U.S. patent application number 16/468569 was filed with the patent office on 2019-12-05 for use of cry14 for the control of nematode pests.
This patent application is currently assigned to BASF Agricultural Solutions Seed US LLC. The applicant listed for this patent is BASF Agricultural Solutions Seed US LLC. Invention is credited to Julia Daum, Axel Elling.
Application Number | 20190364908 16/468569 |
Document ID | / |
Family ID | 61007812 |
Filed Date | 2019-12-05 |
United States Patent
Application |
20190364908 |
Kind Code |
A1 |
Daum; Julia ; et
al. |
December 5, 2019 |
USE OF CRY14 FOR THE CONTROL OF NEMATODE PESTS
Abstract
Compositions and methods for conferring nematicidal activity to
bacteria, plants, plant cells, tissues and seeds are provided. In
particular, methods for killing or controlling a nematode pest
population, particularly a Pratylenchus spp., e.g., Pratylenchus
brachyurus, root knot nematode, reniform nematode, or Lance
nematode population, are provided. The methods include contacting
the nematode pest with a pesticidally-effective amount of a
polypeptide comprising a nematicidal toxin, particularly a
nematicidal toxin active against a Pratylenchus spp. nematode, e.g.
Pratylenchus brachyurus. Further included are methods for
increasing yield in plants by expressing the toxin of the
invention.
Inventors: |
Daum; Julia; (Apex, NC)
; Elling; Axel; (Cary, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF Agricultural Solutions Seed US LLC |
Research Triangle Park |
NC |
US |
|
|
Assignee: |
BASF Agricultural Solutions Seed US
LLC
Research Triangle Park
NC
|
Family ID: |
61007812 |
Appl. No.: |
16/468569 |
Filed: |
December 22, 2017 |
PCT Filed: |
December 22, 2017 |
PCT NO: |
PCT/US2017/068070 |
371 Date: |
June 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62438420 |
Dec 22, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8285 20130101;
Y02A 40/164 20180101; A01N 63/10 20200101; C12N 15/8286 20130101;
C07K 14/325 20130101; A01N 37/46 20130101 |
International
Class: |
A01N 63/02 20060101
A01N063/02; C12N 15/82 20060101 C12N015/82; C07K 14/325 20060101
C07K014/325 |
Claims
1. A method for controlling a Pratylenchus spp. nematode pest
population comprising contacting said population with a
nematicidally-effective amount of a polypeptide comprising an amino
acid sequence having at least 95% sequence identity to the amino
acid sequence of SEQ ID NO: 1 or 2, wherein said polypeptide has
nematicidal activity against the Pratylenchus spp. nematode pest
population.
2. The method of claim 1, wherein said Pratylenchus spp. is
Pratylenchus brachyurus.
3. The method of claim 1, wherein said plant is a soybean
plant.
4. A method for protecting a plant from a Pratylenchus spp.
nematode pest, comprising expressing in a plant or cell thereof a
nucleotide sequence operably linked to a promoter capable of
directing expression of the nucleotide sequence in a plant cell,
wherein said nucleotide sequence is selected from the group
consisting of: a) the nucleotide sequence set forth in SEQ ID NO:3
or 4; and b) a nucleotide sequence that encodes a polypeptide
comprising an amino acid sequence having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 1 or 2, wherein
said polypeptide has nematicidal activity against said Pratylenchus
spp. nematode pest.
5. A method for increasing yield in a plant comprising growing in a
field a plant or a seed thereof having stably incorporated into its
genome a DNA construct comprising a nucleotide sequence operably
linked to a promoter capable of directing expression of the
nucleotide sequence in a plant cell, wherein said nucleotide
sequence is selected from the group consisting of: a) the
nucleotide sequence set forth in SEQ ID NO:3 or 4; and b) a
nucleotide sequence that encodes a polypeptide comprising an amino
acid sequence having at least 95% sequence identity to the amino
acid sequence of SEQ ID NO: 1 or 2, wherein said polypeptide has
nematicidal activity against a Pratylenchus spp. nematode pest;
wherein said field is infested with the Pratylenchus spp. nematode
pest.
6. The method of claim 5, wherein said Pratylenchus spp. is
Pratylenchus brachyurus.
7. The method of claim 4, wherein said plant further comprises one
or more nucleotide sequences encoding one or more insect toxins.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/438,420, filed Dec. 22, 2016, the contents
of which are herein incorporated by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named "APA16-6020WOSEQLIST.txt", created on Oct. 3,
2017, and having a size of 40 kilobytes and is filed concurrently
with the specification. The sequence listing contained in this
ASCII formatted document is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to the field of molecular biology.
Provided are methods for the control of nematode pests using
Cry14.
BACKGROUND OF THE INVENTION
[0004] Nematodes are active, flexible, elongate, organisms that
live on moist surfaces or in liquid environments, including films
of water within soil and moist tissues within other organisms. Many
species of nematodes have evolved to be very successful parasites
of plants and animals and are responsible for significant economic
losses in agriculture and livestock and for morbidity and mortality
in humans (Whitehead (1998) Plant Nematode Control. CAB
International, New York).
[0005] It is estimated that parasitic nematodes cost the
horticulture and agriculture industries in excess of $78 billion
worldwide a year, based on an estimated average 12% annual loss
spread across all major crops. For example, it is estimated that
nematodes cause soybean losses of approximately $3.2 billion
annually worldwide (Barker et al. (1994) Plant and Soil Nematodes:
Societal Impact and Focus for the Future. The Committee on National
Needs and Priorities in Nematology. Cooperative State Research
Service, US Department of Agriculture and Society of
Nematologists). In the high-acreage crop markets, nematode damage
is greatest in soybeans and cotton. There are however, dozens of
additional crops that suffer from significant nematode infestation
including potato, pepper, onion, citrus, coffee, sugarcane,
greenhouse ornamentals and golf course turf grasses.
[0006] Nematodes are known to affect the yield, growth, and health
of crops and plants. The physiological changes in the host plant's
roots caused by larvae and/or adult nematodes can lead to the
formation of galls, which causes a disruption of the vascular
system of the plant's roots. Root elongation can stop completely
and inadequate supply of water and nutrients provided by the
reduced root system can result, causing foliage chlorosis and/or
wilt, as well as stunting of growth, any of which can result in low
yield or death. In addition, nematodes can cause physiological
effects leading to an increase in the susceptibility of plant roots
to bacteria and/or fungi attack, including bacteria and/or fungi
the plant would otherwise resist. Such attack can lead to extensive
secondary decay and rotting.
[0007] The root lesion nematode Pratylenchus brachyurus has become
an increasingly important pathogen of soybean. It has a broad host
range and is widely distributed in tropical and subtropical
regions, especially in Brazil, Africa, and the Southern United
States. Pratylenchus brachyuruns has become a concern among cotton
and soybean growers in the Brazilian Cerrado region and is
considered the main nematode pathogen of soybean in the region. In
soybean, this nematode can reduce yields 30 to 50%, with greater
damage being observed on sandy soils. There are currently no P.
brachyurus-resistant soybean varieties identified to date. Although
several soybean genotypes have been studied for Pratylenchus
brachyuns resistance, and some cultivars identified with increased
tolerance, breeding resistant cultivars against P. brachyurus is
difficult due to the fact that this nematode is polyphagous and
lacks a close interaction with its hosts (Machado (2014) Current
Agricultural Science and Technology 20:26-35; Antonio et al. (2012)
Soil productivity losses in area infested by the nematoid of the
root lesions in Vera, Mont. In: Brazilian Congress of Soy, 6, 2012,
Cuiaba. Abstracts. Londrina: Embrapa Soja, 4pp; Rios et al. (2016)
Ci ncia Rural 46:580-584: Lima et al., 2017, Chapter 6 in the book:
Soybean--The Basis of Yield, Biomass and Productivity: Edited by
Minobu Kasai, ISBN 978-953-51-3118-2, Print ISBN 978-953-51-3117-5,
InTech; Inomoto et al. (2011) Sucessao de culturas sob pivo central
para controle de fitonematoides: variacao populacional,
patogenicidade e estimativa de perdas. Tropical Plant Pathology
36:178-185).
[0008] Methods for controlling infestations by nematodes have been
provided in several forms. Biological and cultural control methods,
including plant quarantines, have been attempted in numerous
instances. Genetic resistance to certain nematodes is available in
some commercial cultivars (e.g., soybeans), but these are
restricted in number and the availability of cultivars with both
desirable agronomic features and resistance is limited.
Furthermore, the production of nematode resistant commercial
varieties by conventional plant breeding based on genetic
recombination through sexual crosses is a slow process and is often
further hampered by a lack of appropriate germplasm.
[0009] Chemical means of controlling plant parasitic nematodes
continue to be essential for many crops which lack adequate natural
resistance. However, chemical agents are often not selective, and
some exert their effects on non-target organisms, effectively
disrupting populations of beneficial microorganisms, for a period
of time following application of the agent. Chemical agents may
persist in the environment and only be slowly metabolized.
[0010] Thus, there exists a need for additional means for
controlling nematode populations in agriculturally-important
plants.
SUMMARY OF INVENTION
[0011] Compositions and methods for conferring nematicidal activity
to plants, plant cells, tissues and seeds are provided. In
particular, methods for killing or controlling a nematode pest
population, particularly a lesion nematode such as Pratylenchus sp,
e.g., Pratylenchus brachyurus, population, are provided. The
invention further provides control of root knot nematode
(Meloidogyne spp. soybean pest nematodes, including but not limited
to Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla,
or Meloidogyne javanica, or any combination thereof), reniform
nematode (Rotylenchulus reniformis) and Lance nematode (Hoplolaimus
spp. such as H. columbus, H, galeatus, and H. magnistylus). The
methods comprise contacting the nematode pest with a
pesticidally-effective amount of a polypeptide comprising a
nematicidal toxin, particularly a nematicidal toxin active against
a Pratylenchus spp. nematode, e.g. Pratylenchus brachyurus, a root
knot nematode, a reniform nematode, or a Lance nematode. In various
embodiments, the nematicidal toxin comprises the amino acid
sequence of SEQ ID NO: 1 or 2, or pesticidally-effective variants
or fragments thereof. In some embodiments, the method for
protecting a plant or cell thereof from a nematode pest population,
particularly a Pratylenchus spp. nematode, e.g. Pratylenchus
brachyurus, a root knot nematode, a reniform nematode, or a Lance
nematode, comprises expressing in a plant or cell thereof a nucleic
acid encoding SEQ ID NO: 1 or 2, or a variant or fragment thereof,
wherein the nucleic acid is operably linked to a promoter capable
of directing expression of the nucleic acid in a plant cell.
[0012] Further comprised are methods for increasing yield in a
plant comprising growing in a field a plant or a seed thereof
having stably incorporated into its genome a DNA construct
comprising a nucleic acid operably linked to a promoter capable of
directing expression of the nucleic acid in a plant cell, wherein
the nucleic acid encodes SEQ ID NO: 1 or 2, or a
pesticidally-effective variant or fragment thereof.
[0013] The compositions and methods of the invention are useful for
the production of organisms with enhanced nematode, e.g.
Pratylenchus spp., root knot nematode, reniform nematode, or Lance
nematode resistance or tolerance. These organisms and compositions
comprising the organisms are desirable for agricultural
purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Pratylenchus resistance greenhouse assay in the
USA
Elite soybean plants with EE-GM4 control Pratylenchus brachyurus in
US greenhouse assays. Soybean plants expressing SEQ ID NO:2
("EE-GM4") were compared to other elite soybean lines: one SCN
susceptible Maturity Group (MG) 3 line ("THORNE"), one MG3 SCN
susceptible line, one MG 6.2 SCN susceptible line and one MG9 SCN
susceptible line ("Susc WT" shows the average for these 3 lines),
one MG3 SCN resistant line (with the rhg1 resistance allele from
P188788, "SCN Res (PI88788)"), and one MG 6.2 SCN resistant line
with the rhg1 and Rhg4 SCN resistance from Peking ("SCN Res
(Peking)"). Plotted are the average numbers of Pratylenchus in
roots 30 days after infestation (5 plants per entry), also showing
the variation observed across variaties (as typically seen in
greenhouse assays). Results show .about.85% control of Pratylenchus
across EE-GM4 lines. Soybean lines with native SCN resistance (from
Peking or PI88788) do not control Pratylenchus brachyurus.
[0015] FIG. 2. Pratylenchus resistance greenhouse assay in
Brazil
Soybean plants with EE-GM4 ("EE-GM4") significantly reduce
Pratylenchus brachyurus in soybean roots. Pratylenchus brachyurus
were isolated from local fields in Brazil. EE-GM4 plants (in two
different US elite lines (both maturity group 6.2, one
SCN-susceptible and one with Peking SCN-resistance ("EE-GM4")) and
five Brazilian soybean lines, with limited Pratylenchus control
("Brazil lines"), one Brazilian line, labeled as low Rf
(reproductive factor) for Pratylenchus ("BRS 7380 (low Rf)"), one
US elite line (maturity group 6.2) that is SCN-susceptible ("SCN
Susc") and one US elite line of MG 6.2 with Peking SCN-resistance
("SCN Res (Peking)") were evaluated for Pratylenchus control in a
greenhouse assay in Brazil. Plotted are the averages of those
entries, also showing the variation observed across varieties (as
typically seen in greenhouse assays). One Brazilian soybean line
(BRS 7380), showed .about..sub.89% reduction of Pratylenchus.
EE-GM4 lines gave .about.79% control of Pratylenchus. Soybean lines
that carry Peking native resistance to SCN do not control
Pratylenchus brachyurus.
DETAILED DESCRIPTION
[0016] The present invention is drawn to methods for regulating
nematode resistance in organisms, particularly plants or plant
cells. By "resistance" is intended that the nematode is killed upon
ingestion or other contact with the polypeptides of the invention
is impaired in the movement, feeding, reproduction, or other
functions of the nematode. Controlling plant-parasitic nematode
populations in a plant or seed thereof will improve nodulation,
germination, root development, emergence, and health, including
resistance to or protection from disease, including bacterial or
fungal disease, which is an important benefit of methods disclosed
and described herein. Thus, methods as described herein are useful
for controlling nematode populations, particularly Pratylenchus
spp. nematode populations, e.g., Pratylenchus brachyurus, root knot
nematode, reniform nematode, or Lance nematode, which provide
improved general plant health, nutrition and/or improved
agronomical benefit of a plant and/or seed. Any benefit related to
nematode population control, such as, for example, reduction in
total number/area of nematodes, reduction in nematode eggs/area, or
reduction in damage to the plant, can be an agronomical benefit of
the present invention. Secondary benefits of controlling the
nematode populations include, without limitation, improved root
development (e.g., improved root or root hair growth), improved
yield, faster emergence, improved plant stress management including
increased stress tolerance and/or improved recovery from stress,
increased mechanical strength, improved drought resistance, reduced
fungal disease infection, and improved plant health. Combinations
of any of these benefits can also be obtained.
[0017] The methods of the present invention involve transformation
of organisms or use of organisms comprising a heterologous
nucleotide sequence encoding a nematicidal protein of the
invention. The methods described herein are useful for controlling
or killing nematode pest populations and for producing compositions
with nematicidal activity against nematode pests.
[0018] By "pesticidal toxin" or "pesticidal protein," or
"nematicidal activity" or "nematicidal toxin" is intended a toxin
that has activity against one or more nematode pests, including,
but not limited to, Pratylenchus spp., including Pratylenchus
alleni, Pratylenchus brachyurus, Pratylenchus coffeae, Pratylenchus
crenatus, Pratylenchus dulscus, Pratylenchus fallax, Pratylenchus
flakkensis, Praylenchus goodeyi, Pratylenchus hexincisus,
Pratylenchus loosi, Pratylenchus minutus, Pratylenchus mulchandi,
Pratylenchus musicola, Pratylenchus neglectus, Pratylenchus
penetrans, Pratylenchus pratensts, Pratylenchus reniformia,
Pratylenchus scribneri, Pratylenchus thornei, Pratylenchus vulnus,
and Pratylenchus zeae, Nematicidal proteins include amino acid
sequences deduced from the full-length nucleotide sequences
disclosed herein, and amino acid sequences that are shorter than
the full-length sequences, either due to the use of an alternate
downstream start site, or due to processing that produces a shorter
protein having nematicidal activity. Processing may occur in the
organism the protein is expressed in, or in the pest after
ingestion of the protein.
[0019] In specific embodiments, the nematicidal protein comprises a
Cry14 protein. In various embodiments, the Cry14 protein is
Cry14Aa1 (GENBANK accession number AAA21516) or Cry14Ab1 (GENBANK
accession number KC 156652). In some embodiments, the Cry14Aa1
protein encompasses the amino acid sequence set forth in SEQ ID NO:
1, as well as variants and fragments thereof. In other embodiments,
the Cry14Ab1 protein encompasses the amino acid sequence set forth
in SEQ ID NO:2, as well as variants and fragments thereof.
Exemplary nucleotide sequences encoding SEQ ID NO: 1 are set forth
in SEQ ID NO:3 and 5. Exemplary nucleotide sequences encoding SEQ
ID NO:2 are set forth in SEQ ID NO:4 and 6.
[0020] Thus, provided herein are methods for killing or controlling
a nematode pest population, e.g. a Pratylenchus spp. population,
e.g., Pratylenchus brachyurus, root knot nematode, reniform
nematode, or Lance nematode, comprising contacting the nematode
pest, or exposing the nematode pest to, a composition comprising
the nematicidal toxin of the invention. In specific embodiments,
the nematicidal protein comprises the Cry14 protein set forth in
SEQ ID NO:1 or 2, as well as variants and fragments thereof.
Isolated Nucleic Acid Molecules, and Variants and Fragments
Thereof
[0021] One aspect of the invention pertains to isolated,
recombinant or chimeric nucleic acid molecules comprising
nucleotide sequences encoding nematicidal proteins and polypeptides
or biologically active portions thereof, as well as nucleic acid
molecules sufficient for use as hybridization probes to identify
nucleic acid molecules encoding proteins with regions of sequence
homology. Also encompassed herein are nucleotide sequences capable
of hybridizing to the nucleotide sequences of the invention under
stringent conditions as defined elsewhere herein. As used herein,
the term "nucleic acid molecule" is intended to include DNA
molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA
molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA. The term "recombinant" encompasses
polynucleotides or polypeptides that have been manipulated with
respect to the native polynucleotide or polypeptide, such that the
polynucleotide or polypeptide differs (e.g., in chemical
composition or structure) from what is occurring in nature. An
"isolated nucleic acid (sequence/molecule)" or "isolated DNA
(sequence/molecule)", as used herein, refers to a nucleic acid or
DNA (sequence/molecule) which is no longer in the natural
environment it was isolated from, e.g., the nucleic acid sequence
in another bacterial host or in a plant genome, or a nucleic acid
or DNA (sequence/molecule) fused to DNA or nucleic acid
(sequence/molecule) from another origin, such as when contained in
a chimeric gene under the control of a (heterologous)
plant-expressible promoter. Any nucleic acid or DNA of this
invention, including any primer, can also be
non-naturally-occurring, such as a nucleic acid or DNA with a
sequence identical to a sequence occurring in nature, but having a
label (missing from the naturally-occurring counterpart), or with a
sequence having at least one nucleotide addition or replacement or
at least one internal nucleotide deletion compared to a
naturally-existing nucleotide, or with a sequence having a sequence
identity below 100% (not identical) to a naturally-existing nucleic
acid or DNA or a fragment thereof, or a nucleic acid or DNA with a
sequence consisting of nucleotide sequences from different origins
that do not occur together in nature (a chimeric or hybrid DNA), or
a man-made synthetic nucleic acid or DNA with a sequence different
from the natural nucleic acid or DNA or a fragment thereof.
[0022] An isolated, recombinant or chimeric nucleic acid (or DNA)
is used herein to refer to a nucleic acid (or DNA) that is no
longer in its natural environment, for example in an in vitro or in
a recombinant bacterial or plant host cell. In some embodiments, an
isolated, recombinant or chimeric nucleic acid is free of sequences
(preferably protein encoding sequences) that naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For purposes of the invention, "isolated"
when used to refer to nucleic acid molecules excludes isolated
chromosomes. For example, in various embodiments, the isolated
delta-endotoxin encoding nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
In various embodiments, a delta-endotoxin protein that is
substantially free of cellular material includes preparations of
protein having less than about 30%, 20%, 10%, or 5% (by dry weight)
of non-delta-endotoxin protein (also referred to herein as a
"contaminating protein"). In some embodiments, the recombinant
nucleic acid of the invention comprises one or more nucleotide
substitutions relative to SEQ ID NO:3-6, or a variant or fragment
thereof.
[0023] Nucleotide sequences encoding the proteins of the present
invention include the sequence set forth in SEQ ID NO:3-6, and
variants, fragments, and complements thereof. By "complement" is
intended a nucleotide sequence that is sufficiently complementary
to a given nucleotide sequence such that it can hybridize to the
given nucleotide sequence to thereby form a stable duplex. The
corresponding amino acid sequences for the nematicidal proteins
encoded by these nucleotide sequences are set forth in SEQ ID NO: 1
and 2.
[0024] Nucleic acid molecules that are fragments of these
nucleotide sequences encoding nematicidal proteins are also
encompassed by the present invention. By "fragment" is intended a
portion of the nucleotide sequence encoding a nematicidal protein.
A fragment of a nucleotide sequence may encode a biologically
active portion of a nematicidal protein, or it may be a fragment
that can be used as a hybridization probe or PCR primer using
methods disclosed below. Nucleic acid molecules that are fragments
of a nucleotide sequence encoding a nematicidal protein comprise at
least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1350, 1400 contiguous nucleotides, or up to the
number of nucleotides present in a full-length nucleotide sequence
encoding a nematicidal protein disclosed herein, depending upon the
intended use. By "contiguous" nucleotides is intended nucleotide
residues that are immediately adjacent to one another. Fragments of
the nucleotide sequences of the present invention will encode
protein fragments that retain the biological activity of the
nematicidal protein and, hence, retain pesticidal activity against
a nematode pest. Thus, biologically-active fragments of the
polypeptides disclosed herein are also encompassed. By "retains
activity" is intended that the fragment will have at least about
30%, at least about 50%, at least about 70%, 80%, 90%, 95% or
higher of the pesticidal activity of the nematicidal protein.
Methods for measuring nematicidal activity are well known in the
art and are also described herein.
[0025] A fragment of a nucleotide sequence encoding a nematicidal
protein that encodes a biologically active portion of a protein of
the invention will encode at least about 15, 25, 30, 50, 75, 100,
125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or 1150 contiguous
amino acids, or up to the total number of amino acids present in a
full-length nematicidal protein of the invention. In some
embodiments, the fragment is a proteolytic cleavage fragment. For
example, the proteolytic cleavage fragment may have an N-terminal
or a C-terminal truncation of at least about 30 amino acids, at
least about 40 amino acids, at least about 50, at least about 100
amino acids, about 120, about 130, about 140, about 150, or about
160 amino acids relative to SEQ ID NO: 1 or 2. In some embodiments,
the fragments encompassed herein result from the removal of the
C-terminal crystallization domain, e.g., by proteolysis, or by
insertion of a stop codon in the coding sequence. In further
embodiments, the fragments encompassed herein comprise an
N-terminal truncation and the N-terminal truncations may comprise a
methionine residue at the truncated N-terminus.
[0026] In various embodiments, the nucleic acid of the invention
comprises a degenerate nucleic acid of SEQ ID NO:3-6, wherein said
degenerate nucleotide sequence encodes the same amino acid sequence
as SEQ ID NO: 1 or 2.
[0027] Preferred nematicidal proteins of the present invention are
encoded by a nucleotide sequence sufficiently identical to the
nucleotide sequence of SEQ ID NO:3-6, or the nematicidal proteins
are sufficiently identical to the amino acid sequence set forth in
SEQ ID NO: 1 or 2. By "sufficiently identical" is intended an amino
acid or nucleotide sequence that has at least about 60% or 65%
sequence identity, about 70% or 75% sequence identity, about 80% or
85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%/0, 96%,
97%, 98%, 99% or greater sequence identity compared to a reference
sequence using one of the alignment programs described herein using
standard parameters. One of skill in the art will recognize that
these values can be appropriately adjusted to determine
corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid
similarity, reading frame positioning, and the like.
[0028] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length. In another embodiment, the percent identity is
calculated across the entirety of the reference sequence (i.e., the
sequence disclosed herein as any of SEQ ID NO: 1-6). The percent
identity between two sequences can be determined using techniques
similar to those described below, with or without allowing gaps. In
calculating percent identity, typically exact matches are counted.
A gap, i.e. a position in an alignment where a residue is present
in one sequence but not in the other, is regarded as a position
with non-identical residues.
[0029] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A nonlimiting
example of a mathematical algorithm utilized for the comparison of
two sequences is the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm
is incorporated into the BLASTN and BLASTX programs of Altschul et
al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to pesticidal-like nucleic
acid molecules of the invention. BLAST protein searches can be
performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to nematicidal protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as
described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationships between molecules. See Altschul
et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and
PSI-Blast programs, the default parameters of the respective
programs (e.g., BLASTX and BLASTN) can be used. Alignment may also
be performed manually by inspection.
[0030] Another non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the ClustalW algorithm
(Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW
compares sequences and aligns the entirety of the amino acid or DNA
sequence, and thus can provide data about the sequence conservation
of the entire amino acid sequence. The ClustalW algorithm is used
in several commercially available DNA/amino acid analysis software
packages, such as the ALIGNX module of the Vector NTI Program Suite
(Invitrogen Corporation, Carlsbad, Calif.). After alignment of
amino acid sequences with ClustalW, the percent amino acid identity
can be assessed. A non-limiting example of a software program
useful for analysis of ClustalW alignments is GENEDOC.TM..
GENEDOC.TM. (Karl Nicholas) allows assessment of amino acid (or
DNA) similarity and identity between multiple proteins. Another
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller (1988)
CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN
program (version 2.0), which is part of the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys, Inc., 9685
Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN
program for comparing amino acid sequences, a PAM 120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0031] Unless otherwise stated. GAP Version 10, which uses the
algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48(3):443-453, will be used to determine sequence identity or
similarity using the following parameters: 0% identity and %
similarity for a nucleotide sequence using GAP Weight of 50 and
Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %
identity or % similarity for an amino acid sequence using GAP
weight of 8 and length weight of 2, and the BLOSUM62 scoring
program. Equivalent programs may also be used. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide residue matches and an identical percent sequence
identity when compared to the corresponding alignment generated by
GAP Version 10.
[0032] The invention also encompasses variant nucleic acid
molecules. "Variants" of the nematicidal protein encoding
nucleotide sequences include those sequences that encode the
nematicidal proteins disclosed herein but that differ
conservatively because of the degeneracy of the genetic code as
well as those that are sufficiently identical as discussed above.
Naturally occurring allelic variants can be identified with the use
of well-known molecular biology techniques, such as polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant nucleotide sequences also include synthetically
derived nucleotide sequences that have been generated, for example,
by using site-directed mutagenesis but which still encode the
nematicidal proteins disclosed in the present invention as
discussed below. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
pesticidal activity against a nematode pest. By "retains activity"
is intended that the variant will have at least about 30%, at least
about 50%, at least about 70%, or at least about 80% of the
pesticidal activity of the native protein. Methods for measuring
pesticidal activity against a nematode pest are well known in the
art and described elsewhere herein.
[0033] The skilled artisan will further appreciate that changes can
be introduced by mutation of the nucleotide sequences of the
invention thereby leading to changes in the amino acid sequence of
the encoded nematicidal proteins, without altering the biological
activity of the proteins. Thus, variant isolated nucleic acid
molecules can be created by introducing one or more nucleotide
substitutions, additions, or deletions into the corresponding
nucleotide sequence disclosed herein, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Such variant nucleotide sequences are also encompassed
by the present invention.
[0034] For example, conservative amino acid substitutions may be
made at one or more, predicted, nonessential amino acid residues. A
"nonessential" amino acid residue is a residue that can be altered
from the wild-type sequence of a nematicidal protein without
altering the biological activity, whereas an "essential" amino acid
residue is required for biological activity. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine).
[0035] Delta-endotoxins generally have five conserved sequence
domains, and three conserved structural domains (see, for example,
de Maagd et al. (2001) Trends Genetics 17:193-199). The first
conserved structural domain consists of seven alpha helices and is
involved in membrane insertion and pore formation. Domain II
consists of three beta-sheets arranged in a Greek key
configuration, and domain III consists of two antiparallel
beta-sheets in "jelly-roll" formation (de Maagd et al., 2001,
supra). Domains II and III are involved in receptor recognition and
binding, and are therefore considered determinants of toxin
specificity.
[0036] Amino acid substitutions may be made in nonconserved regions
that retain function. In general, such substitutions would not be
made for conserved amino acid residues, or for amino acid residues
residing within a conserved motif, where such residues are
essential for protein activity. Examples of residues that are
conserved and that may be essential for protein activity include,
for example, residues that are identical between all proteins
contained in an alignment of similar or related toxins to the
sequences of the invention (e.g., residues that are identical in an
alignment of homologous proteins). Examples of residues that are
conserved but that may allow conservative amino acid substitutions
and still retain activity include, for example, residues that have
only conservative substitutions between all proteins contained in
an alignment of similar or related toxins to the sequences of the
invention (e.g., residues that have only conservative substitutions
between all proteins contained in the alignment homologous
proteins). However, one of skill in the art would understand that
functional variants may have minor conserved or nonconserved
alterations in the conserved residues.
[0037] Alternatively, variant nucleotide sequences can be made by
introducing mutations randomly along all or part of the coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for ability to confer pesticidal activity
against a nematode pest to identify mutants that retain activity.
Following mutagenesis, the encoded protein can be expressed
recombinantly, and the activity of the protein can be determined
using standard assay techniques.
[0038] Using methods such as PCR, hybridization, and the like
corresponding pesticidal sequences can be identified, such
sequences having substantial identity to the sequences of the
invention (e.g., at least about 70%, at least about 75%, 80%, 85%,
90%, 95% or more sequence identity across the entirety of the
reference sequence) and having or conferring pesticidal activity
against a nematode pest. See, for example, Sambrook and Russell
(2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor
Laboratory Press. Cold Spring Harbor, N.Y.) and Innis, et al.
(1990) PCR Protocols: A Guide to Method and Applications (Academic
Press, NY).
[0039] In a hybridization method, all or part of the pesticidal
nucleotide sequence can be used to screen cDNA or genomic
libraries. Methods for construction of such cDNA and genomic
libraries are generally known in the art and are disclosed in
Sambrook and Russell, 2001, supra. The so-called hybridization
probes may be genomic DNA fragments, cDNA fragments, RNA fragments,
or other oligonucleotides, and may be labeled with a detectable
group such as .sup.32P, or any other detectable marker, such as
other radioisotopes, a fluorescent compound, an enzyme, or an
enzyme co-factor. Probes for hybridization can be made by labeling
synthetic oligonucleotides based on the known nematicidal
protein-encoding nucleotide sequence disclosed herein. Degenerate
primers designed on the basis of conserved nucleotides or amino
acid residues in the nucleotide sequence or encoded amino acid
sequence can additionally be used. The probe typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, at least about 25, at least about
50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of
nucleotide sequence encoding a nematicidal protein of the invention
or a fragment or variant thereof. Methods for the preparation of
probes for hybridization are generally known in the art and are
disclosed in Sambrook and Russell, 2001, supra herein incorporated
by reference.
[0040] For example, an entire nematicidal sequence disclosed
herein, or one or more portions thereof, may be used as a probe
capable of specifically hybridizing to corresponding nematicidal
protein-like sequences and messenger RNAs. To achieve specific
hybridization under a variety of conditions, such probes include
sequences that are unique and are preferably at least about 10
nucleotides in length, or at least about 20 nucleotides in length.
Such probes may be used to amplify corresponding pesticidal
sequences from a chosen organism or sample by PCR. This technique
may be used to isolate additional coding sequences from a desired
organism or as a diagnostic assay to determine the presence of
coding sequences in an organism. Hybridization techniques include
hybridization screening of plated DNA libraries (either plaques or
colonies; see, for example, Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press. Cold Spring Harbor, New York).
[0041] Thus, the present invention encompasses probes for
hybridization, as well as nucleotide sequences capable of
hybridization to all or a portion of a nucleotide sequence of the
invention (e.g., at least about 300 nucleotides, at least about
400, at least about 400, 450.500, 1000, 1200, 1500, 2000, 2500,
3000, 3500, or up to the full length of a nucleotide sequence
disclosed herein). Hybridization of such sequences may be carried
out under stringent conditions. By "stringent conditions" or
"stringent hybridization conditions" is intended conditions under
which a probe will hybridize to its target sequence to a detectably
greater degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, preferably less than 500 nucleotides in length.
[0042] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C. Optionally, wash buffers may comprise about 0.1% to
about 1% SDS. Duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours.
[0043] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization, and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with >90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution), it is preferred to increase the SSC concentration so
that a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes. Part I, Chapter 2
(Elsevier, New York), and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience. New York). See Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York).
Isolated Proteins and Variants and Fragments Thereof
[0044] Nematicidal proteins are also encompassed within the present
invention. By "nematicidal protein" is intended a protein having
the amino acid sequence set forth in SEQ ID NO: 1 or 2. Fragments,
biologically active portions, and variants thereof are also
provided, and may be used to practice the methods of the present
invention. An "isolated protein" or a "recombinant protein" is used
to refer to a protein that is no longer in its natural environment,
for example in vitro or in a recombinant bacterial or plant host
cell. In some embodiments, the recombinant protein is a variant of
SEQ ID NO: 1 or 2, wherein the variant comprises at least one amino
acid substitution, deletion, or insertion relative to SEQ ID NO: 1
or 2.
[0045] "Fragments" or "biologically active portions" include
polypeptide fragments comprising amino acid sequences sufficiently
identical to the amino acid sequence set forth in SEQ ID NO: 1 or
2, and that exhibit pesticidal activity against a nematode pest. A
biologically active portion of a nematicidal protein can be a
polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, or more amino
acids in length. Such biologically active portions can be prepared
by recombinant techniques and evaluated for pesticidal activity
against a nematode pest. Methods for measuring pesticidal activity
against a nematode pest are well known in the art (see, for
example, US Patent Application Publication No. US 20160066584) and
described elsewhere herein. As used here, a fragment comprises at
least 8 contiguous amino acids of SEQ ID NO: 1 or 2. The invention
encompasses other fragments, however, such as any fragment in the
protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300,
350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids
in length.
[0046] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 60%, 65%, about 70%,
75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to the amino acid sequence of any of SEQ ID
NO: 1 or 2. Variants also include polypeptides encoded by a nucleic
acid molecule that hybridizes to the nucleic acid molecule of SEQ
ID NO:3-6, or a complement thereof, under stringent conditions.
Variants include polypeptides that differ in amino acid sequence
due to mutagenesis. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
retaining pesticidal activity against a nematode pest. In some
embodiments, the variants have improved activity relative to the
native protein. Methods for measuring pesticidal activity against a
nematode pest are well known in the art (see, for example, US
Patent Application Publication No. US 20160066584) and described
elsewhere herein.
[0047] Bacterial genes quite often possess multiple methionine
initiation codons in proximity to the start of the open reading
frame. Often, translation initiation at one or more of these start
codons will lead to generation of a functional protein. These start
codons can include ATG codons. However, bacteria such as Bacillus
sp. also recognize the codon GTG as a start codon, and proteins
that initiate translation at GTG codons contain a methionine at the
first amino acid. On rare occasions, translation in bacterial
systems can initiate at a TTG codon, though in this event the TTG
encodes a methionine. Furthermore, it is not often determined a
priori which of these codons are used naturally in the bacterium.
Thus, it is understood that use of one of the alternate methionine
codons may also lead to generation of nematicidal proteins. These
nematicidal proteins are encompassed in the present invention and
may be used in the methods of the present invention. It will be
understood that, when expressed in plants, it will be necessary to
alter the alternate start codon to ATG for proper translation.
[0048] In various embodiments of the present invention, nematicidal
proteins include amino acid sequences deduced from the full-length
nucleotide sequences disclosed herein, and amino acid sequences
that are shorter than the full-length sequences due to the use of
an alternate downstream start site.
[0049] Antibodies to the polypeptides of the present invention, or
to variants or fragments thereof, are also encompassed. Methods for
producing antibodies are well known in the art (see, for example,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.; U.S. Pat. No.
4,196,265).
[0050] Thus, one aspect of the invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the invention and their homologs, fusions or
fragments. In a particularly preferred embodiment, the antibody
specifically binds to a protein having the amino acid sequence set
forth in SEQ ID NO: 1 or 2 or a fragment thereof. In another
embodiment, the antibody specifically binds to a fusion protein
comprising an amino acid sequence selected from the amino acid
sequence set forth in SEQ ID NO: 1 or 2 or a fragment thereof. In
various embodiments, the antibody that specifically binds to the
protein of the invention or a fusion protein comprising the protein
of the invention is a non-naturally occurring antibody.
[0051] Antibodies of the invention may be used to quantitatively or
qualitatively detect the protein or peptide molecules of the
invention, or to detect post translational modifications of the
proteins. As used herein, an antibody or peptide is said to
"specifically bind" to a protein or peptide molecule of the
invention if such binding is not competitively inhibited by the
presence of non-related molecules.
[0052] The antibodies of the invention may be contained within a
kit useful for detection of the protein or peptide molecules of the
invention. The invention further comprises a method of detecting
the protein or peptide molecule of the invention (particularly a
protein encoded by the amino acid sequence set forth in SEQ ID NO:
1 or 2, including variants or fragments thereof that are capable of
specifically binding to the antibody of the invention) comprising
contacting a sample with the antibody of the invention and
determining whether the sample contains the protein or peptide
molecule of the invention. Methods for utilizing antibodies for the
detection of a protein or peptide of interest are known in the
art.
Altered or Improved Variants
[0053] It is recognized that DNA sequences of a nematicidal protein
may be altered by various methods, and that these alterations may
result in DNA sequences encoding proteins with amino acid sequences
different than that encoded by a nematicidal protein of the present
invention. This protein may be altered in various ways including
amino acid substitutions, deletions, truncations, and insertions of
one or more amino acids of SEQ ID NO: 1 or 2, including up to about
2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,
about 10, about 15, about 20, about 25, about 30, about 35, about
40, about 45, about 50, about 55, about 60, about 65, about 70,
about 75, about 80, about 85, about 90, about 100, about 105, about
110, about 115, about 120, about 125, about 130, about 135, about
140, about 145, about 150, about 155, or more amino acid
substitutions, deletions or insertions. Methods for such
manipulations are generally known in the art. For example, amino
acid sequence variants of a nematicidal protein can be prepared by
mutations in the DNA. This may also be accomplished by one of
several forms of mutagenesis and/or in directed evolution. In some
aspects, the changes encoded in the amino acid sequence will not
substantially affect the function of the protein. Such variants
will possess the desired pesticidal activity against a nematode
pest. However, it is understood that the ability of a nematicidal
protein to confer pesticidal activity against a nematode pest may
be improved by the use of such techniques upon the compositions of
this invention. For example, one may express a nematicidal protein
in host cells that exhibit high rates of base misincorporation
during DNA replication, such as XL-1 Red (Stratagene, La Jolla,
Calif.). After propagation in such strains, one can isolate the DNA
(for example by preparing plasmid DNA, or by amplifying by PCR and
cloning the resulting PCR fragment into a vector), culture the
nematicidal protein mutations in a non-mutagenic strain, and
identify mutated genes with pesticidal activity against a nematode
pest, for example by performing an assay to test for pesticidal
activity against a nematode pest. Such assays can include
contacting plants with one or more pests and determining the
plant's ability to survive and/or cause the death of the pests.
See, for example, US Patent Application Publication No. US
20160066584).
[0054] Alternatively, alterations may be made to the protein
sequence of many proteins at the amino or carboxy terminus without
substantially affecting activity. This can include insertions,
deletions, or alterations introduced by modern molecular methods,
such as PCR, including PCR amplifications that alter or extend the
protein coding sequence by virtue of inclusion of amino acid
encoding sequences in the oligonucleotides utilized in the PCR
amplification. Alternatively, the protein sequences added can
include entire protein-coding sequences, such as those used
commonly in the art to generate protein fusions. Such fusion
proteins are often used to (1) increase expression of a protein of
interest (2) introduce a binding domain, enzymatic activity, or
epitope to facilitate either protein purification, protein
detection, or other experimental uses known in the art (3) target
secretion or translation of a protein to a subcellular organelle,
such as the periplasmic space of Gram-negative bacteria, or the
endoplasmic reticulum of eukaryotic cells, the latter of which
often results in glycosylation of the protein.
[0055] Variant nucleotide and amino acid sequences of the present
invention also encompass sequences derived from mutagenic and
recombinogenic procedures such as DNA shuffling. With such a
procedure, one or more different nematicidal protein coding regions
can be used to create a new nematicidal protein possessing the
desired properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynuclcotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. For example, using this approach, sequence motifs encoding a
domain of interest may be shuffled between a pesticidal gene of the
invention and other known pesticidal genes to obtain a new gene
coding for a protein with an improved property of interest, such as
an increased insecticidal activity. Strategies for such DNA
shuffling are known in the art. See, for example, Stemmer (1994)
Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature
370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438;
Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997)
Proc. Nat. Acad. Sci. USA 94:4504-4509: Crameri et al. (1998)
Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0056] Domain swapping or shuffling is another mechanism for
generating altered nematicidal proteins. Domains may be swapped
between nematicidal proteins, resulting in hybrid or chimeric
toxins with improved pesticidal activity against a nematode pest or
target spectrum. Methods for generating recombinant proteins and
testing them for pesticidal activity against a nematode pest are
well known in the art (see, for example, Naimov et al. (2001) Appl.
Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl.
Environ. Microbiol. 62:1537-1543: Ge et al. (1991) J. Biol. Chem.
266:17954-17958; Schnepf et al. (1990) J. Biol. Chem.
265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol.
65:2918-2925).
[0057] In yet another embodiment, variant nucleotide and/or amino
acid sequences can be obtained using one or more of error-prone
PCR, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, gene site saturation
mutagenesis, permutational mutagenesis, synthetic ligation
reassembly (SLR), recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, and the like.
Vectors
[0058] A pesticidal sequence of the invention may be provided in an
expression cassette for expression in a host cell of interest, e.g.
a plant cell or a microbe. By "plant expression cassette" is
intended a DNA construct that is capable of resulting in the
expression of a protein from an open reading frame in a plant cell.
Typically these contain a promoter and a coding sequence. Often,
such constructs will also contain a 3' untranslated region. Such
constructs may contain a "signal sequence" or "leader sequence" to
facilitate co-translational or post-translational transport of the
peptide to certain intracellular structures such as the chloroplast
(or other plastid), endoplasmic reticulum, or Golgi apparatus.
[0059] By "signal sequence" is intended a sequence that is known or
suspected to result in cotranslational or post-translational
peptide transport across the cell membrane. In eukaryotes, this
typically involves secretion into the Golgi apparatus, with some
resulting glycosylation. Insecticidal toxins of bacteria are often
synthesized as protoxins, which are protolytically activated in the
gut of the target pest (Chang (1987) Method Enzymol. 153:507-516).
In some embodiments of the present invention, the signal sequence
is located in the native sequence, or may be derived from a
sequence of the invention. By "leader sequence" is intended any
sequence that when translated, results in an amino acid sequence
sufficient to trigger co-translational transport of the peptide
chain to a subcellular organelle. Thus, this includes leader
sequences targeting transport and/or glycosylation by passage into
the endoplasmic reticulum, passage to vacuoles, plastids including
chloroplasts, mitochondria, and the like. Thus, further provided
herein is a polypeptide comprising an amino acid sequence of the
present invention that is operably linked to a heterologous leader
or signal sequence.
[0060] By "plant transformation vector" is intended a DNA molecule
that is necessary for efficient transformation of a plant cell.
Such a molecule may consist of one or more plant expression
cassettes, and may be organized into more than one "vector" DNA
molecule. For example, binary vectors are plant transformation
vectors that utilize two non-contiguous DNA vectors to encode all
requisite cis- and trans-acting functions for transformation of
plant cells (Hellens and Mullineaux (2000) Trends in Plant Science
5:446-451). "Vector" refers to a nucleic acid construct designed
for transfer between different host cells. "Expression vector"
refers to a vector that has the ability to incorporate, integrate
and express heterologous DNA sequences or fragments in a foreign
cell. The cassette will include 5' and/or 3' regulatory sequences
operably linked to a sequence of the invention. By "operably
linked" is intended a functional linkage between a promoter and a
second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in the same reading
frame. In some embodiments, the nucleotide sequence is operably
linked to a heterologous promoter capable of directing expression
of said nucleotide sequence in a host cell, such as a microbial
host cell or a plant host cell. The cassette may additionally
contain at least one additional gene to be cotransformed into the
organism. Alternatively, the additional gene(s) can be provided on
multiple expression cassettes.
[0061] In various embodiments, the nucleotide sequence of the
invention is operably linked to a heterologous promoter, e.g., a
plant promoter. "Promoter" refers to a nucleic acid sequence that
functions to direct transcription of a downstream coding sequence.
The promoter together with other transcriptional and translational
regulatory nucleic acid sequences (also termed "control sequences")
are necessary for the expression of a DNA sequence of interest.
[0062] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the pesticidal sequence to be
under the transcriptional regulation of the regulatory regions.
[0063] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., a promoter), a DNA sequence of the invention, and a
translational and transcriptional termination region (i.e.,
termination region) functional in plants. The promoter may be
native or analogous, or foreign or heterologous, to the plant host
and/or to the DNA sequence of the invention. Additionally, the
promoter may be the natural sequence or alternatively a synthetic
sequence. Where the promoter is "native" or "homologous" to the
plant host, it is intended that the promoter is found in the native
plant into which the promoter is introduced. Where the promoter is
"foreign" or "heterologous" to the DNA sequence of the invention,
it is intended that the promoter is not the native or naturally
occurring promoter for the operably linked DNA sequence of the
invention. The promoter may be inducible or constitutive. It may be
naturally-occurring, may be composed of portions of various
naturally-occurring promoters, or may be partially or totally
synthetic. Guidance for the design of promoters is provided by
studies of promoter structure, such as that of Harley and Reynolds
(1987) Nucleic Acids Res. 15:2343-2361. Also, the location of the
promoter relative to the transcription start may be optimized. See,
e.g., Roberts et al. (1979) Proc. Natl. Acad. Sci. USA, 76:760-764.
Many suitable promoters for use in plants are well known in the
art.
[0064] For instance, suitable constitutive promoters for use in
plants include: the promoters from plant viruses, such as the
peanut chlorotic streak caulimovirus (PCISV) promoter (U.S. Pat.
No. 5,850,019); the 35S promoter from cauliflower mosaic virus
(CaMV) (Odell et al. (1985) Nature 313:810-812); the 35S promoter
described in Kay et al. (1987) Science 236: 1299-1302: promoters of
Chlorella virus methyltransferase genes (U.S. Pat. No. 5,563,328)
and the full-length transcript promoter from figwort mosaic virus
(FMV) (U.S. Pat. No. 5,378,619); the promoters from such genes as
rice actin (McElroy et al. (1990) Plant Cell 2:163-171 and U.S.
Pat. No. 5,641,876); ubiquitin (Christensen et al. (1989) Plant
Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol.
Biol. 18:675-689) and Grefen et al. (2010) Plant J, 64:355-365;
pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588): MAS
(Velten et al. (1984) EABO J. 3:2723-2730 and U.S. Pat. No.
5,510,474); maize H3 histone (Lepetit et al. (1992) Mol. Gen.
Genet. 231:276-285 and Atanassova et al. (1992) Plant J.
2(3):291-300): Brassica napus ALS3 (PCT application WO97/41228); a
plant ribulose-biscarboxylase/oxygenase (RuBisCO) small subunit
gene; the circovirus (AU 689 311) or the Cassava vein mosaic virus
(CsVMV, U.S. Pat. No. 7,053,205); promoters from soybean (Pbdc6 or
Pbdc7, described in WO/2014/150449 or ubiquitin 3 promoter
described in U.S. Pat. Nos. 7,393,948 and 8,395,021): and promoters
of various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002;
5,102,796; 5,182,200; and 5,428,147).
[0065] Suitable inducible promoters for use in plants include: the
promoter from the ACE1 system which responds to copper (Mett et al.
(1993) PNAS 90:4567-4571): the promoter of the maize In2 gene which
responds to benzenesulfonamide herbicide safeners (Hershey et al.
(1991)Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994)Mol.
Gen. Genetics 243:32-38); and the promoter of the Tet repressor
from Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227:229-237).
Another inducible promoter for use in plants is one that responds
to an inducing agent to which plants do not normally respond. An
exemplary inducible promoter of this type is the inducible promoter
from a steroid hormone gene, the transcriptional activity of which
is induced by a glucocorticosteroid hormone (Schena et al. (1991)
Proc. Natl. Acad. Sci. USA 88:10421) or the recent application of a
chimeric transcription activator, XVE, for use in an estrogen
receptor-based inducible plant expression system activated by
estradiol (Zuo et al. (2000) Plant J., 24:265-273). Other inducible
promoters for use in plants are described in EP 332104, PCT WO
93/21334 and PCT WO 97/06269 which are herein incorporated by
reference in their entirety. Promoters composed of portions of
other promoters and partially or totally synthetic promoters can
also be used. See, e.g., Ni et al. (1995) Plant J. 7:661-676 and
PCT WO 95/14098 describing such promoters for use in plants.
[0066] In one embodiment of this invention, a promoter sequence
specific for particular regions or tissues of plants can be used to
express the nematicidal proteins of the invention, such as
promoters specific for seeds (Datla, R. et al., 1997, Biotechnology
Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378
A1), the phaseolin promoter, the glutenin promoter, the
helianthinin promoter (WO92/17580), the albumin promoter
(WV98/45460), the oleosin promoter (WO98/45461), the SAT1 promoter
or the SAT3 promoter (PCT/US98/06978).
[0067] Use may also be made of an inducible promoter advantageously
chosen from the phenylalanine ammonia lyase (PAL), HMG-CoA
reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI),
PR1 family gene, nopaline synthase (nos) and vspB promoters (U.S.
Pat. No. 5,670,349, Table 3), the HMG2 promoter (U.S. Pat. No.
5,670,349), the apple beta-galactosidase (ABG1) promoter and the
apple aminocyclopropane carboxylate synthase (ACC synthase)
promoter (WO98/45445). Multiple promoters can be used in the
constructs of the invention, including in succession.
[0068] The promoter may include, or be modified to include, one or
more enhancer elements. In some embodiments, the promoter may
include a plurality of enhancer elements. Promoters containing
enhancer elements provide for higher levels of transcription as
compared to promoters that do not include them. Suitable enhancer
elements for use in plants include the PCISV enhancer element (U.S.
Pat. No. 5,850,019), the CaMV 35S enhancer element (U.S. Pat. Nos.
5,106,739 and 5,164,316) and the FMV enhancer element (Maiti et al.
(1997) Transgenic Res. 6:143-156), the translation activator of the
tobacco mosaic virus (TMV) described in Application WO87/07644, or
of the tobacco etch virus (TEV) described by Carrington & Freed
1990. J. Virol. 64: 1590-1597, for example, or introns such as the
adh1 intron of maize or intron 1 of rice actin. See also PCT
WO96/23898, WO2012/021794, WO2012/021797, WO2011/084370, and
WO2011/028914.
[0069] Often, such constructs can contain 5' and 3' untranslated
regions. Such constructs may contain a "signal sequence" or "leader
sequence" to facilitate co-translational or post-translational
transport of the peptide of interest to certain intracellular
structures such as the chloroplast (or other plastid), endoplasmic
reticulum, or Golgi apparatus, or to be secreted. For example, the
construct can be engineered to contain a signal peptide to
facilitate transfer of the peptide to the endoplasmic reticulum. By
"signal sequence" is intended a sequence that is known or suspected
to result in cotranslational or post-translational peptide
transport across the cell membrane. In eukarotes, this typically
involves secretion into the Golgi apparatus, with some resulting
glycosylation. By "leader sequence" is intended any sequence that,
when translated, results in an amino acid sequence sufficient to
trigger co-translational transport of the peptide chain to a
sub-cellular organelle. Thus, this includes leader sequences
targeting transport and/or glycosylation by passage into the
endoplasmic reticulum, passage to vacuoles, plastids including
chloroplasts, mitochondria, and the like. It may also be preferable
to engineer the plant expression cassette to contain an intron,
such that mRNA processing of the intron is required for
expression.
[0070] By "3' untranslated region" is intended a polynucleotide
located downstream of a coding sequence. Polyadenylation signal
sequences and other sequences encoding regulatory signals capable
of affecting the addition of polyadenylic acid tracts to the 3' end
of the mRNA precursor are 3' untranslated regions. By "5'
untranslated region" is intended a polynucleotide located upstream
of a coding sequence.
[0071] Other upstream or downstream untranslated elements include
enhancers. Enhancers are polynucleotides that act to increase the
expression of a promoter region. Enhancers are well known in the
art and include, but are not limited to, the SV40 enhancer region
and the 35S enhancer element.
[0072] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, may be native with the plant host,
or may be derived from another source (i.e., foreign or
heterologous to the promoter, the DNA sequence of interest, the
plant host, or any combination thereof). Convenient termination
regions are available from the Ti-plasmid of A. tumefaciens, such
as the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272: Munroe et
al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res.
15:9627-9639.
[0073] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed host cell (synthetic DNA
sequence). That is, the genes can be synthesized using host
cell-preferred codons for improved expression, or may be
synthesized using codons at a host-preferred codon usage frequency.
Expression of the open reading frame of the synthetic DNA sequence
in a cell results in production of the polypeptide of the
invention. Synthetic DNA sequences can be useful to simply remove
unwanted restriction endonuclease sites, to facilitate DNA cloning
strategies, to alter or remove any potential codon bias, to alter
or improve GC content, to remove or alter alternate reading frames,
and/or to alter or remove intron/exon splice recognition sites,
polyadenylation sites. Shine-Delgamo sequences, unwanted promoter
elements and the like that may be present in a native DNA sequence.
Generally, the GC content of the gene will be increased. See, for
example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a
discussion of host-preferred codon usage. Methods are available in
the art for synthesizing plant-preferred genes. See, for example,
U.S. Pat. Nos. 5,380,831, and 5,436,391, U.S. Patent Publication
No. 20090137409, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0074] It is also possible that synthetic DNA sequences may be
utilized to introduce other improvements to a DNA sequence, such as
introduction of an intron sequence, creation of a DNA sequence that
in expressed as a protein fusion to organelle targeting sequences,
such as chloroplast transit peptides, apoplast/vacuolar targeting
peptides, or peptide sequences that result in retention of the
resulting peptide in the endoplasmic reticulum. Thus, in one
embodiment, the nematicidal protein is targeted to the chloroplast
for expression. In this manner, where the nematicidal protein is
not directly inserted into the chloroplast, the expression cassette
will additionally contain a nucleic acid encoding a transit peptide
to direct the nematicidal protein to the chloroplasts. Such transit
peptides are known in the art. See, for example, Von Heijne et al.
(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.
Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant
Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.
Commun. 196:1414-1421: and Shah et al. (1986) Science
233:478-481.
[0075] The pesticidal gene to be targeted to the chloroplast may be
optimized for expression in the chloroplast to account for
differences in codon usage between the plant nucleus and this
organelle. In this manner, the nucleic acids of interest may be
synthesized using chloroplast-preferred codons. See, for example,
U.S. Pat. No. 5,380,831, herein incorporated by reference.
Plant Transformation
[0076] Methods of the invention involve introducing a nucleotide
construct into a plant. By "introducing" is intended to present to
the plant the nucleotide construct in such a manner that the
construct gains access to the interior of a cell of the plant. The
methods of the invention do not require that a particular method
for introducing a nucleotide construct to a plant is used, only
that the nucleotide construct gains access to the interior of at
least one cell of the plant. Methods for introducing nucleotide
constructs into plants are known in the art including, but not
limited to, stable transformation methods, transient transformation
methods, and virus-mediated methods.
[0077] By "plant" is intended whole plants, plant organs (e.g.,
leaves, stems, roots, etc.), seeds, plant cells, propagules,
embryos and progeny of the same. Plant cells can be differentiated
or undifferentiated (e.g. callus, suspension culture cells,
protoplasts, leaf cells, root cells, phloem cells, pollen).
[0078] "Transgenic plants" or "transformed plants" or "stably
transformed" plants or cells or tissues refers to plants that have
incorporated or integrated exogenous nucleic acid sequences or DNA
fragments into the plant cell. These nucleic acid sequences include
those that are exogenous, or not present in the untransformed plant
cell, as well as those that may be endogenous, or present in the
untransformed plant cell. "Heterologous" generally refers to the
nucleic acid sequences that are not endogenous to the cell or part
of the native genome in which they are present, and have been added
to the cell by infection, transfection, microinjection,
electroporation, microprojection, or the like.
[0079] The transgenic plants of the invention express one or more
of the novel toxin sequences disclosed herein. In some embodiments,
the protein or nucleotide sequence of the invention is
advantageously combined in plants with other genes which encode
proteins or RNAs that confer useful agronomic properties to such
plants. Among the genes which encode proteins or RNAs that confer
useful agronomic properties on the transformed plants, mention can
be made of the DNA sequences encoding proteins which confer
tolerance to one or more herbicides, and others which confer
tolerance to certain insects, those which confer tolerance to
certain diseases. DNAs that encodes RNAs that provide nematode or
insect control, and the like. Such genes are in particular
described in published PCT Patent Applications WO91/02071 and
WO95/06128 and in U.S. Pat. No. 7,923,602 and US Patent Application
Publication No. 20100166723, each of which is herein incorporated
by reference in its entirety. In various embodiments, the
transgenic plant further comprises one or more additional genes for
insect resistance (e.g., Cry1, such as members of the Cry1A, Cry1B,
Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as members of
the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C,
Cry9D. Cry9E, and Cry9F families; etc.). It will be understood by
one of skill in the art that the transgenic plant may comprise any
gene imparting an agronomic trait of interest.
[0080] Among the DNA sequences encoding proteins which confer
tolerance to certain herbicides on the transformed plant cells and
plants, mention can be made of a bar or PAT gene or the
Streptomyces coelicolor gene described in WO2009/152359 which
confers tolerance to glufosinate herbicides, a gene encoding a
suitable EPSPS which confers tolerance to herbicides having EPSPS
as a target, such as glyphosate and its salts (U.S. Pat. Nos.
4,535,060, 4,769,061, 5,094,945, 4,940,835, 5,188,642, 4,971,908,
5,145,783, 5,310,667, 5,312,910, 5,627,061, 5,633,435), a gene
encoding glyphosate-n-acetyltransferase (for example, U.S. Pat.
Nos. 8,222,489, 8,088,972, 8,044,261, 8,021,857, 8,008,547,
7,999,152, 7,998,703, 7,863,503, 7,714,188, 7,709,702, 7,666,644,
7,666,643, 7,531,339, 7,527,955, and 7,405,074), a gene encoding
glyphosate oxydoreductase (for example, U.S. Pat. No. 5,463,175),
or a gene encoding an HPPD inhibitor-tolerant protein (for example,
the HPPD inhibitor tolerance genes described in WO 2004/055191, WO
199638567, U.S. Pat. No. 6,791,014, WO2011/068567. WO2011/076345,
WO2011/085221, WO2011/094205, WO2011/068567, WO2011/094199,
WO2011/094205, WO02011/145015, WO2012/056401, and
WO2014/043435).
[0081] Among the DNA sequences encoding a suitable EPSPS which
confer tolerance to the herbicides which have EPSPS as a target,
mention will more particularly be made of the gene which encodes a
plant EPSPS, in particular maize EPSPS, particularly a maize EPSPS
which comprises two mutations, particularly a mutation at amino
acid position 102 and a mutation at amino acid position 106
(WO2004/074443), and which is described in Patent Application U.S.
Pat. No. 6,566,587, hereinafter named double mutant maize EPSPS or
2mEPSPS, or the gene which encodes an EPSPS isolated from
Agrobacterium and which is described by sequence ID No. 2 and
sequence ID No. 3 of U.S. Pat. No. 5,633,435, also named CP4.
[0082] Among the DNA sequences encoding a suitable EPSPS which
confer tolerance to the herbicides which have EPSPS as a target,
mention will more particularly be made of the gene which encodes an
EPSPS GRG23 from Arthrobacter globiformis, but also the mutants
GRG23 ACE 1, GRG23 ACE2, or GRG23 ACE3, particularly the mutants or
variants of GRG23 as described in WO2008/100353, such as
GRG23(ace3)R173K of SEQ ID No. 29 in WO2008/100353.
[0083] In the case of the DNA sequences encoding EPSPS, and more
particularly encoding the above genes, the sequence encoding these
enzymes is advantageously preceded by a sequence encoding a transit
peptide, in particular the "optimized transit peptide" described in
U.S. Pat. No. 5,510,471 or 5,633,448.
[0084] Exemplary herbicide tolerance traits that can be combined
with the nucleic acid sequence of the invention further include at
least one ALS (acetolactate synthase) inhibitor (WO2007/024782); a
mutated Arabidopsis ALS/AHAS gene (U.S. Pat. No. 6,855,533); genes
encoding 2,4-D-monooxygenases conferring tolerance to 2,4-D
(2,4-dichlorophenoxyacetic acid) by metabolization (U.S. Pat. No.
6,153,401); and, genes encoding Dicamba monooxygenases conferring
tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) by
metabolization (US 2008/0119361 and US 2008/0120739).
[0085] In various embodiments, the nucleic acid of the invention is
stacked with one or more herbicide tolerant genes, including one or
more HPPD inhibitor herbicide tolerant genes, and/or one or more
genes tolerant to glyphosate and/or glufosinate.
[0086] Among the DNA sequences encoding proteins concerning
properties of tolerance to insects, mention will more particularly
be made of the Bt proteins widely described in the literature and
well known to those skilled in the art. Mention will also be made
of proteins extracted from bacteria such as Photorhabdus
(WO97/17432 & WO98/08932).
[0087] Among such DNA sequences encoding proteins of interest which
confer novel properties of tolerance to insects, mention will more
particularly be made of the Bt Cry or VIP proteins widely described
in the literature and well known to those skilled in the art. These
include the Cry1F protein or hybrids derived from a Cry1F protein
(e.g., the hybrid Cry1A-Cry1F proteins described in U.S. Pat. Nos.
6,326,169; 6,281,016; 6,218,188, or toxic fragments thereof), the
Cry1A-type proteins or toxic fragments thereof, preferably the
Cry1Ac protein or hybrids derived from the Cry1Ac protein (e.g.,
the hybrid Cry1Ab-Cry1Ac protein described in U.S. Pat. No.
5,880,275) or the Cry1Ab or Bt2 protein or insecticidal fragments
thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Ag
proteins as described in WO2002/057664 or toxic fragments thereof,
the Cry1A. 105 protein described in WO 2007/140256 (SEQ ID No. 7)
or a toxic fragment thereof, the VIP3Aa19 protein of NCBI accession
ABG20428, the VIP3Aa20 protein of NCBI accession ABG20429 (SEQ ID
No. 2 in WO 2007/142840), the VIP3A proteins produced in the COT202
or COT203 cotton events (WO2005/054479 and WO02005/054480,
respectively), the Cry proteins as described in WO2001/47952, the
VIP3Aa protein or a toxic fragment thereof as described in Estruch
et al. (1996). Proc Natl Acad Sci USA. 28; 93(11):5389-94 and U.S.
Pat. No. 6,291,156, the insecticidal proteins from Xenorhabdus (as
described in WO98/50427), Serratia (particularly from S.
entomophila) or Photorhabdus species strains, such as Tc-proteins
from Photorhabdus as described in WO98/08932 (e.g., Waterfield et
al., 2001, Appl Environ Microbiol. 67(11):5017-24; Ffrench-Constant
and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33). Also any
variants or mutants of any one of these proteins differing in some
(1-10, preferably 1-5) amino acids from any of the above sequences,
particularly the sequence of their toxic fragment, or which are
fused to a transit peptide, such as a plastid transit peptide, or
another protein or peptide, is included herein.
[0088] In various embodiments, the nucleic acid of the invention
can be combined in plants with one or more genes conferring a
desirable trait, such as herbicide tolerance, insect tolerance,
drought tolerance, nematode control, water use efficiency, nitrogen
use efficiency, improved nutritional value, disease resistance,
improved photosynthesis, improved fiber quality, stress tolerance,
improved reproduction, and the like.
[0089] Particularly useful transgenic events which may be combined
with the genes of the current invention in plants of the same
species (e.g., by crossing or by re-transforming a plant containing
another transgenic event with a chimeric gene of the invention),
include Event BPS-CV127-9 (soybean, herbicide tolerance, deposited
as NCIMB No. 41603, described in WO2010/080829); Event
DAS21606-3/1606 (soybean, herbicide tolerance, deposited as
PTA-11028, described in WO2012/033794), Event
DAS-44406-6/pDAB8264.44.06.1 (soybean, herbicide tolerance,
deposited as PTA-11336, described in WO2012/075426). Event
DAS-14536-7/pDAB8291.45.36.2 (soybean, herbicide tolerance,
deposited as PTA-11335, described in WO2012/075429), Event DAS68416
(soybean, herbicide tolerance, deposited as ATCC PTA-10442,
described in WO2011/066384 or WO2011/066360); Event DP-305423-1
(soybean, quality trait, not deposited, described in US-A
2008-312082 or WO2008/054747); Event DP-356043-5 (soybean,
herbicide tolerance, deposited as ATCC PTA-8287, described in US-A
2010-0184079 or WO2008/002872); Event FG72 (soybean, herbicide
tolerance, deposited as PTA-11041, described in WO2011/063413),
Event LL27 (soybean, herbicide tolerance, deposited as NCIMB41658,
described in WO2006/108674 or US-A 2008-320616); Event LL55
(soybean, herbicide tolerance, deposited as NCIMB 41660, described
in WO 2006/108675 or US-A 2008-196127); Event MON87701 (soybean,
insect control, deposited as ATCC PTA-8194, described in US-A
2009-130071 or WO2009/064652): Event MON87705 (soybean, quality
trait--herbicide tolerance, deposited as ATCC PTA-9241, described
in US-A 2010-0080887 or WO2010/037016); Event MON87708 (soybean,
herbicide tolerance, deposited as ATCC PTA-9670, described in
WO2011/034704); Event MON87712 (soybean, yield, deposited as
PTA-10296, described in WO2012/051199). Event MON87754 (soybean,
quality trait, deposited as ATCC PTA-9385, described in
WO2010/024976); Event MON87769 (soybean, quality trait, deposited
as ATCC PTA-8911, described in US-A 2011-0067141 or WO2009/102873);
Event MON89788 (soybean, herbicide tolerance, deposited as ATCC
PTA-6708, described in US-A 2006-282915 or WO2006/130436); Event
SYHTOH2/SYN-000H2-5 (soybean, herbicide tolerance, deposited as
PTA-11226, described in WO2012/082548), event EE-GM3/FG72 (soybean,
herbicide tolerance, ATCC Accession No PTA-11041) optionally
stacked with event EE-GM1/LL27 or event EE-GM2/LL55
(WO2011/063413A2); Event DAS-68416-4 (soybean, herbicide tolerance,
ATCC Accession No PTA-10442, WO2011/066360A1); Event DAS-68416-4
(soybean, herbicide tolerance, ATCC Accession No PTA-10442,
WO2011/066384A 1), Event DAS-21606-3 (soybean, herbicide tolerance,
ATCC Accession No. PTA-11028, WO2012/033794A2); Event MON-87712-4
(soybean, quality trait, ATCC Accession NO. PTA-10296,
WO2012/051199A2); Event DAS-44406-6 (soybean, stacked herbicide
tolerance, ATCC Accession NO. PTA-11336, WO2012/075426A1); Event
DAS-14536-7 (soybean, stacked herbicide tolerance, ATCC Accession
NO. PTA-11335, WO2012/075429A1); Event SYN-000H2-5 (soybean,
herbicide tolerance. ATCC Accession NO. PTA-11226,
WO2012/082548A2); Event 8264.44.06.1 (soybean, stacked herbicide
tolerance, Accession No PTA-11336, WO2012075426A2); Event
8291.45.36.2 (soybean, stacked herbicide tolerance, Accession NO.
PTA-11335, WO2012075429A2): Event SYHTOH2 (soybean, ATCC Accession
NO. PTA-11226. WO2012/082548A2); Event pDAB8264.42.32.1 (soybean,
stacked herbicide tolerance, ATCC Accession No PTA-11993,
WO2013/010094A 1).
[0090] Further, provided herein is a method for producing a soybean
plant or seed comprising a nucleotide sequence encoding SEQ ID NO:
1 or 2 combined with another SCN resistance locus/gene, such as by
combining a soybean plant or seed comprising a nucleotide sequence
encoding SEQ ID NO:1 or 2 with another SCN resistance locus/gene
occurring in the same soybean plant/seed, and planting seed
comprising a nucleotide sequence encoding SEQ ID NO:1 or 2 and said
other SCN resistance locus/gene. In one embodiment, the plants,
cells or seeds of the invention contain one or more other SCN
resistance loci/genes that occur in soybean, to get a combination
of different SCN resistance sources in the soybean plants, cells or
seeds of the invention. Several soybean SCN resistance loci or
genes are known and one or more of those can be combined with a
plant comprising SEQ ID NO: 1 or 2 in the same plant, cell or seed,
such as any one of the SCN resistance genes/loci from the
resistance sources PI 88788, PI 548402 (Peking), PI 437654 (Hartwig
or CystX.RTM.), or any combination thereof, or one or more of the
native SCN resistance loci/genes rhg1, rhg1-b, rhg2, rhg3, Rhg4,
Rhg5, qSCN 11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any
of the SCN resistance loci identified on any one of soybean
chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 or any combination thereof (Kim et al. 2016, Theor.
Appl. Genet. 129(12):2295-2311; Kim and Diers 2013, Crop Science
53:775-785; Kazi et al. 2010, Theor. Appl. Gen. 120(3):633-644;
Glover et al. 2004, Crop Science 44(3):936-941: www.soybase.org:
Concibido et al. 2004, Crop Science 44:1121-1131: Webb et al. 1995,
Theor. Appl. Genet. 91:574-581). Also, in one embodiment the plants
or seeds of the invention are combined with one or more SCN
resistance loci in soybean obtained from any one of SCN resistance
sources PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI
88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772,
Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471. PI
633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI
22897, or PI 494182.
[0091] Transformation of plant cells can be accomplished by one of
several techniques known in the art. The pesticidal gene of the
invention may be modified to obtain or enhance expression in plant
cells. Typically a construct that expresses such a protein would
contain a promoter to drive transcription of the gene, as well as a
3' untranslated region to allow transcription termination and
polyadenylation. The organization of such constructs is well known
in the art. In some instances, it may be useful to engineer the
gene such that the resulting peptide is secreted, or otherwise
targeted within the plant cell. For example, the gene can be
engineered to contain a signal peptide to facilitate transfer of
the peptide to the endoplasmic reticulum. It may also be preferable
to engineer the plant expression cassette to contain an intron,
such that mRNA processing of the intron is required for
expression.
[0092] Typically this "plant expression cassette" will be inserted
into a "plant transformation vector". This plant transformation
vector may be comprised of one or more DNA vectors needed for
achieving plant transformation. For example, it is a common
practice in the art to utilize plant transformation vectors that
are comprised of more than one contiguous DNA segment. These
vectors are often referred to in the art as "binary vectors."
Binary vectors as well as vectors with helper plasmids are most
often used for Agrobacterium-mediated transformation, where the
size and complexity of DNA segments needed to achieve efficient
transformation is quite large, and it is advantageous to separate
functions onto separate DNA molecules. Binary vectors typically
contain a plasmid vector that contains the cis-acting sequences
required for T-DNA transfer (such as left border and right border),
a selectable marker that is engineered to be capable of expression
in a plant cell, and a "gene of interest" (a gene engineered to be
capable of expression in a plant cell for which generation of
transgenic plants is desired). Also present on this plasmid vector
are sequences required for bacterial replication. The cis-acting
sequences are arranged in a fashion to allow efficient transfer
into plant cells and expression therein. For example, the
selectable marker gene and the pesticidal gene are located between
the left and right borders. Often a second plasmid vector contains
the trans-acting factors that mediate T-DNA transfer from
Agrobacterium to plant cells. This plasmid often contains the
virulence functions (Vir genes) that allow infection of plant cells
by Agrobacterium, and transfer of DNA by cleavage at border
sequences and vir-mediated DNA transfer, as is understood in the
art (Hellens and Mullineaux (2000) Trends in Plant Science
5:446-451). Several types of Agrobacterium strains (e.g. LBA4404,
GV3101, EHA101, EHA105, etc.) can be used for plant transformation.
The second plasmid vector is not necessary for transforming the
plants by other methods such as microprojection, microinjection,
electroporation, polyethylene glycol, etc.
[0093] In general, plant transformation methods involve
transferring heterologous DNA into target plant cells (e.g.
immature or mature embryos, suspension cultures, undifferentiated
callus, protoplasts, etc.), followed by applying a maximum
threshold level of appropriate selection (depending on the
selectable marker gene) to recover the transformed plant cells from
a group of untransformed cell mass. Explants are typically
transferred to a fresh supply of the same medium and cultured
routinely. Subsequently, the transformed cells are differentiated
into shoots after placing on regeneration medium supplemented with
a maximum threshold level of selecting agent. The shoots are then
transferred to a selective rooting medium for recovering rooted
shoot or plantlet. The transgenic plantlet then grows into a mature
plant and produces fertile seeds (e.g. Hiei et al. (1994) The Plant
Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology
14:745-750). Explants are typically transferred to a fresh supply
of the same medium and cultured routinely. A general description of
the techniques and methods for generating transgenic plants are
found in Ayres and Park (1994) Critical Reviews in Plant Science
13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120.
Since the transformed material contains many cells: both
transformed and non-transformed cells are present in any piece of
subjected target callus or tissue or group of cells. The ability to
kill non-transformed cells and allow transformed cells to
proliferate results in transformed plant cultures. Often, the
ability to remove non-transformed cells is a limitation to rapid
recovery of transformed plant cells and successful generation of
transgenic plants.
[0094] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Generation of transgenic plants may be
performed by one of several methods, including, but not limited to,
microinjection, electroporation, direct gene transfer, introduction
of heterologous DNA by Agrobacterium into plant cells
(Agrobacterium-mediated transformation), bombardment of plant cells
with heterologous foreign DNA adhered to particles, ballistic
particle acceleration, aerosol beam transformation (U.S. Published
Application No. 20010026941; U.S. Pat. No. 4,945,050; International
Publication No. WO 91/00915: U.S. Published Application No.
2002015066), Ledc transformation, and various other non-particle
direct-mediated methods to transfer DNA. Methods for transformation
of chloroplasts are known in the art. See, for example, Svab et al.
(1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga
(1993) Proc. Natl. Acad. Sci. USA 90:913-917: Svab and Maliga
(1993) EMBO J. 12:601-606. The method relies on particle gun
delivery of DNA containing a selectable marker and targeting of the
DNA to the plastid genome through homologous recombination.
Additionally, plastid transformation can be accomplished by
transactivation of a silent plastid-borne transgene by
tissue-preferred expression of a nuclear-encoded and
plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0095] Following integration of heterologous foreign DNA into plant
cells, one then applies a maximum threshold level of appropriate
selection in the medium to kill the untransformed cells and
separate and proliferate the putatively transformed cells that
survive from this selection treatment by transferring regularly to
a fresh medium. By continuous passage and challenge with
appropriate selection, one identifies and proliferates the cells
that are transformed with the plasmid vector. Molecular and
biochemical methods can then be used to confirm the presence of the
integrated heterologous gene of interest into the genome of the
transgenic plant.
[0096] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present invention provides
transformed seed (also referred to as "transgenic seed") having a
nucleotide construct of the invention, for example, an expression
cassette of the invention, stably incorporated into their
genome.
Evaluation of Plant Transformation
[0097] Following introduction of heterologous foreign DNA into
plant cells, the transformation or integration of heterologous gene
in the plant genome is confirmed by various methods such as
analysis of nucleic acids, proteins and metabolites associated with
the integrated gene.
[0098] PCR analysis is a rapid method to screen transformed cells,
tissue or shoots for the presence of incorporated gene at the
earlier stage before transplanting into the soil (Sambrook and
Russell (2001) Molecular Cloning: A Laboratoy Manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor. NY). PCR is carried
out using oligonucleotide primers specific to the gene of interest
or Agrobacterium vector background, etc.
[0099] Plant transformation may be confirmed by Southern blot
analysis of genomic DNA (Sambrook and Russell, 2001, supra). In
general, total DNA is extracted from the transformant, digested
with appropriate restriction enzymes, fractionated in an agarose
gel and transferred to a nitrocellulose or nylon membrane. The
membrane or "blot" is then probed with, for example, radiolabeled
.sup.32P target DNA fragment to confirm the integration of
introduced gene into the plant genome according to standard
techniques (Sambrook and Russell, 2001, supra).
[0100] In Northern blot analysis. RNA is isolated from specific
tissues of transformant, fractionated in a formaldehyde agarose
gel, and blotted onto a nylon filter according to standard
procedures that are routinely used in the art (Sambrook and
Russell, 2001, supra). Expression of RNA encoded by the pesticidal
gene is then tested by hybridizing the filter to a radioactive
probe derived from a pesticidal gene, by methods known in the art
(Sambrook and Russell, 2001, supra).
[0101] Western blot, biochemical assays and the like may be carried
out on the transgenic plants to confirm the presence of protein
encoded by the pesticidal gene by standard procedures (Sambrook and
Russell, 2001, supra) using antibodies that bind to one or more
epitopes present on the nematicidal protein.
Pesticidal Activity in Plants
[0102] In another aspect of the invention, one may generate
transgenic plants expressing a nematicidal protein that has
pesticidal activity against a nematode pest. Methods described
above by way of example may be utilized to generate transgenic
plants, but the manner in which the transgenic plant cells are
generated is not critical to this invention. Methods known or
described in the art such as Agrobacterium-mediated transformation,
biolistic transformation, and non-particle-mediated methods may be
used at the discretion of the experimenter. Plants expressing a
nematicidal protein may be isolated by common methods described in
the art, for example by transformation of callus, selection of
transformed callus, and regeneration of fertile plants from such
transgenic callus. In such process, one may use any gene as a
selectable marker so long as its expression in plant cells confers
ability to identify or select for transformed cells.
[0103] A number of markers have been developed for use with plant
cells, such as resistance to chloramphenicol, the aminoglycoside
G418, hygromycin, or the like. Other genes that encode a product
involved in chloroplast metabolism may also be used as selectable
markers. For example, genes that provide resistance to plant
herbicides such as glyphosate, bromoxynil, or imidazolinone may
find particular use. Such genes have been reported (Stalker et al.
(1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance
nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res.
18:2188 (AHAS imidazolinone resistance gene). Additionally, the
genes disclosed herein are useful as markers to assess
transformation of bacterial or plant cells. Methods for detecting
the presence of a transgene in a plant, plant organ (e.g., leaves,
stems, roots, etc.), seed, plant cell, propagule, embryo or progeny
of the same are well known in the art. In one embodiment, the
presence of the transgene is detected by testing for pesticidal
activity against a nematode pest.
[0104] Fertile plants expressing a nematicidal protein may be
tested for pesticidal activity against a nematode pest, and the
plants showing optimal activity selected for further breeding.
Methods are available in the art to assay for pest activity.
Generally, the protein is mixed and used in feeding assays. See,
for example Marrone et al. (1985) J. of Economic Entomology
78:290-293.
[0105] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (maize), sorghum, wheat, sunflower, tomato, crucifers,
peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,
tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye,
millet, safflower, peanuts, sweet potato, cassava, coffee, coconut,
pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava,
mango, olive, papaya, cashew, macadamia, almond, oats, vegetables,
ornamentals, and conifers.
Use in Pesticidal Control
[0106] General methods for employing strains comprising a
nucleotide sequence of the present invention, or a variant thereof,
in pest control or in engineering other organisms as pesticidal
agents are known in the art. See, for example U.S. Pat. No.
5,039,523 and EP 0480762A2.
[0107] Microorganisms can be genetically altered to contain a
nucleotide sequence encoding SEQ ID NO: 1 or 2, or
nematicidally-active variants or fragments thereof, and protein may
be used for protecting agricultural crops and products from pests.
In one aspect of the invention, whole, i.e., unlysed, cells of a
toxin (pesticide)-producing organism are treated with reagents that
prolong the activity of the toxin produced in the cell when the
cell is applied to the environment of target pest(s).
[0108] Alternatively, the pesticide is produced by introducing a
pesticidal gene into a cellular host. Expression of the pesticidal
gene results, directly or indirectly, in the intracellular
production and maintenance of the pesticide. In one aspect of this
invention, these cells are then treated under conditions that
prolong the activity of the toxin produced in the cell when the
cell is applied to the environment of the target pest(s). The
resulting product retains the toxicity of the toxin. These
naturally encapsulated pesticides may then be formulated in
accordance with conventional techniques for application to the
environment hosting a target pest, e.g., soil, water, and foliage
of plants. See, for example EPA 0192319, and the references cited
therein. Alternatively, one may formulate the cells expressing a
gene of this invention such as to allow application of the
resulting material as a pesticide.
[0109] The active ingredients of the present invention are normally
applied in the form of compositions and can be applied to the crop
area or plant to be treated, simultaneously or in succession, with
other compounds. These compounds can be fertilizers, weed killers,
cryoprotectants, surfactants, detergents, pesticidal soaps, dormant
oils, polymers, and/or time-release or biodegradable carrier
formulations that permit long-term dosing of a target area
following a single application of the formulation. They can also be
selective herbicides, chemical insecticides, virucides,
microbicides, amoebicides, pesticides, fungicides, bacteriocides,
nematocides, molluscicides or mixtures of several of these
preparations, if desired, together with further agriculturally
acceptable carriers, surfactants or application-promoting adjuvants
customarily employed in the art of formulation. Suitable carriers
and adjuvants can be solid or liquid and correspond to the
substances ordinarily employed in formulation technology, e.g.
natural or regenerated mineral substances, solvents, dispersants,
wetting agents, tackifiers, binders or fertilizers. Likewise the
formulations may be prepared into edible "baits" or fashioned into
pest "traps" to permit feeding or ingestion by a target pest of the
pesticidal formulation.
[0110] Methods of applying an active ingredient of the present
invention or an agrochemical composition of the present invention
that contains at least one of the nematicidal proteins disclosed
herein as SEQ ID NO: 1 or 2, or nematicidally-effective variants or
fragments thereof, include leaf application, seed coating and soil
application. The number of applications and the rate of application
depend on the intensity of infestation by the corresponding
pest.
[0111] The composition may be formulated as a powder, dust, pellet,
granule, spray, emulsion, colloid, solution, or such like, and may
be prepared by such conventional means as desiccation,
lyophilization, homogenation, extraction, filtration,
centrifugation, sedimentation, or concentration of a culture of
cells comprising the polypeptide. In all such compositions that
contain at least one such pesticidal polypeptide, the polypeptide
may be present in a concentration of from about 1% to about 99% by
weight.
[0112] Nematode pests may be killed or reduced in numbers in a
given area by the methods of the invention, or may be
prophylactically applied to an environmental area to prevent
infestation by a susceptible pest. Preferably the pest ingests, or
is contacted with, a pesticidally-effective amount of the
polypeptide. By "pesticidally-effective amount" or
"nematicidally-effective amount" is intended an amount of the
pesticide or nematicide that is able to bring about death to at
least one pest, or to noticeably reduce pest growth, feeding, or
normal physiological development of the pest or the host plant in
which the pest infests. This amount will vary depending on such
factors as, for example, the specific target pests to be
controlled, the specific environment, location, plant, crop, or
agricultural site to be treated, the environmental conditions, and
the method, rate, concentration, stability, and quantity of
application of the nematicidally-effective polypeptide composition.
The formulations may also vary with respect to climatic conditions,
environmental considerations, and/or frequency of application
and/or severity of pest infestation.
[0113] The pesticide compositions described may be made by
formulating either the bacterial cell, the crystal and/or the spore
suspension, or the isolated protein component with the desired
agriculturally-acceptable carrier. The compositions may be
formulated prior to administration in an appropriate means such as
lyophilized, freeze-dried, desiccated, or in an aqueous carrier,
medium or suitable diluent, such as saline or other buffer. The
formulated compositions may be in the form of a dust or granular
material, or a suspension in oil (vegetable or mineral), or water
or oil/water emulsions, or as a wettable powder, or in combination
with any other carrier material suitable for agricultural
application. Suitable agricultural carriers can be solid or liquid
and are well known in the art. The term "agriculturally-acceptable
carrier" covers all adjuvants, inert components, dispersants,
surfactants, tackifiers, binders, etc. that are ordinarily used in
pesticide formulation technology; these are well known to those
skilled in pesticide formulation. The formulations may be mixed
with one or more solid or liquid adjuvants and prepared by various
means, e.g., by homogeneously mixing, blending and/or grinding the
pesticidal composition with suitable adjuvants using conventional
formulation techniques. Suitable formulations and application
methods are described in U.S. Pat. No. 6,468,523, herein
incorporated by reference.
Methods for Increasing Plant Yield
[0114] Methods for increasing plant yield are provided. The methods
comprise providing a plant or plant cell expressing a
polynucleotide encoding the nematicidal polypeptide sequence
disclosed herein and growing the plant or a seed thereof in a field
infested with (or susceptible to infestation by) a nematode pest
against which said polypeptide has nematicidal activity. In some
embodiments, the Cry14 polypeptide described herein has nematicidal
activity against a Pratylenchus spp., and said field is infested
with said Pratylenchus spp. In various embodiments, the
Pratylenchus spp. is Pratylenchus brachyurus. In additional
embodiments, the nematode is a root knot nematode, a lesion
nematode, or a Lance nematode. As defined herein, the "yield" of
the plant refers to the quality and/or quantity of biomass produced
by the plant. By "biomass" is intended any measured plant product.
An increase in biomass production is any improvement in the yield
of the measured plant product. Increasing plant yield has several
commercial applications. For example, increasing plant leaf biomass
may increase the yield of leafy vegetables for human or animal
consumption. Additionally, increasing leaf biomass can be used to
increase production of plant-derived pharmaceutical or industrial
products. An increase in yield can comprise any statistically
significant increase including, but not limited to, at least a 1%
increase, at least a 3% increase, at least a 5% increase, at least
a 10% increase, at least a 20% increase, at least a 30%, at least a
50%, at least a 70%, at least a 1000/or a greater increase in yield
compared to a plant not expressing the pesticidal protein described
herein. In specific methods, plant yield is increased as a result
of improved nematode resistance of a plant expressing the
nematicidal protein disclosed herein. Expression of the nematicidal
protein results in a reduced ability of a pest to infest or feed.
In various embodiments, expression of the nematicidal protein
results in improved root development (e.g., improved root or root
hair growth), improved yield, faster emergence, improved plant
stress management including increased stress tolerance and/or
improved recovery from stress, increased mechanical strength,
improved drought resistance, reduced fungal disease infection, and
improved plant health compared to a plant not expressing the
nematicidal protein of the invention.
[0115] The plants can also be treated with one or more chemical
compositions, including one or more herbicide, insecticides, or
fungicides. Exemplary chemical compositions include:
Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron,
Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop,
Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim.
Butafenacil, Halosulfuron, Indaziflam: Fruits/Vegetables
Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl,
Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin,
Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,
Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron,
Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim,
Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad,
Rynaxypyr, Cyazypyr, Triflumuron, Spirotetramat, Imidacloprid,
Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor,
Cyflumetofen, Cyanopyrafen, Clothianidin, Thiamethoxam, Spinotoram,
Thiodicarb, Flonicamid, Mcthiocarb, Emamcctin-benzoate, Indoxacarb.
Fenamiphos, Pyriproxifen, Fenbutatin-oxid; Fruits/Vegetables
Fungicides: Ametoctradin, Azoxystrobin. Benthiavalicarb, Boscalid,
Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamid,
Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole,
Dimetomorph, Dithianon, Fenamidone, Fenhexamid, Fluazinam,
Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluxapyroxad,
Folpet, Fosetyl, Iprodione, Iprovalicarb, Isopyrazam.
Kresoxim-methyl, Mancozeb, Mandipropamid, Metalaxyl/mefenoxam,
Metiram, Metrafenone, Myclobutanil, Penconazole, Penthiopyrad,
Picoxystrobin, Propamocarb, Propiconazole, Propineb, Proquinazid,
Prothioconazole, Pyraclostrobin, Pyrimethanil, Quinoxyfen,
Spiroxamine, Sulphur, Tebuconazole, Thiophanate-methyl,
Trifloxystrobin;
Cereals Herbicides:
[0116] 2,4-D, Amidosulfuron, Bromoxynil, Carfentrazone-E,
Chlorotoluron, Chlorsulfuron, Clodinafop-P, Clopyralid, Dicamba,
Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarbazone-NA,
Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate,
Iodosulfuron, Ioxynil, Isoproturon, MCPA, Mesosulfuron,
Metsulfuron, Pendimethalin, Pinoxaden, Propoxycarbazone,
Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron,
Tralkoxydim. Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron;
Cereals Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim,
Chlorothalonil, Cyflufenamid, Cyproconazole, Cyprodinil,
Dimoxystrobin, Epoxiconazole, Fenpropidin, Fenpropimorph,
Fluopyram. Fluoxastrobin, Fluquinconazole, Fluxapyroxad,
Isopyrazam, Kresoxim-methyl, Metconazole, Metrafenone.
Penthiopyrad, Picoxystrobin, Prochloraz, Propiconazole,
Proquinazid, Prothioconazole, Pyraclostrobin, Quinoxyfen,
Spiroxamine, Tebuconazole, Thiophanate-methyl, Trifloxystrobin
Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin,
alpha-Cypermethrin, -cyfluthrin, Bifenthrin, Imidacloprid,
Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran,
Clorphyriphos, Pirimicarb, Methiocarb, Sulfoxaflor; Maize
Herbicides: Atrazine. Alachlor, Bromoxynil, Acetochlor, Dicamba,
Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate,
Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron,
Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,
Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,
Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,
Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,
Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,
Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, -Cyfluthrin,
Cypermethrin, Bifenthrin, Lufenuron, Tebupirimphos, Ethiprole,
Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avennectin; Maize
Fungicides: Azoxystrobin, Bixafen, Boscalid, Cyproconazole,
Dimoxystrobin, Epoxiconazole, Fenitropan, Fluopyram, Fluoxastrobin,
Fluxapyroxad, Isopyrazam, Metconazole, Penthiopyrad, Picoxystrobin,
Propiconazole, Prothioconazole, Pyraclostrobin. Tebuconazole,
Trifloxystrobin; Rice Herbicides: Butachlor, Propanil,
Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide,
Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,
Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,
Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon,
Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxvsulfuron,
Pretilachlor, Mesotrione, Tefurviltrione, Oxadiazone, Fenoxaprop,
Pyrimisulfan; Rice Insecticides: Diazinon, Fenobucarb, Benfuracarb,
Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb,
Thiacloprid, Chromafenozide, Clothianidin, Ethiprole,
Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,
Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,
Chlorpyriphos, Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor;
Rice Fungicides: Azoxystrobin, Carbendazim, Carpropamid,
Diclocymet, Difenoconazole, Edifenphos, Ferimzone, Gentamycin,
Hexaconazole, Hymexazol, Iprobenfos (IBP), Isoprothiolane,
Isotianil, Kasugamycin, Mancozeb, Metominostrobin, Orysastrobin,
Pencycuron, Probenazole, Propiconazole, Propineb, Pyroquilon,
Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole,
Trifloxystrobin, Validamycin: Cotton Herbicides: Diuron,
Fluometuron, MSMA, Oxyfluorfen, Prometrn, Trifluralin,
Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon,
Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim,
Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides:
Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin,
Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid,
Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb,
Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid
Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,
Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid,
Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma
Cyhalothrin,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,
Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,
Sulfoxaflor: Cotton Fungicides: Azoxystrobin, Bixafen, Boscalid,
Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole,
Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram,
Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil,
Mancozeb, Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin,
Propineb, Prothioconazole, Pyraclostrobin, Quintozene,
Tebuconazole. Tetraconazole, Thiophanate-methyl, Trifloxystrobin;
Soybean Herbicides: Alachlor, Bentazone, Trifluralin,
Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen,
Fluazifop. Glyphosate, Imazamox, Imazaquin, Imazethapyr,
(S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,
Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,
Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,
Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad,
Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin,
-Cyfluthrin, gamma and lambda Cyhalothrin,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,
Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,
beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Bixafen,
Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole,
Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram,
Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione,
Isotianil, Mancozeb, Maneb, Metconazole, Metominostrobin,
Myclobutanil, Penthiopyrad, Picoxystrobin, Propiconazole, Propineb,
Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole.
Thiophanate-methyl, Trifloxystrobin; Sugarbeet Herbicides:
Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,
Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,
Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:
Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,
Dinetofuran, Deltamethrin, -Cyfluthrin, gamma/lambda Cyhalothrin,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,
Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; Canola
Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate,
Glyphosate, Metazachlor, Trifluralin Ethamctsulfuron, Quinmerac,
Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides:
Azoxystrobin, Bixafen, Boscalid, Carbendazim, Cyproconazole,
Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram,
Fluoxastrobin, Flusilazole, Fluxapyroxad, Iprodione, Isopyrazam,
Mepiquat-chloride, Metconazole, Metominostrobin, Paclobutrazole,
Penthiopyrad, Picoxystrobin, Prochloraz, Prothioconazole,
Pyraclostrobin, Tebuconazole, Thiophanate-methyl, Trifloxystrobin,
Vinclozolin; Canola Insecticides: Carbofuran. Thiacloprid,
Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam,
Acetamiprid, Dinetofuran, -Cyfluthrin, gamma and lambda
Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram,
Flubendiamide, Rynaxypyr, Cyazypyr,
4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.
[0117] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL EXAMPLES
Example 1. Cry14Aa1 Expression in Soybean
[0118] Soybean events expressing Cry14Aa1 (SEQ ID NO:3) were
developed through Agrobacterium-mediated transformation of Thorne
soybean plants using a construct containing a gene encoding a
4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor
tolerant herbicide gene (described in WO02014043435) and Cry14Aa1.
Wild-type Thoeme soybean served as the non-nematode resistant
control. Cry14Aa1, when expressed in soybean plants, reduces the
number of Pratylenchus brachyurus that reproduce in the roots
compared with wild-type plants. Three independent event lines
consistently gave the same result showing nematode reduction upon
retest multiple times. In all three events, the number of nematodes
was reduced by 60-85%.
Example 2. Cry14Ab1 Expression in Soybean
[0119] EE-GM4 soybean events expressing Cry14Ab1 (SEQ ID NO:4) were
developed through Agrobacterium-mediated transformation of Thoeme
soybean plants using a construct containing a gene encoding a
4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor
tolerant herbicide gene (described in WO2014043435) and Cry14Ab1.
Wild-type Thorne soybean served as the non-nematode resistant
control. Cry14Ab1, when expressed in soybean plants, reduces the
number of Pratylenchus brachyurus that reproduce in the roots
compared with wild-type plants. Non-transformed Thorne and EE-GM4
seeds were geminated and planted in the greenhouse to check for
control of the lesion nematode, Pratylenchus brachyurus.
Pratylenchus brachyurus nematodes (#1500/plant, different
developmental stages) were applied to the plants when 2 weeks old.
Thirty days after application, Pratylenchus nematodes were
extracted from the roots and counted. The average number of
nematodes found in the roots of plants containing EE-GM4 were
compared with the average number of Pratylenchus nematodes found in
the wild-type Thorne plant roots. On average about 80-90% fewer
Pratylenchus nematodes were found in roots of plants containing
EE-GM4 when compared with the Thorne control roots, indicating
significant control of lesion nematodes by soybean event
EE-GM4.
[0120] FIG. 1 shows results from a Pratylenchus brachyurus
greenhouse assay in the US, comparing elite lines with EE-GM4 in 5
elite soybean lines (one SCN susceptible (MG 1), one SCN resistant
(PI88788, MG 3), one SCN susceptible (MG 6.2), one SCN resistant
(Peking. MG 6.2), and one SCN susceptible (MG 9) to SCN-susceptible
and SCN-resistant US soybean lines. The soybean plants were grown
in small cone pots and kept in greenhouses with temperature varying
between 25-32.degree. C. Pratylenchus brachyurus nematodes,
obtained from South Carolina and increased in the greenhouse were
used to inoculate plants in the V2-V3 development stage.
Approximately 1500 eggs+adults were inoculated per plant and each
entry had 5 plants. Thirty days after infestation, nematodes and
eggs were extracted from the roots and counted. Each entry was run
in two independent experiments. While SCN-susceptible and
SCN-resistant US soybean lines did not show control of
Pratylenchus, plants with EE-GM4 showed about 85% control of
Pratylenchus.
[0121] FIG. 2 shows results from a Pratylenchus brachyurus
greenhouse assay in Brazil, comparing soybean plants with EE-GM4 to
Brazil soybean lines with no resistance and 1 low Rf line, and
SCN-susceptible and -resistant plants. The soybean lines were grown
in small cone pots and kept in greenhouses with temperature varying
between 25-32.degree. C. Pratylenchus brachyurus nematodes,
obtained from Brazil fields and increased in the greenhouse were
used to inoculate plants in the V2-V3 development stage.
Approximately 1000 eggs+adults were inoculated per plant and each
entry had 5 plants. Thirty days after infestation, nematodes and
eggs were extracted from the roots and counted. Results shown are
from a single experiment. One Brazilian soybean line (BRS 7380),
labeled as having a low reproductive factor for Prarylenchus,
showed about 89% reduction of Pratylenchus. Plants with EE-GM4 gave
.about.97% control of Pratylenchus. Soybean lines that carry native
resistance to SCN (rhg1+Rhg4) do not control Pratylenchus
brachyurus.
[0122] Also, plants containing EE-GM4 can be used to control
root-knot nematodes (RKN) such as Meloidogyne incognita. Even
though the population of Meloidogyne incognita does not infest
Thoeme wild-type soybean very well, Thorne plants with EE-GM4 show
a further reduction in the number of RKN eggs/root mass on average,
as compared to untransformed Thorne plants.
Example 3. Vectoring of Genes for Plant Expression
[0123] The coding regions of the invention are connected with
appropriate promoter and terminator sequences for expression in
plants. Such sequences are well known in the art. Techniques for
producing and confirming promoter-gene-terminator constructs also
are well known in the art.
[0124] In one aspect of the invention, synthetic DNA sequences are
designed and generated. These synthetic sequences have altered
nucleotide sequence relative to the parent sequence, but encode
proteins that are essentially identical to the parent sequence. In
some embodiments, the synthetic DNA sequence comprises SEQ ID NO:3
or 4.
[0125] In another aspect of the invention, modified versions of the
synthetic genes are designed such that the resulting peptide is
targeted to a plant organelle, such as the endoplasmic reticulum or
the apoplast. Peptide sequences known to result in targeting of
fusion proteins to plant organelles are known in the art. For
example, the N-terminal region of the acid phosphatase gene from
the White Lupin Lupinus albus (GENBANK1 ID GI: 14276838, Miller et
al. (2001) Plant Physiology 127: 594-606) is known in the art to
result in endoplasmic reticulum targeting of heterologous proteins.
If the resulting fusion protein also contains an endoplasmic
reticulum retention sequence comprising the peptide
N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e., the
"KDEL" motif, SEQ ID NO:7) at the C-terminus, the fusion protein
will be targeted to the endoplasmic reticulum. If the fusion
protein lacks an endoplasmic reticulum targeting sequence at the
C-terminus, the protein will be targeted to the endoplasmic
reticulum, but will ultimately be sequestered in the apoplast.
[0126] Thus, this gene encodes a fusion protein that contains the
N-terminal thirty-one amino acids of the acid phosphatase gene from
the White Lupin Lupinus albus (GENBANK.RTM. ID GI: 14276838, Miller
et al., 2001, supra) fused to the N-terminus of the amino acid
sequence of the invention, as well as the KDEL (SEQ ID NO:7)
sequence at the C-terminus. Thus, the resulting protein is
predicted to be targeted the plant endoplasmic reticulum upon
expression in a plant cell.
[0127] The plant expression cassettes described above are combined
with an appropriate plant selectable marker to aid in the selection
of transformed cells and tissues, and ligated into plant
transformation vectors. These may include binary vectors from
Agrobacterium-mediated transformation or simple plasmid vectors for
aerosol or biolistic transformation.
[0128] In the present invention, an expression cassette including a
synthetic gene encoding Cry14A (SEQ ID NO: 1 or 2) is operably
linked to the promoter region of the sucrose synthase 1 gene of
Oryza sativa (Wang et al. (1992) Plant Molecular Biology, 19,
881-885) or the promoter region of the Cauliflower Mosaic Virus 35S
transcript (Odell et al. (1985) Nature 313, 810-812) and the leader
sequence of the chlorophyll a/b binding protein gene of Petunia
hybrid (Harpster et al. (1988) Molecular and General Genetics 212,
182-190). The expression cassettes further comprised the 3'
untranslated region of the nopaline synthase gene from the T-DNA of
pTiT37 (Depicker et al. (1982) Journal of Molecular and Applied
Genetics 1, 561-573) operably linked to the 3' end of the Cry14
sequence.
Example 4: Soybean Transformation
[0129] Soybean transformation is achieved using methods well known
in the art, such as the one described using the Agrobacterium
tumefaciens mediated transformation soybean half-seed explants
using essentially the method described by Paz et al. (2006), Plant
cell Rep. 25:206. Transformants are identified using tembotrione as
selection marker. The appearance of green shoots was observed, and
documented as an indicator of tolerance to the herbicide
isoxaflutole or tembotrione. The tolerant transgenic shoots will
show normal greening comparable to wild-type soybean shoots not
treated with isoxaflutole or tembotrione, whereas wild-type soybean
shoots treated with the same amount of isoxaflutole or tembotrione
will be entirely bleached. This indicates that the presence of the
HPPD protein enables the tolerance to HPPD inhibitor herbicides,
like isoxaflutole or tembotrione.
[0130] Tolerant green shoots are transferred to rooting media or
grafted. Rooted plantlets are transferred to the greenhouse after
an acclimation period. Plants containing the transgene are then
sprayed with HPPD inhibitor herbicides, as for example with
tembotrione at a rate of 100 g AI/ha or with mesotrione at a rate
of 300 g AI/ha supplemented with ammonium sulfate methyl ester
rapeseed oil. Ten days after the application the symptoms due to
the application of the herbicide are evaluated and compared to the
symptoms observed on wild type plants under the same
conditions.
Example 5: Cotton T0 Plant Establishment and Selection
[0131] Cotton transformation is achieved using methods well known
in the art, especially preferred method in the one described in the
PCT patent publication WO 00/71733. Regenerated plants are
transferred to the greenhouse. Following an acclimation period,
sufficiently grown plants are sprayed with HPPD inhibitor
herbicides as for example tembotrione equivalent to 100 or 200
gAI/ha supplemented with ammonium sulfate and methyl ester rapeseed
oil. Seven days after the spray application, the symptoms due to
the treatment with the herbicide are evaluated and compared to the
symptoms observed on wild type cotton plants subjected to the same
treatment under the same conditions.
[0132] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0133] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
711186PRTBacillus thuringiensis 1Met Asp Cys Asn Leu Gln Ser Gln
Gln Asn Ile Pro Tyr Asn Val Leu1 5 10 15Ala Ile Pro Val Ser Asn Val
Asn Ala Leu Val Asp Thr Ala Gly Asp 20 25 30Leu Lys Lys Ala Trp Glu
Glu Phe Gln Lys Thr Gly Ser Phe Ser Leu 35 40 45Thr Ala Leu Gln Gln
Gly Phe Ser Ala Ser Gln Gly Gly Ala Phe Asn 50 55 60Tyr Leu Thr Leu
Leu Gln Ser Gly Ile Ser Leu Ala Gly Ser Phe Val65 70 75 80Pro Gly
Gly Thr Phe Val Ala Pro Ile Val Asn Met Val Ile Gly Trp 85 90 95Leu
Trp Pro His Lys Asn Lys Thr Ala Asp Thr Glu Asn Leu Ile Lys 100 105
110Leu Ile Asp Glu Glu Ile Gln Lys Gln Leu Asn Lys Ala Leu Leu Asp
115 120 125Gln Asp Arg Asn Asn Trp Thr Ser Phe Leu Glu Ser Ile Phe
Asp Thr 130 135 140Ser Ala Thr Val Ser Asn Ala Ile Ile Asp Ala Gln
Trp Ser Gly Thr145 150 155 160Val Asp Thr Thr Asn Arg Gln Gln Lys
Thr Pro Thr Thr Ser Asp Tyr 165 170 175Leu Asn Val Val Gly Lys Phe
Asp Ser Ala Asp Ser Ser Ile Ile Thr 180 185 190Asn Glu Asn Gln Ile
Met Asn Gly Asn Phe Asp Val Ala Ala Ala Pro 195 200 205Tyr Phe Val
Ile Gly Ala Thr Leu Arg Leu Ser Leu Tyr Gln Ser Tyr 210 215 220Ile
Lys Phe Cys Asn Ser Trp Ile Asp Ala Val Gly Phe Ser Thr Asn225 230
235 240Asp Ala Asn Thr Gln Lys Ala Asn Leu Ala Arg Thr Lys Leu Thr
Met 245 250 255Arg Thr Thr Ile Asn Glu Tyr Thr Gln Arg Val Met Lys
Val Phe Lys 260 265 270Asp Ser Lys Asn Met Pro Thr Ile Gly Thr Asn
Lys Phe Ser Val Asp 275 280 285Ala Tyr Asn Val Tyr Val Lys Gly Met
Thr Leu Asn Val Leu Asp Met 290 295 300Val Ala Ile Trp Ser Ser Leu
Tyr Pro Asn Asp Tyr Thr Ser Gln Thr305 310 315 320Ala Ile Glu Gln
Thr Arg Val Thr Phe Ser Asn Met Val Gly Gln Glu 325 330 335Glu Gly
Thr Asp Gly Thr Leu Lys Ile Tyr Asn Thr Phe Asp Ser Leu 340 345
350Ser Tyr Gln His Ser Leu Ile Pro Asn Asn Asn Val Asn Leu Ile Ser
355 360 365Tyr Tyr Thr Asp Glu Leu Gln Asn Leu Glu Leu Ala Val Tyr
Thr Pro 370 375 380Lys Gly Gly Ser Gly Tyr Ala Tyr Pro Tyr Gly Phe
Ile Leu Asn Tyr385 390 395 400Ala Asn Ser Asn Tyr Lys Tyr Gly Asp
Asn Asp Pro Thr Gly Lys Pro 405 410 415Leu Asn Lys Gln Asp Gly Pro
Ile Gln Gln Ile Asn Ala Ala Thr Gln 420 425 430Asn Ser Lys Tyr Leu
Asp Gly Glu Thr Ile Asn Gly Ile Gly Ala Ser 435 440 445Leu Pro Gly
Tyr Cys Thr Thr Gly Cys Ser Ala Thr Glu Gln Pro Phe 450 455 460Ser
Cys Thr Ser Thr Ala Asn Ser Tyr Lys Ala Ser Cys Asn Pro Ser465 470
475 480Asp Thr Asn Gln Lys Ile Asn Ala Leu Tyr Ala Phe Thr Gln Thr
Asn 485 490 495Val Lys Gly Ser Thr Gly Lys Leu Gly Val Leu Ala Ser
Leu Val Pro 500 505 510Tyr Asp Leu Asn Pro Lys Asn Val Phe Gly Glu
Leu Asp Ser Asp Thr 515 520 525Asn Asn Val Ile Leu Lys Gly Ile Pro
Ala Glu Lys Gly Tyr Phe Pro 530 535 540Asn Asn Ala Arg Pro Thr Val
Val Lys Glu Trp Ile Asn Gly Ala Ser545 550 555 560Ala Val Pro Phe
Tyr Ser Gly Asn Thr Leu Phe Met Thr Ala Thr Asn 565 570 575Leu Thr
Ala Thr Gln Tyr Lys Ile Arg Ile Arg Tyr Ala Asn Pro Asn 580 585
590Ser Asp Thr Gln Ile Gly Val Leu Ile Thr Gln Asn Gly Ser Gln Ile
595 600 605Ser Asn Ser Asn Leu Thr Leu Tyr Ser Thr Thr Asp Ser Ser
Met Ser 610 615 620Ser Asn Leu Pro Gln Asn Val Tyr Val Thr Gly Glu
Asn Gly Asn Tyr625 630 635 640Thr Leu Leu Asp Leu Tyr Ser Thr Thr
Asn Val Leu Ser Thr Gly Asp 645 650 655Ile Thr Leu Lys Leu Thr Gly
Gly Asn Gln Lys Ile Phe Ile Asp Arg 660 665 670Ile Glu Phe Ile Pro
Thr Met Pro Val Pro Ala Pro Thr Asn Asn Thr 675 680 685Asn Asn Asn
Asn Gly Asp Asn Gly Asn Asn Asn Pro Pro His His Gly 690 695 700Cys
Ala Ile Ala Gly Thr Gln Gln Leu Cys Ser Gly Pro Pro Lys Phe705 710
715 720Glu Gln Val Ser Asp Leu Glu Lys Ile Thr Thr Gln Val Tyr Met
Leu 725 730 735Phe Lys Ser Ser Ser Tyr Glu Glu Leu Ala Leu Lys Val
Ser Ser Tyr 740 745 750Gln Ile Asn Gln Val Ala Leu Lys Val Met Ala
Leu Ser Asp Glu Lys 755 760 765Phe Cys Glu Glu Lys Arg Leu Leu Arg
Lys Leu Val Asn Lys Ala Asn 770 775 780Gln Leu Leu Glu Ala Arg Asn
Leu Leu Val Gly Gly Asn Phe Glu Thr785 790 795 800Thr Gln Asn Trp
Val Leu Gly Thr Asn Ala Tyr Ile Asn Tyr Asp Ser 805 810 815Phe Leu
Phe Asn Gly Asn Tyr Leu Ser Leu Gln Pro Ala Ser Gly Phe 820 825
830Phe Thr Ser Tyr Ala Tyr Gln Lys Ile Asp Glu Ser Thr Leu Lys Pro
835 840 845Tyr Thr Arg Tyr Lys Val Ser Gly Phe Ile Gly Gln Ser Asn
Gln Val 850 855 860Glu Leu Ile Ile Ser Arg Tyr Gly Lys Glu Ile Asp
Lys Ile Leu Asn865 870 875 880Val Pro Tyr Ala Gly Pro Leu Pro Ile
Thr Ala Asp Ala Ser Ile Thr 885 890 895Cys Cys Ala Pro Glu Ile Asp
Gln Cys Asp Gly Gly Gln Ser Asp Ser 900 905 910His Phe Phe Asn Tyr
Ser Ile Asp Val Gly Ala Leu His Pro Glu Leu 915 920 925Asn Pro Gly
Ile Glu Ile Gly Leu Lys Ile Val Gln Ser Asn Gly Tyr 930 935 940Ile
Thr Ile Ser Asn Leu Glu Ile Ile Glu Glu Arg Pro Leu Thr Glu945 950
955 960Met Glu Ile Gln Ala Val Asn Arg Lys Asp Gln Lys Trp Lys Arg
Glu 965 970 975Lys Leu Leu Glu Cys Ala Ser Val Ser Glu Leu Leu Gln
Pro Ile Ile 980 985 990Asn Gln Ile Asp Ser Leu Phe Lys Asp Ala Asn
Trp Tyr Asn Asp Ile 995 1000 1005Leu Pro His Val Thr Tyr Gln Thr
Leu Lys Asn Ile Ile Val Pro 1010 1015 1020Asp Leu Pro Lys Leu Lys
His Trp Phe Ile Asp His Leu Pro Gly 1025 1030 1035Glu Tyr His Glu
Ile Glu Gln Lys Met Lys Glu Ala Leu Lys His 1040 1045 1050Ala Phe
Thr Gln Leu Asp Glu Lys Asn Leu Ile His Asn Gly His 1055 1060
1065Phe Ala Thr Asn Leu Ile Asp Trp Gln Val Glu Gly Asp Ala Arg
1070 1075 1080Met Lys Val Leu Glu Asn Asp Ala Leu Ala Leu Gln Leu
Ser Asn 1085 1090 1095Trp Asp Ser Ser Val Ser Gln Ser Ile Asp Ile
Leu Glu Phe Asp 1100 1105 1110Glu Asp Lys Ala Tyr Lys Leu Arg Val
Tyr Ala Gln Gly Ser Gly 1115 1120 1125Thr Ile Gln Phe Gly Asn Cys
Glu Asp Glu Ala Ile Gln Phe Asn 1130 1135 1140Thr Asn Ser Phe Val
Tyr Lys Glu Lys Ile Ile Tyr Phe Asp Thr 1145 1150 1155Pro Ser Ile
Asn Leu His Ile Gln Ser Glu Gly Ser Glu Phe Val 1160 1165 1170Val
Ser Ser Ile Asp Leu Val Glu Leu Ser Asp Asp Glu 1175 1180
118521185PRTBacillus thuringiensis 2Met Asp Cys Asn Leu Gln Ser Gln
Gln Asn Ile Pro Tyr Asn Val Leu1 5 10 15Ala Ile Pro Val Ser Asn Val
Asn Ser Leu Thr Asp Thr Val Gly Asp 20 25 30Leu Lys Lys Ala Trp Glu
Glu Phe Gln Lys Thr Gly Ser Phe Ser Leu 35 40 45Thr Ala Leu Gln Gln
Gly Phe Ser Ala Ser Gln Gly Gly Thr Phe Asn 50 55 60Tyr Leu Thr Leu
Leu Gln Ser Gly Ile Ser Leu Ala Gly Ser Phe Val65 70 75 80Pro Gly
Gly Thr Phe Val Ala Pro Ile Ile Asn Met Val Ile Gly Trp 85 90 95Leu
Trp Pro His Lys Asn Lys Asn Ala Asp Thr Glu Asn Leu Ile Asn 100 105
110Leu Ile Asp Ser Glu Ile Gln Lys Gln Leu Asn Lys Ala Leu Leu Asp
115 120 125Ala Asp Arg Asn Glu Trp Ser Ser Tyr Leu Glu Ser Ile Phe
Asp Ser 130 135 140Ser Asn Asn Leu Asn Gly Ala Ile Val Asp Ala Gln
Trp Ser Gly Thr145 150 155 160Val Asn Thr Thr Asn Arg Thr Leu Arg
Asn Pro Thr Glu Ser Asp Tyr 165 170 175Thr Asn Val Val Thr Asn Phe
Ile Ala Ala Asp Gly Asp Ile Ala Asn 180 185 190Asn Glu Asn His Ile
Met Asn Gly Asn Phe Asp Val Ala Ala Ala Pro 195 200 205Tyr Phe Val
Ile Gly Ala Thr Ala Arg Phe Ala Ala Met Gln Ser Tyr 210 215 220Ile
Lys Phe Cys Asn Ala Trp Ile Asp Lys Val Gly Leu Ser Asp Ala225 230
235 240Gln Leu Thr Thr Gln Lys Ala Asn Leu Asp Arg Thr Lys Gln Asn
Met 245 250 255Arg Asn Ala Ile Leu Asn Tyr Thr Gln Gln Val Met Lys
Val Phe Lys 260 265 270Asp Ser Lys Asn Met Pro Thr Ile Gly Thr Asn
Lys Phe Ser Val Asp 275 280 285Thr Tyr Asn Val Tyr Ile Lys Gly Met
Thr Leu Asn Val Leu Asp Ile 290 295 300Val Ala Ile Trp Pro Ser Leu
Tyr Pro Asp Asp Tyr Thr Ser Gln Thr305 310 315 320Ala Leu Glu Gln
Thr Arg Val Thr Phe Ser Asn Met Val Gly Gln Glu 325 330 335Glu Gly
Thr Asp Gly Ser Leu Arg Ile Tyr Asn Thr Phe Asp Ser Phe
References