Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – is pest or herbicide...
Reexamination Certificate
1999-09-29
2001-03-20
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, is pest or herbicide...
C435S069100, C435S070100, C435S071100, C435S071200, C435S071300, C435S410000, C435S419000, C435S243000, C435S252500, C435S252800, C435S320100, C530S825000, C530S350000, C536S023700, C536S023710, C536S024100, C536S023100, C800S278000, C800S279000, C800S288000, C800S298000, C800S301000, C800S302000
Reexamination Certificate
active
06204057
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to isolated polynucleotides and the proteins encoded thereby and to their use in controlling lamellicorn beetles (Scarabaeidae). In addition, the invention relates to a method of producing those proteins.
BACKGROUND OF THE INVENTION
Lamellicorn beetles, such as the cockchafer (
Melolontha melolontha
) and the wood chafer (
Melolontha hippocastanl
), and especially their larvae (grubs) can cause serious damage to crops in agriculture and forestry. Since their control by means of chemical insecticides is difficult and environmentally harmful, attempts are increasingly being made to control the reproduction and spread of those insects using biological means. For example, EP 633 936 A1 describes a method in which vegetative cells, spores or protein crystals of certain strains of
Bacillus thuringiensis
are used to control lamellicorn beetles. The effectiveness of that method is not satisfactory, however.
WO 87 05928 proposes the use of
Bacillus popilliae
(
B. popilliae
) spores in the biological control of scarabaeid larvae.
B. popilliae
is the causative organism of so-called milky disease in larvae of may bugs and other lamellicorn beetles. The larvae infested with the bacillus have high concentrations of vegetative cells and sporangia of
B. popilliae
in their haemolymph, which result in a milky-white discolouration of the grub.
B. popilliae
was first described by Dutky as a cause of milky disease in the Japanese beetle (
Popillia japonica
) in the U.S.A. (in:
Journal of Agriculftural Research
61 (1940) pages 57-68, “Two new sporeforming bacteria causing milky disease of the Japanese beetle”) and was later identified by Hurpin and Vago and by Wille also in grubs of the cockchafer (
Melolontha melolontha
) (B. Hurpin and C. Vago in:
Entomophaga
3 (1958) pages 285-330, “Les maladies du hanneton commun (
Melolontha melolontha
L. (Col., Scarabaeidae)”; H. Wille in:
Mitteilungen. Schweizerische Entomologische Gesellschaft
29 (1956) pages 271-282:
“Bacillus fribourgensis
n. sp., Erreger einer “milky disease” im Engerling von
Melolontha melolontha
L.”).
Characteristics of the
B. popilliae
bacterium are inter alia that it does not form catalase and that most isolates are resistant to the antibiotic vancomycin and during sporulation form a distinctive protein crystal which is arranged inside the spindle-shaped sporangium next to the actual spore.
In its capacity as a pathogen for scarabaeids,
B. popilliae
has a high degree of specificity. The
B. popilliae
subspecies isolated from different species of scarabaeids differ in some cases considerably in their growth characteristics, in the composition of the protein crystal and in their plasmids.
The infestation of beetle larvae with
B. popilliae
is effected by peroral ingestion of the sporangia. The spores germinate in the gut of the larvae and the vegetative bacterial cells penetrate through the gut epithelium and the basal membrane into the haemolymph where they multiply during the subsequent three to four weeks. The
B. popilliae
cells then sporulate, which ultimately leads to the death of the beetle larva.
In contrast to other Bacillus species, however, under in vitro conditions
B. popilliae
forms predominantly vegetative cells and forms spores only exceptionally. Although WO 87 05 928 describes a method of obtaining the spores in vitro, in which the vegetative cells of
B. popilliae
are cultured in a defined medium and are finally stimulated to sporulate by the addition of a specific adjuvant, that method achieves a sporulation rate of only about 80%. In order to obtain quantities of infectious spore material sufficient for biological control it is therefore necessary to invest considerable resources in terms of equipment, personnel and financial expenditure, which make that method economically unviable and therefore unsuitable for practical purposes.
The aim of the present invention is therefore to provide biological means for controlling scarabaeids that enable those pests to be controlled satisfactorily and that are technically simple and economical to produce also on a large scale. Furthermore, means whose application minimizes the burden on the environment are desirable.
SUMMARY OF THE INVENTION
The present invention addresses the aforementioned needs by providing novel polynucleotide sequences isolated from
B. popilliae
. The proteins encoded by the novel polynucleotide sequences are active against lamellicorn beetles (Scarabaeidae) and can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.
Hence, in one embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a
B. popilliae
crystal protein. In another embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a
B. popilliae
crystal protein that comprises the amino acid sequence set forth in SEQ ID NO:2. In a particularly preferred embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a
B. popilliae
crystal protein, wherein the nucleotide sequence is SEQ ID NO:1.
The present invention is also directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an insecticidal protein comprising an amino acid sequence that is the translation product of a nucleic acid sequence whose complement hybridizes to SEQ ID NO:1 under hybridization conditions of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4
, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or under hybridization conditions of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4
, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.
The present invention is further directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an insecticidal protein, wherein said nucleotide sequence has a complement that hybridizes to a SEQ ID NO:1 under hybridization conditions of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4
, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or under hybridization conditions of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4
, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.
The present invention is still further directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an insecticidal protein, wherein said nucleotide sequence comprises 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 SEQ ID NO:1.
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 transgenic host cell comprising such a chimeric gene. A transgenic host cell according to this aspect of the invention may be a transgenic bacterial cell, a transgenic yeast cell, or a transgenic plant cell, preferably a transgenic plant cell. Even further, the present invention provides a transgenic plant comprising such a transgenic plant cell. Preferably, the transgenic plant is one of the following agronomically important crops: maize, rice, wheat, barley, rye, rape, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugarcane. The present invention also provides seed of such a transgenic plant.
Krieger Lutz
Schnetter Wolfgang
Zhang Jiambing
Bui Phuong T.
Meigs J. Timothy
Syngenta Participations (AG)
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