Cotton cells, plants, and seeds genetically engineered to...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance

Reexamination Certificate

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C800S314000, C800S278000, C800S298000, C800S295000, C800S301000, C800S302000, C435S069100, C435S468000, C435S419000, C536S023100, C536S023600

Reexamination Certificate

active

06710228

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to chimeric genes that express in cotton cells, plants and seeds, and encode insecticides and fungicides having substantially the insect toxicity and fungal toxicity of barley, nettle, and hevein lectins.
BACKGROUND OF THE INVENTION
Chitin-binding proteins (lectins) are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. They are believed to be defense-related, and many exhibit insecticidal and/or anti-fungal activities (Murdock et. al., 1990; Lerner, D. R. and Raikhel, N. V., 1992). Lectins exhibit specific carbohydrate-binding properties. Lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine in susceptible pest species (Schroeder, M. R. and Raikhel, N. V. 1992).
In purified form, barley, nettle, and hevein lectins have shown insecticidal and fungicidal activity against certain species of pests which are known to attack cotton (for example, Heliothis and Fusarium). Various methods are available to utilize lectins to control such pests, but all require providing these proteins sufficiently pure and in sufficient quantity to effect control of the target insect or pathogen. Even when available in sufficient purity or quantity, they must be applied to the crop in such a way so as to effectively reach the target species. Furthermore, because they are proteins, if topically applied to crops they are subject to light and protease inactivation before they can exert their controlling effect. Root associated pathogens are not readily treated with such preparations. Hence, lectins have not been available for use in controlling many serious pests of cotton, even though they might be effective were they available in pure enough and high enough concentrations.
By taking advantage of genetic engineering, a gene responsible for the production of a useful polypeptide can be transferred from a donor cell, in which the gene naturally occurs, to a host cell, in which the gene does not naturally occur; Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464. There are, in fact, few inherent limits to such transfers. Genes can be transferred between viruses, bacteria, plants, and animals. In some cases, the transferred gene is functional, or can be made to be functional, in the host cell. When the host cell is a plant cell, whole plants can sometimes be regenerated from the cell.
Genes typically contain regions of DNA sequences including a promoter and a transcribed region. The transcribed region normally contains a 5′ untranslated region, a coding sequence, and a 3′ untranslated region.
The promoter contains the DNA sequence necessary for the initiation of transcription, during which the transcribed region is converted into mRNA. In eukaryotic cells, the promoter is believed to include a region recognized by RNA polymerase and a region which positions the RNA polymerase on the DNA for the initiation of transcription. This latter region, which is referred to as the TATA box, usually occurs about 30 nucleotides upstream from the site of transcription initiation.
Following the promoter region is a sequence that is transcribed into mRNA but is not translated into polypeptide. This sequence constitutes the so-called 5′ untranslated region and is believed to contain sequences that are responsible for the initiation of translation, such as a ribosome binding site.
The coding region is the sequence that is just downstream from the 5′ untranslated region in the DNA or the corresponding RNA. It is the coding region that is translated into polypeptides in accordance with the genetic code.
Bacillus thuringiensis
, for example, has a gene with a coding sequence that translates into the amino acid sequence of an insecticidal crystal protein.
The coding region is followed by a sequence that is transcribed into mRNA, but is not translated into polypeptide. This sequence is called the 3′ untranslated region and is believed to contain a signal that leads to the termination of transcription and, in eukaryotic mRNA, a signal that causes polyadenylation of the transcribed mRNA strand. Polyadenylation of the mRNA is believed to have processing and transportation functions.
Natural genes can be transferred in their entirety from a donor cell to a host cell. It is often preferable, however, to construct a gene containing the desired coding region with a promoter and, optionally, 5′ and 3′ untranslated regions that do not, in nature, exist in the same gene as the coding region. Such constructs are known as chimeric genes.
Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole (Bednarek, S. Y., and Raikhel, N. V., 1991). The glycosylated carboxyl-terminal propeptide (CTPP) is removed before or concomitant with the deposition of the mature, active protein in vacuoles (Bednarek, et al., 1990). Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides (Wilkins, T. A., Bednarek, S. Y. and Raikhel, S. V., 1990). The nucleotide sequence and deduced amino acid sequence of the barley lectin coding region (barley lectin cDNA clone BLc3) has been reported (see Lemer and Raikhel, 1989; and U.S. Pat. No. 5,276,269, incorporated herein by reference). A chimeric gene construct was created by fusing the BLc3 coding region to the CaMV 35S promoter, and transferring the chimeric gene construct into tobacco plants via
Agrobacterium tumefaciens
mediated transformation (U.S. Pat. No. 5,276,269). Plants were reported to exhibit insecticidal and fungicidal properties.
A full length cDNA clone (HEV1) encoding
Hevea brasiliensis
lectin was isolated from a
H. brasiliensis
latex cDNA library, sequenced, and characterized (see Broekaert et al., 1990; Lee et al., 1991; and U.S. Pat. No. 5,187,262, incorporated herein by reference). Briefly, HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region of 43 amino acids is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. Northern blots, using HEV1 cDNA as a probe, showed that the gene is induced by wounding and the plant hormones abscisic acid and ethylene. Accumulation of these transcripts was seen in leaves, stems, and latex, but not in roots. Chimeric gene constructs fusing the hevein coding region with heterologous promoters were not reported. However, tests with hevein protein showed antifungal activity against Trichoderma, Phycomyces, Botrytis, Septoria, Pyricularia, and Fusarium. The observed activities differed from those of wheat germ aglutinin (another lectin). Furthermore, hevein anti-fungal activity was found to be stable even after heating to 90° C., a condition under which certain chitinase activities are completely destroyed.
A full length cDNA encoding the nettle lectin (Urtica dioica agglutinin) has been cloned, sequenced, and characterized (Lerner and Raikhel, 1992). The protein is made up of 374 amino acids. 21 are a putative signal sequence and 86 amino acids encode the two chitin-binding domains of nettle lectin. These are fused to a 19 amino acid “spacer” domain and a 244 amino acid carboxyl extension with partial identity to a chitinase catalytic domain.
This gene represents another lectin heretofore unavailable as a source for resistance to important cotton insect and fungal pathogens.
The studies noted above underscore the complexity of the biochemistry of plant lectins. These are proteins which must be processed properly and transported into the proper subcellular compartment, usually a vacuole, where they are stored. In order to make use of these proteins in combating cotton pests, o

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