U.S. patent application number 13/893801 was filed with the patent office on 2013-11-21 for toxin genes and methods for their use.
This patent application is currently assigned to Athenix Corporation. The applicant listed for this patent is Athenix Corporation. Invention is credited to Shruti Agarwal, Chris Campbell, Brian McNulty, Kimberly S. Sampson, Daniel J. Tomso.
Application Number | 20130310543 13/893801 |
Document ID | / |
Family ID | 41258971 |
Filed Date | 2013-11-21 |
United States Patent
Application |
20130310543 |
Kind Code |
A1 |
Sampson; Kimberly S. ; et
al. |
November 21, 2013 |
TOXIN GENES AND METHODS FOR THEIR USE
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
polypeptide 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 nucleic acid molecules are
provided. Additionally, amino acid sequences corresponding to the
polynucleotides are encompassed, and antibodies specifically
binding to those amino acid sequences. In particular, the present
invention provides for isolated nucleic acid molecules comprising
nucleotide sequences encoding the amino acid sequence shown in SEQ
ID NO:61-121 and 133-141, or the nucleotide sequence set forth in
SEQ ID NO:1-60, 124-132, and 142-283, as well as variants and
fragments thereof.
Inventors: |
Sampson; Kimberly S.;
(Durham, NC) ; Tomso; Daniel J.; (Bahama, NC)
; Agarwal; Shruti; (Durham, NC) ; McNulty;
Brian; (Chesterfield, MO) ; Campbell; Chris;
(Portland, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Athenix Corporation; |
|
|
US |
|
|
Assignee: |
Athenix Corporation
Morrisville
NC
|
Family ID: |
41258971 |
Appl. No.: |
13/893801 |
Filed: |
May 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12491396 |
Jun 25, 2009 |
8461421 |
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13893801 |
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61158137 |
Mar 6, 2009 |
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61075719 |
Jun 25, 2008 |
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Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
C12N 15/8286 20130101;
Y02A 40/146 20180101; A01N 37/18 20130101; C07K 14/325 20130101;
C07K 16/1278 20130101 |
Class at
Publication: |
530/387.9 |
International
Class: |
C07K 16/12 20060101
C07K016/12 |
Claims
1. An antibody that selectively binds to a polypeptide comprising
the amino acid sequence of SEQ ID NO:96.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/491,396, filed Jun. 25, 2009, which claims the benefit
of U.S. Provisional Application Ser. No. 61/075,719, filed Jun. 25,
2008, and U.S. Provisional Application Ser. No. 61/158,137, filed
Mar. 6, 2009, the contents of which are herein incorporated by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named "APA057US02SEQLIST.txt", created on May 14, 2013,
and having a size of 1,075 kilobytes and is filed concurrently with
the specification. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] 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
[0004] 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
non-targeted 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.
[0005] 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
CryI to CryV based primarily on their insecticidal activity. The
major classes were Lepidoptera-specific (I), Lepidoptera- and
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 Cry1A, Cry1B,
Cry1C, etc. Even more closely related proteins within each division
were given names such as Cry1C1, Cry1C2, etc.
[0006] 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.
[0007] 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
(1989) 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.
[0008] Delta-endotoxins generally have five conserved sequence
domains, and three conserved structural domains (see, for example,
de Maagd et al. (2001) Trends Genetics 17:193-199). 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.
[0009] The intensive use of B. thuringiensis-based insecticides has
already given rise to resistance in field populations of the
diamondback moth, Plutella xylostella (Ferre and Van Rie (2002)
Annu. Rev. Entomol. 47:501-533). The most common mechanism of
resistance is the reduction of binding of the toxin to its specific
midgut receptor(s). This may also confer cross-resistance to other
toxins that share the same receptor (Ferre and Van Rie (2002)).
SUMMARY OF INVENTION
[0010] Compositions and methods for conferring pest resistance to
bacteria, plants, plant cells, tissues and seeds are provided.
Compositions include nucleic acid molecules encoding sequences for
delta-endotoxin polypeptides, vectors comprising those nucleic acid
molecules, and host cells comprising the vectors. Compositions also
include the polypeptide sequences of the endotoxin, 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 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.
[0011] In particular, isolated nucleic acid molecules corresponding
to delta-endotoxin nucleic acid sequences are provided.
Additionally, amino acid sequences corresponding to the
polynucleotides are encompassed. In particular, the present
invention provides for an isolated nucleic acid molecule comprising
a nucleotide sequence encoding the amino acid sequence shown in any
of SEQ ID NO:61-121 and 133-141, or a nucleotide sequence set forth
in any of SEQ ID NO:1-60 and 124-132, 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.
[0012] 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 proteins that have pesticidal activity, or for
detecting the presence of delta-endotoxin proteins or nucleic acids
in products or organisms.
DETAILED DESCRIPTION
[0013] 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 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 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
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,
coleopteran, and nematode pest populations, and for producing
compositions with pesticidal activity.
[0014] 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 or members of the Nematoda phylum, or a
protein that has homology to such a protein. In some cases,
delta-endotoxin proteins have been isolated from other organisms,
including Clostridium bifermentans and Paenibacillus 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.
[0015] Delta-endotoxins include proteins identified as cry1 through
cry43, cyt1 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.
[0016] Provided herein are novel isolated nucleotide sequences that
confer pesticidal activity. Also provided are the amino acid
sequences of the delta-endotoxin proteins. The protein resulting
from translation of this gene allows cells to control or kill pests
that ingest it.
Isolated Nucleic Acid Molecules, and Variants and Fragments
Thereof
[0017] One aspect of the invention pertains to isolated or
recombinant nucleic acid molecules comprising nucleotide sequences
encoding delta-endotoxin proteins and polypeptides or biologically
active portions thereof, as well as nucleic acid molecules
sufficient for use as hybridization probes to identify
delta-endotoxin encoding nucleic acids. As used herein, the term
"nucleic acid molecule" is intended to include DNA molecules (e.g.,
recombinant DNA, 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.
[0018] 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 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 sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
A delta-endotoxin 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 protein
(also referred to herein as a "contaminating protein").
[0019] Nucleotide sequences encoding the proteins of the present
invention include the sequence set forth in SEQ ID NO:1-60 and
124-132, and variants, fragments, 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 sequence for the
delta-endotoxin protein encoded by this nucleotide sequence are set
forth in SEQ ID NO:61-121 and 133-141.
[0020] Nucleic acid molecules that are fragments of these
delta-endotoxin 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. A
fragment of a nucleotide sequence may encode a biologically active
portion of a delta-endotoxin 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 nucleotide sequence comprise at least about 50,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050, 1100,
1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,
1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200,
2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,
2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300,
3350 contiguous nucleotides, or up to the number of nucleotides
present in a full-length delta-endotoxin encoding nucleotide
sequence disclosed herein depending upon the intended use. By
"contiguous" nucleotides is intended nucleotide residues that are
immediately adjacent to one another. Fragments of the nucleotide
sequences of the present invention will encode protein fragments
that retain the biological activity of the delta-endotoxin protein
and, hence, retain pesticidal activity. By "retains activity" is
intended that the fragment will have at least about 30%, at least
about 50%, at least about 70%, 80%, 90%, 95% or higher of the
pesticidal activity of the delta-endotoxin protein. Methods for
measuring pesticidal activity are well known in the art. See, for
example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;
Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.
(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.
5,743,477, all of which are herein incorporated by reference in
their entirety.
[0021] A fragment of a delta-endotoxin encoding 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, 650, 700,
750, 800, 850, 900, 950, 1000, 1050, 1100 contiguous amino acids,
or up to the total number of amino acids present in a full-length
delta-endotoxin protein of the invention.
[0022] Preferred delta-endotoxin proteins of the present invention
are encoded by a nucleotide sequence sufficiently identical to the
nucleotide sequence of SEQ ID NO:1-60 and 124-132. By "sufficiently
identical" is intended an amino acid or nucleotide sequence that
has at least about 60% or 65% sequence identity, about 70% or 75%
sequence identity, about 80% or 85% sequence identity, about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater 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.
[0023] 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).times.100). In one embodiment, the two sequences are the
same length. In another embodiment, the comparison is across the
entirety of the reference sequence (e.g., across the entirety of
one of SEQ ID NO:1-60 and 124-132, or across the entirety of one of
SEQ ID NO:61-121 and 133-141). 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.
[0024] 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 Altschul (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-like
nucleic acid molecules of the invention. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to delta-endotoxin protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as
described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-Blast 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. Alignment may also
be performed manually by inspection.
[0025] 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
(Invitrogen Corporation, Carlsbad, Calif.). 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.TM..
GENEDOC.TM. (Karl Nicholas) allows assessment of amino acid (or
DNA) similarity and identity between multiple proteins. Another
non-limiting 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 Wisconsin Genetics
Software Package, Version 10 (available from Accelrys, Inc., 9685
Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used.
[0026] Unless otherwise stated, GAP Version 10, which uses the
algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48(3):443-453, will be used to determine sequence identity or
similarity using the following parameters: % identity and %
similarity for a nucleotide sequence using GAP Weight of 50 and
Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %
identity or % similarity for an amino acid sequence using GAP
weight of 8 and length weight of 2, and the BLOSUM62 scoring
program. Equivalent programs may also be used. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide residue matches and an identical percent sequence
identity when compared to the corresponding alignment generated by
GAP Version 10.
[0027] The invention also encompasses variant nucleic acid
molecules. "Variants" of the delta-endotoxin encoding nucleotide
sequences include those sequences that encode the delta-endotoxin
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
delta-endotoxin 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%, at least about 50%,
at least about 70%, or at least about 80% of the pesticidal
activity of the native protein. Methods for measuring pesticidal
activity are well known in the art. See, for example, Czapla and
Lang (1990) J. Econ. Entomol. 83: 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. Pat. No. 5,743,477, all of which
are herein incorporated by reference in their entirety.
[0028] The skilled artisan will further appreciate that changes can
be introduced by mutation of the nucleotide sequences of the
invention thereby leading to changes in the amino acid sequence of
the encoded delta-endotoxin 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.
[0029] For example, conservative amino acid substitutions may be
made at one or more predicted, 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 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).
[0030] Delta-endotoxins generally have five conserved sequence
domains, and three conserved structural domains (see, for example,
de Maagd et al. (2001) Trends Genetics 17:193-199). 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.
[0031] 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 amino 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 an alignment of the amino acid sequences of the
present invention and known delta-endotoxin sequences. 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 an alignment of the amino acid sequences of
the present invention and known delta-endotoxin sequences. However,
one of skill in the art would understand that functional variants
may have minor conserved or nonconserved alterations in the
conserved residues.
[0032] 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 delta-endotoxin
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.
[0033] Using methods such as PCR, hybridization, and the like
corresponding delta-endotoxin sequences can be identified, such
sequences having substantial identity to the sequences of the
invention. See, for example, Sambrook and Russell (2001) Molecular
Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A
Guide to Methods and Applications (Academic Press, NY).
[0034] In a hybridization method, all or part of the
delta-endotoxin 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, supra. 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 .sup.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-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, at least about 25, at
least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400
consecutive nucleotides of delta-endotoxin encoding nucleotide
sequence of the invention or a fragment or variant thereof. Methods
for the preparation of probes for hybridization are generally known
in the art and are disclosed in Sambrook and Russell, 2001, supra
herein incorporated by reference.
[0035] For example, an entire delta-endotoxin sequence disclosed
herein, or one or more portions thereof, may be used as a probe
capable of specifically hybridizing to corresponding
delta-endotoxin-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, or at least about 20 nucleotides in
length. Such probes may be used to amplify corresponding
delta-endotoxin 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, Cold Spring Harbor, N.Y.).
[0036] 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.
[0037] 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.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
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.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. 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.
[0038] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. 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 T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, 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 T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) 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.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, 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 T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. 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, Cold Spring Harbor, N.Y.).
Isolated Proteins and Variants and Fragments Thereof
[0039] Delta-endotoxin 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:61-121 and 133-141. Fragments, biologically active portions, and
variants thereof are also provided, and may be used to practice the
methods of the present invention.
[0040] "Fragments" or "biologically active portions" include
polypeptide fragments comprising amino acid sequences sufficiently
identical to the amino acid sequence set forth in any of SEQ ID
NO:61-121 and 133-141 and that exhibit pesticidal activity. A
biologically active portion of a delta-endotoxin 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 pesticidal activity.
Methods for measuring pesticidal activity are well known in the
art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.
83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;
Marrone et al. (1985) J. of Economic Entomology 78:290-293; and
U.S. Pat. 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 of SEQ ID NO:61-121 and 133-141. 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, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or
1300 amino acids.
[0041] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 60%, 65%, about 70%,
75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to the amino acid sequence of any of SEQ ID
NO:61-121 and 133-141. Variants also include polypeptides encoded
by a nucleic acid molecule that hybridizes to the nucleic acid
molecule of SEQ ID NO:1-60 and 124-132, or a complement thereof,
under stringent conditions. 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. 83:2480-2485;
Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.
(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.
5,743,477, all of which are herein incorporated by reference in
their entirety.
[0042] 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. These delta-endotoxin proteins are encompassed
in the present invention and may be used in the methods of the
present invention.
[0043] Antibodies to the polypeptides of the present invention, or
to variants or fragments thereof, are also encompassed. Methods for
producing antibodies are well known in the art (see, for example,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.; U.S. Pat. No.
4,196,265).
Altered or Improved Variants
[0044] It is recognized that DNA sequences of a delta-endotoxin 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 a delta-endotoxin of the present
invention. This protein may be altered in various ways including
amino acid substitutions, deletions, truncations, and insertions of
one or more amino acids of SEQ ID NO:61-121 and 133-141, including
up to about 2, about 3, about 4, about 5, about 6, about 7, about
8, about 9, about 10, about 15, about 20, about 25, about 30, about
35, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 75, about 80, about 85, about 90, about 100, about
105, about 110, about 115, about 120, about 125, about 130 or more
amino acid substitutions, deletions or insertions.
[0045] Methods for such manipulations are generally known in the
art. For example, amino acid sequence variants of a delta-endotoxin
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 to confer pesticidal activity may be improved by
the use of such techniques upon the compositions of this invention.
For example, one may express a delta-endotoxin 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 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 mutations in a
non-mutagenic strain, and identify mutated delta-endotoxin 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.
[0046] 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.
[0047] 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 protein coding
regions can be used to create a new delta-endotoxin 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 a delta-endotoxin gene of the invention and other known
delta-endotoxin 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.
Pat. Nos. 5,605,793 and 5,837,458.
[0048] Domain swapping or shuffling is another mechanism for
generating altered delta-endotoxin 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. (2001) Appl. Environ. Microbiol.
67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol.
62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958;
Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al.
91999) Appl. Environ. Microbiol. 65:2918-2925).
Vectors
[0049] A delta-endotoxin sequence of the invention may be provided
in an expression cassette for expression in a plant of interest. 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.
[0050] 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 co-translational 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.
[0051] 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 trans-acting 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 the ability to incorporate, integrate
and express heterologous DNA sequences or fragments in a foreign
cell. 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.
[0052] "Promoter" refers to a nucleic acid sequence that functions
to direct transcription of a downstream coding sequence. The
promoter together with other transcriptional and translational
regulatory nucleic acid sequences (also termed "control sequences")
are necessary for the expression of a DNA sequence of interest.
[0053] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the delta-endotoxin sequence to
be under the transcriptional regulation of the regulatory
regions.
[0054] 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
translational and transcriptional 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.
[0055] 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; Munroe et
al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res.
15:9627-9639.
[0056] 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 cell-preferred codons for
improved expression, or may be synthesized using codons at a
host-preferred codon usage frequency. Generally, the GC content of
the gene will be increased. See, for example, Campbell and Gowri
(1990) Plant Physiol. 92:1-11 for a discussion of host-preferred
codon usage. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0057] In one embodiment, the delta-endotoxin is targeted to the
chloroplast for expression. In this manner, where the
delta-endotoxin is not directly inserted into the chloroplast, the
expression cassette will additionally contain a nucleic acid
encoding a transit peptide to direct the delta-endotoxin to the
chloroplasts. Such transit peptides are known in the art. See, for
example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126;
Clark et al. (1989) J. 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.
[0058] The delta-endotoxin gene 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. Pat. No. 5,380,831, herein incorporated by reference.
Plant Transformation
[0059] Methods of the invention involve introducing a nucleotide
construct into a plant. By "introducing" is intended to present to
the plant the nucleotide construct in such a manner that the
construct gains access to the interior of a cell of the plant. The
methods of the invention do not require that a particular method
for introducing a nucleotide construct to a plant is used, only
that the nucleotide construct gains access to the interior of at
least one cell of the plant. Methods for introducing nucleotide
constructs into plants are known in the art including, but not
limited to, stable transformation methods, transient transformation
methods, and virus-mediated methods.
[0060] By "plant" is intended whole plants, plant organs (e.g.,
leaves, stems, roots, etc.), seeds, plant cells, propagules,
embryos and progeny of the same. Plant cells can be differentiated
or undifferentiated (e.g. callus, suspension culture cells,
protoplasts, leaf cells, root cells, phloem cells, pollen).
[0061] "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, transfection, microinjection, electroporation,
microprojection, or the like.
[0062] Transformation of plant cells can be accomplished by one of
several techniques known in the art. The delta-endotoxin gene of
the invention may be modified to obtain or enhance 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.
[0063] 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 delta-endotoxin 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 vir-mediated DNA transfer, as is 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, polyethylene glycol, etc.
[0064] 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 plants 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-transformed 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.
[0065] 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. Generation of transgenic plants may be
performed by one of several methods, including, but not limited to,
microinjection, electroporation, direct gene transfer, introduction
of heterologous DNA by Agrobacterium into plant cells
(Agrobacterium-mediated transformation), bombardment of plant cells
with heterologous foreign DNA adhered to particles, ballistic
particle acceleration, aerosol beam transformation (U.S. Published
Application No. 20010026941; U.S. Pat. No. 4,945,050; International
Publication No. WO 91/00915; U.S. Published Application No.
2002015066), Lec1 transformation, and various other non-particle
direct-mediated methods to transfer DNA.
[0066] 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. 12:601-606. The method
relies on particle gun delivery of DNA 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.
[0067] 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. Molecular and
biochemical methods can then be used to confirm the presence of the
integrated heterologous gene of interest into the genome of the
transgenic plant.
[0068] 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.
Evaluation of Plant Transformation
[0069] 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.
[0070] 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) Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried
out using oligonucleotide primers specific to the gene of interest
or Agrobacterium vector background, etc.
[0071] Plant transformation may be confirmed by Southern blot
analysis of genomic DNA (Sambrook and Russell, 2001, supra). 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" is then probed with, for example, radiolabeled
.sup.32P target DNA fragment to confirm the integration of
introduced gene into the plant genome according to standard
techniques (Sambrook and Russell, 2001, supra).
[0072] In Northern blot analysis, RNA is isolated from specific
tissues of transformant, fractionated in a formaldehyde agarose
gel, and blotted onto a nylon filter according to standard
procedures that are routinely used in the art (Sambrook and
Russell, 2001, supra). Expression of RNA encoded by the
delta-endotoxin is then tested by hybridizing the filter to a
radioactive probe derived from a delta-endotoxin, by methods known
in the art (Sambrook and Russell, 2001, supra).
[0073] Western blot, biochemical assays and the like may be carried
out on the transgenic plants to confirm the presence of protein
encoded by the delta-endotoxin gene by standard procedures
(Sambrook and Russell, 2001, supra) using antibodies that bind to
one or more epitopes present on the delta-endotoxin protein.
Pesticidal Activity in Plants
[0074] In another aspect of the invention, one may generate
transgenic plants expressing a delta-endotoxin that has 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, biolistic transformation,
and non-particle-mediated methods may be used at the discretion of
the experimenter. Plants expressing a delta-endotoxin 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.
[0075] 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). Additionally, the
genes disclosed herein are useful as markers to assess
transformation of bacterial or plant cells. Methods for detecting
the presence of a transgene in a plant, plant organ (e.g., leaves,
stems, roots, etc.), seed, plant cell, propagule, embryo or progeny
of the same are well known in the art. In one embodiment, the
presence of the transgene is detected by testing for pesticidal
activity.
[0076] Fertile plants expressing a delta-endotoxin 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.
[0077] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (maize), sorghum, wheat, sunflower, tomato, crucifers,
peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,
tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye,
millet, safflower, peanuts, sweet potato, cassava, coffee, coconut,
pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava,
mango, olive, papaya, cashew, macadamia, almond, oats, vegetables,
ornamentals, and conifers.
[0078] Vegetables include, but are not limited to, tomatoes,
lettuce, green beans, lima beans, peas, and members of the genus
Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals
include, but are not limited to, azalea, hydrangea, hibiscus,
roses, tulips, daffodils, petunias, carnation, poinsettia, and
chrysanthemum. Preferably, plants of the present invention are crop
plants (for example, maize, sorghum, wheat, sunflower, tomato,
crucifers, peppers, potato, cotton, rice, soybean, sugarbeet,
sugarcane, tobacco, barley, oilseed rape, etc.).
Use in Pest Control
[0079] General methods for employing strains comprising a
nucleotide sequence of the present invention, or a variant thereof,
in pesticide control or in engineering other organisms as
pesticidal agents are known in the art. See, for example U.S. Pat.
No. 5,039,523 and EP 0480762A2.
[0080] The Bacillus strains containing a nucleotide sequence of the
present invention, or a variant thereof, or the microorganisms that
have been genetically altered to contain a pesticidal gene and
protein may be used for protecting agricultural crops and products
from pests. In 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).
[0081] Alternatively, the pesticide is produced by introducing a
delta-endotoxin gene into a cellular host. Expression of the
delta-endotoxin 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 a
gene of this invention such as to allow application of the
resulting material as a pesticide.
[0082] Pesticidal Compositions
[0083] 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, molluscicides 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.
[0084] Methods of applying an active ingredient of the present
invention or an agrochemical composition of the present invention
that contains at least one of the pesticidal proteins produced by
the bacterial strains of the present invention include 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.
[0085] The composition may be formulated as a powder, dust, pellet,
granule, spray, emulsion, colloid, solution, or such like, and may
be prepared 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.
[0086] Lepidopteran, coleopteran, or nematode 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.
[0087] 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. Pat. No. 6,468,523, herein
incorporated by reference.
[0088] "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.
[0089] The order Coleoptera includes the suborders Adephaga and
Polyphaga. Suborder Adephaga includes the superfamilies Caraboidea
and Gyrinoidea, while suborder Polyphaga includes the superfamilies
Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea,
Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea,
Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea,
Scarabaeoidea, Cerambycoidea, Chrysomeloidea, and Curculionoidea.
Superfamily Caraboidea includes the families Cicindelidae,
Carabidae, and Dytiscidae. Superfamily Gyrinoidea includes the
family Gyrimidae. Superfamily Hydrophiloidea includes the family
Hydrophilidae. Superfamily Staphylinoidea includes the families
Silphidae and Staphylimidae. Superfamily Cantharoidea includes the
families Cantharidae and Lampyridae. Superfamily Cleroidea includes
the families Cleridae and Dermestidae. Superfamily Elateroidea
includes the families Elateridae and Buprestidae. Superfamily
Cucujoidea includes the family Coccinellidae. Superfamily Meloidea
includes the family Meloidae. Superfamily Tenebrionoidea includes
the family Tenebrionidae. Superfamily Scarabaeoidea includes the
families Passalidae and Scarabaeidae. Superfamily Cerambycoidea
includes the family Cerambycidae. Superfamily Chrysomeloidea
includes the family Chrysomelidae. Superfamily Curculionoidea
includes the families Curculionidae and Scolytidae.
[0090] The order Diptera includes the Suborders Nematocera,
Brachycera, and Cyclorrhapha. Suborder Nematocera includes the
families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae,
Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. Suborder
Brachycera includes the families Stratiomyidae, Tabanidae,
Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae.
Suborder Cyclorrhapha includes the Divisions Aschiza and Aschiza.
Division Aschiza includes the families Phoridae, Syrphidae, and
Conopidae. Division Aschiza includes the Sections Acalyptratae and
Calyptratae. Section Acalyptratae includes the families Otitidae,
Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptratae
includes the families Hippoboscidae, Oestridae, Tachinidae,
Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.
[0091] The order Lepidoptera includes the families Papilionidae,
Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae,
Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae,
Noctuidae, Lymantriidae, Sesiidae, and Tineidae.
[0092] 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 avenae (cereal cyst nematode); and Globodera
rostochiensis and Globodera pailida (potato cyst nematodes). Lesion
nematodes include Pratylenchus spp.
[0093] Insect pests of the invention for the major crops include:
Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon,
black cutworm; Helicoverpa zea, corn earworm; Spodoptera
frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn
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; Cyclocephala borealis, northern masked
chafer (white grub); Cyclocephala immaculata, southern masked
chafer (white grub); Popillia japonica, 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; Agromyza parvicornis, corn blot leafminer;
Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief
ant; Tetranychus urticae, twospotted spider mite; Sorghum: Chilo
partellus, 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; Sipha flava, 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; Frankliniella fusca,
tobacco thrips; Cephus cinctus, wheat stem sawfly; 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 abutilonea, bandedwinged whitefly; Lygus
lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged
grasshopper; Melanoplus differentialis, differential grasshopper;
Thrips tabaci, onion thrips; Franklinkiella fusca, 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; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink 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, seedcorn 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.
Methods for Increasing Plant Yield
[0094] Methods for increasing plant yield are provided. The methods
comprise introducing into a plant or plant cell a polynucleotide
comprising a pesticidal sequence disclosed herein. As defined
herein, the "yield" of the plant refers to the quality and/or
quantity of biomass produced by the plant. By "biomass" is intended
any measured plant product. An increase in biomass production is
any improvement in the yield of the measured plant product.
Increasing plant yield has several commercial applications. For
example, increasing plant leaf biomass may increase the yield of
leafy vegetables for human or animal consumption. Additionally,
increasing leaf biomass can be used to increase production of
plant-derived pharmaceutical or industrial products. An increase in
yield can comprise any statistically significant increase
including, but not limited to, at least a 1% increase, at least a
3% increase, at least a 5% increase, at least a 10% increase, at
least a 20% increase, at least a 30%, at least a 50%, at least a
70%, at least a 100% or a greater increase in yield compared to a
plant not expressing the pesticidal sequence.
[0095] In specific methods, plant yield is increased as a result of
improved pest resistance of a plant expressing a pesticidal protein
disclosed herein. Expression of the pesticidal protein results in a
reduced ability of a pest to infest or feed on the plant, thus
improving plant yield.
[0096] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Discovery of a Novel Pesticidal Genes from Bacillus
thuringiensis
[0097] Novel pesticidal genes were identified from the bacterial
strains listed in Table 1 using the following steps: [0098]
Preparation of extrachromosomal DNA from the strain, which includes
plasmids that typically harbor delta-endotoxin genes [0099]
Mechanical shearing of extrachromosomal DNA to generate
size-distributed fragments [0100] Cloning of .about.2 Kb to
.about.10 Kb fragments of extrachromosomal DNA [0101] Outgrowth of
.about.1500 clones of the extrachromosomal DNA [0102] Partial
sequencing of the 1500 clones using primers specific to the cloning
vector (end reads) [0103] Identification of putative toxin genes
via homology analysis via the MiDAS approach (as described in U.S.
Patent Publication No. 20040014091, which is herein incorporated by
reference in its entirety) [0104] Sequence finishing (walking) of
clones containing fragments of the putative toxin genes of
interest
TABLE-US-00001 [0104] TABLE 1 List of novel genes isolated from
Bacillus thuringiensis Percent Nucleo- Amino identity to tide Acid
closest Closest SEQ ID SEQ ID sequence in sequence Gene Strain NO:
NO: art in art axmi046.sup.1 ATX13026 1 61 54.30% Cry4Aa1
axmi048.sup.2 ATX13026 2 62 56.4% Cry4Ba1 axmi050.sup.3 ATX21049 3
63 17.9% Cry21Ba1 axmi051.sup.4 ATX21049 4 64 21.3% Cry35Ac1
axmi052.sup.5 ATX21049 5 65 19.70% Cry35Aa1 axmi053 ATX21049 6 66
21.9% Cry35Ac1 axmi054 ATX21049 7 67 20.1% Cry35Ac1 axmi055
ATX12976 8 68 42.0% Cry32Ca1 axmi056 ATX12976 9 69 (homology to
BinA/ N-terminus) BinB axmi057 ATX13058 10 70 73.1% Cry32Da1
axmi058 ATX13058 11 71 26.8% Cry6Ba1 axmi059 ATX13058 12 72 56.0%
Cry32Aa1 axmi060 ATX13058 13 73 54.6% Cry32Aa1 axmi061 ATX13058 14
74 29.2% Cry45Aa1 axmi067 ATX12974 15 75 36.3% Cry32Da1 axmi069
ATX12997 17 77 Cry32Ca1 Some N-terminal homology axmi071 ATX12982
18 78 22.9% Cry21Ba1 axmi072 ATX12982 19 79 16.4% Mtx2 axmi073
ATX16042 20 80 15.3% Mtx2 axmi074 ATX12993 21 81 43.8% Cry21Ba1
axmi075 ATX12997 22 82 30.4% Cry32Da1 axmi087 ATX13030 27 87 71.0%
Cry8Aa1 axmi088 ATX13039 28 88 26.2% Cry21Ba1 axmi093 ATX13058 31
91 56.1% Cry32Aa1 .sup.1Potential co-activity when expressed or
paired with another toxin such as Axmi0014 or Axmi008
.sup.2Potential co-activity when expressed or paired with another
toxin such as Axmi0014 or Axmi009 .sup.3An N-terminal domain
homologous to a phospholipase C catalytic domain .sup.4Potential
co-activity when expressed or paired with another toxin such as
Axmi052 .sup.5Potential co-activity when expressed or paired with
another toxin such as Axmi051
Example 2
Discovery of Novel Pesticidal Genes from Bacillus thuringiensis
[0105] Novel pesticidal genes were identified from the strains
listed in Table 2 using the MiDAS approach as described in U.S.
Patent Publication No. 20040014091, which is herein incorporated by
reference in its entirety, using the following steps: [0106]
Preparation of extrachromosomal DNA from the strain.
Extrachromosomal DNA contains a mixture of some or all of the
following: plasmids of various size; phage chromosomes; genomic DNA
fragments not separated by the purification protocol; other
uncharacterized extrachromosomal molecules. [0107] Mechanical or
enzymatic shearing of the extrachromosomal DNA to generate
size-distributed fragments. [0108] Sequencing of the fragmented DNA
[0109] Identification of putative toxin genes via homology and/or
other computational analyses. [0110] When required, sequence
finishing of the gene of interest by one of several PCR or cloning
strategies (e.g. TAIL-PCR).
TABLE-US-00002 [0110] TABLE 2 List of novel genes isolated from
Bacillus thuringiensis Percent Amino identity to Nucleotide Acid
closest Closest SEQ ID SEQ ID sequence sequence Gene Strain NO: NO:
in art in art axmi079 ATX12974 23 83 36.7% Cry32Da1 axmi080
ATX12974 24 84 39.9% Cry42Aa1 axmi081 ATX12974 25 85 68% Orf3,
described as `C- terminal half of a Cry Protein` axmi082 ATX13056
26 86 47.8% Cry32Da1 axmi091 ATX13053 29 89 35.3% Cry8Ba1 axmi092
ATX13053 30 90 74.4% Cry39Orf2 axmi096 ATX13007 32 92 29.6%
Cry32Da1 axmi097 ATX13007 33 93 29.3% Cry32Da1 axmi098 ATX13007 34
94 56% Cry41Ab1 axmi099 ATX13007 35 95 69% axmi081 61% axmi067 60%
axmi079 45% axmi075 45% Cry32Ca1 axmi100 ATX12990 36 96 77% Cry9Ca1
76% Cry9Ea1 74% axmi002 72% Cry9Bb1 axmi101 ATX13035 37 97 65%
Cry7Ba1 62% axmi037 60% axmi029 58% Cry7Ab2 axmi102 ATX13056 38 98
86% axmi082 65% axmi093 58% axmi059 56% Cry32Aa1 axmi103 ATX13056
39 99 64% axmi082 58% Cry32Da1 56% axmi093 52% axmi059 axmi104
ATX13058 40 100 19.3% axmi020 18.3% Cry21Ba1 17.1% Cry5Ba1 17.1%
Cry44Aa axmi107 ATX13007 41 101 35% Vip1Aa2 34% Vip1Da1 axmi108
ATX12984 42 102 90% Cry45Aa1 25% Cry23Aa1 axmi109 ATX12984 43 103
38% Cry45Aa1 axmi110 ATX12984 44 104 43% Cry32Aa1 axmi111 ATX12984
45 105 34% Cry41Ab1 axmi112 ATX12987 46 106 96% Cry1Ab axmi114
ATX14903 47 107 85.8% axmi043 85.8% axmi028 56.7% axmi037 57.2%
Cry7Ca1 56.3% Cry7Ab2 axmi116 ATX12975 48 108 53.5% Cry7Ba1 53.2%
axmi114 53.1% axmi028 53% axmi043 52.3% Cry7Ca1 50.3% Cry7Ab1
axmi117 ATX13029 49 109 92.2% Cry22Ba1 110 48.2% Cry22Aa1 47.1%
Cry22Ab1 axmi118 ATX12989 50 111 25.3% axmi011 22.2% Mtx2 axmi119
ATX13029 51 112 28.8% axmi027 27.8% axmi066 27.5% Cry2Ae1 26.5%
axmi076 26.2% Cry18Aa1 axmi120 ATX13034 52 113 50% Cry8Aa1 53 114
49.5% axmi087 54 115 49.1% Cry8Bb1 47.8% Cry8Bc1 47.8% Cry8Da1 47%
Cry8Ba1 45.8% Cry8Ca1 axmi121 ATX13034 55 116 51.1% axmi013 47.9%
Mtx3 axmi122 ATX13034 56 117 23.1% axmi013 22.8% Mtx2 22.1% Mtx3
21.4% axmi095 axmi123 ATX12989 57 118 26.7% Cry33Aa1 22.7% Cry23Aa1
21.8% Cry15Aa1 19% axmi061 axmi124 ATX9387 58 119 57.6% axmi088
29.1% axmi040 28.4% axmi049 27.1% Cry5Ab1 26.5% Cry21Ba1 26.1%
Cry12Aa1 25.6% axmi074 24.4% axmi031 axmi125.sup.7 ATX13029 59 120
38.4% Cry10Aa1 36.7% Cry10Aa2 31.3% axmi007 31% axmi006
axmi126.sup.8 ATX13029 60 121 82.6% axmi047 81.5% axmi084 80.9%
axmi086 80.9% axmi090 80.5% axmi046 79.2% axmi048 75.3% axmi092 65%
Cry4Ba1 64.1% Cry4Aa1 axmi127 ATX13034 124 133 58% Cry8Da1 axmi129
ATX13015 125 134 63% Cry8Aa1 axmi164 ATX22201 126 135 77% Cry8Aa1
axmi151 ATX12998 127 136 61% Cry7Ba axmi161 ATX12998 128 137 52%
Cry7Ca1 axmi183 ATX14775 129 138 69% Cry9Eb1 axmi132 ATX13029 130
139 55% Cry4Ba axmi138 ATX13027 131 140 47% Cry41Aa1 axmi137
ATX9387 132 141 61% Axmi075 .sup.1This gene is the N-terminal
portion of a split cry gene and is paired in its native context
with Axmi126, which represents the C-terminal portion of the split
cry pair. These genes may act as co-toxins and may show enhanced,
novel, or altered activity when co-expressed or fused. The
intervening region between Axmi125 and the downstream Axmi126 is
set forth in SEQ ID NO: 122. .sup.2This gene is the C-terminal
portion of a split cry gene and is paired in its native context
with Axmi125, which represents the N-terminal portion of the split
cry pair. These genes may act as co-toxins and may show enhanced,
novel, or altered activity when co-expressed or fused.
Example 3
Discovery of a Novel Toxin Gene Axmi068 from Bacillus thuringiensis
Strain ATX13046
[0111] The strain encoding axmi068 was identified as follows:
[0112] Sequence information from known or suspected toxin genes was
used to generate an alignment representing conserved and partially
conserved DNA sequences within a group (family) of toxins. [0113]
Polymerase chain reaction (PCR) primers were designed to
selectively amplify one or more toxin family members based on the
aligned sequence. [0114] DNA isolated from bacterial strains was
screened by PCR to identify strains containing putative homologs to
the target gene family. [0115] PCR products were sequenced to
select a strain containing a gene of interest. The complete gene
sequence was identified from the selected strain via the MiDAS
genomics approach (U.S. Patent Publication No. 20040014091) as
follows: [0116] Preparation of extrachromosomal DNA from the
strain. Extrachromosomal DNA contains a mixture of some or all of
the following: plasmids of various size; phage chromosomes; genomic
DNA fragments not separated by the purification protocol; other
uncharacterized extrachromosomal molecules. [0117] Mechanical or
enzymatic shearing of the extrachromosomal DNA to generate
size-distributed fragments. [0118] Cloning of the extrachromosomal
DNA fragments into a plasmid vector. [0119] Growth and purification
of the cloned of the extrachromosomal DNA. [0120] Partial
sequencing of the clones. [0121] Identification of putative toxin
genes via homology and/or other computational analyses. [0122] When
required, sequence finishing (walking) of clones containing
sequence of the putative toxin genes of interest. [0123] The
nucleotide sequence for axmi068 is set forth in SEQ ID NO:16 and
the amino acid sequence for AXMI068 is set forth in SEQ ID
NO:76.
Gene and Protein Characteristics
[0124] Gene length, DNA base pairs: 1,791 Protein length, amino
acid residues: 597 Estimated protein molecular weight, Da: 66,495
Known homologs and approximate percent identity: Cry1Id1, 71.4%
Example 4
Expression in Bacillus
[0125] The insecticidal gene disclosed herein is amplified by PCR,
and the PCR product is cloned into the Bacillus expression vector
pAX916, or another suitable vector, by methods well known in the
art. The resulting Bacillus strain, containing the vector with axmi
gene is cultured on a conventional growth media, such as CYS media
(10 g/l Bacto-casitone; 3 g/l yeast extract; 6 g/l
KH.sub.2PO.sub.4; 14 g/l K.sub.2HPO.sub.4; 0.5 mM MgSO.sub.4; 0.05
mM MnCl.sub.2; 0.05 mM FeSO.sub.4), until sporulation is evident by
microscopic examination. Samples are prepared and tested for
activity in bioassays.
Example 5
Construction of Synthetic Sequences
[0126] In one aspect of the invention, synthetic axmi sequences
were generated. These synthetic sequences have an altered DNA
sequence relative to the parent axmi sequence, and encode a protein
that is collinear with the parent AXMI protein to which it
corresponds, but lacks the C-terminal "crystal domain" present in
many delta-endotoxin proteins. Synthetic genes are presented in
Table 3.
TABLE-US-00003 TABLE 3 Wildtype Gene Name Synthetic Gene Name SEQ
ID NO: axmi050 axmi050bv01 142 axmi050bv02 143 axmi051 axmi051bv01
144 axmi051bv02 145 axmi052 axmi052bv01 146 axmi052bv02 147 axmi053
axmi053bv01 148 axmi053bv02 149 axmi054 axmi054bv01 150 axmi054bv02
151 axmi055 axmi055bv01 152 axmi055bv02 153 axmi056 axmi056bv01 154
axmi056bv02 155 axmi057 axmi057bv01 156 axmi057bv02 157 axmi058
axmi058bv01 158 axmi058bv02 159 axmi059 axmi059_1bv01 160
axmi059_1bv02 161 axmi059_2bv01 162 axmi059_2bv02 163 axmi060
axmi060bv01 164 axmi060bv02 165 axmi061 axmi061bv01 166 axmi061bv02
167 axmi067 axmi067bv01 168 axmi067bv02 169 axmi069 axmi069bv01 170
axmi069bv02 171 axmi071 axmi071bv01 172 axmi071bv02 173 axmi072
axmi072bv01 174 axmi072bv02 175 axmi073 axmi073bv01 176 axmi073bv02
177 axmi074 axmi074bv01 178 axmi074bv02 179 axmi075 axmi075bv01 180
axmi075bv02 181 axmi079 axmi079bv01 182 axmi079bv02 183 axmi080
axmi080bv01 184 axmi080bv02 185 axmi082 axmi082bv01 186 axmi082bv02
187 axmi087 axmi087_1bv01 188 axmi087_1bv02 189 axmi087_2bv01 190
axmi087_2bv02 191 axmi088 axmi088bv01 192 axmi088bv02 193 axmi091
axmi091bv01 194 axmi091bv02 195 axmi093 axmi093bv01 196 axmi093bv02
197 axmi096 axmi096bv01 198 axmi096bv02 199 axmi097 axmi097_1bv01
200 axmi097_1bv02 201 axmi097_2bv01 202 axmi097_2bv02 203 axmi098
axmi098bv01 204 axmi098bv02 205 axmi100 axmi100bv01 206 axmi100bv02
207 optaxmi100v01 282 optaxmi100v02 283 axmi101 axmi101_1bv01 208
axmi101_1bv02 209 axmi101_2bv01 210 axmi101_2bv02 211 axmi102
axmi102bv01 212 axmi102bv02 213 axmi103 axmi103bv01 214 axmi103bv02
215 axmi104 axmi104bv01 216 axmi104bv02 217 axmi107 axmi107bv01 218
axmi107bv02 219 axmi108 axmi108bv01 220 axmi108bv02 221 axmi109
axmi109bv01 222 axmi109bv02 223 axmi110 axmi110bv01 224 axmi110bv02
225 axmi111 axmi111bv01 226 axmi111bv02 227 axmi112 axmi112bv01 228
axmi112bv02 229 axmi114 axmi114bv01 230 axmi114bv02 231 axmi116
axmi116bv01 232 axmi116bv02 233 axmi117 axmi117bv01 234 axmi117bv02
235 axmi118 axmi118bv01 236 axmi118bv02 237 axmi119 axmi119bv01 238
axmi119bv02 239 axmi120 axmi120_1bv01 240 axmi120_1bv02 241
axmi120_2bv01 242 axmi120_2bv02 243 axmi121 axmi121bv01 244
axmi121bv02 245 axmi122 axmi122bv01 246 axmi122bv02 247 axmi123
axmi123bv01 248 axmi123bv02 249 axmi124 axmi124bv01 250 axmi124bv02
251 axmi125 axmi125bv01 252 axmi125bv02 253 axmi127 axmi127_1bv01
254 axmi127_1bv02 255 axmi127_2bv01 256 axmi127_2bv02 257 axmi129
axmi129_1bv01 258 axmi129_1bv02 259 axmi129_2bv01 260 axmi129_2bv02
261 axmi137 axmi137bv01 262 axmi137bv02 263 axmi138 axmi138bv01 264
axmi138bv02 265 axmi151 axmi151_1bv01 266 axmi151_1bv02 267
axmi151_2bv01 268 axmi151_2bv02 269 axmi161 axmi161_1bv01 270
axmi161_1bv02 271 axmi161_2bv01 272 axmi161_2bv02 273 axmi164
axmi164_1bv01 274 axmi164_1bv02 275 axmi164_2bv01 276 axmi164_2bv02
277 axmi183 axmi183_2bv01 278 axmi183_2bv02 279 axmi183bv01 280
axmi183bv02 281
[0127] In another aspect of the invention, modified versions of
synthetic genes are designed such that the resulting peptide is
targeted to a plant organelle, such as the endoplasmic reticulum or
the apoplast. Peptide sequences known to result in targeting of
fusion proteins to plant organelles are known in the art. For
example, the N-terminal region of the acid phosphatase gene from
the White Lupin Lupinus albus (Genebank ID GI:14276838; Miller et
al. (2001) Plant Physiology 127: 594-606) is known in the art to
result in endoplasmic reticulum targeting of heterologous proteins.
If the resulting fusion protein also contains an endoplasmic
retention sequence comprising the peptide
N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the
"KDEL" motif (SEQ ID NO:123) at the C-terminus, the fusion protein
will be targeted to the endoplasmic reticulum. If the fusion
protein lacks an endoplasmic reticulum targeting sequence at the
C-terminus, the protein will be targeted to the endoplasmic
reticulum, but will ultimately be sequestered in the apoplast.
Example 6
Expression of axmi100 in E. coli and Bacillus
[0128] The complete ORF of axmi100 (3.45 kb which encode 1156 amino
acid long protein) was cloned into an E. coli expression vector
based on pRSF1b (to give pAX5445) and Bacillus vector based on
pAX916 (to give pAX5444). The resulting clones were confirmed by
restriction analysis and finally, by complete sequencing of the
cloned gene.
[0129] For expression in E. coli, BL21*DE3 was transformed with
pAX5445. Single colony was inoculated in LB supplemented with
kanamycin and grown overnight at 37.degree. C. Next day, fresh
medium was inoculated in duplicate with 1% of overnight culture and
grown at 37.degree. C. to logarithmic phase. Subsequently, cultures
were induced with 1 mM IPTG for 3 hours at 37.degree. C. or
overnight at 20.degree. C. Each cell pellet was suspended in 50 mM
sodium carbonate buffer, pH 10.5 supplemented with 1 mM DTT and
sonicated. Analysis by SDS-PAGE detected expression of a 130 kD
protein corresponding to Axmil00.
[0130] For expression in Bacillus, Bacillus thuringiensis was
transformed with pAX5444 and a single colony was grown in CYS-glu
medium for 3 days to sporulation. Cell pellet was then extracted
with 50 mM sodium carbonate buffer, pH 10.5 supplemented with 1 mM
DTT. Soluble fraction showed presence of a 130 kD Axmil00 protein
along with several smaller molecular weight protein bands due to
processing of Axmi100. Trypsinization of Axmi100 gave 2 distinct
protein bands of about 65 kD and 55 kD.
Example 7
Bioassay of Axmi100
Preparation of Samples:
[0131] Cell free extracts from cells expressing AXMI-100 were
typically resuspended in 50 mM sodium carbonate buffer, pH 10.5,
typically with inclusion of 1 mM DTT as a reducing agent. Samples
with and without trypsin were prepared for bioassay testing.
Bioassay Methodology Overview:
[0132] 24-Well tissue culture plates (Corning) were given 1 ml of
multi-species diet (Bio-Serv) and allowed to solidify. Once
solidified, 40 .mu.l of protein sample was placed on the diet
surface of each well and allowed to soak in/dry at room
temperature. Depending upon the experiment, either ECB egg masses,
ten neonate larvae or a single neonate larvae were placed in each
well. Plates were sealed with gas-permeable membranes (Research
Products International) and incubated at 25.degree. C. and 90% RH.
After five or seven days (experiment dependent), samples were
scored visually compared to a buffer only or non-transformed
extract control.
[0133] Strong activity of AXMI-100 was observed on European Corn
Borer and Tobacco Budworm. Activity on black cutworm was observed
at high protein concentrations. Some activity was also observed at
high concentrations on Velvet Bean caterpillar, but the activity of
both black cutworm and velvet bean caterpillar was less pronounced
and more variable than for the other insects tested. Trypsinization
of Axmi100 gave 2 distinct protein bands of about 65 kD and 55 kD,
and did not appear to be required for activity of AXMI-100.
Example 8
Additional Assays for Pesticidal Activity
[0134] 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.
[0135] 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 mouth parts 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.
[0136] 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.
[0137] 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, Fla. 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
Bioassay of Axmi079 and Axmi082
Gene Expression and Purification
[0138] The DNA regions encoding the toxin domains of Axmi079 and
Axmi082 were separately cloned into an E. coli expression vector
pMAL-C4x behind the malE gene coding for Maltose binding protein
(MBP). These in-frame fusions resulted in MBP-Axmi fusion proteins
expression in E. coli. [0139] For expression in E. coli, BL21*DE3
was transformed with individual plasmids. Single colony was
inoculated in LB supplemented with carbenicillin and glucose, and
grown overnight at 37.degree. C. The following day, fresh medium
was inoculated with 1% of overnight culture and grown at 37.degree.
C. to logarithmic phase. Subsequently, cultures were induced with
0.3 mM IPTG for overnight at 20.degree. C. Each cell pellet was
suspended in 20 mM Tris-Cl buffer, pH 7.4+200 mM NaCl+1 mM
DTT+protease inhibitors and sonicated. Analysis by SDS-PAGE
confirmed expression of fusion proteins. [0140] Total cell free
extracts were run over amylose column attached to FPLC for affinity
purification of MBP-axmi fusion proteins. Bound fusion protein was
eluted from the resin with 10 mM maltose solution. Purified fusion
proteins were then cleaved with either Factor Xa or trypsin to
remove the amino terminal MBP tag from the Axmi protein. Cleavage
and solubility of the proteins was determined by SDS-PAGE.
Insect Bioassays
[0140] [0141] Cleaved proteins were tested in insect assays with
appropriate controls. A 5-day read of the plates showed following
activities of these proteins.
TABLE-US-00004 [0141] MBP-Axmi fusion Axmi protein protein cleaved
with Activity on insects Axmi079 Factor Xa, trypsin Diamondback
moth Axmi082 Factor Xa, trypsin Diamondback moth
Additional Insect Bioassay Results:
TABLE-US-00005 [0142] sample Gene tested C. elegans VBC* DBM* SWCB*
CPB* ECB* Hz* Hv* Axmi50 crude 3, 3 extract Axmi52 purified, 1, 0%
digested Axmi58 purified, 4, 100% digested Axmi68 crude 3, 2
extract Axmi88 purified, 1, 0% 1, 0% digested Axmi93 purified, 20%
digested Axmi97 purified, 1, 0% digested Axmi102 crude 4, 100% 3,
75% extract Axmi112 purified, 3, 0% 4, 100% 3, 25% 3, 75% 1, 0% 3,
0% digested Axmi117 purified, 1, 25% digested Axmi100 purified, 4,
100% 4, 100% digested VBC = Velvetbean caterpillar DBM =
diamondback moth SWCB = Southwestern corn borer CPB = Colorado
potato beetle ECB = European corn borer Hz = Helicoverpa zea Hv =
Heliothis virescens *= represented as stunt and mortality percent
where stunting is scored according to the following scale: Score
Definition 0 No Activity 1 Slight, non-uniform stunt 2 Non-uniform
stunt 3 Uniform stunt 4 Uniform stunt with mortality (expressed as
a percentage) 5 Uniform stunt with 100% mortality
Example 10
Vectoring of the Pesticidal Genes of the Invention for Plant
Expression
[0143] Each of the coding regions of the genes of the invention are
connected independently 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 CaMV .sup.35S promoter for expression in dicots, and
the nos or PinII terminators. Techniques for producing and
confirming promoter--gene--terminator constructs also are well
known in the art.
Example 11
Transformation of the Genes of the Invention into Plant Cells by
Agrobacterium-Mediated Transformation
[0144] 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.degree. 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.degree. C. in the dark). After co-cultivation,
explants are transferred to recovery period media for five days (at
25.degree. 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.
Example 12
Transformation of Maize Cells with the Pesticidal Genes of the
Invention
[0145] 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 1000.times. Stock) N6 Vitamins; 800 mg/L
L-Asparagine; 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.degree. C. in the dark.
[0146] 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. Pat. No. 5,240,842).
[0147] DNA constructs designed to express the genes of the
invention 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.degree. 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.degree. 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
TABLE-US-00006 [0148] DN62A5S Media Components per liter Source
Chu'S N6 Basal 3.98 g/L Phytotechnology Labs Salt Mixture (Prod.
No. C 416) Chu's N6 Vitamin 1 mL/L (of 1000x Stock) Phytotechnology
Labs Solution (Prod. No. C 149) L-Asparagine 800 mg/L
Phytotechnology Labs Myo-inositol 100 mg/L Sigma L-Proline 1.4 g/L
Phytotechnology Labs Casaminoacids 100 mg/L Fisher Scientific
Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L (of 1
mg/mL Stock) Sigma D-7299)
[0149] Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N
KCl, add Gelrite (Sigma) to 3 g/L, and autoclave. After cooling to
50.degree. C., add 2 ml/L of a 5 mg/ml stock solution of Silver
Nitrate (Phytotechnology Labs). Recipe yields about 20 plates.
[0150] 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.
[0151] 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.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130310543A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130310543A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References