U.S. patent application number 14/504496 was filed with the patent office on 2015-03-26 for novel antifungal proteins and methods of use.
The applicant listed for this patent is PIONEER HI BRED INTERNATIONAL INC. Invention is credited to JAMES J. ENGLISH, AZALEA S. ONG, NASSER YALPANI.
Application Number | 20150089687 14/504496 |
Document ID | / |
Family ID | 45974147 |
Filed Date | 2015-03-26 |
United States Patent
Application |
20150089687 |
Kind Code |
A1 |
ENGLISH; JAMES J. ; et
al. |
March 26, 2015 |
Novel Antifungal Proteins and Methods of Use
Abstract
Compositions and methods for protecting a plant from a pathogen,
particularly a fungal pathogen, are provided. Compositions include
amino acid sequences, and variants and fragments thereof, for novel
variants of antipathogenic polypeptides generated through DNA
shuffling that exhibit improved antipathogenic activity.
Polynucleotides that encode the antipathogenic polypeptides are
also provided. A method for inducing pathogen resistance in a plant
using the polynucleotides disclosed herein is further provided.
Compositions comprising an antipathogenic polypeptide or a
microorganism comprising an antipathogenic polynucleotide of the
invention in combination with a carrier and methods of using these
compositions to protect a plant from a pathogen are further
provided. Plants, plant cells, seeds, and microorganisms comprising
an antipathogenic polynucleotide or polypeptide of the invention
are also disclosed.
Inventors: |
ENGLISH; JAMES J.; (SAN
RAMON, CA) ; ONG; AZALEA S.; (CASTRO VALLEY, CA)
; YALPANI; NASSER; (JOHNSTON, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI BRED INTERNATIONAL INC |
JOHNSTON |
IA |
US |
|
|
Family ID: |
45974147 |
Appl. No.: |
14/504496 |
Filed: |
October 2, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13240410 |
Sep 22, 2011 |
8853490 |
|
|
14504496 |
|
|
|
|
61406812 |
Oct 26, 2010 |
|
|
|
Current U.S.
Class: |
800/279 ;
424/93.2; 424/93.21; 435/252.3; 435/252.33; 435/254.11; 435/254.2;
435/320.1; 435/325; 435/348; 435/412; 435/419; 530/324;
800/301 |
Current CPC
Class: |
C07K 14/001 20130101;
C07K 14/415 20130101; C12N 15/8282 20130101; A01N 63/00 20130101;
C12N 15/8209 20130101; A01N 63/30 20200101; A01N 65/00 20130101;
A01N 37/46 20130101; A01N 37/46 20130101; A01N 63/10 20200101 |
Class at
Publication: |
800/279 ;
435/320.1; 800/301; 435/412; 435/419; 435/325; 435/348; 435/252.33;
435/252.3; 435/254.11; 435/254.2; 530/324; 424/93.21; 424/93.2 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01N 63/00 20060101 A01N063/00; C07K 14/00 20060101
C07K014/00 |
Claims
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
set forth in SEQ ID NO: 6 or 8; (b) an amino acid sequence having
at least 95% sequence identity to SEQ ID NO: 8, wherein the
polypeptide has antifungal activity; (c) an amino acid sequence
having at least 97% sequence identity to SEQ ID NO: 6, wherein the
polypeptide has antifungal activity; (d) an amino acid sequence
having at least 85% sequence identity to SEQ ID NO: 6, wherein the
amino acid sequence comprises at least one of the amino acid
residues selected from the group consisting of: (i) the arginine
(Arg) residue at the position corresponding to residue 8 of SEQ ID
NO: 6; and (ii) the lysine (Lys) residue at the position
corresponding to residue 42 of SEQ ID NO: 6, wherein the
polypeptide has antifungal activity; and (e) an amino acid sequence
having at least 85% sequence identity to SEQ ID NO: 8, wherein the
amino acid sequence comprises at least one of the amino acid
residues selected from the group consisting of: (i) the arginine
(Arg) residue at the position corresponding to residue 8 of SEQ ID
NO: 8; (ii) the glutamine (Glu) residue at the position
corresponding to residue 41 of SEQ ID NO: 8; and (iii) the lysine
(Lys) residue at the position corresponding to residue 42 of SEQ ID
NO: 8, wherein the polypeptide has antifungal activity.
2. The isolated polypeptide of claim 1, wherein the polypeptide has
antifungal activity against at least one of Colletotrichum
graminocola, Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides.
3. An expression cassette comprising a polynucleotide selected from
the group consisting of: (a) a polynucleotide comprising SEQ ID NO:
5 or 7; (b) polynucleotide encoding an amino acid sequence
comprising SEQ ID NO: 4, 6, or 8; (c) polynucleotide having at
least 85% sequence identity to SEQ ID NO: 5, or 7, wherein the
polynucleotide encodes a polypeptide having antifungal activity;
(d) polynucleotide encoding an amino acid sequence having at least
95% sequence identity to SEQ ID NO: 8, wherein the polynucleotide
encodes a polypeptide having antifungal activity; (e)
polynucleotide encoding an amino acid sequence having at least 97%
sequence identity to SEQ ID NO: 6, wherein the polynucleotide
encodes a polypeptide having antifungal activity; (f)
polynucleotide encoding an amino acid sequence having at least 85%
sequence identity to SEQ ID NO: 6, wherein the amino acid sequence
comprises at least one of the amino acid residues selected from the
group consisting of: (i) the arginine (Arg) residue at the position
corresponding to residue 8 of SEQ ID NO: 6; and (ii) the lysine
(Lys) residue at the position corresponding to residue 42 of SEQ ID
NO: 6, wherein the polynucleotide encodes a polypeptide having
antifungal activity; and (g) polynucleotide encoding an amino acid
sequence having at least 85% sequence identity to SEQ ID NO: 8,
wherein the amino acid sequence comprises at least one of the amino
acid residues selected from the group consisting of: (i) the
arginine (Arg) residue at the position corresponding to residue 8
of SEQ ID NO: 8; (ii) the glutamine (Glu) residue at the position
corresponding to residue 41 of SEQ ID NO: 8; and (iii) the lysine
(Lys) residue at the position corresponding to residue 42 of SEQ ID
NO: 8, wherein the polynucleotide encodes a polypeptide having
antifungal activity; and a heterologous promoter operably linked to
the polynucleotide.
4. The expression cassette of claim 3, wherein the encoded
polypeptide has antifungal activity against at least one of
Colletotrichum graminocola, Diplodia maydis, Fusarium graminearum,
or Fusarium verticillioides.
5. (canceled)
6. A transformed plant comprising the expression cassette of claim
3, wherein the plant has improved fungal resistance over a
non-transformed plant.
7. A method of enhancing fungal resistance in a plant, the method
comprising introducing into the plant the expression cassette of
claim 3.
8. A microorganism comprising the expression cassette of claim
3.
9. An antifungal composition comprising the microorganism of claim
8 and a suitable carrier.
10. A method for protecting a plant from a fungal pathogen, the
method comprising applying a composition according to claim 9 to
the environment of a plant fungal pathogen.
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. A method of enhancing fungal pathogen resistance in a plant,
the method comprising introducing into the plant the expression
cassette of claim 3.
17. (canceled)
18. (canceled)
19. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Non Provisional
application Ser. No. 13/240410 filed Sep. 22, 2011 now granted as
U.S. Pat. No. 8,853,490, which claims the benefit U.S. Provisional
application Ser. No. 61/406,812, filed Oct. 26, 2010; the contents
of which are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to polypeptides having
antipathogenic activity and polynucleotides that encode the same.
Methods of the invention utilize these antipathogenic
polynucleotides and polypeptides to control plant pathogens and to
increase pathogen resistance in plants.
BACKGROUND OF THE INVENTION
[0003] Plant diseases are often a serious limitation on
agricultural productivity and therefore have influenced the history
and development of agricultural practices. A variety of pathogens
are responsible for plant diseases, including fungi, bacteria,
viruses, and nematodes. Among the causal agents of infectious
diseases of crop plants, however, fungi are the most economically
important group of plant pathogens and are responsible for huge
annual losses of marketable food, fiber, and feed.
[0004] 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 more 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
substantial interest in developing 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.
[0005] Many plant diseases, including, but not limited to, maize
stalk rot and ear mold, can be caused by a variety of pathogens.
Stalk rot, for example, is one of the most destructive and
widespread diseases of maize. The disease is caused by a complex of
fungi and bacteria that attack and degrade stalks near plant
maturity. Significant yield loss can occur as a result of lodging
of weakened stalks as well as premature plant death. Maize stalk
rot is typically caused by more than one fungal species, but
Gibberella stalk rot, caused by Gibberella zeae, Fusarium stalk
rot, caused by Fusarium verticillioides, F. proliferatum, or F.
subglutinans, and Anthracnose stalk rot, caused by Colletotrichum
graminicola are the most frequently reported (Smith and White
(1988); Diseases of corn, pp. 701-766 in Corn and Corn Improvement,
Agronomy Series #18 (3rd ed.), Sprague, C. F., and Dudley, J. W.,
eds. Madison, Wis.). Due to the fact that plant diseases can be
caused by a complex of pathogens, broad spectrum resistance is
required to effectively mediate disease control. Thus, a
significant need exists for antifungal compositions that
efficiently target multiple stalk rot and ear mold-causing
pathogens.
[0006] Recently, agricultural scientists have developed crop plants
with enhanced pathogen resistance by genetically engineering plants
to express antipathogenic proteins. A continuing effort to identify
antipathogenic agents and to genetically engineer disease-resistant
plants is underway.
[0007] Thus, in light of the significant impact of plant pathogens,
particularly fungal pathogens, on the yield and quality of crops,
new compositions and methods for protecting plants from pathogens
are needed.
BRIEF SUMMARY OF THE INVENTION
[0008] Compositions and methods for protecting a plant from a
pathogen are provided. The compositions include novel nucleotide
and amino acid sequences for antipathogenic, particularly
antifungal, polypeptides. The presently disclosed polypeptides
display antipathogenic activity against plant fungal pathogens.
Polynucleotides comprising nucleotide sequences that encode the
presently disclosed antipathogenic polypeptides are further
provided. The polypeptides and nucleotide sequences encoding the
same were identified through the use of DNA shuffling. In some
embodiments, the antifungal polypeptides display improved
antipathogenic activity when compared to the parent polypeptide(s)
used in the DNA shuffling event that yielded the novel
antipathogenic polypeptide-encoding sequence. Compositions also
include expression cassettes comprising a polynucleotide that
encodes an antipathogenic polypeptide disclosed herein. Plants,
plant cells, seeds, and microorganisms comprising the presently
disclosed polynucleotides and polypeptides are further
provided.
[0009] The compositions are useful in methods directed to inducing
pathogen resistance, particularly fungal resistance, in plants. In
particular embodiments, the methods comprise introducing into a
plant at least one polynucleotide that encodes an antipathogenic
polypeptide. As a result, the antipathogenic polypeptide is
expressed in the plant, and the pathogen is exposed to the
preferred protein at the site of pathogen attack, thereby leading
to increased pathogen resistance. A tissue-preferred promoter may
be used to drive expression of an antipathogenic protein in
specific plant tissues that are particularly vulnerable to pathogen
attack, such as, for example, the roots, leaves, stalks, vascular
tissues, and seeds. Pathogen-inducible promoters may also be used
to drive the expression of an antipathogenic protein at or near the
site of pathogen infection.
[0010] Further provided are antipathogenic compositions and
formulations and methods for their use in protecting a plant from a
pathogen, particularly a fungal pathogen. In some embodiments,
compositions comprise an antipathogenic polypeptide or a
microorganism comprising a polynucleotide encoding an
antipathogenic polypeptide in combination with a carrier. Methods
of using these compositions to protect a plant from a pathogen
comprise applying the antipathogenic composition to the environment
of the plant pathogen by, for example, spraying, dusting,
broadcasting, or seed coating. The presently disclosed methods and
compositions find use in protecting plants from pathogens,
including fungal pathogens, viruses, nematodes, and the like.
DETAILED DESCRIPTION OF THE INVENTION
[0011] Compositions and methods are provided that are directed to
inducing pathogen resistance, particularly fungal resistance, in
plants. The compositions include novel nucleotide and amino acid
sequences for antipathogenic polypeptides. Specifically, isolated
polypeptides having the amino acid sequence set forth in SEQ ID
NOs: 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, and 26 and variants and
fragments thereof are provided. Isolated polynucleotides, and
variants and fragments thereof, comprising nucleotide sequences
that encode the amino acid sequences shown in SEQ ID NOs: 4, 6, 8,
12, 14, 16, 18, 20, 22, 24, and 26 are further provided.
[0012] The novel antipathogenic polypeptides and nucleotide
sequences encoding the same were generated through DNA shuffling
with known sequences, including sequences encoding the LBNL
antipathogenic polypeptides LBNL-5220 and LBNL-9827-1 that were
previously disclosed in U.S. Pat. No. 7,306,946, herein
incorporated by reference in its entirety. The nucleotide and amino
acid sequences for LBNL-5220 and LBNL-9827-1 are set forth in SEQ
ID NOs: 1 and 2, and 9 and 10, respectively. LBNL-5220 was isolated
from the filamentous fungus Penicillium simplicissimum, while the
species of origin for the LBNL-9827-1 polypeptide was determined to
be the fungus Monascus ruber. The LBNL-5220 and LBNL-9827-1
polypeptides exhibit antifungal activity against the fungi Fusarium
verticillioides (FVE), Fusarium graminearum (FGR), Colletotrichum
graminicola (CGR), and Diplodia maydis (DMA). The presently
disclosed LBNL-5220 and LBNL-9827-1 polypeptide variants (SEQ ID
NOs: 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, and 26) identified
through DNA shuffling exhibit improved activity against at least
one pathogenic target when compared to the parent polypeptide. The
variant LBNL-5220 and LBNL-9827-1 nucleotide sequences are set
forth in SEQ ID NOs: 3, 5, and 7, and 11, 13, 15, 17, 19, 21, and
23, respectively.
[0013] Plants, plant cells, seeds, and microorganisms comprising a
polynucleotide that encodes a presently disclosed antipathogenic
polypeptide of the invention are also disclosed herein.
Antipathogenic compositions comprising an isolated antipathogenic,
particularly an antifungal, polypeptide or a microorganism that
expresses a presently disclosed polypeptide in combination with a
carrier are further provided. The compositions find use in
generating pathogen-resistant plants and in protecting plants from
pathogens, particularly fungal pathogens.
[0014] The polynucleotides and polypeptides of the present
invention find use in methods for inducing pathogen resistance in a
plant. Accordingly, the compositions and methods disclosed herein
are useful in protecting plants against plant pathogens. By "plant
pathogen" is intended any organism that can cause harm to a plant
by inhibiting or slowing the growth of a plant, by damaging the
tissues of a plant, by weakening the immune system of a plant,
reducing the resistance of a plant to abiotic stresses, and/or by
causing the premature death of the plant, etc. Plant pathogens
include fungi, viruses, bacteria, nematodes and the like.
[0015] "Pathogen resistance" or "disease resistance" is intended to
mean that the plant avoids the disease symptoms that are the
outcome of plant-pathogen interactions. That is, pathogens are
prevented from causing plant diseases and the associated disease
symptoms, or alternatively, the disease symptoms caused by the
pathogen are minimized or lessened, such as, for example, the
reduction of stress and associated yield loss.
[0016] "Antipathogenic compositions" or "antipathogenic
polypeptides" is intended to mean that the compositions of the
invention have antipathogenic activity and thus are capable of
suppressing, controlling, and/or killing the invading pathogenic
organism. An antipathogenic polypeptide or composition of the
invention will reduce the disease symptoms resulting from pathogen
challenge by at least about 2%, including but not limited to, about
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or greater. In particular embodiments, the disease
symptoms resulting from pathogen challenge are reduced by an
antipathogenic polypeptide or composition of the invention 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 invention can be utilized to protect plants from disease,
particularly those diseases that are caused by plant pathogens. In
particular embodiments, the antipathogenic activity exhibited by
the polypeptides of the invention is antifungal activity. As used
herein, "antifungal activity" refers to the ability to suppress,
control, and/or kill the invading fungal pathogen. Likewise,
"fungal resistance" refers to enhanced tolerance to a fungal
pathogen when compared to that of an untreated or wild type plant.
Resistance may vary from a slight increase in tolerance to the
effects of the fungal pathogen (e.g., partial inhibition) to total
resistance such that the plant is unaffected by the presence of the
fungal pathogen. An increased level of resistance against a
particular fungal pathogen or against a wider spectrum of fungal
pathogens may both constitute antifungal activity or improved
fungal resistance. Likewise, a polypeptide having "improved
antipathogenic activity" or "improved antifungal activity" can
refer to a polypeptide exhibiting an increase in activity against a
single pathogen or fungus or activity against a wider spectrum of
pathogens or fungi as compared to a reference polypeptide.
[0017] 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 or environment 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.
[0018] 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; each of which
are herein incorporated by reference.
[0019] In some embodiments, the presently disclosed antipathogenic
polypeptides or variants or fragments thereof display an improved
antipathogenic, particularly antifungal, activity when compared to
the parent polypeptide from which it was derived through DNA
shuffling technology (e.g., SEQ ID NO: 2 or 10). In certain
embodiments, the presently disclosed antipathogenic polypeptide
exhibits a 2-fold to 100-fold greater antipathogenic activity
against at least one susceptible pathogen than the parent
polypeptide, including but not limited to, about 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,
12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,
19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold,
70-fold, 80-fold, 90-fold, and 100-fold. The antipathogenic
activity against a particular pathogen can be measured using any
method known in the art, including but not limited to in vitro
assays described above and the antifungal plate assay described in
Example 1. The antifungal plate assay can be performed under either
low or high salt conditions. Low salt conditions include those
conditions in which 1/8.times. of liquid medium (potato dextrose
broth for Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides; Czapek-Dox V8 broth for Colletotrichum
graminocola) plus about 0.25 mM calcium chloride, and about 12.5 mM
potassium chloride is used as the assay medium. High salt
conditions include those conditions in which 1/2.times. liquid
medium (potato dextrose broth for Diplodia maydis, Fusarium
graminearum, and Fusarium verticillioides; Czapek-Dox V8 broth for
Colletotrichum graminocola) plus about 1 mM calcium chloride, and
about 50 mM potassium chloride is used as the assay medium.
[0020] In certain embodiments, a presently disclosed antipathogenic
polypeptide or variant or fragment thereof exhibits improved
antifungal activity against at least one of Colletotrichum
graminocola, Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides. In particular embodiments, the antipathogenic
polypeptide displays about a 4-fold increase in antifungal activity
against the fungus Colletotrichum graminocola in an in vitro
antifungal plate assay (such as that described in Example 1)
performed under high salt conditions when compared to the
polypeptide set forth in SEQ ID NO: 2. In other embodiments, the
antipathogenic polypeptide displays about a 4-fold increase in
antifungal activity against the fungus Colletotrichum graminocola
and about a 2-fold improved antifungal activity against the fungus
Fusarium graminearum in an in vitro antifungal plate assay
performed under high salt conditions when compared to the
polypeptide set forth in SEQ ID NO: 10.
[0021] The compositions disclosed herein comprise isolated
polynucleotides that encode antipathogenic polypeptides, expression
cassettes comprising the presently disclosed antipathogenic
polynucleotides, and isolated antipathogenic polypeptides.
Antipathogenic compositions comprising a presently disclosed
polypeptide in combination with a carrier are also provided. The
invention further discloses plants and microorganisms comprising
polynucleotides that encode antipathogenic proteins.
[0022] As used herein, "polynucleotide" 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.
[0023] The use of the term "polynucleotide" is not intended to
limit the present invention 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 presently disclosed polynucleotides also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0024] 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
invention can be produced either from a polynucleotide disclosed
herein, or by the use of standard molecular biology or biochemical
techniques. For example, a truncated protein of the invention can
be produced by expression of a recombinant polynucleotide of the
invention in an appropriate host cell, or alternatively by a
combination of ex vivo procedures, such as protease digestion and
purification.
[0025] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified polynucleotide mean that the
polynucleotide 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 polynucleotide encoding a protein may comprise
non-translated sequences (e.g., introns) within translated regions
of the polynucleotide or may lack such intervening non-translated
sequences (e.g., as in cDNA).
[0026] The invention encompasses 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 presently disclosed antipathogenic protein 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.
[0027] Fragments and variants of the disclosed polynucleotides and
proteins encoded thereby are also encompassed by the present
invention. By "fragment" is intended a portion of the
polynucleotide or a portion of the amino acid sequence and hence
protein encoded thereby. Fragments of a polynucleotide may encode
protein fragments that retain the biological activity of the native
protein and hence have antipathogenic activity. Alternatively,
fragments of a polynucleotide 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 30 nucleotides,
about 40 nucleotides, about 50 nucleotides, and up to the
full-length polynucleotide encoding the presently disclosed
proteins.
[0028] A fragment of a polynucleotide that encodes a biologically
active portion of a presently disclosed antipathogenic protein will
encode at least 15, 25, 30, or 50 contiguous amino acids, or up to
the total number of amino acids present in a full-length
antipathogenic protein of the invention (for example, 55 amino
acids for SEQ ID NOs: 4, 6, and 8, and 58 amino acids for SEQ ID
NOs: 12, 14, 16, 18, 20, 22, 24, and 26, respectively). Fragments
of a polynucleotide that are useful as hybridization probes or PCR
primers generally need not encode a biologically active portion of
an antipathogenic protein.
[0029] Thus, a fragment of a presently disclosed polynucleotide 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 polynucleotides of
the invention, 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 antipathogenic protein.
Polynucleotides that are fragments of a nucleotide sequence of the
invention comprise at least 15, 20, 50, 75, 100, or 150 contiguous
nucleotides, or up to the number of nucleotides present in a
full-length polynucleotide disclosed herein (for example, 165
nucleotides for SEQ ID NOs: 3, 5, and 7, and 174 nucleotides for
SEQ ID NOs: 12, 14, 16, 18, 20, 22, and 24, respectively).
[0030] "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. 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 invention. 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 elsewhere herein. Variant polynucleotides also include
synthetically derived polynucleotides, such as those generated, for
example, by using site-directed mutagenesis but which still encode
an antipathogenic protein of the invention. Generally, variants of
a particular polynucleotide of the invention 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.
[0031] Variants of a particular polynucleotide of the invention
(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 polypeptide of SEQ ID NO: 4, 6, 8, 12, 14,
16, 18, 20, 22, 24, or 26 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 invention 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.
[0032] "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 present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
antipathogenic 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 invention 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 invention
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.
[0033] In some embodiments, variants of the polypeptides of the
invention retain the amino acid residues that differ from the
parent polypeptide (e.g., SEQ ID NO: 2 and 10) that can contribute
to the enhanced antipathogenic activity of the presently disclosed
polypeptides (those amino acid residues can be determined by
consulting FIGS. 1 and 2). For example, variants of SEQ ID NO: 4
can comprise at least one of the following amino acid residues: the
lysine (Lys) residue at the position corresponding to residue 10 of
SEQ ID NO: 4, the arginine (Arg) residue at the position
corresponding to residue 17 of SEQ ID NO: 4, and the arginine (Arg)
residue at the position corresponding to residue 35 of SEQ ID NO:
4. In some embodiments, variants of SEQ ID NO: 6 can comprise at
least one of the following amino acid residues: the arginine (Arg)
residue at the position corresponding to residue 8 of SEQ ID NO: 6,
and the lysine (Lys) residue at the position corresponding to
residue 42 of SEQ ID NO: 6. As a further example, variants of SEQ
ID NO: 8 can comprise at least one of the following amino acid
residues: the arginine (Arg) residue at the position corresponding
to residue 8 of SEQ ID NO: 8, the glutamine (Glu) residue at the
position corresponding to residue 41 of SEQ ID NO: 8, and the
lysine (Lys) residue at the position corresponding to residue 42 of
SEQ ID NO: 8.
[0034] The proteins of the invention 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.
[0035] Thus, the genes and polynucleotides of the invention include
both the naturally occurring sequences as well as mutant forms.
Likewise, the proteins of the invention encompass both naturally
occurring proteins as well as variations and modified forms
thereof. Such variants will continue to possess the desired
antipathogenic 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
Application Publication No. 75,444.
[0036] 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 antifungal plate
assays. See, for example, Duvick et al. (1992) J. Biol. Chem.
267:18841-18820, herein incorporated by reference.
[0037] 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 presently disclosed
antipathogenic polynucleotides and other known antipathogenic
genes, such as, for example, other LBNL family members, to obtain a
new gene coding for a protein with an improved property of
interest, such as increased antipathogenic 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.
[0038] The polynucleotides of the invention can be used to isolate
corresponding sequences from other organisms, particularly other
plants, more particularly other fungi. 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 present invention. 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 protein and which hybridize under stringent
conditions to the sequences disclosed herein, or to variants or
fragments thereof, are encompassed by the present invention.
[0039] 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.
[0040] 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 invention.
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.).
[0041] 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. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.).
[0042] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended conditions under which a
probe will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, optimally less than 500 nucleotides in length.
[0043] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C. Optionally, wash buffers may comprise about 0.1% to
about 1% SDS. Duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours. The duration of
the wash time will be at least a length of time sufficient to reach
equilibrium.
[0044] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization, and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with .gtoreq.90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution), it is optimal to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
(Elsevier, New York); and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0045] 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."
[0046] (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.
[0047] (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.
[0048] 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.
[0049] 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 at (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 invention. 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 invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (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.
[0050] 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. By "equivalent program" is intended 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.
[0051] 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.
[0052] 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).
[0053] (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.).
[0054] (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.
[0055] In particular aspects, methods for inducing pathogen
resistance in a plant comprise introducing into a plant at least
one polynucleotide, wherein the polynucleotide comprises a
nucleotide sequence encoding an antipathogenic polypeptide of the
invention. The polynucleotide is operably linked to a promoter that
drives expression in the plant. The plant expresses the
antipathogenic polypeptide, thereby exposing the pathogen to the
polypeptide at the site of pathogen attack. In particular
embodiments, the polypeptides have antifungal activity, and the
pathogen is a fungus, such as, for example, Colletotrichum
graminocola, Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides. Expression of an antipathogenic polypeptide of the
invention may be targeted to specific plant tissues where pathogen
resistance is particularly important, such as, for example, the
leaves, roots, stalks, or vascular tissues. 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 invention may also be
targeted to specific subcellular locations within a plant cell or,
alternatively, secreted from the cell, as described herein
below.
[0056] Just as expression of an antipathogenic polypeptide of the
invention 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.
[0057] 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. In particular embodiments, a
nucleotide sequence encoding a barley alpha-amylase (BAA) signal
peptide is joined in frame with a polynucleotide of the invention.
The nucleotide sequence encoding the BAA signal peptide and the
amino acid sequence for the BAA signal peptide are set forth in SEQ
ID NO: 27 and SEQ ID NO: 28, respectively.
[0058] 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 and include, for example, KDEL
(SEQ ID NO: 29), SEKDEL (SEQ ID NO: 30), HDEL (SEQ ID NO: 31), and
HDEF (SEQ ID NO: 32). 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.
[0059] Alternatively, the use of vacuolar targeting labels such as
those described by Raikhel and Chrispeels, supra, in addition to a
signal peptide will result in localization of the peptide in a
vacuolar structure. As described in Raikhel and Chrispeels, supra,
the vacuolar targeting label may be placed in different positions
in the construct. Use of a plastid transit peptide encoding
sequence instead of a signal peptide encoding sequence will result
in localization of the polypeptide in the plastid of the cell type
chosen (Raikhel and Chrispeels, supra). Such transit peptides are
known in the art. See, for example, Von Heijne et al. (1991) Plant
Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.
264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.
84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.
196:1414-1421; and Shah et al. (1986) Science 233:478-481.
Chloroplast targeting sequences that encode such transit peptides
are also known in the art and include the chloroplast small subunit
of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva
Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al.
(1991) J. Biol. Chem. 266(5):3335-3342);
5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et
al. (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan
synthase (Zhao et al. (1995) J. Biol. Chem. 270(11):6081-6087);
plastocyanin (Lawrence et al. (1997) J. Biol. Chem.
272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J.
Biol. Chem. 268(36):27447-27457); and the light harvesting
chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J.
Biol. Chem. 263:14996-14999). A person skilled in the art could
also envision generating transgenic plants in which the
chloroplasts have been transformed to overexpress a gene for an
antipathogenic peptide. See, for example, Daniell (1999) Nature
Biotech 17:855-856; and U.S. Pat. No. 6,338,168.
[0060] One could also envision localizing the antipathogenic
polypeptide in other cellular compartments by addition of suitable
targeting information. (Raikhel and Chrispeels, supra). A useful
site available on the world wide web that provides information and
references regarding recognition of the various targeting sequences
can be found at: psort.nibb.ac.jp/mit. Other references regarding
the state of the art of protein targeting include Silva-Filho
(2003) Curr. Opin. Plant Biol. 6:589-595; Nicchitta (2002) Curr.
Opin. Cell Biol. 14:412-416; Bruce (2001) Biochim Biophys Acta
1541: 2-21; Hadlington & Denecke (2000) Curr. Opin. Plant Biol.
3: 461-468; Emanuelsson et al. (2000) J Mol. Biol. 300: 1005-1016;
Emanuelsson & von Heijne (2001) Biochim Biophys Acta 1541:
114-119, herein incorporated by reference.
[0061] 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. In some embodiments,
the antipathogenic polypeptides of the invention are expressed as
fusion proteins, wherein the propeptide segments (optionally
preceded by a signal peptide) of another antipathogenic polypeptide
is fused to the amino terminal end of the polypeptide of the
invention. For example, the propeptide segments of another LBNL
sequence, such as, for example, LBNL-5200, LBNL-9827-1,
LBNL-5197/8-1, LBNL-5197/8-2, and LB-9827-2 (the sequences of which
were disclosed in U.S. Pat. No. 7,306,946), can be fused to the
amino terminal end of the presently disclosed polypeptides.
[0062] The polynucleotides of the present invention can be
expressed in a host cell, such as a bacterial, fungal, yeast,
insect, mammalian, or preferably plant cells. By "host cell" is
meant a cell which comprises a heterologous polynucleotide of the
invention. Host cells may be prokaryotic cells, such as E. coli, or
eukaryotic cells, such as yeast, insect, amphibian, or mammalian
cells. In some embodiments, host cells are monocotyledonous or
dicotyledonous plant cells. In particular embodiments, the
monocotyledonous host cell is a maize host cell.
[0063] The antipathogenic polynucleotides of the invention can be
provided in expression cassettes for expression in an organism of
interest. The expression cassettes of the invention find use in
generating transformed plants, plant cells, and microorganisms and
in practicing the methods for inducing pathogen resistance
disclosed herein. The cassette will include 5' and 3' regulatory
sequences operably linked to an antipathogenic polynucleotide of
the invention. "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 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.
[0064] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., a promoter), a polynucleotide of the invention, and a
transcriptional and translational termination region (i.e.,
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
invention may be native/analogous to the host cell or to each
other. Alternatively, the regulatory regions and/or the
polynucleotide of the invention 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.
[0065] 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
plant host, or any combination thereof. Convenient termination
regions are available from the Ti-plasmid of A. tumefaciens, such
as the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et
al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.
15:9627-9639. In particular embodiments, the potato proteinase
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.
[0066] 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.
[0067] 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.
[0068] 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) (Virology 154:9-20), 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.
[0069] 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.
[0070] A number of promoters can be used in the practice of the
invention, 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 review of Potenza et al. (2004) In Vitro Cell Dev Biol--Plant
40:1-22, herein incorporated by reference. For example, the nucleic
acids can be combined with constitutive, tissue-preferred, 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) Theon. 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, 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.
[0071] 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.
[0072] Of interest are promoters that are expressed 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. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200) and the inducible maize promoters described
in U.S. Pat. No. 6,429,362 (e.g., Zm-PR1-81 and Zm-PR1-83
promoters), all of which are herein incorporated by reference in
their entirety. The promoters described in U.S. Pat. No. 6,720,480,
such as the Zm-BB11 promoter, may also be used in the practice of
the invention.
[0073] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter, which includes
a pathogen-inducible promoter, may be used in the constructions of
the invention. 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.
[0074] 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 In2-2 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.
[0075] Tissue-preferred promoters can be utilized to target
enhanced expression of the antipathogenic polypeptides of the
invention within a particular plant tissue. For example, a
tissue-preferred promoter may be used to express an antipathogenic
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. Such promoters
can be modified, if necessary, for weak expression.
[0076] 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. Pat.
No. 6,921,815.
[0077] Stalk-preferred promoters may be used to drive expression of
an antipathogenic polypeptide of the invention. 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. In certain embodiments of the
invention, the Zm-419 promoter is used for tissue
preferred-expression in maize stalk tissue. See, for example,
International Publication No. WO 2007/050509 and U.S. Pat. No.
7,538,261.
[0078] 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.
[0079] 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. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus corniculatus,
and in both instances root-specific promoter activity was
preserved. 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). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene, fused to nptII (neomycin phosphotransferase II) showed
similar characteristics. 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.
[0080] "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 an endosperm-specific promoter. Globulin 1 (Glb-1) is
a representative 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, gamma-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.
[0081] 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, 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. Such disclosures are herein
incorporated by reference.
[0082] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention.
[0083] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding sequences,
include such commonly used promoters as the beta lactamase
(penicillinase) and lactose (lac) promoter systems (Chang et al.
(1977) Nature 198:1056), the tryptophan (trp) promoter system
(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda
derived PL promoter and N-gene ribosome binding site (Simatake and
Rosenberg (1981) Nature 292:128). Examples of selection markers for
E. coli include, for example, genes specifying resistance to
ampicillin, tetracycline, or chloramphenicol.
[0084] The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of plasmid
or phage origin. Appropriate bacterial cells are infected with
phage vector particles or transfected with naked phage vector DNA.
If a plasmid vector is used, the bacterial cells are transfected
with the plasmid vector DNA. Expression systems for expressing a
protein of the present invention are available using Bacillus sp.
and Salmonella (Palva et al. (1983) Gene 22:229-235 and Mosbach et
al. (1983) Nature 302:543-545).
[0085] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, a polynucleotide of
the present invention can be expressed in these eukaryotic systems.
In some embodiments, transformed/transfected plant cells, as
discussed infra, are employed as expression systems for production
of the proteins of the instant invention.
[0086] Synthesis of heterologous nucleotide sequences in yeast is
well known. Sherman, F., et al. (1982) Methods in Yeast Genetics,
Cold Spring Harbor Laboratory is a well recognized work describing
the various methods available to produce proteins in yeast. Two
widely utilized yeasts for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like,
as desired.
[0087] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates. The monitoring of the
purification process can be accomplished by using Western blot
techniques, radioimmunoassay, or other standard immunoassay
techniques.
[0088] The sequences of the present invention can also be ligated
to various expression vectors for use in transfecting cell cultures
of, for instance, mammalian, insect, or plant origin. Illustrative
cell cultures useful for the production of the peptides are
mammalian cells. A number of suitable host cell lines capable of
expressing intact proteins have been developed in the art, and
include the HEK293, BHK21, and CHO cell lines. Expression vectors
for these cells can include expression control sequences, such as
an origin of replication, a promoter (e.g. the CMV promoter, a HSV
tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen et al. (1986) Immunol. Rev. 89:49), and necessary processing
information sites, such as ribosome binding sites, RNA splice
sites, polyadenylation sites (e.g., an SV40 large T Ag poly A
addition site), and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection.
[0089] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth and Drosophila cell lines such as a
Schneider cell line (See, Schneider (1987) J. Embryol. Exp.
Morphol. 27:353-365).
[0090] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al. (1983) J. Virol. 45:773-781).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors. Saveria-Campo, M., (1985)
Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA
Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,
Arlington, Va. pp. 213-238.
[0091] Animal and lower eukaryotic (e.g., yeast) host cells are
competent or rendered competent for transfection by various means.
There are several well-known methods of introducing DNA into animal
cells. These include: calcium phosphate precipitation, fusion of
the recipient cells with bacterial protoplasts containing the DNA,
treatment of the recipient cells with liposomes containing the DNA,
DEAE dextrin, electroporation, biolistics, and micro-injection of
the DNA directly into the cells. The transfected cells are cultured
by means well known in the art. Kuchler, R. J. (1997) Biochemical
Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross,
Inc.
[0092] In certain embodiments, the polynucleotides of the present
invention 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 present
invention may be stacked with other antipathogenic genes and the
like. The combinations generated can also include multiple copies
of any one of the polynucleotides of interest. The polynucleotides
of the present invention 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. Pat. No.
6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)); the
disclosures of which are herein incorporated by reference.
[0093] The polynucleotides of the present invention 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; 5,723,756; 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 gene)); 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
present invention 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, and WO 99/25821); the disclosures of which are herein
incorporated by reference.
[0094] These stacked combinations can be created by any method
including, but not limited to, cross-breeding plants by any
conventional or TopCross methodology, or genetic transformation. If
the sequences 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. 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.
[0095] The methods of the invention involve introducing a
polypeptide or polynucleotide into a plant. "Introducing" is
intended to mean presenting to the plant the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell of the plant. The methods of the invention do
not depend on a particular method for introducing a sequence into a
plant, only that the polynucleotide or polypeptides gains access to
the interior of at least one cell of the plant. Methods for
introducing polynucleotide or polypeptides 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.
[0096] "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" 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.
[0097] 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. No. 5,563,055 and U.S. Pat. No. 5,981,840), direct gene
transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and
ballistic particle acceleration (see, for example, U.S. Pat. No.
4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and
U.S. Pat. No. 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.
[0098] In specific embodiments, the antipathogenic sequences of the
invention 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 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 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 its released to become integrated into
the genome is greatly reduced. Such methods include the use
particles coated with polyethylimine (PEI; Sigma #P3143).
[0099] In other embodiments, the polynucleotide of the invention
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 invention within a viral DNA or RNA
molecule. It is recognized that the antipathogenic polypeptide of
the invention 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 invention 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.
[0100] 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 invention 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.
[0101] 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 present invention provides
transformed seed (also referred to as "transgenic seed") having a
polynucleotide of the invention, for example, an expression
cassette of the invention, stably incorporated into their
genome.
[0102] Pedigree breeding starts with the crossing of two genotypes,
such as an elite line of interest and one other elite inbred line
having one or more desirable characteristics (i.e., having stably
incorporated a polynucleotide of the invention, having a modulated
activity and/or level of the polypeptide of the invention, etc)
which complements the elite line of interest. If the two original
parents do not provide all the desired characteristics, other
sources can be included in the breeding population. In the pedigree
method, superior plants are selfed and selected in successive
filial generations. In the succeeding filial generations, the
heterozygous condition gives way to homogeneous lines as a result
of self-pollination and selection. Typically in the pedigree method
of breeding, five or more successive filial generations of selfing
and selection is practiced: F1.fwdarw.F2; F2.fwdarw.F3;
F3.fwdarw.F4; F4.fwdarw.F.sub.5, etc. After a sufficient amount of
inbreeding, successive filial generations will serve to increase
seed of the developed inbred. In specific embodiments, the inbred
line comprises homozygous alleles at about 95% or more of its
loci.
[0103] In addition to being used to create a backcross conversion,
backcrossing can also be used in combination with pedigree breeding
to modify an elite line of interest and a hybrid that is made using
the modified elite line. As discussed previously, backcrossing can
be used to transfer one or more specifically desirable traits from
one line, the donor parent, to an inbred called the recurrent
parent, which has overall good agronomic characteristics yet lacks
that desirable trait or traits. However, the same procedure can be
used to move the progeny toward the genotype of the recurrent
parent but at the same time retain many components of the
non-recurrent parent by stopping the backcrossing at an early stage
and proceeding with selfing and selection. For example, an F1, such
as a commercial hybrid, is created. This commercial hybrid may be
backcrossed to one of its parent lines to create a BC1 or BC2.
Progeny are selfed and selected so that the newly developed inbred
has many of the attributes of the recurrent parent and yet several
of the desired attributes of the non-recurrent parent. This
approach leverages the value and strengths of the recurrent parent
for use in new hybrids and breeding.
[0104] Therefore, an embodiment of this invention is a method of
making a backcross conversion of maize inbred line of interest,
comprising the steps of crossing a plant of maize inbred line of
interest with a donor plant comprising a mutant gene or transgene
conferring a desired trait (i.e., increased pathogen resistance),
selecting an F1 progeny plant comprising the mutant gene or
transgene conferring the desired trait, and backcrossing the
selected F1 progeny plant to the plant of maize inbred line of
interest. This method may further comprise the step of obtaining a
molecular marker profile of maize inbred line of interest and using
the molecular marker profile to select for a progeny plant with the
desired trait and the molecular marker profile of the inbred line
of interest. In the same manner, this method may be used to produce
an F1 hybrid seed by adding a final step of crossing the desired
trait conversion of maize inbred line of interest with a different
maize plant to make F1 hybrid maize seed comprising a mutant gene
or transgene conferring the desired trait.
[0105] Recurrent selection is a method used in a plant breeding
program to improve a population of plants. The method entails
individual plants cross pollinating with each other to form
progeny. The progeny are grown and the superior progeny selected by
any number of selection methods, which include individual plant,
half-sib progeny, full-sib progeny, selfed progeny and topcrossing.
The selected progeny are cross-pollinated with each other to form
progeny for another population. This population is planted and
again superior plants are selected to cross pollinate with each
other. Recurrent selection is a cyclical process and therefore can
be repeated as many times as desired. The objective of recurrent
selection is to improve the traits of a population. The improved
population can then be used as a source of breeding material to
obtain inbred lines to be used in hybrids or used as parents for a
synthetic cultivar. A synthetic cultivar is the resultant progeny
formed by the intercrossing of several selected inbreds.
[0106] Mass selection is a useful technique when used in
conjunction with molecular marker enhanced selection. In mass
selection seeds from individuals are selected based on phenotype
and/or genotype. These selected seeds are then bulked and used to
grow the next generation. Bulk selection requires growing a
population of plants in a bulk plot, allowing the plants to
self-pollinate, harvesting the seed in bulk and then using a sample
of the seed harvested in bulk to plant the next generation. Instead
of self pollination, directed pollination could be used as part of
the breeding program.
[0107] Mutation breeding is one of many methods that could be used
to introduce new traits into an elite line. Mutations that occur
spontaneously or are artificially induced can be useful sources of
variability for a plant breeder. The goal of artificial mutagenesis
is to increase the rate of mutation for a desired characteristic.
Mutation rates can be increased by many different means including
temperature, long-term seed storage, tissue culture conditions,
radiation; such as X-rays, Gamma rays (e.g. cobalt 60 or cesium
137), neutrons, (product of nuclear fission by uranium 235 in an
atomic reactor), Beta radiation (emitted from radioisotopes such as
phosphorus 32 or carbon 14), or ultraviolet radiation (preferably
from 2500 to 2900 nm), or chemical mutagens (such as base analogues
(5-bromo-uracil), related compounds (8-ethoxy caffeine),
antibiotics (streptonigrin), alkylating agents (sulfur mustards,
nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates,
sulfones, lactones), azide, hydroxylamine, nitrous acid, or
acridines. Once a desired trait is observed through mutagenesis the
trait may then be incorporated into existing germplasm by
traditional breeding techniques, such as backcrossing. Details of
mutation breeding can be found in "Principals of Cultivar
Development" Fehr, 1993 Macmillan Publishing Company the disclosure
of which is incorporated herein by reference. In addition,
mutations created in other lines may be used to produce a backcross
conversion of elite lines that comprises such mutations.
[0108] As used herein, the term plant also includes plant cells,
plant protoplasts, plant cell tissue cultures from which plants can
be regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, and the like. 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 invention, provided that these
parts comprise the introduced polynucleotides.
[0109] The present invention 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), cassava (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.
[0110] Vegetables include tomatoes (Lycopersicon esculentum),
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.
[0111] Conifers that may be employed in practicing the present
invention 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 present invention are crop plants (for example, corn, alfalfa,
sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum,
wheat, millet, tobacco, etc.). In other embodiments, corn and
soybean and plants are optimal, and in yet other embodiments corn
plants are optimal.
[0112] 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.
[0113] The compositions of the invention find further use in
methods directed to protecting a plant from a pathogen. "Protecting
a plant from a pathogen" is intended to mean killing the pathogen
or preventing or limiting disease formation on a plant. In some
embodiments, an antipathogenic composition comprising an
antipathogenic polypeptide and a carrier is applied directly to the
environment of a plant pathogen, such as, for example, on a plant
or in the soil or other growth medium surrounding the roots of the
plant, in order to protect the plant from pathogen attack.
Microorganisms comprising a polynucleotide encoding an
antipathogenic protein of the invention and methods of using them
to protect a plant from a pathogen are further provided. In some
embodiments, the transformed microorganism is applied directly to a
plant or to the soil in which a plant grows.
[0114] Antipathogenic compositions, particularly antifungal
compositions, are also encompassed by the present invention.
Antipathogenic compositions may comprise antipathogenic
polypeptides or microorganisms comprising a heterologous
polynucleotide 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.
[0115] The antipathogenic compositions find further use in the
decontamination of plant pathogens during the processing of grain
for animal or human food consumption; during the processing of
feedstuffs, and during the processing of plant material for silage.
In this embodiment, the antipathogenic compositions of the
invention are presented to grain, plant material for silage, or a
contaminated food crop, or during an appropriate stage of the
processing procedure, in amounts effective for antimicrobial
activity.
[0116] A polynucleotide encoding an antipathogenic, particularly
antifungal, polypeptide of the invention 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
rhizoplane) 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.
[0117] 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 vinelandii 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 roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0118] Other illustrative prokaryotes, both Gram-negative and
gram-positive, include Enterobacteriaceae, such as Escherichia,
Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae;
Rhizobiaceae, 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.
[0119] 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.
[0120] Polynucleotides encoding the antipathogenic proteins of the
invention can be introduced into microorganisms that multiply on
plants (epiphytes) to deliver antipathogenic proteins to potential
target pests. Epiphytes, for example, can be gram-positive or
gram-negative bacteria.
[0121] 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). Polynucleotides encoding the
antipathogenic polypeptides of the invention can be introduced into
a root-colonizing Bacillus cereus by standard methods known in the
art.
[0122] Polynucleotides encoding antipathogenic proteins can be
introduced, for example, into the root-colonizing Bacillus by means
of electrotransformation. Specifically, polynucleotides encoding
the antipathogenic 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 antipathogenic 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).
[0123] 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.
[0124] Prior to the application of an antipathogenic composition of
the invention to an area of cultivation, the environment can be
evaluated to determine if the pathogen of interest is present or if
conditions are conducive to pathogen growth or infestation. As used
herein, an "area of cultivation" comprises any region in which one
desires to grow a plant. Such areas of cultivations include, but
are not limited to, a field in which a plant is cultivated (such as
a crop field, a sod field, a tree field, a managed forest, a field
for culturing fruits and vegetables, etc), a greenhouse, a growth
chamber, etc. Evaluation of the environment can aid in determining
the effective amount of the antipathogenic protein or composition
of the invention needed to control a pathogen within an area of
cultivation.
[0125] Environmental conditions that can be evaluated include, but
are not limited to, ground and surface water pollution concerns,
intended use of the crop, crop tolerance, soil residuals, weeds
present in area of cultivation, humidity, soil texture, pH of soil,
amount of organic matter in soil, water content of soil,
application equipment, and tillage practices. Following the
evaluation of the environmental conditions, an effective amount of
an antipathogenic composition of the invention can be applied to
the crop, crop part, seed of the crop or area of cultivation.
[0126] The antipathogenic 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, acaricides, 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.
[0127] 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.
[0128] 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.
[0129] 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 diluent 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.
[0130] 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.).
[0131] 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. 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
antipathogenic polypeptides or transformed microorganisms 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 antipathogenic
polypeptides or transformed microorganisms of the invention.
[0132] 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.
[0133] The time at which an antipathogenic composition is applied
to an area of interest (and any plants therein) may be important in
optimizing pathogen control. The time at which an antipathogenic
composition is applied may be determined with reference to the size
of plants and/or the stage of growth and/or development of plants
in the area of interest. The stages of growth and/or development of
plants are known in the art. For example, soybean plants normally
progress through vegetative growth stages known as VE (emergence),
VC (cotyledon), V1 (unifoliate), and V2 to VN. Soybeans then switch
to the reproductive growth phase in response to photoperiod cues;
reproductive stages include R1 (beginning bloom), R2 (full bloom),
R3 (beginning pod), R4 (full pod), R5 (beginning seed), R6 (full
seed), R7 (beginning maturity), and R8 (full maturity). Corn plants
normally progress through the following vegetative stages VE
(emergence); V1 (first leaf); V2 (second leaf); V3 (third leaf);
V(n) (Nth/leaf); and VT (tasseling). Progression of maize through
the reproductive phase is as follows: R1 (silking); R2
(blistering); R3 (milk); R4 (dough); R5 (dent); and R6
(physiological maturity). Cotton plants normally progress through
VE (emergence), VC (cotyledon), V1 (first true leaf), and V2 to VN.
Then, reproductive stages beginning around V14 include R1
(beginning bloom), R2 (full bloom), R3 (beginning boll), R4
(cutout, boll development), R5 (beginning maturity, first opened
boll), R6 (maturity, 50% opened boll), and R7 (full maturity,
80-90% open bolls). Thus, for example, the time at which an
antipathogenic composition or other chemical is applied to an area
of interest in which plants are growing may be the time at which
some or all of the plants in a particular area have reached at
least a particular size and/or stage of growth and/or development,
or the time at which some or all of the plants in a particular area
have not yet reached a particular size and/or stage of growth
and/or development.
[0134] One of skill in the art will appreciate that the
compositions and methods disclosed herein can be used with other
compositions and methods available in the art for protecting plants
from insect and pathogen attack. For example, methods of the
invention can comprise the use of one or more herbicides,
insecticides, fungicides, nematocides, bactericides, acaricides,
growth regulators, chemosterilants, semiochemicals, repellents,
attractants, pheromones, feeding stimulants or other biologically
active compounds or entomopathogenic bacteria, virus, or fungi to
form a multi-component mixture giving an even broader spectrum of
agricultural protection. General references for these agricultural
protectants include The Pesticide Manual, 13th Edition, C. D. S.
Tomlin, Ed., British Crop Protection Council, Farnham, Surrey,
U.K., 2003 and The BioPesticide Manual, 2nd Edition, L. G. Copping,
Ed., British Crop Protection Council, Farnham, Surrey, U.K.,
2001.
[0135] 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.
[0136] The plant seed of the invention comprising a polynucleotide
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 used alone or
in combination with one of the seed protective coatings customarily
used in seed treatment.
[0137] In an embodiment of the invention, the antipathogenic
compositions of the invention may be used as a pharmaceutical
composition for treatment of fungal and microbial pathogens in
humans and other animals. Diseases and disorders caused by fungal
and microbial pathogens include but are not limited to fungal
meningoencephalitis, superficial fungal infections, ringworm,
Athlete's foot, histoplasmosis, candidiasis, thrush, coccidioidoma,
pulmonary cryptococcus, trichosporonosis, piedra, tinea nigra,
fungal keratitis, onychomycosis, tinea capitis, chromomycosis,
aspergillosis, endobronchial pulmonary aspergillosis, mucormycosis,
chromoblastomycosis, dermatophytosis, tinea, fusariosis,
pityriasis, mycetoma, pseudallescheriasis, and sporotrichosis.
[0138] In some of these embodiments, the antipathogenic polypeptide
is combined with a pharmaceutically acceptable carrier. As used
herein the term "pharmaceutically acceptable carrier" includes
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. Supplementary active
compounds also can be incorporated into the compositions.
[0139] In particular, the antipathogenic polypeptides of the
invention and pharmaceutical compositions comprising the same may
be used to provide treatment for diseases and disorders associated
with, but not limited to, the following fungal pathogens:
Histoplasma capsulatum, Candida spp. (C. albicans, C. tropicalis,
C. parapsilosis, C. guilliermondii, C. glabrata/Torulopsis
glabrata, C. krusei, C. lusitaniae), Aspergillus fumigatus, A.
flavus, A. niger, Rhizopus spp., Rhizomucor spp., Cunninghamella
spp., Apophysomyces spp., Saksenaee spp., Mucor spp., and Absidia
spp. Efficacy of the compositions of the invention as anti-fungal
treatments may be determined through anti-fungal assays known to
one in the art.
[0140] The presently disclosed pharmaceutical compositions may be
administered to a patient through numerous means. Systemic
administration can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. The compounds can also be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0141] In one embodiment, the active compounds are prepared with
pharmaceutically acceptable carriers that will protect the compound
against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0142] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated with each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. Depending on the type and severity of the
disease, about 1 .mu.g/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg)
of active compound is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to about 100 mg/kg
or more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this therapy is easily monitored by
conventional techniques and assays. An exemplary dosing regimen is
disclosed in WO 94/04188. The specification for the dosage unit
forms of the invention are dictated by and directly dependent on
the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an active compound for the
treatment of individuals.
[0143] "Treatment" is herein defined as the application or
administration of a therapeutic agent to a patient, or application
or administration of a therapeutic agent to an isolated tissue or
cell line from a patient, who has a disease, a symptom of disease
or a predisposition toward a disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve or
affect the disease, the symptoms of disease or the predisposition
toward disease. A "therapeutic agent" comprises, but is not limited
to, the polypeptides and pharmaceutical compositions of the
invention.
[0144] The antipathogenic polypeptides of the invention can be used
for any application including coating surfaces to target microbes.
In this manner, target microbes include human pathogens or
microorganisms. Surfaces that might be coated with the
antipathogenic compositions 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.
[0145] The embodiments of the present invention may be effective
against a variety of plant pathogens, particularly fungal
pathogens, such as, for example, Colletotrichum graminocola,
Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides. Pathogens of the invention include, but are not
limited to, viruses or viroids, bacteria, insects, nematodes,
fungi, and the like. Viruses include any plant virus, for example,
tobacco or cucumber mosaic virus, ringspot virus, necrosis virus,
maize dwarf mosaic virus, etc. Fungal pathogens, include but are
not limited to, Colletotrichum graminocola, 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,
Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus,
Tobacco Streak virus, Phakopsora pachyrhizi, Pythium
aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted
wilt virus, Heterodera glycines 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 recondite 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,
Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat
Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak
Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia
tritici, Tilletia laevis, Ustilago tritici, Tilletia indica,
Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola,
Pythium aphanidermatum, High Plains Virus, European wheat striate
virus; Sunflower: Plasmopora halstedii, Sclerotinia sclerotiorum,
Aster Yellows, 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
moniliforme 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, Maize Dwarf Mosaic Virus A & B, Wheat
Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,
Pseudonomas 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, Maize
Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus,
Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus,
Maize Rough Dwarf Virus; 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 circinate, Fusarium
moniliforme, 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, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A
& B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
Sclerophthona macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium
graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium
graminicola, etc.
[0146] Nematodes include, but are not limited to, parasitic
nematodes such as root-knot, cyst, and lesion nematodes, including
Heterodera and Globodera spp.; particularly Globodera rostochiensis
and Globodera pailida (potato cyst nematodes); Heterodera glycines
(soybean cyst nematode); Heterodera schachtii (beet cyst nematode);
and Heterodera avenae (cereal cyst nematode). Additional nematodes
include: Heterodera cajani; Heterodera trifolii; Heterodera oryzae;
Globodera tabacum; Meloidogyne incognita; Meloidogyne javonica;
Meloidogyne hapla; Meloidogyne arenaria; Meloidogyne naasi;
Meloidogyne exigua; Xiphinema index; Xiphinema italiae; Xiphinema
americanum; Xiphinema diversicaudatum; Pratylenchus penetrans;
Pratylenchus brachyurus; Pratylenchus zeae; Pratylenchus coffeae;
Pratylenchus thornei; Pratylenchus scribneri; Pratylenchus vulnus;
Pratylenchus curvitatus; Radopholus similis; Radopholus
citrophilus; Ditylenchus dipsaci; Helicotylenchus multicintus;
Rotylenchulus reniformis; Belonolaimus spp.; Paratrichodorus
anemones; Trichodorus spp.; Primitivus spp.; Anguina tritici; Bider
avenae; Subanguina radicicola; Tylenchorhynchus spp.; Hoplolaimus
seinhorsti; Tylenchulus semipenetrans; Hemicycliophora arenaria;
Belonolaimus langicaudatus; Paratrichodorus xiphinema;
Paratrichodorus christiei; Rhadinaphelenchus cocophilus;
Paratrichodorus minor; Hoplolaimus galeatus; Hoplolaimus columbus;
Criconemella spp.; Paratylenchus spp.; Nacoabbus aberrans;
Aphelenchoides besseyi; Ditylenchus angustus; Hirchmaniella spp.;
Scutellonema spp.; Hemicriconemoides kanayaensis; Tylenchorynchus
claytoni; and Cacopaurus pestis.
[0147] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, "a polypeptide" is
understood to represent one or more polypeptides. As such, the
terms "a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0148] Throughout this specification and the claims, the words
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires
otherwise.
[0149] As used herein, the term "about," when referring to a value
is meant to encompass variations of, in some embodiments .+-.50%,
in some embodiments .+-.20%, in some embodiments .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, in some embodiments
.+-.0.5%, and in some embodiments .+-.0.1% from the specified
amount, as such variations are appropriate to perform the disclosed
methods or employ the disclosed compositions.
[0150] Further, when an amount, concentration, or other value or
parameter is given as either a range, preferred range, or a list of
upper preferable values and lower preferable values, this is to be
understood as specifically disclosing all ranges formed from any
pair of any upper range limit or preferred value and any lower
range limit or preferred value, regardless of whether ranges are
separately disclosed. Where a range of numerical values is recited
herein, unless otherwise stated, the range is intended to include
the endpoints thereof, and all integers and fractions within the
range. It is not intended that the scope of the presently disclosed
subject matter be limited to the specific values recited when
defining a range.
[0151] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which the invention pertains. Although any
methods and materials similar herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are described herein.
[0152] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Antifungal Plate Assay
[0153] LBNL variants were generated through gene shuffling of the
coding sequences for LB-5220 or LB-9827-1 mature peptides (set
forth in SEQ ID NO: 1 and 9, respectively) in which limited
diversity was introduced into the parent sequences through the
spiking of synthetic oligonucleotides into the shuffling
reactions.
[0154] SEQ ID NO: 25 represents the consensus amino acid sequence
of SEQ ID NOs: 2, 4, 6, and 8. SEQ ID NO: 26 represents the
consensus amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18,
20, and 22.
[0155] The antifungal activity of the LBNL variants against the
fungal pathogens Fusarium graminearum (FGR; isolate 73B ISU),
Colletotrichum graminicola (CGR; isolate Carroll-IA-99), Fusarium
verticillioides (FVE; isolate MO33), and Diplodia maydis (DMA;
isolate Warren-IN-96) was assessed using a standard plate
assay.
Preparation of Cultures for Spore Production
[0156] Cultures of FVE were prepared using V8 agar plates. FGR,
CGR, and DMA cultures were prepared using 1/2.times. oatmeal agar.
Media recipes are provided below.
[0157] Specifically, tubes containing silica-gel fungal stocks
stored at -20.degree. C. were briefly flamed, and approximately 5
crystals were sprinkled onto the agar surface. 2-3 plates of each
fungal isolate were prepared. The newly plated cultures were stored
in a plastic box to prevent the cultures from drying out. FVE
cultures were grown in the dark at room temperature. CGR cultures
were grown in ambient light at room temperature. FGR and DMA
cultures were grown in an illuminated growth chamber at 27.degree.
C. New cultures were prepared every other week to maintain a
consistent supply of spores.
Spore Preparation
[0158] Spores were prepared from 2-4 week old cultures of FVE, FGR,
CGR, and DMA. For FGR, FVE, and DMA, a portion of the culture plate
was rinsed with a small amount of assay medium. The rinse solution
was permitted to remain on the DMA plates for a time sufficient to
allow the pycnidia rupture. The assay medium was then transferred
to a sterile tube. Samples were vortexed, and spores were
quantified using a hemacytometer.
[0159] For CGR, a sterile loop was gently dragged across orange
areas of the culture plate. The loop was then inserted into a small
volume of assay media, and the media was mixed with the loop to
suspend the spores. Samples were vortexed, and spores were
quantified using a hemacytometer.
[0160] Spores were diluted to the desired concentration with assay
medium (4,000 spores per mL for FGR, FVE, and CGR, and 6,000 spores
per mL for DMA) and kept on ice prior to beginning the antifungal
activity assay.
Assay Plate Preparation Details
[0161] Standard non-tissue culture treated 96 well flat bottom
plates or 1/2 area non-treated plates (Costar) were used in the
antifungal plate assays. Assay medium was potato dextrose broth for
FVE, FGR and DMA, and Czapec-Dox V8 was used for CGR. For low salt
conditions, 1/8.times. of liquid medium (potato dextrose broth for
Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides; Czapek-Dox V8 broth for Colletotrichum
graminocola) plus about 0.25 mM calcium chloride, and about 12.5 mM
potassium chloride was used as the assay medium. For high salt
conditions, 1/2.times. liquid medium (potato dextrose broth for
Diplodia maydis, Fusarium graminearum, and Fusarium
verticillioides; Czapek-Dox V8 broth for Colletotrichum
graminocola) plus about 1 mM calcium chloride, and about 50 mM
potassium chloride was used as the assay medium.
[0162] Antifungal polypeptides at various concentrations were added
to the plates at 50 .mu.L/well for a standard assay plate or 25
.mu.L/well for a half area plate. An equal volume of media with
fungal spores at 2 times the above concentrations was then added to
start the assay. The plates were sealed with a gas permeable
membrane (Breathe-Easy.RTM., Cat. No. BEM-1, Diversified Biotech,
Boston, Mass.), and the assay was allowed to develop in the dark at
28.degree. C. for 24 to 48 hours.
[0163] After the incubation period, the plates were placed on an
inverted microscope, and each well was examined and scored to
determine the 1050 of the antifungal polypeptide.
Results
[0164] Tables 1 and 2 provide the results of antifungal activity
assays with the LBNL variants.
TABLE-US-00001 TABLE 1 In vitro antifungal activity (IC50 in ppm)
of LBNL variants against F. graminearum (FGR), C. graminicola
(CGR), F. verticillioides (FVE), and D. maydis (DMA) under low salt
conditions. Fungus LBNL protein CGR FGR FVE DMA LB-5220 0.4-0.2
>10 0.7 <0.1 LB-5220 (ps 1B4) 0.4-0.2 >10 0.4 0.1 LB-5220
(r2 5G7) 0.4-0.2 >10 0.4 0.1 LB-5220 (r2 6F2) 0.4 >10 1.5 0.1
LB-9827-1 1.5 <0.1 0.7 1.5 LB-9827-1 (2F3) 0.7 <0.1 0.4 0.7
LB-9827-3A-11E4 N/D N/D N/D N/D LB-9827-3B-21E10 N/D N/D N/D N/D
LB-9827-3B-25G5 N/D N/D N/D N/D LB-9827-3B-25F2 N/D N/D N/D N/D
LB-9827-3B-26G11 N/D N/D N/D N/D N/D: Not Determined
TABLE-US-00002 TABLE 2 In vitro antifungal activity (IC50 in ppm)
of LBNL variants against F. graminearum (FGR), C. graminicola
(CGR), F. verticillioides (FVE), and D. maydis (DMA) under high
salt conditions. Fungus LBNL protein CGR FGR FVE DMA LB-5220 50-25
>100 >100 50 LB-5220 (ps 1B4) 12-6 >100 50-25 12 LB-5220
(r2 5G7) 6 >100 25-12 12 LB-5220 (r2 6F2) 12 >100 25 6
LB-9827-1 3 1.5 6 12 LB-9827-1 (2F3) 3 50 12-6 12 LB-9827-3A-11E4
1.25 0.63 15 0.94 LB-9827-3B-21E10 1.25 1.25 30 30 LB-9827-3B-25G5
1.25 2.5 3.8 0.47 LB-9827-3B-25F2 1.25 2.5 15 7.5 LB-9827-3B-26G11
0.63 N/D N/D N/D N/D: Not determined
Media Recipes
1.times. Czapek-Dox V8 Broth:
[0165] For each liter, suspend 35 grams Difco Czapek-Dox Broth
(#233810)* in dH.sub.2O and add 180 milliliters V8.RTM. juice that
has been clarified by centrifugation (3,000.times.g is plenty).
Raise final volume to 1 liter and autoclave at 121.degree. C. for
20 minutes. The media is filter sterilized to remove any remaining
debris.
*The approximate formula of Difco Czapek-Dox Broth (#233810) per
liter is 30.0 g saccharose, 3.0 g sodium nitrate, 1.0 g dipotassium
phosphate, 0.5 g magnesium sulfate, 0.5 g potassium chloride, and
0.01 g ferrous sulfate.
1.times. Potato Dextrose Broth:
[0166] For each liter, suspend 24 grams Difco Potato Dextrose Broth
(#0549-17-9)** in dH.sub.2O and raise final volume to 1 liter and
autoclave at 121.degree. C. for 20 minutes. The media is filter
sterilized to remove any remaining debris.
**The approximate formula of Difco Potato Dextrose Broth
(#0549-17-9) per liter is 4.0 g potato starch and 20.0 g
dextrose.
V8.RTM. Agar:
[0167] For each liter, dissolve 180 mL V8.RTM. juice and 3 grams
calcium carbonate in 820 mL deionized water and then add 17 grams
Bacto-agar in dH.sub.2O in a 4 liter vessel. 10 drops of 5%
antifoam A may be optionally added per liter prepared. Cover and
autoclave at 121.degree. C. for 20 minutes. Pour plates in sterile
hood.
Oatmeal Agar:
[0168] For each liter, suspend 36.24 grams of Difco Oatmeal Agar
(#0552-17-3) and 4.25 grams agar in dH.sub.2O in a 4 liter vessel,
cover and autoclave at 121.degree. C. for 20 minutes. Pour plates
in sterile hood.
TABLE-US-00003 FVE FGR CGR DMA Isolate name MO33 73B ISU
Carroll-IA-99 Warren-IN-96 Medium for V8 Agar 1/2X Oatmeal 1/2X
Oatmeal 1/2X Oatmeal sporulation Agar Agar Agar Agar culture age
2-4 weeks 2-4 weeks 2-4 weeks 2-4 weeks range for in old old old
old vitro assay Suggested Every other Every other Every other Every
other schedule for week week week week starting agar cultures
Liquid medium Potato Potato Czapec-Dox Potato for in vitro assay
dextrose broth dextrose broth V8 broth dextrose broth Spore Density
4,000 4,000 4,000 6,000 for in vitro assay (spores/mL)
Example 2
Transformation and Regeneration of Transgenic Maize Plants
[0169] 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: 4, 6, 8, 12,
14, 16, 18, 20, 22, or 24 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.RTM.).
Alternatively, the selectable marker gene is provided on a separate
plasmid.
[0170] Transformation is Performed as Follows. Media Recipes Follow
Below.
Preparation of Target Tissue
[0171] The ears are husked and surface sterilized in 30%
Clorox.RTM. 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
[0172] A plasmid vector comprising a nucleotide sequence encoding
the antipathogenic polypeptide set forth in SEQ ID NO: 4, 6, 8, 12,
14, 16, 18, 20, 22, or 24 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: [0173] 100 .mu.L prepared
tungsten particles in water [0174] 10 .mu.L (1 .mu.g) DNA in Tris
EDTA buffer (1 .mu.g total DNA) [0175] 100 .mu.L 2.5 M CaCl.sub.2
[0176] 10 .mu.L 0.1 M spermidine
[0177] 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
[0178] The sample plates are bombarded at level #4 in a particle
gun. 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
[0179] 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
[0180] Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts
(SIGMA C-1416), 1.0 mL/LEriksson'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-I H.sub.2O
following adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite.RTM.
(added after bringing to volume with D-I H2O); 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.RTM. (added after bringing to
volume with D-I H2O); and 0.85 mg/L silver nitrate and 3.0 mg/L
bialaphos (both added after sterilizing the medium and cooling to
room temperature).
[0181] 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 H2O after
adjusting to pH 5.6); 3.0 g/L Gelrite.RTM. (added after bringing to
volume with D-I H2O); and 1.0 mg/L indoleacetic acid and 3.0 mg/L
Bialaphos.RTM. (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 3
Agrobacterium-Mediated Transformation of Maize and Regeneration of
Transgenic Plants
[0182] For Agrobacterium-mediated transformation of maize with a
nucleotide sequence encoding the polypeptide of SEQ ID NO: 4, 6, 8,
12, 14, 16, 18, 20, 22, or 24, 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 4
Transformation of Soybean Embryos
Culture Conditions
[0183] Soybean embryogenic suspension cultures (cv. Jack) are
maintained in 35 ml liquid medium SB196 (see recipes below) on
rotary shaker, 150 rpm, 26.degree. C. with cool white fluorescent
lights on 16:8 hr day/night photoperiod at light intensity of 60-85
.mu.E/m2/s. Cultures are subcultured every 7 days to two weeks by
inoculating approximately 35 mg of tissue into 35 ml of fresh
liquid SB196 (the preferred subculture interval is every 7
days).
[0184] Soybean embryogenic suspension cultures are transformed with
the plasmids and DNA fragments described in the following examples
by the method of particle gun bombardment (Klein et al. (1987)
Nature, 327:70).
Soybean Embryogenic Suspension Culture Initiation
[0185] Soybean cultures are initiated twice each month with 5-7
days between each initiation.
[0186] Pods with immature seeds from available soybean plants 45-55
days after planting are picked, removed from their shells and
placed into a sterilized magenta box. The soybean seeds are
sterilized by shaking them for 15 minutes in a 5% Clorox.RTM.
solution with 1 drop of ivory soap (95 ml of autoclaved distilled
water plus 5 ml Clorox.RTM. and 1 drop of soap). Mix well. Seeds
are rinsed using 2 1-liter bottles of sterile distilled water and
those less than 4 mm are placed on individual microscope slides.
The small end of the seed is cut and the cotyledons pressed out of
the seed coat. Cotyledons are transferred to plates containing SB1
medium (25-30 cotyledons per plate). Plates are wrapped with fiber
tape and stored for 8 weeks. After this time secondary embryos are
cut and placed into SB 196 liquid media for 7 days.
Preparation of DNA for Bombardment
[0187] Either an intact plasmid or a DNA plasmid fragment
containing the genes of interest and the selectable marker gene are
used for bombardment. Plasmid DNA for bombardment are routinely
prepared and purified using the method described in the Promega.TM.
Protocols and Applications Guide, Second Edition (page 106).
Fragments of the plasmids carrying the antifungal protein coding
sequence are obtained by gel isolation of double digested plasmids.
In each case, 100 ug of plasmid DNA is digested in 0.5 ml of the
specific enzyme mix that is appropriate for the plasmid of
interest. The resulting DNA fragments are separated by gel
electrophoresis on 1% SeaPlaque.RTM. GTG.RTM. agarose (BioWhitaker
Molecular Applications) and the DNA fragments containing the
antifungal protein coding sequence are cut from the agarose gel.
DNA is purified from the agarose using the GELase digesting enzyme
following the manufacturer's protocol.
[0188] A 50 .mu.l aliquot of sterile distilled water containing 3
mg of gold particles (3 mg gold) is added to 5 .mu.l of a 1
.mu.g/.mu.l DNA solution (either intact plasmid or DNA fragment
prepared as described above), 50 .mu.l 2.5M CaCl.sub.2 and 20 .mu.l
of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a
vortex shaker and spun for 10 sec in a bench microfuge. After a
wash with 400 .mu.l 100% ethanol the pellet is suspended by
sonication in 40 .mu.l of 100% ethanol. Five .mu.l of DNA
suspension is dispensed to each flying disk of the Biolistic
PDS1000/HE instrument disk. Each 5 .mu.l aliquot contains
approximately 0.375 mg gold per bombardment (i.e. per disk).
Tissue Preparation and Bombardment with DNA
[0189] Approximately 150-200 mg of 7 day old embryonic suspension
cultures are placed in an empty, sterile 60.times.15 mm petri dish
and the dish covered with plastic mesh. Tissue is bombarded 1 or 2
shots per plate with membrane rupture pressure set at 1100 PSI and
the chamber evacuated to a vacuum of 27-28 inches of mercury.
Tissue is placed approximately 3.5 inches from the
retaining/stopping screen.
Selection of Transformed Embryos
[0190] Transformed embryos were selected either using hygromycin
(when the hygromycin phosphotransferase, HPT, gene was used as the
selectable marker) or chlorsulfuron (when the acetolactate
synthase, ALS, gene was used as the selectable marker).
Hygromycin (HPT) Selection
[0191] Following bombardment, the tissue is placed into fresh SB196
media and cultured as described above. Six days post-bombardment,
the SB196 is exchanged with fresh SB196 containing a selection
agent of 30 mg/L hygromycin. The selection media is refreshed
weekly. Four to six weeks post selection, green, transformed tissue
may be observed growing from untransformed, necrotic embryogenic
clusters. Isolated, green tissue is removed and inoculated into
multiwell plates to generate new, clonally propagated, transformed
embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection
[0192] Following bombardment, the tissue is divided between 2
flasks with fresh SB196 media and cultured as described above. Six
to seven days post-bombardment, the SB196 is exchanged with fresh
SB196 containing selection agent of 100 ng/ml Chlorsulfuron. The
selection media is refreshed weekly. Four to six weeks post
selection, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated, green
tissue is removed and inoculated into multiwell plates containing
SB196 to generate new, clonally propagated, transformed embryogenic
suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants
[0193] In order to obtain whole plants from embryogenic suspension
cultures, the tissue must be regenerated.
Embryo Maturation
[0194] Embryos are cultured for 4-6 weeks at 26.degree. C. in SB196
under cool white fluorescent (Phillips cool white Econowatt
F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a
16:8 hr photoperiod with light intensity of 90-120 uE/m2 s. After
this time embryo clusters are removed to a solid agar media, SB
166, for 1-2 weeks. Clusters are then subcultured to medium SB103
for 3 weeks. During this period, individual embryos can be removed
from the clusters and screened for fungal resistance.
Embryo Desiccation and Germination
[0195] Matured individual embryos are desiccated by placing them
into an empty, small petri dish (35.times.10 mm) for approximately
4-7 days. The plates are sealed with fiber tape (creating a small
humidity chamber). Desiccated embryos are planted into SB71-4
medium where they were left to germinate under the same culture
conditions described above. Germinated plantlets are removed from
germination medium and rinsed thoroughly with water and then
planted in Redi-Earth in 24-cell pack tray, covered with clear
plastic dome. After 2 weeks the dome is removed and plants hardened
off for a further week. If plantlets looked hardy they are
transplanted to 10'' pot of Redi-Earth with up to 3 plantlets per
pot. After 10 to 16 weeks, mature seeds are harvested, chipped and
analyzed for proteins.
Media Recipes
[0196] SB 196--FN Lite liquid proliferation medium (per
liter)--
TABLE-US-00004 MS FeEDTA - 100x Stock 1 10 ml MS Sulfate - 100x
Stock 2 10 ml FN Lite Halides - 100x Stock 3 10 ml FN Lite P, B, Mo
- 100x Stock 4 10 ml B5 vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L
final concentration) 1.0 ml KNO3 2.83 gm (NH4)2 SO 4 0.463 gm
Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00005 [0197] Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x
Stock Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g
1.392 g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g
MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g
0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides
100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x
Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve in
dark bottle while stirring
[0198] SB1 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 31.5
g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8
g TC agar.
[0199] SB 166 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60 g
maltose; 750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH 5.7;
and, 2 g gelrite.
[0200] SB 103 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60 g
maltose; 750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.
[0201] SB 71-4 solid medium (per liter) comprises: 1 bottle
Gamborg's B5 salts w/ sucrose (Gibco/BRL-Cat#21153-036); pH 5.7;
and, 5 g TC agar.
[0202] 2,4-D stock is obtained premade from Phytotech cat# D
295--concentration is 1 mg/ml.
[0203] B5 Vitamins Stock (per 100 ml) which is stored in aliquots
at -20 C comprises: 10 g myo-inositol; 100 mg nicotinic acid; 100
mg pyridoxine HCl; and, 1 g thiamine. If the solution does not
dissolve quickly enough, apply a low level of heat via the hot stir
plate. Chlorsulfuron Stock comprises 1 mg/ml in 0.01 N Ammonium
Hydroxide
[0204] 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.
[0205] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the foregoing list of embodiments and appended
claims. Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation.
Sequence CWU 1
1
321168DNAPenicillium simplicissimumCDS(1)...(168) 1ctc aag tac acc
ggc acc tgc acc cgc gcc aac aac cag tgc aag tat 48Leu Lys Tyr Thr
Gly Thr Cys Thr Arg Ala Asn Asn Gln Cys Lys Tyr1 5 10 15aag ggc cag
aac gat cgc gac aca ttc gtc aaa tgc ccg act ttt gcg 96Lys Gly Gln
Asn Asp Arg Asp Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30aac aag
aag tgt aca agg gat ggc gct cct tgc tcc ttc gac agc tac 144Asn Lys
Lys Cys Thr Arg Asp Gly Ala Pro Cys Ser Phe Asp Ser Tyr 35 40 45tct
aga gca gtg act tgc gat tag 168Ser Arg Ala Val Thr Cys Asp 50
55255PRTPenicillium simplicissimum 2Leu Lys Tyr Thr Gly Thr Cys Thr
Arg Ala Asn Asn Gln Cys Lys Tyr1 5 10 15 Lys Gly Gln Asn Asp Arg
Asp Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30 Asn Lys Lys Cys
Thr Arg Asp Gly Ala Pro Cys Ser Phe Asp Ser Tyr 35 40 45 Ser Arg
Ala Val Thr Cys Asp 50 55 3168DNAArtificial SequenceSynthetic
oligonucleotide 3ctg aag tac acc ggt act tgt acc cgc aaa aac aac
cag tgt aaa tat 48Leu Lys Tyr Thr Gly Thr Cys Thr Arg Lys Asn Asn
Gln Cys Lys Tyr1 5 10 15cgt ggc cag aac aat cgc gat act ttt gtg aag
tgc ccg acc ttc gcc 96Arg Gly Gln Asn Asn Arg Asp Thr Phe Val Lys
Cys Pro Thr Phe Ala 20 25 30aac aaa cgt tgc acc cgt gac ggc gct cca
tgc tcc ttc gac tct tac 144Asn Lys Arg Cys Thr Arg Asp Gly Ala Pro
Cys Ser Phe Asp Ser Tyr 35 40 45tct cgt gcg gtt act tgc gat tag
168Ser Arg Ala Val Thr Cys Asp 50 55455PRTArtificial
SequenceSynthetic peptide LB-5220 (ps-1B4) 4Leu Lys Tyr Thr Gly Thr
Cys Thr Arg Lys Asn Asn Gln Cys Lys Tyr1 5 10 15 Arg Gly Gln Asn
Asn Arg Asp Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30 Asn Lys
Arg Cys Thr Arg Asp Gly Ala Pro Cys Ser Phe Asp Ser Tyr 35 40 45
Ser Arg Ala Val Thr Cys Asp 50 55 5168DNAArtificial
SequenceSynthetic oligonucleotide 5ctg aag tac acc ggt acc tgt cgc
cgc gcc aac aac cag tgt aaa tat 48Leu Lys Tyr Thr Gly Thr Cys Arg
Arg Ala Asn Asn Gln Cys Lys Tyr1 5 10 15aaa ggc cag aac aat cgc gat
acc ttt gtg aag tgc ccg acc ttc gcc 96Lys Gly Gln Asn Asn Arg Asp
Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30aac aaa aaa tgc acc cgt
gac ggc gcg aag tgc tcc ttc gac tct tac 144Asn Lys Lys Cys Thr Arg
Asp Gly Ala Lys Cys Ser Phe Asp Ser Tyr 35 40 45tct cgt gcg gtt act
tgc gat tag 168Ser Arg Ala Val Thr Cys Asp 50 55655PRTArtificial
SequenceSynthetic peptide LB-5220 (r2-5G7) 6Leu Lys Tyr Thr Gly Thr
Cys Arg Arg Ala Asn Asn Gln Cys Lys Tyr1 5 10 15 Lys Gly Gln Asn
Asn Arg Asp Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30 Asn Lys
Lys Cys Thr Arg Asp Gly Ala Lys Cys Ser Phe Asp Ser Tyr 35 40 45
Ser Arg Ala Val Thr Cys Asp 50 55 7168DNAArtificial
SequenceSynthetic oligonucleotide 7ctg aag tac acc ggt acc tgt cgc
cgc gcc aac aac cag tgt aaa tat 48Leu Lys Tyr Thr Gly Thr Cys Arg
Arg Ala Asn Asn Gln Cys Lys Tyr1 5 10 15aaa ggc cag aac aat cgc gat
acc ttt gtg aag tgc ccg acc ttc gcc 96Lys Gly Gln Asn Asn Arg Asp
Thr Phe Val Lys Cys Pro Thr Phe Ala 20 25 30aac aaa aaa tgc acc cgt
gac ggc gag aag tgc tcc ttc gac tct tac 144Asn Lys Lys Cys Thr Arg
Asp Gly Glu Lys Cys Ser Phe Asp Ser Tyr 35 40 45tct cgt gcg gtt act
tgc gat tag 168Ser Arg Ala Val Thr Cys Asp 50 55855PRTArtificial
SequenceLB-5220 (r2-6F2) 8Leu Lys Tyr Thr Gly Thr Cys Arg Arg Ala
Asn Asn Gln Cys Lys Tyr1 5 10 15 Lys Gly Gln Asn Asn Arg Asp Thr
Phe Val Lys Cys Pro Thr Phe Ala 20 25 30 Asn Lys Lys Cys Thr Arg
Asp Gly Glu Lys Cys Ser Phe Asp Ser Tyr 35 40 45 Ser Arg Ala Val
Thr Cys Asp 50 55 9177DNAMonascus ruberCDS(1)...(177) 9ctc agc aag
ttc ggc ggc gag tgc agc ctc aag cac aac acc tgc acc 48Leu Ser Lys
Phe Gly Gly Glu Cys Ser Leu Lys His Asn Thr Cys Thr1 5 10 15tac ctc
aag ggc ggc aag aat cat gtg gtc aac tgc gga tct gcc gct 96Tyr Leu
Lys Gly Gly Lys Asn His Val Val Asn Cys Gly Ser Ala Ala 20 25 30aac
aag aag tgt aag tca gac agg cat cat tgt gaa tac gat gaa cac 144Asn
Lys Lys Cys Lys Ser Asp Arg His His Cys Glu Tyr Asp Glu His 35 40
45cac aag cga gtt gac tgc cag acc cca gtc tag 177His Lys Arg Val
Asp Cys Gln Thr Pro Val 50 551058PRTMonascus ruber 10Leu Ser Lys
Phe Gly Gly Glu Cys Ser Leu Lys His Asn Thr Cys Thr1 5 10 15 Tyr
Leu Lys Gly Gly Lys Asn His Val Val Asn Cys Gly Ser Ala Ala 20 25
30 Asn Lys Lys Cys Lys Ser Asp Arg His His Cys Glu Tyr Asp Glu His
35 40 45 His Lys Arg Val Asp Cys Gln Thr Pro Val 50 55
11174DNAArtificial SequenceSynthetic oligonucleotide 11ctg tcc aaa
tat ggc ggc gaa tgc tct ctg aaa cat aat act tgc acg 48Leu Ser Lys
Tyr Gly Gly Glu Cys Ser Leu Lys His Asn Thr Cys Thr1 5 10 15tac cgg
aag ggt ggc aag aac caa gtt gta aat tgc ggg acg gcc gcg 96Tyr Arg
Lys Gly Gly Lys Asn Gln Val Val Asn Cys Gly Thr Ala Ala 20 25 30aat
aaa aaa tgc aag acg gat cgt cac cac tgt gaa tac gat gaa tat 144Asn
Lys Lys Cys Lys Thr Asp Arg His His Cys Glu Tyr Asp Glu Tyr 35 40
45cac aaa aga gtt gat tgt cag acc ccg gtg 174His Lys Arg Val Asp
Cys Gln Thr Pro Val 50 551258PRTArtificial SequenceSynthetic
peptide LB-9827-1-2F3 12Leu Ser Lys Tyr Gly Gly Glu Cys Ser Leu Lys
His Asn Thr Cys Thr1 5 10 15 Tyr Arg Lys Gly Gly Lys Asn Gln Val
Val Asn Cys Gly Thr Ala Ala 20 25 30 Asn Lys Lys Cys Lys Thr Asp
Arg His His Cys Glu Tyr Asp Glu Tyr 35 40 45 His Lys Arg Val Asp
Cys Gln Thr Pro Val 50 55 13174DNAArtificial SequenceSynthetic
oligonucleotide 13ctg tcc aaa tat ggc ggc gaa tgc tct cgc aaa cat
aat acc tgc acg 48Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg Lys His
Asn Thr Cys Thr1 5 10 15tac aaa aag ggt ggc aag aat caa atc gta aat
tgc cct acg gcc gcg 96Tyr Lys Lys Gly Gly Lys Asn Gln Ile Val Asn
Cys Pro Thr Ala Ala 20 25 30aat aaa cgc tgc aag acg gac cgt cac cac
tgt gaa tac gat gaa tat 144Asn Lys Arg Cys Lys Thr Asp Arg His His
Cys Glu Tyr Asp Glu Tyr 35 40 45cac cgt cgt gtt gat tgt caa acc ccg
gtg 174His Arg Arg Val Asp Cys Gln Thr Pro Val 50
551458PRTArtificial SequenceLB-9827-3A-11E4 14Leu Ser Lys Tyr Gly
Gly Glu Cys Ser Arg Lys His Asn Thr Cys Thr1 5 10 15 Tyr Lys Lys
Gly Gly Lys Asn Gln Ile Val Asn Cys Pro Thr Ala Ala 20 25 30 Asn
Lys Arg Cys Lys Thr Asp Arg His His Cys Glu Tyr Asp Glu Tyr 35 40
45 His Arg Arg Val Asp Cys Gln Thr Pro Val 50 55 15174DNAArtificial
SequenceSynthetic oligonucleotide 15ctg tcc aaa tat ggc ggc gaa tgc
tct cgc gca cat aat act tgc acg 48Leu Ser Lys Tyr Gly Gly Glu Cys
Ser Arg Ala His Asn Thr Cys Thr1 5 10 15tac cgt aag ggt ggc aag aac
caa gtt gta aac tgc cca agc gcc gcg 96Tyr Arg Lys Gly Gly Lys Asn
Gln Val Val Asn Cys Pro Ser Ala Ala 20 25 30aat aaa aaa tgc aag agc
gat cgt cac cac tgt gaa tac gat gaa tat 144Asn Lys Lys Cys Lys Ser
Asp Arg His His Cys Glu Tyr Asp Glu Tyr 35 40 45cac aaa cgc gtt gat
tgt caa acc ccg gtg 174His Lys Arg Val Asp Cys Gln Thr Pro Val 50
551658PRTArtificial SequenceSynthetic peptide LB-9827-3B-21E10
16Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg Ala His Asn Thr Cys Thr1
5 10 15 Tyr Arg Lys Gly Gly Lys Asn Gln Val Val Asn Cys Pro Ser Ala
Ala 20 25 30 Asn Lys Lys Cys Lys Ser Asp Arg His His Cys Glu Tyr
Asp Glu Tyr 35 40 45 His Lys Arg Val Asp Cys Gln Thr Pro Val 50 55
17174DNAArtificial SequenceSynthetic oligonucleotide 17ctg tcc aaa
tat ggc ggc gaa tgc tct cgc gaa cat aat act tgc acg 48Leu Ser Lys
Tyr Gly Gly Glu Cys Ser Arg Glu His Asn Thr Cys Thr1 5 10 15tac aag
aag ggt ggc aag aac caa gtt gta gcc tgc ggt aag gcc gcg 96Tyr Lys
Lys Gly Gly Lys Asn Gln Val Val Ala Cys Gly Lys Ala Ala 20 25 30aat
aaa aaa tgc aag acg gat cgt cac cac tgt gaa tac gat agc tat 144Asn
Lys Lys Cys Lys Thr Asp Arg His His Cys Glu Tyr Asp Ser Tyr 35 40
45cac aaa aaa gtt gat tgt caa acc ccg gtg 174His Lys Lys Val Asp
Cys Gln Thr Pro Val 50 551858PRTArtificial SequenceSynthetic
peptide LB-9827-3B-21F9 18Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg
Glu His Asn Thr Cys Thr1 5 10 15 Tyr Lys Lys Gly Gly Lys Asn Gln
Val Val Ala Cys Gly Lys Ala Ala 20 25 30 Asn Lys Lys Cys Lys Thr
Asp Arg His His Cys Glu Tyr Asp Ser Tyr 35 40 45 His Lys Lys Val
Asp Cys Gln Thr Pro Val 50 55 19174DNAArtificial SequenceSynthetic
oligonucleotide 19ctg tcc aaa tat ggc ggc gaa tgc tct cgc gaa cat
aat act tgc acg 48Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg Glu His
Asn Thr Cys Thr1 5 10 15tac ctg aag ggt ggc aag aac caa gtt gta gcc
tgc ggt agc gcc gcg 96Tyr Leu Lys Gly Gly Lys Asn Gln Val Val Ala
Cys Gly Ser Ala Ala 20 25 30aat aaa aaa tgc aag cgc gat cgt cac cac
tgt gaa tac gat gac tat 144Asn Lys Lys Cys Lys Arg Asp Arg His His
Cys Glu Tyr Asp Asp Tyr 35 40 45cac aaa acc gtt gat tgt caa acc ccg
gtg 174His Lys Thr Val Asp Cys Gln Thr Pro Val 50
552058PRTArtificial SequenceSynthetic peptide LB-9827-3B-25F2 20Leu
Ser Lys Tyr Gly Gly Glu Cys Ser Arg Glu His Asn Thr Cys Thr1 5 10
15 Tyr Leu Lys Gly Gly Lys Asn Gln Val Val Ala Cys Gly Ser Ala Ala
20 25 30 Asn Lys Lys Cys Lys Arg Asp Arg His His Cys Glu Tyr Asp
Asp Tyr 35 40 45 His Lys Thr Val Asp Cys Gln Thr Pro Val 50 55
21174DNAArtificial SequenceSynthetic oligonucleotide 21ctg tcc aaa
tat ggc ggc gaa tgc tct cgc gca cat aat act tgc acg 48Leu Ser Lys
Tyr Gly Gly Glu Cys Ser Arg Ala His Asn Thr Cys Thr1 5 10 15tac cgt
aag ggt ggc aag aac caa gtt gta aag tgc ggt acc gcc gcg 96Tyr Arg
Lys Gly Gly Lys Asn Gln Val Val Lys Cys Gly Thr Ala Ala 20 25 30aat
aaa aaa tgc aag agc gat cgt cac cac tgt gaa tac gat gac tat 144Asn
Lys Lys Cys Lys Ser Asp Arg His His Cys Glu Tyr Asp Asp Tyr 35 40
45cac aaa cgc gtt gat tgt caa acc ccg gtg 174His Lys Arg Val Asp
Cys Gln Thr Pro Val 50 552258PRTArtificial SequenceSynthetic
peptide LB-9827-3B-25G5 22Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg
Ala His Asn Thr Cys Thr1 5 10 15 Tyr Arg Lys Gly Gly Lys Asn Gln
Val Val Lys Cys Gly Thr Ala Ala 20 25 30 Asn Lys Lys Cys Lys Ser
Asp Arg His His Cys Glu Tyr Asp Asp Tyr 35 40 45 His Lys Arg Val
Asp Cys Gln Thr Pro Val 50 55 23174DNAArtificial SequenceSynthetic
oligonucleotide 23ctg tcc aaa tat ggc ggc gaa tgc tct aag gaa cat
aat act tgc aag 48Leu Ser Lys Tyr Gly Gly Glu Cys Ser Lys Glu His
Asn Thr Cys Lys1 5 10 15tac ctg aag ggt ggc aag aac caa gtt gta gcc
tgc cca aag gcc gcg 96Tyr Leu Lys Gly Gly Lys Asn Gln Val Val Ala
Cys Pro Lys Ala Ala 20 25 30aat aaa aaa tgc aag acg gat cgt cac cac
tgt gaa tac gat gaa tat 144Asn Lys Lys Cys Lys Thr Asp Arg His His
Cys Glu Tyr Asp Glu Tyr 35 40 45cac aaa acc gtt gat tgt caa acc ccg
gtg 174His Lys Thr Val Asp Cys Gln Thr Pro Val 50
552458PRTArtificial SequenceSynthetic peptide LB-9827-3B-26G11
24Leu Ser Lys Tyr Gly Gly Glu Cys Ser Lys Glu His Asn Thr Cys Lys1
5 10 15 Tyr Leu Lys Gly Gly Lys Asn Gln Val Val Ala Cys Pro Lys Ala
Ala 20 25 30 Asn Lys Lys Cys Lys Thr Asp Arg His His Cys Glu Tyr
Asp Glu Tyr 35 40 45 His Lys Thr Val Asp Cys Gln Thr Pro Val 50 55
2555PRTArtificial SequenceSynthetic peptide; Consensus sequence
25Leu Lys Tyr Thr Gly Thr Cys Thr Arg Ala Asn Asn Gln Cys Lys Tyr1
5 10 15 Lys Gly Gln Asn Asn Arg Asp Thr Phe Val Lys Cys Pro Thr Phe
Ala 20 25 30 Asn Lys Lys Cys Thr Arg Asp Gly Ala Pro Cys Ser Phe
Asp Ser Tyr 35 40 45 Ser Arg Ala Val Thr Cys Asp 50 55
2658PRTArtificial SequenceSynthetic peptide; Consensus sequence
26Leu Ser Lys Tyr Gly Gly Glu Cys Ser Arg Xaa His Asn Thr Cys Thr1
5 10 15 Tyr Arg Lys Gly Gly Lys Asn Gln Val Val Asn Cys Gly Ser Ala
Ala 20 25 30 Asn Lys Lys Cys Lys Ser Asp Arg His His Cys Glu Tyr
Asp Glu Tyr 35 40 45 His Lys Arg Val Asp Cys Gln Thr Pro Val 50 55
2772DNAHordeum vulgareCDS(1)...(72) 27atg gcc aac aag cac ctg tcc
ctc tcc ctc ttc ctc gtg ctc ctc ggc 48Met Ala Asn Lys His Leu Ser
Leu Ser Leu Phe Leu Val Leu Leu Gly1 5 10 15ctc tcc gcc tcc ctc gcc
tcc gga 72Leu Ser Ala Ser Leu Ala Ser Gly 202824PRTHordeum vulgare
28Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe Leu Val Leu Leu Gly1
5 10 15 Leu Ser Ala Ser Leu Ala Ser Gly 20 294PRTArtificial
SequenceSynthetic peptide 29Lys Asp Glu Leu1 306PRTArtificial
SequenceSynthetic peptide 30Ser Glu Lys Asp Glu Leu1 5
314PRTArtificial SequenceSynthetic peptide 31His Asp Glu Leu1
324PRTArtificial SequenceSynthetic peptide 32His Asp Glu Phe1
* * * * *
References