations of enzymes from several different sources are available from Sigma Chemical Company (St. Louis, Mo.) and other suppliers. Amino acid oxidases can also be obtained from sources including, but not limited to snake venom, mammalian, and avian sources (Bright and Porter, 1975). Lysine and other amino acid oxidases (E.C. 1.4.3.2) are naturally produced by micro-organisms such as Trichoderma sp., Neurospora sp., Penicillium sp., and Proteus sp. (Kusakabe et al., 1979; 1980; Niederman and Lerch, 1990; Knight, 1948; Stumpf and Green, 1944). Although lysine oxidase has been shown to have antitumor activity (Kusakabe et al., 1979; Id., 1980), there have been no reports of insecticidal activity associated with this enzyme. Also, there have been no reports of insecticidal activity being associated with an amino acid oxidase enzyme when combined with any other compound. However, we have unexpectedly found that a composition comprising a lysine oxidase and a previously unidentified M
r
50,000 protein yield potent insecticidal activity when combined in a mixture and ingested by an insect. The M
r
50,000 protein is described herein as a tedanalactam synthase shown herein to have at least one enzyme activity in which &Dgr;1-piperideine-2-carboxylate is converted to tedanalactam. Described herein are methods for using a combination of lysine oxidase and tedanalactam synthase to control infestation of plants by insect pests.
SUMMARY OF THE INVENTION
This invention relates generally to novel compositions and methods for the control of undesired insects. It is therefore a particular object of the present invention to present materials and methods used in the preparation of compositions and plants capable of controlling insect infestation when ingested by the insect. It is also an object of the present invention to provide protein compositions capable of controlling BWV, CRW, WW, CPB or other insect pests, and genes useful in producing such proteins. It is a further object of the present invention to provide genetic constructs for and methods of inserting such genetic material into microorganisms and plant cells. It is another object of the present invention to provide transformed microorganisms and plants containing such genetic material. Still another object of this invention relates to methods and reagents such as polynucleotides and antibodies, and the use of such methods and reagents in kit form, for detecting the individual molecules which comprise the active compositions as noted herein. In addition, variants of the molecules which comprise the active compositions are also contemplated by this invention.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a composition containing at least two proteins, lysine oxidase enzyme and tedanalactam synthase enzyme, which is capable of controlling insects, particularly coleopteran insects. These two proteins cause mortality and stunting of larvae of coleopteran insects when co-ingested. The proteins may be applied directly to plants or introduced in other ways such as through the application of plant-colonizing microorganisms or by transformed plants generated using recombinant DNA methods wherein the recombinant plants express genes encoding these enzymes.
In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of controlling insect infestation of plants comprising providing a composition containing at least a lysine or amino acid oxidase along with a second enzyme, which is preferably a tedanalactam synthase, for ingestion by the insect. It is apparent that neither protein alone is able to confer any insecticidal activity. However, it is the combination of the amino acid oxidase along with the second enzyme which is effective in confering insecticidal activity upon ingestion of such a composition by an insect. The composition, upon ingestion by the insect, contains a sufficient insecticidal amount of the proteins, such that the insect is unable to survive or is rendered incapable of causing further damage to a plant to which the composition has been applied. The composition contains a first enzyme which is a lysine oxidase enzyme, and a second enzyme which is capable of converting &Dgr;1-piperideine-2-carboxylate to tedanalactam. The proteins in the composition are preferably isolated from extracts of fungal species fermentations in which the extracts have been shown to exhibit insecticidal activity. The fungal species herein which produces an insecticidally effective extract composition was determined to be a Trichoderma species of fungi, and in particular a
Trichoderma viride.
Another Monsanto Company fungal isolate was designated as Trichoderma sp. F22844 also produces an insecticidally effective extract composition. The genes encoding the proteins in the illustrative composition are therefore preferably isolated from a Trichoderma species of fungi, however, other uncharacterized fungal species are believed to contain at least a lysine oxidase gene and a second protein which, in combination provide efficacious insecticidal activity.
The composition can contain as the second enzyme a protein which is approximately 50,000 Da, which is also recognized by one skilled in the art as a protein or enzyme which is approximately M
r
50,000. It is believed that the second enzyme can be isolated from any number of species, however it is preferably isolated from a species which produces compounds which exhibit coleopteran insecticidal activity, and more preferably isolated from a fungal species. It is also believed that any fungal species which exhibits coleopteran insecticidal activity and also produces a lysine oxidase may also produce a second enzyme which in combination with the lysine oxidase confers effective insecticidal activity when ingested by target insect. Furthermore, any species which produces a lysine oxidase and which also contains a gene which hybridizes under stringent conditions to a Trichoderma species gene encoding an approximately M
r
50,000 Da protein which converts &Dgr;1-piperideine-2-carboxylate to tedanalactam may confer effecti
Greenplate John T.
Isaac Barbara G.
Purcell John P.
Romano Charles P.
Ball T. K.
Howrey Simon Arnold & White , LLP
Monsanto Technolgy LLC
Witz Jean C.
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