CA2594294C - Plant-optimized genes encoding pesticidal toxins - Google Patents
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Abstract
The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimized, polynucleotide sequences that encode pesticidal toxins (full-length and truncated). 'Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transfer plants in order to confer pest resistance upon said plants. The subject invention further provides plant-optimized, polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. In addition, the subject invention provides preferred amino acid sequences that are encoded by the plant-optimized polynucleotide sequences disclosed herein.
Description
DEMANDES OU BREVETS VOLUMINEUX
LA PRtSENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.
THIS IS VOLUME OF _2 NOTE: For additional volumes please contact the Canadian Patent Office.
l DESCRIPTION
PLANT-OPTIMIZED GENES ENCODING
PESTICIDAL TOXINS
Backo ound of the Invention Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Chemical pesticides have provided an effective method of pest control:
however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being inereasingly scrutinized, and correctly so, for their potential toxic environmental consequences.
Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result oftunoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed.
Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb. and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates.
Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.
Because of the problems associated with the use of synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control I
agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment.
A biological pesticidal agent that is enjoying increasing populanty is the soil microbe Bacillus thuringiensis (B.t_). The soil microbe Bacillus thuringiensis (B.t. ) is a Gram-positive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity. Some B.I.
strains produce, and can be characterized by, parasporal crystalline protein inclusions. These "d-endotoxins," which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highlv toxic to pests and are specific in their toxic activity.
Preparations of the spores and crystals of B. tlturingiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example.
B_ t/zuringiensis var. kurstaki HD- I produces a crystalline 6-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
The cloning and expression of a B.r. crystai protein gene in Escizerichia coii was described in the pubiished literature more than 15 vears ago (Schnepf, H.E..
H.R. Whiteley [1981] Proc. ,Vatl..4cad. Sci. USA 78:2893-2897.). U.S. Patent No. 4,448,885 and U.S. Patent No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coti.
Recombinant DNA-based B.t. products have been produced and approved for use.
Commercial use of B.I. pesticides was originally restricted to a narrow range of lepidopteran (caterpillar) pests. More recently, however, investigators have discovered B.t.
pesticides with specificities for a much broader range of pests_ For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.I. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectivelv (Gaertner, F.H. [1989) "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms." in Controlled Delivery of Crop Protection Agents, R.M.
Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255).
New subspecies of B.I. have now been identified, and genes responsible for active 6-endotoxin proteins have been isolated and sequenced (Hofte, H., H.R. Whiteley [1989]
Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t.
crvstal protein genes into four major classes. The classes were crvI (Lepidoptera-specific), crvll (Lepidoptera-and Diptera-specific), cryIII (Coleoptera-specific), and crvIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J.S., J. Pavne, L. Kim [1992] Bio/Technolo,gy 10:271-275). For example, the designations CryV and CryVi have been proposed for two new groups of nematode-active toxins.
Many Bacillus thuringiensis 6-endotoxin crystal protein molecules are composed of two functional segments. For these proteins, the protease-resistant core toxin is the first segment and corresponds to about the first half of the protein molecule. The three-dimensional structure of a core segment of a CryllIA B.t. S-endotoxin is known, and it was proposed that all related toxins have that same overall structure (Li, J., J. Carroll, D.J. Ellar [19911 Nature 353:81 5-821).
The second half of the molecule is often referred to as the "protoxin segment." The protoxin segment is believed to participate in toxin crystal formation (Arvidson, H., P.E. Dunn, S. Strand, A.I. Aronson [1989] iVfolecularMicrodiology 3:1533-1534; Choma, C.T., W.K.
Surewicz, P.R.
Carey, M. Pozsgay, T. Raynor, H. Kaplan [1990] Eur. J. Biochem. 189:523-527).
The :ull 130 kDa toxin molecule is typically processed to the resistant core segment by proteases in the insect gut. The protoxin segment may thus convey a partial insect specificity for the toxin by limiting the accessibility of the core to the insect bv reducing the protease processing of the toxin molecule (Haider, M.Z., B.H. Knowles. D.J. Ellar [1986] Eur. J. Biochem.
156:531-540) or by reducing toxin solubility (Aronson, A.L. E.S. Han. W. McGaughey, D. Johnson [
1991 ].-lppl.
Environ. Microbiol. 57:981-986).
The 1989 nomenclature and classification scheme of Hofte and Whiteiey was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. The number of sequenced Bacillus thuringiensis crystal protein genes currently stands at more than 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Criclanore et al. [ 19961 Society for lnvertebrate Pathology, 29th Annual Meeting, IIIrd International Colloquium on Bacillus thuringiensis, University of Cordoba.
Cordoba. Spain, September 1-6, 1996, abstract). The mnemonic "cry" has been retained for all of the toxin genes except cvtA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, although a number have been reclassified.
With the use of genetic engineering techniques, new approaches for delivering B.t.
toxins to agricultural environments are under development, including the use of plants genetically engineered with B.t. toxin genes for insect resistance and the use of stabilized, microbial cells as delivery vehicles of B.t. toxins (Gaertner, F.H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B_t. endotoxin genes are becoming commercially valuable.
Vanous improvements have been achieved by modifying B.I. toxins and/or their genes.
For example, U.S. Patent Nos. 5,380,831 and 5.567,862 relate to the production of synthetic insecticidal crystal protein genes having improved expression in plants.
Obstacles to the successful agricultural use of B.t. toxins include the deveioprnent of resistance to B.t. toxins by insects. In addition, certain insects can be refractory to the effects of B.t. The latter includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.I.
S-endotoxins.
Thus, resistance management strategies in B.t. plant technology have become of great interest, and there remains a great need for new toxin genes. As a result of extensive research and resource investment, other patents have issued for new B.t. isolates, toxins, and genes, and for new uses of B_t. isolates. See Feitelson el al., supra, for a review.
Additional examples include the following:
B.t. Isolate, Toxin, and/or Exemplified Pesticidal U.S. Patent Lo. (unless Gene Activitv of Toxin otherwise indicated) PS81I, 81IA, 8l IB2 lepidopteran 5,126,133; 5,188.960 CrylAc lepidopteran Adang et al.. GENBANK Acc.
No. M 11068 IC / IA(b) chimeric toxin lepidopteran 5.593,881 IF / IA(b) chimeric toxin lepidopteran 5,527,883 PS 158C, 158C2c lepidopteran 5,268,172; 5,723,758 PS31 G 1, 31 G 1 a lepidopteran WO 98/00546 (published PCT
application) However, the discovery of new B.t. isolates and new uses of known B.t.
isolates remains an empirical, unpredictable art.
There remains a great need for new toxin genes that can be successfully expressed at adequate levels in plants in a manner that will result in the effective control of insects and other pests.
Brief Summary of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimized poiynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform plants in order to confer pest resistance upon said plants.
In one preferred embodiment, the subject invention provides plant-optimized genes that 5 encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, 1 AC-TB-PO, 1 AC-TBX-PO, 1 C-T-PO, l C I AB-PO, 15 8C2c-PO, 15 8C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide sequences which encode Cry 1 F toxins that are active against lepidopteran insects. These polynucleotide sequences include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimized protoxins are designated PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for pesticidal toxins. These toxins are encoded by the genes designated 1F1AB-PO; 1F-T-PO, 1F-7G-PO, and 1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO; 15 8C2c-PO;
158C2c-T-PO; and 31 G 1 a-T-PO. Furthermore, the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-IAB-PO and PT-lAB-2-PO.
Brief Description of the Sequences SEQ ID NO. 1 is a polynucleotide sequence for a full-length, plant-optimized crylFlcrylA(b) hybrid gene designated 1 F 1 AB-PO.
SEQ ID NO. 2 is an amino acid sequence for a full-length, plant-optimized CryIF/CryIA(b) chimeric toxin. The 1F1AB-PO gene encodes this toxin.
SEQ ID NO. 3 is a polynucleotide sequence for a truncated, plant-optimized crylF gene designated IF-T-PO.
SEQ ID NO. 4 is an amino acid sequence for a truncated, plant-optimized CryIF
toxin.
The genes designated 1F-T-PO, 1F-7G-PO, and 1F-7Z-PO encode this toxin.
SEQ ID NO. 5 is the native polynucleotide sequence of the wild-type, full length B.t.
toxin gene designated 81 IA (crylF).
SEQ ID NO. 6 is the amino acid sequence of the full length, wild-type B.t.
toxin designated 81 IA (Cry1F).
SEQ ID NO. 7 is a polynucleotide sequence for a gene designated 1 F-7G-PO, which is optimized for expression in cotton.
SEQ ID NO. 8 is a polynucleotide sequence for a gene designated 1 F-7Z-PO, which is optimized for expression in maize.
SEQ ID NO. 9 is a polynucleotide sequence designated PT-1 AB-PO, wiuch is optimized for expression in plants. This gene, which encodes a Cry 1Ab protoxin portion, can be used in conjunction with truncated genes (genes encoding truncated, core toxins) to make full-length toxins. Unless otherwise indicated, the chimeric genes exemplified herein are shown with this polynucleotide sequence (PT-1 AB-PO).
SEQ ID NO. 10 is a polynucleotide sequence designated PT-IAB-2-PO, which is optimized for expression in cotton. This polvnucleotide sequence is an alternative to PT-1 AB-PO (and also encodes a CrylAb protoxin portion) and can also be used in conjunction with uvncated genes (genes encoding truncated, core toxins) to make full-length toxins. PT- i AB-?-PO is preferred for use in a host that is transformed with more than one twe of endotoxin transgene.
SEQ ID NO. 11 is an amino acid sequence of a protoxin portion encoded by the genes designated PT-IAB-PO and PT-lAB-2-PO.
SEQ ID NO. 12 is a polynucleotide sequence for a gene designated 1 AC 1.-a.B-N-PO, which is optimized for expression in plants. This gene encodes a chimenc Cry 1 Ac ( N-terminal) : Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 13 is a polynucleotide sequence for a gene designated I AC I AB-PO, which is optimized for expression in plants_ This gene encodes a chimenc CrvlAc (N-terminal) %
Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 14 is a polynucleotide sequence for a gene designated 1 AC 1 AB-B-PO, which is optimized for expression in plants. This gene encodes a chimeric Cry I Ac (N-terminal) ! Cry I Ab (protoxin) toxin.
SEQ ID NO. 15 is an amino acid sequence of a toxin encoded by the genes designated I AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO.
SEQ ID NO. 16 is a polynucleotide sequence for a gene designated 1 AC-T-PO, which is optimized for expression in plants. This plant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry lAc toxin described by Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 17 is a polynucleotide sequence for a gene designated I AC-TB-PO, which is optimized for expression in plants. This piant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry I Ac toxin descnbed by Adang et al, in GENBAINK (Acc. No. M 11068)_ SEQ ID NO. 18 is an alternative polynucleotide sequence for a gene designated I AC-TBX-PO, which is optimized for expression in plants. This plant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry 1 Ac toxin described by Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 19 is a polynucleotide sequence, optimized for expression in dicots, for a gene designated 1 C-T-PO, which encodes the truncated form of a Cry1 C toxin designated 811B2 in U.S. Patent No. 5,246,852.
SEQ ID NO. 20 is a polynucleotide sequence for a gene designated 1 C 1 AB-PO, which is optimized for expression in plants. This gene encodes a chimeric Crv1C (N-terminal) i Cry 1Ab (protoxin) toxin.
SEQ ID NO. 21 is an amino acid sequence of a toxin encoded by the gene designated 1 C 1 AB-PO.
SEQ LD NO. 22 is a polynucleotide sequence for a gene designated 158C2c-PO.
SEQ ID'1O. 23 is an amino acid sequence for a full-length toxin encoded bv the ;ene desimated 158C2c-PO.
SEQ ID NO. 24 is a polynucleotide sequence for a gene designated 158C2c-T'-PO.
SEQ LD NO. 25 is an amino acid sequence for a truncated toxin encoded by the gene designated 158C2c-T-PO.
SEQ ID NO. 26 is a poiynucleotide sequence for a gene designated 31 G I a-T-PO, which is optimized for expression in maize.
SEQ m NO. 27 is an amino acid sequence for a truncated toxin encoded by the gene desienated 31 G 1 a-T-PO.
Detailed Disclosure of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimized polynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform ptants in order to confer pest resistance upon said plants.
In one preferred embodiment, the subject invention provides plant-optimized genes that encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1 AC 1 AB-N-PO, 1 AC I AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, IAC-TB-PO, IAC-TBX-PO, IC-T-PO, 1 C 1 AB-PO, 158C2c-PO, 158C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide sequences which encode Cryl F toxins that are active against lepidopteran insects. These polynucleotide sequences include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimized protoxins are designated PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for pesticidal toxins. These toxins are encoded by the genes designated 1 F 1 AB-PO; 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO;
158C2c-PO;
158C2c-T-PO; and 31GIa-T-PO. Furthermore, the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-IAB-PO and PT-1AB-2-PO.
In one embodiment the subject invention provides genes which express a CryIF
toxin that is truncated compared to the full length CryIF toxin. The truncated toxins of the subject invention are typically missing all or a portion of the protoxin segment. Also, the truncated genes of the subject invention can be used for the production of fusion (or chimeric) genes and proteins. One example is the plant-optimized gene comprising a crylF portion and a cryIA(b) portion, wherein the hybrid gene encodes a chimeric toxin. In a preferred embodiment, the CryIF
portion of the chimeric toxin is itself pesticidal.
More specifically, one example of a chimeric DNA molecule of the subject invention is shown in SEQ IF NO. 1, which as a crylF 5' portion and a 3' crylA(b) portion of the DNA
molecule. The chimeric toxin encoded by SEQ IF NO. I is shown in SEQ ID NO. 2.
The chimeric toxin encoded by SEQ ID NO. 1 comprises a Cryl F core toxin comprising approximately the first 605 amino acids encoded by the nucleotides from approximately 1 to approximately 1815. This chimeric gene also comprises a crylAb protoxin portion, which encodes amino acids from approximately 606 to approximately 1148. The CrylAb protoxin portion is encoded by the nucleotides from approximately 1816 to approximately 3444.
The sequence of a preferred, truncated crylF gene of the subject invention (1815 nucleotides) is shown in SEQ ID NO. 3. This truncated gene corresponds to nucleotides 1-1815 of the chimenc gene of SEQ ID NO. 1. A stop codon, such as TAA or TAG, can be added to this sequence at positions 1816-1818. for example, if the use of a truncated toxin, without a protoxin portion, is desired. Other polvnucleotide sequences and genes of the subject invention can be similarly modified, as would be recognized by one skilled in the art.
The synthetic, truncated Cry iF toxin (encoded by SEQ ID NO. 3) is shown in SEQ ID NO. 4.
As can be seen by comparing, for example. SEQ ID NOS. l and 2 with SEQ ID NOS.
3 and 4, and .vith SEQ ID NOS. 9 and 10, there can be some overlap between the sequences for the "truncated genes" and the sequences for the "protoxin portions"
exemplified herein.
PT-IAB-PO can be used in preferred embodiments in combination with other truncated oenes of the subject invention, such as the 1 C-T-PO gene, in order to form other hybrid genes that encode full-length toxins. PT-1 AB-2-PO (an altemative polynucleotide sequence that encodes a protoxin portion) can also be used with truncated genes (which are smailer than full-length toxin genes, so long as the protein encoded bv the truncated gene retains pesticidal activity) to encode chimeric or hybrid toxins. Preferred uses of PT-lAB-2-PO
are described above in the section entitled "Description of the Sequences."
Using techniques such as computer- or software-assisted sequence alinments, differences can be noted in the nucleotide sequence of the subject plant-optimized "enes as compared to the wild-type genes or to previously lmown genes. For example, SEQ
ID NO. I
or SEQ ID NO 3 can be compared to SEQ ID NO. 5, which is the 3522-basepair, wild-type crvlF
gene. Similarly, differences in the unique amino acid sequences of the subject invention can be noted as compared to wild-type toxins or to previously known toxins.
It should be apparent to a person skilled in this art that, given the sequences of the genes as set forth herein, the genes of the subject invention can be obtained through several means.
In preferred embodiments, the subject genes may be constructed synthetically by using a gene synthesizer, for example. The specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by point-mutation techniques) from certain isolates deposited at a culture depository as discussed below. For example, a wild-type crvIF gene can be obtained from B.t. isolate PS81I. Likewise, the crylA(b) portions of the hybrid genes of the subject invention can be produced synthetically or can be derived by modifying wild-type genes.
CryIA(b) toxins and genes have been described in, for example, H6fte et al. (1986) Eur. J.
Biochem. 161:273; Geiser et al. (1986) Gene 48:109; and Haider et al. (1988) Nucleic,4cids Res_ 16:10927. Clones and additional wild-type isolates are discussed in more detail, above, in the section entitled "Background of the Invention" and in the list, below.
Cultures discussed in this application have becn deposited in the Agricultural IZcsearch Service Patent Culture Collection (NRRL), Nortllern Regional Research Center, 1815 North University Street, Peona, Illinois 61604, USA. "I'he deposited strains listed below are disclosed in the patent references as discussed above in the section entitled "Background of the lnventron."
Subculture Accession Number Deposit Date B. PS81I NRRL B-18484 April 19. 1989 E. coli (NM522) (pMYC1603) (81IA) NRRI. B-185 17 June 30, 1989 10 E. coli (NM522) (pMYC394) (81IB22) NRRL B-18500 iMav 17. 1989 B.t. PS158C2 NRRL B-18872 Aug. 27, I991 E. coli (NM522) (pMYC2383) (158C2c) NRRI. B-21428 April 11, 1995 B_t. PS31G1 NRRL B-21560 April 18, 1996 E. coli (N'Vf522) (pMYC2454) c.:lGlal NRRL B-21796N Sept. 30, 1997 It should be understood that the availabilitv of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by govesnmental action.
Genes and toxins. The polvnucleotides of the subject invention can be used to form complete "genes" to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, the polynucleotides of the subject invention are shown without stop codons. Also, the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest. as is readily lmown in the art.
As the skilled artisan would readily recognize, DNA can exist in a double-stranded form.
In this arrangement, one strand is complementary to the other strand and vice versa. "I tle "coding strand" is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest.
In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA
which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exeniplified DNA are included in the subject invention.
Certain DNA sequences of the subject rnverltion have been specifically exerriplified iierein_ These sequences are exemplary of the subject inventron. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof(such as mutants, fusions, chimenes, truncations, fragments, and smaller genes) that exhibit the same or similar charactenstics relating to expressing toxins in plants, as compared to those specifically disclosed herein. As used herein, "variants" and "equivalents" refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity, ;n plants. Fragments retaining pesticidal activity are also included in this deftnitiori. Thus, polynucleotides that are smaller than those specifically exemplified are included in the subject invention, so long as the polvnucleotide encodes a pesticidal toxin.
Genes can be modified, and variations of genes may be readilv constructed, usin"
standard techniques. For example, techniques for malcing point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used accordinc, to standard procedures, and enzymes such as Ba131 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.
It shouid be noted that equivalent genes will encode toxins that have high amino acid identity or homology with the toxins encoded by the subject genes. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table I provides a listing of examples of amino acids belonging to each class.
Table 1.
Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Vat, Leu. Ile, Pro, Met. Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Asp, Glu Basic Lys, Arg, His In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the biological activity of the toxin.
As used herein, reference to "isolated" polynucleotides and / or "puritied"
toxins refers to these molecuies when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to "isolated and purified" signifies the involvement of the "hand of man" as described herein.
Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preiiminary steps for preparing precursors, for example, that will eventually be used to transform. in preferred embodiments, plant cells and plants so that they express the toxins encoded by the genes of the subject invention. Microbes transformed and used in this manner are within the scope of the subject invennon. Recombinant microbes may be, for exampie, B.t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques_ Materials necessary for these transformations are disclosed herein or are otherwise readily available to the slcilled artisan.
Thus, in preferred embodiments, expression of the toxin gene results, directly or indirectiy, in the intracellular production and maintenance of the pesticide.
When transformed plants are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest.
The B.t. toxin gene can be introduced via a suitable vector into a host, preferably a plant host. There are many crops of interest, such as corn, wheat, rice, cotton, soybeans, and sunflowers. The genes of the subject invention are particulariv well suited for providing stable maintenance and expression, in the transfocmed plant, of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
While the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,83 1.
Following is an example which illustrates procedures for practicing the invention. This example should not be construed as limiting.
Example l- Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with the subject polynucleotide sequences encoding insecticidal toxins. The transformed plants are resistant to attack by the target pest. The genes of the subject invention are optimized for use in plants.
Obviously, a promoter region capable of expressing the gene in a plant is needed. Thus, for in planta expression, the DNA of the subject invention is under the control of an appropriate promoter region. Techniques for obtaining in planta expression by using such constructs is known in the art.
Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well 4atown in the art. For example, a large number of cloning vectors compnsing a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M l 3mp senes.
pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli.
The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis. and other biochemical-molecular biological methods are generally carried out as methods of analysis.
After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA
sequence. Each plasmid sequence can be cloned in the same or other plasmids.
Depending on the method of inserting desired genes into the plant, other DNA
sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transfot7nation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensiveiy researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Planr Vector Sysieni, Offset-durkkenj Kanters B.V., Aiblasserdam, Chapter 5; Fraley et al., (1986) Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) FAIBO J. 4:277-287.
Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamvcin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallv emploved marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
A large number of techniques are available for inserting DNA into a plant host cell.
Those techniques include transformation with T-DNA using 4grobacteriuni tunzefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cioned into special piasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA_ The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cartnot repiicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tinnefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polvlinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [ 1978] rLlol. Gen. Genet. 163:181-187). The Agrobacteriunl used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium rumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant ceil. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation.
lt is possible to use ordinary plasmids, such as, for example. pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
It should be understood that the exampies and embodiments described herein are for ~ illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
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l DESCRIPTION
PLANT-OPTIMIZED GENES ENCODING
PESTICIDAL TOXINS
Backo ound of the Invention Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Chemical pesticides have provided an effective method of pest control:
however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being inereasingly scrutinized, and correctly so, for their potential toxic environmental consequences.
Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result oftunoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed.
Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb. and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates.
Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.
Because of the problems associated with the use of synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control I
agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment.
A biological pesticidal agent that is enjoying increasing populanty is the soil microbe Bacillus thuringiensis (B.t_). The soil microbe Bacillus thuringiensis (B.t. ) is a Gram-positive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity. Some B.I.
strains produce, and can be characterized by, parasporal crystalline protein inclusions. These "d-endotoxins," which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highlv toxic to pests and are specific in their toxic activity.
Preparations of the spores and crystals of B. tlturingiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example.
B_ t/zuringiensis var. kurstaki HD- I produces a crystalline 6-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
The cloning and expression of a B.r. crystai protein gene in Escizerichia coii was described in the pubiished literature more than 15 vears ago (Schnepf, H.E..
H.R. Whiteley [1981] Proc. ,Vatl..4cad. Sci. USA 78:2893-2897.). U.S. Patent No. 4,448,885 and U.S. Patent No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coti.
Recombinant DNA-based B.t. products have been produced and approved for use.
Commercial use of B.I. pesticides was originally restricted to a narrow range of lepidopteran (caterpillar) pests. More recently, however, investigators have discovered B.t.
pesticides with specificities for a much broader range of pests_ For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.I. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectivelv (Gaertner, F.H. [1989) "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms." in Controlled Delivery of Crop Protection Agents, R.M.
Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255).
New subspecies of B.I. have now been identified, and genes responsible for active 6-endotoxin proteins have been isolated and sequenced (Hofte, H., H.R. Whiteley [1989]
Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t.
crvstal protein genes into four major classes. The classes were crvI (Lepidoptera-specific), crvll (Lepidoptera-and Diptera-specific), cryIII (Coleoptera-specific), and crvIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J.S., J. Pavne, L. Kim [1992] Bio/Technolo,gy 10:271-275). For example, the designations CryV and CryVi have been proposed for two new groups of nematode-active toxins.
Many Bacillus thuringiensis 6-endotoxin crystal protein molecules are composed of two functional segments. For these proteins, the protease-resistant core toxin is the first segment and corresponds to about the first half of the protein molecule. The three-dimensional structure of a core segment of a CryllIA B.t. S-endotoxin is known, and it was proposed that all related toxins have that same overall structure (Li, J., J. Carroll, D.J. Ellar [19911 Nature 353:81 5-821).
The second half of the molecule is often referred to as the "protoxin segment." The protoxin segment is believed to participate in toxin crystal formation (Arvidson, H., P.E. Dunn, S. Strand, A.I. Aronson [1989] iVfolecularMicrodiology 3:1533-1534; Choma, C.T., W.K.
Surewicz, P.R.
Carey, M. Pozsgay, T. Raynor, H. Kaplan [1990] Eur. J. Biochem. 189:523-527).
The :ull 130 kDa toxin molecule is typically processed to the resistant core segment by proteases in the insect gut. The protoxin segment may thus convey a partial insect specificity for the toxin by limiting the accessibility of the core to the insect bv reducing the protease processing of the toxin molecule (Haider, M.Z., B.H. Knowles. D.J. Ellar [1986] Eur. J. Biochem.
156:531-540) or by reducing toxin solubility (Aronson, A.L. E.S. Han. W. McGaughey, D. Johnson [
1991 ].-lppl.
Environ. Microbiol. 57:981-986).
The 1989 nomenclature and classification scheme of Hofte and Whiteiey was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. The number of sequenced Bacillus thuringiensis crystal protein genes currently stands at more than 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Criclanore et al. [ 19961 Society for lnvertebrate Pathology, 29th Annual Meeting, IIIrd International Colloquium on Bacillus thuringiensis, University of Cordoba.
Cordoba. Spain, September 1-6, 1996, abstract). The mnemonic "cry" has been retained for all of the toxin genes except cvtA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, although a number have been reclassified.
With the use of genetic engineering techniques, new approaches for delivering B.t.
toxins to agricultural environments are under development, including the use of plants genetically engineered with B.t. toxin genes for insect resistance and the use of stabilized, microbial cells as delivery vehicles of B.t. toxins (Gaertner, F.H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B_t. endotoxin genes are becoming commercially valuable.
Vanous improvements have been achieved by modifying B.I. toxins and/or their genes.
For example, U.S. Patent Nos. 5,380,831 and 5.567,862 relate to the production of synthetic insecticidal crystal protein genes having improved expression in plants.
Obstacles to the successful agricultural use of B.t. toxins include the deveioprnent of resistance to B.t. toxins by insects. In addition, certain insects can be refractory to the effects of B.t. The latter includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.I.
S-endotoxins.
Thus, resistance management strategies in B.t. plant technology have become of great interest, and there remains a great need for new toxin genes. As a result of extensive research and resource investment, other patents have issued for new B.t. isolates, toxins, and genes, and for new uses of B_t. isolates. See Feitelson el al., supra, for a review.
Additional examples include the following:
B.t. Isolate, Toxin, and/or Exemplified Pesticidal U.S. Patent Lo. (unless Gene Activitv of Toxin otherwise indicated) PS81I, 81IA, 8l IB2 lepidopteran 5,126,133; 5,188.960 CrylAc lepidopteran Adang et al.. GENBANK Acc.
No. M 11068 IC / IA(b) chimeric toxin lepidopteran 5.593,881 IF / IA(b) chimeric toxin lepidopteran 5,527,883 PS 158C, 158C2c lepidopteran 5,268,172; 5,723,758 PS31 G 1, 31 G 1 a lepidopteran WO 98/00546 (published PCT
application) However, the discovery of new B.t. isolates and new uses of known B.t.
isolates remains an empirical, unpredictable art.
There remains a great need for new toxin genes that can be successfully expressed at adequate levels in plants in a manner that will result in the effective control of insects and other pests.
Brief Summary of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimized poiynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform plants in order to confer pest resistance upon said plants.
In one preferred embodiment, the subject invention provides plant-optimized genes that 5 encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, 1 AC-TB-PO, 1 AC-TBX-PO, 1 C-T-PO, l C I AB-PO, 15 8C2c-PO, 15 8C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide sequences which encode Cry 1 F toxins that are active against lepidopteran insects. These polynucleotide sequences include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimized protoxins are designated PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for pesticidal toxins. These toxins are encoded by the genes designated 1F1AB-PO; 1F-T-PO, 1F-7G-PO, and 1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO; 15 8C2c-PO;
158C2c-T-PO; and 31 G 1 a-T-PO. Furthermore, the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-IAB-PO and PT-lAB-2-PO.
Brief Description of the Sequences SEQ ID NO. 1 is a polynucleotide sequence for a full-length, plant-optimized crylFlcrylA(b) hybrid gene designated 1 F 1 AB-PO.
SEQ ID NO. 2 is an amino acid sequence for a full-length, plant-optimized CryIF/CryIA(b) chimeric toxin. The 1F1AB-PO gene encodes this toxin.
SEQ ID NO. 3 is a polynucleotide sequence for a truncated, plant-optimized crylF gene designated IF-T-PO.
SEQ ID NO. 4 is an amino acid sequence for a truncated, plant-optimized CryIF
toxin.
The genes designated 1F-T-PO, 1F-7G-PO, and 1F-7Z-PO encode this toxin.
SEQ ID NO. 5 is the native polynucleotide sequence of the wild-type, full length B.t.
toxin gene designated 81 IA (crylF).
SEQ ID NO. 6 is the amino acid sequence of the full length, wild-type B.t.
toxin designated 81 IA (Cry1F).
SEQ ID NO. 7 is a polynucleotide sequence for a gene designated 1 F-7G-PO, which is optimized for expression in cotton.
SEQ ID NO. 8 is a polynucleotide sequence for a gene designated 1 F-7Z-PO, which is optimized for expression in maize.
SEQ ID NO. 9 is a polynucleotide sequence designated PT-1 AB-PO, wiuch is optimized for expression in plants. This gene, which encodes a Cry 1Ab protoxin portion, can be used in conjunction with truncated genes (genes encoding truncated, core toxins) to make full-length toxins. Unless otherwise indicated, the chimeric genes exemplified herein are shown with this polynucleotide sequence (PT-1 AB-PO).
SEQ ID NO. 10 is a polynucleotide sequence designated PT-IAB-2-PO, which is optimized for expression in cotton. This polvnucleotide sequence is an alternative to PT-1 AB-PO (and also encodes a CrylAb protoxin portion) and can also be used in conjunction with uvncated genes (genes encoding truncated, core toxins) to make full-length toxins. PT- i AB-?-PO is preferred for use in a host that is transformed with more than one twe of endotoxin transgene.
SEQ ID NO. 11 is an amino acid sequence of a protoxin portion encoded by the genes designated PT-IAB-PO and PT-lAB-2-PO.
SEQ ID NO. 12 is a polynucleotide sequence for a gene designated 1 AC 1.-a.B-N-PO, which is optimized for expression in plants. This gene encodes a chimenc Cry 1 Ac ( N-terminal) : Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 13 is a polynucleotide sequence for a gene designated I AC I AB-PO, which is optimized for expression in plants_ This gene encodes a chimenc CrvlAc (N-terminal) %
Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 14 is a polynucleotide sequence for a gene designated 1 AC 1 AB-B-PO, which is optimized for expression in plants. This gene encodes a chimeric Cry I Ac (N-terminal) ! Cry I Ab (protoxin) toxin.
SEQ ID NO. 15 is an amino acid sequence of a toxin encoded by the genes designated I AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO.
SEQ ID NO. 16 is a polynucleotide sequence for a gene designated 1 AC-T-PO, which is optimized for expression in plants. This plant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry lAc toxin described by Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 17 is a polynucleotide sequence for a gene designated I AC-TB-PO, which is optimized for expression in plants. This piant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry I Ac toxin descnbed by Adang et al, in GENBAINK (Acc. No. M 11068)_ SEQ ID NO. 18 is an alternative polynucleotide sequence for a gene designated I AC-TBX-PO, which is optimized for expression in plants. This plant-optimized gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a Cry 1 Ac toxin described by Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 19 is a polynucleotide sequence, optimized for expression in dicots, for a gene designated 1 C-T-PO, which encodes the truncated form of a Cry1 C toxin designated 811B2 in U.S. Patent No. 5,246,852.
SEQ ID NO. 20 is a polynucleotide sequence for a gene designated 1 C 1 AB-PO, which is optimized for expression in plants. This gene encodes a chimeric Crv1C (N-terminal) i Cry 1Ab (protoxin) toxin.
SEQ ID NO. 21 is an amino acid sequence of a toxin encoded by the gene designated 1 C 1 AB-PO.
SEQ LD NO. 22 is a polynucleotide sequence for a gene designated 158C2c-PO.
SEQ ID'1O. 23 is an amino acid sequence for a full-length toxin encoded bv the ;ene desimated 158C2c-PO.
SEQ ID NO. 24 is a polynucleotide sequence for a gene designated 158C2c-T'-PO.
SEQ LD NO. 25 is an amino acid sequence for a truncated toxin encoded by the gene designated 158C2c-T-PO.
SEQ ID NO. 26 is a poiynucleotide sequence for a gene designated 31 G I a-T-PO, which is optimized for expression in maize.
SEQ m NO. 27 is an amino acid sequence for a truncated toxin encoded by the gene desienated 31 G 1 a-T-PO.
Detailed Disclosure of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimized polynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform ptants in order to confer pest resistance upon said plants.
In one preferred embodiment, the subject invention provides plant-optimized genes that encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1 AC 1 AB-N-PO, 1 AC I AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, IAC-TB-PO, IAC-TBX-PO, IC-T-PO, 1 C 1 AB-PO, 158C2c-PO, 158C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide sequences which encode Cryl F toxins that are active against lepidopteran insects. These polynucleotide sequences include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimized protoxins are designated PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for pesticidal toxins. These toxins are encoded by the genes designated 1 F 1 AB-PO; 1 F-T-PO, 1 F-7G-PO, and 1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO;
158C2c-PO;
158C2c-T-PO; and 31GIa-T-PO. Furthermore, the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-IAB-PO and PT-1AB-2-PO.
In one embodiment the subject invention provides genes which express a CryIF
toxin that is truncated compared to the full length CryIF toxin. The truncated toxins of the subject invention are typically missing all or a portion of the protoxin segment. Also, the truncated genes of the subject invention can be used for the production of fusion (or chimeric) genes and proteins. One example is the plant-optimized gene comprising a crylF portion and a cryIA(b) portion, wherein the hybrid gene encodes a chimeric toxin. In a preferred embodiment, the CryIF
portion of the chimeric toxin is itself pesticidal.
More specifically, one example of a chimeric DNA molecule of the subject invention is shown in SEQ IF NO. 1, which as a crylF 5' portion and a 3' crylA(b) portion of the DNA
molecule. The chimeric toxin encoded by SEQ IF NO. I is shown in SEQ ID NO. 2.
The chimeric toxin encoded by SEQ ID NO. 1 comprises a Cryl F core toxin comprising approximately the first 605 amino acids encoded by the nucleotides from approximately 1 to approximately 1815. This chimeric gene also comprises a crylAb protoxin portion, which encodes amino acids from approximately 606 to approximately 1148. The CrylAb protoxin portion is encoded by the nucleotides from approximately 1816 to approximately 3444.
The sequence of a preferred, truncated crylF gene of the subject invention (1815 nucleotides) is shown in SEQ ID NO. 3. This truncated gene corresponds to nucleotides 1-1815 of the chimenc gene of SEQ ID NO. 1. A stop codon, such as TAA or TAG, can be added to this sequence at positions 1816-1818. for example, if the use of a truncated toxin, without a protoxin portion, is desired. Other polvnucleotide sequences and genes of the subject invention can be similarly modified, as would be recognized by one skilled in the art.
The synthetic, truncated Cry iF toxin (encoded by SEQ ID NO. 3) is shown in SEQ ID NO. 4.
As can be seen by comparing, for example. SEQ ID NOS. l and 2 with SEQ ID NOS.
3 and 4, and .vith SEQ ID NOS. 9 and 10, there can be some overlap between the sequences for the "truncated genes" and the sequences for the "protoxin portions"
exemplified herein.
PT-IAB-PO can be used in preferred embodiments in combination with other truncated oenes of the subject invention, such as the 1 C-T-PO gene, in order to form other hybrid genes that encode full-length toxins. PT-1 AB-2-PO (an altemative polynucleotide sequence that encodes a protoxin portion) can also be used with truncated genes (which are smailer than full-length toxin genes, so long as the protein encoded bv the truncated gene retains pesticidal activity) to encode chimeric or hybrid toxins. Preferred uses of PT-lAB-2-PO
are described above in the section entitled "Description of the Sequences."
Using techniques such as computer- or software-assisted sequence alinments, differences can be noted in the nucleotide sequence of the subject plant-optimized "enes as compared to the wild-type genes or to previously lmown genes. For example, SEQ
ID NO. I
or SEQ ID NO 3 can be compared to SEQ ID NO. 5, which is the 3522-basepair, wild-type crvlF
gene. Similarly, differences in the unique amino acid sequences of the subject invention can be noted as compared to wild-type toxins or to previously known toxins.
It should be apparent to a person skilled in this art that, given the sequences of the genes as set forth herein, the genes of the subject invention can be obtained through several means.
In preferred embodiments, the subject genes may be constructed synthetically by using a gene synthesizer, for example. The specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by point-mutation techniques) from certain isolates deposited at a culture depository as discussed below. For example, a wild-type crvIF gene can be obtained from B.t. isolate PS81I. Likewise, the crylA(b) portions of the hybrid genes of the subject invention can be produced synthetically or can be derived by modifying wild-type genes.
CryIA(b) toxins and genes have been described in, for example, H6fte et al. (1986) Eur. J.
Biochem. 161:273; Geiser et al. (1986) Gene 48:109; and Haider et al. (1988) Nucleic,4cids Res_ 16:10927. Clones and additional wild-type isolates are discussed in more detail, above, in the section entitled "Background of the Invention" and in the list, below.
Cultures discussed in this application have becn deposited in the Agricultural IZcsearch Service Patent Culture Collection (NRRL), Nortllern Regional Research Center, 1815 North University Street, Peona, Illinois 61604, USA. "I'he deposited strains listed below are disclosed in the patent references as discussed above in the section entitled "Background of the lnventron."
Subculture Accession Number Deposit Date B. PS81I NRRL B-18484 April 19. 1989 E. coli (NM522) (pMYC1603) (81IA) NRRI. B-185 17 June 30, 1989 10 E. coli (NM522) (pMYC394) (81IB22) NRRL B-18500 iMav 17. 1989 B.t. PS158C2 NRRL B-18872 Aug. 27, I991 E. coli (NM522) (pMYC2383) (158C2c) NRRI. B-21428 April 11, 1995 B_t. PS31G1 NRRL B-21560 April 18, 1996 E. coli (N'Vf522) (pMYC2454) c.:lGlal NRRL B-21796N Sept. 30, 1997 It should be understood that the availabilitv of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by govesnmental action.
Genes and toxins. The polvnucleotides of the subject invention can be used to form complete "genes" to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, the polynucleotides of the subject invention are shown without stop codons. Also, the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest. as is readily lmown in the art.
As the skilled artisan would readily recognize, DNA can exist in a double-stranded form.
In this arrangement, one strand is complementary to the other strand and vice versa. "I tle "coding strand" is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest.
In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA
which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exeniplified DNA are included in the subject invention.
Certain DNA sequences of the subject rnverltion have been specifically exerriplified iierein_ These sequences are exemplary of the subject inventron. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof(such as mutants, fusions, chimenes, truncations, fragments, and smaller genes) that exhibit the same or similar charactenstics relating to expressing toxins in plants, as compared to those specifically disclosed herein. As used herein, "variants" and "equivalents" refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity, ;n plants. Fragments retaining pesticidal activity are also included in this deftnitiori. Thus, polynucleotides that are smaller than those specifically exemplified are included in the subject invention, so long as the polvnucleotide encodes a pesticidal toxin.
Genes can be modified, and variations of genes may be readilv constructed, usin"
standard techniques. For example, techniques for malcing point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used accordinc, to standard procedures, and enzymes such as Ba131 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.
It shouid be noted that equivalent genes will encode toxins that have high amino acid identity or homology with the toxins encoded by the subject genes. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table I provides a listing of examples of amino acids belonging to each class.
Table 1.
Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Vat, Leu. Ile, Pro, Met. Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Asp, Glu Basic Lys, Arg, His In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the biological activity of the toxin.
As used herein, reference to "isolated" polynucleotides and / or "puritied"
toxins refers to these molecuies when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to "isolated and purified" signifies the involvement of the "hand of man" as described herein.
Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preiiminary steps for preparing precursors, for example, that will eventually be used to transform. in preferred embodiments, plant cells and plants so that they express the toxins encoded by the genes of the subject invention. Microbes transformed and used in this manner are within the scope of the subject invennon. Recombinant microbes may be, for exampie, B.t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques_ Materials necessary for these transformations are disclosed herein or are otherwise readily available to the slcilled artisan.
Thus, in preferred embodiments, expression of the toxin gene results, directly or indirectiy, in the intracellular production and maintenance of the pesticide.
When transformed plants are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest.
The B.t. toxin gene can be introduced via a suitable vector into a host, preferably a plant host. There are many crops of interest, such as corn, wheat, rice, cotton, soybeans, and sunflowers. The genes of the subject invention are particulariv well suited for providing stable maintenance and expression, in the transfocmed plant, of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
While the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,83 1.
Following is an example which illustrates procedures for practicing the invention. This example should not be construed as limiting.
Example l- Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with the subject polynucleotide sequences encoding insecticidal toxins. The transformed plants are resistant to attack by the target pest. The genes of the subject invention are optimized for use in plants.
Obviously, a promoter region capable of expressing the gene in a plant is needed. Thus, for in planta expression, the DNA of the subject invention is under the control of an appropriate promoter region. Techniques for obtaining in planta expression by using such constructs is known in the art.
Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well 4atown in the art. For example, a large number of cloning vectors compnsing a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M l 3mp senes.
pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli.
The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis. and other biochemical-molecular biological methods are generally carried out as methods of analysis.
After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA
sequence. Each plasmid sequence can be cloned in the same or other plasmids.
Depending on the method of inserting desired genes into the plant, other DNA
sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transfot7nation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensiveiy researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Planr Vector Sysieni, Offset-durkkenj Kanters B.V., Aiblasserdam, Chapter 5; Fraley et al., (1986) Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) FAIBO J. 4:277-287.
Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamvcin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallv emploved marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
A large number of techniques are available for inserting DNA into a plant host cell.
Those techniques include transformation with T-DNA using 4grobacteriuni tunzefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cioned into special piasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA_ The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cartnot repiicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tinnefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polvlinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [ 1978] rLlol. Gen. Genet. 163:181-187). The Agrobacteriunl used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium rumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant ceil. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation.
lt is possible to use ordinary plasmids, such as, for example. pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
It should be understood that the exampies and embodiments described herein are for ~ illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
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Claims (8)
1. ~A polynucleotide optimized for expression in a plant cell wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 12, SEQ ID
NO. 13, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18.
NO. 13, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18.
2. ~A plant cell transformed by a polynucleotide wherein said polynucleotide is optimized for expression in a plant cell and wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 17 and SEQ ID NO. 18.
ID NO. 17 and SEQ ID NO. 18.
3. ~A DNA construct which comprises a polynucleotide optimized for expression in a plant cell wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18.
4. ~A method of controlling a pest wherein said method comprises contacting said pest with a toxin produced by a transformed plant cell wherein said plant cell is transformed with a polynucleotide optimized for expression in a plant wherein said polynucleotide is selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ
ID NO. 17 and SEQ ID NO. 18.
NO. 16, SEQ
ID NO. 17 and SEQ ID NO. 18.
5. ~A polynucleotide optimized for expression in a plant cell wherein said polynucleotide is SEQ ID NO. 14.
6. ~A plant cell transformed by a polynucleotide wherein said polynucleotide is optimized for expression in a plant cell and wherein said polynucleotide is SEQ ID NO. 14.
7. ~A DNA construct which comprises a polynucleotide optimized for expression in a plant cell wherein said polynucleotide is SEQ ID NO. 14.
8. ~A method of controlling a pest wherein said method comprises contacting said pest with a toxin produced by a transformed plant cell wherein said plant cell is transformed with a polynucleotide optimized for expression in a plant wherein said polynucleotide is SEQ ID NO.
14.
14.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6521597P | 1997-11-12 | 1997-11-12 | |
| US60/065,215 | 1997-11-12 | ||
| US7644598P | 1998-03-02 | 1998-03-02 | |
| US60/076,445 | 1998-03-02 | ||
| US09/178,252 | 1998-10-23 | ||
| US09/178,252 US6218188B1 (en) | 1997-11-12 | 1998-10-23 | Plant-optimized genes encoding pesticidal toxins |
| CA002309131A CA2309131C (en) | 1997-11-12 | 1998-11-04 | Plant-optimized genes encoding pesticidal toxins |
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| CA002309131A Division CA2309131C (en) | 1997-11-12 | 1998-11-04 | Plant-optimized genes encoding pesticidal toxins |
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| CA2594294C true CA2594294C (en) | 2011-01-18 |
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