Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-09-22
2001-08-21
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C043S132100, C424S405000, C536S023700
Reexamination Certificate
active
06277823
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel toxins from
Xenorhabdus nematophilus, Xenorhabdus poinarii
, and
Photorhabdus luminescens
, nucleic acid sequences whose expression results in said toxins, and methods of making and methods of using the toxins and corresponding nucleic acid sequences to control insects.
BACKGROUND OF THE INVENTION
Insect pests are a major cause of crop losses. Solely in the US, about $7.7 billion are lost every year due to infestation by various genera of insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to gardeners and home owners.
Insect pests are mainly controlled by intensive applications of chemical insecticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or death of the insects. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management strategies, but there is an increasing need for alternative pest control agents. Biological insect control agents, such as
Bacillus thuringiensis
strains expressing insecticidal toxins like &dgr;-endotoxins, have also been applied with satisfactory results, offering an alternative or a complement to chemical insecticides. Recently, the genes coding for some of these &dgr;-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of insecticidal toxins in transgenic plants, such as
Bacillus thuringiensis
&dgr;-endotoxins, has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents. Yet, even in this case, the development of resistance remains a possibility and only a few specific insect pests are controllable. Consequently, there remains a long-felt but unfulfilled need to discover new and effective insect control agents that provide an economic benefit to farmers and that are environmentally acceptable.
SUMMARY OF THE INVENTION
The present invention addresses the long-standing need for novel insect control agents. Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.
In the search for novel insect control agents, certain classes of nematodes from the genera Heterorhabdus and Steinemema are of particular interest because of their insecticidal properties. They kill insect larvae and their offspring feed in the dead larvae. Indeed, the insecticidal activity is due to symbiotic bacteria living in the nematodes. These symbiotic bacteria are Photorhabdus in the case of Heterorhabdus and Xenorhabdus in the case of Steinemema.
The present invention is drawn to nucleotide sequences isolated from
Xenorhabdus nematophilus
, and nucleotide sequences substantially similar thereto, whose expression result in insecticidal toxins that are highly toxic to economically important pests, particularly plant pests. The invention is further drawn to the insecticidal toxin resulting from the expression of the nucleotide sequence, and to compositions and formulations containing the insecticidal toxin, that are capable of inhibiting the ability of insect pests to survive, grow or reproduce, or of limiting insect-related damage or loss in crop plants. The invention is further drawn to a method of making the toxin and to methods of using the nucleotide sequence, for example in microorganisms to control insects or in transgenic plants to confer insect resistance, and to a method of using the toxin, and compositions and formulations comprising the toxin, for example applying the toxin, composition or formulation to insect infested areas, or to prophylactically treat insect susceptible areas or plants to confer protection or resistance against harmful insects.
The novel toxin is highly insecticidal against
Plutella xylostella
(diamondback moth), an economically important insect pest. The toxin can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.
According to one aspect, the present invention provides an isolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects. In one embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. Preferably, the nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 3, 5, 7, 9, 11, 13, and 15. Most preferably, the nucleotide sequence comprises nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. In another embodiment, the nucleotide sequence comprises the approximately 3.0 kb DNA fragment comprised in pCIB9369 (NRRL B-21883).
According to a preferred embodiment, the toxins resulting from expression of the nucleic acid molecules of the invention have activity against
Plutella xylostella.
In another aspect, the present invention provides an isolated nucleic acid molecule comprising a 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.
The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene. Still further, the present invention provides a host cell comprising such a chimeric gene. A host cell according to this aspect of the invention may be a bacterial cell, a yeast cell, or a plant cell, preferably a plant cell. Even further, the present invention provides a plant comprising such a plant cell. Preferably, the plant is maize.
In yet another aspect, the present invention provides toxins produced by the expression of DNA molecules of the present invention. According to a preferred embodiment, the toxins of the invention have activity against
Plutella xylostella.
In one embodiment, the toxins are produced by the
E. coli
strain designated as NRRL accession number B-21883.
In another embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 3, 5, 7, 9, 11, 13, and 15
Anderson Arne Robert
Kramer Vance Cary
Morgan Michael Kent
Fronda Christian L.
Holmes Jennifer L.
Meigs J. Timothy
Nashed Nashaat T.
Stults Larry W.
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