Title of Invention

"A RECOMBINANT NUCLEIC ACID MOLECULE"

Abstract Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a delta-endotoxin and delta-endotoxin-associated polypeptides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated delta-endotoxin and delta-endotoxin-associated nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in in SEQ ID NOS:3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 27, and 29, and the nucleotide sequences set forth in SEQ ID NOS:l, 2, 4, 6, 8, 10, 12, 13, 15, 17, 19, 21, 23, 25, 26, and 28, as well as variants and fragments thereof.
Full Text DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE
FIELD OF THE INVENTION
This invention relates to the field of molecular biology. Provided are novel
genes that encode pesticidal proteins. These proteins and the nucleic acid sequences
that encode them are useful in preparing pesticidal formulations and in the production
of transgenic pest-resistant plants.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis is a Gram-positive spore forming soil bacterium
characterized by its ability to produce crystalline inclusions that are specifically toxic
to certain orders and species of insects, but are harmless to plants and other nontargeted
organisms. For this reason, compositions including Bacillus thuringiensis
strains or their insecticidal proteins can be used as environmentally acceptable
insecticides to control agricultural insect pests or insect vectors for a variety of human
or animal diseases.
Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have
potent insecticidal activity against predominantly Lepidopteran, Dipteran, and
Coleopteran larvae. These proteins also have shown activity against Hymenoptera,
Homoptera, Phthiraptera, Mallophaga, and Acari pest orders, as well as other
invertebrate orders such as Nemathelminthes, Platyhelminthes, and
Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensis family tree. In
Advanced Engineered Pesticides. Marcel Dekker, Inc., New York, N.Y.) These
proteins were originally classified as Cryl to CryV based primarily on their
insecticidal activity. The major classes were Lepidoptera-specific (I), Lepidopteraand
Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and
nematode-specific (V) and (VI). The proteins were further classified into subfamilies;
more highly related proteins within each family were assigned divisional letters such
as Cryl A, Cry IB, Cry 1C, etc. Even more closely related proteins within each
division were given names such as CrylCl, CrylC2, etc.
A new nomenclature was recently described for the Cry genes based upon
amino acid sequence homology rather than insect target specificity (Crickmore et al.
(1998) Microbiol. Mol. Biol. Rev. 62:807-813). In the new classification, each toxin
is assigned a unique name incorporating a primary rank (an Arabic number), a
secondary rank (an uppercase letter), a tertiary rank (a lowercase letter), and a
quaternary rank (another Arabic number). In the new classification, Roman numerals
have been exchanged for Arabic numerals in the primary rank. Proteins with less than
45% sequence identity have different primary ranks, and the criteria for secondary
and tertiary ranks are 78% and 95%, respectively.
The crystal protein does not exhibit insecticidal activity until it has been
ingested and solubilized in the insect midgut. The ingested protoxin is hydrolyzed by
proteases in the insect digestive tract to an active toxic molecule. (Hofte and
Whiteley ( 1 989) Microbiol. Rev. 53:242-255). This toxin binds to apical brush border
receptors in the midgut of the target larvae and inserts into the apical membrane
creating ion channels or pores, resulting in larval death.
Delta-endotoxins generally have five conserved sequence domains, and three
conserved structural domains (see, for example, de Maagd et al. (2001) Trends
Genetics 1 7: 193- 1 99). The first conserved structural domain consists of seven alpha
helices and is involved in membrane insertion and pore formation. Domain II
consists of three beta-sheets arranged in a Greek key configuration, and domain III
consists of two antiparallel beta-sheets in 'jelly-roll' formation (de Maagd et al.
(2001 ) supra). Domains II and III are involved in receptor recognition and binding,
and are therefore considered determinants of toxin specificity.
Because of the devastation that insects can confer, there is a continual need to
discover new forms of Bacillus thuringiensis delta-endotoxins.
SUMMARY OF INVENTION
Compositions and methods for conferring pesticide resistance to bacteria,
plants, plant cells, tissues, and seeds are provided. Compositions include isolated
nucleic acid molecules encoding sequences for delta-endotoxin and delta-endotoxinassociated
polypeptides, vectors comprising those nucleic acid molecules, and host
cells comprising the vectors. Compositions also include isolated or recombinant
polypeptide sequences of the endotoxin, compositions comprising these polypeptides,
and antibodies to those polypeptides. The nucleotide sequences can be used in DNA
constructs or expression cassettes for transformation and expression in organisms,
including microorganisms and plants. The nucleotide or amino acid sequences may
be synthetic sequences that have been designed for optimum expression in an
organism, including, but not limited to, a microorganism or a plant. Compositions
also comprise transformed bacteria, plants, plant cells, tissues, and seeds.
In particular, the present invention provides for isolated nucleic acid
molecules comprising a nucleotide sequence encoding an amino acid sequence shown
inSEQIDNO:3,5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 27, or 29, or a nucleotide
sequence set forth in SEQ ID NO:1, 2, 4, 6, 8, 10, 12, 13, 15, 17, 19, 21, 23, 25, 26, or
28, as well as variants and fragments thereof. Nucleotide sequences that are
complementary to a nucleotide sequence of the invention, or that hybridize to a
sequence of the invention, are also encompassed.
Methods are provided for producing the polypeptides of the invention, and for
using those polypeptides for controlling or killing a lepidopteran or coleopteran pest.
The compositions and methods of the invention are useful for the production
of organisms with pesticide resistance, specifically bacteria and plants. These
organisms and compositions derived from them are desirable for agricultural
purposes. The compositions of the invention are also useful for generating altered or
improved delta-endotoxin or delta-endotoxin-associated proteins that have pesticidal
activity, or for detecting the presence of delta-endotoxin or delta-endotoxin-associated
proteins or nucleic acids in products or organisms.
DESCRIPTION OF FIGURES
Figure 1 shows an alignment of AXMI-004 (SEQ ID NO:3) with cry 1 Ac (SEQ ID
NO:31), crylCa (SEQ ID NO:32), cry2Aa (SEQ ID NO:34), cry3Aal (SEQ ID
NO:35), cry] la (SEQ ID NO:33), and cry7Aa (SEQ ID NO:41). Toxins having Cterminal
non-toxic domains were artificially truncated as shown. The alignment shows the most highly conserved amino acid residues highlighted in black, and highly conserved amino acid residues highlighted in gray. Conserved group 1 is found from about amino acid residue 196 to about 217 of SEQ ID NO:20. Conserved group 2 is found from about amino acid residue 269 to about 311 of SEQ ID NO:20. Conserved group 3 is found from about amino acid residue 514 to about 556 of SEQ ID NO:20. Conserved group 4 is found from about amino acid residue 574 to about 584 of SEQ ID NO:20. Conserved group 5 is found from about amino acid residue 651 to about 661 of SEQ ID NO:20.
Figures 7 A, B, and C show an alignment of AXMI-014 (SEQ ID NO:27) with cryl Aa (SEQ ID NO:30), cryl Ac (SEQ ID NO:31), crylla (SEQ ID NO:33), cry2Aa (SEQ ID NO:34), cry3Aal (SEQ ID NO:35), cry3Bb (SEQ ID NO:37), cry4Aa (SEQ ID NO:38), cry4Ba (SEQ ID NO:39), cry6Aa (SEQ ID NO:40), cry7Aa (SEQ ID NO:41), cry8Aa (SEQ ID NO:42), crylOAa (SEQ ID NO:43), cryl6Aa (SEQ ID NO:44), cryl9Ba (SEQ ID NO:45), cry24Aa (SEQ ID NO:47), cry25Aa (SEQ ID NO:48), cry39Aal (SEQ ID NO:49), and cry40Aal (SEQ ID NO:51). Toxins having C-terminal non-toxic domains were artificially truncated as shown. The alignment shows the most highly conserved amino acid residues highlighted in black, and highly conserved amino acid residues highlighted in gray. Conserved group 1 is found from about amino acid residue 177 to about 188 of SEQ ID NO:27. Conserved group 2 is found from about amino acid residue 251 to about 293 of SEQ ID NO:27. Conserved group 3 is found from about amino acid residue 483 to about 533 of SEQ ID NO:27. Conserved group 4 is found from about amino acid residue 552 to about 562 of SEQ ID NO:27.
Figure 8 shows a photograph of a 4-20% gradient SDS acrylamide gel. Lanes 1-4 contain various concentrations of sporulated Bacillus cell culture expressing 69 kD AXMI-004 protein. Lanes 5-8 contain various concentrations of BSA. Lane 9 contains a size marker. An arrow indicates the 69 kD band.STATEMENT OF INVENTION:
Accordingly, the invention relates to a recombinant nucleic acid molecule selected from the group consisting of:
a. a nucleic acid molecule comprising the nucleotide sequence selected from SEQ ID
NO. 1, SEQ ID NO. 2 and SEQ ID NO. 4;
b. a nucleic acid molecule comprising a nucleotide sequence having at least 95%
sequence identity to the sequence of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID
NO. 4; and
c. a complement of any of the nucleic acid molecule of (a) and (b);
wherein, the nucleic acid molecule encodes a delta-endotoxin polypeptide having pesticidal activity.
d. a nucleic acid molecule comprising a nucleotide sequence which encodes a
polypeptide having at least 95% amino acid sequence identity to the amino
acid sequence of SEQ ID NO. 3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 27 or 29;
and
e. a complement of any of the nucleic acid molecule of (a) to (d);
wherein, the nucleic acid molecule encodes a polypeptide having pesticidal activity.
DETAILED DESCRIPTION
The present invention is drawn to compositions and methods for regulating pest resistance in organisms, particularly plants or plant cells. The methods involve

transforming organisms with a nucleotide sequence encoding a delta-endotoxin or
delta-endotoxin-associated protein of the invention. In particular, the nucleotide
sequences of the invention are useful for preparing plants and microorganisms that
possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant
tissues and seeds are provided. Compositions are delta-endotoxin or delta-endotoxinassocialed
nucleic acids and proteins of Bacillus thuringiensis. The sequences find
use in the construction of expression vectors for subsequent transformation into
organisms of interest, as probes for the isolation of other delta-endotoxin or deltaendotoxin-
associated genes, and for the generation of altered pesticidal proteins by
methods known in the art, such as domain swapping or DNA shuffling. The proteins
find use in controlling or killing lepidopteran or coleopteran pest populations and for
producing compositions with pesticidal activity.
Definitions
By "delta-endotoxin" is intended a toxin from Bacillus thuringiensis that has
toxic activity against one or more pests, including, but not limited to, members of the
Lepidoptera, Diptera, and Coleoptera orders, hi some cases, delta-endotoxin proteins
have been isolated from other organisms, including Clostridium bifermentans and
Paenihacillus popilliae. Delta-endotoxin proteins include amino acid sequences
deduced from the full-length nucleotide sequences disclosed herein, and amino acid
sequences that are shorter than the full-length sequences, either due to the use of an
alternate downstream start site, or due to processing that produces a shorter protein
having pesticidal activity. Processing may occur in the organism the protein is
expressed in, or in the pest after ingestion of the protein. Delta-endotoxins include
proteins identified as cryl through cry43, cytl and cyt2, and Cyt-like toxin. There are
currently over 250 known species of delta-endotoxins with a wide range of
specificities and toxicities. For an expansive list see Crickmore et al. (1998),
Microbiol. Mol. Biol. Rev. 62:807-813, and for regular updates see Crickmore et al.
(2003) "Bacillus thuringiensis toxin nomenclature," at
www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.
Bacterial genes, such as the AXMI genes of this invention, quite often possess
multiple methionine initiation codons in proximity to the start of the open reading
frame. Often, translation initiation at one or more of these start codons will lead to
generation of a functional protein. These start codons can include ATG codons.
However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon,
and proteins that initiate translation at GTG codons contain a methionine at the first
amino acid. Furthermore, it is not often determined a priori which of these codons are
used naturally in the bacterium. Thus, it is understood that use of one of the alternate
methionine codons may also lead to generation of delta-endotoxin proteins that
encode pesticidal activity. For example, an alternate start site for an AXMI-004 deltaendotoxin
protein of the invention is at base pair 385 of SEQ ID NO: 1. Translation
from this alternate start site results in the amino acid sequence found in SEQ ID
NO:5. An alternate start site for an AXMI-007 delta-endotoxin protein of the
invention may be at base pair 151 of SEQ ID NO:8. Translation from this alternate
start site results in the amino acid sequence found in SEQ ID NO: 11. An alternate
start site for an AXMI-008 delta-endotoxin protein of the invention may be at
nucleotide 177 of SEQ ID NO: 12. Translation from this alternate start site results in
the amino acid sequence found in SEQ ID NO: 16. An alternate start site for an
AXM1-009 delta-endotoxin protein of the invention may be at nucleotide 34 of SEQ
ID NO: 19. Translation from this alternate start site results in the amino acid sequence
found in SEQ ID NO:22. An additional alternate start site for an AXMI-009 deltaendotoxin
protein of the invention maybe at nucleotide 64 of SEQ ID NO:1.
Translation from this alternate start site results in the amino acid sequence found in
SEQ ID NO:24. An alternate start site for an AXMI-014 delta-endotoxin protein of
the invention may be at base pair 136 of SEQ ID NO:25. Translation from this
alternate start site results in the amino acid sequence found in SEQ ID NO:29. These
delta-endotoxin proteins are encompassed in the present invention and may be used in
the methods of the present invention.
In addition, there may be one or more additional open reading frames in the
disclosed nucleotide sequences that encode one or more delta-endotoxin-associated
proteins. By "delta-endotoxin-associated protein" is intended a protein encoded by a
nucleotide sequence disclosed herein using an alternate open reading frame than that
used by the delta-endotoxins of the present invention. Proteins such as these are
known in the art as helper proteins, stabilizing sequences, or delta-endotoxinassociated
proteins. These delta-endotoxin-associated proteins may have pesticidal
activity, or may be important in facilitating expression of delta-endotoxin proteins.
Methods are known in the art for measuring pesticidal activity and for determining the
effects of delta-endotoxin-associated proteins on delta-endotoxin protein expression
and crystal formation (see, for example, Park et al. (1999) FEMS Microbiol. Lett.
181:319-327; Ge et al. (1998) FEMS Microbiol. Lett. 165:35-41; Rosso and Delecluse
(1997) Appl. Environ. Microbiol. 63:4449-4455). These delta-endotoxin-associated
proteins are encompassed by the present invention, and may be used in the methods
disclosed herein, either alone or in combination with known delta-endotoxin proteins.
In one embodiment, the delta-endotoxin-associated protein has the amino acid
sequence found in SEQ ID NO: 18 and is encoded by the nucleotide sequence of SEQ
ID NO: 17.
By "plant cell" is intended all known forms of plant, including
undifferentiated tissue (e.g. callus), suspension culture cells, protoplasts, leaf cells,
root cells, phloem cells, plant seeds, pollen, propagules, embryos and the like. By
"plant expression cassette" is intended a DNA construct that is capable of resulting in
the expression of a protein from an open reading frame in a plant cell. Typically these
contain a promoter and a coding sequence. Often, such constructs will also contain a
3' untranslated region. Such constructs may contain a 'signal sequence' or 'leader
sequence' to facilitate co-translational or post-translational transport of the peptide to
certain intracellular structures such as the chloroplast (or other plastid), endoplasmic
reticulum, or Golgi apparatus.
By "signal sequence" is intended a sequence that is known or suspected to
result in cotranslational or post-translational peptide transport across the cell
membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus,
with some resulting glycosylation. By "leader sequence" is intended any sequence
that when translated, results in an amino acid sequence sufficient to trigger cotranslational
transport of the peptide chain to a sub-cellular organelle. Thus, this
includes leader sequences targeting transport and/or glycosylation by passage into the
endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts,
mitochondria, and the like.
By "plant transformation vector" is intended a DNA molecule that is
necessary for efficient transformation of a plant cell. Such a molecule may consist of
one or more plant expression cassettes, and may be organized into more than one
'vector' DNA molecule. For example, binary vectors are plant transformation vectors
that utilize two non-contiguous DNA vectors to encode all requisite cis- and transacting
functions for transformation of plant cells (Hellens and Mullineaux (2000)
Trends in Plant Science 5:446-451). "Vector" refers to a nucleic acid construct
designed for transfer between different host cells. "Expression vector" refers to a
vector that has ability to incorporate, integrate and express heterologous DNA
sequences or fragments in a foreign cell.
"Transgenic plants" or "transformed plants" or "stably transformed plants or
cells or tissues" refers to plants that have incorporated or integrated exogenous
nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid
sequences include those that are exogenous, or not present in the untransformed plant
cell, as well as those that may be endogenous, or present in the untransformed plant
cell. "Heterologous" generally refers to the nucleic acid sequences that are not
endogenous to the cell or part of the native genome in which they are present, and
have been added to the cell by infection, trans fection, microinjection, electroporation,
microprojection, or the like.
"Promoter" refers to a nucleic acid sequence that functions to direct
transcription of a downstream coding sequence. The promoter together with other
transcripti onal and translational regulatory nucleic acid sequences (also termed
"control sequences") are necessary for the expression of a DNA sequence of interest.
Provided herein are novel isolated nucleotide sequences that confer pesticidal
activity. Also provided are the amino acid sequences for the delta- endo toxin and
delta-endotoxin-associated proteins. The protein resulting from translation of this
gene allows cells to control or kill pests that ingest it.
An "isolated" or "purified" nucleic acid molecule or protein, or biologically
active portion thereof, is substantially free of other cellular material, or culture
medium when produced by recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized. Preferably, an "isolated"
nucleic acid is free of sequences (preferably protein encoding sequences) that
naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the nucleic acid is
derived. For purposes of the invention, "isolated" when used to refer to nucleic acid
molecules excludes isolated chromosomes. For example, in various embodiments, the
isolated delta-endotoxin or delta-endotoxin-associated-encoding nucleic acid
molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequence that naturally flanks the nucleic acid molecule in genomic DNA
of the cell from which the nucleic acid is derived. A delta-endotoxin or deltaendotoxin-
associated protein that is substantially free of cellular material includes
preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight)
of non-delta-endotoxin or non-delta-endotoxin-associated protein (also referred to
herein as a "contaminating protein"). Various aspects of the invention are described
in further detail in the following subsections.
Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof
One aspect of the invention pertains to isolated nucleic acid molecules
comprising nucleotide sequences encoding delta-endotoxin or delta-endotoxinassociated
proteins and polypeptides or biologically active portions thereof, as well as
nucleic acid molecules sufficient for use as hybridization probes to identify deltaendotoxin
or delta-endotoxin-associated-encoding nucleic acids. As used herein, the
term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. The nucleic acid molecule can be single-stranded
or double-stranded, but preferably is double-stranded DNA.
Nucleotide sequences encoding the proteins of the present invention include
the sequences set forth in SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 13, 15, 17, 19, 21, 23, 25,
26, and 28, and complements thereof. By "complement" is intended a nucleotide
sequence that is sufficiently complementary to a given nucleotide sequence such that
it can hybridize to the given nucleotide sequence to thereby form a stable duplex. The
corresponding amino acid sequences for the delta-endotoxin or delta-endotoxinassociated
proteins encoded by these nucleotide sequences are set forth in SEQ ID
NOS:3, 5, 7, 9, 1 1, 14, 16, 18, 20, 22, 24, 27, and 29.
Nucleic acid molecules that are fragments of these delta-endotoxin or deltaendotoxin-
associated protein-encoding nucleotide sequences are also encompassed by
the present invention. By "fragment" is intended a portion of the nucleotide sequence
encoding a delta-endotoxin protein or delta-endotoxin-associated protein. A fragment
of a nucleotide sequence may encode a biologically active portion of a deltaendotoxin
or delta-endotoxin-associated protein, or it may be a fragment that can be
used as a hybridization probe or PCR primer using methods disclosed below. Nucleic
acid molecules that are fragments of a delta-endotoxin or a delta-endotoxin-associated
nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 3500,
4000, 4500, 5000, 5500 nucleotides, or up to the number of nucleotides present in a
full-length delta-endotoxin or delta-endotoxin-associated protein-encoding nucleotide
sequence disclosed herein (for example, 2190 nucleotides for SEQ ID NO:1, 1890 for
SEQ ID NO:2, etc.), depending upon the intended use.
Fragments of the nucleotide sequences of the present invention will encode
protein fragments that retain the biological activity of the delta endotoxin or deltaendotoxin-
associated protein and, hence, retain pesticidal activity or delta-endotoxinassociated
protein activity, respectively. By "delta-endotoxin activity" is intended
pesticidal activity. By "delta-endotoxin-associated protein activity" is intended that
the protein have pesticidal activity, or that the protein improves expression of a deltaendotoxin
protein. This improvement in protein expression can happen by any
mechanism. By "retains activity" is intended that the fragment will have at least about
30%, preferably at least about 50%, more preferably at least about 70%, even more
preferably at leas! about 80% of the activity of the delta-endotoxin or delta-endotoxinassociated
protein. Methods are known in the art for determining the effects of deltaendotoxin-
associated proteins on delta-endotoxin protein expression and crystal
formation (see, for example, Park et al. (1999) FEMS Microbiol. Lett. 181:319-327;
Ge et al. (1998) FEMS Microbiol. Lett. 165:35-41; Rosso and Delecluse (1997) Appl.
Environ. Microbiol, 63:4449-4455). Methods for measuring pesticidal activity are
well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.
83(6): 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.
(1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477, all of
which are herein incorporated by reference in their entirety.
A fragment of a delta-endotoxin or delta-endotoxin-associated proteinencoding
nucleotide sequence that encodes a biologically active portion of a protein
of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200,
250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous amino acids, or up to the
total number of amino acids present in a full-length delta-endotoxin or delta-
- Wendotoxin-
associated protein of the invention (for example, 629 amino acids for SEQ
ID NO:3, 601 amino acids for SEQ ID NO:5, etc.).
Preferred delta-endotoxin or delta-endotoxin-associated proteins of the present
invention are encoded by a nucleotide sequences sufficiently identical to the
nucleotide sequences of in SEQ ID NO:3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 27, or 29.
By "sufficiently identical" is intended an amino acid or nucleotide sequence that has
at least about 60% or 65% sequence identity, preferably about 70% or 75% sequence
identity, more preferably about 80% or 85% sequence identity, most preferably about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity
compared to a reference sequence using one of the alignment programs described
herein using standard parameters. One of skill in the art will recognize that these
values can be appropriately adjusted to determine corresponding identity of proteins
encoded by two nucleotide sequences by taking into account codon degeneracy,
amino acid similarity, reading frame positioning, and the like.
To determine the percent identity of two amino acid sequences or of two
nucleic acids, the sequences are aligned for optimal comparison purposes. The
percent identity between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., percent identity = number of identical
positions/total number of positions (e.g., overlapping positions) x 100). In one
embodiment, the two sequences are the same length. The percent identity between
two sequences can be determined using techniques similar to those described below,
with or without allowing gaps. In calculating percent identity, typically exact matches
are counted.
The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A nonlimiting example of a
mathematical algorithm utilized for the comparison of two sequences is the algorithm
of Karlin and AHschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an
algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al.
(1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the
BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences
homologous to delta-endotoxin or delta-endotoxin-associated nucleic acid molecules
of the invention. BLAST protein searches can be performed with the BLASTX
-TTprogram,
score --•- 50, wordlength = 3, to obtain amino acid sequences homologous to
delta-endotoxin or delta-endotoxin-associated protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSIBlast
can be used to perform an iterated search that detects distant relationships
between molecules. See, Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, and PSI-Blast programs, the default parameters of the respective
programs (e.g., BLASTX and BLASTN) can be used. See, www.ncbi.nlm.nih.gov.
Another non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic
Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of
the amino acid or DNA sequence, and thus can provide data about the sequence
conservation of the entire amino acid sequence. The ClustalW algorithm is used in
several commercially available DNA/amino acid analysis software packages, such as
the ALIGNX module of the vector NTi Program Suite (Informax, Inc). After
alignment of amino acid sequences with ClustalW, the percent amino acid identity
can be assessed. A non-limiting example of a software program useful for analysis of
ClustalW alignments is GeneDoc™. Genedoc™ (Karl Nicholas) allows assessment of
amino acid (or DNA) similarity and identity between multiple proteins. Another nonlimiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an
algorithm is incorporated into the ALIGN program (version 2.0), which is part of the
GCG sequence alignment software package (available from Accelrys, Inc., 9865
Scranton Rd., San Diego, California, USA). When utilizing the ALIGN program for
comparing amino acid sequences, a PAM 120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used.
The invention also encompasses variant nucleic acid molecules. "Variants" of
the delta-endotoxin or delta-endotoxin-associated protein-encoding nucleotide
sequences include those sequences that encode the delta-endotoxin or deltaendotoxin-
associated proteins disclosed herein but that differ conservatively because
of the degeneracy of the genetic code as well as those that are sufficiently identical as
discussed above. Naturally occurring allelic variants can be identified with the use of
well-known molecular biology techniques, such as polymerase chain reaction (PCR)
and hybridization techniques as outlined below. Variant nucleotide sequences also
include synthetically derived nucleotide sequences that have been generated, for
example, by using site-directed mutagenesis but which still encode the deltaendotoxin
or delta-endotoxin-associated proteins disclosed in the present invention as
discussed below. Variant proteins encompassed by the present invention are
biologically active, that is they continue to possess the desired biological activity of
the native protein, that is, retaining pesticidal activity. By "retains activity" is
intended that the variant will have at least about 30%, preferably at least about 50%,
more preferably at least about 70%, even more preferably at least about 80% of the
activity of the delta-endo toxin or delta-endotoxin-associated protein. Methods for
measuring pesticidal activity are well known in the art. See, for example, Czapla and
Lang (1990) J. Econ. Entomol. 83(6): 2480-2485; Andrews et al. (1988) Biochem. J.
252:199-206; Marrone et al. (1 985) J. of Economic Entomology 78:290-293; and U.S.
Patent No. 5,743,477, all of which are herein incorporated by reference in their
entirety.
The invention also encompasses variant nucleic acid molecules. "Variants" of
the delta-endotoxin or delta-endotoxin-associated-encoding nucleotide sequences
include those sequences that encode the delta-endotoxin or delta-endotoxin-associated
proteins disclosed herein but that differ conservatively because of the degeneracy of
the genetic code as well as those that are sufficiently identical as discussed above.
Naturally occurring allelic variants can be identified with the use of well-known
molecular biology techniques, such as polymerase chain reaction (PCR) and
hybridization techniques as outlined below. Variant nucleotide sequences also
include synthetically derived nucleotide sequences that have been generated, for
example, by using site-directed mutagenesis but which still encode the deltaendotoxin
or delta-endotoxin-associated proteins disclosed in the present invention as
discussed below.
The skilled artisan will further appreciate that changes can be introduced by
mutation into the nucleotide sequences of the invention thereby leading to changes in
the amino acid sequence of the encoded delta-endotoxin or delta-endotoxin-associated
proteins, without altering the biological activity of the proteins. Thus, variant isolated
nucleic acid molecules can be created by introducing one or more nucleotide
substitutions, additions, or deletions into the corresponding nucleotide sequence
disclosed herein, such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be introduced by
standard techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Such variant nucleotide sequences are also encompassed by the present
invention.
For example, preferably, conservative amino acid substitutions may be made
at one or more predicted, preferably nonessential amino acid residues. A
"nonessential" amino acid residue is a residue that can be altered from the wild-type
sequence of a delta-endotoxin or delta-endotoxin-associated protein without altering
the biological activity, whereas an "essential" amino acid residue is required for
biological activity. A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have been defined in the
art. These families include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine).
There are generally five highly conserved regions among the delta-endotoxin
proteins, concentrated largely in the center of the domain or at the junction between
domains (Rajamohan et al. (1998) Prog. Nucleic Acid Res. Mol. Biol. 60:1 -23). The
blocks of conserved amino acids for various delta-endotoxins as well as consensus
sequences may be found in Schnepf et al. (1998) Microbio. Mol. Biol. Rev. 62:775-
806 and Lereclus et al. (1989) Role, Structure, and Molecular Organization of the
Genes Coding for the Parasporal d-endotoxins of Bacillus thuringiensis. In Regulation
of Procaryotic Development. Issar Smit, Slepecky, R.A., Setlow, P. American Society
for Microbiology, Washington, D.C. 20006. It has been proposed that deltaendotoxins
having these conserved regions may share a similar structure, consisting of
three domains (Li et al. (1991) Nature 353: 815-821). Domain I has the highest
similarity between delta-endotoxins (Bravo (1997) /. Bacterial. 179:2793-2801).
Amino acid substitutions may be made in nonconserved regions that retain
function. In general, such substitutions would not be made for conserved amino acid
residues, or for arnino acid residues residing within a conserved motif, where such
residues are essential for protein activity. Examples of residues that are conserved
and that may be essential for protein activity include, for example, residues that are
identical between all proteins contained in the alignments provided. Examples of
residues that are conserved but that may allow conservative amino acid substitutions
and still retain activity include, for example, residues that have only conservative
substitutions between all proteins contained in the alignments provided. However,
one of skill in the art would understand that functional variants may have minor
conserved or nonconserved alterations in the conserved residues.
Alternatively, variant nucleotide sequences can be made by introducing
mutations randomly along all or part of the coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for ability to confer deltaendotoxin
or delta-endotoxin-associated activity to identify mutants that retain
activity. Following mutagenesis, the encoded protein can be expressed
recombinantly, and the activity of the protein can be determined using standard assay
techniques.
Using methods such as PCR, hybridization, and the like corresponding deltaendotoxin
or delta-endotoxin-associated sequences can be identified, such sequences
having substantial identity to the sequences of the invention. See, for example,
Sambrook J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).
In a hybridization method, all or part of the delta-endotoxin or deltaendotoxin-
associated nucleotide sequence can be used to screen cDNA or genomic
libraries. Methods for construction of such cDNA and genomic libraries are generally
known in the art and are disclosed in Sambrook and Russell, 2001. The so-called
hybridization probes may be genomic DNA fragments, cDNA fragments, RNA
fragments, or other oligonucleotides, and may be labeled with a detectable group such
as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent
compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made
by labeling synthetic oligonucleotides based on the known delta-endotoxin or deltaendotoxin-
associated-encoding nucleotide sequence disclosed herein. Degenerate
primers designed on the basis of conserved nucleotides or amino acid residues in the
nucleotide sequence or encoded amino acid sequence can additionally be used. The
probe typically comprises a region of nucleotide sequence that hybridizes under
stringent conditions to at least about 12, preferably about 25, more preferably at least
about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides
of delta-endotoxin or delta-endotoxin-associated-encoding nucleotide sequence of the
invention or a fragment or variant thereof. Preparation of probes for hybridization is
generally known in the art and is disclosed in Sambrook and Russell, 2001, herein
incorporated by reference.
In hybridization techniques, all or part of a known nucleotide sequence is used
as a probe that selectively hybridizes to other corresponding nucleotide sequences
present in a population of cloned genomic DNA fragments or cDNA fragments (i.e.,
genomic or cDNA libraries) from a chosen organism. The hybridization probes may
be genomic DNA fragments, cDNA fragments, RNA fragments, or other
oligonucleotides, and may be labeled with a detectable group such as 32P, or any other
detectable marker. Thus, for example, probes for hybridization can be made by
labeling synthetic oligonucleotides based on the delta-endotoxin or delta-endotoxinassociated
sequence of the invention. Methods for preparation of probes for
hybridization and for construction of cDNA and genomic libraries are generally
known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New
York).
For example, the entire delta-endotoxin or delta-endotoxin-associated
sequence disclosed herein, or one or more portions thereof, may be used as a probe
capable of specifically hybridizing to corresponding delta-endotoxin or deltaendotoxin-
associated-like sequences and messenger RNAs. To achieve specific
hybridization under a variety of conditions, such probes include sequences that are
unique and are preferably at least about 10 nucleotides in length, and most preferably
at least about 20 nucleotides in length. Such probes may be used to amplify
corresponding delta-endotoxin or delta-endotoxin-associated sequences from a chosen
organism by PCR. This technique may be used to isolate additional coding sequences
from a desired organism or as a diagnostic assay to determine the presence of coding
sequences in an organism. Hybridization techniques include hybridization screening
of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, New York).
Hybridization of such sequences may be carried out under stringent
conditions. By "stringent conditions" or "stringent hybridization conditions" is
intended conditions under which a probe will hybridize to its target sequence to a
detectably greater degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will be different in
different circumstances. By controlling the stringency of the hybridization and/or
washing conditions, target sequences that are 100% complementary to the probe can
be identified (homologous probing). Alternatively, stringency conditions can be
adjusted to allow some mismatching in sequences so that lower degrees of similarity
are detected (heterologous probing). Generally, a probe is less than about 1000
nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is
less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes
(e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than
50 nucleotides). Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1%
SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX to 2X SSC (20X SSC - 3.0
M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency
conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency
conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and
a wash in 0.1 X SSC at 60 to 65°C. Optionally, wash buffers may comprise about
0.1% to about 1% SDS. Duration of hybridization is generally less than about 24
hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash solution. For DNADNA
hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) -
0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form is the
percentage of formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH)
at which 50% of a complementary target sequence hybridizes to a perfectly matched
probe. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm,
hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the
desired identity. For example, if sequences with >90% identity are sought, the Tm can
be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower
than the thermal melting point (Tm) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm);
moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or
10°C lower than the thermal melting point (Tm); low stringency conditions can utilize
a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal
melting point (Tm). Using the equation, hybridization and wash compositions, and
desired Tm, those of ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If the desired degree of
mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide
solution), it is preferred to increase the SSC concentration so that a higher temperature
can be used. An extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—
Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and
Ausubel et al, eds. (1995) Current Protocols in Molecular Biology, Chapter 2
(Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, New York).
Isolated Proteins and Variants and Fragments Thereof
Delta-endotoxin and delta-endotoxin-associated proteins are also encompassed
within the present invention. By "delta-endotoxin protein" is intended a protein
having the amino acid sequence set forth in SEQ ID NO:3, 5, 7, 9, 11, 14, 16, 20, 22,
24, 27, or 29. By "delta-endotoxin-associated protein" is intended a protein having the
amino acid sequence set forth in SEQ ID NO: 18. Fragments, biologically active
portions, and variants thereof are also provided, and may be used to practice the
methods of the present invention.
"Fragments" or "biologically active portions" include polypeptide fragments
comprising a portion of an amino acid sequence encoding a delta-endotoxin or deltaendotoxin-
associated protein as set forth in SEQ ID NO:3, 5, 7, 9, 11, 14, 16, 18, 20,
22, 24, 27, or 29, and that retain delta-endotoxin activity or delta-endotoxinassociated
activity. A biologically active portion of a delta-endotoxin or deltaendotoxin-
associated protein can be a polypeptide that is, for example, 10, 25, 50, 100
or more amino acids in length. Such biologically active portions can be prepared by
recombinant techniques and evaluated for delta-endotoxin or delta-endotoxinassociated
activity. Methods for measuring pesticidal activity are well known in the
art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83(6): 2480-2485;
Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic
Entomology 78:290-293; and U.S. Patent No. 5,743,477, all of which are herein
incorporated by reference in their entirety. As used here, a fragment comprises at
least 8 contiguous amino acids SEQ ID NO:3, 5, 7, 9, 11, 14, 16, 18, 20, 22, 24, 27, or
29. The invention encompasses other fragments, however, such as any fragment in
the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, and 650 amino acids.
By "variants" is intended proteins or polypeptides having an amino acid
sequence that is at least about 60%, 65%, preferably about 70%, 75%, more
preferably about 80%, 85%, most preferably about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:3, 5, 7,
9, 11, 14, 16, 18, 20, 22, 24, 27, or 29. Variants also include polypeptides encoded by
a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:1,
2,4,6,8, 10, 12, 13, 15, 17, 19, 21, 23, 25, 26, or 28, or a complement thereof, under
stringent conditions. Such variants generally retain delta-endotoxin or deltaendotoxin-
associated activity. Variants include polypeptides that differ in amino acid
sequence due to mutagenesis. Variant proteins encompassed by the present invention
are biologically active, that is they continue to possess the desired biological activity
of the native protein, that is, retaining pesticidal activity. Methods for measuring
pesticidal activity are well known in the art. See, for example, Czapla and Lang
(1990) J. Econ. Entomol. 83(6): 2480-2485; Andrews et al. (1988) Biochem. J.
252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.
Patent No. 5,743,477, all of which are herein incorporated by reference in their
entirety.
Altered or Improved Variants
It is recognized that DNA sequences of a delta-endotoxin or delta-endotoxinassociated
protein may be altered by various methods, and that these alterations may
result in DNA sequences encoding proteins with amino acid sequences different than
that encoded by the delta-endotoxin or delta-endotoxin-associated protein of the
present invention. This protein may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for such manipulations
are generally known in the art. For example, amino acid sequence variants of the
delta-endotoxin or delta-endotoxin-associated protein can be prepared by mutations in
the DNA. This may also be accomplished by one of several forms of mutagenesis
and/or in directed evolution. In some aspects, the changes encoded in the amino acid
sequence will not substantially affect the function of the protein. Such variants will
possess the desired pesticidal activity. However, it is understood that the ability of a
delta-endotoxin or delta-endotoxin-associated protein to confer pesticidal activity may
be improved by the use of such techniques upon the compositions of this invention.
For example, one may express the delta-endotoxin or delta-endotoxin-associated
protein in host cells that exhibit high rates of base misincorporation during DNA
replication, such as XL-1 Red (Stratagene). After propagation in such strains, one can
isolate the delta-endotoxin or delta-endotoxin-associated DNA (for example by
preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR
fragment into a vector), culture the delta-endotoxin or delta-endotoxin-associated
mutations in a non-mutagenic strain, and identify mutated delta-endotoxin or deltaendotoxin-
associated genes with pesticidal activity, for example by performing an
assay to test for pesticidal activity. Generally, the protein is mixed and used in
feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology
78:290-293. Such assays can include contacting plants with one or more pests and
determining the plant's ability to survive and/or cause the death of the pests.
Examples of mutations that result in increased toxicity are found in Schnepf et al.
(1998) Microbiol. Mol. Biol. Rev. 62:775-806.
Alternatively, alterations may be made to the protein sequence of many
proteins at the amino or carboxy terminus without substantially affecting activity.
This can include insertions, deletions, or alterations introduced by modern molecular
methods, such as PCR, including PCR amplifications that alter or extend the protein
coding sequence by virtue of inclusion of amino acid encoding sequences in the
oligonucleotides utilized in the PCR amplification. Alternatively, the protein
sequences added can include entire protein-coding sequences, such as those used
commonly in the art to generate protein fusions. Such fusion proteins are often used to
(1) increase expression of a protein of interest (2) introduce a binding domain,
enzymatic activity, or epitope to facilitate either protein purification, protein
detection, or other experimental uses known in the art (3) target secretion or
translation of a protein to a subcellular organelle, such as the periplasmic space of
Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of
which often results in glycosylation of the protein.
Variant nucleotide and amino acid sequences of the present invention also
encompass sequences derived from mutagenic and recombinogenic procedures such
as DNA shuffling. With such a procedure, one or more different delta-endotoxin or
delta-endotoxin-associated protein coding regions can be used to create a new deltaendotoxin
or delta-endotoxin-associated protein possessing the desired properties. In
this manner, libraries of recombinant polynucleotides are generated from a population
of related sequence polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in vivo. For
example, using this approach, sequence motifs encoding a domain of interest may be
shuffled between the delta-endotoxin or delta-endotoxin-associated gene of the
invention and other known delta-endotoxin or delta-endotoxin-associated genes to
obtain a new gene coding for a protein with an improved property of interest, such as
an increased insecticidal activity. Strategies for such DNA shuffling are known in the
art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;
Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-
438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.
Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Patent Nos. 5,605,793 and 5,837,458.
Domain swapping or shuffling is another mechanism for generating altered
delta-endotoxin or delta-endotoxin-associated proteins. Domains II and III may be
swapped between delta-endotoxin proteins, resulting in hybrid or chimeric toxins with
improved pesticidal activity or target spectrum. Methods for generating recombinant
proteins and testing them for pesticidal activity are well known in the art (see, for
example, Naimov et al. (2QQl)Appl. Environ. Microbiol. 67:5328-5330; de Maagd et
al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Geetal. (1991) J. Biol. Chem.
266:17954-17958; Schnepf et al. (1990> /. Biol. Chem. 265:20923-20930; Rang et al.
91999) Appl. Environ. Micriobiol. 65:2918-2925).
Plant Transformation
Transformation of plant cells can be accomplished by one of several
techniques known in the art. First, one engineers the delta-endotoxin or deltaendotoxin-
associated gene in a way that allows its expression in plant cells. Typically
a construct that expresses such a protein would contain a promoter to drive
transcription of the gene, as well as a 3' untranslated region to allow transcription
termination and polyadenylation. The organization of such constructs is well known
in the art. In some instances, it may be useful to engineer the gene such that the
resulting peptide is secreted, or otherwise targeted within the plant cell. For example,
the gene can be engineered to contain a signal peptide to facilitate transfer of the
peptide to the endoplasmic reticulum. It may also be preferable to engineer the plant
expression cassette to contain an intron, such that mRNA processing of the intron is
required for expression.
Typically this 'plant expression cassette' will be inserted into a 'plant
transformation vector'. This plant transformation vector may be comprised of one or
more DNA vectors needed for achieving plant transformation. For example, it is a
common practice in the art to utilize plant transformation vectors that are comprised
of more than one contiguous DNA segment. These vectors are often referred to in the
art as 'binary vectors'. Binary vectors as well as vectors with helper plasmids are
most often used for Agrobacterium-mediated transformation, where the size and
complexity of DNA segments needed to achieve efficient transformation is quite
large, and it is advantageous to separate functions onto separate DNA molecules.
Binary vectors typically contain a plasmid vector that contains the cis-acting
sequences required for T-DNA transfer (such as left border and right border), a
selectable marker that is engineered to be capable of expression in a plant cell, and a
'gene of interest' (a gene engineered to be capable of expression in a plant cell for
which generation of transgenic plants is desired). Also present on this plasmid vector
are sequences required for bacterial replication. The cis-acting sequences are arranged
in a fashion to allow efficient transfer into plant cells and expression therein. For
example, the selectable marker gene and the gene of interest are located between the
left and right borders. Often a second plasmid vector contains the trans-acting factors
that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often
contains the virulence functions (Vir genes) that allow infection of plant cells by
Agrobacterium, and transfer of DNA by cleavage at border sequences and virmediated
DNA transfer, as in understood in the art (Hellens and Mullineaux (2000)
Trends in Plant Science, 5:446-451). Several types of Agrobacterium strains (e.g.
LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation.
The second plasmid vector is not necessary for transforming the plants by other
methods such as microprojection, microinjection, electroporation, polyethelene
glycol, etc.
In general, plant transformation methods involve transferring heterologous
DNA into target plant cells (e.g. immature or mature embryos, suspension cultures,
undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold
level of appropriate selection (depending on the selectable marker gene) to recover the
transformed plant cells from a group of untransformed cell mass. Explants are
typically transferred to a fresh supply of the same medium and cultured routinely.
Subsequently, the transformed cells are differentiated into shoots after placing on
regeneration medium supplemented with a maximum threshold level of selecting
agent. The shoots are then transferred to a selective rooting medium for recovering
rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and
produces fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6: 271-282; Ishida et
al. (1996) Nature Biotechnology 14: 745-750). Explants are typically transferred to a
fresh supply of the same medium and cultured routinely. A general description of the
techniques and methods for generating transgenic plantlets are found in Ayres and
Park, 1994 (Critical Reviews in Plant Science 13: 219-239) and Bommineni and
Jauhar, 1997 (Maydica 42: 107-120). Since the transformed material contains many
cells; both transformed and non-trans formed cells are present in any piece of
subjected target callus or tissue or group of cells. The ability to kill non-transformed
cells and allow transformed cells to proliferate results in transformed plant cultures.
Often, the ability to remove non-transformed cells is a limitation to rapid recovery of
transformed plant cells and successful generation of transgenic plants.
Generation of transgenic plants may be performed by one of several methods,
including but not limited to introduction of heterologous DNA by Agrobacterium into
plant cells (Agrobacterium-mediaied transformation), bombardment of plant cells
with heterologous foreign DNA adhered to particles, and various other non-particle
direct-mediated methods (e.g. Hiei et al. (1994) The Plant Journal 6: 271-282; Ishida
et al. (1996) Nature Biotechnology 14: 745-750; Ayres and Park (1994) Critical
Reviews in Plant Science 13: 219-239; Bommineni and Jauhar (1997) Maydica 42:
107-120) to transfer DNA.
Transformation protocols as well as protocols for introducing nucleotide
sequences into plants may vary depending on the type of plant or plant cell, i.e.,
monocot or dicot, targeted for transformation. Suitable methods of introducing
nucleotide sequences into plant cells and subsequent insertion into the plant genome
include microinjection (Crossway et al. (1986) Biotechniques 4:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,
Agrohacterium-mediated transformation (U.S. Patent No. 5,563,055; U.S. Patent No.
5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBOJ. 3:2717-2722),
and ballistic particle acceleration (see, for example, U.S. Patent No. 4,945,050; U.S.
Patent No. 5,879,918; U.S. Patent No. 5,886,244; U.S. Patent No. 5,932,782; Tomes
et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed.
Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology
6:923-926); aerosol beam transformation (U.S. Published Application No.
20010026941; U.S. Patent No. 4,945,050; International Publication No. WO
91/00915; U.S. Published Application No. 2002015066); and Lecl transformation
(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477;
Sanford et al. (1987) Paniculate Science and Technology 5:27-37; Christou et al.
(1988) Plant Physiol. 87:671-674; McCabe et al. (1988) Bio/Technology 6:923-926;
Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; Singh et al. (1998)
Thcor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology
8:736-740; Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309; U.S. Patent
No. 5,240,855; U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)
'Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in
Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg
(Springer-Verlag, Berlin); Klein et al. (1988) Plant Physiol. 91:440-444; Hooykaas-
Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Patent No. 5,736,369;
Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet
et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al.
(Longman, New York), pp. 197-209; Kaeppler et al. (1990) Plant Cell Reports 9:415-
418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566; D'Halluin et al.
(1992) Plant Cell 4:1495-1505; Li et al. (1993) Plant Cell Reports 12:250-255 and
Christou and Ford (1995) Annals of Botany 75:407-413; Osjoda et al. (1996) Nature
Biotechnology 14:745-750; all of which are herein incorporated by reference.
Following integration of heterologous foreign DNA into plant cells, one then
applies a maximum threshold level of appropriate selection in the medium to kill the
untransformed cells and separate and proliferate the putatively transformed cells that
survive from this selection treatment by transferring regularly to a fresh medium. By
continuous passage and challenge with appropriate selection, one identifies and
proliferates the cells that are transformed with the plasmid vector. Then molecular
and biochemical methods will be used for confirming the presence of the integrated
heterologous gene of interest in the genome of transgenic plant.
The cells that have been transformed may be grown into plants in accordance
with conventional ways. See, for example, McCormick et al. (1986) Plant Cell
Reports 5:81-84. These plants may then be grown, and either pollinated with the
same transformed strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic identified. Two or
more generations may be grown to ensure that expression of the desired phenotypic
characteristic is stably maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been achieved. In this manner,
the present invention provides transformed seed (also referred to as "transgenic seed")
having a nucleotide construct of the invention, for example, an expression cassette of
the invention, stably incorporated into their genome.
The delta-endotoxin or delta-endotoxin-associated sequences of the invention
may be provided in expression cassettes for expression in the plant of interest. The
cassette will include 5' and 3' regulatory sequences operably linked to a sequence of
the invention. By "operably linked" is intended a functional linkage between a
promoter and a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences being linked are
contiguous and, where necessary to join two protein coding regions, contiguous and in
the same reading frame. The cassette may additionally contain at least one additional
gene to be cotransformed into the organism. Alternatively, the additional gene(s) can
be provided on multiple expression cassettes.
Such an expression cassette is provided with a plurality of restriction sites for
insertion of the delta-endotoxin or delta-endotoxin-associated sequence to be under
the transcriptional regulation of the regulatory regions.
The expression cassette will include in the 5'-3' direction of transcription, a
transcriptional and translational initiation region (i.e., a promoter), a DNA sequence
of the invention, and a transcriptional and translational termination region (i.e.,
termination region) functional in plants. The promoter may be native or analogous, or
foreign or heterologous, to the plant host and/or to the DNA sequence of the
invention. Additionally, the promoter may be the natural sequence or alternatively a
synthetic sequence. Where the promoter is "native" or "homologous" to the plant
host, it is intended that the promoter is found in the native plant into which the
promoter is introduced. Where the promoter is "foreign" or "heterologous" to the
DNA sequence of the invention, it is intended that the promoter is not the native or
naturally occurring promoter for the operably linked DNA sequence of the invention.
The termination region may be native with the transcriptional initiation region,
may be native with the operably-linked DNA sequence of interest, may be native with
the plant host, or may be derived from another source (i.e., foreign or heterologous to
the promoter, the DNA sequence of interest, the plant host, or any combination
thereof)- Convenient termination regions are available from the Ti-plasmid of A.
tumefaciens, such as the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al (1991) Mol Gen. Genet. 262:141-144; Proudfoot (1991)
Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990)
Plant Cell 2:1261-1272; Uunroeetal. (1990) Gene 91:151-158; Ballas et al. (1989)
Nucleic Acids Res. 17:7891-7903; and ioshietal. (1987) Nucleic Acid Res. 15:9627-
9639.
Where appropriate, the gene(s) may be optimized for increased expression in
the transformed host cell. That is, the genes can be synthesized using host cellpreferred
codons for improved expression, or may be synthesized using codons at a
host-preferred codon usage frequency. See, for example, Campbell and Gowri (1990)
Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are
known in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent
Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published Application
Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
In one embodiment, the nucleic acids of interest are targeted to the chloroplast
for expression. In this manner, where the nucleic acid of interest is not directly
inserted into the chloroplast, the expression cassette will additionally contain a nucleic
acid encoding a transit peptide to direct the gene product of interest to the
chloroplasts. Such transit peptides are known in the art. See, for example, Von
Reijneetal. (1991) Plant Mol. Biol Rep. 9:104-126; Clarketal. (1989) /. Biol.
Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;
Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al.
(1986) Science 233:478-481.
Methods for transformation of chloroplasts are known in the art. See, for
example, Svab et al. (1990) Proc, Natl. Acad. Sci. USA 87:8526-8530; Svab and
Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993)
EMBO J. \ 2:601-606. The method relies on particle gun delivery of DMA containing
a selectable marker and targeting of the DNA to the plastid genome through
homologous recombination. Additionally, plastid transformation can be
accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred
expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a
system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA
91:7301-7305.
The nucleic acids of interest to be targeted to the chloroplast may be optimized
for expression in the chloroplast to account for differences in codon usage between
the plant nucleus and this organelle. In this manner, the nucleic acids of interest may
be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No.
5,380,831, herein incorporated by reference.
Evaluation of Plant Transformation
Following introduction of heterologous foreign DNA into plant cells, the
transformation or integration of heterologous gene in the plant genome is confirmed
by various methods such as analysis of nucleic acids, proteins and metabolites
associated with the integrated gene.
PCR Analysis: PCR analysis is a rapid method to screen transformed cells, tissue or
shoots for the presence of incorporated gene at the earlier stage before transplanting
into the soil (Sambrook and Russell, 2001). PCR is carried out using oligonucleotide
primers specific to the gene of interest or Agrobacterium vector background, etc.
Southern Analysis: Plant transformation is confirmed by Southern blot analysis of
genomic DNA (Sambrook and Russell, 2001). In general, total DNA is extracted
from the transformant, digested with appropriate restriction enzymes, fractionated in
an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane
or "blot" then is probed with, for example, radiolabeled 32P target DNA fragment to
confirm the integration of introduced gene in the plant genome according to standard
techniques (Sambrook and Russell, 2001. Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Northern Analysis: RNA is isolated from specific tissues of transformant, fractionated
in a formaldehyde agarose gel, blotted onto a nylon filter according to standard
procedures that are routinely used in the art (Sambrook, J., and Russell, D.W. 2001.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY). Expression of RNA encoded by the delta-endotoxin or
delta-endotoxin-associated is then tested by hybridizing the filter to a radioactive
probe derived from a delta-endotoxin or delta-endotoxin-associated protein , by
methods known in the art (Sambrook and Russell, 2001).
Western blot and Biochemical assays: Western blot and biochemical assays and the
like may be carried out on the transgenic plants to confirm the presence of protein
encoded by the delta-endotoxin or delta-endotoxin-associated gene by standard
procedures (Sambrook, J., and Russell, D.W. 2001. Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) using
antibodies that bind to one or more epitopes present on the delta-endotoxin or deltaendotoxin-
associated protein.
Pesticidal activity in plants
In another aspect of the invention, one may generate transgenic plants
expressing delta-endotoxin or delta-endotoxin-associated proteins that have pesticidal
activity. Methods described above by way of example may be utilized to generate
transgenic plants, but the manner in which the transgenic plant cells are generated is
not critical to this invention. Methods known or described in the art such as
Agrobacterium-mediated transformation, aerosol beam, biolistic transformation, and
non-particle-mediated methods may be used at the discretion of the experimenter.
Plants expressing delta-endotoxin or delta-endotoxin-associated proteins may be
isolated by common methods described in the art, for example by transformation of
callus, selection of transformed callus, and regeneration of fertile plants from such
transgenic callus. In such process, one may use any gene as a selectable marker so
long as its expression in plant cells confers ability to identify or select for transformed
cells.
A number of markers have been developed for use with plant cells, such as
resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like.
Other genes that encode a product involved in chloroplast metabolism may also be
used as selectable markers. For example, genes that provide resistance to plant
herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use.
Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314
(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res.
18:2188 (AHAS imidazolinone resistance gene).
Fertile plants expressing a delta-endotoxin or a delta-endotoxin-associated
protein may be tested for pesticidal activity, and the plants showing optimal activity
selected for further breeding. Methods are available in the art to assay for pest
activity. Generally, the protein is mixed and used in feeding assays. See, for example
Marrone et al. (1985) J. of Economic Entomology 78:290-293.
Use in Pesticidal Control
General methods for employing the strains of the invention in pesticide control
or in engineering other organisms as pesticidal agents are known in the art. See, for
example U.S. Patent No. 5,039,523 and EP 0480762A2.
The Bacillus strains of the invention or the microorganisms which have been
genetically altered to contain the pesticidal gene and protein may be used for
protecting agricultural crops and products from pests, hi one aspect of the invention,
whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with
reagents that prolong the activity of the toxin produced in the cell when the cell is
applied to the environment of target pest(s).
Alternatively, the pesticide is produced by introducing a heterologous gene
into a cellular host. Expression of the heterologous gene results, directly or indirectly,
in the intracellular production and maintenance of the pesticide. In one aspect of this
invention, these cells are then treated under conditions that prolong the activity of the
toxin produced in the cell when the cell is applied to the environment of target pest(s).
The resulting product retains the toxicity of the toxin. These naturally encapsulated
pesticides may then be formulated in accordance with conventional techniques for
application to the environment hosting a target pest, e.g., soil, water, and foliage of
plants. See, for example EPA 0192319, and the references cited therein.
Alternatively, one may formulate the cells expressing the genes of this invention such
as to allow application of the resulting material as a pesticide.
The active ingredients of the present invention are normally applied in the
form of compositions and can be applied to the crop area or plant to be treated,
simultaneously or in succession, with other compounds. These compounds can be
fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps,
dormant oils, polymers, and/or time-release or biodegradable carrier formulations that
permit long-term dosing of a target area following a single application of the
formulation. They can also be selective herbicides, chemical insecticides, virucides,
microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides,
mollusocides or mixtures of several of these preparations, if desired, together with
further agriculturally acceptable carriers, surfactants or application-promoting
adjuvants customarily employed in the art of formulation. Suitable carriers and
adjuvants can be solid or liquid and correspond to the substances ordinarily employed
in formulation technology, e.g. natural or regenerated mineral substances, solvents,
dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise the formulations
may be prepared into edible "baits" or fashioned into pest "traps" to permit feeding or
ingestion by a target pest of the pesticidal formulation.
Preferred methods of applying an active ingredient of the present invention or
an agrochemical composition of the present invention which contains at least one of
the pesticidal proteins produced by the bacterial strains of the present invention are
leaf application, seed coating and soil application. The number of applications and
the rate of application depend on the intensity of infestation by the corresponding
pest.
The composition may be formulated as a powder, dust, pellet, granule, spray,
emulsion, colloid, solution, or such like, and may be preparable by such conventional
means as desiccation, lyophilization, homogenation, extraction, filtration,
centrifugation, sedimentation, or concentration of a culture of cells comprising the
polypeptide. In all such compositions that contain at least one such pesticidal
polypeptide, the polypeptide may be present in a concentration of from about 1% to
about 99% by weight.
Lepidopteran or coleopteran pests may be killed or reduced in numbers in a
given area by the methods of the invention, or may be prophylactically applied to an
environmental area to prevent infestation by a susceptible pest. Preferably the pest
ingests, or is contacted with, a pesticidally-effective amount of the polypeptide. By
"pesticidally-effective amount" is intended an amount of the pesticide that is able to
bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or
normal physiological development. This amount will vary depending on such factors
as, for example, the specific target pests to be controlled, the specific environment,
location, plant, crop, or agricultural site to be treated, the environmental conditions,
and the method, rate, concentration, stability, and quantity of application of the
pesticidally-effective polypeptide composition. The formulations may also vary with
respect to climatic conditions, environmental considerations, and/or frequency of
application and/or severity of pest infestation.
The pesticide compositions described may be made by formulating either the
bacterial cell, crystal and/or spore suspension, or isolated protein component with the
desired agriculturally-acceptable carrier. The compositions may be formulated prior to
administration in an appropriate means such as lyophilized, freeze-dried, desiccated,
or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer.
The formulated compositions may be in the form of a dust or granular material, or a
suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a
wettable powder, or in combination with any other carrier material suitable for
agricultural application. Suitable agricultural carriers can be solid or liquid and are
well known in the art. The term "agriculturally-acceptable carrier" covers all
adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are
ordinarily used in pesticide formulation technology; these are well known to those
skilled in pesticide formulation. The formulations may be mixed with one or more
solid or liquid adjuvants and prepared by various means, e.g., by homogeneously
mixing, blending and/or grinding the pesticidal composition with suitable adjuvants
using conventional formulation techniques. Suitable formulations and application
methods are described in U.S. Patent No. 6,468,523, herein incorporated by reference.
"Pest" includes but is not limited to, insects, fungi, bacteria, nematodes, mites,
ticks, and the like. Insect pests include insects selected from the orders Coleoptera,
Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,
Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,
Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera.
Insect pests include insects selected from the orders Coleoptera, Diptera,
Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera,
Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc.,
particularly Coleoptera and Lepidoptera. Insect pests of the invention for the major
crops include: Maize: Ostrinia nubilalis, European com borer; Agrotis ipsilon, black
cutworm; Helicoverpa zea, corn earworm; Spodopterafrugiperda, fall armyworm;
Diatraea grandiosella, southwestern com borer; Elasmopalpus lignosellus, lesser
cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western
corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica
undecimpunctata howardi, southern corn rootworm; Melanotus spp., wireworms;
Cydocephala bo real is, northern masked chafer (white grub); Cyclocephala
immaculata, southern masked chafer (white grub); Popilliajaponica, Japanese beetle;
Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug;
Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid;
Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged
grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura,
seedcorn maggot; Agromyzaparvicornis, corn blot leafminer; Anaphothrips
obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted
spider mite; Sorghum: Chilopartellus, sorghum borer; Spodoptera frugiperda, fall
armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser
cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white
grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal
leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize
billbug; Rhopalosiphum maidis; corn leaf aphid; Siphaflava, yellow sugarcane aphid;
Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted
spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda,
fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia,
western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema
melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica
undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis
graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus
femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential
grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor,
Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem
maggot; Hylemya coarctata, wheat bulb fly; Frankliniellafusca, tobacco thrips;
Cephus cinctus, wheat stem sawfiy; Aceria tulipae, wheat curl mite; Sunflower:
Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth;
zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens,
cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet
armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, boll
weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;
Trialeurodes ahutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant
bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis,
differential grasshopper; Thrips (abaci, onion thrips; Franklinkiellafusca, tobacco
thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,
twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera
frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape
colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil;
Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, chinch bug;
Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean
looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green
cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;
Spodoptera exigua, beet armyworm; Heliothis virescens, cotton budworm;
Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus
persicae, green peach aphid; Empoascafabae, potato leamopper; Acrosternum hilare,
green slink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus
differentialis, differential grasshopper; Hylemya platura, seedcorn maggot;
Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus
turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite;
Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;
Schizaphis graminum, greenbug; Blissus leucopterus leucopterus, chinch bug;
Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Delia
platura, seedcom maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown
wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta
cruciferae, Flea beetle; Mamestra configurata, Bertha armyworm; Plutella xylostella,
Diamond-back moth; Delia ssp., Root maggots.
Nematodes include parasitic nematodes such as root-knot, cyst, and lesion
nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.;
particularly members of the cyst nematodes, including, but not limited to, Heterodera
glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode);
Heterodera avenue (cereal cyst nematode); and Globodera rostochiensis and
Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus
spp.
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
Example 1. Extraction of Plasmid DNA
Plasmid DNA from strains ATX 13026 or ATX 13002 were prepared in the
following way. A pure culture of strain ATX 13026 or strain ATX 13002 was grown in
large quantities of rich media. The culture was centrifuged to harvest the cell pellet.
The cell pellet was then prepared by treatment with SDS by methods known in the art,
resulting in breakage of the cell wall and release of DNA. Proteins and large genomic
DNA was then precipitated by a high salt concentration. The plasmid DNA was
precipitated by standard ethanol precipitation. The plasmid DNA was separated from
any remaining chromosomal DNA by high-speed centrifugation through a cesium
chloride gradient. The DNA was visualized in the gradient by UV light and the band
of lower density (i.e. the lower band) was extracted using a syringe. This band
contained the plasmid DNA from the strain (either ATX 13026 or ATX 13002) The
quality of the DNA was checked by visualization on an agarose gel by methods
known in the art.
Example 2. Cloning of Genes
The purified plasmid DNA was sheared into 5-10 kb sized fragments and the
5' and 3' single stranded overhangs repaired using T4 DNA polymerase and Klenow
fragment in the presence of all four dNTPs, as known in the art. Phosphates were
then attached to the 5' ends by treatment with T4 polynucleotide kinase, as known in
the art. The repaired DNA fragments were ligated overnight into a standard high
copy vector (i.e. pBluescript SK+), suitably prepared to accept the inserts as known in
the art (for example by digestion with a restriction enzyme producing blunt ends).
The quality of the library was analyzed by digesting a subset of clones with a
restriction enzyme known to have a cleavage site flanking the cloning site. A high
percentage of clones were determined to contain inserts, with an average insert size of
5-6 kb.
Example 3. High Throughput Sequencing of Library Plates
The libraries prepared by the methods above were plated onto rich media
containing the appropriate antibiotic to maintain the plasmids clones, and colonies
were individually picked into 96-well blocks containing 2 mis of media containing the
appropriate antibiotic. These blocks were grown overnight at 37°C at a shaking speed
of 350 rpm. The blocks were centrifuged to harvest the cells to the bottom of the
block. Plasmid DNA was isolated from these cultures by standard alkaline lysis prep
in a high throughput format.
The end sequences of clones from this library were determined for a large
number of clones from each block in the following way: The DNA sequence of each
clone chosen for analysis was determined using the fluorescent dye terminator
sequencing technique (Applied Biosystems) and standard primers flanking each side
of the cloning site. Once the reactions had been carried out in the thermocycler, the
DNA was precipitated using standard ethanol precipitation. The DNA was
resuspended in water and loaded onto a capillary sequencing machine. Each library
plate of DNA was sequenced from either end of the cloning site, yielding two reads
per plate over each insert.
Example 4. Assembly and Screening of Sequencing Data
DNA sequences obtained were compiled into an assembly project and aligned
together to form contigs. This can be done efficiently using a computer program, such
as Vector NTi, or alternatively by using the Pred/Phrap suite of DNA alignment and
analysis programs. These contigs, along with any individual read that may not have
been added to a contig, were compared to a compiled database of all classes of known
pesticidal genes. Contigs or individual reads identified as having identity to a known
endotoxin or pesticidal gene were analyzed further. Among the sequences obtained,
clones pAX004, pAX006, pAXOOV, pAXOOS, pAX009, and pAX014 contained DNA
identified as having homology to known endotoxin genes. Therefore, these clones
were selected for further sequencing.
Example 5. Sequencing and Identification of Delta-Endotoxin Genes
Primers were designed to anneal to sequences with homology to endotoxin
genes, in a manner such that DNA sequences generated from such primers would
overlap existing DNA sequence of the clone(s). This process, known as "oligo
walking," is well known in the art. This process was utilized to determine the entire
DNA sequence of the region exhibiting homology to a known endotoxin gene. In the
case of the clones mentioned above, this process was used to determine the DNA
sequence of the entire clone, resulting in a single nucleotide sequence for each gene.
The completed DNA sequence was then placed back into the original large assembly
for further validation. This allowed incorporation of more DNA sequence reads into
the contig, resulting in multiple reads of coverage over the entire region.
Analysis of the DNA sequence of each region with homology to a known
endotoxin gene identified an open reading frame with homology to a known deltaendotoxin
gene. The open reading frame identified from pAX004 is designated as
AXM1-004. The open reading frame identified from pAX006 is designated AXMI-
006. The open reading frame identified from pAXOO? is designated AXMI-007. The
open reading frame identified from pAXOOS is designated AXMI-008. The open
reading frame identified from pAX009 is designated AXMI-009. The open reading
frame identified from pAX014 is designated AXMI-014. The DNA sequence of
AXMI-004 is provided as SEQ II) NOS:1 and 2, and the amino acid sequence of the
predicted AMX1-004 protein is provided as SEQ ID NO:3. An alternate start site for
AXMI-004 at nucleotide 385 of SEQ ID NO:1 generates the amino acid sequence
provided as SEQ ID NO:5. The DNA sequence of AXMI-006 is provided as SEQ ID
NO:6, and the amino acid sequence of the predicted AMXI-006 protein is provided in
SEQ ID NO:7. The DNA sequence of AXMI-007 is provided as SEQ ID NO:8, and
the amino acid sequence of the predicted AMXI-007 protein is provided in SEQ ID
NO:9. An alternate start site for AXMI-007 at nucleotide 151 of SEQ ID NO:8
generates the amino acid sequence provided as SEQ ID NO: 11. The DNA sequence
of AXMI-008 is provided as SEQ ID NOS:12 and 13, and the amino acid sequence of
the predicted AMXI-008 protein is provided in SEQ ID NO: 14. An alternate start site
for AXMI-008 at nucleotide 177 of SEQ ID NO: 12 generates the amino acid
sequence provided as SEQ ID NO: 16. Further analysis identified an open reading
frame immediately 3' to the end of the AXMI-008 open reading frame. This predicted
amino acid sequence of this orf, referred to herein as AXMI-008orf2, is provided in
SEQ ID NO:18. The DNA sequence of AXMI-009 is provided as SEQ ID NO:19, and
the amino acid sequence of the predicted AMXI-009 protein is provided in SEQ ID
NO:20. An alternate start site for AXMI-009 at nucleotide 34 of SEQ ID NO: 19
generates the amino acid sequence provided as SEQ ID NO:22. Another alternate start
site for AXMI-009 at nucleotide 64 of SEQ ID NO: 19 generates the amino acid
sequence provided as SEQ ID NO:24. The DNA sequence of AXMI-014 is provided
as SEQ ID NOS:25 and 26, and the amino acid sequence of the predicted AMXI-008
protein is provided as SEQ ID NO:27. An alternate start site for AXMI-014 at
nucleotide 136 of SEQ ID NO:25 generates the amino acid sequence provided as SEQ
ID NO:29.
Example 6. Homology of Isolated Genes to Known Endotoxin Genes
Searches of DNA and protein databases with the DNA sequences and amino
acid sequences of the present invention reveal that these sequences are homologous to
known endo toxins.
AXMI-004
Figure 1 shows an alignment of AXMI-004 with several endotoxins. Blast
searches identify crylCa as having the strongest block of homology, with an overall
sequence identity in the toxic domain of 43% (see Table 1).
Table 1. Amino Acid Identity of AXMI-004 with Exemplary Endotoxin Classes
(Table Removed)
Figure 2 shows an alignment of AXMI-006 with several endotoxins. Blast
searches identify cry4Aa as having the strongest block of homology, though
alignment of AMXI-006 protein (SEQ ID NO:7) to a large set of endotoxin proteins
shows that the most homologous protein is crylOAa. The overall amino acid identity
of crylOAa to AXMI-006 is 25% (see Table 2). Inspection of the amino acid
sequence of AXMI-006 suggests that it does not contain a C-terminal non-toxic
domain as is present in several endotoxin families. By removing this C-terminal
protein of the toxins from the alignment, the alignment reflects the amino acid identity
present solely in the toxin domains (see Table 2, column three). This 'trimmed'
alignment is shown in Figure 2.
Table 2. Amino Acid Identity of AXMI-006 with Exemplary Endotoxin Classes
(Table Removed)
Blast searches identify cry4Aa as having the strongest block of homology to
AXMI-007, though alignment of AMXI-007 protein (SEQ ID NO:9) to a large set of
endotoxin proteins shows that the most homologous protein is crylOAa. The overall
amino acid identity ofcrylOAa to AXMI-007 is 25% (see Table 3). Inspection of the
amino acid sequence of AXMI-007 suggests that it does not contain a C-terminal nontoxic
domain as is present in several endotoxin families. By removing this C-terminal
protein of the toxins from the alignment, the alignment reflects the amino acid
identify present solely in the toxin domains (see Table 3, column three). This
'trimmed' alignment is shown in Figure 3.
Table 3. Ammo Acid Identity of AXMI-007 with Exemplary Endotoxin Classes
(Table Removed)
Blast searches identify cry40Aa as having the strongest block of homo logy to
AXMI-008, and alignment of AMXI-008 protein (SEQ ID NO: 14) to a large set of
endotoxin proteins shows that the most homologous protein is cry40Aa. The overall
amino acid identity ofcry40Aa to AXMI-008 is 66% (see Table 4). Inspection of the
amino acid sequence of AXMI-008 suggests that it does not contain a C-terminal nontoxic
domain as is present in several endotoxin families. By removing this C-terminal
protein of the toxins from the alignment, the alignment reflects the amino acid
identify present solely in the toxin domains (see Table 4, column three). This
'trimmed' alignment is shown in Figure 4.
Table 4. Amino Acid Identity of AXMI-008 with Exemplary Endotoxin Classes
(Table Removed)
The open reading frame immediately downstream (3") to the AXMI-008
coding region has homology to known endotoxin-related proteins. Blast searches
identify crybun3orf2 (the downstream orf of cry40Aa) as having the strongest block
of homology. Several other orf-2 like proteins are present in databases, and an
alignment of AMXI-008-orf2 protein (SEQ ID NO: 18) to a set of these proteins is
shown in Figure 5. These proteins also share homology to the C-terminal non-toxic
domain ofcry4Aa and cry4Ba. The overall amino acid identity of AXMI-8-or£2 to
cry40Aaorf2 is 86% (see Table 5).
Table 5. Amino acid identity of AXMI-008-orf2 to related proteins
(Table Removed)
Blast searches identify cryBAa as having the strongest block of homology to
AXMI-009, and alignment of AMXI-009 protein (SEQ ID NO:20) to a large set of
endotoxin proteins shows that the most homologous proteins are crySBa and cry!6Aa.
The overall amino acid identity of crySBa and cry!6Aa to AXMI-009 is 26% (see
Table 6). Inspection of the amino acid sequence of AXMI-009 suggests that it does
not contain a C-terminal non-toxic domain as is present in several endotoxin families.
By removing this C-terminal protein of the toxins from the alignment, the alignment
reflects the amino acid identify present solely in the toxin domains (see Table 6,
column three). This 'trimmed' alignment is shown in Figure 6.
Table 6. Amino Acid Identity of AXMI-009 with Exemplary Endotoxin Classes
(Table Removed)
Blast searches identify cry40Aa as having the strongest block of homology to
AXMI-014, and alignment of AMXI-0014 protein (SEQ ID NO:27) to a large set of
endotoxin proteins shows that the most homologous protein is cry40Aa. The overall
amino acid identity ofcry40Aa to AXMI-014 is 55% (see Table 7). Inspection of the
amino acid sequence of AXMI-014 suggests that it does not contain a C-terminal nontoxic
domain as is present in several endotoxin families. By removing this C-terminal
protein of the toxins from the alignment, the alignment reflects the amino acid
identify present solely in the toxin domains (see Table 7, column three). This
'trimmed' alignment is shown in Figure 7.
Table 7. Amino Acid Identity of AXMI-014 with Exemplary Endotoxin
(Table Removed)
Example 7. Homology between AXMI-006 and AXMI-007
Comparison of the amino acid sequences of AXMI-007 with AXMI-006
shows that the two toxins share significant amino acid homo logy. Alignment of the
amino acid sequence of AXMI-006 (SEQ ID NO:7) and AXMI-007 (SEQ ID NO:9)
show the proteins to be 85 % identical at the amino acid level. Thus AXMI-006 and
AXMI-007 constitute a new class of related endotoxins.
Example 8. Assays for Pesticidal Activity
The ability of a pesticidal protein to act as a pesticide upon a pest is often
assessed in a number of ways. One way well known in the art is to perform a feeding
assay. In such a feeding assay, one exposes the pest to a sample containing either
compounds to be tested, or control samples. Often this is performed by placing the
material to be tested, or a suitable dilution of such material, onto a material that the
pest will ingest, such as an artificial diet. The material to be tested may be composed
of a liquid, solid, or slurry. The material to be tested may be placed upon the surface
and then allowed to dry. Alternatively, the material to be tested may be mixed with a
molten artificial diet, then dispensed into the assay chamber. The assay chamber may
be, for example, a cup, a dish, or a well of a microtiter plate.
Assays for sucking pests (for example aphids) may involve separating the test
material from the insect by a partition, ideally a portion that can be pierced by the
sucking mouthparts of the sucking insect, to allow ingestion of the test material. Often
the test material is mixed with a feeding stimulant, such as sucrose, to promote
ingestion of the test compound.
Other types of assays can include microinjection of the test material into the
mouth, or gut of the pest, as well as development of transgenic plants, followed by
test of the ability of the pest to feed upon the transgenic plant. Plant testing may
involve isolation of the plant parts normally consumed, for example, small cages
attached to a leaf, or isolation of entire plants in cages containing insects.
Other methods and approaches to assay pests are known in the art, and can be
found, for example in Robertson, J. L. & H. K. Preisler. 1992. Pesticide bioassays
with arthropods. CRC, Boca Raton, FL. Alternatively, assays are commonly
described in
the journals "Arthropod Management Tests" and "Journal of Economic Entomology"
or by discussion with members of the Entomological Society of America (ESA).
Example 9. Cloning of AXMI-006 for Protein Expression
AXMI-006 was cloned into a vector for E. coli expression as follows.
pAX480 contains the kanamycin resistance gene for selection of transformants, and
the tac promoter which is inducible by EPTG for regulated protein expression.
pAX480 was modified by inserting a DNA segment encoding a 6xHis-tag region
immediately upstream of the insert cloning region, such that resulting clones contain a
6xHis-tag at the N-terminus of the expressed protein. Methods for expressing proteins
with 6xHis-tag fusions, and their use for purification and analysis of protein
expression are well known in the art.
The coding sequence for AXMI-006 was PCR-amplified using PfuUltra™
High-Fidelity DNA Polymerase (Stratagene). Oligonucleotide primers were designed
such that the resulting PCR product contained desired restriction sites near each end,
to facilitate cloning. The resulting PCR product (approximately 2.2 kb) was digested
with the appropriate restriction enzyme, and subcloned into the modified pAX480.
Insert-containing clones were identified by restriction analysis. The resulting clone,
pAX906, contained the AXMI-006 open reading frame fused to the six his tag, such
that transcription and translation resulted in production of a 'fusion protein' with a
stretch of six histidines. The DNA sequence of pAX906 was confirmed by DNA
sequence analysis and subsequently transformed into chemically competent E. coli
BL21, as described by the manufacturer (Stratagene, La Jolla, CA).
A single colony of pAX906 in BL21 was inoculated into LB media
supplemented with kanamycin and grown for several hours at 37°C with vigorous
agitation. These cultures were grown to an OD6oo ranging from 0.6-0.8; then protein
production was induced by addition of 0.1 mM IPTG. Cultures were grown under
inducing conditions for 3 hours, and then the cells were pelleted by centrifugation and
resuspended in PBS. Cells were sonicated using a Misonix Sonicator 3000 for a total
of 30 seconds using 10-second sonication intervals and incubation on ice for one
minute.
Example 10. Bioassay of AXMI-006 Activity on Heliothis virescens and Spodoptera
frugiperda
Bioassays of sonicated pAX906 cultures were performed using artificial diet
(Multiple Species Diet, Southland Products, Lake Village, Arkansas). Bioassays were
carried out by applying sonicated pAX906 cells, or cells of the E.coli strain as a
control, to the diet surface and allowing the diet surface to dry. Bioassays were
performed in 24-well tissue culture plates. The bioassays were held in the dark at 25°
C and 65% relative humidity. Trays were sealed with Breathe Easy Sealing Tape
(Diversified Biotech, Boston, MA). Results were recorded at 5 days.
Table 8. Bioassay of AXMI-006 on Spodoptem frugiperda
Sample
AXMI-006
Negative control
Bioassay Result
Stunting
No Stunting
Table 9. Bioassay of AXMI-006 on Heliothis virescens
Sample
AXMI-006
Negative Control
Bioassay Result
Stunting
No Stunting
Stunting is defined reduced insect size, and severally reduced larval feeding.
Stunted insects may also demonstrate avoidance of the treated diet compared to the
untreated diet.
Example 11. Pesticidal Activity of AXMI-008. AXMI-009. and AXMI-014 on
Trichoplusia ni (Cabbage Looper)
Escherichia coli strains containing either pAXOOS, pAX009 or pAX-014, as
well as a culture of untransformed Escherichia coli were grown in 2 ml of LB Broth
(Luria-Bertani Broth, Becton Dickinson & Company, Sparks, Md.) for 24 hours at 37C
C with agitation at 250 rpm. Plasmid-containing strains were grown in grown in LB
containing the appropriate antibiotic to select for maintenance of the plasmid in E.
coli.
Bioassays were performed using artificial diet (Multiple Species Diet,
Southland Products, Lake Village, Arkansas) in 24 well tissue culture plates.
Bioassays were carried out by applying the Escherichia coli culture containing pAX-
014 to the diet surface and allowing the diet surface to dry. The strains were applied
as whole cultures to the diet at a concentration of 40 ul of culture per well. The
bioassays were held in the dark at 25° C and 65% relative humidity. Trays were
sealed with Breathe Easy Sealing Tape (Diversified Biotech, Boston, MA). Results
were recorded at 5 days.
-*srTable
10. Pesticidal Activity on T. ni
Sample
AXMI-014
AXMI-008
AXMI-009
Negative Control
# Dead/ Total
13/13
6/6
17/17
0/13
% Mortality
100%
100%
100%
0%
Example 12. Expression of Delta-Endotoxin Genes in Bacillus
AXMl-004, AXMI-006, AXMI-007, AXMI-008, and AXMI-009 were
amplified by PCR from the clones from Example 4, and cloned into the Bacillus
Expression vector pAX916 by methods well known in the art. For AXMI-004 the
resulting clone was designated pAX920. For AXMI-006 the resulting clone was
designated pAX921. For AXMI-007 the resulting clone was designated pAX919. For
AXMI-008 the resulting clone was designated pAX922. For AXMI-009 the resulting
clone was designated pAX917. The resulting clones expressed the relevant protein
when transformed into cells of a cry(-) Bacillus thuringiensis strain. The Bacillus
strains containing delta-endotoxin genes and expressing the delta-endotoxin proteins
may be cultured on a variety of conventional growth media. A Bacillus strain
containing the desired gene was grown in CYS media (10 g/1 Bacto-casitone; 3 g/1
yeast extract; 6 g/1 KH2PO4; 14 g/1 K2HPO4; 0.5 mM MgSO4; 0.05 mM MnCl2; 0.05
mM FeSO4), until sporulation was evident by microscopic examination. Samples were
prepared, and delta-endotoxin proteins were tested for insecticidal activity in
bioassays against important insect pests.
Methods
To prepare CYS media: 10 g/1 Bacto-casitone; 3 g/1 yeast extract; 6 g/1
KH2PO4; 14 g/1 K2HPO4; 0.5 mM MgSO4; 0.05 mM MnCl2; 0.05 mM FeSO4. The
CYS mix should be pH 7, if adjustment is necessary. NaOH or HC1 are preferred.
The media is then autoclaved and 100 ml of 10X filtered glucose is added after
autoclaving. If the resultant solution is cloudy it can be stirred at room temperature to
clear.
Example 13- N-terniinal Amino Acid Sequence of AXMI-004 Expressed in Bacillus
Analysis of AXMI-004 expressed in Bacillus suggested that the protein
product detected in these cultures may be reduced in size relative to the full-length
AXMI-004 protein. Since many endotoxin proteins are cleaved at the N-terminus after
expression in Bacillus, we determined the N-terminus of the AXMI-004 protein
resulting from Bacillus expression. Protein samples from AXMI-004 were separated
on PAGE gels, and the protein transferred to PVDF membrane by methods known in
the art. The protein band corresponding to AXMI-004 was excised. The N-terminal
amino acid sequence of this protein was determined by serial Edman degradation as
known in the art. The sequence obtained was as follows:
ERFDKNDALE
Comparison of this amino acid sequence with the sequence of the full length
AXMI-004 (SEQ ID NO:3) demonstrates that this amino sequence results from
internal cleavage of the AXMI-004 after expression in Bacillus, resulting in a protein
with an N-terminus corresponding to amino acid 28 of SEQ ID NO:3 (disclosed as
SEQ ID NO:5).
Example 14. Bioassay of AXMI-004 on Insect Pests
Insecticidal activity of AXMI-004 was established utilizing accepted bioassay
procedures using a sporulated Bacillus cell culture lysate expressing AXMI-004. The
Bacillus culture was grown in 50 ml CYS media for both standard bioassay and LCso
bioassays. The cultures were then grown for 2 to 3 days at 30°C, 250 rpm until the
cells were sporulated. Sporulation was determined by examining microscopically for
the presence of spores. AXMI-004 protein samples were prepared by centrifugation of
the sporulated cultures at 12,000 x g for 10 min. The pellet was collected and
resuspended in 4 ml 20 mM Tris-HCl, pH 8.0. The suspension was sonicated for 20
seconds (at top power using a micro probe) while placing the tube on ice. The protein
concentration of the sample was determined by electrophoresis on an SDS 4-20%
gradient acrylamide gel along with a known quantity of bovine serum albumin (BSA)
(Figure 8). The concentration of AXMI-004 was determined to be 0.4 jag/ul.
AXMI-004 insecticidal activity was tested using a surface treatment bioassay
with artificial diet (Multiple Species diet, Southland Products, Lake Village,
Arkansas) prepared as known in the art. Bioassays were carried out by applying the
Bacillus culture expressing AXMI-004 to the diet surface and allowing the surface to
air-dry. Standard bioassays utilized five eggs per well and LCjo bioassays utilized ten
neonate insect larvae per well. The eggs or larvae were applied using a fine tip
paintbrush. Standard surface bioassays were carried out in 24 well tissue culture
plates. 40ul of each sample was applied to each well. Since each well has a surface
area of 2 cm2 (plate source), a 40 ul cell lysate sample contained approximately 0.4
ug/ul AXMI-004. Bioassays where the LCso was determined were done in 48 well
tissue culture plates, each well representing a surface area of 1 cm2 (source) using
approximately 20ul of 0.4 jag/ul AXMI-004 per well. The final amount of AXMI-004
protein in each bioassay was approximately 8 |o.g/cm2. Bioassay trays were sealed
with Breathe Easy Sealing Tape (Diversified Biotech, Boston MA). Control samples
included media only samples, and wells that were not treated with samples. Bioassays
were then held for five days in the dark at 25° C and 65% relative humidity and
results recorded.
Table 11. Insecticidal Activity of AXMI-004
Insect (Latin Name)
Ostrinia nubilalis
Agrotis ipsilon
Heliothis zea
Spodoptera frugiperda
Heliothis vires cens
Pectinophora gossypiella
Manduca sexta
Trichoplusia ni
Common Name
European Corn Borer
Black Cutworm
Corn Earworm
Fall Armyworm
Tobacco Budworm
Pink Bollworm
Tobacco Hornworm
Cabbage Looper
Activity of AXMI-004
100% mortality
Stunted
Stunted
Stunted
100% mortality
75% mortality
100% mortality
100% mortality
AXM1-004 showed strong insecticidal activity (100% mortality) against
Ostrinia nubilalis and Heliothis virescens. AXMI-004 also showed insecticidal
activity of 50-75% mortality against Pectinophora gossypiella. A concentration of 43
(ig/cm2 AXMI-004 gave 70% mortality against Pectinophora gossypiella. AXMI-004
severely stunted the growth ofAgrotis ipsilon, Heliothis zea, and Spodoptera
frugiperda.
Example 15. Quantitation of AXMI-Q04 Insecticidal Activity against Heliothis
virescens and Ostrinia nubilalis
The LCso of AXMI-004 protein on Ostrinia nubilalis and Heliothis virescens
larvae were determined by testing a range of AXMI-004 protein concentrations in
insect bioassays, and applying these protein samples to the surface of insect diet.
Mortality was recorded at each protein concentration and analyzed using a Probit
analysis program. Results were significant at the 95% confidence interval. Since
assays were performed by surface contamination, LC50s were determined assuming
that the entire protein sample remained at the surface during the assay, with little
diffusion below the level ingested by the insects. Thus, the values determined may
somewhat underestimate the toxicity of the AXMI-004 protein on the tested insects.
Table 12. LC50 of AXMI-004 on Ostrinia nubilalis
(Table Removed)
LC50= 2874 ng/cm2; 95% CI = 2189-3933
Example 16. Quantitation of AXMI-OQ4, AXM1-Q06, AXMI-007, and AXMI-009
Insecticidal Activity against Lygus lineolaris
Bacterial lysates were prepared by growing the Bacillus in 50 ml of CYS
media for 60 hours. The Bacillus culture was then centrifuged at 12,000 rpm for ten
minutes and the supernatant discarded. The pellet was resuspended in 5 ml of 20 mM
Tris HCI at pH 8.
Bioassays were performed by cutting both the tip and the cap off an Eppendorf
tube to form a feeding chamber. The insecticidal protein or control was presented to
the insect in a solution that was poured into the cap and covered with parafilm
(Pechiney Plastic Packaging, Chicago 1L) that the insect could pierce upon feeding.
The Eppendorf tube was placed back on the cap top down and 1st or 2nd instar Lygus
nymphs were placed into the Eppendorf chamber with a fine tip brush. The cut
Eppendorf tube tip was sealed with parafilm creating an assay chamber. The resultant
assay chamber was incubated at ambient temperature cap side down. Insecticidal
proteins were tested in a solution of 15% glucose at a concentration of 6.6 ug/ml.
Table 14. Insecticidal Activity on Lygus lineolaris
Protein
AXMI-004
AXMI-006
AXMI-007
AXMI-009
Control
No. Dead/Total
2/4
1/6
3/6
2/4
0/9
% Mortality
50%
16.7%
50%
50%
0%
Example 17. Bioassay of AXM1-008 on Tenebrio molitor
Samples of Bacillus cultures expressing AXMI-008 were prepared and tested
for pesticidal activity on Tenebrio molitor. When pAX 922 is prepared as an insoluble
fraction at pH 4.0 it showed activity against commonly called the yellow mealworm.
Samples of AXMI-008 were prepared from a culture of a Bacillus strain
containing pAX 922. The bioassay sample was prepared by growing a culture in CYS
media for 4 days, until sporulation. The sample was centrifuged at 10,000 rpm for 10
minutes and the supernatant discarded. The pellet was washed in 20mM Tris pH 8 and
spun at 10,000 rpm for 10 minutes. The supernatant was discarded and the pellet
resuspended in 3 mis of 50 mM Sodium Citrate and 25 mM Sodium Chloride with
2mM DTT at pH 4. The sample was incubated at 37° C for 1 hour. After incubation
the sample was spun at 13,000 rpm for 10 minutes and the supernatant discarded. The
pellet was resuspended in 50 mM Sodium Citrate and 25 mM Sodium Chloride at pH
4.
Bioassays of samples on Tenebrio molitor were performed on an artificial diet
(Southern Com Rootworm Diet, Bioserv, Frenchtown, NJ, #F9757B) in 24 well tissue
culture plates. The sample was applied as a surface treatment with a concentration of
AxmiOOS at 8 ug/cm2 and allowed to air dry. The insects were applied using a fine tip
brush. Bioassay trays were sealed with Breathe Easy Sealing Tape (Diversified
Biotech, Boston, MA) and incubated without light at 65% relative humidity, 25° C.
for seven days and results recorded.
Table 15. Pesticidal Activity of AXMI-008 on T. molitor
Sample # Dead/ Total
3 of 4
% Mortality
AXMI-008 75%*
* Remaining Tenebrio molitor was stunted. Stunting is observed as reduced larval
size and growth, and severely reduced or minimal feeding. The insect may also
demonstrate avoidance of the treated diet compared to the untreated diet.
Example 18. Bioassay of AXMI-009 Protein on Coleopteran Pests
Bioassays of AXMI-009 protein preparations were performed by pipetting 40
\\\ of insoluble fraction onto a 2 cm2 diet surface for a final total protein concentration
of Sfig/cm". Diahrotica virgifera virgifera and Diabrotica undecimpunctata were
tested using Southern Com Rootworrn Diet (Bioserv, Frenchtown, NJ, #F9757B).
Bioassays were carried out by applying the Bacillus culture expressing AXMI-009 to
the diet surface and allowing the diet surface to dry. Bioassays were performed in 24
well tissue culture plates. Standard bioassays utilized 25 eggs per well. The eggs were
applied in a solution containing 0.1% agar and 30 ug/ml nystatin. Trays were sealed
with Breathe Easy Sealing Tape (Diversified Biotech, Boston, MA) and the lids
placed back on the trays. Bioassays were incubated without light at 65% Relative
Humidity (RH), 25° C for seven days. Activity was seen with the insoluble fraction
for both Diahrotica virgifera virgifera and Diabrotica undecimpunctata. Controls
were a media only, buffer of 1 mM Tris at pH 10.5, and the Bacillus expression vector
pAX916.
Table_l 6._ Western Corn Rootworrn (Diabrotica virgifera virgifera)
(Table Removed)
_ Table 17. Southern Corn Rootworm (Diabrotica undecimpunctatd)
(Table Removed)
Example 19. Vectoring for Plant Expression
The delta-endotoxin coding region DNA is operably connected with
appropriate promoter and terminator sequences for expression in plants. Such
sequences are well known in the art and may include the rice actin promoter or maize
ubiquitin promoter for expression in monocots, the Arabidopsis UBQ3 promoter or
CaM V 35S promoter for expression in dicots, and the nos or Pinll terminators.
Techniques for producing and confirming promoter - gene - terminator constructs
also are well known in the art.
The plant expression cassettes described above are combined with an
appropriate plant selectable marker to aid in the selections of transformed cells and
tissues, and Hgated into plant transformation vectors. These may include binary
vectors from Agrobacterium-medialQd transformation or simple plasmid vectors for
aerosol or biolistic transformation.
Example 20. Transformation of Maize Cells
Maize ears are collected 8-12 days after pollination. Embryos are isolated
from the ears, and those embryos 0.8-1.5 mm in size are used for transformation.
Embryos are plated scutellum side-up on a suitable incubation media, such as
DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of lOOOx Stock) N6 Vitamins; 800 mg/L LAsparagme;
100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L
sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D), and incubated overnight at 25°C in the dark.
The resulting explants are transferred to mesh squares (30-40 per plate),
transferred onto osmotic media for 30-45 minutes, then transferred to a beaming plate
(see, for example, PCT Publication No. WO/0138514 and U.S. Patent No. 5,240,842).
DNA constructs designed to express the delta-endotoxin in plant cells are
accelerated into plant tissue using an aerosol beam accelerator, using conditions
essentially as described in PCT Publication No. WO/0138514. After beaming,
embryos are incubated for 30 min on osmotic media, then placed onto incubation
media overnight at 25°C in the dark. To avoid unduly damaging beamed explants,
they are incubated for at least 24 hours prior to transfer to recovery media. Embryos
are then spread onto recovery period media, for 5 days, 25°C in the dark, then
transferred to a selection media. Explants are incubated in selection media for up to
eight weeks, depending on the nature and characteristics of the particular selection
utilized. After the selection period, the resulting callus is transferred to embryo
maturation media, until the formation of mature somatic embryos is observed. The
resulting mature somatic embryos are then placed under low light, and the process of
regeneration is initiated by methods known in the art. The resulting shoots are allowed
to root on rooting media, and the resulting plants are transferred to nursery pots and
propagated as transgenic plants.
Materials
DN62A5S Media
Components
Chu'S N6 Basal
Salt Mixture
(Prod. No. C
416) ,
Chu'sN6
Vitamin
Solution (Prod.
No. C 149)
L-Asparagine
Myo-inositol
L-Proline
Casaminoacids
Sucrose
2,4-D (Prod. No.
D-7299)
per liter
3.98 g/L
1 mL/L (of lOOOx Stock)
800 mg/L
100 mg/L
1.4 g/L
100 mg/L
50 g/L
1 mL/L (of 1 mg/mL Stock)
Source
Phytotechnology Labs
Phytotechnology Labs
Phytotechnology Labs
Sigma
Phytotechnology Labs
Fisher Scientific
Phytotechnology Labs
Sigma
Adjust the pH of the solution to pH to 5.8 with IN KOH/1N KC1, add Gelrite
(Sigma) to 3g/L, and autoclave. After cooling to 50°C, add 2 ml/L of a 5 mg/ml stock
solution of Silver Nitrate (Phytotechnology Labs). Recipe yields about 20 plates.
Example 21. Transformation into Plant Cells by Agrobacterium-Mediated
Transformation
Ears are collected 8-12 days after pollination. Embryos are isolated from the
ears, and those embryos 0.8-1.5 mm in size are used for transformation. Embryos are
plated scutellum side-up on a suitable incubation media, and incubated overnight at
25°C in the dark. However, it is not necessary per se to incubate the embryos
overnight. Embryos are contacted with an Agrobacterium strain containing the
appropriate vectors for Ti plasmid mediated transfer for 5-10 min, and then plated
onto co-cultivation media for 3 days (25°C in the dark). After co-cultivation, explants
are transferred to recovery period media for five days (at 25°C in the dark). Explants
are incubated in selection media for up to eight weeks, depending on the nature and
characteristics of the particular selection utilized. After the selection period, the
resulting callus is transferred to embryo maturation media, until the formation of
mature somatic embryos is observed. The resulting mature somatic embryos are then
placed under low light, and the process of regeneration is initiated as known in the art.
The resulting shoots are allowed to root on rooting media, and the resulting plants are
transferred to nursery pots and propagated as transgenic plants.
Conclusions
The delta-endotoxin proteins of the present invention have activity against
numerous pests, as shown in the examples above. AXMI-004 has pesticidal activity
against pests including Ostrinia nubilalis, Agrotis ipsilon, Heliothis zea, Spodoptera
frugiperda, Heliothis virescens, Pectinophora gossypiella, Manduca Sexta,
Trichoplasia ni, and Lygus lineolaris. AXMI-006 has pesticidal activity against pests
including Heliothis virescens, Spodoptera frugiperda, and Lygus lineolaris. AXMI-
007 has pesticidal activity against pests including Lygus lineolaris. AXMI-008 has
pesticidal activity against pests including Tenebrio molitor and Trichoplusia ni.
AXMI-009 has pesticidal activity against pests including Lygus lineolaris,
Trichoplusia ni, Diabrotica virgifera virgifera, and Diabrotica undecimpunctata.
AXMI-014 has pesticidal activity against pests including Trichoplusia ni.
All publications and patent applications mentioned in the specification are
indicative of the level of skill of those skilled in the art to which this invention
pertains. All publications and patent applications are herein incorporated by reference
to the same extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be obvious
that certain changes and modifications may be practiced within the scope of the
appended claims.










WE CLAIM:
1. A recombinant nucleic acid molecule selected from the group consisting of:
a. a nucleic acid molecule comprising the nucleotide sequence selected from
SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 4;
b. a nucleic acid molecule comprising a nucleotide sequence having at least 95%
sequence identity to the sequence of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ
ID NO. 4; and
c. a complement of any of the nucleic acid molecule of (a) and (b);
wherein, the nucleic acid molecule encodes a delta-endotoxin polypeptide having pesticidal activity.
2. A nucleic acid molecule as claimed in claim 1, wherein the nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.
3. A nucleic acid molecule as claimed in claim 2, wherein the synthetic sequence has increased GC content.
4. A vector comprising the nucleic acid molecule as claimed in claim 1, wherein the vector is a copy vector and/or expression vector.
5. A vector as claimed in claim 4, wherein the copy vector is pBluescript vector and expression vector is pAX916.
6. A vector as claimed in claim 4, wherein the nucleic acid molecule encodes a heterologous polypeptide.
7. A bacterial host cell comprising the vector as claimed in claim 4.
8. A bacterium host cell as claimed in claim 7, wherein the cell is Bacillus and Agrobacterium.
9. An isolated polypeptide encoded by the nucleic acid molecule as claimed in claim 1 selected from the group consisting of:
a. a polypeptide comprising the amino acid sequence selected from SEQ ID NO. 3 and SEQ ID NO. 5;
b. a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 3 and SEQ ID NO. 5; and
wherein, said polypeptide has pesticidal activity.
10. A polypeptide as claimed in claim 9, wherein the polypeptide comprises a heterologous amino acid sequence.
11. A pesticidally-effective polypeptide composition as and when prepared with the polypeptide encoded by the nucleic acid molecule as claimed in claim 1, wherein the concentration of the polypeptide ranges from 1% to 99% by weight.
12. A composition as claimed in claim 11, wherein the composition is selected from the group consisting of powder, dust, pellet, granule, spray, emulsion, colloid and solution.
13. A nucleic acid molecule, a vector, a bacterial host cell, a polypeptide and a composition substantially such as herein described with reference to the accompanying drawings and as illustrated in the foregoing examples.


Documents:


Patent Number 237910
Indian Patent Application Number 3665/DELNP/2005
PG Journal Number 4/2010
Publication Date 22-Jan-2010
Grant Date 12-Jan-2010
Date of Filing 18-Aug-2005
Name of Patentee ATHENIX CORPORATION
Applicant Address ELLIS ROAD, SUITE B,DURHAM, NC 27703, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 CAROZZI, NADINE 8303 MEADOW RIDGE COURT, RALEIGH, NC 27615, U.S.A.
2 HARGISS, TRACY 204 BILLINGRATH TURN LANE, CARY, NC 27519, U.S.A.
3 KOZIEL, MICHAEL, G. 1601 LIATRIS LANE, RALEIGH, NC 27613, U.S.A.
4 DUCK, NICHOLAS, B. 1227 TARTARIAN TRAIL, APEX, NC 27502, U.S.A.
5 CARR, BRIAN 3103 SHINLEAF COURT, RALEIGH, NC 27613, U.S.A.
PCT International Classification Number C12N 15/32
PCT International Application Number PCT/US2004/005829
PCT International Filing date 2004-02-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/448,633 2003-02-20 U.S.A.
2 60/448,806 2003-02-20 U.S.A.
3 60/448,810 2003-02-20 U.S.A.
4 60/448,812 2003-02-20 U.S.A.
5 60/448,141, 2004-02-19 U.S.A.