Note: Descriptions are shown in the official language in which they were submitted.
AXMI277 NEMATODE TOXIN AND METHODS FOR ITS USE
10
FIELD OF THE INVENTION
This invention relates to the field of molecular biology. Provided are novel
genes that
encode pesticidal proteins. These proteins and the nucleic acid sequences that
encode them are
useful in preparing pesticidal formulations and in the production of
transgenic pest-resistant
plants.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis is a Gram-positive spore forming soil bacterium
characterized by
its ability to produce crystalline inclusions that are specifically toxic to
certain orders and species
of insects, but are harmless to plants and other 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.
Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have
potent
insecticidal activity against predominantly Lepidopteran, Hemipteran,
Dipteran, and Coleopteran
larvae. These proteins also have shown activity against Hymenoptera,
Homoptera, Phthiraptera,
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ilallophaga, 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 (1), Lepidoptera- and Diptera-specific
(11), 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 Ciy1C1,Cry1C2, etc.
A new nomenclature was recently described for the Cry genes based upon amino
acid
sequence homology rather than insect target specificity (Crickmore et al.
(1998) Microbiol. Mol.
Biol. Rev. 62:807-813). In the new classification, each toxin is assigned a
unique name
incorporating a primary rank (an Arabic number), a secondary rank (an
uppercase letter), a
tertiary rank (a lowercase letter), and a quaternary rank (another Arabic
number). In the new
classification, Roman numerals have been exchanged for Arabic numerals in the
primary rank.
Proteins with less than 45% sequence identity have different primary ranks,
and the criteria for
secondary and tertiary ranks are 78% and 95%, respectively.
The crystal protein does not exhibit insecticidal activity until it has been
ingested and
solubilized in the insect midgut. The ingested protoxin is hydrolyzed by
proteases in the insect
digestive tract to an active toxic molecule. (Hofte and Whiteley (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.
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 111 consists of two antiparallel beta-sheets in
"jelly-roll" formation (dc
Maagd et al., 2001, supra). Domains II and III are involved in receptor
recognition and binding,
and are therefore considered determinants of toxin specificity.
Because of the devastation that insects can confer, and the improvement in
yield by
controlling insect pests, there is a continual need to discover new forms of
pesticidal toxins.
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SUMMARY OF INVENTION
Compositions and methods for conferring pesticidal activity to bacteria,
plants, plant
cells, tissues and seeds are provided. Compositions include nucleic acid
molecules encoding
sequences for pesticidal and insectidal polypeptides, vectors comprising those
nucleic acid
molecules, and host cells comprising the vectors. Compositions also include
the pesticidal
polypeptide sequences 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 bacteria, plants, plant
cells, tissues, and
seeds comprising the nucleotide sequence of the invention.
In particular, isolated nucleic acid molecules are provided that encode a
pesticidal
protein. Additionally, amino acid sequences corresponding to the pesticidal
protein are
encompassed. In particular, the present invention provides for an isolated or
recombinant
nucleic acid molecule comprising a nucleotide sequence encoding the amino acid
sequence
shown in SEQ ID NO:2 or 3 or a nucleotide sequence set forth in SEQ ID NO:1,
as well as
biologically-active 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 or a complement thereof are also encompassed. Further provided are
vectors, host
cells, plants, and seeds comprising the nucleotide sequences of the invention,
or nucleotide
sequences encoding the amino acid sequences of the invention, as well as
biologically-active
variants and fragments thereof.
Methods are provided for producing the polypeptides of the invention, and for
using
those polypeptides for controlling or killing a lepidopteran, hemipteran,
coleopteran, nematode,
or dipteran pest. Methods and kits for detecting the nucleic acids and
polypeptides of the
invention in a sample are also included.
The compositions and methods of the invention are useful for the production of
organisms with enhanced pest resistance or tolerance. These organisms and
compositions
comprising the organisms are desirable for agricultural purposes. The
compositions of the
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invention are also useful for generating altered or improved proteins that
have pesticidal activity,
or for detecting the presence of pesticidal proteins or nucleic acids in
products or organisms.
DETAILED DESCRIPTION
The present invention is drawn to compositions and methods for regulating pest
resistance or tolerance in organisms, particularly plants or plant cells. By
"resistance" is
intended that the pest (e.g., insect) is killed upon ingestion or other
contact with the polypeptides
of the invention. By "tolerance" is intended an impairment or reduction in the
movement,
feeding, reproduction, or other functions of the pest. The methods involve
transforming
organisms with a nucleotide sequence encoding a pesticidal 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 pesticidal nucleic
acids and proteins of
Bacillus or other species. The sequences find use in the construction of
expression vectors for
subsequent transformation into organisms of interest, as probes for the
isolation of other
homologous (or partially homologous) genes, and for the generation of altered
pesticidal proteins
by methods known in the art, such as domain swapping or DNA shuffling, for
example, with
members of the Cryl, Cry2, and Cry9 families of endotoxins. The proteins find
use in
controlling or killing lepidopteran, hemipteran, coleopteran, dipteran, and
nematode pest
populations and for producing compositions with pesticidal activity.
By "pesticidal toxin" or "pesticidal protein" is intended a toxin that has
toxic activity
against one or more pests, including, but not limited to, members of the
Lepidoptera, Diptera,
and Coleoptera orders, or the Nematoda phylum, or a protein that has homology
to such a
protein. Pesticidal proteins have been isolated from organisms including, for
example, Bacillus
sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal 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.
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Pesticidal proteins encompass delta-endotoxins. Delta-endotoxins include
proteins
identified as cry] through cry43, cytl and cyt2, and Cyt-like toxin. There are
currently over 250
known species of delta-endotoxins with a wide range of specificities and
toxicities. For an
expansive list see Crickmore et al. (1998), Microbiol. MoL Biol. Rev. 62:807-
813, and for
regular updates see Crickmore et al. (2003) "Bacillus thuringiensi.s toxin
nomenclature,".
Thus, provided herein are novel isolated or recombinant nucleotide sequences
that confer
pesticidal activity. These nucleotide sequences encode polypeptides with
homology to known
delta-endotoxins or binary toxins. Also provided are the amino acid sequences
of the pesticidal
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
One aspect of the invention pertains to isolated or recombinant nucleic acid
molecules
comprising nucleotide sequences encoding pesticidal proteins and polypeptides
or biologically
active portions thereof, as well as nucleic acid molecules sufficient for use
as hybridization
probes to identify nucleic acid molecules encoding proteins with regions of
sequence homology.
Also encompassed herein are nucleotide sequences capable of hybridizing to the
nucleotide
sequences of the invention under stringent conditions as defined elsewhere
herein. 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.
An "isolated" or "recombinant" nucleic acid sequence (or DNA) is used herein
to refer to
a nucleic acid sequence (or DNA) that is no longer in its natural environment,
for example in an
in vitro or in a recombinant bacterial or plant host cell. In some
embodiments, an isolated or
recombinant 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
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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. In various embodiments, 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").
Nucleotide sequences encoding the proteins of the present invention include
the sequence
set forth in SEQ ID NO:1, 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 sequences for the pesticidal proteins
encoded by these
nucleotide sequences are set forth in SEQ ID NO:2 or 3.
Nucleic acid molecules that are fragments of these nucleotide sequences
encoding
pesticidal proteins are also encompassed by the present invention. By
"fragment" is intended a
portion of the nucleotide sequence encoding a pesticidal protein. A fragment
of a nucleotide
sequence may encode a biologically active portion of a pesticidal 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 nucleotide sequence
encoding a pesticidal
protein comprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 1100,
1200, 1300, 1350, 1400 contiguous nucleotides, or up to the number of
nucleotides present in a
full-length nucleotide sequence encoding a pesticidal protein 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 pesticidal protein
and, hence, retain pesticidal activity. Thus, biologically-active fragments of
the polypeptides
disclosed herein are also encompassed. 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 pesticidal protein. In one embodiment, the
pesticidal activity is
coleoptericidal activity. In another embodiment, the pesticidal activity is
lepidoptericidal
activity. In another embodiment, the pesticidal activity is nematocidal
activity. In another
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embodiment, the pesticidal activity is diptericidal activity. In another
embodiment, the pesticidal
activity is hemiptericidal activity. Methods for measuring pesticidal activity
are well known in
the art. See, for example, Czapla and Lang (1990)1 Econ. Entomol. 83:2480-
2485; Andrews et
al. (1988) Biochem. 1 252:199-206; Marrone etal. (1985)/ of Economic
Entomology 78:290-
293; and U.S. Patent No. 5,743,477,
A fragment of a nucleotide sequence encoding a pesticidal protein 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 contiguous amino acids,
or up to the total
number of amino acids present in a full-length pesticidal protein of the
invention. In some
embodiments, the fragment is a proteolytic cleavage fragment. For example, the
proteolytic
cleavage fragment may have an N-terminal or a C-terminal truncation of at
least about 100
amino acids, about 120, about 130, about 140, about 150, or about 160 amino
acids relative to
SEQ ID NO:2 or 3. In some embodiments, the fragments encompassed herein result
from the
removal of the C-terminal crystallization domain. e.g., by proteolysis or by
insertion of a stop
codon in the coding sequence.
Preferred pesticidal proteins of the present invention are encoded by a
nucleotide
sequence sufficiently identical to the nucleotide sequence of SEQ ID NO:1, or
the pesticidal
proteins are sufficiently identical to the amino acid sequence set forth in
SEQ ID NO:2 or 3. 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.
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
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positions) x 100). In one embodiment, the two sequences are the same length.
In another
embodiment, the percent identity is calculated across the entirety of the
reference sequence (i.e.,
the sequence disclosed herein as any of SEQ ID NO:1-3). 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. A gap, i.e. a
position in an alignment where a residue is present in one sequence but not in
the other, is
regarded as a position with non-identical residues.
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.
Sc!. USA
90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX
programs of
Altschul etal. (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 pesticidal-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 pesticidal 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 etal. (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 etal. (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.
Another non-limiting example of a mathematical algorithm utilized for the
comparison of
sequences is the ClustalW algorithm (Higgins etal. (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, CA). 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
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analysis of ClustalW alignments is GENEDOCTM. GENEDOCTM (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) CA BIOS 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, CA, 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.
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.
The invention also encompasses variant nucleic acid molecules. "Variants" of
the
pesticidal protein encoding nucleotide sequences include those sequences that
encode the
pesticidal 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 pesticidal 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, 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
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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. Patent No. 5,743,477,
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 pesticidal 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 arc
introduced into the
encoded protein. Mutations can be introduced by standard techniques, such as
site-directed
mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences
are also
encompassed by the present invention.
For example, 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 pesticidal 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, argininc, histidinc), acidic side chains (e.g., aspartic
acid, glutamic acid),
uncharged polar side chains (e.g., glycinc, asparaginc, glutamine, scrine,
threoninc, 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).
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
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Maagd et al., 2001, supra). Domains II and III are involved in receptor
recognition and binding,
and are therefore considered determinants of toxin specificity.
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
similar or related toxins to the sequences of the invention (e.g., residues
that are identical in an
alignment of homologous proteins). 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 similar or
related toxins to the sequences of the invention (e.g., residues that have
only conservative
substitutions between all proteins contained in the alignment homologous
proteins). However,
one of skill in the art would understand that functional variants may have
minor conserved or
nonconserved alterations in the conserved residues.
Alternatively, variant nucleotide sequences can be made by introducing
mutations
randomly along all or part of the coding sequence, such as by saturation
mutagenesis, and the
resultant mutants can be screened for ability to confer pesticidal activity to
identify mutants that
retain activity. Following mutagenesis, the encoded protein can be expressed
recombinantly, and
.. the activity of the protein can be determined using standard assay
techniques.
Using methods such as PCR, hybridization, and the like corresponding
pesticidal
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, NY) and
Innis, et al.
.. (1990) PR Protocols: A Guide to Methods and Applications (Academic Press,
NY).
In a hybridization method, all or part of the pesticidal nucleotide sequence
can be used to
screen cDNA or genomic libraries. Methods for construction of such cDNA and
gnomic
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 32P, or
any other detectable marker, such as other radioisotopes, a fluorescent
compound, an enzyme, or
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an enzyme co-factor. Probes for hybridization can be made by labeling
synthetic
oligonucleotides based on the known pesticidal protein-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, or 200
consecutive nucleotides of nucleotide sequence encoding a pesticidal protein
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
For example, an entire pesticidal sequence disclosed herein, or one or more
portions
thereof, may be used as a probe capable of specifically hybridizing to
corresponding pesticidal
protein-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 pesticidal 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; sec, for
example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York).
Thus, the present invention encompasses probes for hybridization, as well as
nucleotide
sequences capable of hybridization to all or a portion of a nucleotide
sequence of the invention
(e.g., at least about 300 nucleotides, at least about 400, at least about 500,
1000, 1200, 1500,
2000, 2500, 3000, 3500, or up to the full length of a nucleotide sequence
disclosed herein).
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
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identified (homologous probing). Alternatively, stringency conditions can be
adjusted to allow
some mismatching in sequences so that lower degrees of similarity are detected
(heterologous
probing). Generally, a probe is less than about 1000 nucleotides in length,
preferably less than
500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration
is less than
about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or
other salts) at pH 7.0
to 8.3 and the temperature is at least about 30 C for short probes (e.g., 10
to 50 nucleotides) and
at least about 60 C for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may
also be achieved with the addition of destabilizing agents such as formamide.
Exemplary low
stringency conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M
NaCl, 1% SDS (sodium dodecyl sulphate) at 37 C, and a wash in 1X to 2X SSC
(20X SSC = 3.0
M NaCl/0 .3 M trisodium citrate) at 50 to 55 C. Exemplary moderate stringency
conditions
include hybridization in 40 to 45% formamide, 1.0 M NaC1, 1% SDS at 37 C, and
a wash in
0.5X to lx SSC at 55 to 60 C. Exemplary high stringency conditions include
hybridization in
50% formamide, 1 M NaCl, 1% SDS at 37 C, and a wash in 0.1X SSC at 60 to 65 C.
Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of
hybridization
is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the
critical factors
being the ionic strength and temperature of the final wash solution. For DNA-
DNA hybrids, the
Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal.
Biochem.
138:267-284: Tm = 81.5 C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L;
where M is
the molarity of monovalent cations, %GC is the percentage of guanosine and
cytosine
nucleotides in the DNA, % form is the percentage of formamide in the
hybridization solution,
and L is the length of the hybrid in base pairs. The Tm is the temperature
(under defined ionic
strength and pH) at which 50% of a complementary target sequence hybridizes to
a perfectly
matched probe. Tm is reduced by about 1 C for each 1% of mismatching; thus,
Tm,
hybridization, and/or wash conditions can be adjusted to hybridize to
sequences of the desired
identity. For example, if sequences with >90% identity are sought, the Tm can
be decreased
10 C. Generally, stringent conditions are selected to be about 5 C lower than
the thermal
melting point (Tm) for the specific sequence and its complement at a defined
ionic strength and
pH. However, severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3, or
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4 C lower than the thermal melting point (Tm); moderately stringent conditions
can utilize a
hybridization and/or wash at 6, 7, 8, 9, or 10 C lower than the thermal
melting point (T,,); low
stringency conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 15, or 20 C lower
than the thermal melting point (Tm). Using the equation, hybridization and
wash compositions,
and desired Tm, those of ordinary skill will understand that variations in the
stringency of
hybridization and/or wash solutions are inherently described. If the desired
degree of
mismatching results in a Tm of less than 45 C (aqueous solution) or 32 C
(formamide solution),
it is preferred to increase the SSC concentration so that a higher temperature
can be used. An
extensive guide to the hybridization of nucleic acids is found in Tijssen
(1993) Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic
Acid Probes,
Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995)
Current Protocols in
Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New
York). See
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, New York).
Isolated Proteins and Variants and Fragments Thereof
Pesticidal proteins are also encompassed within the present invention. By
"pesticidal
protein" is intended a protein having the amino acid sequence set forth in SEQ
ID NO:2 or 3.
Fragments, biologically active portions, and variants thereof are also
provided, and may be used
to practice the methods of the present invention. An "isolated protein" or a
"recombinant
protein" is used to refer to a protein that is no longer in its natural
environment, for example in
vitro or in a recombinant bacterial or plant host cell.
"Fragments" or "biologically active portions" include polypeptide fragments
comprising
amino acid sequences sufficiently identical to the amino acid sequence set
forth in SEQ ID NO:2
or 3, and that exhibit pesticidal activity. A biologically active portion of a
pesticidal protein can
be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300,
350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,
1250, 1300, 1350, 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)1 Econ.
Entomol. 83:2480-
2485; Andrews etal. (1988) Biochem. 1252:199-206; Marrone et al. (1985) / of
Economic
-14-
Entomology 78:290-293; and U.S. Patent No. 5,743,477>
As used here, a fragment comprises at least 8 contiguous amino acids
of SEQ ID NO:2 or 3. The invention encompasses other fragments, however, such
as any
fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250,
300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300,
1350 or more amino acids in length.
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:2 or 3.
Variants also include polypeptides encoded by a nucleic acid molecule that
hybridizes to the
nucleic acid molecule of SEQ ID NO:!, or a complement thereof, under stringent
conditions.
Variants include polypeptides that differ in amino acid sequence due to
mutagencsis. 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.
In some embodiments, the variants have improved activity relative to the
native protein.
Methods for measuring pesticidal activity are well known in the art. See, for
example, Czapla
and Lang (1990)1 Econ. Entomol. 83:2480-2485; Andrews etal. (1988) Biochem. J.
252:199-
206; Marrone etal. (1985)J. of Economic Entomology 78:290-293; and U.S. Patent
No.
5,743,477.
Bacterial genes, such as the canti 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. On rare occasions, translation
in bacterial systems
can initiate at a TTG codon, though in this event the TTG encodes a
methionine. 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
pesticidal proteins. These pesticidal proteins are encompassed in the present
invention and may
be used in the methods of the present invention. It will be understood that,
when expressed in
plants, it will be necessary to alter the alternate start codon to ATG for
proper translation.
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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, NY; U.S. Patent No. 4,196,265).
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, NY; U.S. Patent No. 4,196,265).
Thus, one aspect of the invention concerns antibodies, single-chain antigen
binding
molecules, or other proteins that specifically bind to one or more of the
protein or peptide
molecules of the invention and their homologs, fusions or fragments. In a
particularly preferred
embodiment, the antibody specifically binds to a protein having the amino acid
sequence set
forth in SEQ ID NO:2 or 3, or a fragment thereof In another embodiment, the
antibody
specifically binds to a fusion protein comprising an amino acid sequence
selected from the amino
acid sequence set forth in SEQ ID NO:2 or 3, or a fragment thereof.
Antibodies of the invention may be used to quantitatively or qualitatively
detect the
protein or peptide molecules of the invention, or to detect post translational
modifications of the
proteins. As used herein, an antibody or peptide is said to "specifically
bind" to a protein or
peptide molecule of the invention if such binding is not competitively
inhibited by the presence
of non-related molecules.
Altered or Improved Variants
It is recognized that DNA sequences of a pesticidal protein may be altered by
various
methods, and that these alterations may result in DNA sequences encoding
proteins with amino
.. acid sequences different than that encoded by a pesticidal protein 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:2 or 3, 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, about 135, about 140, about 145, about 150, about 155, or more
amino acid
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substitutions, deletions or insertions. Methods for such manipulations are
generally known in the
art. For example, amino acid sequence variants of a pesticidal 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
pesticidal protein to confer
pesticidal activity may be improved by the use of such techniques upon the
compositions of this
invention. For example, one may express a pesticidal protein in host cells
that exhibit high rates
of base misincorporation during DNA replication, such as XL-1 Red (Stratagene,
La Jolla, CA).
After propagation in such strains, one can isolate the DNA (for example by
preparing plasmid
DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a
vector), culture
the pesticidal protein mutations in a non-mutagenic strain, and identify
mutated genes with
pesticidal activity, for example by performing an assay to test for pesticidal
activity. Generally,
the protein is mixed and used in feeding assays. See, for example Marrone et
al. (1985)J. of
Economic Entomology 78:290-293. Such assays can include contacting plants with
one or more
pests and determining the plant's ability to survive and/or cause the death of
the pests. Examples
of mutations that result in increased toxicity are found in Schnepf et al.
(1998) Microbiol. Mol.
Biol. Rev. 62:775-806.
Alternatively, alterations may be made to the protein sequence of many
proteins at the
amino or carboxy terminus without substantially affecting activity. This can
include insertions,
deletions, or alterations introduced by modem 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.
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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 pesticidal protein coding regions
can be used to
create a new pesticidal 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 pesticidal gene of the
invention and
other known pesticidal 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.11/IoL Biol. 272:336-347; Zhang et al. (1997) Proc.
Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patent Nos.
5,605,793 and
5,837,458.
Domain swapping or shuffling is another mechanism for generating altered
pesticidal
proteins. Domains may be swapped between pesticidal 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
A pesticidal 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
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intracellular structures such as the chloroplast (or other plastid),
endoplasmic reticulum, or Golgi
apparatus.
By "signal sequence" is intended a sequence that is known or suspected to
result in
cotranslational or post-translational peptide transport across the cell
membrane. In eukaryotes,
this typically involves secretion into the Golgi apparatus, with some
resulting glycosylation.
Insecticidal toxins of bacteria are often synthesized as protoxins, which are
protolytically
activated in the gut of the target pest (Chang (1987) Methods Enzymol. 153:507-
516). In some
embodiments of the present invention, the signal sequence is located in the
native sequence, or
may be derived from a sequence of the invention. 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 subcellular organelle. Thus,
this includes leader
sequences targeting transport and/or glycosylation by passage into the
endoplasmic reticulum,
passage to vacuoles, plastids including chloroplasts, mitochondria, and the
like.
By "plant transformation vector" is intended a DNA molecule that is necessary
for
efficient transformation of a plant cell. Such a molecule may consist of one
or more plant
expression cassettes, and may be organized into more than one "vector" DNA
molecule. For
example, binary vectors are plant transformation vectors that utilize two non-
contiguous DNA
vectors to encode all requisite cis- and 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/or
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.
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In various embodiments, the nucleotide sequence of the invention is operably
linked to a
promoter, e.g., a plant promoter. "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.
Such an expression cassette is provided with a plurality of restriction sites
for insertion of
the pesticidal sequence to be under the transcriptional regulation of the
regulatory regions.
The expression cassette will include in the 5'-3' direction of transcription,
a
transcriptional and translational initiation region (i.e., a promoter), a DNA
sequence of the
invention, and a 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.
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; San facon 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
etal. (1989)
Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res.
15:9627-9639.
Where appropriate, the gene(s) may be optimized for increased expression in
the
transformed host cell. That is, the genes can be synthesized using host 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 Gown i (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred
codon usage.
-20-
Methods are available in the art for synthesizing plant-preferred genes. See,
for example, U.S.
Patent Nos. 5,380,831, and 5,436,391, U.S. Patent Publication No. 20090137409,
and Murray et
al. (1989) Nucleic Acids Res. 17:477-498.
In one embodiment, the pesticidal protein is targeted to the chloroplast for
expression. In
this manner, where the pesticidal protein is not directly inserted into the
chloroplast, the
expression cassette will additionally contain a nucleic acid encoding a
transit peptide to direct the
pesticidal protein 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)1.
Chem. 264:17544-17550; Della-Cioppa et al. (1987)P/cult Phy.slo/. 84:965-968;
Romer et al.
(1993) Biochem. Biophys. Res. Conunun. 196:1414-1421; and Shah et al. (1986)
Science
233:478-481.
The pesticidal 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. Patent No. 5,380,831.
Plant Transformation
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.
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).
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"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.
The transgenic plants of the invention express one or more of the novel toxin
sequences
disclosed herein. In various embodiments, the transgenic plant further
comprises one or more
additional genes for insect resistance (e.g., Cryl, such as members of the
Cry1A, Cryl B, Cry1C,
Cryl D, Cryl E, and CrylF families; Cry2, such as members of the Cry2A family;
Cry9, such as
members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.).
It will be
understood by one of skill in the art that the transgenic plant may comprise
any gene imparting
an agronomic trait of interest.
Transformation of plant cells can be accomplished by one of several techniques
known in
the art. The pesticidal 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.
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
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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 pesticidal gene 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.
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 plantict then grows into a mature
plant and produces
fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; lshida 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) Mayalica 42:107-120. Since
the
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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.
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.
Patent No. 4,945,050; International Publication No. WO 91/00915; U.S.
Published Application
No. 2002015066), Led l transformation, and various other non-particle direct-
mediated methods
to transfer DNA.
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 at. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
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.
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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
Following introduction of heterologous foreign DNA into plant cells, the
transformation
or integration of heterologous gene in the plant genome is confirmed by
various methods such as
analysis of nucleic acids, proteins and metabolites associated with the
integrated gene.
PCR analysis 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, NY). PCR is carried out using oligonucleotide
primers specific to the
gene of interest or Agrobacterium vector background, etc.
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 32P target DNA fragment to confirm the integration of introduced
gene into the plant
genome according to standard techniques (Sambrook and Russell, 2001, supra).
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 pesticidal gene is then tested by hybridizing the filter to
a radioactive probe
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derived from a pesticidal gene, by methods known in the art (Sambrook and
Russell, 2001,
supra).
Western blot, biochemical assays and the like may be carried out on the
transgenic plants
to confirm the presence of protein encoded by the pesticidal gene by standard
procedures
(Sambrook and Russell, 2001, supra) using antibodies that bind to one or more
epitopcs present
on the pesticidal protein.
Pesticidal Activity in Plants
In another aspect of the invention, one may generate transgenic plants
expressing a
pesticidal protein 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
pesticidal protein
may be isolated by common methods described in the art, for example by
transformation of
callus, selection of transformed callus, and regeneration of fertile plants
from such transgenic
callus. In such process, one may use any gene as a selectable marker so long
as its expression in
plant cells confers ability to identify or select for transformed cells.
A number of markers have been developed for use with plant cells, such as
resistance to
chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes
that encode a
product involved in chloroplast metabolism may also be used as selectable
markers. For
example, genes that provide resistance to plant herbicides such as glyphosate,
bromoxynil, or
imidazolinone may find particular use. Such genes have been reported (Stalker
et at. (1985)J.
Biol. Chem. 263:6310-6314 (bromoxynil resistance nitrilase gene); and
Sathasivan et at. (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.
Fertile plants expressing a pesticidal protein may be tested for pesticidal
activity, and the
plants showing optimal activity selected for further breeding. Methods are
available in the art to
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assay for pest activity. Generally, the protein is mixed and used in feeding
assays. See, for
example Marrone etal. (1985)J. of Economic Entomology 78:290-293.
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.
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 Pesticidal Control
General methods for employing strains comprising a nucleotide sequence of the
present
invention, or a variant thereof, in pest control or in engineering other
organisms as pesticidal
agents are known in the art. See, for example U.S. Patent No. 5,039,523 and EP
0480762A2.
The Bacillus strains 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 of the invention 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).
Alternatively, the pesticide is produced by introducing a pesticidal gene into
a cellular
host. Expression of the pesticidal 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 the target pest(s). The resulting product
retains the toxicity of
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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.
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.
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.
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.
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Lepidopteran, hemipteran, dipteran, or coleopteran pests may be killed or
reduced in
numbers in a given area by the methods of the invention, or may be
prophylactically applied to
an environmental area to prevent infestation by a susceptible pest. Preferably
the pest ingests, or
is contacted with, a pesticidally-effective amount of the polypeptide. By
"pesticidally-effective
amount" is intended an amount of the pesticide that is able to bring about
death to at least one
pest, or to noticeably reduce pest growth, feeding, or normal physiological
development. This
amount will vary depending on such factors as, for example, the specific
target pests to be
controlled, the specific environment, location, plant, crop, or agricultural
site to be treated, the
environmental conditions, and the method, rate, concentration, stability, and
quantity of
application of the pesticidally-effective polypeptide composition. The
formulations may also
vary with respect to climatic conditions, environmental considerations, and/or
frequency of
application and/or severity of pest infestation.
The pesticide compositions described may be made by formulating either the
bacterial
cell, the crystal and/or the spore suspension, or the isolated protein
component with the desired
agriculturally-acceptable carrier. The compositions may be formulated prior to
administration in
an appropriate means such as lyophilized, freeze-dried, desiccated, or in an
aqueous carrier,
medium or suitable diluent, such as saline or other buffer. The formulated
compositions may be
in the form of a dust or granular material, or a suspension in oil (vegetable
or mineral), or water
or oil/water emulsions, or as a wettable powder, or in combination with any
other carrier material
suitable for agricultural application. Suitable agricultural carriers can be
solid or liquid and are
well known in the art. The term "agriculturally-acceptable carrier" covers all
adjuvants, inert
components, dispersants, surfactants, tackifiers, binders, etc. that are
ordinarily used in pesticide
formulation technology; these are well known to those skilled in pesticide
formulation. The
formulations may be mixed with one or more solid or liquid adjuvants and
prepared by various
means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal
composition with
suitable adjuvants using conventional formulation techniques. Suitable
formulations and
application methods are described in U.S. Patent No. 6,468,523,,
"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,
Lepicloptera, Mallophaga, Homoptera, Hetniptera, Orthroptera, Thysanoptera,
Dermaptera,
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Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera,
Lepidoptera, and
Diptera.
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, Diyopoidea, 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 Gyriniclae. Superfamily
Hydrophiloidea includes the family Hydrophilidae. Superfamily Staphylinoidea
includes the
families Silphidae and Staphylinidae. 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 Pas salidae and Scarabaeidae. Superfamily
Cerambycoidea
includes the family Ceranzbycidae. Superfamily Chrysomeloidea includes the
family
Chrysomelidae. Superfamily Curculionoidea includes the families Cztrculionidae
and
Scolytidae.
The order Diptera includes the Suborders Nematocera, Brachycera, and
Cyclorrhapha.
Suborder Nenzatocera 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 Phoridcte, 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.
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The order Lepidoptera includes the families Papilionidae, Pieridae,
Lycaenidae,
Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae,
Geometridae,
Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.
Insect pests of the invention for the major crops include: Maize: Ostrinia
nubilalis,
European corn borer; Agrotis ipsilon, black cutworm; Relicoverpa zea, corn
earworm;
Spodoptera frugeperda, fall armyworm; Diatraea grandiose/la, southwestern corn
borer;
Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis,
surgarcane borer;
Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi,
northern corn
rootworm; Diabrotica undeeimpunctata 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;
Chaetocneina pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug;
Rhopalosiphum
maidis, corn leaf aphid; Anuraphis maidimdicis, 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; Solenopseis
milesta, thief ant;
Tetranychus urticae, twospotted spider mite; Sorghum: Chilo partellus, sorghum
borer;
Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm;
Elastnopalpus
lignosellus, lesser cornstalk borer; Feltia subterranea, granulate cutworm;
Phyllophaga crinita,
white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulerna 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; Con tarinia sorghicola, sorghum midge; Tetranychus
cinnabarinus,
carmine spider mite; Tetranychus urticae, twospotted spider mite; Wheat:
Pseudaletia
unipunctata, army worm; Spodopterafrugiperda, fall armyworm; Elasmopalpus
lignosellus,
lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus
lignosellus, lesser
cornstalk borer; Ouleena melanopus, cereal leaf beetle; Hypera punctata,
clover leaf weevil;
Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat
aphid; Schizaphis
grain mum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus
femurrubrum,
redlegged grasshopper; Melanoplus differentiate's, differential grasshopper;
Melanoplus
sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly;
Sitodiplos is masellana,
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wheat midge; kleronzyza 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;
Ifelanoplus 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 jrugiperda,
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 hi/are,
green stink bug;
Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemnzatalis,
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
hi/are, green
stink bug; Melanoplus .femurrubrum, redlegged grasshopper; Melanoplus
differentialis,
differential grasshopper; Hylemya platura, seedcom maggot; Sericothrips
variabilis, soybean
thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider
mite; Tetranychus
urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn
borer; Agrotis ipsilon,
black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus,
chinch bug;
Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Delia
platura,
seedcom maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown
wheat mite; Oil
Seed Rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, Flea
beetle;
Mamestra configurata, Bertha armyworm; Plutella xylostella, Diamond-back moth;
Delia ssp.,
Root maggots.
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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.
Methods for Increasing Plant Yield
Methods for increasing plant yield are provided. The methods comprise
providing a plant
.. or plant cell expressing a polynucleotide encoding the pesticidal
polypeptide sequence disclosed
herein and growing the plant or a seed thereof in a field infested with (or
susceptible to
infestation by) a pest against which said polypeptide has pesticidal activity.
In some
embodiments, the polypeptide has pesticidal activity against a lepidopteran,
coleopteran,
dipteran, hemipteran, or nematode pest, and said field is infested with a
lepidopteran,
hcmipteran, coleopteran, diptcran, or nematode pest. 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. 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.
The plants can also be treated with one or more chemical compositions,
including one or
more herbicide, insecticides, or fungicides. Exemplary chemical compositions
include:
Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron,
Metribuzin,
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Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat,
Propyzamide,
Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables
Insecticides:
Aldicarb , Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos,
Cypermethrin,
Dcltamethrin, Abamcctin, Cyfluthrin/beta-cyfluthrin, Esfenvaleratc, Lambda-
cyhalothrin,
Accquinocyl, Bifcnazate, Methoxyfcnozide, Novaluron, Chromafenozidc,
Thiacloprid,
Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen,
Spinosad,
Rynaxypyr, Cyazypyr, Triflumuron,Spirotetramat, Imidacloprid, Flubendiamide,
Thiodicarb,
Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin,
Thiamethoxam,
Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate,
Indoxacarb,
Fenamiphos, Pyriproxifen, Fenbutatin-oxid; Fruits/Vegetables Fungicides:
Ametoctradin,
Azoxystrobin, Benthiavalicarb, Boscalid, Captan, Carbendazim, Chlorothalonil,
Copper,
Cyazofamid, Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil,
Difenoconazole,
Dimetomorph, Dithianon, Fenamidone, Fenhexamid, Fluazinam, Fludioxonil,
Fluopicolide,
Fluopyram, Fluoxastrobin, Fluxapyroxad, Folpet, Fosetyl, Iprodione,
Iprovalicarb, Isopyrazam,
Krcsoxim-methyl, Mancozeb, Mandipropamid, Metalaxyl/mefenoxam, Mctiram,
Metrafcnone,
Myclobutanil, Penconazole, Penthiopyrad, Picoxystrobin, Propamocarb,
Propiconazole,
Propineb, Proquinazid, Prothioconazole, Pyraclostrobin, Pyrimethanil,
Quinoxyfen,
Spiroxamine, Sulphur, Tebuconazole, Thiophanate-methyl, Trifloxystrobin;
Cereals Herbicides:
2.4-D, Amidosulfuron, Bromoxynil, Carfentrazone-E, Chlorotoluron,
Chlorsulfuron, Clodinafop-
P, Clopyralid, Dicamba, Diclofop-M, Diflufenican, Fenoxaprop, Florasulam,
Flucarbazone-NA,
Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate,
Iodosulfuron, Ioxynil,
Isoproturon, MCPA, Mesosulfuron, Metsulfuron, Pendimethalin, Pinoxaden,
Propoxycarbazone,
Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron, Tralkoxydim,
Triasulfuron,
Tribenuron, Trifluralin, Tritosulfuron; Cereals Fungicides: Azoxystrobin,
Bixafen, Boscalid,
.. Carbendazim, Chlorothalonil, Cyflufenamid, Cyproconazole, Cyprodinil,
Dimoxystrobin,
Epoxiconazolc, Fenpropidin, Fenpropimorph, Fluopyram, Fluoxastrobin,
Fluquinconazolc,
Fluxapyroxad, Isopyrazam, Kresoxim-methyl, Metconazolc, Metrafenone,
Penthiopyrad,
Picoxystrobin, Prochloraz, Propiconazole, Proquinazid, Prothioconazole,
Pyraclostrobin,
Quinoxyfen, Spiroxamine, Tebuconazole, Thiophanate-methyl , Trifloxystrobin;
Cereals
Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-
Cypermethrin,13-cyfluthrin,
Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid,
Acetamiprid, Dinetofuran,
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Clorphyriphos, Pirimicarb, Methiocarb, Sulfoxaflor; Maize Herbicides:
Atrazine, Alachlor,
Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate,
Glyphosate,
Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron,
Rimsulfuron,
Sulcotrionc, Foramsulfuron, Topramczone, Tembotrione, Saflufenacil,
Thicncarbazone,
Flufenacct, Pyroxasuffon; Maize Insecticides: Carbofuran, Chlorpyrifos,
Bifenthrin, Fipronil,
Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam,
Clothianidin,
Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb,
13-Cyfluthrin,
Cypermethrin, Bifenthrin, Lufenuron, Tebupirimphos, Ethiprole, Cyazypyr,
Thiacloprid,
Acetamiprid, Dinetofuran, Avermectin; Maize Fungicides: Azoxystrobin, Bixafen,
Boscalid,
Cyproconazole, Dimoxystrobin, Epoxiconazole, Fenitropan, Fluopyram,
Fluoxastrobin,
Fluxapyroxad, Isopyrazam, Metconazole, Penthiopyrad, Picoxystrobin,
Propiconazole,
Prothioconazole, Pyraclostrobin, Tebuconazole, Trifloxystrobin; Rice
Herbicides: Butachlor,
Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide,
Imazosulfuron,
Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,
Thiobencarb, Indanofan,
Flufenacct, Fentrazamide, Halosulfuron, Oxaziclomcfone, Benzobicyclon,
Pyriftalid,
Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione,
Tefuryltrione,
Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,
Fenobucarb, Benfuracarb,
Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,
Chromafenozide,
Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid,
Thiamethoxam,
Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,
Chlorpyriphos,
Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor; Rice Fungicides:
Azoxystrobin,
Carbendazim, Carpropamid, Diclocymet, Difenoconazo1e, Edifenphos, Ferimzone,
Gentamycin,
Hexaconazole, Hymexazo1, Iprobenfos (IBP), Isoprothiolane, Isotianil,
Kasugamycin,
Mancozeb, Metominostrobin, Orysastrobin, Pencycuron, Probenazole,
Propiconazole, Propineb,
Pyroquilon, Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole,
Trifloxystrobin,
Validamycin; Cotton Herbicides: Diuron, Fluomcturon, MSMA, Oxyfluorfen,
Prometryn,
Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,
Norflurazon, Pendimethalin,
Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin,
Thidiazuron;
Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,
Deltamethrin, Abamectin,
Acetamiprid, Ernamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-
Cyhalothrin, Spinosad,
Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid
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Flubendiamide, Triflumuron,Rynaxypyr,Beta-Cyfluthrin,Spirotetramat,
Clothianidin,
Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad,
Spinotoram,
gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yOmethyl](2,2-difluorethyDamino]furan-
2(5H)-on,
Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromcsifen, Sulfoxaflor;
Cotton Fungicides:
Azoxystrobin, Bixafcn, Boscalid, Carbcndazim, Chlorothalonil, Copper,
Cyproconazolc,
Di fenoconazole, Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam,
Fluopyram,
Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb,
Maneb,
Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole,
Pyraclostrobin,
Quintozene, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin;
Soybean
Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-
Methyl,
Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,
Imazethapyr, (S-
)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean
Insecticides:
Lambda-cyhalothrin, Methomyl, Imidacloprid, Clothianidin, Thiamethoxam,
Thiacloprid,
Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad,
Spinotoram,
Emamectin-Benzoate, Fipronil, Ethiprolc, Deltamethrin, B-Cyfluthrin, gamma and
lambda
Cyhalothrin, 4-[[(6-Chlorpyridin-3-yOmethyli(2,2-difluorethyl)amino]furan-
2(5H)-on,
Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-
Cyfluthrin; Soybean
Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil,
Copper,
Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam,
Fluopyram,
Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil,
Mancozeb, Maneb,
Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad, Picoxystrobin,
Propiconazole,
Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole,
Thiophanate-methyl,
Trifloxystrobin; Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate,
Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron,
Quinmerac, Cycloxydim,
.. Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:
Imidacloprid, Clothianidin,
Thiamethoxam, Thiacloprid, Acctamiprid, Dinetofuran, Deltamethrin, 13-
Cyfluthrin,
gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-
difluorethyl)amino]furan-
2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; Canola
Herbicides:
Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor,
Trifluralin
Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola
Fungicides:
Azoxystrobin, Bixafen, Boscalid, Carbendazim, Cyproconazole, Difenoconazole,
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Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin,
Flusilazole,
Fluxapyroxad, Iprodione, Isopyrazam, Mepiquat-chloride, Metconazole,
Metominostrobin,
Paclobutrazole, Penthiopyrad., Picoxystrobin, Prochloraz, Prothioconazole,
Pyraclostrobin,
Tebuconazole, Thiophanate-methyl, Trifloxystrobin, Vinelozolin; Canola
Insecticides:
Carbofuran, Thiacloprid, Deltamethrin, lmidacloprid, Clothianidin,
Thiamethoxam, Acetamiprid,
Dinetofuran, B-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate,
Ethiprole,
Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-
3-
yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL EXAMPLES
Example 1. Discovery of novel pesticidal genes from Bacillus thuringiensis
A novel pesticidal gene was identified from bacterial strain ATX47290 (Table
1).
Table 1. Novel gene identified from strain ATX47290
Amino
Molecular Nucleotide
acid
Gene name weight Closest homolog SEQ ID
SEQ
(kD) NO
ID NO
Axmi277 133.1 73.5% Axmi031 1 2
Axmi277(trun) 74.4% Cry14Aa (trun) 3
Axmi277 is amplified by PCR from pAX980, 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 axtni gene is
cultured on a
conventional growth media, such as CYS media (10 g/1 Bacto-casitone; 3 g/1
yeast extract; 6
KH2PO4; 14 g/1 K2HPO4; 0.5 mM MgSO4; 0.05 mM MnC12; 0.05 mM FeSO4), until
sporulation
is evident by microscopic examination. Samples are prepared and tested for
activity in bioassays.
Example 2. Activity of Axmi277 against C. elegans
The full-length and truncated genes were overexpressed in E. coil BL21(DE3)
star strain
behind the leader sequence that encodes for maltose binding protein (pMAL
expression vector).
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The cultures were grown at 37 C, 250 rpm in LB medium until the cultures
reached an optical
density (0D600) of 0.4 to 0.6, then cultures were switched to 20 C and protein
production was
induced by addition of 0.2 to 0.3 mM IPTG. The cells were harvested after 16
hours and the cell
pellet was stored for protein purification. The heterologous overexpressed
proteins were purified
using an AKTA FPLC system by affinity chromatography using amylose/agarose
beads. The
resin was used for isolation of proteins fused to maltose binding proteins.
The proteins were
eluted out of the column in the first 1 to 2 column volumes with buffer
containing 10mM
maltose. The purity and characterization of the proteins were done by SDS-PAGE
analysis.
Axmi277 showed very strong activity against C. elegans (80% average mortality
compared with controls). Both forms of the proteins, full length and truncated
version, showed
strong activity at low concentration, and the activities were heat labile.
Before submission to
bioassays, the proteins were processed with Factor Xa for removal of MBP
fragment.
Example 3. Additional Assays for Pesticidal Activity
The nucleotide sequences of the invention can be tested for their ability to
produce
pesticidal proteins. 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, and then dispensed into the assay
chamber. The assay
chamber may be, for example, a cup, a dish, or a well of a microtiter plate.
Assays for sucking pests (for example aphids) may involve separating the test
material
from the insect by a partition, ideally a portion that can be pierced by the
sucking 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.
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
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normally consumed, for example, small cages attached to a leaf, or isolation
of entire plants in
cages containing insects.
Other methods and approaches to assay pests are known in the art, and can be
found, for
example in Robertson and Preisler, eds. (1992) Pesticide bioassays with
arthropods, CRC, Boca
Raton, FL. Alternatively, assays arc 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).
In some embodiments, the DNA regions encoding the toxin region of the
pesticidal
proteins disclosed herein are cloned into the E. coli expression vector pMAL-
C4x behind the
malE gene coding for Maltose binding protein (MBP). These in-frame fusions
result in MBP-
Axmi fusion proteins expression in E. co/i.
For expression in E. coli, BL21*DE3 are transformed with individual plasmids.
Single
colonies are inoculated in LB supplemented with carbenicillin and glucose, and
grown overnight
at 37 C. The following day, fresh medium is inoculated with 1% of overnight
culture and grown
at 37 C to logarithmic phase. Subsequently, cultures are induced with 0.3mM
IPTG overnight at
C. Each cell pellet is suspended in 20mM Iris-Cl buffer, pH 7.4 + 200m1VI
NaC1+ 1mM
DTT + protease inhibitors and sonicated. Analysis by SDS-PAGE can be used to
confirm
expression of the fusion proteins.
Total cell free extracts are then run over amylose column attached to fast
protein liquid
20 chromatography (FPLC) for affinity purification of MBP-axmi fusion
proteins. Bound fusion
proteins are eluted from the resin with 10mM maltose solution. Purified fusion
proteins are 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 can be determined by SDS-PAGE
Example 4. Vectoring of Genes for Plant Expression
The coding regions of the invention are connected with appropriate promoter
and
terminator sequences for expression in plants. Such sequences arc 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 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.
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In one aspect of the invention, synthetic DNA sequences are designed and
generated.
These synthetic sequences have altered nucleotide sequence relative to the
parent sequence, but
encode proteins that are essentially identical to the parent sequence.
In another aspect of the invention, modified versions of the synthetic genes
arc 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 Lupinu.s albus (GENBANK 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 reticulum
retention sequence comprising the peptide N-terminus-lysine-aspartic acid-
glutamic acid-leucine
(i.e., the "KDEL" motif, SEQ ID NO:4) 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.
Thus, this gene encodes a fusion protein that contains the N-terminal thirty-
one amino
acids of the acid phosphatase gene from the White Lupin Lupinus alhus
(GENBANK(R) ID
GI:14276838 , Miller et al., 2001, supra) fused to the N-terminus of the amino
acid sequence of
the invention, as well as the KDEL sequence at the C-terminus. Thus, the
resulting protein is
.. predicted to be targeted the plant endoplasmic reticulum upon expression in
a plant cell.
The plant expression cassettes described above are combined with an
appropriate plant
selectable marker to aid in the selection of transformed cells and tissues,
and ligated into plant
transformation vectors. These may include binary vectors from Agrobacterium-
mediated
transformation or simple plasmid vectors for aerosol or biolistic
transformation.
Example 5. Transformation of Maize Cells with the pesticidal protein genes
described herein
Maize ears are best collected 8-12 days after pollination. Embryos are
isolated from the
ears, and those embryos 0.8-1.5 mm in size are preferred for use in
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 1000x Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L
Myo-inositol;
1.4 g/L L-Proline; 100 mg/L Casamino acids; 50 g/L sucrose; 1 mL/L (of 1
mg/nit Stock) 2,4-
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D). However, media and salts other than DN62A5S are suitable and are known in
the art.
Embryos are incubated overnight at 25 C in the dark. However, it is not
necessary per se to
incubate the embryos overnight.
The resulting explants arc transferred to mesh squares (30-40 per plate),
transferred onto
osmotic media for about 30-45 minutes, then transferred to a beaming plate
(see, for example,
PCT Publication No. WO/0138514 and U.S. Patent No. 5,240,842).
DNA constructs designed to 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 about 30
min on
osmotic media, and placed onto incubation media overnight at 25 C in the dark.
To avoid
unduly damaging beamed explants, they are incubated for at least 24 hours
prior to transfer to
recovery media. Embryos are then spread onto recovery period media, for about
5 days, 25 C in
the dark, then transferred to a selection media. Explants are incubated in
selection media for up
to eight weeks, depending on the nature and characteristics of the particular
selection utilized.
After the selection period, the resulting callus is transferred to embryo
maturation media, until
the formation of mature somatic embryos is observed. The resulting mature
somatic embryos are
then placed under low light, and the process of regeneration is initiated by
methods known in the
art. The resulting shoots are allowed to root on rooting media, and the
resulting plants are
transferred to nursery pots and propagated as transgenic plants.
Materials
DN62A5S Media
Components Per Liter Source
Chu's N6 Basal Salt Mixture
(Prod. No. C 416) 3.98 g/L Phytotechnology Labs
Chu's N6 Vitamin Solution
1 mL/L (of 1000x Stock) Phytotechnology Labs
(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
Casamino acids 100 mg/L Fisher Scientific
Sucrose 50 g/L Phytotechnology Labs
2,4-D (Prod. No. D-7299) 1 mL/L (of 1 mg/mL Stock) Sigma
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The pH of the solution is adjusted to pH 5.8 with IN KOH/1N KC1, Gelrite
(Sigma) is
added at a concentration up to 3g/L, and the media is autoclaved. After
cooling to 50 C, 2 ml/L
of a 5 mg/m1 stock solution of silver nitrate (Phytotechnology Labs) is added.
Example 6. Transformation of genes of the invention in Plant Cells by
Agrobacterium-Mediated
Transformation
Ears are best collected 8-12 days after pollination. Embryos are isolated from
the ears,
and those embryos 0.8-1.5 mm in size are preferred for use in transformation.
Embryos are
plated scutellum side-up on a suitable incubation media, and incubated
overnight at 25 C in the
dark. However, it is not necessary per se to incubate the embryos overnight.
Embryos are
contacted with an Agrobacterium strain containing the appropriate vectors for
Ti plasmid
mediated transfer for about 5-10 min, and then plated onto co-cultivation
media for about 3 days
(25 C in the dark). After co-cultivation, explants are transferred to recovery
period media for
about five days (at 25 C in the dark). Explants are incubated in selection
media for up to eight
weeks, depending on the nature and characteristics of the particular selection
utilized. After the
selection period, the resulting callus is transferred to embryo maturation
media, until the
formation of mature somatic embryos is observed. The resulting mature somatic
embryos are
then placed under low light, and the process of regeneration is initiated as
known in the art.
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.
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.
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