U.S. patent application number 11/874941 was filed with the patent office on 2008-04-17 for methods of protecting plants from pathogenic fungi and nematodes.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Hana S. Ali, Robert J. Keenan, Michael Lassner, Mathias L. Muller, Gowri Shah, Jun-Zhi Wei, Gusui Wu.
Application Number | 20080090293 11/874941 |
Document ID | / |
Family ID | 36793504 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090293 |
Kind Code |
A1 |
Ali; Hana S. ; et
al. |
April 17, 2008 |
Methods of Protecting Plants from Pathogenic Fungi and
Nematodes
Abstract
Methods for protecting a plant from a pathogen, particularly a
pathogenic fungus or nematode, are provided. A method for enhancing
pathogen resistance in a plant using the nucleotide sequences
disclosed herein is further provided. The method comprises
introducing into a plant an expression cassette comprising a
promoter operably linked to a nucleotide sequence that encodes an
antipathogenic polypeptide of the invention. Transformed plants,
plant cells, seeds, and microorganisms comprising a nucleotide
sequence that encodes an antipathogenic polypeptide of the
embodiments, or variant or fragment thereof, are also
disclosed.
Inventors: |
Ali; Hana S.; (San
Francisco, CA) ; Keenan; Robert J.; (Chicago, IL)
; Lassner; Michael; (Foster City, CA) ; Muller;
Mathias L.; (Santa Cruz, CA) ; Shah; Gowri;
(Fremont, CA) ; Wei; Jun-Zhi; (Palo Alto, CA)
; Wu; Gusui; (Palo Alto, CA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE
P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
36793504 |
Appl. No.: |
11/874941 |
Filed: |
October 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11172536 |
Jun 30, 2005 |
7301069 |
|
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11874941 |
Oct 19, 2007 |
|
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60584729 |
Jun 30, 2004 |
|
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Current U.S.
Class: |
435/468 ;
800/301 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8282 20130101; Y02A 40/164 20180101; C07K 14/415 20130101;
C12N 15/8285 20130101 |
Class at
Publication: |
435/468 ;
800/301 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/82 20060101 C12N015/82 |
Claims
1. A transgenic plant having stably incorporated into its genome a
polynucleotide sequence encoding a polypeptide that confers
resistance to at least one plant pathogenic nematode, wherein said
polynucleotide sequence is selected from the group consisting of:
a) a polynucleotide sequence encoding a polypeptide having at least
95% sequence identity to SEQ ID NO: 4; b) a polynucleotide sequence
encoding a polypeptide having at least 95% sequence identity to SEQ
ID NO: 5; and c) a polynucleotide sequence having at least 95%
identity to SEQ ID NO: 6; wherein said plant has improved
resistance to at least one plant pathogenic fungus, as compared to
a control plant.
2. The plant according to claim 1, wherein said plant is a
monocot.
3. The plant according to claim 1, wherein said plant is a
dicot.
4. Transformed seed of the plant according to claim 1, wherein the
seed comprise the polynucleotide.
5. The plant according to claim 1, wherein said polynucleotide is
operably linked to a promoter that drives expression in a cell of
said plant, wherein said promoter is selected from the group
consisting of: a) a constitutive promoter; b) a tissue-specific
promoter; and c) an inducible promoter.
6. The plant according to claim 1, wherein the polypeptide further
comprises a heterologous signal sequence.
7. The plant according to claim 6, wherein the signal sequence is a
secretion signal sequence.
8. The plant according to claim 6, wherein the signal sequence is
an organelle signal sequence.
9. The plant according to claim 8, wherein the signal sequence is a
plastid signal sequence.
10. The plant according to claim 1, wherein said polypeptide is SEQ
ID NO: 4.
11. The plant according to claim 1, wherein said polypeptide is SEQ
ID NO: 5.
12. The plant according to claim 1, wherein said polynucleotide is
SEQ ID NO: 6.
13. The plant according to claim 1, wherein said pathogenic fungus
is selected from the group consisting of: (a) Fusarium oxysporum;
and (b) Verticillium dahliae.
14. A method for enhancing resistance of a plant to at least one
plant pathogenic fungus, said method comprising: (a) stably
transforming a plant cell with at least one expression cassette
comprising a polynucleotide sequence operably linked to a promoter
that drives expression in a cell of said plant, wherein said
polynucleotide sequence encodes a polypeptide that confers
resistance to at least one plant pathogenic fungus, and wherein
said polynucleotide sequence is selected from the group consisting
of: i. a polynucleotide sequence encoding a polypeptide having at
least 95% sequence identity to SEQ ID NO: 4; ii. a polynucleotide
sequence encoding a polypeptide having at least 95% sequence
identity to SEQ ID NO: 5; and iii. a polynucleotide sequence having
at least 95% identity to SEQ ID NO: 6; and (b) regenerating a
transformed plant from said plant cell, wherein the plant has
enhanced resistance to said pathogenic fungus as compared to a
plant that does not comprise said expression cassette.
15. The method of claim 14, wherein said pathogenic fungus is
selected from the group consisting of: (a) Fusarium oxysporum; and
(b) Verticillium dahliae.
16. The method of claim 14, wherein said polynucleotide sequence
encodes the polypeptide set forth in SEQ ID NO: 4.
17. The method of claim 14, wherein said polynucleotide sequence
encodes the polypeptide set forth in SEQ ID NO: 5.
18. The method of claim 14, wherein said polynucleotide sequence is
SEQ ID NO:6.
19. The method of claim 14, wherein said promoter is selected from
the group consisting of: a) a constitutive promoter; b) a
tissue-specific promoter; and c) an inducible promoter.
20. The method of claim 14, wherein the polypeptide further
comprises a heterologous signal sequence.
21. The method of claim 20, wherein the signal sequence is a
secretion signal sequence.
22. The method of claim 20, wherein the signal sequence is an
organelle signal sequence.
23. The method of claim 22, wherein the signal sequence is a
plastid signal sequence.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
11/172,536, filed on Jun. 30, 2005, which claims the benefit of
U.S. Provisional Application No. 60/584,729, filed on Jun. 30,
2004, both of which are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of protecting
plants from pathogens through the use of polypeptides having
antifungal and/or nematicidal activity and the nucleic acid
sequences that encode them. Methods of the invention utilize these
polypeptides and nucleic acid sequences to control plant pathogens
and to increase pathogen resistance in plants. Transgenic plants
and seeds are also included.
BACKGROUND OF THE INVENTION
[0003] Disease in plants results from biotic and abiotic causes.
Biotic causes include, but are not limited to, fungi and nematodes.
A host of cellular processes enables plants to defend themselves
from disease caused by pathogenic agents. These processes
apparently form an integrated set of resistance mechanisms that is
activated by initial infection and then limits further spread of
the invading pathogenic organism.
[0004] Subsequent to recognition of a plant pathogen, plants can
activate an array of biochemical responses. Generally, the plant
responds by inducing several local responses in the cells
immediately surrounding the infection site. The most common
resistance response observed in both nonhost and race-specific
interactions is termed the "hypersensitive response" (HR). In the
hypersensitive response, cells contacted by the pathogen, and often
neighboring cells, rapidly collapse and dry in a necrotic fleck.
Other responses include the deposition of callose, the physical
thickening of cell walls by lignification, and the synthesis of
various antibiotic small molecules and proteins. Genetic factors in
both the host and the pathogen determine the specificity of these
local responses, which can be very effective in limiting the spread
of infection.
[0005] Incidence of plant diseases has traditionally been
controlled by agronomic practices that include crop rotation, the
use of agrochemicals, and conventional breeding techniques. The use
of chemicals to control plant pathogens, however, increases costs
to farmers and causes harmful effects on the ecosystem. Consumers
and government regulators alike are becoming increasingly concerned
with the environmental hazards associated with the production and
use of synthetic agrochemicals for protecting plants from
pathogens. Because of such concerns, regulators have banned or
limited the use of some of the most hazardous chemicals. The
incidence of fungal diseases has been controlled to some extent by
breeding resistant crops. Traditional breeding methods, however,
are time-consuming and require continuous effort to maintain
disease resistance as pathogens evolve. See, for example, Grover
and Gowthaman (2003) Curr. Sci. 84:330-340. Thus, there is a
significant need for novel alternatives for the control of plant
pathogens that possess a lower risk of pollution and environmental
hazards than is characteristic of traditional agrochemical-based
methods and that are less cumbersome than conventional breeding
techniques.
[0006] Recently, agricultural scientists have developed crop plants
with enhanced pathogen resistance by genetically engineering plants
to express antipathogenic proteins. For example, potatoes and
tobacco plants genetically engineered to produce an antifungal
endochitinase protein were shown to exhibit increased resistance to
foliar and soil-borne fungal pathogens. See Lorito et al. (1998)
Proc. Natl. Acad. Sci. 95:7860-7865. Moreover, transgenic barley
that is resistant to the stem rust fungus has also been developed.
See Horvath et al. (2003) Proc. Natl. Acad. Sci. 100:364-369. A
continuing effort to identify antipathogenic agents and to
genetically engineer disease-resistant plants is underway.
[0007] Nematode infection is a significant problem in the farming
of many agriculturally significant crops. For example, soybean cyst
nematode (Heterodera glycines) is a widespread pathogen that is
believed to be responsible for yield losses in soybeans, estimated
to be in excess of $1 billion per year in North America. Such
damage is the result of the stunting of the soybean plant caused by
the cyst nematode. The stunted plants have smaller root systems,
show symptoms of mineral deficiencies in their leaves, and wilt
easily. Other pathogenic nematodes of significance to agriculture
include the potato cyst nematodes Globodera rostochiensis and
Globodera pallida, which are key pathogens of the potato, the
cotton cyst nematode, Meloidogyne incognita, and the beet cyst
nematode Heterodera schachtii, which is a major problem for sugar
beet growers in Europe and the United States.
[0008] The primary method of controlling nematodes has been through
the application of highly toxic chemical compounds. The widespread
use of chemical compounds poses many problems with regard to the
environment because of the non-selectivity of the compounds and the
development of insect resistance to the chemicals. Nematicides such
as Aldicarb and its breakdown products are known to be highly toxic
to mammals. As a result, government restrictions have been imposed
on the use of these chemicals. The most widely used nematicide,
methyl bromide, is scheduled to be soon retired from use, and at
present, there is no promising candidate to replace this treatment.
Thus, there is a great need for effective, non-chemical methods and
compositions for nematode control.
[0009] Various approaches to pathogen control have been tried
including the use of biological organisms which are typically
"natural predators" of the species sought to be controlled. Such
predators may include other insects, fungi, and bacteria such as
Bacillus thuringiensis. Alternatively, large colonies of insect
pests have been raised in captivity, sterilized and released into
the environment in the hope that mating between the sterilized
insects and fecund wild insects will decrease the insect
population. While these approaches have had some success, they
entail considerable expense and present several major difficulties.
For example, it is difficult both to apply biological organisms to
large areas and to cause such living organisms to remain in the
treated area or on the treated plant species for an extended time.
Predator insects can migrate and fungi or bacteria can be washed
off of a plant or removed from a treated area by rain.
Consequently, while the use of such biological controls has
desirable characteristics and has met with some success, in
practice these methods have not achieved the goal of controlling
pathogen damage to crops.
[0010] Advances in biotechnology have presented new opportunities
for pathogen control through genetic engineering. In particular,
advances in plant genetics coupled with the identification of
naturally-occurring plant defensive compounds or agents offer the
opportunity to create transgenic crop plants capable of producing
such defensive agents and thereby protect the plants against
disease.
[0011] One promising method for nematode control is the production
of transgenic plants that are resistant to nematode infection and
reproduction. For example, with the use of nematode-inducible
promoters, plants can be genetically altered to express nematicidal
proteins in response to exposure to nematodes. See, for example,
U.S. Pat. No. 6,252,138, herein incorporated by reference.
Similarly, with the use of proteins having a deleterious effect on
nematodes, plants can be genetically altered to express such
deleterious proteins in response to nematode attack.
[0012] Thus, in light of the significant impact of plant pathogens,
particularly fungal and nematode pathogens, on the yield and
quality of crops, new methods for protecting plants from such
pathogens are needed.
BRIEF SUMMARY OF THE INVENTION
[0013] The embodiments of the invention provide transgenic plants
with enhanced resistance to pathogens, each plant comprising a
polynucleotide encoding a polypeptide comprising an amino acid
sequence at least 95% identical to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10
or 11, wherein said plant has improved pathogen resistance to at
least one pathogen selected from the group consisting of plant
pathogenic fungi and nematodes. The plant may be a monocot or a
dicot. Seeds of such transgenic plants are also provided for.
Similarly, the embodiments provide monocot or dicot transgenic
plants and seeds with enhanced resistance to pathogens wherein the
plant comprises a polynucleotide sequence at least 95% identical to
SEQ ID NOs: 3, 6, 9, 12, 13, or 14, wherein said plant has improved
pathogen resistance to at least one pathogen selected from the
group consisting of plant pathogenic fungi and nematodes. The
polypeptides expressed in the transgenic plants may or may not
comprise a signal sequence.
[0014] The embodiments of the invention also provide methods of
enhancing resistance of a plant to a pathogen, the methods
comprising introducing into a plant cell an expression cassette
comprising a nucleotide sequence operably linked to a promoter,
wherein the nucleotide sequence has at least 95% identity to SEQ ID
NOs: 3, 6, 9, 12, 13 or 14, or wherein the nucleotide sequence
encodes a polypeptide comprising an amino acid sequence identical
or substantially identical to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10 or
11, and wherein the polypeptide has activity against at least one
pathogen selected from the group consisting of plant pathogenic
fungi and nematodes. The plant cell is used to regenerate a
transformed plant wherein the level of pathogen resistance in the
transformed plant is increased in comparison to a plant that does
not comprise the expression cassette. The polypeptides of these
embodiments may or may not comprise a signal sequence.
[0015] The promoters used in the expression cassettes of the
embodiments are selected from the group consisting of constitutive,
tissue-specific, root-specific, inducible and pathogen-inducible
promoters. In some embodiments, the polypeptide with activity
against plant pathogens comprises a signal sequence. In some
embodiments, the polypeptide lacks a signal sequence. In some
embodiments, the signal sequence is a secretion signal sequence,
while in others it is an organelle and/or plastid signal
sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 depicts results from a Meloidogyne incognita
infection assay in Astragalus hairy roots.
[0017] FIG. 2 depicts results from a Meloidogyne incognita
infection assay in transgenic tobacco plants.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Embodiments of the invention provide compositions and
methods directed to enhancing pathogen resistance, particularly
nematode and fungal resistance, in plants. The embodiments provide
polynucleotides encoding amino acid sequences for nematicidal and
antifungal polypeptides. Specifically, the embodiments provide
nematicidal and antifungal polypeptides having the amino acid
sequences set forth in SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10 and 11 and
variants and fragments thereof. Isolated nucleic acid molecules,
and variants and fragments thereof, comprising nucleotide sequences
that encode the amino acid sequences shown in SEQ ID NOs: 1, 2, 4,
5, 7, 8, 10 and 11 are further provided.
[0019] Nucleotide sequences that encode the polypeptides of SEQ ID
NOs: 1, 2, 4, 5, 7, 8, 10 and 11 are provided. These nucleotide
sequences are set forth in SEQ ID NOs:3, 6, 9, 12, 13, and 14. Some
of these nucleotide sequences have been optimized for expression in
E. coli. Plants, plant cells, seeds, and microorganisms comprising
a nucleotide sequence that encodes a nematicidal and/or antifungal
polypeptide of the embodiments are also disclosed herein.
Nematicidal and antifungal compositions comprising an isolated
nematicidal polypeptide or antifungal polypeptide or a
microorganism that expresses a polypeptide of the embodiments are
further provided. The compositions of the embodiments find use in
generating nematode-resistant and fungal-resistant plants and in
protecting plants from nematodes and fungi.
[0020] The polypeptides disclosed herein display activity against
nematodes, such as, for example, Caenorhabditis elegans and
Meloidogyne incognita. The polypeptides disclosed herein also
display antifungal activity against plant pathogenic fungi, such
as, for example, Fusarium oxysporum and Verticillium dahliae. The
species of origin of these nematicidal and antifungal polypeptides
are plant species. In particular, the source of the polypeptides of
SEQ ID NOs:1, 2, 7, and 8 is Arabidopsis thaliana. The source of
the polypeptides of SEQ ID NOs: 4 and 5 is Picea mariana (black
spruce), and the source of the polypeptides of SEQ ID NOs: 10 and
11 is Petunia x hybrida.
[0021] "Nematicidal compositions" or "nematicidal polypeptides" is
intended to mean that the compositions or polypeptides of the
embodiments have nematicidal activity and thus are capable of
suppressing, controlling, and/or killing the invading nematode.
"Antifungal compositions" or "antifungal polypeptides" is intended
to mean that the compositions or polypeptides of the embodiments
have antifungal activity and thus are capable of suppressing,
controlling, and/or killing the invading fungus. A nematicidal or
antifungal polypeptide of the embodiments will reduce the disease
symptoms resulting from nematode or fungal challenge by at least
about 5% to about 50%, at least about 10% to about 60%, at least
about 30% to about 70%, at least about 40% to about 80%, or at
least about 50% to about 90% or greater. Hence, the methods of the
embodiments can be utilized to protect plants from fungi and
nematodes.
[0022] The polynucleotides and polypeptides of the embodiments find
use in methods for inducing pathogen resistance in a plant.
Accordingly, the compositions and methods disclosed herein are
useful in protecting plants against nematodes and fungi. "Pathogen
resistance" is intended to mean that the plant avoids the disease
symptoms that are the outcome of plant-nematode interactions or
plant-fungus interactions. A plant with "improved pathogen
resistance" or "enhanced pathogen resistance" is intended to mean
that a plant, which has been transformed with a nucleic acid
molecule of the embodiments, and which is expressing a polypeptide
of the embodiments, exhibits a level of resistance or tolerance to
a fungal or nematode pathogen that is increased in comparison to a
plant that does not comprise said nucleic acid molecule, such as a
wild type plant. That is, nematodes or fungi are prevented from
causing plant disease and the associated disease symptoms in the
transformed plant, or alternatively, the disease symptoms caused by
the nematode or fungus are minimized or lessened, such as, for
example, the reduction of stress and associated yield loss.
Resistance may vary from a slight increase in tolerance to the
effects of the pathogen to total resistance such that the plant is
unaffected by the presence of the pathogen. An increased level of
resistance against a particular fungus or nematode or against a
wider spectrum of fungi or nematodes may both constitute antifungal
and nematicidal activity or improved nematode or fungus resistance.
The plants of the embodiments exhibit an improvement of at least
5%, at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or up to 100% improvement compared to an untransformed plant.
Such improvement may be measured by any suitable means known in the
art, such as, but not limited to, counting cysts or eggs of
nematodes, counting fungal lesions on plants, measuring fungal
biomass, comparing plant yields, and other methods described in the
following paragraphs.
[0023] Assays that measure antipathogenic activity are commonly
known in the art, as are methods to quantitate disease resistance
in plants following pathogen infection. See, for example, U.S. Pat.
No. 5,614,395, herein incorporated by reference. Such techniques
include, measuring over time, the average lesion diameter, the
pathogen biomass, and the overall percentage of decayed plant
tissues. For example, a plant either expressing an antipathogenic
polypeptide or having an antipathogenic composition applied to its
surface shows a decrease in tissue necrosis (i.e., lesion diameter)
or a decrease in plant death following pathogen challenge when
compared to a control plant that was not exposed to the
antipathogenic composition. Alternatively, antipathogenic activity
can be measured by a decrease in pathogen biomass. For example, a
plant expressing an antipathogenic polypeptide or exposed to an
antipathogenic composition is challenged with a pathogen of
interest. Over time, tissue samples from the pathogen-inoculated
tissues are obtained and RNA is extracted. The percent of a
specific pathogen RNA transcript relative to the level of a plant
specific transcript allows the level of pathogen biomass to be
determined. See, for example, Thomma et al. (1998) Plant Biology
95:15107-15111, herein incorporated by reference.
[0024] Furthermore, in vitro antipathogenic assays include, for
example, the addition of varying concentrations of the
antipathogenic composition to paper disks and placing the disks on
agar containing a suspension of the pathogen of interest. Following
incubation, clear inhibition zones develop around the discs that
contain an effective concentration of the antipathogenic
polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892, herein
incorporated by reference). Additionally, microspectrophotometrical
analysis can be used to measure the in vitro antipathogenic
properties of a composition (Hu et al. (1997) Plant Mol. Biol.
34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,
both of which are herein incorporated by reference). Assays that
specifically measure antifungal activity are also well known in the
art. See, for example, Duvick et al. (1992) J. Biol. Chem.
267:18814-18820; Lacadena et al. (1995) Arch. Biochem. Biophys.
324:273-281; Xu et al. (1997) Plant Mol. Biol. 34: 949-959; Lee et
al. (1999) Biochem. Biophys. Res. Comm. 263:646-651; Vila et al.
(2001) Mol. Plant. Microbe Interact. 14:1327-1331; Moreno et al.
(2003) Phytpathol. 93:1344-1353; Kaiserer et al. (2003) Arch.
Microbiol. 180:204-210; and U.S. Pat. No. 6,015,941.
[0025] Also contemplated are antipathogenic assays directed at
nematode pathogens. Such assays are known to the skilled artisan,
and may include assays directed at specific characteristics of
nematode pathogen infections, such as assays directed at nematode
feeding site formation. Such assays include those disclosed in U.S.
Pat. Nos. 6,008,436; and 6,252,138; herein incorporated by
reference.
[0026] The embodiments disclose plants transformed with nucleic
acid molecules that encode nematicidal and antifungal proteins. The
compositions find use in methods for inducing pathogen resistance
in a plant and for protecting a plant from a fungus or a nematode.
One of skill in the art will appreciate that the compositions and
methods disclosed herein can be used in combination with other
compositions and methods available in the art for protecting plants
from pathogen attack.
[0027] In particular aspects, methods for inducing nematode or
fungal resistance in a plant comprise introducing into a plant at
least one expression cassette, wherein the expression cassette
comprises a nucleotide sequence encoding a nematicidal or
antifungal polypeptide of the embodiments operably linked to a
promoter that drives expression in the plant. The plant expresses
the polypeptide, thereby exposing the nematode or fungus to the
polypeptide at the site of attack. Expression of a polypeptide of
the embodiments may be targeted to specific plant tissues where
nematode or fungal resistance is particularly important, such as,
for example, roots, leaves, or stems. Such tissue-preferred
expression may be accomplished by root-preferred, leaf-preferred,
vascular tissue-preferred, stalk-preferred, or seed-preferred
promoters. Moreover, the polypeptides of the embodiments may also
be targeted to specific subcellular locations within a plant cell
or, alternatively, secreted from the cell, as described herein
below.
[0028] Just as expression of a polypeptide of the embodiments may
be targeted to specific plant tissues or cell types through the use
of appropriate promoters, it may also be targeted to different
locations within the cell through the use of targeting information
or "targeting labels." Unlike the promoter, which acts at the
transcriptional level, such targeting information is part of the
initial translation product. Depending on the mode of infection of
the pathogen or the metabolic function of the tissue or cell type,
the location of the protein in different compartments of the cell
may make it more efficacious against a given pathogen or make it
interfere less with the functions of the cell. For example, one may
produce a protein preceded by a signal peptide, which directs the
translation product into the endoplasmic reticulum, by including in
the construct (i.e. expression cassette) sequences encoding a
signal peptide (such sequences may also be called the "signal
sequence"). The signal sequence used could be, for example, one
associated with the gene encoding the polypeptide, or it may be
taken from another gene.
[0029] There are many signal peptides described in the literature,
and they are largely interchangeable (Raikhel and Chrispeels,
"Protein sorting and vesicle traffic" in Buchanan et al., eds,
(2000) Biochemistry and Molecular Biology of Plants (American
Society of Plant Physiologists, Rockville, Md.), herein
incorporated by reference). The addition of a signal peptide will
result in the translation product entering the endoplasmic
reticulum (in the process of which the signal peptide itself is
removed from the polypeptide), but the final intracellular location
of the protein depends on other factors, which may be manipulated
to result in localization most appropriate for the pathogen and
cell type. The default pathway, that is, the pathway taken by the
polypeptide if no other targeting labels are included, results in
secretion of the polypeptide across the cell membrane (Raikhel and
Chrispeels, supra) into the apoplast. The apoplast is the region
outside the plasma membrane system and includes cell walls,
intercellular spaces, and the xylem vessels that form a continuous,
permeable system through which water and solutes may move. This
will often be a suitable location.
[0030] Other pathogens may be more effectively combated by locating
the peptide within the cell rather than outside the cell membrane.
This can be accomplished, for example, by adding an endoplasmic
reticulum retention signal encoding sequence to the sequence of the
gene. Methods and sequences for doing this are described in Raikhel
and Chrispeels, supra; for example, adding sequences encoding the
amino acids K, D, E and L in that order, or variations thereof
described in the literature, to the end of the protein coding
portion of the polypeptide will accomplish this. ER retention
sequences are well known in the art. See, for example, Denecke et
al. (1992). EMBO J. 11:2345-2355; Wandelt et al. (1992) Plant J.
2:181-192; Denecke et al. (1993) J. Exp. Bot. 44:213-221; Vitale et
al. (1993) J. Exp. Bot. 44:1417-1444; Gomord et al. (1996) Plant
Physiol. Biochem. 34:165-181; Lehmann et al. (2001) Plant Physiol.
127 (2): 436-449.
[0031] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues (e.g., peptide nucleic acids) having the essential
nature of natural nucleotides in that they hybridize to
single-stranded nucleic acids in a manner similar to naturally
occurring nucleotides.
[0032] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. Polypeptides of the
embodiments can be produced either from a nucleic acid molecule
disclosed herein, or by the use of standard molecular biology
techniques. For example, a truncated protein of the embodiments can
be produced by expression of a recombinant nucleic acid molecule of
the embodiments in an appropriate host cell, or alternatively by a
combination of ex vivo procedures, such as protease digestion and
purification.
[0033] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified nucleic acid molecule mean that the
nucleic acid molecule comprises the requisite information to direct
translation of the nucleotide sequence into a specified protein.
The information by which a protein is encoded is specified by the
use of codons. A nucleic acid molecule encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid sequence or may lack such intervening
non-translated sequences (e.g., as in cDNA).
[0034] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, y-carboxyglutamate, and
O-phosphoserine. Amino acids may be referred to herein by either
the commonly known three letter symbols or by the one-letter
symbols recommended by the IUPAC-IUB Biochemical Nomenclature
Commission. Nucleotides, likewise, may be referred to by their
commonly accepted single-letter codes.
[0035] The embodiments encompass methods of using isolated or
substantially purified polynucleotide or protein compositions. An
"isolated" or "purified" polynucleotide or protein, or biologically
active portion thereof, is substantially or essentially free from
components that normally accompany or interact with the
polynucleotide or protein as found in its naturally occurring
environment. Thus, an isolated or purified polynucleotide or
protein is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. Optimally, an "isolated" polynucleotide is
free of sequences (optimally protein encoding sequences) that
naturally flank the polynucleotide (i.e., sequences located at the
5' and 3' ends of the polynucleotide) in the genomic DNA of the
organism from which the polynucleotide is derived. For example, in
various embodiments, the isolated polynucleotide can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequence that naturally flank the polynucleotide in
genomic DNA of the cell from which the polynucleotide is derived. A
protein that is substantially free of cellular material includes
preparations of protein having less than about 30%, 20%, 10%, 5%,
or 1% (by dry weight) of contaminating protein. When the protein of
the embodiments or biologically active portion thereof is
recombinantly produced, optimally culture medium represents less
than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical
precursors or non-protein-of-interest chemicals.
[0036] Fragments and variants of the disclosed nucleotide sequences
and proteins encoded thereby are also encompassed by the
embodiments. "Fragment" is intended to mean a portion of the
nucleotide sequence or a portion of the amino acid sequence and
hence protein encoded thereby. Fragments of a nucleotide sequence
may encode protein fragments that retain the biological activity of
the native protein and hence have antipathogenic activity, more
particularly nematicidal or antifungal activity. Alternatively,
fragments of a nucleotide sequence that are useful as hybridization
probes generally do not encode fragment proteins retaining
biological activity. Thus, fragments of a nucleotide sequence may
range from at least about 20 nucleotides, about 50 nucleotides,
about 100 nucleotides, and up to the full-length nucleotide
sequence encoding the polypeptides of the embodiments.
[0037] A fragment of a nucleotide sequence that encodes a
biologically active portion of an antipathogenic polypeptide of the
embodiments will encode at least 15, 25, 30, 40, or 50 contiguous
amino acids, or up to the total number of amino acids present in a
full-length antifungal polypeptide of the embodiments (for example,
66 amino acids for SEQ ID NO:1). Fragments of a nucleotide sequence
that are useful as hybridization probes or PCR primers generally
need not encode a biologically active portion of an antipathogenic
protein.
[0038] As used herein, "full-length sequence" in reference to a
specified polynucleotide means having the entire nucleic acid
sequence of a native sequence. "Native sequence" is intended to
mean an endogenous sequence, i.e., a non-engineered sequence found
in an organism's genome.
[0039] Thus, a fragment of a nucleotide sequence of the embodiments
may encode a biologically active portion of an antipathogenic
polypeptide, or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed below. A
biologically active portion of an antipathogenic polypeptide can be
prepared by isolating a portion of one of the nucleotide sequences
of the embodiments, expressing the encoded portion of the
antipathogenic protein (e.g., by recombinant expression in vitro),
and assessing the activity of the encoded portion of the
nematicidal or antifungal protein. Nucleic acid molecules that are
fragments of a nucleotide sequence of the embodiments comprise at
least 15, 20, 50, 75, 100, or 150 contiguous nucleotides, or up to
the number of nucleotides present in a full-length nucleotide
sequence disclosed herein.
[0040] "Variants" is intended to mean substantially similar
sequences. For polynucleotides, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more internal
sites within the native polynucleotide and/or a substitution of one
or more nucleotides at one or more sites in the native
polynucleotide. As used herein, a "native" polynucleotide or
polypeptide comprises a naturally occurring nucleotide sequence or
amino acid sequence, respectively. One of skill in the art will
recognize that variants of the nucleic acid sequences of the
embodiments will be constructed such that the open reading frame is
maintained. For polynucleotides, conservative variants include
those sequences that, because of the degeneracy of the genetic
code, encode the amino acid sequence of one of the antipathogenic
polypeptides of the embodiments. Naturally occurring allelic
variants such as these can be identified with the use of well-known
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant polynucleotides also include synthetically derived
polynucleotide, such as those generated, for example, by using
site-directed mutagenesis but which still encode an antipathogenic
protein of the embodiments. Generally, variants of a particular
polynucleotide of the embodiments will have at least about 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that
particular polynucleotide as determined by sequence alignment
programs and parameters described elsewhere herein.
[0041] Variants of a particular polynucleotide of the embodiments
(i.e., the reference polynucleotide) can also be evaluated by
comparison of the percent sequence identity between the polypeptide
encoded by a variant polynucleotide and the polypeptide encoded by
the reference polynucleotide. Thus, for example, an isolated
polynucleotide that encodes a polypeptide with a given percent
sequence identity to the polypeptides of SEQ ID NOs: 1, 3, 5, 7,
and 9 are disclosed. Percent sequence identity between any two
polypeptides can be calculated using sequence alignment programs
and parameters described elsewhere herein. Where any given pair of
polynucleotides of the embodiments is evaluated by comparison of
the percent sequence identity shared by the two polypeptides they
encode, the percent sequence identity between the two encoded
polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity.
[0042] "Variant" protein is intended to mean a protein derived from
the native protein by deletion or addition of one or more amino
acids at one or more internal sites in the native protein and/or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the embodiments are
biologically active, that is they continue to possess the desired
biological activity of the native protein, that is, antipathogenic,
particularly nematicidal or antifungal activity as described
herein. Such variants may result from, for example, genetic
polymorphism or from human manipulation. Biologically active
variants of a native antipathogenic protein of the embodiments will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the amino acid sequence for the native protein
as determined by sequence alignment programs and parameters
described elsewhere herein. A biologically active variant of a
protein of the embodiments may differ from that protein by as few
as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few
as 5, as few as 4, 3, 2, or even 1 amino acid residue.
[0043] The proteins of the embodiments may be altered in various
ways including amino acid substitutions, deletions, truncations,
and insertions. Methods for such manipulations are generally known
in the art. For example, amino acid sequence variants and fragments
of the antipathogenic proteins can be prepared by mutations in the
DNA. Methods for mutagenesis and polynucleotide alterations are
well known in the art. See, for example, Kunkel (1985) Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.
154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.
(1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff et al. (1978) Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by reference. Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be
optimal.
[0044] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine) can be modified to yield a
functionally identical molecule. Accordingly, each silent variation
of a nucleic acid which encodes a polypeptide is implicit in each
described sequence.
[0045] As to amino acid sequences, one of skill will recognize that
individual substitutions, in a nucleic acid, peptide, polypeptide,
or protein sequence which alters a single amino acid or a small
percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art.
[0046] The following six groups each contain amino acids that are
conservative substitutions for one another: [0047] 1) Alanine (A),
Serine (S), Threonine (T); [0048] 2) Aspartic acid (D), Glutamic
acid (E); [0049] 3) Asparagine (N), Glutamine (Q); [0050] 4)
Arginine (R), Lysine (K); [0051] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and [0052] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W). [0053] (see, e.g., Creighton,
Proteins (1984)).
[0054] Thus, the genes and polynucleotides of the embodiments
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the embodiments encompass
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
antipathogenic, particularly nematicidal or antifungal, activity.
Obviously, the mutations that will be made in the DNA encoding the
variant must not place the sequence out of reading frame and
optimally will not create complementary regions that could produce
secondary mRNA structure. See, EP Patent No. 0075444.
[0055] In nature, some polypeptides are produced as complex
precursors which, in addition to targeting labels such as the
signal peptides discussed elsewhere in this application, also
contain other fragments of peptides which are removed (processed)
at some point during protein maturation, resulting in a mature form
of the polypeptide that is different from the primary translation
product (aside from the removal of the signal peptide). "Mature
protein" refers to a post-translationally processed polypeptide;
i.e., one from which any pre- or propeptides present in the primary
translation product have been removed. "Precursor protein" or
"prepropeptide" or "preproprotein" all refer to the primary product
of translation of mRNA; i.e., with pre- and propeptides still
present. Pre- and propeptides may include, but are not limited to,
intracellular localization signals. "Pre" in this nomenclature
generally refers to the signal peptide. The form of the translation
product with only the signal peptide removed but no further
processing yet is called a "propeptide" or "proprotein." The
fragments or segments to be removed may themselves also be referred
to as "propeptides." A proprotein or propeptide thus has had the
signal peptide removed, but contains propeptides (here referring to
propeptide segments) and the portions that will make up the mature
protein. The skilled artisan is able to determine, depending on the
species in which the proteins are being expressed and the desired
intracellular location, if higher expression levels might be
obtained by using a gene construct encoding just the mature form of
the protein, the mature form with a signal peptide, or the
proprotein (i.e., a form including propeptides) with a signal
peptide. For optimal expression in plants or fungi, the pre- and
propeptide sequences may be needed. The propeptide segments may
play a role in aiding correct peptide folding.
[0056] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays. That is, the activity can be evaluated by assays
that measure antipathogenic activity such as, for example,
antifungal plate assays and other methods described elsewhere in
this disclosure. See, for example, Duvick et al. (1992) J. Biol.
Chem. 267:18841-18820, herein incorporated by reference.
[0057] Variant polynucleotides and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different antipathogenic protein coding sequences can be
manipulated to create a new antipathogenic protein possessing the
desired properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. For example, using this approach, sequence motifs encoding a
domain of interest may be shuffled between the antipathogenic
protein gene of the embodiments and other known antipathogenic
protein genes to obtain a new gene coding for a protein with an
improved property of interest, such as increased antifungal or
nematicidal activity. Strategies for such DNA shuffling are known
in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci.
USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et
al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0058] The polynucleotides of the embodiments can be used to
isolate corresponding sequences from other organisms, particularly
other plants. In this manner, methods such as PCR, hybridization,
and the like can be used to identify such sequences based on their
sequence homology to the sequences set forth herein. Sequences
isolated based on their sequence identity to the entire sequences
set forth herein or to variants and fragments thereof are
encompassed by the embodiments. Such sequences include sequences
that are orthologs of the disclosed sequences. "Orthologs" is
intended to mean genes derived from a common ancestral gene and
which are found in different species as a result of speciation.
Genes found in different species are considered orthologs when
their nucleotide sequences and/or their encoded protein sequences
share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of
orthologs are often highly conserved among species. Thus, isolated
polynucleotides that encode for an antipathogenic, particularly
antifungal or nematicidal protein and which hybridize under
stringent conditions to the sequences disclosed herein, or to
variants or fragments thereof, are encompassed by the
embodiments.
[0059] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to; methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0060] In hybridization techniques, all or part of a known
polynucleotide is used as a probe that selectively hybridizes to
other corresponding polynucleotides present in a population of
cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The 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. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the polynucleotides of the embodiments.
Methods for preparation of probes for hybridization and for
construction of cDNA and genomic libraries are generally known in
the art and are disclosed in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0061] For example, an entire polynucleotide disclosed herein, or
one or more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding polynucleotides and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique among
antipathogenic polynucleotide sequences and are optimally at least
about 10 nucleotides in length, and most optimally at least about
20 nucleotides in length. Such probes may be used to amplify
corresponding polynucleotides from a chosen organism by PCR. This
technique may be used to isolate additional coding sequences from a
desired organism or as a diagnostic assay to determine the presence
of coding sequences in an organism. Hybridization techniques
include hybridization screening of plated DNA libraries (either
plaques or colonies; see, for example, Sambrook et al. supra).
[0062] Hybridization of such sequences may be carried out under
stringent conditions. "Stringent conditions" or "stringent
hybridization conditions" is intended to mean 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, optimally less than 500 nucleotides in length.
[0063] 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 final wash in 0.1.times.SSC at
60 to 65.degree. C. for at least 30 minutes. 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. The duration of the wash time will be at least
a length of time sufficient to reach equilibrium.
[0064] 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
thermal melting point (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
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 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 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 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
optimal to increase the SSC concentration so that a higher
temperature can be used. An extensive guide to the hybridization of
nucleic acid sequences 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. supra.
[0065] The following terms are used to describe the sequence
relationships between two or more polynucleotides or polypeptides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", and, (d) "percentage of sequence identity."
[0066] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0067] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two polynucleotides. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0068] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local
alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0069] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the embodiments. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the embodiments. 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 (in BLAST 2.0) 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, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also
be performed manually by inspection.
[0070] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using Gap Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using Gap Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. "Equivalent program" is intended to mean any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0071] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the GCG Wisconsin
Genetics Software Package for protein sequences are 8 and 2,
respectively. For nucleotide sequences the default gap creation
penalty is 50 while the default gap extension penalty is 3. The gap
creation and gap extension penalties can be expressed as an integer
selected from the group of integers consisting of from 0 to 200.
Thus, for example, the gap creation and gap extension penalties can
be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65 or greater.
[0072] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the Quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
[0073] (c) As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0074] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0075] The use of the term "polynucleotide" is not intended to
limit the embodiments to polynucleotides comprising DNA. Those of
ordinary skill in the art will recognize that polynucleotides, can
comprise ribonucleotides and combinations of ribonucleotides and
deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides
include both naturally occurring molecules and synthetic analogues.
The polynucleotides of the embodiments also encompass all forms of
sequences including, but not limited to, single-stranded forms,
double-stranded forms, and the like.
[0076] In some embodiments, expression cassettes comprising a
promoter operably linked to a heterologous nucleotide sequence of
the embodiments that encodes an antipathogenic polypeptide are
further provided. The expression cassettes of the embodiments find
use in generating transformed plants, plant cells, and
microorganisms and in practicing the methods for inducing pathogen
resistance disclosed herein. The expression cassette will include
5' and 3' regulatory sequences operably linked to a polynucleotide
of the embodiments. "Operably linked" is intended to mean a
functional linkage between two or more elements. For example, an
operable linkage between a polynucleotide of interest and a
regulatory sequence (i.e., a promoter) is a functional link that
allows for expression of the polynucleotide of interest. Operably
linked elements may be contiguous or non-contiguous. When used to
refer to the joining of two protein coding regions, by operably
linked it is intended that the coding regions are in the same
reading frame. The cassette may additionally contain at least one
additional gene to be cotransformed into the organism.
Alternatively, the additional gene(s) can be provided on multiple
expression cassettes. Such an expression cassette is provided with
a plurality of restriction sites and/or recombination sites for
insertion of the polynucleotide that encodes an antipathogenic
polypeptide to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes.
[0077] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional initiation region (i.e., a
promoter), translational initiation region, a polynucleotide of the
embodiments, a translational termination region and, optionally, a
transcriptional termination region functional in the host organism.
The regulatory regions (i.e., promoters, transcriptional regulatory
regions, and translational termination regions) and/or the
polynucleotide of the embodiments may be native/analogous to the
host cell or to each other. Alternatively, the regulatory regions
and/or the polynucleotide of the embodiments may be heterologous to
the host cell or to each other. As used herein, "heterologous" in
reference to a sequence is a sequence that originates from a
foreign species, or, if from the same species, is substantially
modified from its native form in composition and/or genomic locus
by deliberate human intervention. For example, a promoter operably
linked to a heterologous polynucleotide is from a species different
from the species from which the polynucleotide was derived, or, if
from the same/analogous species, one or both are substantially
modified from their original form and/or genomic locus, or the
promoter is not the native promoter for the operably linked
polynucleotide.
[0078] The optionally included termination region may be native
with the transcriptional initiation region, may be native with the
operably linked polynucleotide 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 polynucleotide of interest, the
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 Acids Res.
15:9627-9639. In particular embodiments, the potato protease
inhibitor II gene (PinII) terminator is used. See, for example,
Keil et al. (1986) Nucl. Acids Res. 14:5641-5650; and An et al.
(1989) Plant Cell 1:115-122, herein incorporated by reference in
their entirety.
[0079] Where appropriate, the polynucleotides may be optimized for
increased expression in the transformed organism. For example, the
polynucleotides can be synthesized using plant-preferred codons for
improved expression. See, for example, Campbell and Gowri (1990)
Plant Physiol. 92:1-11 for a discussion of host-preferred codon
usage. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0080] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0081] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238),
MDMV leader (Maize Dwarf Mosaic Virus), and human immunoglobulin
heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature
353:90-94); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature
325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al.
(1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.
237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et
al. (1991) Virology 81:382-385). See also, Della-Cioppa et al.
(1987) Plant Physiol. 84:965-968.
[0082] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0083] The expression cassette can also comprise a selectable
marker gene for the selection of transformed cells. Selectable
marker genes are utilized for the selection of transformed cells or
tissues. Marker genes include genes encoding antibiotic resistance,
such as those encoding neomycin phosphotransferase II (NEO) and
hygromycin phosphotransferase (HPT), as well as genes conferring
resistance to herbicidal compounds, such as glufosinate ammonium,
bromoxynil, imidazolinones, glyphosate and
2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers
include phenotypic markers such as .beta.-galactosidase and
fluorescent proteins such as green fluorescent protein (GFP) (Su et
al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004)
Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al.
(2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant
Physiol 129:913-42), and yellow florescent protein (PhiYFP.TM. from
Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For
additional selectable markers, see generally, Yarranton (1992)
Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc.
Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72;
Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980)
in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown
et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722;
Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404;
Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553;
Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D.
Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.
Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.
10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA
89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA
88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.
19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.
10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.
35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;
Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)
Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985)
Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag,
Berlin); Gill et al. (1988) Nature 334:721-724, and WO 02/36782.
Such disclosures are herein incorporated by reference.
[0084] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the
embodiments.
[0085] The term "promoter" refers to regions or sequence located
upstream and/or downstream from the start of transcription that are
involved in recognition and binding of RNA polymerase and other
proteins to initiate transcription. Promoters include nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A promoter
also optionally includes distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0086] A number of promoters can be used in the practice of the
embodiments, including the native promoter of the polynucleotide
sequence of interest. The promoters can be selected based on the
desired outcome. A wide range of plant promoters are discussed in
the recent review of Potenza et al. (2004) In Vitro Cell Dev
Biol--Plant 40:1-22, herein incorporated by reference. For example,
the nucleic acid molecules can be combined with constitutive,
tissue-preferred, pathogen-inducible, or other promoters for
expression in plants. Such constitutive promoters include, for
example, the core promoter of the Rsyn7 promoter and other
constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.
6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature
313:810-812); rice actin (McElroy et al. (1990) Plant Cell
2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.
12:619-632 and Christensen et al. (1992) Plant Mol. Biol.
18:675-689); PEMU (Last et al. (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. Pat. No. 5,659,026), and the like. Other
constitutive promoters include, for example, those disclosed in
U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0087] Generally, it will be beneficial to express the gene from an
inducible promoter, particularly from a pathogen-inducible
promoter. Such promoters include those from pathogenesis-related
proteins (PR proteins), which are induced following infection by a
pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,
chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J.
Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656;
and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO
99/43819, herein incorporated by reference.
[0088] Of interest are promoters that result in expression of a
protein locally at or near the site of pathogen infection. See, for
example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton
et al. (1989) Molecular Plant-Microbe Interactions 2:325-331;
Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430;
Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996)
Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al.
(1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.
Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201;
Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No.
5,750,386 (nematode-inducible); and the references cited therein. A
further example is the inducible promoter for the maize PRms gene,
whose expression is induced by the pathogen Fusarium
verticillioides (see, for example, Cordero et al. (1992)Physiol.
Mol. Plant. Path. 41:189-200).
[0089] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the embodiments. Such wound-inducible
promoters include potato proteinase inhibitor (pin II) gene (Ryan
(1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature
Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148;
win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet.
215:200-208); systemin (McGurl et al. (1992) Science
225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol.
22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI
gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,
herein incorporated by reference.
[0090] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize ln2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0091] Tissue-preferred promoters can be utilized to target
enhanced expression of the antipathogenic polypeptides of the
embodiments within a particular plant tissue. For example, a
tissue-preferred promoter may be used to express a nematicidal or
antifungal polypeptide in a plant tissue where disease resistance
is particularly important, such as, for example, the roots or the
leaves. Tissue-preferred promoters include Yamamoto et al. (1997)
Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.
38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet.
254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;
Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et
al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996)
Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.
20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590;
and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.
[0092] Such promoters can be modified, if necessary, for weak
expression.
[0093] Vascular tissue-preferred promoters are known in the art and
include those promoters that selectively drive protein expression
in, for example, xylem and phloem tissue. Vascular tissue-preferred
promoters include, but are not limited to, the Prunus serotina
prunasin hydrolase gene promoter (see, e.g., International
Publication No. WO 03/006651), and also those found in U.S. patent
application Ser. No. 10/109,488.
[0094] Stalk-preferred promoters may be used to drive expression of
an antipathogenic polypeptide of the embodiments. Exemplary
stalk-preferred promoters include the maize MS8-15 gene promoter
(see, for example, U.S. Pat. No. 5,986,174 and International
Publication No. WO 98/00533), and those found in Graham et al.
(1997) Plant Mol Biol 33(4): 729-735.
[0095] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0096] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. Leach and Aoyagi (1991) describe their
analysis of the promoters of the highly expressed rolC and rolD
root-inducing genes of Agrobacterium rhizogenes (see Plant Science
(Limerick) 79(1):69-76). Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179.
[0097] "Seed-preferred" promoters include both "seed-specific"
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10:108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); milps (myo-inositol-1-phosphate synthase) (see WO 00/11177
and U.S. Pat. No. 6,225,529; herein incorporated by reference).
Gamma-zein is a preferred endosperm-specific promoter. Glob-1 is a
preferred embryo-specific promoter. For dicots, seed-specific
promoters include, but are not limited to, bean .beta.-phaseolin,
napin, .beta.-conglycinin, soybean lectin, cruciferin, and the
like. For monocots, seed-specific promoters include, but are not
limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein,
waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO
00/12733, where seed-preferred promoters from end1 and end2 genes
are disclosed; herein incorporated by reference.
[0098] In certain embodiments the nucleic acid sequences of the
embodiments can be stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
phenotype. For example, the polynucleotides of the embodiments may
be stacked with any other polynucleotides of the embodiments, such
as any combination of SEQ ID NOS: 3, 6, 9, 12, 13, or 14, or with
other nematicidal or antifungal genes and the like. The
combinations generated can also include multiple copies of any one
of the polynucleotides of interest. The polynucleotides of the
embodiments can also be stacked with any other gene or combination
of genes to produce plants with a variety of desired trait
combinations including but not limited to traits desirable for
animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);
balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;
5,885,801; 5,885,802; and 5,703,409); barley high lysine
(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO
98/20122); and high methionine proteins (Pedersen et al. (1986) J.
Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and
Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased
digestibility (e.g., modified storage proteins (U.S. application
Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S.
application Ser. No. 10/005,429, filed Dec. 3, 2001)), the
disclosures of which are herein incorporated by reference. The
polynucleotides of the embodiments can also be stacked with traits
desirable for insect, disease or herbicide resistance (e.g.,
Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser et al (1986) Gene
48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825);
fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones et al. (1994)
Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos
et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants
that lead to herbicide resistance such as the S4 and/or Hra
mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS genes, GAT genes such as those disclosed in U.S.
Patent Application Publication US2004/0082770, also WO02/36782 and
WO03/092360)); and traits desirable for processing or process
products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified
oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544;
WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases
(AGPase), starch synthases (SS), starch branching enzymes (SBE) and
starch debranching enzymes (SDBE)); and polymers or bioplastics
(e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,
polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase
(Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate
expression of polyhydroxyalkanoates (PHAs)), the disclosures of
which are herein incorporated by reference. One could also combine
the polynucleotides of the embodiments with polynucleotides
providing agronomic traits such as male sterility (e.g., see U.S.
Pat. No. 5,583,210), stalk strength, flowering time, or
transformation technology traits such as cell cycle regulation or
gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the
disclosures of which are herein incorporated by reference.
[0099] These stacked combinations can be created by any method
including but not limited to cross breeding plants by any
conventional or TopCross.RTM. methodology, or genetic
transformation. If the traits are stacked by genetically
transforming the plants, the polynucleotide sequences of interest
can be combined at any time and in any order. For example, a
transgenic plant comprising one or more desired traits can be used
as the target to introduce further traits by subsequent
transformation. The traits can be introduced simultaneously in a
co-transformation protocol with the polynucleotides of interest
provided by any combination of transformation cassettes. For
example, if two sequences will be introduced, the two sequences can
be contained in separate transformation cassettes (trans) or
contained on the same transformation cassette (cis). Expression of
the sequences can be driven by the same promoter or by different
promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of the
polynucleotide of interest. This may be combined with any
combination of other suppression cassettes or overexpression
cassettes to generate the desired combination of traits in the
plant. It is further recognized that polynucleotide sequences can
be stacked at a desired genomic location using a site-specific
recombination system. See, for example, WO99/25821, WO99/25854,
WO99/25840, WO99/25855, and WO99/25853, all of which are herein
incorporated by reference.
[0100] The methods of the embodiments involve introducing a
polypeptide or polynucleotide into a plant. "Introducing" is
intended to mean presenting to the plant the polynucleotide. In
some embodiments, the polynucleotide will be presented in such a
manner that the sequence gains access to the interior of a cell of
the plant, including its potential insertion into the genome of a
plant. The methods of the embodiments do not depend on a particular
method for introducing a sequence into a plant, only that the
polynucleotide gains access to the interior of at least one cell of
the plant. Methods for introducing polynucleotides into plants are
known in the art including, but not limited to, stable
transformation methods, transient transformation methods, and
virus-mediated methods. Polypeptides can also be introduced to a
plant in such a manner that they gain access to the interior of the
plant cell or remain external to the cell but in close contact with
it.
[0101] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof. "Transient transformation" or "transient
expression" is intended to mean that a polynucleotide is introduced
into the plant and does not integrate into the genome of the plant
or a polypeptide is introduced into a plant.
[0102] Transformation protocols as well as protocols for
introducing polypeptides or polynucleotide sequences into plants
may vary depending on the type of plant or plant cell, i.e.,
monocot or dicot, targeted for transformation. Suitable methods of
introducing polypeptides and polynucleotides into plant cells
include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S.
Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, Sanford et al., U.S. Pat.
Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al.
(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe
et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO
00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926
(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.
27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S.
Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988)
Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.
(1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.),
pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports
9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566
(whisker-mediated transformation); D'Halluin et al. (1992) Plant
Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell
Reports 12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology
14:745-750 (maize via Agrobacterium tumefaciens); all of which are
herein incorporated by reference.
[0103] In specific embodiments, the antipathogenic sequences of the
embodiments can be provided to a plant using a variety of transient
transformation methods. Such transient transformation methods
include, but are not limited to, the introduction of the
antipathogenic protein or variants and fragments thereof directly
into the plant or the introduction of the antipathogenic protein
transcript into the plant. Such methods include, for example,
microinjection or particle bombardment. See, for example, Crossway
et al. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al. (1986)
Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci.
91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science
107:775-784, all of which are herein incorporated by reference.
Alternatively, the polynucleotide can be transiently transformed
into the plant using techniques known in the art. Such techniques
include a viral vector system and the precipitation of the
polynucleotide in a manner that precludes subsequent release of the
DNA. Thus, the transcription from the particle-bound DNA can occur,
but the frequency with which it's released to become integrated
into the genome is greatly reduced. Such methods include the use of
particles coated with polyethylimine (PEI; Sigma #P3143).
[0104] In other embodiments, the polynucleotides of the embodiments
may be introduced into plants by contacting plants with a virus or
viral nucleic acids. Generally, such methods involve incorporating
a nucleotide construct of the embodiments within a viral DNA or RNA
molecule. It is recognized that the an antipathogenic polypeptide
of the embodiments may be initially synthesized as part of a viral
polyprotein, which later may be processed by proteolysis in vivo or
in vitro to produce the desired recombinant protein. Further, it is
recognized that promoters of the embodiments also encompass
promoters utilized for transcription by viral RNA polymerases.
Methods for introducing polynucleotides into plants and expressing
a protein encoded therein, involving viral DNA or RNA molecules,
are known in the art. See, for example, U.S. Pat. Nos. 5,889,191,
5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996)
Molecular Biotechnology 5:209-221; herein incorporated by
reference.
[0105] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the embodiments can be
contained in a transfer cassette flanked by two non-recombinogenic
recombination sites. The transfer cassette is introduced into a
plant having stably incorporated into its genome a target site
which is flanked by two non-recombinogenic recombination sites that
correspond to the sites of the transfer cassette. An appropriate
recombinase is provided and the transfer cassette is integrated at
the target site. The polynucleotide of interest is thereby
integrated at a specific chromosomal position in the plant
genome.
[0106] 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 progeny 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 embodiments provide transformed seed
(also referred to as "transgenic seed") having a nucleotide
construct of the embodiments, for example, an expression cassette
of the embodiments, stably incorporated into their genome.
[0107] As used herein, the term "plant" includes whole plants,
plant cells, plant protoplasts, plant cell tissue cultures from
which a maize plant can be regenerated, plant calli, plant clumps,
and plant cells that are intact in plants or parts of plants such
as embryos, pollen, seeds, endosperm, seed coat, leaves, flowers,
floral organs/structures (e.g. bracts, sepals, petals, stamens,
carpels, anthers and ovules) branches, fruit, kernels, ears, cobs,
husks, stalks, tubers, roots, root tips, anthers, plant tissue
(e.g. vascular tissue, ground tissue, and the like) and cells (e.g.
guard cells, egg cells, trichomes and the like) and progeny of
same. Grain is intended to mean the mature seed produced by
commercial growers for purposes other than growing or reproducing
the species. Progeny, variants, and mutants of the regenerated
plants are also included within the scope of the embodiments,
provided that these parts comprise the introduced polynucleotides.
The class of plants that can be used in the method of the
embodiments is generally as broad as the class of higher and lower
plants amenable to transformation techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns,
and multicellular algae. It includes plants of a variety of ploidy
levels, including aneuploid, polyploid, diploid, haploid and
hemizygous.
[0108] The methods of the embodiments may be used to induce
pathogen resistance or protect from pathogen attack any plant
species, including, but not limited to, monocots and dicots.
Examples of plant species of interest include, but are not limited
to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
juncea), particularly those Brassica species useful as sources of
seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye
(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),
millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana)), sunflower (Helianthus annuus),
safflower (Carthamus tinctorius), wheat (Triticum aestivum),
soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0109] Vegetables include tomatoes (Lycopersicon lycopersicon),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum.
[0110] Conifers that may be employed in practicing the embodiments
include, for example, pines such as loblolly pine (Pinus taeda),
slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),
lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata);
Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga
canadensis); Sitka spruce (Picea glauca); redwood (Sequoia
sempervirens); true firs such as silver fir (Abies amabilis) and
balsam fir (Abies balsamea); and cedars such as Western red cedar
(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis
nootkatensis). In specific embodiments, plants of the embodiments
are crop plants (for example, corn, alfalfa, sunflower, Brassica,
soybean, cotton, safflower, peanut, sorghum, wheat, millet,
tobacco, etc.). In other embodiments, corn and soybean plants are
optimal, and in yet other embodiments corn plants are optimal.
[0111] Other plants of interest include grain plants that provide
seeds of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,
lima bean, fava bean, lentils, chickpea, etc.
[0112] Antipathogenic compositions, particularly antifungal
compositions, are also encompassed by the present invention.
Antipathogenic compositions may comprise antipathogenic
polypeptides or transformed microorganisms comprising a nucleotide
sequence that encodes an antipathogenic polypeptide. The
antipathogenic compositions of the invention may be applied to the
environment of a plant pathogen, as described herein below, thereby
protecting a plant from pathogen attack. Moreover, an
antipathogenic composition can be formulated with an acceptable
carrier that is, for example, a suspension, a solution, an
emulsion, a dusting powder, a dispersible granule, a wettable
powder, and an emulsifiable concentrate, an aerosol, an impregnated
granule, an adjuvant, a coatable paste, and also encapsulations in,
for example, polymer substances.
[0113] A gene encoding an antipathogenic, particularly nematicidal
or antifungal, polypeptide of the embodiments may be introduced
into any suitable microbial host according to standard methods in
the art. For example, microorganism hosts that are known to occupy
the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or
rhizoplana) of one or more crops of interest may be selected. These
microorganisms are selected so as to be capable of successfully
competing in the particular environment with the wild-type
microorganisms, and to provide for stable maintenance and
expression of the gene expressing the antipathogenic protein.
[0114] Such microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms such as bacteria, e.g.,
Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly
yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas
syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter
xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas
campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter
xyli and Azotobacter vinlandir and phytosphere yeast species such
as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0115] Other illustrative prokaryotes, both Gram-negative and
gram-positive, include Enterobacteriaceae, such as Escherichia,
Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae;
Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae and
Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes
and Ascomycetes, which includes yeast, such as Saccharomyces and
Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,
Aureobasidium, Sporobolomyces, and the like.
[0116] Microbial host organisms of particular interest include
yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces
spp., and Sporobolomyces spp., phylloplane organisms such as
Pseudomonas spp., Erwinia spp., and Flavobacterium spp., and other
such organisms, including Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
[0117] Genes encoding the antipathogenic proteins of the
embodiments can be introduced into microorganisms that multiply on
plants (epiphytes) to deliver antipathogenic proteins to potential
target pathogens. Epiphytes, for example, can be gram-positive or
gram-negative bacteria.
[0118] Root-colonizing bacteria, for example, can be isolated from
the plant of interest by methods known in the art. Specifically, a
Bacillus cereus strain that colonizes roots can be isolated from
roots of a plant (see, for example, Handelsman et al. (1991) Appl.
Environ. Microbiol. 56:713-718). Genes encoding the antifungal or
nematicidal polypeptides of the embodiments can be introduced into
a root-colonizing Bacillus cereus by standard methods known in the
art.
[0119] Genes encoding antifungal or nematicidal proteins can be
introduced, for example, into the root-colonizing Bacillus by means
of electrotransformation. Specifically, genes encoding the proteins
can be cloned into a shuttle vector, for example, pHT3101 (Lerecius
et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The shuttle
vector pHT3101 containing the coding sequence for the particular
protein can, for example, be transformed into the root-colonizing
Bacillus by means of electroporation (Lerecius et al. (1989) FEMS
Microbiol. Letts. 60: 211-218).
[0120] Methods are provided for protecting a plant from a pathogen
comprising applying an effective amount of an antipathogenic
protein or composition of the invention to the environment of the
pathogen. "Effective amount" is intended to mean an amount of a
protein or composition sufficient to control a pathogen. The
antipathogenic proteins and compositions can be applied to the
environment of the pathogen by methods known to those of ordinary
skill in the art.
[0121] The antifungal compositions of the invention may be obtained
by the addition of a surface-active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV
protective, a buffer, a flow agent or fertilizers, micronutrient
donors, or other preparations that influence plant growth. One or
more agrochemicals including, but not limited to, herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides,
acaracides, plant growth regulators, harvest aids, and fertilizers,
can be combined with carriers, surfactants or adjuvants customarily
employed in the art of formulation or other components to
facilitate product handling and application for particular target
pathogens. 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. The active ingredients of the present invention are
normally applied in the form of compositions and can be applied to
the crop area, plant, or seed to be treated. For example, the
compositions of the present invention may be applied to grain in
preparation for or during storage in a grain bin or silo, etc. The
compositions of the present invention may be applied simultaneously
or in succession with other compounds. 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 antipathogenic proteins, more particularly antifungal proteins,
of the present invention include, but are not limited to, foliar
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 or pathogen.
[0122] Suitable surface-active agents include, but are not limited
to, anionic compounds such as a carboxylate of, for example, a
metal; carboxylate of a long chain fatty acid; an
N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty
alcohol ethoxylates or salts of such esters; fatty alcohol sulfates
such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium
cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated
alkylphenol sulfates; lignin sulfonates; petroleum sulfonates;
alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower
alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of
sulfonated phenol-formaldehyde condensates; more complex sulfonates
such as the amide sulfonates, e.g., the sulfonated condensation
product of oleic acid and N-methyl taurine; or the dialkyl
sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate.
Non-ionic agents include condensation products of fatty acid
esters, fatty alcohols, fatty acid amides or fatty-alkyl- or
alkenyl-substituted phenols with ethylene oxide, fatty esters of
polyhydric alcohol ethers, e.g., sorbitan fatty acid esters,
condensation products of such esters with ethylene oxide, e.g.,
polyoxyethylene sorbitar fatty acid esters, block copolymers of
ethylene oxide and propylene oxide, acetylenic glycols such as
2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic
glycols. Examples of a cationic surface-active agent include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate or oleate; or oxygen-containing amine such as an amine
oxide of polyoxyethylene alkylamine; an amide-linked amine prepared
by the condensation of a carboxylic acid with a di- or polyamine;
or a quaternary ammonium salt.
[0123] Examples of inert materials include but are not limited to
inorganic minerals such as kaolin, phyllosilicates, carbonates,
sulfates, phosphates, or botanical materials such as cork, powdered
corncobs, peanut hulls, rice hulls, and walnut shells.
[0124] The antipathogenic compositions of the present invention can
be in a suitable form for direct application or as a concentrate of
primary composition that requires dilution with a suitable quantity
of water or other diluant before application. The concentration of
the antipathogenic polypeptide will vary depending upon the nature
of the particular formulation, specifically, whether it is a
concentrate or to be used directly. The composition contains 1 to
98% of a solid or liquid inert carrier, and 0 to 50%, optimally 0.1
to 50% of a surfactant. These compositions will be administered at
the labeled rate for the commercial product, optimally about 0.01
lb-5.0 lb. per acre when in dry form and at about 0.01 pts.-10 pts.
per acre when in liquid form.
[0125] In a further embodiment, the compositions, as well as the
transformed microorganisms and antipathogenic proteins, of the
invention can be treated prior to formulation to prolong the
antipathogenic, particularly antifungal, activity when applied to
the environment of a target pathogen as long as the pretreatment is
not deleterious to the activity. Such treatment can be by chemical
and/or physical means as long as the treatment does not
deleteriously affect the properties of the composition(s). Examples
of chemical reagents include but are not limited to halogenating
agents; aldehydes such a formaldehyde and glutaraldehyde;
anti-infectives, such as zephiran chloride; alcohols, such as
isopropanol and ethanol; and histological fixatives, such as
Bouin's fixative and Helly's fixative (see, for example, Humason
(1967) Animal Tissue Techniques (W.H. Freeman and Co.).
[0126] The antipathogenic compositions of the invention can be
applied to the environment of a plant pathogen by, for example,
spraying, atomizing, dusting, scattering, coating or pouring,
introducing into or on the soil, introducing into irrigation water,
by seed treatment or general application or dusting at the time
when the pathogen has begun to appear or before the appearance of
pathogens as a protective measure. For example, the antipathogenic
protein and/or transformed microorganisms of the invention may be
mixed with grain to protect the grain during storage. It is
generally important to obtain good control of pathogens in the
early stages of plant growth, as this is the time when the plant
can be most severely damaged. The compositions of the invention can
conveniently contain an insecticide if this is thought necessary.
In one embodiment of the invention, the composition is applied
directly to the soil, at a time of planting, in granular form of a
composition of a carrier and dead cells of a Bacillus strain or
transformed microorganism of the invention. Another embodiment is a
granular form of a composition comprising an agrochemical such as,
for example, a herbicide, an insecticide, a fertilizer, an inert
carrier, and dead cells of a Bacillus strain or transformed
microorganism of the invention.
[0127] Compositions of the invention find use in protecting plants,
seeds, and plant products in a variety of ways. For example, the
compositions can be used in a method that involves placing an
effective amount of the antipathogenic, more particularly,
antifungal, composition in the environment of the pathogen by a
procedure selected from the group consisting of spraying, dusting,
broadcasting, or seed coating.
[0128] Before plant propagation material (fruit, tuber, bulb, corm,
grains, seed), but especially seed, is sold as a commercial
product, it is customarily treated with a protective coating
comprising herbicides, insecticides, fungicides, bactericides,
nematicides, molluscicides, or mixtures of several of these
preparations, if desired together with further carriers,
surfactants, or application-promoting adjuvants customarily
employed in the art of formulation to provide protection against
damage caused by bacterial, fungal, or animal pests. In order to
treat the seed, the protective coating may be applied to the seeds
either by impregnating the tubers or grains with a liquid
formulation or by coating them with a combined wet or dry
formulation. In addition, in special cases, other methods of
application to plants are possible, e.g., treatment directed at the
buds or the fruit.
[0129] The plant seed of the invention comprising a DNA molecule
comprising a nucleotide sequence encoding an antipathogenic
polypeptide of the invention may be treated with a seed protective
coating comprising a seed treatment compound, such as, for example,
captan, carboxin, thiram, methalaxyl, pirimiphos-methyl, and others
that are commonly used in seed treatment. Alternatively, a seed of
the invention comprises a seed protective coating comprising an
antipathogenic, more particularly antifungal, composition of the
invention is used alone or in combination with one of the seed
protective coatings customarily used in seed treatment.
[0130] The antifungal polypeptides of the invention can be used for
any application including coating surfaces to target microbes. In
this manner, the target microbes include human pathogens or
microorganisms. Surfaces that might be coated with the antifungal
polypeptides of the invention include carpets and sterile medical
facilities. Polymer bound polypeptides of the invention may be used
to coat surfaces. Methods for incorporating compositions with
antimicrobial properties into polymers are known in the art. See
U.S. Pat. No. 5,847,047, herein incorporated by reference.
[0131] The methods of the embodiments may be effective against a
variety of plant fungal pathogens, such as, for example,
Colletotrichum graminocola, Diplodia maydis, Verticillium dahliae,
Fusarium graminearum, Fusarium oxysporum and Fusarium
verticillioides. Pathogens of the embodiments include nematodes and
fungi. Fungal pathogens, include but are not limited to,
Colletotrichum graminicola, Diplodia maydis, Fusarium graminearum,
and Fusarium verticillioides. Specific pathogens for the major
crops include: Soybeans: Phytophthora megasperma fsp. glycinea,
Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia
sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae
(Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium
rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora
manshurica, Colletotrichum dematium (Colletotichum truncatum),
Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola,
Alternaria alternata, Pseudomonas syringae p.v. glycinea,
Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,
Fusarium semitectum, Phialophora gregata, Glomerella glycines,
Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum,
Pythium debaryanum, Fusarium solani; Canola: Albugo candida,
Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani,
Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium
ultimum, Peronospora parasitica, Fusarium roseum, Alternaria
alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum,
Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium
debaryanum, Pythium aphanidermatum, Phytophthora megasperma,
Peronospora trifoliorum, Phoma medicaginis var. medicaginis,
Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila
medicaginis, Fusarium oxysporum, Verticillium albo-atrum,
Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,
Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum
trifolii, Leptosphaerulina briosiana, Uromyces striatus,
Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium
botryosum, Leptotrichila medicaginis; Wheat: Pseudomonas syringae
p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.
translucens, Pseudomonas syringae p.v. syringae, Alternaria
alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium
avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici,
Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe
graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia
recondita f.sp. tritici, Puccinia striiformis, Pyrenophora
tritici-repentis, Septoria nodorum, Septoria tritici, Septoria
avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani,
Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium
aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris
sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis,
Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium
arrhenomannes, Pythium gramicola, Pythium aphanidermatum,
Sunflower: Plasmopora halstedii, Sclerotinia sclerotiorum, Septoria
helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria
zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina
phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus
arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium
dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium
acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn:
Colletotrichum graminicola, Fusarium verticillioides var.
subglutinans, Erwinia stewartii, F. verticillioides, Gibberella
zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis),
Pythium irregulare, Pythium debaryanum, Pythium graminicola,
Pythium splendens, Pythium ultimum, Pythium aphanidermatum,
Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus
heterostrophus), Helminthosporium carbonum I, II & III
(Cochliobolus carbonum), Exserohilum turcicum I, II & III,
Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta
maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis,
Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina,
Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum,
Curvularia lunata, Curvularia inaequalis, Curvularia pallescens,
Clavibacter michiganense subsp. nebraskense, Trichoderma viride,
Claviceps sorghi, Pseudomonas avenae, Erwinia chrysanthemi pv. zea,
Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora,
Sclerophthora macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Peronosclerospora maydis,
Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella
zeae, Cephalosporium maydis, Cephalosporium acremonium, Sorghum:
Exserohilum turcicum, C. sublineolum, Cercospora sorghi,
Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae
p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas
andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia
circinata, Fusarium verticillioides, Alternaria alternata,
Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia
lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas
alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,
Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca
reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps
sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona
macrospora, Peronosclerospora sorghi, Peronosclerospora
philippinensis, Sclerospora graminicola, Fusarium graminearum,
Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola,
etc.
[0132] The methods of the embodiments may also be effective against
a variety of plant nematode pathogens, such as, for example,
parasitic nematodes such as root-knot, cyst, and lesion nematodes,
including Heterodera spp., Meloidogyne spp., and Globodera spp.;
particularly members of the cyst nematodes, including, but not
limited to, Heterodera glycines (soybean cyst nematode); Heterodera
schachtii (beet cyst nematode); Heterodera avenae (cereal cyst
nematode); Meloidogyne incognita (cotton cyst nematode) and
Globodera rostochiensis and Globodera pailida (potato cyst
nematodes). Lesion nematodes include Pratylenchus spp.
[0133] The article "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
element.
[0134] Units, prefixes, and symbols may be denoted in their Si
accepted form. Unless otherwise indicated, nucleic acid sequences
are written left to right in 5' to 3' orientation; amino acid
sequences are written left to right in amino to carboxy
orientation, respectively. Numeric ranges are inclusive of the
numbers defining the range. Amino acids may be referred to herein
by either their commonly known three letter symbols or by the
one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to
by their commonly accepted single-letter codes. The above-defined
terms are more fully defined by reference to the specification as a
whole.
[0135] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
EXPERIMENTAL
Example 1
C. elegans Feeding Bioassays
[0136] A number of polypeptides were recombinantly expressed in E.
coli and then screened in a nematode bioassay.
[0137] C. elegans feeding bioassays. Screening was performed in
high throughput using E. coli expressed material, corresponding to
the predicted mature regions of various small peptides. Expression
of biologically functional polypeptides involved producing a fusion
protein that included a maltose-binding protein (MBP) and a
polypeptide of interest and subsequently cleaving the fusion
protein at a protease recognition sequence to release the peptide
of interest. DNA encoding the polypeptide of interest was fused to
the C-terminus of the MelE gene in the E. coli expression vector
PMAL (New England Biolabs; see, Guan et al., Gene 67:21-30 (1987);
and Maina et al., Gene 74:365-73 (1988)). Sequences encoding the
cleavage site of proteases Factor Xa or Genenase I were
incorporated between the genes of MBP and the polypeptide of
interest. A histidine tag was also added to the N-terminus of
MBP.
[0138] The constructed plasmid vector was transformed into cells of
E. coli XL-1 Blue and transformants were grown in 2YT medium
containing 50 .mu.g/mL carbenicillin to a cell density of
OD.sub.600=0.6-0.9. Expression of the fusion protein was induced by
addition of IPTG into the culture to a final concentration of 1 mM.
Cells were grown for 4-16 hours to saturation before harvesting.
Cells were harvested by centrifugation and then lysed with B-PER
reagent (Pierce Chemicals, Rockford, Ill.) to obtain the fraction
of soluble proteins. The fusion protein was purified from the cell
lysate supernatant utilizing the histidine tag by incubating the
cell lysate with Ni-NTA agarose resins for 20 minutes to 1 hour.
The resins were washed with Tris buffer to remove all unbound
proteins. Two-mercaptoethanol (10 mM) was included in the lysis and
washing buffers to allow partial refolding of the proteins. Elution
of the bound fusion protein was performed with buffer containing
20-40 mM histidine. To release the polypeptide of interest, the
purified fusion protein was incubated with Factor Xa or Genease I
(RT, 8-24 hours). The cleaved protein sample was then used in
nematode activity assays.
[0139] Hit characterization. Approximate IC.sub.50's (in .mu.g/mL)
for 4 hits determined from C. elegans bioassay using cleaved
peptides. Concentrations were determined based on Bradford (BSA
standard) and assume 100% cleavage of the MBP-Kistrin fusion.
TABLE-US-00001 Kis2 SEQ ID NO: 1 15 .mu.g/mL Kis5 SEQ ID NO: 4 75
.mu.g/mL Kis6 SEQ ID NO: 7 150 .mu.g/mL Kis10 SEQ ID NO: 10 30
.mu.g/mL
Example 2
M. incognita Feeding Bioassays in Hairy Roots and Transgenic
Tobacco
[0140] Kistrins in Astragalus hairy roots. Two of the polypeptides
described in Example 1 (Kis2 (SEQ ID NO: 1) and Kis10 (SEQ ID NO:
10)) were used for in planta testing against root knot nematode
(Meloidogyne incognita). For in planta expression, four different
gene constructs were prepared. All constructs are in PMAXY 4764
binary vector. A double MMV promoter in this vector drove the
expression of the kistrins.
[0141] Kis2 is an Arabidopsis gene and Kis10 is a Petunia gene. The
sequence of the full-length cDNA containing an N-terminal extension
sequence coding for the pre-pro and signal peptide is available for
these two genes in GenBank. A total of four expression cassettes
were prepared for in planta analysis. The designation of the binary
vector constructs is as follows:
PVER 007--SEQ ID NO:3, encoding SEQ ID NO: 1--Kis2 (Mature
peptide)
PVER 008--SEG ID NO:13, encoding SEQ ID NO: 2--Kis2
(Full-length)
PVER 009--SEQ ID NO:12, encoding SEQ ID NO: 10--Kis10 (Mature
peptide)
PVER 010--SEQ ID NO:14, encoding SEQ ID NO: 11--Kis10
(Full-length)
[0142] Agrobacterium rhizogenes strain (K599) was transformed with
the above constructs. PMAXY 4764 empty vector served as a negative
control. Astragalus sinicus hypocotyls were co-cultivated with A.
rhizogenes transformants following standard tissue culture
protocols. Transformed hairy roots were regenerated on nutrient
media plates containing kanamycin as a plant selectable marker.
[0143] After regeneration, the hairy roots were transferred to
multiple petri plates for inoculation. Meloidogyne incognita stocks
are maintained on MiroTom tomato plants in growth rooms. Nematode
eggs were harvested from infected plants and hairy roots were
inoculated under sterile conditions following standard protocols.
The hairy roots were scored approximately five weeks
post-inoculation. Scoring for resistance/susceptibility was
performed by extracting and counting five different aliquots of the
eggs from each plate. The final data is expressed as percentage of
eggs relative to the empty vector control. A summary of the results
is presented in FIG. 1.
[0144] Agrobacterium tumefaciens strain (EHA105) was transformed
with the above constructs. pMAXY4764 empty vector served as a
negative control. Tobacco (variety Petit Havana) leaf explants were
co-cultivated with Agrobacterium tumefaciens transformants
following standard tissue culture protocols. Ten independent
transgenic lines were generated for each construct. For each line
of stable transformant, two clones were prepared. Plants were
inoculated with Meloidogyne inocognita eggs as explained for hairy
roots. Scoring for resistance/susceptibility was performed by
collecting roots from each plant, extracting eggs and counting the
number of eggs as explained for hairy roots. The final data is
expressed as percentage of eggs relative to the empty vector
control. A summary of the results is presented in FIG. 2.
Example 3
Fungal Bioassays
[0145] The same polypeptides of Example 1 which were recombinantly
expressed in E. coli were also screened in an anti-fungal bioassay
against five selected fungal pathogens. Table 1 illustrates the
results of the anti-fungal screening assay.
[0146] All fungal strains were grown and maintained on potato
dextrose agar (PDA) plates, in a 30.degree. C. incubator. These
plates were kept in smaller secondary containers (per fungal
strain), with moist paper towels to maintain high humidity. Spores
were harvested in a quarter strength of potato dextrose broth (PDB)
after about 2 weeks of growth, counted using a hemacytometer, and
subsequently stored in small aliquots at -80.degree. C.
[0147] The frozen spores were diluted to the working concentration
(determined empirically for each fungal strain), in a quarter
strength of PDB, and 50 .mu.L (per well) were added to sterile,
flat-bottomed 96-well assay plates. The assay plates were incubated
in the humid boxes at room temperature for 5-7 hours to allow the
spores to germinate. Serial dilutions of purified, protease-cleaved
fusion protein samples were then added to the assay plates, in 50
.mu.L volumes, for a final assay volume of 100 .mu.L per well. The
assay plates were allowed to incubate overnight, in a humid box, at
30.degree. C. Antifungal activity was scored after 18 to 48 hours,
depending upon the strain of fungus. TABLE-US-00002 TABLE 1 Primary
screening (score: 0 = no effect; 1 = partial growth inhibition; 2 =
strong inhibition): Alternaria Fusarium Fusarium Botrytis
Verticillium SEQ ID NO: brassicicola verticillioides oxysporum
cinerea dahliae Kis2 1 0 0 0 0 0 Kis5 4 0 0 1 0 2 Kis6 7 0 0 0 0 1
Kis10 10 0 0 0 0 0
Example 4
Transformation and Regeneration of Transgenic Maize Plants
[0148] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing a nucleotide sequence encoding
the antipathogenic polypeptide set forth in SEQ ID NO:1 operably
linked to a promoter that drives expression in a maize plant cell
and a selectable marker (e.g., the selectable marker gene PAT
(Wohlleben et al. (1988) Gene 70:25-37), which confers resistance
to the herbicide Bialaphos). Alternatively, the selectable marker
gene is provided on a separate plasmid. Transformation is performed
as follows. Media recipes follow below.
Preparation of Target Tissue
[0149] The ears are husked and surface sterilized in 30% Clorox
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
Preparation of DNA
[0150] A plasmid vector comprising a nucleotide sequence encoding
the antipathogenic polypeptide set forth in SEQ ID NO:1 operably
linked to a promoter that drives expression in a maize cell is
made. This plasmid DNA plus plasmid DNA containing a selectable
marker (e.g., PAT) is precipitated onto 1.1 .mu.m (average
diameter) tungsten pellets using a CaCl.sub.2 precipitation
procedure as follows:
[0151] 100 .mu.L prepared tungsten particles in water
[0152] 10 .mu.L (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0153] 100 .mu.L2.5M CaCl.sub.2
[0154] 10 .mu.L 0.1 M spermidine
[0155] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 mL 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.L 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.L spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
Particle Gun Treatment
[0156] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
Subsequent Treatment
[0157] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/L Bialaphos, and subcultured every 2 weeks. After approximately
10 weeks of selection, selection-resistant callus clones are
transferred to 288J medium to initiate plant regeneration.
Following somatic embryo maturation (2-4 weeks), well-developed
somatic embryos are transferred to medium for germination and
transferred to the lighted culture room. Approximately 7-10 days
later, developing plantlets are transferred to 272V hormone-free
medium in tubes for 7-10 days until plantlets are well established.
Plants are then transferred to inserts in flats (equivalent to
2.5'' pot) containing potting soil and grown for 1 week in a growth
chamber, subsequently grown an additional 1-2 weeks in the
greenhouse, then transferred to classic 600 pots (1.6 gallon) and
grown to maturity. Plants are monitored and scored for fungal
resistance.
Bombardment and Culture Media
[0158] Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts
(SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000.times.
SIGMA-1511), 0.5 mg/L thiamine HCl, 120.0 g/L sucrose, 1.0 mg/L
2,4-D, and 2.88 g/L L-proline (brought to volume with D-1 H.sub.2O
following adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite (added
after bringing to volume with D-I H.sub.2O); and 8.5 mg/L silver
nitrate (added after sterilizing the medium and cooling to room
temperature). Selection medium (560R) comprises 4.0 g/L N6 basal
salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose,
and 2.0 mg/L 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/L Gelrite (added after
bringing to volume with D-I H.sub.2O); and 0.85 mg/L silver nitrate
and 3.0 mg/L bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0159] Plant regeneration medium (288J) comprises 4.3 g/L MS salts
(GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and
0.40 g/L glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/L
myo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose, and 1.0 mL/L of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/L Gelrite (added after bringing
to volume with D-I H.sub.2O); and 1.0 mg/L indoleacetic acid and
3.0 mg/L bialaphos (added after sterilizing the medium and cooling
to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/L MS
salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100
g/L nicotinic acid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL,
and 0.40 g/L glycine brought to volume with polished D-I H.sub.2O),
0.1 g/L myo-inositol, and 40.0 g/L sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/L
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Example 5
Agrobacterium-Mediated Transformation of Maize and Regeneration of
Transgenic Plants
[0160] For Agrobacterium-mediated transformation of maize with the
polynucleotide construct containing SEQ ID NO: 1, the method of
Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent
publication WO98/32326; the contents of which are hereby
incorporated by reference). Briefly, immature embryos are isolated
from maize and the embryos contacted with a suspension of
Agrobacterium, where the bacteria are capable of transferring the
polynucleotide construct to at least one cell of at least one of
the immature embryos (step 1: the infection step). In this step the
immature embryos are immersed in an Agrobacterium suspension for
the initiation of inoculation. The embryos are co-cultured for a
time with the Agrobacterium (step 2: the co-cultivation step). The
immature embryos are cultured on solid medium following the
infection step. Following this co-cultivation period an optional
"resting" step is performed. In this resting step, the embryos are
incubated in the presence of at least one antibiotic known to
inhibit the growth of Agrobacterium without the addition of a
selective agent for plant transformants (step 3: resting step). The
immature embryos are cultured on solid medium with antibiotic, but
without a selecting agent, for elimination of Agrobacterium and for
a resting phase for the infected cells. Next, inoculated embryos
are cultured on medium containing a selective agent and growing
transformed callus is recovered (step 4: the selection step). The
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step), and calli grown on selective medium are cultured on solid
medium to regenerate the plants.
Example 6
Transformation of Somatic Soybean Embryo Cultures and Regeneration
of Soybean Plants
[0161] The following stock solutions and media are used for
transformation and regeneration of soybean plants:
Stock solutions
[0162] Sulfate 100.times. Stock: 37.0 g MgSO.sub.4.7H.sub.2O, 1.69
g MnSO.sub.4.H.sub.2O, 0.86 g ZnSO.sub.4.7H.sub.2O, 0.0025 g
CuSO.sub.4.5H.sub.2O. [0163] Halides 100.times. Stock: 30.0 g
CaCl.sub.2.2H.sub.2O, 0.083 g KI, 0.0025 g CoCl.sub.2.6H.sub.2O,
[0164] P, B, Mo 100.times. Stock: 18.5 g KH.sub.2PO.sub.4, 0.62 g
H.sub.3BO.sub.3, 0.025 g Na.sub.2MoO.sub.4.2H.sub.2O [0165] Fe EDTA
100.times. Stock: 3.724 g Na.sub.2EDTA, 2.784 g
FeSO.sub.4.7H.sub.2O. [0166] 2,4-D Stock: 10 mg/mL. [0167] Vitamin
B5 1000.times. Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,
0.10 g pyridoxine HCl, 1 g thiamine. Media (per Liter) [0168]
SB196: 10 mL of each of the above stock solutions, 1 mL B5 vitamin
stock, 0.463 g (NH.sub.4).sub.2 SO.sub.4, 2.83 g KNO.sub.3, 1 mL
2,4-D stock, 1 g asparagine, 10 g sucrose, pH 5.7. [0169] SB103: 1
pk. Murashige & Skoog salts mixture, 1 mL B5 vitamin stock, 750
mg MgCl.sub.2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.
[0170] SB166: SB103 supplemented with 5 g per liter activated
charcoal. [0171] SB71-4: Gamborg's B5 salts (Gibco-BRL catalog No.
21153-028), 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH
5.7.
[0172] Soybean embryogenic suspension cultures are maintained in 35
mL liquid medium (SB196) on a rotary shaker (150 rpm) at 28.degree.
C. with fluorescent lights providing a 16 hour day/8 hour night
cycle. Cultures are subcultured every 2 weeks by inoculating
approximately 35 mg of tissue into 35 mL of fresh liquid media.
[0173] Soybean embryogenic suspension cultures are transformed by
the method of particle gun bombardment (see Klein et al. (1987)
Nature 327:70-73) using a DuPont Biolistic PDS1000/He
instrument.
[0174] In particle gun bombardment procedures it is possible to use
purified 1) entire plasmid DNA or, 2) DNA fragments containing only
the recombinant DNA expression cassette(s) of interest. For every
eight bombardment transformations, 30 .mu.l of suspension is
prepared containing 1 to 90 picograms (pg) of DNA fragment per base
pair of DNA fragment. The recombinant DNA plasmid or fragment used
to express the antifungal gene is on a separate recombinant DNA
plasmid or fragment from the selectable marker gene. Both
recombinant DNA plasmids or fragments are co-precipitated onto gold
particles as follows. The DNAs in suspension are added to 50 .mu.L
of a 20-60 mg/mL 0.6 .mu.m gold particle suspension and then
combined with 50 .mu.L CaCl.sub.2 (2.5 M) and 20 .mu.L spermidine
(0.1 M) The mixture is pulse vortexed 5 times, spun in a microfuge
for 10 seconds, and the supernatant removed. The DNA-coated
particles are then washed once with 150 .mu.L of 100% ethanol,
pulse vortexed and spun in a microfuge again, and resuspended in 85
.mu.L of anhydrous ethanol. Five .mu.L of the DNA-coated gold
particles are then loaded on each macrocarrier disk.
[0175] Approximately 150 to 250 mg of two-week-old suspension
culture is placed in an empty 60 mm.times.15 mm petri plate and the
residual liquid is removed from the tissue using a pipette. The
tissue is placed about 3.5 inches away from the retaining screen
and each plate of tissue is bombarded once. Membrane rupture
pressure is set at 650 psi and the chamber is evacuated to -28
inches of Hg. Eighteen plates are bombarded, and, following
bombardment, the tissue from each plate is divided between two
flasks, placed back into liquid media, and cultured as described
above.
[0176] Seven days after bombardment, the liquid medium is exchanged
with fresh SB196 medium supplemented with 50 mg/mL hygromycin or
100 ng/mL chlorsulfuron, depending on the selectable marker gene
used in transformation. The selective medium is refreshed weekly or
biweekly. Seven weeks post-bombardment, green, transformed tissue
is observed growing from untransformed, necrotic embryogenic
clusters. Isolated green tissue is removed and inoculated into
individual flasks to generate new, clonally-propagated, transformed
embryogenic suspension cultures. Thus, each new line is treated as
independent transformation event. These suspensions can then be
maintained as suspensions of embryos clustered in an immature
developmental stage through subculture or can be regenerated into
whole plants by maturation and germination of individual somatic
embryos.
[0177] Transformed embryogenic clusters are removed from liquid
culture and placed on solid agar medium (SB166) containing no
hormones or antibiotics for one week. Embryos are cultured at
26.degree. C. with mixed fluorescent and incandescent lights on a
16 hour day:8 hour night schedule. After one week, the cultures are
then transferred to SB103 medium and maintained in the same growth
conditions for 3 additional weeks. Prior to transfer from liquid
culture to solid medium, tissue from selected lines is assayed by
PCR or Southern analysis for the presence of the antifungal
gene.
[0178] Somatic embryos become suitable for germination after 4
weeks and are then removed from the maturation medium and dried in
empty petri dishes for 1 to 5 days. The dried embryos are then
planted in SB71-4 medium where they are allowed to germinate under
the same light and germination conditions described above.
Germinated embryos are transferred to sterile soil and grown to
maturity.
[0179] All publications and patent applications mentioned in the
specification are indicative of the level 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.
[0180] 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
14 1 66 PRT Arabidopsis thaliana PEPTIDE (0)...(0) Kis2 mature
peptide 1 Gly Lys Leu Lys Pro Gln Gln Cys Asn Ser Lys Cys Ser Phe
Arg Cys 1 5 10 15 Ser Ala Thr Ser His Lys Lys Pro Cys Met Phe Phe
Cys Leu Lys Cys 20 25 30 Cys Lys Lys Cys Leu Cys Val Pro Pro Gly
Thr Phe Gly Asn Lys Gln 35 40 45 Thr Cys Pro Cys Tyr Asn Asn Trp
Lys Thr Lys Glu Gly Arg Pro Lys 50 55 60 Cys Pro 65 2 97 PRT
Arabidopsis thaliana PEPTIDE (0)...(0) Kis 2 (NP_566186) 2 Met Ala
Asn Cys Ile Arg Arg Asn Ala Leu Phe Phe Leu Thr Leu Leu 1 5 10 15
Phe Leu Leu Ser Val Ser Asn Leu Val Gln Ala Ala Arg Gly Gly Gly 20
25 30 Lys Leu Lys Pro Gln Gln Cys Asn Ser Lys Cys Ser Phe Arg Cys
Ser 35 40 45 Ala Thr Ser His Lys Lys Pro Cys Met Phe Phe Cys Leu
Lys Cys Cys 50 55 60 Lys Lys Cys Leu Cys Val Pro Pro Gly Thr Phe
Gly Asn Lys Gln Thr 65 70 75 80 Cys Pro Cys Tyr Asn Asn Trp Lys Thr
Lys Glu Gly Arg Pro Lys Cys 85 90 95 Pro 3 198 DNA Artificial
Sequence optimized for expression in E. coli misc_feature (0)...(0)
Sequence encoding SEQ ID NO1 as expressed in E. coli for C. elegans
bioassays 3 ggcaaactga aaccgcaaca gtgtaactct aaatgcagct tccgttgctc
cgcaactagc 60 cataagaagc cgtgcatgtt cttttgcctg aaatgttgta
aaaagtgcct gtgcgtaccg 120 ccgggcacct tcggcaataa gcagacgtgt
ccgtgctaca acaactggaa aactaaagaa 180 ggtcgcccga aatgtcca 198 4 66
PRT Picea mariana PEPTIDE (0)...(0) Kis5 mature peptide 4 Gly Ser
Leu Arg Pro Ser Glu Cys Gly Gln Arg Cys Ser Tyr Arg Cys 1 5 10 15
Ser Ala Thr Ser His Lys Lys Pro Cys Met Phe Phe Cys Gln Lys Cys 20
25 30 Cys Ala Lys Cys Leu Cys Val Pro Pro Gly Thr Phe Gly Asn Lys
Gln 35 40 45 Val Cys Pro Cys Tyr Asn Asn Trp Lys Thr Gln Gln Gly
Gly Pro Lys 50 55 60 Cys Pro 65 5 110 PRT Picea mariana PEPTIDE
(0)...(0) AAC32128 (Kis5) 5 Met Ala Arg Leu Gln Ser Phe Ala Val Leu
Leu Ile Thr Ile Phe Ala 1 5 10 15 Leu Phe Ile Trp Asn Ile Glu Ala
Ala Leu Pro His Ser Asn Val Asp 20 25 30 Pro Phe Met Glu Gln Lys
Gln Gly Gln Tyr Gly Glu Gly Ser Leu Arg 35 40 45 Pro Ser Glu Cys
Gly Gln Arg Cys Ser Tyr Arg Cys Ser Ala Thr Ser 50 55 60 His Lys
Lys Pro Cys Met Phe Phe Cys Gln Lys Cys Cys Ala Lys Cys 65 70 75 80
Leu Cys Val Pro Pro Gly Thr Phe Gly Asn Lys Gln Val Cys Pro Cys 85
90 95 Tyr Asn Asn Trp Lys Thr Gln Gln Gly Gly Pro Lys Cys Pro 100
105 110 6 198 DNA Artificial Sequence optimized for expression in
E. coli misc_feature (0)...(0) Sequence encoding SEQ ID NO4 as
expressed in E. coli for C. elegans bioassays 6 ggctctctgc
gtccgtctga atgtggtcag cgttgcagct accgttgctc cgcaactagc 60
cataagaagc cgtgcatgtt cttttgccaa aaatgttgtg caaagtgcct gtgcgtaccg
120 ccgggcacct tcggcaataa gcaggtttgt ccgtgctaca acaactggaa
aactcagcag 180 ggtggtccga aatgtcca 198 7 66 PRT Arabidopsis
thaliana PEPTIDE (0)...(0) Kis6 mature peptide 7 Gly Tyr Ala Lys
Lys Ile Asp Cys Gly Ser Ala Cys Val Ala Arg Cys 1 5 10 15 Arg Leu
Ser Arg Arg Pro Arg Leu Cys His Arg Ala Cys Gly Thr Cys 20 25 30
Cys Tyr Arg Cys Asn Cys Val Pro Pro Gly Thr Tyr Gly Asn Tyr Asp 35
40 45 Lys Cys Gln Cys Tyr Ala Ser Leu Thr Thr His Gly Gly Arg Arg
Lys 50 55 60 Cys Pro 65 8 98 PRT Arabidopsis thaliana PEPTIDE
(0)...(0) S60229 (Kis6) 8 Met Ala Ile Ser Lys Ala Leu Ile Ala Ser
Leu Leu Ile Ser Leu Leu 1 5 10 15 Val Leu Gln Leu Val Gln Ala Asp
Val Glu Asn Ser Gln Lys Lys Asn 20 25 30 Gly Tyr Ala Lys Lys Ile
Asp Cys Gly Ser Ala Cys Val Ala Arg Cys 35 40 45 Arg Leu Ser Arg
Arg Pro Arg Leu Cys His Arg Ala Cys Gly Thr Cys 50 55 60 Cys Tyr
Arg Cys Asn Cys Val Pro Pro Gly Thr Tyr Gly Asn Tyr Asp 65 70 75 80
Lys Cys Gln Cys Tyr Ala Ser Leu Thr Thr His Gly Gly Arg Arg Lys 85
90 95 Cys Pro 9 198 DNA Artificial Sequence optimized for
expression in E. coli. misc_feature (0)...(0) Sequence encoding SEQ
ID NO7 as expressed in E. coli for C. elegans bioassays 9
ggctacgcaa aaaaaattga ttgtggttct gcttgcgttg cgcgttgccg tctgtctcgt
60 cgcccacgtc tgtgccaccg cgcctgcggc acttgttgtt accgctgcaa
ctgcgtaccg 120 ccgggcacct atggcaatta cgataaatgt cagtgctacg
ccagcctgac cactcacggc 180 ggtcgccgca aatgtcca 198 10 66 PRT Petunia
x hybrida PEPTIDE (0)...(0) Kis10 mature peptide 10 Gly Ser Leu Lys
Pro Ser Gln Cys Leu Pro Gln Cys Thr Arg Arg Cys 1 5 10 15 Ser Gln
Thr Gln Tyr His Asn Ala Cys Met Leu Phe Cys Gln Lys Cys 20 25 30
Cys Asn Lys Cys Leu Cys Val Pro Pro Gly Phe Tyr Gly Asn Lys Gly 35
40 45 Val Cys Pro Cys Tyr Asn Asn Trp Lys Thr Lys Glu Gly Gly Pro
Lys 50 55 60 Cys Pro 65 11 105 PRT Petunia x hybrida PEPTIDE
(0)...(0) CAD10105 (Kis10) 11 Met Ala Lys Leu Val Pro Ile Phe Leu
Leu Ala Leu Phe Val Ile Ser 1 5 10 15 Met Phe Ala Thr Thr Val Leu
Ala Ser His Asp Pro Lys Arg Gly His 20 25 30 His His Lys Gly Tyr
Gly Pro Gly Ser Leu Lys Pro Ser Gln Cys Leu 35 40 45 Pro Gln Cys
Thr Arg Arg Cys Ser Gln Thr Gln Tyr His Asn Ala Cys 50 55 60 Met
Leu Phe Cys Gln Lys Cys Cys Asn Lys Cys Leu Cys Val Pro Pro 65 70
75 80 Gly Phe Tyr Gly Asn Lys Gly Val Cys Pro Cys Tyr Asn Asn Trp
Lys 85 90 95 Thr Lys Glu Gly Gly Pro Lys Cys Pro 100 105 12 198 DNA
Artificial Sequence optimized for expression in E. coli
misc_feature (0)...(0) Sequence encoding SEQ ID NO10 as expressed
in E. coli for C. elegans bioassays 12 ggctctctga aaccgtctca
gtgtctgcca cagtgcaccc gtcgttgctc ccaaactcaa 60 taccataacg
catgcatgct gttttgccaa aaatgttgta acaagtgcct gtgcgtaccg 120
ccgggcttct atggcaataa gggcgtttgt ccgtgctaca acaactggaa aactaaagaa
180 ggtggtccga aatgtcca 198 13 294 DNA Artificial Sequence
optimized for expression in E. coli misc_difference (0)...(0)
Sequence encoding SEQ ID NO2 as expressed in plant hairy root for
anti-nematode bioassays 13 atggcgaatt gtatcagaag aaatgctctt
ttcttcttga ctcttctctt tttattgtca 60 gtctccaacc tcgttcaggc
tgctcgtggt ggtggcaaac tgaaaccgca acagtgtaac 120 tctaaatgca
gcttccgttg ctccgcaact agccataaga agccgtgcat gttcttttgc 180
ctgaaatgtt gtaaaaagtg cctgtgcgta ccgccgggca ccttcggcaa taagcagacg
240 tgtccgtgct acaacaactg gaaaactaaa gaaggtcgcc cgaaatgtcc ataa 294
14 318 DNA Artificial Sequence optimized for expression in E. coli
misc_feature (0)...(0) Sequence encoding SEQ ID NO11 as expressed
in plant hairy root for anti-nematode bioassays 14 atggccaagc
ttgttcccat ttttcttttg gccctatttg tcatctccat gtttgctacc 60
acagttcttg cttcacatga tccaaagcgc gggcaccatc ataaagggta tggaccaggc
120 tctctgaaac cgtctcagtg tctgccacag tgcacccgtc gttgctccca
aactcaatac 180 cataacgcat gcatgctgtt ttgccaaaaa tgttgtaaca
agtgcctgtg cgtaccgccg 240 ggcttctatg gcaataaggg cgtttgtccg
tgctacaaca actggaaaac taaagaaggt 300 ggtccgaaat gtccataa 318
* * * * *
References