Note: Descriptions are shown in the official language in which they were submitted.
CA 02763879 2012-01-05
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CA 02763879 2012-01-05
NOVEL BACILLUS TIIURINGIENSIS CRYSTAL POLYPEPTIDES,
I'OLYNUCLEO'I'IDES, AND COMPOSITIONS THEREOF
David Cerf
Ruth Cong
Michael Freeman
Kevin McBride
'Takashi Yamamoto
I') 101)011
FIELD OF THE INVENTION
I i 100021 The present invention relates generally to the field of pest
control and provides
insecticidal polypeptides related to Bacillus thuringiensis Cry] polypeptides
and the
polynucleotides that encode them. The present invention also relates to
methods and
compositions for altering resistance of plants to insect predation including,
but not limited to,
transgenic plant production.
t)
BACKGROUND OF THE INVENTION
100031 Numerous commercially valuable plants, including common agricultural
crops,
are susceptible to attack by insect and nematode pests. These pests can cause
substantial
reductions in crop yield and quality. Traditionally, farmers have relied
heavily on chemical
pv,,ticidcs to combat pest damage. However, the use of chemical pesticides
raises its own set
ol'pri>blems, including the cost and inconvenience of applying the pesticides.
Furthermore,
::i>emical residues raise environmental and health concerns. For these and
other reasons there
is a demand for alternative insecticidal agents.
(0004) An environmentally friendly approach to controlling pests is the use of
pesticidal
C crystal proteins derived from the soil bacterium Bacillus thuringiensis
("Bt"), commonly
referred to as "Cry proteins." The Cry proteins are globular protein molecules
which
.>ccutnn>late as protoxins in crystalline form during late stage of the
sporulation of Bacillus
hwruvglensis. After ingestion by the pest, the crystals are solubilized to
release protoxins in
ilic alkaline inidut environment of the larvae. Protoxins (-130 kDa) are
converted into
mature toxic fragments (-66 kDa N terminal region) by gut proteases. Many of
these
I
CA 02763879 2012-01-05
proteins are quite toxic to specific target insects, but harmless to plants
and other non-
targeted organisms. Some Cry proteins have been recombinantly expressed in
crop plants to
provide pest-resistant transgenic plants. Among those, Bt-transgenic cotton
and corn have
been widely cultivated.
100051 A large number of Cry proteins have been isolated, characterized and
classified
based on amino acid sequence homology (Crickmore et al., 1998, Microbiol. Mol.
BioL Rev.,
62: 807-813). This classification scheme provides a systematic mechanism for
naming and
categorizing newly discovered Cry proteins. The Cry l classification is the
best known and
contains the highest number of cry genes which currently totals over 130.
[00061 It has generally been found that individual Cry proteins possess
relatively narrow
activity spectra. For example, CrylAc was the first toxin to be deployed in
transgenie cotton
for control of H. virescens and H. zea insect pests. This toxin is known for
its high level
toxicity to H. virescens. However, it is slightly deficient in its ability to
control H. zea and
has almost no activity on Spodoptera species. Additionally, CrylAb toxin has
slightly less
activity on H. zea than Cry I Ac but has far superior activity against S.
exigua.
(00071 Second generation transgenic crops could be more resistant to insects
if they are
able to express multiple and/or novel Bt genes. Accordingly, new insecticidal
proteins
having broad activity spectra would be highly desirable.
SUMMARY OF THE INVENTION
100081 The present invention relates to Cry polypeptides derived from Bacillus
thuringiensis Cryl polypeptides (e.g., CrylAa, CrylAb, CrylAc, CrylAd, CrylAe,
CrylA&
and CrylCa) including, but not limited to, the Cryl-derived polypeptides of
SEQ ID NOS:2,
4,6,8,10,12, 14, 16, 18, 20, 22, 24, 26, and 28. In addition to the
polypeptide sequence of
Cryl-derived polypeptides, it will be appreciated that polypeptides of the
invention also
encompass variants thereof, including, but not limited to, any fragment
including the gut
activated mature toxin fragment, analog, homolog, naturally occurring allele,
or mutant
thereof. Polypeptides of the invention also encompass those polypeptides that
are encoded by
any Cryl -derived nucleic acid of the invention. In one embodiment, shuffled
polypeptides
that have at least one Cryl functional activity (e.g., insecticidal activity)
and are at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 /., or 99.5% identical to the
mature toxin
portion of polypeptide sequence of any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22,
24, 26, 28, or variants thereof. In another embodiment, polypeptides that have
at least one
Cryl functional activity (e.g., insecticidal activity) and are at least 99% or
99.5% identical to
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CA 02763879 2012-01-05
the mature toxin portion of polypeptide sequence of any of SEQ ID NOS: 2, 4,
6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, or variants thereof. Methods of production of
the polypeptides
of the invention, e.g., by recombinant means, are also provided. Compositions
comprising
one or more polypeptides of the invention are also encompassed.
[00091 The present invention also relates to Cryl-derived nucleic acid
molecules of SEQ
ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. Also
encompassed by the present
invention are fragments and analogs which encode polypeptides that are at
least partially
functionally active, i.e., they are capable of displaying one or more known
functional
activities associated with a wild type Cryl polypeptide. In one embodiment, it
encompasses
an isolated shuffled nucleic acid molecule that is at least at least 90%,
910/0, 920/0, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 99.5% identical to any of SEQ ID NOS: 1, 3, 5, 7,
9, 11, 13,
15, 21, 23, 25, 27, or a compliment thereof. In another embodiment, it
encompasses
an isolated nucleic acid molecule that is are at least 99% or 99.5% identical
to the mature
toxin portion of polypeptide sequence of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11,
13, 15, 17,
19, 21, 23, 25, 27, or a compliment thereof. Vectors comprising nucleic acids
of the
invention are also encompassed. Cells or plants comprising the vectors of the
invention are
also encompassed.
[0010) The present invention also relates to transgenic plants expressing a
nucleic acid
and/or polypeptide of the invention. The transgenic plants can express the
transgene in any
way known in the art including, but not limited to, constitutive expression,
developmentally
regulated expression, tissue specific expression, etc. Seed obtained from a
transgenic plant of
the invention is also encompassed.
BRIEF DESCRIPTION OF THE FIGURES
[0011) FIG.1 shows insecticidal activity of variants isolated from single gene
shuffling
of CrylAb against Helicorverpa zea. Each of the purified protoxins was
introduced into the
diet of an insect and the EC 0 of each was determined. The EC50 values were
then converted
to relative inverse values. The ECso of wild type Cryl Ca against H. zea was
given a value of
1Ø The EC50 of the remaining protoxins were assigned a relative value.
[0012) FIG. 2 shows a comparison of relative activity of protoxin encoded by
shuffled
variant AR6 with that of wild type CrylAb, CrylAc, and CrylCa on Hellothis
virescens,
Hellcoverpa zea, and Spodoptera exigua. Each of the purified protoxins was
introduced into
the diet of an insect and the EC50 of each was determined. The EC50 values
were then
converted to relative inverse values. The protoxin showing the lowest EC50
(highest specific
3
CA 02763879 2012-01-05
activity) for each insect type was given a value of 1Ø The EC50 of the
remaining protoxins
were assigned a lower relative value.
100131 FIG. 3 shows the relative efficacy of Cry1Ca shuffled variants against
Spodoptera
exigua. Each of the purified protoxins was introduced into the diet of an
insect and the EC,o
of each was determined. The EC50 values were then converted to relative
inverse values.
The EC50 of wild type CrylCa against Spodoptera exigua was given a value of
1Ø The EC50
of the remaining protoxins were assigned a relative value.
100141 FIG. 4 shows the expression of synthetic AR6 (SEQ ID NO: 5), MR8' (SEQ
ID
NO: 11, and CR62 (SEQ ID NO: 9) genes in a transient leaf assay. The synthetic
genes were
expressed in Nicotiana benthanniana leaves using an Agrobacterium leaf
infiltration assay. A
western blot of resulting leaf extracts demonstrates the production of
protoxin from the AR6,
MR8', and CR62 synthetic genes. Lanes are as follows: molecular weight marker,
100 ng
CrylCa protoxin standard, 200 ng CrylCa protoxin standard, extract from leaf
expressing
synthetic MR8', extract from leaf expressing synthetic AR6, extract from leaf
expressing
synthetic CR62. A rabbit polyclonal antiserum raised against purified CrylCa
protein was
used to probe the western blot (it had been predetermined that the CrylCa
polyclonal
antiserum cross-reacts strongly to AR6, CR62, and MRS' proteins).
100151 FIGS. 5A-5B show in planta insecticidal activity of synthetic AR6,
MR8', and
CR62 genes. Each variant was expressed in N. bentbamtana using Agrobacterium
infiltration. Each leaf disk was fed to (A) H. zea or (B) S exigua. Following
a 24-hour
incubation period, the feeding activity was determined by visual observation.
Positive
controls for H. zea activity and S. exigua activity were a Cry2Ab-like
polypeptide (SEQ ID
NO: 35) and CrylCa shuffled gene CR62, respectively. The ratio shown for each
panel
refers to the relative amount of test Agrobacterium containing the gene of
interest to
Agrobacterium not containing a test gene. This dilution effectively reduces
the level of test
protein produced It should be noted that negative control leaves infiltrated
with
Agrobacterium not containing a test gene were completely consumed by the
insect larvae
during the assay period (not shown). .
[0016j FIG. 6 shows in planta activity of MR8' shuffled variants against H.
zea. The
indicated variant was expressed in N. benthamiana leaves using Agrobacterium
infiltration
followed by a four day co-cultivation period. Each resulting leaf disk was fed
to H. zea.
Following a 24-hour incubation period, the feeding activity was determined by
video capture
of the leaf disk. The y-axis is the number of pixels present in the captured
leaf disk image.
The greater the number of pixels, the greater the amount of uneaten
(protected) leaf
4
CA 02763879 2012-01-05
remaining. The x-axis is the variant tested. The assay was repeated two to
four times as
indicated for each variant.
100171 FIG. 7 shows in planta activity of MR8' shuffled variants against S.
exigua. The
indicated variant was expressed in N. benthamiana leaves using Agrobacterium
infiltration
followed by a four day co-cultivation period. Each resulting leaf disk was fed
to S. erigua.
Following a 24-hour incubation period, the feeding activity was determined by
video capture
of the leaf disk. The y-axis is the number of pixels present in the captured
leaf disk image.
The greater the number of pixels, the greater the amount of uneaten
(protected) leaf
remaining. The x-axis is the variant tested. The experiment was repeated 3
times.
DETAILED DESCRIPTION
[00181 The present invention provides insecticidal polypeptides related to
Bacillus Cryl
polypeptides (e.g., CrylAa, CrylAb, CrylAc, CrylAd, CrylAe, CrylAg, and
CrylCa).
Nucleic acid molecules encoding the polypeptides of the invention are also
provided.
Methods for using the polypeptides and nucleic acids of the invention to
enhance resistance
of plants to insect predation are encompassed.
Poles tides of the Invention
[00191 The present invention relates to Cry polypeptides derived from Bacillus
thuringiensis Cryl polypeptides (e.g., CrylAa, CrylAb, CrylAc, CrylAd, CrylAe,
CrylAg,
and Cryl Ca). In preferred embodiments, the Cryl -derived polypeptides
represent the mature
6-endotoxin region and are selected from the group consisting of SEQ ID NOS:
2, 4, 6, 8, 10,
12, 14,16, 18, 20, 22, 24, 26, 28. Polypeptides of the invention also
encompass those
polypeptides that are encoded by any Cryl -derived nucleic acid of the
invention.
[00201 In addition to the polypeptide sequence of Cryl-derived polypeptides,
it will be
appreciated that polypeptides of the invention also encompass variants
thereof, including, but
not limited to, any substantially similar sequence, any fragment, analog,
homolog, naturally
occurring allele, or mutant thereof. Variants encompassed by the invention are
polypeptides
that are at least partially functionally active, i.e., they are capable of
displaying one or more
known functional activities associated with a wild type Cryl polypeptide. Such
functional
activities include, but are not limited to, biological activities, such as
insecticidal activity,
antigenicity, i.e., an ability to bind or compete with a wild type Cryl for
binding to an anti-
Cry l antibody, immunogenicity, i. e., an ability to generate antibody which
binds to a wild
5
CA 02763879 2012-01-05
type Cryl polypeptide. In some embodiments, the variants have at least one
functional
activity that is substantially similar to its parent polypeptide (e.g., a
variant of Cryl -derived
polypeptide will have at least one functional activity that is substantially
similar to the Cryl -
derived polypeptide to which it is most similar). As used herein, the
functional activity of the
variant will be considered "substantially similar" to its parent polypeptide
if it is within one
standard deviation of the parent.
[0021[ In one embodiment, shuffled mature S-endotoxin polypeptides that have
at least
one Cryl functional activity (e.g., insecticidal activity) and are at least at
least 90%, 91 %,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the polypeptide
sequence
of any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 are
encompassed by
the invention.
[0022) As used herein, where a sequence is defined as being "at least X%
identical" to a
reference sequence, e.g., "a polypeptide at least 95% identical to SEQ ID NO:
2," it is to be
understood that "X% identical" refers to absolute percent identity, unless
otherwise indicated.
The term "absolute percent identity" refers to a percentage of sequence
identity determined
by scoring identical amino acids or nucleic acids as one and any substitution
as zero,
regardless of the similarity of mismatched amino acids or nucleic acids. In a
typical sequence
alignment the "absolute percent identity" of two sequences is presented as a
percentage of
amino acid or nucleic acid "identities." In cases where an optimal alignment
of two
sequences requires the insertion of a gap in one or both of the sequences, an
amino acid
residue in one sequence that aligns with a gap in the other sequence is
counted as a mismatch
for purposes of determining percent identity. Gaps can be internal or
external, i.e., a
truncation. Absolute percent identity can be readily determined using, for
example, the
Clustal W program, version 1.8, June 1999, using default parameters (Thompson
et al., 1994,
Nucleic Acids Research 22: 4673-4680).
[00231 In another embodiment, mature S-endotoxin polypeptides that have at
least one
Cryl functional activity (e.g., insecticidal activity), are at least 99 x6 or
99.5% identical to the
polypeptide sequence of any of SEQ ID NOS: 2,4,6, 8, 10,12, 14, 16, 18,
20,22,24,26,28
and are encoded by a polynucleotide that hybridizes under stringent conditions
to a nucleic
acid that encodes any of SEQ ID NOS: 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28.
[00241 In a specific embodiment, a fragment of the invention corresponds to
the length of
the processed pro-toxin. The toxin corresponds to the N-terminal portion of
the full length
Cryl polypeptide. In preferred embodiments, the N-terminal -50kDa-75kDa
fragment
corresponds to the toxin. In more preferred embodiments, the N-terminal -66kDa
fragment
6
CA 02763879 2012-01-05
corresponds to the toxin. Polypeptides that correspond to this processed Cryl
fragment can
be provided in the methods of the present invention directly to circumvent the
need for pro-
toxin processing.
[0025) The full protoxin nucleotide or polypeptide sequences are made up of
the domain
1, II, and III toxin regions in the context of the protoxin 5' or N-terminal
and 3' or C-terminal
protoxin regions. In some cases the protoxin and toxin regions are derived
from the same
Cryl-type molecule, such as CR62 being fully derived from Cry 1Ca. In other
cases the 5' or
N-terminal region is derived primarily from one molecule while the C-terminal
protoxin
region is derived from another such as with AR6, MR8' and derivatives in which
the 5' or N-
terminal region is predominantly derived from Cryl Ab while the 3' or C-
terminal region
corresponding to the protoxin region is from CrylCa. It is recognized that the
active 8-
endotoxin region of the molecules could retain the exact activity in the
context of a different
set of protoxin sequences derived from other Cryl molecules.
[0026) In another specific embodiment, a fragment of the invention corresponds
to a
Cryl domain. Mature Cryl toxin polypeptides have three domains including i)
domain I
which is involved in insertion into the insect apical midgut membrane and
affects ion channel
function, ii) domain II which is involved in receptor binding on the insect
midgut epithelial
cell membrane, and iii) domain III which is involved in ion channel function,
receptor
binding, and insertion into the membrane (Schnepf eta!., 1998, MicrobioL Moles
Biol. Rev.
62:775-806).
100271 In another embodiment, analog polypeptides are encompassed by the
invention.
Analog polypeptides may possess residues that have been modified, Le., by the
covalent
attachment of any type of molecule to the Cryl -derived polypeptides. For
example, but not
by way of limitation, an analog polypeptide of the invention may be modified,
e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known
protectingiblocking groups, proteolytic cleavage, linkage to a cellular ligand
or other protein,
etc. An analog polypeptide of the invention may be modified by chemical
modifications
using techniques known to those of skill in the art, including, but not
limited to specific
chemical cleavage, acetylation, formylation, synthesis in the presence of
tunicamycin (an
inhibitor of N-linked glycosylation and the formation of N-glycosidic protein-
carbohydrate
linkages), etc. Furthermore, an analog of a polypeptide of the invention may
contain one or
more non-classical amino acids.
[0028) Methods of production of the polypeptides of the invention, e.g., by
recombinant
means, are also provided.
7
CA 02763879 2012-01-05
100291 Compositions comprising one or more polypeptides of the invention are
also
encompassed. The compositions of the invention can further comprise additional
agents
including, but not limited to, spreader-sticker adjuvants, stabilizing agents,
other insecticidal
additives, diluents, agents that optimize the theological properties or
stability of the
composition, such as, for example, surfactants, emulsifiers, dispersants,
and/or polymers.
Nucleic Acids of the Invention
100301 The present invention also relates to Cryi-derived nucleic acid
molecules. In
preferred embodiments, the Cryl-derived nucleic acid molecules are selected
from the group
consisting of SEQ ID NOS:I, 3, 5, 7, 9, 11, 13,15,17, 19, 21, 23, 25, and 27:
Nucleic acid
molecules of the invention also encompass those nucleic acid molecules that
encode any
Cryl-derived polypeptide of the invention.
[00311 In addition to the nucleic acid molecule of Cryl -derived nucleic acid
molecules, it
will be appreciated that nucleic acids of the invention also encompass
variants thereof,
including, but not limited to any substantially similar sequence, any fragment
including the
toxin fragment, homolog, naturally occurring allele, or mutant thereof.
Variant nucleic acid
molecules encompassed by the present invention encode polypeptides that are at
least
partially functionally active, i.e., they are capable of displaying one or
more known
functional activities associated with a wild type Cryl polypeptide. Such
functional activities
include, but are not limited to, biological activities, such as insecticidal
activity; antigenicity,
i.e., an ability to bind or compete with a wild type Cryl for binding to an
anti-Cryl antibody,
immunogenicity, i.e., an ability to generate antibody which binds to a wild
type Cryl
polypeptide. In some embodiments, the variants have at least one functional
activity that is
substantially similar to its parent nucleic acid molecule (e.g., a variant of
a Cryl-derived
nucleic acid molecule will encode a polypeptide that has at least one
functional activity that is
substantially similar to the polypeptide encoded for by the Cry 1-derived
nucleic acid
molecule to which it most similar). As used herein, the functional activity of
the variant will
be considered "substantially similar" to its parent polypeptide if it is
within one standard
deviation of the parent.
100321 In one embodiment, shuffled nucleic acid molecules that are at least
90%, 918/6,
92%, 93%, 94%, 95%, 96%, 97%, 98 /., 99%, or 99.5% identical to any of the
nucleic acid
molecules of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 are
encompassed
by the invention. In another embodiment, nucleic acid molecules that are at
least 9996 or
8
CA 02763879 2012-01-05
99.5% identical to any of the nucleic acid molecules of SEQ ID NOS: 1, 3, 5,
7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27 are encompassed by the invention.
(0033) To determine the percent identity of two nucleic acid molecules, the
sequences are
aligned for optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a
first nucleic acid molecule for optimal alignment with a second or nucleic
acid molecule).
The nucleotides at corresponding nucleotide positions are then compared. When
a position in
the first sequence is occupied by the same nucleotide as the corresponding
position in the
second sequence, then the molecules are identical at that position. The
percent identity
between the two sequences is a function of the number of identical positions
shared by the
sequences (L e., % identity s number of identical overlapping positions/total
number of
positions x 100%). In one embodiment, the two sequences are the same length.
(0034( The determination of percent identity between two sequences can also be
accomplished using a mathematical algorithm. A non-limiting example of a
mathematical
algorithm utilized for the comparison of two sequences is the algorithm of
Karlin and
Altschul (Karlin and Altschul, 1990, Prof. Natl. Acad ScL 87:2264-2268,
modified as in
Karlin and Altschul, 1993, Pros Natl. Acad Sd. 90:5873-5877). Such an
algorithm is
incorporated into the NBLAST and XBLAST programs (Altschul et al., 1990, J.
Mod Biol.
215:403 and Altschul et al., 1997, Nucleic Add Res. 25:3389-3402). Software
for
performing BLAST analyses is publicly available, e.g., through the National
Center for
Biotechnology Information. This algorithm involves first identifying high
scoring sequence
pairs (HSPs) by identifying short words of length Win the query sequence,
which either
match or satisfy some positive-valued threshold score T when aligned with a
word of the
same length in a database sequence. T is referred to as the neighborhood word
score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or
more negative-
scoring residue alignments; or the end of either sequence is reached. The
BLAST algorithm
parameters W, T, and X determine the sensitivity and speed of the alignment.
The BLASTN
9
CA 02763879 2012-01-05
program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation
(E) of 10, a cutoff of 100, M = 5, N -"4, and a comparison of both strands.
For amino acid
sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an
expectation (E)
of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, PNAS,
89:10915).
[0035) The Clustal V method of alignment can also be used to determine percent
identity
(Higgins and Sharp, 1989, CABIOS. 5:151-153) and found in the Megalign program
of the
LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). The
"default
parameters" are the parameters pro-set by the manufacturer of the program and
for multiple
alignments they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10,
while for pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW-5 and
DIAGONALS SAVED=5. After alignment of the sequences, using the Clustal V
program, it
is possible to obtain a "percent identity" by viewing the "sequence distances"
table on the
same program.
[0036) The percent identity between two sequences can be determined using
techniques
similar to those described above, with or without allowing gaps. In
calculating percent
identity, typically only exact matches are counted.
100371 In another embodiment, nucleic acid molecules incorporating any of the
herein-
described nucleic acid molecules of Cryi-derived nucleic acid molecules are
encompassed by
the invention. Nucleic acid molecules are encompassed that have at least one
Cry l functional
activity (e.g., insecticidal activity). In this regard, the described
sequences encoding the toxin
may be combined with domains from other Cry proteins to form the complete Cry
protein.
[0038) In a specific embodiment, the combination corresponds to a nucleic acid
molecule
that encodes a complete Cry protein. The toxin corresponds to the N-terminal
portion of the
full length Cryl polypeptide. Nucleic acid molecules encoding domain I and
nucleic acid
molecules encoding domain II may then be combined with the described nucleic
acid
molecules to form a nucleic acid molecule encoding a mature Cry protein.
100391 In another specific embodiment, a fragment of the invention encodes a
polypeptide that corresponds to any of domains 1,11 or III of a mature Cryl
toxin.
100401 In another embodiment, a nucleic acid molecule that hybridizes under
stringent
conditions to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27 is
encompassed by the invention. The phrase "stringent conditions" refers to
hybridization
conditions under which a nucleic acid will hybridize to its target nucleic
acid, typically in a
complex mixture of nucleic acid, but to essentially no other nucleic acids.
Stringent
CA 02763879 2012-01-05
conditions are sequence-dependent and will be different in different
circumstances. Longer
nucleic acids hybridize specifically at higher temperatures. Extensive guides
to the
hybridization of nucleic acids can be found in the art (e.g., Tijssen,
Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Probes.
"Overview of
principles of hybridization and the strategy of nucleic acid assays" (1993)).
Generally, highly
stringent conditions are selected to be about 5-10 C lower than the thermal
melting point
(Tm) for the specific nucleic acid at a defined ionic strength and pH. Low
stringency
conditions are generally selected to be about 15-30 C below the Tm. The T. is
the
temperature (under defined ionic strength, pH, and nucleic acid concentration)
at which 50%
of the probes complementary to the target hybridize to the target nucleic acid
at equilibrium
(as the target nucleic acids are present in excess, at Tm, 5(% of the probes
are occupied at
equilibrium). Hybridization conditions are typically those in which the salt
concentration is
less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion
concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for
short probes (e.g.,
10 to 50 nucleotides) and at least about 60 C for long probes (e.g., greater
than 50
nucleotides). Stringent conditions may also be achieved with the addition of
destabilizing
agents such as formamide. For selective or specific hybridization, a positive
signal is at least
two times background, and preferably 10 times background hybridization. In one
embodiment, stringent conditions include at least one wash (usually 2) in 0.2X
SSC at a
temperature of at least about 50 C, usually about 55 C, or sometimes 60 C
or 65 C. for 20
minutes, or substantially equivalent conditions. In a specific embodiment, the
nucleic acid
molecule of the invention specifically hybridizes following at least one wash
in 0.2X SSC at
55 C for 20 minutes to a polynucleotide encoding the polypeptide of any of
SEQ ID NOS:2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28. In another embodiment,
stringent conditions
include hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45
C followed by
one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C.
(00411 The phrase "specifically hybridizes" refers to the binding, duple ring,
or
hybridizing of a molecule only to a particular nucleotide sequence under
stringent
hybridization conditions when that sequence is present in a complex mixture
(e.g., total
cellular or library DNA or RNA).
(00421 Vectors comprising nucleic acids of the invention are also encompassed.
Cells or
plants comprising the vectors of the invention are also encompassed.
(00431 The term "nucleic acid" or "nucleic acid molecule" herein refer to a
single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read
from the 5' to
11
CA 02763879 2012-01-05
the 3' end. It includes chromosomal DNA, self-replicating plasmids and DNA or
RNA that
performs a primarily structural role. The term "encoding" refers to a
polynucleotide sequence
encoding one or more amino acids. The term does not require a start or stop
codon. An
amino acid sequence can be encoded in any one of six different reading frames
provided by a
polynucleotide sequence and its complement.
100441 Table I discloses Cry l-derived sequences and the corresponding
sequence
identity number.
Crvl -Derived Sequences
100451 Cry l-derived polypeptides and nucleic acid molecules of the invention
can be
created by introducing one or more nucleotide substitutions, additions and/or
deletions into
the nucleotide sequence of a wild type Cryl (e.g., CrylAa, CrylAb, CrylAc,
CrylAd,
CrylAe, CrylAg, and Cry!Ca) or related nucleic acids, such that one or more
amino acid
substitutions, additions and/or deletions are introduced into the encoded
protein. Generally,
Cryl -derived sequences are created in order to accentuate a desirable
characteristic or reduce
an undesirable characteristic of a wild type Cryl polypeptide. In one
embodiment, Cryl-
derived polypeptides have improved insecticidal activity over the
corresponding wild type
Cryl including, but not limited to, greater potency and/or increased insect
pest range. In
another embodiment, Cryl-derived polypeptides are expressed better than the
corresponding
wild type Cryl in a microbial host or a plant host including, but not limited
to, increased half
life, less susceptible to degradation, and/or more efficient transcription or
translation.
[00461 In one embodiment, Bacillus thuringiensis derived CrylAb (SEQ ID NO:
33) or
Cry I Ca (SEQ ID NO: 29,, coding region: 47-3616) nucleic acid molecules were
used as a
templates to create shuffled cryl nucleotide fragments. In another embodiment,
variants
isolated from one round of alteration can be used as template for further
rounds of alteration
(e.g., AR6, CR62, or MR8'). In another embodiment, templates encoding Cryl
proteins to be
altered or shuffled can be re-synthesized to have a different nucleic acid
sequence to provide
improved expression in host cells for screening and / or commercialization
purposes. Each of
the Cryl-type molecules described herein whether derived from the 5' or N-
terminal region of
CrylAb or CrylCa contain the protoxin 3' or C-terminal region of CrylCa.
100471 Sequence alterations can be introduced by standard techniques such as
directed
molecular evolution techniques e.g., DNA shuffling methods (see e.g.,
Christians at al:, 1999,
Nature Biotechnology 17:259-264; Crameri et al., 1998, Nature, 391:288-291;
Crameri, et
al., 1997, Nature Biotechnology 15:436-438; Crameri et al., 1996, Nature
Biotechnology
12
CA 02763879 2012-01-05
14:315-319; Stemmer, 1994, Nature 370:389-391; Stemmer et al., 1994, Proc.
Na:!. Acad.
Sci., 91:10747-10751; United States Patent Nos. 5,605,793;
6,117,679,,6,132,970,,5,939,250;
5,965,408; 6,171,820; International Publication Nos. WO 95/22625; WO 97/0078;
WO
97/35966; WO 98/27230; WO 00/42651; and WO 01/75767); site directed
mutagenesis (see
e.g., Kunkel, 1985, Proc. NatL Acad. Sci., 82:488-492; Oliphant et al., 1986,
Gene 44:177-
183); oligonuclootide-directed mutagenesis (see e.g., Reidhaar-Olson et aL,
1988, Science
241:53-57); chemical mutagenesis (see e.g., Eckert at aL, 1987, Mutat. Res.
178:1-10); error
prone PCR (see e.g., Caldwell & Joyce, 1992, PCR Methods Applic. 2:28-33); and
cassette
mutagenesis (see e.g., Arkin et aL, Proc. Natl. Acad. Sci., 1992, 89:7871-
7815); (see
generally, e.g., Arnold, 1993, Curr. Opinion BiotechnoL 4:450-455; Ling et aL,
1997, Anal.
Biochem., 254(2):157-78; Dale et aL, 1996, Methods MoL Biol. 57:369-74; Smith,
1985,
Ann. Rev. Genet. 19:423-462; Botstein et al., 1985, Science, 229:1193-1201;
Carter, 1986,
Bioehem. J. 237:1-7; Kramer et al., 1984, Cell 38:879-887; Wells et al., 1985,
Gene 34:315-
323; Minshull et aL, 1999, Current Opinion in Chemical Biology 3:284-290).
[0048) In one embodiment, DNA shuffling is used to create Cryl-derived nucleic
acid
molecules. DNA shuffling can be accomplished in vitro, in vivo, in silico, or
a combination
thereof. In silico methods of recombination can be performed in which genetic
algorithms
are used in a computer to recombine sequence strings which correspond to
homologous (or
even non-homologous) nucleic acids. The resulting recombined sequence strings
are
optionally converted into nucleic acids by synthesis of nucleic acids which
correspond to the
recombined sequences, e.g., in concert with oligonucleotide synthesis gene
reassembly
techniques. This approach can generate random, partially random or designed
alterations.
Many details regarding in silico recombination, including the use of genetic
algorithms,
genetic operators and the like in computer systems, combined with generation
of
corresponding nucleic acids as well as combinations of designed nucleic acids
(e.g., based on
cross-over site selection) as well as designed, pseudo-random or random
recombination
methods are described in the art (see e.g., International Publication Nos. WO
00/42560 and
WO 00/42559).
100491 In another embodiment, targeted mutagenesis is used to create Cryl -
derived
nucleic acid molecules by choosing particular nucleotide sequences or
positions of the
corresponding wild type Cry l or related nucleic acid molecules for
alteration. Such targeted
mutations can be introduced at any position in the nucleic acid. For example,
one can make
nucleotide substitutions leading to amino acid substitutions at "non-
essential" or "essential"
amino acid residues. A "non-essential" amino acid residue is a residue that
can be altered
13
CA 02763879 2012-01-05
from the wild-type sequence without altering the biological activity, whereas
an "essential"
amino acid residue is required for at least one biological activity of the
polypeptide. For
example, amino acid residues that are not conserved or only semi-conserved
among
homologs of various species may be non-essential for activity. Alternatively,
amino acid
residues that are conserved among the homologs of various species may be
essential for
activity. Ems..
[00501 Such targeted mutations can be conservative or non-conservative. A "non-
conservative amino acid substitution" is one in which the amino acid residue
is replaced with
an amino acid residue having a dissimilar side chain. Families of amino acid
residues having
similar side chains have been defined in the art. These families include amino
acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid,
glutarnic acid, asparagine, glutamine), uncharged polar side chains (e.g.,
glycine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine,
praline, phenylalanine, methionine, tryptophan), a-branched side chains (e.g.,
threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan,
histidine).
[00511 Alternatively or in addition to non-conservative amino acid residue
substitutions,
such targeted mutations can be conservative. A "conservative amino acid
substitution" is one
in which the amino acid residue is replaced with an amino acid residue having
a similar side
chain. Following mutagenesis, the encoded protein can be expressed
recombinantly and the
activity of the protein can be determined.
[00521 In another embodiment, random mutagenesis is used to create Cryl-
derived
nucleotides. Mutations can be introduced randomly along all or part of the
coding sequence
(e.g., by saturation mutagenesis or by error prone PCR). In certain
embodiments, nucleotide
sequences encoding other related polypeptides that have similar domains,
structural motifs,
active sites, or that align with a portion of the Cryl of the invention with
mismatches or
imperfect matches, can be used in the mutagenesis process to generate
diversity of sequences.
[00531 It should be understood that for each mutagenesis step in some of the
techniques
mentioned above, a number of iterative cycles of any or all of the steps may
be performed to
optimize the diversity of sequences. The above-described methods can be used
in
combination in any desired order. In many instances, the methods result in a
pool of altered
nucleic acid sequences or a pool of recombinant host cells comprising altered
nucleic acid
sequences. The altered nucleic acid sequences or host cells expressing an
altered nucleic acid
sequence with the desired characteristics can be identified by screening with
one or more
14
CA 02763879 2012-01-05
assays known in the art. The assays may be carried out under conditions that
select for
polypeptides possessing the desired physical or chemical characteristics. The
alterations in
the nucleic acid sequence can be determined by sequencing the nucleic acid
molecule
encoding the altered polypeptide in the variants.
100541 Additionally, Cryl -derived nucleic acid molecules can be codon
optimized, either
wholly or in part. Because any one amino acid (except for methionine and
tryptophan) is
encoded by a number of codons (Table 2), the sequence of the nucleic acid
molecule may be
changed without changing the encoded amino acid. Codon optimization is when
one or more
codons are altered at the nucleic acid level such that the amino acids are not
changed but
expression in a particular host organism is increased. Those having ordinary
skill in the art
will recognize that codon tables and other references providing preference
information for a
wide range of organisms are available in the an.
Methods of Assaying Insecticidal Activity
(00551 As used herein, the term "insecticidal activity" refers to the ability
of a
polypeptide to decrease or inhibit insect feeding and/or to increase insect
mortality upon
ingestion of the polypeptide. Although any insect may be affected, preferably
insects of the
Lepidopteran order including the Helicoverpa, Heltothis, or Spodoptera genera
of insects are
affected.
100561 A variety of assays can be used to determine whether a particular
polypeptide of
the invention has insecticidal activity and, if so, to what degree. Generally,
an insect pest is
provided a polypeptide of the invention in any form that can be ingested. The
reaction of the
insect pest to ingestion of the polypeptide of the invention is observed
(e.g., for about one to
three days). A decrease or inhibition of feeding and/or an increase in insect
pest mortality
after ingestion of the polypeptide of the invention are indicators of
insecticidal activity. A
polypeptide of the invention with unknown insecticidal activity should be
compared to a
positive and/or negative control to assess more accurately the outcome of the
assay.
[00571 In one embodiment, a polypeptide of the invention is purified (either
in soluble
form or in crystal form) and added to the insect diet.
100581 In another embodiment, a polypeptide of the invention is expressed in a
recombinant microbe (e.g., E. coli). The recombinant microbe is fed directly
to the insect
pests (see Moellenbeck et aL, 2001, Nat. Biotechnol. 19:668).
[00591 In another embodiment, the polypeptide of the invention is expressed in
a plant
and the plant is fed to the insect pest. Following the incubation period, the
feeding activity of
CA 02763879 2012-01-05
the insect pest can be determined by visual observation (e.g., of approximate
fraction of leaf
area remaining) or video capture (e.g., number of pixels in a leaf area
remaining) of the plant
parts that would normally have been eaten by the insect pest. In a specific
embodiment,
expression of the polypeptide of the invention in the plant is transient. In
such embodiments,
a nucleic acid encoding a polypeptide of the invention is cloned into a plant
expression vector
and transfected into Agrobacterium tumefaciens. The transformed bacterial
culture is co-
cultivated with a leaf from N. benthamiana and, using forced infiltration, the
leaf expresses
the polypeptide of the invention. However, expression of the polypeptide is
variable between
leaf co-cultures. In another specific embodiment, expression of the
polypeptide of the
invention in the plant is stable. In such embodiments, a transgenic plant is
made that
expresses a polypeptide of the invention.
[0060) In another embodiment, insecticidal activity of a polypeptide of the
invention can
be assayed by measuring cell death and/or cell growth using cultured cells.
Such assays
typically involve the use of cultured insect cells that are susceptible to the
particular toxin
being screened, or cells that express a receptor for the particular toxin,
either naturally or as a
result of expression of a heterologous gene. Thus, in addition to insect
cells, mammalian,
bacterial, and yeast cells are among those cells useful in the in vitro
assays. In vitro
bioassays which measure toxicity against cultured cells are described in the
art (e.g., Johnson,
1994, J. Invertebr. Pathol. 63:123-129).
[00611 In another embodiment, insecticidal activity of a polypeptide of the
invention can
be assayed by measuring pore formation in target insect-derived midgut
epithelial membrane
vesicles (Juttner and Ebel, 1998, Biochim. Biophys. Acta 1370:51-63.; English
et a!.,
1991, Insect Biochem. 21:177-184). Such an assay may constitute toxin
conditional release
of a ligand activated substrate from the lumen of the membrane vesicles. This
requires that
the ligand be on the outside of the vesicle. Alternatively the reverse
scenario may be utilized
whereby the ligand is in the vesicle lumen and the ready to be activated
substrate is located
on the outside of the vesicle. The higher the toxin activity the greater the
number or size of
pores formed.
Methos of Enhancing Insect Resistance in Plants
100621 The present invention provides methods of enhancing plant resistance to
insect
pests including, but not limited to, members of the Helicoverpa ssp.(e.g.,
Helicoverpa Zea
and Heliothis virescens) and/or Spodoptera ssp. (e.g., Spodoptera exigua.
Spodoptera
frugiperda) through the use of Cryl-derived insecticidal polypeptides. Any
method known in
16
CA 02763879 2012-01-05
the art can be used to cause the insect pests to ingest one or more
polypeptides of the
invention during the course of feeding on the plant. As such, the insect pest
will ingest
insecticidal amounts of the one or more polypeptides of the invention and may
discontinue
feeding on the plant. In some embodiments, the insect pest is killed by
ingestion of the one
or more polypeptides of the invention. In other embodiments, the insect pests
are inhibited or
discouraged from feeding on the plant without being killed.
100631 In one embodiment, transgenic plants can be made to express one or more
polypeptides of the invention. The transgenic plant may express the one or
more
polypeptides of the invention in all tissues (e.g., global expression).
Alternatively, the one or
more polypeptides of the invention may be expressed in only a subset of
tissues (e.g., tissue
specific expression), preferably those tissues consumed by the insect pest.
Polypeptides of
the invention can be expressed constitutively in the plant or be under the
control of an
inducible promoter. Polypeptides of the invention may be expressed in the
plant cytosol or in
the plant chloroplast either by protein targeting or by transformation of the
chloroplast
genome.
100641 In another embodiment, a composition comprising one or more
polypeptides of
the invention can be applied externally to a plant susceptible to the insect
pests. External
application of the composition includes direct application to the plant,
either in whole or in
part, and/or indirect application, e.g., to the environment surrounding the
plant such as the
soil. The composition can be applied by any method known in the art including,
but not
limited to, spraying, dusting, sprinkling; or the like. In general, the
composition can be
applied at any time during plant growth. One skilled in the art can use
methods known in the
art to determine empirically the optimal time for administration of the
composition. Factors
that affect optimal administration time include, but are not limited to, the
type of susceptible
plant, the type of insect pest, which one or more polypeptides of the
invention are
administered in the composition.
100651 The composition comprising one or more polypeptides of the invention
may be
substantially purified polypeptides, a cell suspension, a cell pellet, a cell
supernatant, a cell
extract, or a spore-crystal complex of Bacillus thuringiensis cells. The
composition
comprising one or more polypeptides of the invention may be in the form of a
solution, an
emulsion, a suspension, or a powder. Liquid formulations may be aqueous or non-
aqueous
based and may be provided as foams, gels, suspensions, emulsifiable
concentrates, or the like.
The formulations may include agents in addition to the one or more
polypeptides of the
invention. For example, compositions may further comprise spreads sticker
adjuvants,
17
CA 02763879 2012-01-05
stabilizing agents, other insecticidal additives, diluents, agents that
optimize the Theological
properties or stability of the composition, such as, for example, surfactants,
emulsifiers,
dispersants, or polymers.
[0066) In another embodiment, recombinant hosts that express one or more
polypeptides
of the invention are applied on or near. a plant susceptible to attack by an
insect pest. The
recombinant hosts include, but are not limited to, microbial hosts and insect
viruses that have
been transformed with and express one or more nucleic acid molecules (and thus
polypeptides) of the invention. In some embodiments, the recombinant host
secretes the
polypeptide of the invention into its surrounding environment so as to contact
an insect pest.
In other embodiments, the recombinant hosts colonize one or more plant tissues
susceptible
to insect infestation.
Recombinant Exuression
[0067) Nucleic acid molecules and polypeptides of the invention can be
expressed
recombinantly using standard recombinant DNA and molecular cloning techniques
that are
well known in the-art (e.g., Sambrook, Fritsch, and Maniatis, Molecular
Cloning; A
Laboratory Manual: Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
1989).
Additionally, recombinant DNA techniques may be used to create nucleic acid
constructs
suitable for use in making transgenic plants.
100681 Accordingly, an aspect of the invention pertains to vectors, preferably
expression
vectors, comprising a nucleic acid molecule of the invention, or a variant
thereof. As used
herein, the term "vector" refers to a polynucleotide capable of transporting
another nucleic
acid to which it has been linked. One type of vector is a "plasmid", which
refers to a circular
double stranded DNA loop into which additional DNA segments can be introduced.
Another
type of vector is a viral vector, wherein additional DNA segments can be
introduced into the
viral genoma
[00691 Certain vectors are capable of autonomous replication in a host cell
into which
they are introduced (e.g., bacterial vectors having a bacterial origin of
replication and
episomal vectors). Other vectors (e.g., non-episomal vectors) are integrated
into the genome
of a host cell upon introduction into the host cell, and thereby are
replicated along with the
host genome. In general, expression vectors of utility in recombinant DNA
techniques are
often in the form of plasmids (vectors). However, the invention is intended to
include such
18
CA 02763879 2012-01-05
other forms of expression vectors, such as viral vectors (e.g., replication
defective
retroviruses).
100701 The recombinant expression vectors of the invention comprise a nucleic
acid
molecule of the invention in a form suitable for expression of the nucleic
acid molecule in a
host cell. This means that the recombinant expression vectors include one or
more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is operably
associated with the polynucleotide to be expressed. Within a recombinant
expression vector,
"operably associated" is intended to mean that the nucleotide sequence of
interest is linked to
the regulatory sequence(s) in a manner which allows for expression of the
nucleotide
sequence (e.g., in an in vitro transcription/translation system or in a host
cell when the vector
is introduced into the host cell). The term "regulatory sequence" is intended
to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals).
Such regulatory sequences are described in the art (e.g., Goeddel, Gene
Exvression
Technology: Methods in Enzvmolosv. 1990, Academic Press, San Diego, CA).
Regulatory
sequences include those which direct constitutive expression of a nucleotide
sequence in '
many types of host cells and those which direct expression of the nucleotide
sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by those
skilled in the art that the design of the expression vector can depend on such
factors as the
choice of the host cell to be transformed, the level of expression of protein
desired, the area
of the organism in which expression is desired, etc. The expression vectors of
the invention
can be introduced into host cells to thereby produce proteins or peptides,
including fusion
proteins or peptides, encoded by nucleic acids molecules as described herein.
[00711 In some embodiments, isolated nucleic acids which serve as promoter or
enhancer
elements can be introduced in the appropriate position (generally upstream) of
a non-
heterologous form of a polynucleotide of the present invention so as to up or
down regulate
expression of a polynucleotide of the present invention. For example,
endogenous promoters
can be altered in vivo by mutation, deletion, and/or substitution (see, U.S.
Patent No.
5,565,350; International Patent Application No. PCT/US93/03868), or isolated
promoters can
be introduced into a plant cell in the proper orientation and distance from a
cognate gene of a
polynucleotide of the present invention so as to control the expression of the
gene. Gene
expression can be modulated under conditions suitable for plant growth so as
to alter the total
concentration and/or alter the composition of the polypeptides of the present
invention in
plant cell.
19
CA 02763879 2012-01-05
[00721 If polypeptide expression is desired in a eukaryotic system, it is
generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region.
The polyadenylation region for plant expression can be derived from the
natural gene, from a
variety of plant genes, or from Agrobacterium T-DNA. The 3' end sequence to be
added can
be derived from, for example, the nopaline synthase or octopine synthase
genes, or
alternatively from another plant gene, or less preferably from any other
eukaryotic gene.
100731 The recombinant expression vectors of the invention can be designed for
expression of a polypeptide of the invention in prokaryotic (e.g.,
Enterobacteriaceae, such as
Escherichia; Bacillaceae; Rhizoboceae, such as Rhizobium and Rhizobacter,
Spirillaceae,
such as photobacterium; Zymomonas; Serratia; Aeromonas; Vibrio; Desulfovibrio;
Spirillum;
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter,
Azotobacteraceae and Nitrobacteraceae) or eukaryotic cells (e.g., insect cells
using
baculovirus expression vectors, yeast cells, plant cells, or mammalian cells)
(see Goeddel,
supra. For a discussion on suitable host cells). Alternatively, the
recombinant expression
vector can be transcribed and translated in vitro, for example using T7
promoter regulatory
sequences and T7 polymerase.
100741 Expression of proteins in'prokaryotes is most often carried out in E.
coil with
vectors comprising constitutive or inducible promoters directing the
expression of either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein
encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion
vectors typically serve at least three purposes: 1) to increase expression of
the recombinant
protein; 2) to increase the solubility of the recombinant protein; and/or 3)
to aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification. Often, in
fusion expression vectors, a proteolytic cleavage site is introduced at the
junction of the
fusion moiety and the recombinant protein to enable separation of the
recombinant protein
from the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical
fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and
Johnson, 1988,
Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pR1T5 (Pharmacia,
Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding
protein, or
protein A, respectively, to the target recombinant protein.
[00751 In another embodiment, the expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast S. cerevisiae include pYepSecl
(Baldari et a!.,
1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943),
pJRY88
CA 02763879 2012-01-05
(Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen Corp., San Diego,
CA), and
pPicZ (Invitrogen Corp., San Diego, CA).
[0076) Alternatively, the expression vector is a baculovirus expression
vector.
Baculovirus vectors available for expression of proteins in cultured insect
cells (e.g., Sf 9
cells) include the,pAc series (Smith et aL, 1983, Mol. Cell Biol. 3:2156-2165)
and the pVL
series (Lucklow and Summers, 1989, Virology 170:31-39).
(00771 In yet another embodiment, a nucleic acid of the invention is expressed
in plant
cells using a plant expression vector including, but not limited to, tobacco
mosaic virus and
potato virus expression vectors.
[0078) Other suitable expression systems for both prokaryotic and eukaryotic
cells are
known in the art (see, e.g., chapters 16 and 17 of Sambrook et al. 1990,
Molecular Cloning A
Laboratory Manual. 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY).
(0079) A number of promoters can be used in the practice of the invention. The
promoters can be selected based on the desired outcome. The nucleic acids can
be combined
with constitutive, tissue-specific, inducible, or other promoters for
expression in the host
organism.
[0080) A "tissue-specific promoter" may direct expression of nucleic acids of
the present
invention in a specific tissue, organ or cell type. Tissue-specific promoters
can be inducible.
Similarly, tissue-specific promoters may only promote transcription within a
certain time
frame or developmental stage within that tissue. Other tissue specific
promoters may be
active throughout the life cycle of a particular tissue. One of ordinary skill
in the art will
recognize that a tissue-specific promoter may drive expression of operably
linked sequences
in tissues other than the target tissue. Thus, as used herein, a tissue-
specific promoter is one
that drives expression preferentially in the target tissue or cell type, but
may also lead to some
expression in other tissues as well. A number of tissue-specific promoters can
be used in the
present invention. With the appropriate promoter, any organ can be targeted,
such as shoot
vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers
and floral
organsistructures (e.g., bracts, sepals, petals, stamens, carpels, anthers and
ovules), seed
(including embryo, endosperm, and seed coat) and fruit. For instance,
promoters that direct
expression of nucleic acids in leaves, roots or flowers are useful for
enhancing resistance to
pests that infect those organs. For expression of a polynucleotide of the
present invention in
the aerial vegetative organs of a plant, photosynthetic organ-specific
promoters, such as the
RBCS promoter (Khoudi et al., Gene 197:343, 1997), can be used. Root-specific
expression
of polynucleotides of the present invention can be achieved under the control
of a root-
21
CA 02763879 2012-01-05
specific promoter, such as, for example, the promoter from the ANRI gene
(Zhang and Fordo,
Science, 279:407, 1998). Other exemplary promoters include the root-specific
glutamine
synthetase gene from soybean (Hirel et al., 1992, Plant Molecular Biology
20:207-218) and
the root-specific control element in the GRP 1.8 gene of French bean (Keller
et al., 1991, The
Plant Cell 3:1051-1061).
[00811 A "constitutive promoter" is defined as a promoter which will direct
expression of
a gene in all tissues and are active under most environmental conditions and
states of
development or cell differentiation. Examples of constitutive promoters
include the
cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'-
or 2'- promoter
derived from T-DNA ofAgrobacterium tumafaciens, and other transcription
initiation
regions from various plant genes known to those of ordinary skill in the an.
Such genes
include for example, ACT11 from Arabidopsis (Huang et a!. 1996, Plant Mol.
BioL 33:125-
139), Cat3 from Arabidopsis (GenBank Accession No. U43147, Zhong et al., 1996,
Mol.
Gen. Genet. 251:196-203), the gene encoding stearoyl-acyl carrier protein
desaturase from
Brassica napus (Genbank Accession No. X74782, Solocombe eta!. 1994, Plant
PhysioL
104:1167-1176), GPcI from maize (GenBank Accession No. X15596, Martinez et
al., 1989,
J. Mot Biol. 208:551-565), and Gpc2 from maize (GenBank Accession No. U45855,
Manjunath at al., 1997, Plant Mo!. Biol. 33:97-112). Any strong, constitutive
promoter, such
as the CaMV 35S promoter, can be used for the expression of polynucleotides of
the present
invention throughout the plant.
100821 The term "inducible promoter" refers to a promoter that is under
precise
environmental or developmental control. Examples of environmental conditions
that may
effect transcription by inducible promoters include anaerobic conditions,
elevated
temperature, the presence of light, or spraying with chemicals/hormones.
(00831 Suitable constitutive promoters for use in a plant host cell include,
for example,
the core promoter of the Rsyn7 promoter and other related constitutive
promoters
(International Publication No. WO 99/43838 and U.S. Patent No. 6,072,050); the
core CaMV
35S promoter (Odell et al., 1985, Nature 313:810-812); rice actin (McElroy et
a!., 1990,
Plant Cell 2:163-171); ubiquitin (Christensen et al., 1989, Plant Mal. Biol.
12:619-632 and
Christensen eta!., 1992, Plant Mol. Biol. 18:675-689); pEMU (Last et al.,
1991, Theor. AppL
Genet. 81:581-588); MAS (Velten et a!., 1984, EMBOJ. 3:2723-2730); ALS
promoter (U.S.
Patent No. 5,659,026), and the like (e.g., U.S. Patent Nos. 5,608,149;
5,608,144; 5,604,121;
5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611).
22
CA 02763879 2012-01-05
[00841 Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms
refer not only to the particular subject cell but to the progeny or potential
progeny of such a
cell. Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to the
parent cell, but are still included within the scope of the term as used
herein.
100851 Accordingly, the present invention provides a host cell having an
expression
vector comprising a nucleic acid of the invention, or a variant thereof. A
host cell can be any
prokaryotic (e.g., E. col4 Bacillus thuringiensis or other Bacillus spp.) or
eukaryotic cell
(e.g., insect cells, yeast or plant cells). The invention also provides a
method for expressing a
nucleic acid of the invention thus making the encoded polypeptide comprising
the steps of i)
culturing a cell comprising a nucleic acid molecule of the invention under
conditions that
allow production of the encoded polypeptide; and ii) isolating the expressed
polypeptide.
[00861 Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid molecules into a host cell,
including calcium
phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection,
lipofection, or electroporation. Suitable methods for transforming or
transfecting host cells
can be found in the art (e.g., Sambrook, et al. supra.).
100871 Additionally, it is possible to target expression of the particular DNA
into a
particular location in a plant. For example, the genes in plants encoding the
small subunit of
RUBISCO (SSU) are often highly expressed, light regulated and sometimes show
tissue
specificity. These expression properties are largely due to the promoter
sequences of these
genes. It has been possible to use SSU promoters to express heterologous genes
in
transformed plants. Typically a plant will contain multiple SSU genes, and the
expression
levels and tissue specificity of different SSU genes will be different. The
SSU proteins are
encoded in the nucleus and synthesized in the cytoplasm as precursors that
contain an N-
terminal extension known as the chloroplast transit peptide (CTP). The CTP
directs the
precursor to the chloroplast and promotes the uptake of the SSU protein into
the chloroplast.
In this process, the CTP is cleaved from the SSU protein. These CTP sequences
have been
used to direct heterologous proteins into chloroplasts of transformed plants.
23
CA 02763879 2012-01-05
100881 The SSU promoters might have several advantages for expression of
heterologous
genes in plants. Some SSU promoters are very highly expressed and could give
rise to
expression levels as high as or higher than those observed with other
promoters. Because of
the differing the tissue distribution of expression from SSU promoters, for
control of some
insect pests, it may be advantageous to direct the expression of crystal
proteins to those cells
in which SSU is most highly expressed.
100891 For example, although relatively constitutive, in the leaf the CaMV35S
promoter
is more highly expressed in vascular tissue than in some other parts of the
leaf, while most
SSU promoters are most highly expressed in the mesophyll cells of the leaf.
Some SSU
promoters also are more highly tissue specific, so it could be possible to
utilize a specific
SSU promoter to express the protein of the present invention in only a subset
of plant tissues,
if for example expression of such a protein in certain cells was found to be
deleterious to
those cells. For example, for control of Colorado potato beetle in potato, it
may be
advantageous to use SSU promoters to direct crystal protein expression to the
leaves but not
to the edible tubers.
[00901 Utilizing SSU CTP sequences to localize crystal proteins to the
chloroplast might
also be advantageous. Localization of the B. thuringiensis crystal proteins to
the chloroplast
could protect these from proteases found in the cytoplasm. This could
stabilize the proteins
and lead to higher levels of accumulation of active toxin. cry genes
containing the CTP may
be used in combination with the SSU promoter or with other promoters such as
CaMV35S.
100911 It may also be advantageous for some purposes to direct the Cry
proteins to other
compartments of the plant cell, as such may result in reduced exposure of the
proteins to
cytoplasmic proteases, in turn leading to greater accumulation of the protein,
which could
yield enhanced insecticidal activity. Extracellular localization could lead to
increased
exposure of certain insects to the Cry proteins, which could also lead to
enhanced insecticidal
activity. If a particular Cry protein was found to harm plant cell fimction,
then localization to
a noncytoplasmic compartment could protect these cells from the protein.
100921 By way of example, in plants as well as other eukaryotes, proteins that
are to be
localized either extracellularly or in several specific compartments are
typically synthesized
with an N-terminal amino acid extension known as the signal peptide. This
signal peptide
directs the protein to enter the compartmentalization pathway, and it is
typically cleaved from
the mature protein as an early step in compartmentalization. For an
extracellular protein, the
secretory pathway typically involves cotranslational insertion into the
endoplasmic reticulum
with cleavage of the signal peptide occurring at this stage. The mature
protein then passes
24
CA 02763879 2012-01-05
through the Golgi body into vesicles that ft m with the plasma membrane thus
releasing the
protein into the extracellular space. Proteins destined for other compartments
follow a
similar pathway. For example, proteins that are destined for the endoplasmic
reticulum or the
Golgi body follow this scheme, but they are specifically retained in the
appropriate
compartment. In plants, some proteins are also targeted to the vacuole,
another membrane
bound compartment in the cytoplasm of many plant cells. Vacuole targeted
proteins diverge
from the above pathway at the Golgi body where they enter vesicles that fuse
with the
vacuole.
(0093] A common feature of this protein targeting is the signal peptide that
initiates the
compartmentalization process. Fusing a signal peptide to a protein will in
many cases lead to
the targeting of that protein to the endoplasmic reticulum. The efficiency of
this step may
depend on the sequence of the mature protein itself as well. The signals that
direct a protein
to a specific compartment rather than to the extracellular space are not as
clearly defined. It
appears that many of the signals that direct the protein to specific
compartments are contained
within the amino acid sequence of the mature protein. This has been shown for
some vacuole
targeted proteins, but it is not yet possible to define these sequences
precisely. It appears that
secretion into the extracellular space is the "default" pathway for a protein
that contains a
signal sequence but no other compartmentalization signals. Thus, a strategy to
direct Cry
proteins out of the cytoplasm is to fuse the genes for synthetic Cry proteins
to DNA
sequences encoding known plant signal peptides. These fusion genes will give
rise to cry
proteins that enter the secretory pathway, and lead to extracellular secretion
or targeting to
the vacuole or other compartments.
(0094] Signal sequences for several plant genes have been described. One such
sequence
is for the tobacco pathogenesis related protein PRIb has been previously
described
(Cornelissen et al., 1986). The PRIb protein is normally localized to the
extracellular space.
Another type of signal peptide is contained on seed storage proteins of
legumes. These
proteins are localized to the protein body of seeds, which is a vacuole like
compartment
found in seeds. A signal peptide DNA sequence for the .bets.-subunit of the 7S
storage
protein of common been (Phaseolus vulgaris), PvuB has been described (Doyle at
al., 1986).
Based on the published these published sequences, genes may be synthesized
chemically
using oligonuclootides that encode the signal peptides for PRIb and PvuB. In
some cases to
achieve secretion or compartmentalization of heterologous proteins, it may be
necessary to
include some amino acid sequence beyond the normal cleavage site of the signal
peptide.
This may be necessary to insure proper cleavage of the signal peptide.
CA 02763879 2012-01-05
Production of Transgenic Plants
[0095] Any method known in the an can be used for transforming a plant or
plant cell
with a nucleic acid molecule of the present invention. Nucleic acid molecules
can be
incorporated into plant DNA (e.g., genomic DNA or chloroplast DNA) or be
maintained
without insertion into the plant DNA (e.g., through the use of artificial
chromosomes).
Suitable methods of introducing nucleic acid molecules into plant cells
include
microinjection (Crossway et al., 1986, Biotechniques 4:320-334);
electroporation (Riggs at
al., 1986, Proc. Natl. Acad. Sci. 83:5602-5606; D'Halluin et al., 1992, Plant
Cell 4:1495-
1505); Agrobacterium-mediated transformation (U.S. Patent Nos. 5,563,055 and
5,981,840,
Osjoda et al., 1996, Nature Biotechnology 14:745-750; Horsch at al., 1984,
Science 233:496-
498, Fraley et al., 1983, Proc. Natl. Acad. Sci. 80:4803, and Gene Transfer to
Plants,
Potrykus, ed., Springer-Verlag, Berlin 1995); direct gene transfer (Paszkowski
et al., 1984,
EMBO J. 3:2717-2722); ballistic particle acceleration (U.S. Patent Nos.
4,945,050;
5,879,918; 5,886,244; 5,932,782; Tomes at aL, 1995, "Direct DNA Transfer into
Intact Plant
Cells via Microprojectile Bombardment, in Plant Cell. Tissue. and Oraan
Culture:
Fundamental Methods. ed. Gamborg and Phillips, Springer-Verlag, Berlin; and
McCabe et
al., 1988, Biotechnology 6:923-926); virus-mediated transformation (U.S.
Patent Nos.
5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931); pollen
transformation (De Wet
at al., 1985, in The Experimental Manipulation of Ovule Tissues. ed. Chapman
et al.,
Longman, New York, pp. 197-209); Lec 1 transformation (U.S. Patent Application
Ser. No.
09/435,054; International Publication No. WO 00/28058); whisker-mediated
transformation
(Kaeppler et al., 1990, Plant Cell Reports 9:415-418; Kaeppler et al., 1992,
Tlieor. AppL
Genet. 84:560-566); and chloroplast transformation technology (Bogorad, 2000,
Trends in
Biotechnology 18: 257-263; Ramesh et al., 2004, Methods Mal Biol. 274:301-7;
Hou et aL,
2003, Transgenic Res. 12:111-4; Kindle et aL, 1991, Pros Natl. Acad. Sci.
88:1721-5;
Bateman and Purton, 2000, Mo! Gen Genet. 263:404-10; Sidorov et al., 1999,
Plant J.
19:209-216).
[00961 The choice of transformation protocols used for generating transgenic
plants and
plant cells can vary depending on the type of plant or plant cell, i. e.,
monocot or dicot,
targeted for transformation. Examples of transformation protocols particularly
suited for a
particular plant type include those for: potato (Tu et a!., 1998, Plant
Molecular Biology
37:829-838; Chong et al., 2000, Transgenic Research 9:71-78); soybean
(Christou et al.,
26
CA 02763879 2012-01-05
1988, Plant Physiol. 87:671-674; McCabe et al., 1988, BloTechnology 6:923-926;
Finer and
McMullen, 1991, In Vitro Cell Dev. Biol. 27P: 175-182; Singh et al., 1998,
Theor. Apps
Genet. 96:319-324); maize (Klein et al., 1988, Proe. NatL Acad Sc1. 85:4305-
4309; Klein et
al., 1988, Biotechnology 6:559-563; Klein et al., 1988, Plant Physiol. 91:440-
444; Fromm et
al., 1990, Biotechnology 8:833-839; Tomes et al., 1995, "Direct DNA Transfer
into Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell. Tissue, and Organ
Culture:
Fundamental Methods. ed. Gamborg (Springer-Verlag, Berlin)); cereals (Hooykaas-
Van
Slogteren et al., 1984, Nature 311:763-764; U.S. Patent No. 5,736,369).
100971 In some embodiments, more than one construct is used for transformation
in the
generation of transgenic plants and plant cells. Multiple constructs may be
included in cis or
trans positions. In preferred embodiments, each construct has a promoter and
other
regulatory sequences.
(00981 Transformed plant cells which are derived by any of the above
transformation
techniques can be cultured to regenerate a whole plant which possesses the
transformed
genotype and thus the desired phenotype. Such regeneration techniques rely on
manipulation
of certain phytohormones in a tissue culture growth medium, typically relying
on a biocide
and/or herbicide marker that has been introduced together with the desired
nucleotide
sequences. Plant regeneration from cultured protoplasts is described in the
art (e.g., Evans et
al., Protovlasts Isolation and Cul_tu*Y" Handbook of Plant Colt Culture. pp.
124-176,
MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of
Plants,
Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985). Regeneration can
also be
oBtained from plant callus, explants, organs, or parts thereof. Such
regeneration techniques
are also described in the art (e.g., Klee at al. 1987, Ann. Rev. of Plant
Phys. 38:467-486).
(00991 The term "plant" includes whole plants, shoot vegetative
organs/structures (e.g.
leaves, stems and tubers), roots, flowers and floral organs/structures (e.g.
bracts, sepals,
petals, stamens, carpels, anthers and ovules), seed (including embryo,
endosperm, and seed
coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground
tissue, and the
like) and cells (e.g. guard cells, egg cells, trichomes and the like), and
progeny of same. The
class of plants that can be used in methods of the present invention includes
the class of
higher and lower plants amenable to transformation techniques, including
angiosperms
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and
multicellular algae.
Plants of a variety of ploidy levels, including ancuploid, polyploid, diploid,
haploid and
hemizygous plants are also included.
27
CA 02763879 2012-01-05
1001001 The nucleic acid molecules of the invention can be used to confer
desired traits on
essentially any plant. Thus, the invention has use over a broad range of
plants, including
species from the genera Agrotts, Allium, Ananas, Anacardium, Apium, Arachis,
Asparagus,
Athamantha, Atropa, Avena, Bambusa, Beta, Brassies, Bromus, Browaalia,
Camellia,
Cannabis, Carica, Ceratonia. Cicer, Chenopodium, Chicorium, Citrus, Citrullus,
Capsicum,
Carthamus, Cocos, Coffee, Coix, Cucumis, Cucurbita, Cynodon, Dactylts, Datura,
Daucus,
Dianthus, Digitalis, Dioscorea, Elaeis. Eliusine, Euphorbia, Festuca, Ficus.
Fragaria,
Geranium, Glycine, Graminae, Gossypium, Hellanthus, Heterocallis, Hevea,
Hibiscus,
Hordeum, Hyoscyamus,1pomoea, Lactuca. Lathyrus, Lens, Lilium, Linum, Lolium,
Lotus.
Lupinus, Lycopersicon, Macadamia, Macrophylla, Malus, Mangifera, Manihot,
Malorana,
Medicago, Musa, Narcissus, Nemesia, Nicotiana, Onobrychis, Olea, Olyreae,
Oryza,
Panicum, Panicum, Panieum, Pannisetum, Pennisetum,, Petunia, Pelargonium,
Persea,
Pharoideae, Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum, Populus,
Pseudotsuga,
Pyrus, Prunus, Pseutotsuge, Psidium, Quercus, Ranunculus, Raphanus, Ribes,
Ricinus,
Rhododendron, Rosa, Saceharum, Salpiglossis, Secale, Senecio, Setarla,
Sequoia, Sinapis,
Solanum, Sorghum, Stenotaphrum, Theobromus, Trigonella, Trifolium, Trigonella,
Triticum,
Tsuga, Tulips, Vicia, Vitis, Vigna, and Zea.
1001011 In specific embodiments, transgenic plants are maize, potato, rice,
soybean,
alfalfa, sunflower, canola, or cotton plants.
1001021 Transgenic plants may be grown and pollinated with either the same
transformed
strain or different strains. Two or more generations of the plants may be
grown to ensure that
expression of the desired nucleic acid molecule, polypeptide and/or phenotypic
characteristic
is stably maintained and inherited. One of ordinary skill in the art will
recognize that after
the nucleic acid molecule of the present invention is stably incorporated in
transgenic plants
and confirmed to be operable, it can be introduced into other plants by sexual
crossing. Any
of a number of standard breeding techniques can be used, depending upon the
species to be
crossed.
1001031 In certain embodiments the polynucleotides of the embodiments can be
stacked
with any combination of polynucleotide sequences of interest in order to
create plants with a
desired trait. For example, the polynucleotides of the embodiments may be
stacked with any
other polynucleotides encoding polypeptides having pesticidal and/or
insecticidal activity,
such as other Bt toxic proteins (described in, for example, U.S. Pat. Nos.
5,366,892;
5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene
48:109), lectins
(Van Damme at al. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S.
Pat No.
28
CA 02763879 2012-01-05
5,981,722), and the like. The combinations generated can also include multiple
copies of any
one of the polynucleotides of interest. The polynucleotides of the embodiments
can also be
stacked with any other gene or combination of genes to produce plants with a
variety of
desired trait combinations including, but not limited to, traits desirable for
animal feed such
as high oil genes (e.g,, U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins
(U.S. Pat. Nos. 5,990,389; 5,885,801; 5.335,802; and 5,703,409); barley high
lysine
(Williamson ct al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122) and
high
methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara
et al. (1988)
6ne '71:359: and Musumura et al. (1989) Plant Mol. Biol. 12:123)); increased
digestibility
I') (e.g., modified storage proteins (U.S. Patent 6,858,778. filed Nov. 7,
2001);
and thioredoxins (U.S. Patent 7.009.087, tiled Dec. 3, 2001)).
100104J The polynucleotides of the embodiments can also be stacked with traits
desirable
for disease or herbicide resistance (e.g., fumonisin detoxification genes
(U.S. Pat. No.
", 92,931); avirulence and disease resistance genes (Jones et al. (1994)
Science 266:789;
?rl:trtin et al. (H93) Science 262:1432; Mindrinos et al. (1994) Cell
78:1089); acetolactate
synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or
Hra mutations;
genes encoding resistance to inhibitors of glutamine synthase such as
phosphinothricin or
basta (e.g., bar or PAT genes); and glyphosate resistance (EPSPS and GAT
(glyphosate
2,) acetyl transti;rase) genes (Castle et al. (2004) Science 304:1151)); and
traits desirable for
processing or process products such as high oil (e.g., U.S. Pat. No.
6,232,529); modified oils
(e.g fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516));
modified starches
(e.t''., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes
(S 13E.), and starch debranching enzymes (SDBE)); and polymers or bioplastics
(e.g., U.S. Pat.
2S No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA
rcductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate
expression of
polyltydroxvalkanoates (I'HAs)).
One could also combine the polynucleotides of the embodiments with
polvitucleotides providing agronomic traits such as male sterility (see, e.g.,
U.S. Pat. No.
3(' 5,583,2 10), stalk strength, flowering time, or transformation technology
traits such as cell
cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO
99/25821).
1001051 These stacked combinations can be created by any method including, but
not
limited to, cross-breeding plants by any conventional or TopCross methodology,
or genetic
29
CA 02763879 2012-01-05
transformation. If the sequences are stacked by genetically transforming the
plants, the
pol)nucleotide sequences of interest can he combined at any time and in any
order. For
,example, a transgenic plant comprising one or more desired traits can be used
as the target to
introduce li,rther traits by subsequent transformation. The traits can be
introduced
simultaneously in a co-transformation protocol with the polynucleotides of
interest provided
by any combination of transformation cassettes. For example, if two sequences
will be
introduced, the two sequences can be contained in separate transformation
cassettes (trans) or
contained on the same transformation cassette (cis). Expression of the
sequences can be
driven by the same promoter or by different promoters. In certain cases, it
may be desirable to
I ) introduce a transformation cassette that will suppress the expression of
the polynucleotide of
interest. l'his may be combined with any combination of other suppression
cassettes or
overexpression cassettes to generate the desired combination of traits in the
plant. It is further
recognized that polynucleotide sequences can be stacked at a desired genomic
location using
a site-specific recombination system. See, e.g., WO 99/25821, WO 99/25854, WO
99/25840,
1 5 4VO 99/25855, and WO 99/25853.
i)etcnninatiun of Expression in Transgenic Plants
1001061 Any method known in the art can be used for determining the level of
expression
in a plant of a nucleic acid molecule of the invention or polypeptide encoded
therefrom. For
2() example, the expression level in a plant of a polypeptide encoded by a
nucleic acid molecule
of the invention can be determined by immunoassay, quantitative gel
electrophoresis, etc.
Expression of nucleic acid molecules of the invention can be measured directly
by reverse
transcription quantitative PCR (qRT-PCR) of isolated RNA form the plant.
Additionally, the
expression level in a plant of a polypeptide encoded by a nucleic acid
molecule of the
2 invention can be determined by the degree to which the plant phenotype is
altered. In a
specific embodiment, enhanced insect resistance is the phenotype to be
assayed.
1001071 As used herein, "enhanced insect resistance" refers to increased
resistance of a
transgenic plant expressing a polypeptide of the invention to consumption
and/or infestation
by an insect pest as compared to a plant not expressing a polypeptide of the
invention.
>t Enhanced resistance can be measured in a number of ways. In one embodiment,
enhanced
resistance is measured by decreased damage to a plant expressing a polypeptide
of the
invcr.tion as compared to a plant not expressing a polypcptide of the
invention after the same
period of insect incubation. Insect damage can be assessed visually. For
example in cotton
plants, damage after infestation can be measured by looking directly at cotton
plant bolls for
CA 02763879 2012-01-05
signs of consumption by insects. In another embodiment, enhanced resistance is
measured by
increased crop yield from a plant expressing a polypeptide of the invention as
compared to a
plant not expressing a polypeptide of the invention after the same period of
insect incubation.
In particular embodiments, the insect pests are from the order of Lepidopteran
insects
including 1!e!iothine, ilgrotis, Pseudoplusia, Chilo, Spodoptera spp and
others.
1001081 Determinations can be made using whole plants, tissues thereof, or
plant cell
culture.
1001091 The scope of the claims should not be limited by the preferred
embodiments set forth in these examples, but should be given the broadest
interpretation
consistent with the description as a whole.
X001101
lFXAMPLES
Example 1: Single Gene Shuffling
1001111 Cry I Ac toxin is currently the most potent toxin known for control of
Heliothis
2() insects in cotton. However, Cryl Ac has very little activity on secondary
pests of the
Spodoptera class. Cryl Ab toxin is an excellent starting activity for cotton
insect pest control
,since it has slightly less activity on H. zea than CrylAc but far superior S.
exigua activity. To
meet this product deficiency, a CrylAb-like gene was shuffled to obtain Cryl-
derived
polypeptides that have improved Heliothine activity while retaining
essentially full
Spodoptera potency. One method used to generate Cry 1-derived polypeptides was
'single
gene snuffling' (mutagenesis combined with shuffling). Shuffling of CrylAb was
done as
follows. f%vo overlapping fragments of a 5' portion of the CrylAb gene from
the translation
start to the konl site were amplified by two separate PCR reactions from a Bt
kurstaki strain
that contains a Cry 1 Ab 1 gene. These fragments were further fragmented by
endonuclease and
assembled under certain mutational conditions to create a series or library of
shuffled genes.
Phis shuffled portion contains the region coding for the mature toxin. In
order to clone and
express the shuffled gene library, we constructed an E. coli-Bt shuttle vector
that contains a
!.etracyclinc-resistant gene and two replicons for both hosts. The vector also
contains the
31
CA 02763879 2012-01-05
remaining (not shuffled) 3' portion of the cry] Ca gene from the Kpnl site to
the translation
end along with the cry] Ca transcription promoter and cry/Ac terminator. When
the shuffled
gene library was cloned in this vector, the full-length 135-kDa proteins were
produced. The
shuffled gene library was expressed in a cry-minus Bt host called BIG8, which
was derived
from the HD1 strain by plasmid curing. A selection was made to assure a high
transformation
competency by electroporation which is required for making a diversified
shuffled library.
The selected host, BtG8, showed a level of competency over 106 transformants
per I ug
DNA. A shuffled gene library was made by sequentially transforming E. cola XL-
I Blue, E.
colt GM2163 and B1G8. XL- I Blue was used for the high transformation
efficiency. The
plasmid was prepared from transformed XL-1 Blue calls, and a small portion was
examined
by gel electrophoresis to ensure no visible amount of vector molecules without
the shuffled
DNA. GM2163 was used to prepare unmethylated DNA for electroporation
transformation of
BtG8. The transformed BIG8 that grew on tetracycline plates were picked onto
96-well plates
by robot. These plates were incubated until sporulation and cultures used as
seeds for assay
sample production. We used two-tier insect screening to obtain high throughput
The first tier
was to eliminate variants without any detectable activity. The first tier
assay samples were
produced in CYS liquid medium as described in a publication by Yamamoto
(Identification
of entomocidal toxins of Bacillus thuringiensis by high-performance liquid
chromatography.
in Analytical chemistry of Bacillus thuringiensis. ed. Hickle, LA. and Fitch,
W.L., American
Chemical Society, Washington DC, USA, 46-60,1990) in shallow, 96-well plates.
At this
stage, culture broth containing crystals and spores was assayed with neonate
H. zea larvae in
96-well plates containing an artificial insect diet. Those variants showing
the activity were
selected for the next step. For the second tier screening, the crystal
proteins were purified
from I ml culture broth produced in deep 96-well plates by differential
solubilization
between pH 10.5 and pH 4.4. The crystals were solubilized at pH 10.5 with 2% 2-
mercaptoethanol, and the solubilized crystal proteins were precipitated at pH
4.4. After
protein concentrations were determined, serial dilutions were made and assayed
against H.
zea larvae using the insect diet incorporation assay. After screening several
thousand variants,
we found a substantial number of proteins showing improved H. zea activity
over the parent
CrylAb. These improved variants were then tested against Spodoptera exigua.
[00112) Polypeptides that resulted from the single gene shuffling were
screened for
increased H. zea activity relative to wild type CrylAb. AR2 (SEQ ID NOS:1 and
2) and
AR6 (SEQ ID NOS:3 and 4) were identified as Cryl -derived polypeptides that
showed
32
CA 02763879 2012-01-05
improved activity against H. zea (Fig. 1). Activity of AR6 was further
investigated by
comparing relative inverse ECso values for protoxins of AR6, Cryl Ab, Cry 1
Ac, and Cryl Ca
on Heliothis virescens, Helicoverpa zea, and Spodoptera exigua (Fig. 2).
Purified Cryl Ab,
AR6, Cry] Ac, and CrylCa protoxins were introduced into the artificial diet at
six doses and
in 24 replicates to determine the EC5o of each protoxin against the three
insects. The
experiment was repeated three times and ECso values were expressed as an
average of the
three trials. The EC50 values were then converted to relative inverse values.
Since Cry] Ac
had the lowest EC5o (highest specific activity) on Heliothis virescens and
Helicoverpa zea it
was given a value of 1.0 for each of those respective insect pests. Other
protoxin samples
had higher EC50 values for both H. virescens and H. zea (lower specific
activity) and were
converted to values relative to that of CrylAc. Likewise CrylCa had the lowest
EC50 value
for Spodoptera exigua and so was given a relative value of 'I.0' on that pest.
EC50 values of
other protoxins were higher (lower specific activity) and were assigned a
lower relative value
for this pest. These data showed that AR6 has nearly twice the specific
activity as wild type
CrylAb for both H. zea and S. exigua (Fig. 2). A description of the amino acid
sequence
differences between the parent toxin Cryl Ab and the shuffled clones is
described in Table 3.
[001131 An additional single gene shuffling experiment was carried out to
improve the
Spodoptera activity of CrylCa. As was done for shuffling the crylAb gene, a
crylCa DNA
template was subjected to mutagenesis and DNA shuffling. Protein produced from
the
shuffled variants was screened for improved S. exigua activity. One of the
variants, CR62
(SEQ ID NOS: 7 and 8), was found to have a -3-fold improved ECso compared to
the wild
type CrylCa protein (Fig. 3).
Ex a 2: Construction of Synthetic CR62 Gene
[001141 The DNA sequences of CR62 and the parental gene, CrylCa, were modified
using
random codon usage to create fully synthetic plant expressible genes (SEQ ID
NO: 9 and
SEQ ID NO:3 1, respectively. Table 4 provides a description of the encoded
amino acid
sequence differences between these genes. Following construction of synthetic
CR62 and
CrylCa genes, the coding regions were cloned into binary vector behind a
strong constitutive
plant viral promoter and the subsequent plasmids transformed into
Agrobacterium
tumefaciens C58. These strains were tested for efficacy in planta using an
Agrobacterium
leaf infiltration based transient expression system followed by leaf disk
bioassays with
Spodoptera exigua. Using this assay it was shown that both genes expressed
insecticidal
activity although the shuffled CR62 gene performed better than the non-
shuffled wild type
33
CA 02763879 2012-01-05
parent (data not shown).
Example 3: Construction of Synthetic MR8' and AR6 Genes
1001151 The DNA sequence of AR6 was targeted for modification to create a
synthetic
version of the AR6 coding region (SEQ ID NOS: 5 and 6) as described for CR62
in section
6.2. However, in this instance only the 5' end of AR6 encoding the N-terminal
protoxin and
toxin domains were targeted for re-synthesis. This N-terminal encoding region
was spliced to
the already existing synthetic C-terminal protoxin encoding region from the
synthetic CR62
gene to form a complete protoxin gene for plant expression. In the process of
producing a
synthetic AR6 gene a precursor gene was constructed. This gene, termed
MR8'(SEQ ID
NO: 11), encodes eight amino acid residue differences from that of AR6 (SEQ ID
NO:6) in
the toxin portion and four amino acid differences in the protoxin portion of
the protein (Table
3).
Example 4: In plants testing of the Synthetic AR6 Gene
1001161 Following construction of synthetic MR8' and AR6 genes, the coding
regions
were cloned into a binary vector with a strong constitutive plant viral
promoter and the
subsequent plasmids transformed into Agrobacterium tumefaciena C58. These
strains were
tested for efficacy in planta using an Agrobacterium leaf infiltration based
transient
expression system followed by leaf disk insect bioassays. Both synthetic AR6
and MR8'
were expressed in the transient leaf assay as shown by Western Blot analysis
(Fig. 4).
1001171 To test for in planta activity, a leaf disk expressing a polypeptide
of interest was
provided to a pest. Following a 24-hour incubation period, the feeding
activity of the pest on
the leaf disk was determined by visual observation. Positive controls for H.
zea activity and
S. exigua activity were genes encoding Cry2Ab-like (*) polypeptide and CR62,
respectively.
The results showed that both synthetic AR6 and MRS' confer high-level
resistance to both H.
zea (Fig. 5A) and S. exigua (Fig. 5B). Leaf disks infiltrated with
Agrobacterium lacking a
Cry gene were completely consumed by the insect larvae during the assay period
(not
shown).
Example 5: Further Shuffling Using MR8' as Parent
1001181 To further improve the activity of MR8', a second round of DNA
shuffling was
performed using MRS' as the parent clone. Shuffling was performed on a
fragmented MR8'
DNA template by directing added sequence diversity with oligonucleotides. As
the MR8'
34
CA 02763879 2012-01-05
gene encodes a protoxin, shuffling was limited to the active toxin region that
is responsible
for the insecticidal properties. Two kinds of sequence diversity were used to
incorporate into
the shuffling reactions: phylogenetic and computer generated random diversity.
Phylogenetic
diversity originated from aligning first round hits AR6, MR8', and wild type
CrylAa,
CrylAb, CrylAc, CrylAd, CrylAe, and CrylAg polypeptides. Random diversity was
generated by choosing random amino acid positions and directing either
conservative or non-
conservative amino acid changes at those positions. Both kinds of diversity
were
incorporated into the parent MR8' gene and encoded protein on a domain by
domain basis.
Several libraries were constructed, each focusing on a selected type of
diversity and applied
to isolated toxin domain regions or the entire toxin region. Following DNA
shuffling each
PCR amplified library fragment was reintroduced into the remaining MR8'
protoxin fragment
by PCR stitching. The library of reconstructed protoxins was then cloned into
a pUC like
vector such that the Cry i-derived polypeptides were expressed in E. cola from
the LacZ
promoter.
1001191 In order to assess the activity of the Cryl-derived polypeptides
against H. zea,
high throughput screening using an artificial diet containing whole E. colt
cells expressing
each of the Cryl-derived polypeptides in an array format was performed (data
not shown).
Those variants having a high level of activity were then tested for in planta
activity. The
amino acid diversity present in the variants tested is shown in Table 5. The
amino acid
sequences of the shuffled toxin regions as well as nucleotide sequences
encoding each
protoxin are provided by SEQ ID NOS: 11-28.
1001201 To initiate the in planta assays, all highly active Cryl-derived
variants were
cloned into an Agrobacterium tumefaciens based plant expression vector. The
binary
plasmids were then transformed into a host Agrobacterium. The Cryl-derived
polypeptides
were then screened by co-cultivating each in four replicates with N.
benthamiana leaves
(using forced infiltration of each respective culture). Leaf disks were
excised from the
infiltrated leaf areas and infested with individual P instar H. zea or 4'b
instar S. exigua
larvae. After 24 hours feeding activity was determined by video capture of the
remaining leaf
area expressed in pixels.
1001211 Figure 6 shows the activity of the indicated Cryl-derived polypeptides
on H. zea.
Figure 7 shows the activity of the indicated Cryl-derived polypeptides on S.
exigua. All of
the tested Cry-1 derived polypeptides show improved activity against H. zea as
compared to
parent polypeptide MR8' while retaining activity against S. exigua that is at
least as good as
MR8'.
CA 02763879 2012-01-05
Table I : Cryl and Cry I-derived sequences
Variant name Full Protoxin Shuffled Mature Sequence Type SEQ ID NO
Region Region Toxin Region
AR2 1-3543 bp 1-2175 bp 85-1857 6 nucleic acid I
AR2 1-1181 as 1-725 as 29-619 as of a tide 2
AR6 1-3543 bp 1-2175 bp 85-1857 bp nucleic acid 3
1 AR6 1-1181 as 1-725 as 29-619 of a tide 4
Synthetic AR6 -3546 bp 1-2178 bp 88-1860 bp nucleic acid 5
Synthetic ARG 1-1182 as 1-726 as 30-620 as polypeptide 6
CR62 1-3567 bp 1-2199 bp 82-1590 bp nucleic acid 7
CR62 1-1189 as 1-733 as 28-630 as polypeptide 8
Svnthetic CR62 1-3567 bp 1-2199 bp 82-1890 bp nucleic acid 9
Synthetic CR62 1-1189 as 1-733 as 28-630 as polypeptide 10
MR8' 1-3546 b 88-1360 6 88-1860 bp nucleic acid I I
MR8' 1-1 182 as 30-620 au 30-620 as of a tide 12
giant =11 1-35,16 bp 88-1860 b 88-1860 1)13 nucleic: acid 13
Variant 41 1-1182 as 30-620 as 30-620 as of a tide 14
Variant 75 1-3516 bp 88-1860 bp 88-1860 hp nucleic acid 15
1 Variant 75 1-1 182 as 30-620 as 30-620 as of tide 16
Variant 80 1-3546 bp 88-1860 bp 88-1860 bp nucleic acid 17
Variant 80 I - 1182 as 30-620 as 30-620 as of a tide 18
Vnriant 85 1-3546 bp 88-1860 bp 88-1860 bp nucleic acid 19
Variant 85 I-1 182 as 30-620 as 30-620 as of a tide 20
Variant 88 1-3546 bp 88-1860 bp 88-1860 b .p acid 21
variant 88 1-I 182 as 30-620 as 30-620 as of a tide 22
Variant 90 1-3546 b 88-1860 bp 88-1860 bp nucleic acid 23
\t:tiant 90 1 1-1 182 as 30-620 as 30-620 as of a tide 24
V, runt 5-10 1-3546 bp 88-1860 bp 88-1860 bp nucleic acid 25
Variant 5-40 1-1182 as 30-620 as 30-620 as polypeptide 26
Variant 5-44 1-3546 bp 88-1860 b 88-1860 6 nucleic acid 27
Variant 5-44 1-1 182 as 30-620 as 30-620 as polypeptide 28
Crii(;a reference -- nucleic acid 29
Cr., I Ca reference of tide 30
Sy:;thetic Cry ICa 1-3567 bp i -- 32-1890 bp nucleic acid 31
S r,thatic ry ICa 1-1 189 as -- 28-630 as of a tide 32
Crvl Ab refcre:ice -- 85-1866 bp nucleic acid 33
Crv1 Ab reference 1-1 155 as -- 29-622 as of a tide 34
Crv2Ab-like (') 1-633 as -- polypeptide 35
rcl.;rcncc
1 CrvIAc refcreuce 1-1178 as I -- 29-623 of a tide 36
Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal
Proteins
N. Crickmore, D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus,
J. Baum, and
1).11. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813
36
CA 02763879 2012-01-05
Table : Codon Table
Amino acids Codon
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGO CGA CGC COG CGU
Serino Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACO ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine T Y UAC UAU
Table 3: Comparison of amino acid sequence differences between CrvlAb and 1st
round
shuffled hits
Amino
nos oiS"~~^ g>;I.'
Cryt ( 33 I V N P K L
AR2(SEQIONO:1) HTa- - - - - VD - TOPERNF - S- L- - E- - -
ONO:3) H T - - - - - - V 0 D T D P E R N - - S - - - - - - 0 - -
ARe (SEQI -
SynlhNloARa(SEOIONO:S) O H T- - - - - - V O D T D P E RN- - S- - D R. - - P P
MRa SEQIONO:11 - 0H- - V KT H A - - - T 0 P E R N - V - A - 0 R - - - P P
Amino acid alignments derived from translation of listed DNA sequences. A gap
at position 2
is inserted into non-synthetically derived amino acid sequences to accommodate
insertion of
a glycine residue at that position in the synthetically derived polypeptide
sequences. Thus, the
matching amino acid positions in SEQ IDNOs: 1, 3, and 33 would be one less
than each of
the above alignment coordinates beyond position 1.
Table 4: Comparison of amino acid sequence differences between Crvl Ca and
shuffled hit
clone CR62
Amino Acid n:
mno 124 248 294 312 485
Synthetic ry1Ca(SEQIDN0:31) I -T R D D I I
Synthetic CR62 (SEQIDN0:9) A A A G. L H V T
CR62 (SEQIDNO:T A A A G L H V T
Amino acid alignments derived from translation of listed DNA sequences.
37
CA 02763879 2012-01-05
Table 5: Comparison of amino acid sequence differences between 8-endotoxin
region for
CrylAb and 2nd round shuffled hits
Amity Add sition:
S uenc&Nams - - ! $
CrYIAD ( N R I Y Y I Y V N I P 9 V
MRr (SEOIONO:12) . - . . V = K T . - H - - A - . - - - - - - - . T 0 P E R N
a E41(3EOIONO.14) . . S . V = K . . . . . . . V . A - . = = - = - T 0 P E R N
a s d 7e (SEOION0:1S) V... V- K T.- H 0- A- -. N. ..... T 0 P E R N
aa* 00 (SEQIO"QIq . = . = V - K T = F H - I A = = - . = . = = = . T 0 P E R N -
wWA86(SHQION070) = V . . V V K T - - H - I A = - = . = . = = = . T O P E R N
WM as (SEOIONO:~ V K T H I A . = = - . - - . = - T 0 P E R N -
a
VaWM 90 (SEOIONO'34) =.-= V K T F F H I A -- - - -- T D P E R N-
VaIsl 5.40(SEOINO:2E) -.... V K T -- H . A - = V I V T O P E R N
a. $44 SEOION" = V = K T - - H - = A F=- Y V T 0 P E R N
Amino acid positions are relative to +1 being the first residue of the mature
toxin.
38
CA 02763879 2012-01-05
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