Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL INSECTICIDAL TOXINS FROM XENORHABDUS NEMATOPHILUS AND
NUCLEIC ACID SEQUENCES CODING THEREFOR
The invention relates to novel toxins from Xenorhabdus nematophilus,
Xenorhabdus
poinarii, and Phoforhabdus luminescens, nucleic acid sequences whose
expression results
in said toxins, and methods of making and methods of using the toxins and
corresponding
nucleic acid sequences to control insects.
Insect pests are a major cause of crop losses. Solely in the US, about $7.7
billion
are lost every year due to infestation by various genera of insects. In
addition to losses in
field crops, insect pests are also a burden to vegetable and fruit growers, to
producers of
ornamental flowers, and they are a nuisance to gardeners and home owners.
Insect pests are mainly controlled by intensive applications of chemical
insecticides,
which are active through inhibition of insect growth, prevention of insect
feeding or
reproduction, or death of the insects. Good insect control can thus be
reached, but these
chemicals can sometimes also affect other, beneficial insects. Another problem
resulting
from the wide use of chemical pesticides is the appearance of resistant insect
varieties.
This has been partially alleviated by various resistance management
strategies, but there is
an increasing need for alternative pest control agents. Biological insect
control agents,
such as Bacillus thuringiensis strains expressing insecticidal toxins like 8-
endotoxins, have
also been applied with satisfactory results, offering an alternative or a
complement to
chemical insecticides. Recently, the genes coding for some of these 8-
endotoxins have
been isolated and their expression in heterologous hosts have been shown to
provide
another tool for the control of economically important insect pests. In
particular, the
expression of insecticidal toxins in transgenic plants, such as Bacillus
thuringiensis 8-
endotoxins, has provided efficient protection against selected insect pests,
and transgenic
plants expressing such toxins have been commercialized, allowing farmers to
reduce
applications of chemical insect control agents. Yet, even in this case, the
development of
resistance remains a possibility and only a few specific insect pests are
controllable.
Consequently, there remains a long-felt but unfulfilled need to discover new
and effective
insect control agents that provide an economic benefit to farmers and that are
environmentally acceptable.
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The present invention addresses the long-standing need for novel insect
control
agents. Particularly needed are control agents that are targeted to
economically important
insect pests and that efficiently control insect strains resistant to existing
insect control
agents. Furthermore, agents whose application minimizes the burden on the
environment
are desirable.
In the search for novel insect control agents, certain classes of nematodes
from the
genera Heferorhabdus and Steinemema are of particular interest because of
their
insecticidal properties. They kill insect larvae and their offspring feed in
the dead larvae.
Indeed, the insecticidal activity is due to symbiotic bacteria living in the
nematodes. These
symbiotic bacteria are Photorhabdus in the case of Heterorhabdus and
Xenorhabdus in the
case of Steinernema.
The present invention. is drawn to nucleotide sequences isolated from
Xenorhabdus
nemafophilus, and nucleotide sequences substantially similar thereto, whose
expression
result in insecticidal toxins that are highly toxic to economically important
pests, particularly
plant pests. The invention is further drawn to the insecticidal toxin
resulting from the
expression of the nucleotide sequence, and to compositions and formulations
containing
the insecticidal toxin, that are capable of inhibiting the ability of insect
pests to survive, grow
or reproduce, or of limiting insect-related damage or loss in crop plants. The
invention is
further drawn to a method of making the toxin and to methods of using the
nucleotide
sequence, for example in microorganisms to control insects or in transgenic
plants to confer
insect resistance, and to a method of using the toxin, and compositions and
formulations
comprising the toxin, for example applying the toxin, composition or
formulation to insect
infested areas, or to prophylactically treat insect susceptible areas or
plants to confer
protection or resistance against harmful insects.
The novel toxin is highly insecticidal against Plutella xylosfella
(diamondback moth),
an economically important insect pest. The toxin can be used in multiple
insect control
strategies, resulting in maximal efficiency with minimal impact on the
environment.
According to one aspect, the present invention provides an isolated nucleic
acid
molecule comprising: (a) a nucleotide sequence substantially similar to a
nucleotide
sequence selected from the group consisting of: nucleotides 569-979 of SEa ID
N0:1,
nucleotides 1045-2334 of SEQ ID N0:1, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8,
SEQ
ID N0:10, SEQ ID N0:12, and SEQ ID N0:14; or (b) a nucleotide sequence
isocoding with
the nucleotide sequence of (a); wherein expression of said nucleic acid
molecule results in
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at least one toxin that is active against insects. In one embodiment of this
aspect, the
nucleotide sequence is isocoding with a nucleotide sequence substantially
similar to
nucleotides 569-979 of SEO ID N0:1, nucleotides 1045-2334 of SEQ ID N0:1, SEQ
ID
N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ ID N0:10, SEQ ID N0:12, or SEQ ID N0:14.
Preferably, the nucleotide sequence is substantially similar to nucleotides
569-979 of SEQ
ID N0:1, nucleotides 1045-2334 of SEQ ID N0:1, SEQ ID N0:4, SEQ ID N0:6, SEQ
ID
N0:8, SEO ID NO:10, SEQ ID N0:12, or SEQ ID N0:14. More preferably, the
nucleotide
sequence encodes an amino acid sequence selected from the group consisting of
SEQ ID
NOs:2, 3, 5, 7, 9, 11, 13, and 15. Most preferably, the nucleotide sequence
comprises
nucleotides 569-979 of SEO ID N0:1, nucleotides 1045-2334 of SEQ ID N0:1, SEQ
ID
N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ !D N0:10, SEQ ID N0:12, or SEQ ID N0:14.
In
another embodiment, the nucleotide sequence comprises the approximately 3.0 kb
DNA
fragment comprised in pCIB9369 (NRRL B-21883).
According to a preferred embodiment, the toxins resulting from expression of
the
nucleic acid molecules of the invention have activity against Plutella
xylostella.
In another aspect, the present invention provides an isolated nucleic acid
molecule
comprising a 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair
nucleotide portion
identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or
50 (preferably
20) base pair nucleotide portion of a nucleotide sequence selected from the
group
consisting of: nucleotides 569-979 of SEQ ID N0:1, nucleotides 1045-2334 of
SEQ ID
N0:1, SEQ ID N0:4, SE4 ID N0:6, SEQ ID N0:8, SEQ ID N0:10, SEQ ID N0:12, and
SEO
lD N0:14, wherein expression of said nucleic acid molecule results in at least
one toxin that
is active against insects.
The present invention also provides a chimeric gene comprising a heterologous
promoter sequence operatively linked to a nucleic acid molecule of the
invention. Further,
the present invention provides a recombinant vector comprising such a chimeric
gene. Still
further, the present invention provides a host cell comprising such a chimeric
gene. A host
cell according to this aspect of the invention may be a bacterial cell, a
yeast cell, or a plant
cell, preferably a plant cell. Even further, the present invention provides a
plant comprising
such a plant cell. Preferably, the plant is maize.
In yet another aspect, the present invention provides toxins produced by the
expression of DNA molecules of the present invention. According to a preferred
embodiment, the toxins of the invention have activity against Plutella
xylostella.
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In one embodiment, the toxins are produced by the E. coli strain designated as
NRRL
accession number B-21883.
fn another embodiment, a toxin of the invention comprises an amino acid
sequence
selected from the group consisting of: SEQ ID NOs:2, 3, 5, 7, 9, 11, 13, and
15.
Also provided is a composition comprising an insecticidally effective amount
of a toxin
of the invention. In another aspect, the present invention provides a method
of producing a
toxin that is active against insects, comprising: (a) obtaining a host cell
comprising a
chimeric gene, which itself comprises a heterologous promoter sequence
operatively linked
to the nucleic acid molecule of the invention; and (b) expressing the nucleic
acid molecule in
the cell, which results in at least one toxin that is active against insects.
in a further aspect, the present invention provides a method of producing an
insect-
resistant plant, comprising introducing a nucleic acid molecule of the
invention into the
plant, wherein the nucleic acid molecule is expressible in the plant in an
effective amount to
control insects. According to a preferred embodiment, the insects are Plutella
xylosrella.
In a still further aspect, the present invention provides a method of
controlling insects
comprising delivering to the insects an effective amount of a toxin according
to the present
invention. According to a preferred embodiment, the insects are Plutella
xylostella.
Preferably, the toxin is delivered to the insects orally.
Yet another aspect of the present invention is the provision of a method for
mutagenizing a nucleic acid molecule according to the present invention,
wherein the
nucleic acid molecule has been cleaved into population of double-stranded
random
fragments of a desired size, comprising: (a) adding to the population of
double-stranded
random fragments one or more single- or double-stranded oligonucleotides,
wherein the
oligonucleotides each comprise an area of identity and an area of heterology
to a double-
stranded template pofynucleotide; (b) denaturing the resultant mixture of
double-stranded
random fragments and oligonucleotides into single-stranded fragments; (c)
incubating the
resultant population of single-stranded fragments with a polymerase under
conditions which
result in the annealing of the single-stranded fragments at the areas of
identity to form pairs
of annealed fragments, the areas of identity being sufficient for one member
of a pair to
prime replication of the other, thereby forming a mutagenized double-stranded
polynucleotide; and (d) repeating the second and third steps for at least two
further cycles,
wherein the resultant mixture in the second step of a further cycle includes
the mutagenized
double-stranded polynucleotide from the third step of the previous cycle, and
wherein the
further cycle forms a further mutagenized double-stranded polynucleotide.
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Other aspects and advantages of the present invention will become apparent to
those
skilled in the art from a study of the description of the invention and non-
limiting examples.
DEFINITIONS
Activity" of the toxins of the invention is meant that the toxins function as
orally
active insect control agents, have a toxic effect, or are able to disrupt or
deter insect
feeding, which may or may not cause death of the insect. When a toxin of the
invention is
delivered to the insect, the result is typically death of the insect, or the
insect does not feed
upon the source that makes the toxin available to the insect.
"Associated with / operatively linked" refer to two nucleic acid sequences
that are
related physically or functionally. For example, a promoter or regulatory DNA
sequence is
said to be "associated with" a DNA sequence that codes for an RNA or a protein
if the two
sequences are operatively linked, or situated such that the regulator DNA
sequence will
affect the expression level of the coding or structural DNA sequence.
A "chimeric gene° is a recombinant nucleic acid sequence in which a
promoter or
regulatory nucleic acid sequence is operatively linked to, or associated with,
a nucleic acid
sequence that codes for an mRNA or which is expressed as a protein, such that
the
regulator nucleic acid sequence is able to regulate transcription or
expression of the
associated nucleic acid sequence. The regulator nucleic acid sequence of the
chimeric
gene is not normally operatively linked to the associated nucleic acid
sequence as found in
nature.
A "coding sequence" is a nucleic acid sequence that is transcribed into RNA
such as
mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is
then
translated in an organism to produce a protein.
To "control" insects means to inhibit, through a toxic effect, the ability of
insect pests
to survive, grow, feed, and/or reproduce, or to limit insect-related damage or
loss in crop
plants. To "control" insects may or may not mean killing the insects, although
it preferably
means killing the insects.
To "deliver" a toxin means that the toxin comes in contact with an insect,
resulting in
toxic effect and control of the insect. The toxin can be delivered in many
recognized ways,
e.g., orally by ingestion by the insect or by contact with the insect via
transgenic plant
expression, formulated protein composition(s), sprayable protein
composition(s), a bait
matrix, or any other art-recognized toxin delivery system.
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"Expression cassette as used herein means a nucleic acid sequence capable of
directing expression of a particular nucleotide sequence in an appropriate
host cell,
comprising a promoter operably linked to the nucleotide sequence of interest
which is
operably linked to termination signals. It also typically comprises sequences
required for
proper translation of the nucleotide sequence. The expression cassette
comprising the
nucleotide sequence of interest may be chimeric, meaning that at least one of
its
components is heterologous with respect to at least one of its other
components. The
expression cassette may also be one which is naturally occurring but has been
obtained in
a recombinant form useful for heterologous expression. Typically, however, the
expression
cassette is heterologous with respect to the host, i.e., the particular
nucleic acid sequence
of the expression cassette does not occur naturally in the host cell and must
have been
introduced into the host cell or an ancestor of the host cell by a
transformation event. The
expression of the nucleotide sequence in the expression cassette may be under
the control
of a constitutive promoter or of an inducible promoter which initiates
transcription only when
the host cell is exposed to some particular external stimulus. In the case of
a multicellular
organism, such as a plant, the promoter can also be specific to a particular
tissue, or organ,
or stage of development.
A "gene" is a defined region that is located within a genome and that, besides
the
aforementioned coding nucleic acid sequence, comprises other, primarily
regulatory, nucleic
acid sequences responsible for the control of the expression, that is to say
the transcription
and translation, of the coding portion. A gene may also comprise other 5' and
3'
untranslated sequences and termination sequences. Further elements that may be
present
are, for example, introns.
"Gene of interest" refers to any gene which, when transferred to a plant,
confers upon
the plant a desired characteristic such as antibiotic resistance, virus
resistance, insect
resistance, disease resistance, or resistance to other pests, herbicide
tolerance, improved
nutritional value, improved performance in an industrial process or altered
reproductive
capability. The "gene of interest° may also be one that is transferred
to plants for the
production of commercially valuable enzymes or metabolites in the plant.
A "heterologous" nucleic acid sequence is a nucleic acid sequence not
naturally
associated with a host cell into which it is introduced, including non-
naturally occurring
multiple copies of a naturally occurring nucleic acid sequence.
A "homologous" nucleic acid sequence is a nucleic acid sequence naturally
associated with a host cell into which it is introduced.
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"Homologous recombination" is the reciprocal exchange of nucleic acid
fragments
between homologous nucleic acid molecules.
"Insecticidal" is defined as a toxic biological activity capable of
controlling insects,
preferably by killing them.
A nucleic acid sequence is "isocoding with" a reference nucleic acid sequence
when
the nucleic acid sequence encodes a polypeptide having the same amino acid
sequence as
the polypeptide encoded by the reference nucleic acid sequence.
An "isolated" nucleic acid molecule or an isolated enzyme is a nucleic acid
molecule
or enzyme that, by the hand of man, exists apart from its native environment
and is
therefore not a product of nature. An isolated nucleic acid molecule or enzyme
may exist in
a purified form or may exist in a non-native environment such as, for example,
a
recombinant host cell.
A "nucleic acid molecule" or "nucleic acid sequence" is a linear segment of
single- or
double-stranded DNA or RNA that can be isolated from any source. In the
context of the
present invention, the nucleic acid molecule is preferably a segment of DNA.
"ORF' means open reading frame.
A "plant" is any plant at any stage of development, particularly a seed plant.
A "plant cell" is a structural and physiological unit of a plant, comprising a
protoplast
and a cell wall. The plant cell may be in form of an isolated single cell or a
cultured cell, or
as a part of higher organized unit such as, for example, plant tissue, a plant
organ, or a
whole plant.
"Plant cell culture" means cultures of plant units such as, for example,
protoplasts,
cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules,
embryo sacs, zygotes
and embryos at various stages of development.
"Plant material" refers to leaves, stems, roots, flowers or flower parts,
fruits, pollen,
egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other
part or product of a
plant.
A "plant organ" is a distinct and visibly structured and differentiated part
of a plant
such as a root, stem, leaf, flower bud, or embryo.
"Plant tissue" as used herein means a group of plant cells organized into a
structural
and functional unit. Any tissue of a plant in plants or in culture is
included. This term
includes, but is not limited to, whole plants, plant organs, plant seeds,
tissue culture and
any groups of plant cells organized into structural and/or functional units.
The use of this
term in conjunction with, or in the absence of, any specific type of plant
tissue as listed
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above or otherwise embraced by this definition is not intended to be exclusive
of any other
type of plant tissue.
A "promoter" is an untranslated DNA sequence upstream of the coding region
that
contains the binding site for RNA polymerase II and initiates transcription of
the DNA. The
promoter region may also include other elements that act as regulators of gene
expression.
A "protoplast" is an isolated plant cell without a cell wall or with only
parts of the cell
wall.
"Regulatory elements" refer to sequences involved in controlling the
expression of a
nucleotide sequence. Regulatory elements comprise a promoter operably linked
to the
nucleotide sequence of interest and termination signals. They also typically
encompass
sequences required for proper translation of the nucleotide sequence.
In its broadest sense, the term "substantially similar", when used herein with
respect
to a nucleotide sequence, means a nucleotide sequence corresponding to a
reference
nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having
substantially the same structure and function as the polypeptide encoded by
the reference
nucleotide sequence, e.g. where only changes in amino acids not affecting the
polypeptide
function occur. Desirably the substantially similar nucleotide sequence
encodes the
polypeptide encoded by the reference nucleotide sequence. The percentage of
identity
between the substantially similar nucleotide sequence and the reference
nucleotide
sequence desirably is at least 80%, more desirably at least 85%, preferably at
least 90%,
more preferably at least 95%, still more preferably at least 99%. A nucleotide
sequence
"substantially similar" to reference nucleotide sequence hybridizes to the
reference
nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA
at
50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more desirably
in 7% sodium dodecyl
sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC,
0.1 % SDS at
50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaP04, 1 mM EDTA at
50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C, preferably in
7% sodium dodecyl
sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 X
SSC, 0.1 % SDS at
50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04,
1 mM EDTA at
50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C.
"Synthetic" refers to a nucleotide sequence comprising structural characters
that are
not present in the natural sequence. For example, an artificial sequence that
resembles
more closely the G+C content and the normal codon distribution of dicot and/or
monocot
genes is said to be synthetic.
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'Transformation" is a process for introducing heterologous nucleic acid into a
host
cell or organism. In particular, "transformation" means the stable integration
of a DNA
molecule into the genome of an organism of interest.
'Transformed / transgenic / recombinant" refer to a host organism such as a
bacterium or a plant into which a heterologous nucleic acid molecule has been
introduced.
The nucleic acid molecule can be stably integrated into the genome of the host
or the
nucleic acid molecule can also be present as an extrachromosomal molecule.
Such an
extrachromosomal molecule can be auto-replicating. Transformed cells, tissues,
or plants
are understood to encompass not only the end product of a transformation
process, but
also transgenic progeny thereof. A "non-transformed", "non-transgenic", or
"non-
recombinant" host refers to a wild-type organism, e.g., a bacterium or plant,
which does not
contain the heterologous nucleic acid molecule.
Nucleotides are indicated by their bases by the following standard
abbreviations:
adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are
likewise indicated
by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R),
asparagine (Asn;
N), aspartic acid (Asp; D}, cysteine (Cys; C), glutamine (Gln; Q), glutamic
acid (Glu; E),
' glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L),
lysine (Lys; K),
methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;
S), threonine
(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
Furthermore, (Xaa; X)
represents any amino acid.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID N0:1 is the sequence of the approximately 3.0 kb DNA fragment comprised
in
Xenorhabdus nematophilus clone pCIB9369, which comprises the following ORFs at
the
specified nucleotide positions:
Name Start End
orf1 569 979
orf2 1045 2334
SEQ ID N0:2 is the sequence of the -15 kDa protein encoded by orf1 of clone
pCIB9369.
SEQ ID N0:3 is the sequence of the 47.7 kDa Juvenile Hormone Esterase-like
protein
encoded by orf2 of clone pCIB9369.
SEQ ID N0:4 is the DNA sequence of orf1 of Xenorhabdus nematophilus clone
pCIB9381.
SEQ ID N0:5 is the sequence of the protein encoded by orf1 of clone pCIB9381.
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SEQ ID N0:6 is the DNA sequence of orf2 of Xenorhabdus nematophilus clone
pCIB9381.
SEQ ID N0:7 is the sequence of the Juvenile Hormone Esterase-like protein
encoded by
orf2 of clone pCIB9381.
SEQ ID N0:8 is the DNA sequence of orf1 of Xenorhabdus poinarii clone
pCIB9354.
SEO ID N0:9 is the sequence of the protein encoded by orf1 of clone pCIB9354.
SEO ID N0:10 is the DNA sequence of orf2 of Xenorhabdus poinarii clone
pCIB9354.
SEQ ID N0:11 is the sequence of the Juvenile Hormone Esterase-like protein
encoded by
orf2 of clone pCIB9354.
SEQ ID N0:12 is the DNA sequence of orf1 of Photorhabdus luminescens clone
pC1B9383-21.
SEO ID N0:13 is the sequence of the protein encoded by orfl of clone pCIB9383-
21.
SEQ ID N0:14 is the DNA sequence of orf2 of Photorhabdus luminescens clone
pCIB9383-21.
SEQ ID N0:15 is the sequence of the Juvenile Hormone Esterase-like protein
encoded by
orf2 of clone pCIB9383-21.
DEPOSITS
The following material has been deposited with the Agricultural Research
Service,
Patent Culture Collection (NRRL), 1815 North University Street, Peoria,
Illinois 61604, under
the terms of the Budapest Treaty on the International Recognition of the
Deposit of
Microorganisms for the Purposes of Patent Procedure. All restrictions on the
availability of
the deposited material will be irrevocably removed upon the granting of a
patent.
Clone Accession Number Date of Deposit
pCIB9369 NRRL B-21883 November 12, 1997
pCIB9354 NRRL B-30109 February 25, 1999
pCIB9381 NRRL B-30110 February 25, 1999
pCIB9383-21 NRRL B-30111 February 25, 1999
Novel Nucleic Acid Se4uences whose Expression Results in Insecticidal Toxins
This invention relates to nucleic acid sequences whose expression results in
novel
toxins, and to the making and using of the toxins to control insect pests. The
nucleic acid
sequences are isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii,
and
Photorhabdus luminescens, members of the Enterobacteriaceae family.
Xenorhabdus are
symbiotic bacteria of nematodes of the genus Steinernema. Photorhabdus are
symbiotic
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bacteria of nematodes of the genus Heterorhabditis. The nematodes colonize
insect lama,
kill them, and their offspring feed on the dead larvae. The insecticidal
activity is actually
produced by the symbiotic Xenorhabdus and Photorhabdus bacteria. The inventors
are the
first to isolate the nucleic acid sequences of the present invention. The
expression of the
nucleic acid sequences of the present invention results in toxins that can be
used to control
Lepidopteran insects such as Plufella xylostella (Diamondback Moth).
A nucleotide sequence of the present invention in clone pCIB9369 is
characterized
by an approximately 3.0 kb DNA fragment deposited pursuant to the Budapest
Treaty for
Patent Deposits under Accession Number NRRL B-21883. The sequence of this DNA
fragment is set forth in SEo ID N0:1. Two open reading frames (ORF) are
present in SEQ
ID N0:1 (nucleotides 569-979 and nucleotides 1045-2334, respectively), coding
for proteins
of predicted sizes of 15 kDa and 47.7 kDa (SEQ ID NOs:2 and 3, respectively).
The two
ORFs are arranged in an operon-like structure. A search for known sequences
showing
homology to each individual ORF using the UWGCG Blast and Gap programs does
not
reveal any significant match for ORF #1 and reveals 21 % identity between ORF
#2 and
Bacillus thuringensis cry3A protein, which is not considered to be significant
in the art. A
Gap analysis of the protein encoded by ORF #2 of pCIB9369 by the Blast program
identifies 30.6% AA identity and 44.1 % AA similarity to a juvenile hormone
esterase-related
protein (GenBank accession 2921553; Henikoff et al., PNAS USA 89: 10915-10919
(1992)). The nucleotide sequence of the present invention is also compared to
known
Xenorhabdus nematophilus sequences encoding the insecticidal toxin toxb4 (WO
95/00647), but no significant homology is found. The 3.0 kb DNA fragment is
also
compared to the nucleotide sequence published in WO 98/08388. Twenty-two
sequences
of 60 nucleotides each (60-mers) from the 38.2 kb DNA fragment described in WO
98/08388 are compared to the 3.0 kb DNA fragment of the present invention
using the
UWGCG Gap program. The nucleotide sequence of the first 60-mer starts at base
1 of the
38.2 kb DNA fragment and the other 60-mers are located at approximately 2.0 kb
intervals.
Each of the 22 sequences as well as their complementary sequences are tested.
The
highest percent of identity between the 3.0 kb DNA fragment of the present
invention and
one of these 60-mers is 53%, which is not a significant homology. Furthermore,
five
different DNA fragments of the 38.2 kb sequence are tested for hybridization
to the 3.0 kb
fragment of the present invention by Southern blot analysis. None of them
reveal a positive
hybridization signal.
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The nucleotide sequences of pCIB9381, pCIB9354, and pCIB9383-21 also reveal
two open reading frames in each of these clones. The nucleotide sequences of
the two
ORFs in each of pC189381 and pCIB9383-21 are highly homologous to those in
pCIB9369.
Hence, the ORF #2 proteins of pCIB9381 and pCIB9383-21 have essentially the
same
homology to the juvenile hormone esterase-related protein as does the ORF #2
protein of
pCIB9369. The nucleotide sequence of ORF #1 of pCIB9354 is 77% identical to
the
nucleotide sequence of ORF #1 of pCIB9369, and the nucleotide sequence of ORF
#2 of
pCIB9354 is 79% identical to the nucleotide sequence of ORF #2 of pCIB9369.
The ORF
#2 protein of pCIB9354 also has homology to the juvenile hormone esterase-
related protein
(29.2% AA identity and 42.2% AA similarity).
In a preferred embodiment, the invention encompasses a nucleotide sequence
substantially similar to nucleotides 569-979 of SEQ ID N0:1, nucleotides 1045-
2334 of SEQ
ID N0:1, SEO ID N0:4, SEQ ID NO:fi, SEQ ID N0:8, SEQ ID N0:10, SEQ ID N0:12,
and
SEQ ID N0:14, whose expression results in an insecticidal toxin. The present
invention also
encompasses recombinant vectors comprising the nucleic acid sequences of this
invention.
In such vectors, the nucleic acid sequences are preferably comprised in
expression
cassettes comprising regulatory elements for expression of the nucleotide
sequences in a
host cell capable of expressing the nucleotide sequences. Such regulatory
elements usually
comprise promoter and termination signals and preferably also comprise
elements allowing
efficient translation of polypeptides encoded by the nucleic acid sequences of
the present
invention. Vectors comprising the nucleic acid sequences are usually capable
of replication
in particular host cells, preferably as extrachromosomal molecules, and are
therefore used
to amplify the nucleic acid sequences of this invention in the host cells. In
one embodiment,
host cells for such vectors are microorganisms, such as bacteria, in
particular E.coli. In
another embodiment, host cells for such recombinant vectors are endophytes or
epiphytes.
A preferred host cell for such vectors is a eukaryotic cell, such as a yeast,
a plant cell, or an
insect cell. Plant cells such as maize cells are most preferred host cells. In
another
preferred embodiment, such vectors are viral vectors and are used for
replication of the
nucleotide sequences in particular host cells, e.g. insect cells or plant
cells. Recombinant
vectors are also used for transformation of the nucleotide sequences of this
invention into
host cells, whereby the nucleotide sequences are stably integrated into the
DNA of such
host cells. In one, such host cells are prokaryotic cells. In a preferred
embodiment, such
host cells are eukaryotic cells, such as yeast cells, insect cells, or plant
cells. In a most
preferred embodiment, the host cells are plant cells, such as maize cells.
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The nucleotide sequences of the invention can be isolated using the techniques
described in the examples below, or by PCR using the sequences set forth in
the sequence
listing as the basis for constructing PCR primers. For example,
oligonucleotides having the
sequence of approximately the first and last 20-25 consecutive nucleotides of
orfl of SEQ
ID NO:i (e.g., nucleotides 569-588 and 957-976 of SEQ ID N0:1 ) can be used as
PCR
primers to amplify the orfl coding sequence (nucleotides 569-976 of SEQ ID
N0:1) directly
from the source strain (Xenorhabdus nemafophilus strain ATCC 19061 ). The
other gene
sequences of the invention can likewise be amplified by PCR from the
respective source
strains using the ends of the coding sequences set forth in the sequence
listing as the basis
for PCR primers.
In another preferred embodiment, the insecticidal toxins comprise at least one
polypeptide encoded by a nucleotide sequence of the invention. The molecular
weight of an
insecticidal toxin according to the invention is larger than 6,000, as
determined by size
fractionation experiments. After treatment with proteinase K, only a minimal
decrease in
insecticidal activity is observed in the insect bioassay, indicating that the
insecticidal toxins
are substantially resistant to proteinase K treatment. The insecticidal toxins
retain their
insecticidal activity after being stored at 22°C or at 4°C for 2
weeks. They also retains their
insecticidal activity after being freeze dried and stored at 22°C for 2
weeks. The insecticidal
toxins are also still active after incubation for 5 minutes at 60°C,
but they loses their
insecticidal activity after incubation for 5 minutes at 100°C or
80°C.
In further embodiments, the nucleotide sequences of the invention can be
modified
by incorporation of random mutations in a technique known as in-vitro
recombination or
DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-
391 (1994)
and lJS Patent 5,605,793, which are incorporated herein by reference. Millions
of mutant
copies of a nucleotide sequence are produced based on an original nucleotide
sequence of
this invention and variants with improved properties, such as increased
insecticidal activity,
enhanced stability, or different specificity or range of target insect pests
are recovered. The
method encompasses forming a mutagenized double-stranded polynucleotide from a
template double-stranded polynucleotide comprising a nucleotide sequence of
this
invention, wherein the template double-stranded polynucleotide has been
cleaved into
double-stranded-random fragments of a desired size, and comprises the steps of
adding to
the resultant population of double-stranded random fragments one or more
single or
double-stranded oligonucleotides, wherein said oligonucleotides comprise an
area of
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identity and an area of heterology to the double-stranded template
polynucleotide;
denaturing the resultant mixture of double-stranded random fragments and
oligonucleotides
into single-stranded fragments; incubating the resultant population of single-
stranded
fragments with a polymerase under conditions which result in the annealing of
said single-
stranded fragments at said areas of identity to form pairs of annealed
fragments, said areas
of identity being sufficient for one member of a pair to prime replication of
the other, thereby
forming a mutagenized double-stranded polynucleotide; and repeating the second
and third
steps for at least two further cycles, wherein the resultant mixture in the
second step of a
further cycle includes the mutagenized double-stranded polynucleotide from the
third step
of the previous cycle, and the further cycle forms a further mutagenized
double-stranded
polynucleotide. In a preferred embodiment, the concentration of a single
species of doubfe-
stranded random fragment in the population of double-stranded random fragments
is less
than 1 % by weight of the total DNA. In a further preferred embodiment, the
template
double-stranded polynucleotide comprises at least about 100 species of
polynucleotides. In
another preferred embodiment, the size of the double-stranded random fragments
is from
about 5 by to 5 kb. In a further preferred embodiment, the fourth step of the
method
comprises repeating the second and the third steps for at least 10 cycles.
Expression of the Nucleotide Sequences in Heterologious Microbial Hosts
As biological insect control agents, the insecticidal toxins are produced by
expression
of the nucleotide sequences in heterologous host cells capable of expressing
the nucleotide
sequences. In a first embodiment, Xenorhabdus nematophilus, Xenorhabdus
poinarii, or
Phoforhabdus luminescens cells comprising modifications of at least one
nucleotide
sequence of this invention at its chromosomal location are described. Such
modifications
encompass mutations or deletions of existing regulatory elements, thus leading
to altered
expression of the nucleotide sequence, or the incorporation of new regulatory
elements
controlling the expression of the nucleotide sequence. In another embodiment,
additional
copies of one or more of the nucleotide sequences are added to Xenorhabdus
nematophilus, Xenorhabdus poinarii, or Photorhabdus luminescens cells either
by insertion
into the chromosome or by introduction of extrachromosomally replicating
molecules
containing the nucleotide sequences.
In another embodiment, at least one of the nucleotide sequences of the
invention is
inserted into an appropriate expression cassette, comprising a promoter and
termination
signals. Expression of the nucleotide sequence is constitutive, or an
inducible promoter
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responding to various types of stimuli to initiate transcription is used. In a
preferred
embodiment, the cell in which the toxin is expressed is a microorganism, such
as a virus, a
bacteria, or a fungus. In a preferred embodiment, a virus, such as a
baculovirus, contains a
nucleotide sequence of the invention in its genome and expresses large amounts
of the
corresponding insecticidal toxin after infection of appropriate eukaryotic
cells that are
suitable for virus replication and expression of the nucleotide sequence. The
insecticidal
toxin thus produced is used as an insecticidal agent. Alternatively,
baculoviruses
engineered to include the nucleotide sequence are used to infect insects in-
vivo and kill
them either by expression of the insecticidal toxin or by a combination of
viral infection and
expression of the insecticidal toxin.
Bacterial cells are also hosts for the expression of the nucleotide sequences
of the
invention. In a preferred embodiment, non-pathogenic symbiotic bacteria, which
are able to
live and replicate within plant tissues, so-called endophytes, or non-
pathogenic symbiotic
bacteria, which are capable of colonizing the phyllosphere or the rhizosphere,
so-called
epiphytes, are used. Such bacteria include bacteria of the genera
Agrobacterium,
Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enferobacter,
Erwinia,
Flavobacter, Klebsiella, Pseudomonas, Fihizobium, Serratia, Streptomyces and
Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium are also
possible
hosts for expression of the inventive nucleotide sequences for the same
purpose.
Techniques for these genetic manipulations are specific for the different
available
hosts and are known in the art. For example, the expression vectors pKK223-3
and
pKK223-2 can be used to express heterologous genes in E. coli, either in
transcriptional or
translational fusion, behind the tac or trc promoter. For the expression of
operons encoding
multiple ORFs, the simplest procedure is to insert the operon into a vector
such as pKK223-
3 in transcriptional fusion, allowing the cognate ribosome binding site of the
heterologous
genes to be used. Techniques for overexpression in gram-positive species such
as Bacillus
are also known in the art and can be used in the context of this invention
(Quax et al. In.:
Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et
al.,
American Society for Microbiology, Washington (1993)). Alternate systems for
overexpression rely for example, on yeast vectors and include the use of
Pichia,
Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrial microorganisms:
basic and
applied molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society
for
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Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12:173-177
(1994); van
den Berg et al., Biotechnology 8:135-139 (1990)).
In another preferred embodiment, at least one of the described nucleotide
sequences is transferred to and expressed in Pseudomonas fluorescens strain
CGA267356
(described in the published application EU 0 472 494 and in WO 94/01561) which
has
biocontrol characteristics. In another preferred embodiment, a nucleotide
sequence of the
invention is transferred to Pseudomonas aureofaciens strain 30-84 which also
has
biocontrol characteristics. Expression in heterologous biocontrol strains
requires the
selection of vectors appropriate for replication in the chosen host and a
suitable choice of
promoter. Techniques are well known in the art for expression in gram-negative
and gram-
positive bacteria and fungi.
Expression of the Nucleotide Seguences in Plant Tissue
In a particularly preferred embodiment, at least one of the insecticidal
toxins of the
invention is expressed in a higher organism, e.g., a plant. In this case,
transgenic plants
expressing effective amounts of the toxins protect themselves from insect
pests. When the
insect starts feeding on such a transgenic plant, it also ingests the
expressed toxins. This
will deter the insect from further biting into the plant tissue or may even
harm or kill the
insect. A nucleotide sequence of the present invention is inserted into an
expression
cassette, which is then preferably stably integrated in the genome of said
plant. In another
preferred embodiment, the nucleotide sequence is included in a non-pathogenic
self-
replicating virus. Plants transformed in accordance with the present invention
may be
monocots or dicots and include, but are not limited to, maize, wheat, barley,
rye, sweet
potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip,
radish, spinach,
asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini,
apple, pear,
quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape,
raspberry,
blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,
sorghum,
sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton,
alfalfa, rice,
potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous
and
deciduous trees.
Once a desired nucleotide sequence has been transformed into a particular
plant
species, it may be propagated in that species or moved into other varieties of
the same
species, particularly including commercial varieties, using traditional
breeding techniques.
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A nucleotide sequence of this invention is preferably expressed in transgenic
plants,
thus causing the biosynthesis of the corresponding toxin in the transgenic
plants. In this
way, transgenic plants with enhanced resistance to insects are generated. For
their
expression in transgenic plants, the nucleotide sequences of the invention may
require
modification and optimization. Although in many cases genes from microbial
organisms can
be expressed in plants at high levels without modification, low expression in
transgenic
plants may result from microbial nucleotide sequences having codons that are
not preferred
in plants. It is known in the art that all organisms have specific preferences
for codon
usage, and the codons of the nucleotide sequences described in this invention
can be
changed to conform with plant preferences, while maintaining the amino acids
encoded
thereby. Furthermore, high expression in plants is best achieved from coding
sequences
that have at least 35% about GC content, preferably more than about 45%, more
preferably
more than about 50%, and most preferably more than about 60%. Microbial
nucleotide
sequences which have low GC contents may express poorly in plants due to the
existence
of ATTTA motifs which may destabilize messages, and AATAAA motifs which rnay
cause
inappropriate polyadenylation. Although preferred gene sequences may be
adequately
expressed in both monocotyledonous and dicotyledonous plant species, sequences
can be
modified to account for the specific codon preferences and GC content
preferences of
monocotyledons or dicotyledons as these preferences have been shown to differ
(Murray et
al. Nucl. Acids Res. 17: 477-498 (1989)). In addition, the nucleotide
sequences are
screened for the existence of illegitimate splice sites that may cause message
truncation.
All changes required to be made within the nucleotide sequences such as those
described
above are made using well known techniques of site directed mutagenesis, PCR,
and
synthetic gene construction using the methods described in the published
patent
applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, and WO
93/07278 (to
Ciba-Geigy).
For efficient initiation of translation, sequences adjacent to the initiating
methionine
may require modification. For example, they can be modified by the inclusion
of sequences
known to be effective in plants. Joshi has suggested an appropriate consensus
for plants
(NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus
translation
initiator (1993/1994 catalog, page 210). These consensuses are suitable for
use with the
nucleotide sequences of this invention. The sequences are incorporated into
constructions
comprising the nucleotide sequences, up to and including the ATG (whilst
leaving the
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second amino acid unmodified), or alternatively up to and including the GTC
subsequent to
the ATG (with the possibility of modifying the second amino acid of the
transgene).
Expression of the nucleotide sequences in transgenic plants is driven by
promoters
shown to be functional in plants. The choice of promoter will vary depending
on the
temporal and spatial requirements for expression, and also depending on the
target
species. Thus, expression of the nucleotide sequences of this invention in
leaves, in ears, in
inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings
is preferred. In
many cases, however, protection against more than one type of insect pest is
sought, and
thus expression in multiple tissues is desirable. Although many promoters from
dicotyledons have been shown to be operational in monocotyledons and vice
versa, ideally
dicotyledonous promoters are selected for expression in dicotyledons, and
monocotyledonous promoters for expression in monocotyledons. However, there is
no
restriction to the provenance of selected promoters; it is sufficient that
they are operational
in driving the expression of the nucleotide sequences in the desired cell.
Preferred promoters that are expressed constitutively include promoters from
genes
encoding actin or ubiquitin and the CaMV 35S and 19S promoters. The nucleotide
' sequences of this invention can also be expressed under the regulation of
promoters that
are chemically regulated. This enables the insecticidal toxins to be
synthesized only when
the crop plants are treated with the inducing chemicals. Preferred technology
for chemical
induction of gene expression is detailed in the published application EP 0 332
104 (to Ciba-
Geigy) and US patent 5,614,395. A preferred promoter for chemical induction is
the
tobacco PR-1a promoter.
A preferred category of promoters is that which is wound inducible. Numerous
promoters have been described which are expressed at wound sites and also at
the sites of
phytopathogen infection. Ideally, such a promoter should only be active
locally at the sites
of infection, and in this way the insecticidal toxins only accumulate in cells
which need to
synthesize the insecticidal toxins to kill the invading insect pest. Preferred
promoters of this
kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208
(1989), Xu et
al. Plant Molec. Biol. 22: 573-588 (1993), Logemann ef aL Plant Cell 1: 151-
158 (1989),
Rohrmeier 8~ Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant
Molec. Biol. 22:
129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).
Preferred tissue specific expression patterns include green tissue specific,
root
specific, stem specific, and flower specific. Promoters suitable for
expression in green
CA 02326067 2000-10-20
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-is-
tissue include many which regulate genes involved in photosynthesis and many
of these
have been cloned from both monocotyledons and dicotyledons. A preferred
promoter is the
maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula,
Plant
Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific
expression is that
described by de Framond (FEES 290: 103-106 (1991 ); EP 0 452 269 to Ciba-
Geigy). A
preferred stem specific promoter is that described in US patent 5,625,136 (to
Ciba-Geigy)
and which drives expression of the maize trpA gene.
Especially preferred embodiments of the invention are transgenic plants
expressing
at least one of the nucleotide sequences of the invention in a root-preferred
or root-specific
fashion. Further preferred embodiments are transgenic plants expressing the
nucleotide
sequences in a wound-inducible or pathogen infection-inducible manner.
In addition to the selection of a suitable promoter, constructions for
expression of an
insecticidal toxin in plants require an appropriate transcription terminator
to be attached
downstream of the heterologous nucleotide sequence. Several such terminators
are
available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any
available
terminator known to function in plants can be used in the context of this
invention.
' Numerous other sequences can be incorporated into expression cassettes
described in this invention. These include sequences which have been shown to
enhance
expression such as intron sequences (e.g. from Adh1 and bronzel) and viral
leader
sequences (e.g. from TMV, MCMV and AMV).
It may be preferable to target expression of the nucleotide sequences of the
present
invention to different cellular localizations in the plant. In some cases,
localization in the
cytosol may be desirable, whereas in other cases, localization in some
subcellufar organelle
may be preferred. Subcellular localization of transgene encoded enzymes is
undertaken
using techniques well known in the art. Typically, the DNA encoding the target
peptide from
a known organelle-targeted gene product is manipulated and fused upstream of
the
nucleotide sequence. Many such target sequences are known for the chloroplast
and their
functioning in heterologous constructions has been shown. The expression of
the
nucleotide sequences of the present invention is also targeted to the
endoplasmic reticulum
or to the vacuoles of the host cells. Techniques to achieve this are well-
known in the art.
Vectors suitable for plant transformation are described elsewhere in this
specification. For Agrobacterium-mediated transformation, binary vectors or
vectors
carrying at least one T-DNA border sequence are suitable, whereas for direct
gene transfer
any vector is suitable and linear DNA containing only the construction of
interest may be
CA 02326067 2000-10-20
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preferred. In the case of direct gene transfer, transformation with a single
DNA species or
co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096
(1986)). For
both direct gene transfer and Agrobacterium-mediated transfer, transformation
is usually
(but not necessarily) undertaken with a selectable marker which may provide
resistance to
an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta).
Examples of
such markers are neomycin phosphotransferase, hygromycin phosphotransferase,
dihydrofolate reductase, phosphinothricin acetyltransferase, 2, 2-
dichloroproprionic acid
dehalogenase, acetohydroxyacid synthase, 5-enoipyruvyl-shikimate-phosphate
synthase,
haloarylnitrilase, protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase,
dihydropteroate synthase, chloramphenicol acetyl transferase, and p-
glucuronidase. The
choice of selectable or screenable marker for plant transformation is not,
however, critical to
the invention.
The recombinant DNA described above can be introduced into the plant cell in a
number of art-recognized ways. Those skilled in the art will appreciate that
the choice of
method might depend on the type of plant targeted for transformation. Suitable
methods of
transforming plant cells include microinjection (Crossway et al.,
BioTechniques 4320-334
(1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-
5606 (1986),
Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6:915-921
(1988);
See also, Ishida et al., Nature Biotechnology 14:745-750 (June 1996) for maize
transformation), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722
(1984);
Hayashimoto et al., Plant PhysioL 93:857-863 (1990)(rice)), and ballistic
particle
acceleration using devices available from Agracetus, Inc., Madison, Wisconsin
and Dupont,
Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent
4,945,050; and
McCabe et al., Biotechnology 6923-926 (1988)). See also, Weissinger et al.,
Annual Rev.
Genet. 22:421-477 {1988); Sanford et al., Particulate Science and Technology
527-37
91987)(onion); Svab et aL, Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990)
(tobacco
chloroplast); Christou et al., Plant PhysioL 87:671-674 (1988)(soybean);
McCabe et al.,
BiolTechnology 6:923-926 (1988)(soybean); Kfein et al., Proc. Natl. Acad. Sci.
USA,
85:4305-4309 (1988)(maize); Klein et al., BiolTechnology 6:559-563 (1988)
(maize); Klein
et al., Plant Physiol. 91:440-444 (1988) (maize); Fromm et al., BiolTechnology
8:833-839
(1990); and Gordon-Kamm et al., Planf Cell 2: 603-618 (1990) {maize); Koziel
et al.,
Biotechnology 11: 194-200 (1993) (maize); Shimamoto et al., Nature 338: 274-
277 (i 989)
(rice); Christou et al., Biotechnology 9: 957-962 (1991 ) (rice); Datta et
al., BiolTechnology
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
-21 -
8:736-740 (1990) (rice); European Patent Application EP 0 332 581
(orchardgrass and
other Pooideae); Vasil et al., Biotechnology 1 i: 1553-1558 (1993) (wheat);
Weeks et al.,
Planf Physiol. i02: 1077-1084 (1993) (wheat); Wan et al., Planf Physiol. i04:
37-48 (1994)
(barley); Jahne et al., Theor. Appl. Genet. 89:525-533 (1994)(barley); Umbeck
et al.,
BiolTechnology 5: 263-266 (1987) (cotton); Casas et al., Proc. Nafl. Acad.
Sci. USA
90:11212-11216 {Dec. 1993) (sorghum); Somers ef al., BiolTechnology 10:1589-
1594 (Dec.
1992) (oat); Torbert et al., Planf Cell Reports 14:635-640 (1995) (oat); Weeks
et al., Planf
Physiol. 102:1077-1084 (1993) (wheat); Chang et al., WO 94/13822 (wheat) and
Nehra et
al., The Plant Journal 585-297 {1994) (wheat). A particularly preferred set of
embodiments for the introduction of recombinant DNA molecules into maize by
microprojectile bombardment can be found in Koziel et al., Biotechnology 11:
194-200
(1993), Hiil et al., Euphytica 85:119-123 (1995) and Koziel et al., Annals of
fhe New York
Academy of Sciences 792:164-171 (1996). An additional preferred embodiment is
the
protoplast transformation method for maize as disclosed in EP 0 292 435.
Transformation of
plants can be undertaken with a single DNA species or multiple DNA species
(i.e.
co-transformation) and both these techniques are suitable for use with the
peroxidase
coding sequence.
In another preferred embodiment, a nucleotide sequence of the present
invention is
directly transformed into the plastid genome. A major advantage of plastid
transformation is
that plastids are generally capable of expressing bacterial genes without
substantial
modification, and plastids are capable of expressing multiple open reading
frames under
control of a single promoter. Plastid transformation technology is extensively
described in
U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no.
WO
95/16783, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-
7305. The basic
technique for chloroplast transformation involves introducing regions of
cloned plastid DNA
flanking a selectable marker together with the gene of interest into a
suitable target tissue,
e.g., using biolistics or protoplast transformation (e.g., calcium chloride or
PEG mediated
transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences,
facilitate
homologous recombination with the plastid genome and thus allow the
replacement or
modification of specific regions of the plastome. Initially, point mutations
in the chloroplast
16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or
streptomycin are
utilized as selectable markers for transformation {Svab, Z., Hajdukiewicz, P.,
and Maliga, P.
{1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P.
(1992) Plant
CA 02326067 2000-10-20
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Cell 4, 39-45). This resulted in stable homoplasmic transformants at a
frequency of
approximately one per 100 bombardments of target leaves. The presence of
cloning sites
between these markers allowed creation of a plastid targeting vector for
introduction of
foreign genes (Staub, J.M., and Maliga, P. (1993) EM80 J. 12, 601-606).
Substantial
increases in transformation frequency are obtained by replacement of the
recessive rRNA
or r-protein antibiotic resistance genes with a dominant selectable marker,
the bacterial
aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-
adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA
90, 913-917).
Previously, this marker had been used successfully for high-frequency
transformation of the
plastid genome of the green alga Chiamydomonas reinhardfii (Goldschmidt-
Clermont, M.
(1991 ) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for
plastid
transformation are known in the art and encompassed within the scope of the
invention.
Typically, approximately 15-20 cell division cycles following transformation
are required to
reach a homoplastidic state. Plastid expression, in which genes are inserted
by homologous
recombination into all of the several thousand copies of the circular plastid
genome present
in each plant cell, takes advantage of the enormous copy number advantage over
nuclear-
expressed genes to permit expression levels that can readily exceed 10% of the
total
soluble plant protein. In a preferred embodiment, a nucleotide sequence of the
present
invention is inserted into a plastid targeting vector and transformed into the
plastid genome
of a desired plant host. Plants homoplastic for plastid genomes containing a
nucleotide
sequence of the present invention are obtained, and are preferentially capable
of high
expression of the nucleotide sequence.
Formulation of Insecticidal Compositions
The invention also includes compositions comprising at least one of the
insecticidal
toxins of the present invention. In order to effectively control insect pests
such compositions
preferably contain sufficient amounts of toxin. Such amounts vary depending on
the crop to
be protected, on the particular pest to be targeted, and on the environmental
conditions,
such as humidity, temperature or type of soil. In a preferred embodiment,
compositions
comprising the insecticidal toxins comprise host cells expressing the toxins
without
additional purification. In another preferred embodiment, the cells expressing
the
insecticidal toxins are lyophilized prior to their use as an insecticidal
agent. In another
embodiment, the insecticidal toxins are engineered to be secreted from the
host cells. In
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cases where purification of the toxins from the host cells in which they are
expressed is
desired, various degrees of purification of the insecticidal toxins are
reached.
The present invention further embraces the preparation of compositions
comprising
at least one insecticidal toxin of the present invention, which is
homogeneously mixed with
one or more compounds or groups of compounds described herein. The present
invention
also relates to methods of treating plants, which comprise application of the
insecticidal
toxins or compositions containing the insecticidal toxins, to plants. The
insecticidal toxins
can be applied to the crop area in the form of compositions or plant to be
treated,
simultaneously or in succession, with further compounds. These compounds can
be both
fertilizers or micronutrient donors or other preparations that influence plant
growth. They
can also be seiective herbicides, insecticides, fungicides, bactericides,
nematicides,
molluscicides or mixtures of several of these preparations, if desired
together with further
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.
A preferred method of applying insecticidal toxins of the present invention is
by
spraying to the environment hosting the insect pest like the soil, water, or
foliage of plants.
The number of applications and the rate of application depend on the type and
intensity of
infestation by the insect pest. The insecticidal toxins can also penetrate the
plant through
the roots via the soil (systemic action) by impregnating the locus of the
plant with a liquid
composition, or by applying the compounds in solid form to the soil, e.g. in
granular form
(soil application). The insecticidal toxins may also be applied to seeds
(coating) by
impregnating the seeds either with a liquid formulation containing
insecticidal toxins, or
coating them with a solid formulation. In special cases, further types of
application are also
possible, for example, selective treatment of the plant stems or buds. The
insecticidal
toxins can also be provided as bait located above or below the ground.
The insecticidal toxins are used in unmodified form or, preferably, together
with the
adjuvants conventionally employed in the art of formulation, and are therefore
formulated in
known manner to emulsifiabie concentrates, coatable pastes, directly sprayable
or dilutable
solutions, dilute emulsions, wettable powders, soluble powders, dusts,
granulates, and also
encapsulations, for example, in polymer substances. Like the nature of the
compositions,
the methods of application, such as spraying, atomizing, dusting, scattering
or pouring, are
chosen in accordance with the intended objectives and the prevailing
circumstances.
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The formulations, compositions or preparations containing the insecticidal
toxins
and, where appropriate, a solid or liquid adjuvant, are prepared in known
manner, for
example by homogeneously mixing and/or grinding the insecticidal toxins with
extenders,
for example solvents, solid carriers and, where appropriate, surface-active
compounds
(surfactants).
Suitable solvents include aromatic hydrocarbons, preferably the fractions
having 8 to
12 carbon atoms, for example, xylene mixtures or substituted naphthalenes,
phthalates
such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as
cyclohexane
or paraffins, alcohols and glycols and their ethers and esters, such as
ethanol, ethylene
glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly
polar
solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl
formamide, as well
as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or
water.
The solid carriers used e.g. for dusts and dispersible powders, are normally
natural
mineral fillers such as calcite, talcum, kaolin, montmorillonite or
attapulgite. in order to
improve the physical properties it is also possible to add highly dispersed
silicic acid or
highly dispersed absorbent polymers. Suitable granulated adsorptive carriers
are porous
types, for example pumice, broken brick, sepiolite or bentonite; and suitable
nonsorbent
carriers are materials such as calcite or sand. In addition, a great number of
pregranulated
materials of inorganic or organic nature can be used, e.g. especially dolomite
or pulverized
plant residues.
Suitable surface-active compounds are nonionic, cationic and/or anionic
surfactants
having good emulsifying, dispersing and wetting properties. The term
"surfactants" will also
be understood as comprising mixtures of surfactants. Suitable anionic
surfactants can be
both water-soluble soaps and water-soluble synthetic surface-active compounds.
Suitable soaps are the alkali metal salts, alkaline earth metal salts or
unsubstituted
or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon
atoms), for
example the sodium or potassium salts of oleic or stearic acid, or of natural
fatty acid
mixtures which can be obtained for example from coconut oil or tallow oil. The
fatty acid
methyltaurin salts may also be used.
More frequently, however, so-called synthetic surfactants are used, especially
fatty
sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or
alkylarylsulfonates.
The fatty sulfonates or sulfates are usually in the form of alkali metal
salts, alkaline
earth metal salts or unsubstituted or substituted ammonium salts and have a 8
to 22 carbon
alkyl radical which also includes the alkyl moiety of alkyl radicals, for
example, the sodium
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or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of
fatty alcohol
sulfates obtained from natural fatty acids. These compounds also comprise the
salts of
sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide
adducts. The
sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups
and one fatty
acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates
are the
sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid,
dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde
condensation product. Also suitable are corresponding phosphates, e.g. salts
of the
phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of
ethylene oxide.
Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic
or
cycioaliphatic alcohols, or saturated or unsaturated fatty acids and
alkylphenols, said
derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in
the (aliphatic)
hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the
alkylphenols.
Further suitable non-ionic surfactants are the water-soluble adducts of
polyethylene
oxide with polypropylene glycol, ethylenediamine propylene glycol and
alkylpolypropylene
glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts
contain 20 to 250
ethylene glycol ether groups and 10 to 100 propylene glycol ether groups.
These
compounds usually contain 1 to 5 ethylene glycol units per propylene glycol
unit.
Representative examples of non-ionic surfactants are
nonylphenolpolyethoxyethanols, castor oil polyglycol ethers,
polypropylene/polyethylene
oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and
octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and
polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which have, as N-
substituent, at least one C8-C22 alkyl radical and, as further substituents,
lower
unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The
salts are
preferably in the form of halides, methylsulfates or ethylsulfates, e.g.
stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium
bromide.
The surfactants customarily employed in the art of formulation are described,
for
example, in "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing
Corp.
Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface
Active
Agents," Chemical Publishing Co., Inc. New York, 1980.
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EXAMPLES
The invention will be further described by reference to the following detailed
examples. These examples are provided for purposes of illustration only, and
are not
intended to be limiting unless otherwise specified. Standard recombinant DNA
and
molecular cloning techniques used here are well known in the art and are
described by
Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons,
Inc. (1994); T.
Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory
Manual, Cold
Spring Harbor laboratory, Cold Spring Harbor, NY (1989); and by T.J. Silhavy,
M.L. Berman,
and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold
Spring Harbor, NY (1984).
A Isolation Of Nucleotide Seguences Whose Expression Results In Toxins Active
A a4 inst
Le~dopteran insects
Example 1: Growth of Xenorhabdus and Photorhabdus Strains
For insect bioassays, the following strains are grown in nutrient broth at
25°C for 3
days in the growth media recommended by ATCC. For DNA isolation, the cultures
are
grown for 24 hr under the same conditions.
Xenorhabdus nematophilus strain ATCC 19061
Xenorhabdus nematophilus strain Ps1, a USDA isolate
Xenorhabdus poinarii strain ATCC 49122
Photorhabdus luminescens strain PsS, a USDA isolate
Example 2: Insect Bioassay
Plutella xylostella (Px) bioassays are performed by aliquoting 50w1 of each
E.coli
culture on the solid artificial P. xylostella diet (Biever and Boldt, Annals
of Entomological
Society of America,1971; Shelton, et al J. Ent. Sci 26:17). 4 ml of the diet
is poured into 1
oz. clear plastic cups (Bioserve product #9051 ). 5 neonate P. xylostella from
a diet
adapted lab colony are placed in each diet containing cup and then covered
with a white
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paper lid (Bioserve product #9049). 10 larvae are assayed per concentration.
Trays of cups
are placed in an incubator for 3 days at 72°F with a 14:10 (hours)
light: dark cycle. The
number of live larvae in each cup is recorded.
Example 3: Results of Bioassays
The broth of Xenorhabdus nematophilus strain ATCC i 9061 gives 100% mortality
against Plutella xylostella (Px) in the insect bioassay of Example 2. Broth of
each of
Xenorhabdus nematophilus strain Psi , Xenorhabdus poinarii strain ATCC 49122,
and
Photorhabdus luminescens strain PsS, likewise gives 100% mortality against
Plutella
xylostella (Px) in the insect bioassay of Example 2.
Example 4: Construction of Cosmid Libraries
Total DNA is isolated from Xenorhabdus nematophilus strain ATCC 19061 by
treating
freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg/ml
lysozyme
for 30 minutes at 37°C. Proteinase K is added to a final concentration
of 100 p.g/ml in 0.5%
SDS and incubated at 45°C. The solution clears and becomes very
viscous. The SDS
concentration is increased to 1 % and 300 mM NaCI and equal volume of phenol-
chloroform-isoamyl alcohol are added. The sample is gently mixed for 5 minutes
and
centrifuged at 3K. This is repeated twice. The aqueous phase is then mixed
with 0.7
volumes isopropanol and centrifuged. The DNA pellet is washed three times with
70%
ethanol and gently resuspended in 0.5X TE. 6 p.g of DNA are treated with 0.3
unit of Sau3A
per pg of DNA at 37°C for 3.5 minutes in a volume of 100 pl. The sample
is then heated for
30 minutes at 65°C to inactivate the enzyme, then incubated with 2
units of calf intestinal
alkaline phosphatase for 30 minutes at 37°C. The sample is mixed with
an equal volume of
phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is
removed and
mixed with 0.7 volumes isopropanol and centrifuged. The pellet is resuspended
in 0.5X TE
at a concentration of 100 ng/ml.
SuperCos cosmid vector (Stratagene, La Jolla, CA) is prepared as described by
the
supplier utilizing the BamHl cloning site. Prepared SuperCos at 100 rlg/ml is
ligated with the
X. nematophilus DNA previously digested with Sau3A at a ratio of 2:1 in a 5
p.l volume
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overnight at 6°C. The ligation mixture is packaged using Gigapack XL
III (Stratagene) as
described by the supplier. Packaged phages are infected into XL-1 MR E. coli
cells
(Stratagene) as described by the supplier. The cosmid library is plated on L-
agar with 50
p,g/ml kanamycin and incubated 16 hours at 37°C. 500 colonies are
patched to fresh L-kan
plates at a density of 50/plate. The cells are washed off with L broth and
mixed with 20%
glycerol and frozen at -80°C.
In the case of the 4.2 Mb large genome of X. nematophilus, 450 clones with an
average size of 40 Kb correspond to a 4-fold coverage of the genome.
Therefore, screening
of 450 clones should result in a 99% probability of finding any gene.
Cosmid libraries from Xenorhabdus nematophilus strain Psl, Xenorhabdus
poinarii
strain ATCC 49122, and Photorhabdus luminescens strain Ps5 are constructed in
a like
manner.
Example 5: Results of the Cosmids Bioassays and Identification of Clones
Having
Insecticidal Activity
400 E.coli clones from each cosmid library are screened by insect bioassay
yielding
clones with activity against Plutella xylostella. An insecticidal cosmid clone
from
Xenorhabdus nematophilus strain ATCC 19061 is identified as pC189362. A 42 kb
insecticidal cosmid clone from Xenorhabdus nemafophilus strain Ps1 is
identified as
pCIB9379. A 42 kb insecticidal cosmid clone from Xenorhabdus poinarii strain
ATCC 49122
is identified as pCIB9354. A 7 kb insecticidal cosmid clone from Photorhabdus
luminescens
strain Ps5 is identified as pCIB9383-21.
Example 6: Isolation of Subclones Having Insecticidal Activity
Clone pCIB9362 is digested with Sacll and a 9 kb DNA fragment is isolated.
This
fragment is ligated into Bluescript cut with Sacll. The ligation mixture is
transformed into
DHSa E, coli cells as described in Molecular Cloning, second edition (Sambrook
et al.). The
transformation mixture is plated on L agar plus 100 wglml ampillicin and
incubated at 37°C
overnight. Isolated colonies are grown up in L broth with 100 pg/ml ampillicin
and plasmid
DNA isolated by alkaline minipreps as described in Molecular Cloning, second
edition. The
9 kb Sacll clone is identified as pCIB9362-3 and gives 100% mortality when
bioassayed
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against Plutella xylostella. 3 p.g of pCIB9362-3 is isolated, digested with
0.3 unit of Sau3A
per pg DNA for 4 , 6 and 8 min. at 37°C and heated at 75°C for
15 min. Samples are pooled
and ligated into pUCl9 previously digested with BamHl and treated with calf
intestinal
alkaline phosphatase. The ligation is transformed into DHSa E, coli cells,
plated on L agar
with Xgal/Amp as described in Molecular Cloning and grown overnight at
37°C. White
colonies are picked and grown up in L broth with 100 p.glml ampicillin and
plasmid DNA is
isolated as previously described. DNA is digested with Ecoj~llHindlll and
novel restriction
patterns are sequenced. Sequencing primers are ordered from Genosys
Biotechnologies
(Woodlands, Tx). Sequencing is performed using the dideoxy chain-termination
method
and is completed using Applied Biosystems Inc. model 377 automated DNA
sequencer
(Foster City, CA). The sequence is assembled using Sequencher 3.0 from Gene
Codes
Corporation ( Ann Arbor, Michgan).
After identification of restriction sites and of potential ORF's, pCIB9362-3
is digested
with Clal and a 3.0 kb fragment is isolated cloned into Bluescript and
transformed into
DHSa E. coli cells. The isolated colonies are grown as previously described
and plasmid
DNA isolated by the alkaline method. The subclone is identified as pCIB9369
and gives
100% mortality against Plutella xylostella in a bioassay. pCIB9369 has been
deposited on
November 12, 1997 and identified by NRRL Number B-21883 at the USDA ARS Patent
Culture Collection.
A 20 kb fragment identified as pCIB9381 is subcloned from cosmid pCIB9379
using a
Notl digest.
Example 7: Bioassay Results
Insect Bioassays are conducted for pCIB9369 for a variety of insects.
Insect CIB9369
Plutella xylostella +++
Heliothis virescens na
Helioverpa zea na
Spodoptera exguia na
C. ele ans na
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na = not active
+ = significant growth inhibition
++ _ >40% mortality, but less than 100%
+++ = 100% mortality
These results show that the insecticidal toxin resulting from expression of
the 3.0 kb
nucleotide sequence in pCIB9369 is highly active against Plutella xylostella.
Bioassays for pC1B9381, pCIB9354, and pCIB9383-21 also show high insecticidal
activity against Plutella xylosfella.
Example 8: Size Fraction of the Insecticidal Activity
Xenorhabdus nematophilus cosmid clone pCIB9369 and Stratagene's Blue Script
vector in E. coli host DH5 are grown in media consisting of 50% Terrific broth
and 50%
Luria broth, supplemented with 50 pg/ml of ampicilin. Shake flask cultures (
300 ml in 1000
ml baffled flasks ) are grown at 250 RPM, overnight at 37°C. Cultures
of each strain are
centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4°C. The pelleted
cells are
' resuspended in 30 ml of 50mM NaCI, 25 mM Tris base, pH 7Ø The concentrated
cells are
disrupted by sonication using a Branson Model 450 Sonicator for approximately
eight 10
second cycles with cooling on ice between cycles. The sonicates are
centrifuged in a
Sorvall SS34 rotor at 6,000 RPM for 10 minutes at 4°C. The resultant
supernatants are
filtered through a 0.2 N filter. The pellets from the centrifuged sonicates
are resuspended in
30 ml of 50mM NaCI, 25 mM Tris base, pH 7Ø
The 3 ml fractions of the filtrates are applied to Bio-Rad Econo-Pac 10DG
columns
that had been previously equilibrated with 10 ml of 50mM NaCI, 25 mM Tris
base, pH 7Ø
The flow through collected during sample loading is discarded. The samples are
fractionated with two subsequent additions of 4 ml each of the NaCI - Tris
equilibration
buffer. The first three fractions are saved for testing. The first fraction
should contain all
material above about 6,000 mol. wt. The subsequent fractions should contain
material
smaller than 6,000 mol. wt.
A sample of the sonicated filtrate and the resuspended pellet following
sonication,
are tested along with the three fractions from the 10DG column for activity on
P. xylostella
neonates in surface contamination assays. The filtered supernatant of the
sonicate and the
first column fraction from the 9369 sample is highly active on P, xylostella.
The second and
third fractions from the 9369 sample are not active. None of the samples from
the DHSa
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with the Blue Script pasmid culture have activity. These results suggest that
the insecticidal
activity encoded on X. nematophilus clone pCIB9369 as well as the homologues
from
pCIB9381, pCIB9354, and pCIB9383-21 are larger than 6,000 molecular weight.
Example 9: Stability of the Insecticidal Activity
A 300 ml of Lucia broth supplemented with 100 p.g/ml ampicillin is inoculated
with
pCIB9369 and grown over night at 37°C. The samples are placed in
sterile 15 ml screw cap
tubes and stored at 22°C and 4°C. One sample is centrifuged and
the supernatant
removed, freeze dried and stored at 22°C. These samples are stored in
these conditions for
2 weeks and then bioassayed against Px. The freeze dried material is
resuspended in the
same volume as prior to freeze drying the sample. All samples are resuspended
by
vortexing. Another sample is treated at 100°C for 5 minutes.
Treatment Result
22°C (2 weeks) ++
4°C (2 weeks) ++
Freeze Dried (2 weeks) ++
100°C for 5 minutes na
na = not active.
Example 10: Heat Inactivation of the Insecticidal Activity
The heat stability of the toxin is determined. Overnight cultures of the E.
coli strain
pCIB9369 (E. coli host DHSa, carrying the 3.0 kb DNA of Xenorhabdus
nematophilus ) are
grown in a 50:50 mixture of Lucia broth and terrific broth. Cultures are grown
at 37°C in
culture tubes on a tube roller. Samples of one ml each of the culture are
placed in a 1.5 ml
eppendorf tube and placed at 60°C, 80°C. The samples are removed
after five minutes and
allowed to cool to room temperature. This sample along with an untreated
portion of the
culture is assayed on P. xylostella. 50N1 of sample of sample is spread on
diet, allowed to
dry and neonate larvae P. xylostelia applied to the surface. The assay is
incubated for 5
days at room temperature.
The untreated sample and the sample treated at 60°C cause 100%
mortality. The
sample treated at 80°C and a diet alone control do not cause observable
mortality.
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Example 11: Protease Treatment of the Insecticidal Activity
Xenorhabdus nematophilus cosmid clone pCIB9369 and Stratagene's Biue Script
vector in E. coli host DH5 are grown in media consisting of 50% Terrific broth
and 50%
Luria broth, supplemented with 50 ug/ml of ampicilin. Shake flask cultures (
300 ml in 1000
ml baffled flasks ) are grown at 250 RPM, overnight at 37°C. Cultures
of each strain are
centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4°C. The pelleted
cells are resuspended
in 30 ml of 50mM NaCI, 25 mM Tris base, pH 7Ø The concentrated cells are
disrupted by
sonication using a Branson Model 450 Sonicator for approximately eight 10
second cycles
with cooling on ice between cycles. The sonicates are centrifuged in a Sorvall
SS34 rotor at
6,000 RPM for i 0 minutes at 4°C. The resultant supernatants are
filtered through a 0.2 p
filter. One ml samples of the supernatants are adjusted to 5mM Ca++ with the
addition of
CaCl2. Protease K (Gibco BRL; Gaithersburg, MD ) is added to 500 Ng / ml.
Samples are
incubated for 2 and 24 hr at 37°C. Control samples are prepared and
incubated with added
Ca++ but no protease.
The sonicate filtrate from pCIB9369 and the pCIB9369 samples incubated in the
presence of 5mM Ca++ produce 100% mortality on Plufella xylosrella neonate
larvae. The
pCIB9369 samples incubated with protease K in addition to Ca++ show slightly
less
mortality, approximately 90% mortality on Plutella.
Example 12: Sequence Comparisons with the Protein Sequence of cry3A and the
Protein
Sequence of a Juvenile Hormone Esterase
The nucleotide sequence of pCIB9369 (SEQ ID N0:1 ) reveals two open reading
frames (ORFs) at nucleotides 569-976 and 1045-2334. ORF #1 has no homology to
any
sequences in Genbank after a search with the UWGCG Blast and Gap programs. A
Gap
analysis of the protein encoded by ORF #2 of pCIB9369 by the Blast program
identifies
some insignificant homology (21 % identity) to Bacillus fhuringensis cry3A
protein. However,
a Gap analysis of the protein encoded by ORF #2 of pCIB9369 (SEQ ID N0:3) by
the Blast
program does identify homology (30.6% AA identity and 44.1 % AA similarity) to
a juvenile
hormone esterase-related protein (GenBank accession 2921553; Henikoff et al.,
PNAS
USA 89: 10915-10919 (1992)).
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The nucleotide sequences of pCIB9381, pCIB9354, and pCIB9383-21 also reveal
two
open reading frames in each of these clones. The nucleotide sequences of the
two ORFs
in each of pCIB938i and pCIB9383-21 are all over 90% identical to those in
pCIB9369.
Hence, the ORF #2 proteins of pCIB9381 and pCIB9383-21 have essentially the
same
homology to the juvenile hormone esterase-related protein as does the ORF #2
protein of
pCIB9369. The nucleotide sequence of ORF #1 of pCIB9354 is 77% identical to
the
nucleotide sequence of ORF #1 of pCIB9369, and the nucleotide sequence of ORF
#2 of
pCIB9354 is 79% identical to the nucleotide sequence of ORF #2 of pCIB9369.
The ORF
#2 protein of pCIB9354 also has homology to the juvenile hormone esterase-
related protein
(29.2% AA identity and 42.2% AA similarity).
Example 13: Sequence Comparison of pCIB9369 and Sequences from WO 98108388
Twenty-two sequences of 60 nucleotides each (60-mers) are derived from the
38.2 kb
DNA fragment whose nucleotide sequence is described in WO 98/08388 and are
compared
to the nucleotide sequence of pCIB9362-3, which comprises pCIB9369. The first
60-mer
starts at base 1 in the 38.2 kb DNA fragment, while the other 60-mers are
located at
approximately 2 kb intervals on the DNA fragment. Their positions on the 38.2
kb DNA
fragment are listed below:
1-60; 2,041-2,100; 4,021-4,080; 6,001-6,060; 8,041-8,100; 10,021-10,080;
12,001-
12,060; 14,041-14,100; 16,021-16,080; 18,001-18,060; 20,041-20,100; 22,021-
22,080;
24,001-24,060; 26,041-26,100; 28,021-28,080; 30,001-30,060; 32,041-32,100;
34,021-
34,080; 36,001-36,060; 38,041-38,100; 38,161-38,220.
The sequences are compared using UWGCG Gap program and each of the 22 60-
mer sequences as well as their complementary sequences are tested. The results
of these
alignments indicate that the highest percentage of identity is 53%, which is
not considered
to be a significant homology in the art.
Example 14: Southern Blot Analysis using Probes Derived from WO 98/08388
Sequences
Pairs of oligonucleotides are designed to amplify DNA fragments of the 38.2 kb
DNA
fragment published in WO 98/08388. The oligonucleotides are ordered from
Genosys
Biotechnologies (The Woodlands, Texas) and their positions in the 38.2 kb DNA
fragment
are indicated below. Also listed are their and the sizes of the amplified PCR
fragments:
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VK1046: positions 20-40
VK1047: positions 2,078-2,100
Size of the PCR fragment amplified using VK1046 and VK1047: 2,080 by
VK1048: positions 11,221-11,241
VK1049: positions 13,360-13,380
Size of the PCR fragment amplified using VK1048 and VK1049: 2,120 by
VK1050: positions 26,581-26,601
VK1051: positions 28,537-28,560
Size of the PCR fragment amplified using VK1050 and VK1051: 1,979 by
VK1052: positions 18,901-18,921
VK1053: positions 20,321-20,340
Size of the PCR fragment amplified using VK1052 and VK1053: 1,439 by
VK1054: positions 34,261-34,281
VK1055: positions 35,320-35,340 BP
Size of the PCR fragment amplified using VK1054 and VK1055: 1,079 by
The PCR reactions are completed using a Perkin-Elmer 9600 Thermo-Cycler with
the following conditions: 94°C, 2 min.; then 30 cycles at 94°C,
30 sec; 54°C, 30 sec; 72°C, 4
min. The samples contain 800 ng of Xenorhabdus nematophilus DNA, 0.1-0.5 pM of
each
pair of oligonucleotides, 250 p.M dNTP, 5U Taq Polymerase and 1 X buffer
(Perkin-Elmer) in
a final volume of 100 p,l. The completed reactions are precipitated in
ethanol, resuspended
in TE and loaded on a 1 % SeaPlaque (FMC, Rockland, Maine) TBE gei. After
electrophoresis, the fragments are cut out from the gel after ethidium bromide
staining and
visualization under UV light. The gel slices are melted at 65°C and 10
pl aliquots are mixed
with 10 p! distilled water, boiled for 5 min. and placed on ice. Then, 15 pl
of Random
Priming label buffer ( GIBCO-BRL, Gaithersburg, MD), 6 p.l dNTP mix (without
dCTP) , 80
pCi a-dCT32P and 1 p,l Klenow are mixed. The labeling reaction is carried out
during 60 min.
at room temperature. The samples are cleaned up on Nick columns (Pharmacia
Biotech)
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according to the supplier's recommendations. The probes are boiled for 5 min.
and placed
on ice.
A Southern blot is performed by digesting Xenorhabdus nematophilus total DNA,
DNA derived from cosmids pCIB9362 and pCIB9363 (these cosmids overlap over 25
kb and
both contain the DNA fragment of pCIB9369; pCIB9362 was used for subcloning),
DNA
derived from subclones pCIB9362-3 (9 kb Sacll fragment) and pCIB9369 (2.96 kb
Clal
fragment), digested with Clal, Sacll or Hindlll. The digestion reactions are
loaded on a
0.75% agarose TBE gel and run overnight. A picture is taken and the gel is
treated as
described by Bio-Rad for blotting to a Zeta-Probe hybridization membrane.
After blotting,
the membrane is baked at 80°C for 30 min. The membrane is then placed
in 7% SDS, 250
mM sodium phosphate, pH 7.2 and incubated at 67°C for 30 min. Fresh
solution is added
and after equilibration to 67°C, the radioactive probes described above
are added and
allowed to hybridize overnight. The membrane is washed in 2X SSC, 0.5% SDS for
30 min.
at 67°C and then 0.5X SSC, 0.5% SDS for 30 min. at 67°C. The
membrane is exposed on
to a film for 1 hr and 3 hr. The film is developed and the results show that
the PCR probes
from the WO 98/08388 sequence do not hybridize to the DNA of the cosmids or
the DNA of
the subclones described in this invention. However, a strong hybridization
signal is
observed with X, nematophilus DNA.
These results corroborate the results of the sequence comparisons and show
that
clone pCIB9369 is different from the nucleotide sequence described in WO
98/08388.
B Expression of the Nucleic Acid Seguences of the Invention in Heterolo4ous
Microbial
Hosts
Microorganisms which are suitable for the heterologous expression of the
nucleotide
sequences of the invention are all microorganisms which are capable of
colonizing plants or
the rhizosphere. As such they will be brought into contact with insect pests.
These include
gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia,
the
gram-positive microorganism Bacillus and the fungi Trichoderma, Gliocladium,
and
Saccharomyces cerevisiae. Particularly preferred heterologous hosts are
Pseudomonas
fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas
aureofaciens,
Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus
subtilis,
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Bacillus cereus, Trichoderma wide, Trichoderma harzianum, Gliocladium wens,
and
Saccharomyces cerevisiae.
Example 19: Expression of the Nucleotide Sequences in E, toll and Other Gram-
Negative
Bacteria
Many genes have been expressed in gram-negative bacteria in a heterologous
manner. Expression vector pKK223-3 (Pharmacia catalogue # 27-4935-01 ) allows
expression in E. toll. This vector has a strong fat promoter (Brosius, J. et
al., Proc. Natl.
Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A
number of other
expression systems have been developed for use in E. toll. The thermoinducible
expression vector pPL (Pharmacia #27-4946-01 ) uses a tightly regulated
bacteriophage ~.
promoter which allows for high level expression of proteins. The lac promoter
provides
another means of expression but the promoter is not expressed at such high
levels as the
tat promoter. With the addition of broad host range replicons to some of these
expression
system vectors, expression of the nucleotide sequence in closely related gram
negative-
bacteria such as Pseudomonas, Enferobacter, Serratia and Erwinia is possible.
For
example, pLRKD211 (Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820
(1984))
contains the broad host range repficon on T which allows replication in many
gram-negative
bacteria.
In E. toll, induction by IPTG is required for expression of the tat (i.e. trp-
lac)
promoter. When this same promoter (e.g. on wide-host range plasmid pLRKD211)
is
introduced into Pseudomonas it is constitutively active without induction by
IPTG. This trp-
lac promoter can be placed in front of any gene or operon of interest for
expression in
Pseudomonas or any other closely related bacterium for the purposes of the
constitutive
expression of such a gene. Thus, a nucleotide sequence whose expression
results in an
insecticidal toxin can therefore be placed behind a strong constitutive
promoter, transferred
to a bacterium which has plant or rhizosphere colonizing properties turning
this organism to
an insecticidal agent. Other possible promoters can be used for the
constitutive expression
of the nucleotide sequence in gram-negative bacteria. These include, for
example, the
promoter from the Pseudomonas regulatory genes gafA and IemA (WO 94/01561 )
and the
Pseudomonas savastanoi IAA operon promoter (Gaffney et al., J. Bacteriol. 172:
5593-5601
(1990).
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Example 20: Expression of the Nucleotide Sequences in Gram-Positive Bacteria
Heterologous expression of the nucleotides sequence in gram-positive bacteria
is
another means of producing the insecticidal toxins. Expression systems for
Bacillus and
Strepi'omyces are the best characterized. The promoter for the erythromycin
resistance
gene (ermFt) from Streptococcus pneumoniae has been shown to be active in gram-
positive
aerobes and anaerobes and also in E.coli (Trieu-Cuot et al., Nucl Acids Res
18: 3660
(1990)). A further antibiotic resistance promoter from the thiostreptone gene
has been used
in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). The
shuttle
vector pHT3101 is also appropriate for expression in Bacillus (Lereclus, FEMS
Microbiol
Lett 60: 211-218 (1989)). A significant advantage of this approach is that
many gram-
positive bacteria produce spores which can be used in formulations that
produce
insecticidal agents with a longer shelf life. Bacillus and Streptomyces
species are
aggressive colonizers of soils
Example 21: Expression of the Nucleotide Sequences in Fungi
Trichoderma harzianum and Gliocladium wens have been shown to provide varying
levels of biocontrol in the field (US 5,165,928 and US 4,996,157, both to
Cornell Research
Foundation). A nucleotide sequence whose expression results in an insecticidal
toxin could
be expressed in such a fungus. This could be accomplished by a number of ways
which are
well known in the art. One is protoplast-mediated transformation of the fungus
by PEG or
electroporation-mediated techniques. Alternatively, particle bombardment can
be used to
transform protoplasts or other fungal cells with the ability to develop into
regenerated
mature structures. The vector pAN7-1, originally developed for Aspergillus
transformation
and now used widely for fungal transformation (Curragh et al., Mycol. Res.
97(3): 313-317
(1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 56:
117-124 (1987))
is engineered to contain the nucleotide sequence. This plasmid contains the E,
coli the
hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter
and the
trpC terminator (Punt et al., Gene 56: 117-124 (1987)).
In a preferred embodiment, the nucleic acid sequences of the invention are
expressed in the yeast Saccharomyces cerevisiae. For example, each of the two
ORF's
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from pCIB9369, pCIB9381, pCIB9354, or pCIB9383 are cloned into individual
vectors with
the GAL1 inducible promoter and the CYC1 terminator. Each vector has
ampicillin
resistance and the 2 micron replicon. The vectors preferably differ in their
yeast growth
markers. The constructs are transformed into S. cerevisiae independently and
together.
The ORFs are expressed together and tested for protein expression and
insecticidal
activity.
C. Formulation of the Insecticidal Toxin
lnsecticidal formulations are made using active ingredients which comprise
either the
isolated toxin or alternatively suspensions or concentrates of cells which
produce it and
which are described in the examples above. For example, E. coli cells
expressing the
insecticidal toxin may be used for the control of the insect pests.
Formulations are made in
liquid or solid form and are described below.
Example 18: Liquid Formulation of Insecticidai Compositions
In the following examples, percentages of composition are given by weight:
1. Emulsifiable concentrates:a b c
Active ingredient 20% 40% 50%
Calcium dodecylbenzenesulfonate5% 8% 6%
Castor oil polyethlene glycol5% - -
ether (36 moles of ethylene
oxide)
Tributylphenol polyethylene - 12% 4%
glyco
ether (30 moles of ethylene
oxide)
Cyclohexanone - 15% 20%
Xylene mixture 70% 25 % 20%
Emulsions of any required concentration can be produced from such concentrates
by dilution with water.
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2. Solutions: a b c d
Active ingredient 80% 10% 5% 95%
Ethylene glycol monomethyl 20% - - -
ether
Polyethylene glycol 400 - 70% - -
N-methyl-2-pyrrolidone - 20 % - -
Epoxidised coconut oil - - 1 % 5%
Petroleum distillate - - 94% -
(boiling range 160-190°
These solutions are suitable for application in the form of microdrops.
3. Granulates: a b
Active ingredient 5% 10%
Kaolin 94% -
Highly dispersed silicic acid 1
Attapulgit - 90%
The active ingredient is dissolved in methylene chloride, the solution is
sprayed onto
the carrier, and the solvent is subsequently evaporated off in vacuo.
4. Dusts: a b
Active ingredient 2% 5%
Highly dispersed silicic 1 % 5%
acid
Talcum 97% -
Kaolin - 90%
Ready-to-use dusts are obtained by intimately mixing the carriers with the
active
ingredient.
Example 19: Solid Formulation of Insecticidal Compositions
In the following examples, percentages of compositions are by weight.
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1. Wettable owders: a b c
Active ingredient 20% 60% 75%
Sodium lignosulfonate 5% 5% -
Sodium lauryl sulfate 3% - 5%
Sodium diisobutylnaphthalene - 6% 10
sulfonate
Octylphenol polyethylene glycol- 2% -
ether
(7-8 moles of ethylene oxide)
Highly dispersed silicic acid 5% 27% 10%
Kaolin 67% - _
The active ingredient is thoroughly mixed with the adjuvants and the mixture
is
thoroughly ground in a suitable mill, affording wettable powders which can be
diluted with
water to give suspensions of the desired concentrations.
2. Emulsifiable concentrate:
Active ingredient 10%
Octyfphenol polyethylene glycol ether 3%
(4-5 moles of ethylene oxide)
Calcium dodecylbenzenesulfonate 3%
Castor oil polyglycol ether 4%
(36 moles of ethylene oxide)
Cyclohexanone 30%
Xylene mixture 50%
Emulsions of any required concentration can be obtained from this concentrate
by
dilution with water.
3. Dusts: a b
Active ingredient 5% 8%
Talcum 95% -
Kaolin - 92%
Ready-to-use dusts are obtained by mixing the active ingredient with the
carriers,
and grinding the mixture in a suitable mill.
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4. Extruder granulate:
Active ingredient 10%
Sodium lignosulfonate 2%
Carboxymethylcellulose 1
Kaolin 87%
The active ingredient is mixed and ground with the adjuvants, and the mixture
is
subsequently moistened with water. The mixture is extruded and then dried in a
stream of
ai r.
5. Coated q~ranulate:
Active ingredient 3%
Polyethylene glycol 200 3%
Kaolin 94%
The finely ground active ingredient is uniformly applied, in a mixer, to the
kaolin
moistened with polyethylene glycol. Non-dusty coated granulates are obtained
in this
manner.
6. Suspension concentrate:
Active ingredient 40%
Ethylene glycol 10%
Nonylphenol polyethylene glycol6%
(15 moles of ethylene oxide)
Sodium lignosulfonate 10%
Carboxymethylcellulose 1
37 % aqueous formaldehyde 0.2%
solution
Silicone oil in 75 % aqueous 0.8%
emulsion
W ate r 32%
The finely ground active ingredient is intimately mixed with the adjuvants,
giving a
suspension concentrate from which suspensions of any desire concentration can
be
obtained by dilution with water.
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The insecticidal formulations described above are applied to the plants
according to
methods well known in the art, in such amounts that the insect pests are
controlled by the
insecticidal toxin.
D Expression of the Nucleotide Sequences in Trans4enic Plants
The nucleic acid sequences described in this application can be incorporated
into
plant cells using conventional recombinant DNA technology. Generally, this
involves
inserting a coding sequence of the invention into an expression system to
which the coding
sequence is heterologous (i.e., not normally present) using standard cloning
procedures
known in the art. The vector contains the necessary elements for the
transcription and
translation of the inserted protein-coding sequences. A large number of vector
systems
known in the art can be used, such as plasmids, bacteriophage viruses and
other modified
viruses. Suitable vectors include, but are not limited to, viral vectors such
as lambda vector
systems ~,gtl1, ~.gtl0 and Charon 4; plasmid vectors such as p81121, pBR322,
pACYC177,
pACYC184, pAR series, pKK223-3, pUCB, pUC9, pUCl8, pUCl9, pLG339, pRK290,
pKC37, pKC101, pCDNAII; and other similar systems. The components of the
expression
system may also be modified to increase expression. For example, truncated
sequences,
nucleotide substitutions or other modifications may be employed. The
expression systems
described herein can be used to transform virtually any crop plant cell under
suitable
conditions. Transformed cells can be regenerated into whole plants such that
the
nucleotide sequence of the invention confer insect resistance to the
transgenic plants.
Example 22: Modification of Coding Sequences and Adjacent Sequences
The nucleotide sequences described in this application can be modified for
expression in transgenic plant hosts. A host plant expressing the nucleotide
sequences and
which produces the insecticidal toxins in its cells has enhanced resistance to
insect attack
and is thus better equipped to withstand crop losses associated with such
attack.
The transgenic expression in plants of genes derived from microbial sources
may
require the modification of those genes to achieve and optimize their
expression in plants.
In particular, bacterial ORFs which encode separate enzymes but which are
encoded by the
same transcript in the native microbe are best expressed in plants on separate
transcripts.
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To achieve this, each microbial ORF is isolated individually and cloned within
a cassette
which provides a plant promoter sequence at the 5' end of the ORF and a plant
transcriptional terminator at the 3' end of the ORF. The isolated ORF sequence
preferably
includes the initiating ATG codon and the terminating STOP codon but may
include
additional sequence beyond the initiating ATG and the STOP codon. In addition,
the ORF
may be truncated, but still retain the required activity; for particularly
long ORFs, truncated
versions which retain activity may be preferable for expression in transgenic
organisms. By
"plant promote" and "plant transcriptional terminator" it is intended to mean
promoters and
transcriptional terminators which operate within plant cells. This includes
promoters and
transcription terminators which may be derived from non-plant sources such as
viruses (an
example is the Cauliflower Mosaic Virus).
In some cases, modification to the ORF coding sequences and adjacent sequence
is not required. It is sufficient to isolate a fragment containing the ORF of
interest and to
insert it downstream of a plant promoter. For example, Gaffney et at. (Science
261: 754-
756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants
under the
control of the CaMV 35S promoter and the CaMV tml terminator successfully
without
modification of the coding sequence and with x by of the Pseudomonas gene
upstream of
the ATG still attached, and y by downstream of the STOP codon still attached
to the nahG
ORF. Preferably as little adjacent microbial sequence should be left attached
upstream of
the ATG and downstream of the STOP codon. In practice, such construction may
depend
on the availability of restriction sites.
fn other cases, the expression of genes derived from microbial sources may
provide
problems in expression. These problems have been well characterized in the art
and are
particularly common with genes derived from certain sources such as Bacillus.
These
problems may apply to the nucleotide sequence of this invention and the
modification of
these genes can be undertaken using techniques now well known in the art. The
following
problems may be encountered:
1. Codon Usage.
The preferred codon usage in plants differs from the preferred codon usage in
certain microorganisms. Comparison of the usage of codons within a cloned
microbial ORF
to usage in plant genes (and in particular genes from the target plant) will
enable an
identification of the codons within the ORF which should preferably be
changed. Typically
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plant evolution has tended towards a strong preference of the nucleotides C
and G in the
third base position of monocotyledons, whereas dicotyledons often use the
nucleotides A or
T at this position. By modifying a gene to incorporate preferred codon usage
for a particular
target transgenic species, many of the problems described below for GC/AT
content and
illegitimate splicing will be overcome.
2. GC/AT Content.
Plant genes typically have a GC content of more than 35%. ORF sequences which
are rich in A and T nucleotides can cause several problems in plants. Firstly,
motifs of
ATTTA are believed to cause destabilization of messages and are found at the
3' end of
many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals
such as
AATAAA at inappropriate positions within the message is believed to cause
premature
truncation of transcription. In addition, monocotyledons may recognize AT-rich
sequences
as splice sites (see below).
3. Sequences Adjacent to the initiating Methionine.
Plants differ from microorganisms in that their messages do not possess a
defined
ribosome binding site. Rather, it is believed that ribosomes attach to the 5'
end of the
message and scan for the first available ATG at which to start translation.
Nevertheless, it
is believed that there is a preference for certain nucleotides adjacent to the
ATG and that
expression of microbial genes can be enhanced by the inclusion of a eukaryotic
consensus
translation initiator at the ATG. Clontech (1993/1994 catalog, page 210,
incorporated
herein by reference) have suggested one sequence as a consensus translation
initiator for
the expression of the E. coli uidA gene in plants. Further, Joshi (NAR 15:
6643-6653
(1987), incorporated herein by reference) has compared many plant sequences
adjacent to
the ATG and suggests another consensus sequence. In situations where
difficulties are
encountered in the expression of microbial ORFs in plants, inclusion of one of
these
sequences at the initiating ATG may improve translation. In such cases the
last three
nucleotides of the consensus may not be appropriate for inclusion in the
modified sequence
due to their modification of the second AA residue. Preferred sequences
adjacent to the
initiating methionine may differ between different plant species. A survey of
14 maize
genes located in the GenBank database provided the following results:
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Position Before theInitiatina ATG in 14 Maize Genes:
-10 9 -8 -7 -6 -5 -4 -3 -2 -1
-
C 3 8 4 6 2 5 6 0 10 7
T 3 0 3 4 3 2 1 1 1 0
A 2 3 1 4 3 2 3 7 2 3
G 6 3 6 0 6 5 4 6 1 5
This analysis can be done for the desired plant species into which the
nucleotide sequence
is being incorporated, and the sequence adjacent to the ATG modified to
incorporate the
preferred nucleotides.
4. Removal of Illegitimate Splice Sites.
Genes cloned from non-plant sources and not optimized for expression in plants
may also contain motifs which may be recognized in plants as 5' or 3' splice
sites, and be
cleaved, thus generating truncated or deleted messages. These sites can be
removed
using the techniques well known in the art.
Techniques for the modification of coding sequences and adjacent sequences are
well known in the art. In cases where the initial expression of a microbial
ORF is low and it
is deemed appropriate to make alterations to the sequence as described above,
then the
construction of synthetic genes can be accomplished according to methods well
known in
the art. These are, for example, described in the published patent disclosures
EP 0 385
962 (to Monsanto), EP 0 359 472 (to Lubrizol) and WO 93/07278 (to Ciba-Geigy),
all of
which are incorporated herein by reference. In most cases it is preferable to
assay the
expression of gene constructions using transient assay protocols (which are
well known in
the art) prior to their transfer to transgenic plants.
Example 23: Construction of Plant Expression Cassettes
Coding sequences intended for expression in transgenic plants are first
assembled in
expression cassettes behind a suitable promoter expressible in plants. The
expression
cassettes may also comprise any further sequences required or selected for the
expression
of the transgene. Such sequences include, but are not restricted to,
transcription
terminators, extraneous sequences to enhance expression such as introns, vital
sequences,
and sequences intended for the targeting of the gene product to specific
organelles and cell
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compartments. These expression cassettes can then be easily transferred to the
plant
transformation vectors described below. The following is a description of
various
components of typical expression cassettes.
1. Promoters
The selection of the promoter used in expression cassettes will determine the
spatial
and temporal expression pattern of the transgene in the transgenic plant.
Selected
promoters will express transgenes in specific cell types (such as leaf
epidermal cells,
mesophyll cells, root cortex cells) or in specific tissues or organs (roots,
leaves or flowers,
for example) and the selection will reflect the desired location of
accumulation of the gene
product. Alternatively, the selected promoter may drive expression of the gene
under
various inducing conditions. Promoters vary in their strength, i.e., ability
to promote
transcription. Depending upon the host cell system utilized, any one of a
number of suitable
promoters can be used, including the gene's native promoter. The following are
non-
limiting examples of promoters that may be used in expression cassettes.
a. Constitutive Expression, the Ubiquitin Promoter:
Ubiquitin is a gene product known to accumulate in many cell types and its
promoter
has been cloned from several species for use in transgenic plants (e.g.
sunflower - Binet et
al. Plant Science 79: 87-94 (1991 ); maize - Christensen et al. Plant Molec.
Biol. 12: 619-
632 (1989); and Arabidopsis - Norris et al., Plant Mol. Biol. 21:895-906
(1993)). The maize
ubiquitin promoter has been developed in transgenic monocot systems and its
sequence
and vectors constructed for monocot transformation are disclosed in the patent
publication
EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor
et al. (Plant
Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) that comprises the
maize
ubiquitin promoter and first intron and its high activity in cell suspensions
of numerous
monocotyledons when introduced via microprojectile bombardment. The
Arabidopsis
ubiquitin promoter is ideal for use with the nucleotide sequences of the
present invention.
The ubiquitin promoter is suitable for gene expression in transgenic plants,
both
monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or
any of
the transformation vectors described in this application, modified by the
introduction of the
appropriate ubiquitin promoter and/or intron sequences.
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b. Constitutive Expression, the CaMV 35S Promoter:
Construction of the plasmid pCGN1761 is described in the published patent
application EP 0 392 225 (Example 23), which is hereby incorporated by
reference.
pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional
terminator
with a unique EcoRl site between the promoter and the terminator and has a pUC-
type
backbone. A derivative of pCGN1761 is constructed which has a modified
polylinker which
includes Notl and Xhol sites in addition to the existing EcoRl site. This
derivative is
designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA
sequences
or coding sequences (including microbial ORF sequences) within its polylinker
for the
purpose of their expression under the control of the 35S promoter in
transgenic plants. The
entire 35S promoter-coding sequence-tml terminator cassette of such a
construction can be
excised by Hindlll, Sphl, Sall, and Xbal sites 5' to the promoter and Xbal,
BamHl and Bgll
sites 3' to the terminator for transfer to transformation vectors such as
those described
below. Furthermore, the double 35S promoter fragment can be removed by 5'
excision with
Hindlll, Sphl, Sall, Xbal, or Pstl, and 3' excision with any of the
polyiiinker restriction sites
(EcoRl, Notl or Xho~ for replacement with another promoter. If desired,
modifications
around the cloning sites can be made by the introduction of sequences that may
enhance
translation. This is particularly useful when overexpression is desired. For
example,
pCGN1761 ENX may be modified by optimization of the translational initiation
site as
described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by
reference.
c. Constitutive Expression, the Actin Promoter:
Several isoforms of actin are known to be expressed in most cell types and
consequently the actin promoter is a good choice for a constitutive promoter.
In particular,
the promoter from the rice Actl gene has been cloned and characterized
(McElroy et al.
Plant Cell 2: 163-171 (1990)). A l.3kb fragment of the promoter was found to
contain all
the regulatory elements required for expression in rice protoplasts.
Furthermore, numerous
expression vectors based on the Actl promoter have been constructed
specifically for use in
monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991 )). These
incorporate
the Actl-intron 1, Adh! 5' flanking sequence and Adhl-intron 1 (from the maize
alcohol
dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing
highest expression were fusions of 35S and Actl intron or the Actl 5' flanking
sequence and
the Actl intron. Optimization of sequences around the initiating ATG (of the
GUS reporter
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gene) also enhanced expression. The promoter expression cassettes described by
McElroy
et al. (Mol. Gen. Genet. 231: 150-i 60 (1991 )) can be easily modified for
gene expression
and are particularly suitable for use in monocotyledonous hosts. For example,
promoter-
containing fragments is removed from the McElroy constructions and used to
replace the
double 35S promoter in pCGN1761 ENX, which is then available for the insertion
of specific
gene sequences. The fusion genes thus constructed can then be transferred to
appropriate
transformation vectors. In a separate report, the rice Act! promoter with its
first intron has
also been found to direct high expression in cultured barley cells (Chibbar et
al. Plant Cell
Rep. 12: 506-509 (1993)).
d. Inducible Expression, the PR-1 Promoter:
The double 35S promoter in pCGN1761 ENX may be replaced with any other
promoter
of choice that will result in suitably high expression levels. By way of
example, one of the
chemically regulatable promoters described in U.S. Patent No. 5,614,395 may
replace the
double 35S promoter. The promoter of choice is preferably excised from its
source by
restriction enzymes, but can alternatively be PCR-amplified using primers that
carry
appropriate terminal restriction sites. Should PCR-amplification be
undertaken, then the
promoter should be re-sequenced to check for amplification errors after the
cloning of the
amplified promoter in the target vector. The chemically/pathogen regulatable
tobacco PR-
1 a promoter is cleaved from plasmid pCIB1004 (for construction, see example
21 of
EP 0 332 104, which is hereby incorporated by reference) and transferred to
pfasmid
pCGN1761 ENX (Uknes et al., 1992). pCIB1004 is cleaved with Ncol and the
resultant 3'
ovefiang of the linearized fragment is rendered blunt by treatment with T4 DNA
polymerase. Tile fragment is then cleaved with Hindlll and the resultant PR-1
a promoter-
containing fragment is gel purified and cloned into pCGN1761 ENX from which
the double
35S promoter has been removed. This is done by cleavage with Xhol and blunting
with T4
polymerase, followed by cleavage with Hindlil and isolation of the larger
vector-terminator
containing fragment into which the pCIB1004 promoter fragment is cloned. This
generates
a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and
an
intervening polylinker with unique EcoRi and Notl sites. The selected coding
sequence can
be inserted into this vector, and the fusion products (i.e. promoter-gene-
terminator) can
subsequently be transferred to any selected transformation vector, including
those
described infra. Various chemical regulators may be employed to induce
expression of the
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selected coding sequence in the plants transformed according to the present
invention,
including the benzothiadiazole, isonicotinic acid, and salicylic acid
compounds disclosed in
U.S. Patent Nos. 5,523,311 and 5,614,395.
e. Inducible Expression, an Ethanol-Inducible Promoter:
A promoter inducible by certain alcohols or ketones, such as ethanol, may also
be
used to confer inducible expression of a coding sequence of the present
invention. Such a
promoter is for example the alcA gene promoter from Aspergillus nidulans
(Caddick et al.
(1998) Nat. Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes
alcohol
dehydrogenase I, the expression of which is regulated by the AIcR
transcription factors in
presence of the chemical inducer. For the purposes of the present invention,
the CAT
coding sequences in plasmid paIcA:CAT comprising a alcA gene promoter sequence
fused
to a minimal 35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180)
are replaced
by a coding sequence of the present invention to form an expression cassette
having the
coding sequence under the control of the alcA gene promoter. This is carried
out using
methods well known in the art.
f. Inducible Expression, a Glucocorticoid-Inducible Promoter:
Induction of expression of a nucleic acid sequence of the present invention
using
systems based on steroid hormones is also contemplated. For example, a
glucocorticoid-
mediated induction system is used (Aoyama and Chua (1997) The Plant Journal
11: 605-
612) and gene expression is induced by application of a glucocorticoid, for
example a
synthetic gfucocorticoid, preferably dexamethasone, preferably at a
concentration ranging
from 0.1 mM to 1 mM, more preferably from 1 OmM to 1 OOmM. For the purposes of
the
present invention, the luciferase gene sequences are replaced by a nucleic
acid sequence
of the invention to form an expression cassette having a nucleic acid sequence
of the
invention under the control of six copies of the GAL4 upstream activating
sequences fused
to the 35S minimal promoter. This is carried out using methods well known in
the art. The
traps-acting factor comprises the GAL4 DNA-binding domain (Keegan et al.
(1986) Science
231: 699-704) fused to the transactivating domain of the herpes viral protein
VP16
(Triezenberg et al. (1988) Genes DeveL 2: 718-729) fused to the hormone-
binding domain
of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080).
The expression
of the fusion protein is controlled by any promoter suitable for expression in
plants known in
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the art or described here. This expression cassette is also comprised in the
plant comprising
a nucleic acid sequence of the invention fused to the 6xGAL4/minimal promoter.
Thus,
tissue- or organ-specificity of the fusion protein is achieved leading to
inducible tissue- or
organ-specificity of the insecticidal toxin.
g. Root Specific Expression:
Another pattern of gene expression is root expression. A suitable root
promoter is
described by de Framond (FEES 290: 103-106 (1991 )) and also in the published
patent
application EP 0 452 269, which is herein incorporated by reference. This
promoter is
transferred to a suitable vector such as pCGN1761 ENX for the insertion of a
selected gene
and subsequent transfer of the entire promoter-gene-terminator cassette to a
transformation
vector of interest.
h. Wound-Inducible Promoters:
Wound-inducible promoters may also be suitable for gene expression. Numerous
such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-
588 (1993),
' Logemann -et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant
Molec. Biol. 22:
783-792 (1993), Firek et al. Plant Molec. _Biol. 22: 129-142 (1993), Warner ef
al. Plant J. 3:
191-201 (i993)) and all are suitable for use with the instant invention.
Logemann et a!.
describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu
et al.
show that a wound-inducible promoter from the dicotyledon potato (pint) is
active in the
monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the
maize Wipl
cDNA which is wound induced and which can be used to isolate the cognate
promoter using
standard techniques. Similar, Firek ef al. and Warner et al, have described a
wound-
induced gene from the monocotyledon Asparagus officinalis, which is expressed
at local
wound and pathogen invasion sites. Using cloning techniques well known in the
art, these
promoters can be transferred to suitable vectors, fused to the genes
pertaining to this
invention, and used to express these genes at the sites of plant wounding.
i. Pith-Preferred Expression:
Patent Application WO 93/07278, which is herein incorporated by reference,
describes the isolation of the maize trpA gene, which is preferentially
expressed in pith
cells. The gene sequence and promoter extending up to -1726 by from the start
of
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transcription are presented. Using standard molecular biological techniques,
this promoter,
or parts thereof, can be transferred to a vector such as pCGN1761 where it can
replace the
35S promoter and be used to drive the expression of a foreign gene in a pith-
preferred
manner. In fact, fragments containing the pith-preferred promoter or parts
thereof can be
transferred to any vector and modified for utility in transgenic plants.
j. Leaf-Specific Expression:
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by
Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard
molecular
biological techniques the promoter for this gene can be used to drive the
expression of any
gene in a leaf-specific manner in transgenic plants.
k. Pollen-Specific Expression:
WO 93/07278 describes the isolation of the maize calcium-dependent protein
kinase
(CDPK) gene which is expressed in pollen cells. The gene sequence and promoter
extend
up to 1400 by from the start of transcription. Using standard molecular
biological
techniques, this promoter or parts thereof, can be transferred to a vector
such as
pCGN1761 where it can replace the 35S promoter and be used to drive the
expression of a
nucleic acid sequence of the invention in a pollen-specific manner.
2. Transcriptiona! Terminators
A variety of transcriptional terminators are available for use in expression
cassettes.
These are responsible for the termination of transcription beyond the
transgene and its
correct polyadenylation. Appropriate transcriptional terminators are those
that are known to
function in plants and include the CaMV 35S terminator, the tml terminator,
the nopaline
synthase terminator and the pea rbcS E9 terminator. These can be used in both
monocotyledons and dicotyledons. In addition, a gene's native transcription
terminator may
be used.
3. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the
genes of this
invention to increase their expression in transgenic plants.
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Various intron sequences have been shown to enhance expression, particularly
in
monocotyledonous cells. For example, the introns of the maize Adhl gene have
been found
to significantly enhance the expression of the wild-type gene under its
cognate promoter
when introduced into maize cells. Intron 1 was found to be particularly
effective and
enhanced expression in fusion constructs with the chloramphenicol
acetyltransferase gene
(Callis et al., Genes Develop. 1: 1183-1200 (1987)). in the same experimental
system, the
intron from the maize bronze i gene had a similar effect in enhancing
expression. Intron
sequences have been routinely incorporated into plant transformation vectors,
typically
within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also
known to
enhance expression, and these are particularly effective in dicotyledonous
cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-
sequence"), Maize
Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown
to be
effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:
8693-8711 (1987);
Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).
' 4. Targeting of the Gene Product Within the Cell
Various mechanisms for targeting gene products are known to exist in plants
and the
sequences controlling the functioning of these mechanisms have been
characterized in
some detail. For example, the targeting of gene products to the chloroplast is
controlled by
a signal sequence found at the amino terminal end of various proteins which is
cleaved
during chloroplast -import to yield the mature protein (e.g. Comai et al. J.
Biol. Chem. 263:
15104-15109 (1988)). These signal sequences can be fused to heterologous gene
products to effect the import of heterologous products into the chloroplast
(van den Broeck,
et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal
sequences can be
isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB
protein, the
EPSP synthase enzyme, the GS2 protein and many other proteins which are known
to be
chforoplast localized. See also, the section entitled "Expression With
Chloroplast Targeting"
in Example 37 of U.S. Patent No. 5,639,949.
Other gene products are localized to other organelles such as the
mitochondrion and
the peroxisome (e.g. Unger ef al. Plant Molec. Biol. 13: 411-418 (1989)). The
cDNAs
encoding these products can also be manipulated to effect the targeting of
heterologous
gene products to these organelles. Examples of such sequences are the nuclear-
encoded
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ATPases and specific aspartate amino transferase isoforms for mitochondria.
Targeting
cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad.
Sci. USA 82:
6512-6516 (1985)).
In addition, sequences have been characterized which cause the targeting of
gene
products to other cell compartments. Amino terminal sequences are responsible
for
targeting to the ER, the apoplast, and extracellular secretion from aleurone
cells (Koehler &
Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in
conjunction
with carboxy terminal sequences are responsible for vacuolar targeting of gene
products
{Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above to
transgene
sequences of interest it is possible to direct the transgene product to any
organelle or cell
compartment. For chloroplast targeting, for example, the chloroplast signal
sequence from
the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is
fused in
frame to the amino terminal ATG of the transgene. The signal sequence selected
should
include the known cleavage site, and the fusion constructed should take into
account any
amino acids after the cleavage site which are required for cleavage. In some
cases this
requirement may be fulfilled by the addition of a small number of amino acids
between the
cleavage site and the transgene ATG or, alternatively, replacement of some
amino acids
within the transgene sequence. Fusions constructed for chloroplast import can
be tested
for efficacy of chloroplast uptake by in vitro translation of in vitro
transcribed constructions
followed by in vitro chloroplast uptake using techniques described by Bartlett
et al. In:
Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp
1081-1091
(1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These
construction
techniques are well known in the art and are equally applicable to
mitochondria and
peroxisomes.
The above-described mechanisms for cellular targeting can be utilized not only
in
conjunction with their cognate promoters, but also in conjunction with
heterologous
promoters so as to effect a specific cell-targeting goal under the
transcriptional regulation of
a promoter that has an expression pattern different to that of the promoter
from which the
targeting signal derives.
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Example 24: Construction of Plant Transformation Vectors
Numerous transformation vectors available for plant transformation are known
to
those of ordinary skill in the plarit transformation arts, and the genes
pertinent to this
invention can be used in conjunction with any such vectors. The selection of
vector will
depend upon the preferred transformation technique and the target species for
transformation. For certain target species, different antibiotic or herbicide
selection markers
may be preferred. Selection markers used routinely in transformation include
the nptll
gene, which confers resistance to kanamycin and related antibiotics (Messing &
Vierra.
Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar
gene, which
confers resistance to the herbicide phosphinothricin (White et al., Nucl.
Acids Res 18: 1062
(1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene,
which confers
resistance to the antibiotic hygromycin _(Blochinger & Diggelmann, Mol Cell
Biol 4: 2929-
2931 ), and the dhfr gene, which confers resistance to methatrexate (Bourouis
et al., EMBO
J. 2 7 : 1099-1104 (1983)), and the EPSPS gene, which confers resistance to
glyphosate
(U.S. Patent Nos. 4,940,935 and 5,188,642).
1. Vectors Suitable for Agrobacterium Transformation
Many vectors are available for transformation using Agrobact~erium
tumefaciens.
These typically carry at least one T-DNA border sequence and include vectors
such as
pBINl9 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of
two typical
vectors suitable for Agrobacterium transformation is described.
a. pCIB200 and pCIB2001:
The binary vectors pcIB200 and pC182001 are used for the construction of
recombinant vectors for use with Agrobacterium and are constructed in the
following
manner. pTJS75kan is created by IVarl digestion of pTJS75 (Schmidhauser &
Helinski, J.
Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-
resistance gene,
followed by insertion of an Acci fragment from pUC4K carrying an NPTII
(Messing & Vierra,
Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et
al., Plant
Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the EcoRV
fragment of
PCIB7 which contains the left and right T-DNA borders, a plant selectable
noslnpfil chimeric
gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and
the Xhol
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digested fragment are cloned into Sall-digested pTJS75kan to create pCIB200
(see also EP
0 332 104, example 19). pCIB200 contains the following unique polylinker
restriction sites:
EcoF?l, Sstl, Kpnl, Bglll, Xbal, and Sall. pCIB2001 is a derivative of pCIB200
created by the
insertion into the polylinker of additional restriction sites. Unique
restriction sites in the
polylinker of pCIB2001 are EcoRl, Sstl, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll,
Avrll, Apal, Hpal,
and Stul. pCIB2001, in addition to containing these unique restriction sites
also has plant
and bacterial kanamycin selection, left and right T-DNA borders for
Agrobacterium-mediated
transformation, the RK2-derived trfA function for mobilization between E. coli
and other
hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker
is suitable
for the cloning of plant expression cassettes containing their own regulatory
signals.
b. pCIBlO and Hygromycin Selection Derivatives thereof:
The binary vector pCIBlO contains a gene encoding kanamycin resistance for
selection in plants and T-DNA right and left border sequences and incorporates
sequences
from the wide host-range plasmid pRK252 allowing it to replicate in both E.
coli and
Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-
161 (1987)).
Various derivatives of pCIBlO are constructed which incorporate the gene for
hygromycin B
phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These
derivatives
enable selection of transgenic plant cells on hygromycin only (pCIB743), or
hygromycin and
kanamycin (pCIB715, pCIB717).
2. Vectors Suitable for non-Agrobacterium Transformation
Transformation without the use of Agrobacterium tumefaciens circumvents the
requirement for T-DNA sequences in the chosen transformation vector and
consequently
vectors lacking these sequences can be utilized in addition to vectors such as
the ones
described above which contain T-DNA sequences. Transformation techniques that
do not
rely on Agrobacterium include transformation via particle bombardment,
protoplast uptake
(e.g. PEG and electroporation) and microinjection. The choice of vector
depends largely on
the preferred selection for the species being transformed. Below, the
construction of typical
vectors suitable for non-Agrobacterium transformation is described.
a. pCIB3064:
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pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques
in
combination with selection by the herbicide basta (or phosphinothricin). The
plasmid
pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. toll
GUS gene
and the CaMV 35S transcriptional terminator and is described in the PCT
published
application WO 93/07278. The 35S promoter of this vector contains two ATG
sequences 5'
of the start site. These sites are mutated using standard PCR techniques in
such a way as
to remove the ATGs and generate the restriction sites Sspl and Pvull. The new
restriction
sites are 96 and 37 by away from the unique Sall site and 101 and 42 by away
from the
actual start site. The resultant derivative of pCIB246 is designated pCIB3025.
The GUS
gene is then excised from pCIB3025 by digestion with Sall and Sacl, the
termini rendered
blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is
obtained from the
John fnnes Centre, Norwich and the a 400 by Smal fragment containing the bar
gene from
Streptomyces viridochromogenes is excised and inserted into the Hpal site of
pCIB3060
{Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which
comprises the bar gene under the control of the CaMV 35S promoter and
terminator for
herbicide selection, a gene for ampicillin resistance (for selection in E.
coh) and a polylinker
with the unique sites Sphl, Pstl, Hindlll, and BamHl. This vector is suitable
for the cloning
of plant expression cassettes containing their own regulatory signals.
b. pSOGl9 and pSOG35:
pSOG35 is a transformation vector that utilizes the E. toll gene dihydrofolate
reductase (DFR) as a selectable marker conferring resistance to methotrexate.
PCR is
used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene
(-550 bp)
and 18 by of the GUS untranslated leader sequence from pSOGlO. A 250-by
fragment
encoding the E. toll dihydrofolate reductase type II gene is also amplified by
PCR and
these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221
(Ciontech)
which comprises the pUCl9 vector backbone and the nopaline synthase
terminator.
Assembly of these fragments generates pSOGl9 which contains the 35S promoter
in fusion
with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline
synthase
terminator. Replacement of the GUS leader in pSOGl9 with the leader sequence
from
Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOGl9 and
pSOG35
carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Psfl and
EcoRl sites
available for the cloning of foreign substances.
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3. Vector Suitable for Chloroplast Transformation
For expression of a nucleotide sequence of the present invention in plant
plastids,
plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The
nucleotide
sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence.
This
vector is then used for plastid transformation and selection of transformants
for
spectinomycin resistance. Alternatively, the nucleotide sequence is inserted
in pPH143 so
that it replaces the aadH gene. In this case, transformants are selected for
resistance to
PROTOX inhibitors.
Example 25: Transformation
Once a nucleic acid sequence of the invention has been cloned into an
expression
system, it is transformed into a plant cell. Methods for transformation and
regeneration of
plants are well known in the art. For example, Ti plasmid vectors have been
utilized for the
delivery of foreign DNA, as well as direct DNA uptake, liposomes,
electroporation, micro-
injection, and microprojectiles. In addition, bacteria from the genus
Agrobacterium can be
utilized to transform plant cells. Below are descriptions of representative
techniques for
transforming both dicotyledonous and monocotyledonous plants, as well as a
representative plastid transformation technique.
1. Transformation of Dicotyfedons
Transformation techniques for dicotyledons are well known in the art and
include
Agrobacterium-based techniques and techniques that do not require
Agrobacterium. Non-
Agrobacterium techniques involve the uptake of exogenous genetic material
directly by
protoplasts or cells. This can be accomplished by PEG or electroporation
mediated uptake,
particle bombardment-mediated delivery, or microinjection. Examples of these
techniques
are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et
al., Mol. Gen.
Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986),
and Klein et
al., Nature 327: 70-73 (1987). In each case the transformed cells are
regenerated to whole
plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for
transformation of
dicotyledons because of its high efficiency of transformation and its broad
utility with many
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different species. Agrobacterium transformation typically involves the
transfer of the binary
vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an
appropriate
Agrobacterium strain which may depend of the complement of vir genes carried
by the host
Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g.
strain
C18542 _for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)).
The
transfer of the recombinant binary vector to Agrobacterium is accomplished by
a triparental
mating procedure using E. coli carrying the recombinant binary vector, a
helper E. coli strain
which carries a plasmid such as pRK2013 and which is able to mobilize the
recombinant
binary vector to the target Agrobacterium strain. Alternatively, the
recombinant binary
vector can be transferred to Agrobacterium by DNA transformation (Hofgen &
Willmitzer,
Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium
usually
involves co-cultivation of the Agrobacterium with explants from the plant and
follows
protocols well known in the art. Transformed tissue is regenerated on
selectable medium
carrying the antibiotic or herbicide resistance marker present between the
binary plasmid T-
DNA borders.
Another approach to transforming plant cells with a gene involves propelling
inert or
biologically active particles at plant tissues and cells. This technique is
disclosed in U.S.
Patent Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al.
Generally, this
procedure involves propelling inert or biologically active particles at the
cells under
conditions effective to penetrate the outer surface of the cell and afford
incorporation within
the interior thereof. When inert particles are utilized, the vector can be
introduced into the
cell by coating the particles with the vector containing the desired gene.
Alternatively, the
target cell can be surrounded by the vector so that the vector is carried into
the cell by the
wake of the particle. Biologically active particles (e.g., dried yeast cells,
dried bacterium or a
bacteriophage, each containing DNA sought to be introduced) can also be
propelled into
plant cell tissue.
2. Transformation of Monocotyledons
Transformation of most monocotyledon species has now also become routine.
Preferred techniques include direct gene transfer into protoplasts using PEG
or
electroporation techniques, and particle bombardment into callus tissue.
Transformations
can be undertaken with a single DNA species or multiple DNA species (i.e. co-
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transformation) and both these techniques are suitable for use with this
invention. Co-
transformation may have the advantage of avoiding complete vector construction
and of
generating transgenic plants with unlinked loci for the gene of interest and
the selectable
marker, enabling the removal of the selectable marker in subsequent
generations, should
this be regarded desirable. However, a disadvantage of the use of co-
transformation is the
less than 100% frequency with which separate DNA species are integrated into
the genome
(Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe
techniques for the preparation of callus and protoplasts from an elite inbred
line of maize,
transformation of protoplasts using PEG or electroporation, and the
regeneration of maize
plants from transformed protoplasts. Gordon-Kamm ef al. (Plant Cell 2: 603-618
(1990))
and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques
for
transformation of A188-derived maize line using particle bombardment.
Furthermore,
WO 93/07278 and Koziel et al. {Biotechnology 11: 194-200 (1993)) describe
techniques for
the transformation of elite inbred lines of maize by particle bombardment.
This technique
utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear
14-15 days
' after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer
techniques
utilizing protoplasts or particle bombardment. Protoplast-mediated
transformation has been
described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7:
379-384
(1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al.
Biotechnology 8: 736-740
(1990)). Both types are also routinely transformable using particle
bombardment (Christou
et al. Biotechnology 9_: 957-962 (199i)). Furthermore, WO 93121335 describes
techniques
for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the generation,
transformation and regeneration of Pooideae protoplasts. These techniques
allow the
transformation of Dactylis and wheat. Furthermore, wheat transformation has
been
described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle
bombardment
into cells of type C long-term regenerable callus, and also by Vasil et al.
(Biotechnology 11:
1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993))
using particle
bombardment of immature embryos and immature embryo-derived callus. A
preferred
technique for wheat transformation, however, involves the transformation of
wheat by
particle bombardment of immature embryos and includes either a high sucrose or
a high
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maltose step prior to gene delivery. Prior to bombardment, any number of
embryos (0.75-1
mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog,
Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/I 2,4-D for induction of
somatic
embryos, which is allowed to proceed in the dark. On the chosen day of
bombardment,
embryos are removed from the induction medium and placed onto the osmoticum
(Le.
induction medium with sucrose or maltose added at the desired concentration,
typically
15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded.
Twenty
embryos per target plate is typical, although not critical. An appropriate
gene-carrying
plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold
particles
using standard procedures. Each plate of embryos is shot with the DuPont
Biolistics0
helium device using a burst pressure of -1000 psi using a standard 80 mesh
screen. After
bombardment, the embryos are placed back into the dark to recover for about 24
h (still on
osmoticum). After 24 hrs, the embryos are removed from the osmoticum and
placed back
onto induction medium where they stay for about a month before regeneration.
Approximately one month later the embryo explants with developing embryogenic
callus are
transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA),
further containing
' the appropriate selection agent (10 mg/I basta in the case of pCIB3064 and 2
mg/I
methotrexate in the case of pSOG35). After approximately one month, developed
shoots
are transferred to larger sterile containers known as "GA7s" which contain
half-strength MS,
2% sucrose, and the same concentration of selection agent.
Tranformation of monocotyledons using Agrobacterium has also been described.
See, WO 94/00977 and U.S. Patent No. 5,591,616, both of which are incorporated
herein
by reference.
3. Transformation of Plastids
Seeds of Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in
a 1"
circular array on T agar medium and bombarded 12-14 days after sowing with 1
irtn
tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids
pPH143 and
pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-
9i7).
Bombarded seedlings are incubated on T medium for two days after which leaves
are
excised and placed abaxial side up in bright light (350-500 Nmol photons/m2/s)
on plates of
RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8528-
8530)
containing 500 Ng/ml spectinomycin dihydrochloride (Sigma, St. Louis, MO).
Resistant
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shoots appearing underneath the bleached leaves three to eight weeks after
bombardment
are subcloned onto the same selective medium, allowed to form callus, and
secondary
shoots isolated and subcloned. Complete segregation of transformed plastid
genome
copies (homoplasmicity) in independent subclones is assessed by standard
techniques of
Southern blotting (Sambrook et al., (1989) Molecular Clo- nina: A Laboratory
Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor). BamHl/EcoRl-digested total
cellular DNA
(Mettler, I. J. (1987) Plant Mol Biol Reporter 5, 346-349) is separated on 1 %
Tris-borate
(TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with
32P-
labeled random primed DNA sequences corresponding to a 0.7 kb BamHl/Hindlll
DNA
fragment from pC8 containing a portion of the rps7/12 plastid targeting
sequence.
Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA
medium
(McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the
greenhouse.
E. Breeding and Seed Production
Example 26: Breeding
The plants obtained via tranformation with a nucleic acid sequence of the
present
invention can be any of a wide variety of plant species, including those of
monocots and
dicots; however, the plants used in the method of the invention are preferably
selected from
the list of agronomically important target crops set forth supra. The
expression of a gene of
the present invention in combination with other characteristics important for
production and
quality can be incorporated into plant lines through breeding. Breeding
approaches and
techniques are known in the art. See, for example, Welsh J. R., Fundamentals
of Planf
Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R.
(Ed.)
American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of
Planf
Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D.P.,
Breeding for
Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and
Wricke and
Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter
and Co.,
Berlin (1986).
The genetic properties engineered into the transgenic seeds and plants
described
above are passed on by sexual reproduction or vegetative growth and can thus
be
maintained and propagated in progeny plants. Generally said maintenance and
propagation
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make use of known agricultural methods developed to fit specific purposes such
as tilling,
sowing or harvesting. Specialized processes such as hydroponics or greenhouse
technologies can also be applied. As the growing crop is vulnerable to attack
and damages
caused by insects or infections as well as to competition by weed plants,
measures are
undertaken to control weeds, plant diseases, insects, nematodes, and other
adverse
conditions to improve yield. These include mechanical measures such a tillage
of the soil or
removal of weeds and infected plants, as well as the application of
agrochemicals such as
herbicides, fungicides, gametocides, nematicides, growth regulants, ripening
agents and
insecticides.
Use of the advantageous genetic properties of the transgenic plants and seeds
according to the invention can further be made in plant breeding, which aims
at the
development of plants with improved properties such as tolerance of pests,
herbicides, or
stress, improved nutritional value, increased yield, or improved structure
causing less loss
from lodging or shattering. The various breeding steps are characterized by
well-defined
human intervention such as selecting the lines to be crossed, directing
pollination of the
parental lines, or selecting appropriate progeny plants. Depending on the
desired
properties, different breeding measures are taken. The relevant techniques are
well known
in the art and include but are not limited to hybridization, inbreeding,
backcross breeding,
multiline breeding, variety blend, interspecific hybridization, aneuploid
techniques, etc.
Hybridization techniques also include the sterilization of plants to yield
male or female
sterile plants by mechanical, chemical, or biochemical means. Cross
pollination of a male
sterile plant with pollen of a different line assures that the genome of the
male sterile but
female fertile plant will uniformly obtain properties of both parental lines.
Thus, the
transgenic seeds and plants according to the invention can be used for the
breeding of
improved plant lines, that for example, increase the effectiveness of
conventional methods
such as herbicide or pestidice treatment or allow one to dispense with said
methods due to
their modified genetic properties. Alternatively new crops with improved
stress tolerance can
be obtained, which, due to their optimized genetic "equipment, yield harvested
product of
better quality than products that were not able to tolerate comparable adverse
developmental conditions.
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Example 27: Seed Production
In seed production, germination quality and uniformity of seeds are essential
product
characteristics, whereas germination quality and uniformity of seeds harvested
and sold by
the farmer is not important. As it is difficult to keep a crop free from other
crop and weed
seeds, to control seedbome diseases, and to produce seed with good
germination, fairly
extensive and well-defined seed production practices have been developed by
seed
producers, who are experienced in the art of growing, conditioning and
marketing of pure
seed. Thus, it is common practice for the farmer to buy certified seed meeting
specific
quality standards instead of using seed harvested from his own crop.
Propagation material
to be used as seeds is customarily treated with a protectant coating
comprising herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides, or
mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan,
carboxin,
thiram (TMTD~), methalaxyl (Apron~), and pirimiphos-methyl (Actellic~). If
desired, these
compounds are formulated together with further carriers, surfactants or
application-
promoting adjuvants customarily employed in the art of formulation to provide
protection
against damage caused by bacterial, fungal or animal pests. The protectant
coatings may
be applied by impregnating propagation material with a liquid formulation or
by coating with
a combined wet or dry formulation. Other methods of application are also
possible such as
treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural
methods,
such as the methods examplified above, which are characterized by the use of
transgenic
plants, transgenic plant material, or transgenic seed according to the present
invention.
The seeds may be provided in a bag, container or vessel comprised of a
suitable
packaging material, the bag or container capable of being closed to contain
seeds. The
bag, container or vessel may be designed for either short term or long term
storage, or both,
of the seed. Examples of a suitable packaging material include paper, such as
kraft paper,
rigid or pliable plastic or other polymeric material, glass or metal.
Desirably the bag,
container, or vessel is comprised of a plurality of layers of packaging
materials, of the same
or differing type. In one embodiment the bag, container or vessel is provided
so as to
exclude or limit water and moisture from contacting the seed. In one example,
the bag,
container or vessel is sealed, for example heat sealed, to prevent water or
moisture from
entering. In another embodiment water absorbent materials are placed between
or
adjacent to packaging material layers. 1n yet another embodiment the bag,
container or
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vessel, or packaging material of which it is comprised is treated to limit,
suppress or
prevent disease, contamination or other adverse affects of storage or
transport of the seed.
An example of such treatment is sterilization, for example by chemical means
or by
exposure to radiation. Comprised by the present invention is a commercial bag
comprising
seed of a transgenic plant comprising a gene of the present invention that is
expressed in
said transformed plant at higher levels than in a wild type plant, together
with a suitable
carrier, together with label instructions for the use thereof for conferring
broad spectrum
disease resistance to plants.
The above disclosed embodiments are illustrative. This disclosure of the
invention will
place one skilled in the art in possession of many variations of the
invention. All such
obvious and foreseeable variations are intended to be encompassed by the
appended
claims.
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SEQUENCE LISTING
<110> Novartis AG
<120> NOVEL INSECTICIDAL TOXINS FROM XENORHABDUS NEMATOPHILUS
AND NUCLEIC ACID SEQUENCES CODING THEREFOR
<130> 30472/A/CGC1992
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1/19
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aca ata gaa cct cat tca tct gtt gag gcg ata aga acc gat aca ccg 736
Thr Ile Giu Pro His Ser Ser Val Glu Ala Ile Arg Thr Asp Thr Pro
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att att cct gaa tca cga cca aat tac tat gtt get aat tct ggc ccg 784
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gcc tca tca gtc aga get gtt ttc tat tgg tcc cac tct ttt aca tca 832
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tat aac gat aca gac aaa cgt get ttt gtt acc ggc tac aat cta taa 976
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att tct aca cct aat tat gaa tgg gat atg tca tca ata ata aaa gat 1182
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tca ttt ttg tta ggg tta ttt tgg cca caa caa acc gac aat act tgg 1278
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aaa atg gaa cat gtg caa ttc atg cta gaa tcc tca cct ggc act caa 1422
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gac gaa aaa ttc aag tct ttt gat aac aaa aca aat tat caa att ctt 1518
Asp Glu Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu
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2/19
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ccc atg tat acc aat acg att atg tta caa gcc cct tat tgg aaa atg 1566
Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met
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ggt ata gag aga aaa gat gag atc aaa cta aca gat ata gaa gtt aat 1614
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gaa tta aaa gag ctg ata gga aaa tta tct acc agc gcc gat aaa tat 1662
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att cat gat gtc tat act cgt gaa tat gat aat gcg atg aac act tca 1710
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aca gca gca aat atc acc aat aat tta tta tct gta aga ggc tat tgt 1758
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360 365 370
tta tta cat ggt tta gaa tgt ctc gaa gtc att aac cat ata caa aat 1806
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375 380 385 390
aat agc ctt gag caa agt ttt tat cct aaa act atc agc tac tcc acc 1854
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gta ttc gat cgc cag aca aat aaa aca agg gtt caa gcc ctg aca gaa 1902
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gca ccc aga gtt gga ggc att aaa gtc aca ttt gca aac gat gca tct 2046
Ala Pro Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser
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tat acc ctc ggt aca gta act tca gaa gta aac tca att gaa ctg aat 2094
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cgc tat gcc agt aac tat aga aaa tat get gtc gat aac cac tat att 2238
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3/19
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gca ggc att gca gtt tca tac cat atg ata get gat aaa aaa tca 2331
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Phe Val Thr Gly Tyr Asn Leu
130 135
<210> 3
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<212> PRT
<213> Xenorhabdus nematophilus
4/19
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<400> 3
Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile
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Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp Ala Ile
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Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe
5C 55 60
Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln
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Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln Ala Asn Leu Lys Thr
85 90 95
Ile Glr_ Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly Lys Met
100 105 110
Glu His Val Gln Phe Met Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser
115 120 125
His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu
130 135 140
Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met
145 150 155 160
Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile
165 170 175
Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile Glu Val Asn Glu Leu
180 185 190
Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His
195 200 205
Asp Va'._ Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala
210 215 220
Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu
225 230 235 240
His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser
245 250 255
Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe
260 265 270
Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp
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Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn
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305 310 315 320
Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr
325 330 335
5/19
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Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile Glu Leu Asn Asp Ser
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Val Iie Thr Ser Leu Glu Val Trp Gly Asn Gly Ala Ile Asp Glu Ala
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Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr
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Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn His Tyr Ile Ser Gly
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Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly
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Ile Aia Val Ser Tyr His Met Ile Ala Asp Lys Lys Ser
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<210>
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<221> CDS
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ata gca aca gaa acc gac ctc caa aat aca cct get tca gca ccc tta 96
Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20 25 30
tca att att aat ttt agg gat atg aca ata gaa cct cat tca tct gtt 144
Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val
35 40 45
gag gcg ata aga acc gat aca ccg att att cct gaa tca cga cca aat 192
Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn
50 55 60
tac tat gtt get aat tct ggc ccg gcc tca tca gtc aga get gtt ttc 240
Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe
65 70 75 80
tat tgg tcc cac tct ttt aca tca gaa tgg ttt gaa tct tcc tct att 288
Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile
85 90 95
att gta aaa gca ggc gaa gac gga gtc tta cat tca ccg ggt aat tct 336
Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser
100 105 110
tta tat tac agc aag gtt gta att tat aac gat aca gac aaa cgt get 384
Leu Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala
115 120 125
ttt gtt acc ggc tac aat cta taa 408
Phe Val Thr Gly Tyr Asn Leu
130 135
6/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
<210>
<211> i35
<212> PRT
<213> Xenorhabdus nematophilus
<400> 5
Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn
1 5 10 15
Ile A'_a Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20 25 30
Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val
35 40 45
Glu A-ia Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn
5C 55 60
Tyr Ty= Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe
65 70 75 80
Tyr T=p Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile
85 90 95
Ile Va'_ Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser
100 105 110
Leu Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala
115 120 125
Phe Va_ Thr Gly Tyr Asn Leu
130 135
<210> 6
<211> 1290
<212> DNA
<213> Xenorhabdus us
nematophil
<220>
<221> CDS
<222> (1)..(1287)
<223> JHE-like of
orf2 pCIB9381
<400> 6
atg aat gaaccgatg aatactaat gaatcacaa gtttcagag ata 48
aat
Met Asn GluProMet AsnThrAsn GluSerGln ValSerGlu Ile
Asn
1 5 10 15
gta ccc atgaatgaa tctatatta gcagcacct tattcaatt tct 96
tca
Val Pro MetAsnGlu SerIleLeu AlaAlaPro TyrSerIle Ser
Ser
20 25 30
aca cc. tatgaatgg gatatgtca tcaataata aaagatgcc att 144
aat
Thr Pro TyrGluTrp AspMetSer SerIleIle LysAspAla Ile
Asn
35 40 45
att gg:. ataggcttt attcctggt ccgggctca gcaatatca ttt 192
ggt
Ile Gly IleGlyPhe IleProGly ProGlySer AlaIleSer Phe
Gly
5f 55 60
ttg tta ttattttgg ccacaacaa accgacaat acttgggag caa 240
ggg
Leu Leu LeuPheTrp ProGlnGln ThrAspAsn ThrTrpGlu Gln
Gly
7/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
65 70 75 80
att ctc caa aaa gta gaa caa atg atc gag caa gcc aat ctc aaa act 288
Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln Ala Asn Leu Lys Thr
85 90 95
att caa gga ata ttg aac ggc gat ata caa gaa att aaa ggc aaa atg 336
Ile Gln Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly Lys Met
100 105 110
gaa cat gtg caa ttc atg cta gaa tcc tca cct ggc act caa gaa agc 384
Glu His Val Gln Phe Met Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser
115 120 125
cat gac gca tac atg ttt ctg gcg aga tat ctg gtc agt ata gac gaa 432
His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu
130 135 140
aaa ttc aag tct ttt gat aac aaa aca aat tat caa att ctt ccc atg 480
Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met
145 150 155 160
tat acc aat acg att atg tta caa gcc cct tat tgg aaa atg ggt ata 528
Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile
165 170 175
gag aga aaa gat gag ata aaa cta aca gat ata gaa gtt aat gaa tta 576
Glu Arg Lys Asp Glu Iie Lys Leu Thr Asp Ile Glu Val Asn Glu Leu
180 185 190
aaa gag ctg ata gga aaa tta tct acc agc gcc gat aaa tat att cat 624
Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His
195 200 205
gat gtc tat act cgt gaa tat gat aat gcg atg aac act tca aca gca 672
Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala
210 215 220
gca aat atc acc aat aat tta tta tct gta aga ggc tat tgt tta tta 720
Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu
225 230 235 240
cat ggt tta gaa tgt ctc gaa gtc att aac cat ata caa aat aat agc 768
His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser
245 250 255
ctt gag caa agt ttt tat cct aaa act atc agc tac tcc acc gta ttc 816
Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe
260 265 270
gat cgc cag aca aat aaa aca agg gtt caa gcc ctg aca gaa gac gat 864
Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp
275 280 285
caa atg caa gag cca ttc aag cct get tta att aat ggg aag tac aac 912
Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn
290 295 300
aaa ata aaa tca ttg att ggg tat gta caa aga atc gga aac gca ccc 960
Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro
305 310 315 320
aga gtt gga ggc att aaa gtc aca ttt gca aac gat gca tct tat acc 1008
Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr
325 330 335
8/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
ctcggtaca gtaacttca gaagtaaac tcaattgaa ctgaatgac agc 1056
LeuGlyThr ValThrSer GluValAsn SerIleGlu LeuAsnAsp Ser
340 345 350
gttataacc agcctggaa gtatgggga aatggcget gttgatgag gca 1104
ValIleThr SerLeuGlu ValTrpGly AsnGlyAla ValAspGlu Ala
355 360 365
ttctttaca ttaagtgac ggacgtcaa tttaggctt ggccaacgc tat 1152
PhePheThr LeuSerAsp GlyArgGln PheArgLeu GlyGlnArg Tyr
370 375 380
gccag~aac tatagaaaa tatgetgtc gataaccac tatatttca gga 1200
AlaSerAsn TyrArgLys TyrAlaVal AspAsnHis TyrIleSer Gly
385 390 395 400
ttgtactta gccagtgat gaaccttca ttggcaggt caagcagca ggc 1248
LeuTy~Leu AlaSerAsp GluProSer LeuAlaGly GlnAlaAla Gly
405 410 415
attgcagtt tcataccat atgataget gataaaaaa tcatag 1290
IleAiaVal SerTyrHis MetIleAla AspLysLys Ser
420 425
<210>
7
<211> 9
42
<212> T
PR
<213> norhabdu s matophilus
xe ne
<400>
7
MetAsn AsnGluPro MetAsnThr AsnGluSerGln ValSer GluIle
1 5 10 15
ValPro SerMetAsn GluSerIle LeuAlaAlaPro TyrSer IleSer
20 25 30
ThrPro AsnTyrGlu TrpAspMet SerSerIleIle LysAsp AlaIle
35 40 45
IleGly GlyIleGly PheIlePro GlyProGlySer AlaIle SerPhe
50 55 60
LeuLeu GlyLeuPhe TrpProGln GlnThrAspAsn ThrTrp GluGln
65 70 75 80
IleLeu GlnLysVal GluGlnMet IleGluGlnAla AsnLeu LysThr
85 90 95
IleGin GlyIleLeu AsnGlyAsp IleGlnGluIle LysGly LysMet
100 105 110
GluHis ValGlnPhe MetLeuGlu SerSerProGly ThrGln GluSer
115 120 125
HisAsp AlaTyrMet PheLeuAla ArgTyrLeuVal SerIle AspGlu
13C 135 140
LysPhe LysSerPhe AspAsnLys ThrAsnTyrGln IleLeu ProMet
145 150 155 160
TyrThr AsnThrIle MetLeuGln AlaProTyrTrp LysMet GlyIle
165 170 175
9/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile Glu Val Asn Glu Leu
180 185 190
Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His
195 200 205
Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala
210 215 220
Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu
225 230 235 240
His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser
245 250 255
Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe
260 265 270
Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp
275 280 285
Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn
290 295 300
Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro
305 310 315 320
Arg Vai Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr
325 330 335
Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile Glu Leu Asn Asp Ser
340 345 350
Val Ile Thr Ser Leu Glu Val Trp Gly Asn Gly Ala Val Asp Glu Ala
355 360 365
Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr
370 375 380
Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn His Tyr Ile Ser Gly
385 390 395 400
Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly
405 410 415
Ile Ala Val Ser Tyr His Met Ile Ala Asp Lys Lys Ser
420 425
<210> 8
<211> 408
<212> DNA
<213> Xenorhabdus poinarii
<220>
<221> CDS
<222> (1)..(405)
<223> orfl of pCIB9354
<400> 8
atg atc aca atc aat atc agt ggt ggt aat gta aca att aat aac aat 48
Met Ile Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn
1 5 10 15
atc agt tca gta acg gat atc caa aaa ccc ctt gat gca gaa ccc ctc 96
/ 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
IleSe. SerValThr AspIleGln LysProLeuAsp AlaGlu ProLeu
20 25 30
tcagtc acgaattat agagatctg acaatagagccg cactca tctatt 144
SerVa- ThrAsnTyr ArgAspLeu ThrIleGluPro HisSer SerIle
35 40 45
caagca gacagaacg gacaccccc attattcctgaa acacgc cctgat 192
GlnAiG AspArgThr AspThrPro IleIleProGlu ThrArg ProAsp
5r 55 60
tattai atcgetaac tcaggccct gettcatcagtc aaaget gtgttt 240
TyrTy~ IleAlaAsn SerGlyPro AlaSerSerVal LysAla ValPhe
65 70 75 80
tattgc tcgcattcg tttacatcg gaatggttcgag tattca tctatc 288
TyrTry SerHisSer PheThrSer GluTrpPheGlu TyrSer SerIle
85 90 95
acgg~a aaagcagga gaagatgga atattaaaatea ccgagt aatget 336
ThrVa--LysAlaGly GluAspGly IleLeuLysSer ProSer AsnAla
100 105 110
gtatai tacagtaaa gtagtcatt tataatgataca gataag cggget 384
ValTy= TyrSerLys ValValIle TyrAsnAspThr AspLys ArgAla
115 120 125
tttgtc actggatat aacatgtaa 408
PheVal ThrGlyTyr AsnMet
13G 135
<210> 9
<211> i35
<212> PRT
<213> Xenorhabdus poinarii
<400> 9
Met Iie Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn
1 5 10 15
Ile Se. Ser Val Thr Asp Ile Gln Lys Pro Leu Asp Ala Glu Pro Leu
20 25 30
Ser Va'_ Thr Asn Tyr Arg Asp Leu Thr Ile Glu Pro His Ser Ser Ile
35 40 45
Gln Ala Asp Arg Thr Asp Thr Pro Ile Ile Pro Glu Thr Arg Pro Asp
5p 55 60
Tyr Ty~ Ile Ala Asn Ser Gly Pro Ala Sex Ser Val Lys Ala Val Phe
65 70 75 80
Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Tyr Ser Ser Ile
85 90 95
Thr Vai Lys Ala Gly Glu Asp Gly Ile Leu Lys Ser Pro Ser Asn Ala
100 105 110
Val Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala
115 120 125
Phe Va'_ Thr Gly Tyr Asn Met
13~ 135
11 / 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
<210> 10
<211> 1056
<212> DNA
<213> Xenorhabdus poinarii
<220>
<221> CDS
<222> (i)..(1053)
<223> JHL'-like orf2 of pCIB9354
<400> 10
atg aat aat agt cca atg aat gat cag tta tca aca gcg cct tat tca 48
Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser
1 5 10 15
att tcg aca ccc aat tat gaa tgg gat atg tca tca atc ata aaa gat 96
Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp
20 25 30
gcc att atc ggt ggc ata gga ttt att ccc gga cca ggc cct gca atc 144
Ala Ile Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Pro Ala Ile
35 40 45
tct ttt tta tta gga ctg ttc tgg cca caa cag aca gac aat acc tgg 192
Ser Phe Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp
50 55 60
gat caa atc ctc caa aaa atc gaa caa atg ata gaa gaa gcg aat tta 240
Asp Gln Ile Leu Gln Lys Ile Glu Gln Met Ile Glu Glu Ala Asn Leu
65 70 75 80
aaa acc att aaa ggt ata tta aat gga gat ata caa gaa att aaa gga 288
Lys Thr Ile Lys Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly
85 90 95
aaa atg gac cat gtg aaa tct atg cta gag aat tct cct ggc agc cag 336
Lys Met Asp His Val Lys Ser Met Leu Glu Asn Ser Pro Gly Ser Gln
100 105 110
gaa agc cat gat get tat atg ttt ctg gca agg ttt ttg gtc agt att 3B4
Glu Ser His Asp Ala Tyr Met Phe Leu Ala Arg Phe Leu Val Ser Ile
115 120 125
gat gaa aaa ttc aaa tct ttc gat gat aga aca aat tat caa att ctt 432
Asp Glu Lys Phe Lys Ser Phe Asp Asp Arg Thr Asn Tyr Gln Ile Leu
130 135 140
ccc atg tac acg aat aca att atg tta caa gcg cct tat tgg aaa atg 480
Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met
145 150 155 160
ggc atc gaa aag aaa gag gat atc ggt tta acc gat att gaa gtt ggt 528
Gly Ile Glu Lys Lys Glu Asp Ile Gly Leu Thr Asp Ile Glu Val Gly
165 170 175
gaa tta aaa gaa ctt atc gat aaa tta tat act aaa tca tat gat tat 576
Glu Leu Lys Glu Leu Ile Asp Lys Leu Tyr Thr Lys Ser Tyr Asp Tyr
180 185 190
atc aat aat acg tat aat cgt gaa tat aat aat gca atc aat acg tca 524
Ile Asn Asn Thr Tyr Asn Arg Glu Tyr Asn Asn Ala Ile Asn Thr Ser
195 200 205
acc gca gag agt atc acc aat aat tta ttg tct gtc aga gga tat tgt 672
12 / 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
ThrAlaGlu SerIleThr AsnAsnLeu LeuSerVal ArgGlyTyr Cys
210 215 220
ttattacat ggttgtgaa tgccttgaa gttattgcg catatacaa aac 720
LeuLeuHis GlyCysGlu CysLeuGlu ValIleAla HisIleGln Asn
225 230 235 240
aatagtctt gataaaggc ttctaccct aaaacgatc agctattcg agt 768
AsnSerLeu AspLysGly PheTyrPro LysThrIle SerTyrSer Ser
245 250 255
gttttcgat cgtcctaca aacaaaatg agaattcag gcgcttaca gaa 816
ValPheAsp ArgProThr AsnLysMet ArgIleGln AlaLeuThr Glu
260 265 270
gatgaccaa atgcaagaa ccgttcaaa ccttctttc gtcaatggt caa 864
AspAspGln MetGlnGlu ProPheLys ProSerPhe ValAsnGly Gln
275 280 285
tataataaa ataaaatca ttggagggt tatgtcaca aggatcggc aat 912
TyrAsnLys IleLysSer LeuGluGly TyrValThr ArgIleGly Asn
29G 295 300
gccccccga gtcggcgga attaaaatc acatttgaa aacaacgca tct 960
AlaProArg ValGlyGly IleLysIle ThrPheGlu AsnAsnAla Ser
305 310 315 320
tatactctt ggcactgta acttcagaa acaacctct attgaactc aat 1008
TyrThrLeu GlyThrVal ThrSerGlu ThrThrSer IleGluLeu Asn
325 330 335
gagagtgtt ataaccagc atagaagtg tggggagag tggtgccgt tga 1056
GluSerVal IleThrSer IleGluVal TrpGlyGlu TrpCysArg
340 345 350
<210> 11
<211> 351
<212> PRT
<213> Xenorhabdus poinarii
<400> 11
Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser
1 5 10 15
Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp
20 25 30
Ala Ile Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Pro Ala Ile
35 40 45
Ser Phe Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp
50 55 60
Asp Gln Ile Leu Gln Lys Ile Glu Gln Met Ile Glu Glu Ala Asn Leu
65 70 75 80
Lys Thr Ile Lys Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly
85 90 95
Lys Met Asp His Val Lys Ser Met Leu Glu Asn Ser Pro Gly Ser Gln
100 105 110
Glu Ser His Asp Ala Tyr Met Phe Leu Ala Arg Phe Leu Val Ser Ile
115 120 125
13 / 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
Asp Glu Lys Phe Lys Ser Phe Asp Asp Arg Thr Asn Tyr Gln Ile Leu
130 135 140
Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met
145 150 155 160
Gly Ile Glu Lys Lys Glu Asp Ile Gly Leu Thr Asp Ile Glu Val Gly
165 170 175
Glu Leu Lys Glu Leu Ile Asp Lys Leu Tyr Thr Lys Ser Tyr Asp Tyr
180 185 190
Ile Asn Asn Thr Tyr Asn Arg Glu Tyr Asn Asn Ala Ile Asn Thr Ser
195 200 205
Thr Ala Glu Ser Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys
210 215 220
Leu Leu His Gly Cys Glu Cys Leu Glu Val Ile Ala His Ile Gln Asn
225 230 235 240
Asn Ser Leu Asp Lys Gly Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Ser
245 250 255
Val Phe Asp Arg Pro Thr Asn Lys Met Arg Ile Gln Ala Leu Thr Glu
260 265 270
Asp Asp Gln Met Gln Glu Pro Phe Lys Pro Ser Phe Val Asn Gly Gln
275 280 285
Tyr Asn Lys Ile Lys Ser Leu Glu Gly Tyr Val Thr Arg Ile Gly Asn
290 295 300
Ala Pro Arg Val Gly Gly Ile Lys Ile Thr Phe Glu Asn Asn Ala Ser
305 310 315 320
Tyr Thr Leu Gly Thr Val Thr Ser Glu Thr Thr Ser Ile Glu Leu Asn
325 330 335
Glu Ser Val Ile Thr Ser Ile Glu Val Trp Gly Glu Trp Cys Arg
340 345 350
<210> 12
<211> 408
<212> DNA
<213> Photorhabdus luminescens
<220>
<221> CDS
<222> (1)..(405)
<223> orfl of pCIB9383-21
<400> 12
atg att aca atc aat atc act ggt gat aat gta aga gtt aat aac aat 48
Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn
1 5 10 15
ata gca aca gaa acc gac ctc caa aat aca cct get tca gca ccc tta 96
Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20 25 30
tca att att aat ttt agg gat atg aca ata gaa cct cat tca tct gtt 144
Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val
14/19
CA 02326067 2000-10-20
WO 99!54472 PCT/EP99102629
35 40 45
gaggcg ataagaacc gatacaccg attattcctgaa tcacga ccaaat 192
GluAla IleArgThr AspThrPro IleIleProGlu SerArg ProAsn
50 55 60
tactat gttgetaat tctggcccg gectcatcagtc agaget gttttc 240
TyrTyr ValAlaAsn SerGlyPro AlaSerSerVal ArgAla ValPhe
65 70 75 80
tattgg tcccactct tttacatca gaatggtttgaa tcttcc tctatt 288
TyrTrp SerHisSer PheThrSer GluTrpPheGlu SerSer SerIle
85 90 95
attgta aaagcaggc gaagacgga gtcttacattca ccgggt aattct 336
IleVa'~LysAlaGly GluAspGly ValLeuHisSer ProGly AsnSer
100 105 110
ttatat tacageaag gttgtaatt tataacgataea gacaaa cgtget 384
LeuTy~ TyrSerLys ValValIle TyrAsnAspThr AspLys ArgAla
115 120 125
tttgtt accggctac aatctataa 408
PheVal ThrGlyTyr AsnLeu
130 135
<210> 13
<211> 135
<212> PRT
<213> Photorhabdus luminescens
<400> 13
Met Iie Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn
1 5 10 15
Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20 25 30
Ser Iie Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val
35 40 45
Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn
50 55 60
Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe
65 70 75 80
Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile
85 90 95
Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser
100 105 110
Leu Ty= Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala
115 120 125
Phe Val Thr Gly Tyr Asn Leu
130 135
<210> 14
<211> 1320
<212> DNA
<213> Photorhabdus luminescens
15/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
<220>
<221> CDS
<222> (1)..(1317)
<223> JHE-like orf2 of pCIB9383-21
<400> 14
atg aat aat gaa ccg atg aat act aat gaa tca caa get tca gag ata 48
Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile
1 5 10 15
gta ccc tca atg aat gaa tct ata tta aat gaa tct ata tta aat gaa 96
Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu
20 25 30
tct ata tta gca gca cct tat tca att tct aca cct aat tat gaa tgg 144
Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser Thr Pro Asn Tyr Glu Trp
35 40 45
gat atg tca tca ata ata aaa gat gcc att att ggt ggt ata ggc ttt 192
Asp Met Ser Ser Ile Ile Lys Asp Ala Ile Ile Gly Gly Ile Gly Phe
50 55 60
att cct ggt ccg ggc tca gca ata tca ttt ttg tta ggg tta ttt tgg 240
Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe Leu Leu Gly Leu Phe Trp
65 70 75 80
cca caa caa acc gac aat act tgg gag caa att ctc caa aaa gta gaa 288
Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln Ile Leu Gln Lys Val Glu
85 90 95
caa atg atc gag caa gcc aat ctc aaa act att caa gga ata ttg aac 336
Gln Met Ile Glu Gln Ala Asn Leu Lys Thr Ile Gln Gly Ile Leu Asn
100 105 110
ggc gat ata caa gaa att aaa ggc aaa atg gaa cat gtg caa ttc atg 384
Gly Asp Ile Gln Glu Ile Lys Gly Lys Met Glu His Val Gln Phe Met
115 120 125
cta gaa tcc tca cct ggc act caa gaa agc cat gac gca tac atg ttt 432
Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser His Asp Ala Tyr Met Phe
130 135 140
ctg gcg aga tat ctg gtc agt ata gac gaa aaa ttc aag tct ttt gat 480
Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu Lys Phe Lys Ser Phe Asp
145 150 155 160
aac aaa aca aat tat caa att ctt ccc atg tat acc aat acg att atg 528
Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met Tyr Thr Asn Thr Ile Met
165 170 175
tta caa gcc cct tat tgg aaa atg ggt ata gag aga aaa gat gag ata 576
Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile Glu Arg Lys Asp Glu Ile
180 185 190
aaa cta aca gat ata gaa gtt aat gaa tta aaa gag ctg ata gga aaa 624
Lys Leu Thr Asp Ile Glu Val Asn Glu Leu Lys Glu Leu Ile Gly Lys
195 200 205
tta tct acc agc gcc gat aaa tat att cat gat gtc tat act cgt gaa 672
Leu Ser Thr Ser Ala Asp Lys Tyr Ile His Asp Val Tyr Thr Arg Glu
210 215 220
tat gat aat gcg atg aac act tca aca gca gca aat atc acc aat aat 720
Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala Ala Asn Ile Thr Asn Asn
16 / 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
225 230 235 240
tta tta tct gta aga ggc tat tgt tta tta cat ggt tta gaa tgt ctc 768
Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu His Gly Leu Glu Cys Leu
245 250 255
gaa gtc att aac cat ata caa aat aat agc ctt gag caa agt ttt tat 816
Glu Val Ile Asn His Ile Gln Asn Asn Ser Leu Glu Gln Ser Phe Tyr
260 265 270
cct aaa act atc agc tac tcc acc gta ttc gat cgc cag aca aat aaa 864
Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe Asp Arg Gln Thr Asn Lys
275 280 285
aca agg gtt caa gcc ctg aca gaa gac gat caa atg caa gag cca ttc 912
Thr Arg Val Gln Ala Leu Thr Glu Asp Asp Gln Met Gln Glu Pro Phe
290 295 300
aag cct get tta att aat ggg aag tac aac aaa ata aaa tca ttg att 960
Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn Lys Ile Lys Ser Leu Ile
305 310 315 320
ggg tat gta caa aga atc gga aac gca ccc aga gtt gga ggc att aaa 1008
Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro Arg Val Gly Gly Ile Lys
325 330 335
gtc aca ttt gca aac gat gca tct tat acc ctc ggt aca gta act tca 1056
Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr Leu Gly Thr Val Thr Ser
340 345 350
gaa gta aac tca att gaa ctg aat gac agc gtt ata acc agc ctg gaa 1104
Glu Val Asn Ser Ile Glu Leu Asn Asp Ser Val Ile Thr Ser Leu Glu
355 360 365
gta tgg gga aat ggc get gtt gat gag gca ttc ttt aca tta agt gac 1152
Val Trp Gly Asn Gly Ala Val Asp Glu Ala Phe Phe Thr Leu Ser Asp
370 375 380
gga cgt caa ttt agg ctt ggc caa cgc tat gcc agt aac tat aga aaa 1200
Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr Ala Ser Asn Tyr Arg Lys
385 390 395 400
tat get gtc gat aac cac tat att tca gga ttg tac tta gcc agt gat 1248
Tyr Ala Val Asp Asn His Tyr Ile Ser Gly Leu Tyr Leu Ala Ser Asp
405 410 415
gaa cct tca ttg gca ggt caa gca gca ggc att gca gtt tca tac cat 1296
Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly Ile Ala Val Ser Tyr His
420 425 430
atg ata get gat aaa aaa tca tag 1320
Met Ile Ala Asp Lys Lys Ser
435
<210> 15
<211> 439
<212> PRT
<213> Photorhabdus luminescens
<400> 15
Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile
1 5 10 15
Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu
17 / 19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
20 25 30
Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser Thr Pro Asn Tyr Glu Trp
35 40 45
Asp Met Ser Ser Ile Ile Lys Asp Ala Ile Ile Gly Gly Ile Gly Phe
50 55 60
Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe Leu Leu Gly Leu Phe Trp
65 70 75 80
Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln Ile Leu Gln Lys Val Glu
85 90 95
Gln Met Ile Glu Gln Ala Asn Leu Lys Thr Ile Gln Gly Ile Leu Asn
100 105 110
Gly Asp Ile Gln Glu Ile Lys Gly Lys Met Glu His Val Gln Phe Met
115 120 125
Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser His Asp Ala Tyr Met Phe
130 135 140
Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu Lys Phe Lys Ser Phe Asp
145 150 155 160
Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met Tyr Thr Asn Thr Ile Met
165 170 175
Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile Glu Arg Lys Asp Glu Ile
180 185 190
Lys Leu Thr Asp Ile Glu Val Asn Glu Leu Lys Glu Leu Ile Gly Lys
195 200 205
Leu Ser Thr Ser Ala Asp Lys Tyr Ile His Asp Val Tyr Thr Arg Glu
210 215 220
Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala Ala Asn Ile Thr Asn Asn
225 230 235 240
Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu His Gly Leu Glu Cys Leu
245 250 255
Glu Val Ile Asn His Ile Gln Asn Asn Ser Leu Glu Gln Ser Phe Tyr
260 265 270
Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe Asp Arg Gln Thr Asn Lys
275 280 285
Thr Arg Val Gln Ala Leu Thr Glu Asp Asp Gln Met Gln Glu Pro Phe
290 295 300
Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn Lys Ile Lys Ser Leu Ile
305 310 315 320
Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro Arg Val Gly Gly Ile Lys
325 330 335
Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr Leu Gly Thr Val Thr Ser
340 345 350
Glu Val Asn Ser Ile Glu Leu Asn Asp Ser Val Ile Thr Ser Leu Glu
355 360 365
18/19
CA 02326067 2000-10-20
WO 99/54472 PCT/EP99/02629
Val Trp Gly Asn Gly Ala Val Asp Glu Ala Phe Phe Thr Leu Ser Asp
370 375 380
Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr Ala Ser Asn Tyr Arg Lys
385 390 395 400
Tyr Ala Val Asp Asn His Tyr ile Ser Gly Leu Tyr Leu Ala Ser Asp
405 410 415
Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly Ile Ala Val Ser Tyr His
420 425 430
Met Ile Ala Asp Lys Lys Ser
435
19 / 19
