U.S. patent application number 17/304156 was filed with the patent office on 2021-10-14 for antifungal plant proteins, peptides, and methods of use.
The applicant listed for this patent is Donald Danforth Plant Science Center. Invention is credited to Dilip Shah.
Application Number | 20210317470 17/304156 |
Document ID | / |
Family ID | 1000005678901 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317470 |
Kind Code |
A1 |
Shah; Dilip |
October 14, 2021 |
ANTIFUNGAL PLANT PROTEINS, PEPTIDES, AND METHODS OF USE
Abstract
Provided are transgenic plants expressing MtDef5 antifungal
proteins and peptides exhibiting high levels of resistance to
susceptible fungi. Such transgenic plants contain a recombinant DNA
construct comprising a natural or heterologous signal peptide
sequence operably linked to a nucleic acid sequence encoding these
molecules. Also provided are methods of producing such plants,
methods of protecting plants against susceptible fungal infection
and damage, as well as compositions that can be applied to the
locus of plants, comprising microorganisms expressing these
molecules, or these molecules themselves, as well as pharmaceutical
compositions containing these molecules. Human and veterinary
therapeutic use of MtDef5 antifungal proteins and peptides to treat
susceptible fungal infections are also encompassed by the
invention.
Inventors: |
Shah; Dilip; (St. Louis,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Donald Danforth Plant Science Center |
ST. LOUIS |
MO |
US |
|
|
Family ID: |
1000005678901 |
Appl. No.: |
17/304156 |
Filed: |
June 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16277157 |
Feb 15, 2019 |
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17304156 |
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14888011 |
Oct 29, 2015 |
10253328 |
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PCT/US2014/035786 |
Apr 29, 2014 |
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16277157 |
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61817415 |
Apr 30, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 65/20 20130101;
C07K 14/415 20130101; C12N 15/8282 20130101; A01H 5/00
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/00 20060101 A01H005/00; C07K 14/415 20060101
C07K014/415; A01N 65/20 20060101 A01N065/20 |
Claims
1.-20. (canceled)
21. A peptide comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO: 58,
wherein the peptide comprises a fragment of a mature MtDef5
protein, wherein one or more amino acids are deleted from the
N-terminal end, C-terminal end, the middle of the mature MtDef5
protein, or combinations thereof, and possesses antifungal
activity.
22. The peptide of claim 21, wherein the peptide comprises the
gamma-core motif set forth as SEQ ID NO:81.
23. The peptide of claim 21, wherein the peptide contains at least
one conservative amino acid substitutions wherein one or more amino
acids in the SEQ ID NO: 58 sequence are substituted with another
amino acid having a charge and polarity which is similar to that of
the amino acid of SEQ ID NO: 58.
24. The peptide of claim 23, wherein one or more basic amino acids
of SEQ ID NO: 58 are substituted with another basic amino acid.
25. The peptide of claim 23, wherein one or more neutral nonpolar
amino acids of SEQ ID NO: 58 are substituted with another neutral
nonpolar amino acid.
26. The peptide of claim 21, wherein the peptide comprises the
gamma-core motif set forth as SEQ ID NO:81 and wherein any amino
acid substitutions SEQ ID NO: 58 are conservative amino acid
substitutions wherein one or more amino acids in the SEQ ID NO: 58
sequence are substituted with another amino acid having a charge
and polarity which is similar to that of the native amino acid of
SEQ ID NO: 58 which is substituted.
27. The peptide of claim 21, wherein the fragment is a synthetic
mutant of the mature MtDef5 protein.
28. A composition comprising the peptide of claim 21 and an
agriculturally, pharmaceutically, or veterinarily acceptable
carrier, diluent, or excipient.
29. The composition of claim 28, wherein the composition further
comprises a surfactant and/or a fungicide, wherein the fungicide is
optionally a polyoxine, nikkomycine, carboxyamide, aromatic
carbohydrates, carboxine, morpholine, an inhibitors of sterol
biosynthesis, or an organophosphorus compound.
30. The composition of claim 27, wherein the composition further
comprises an antifungal compound, wherein the antifungal compound
is a polyene, imidazole, triazole, thiazole, allylamine, or
echinocandin.
31. A DNA construct encoding the peptide of claim 21, wherein the
nucleic acid is operably linked to a heterologous promoter.
32. A cell comprising the DNA construct of claim 31.
33. The cell of claim 32, wherein the cell is a plant, bacterial,
or yeast cell.
34. A method of treating a susceptible fungal infection in a
patient or other subject in need thereof, comprising administering
to said patient an antifungal effective amount of the peptide of
claim 21.
35. The method of claim 34, wherein said administering is performed
topically.
36. A method of preventing, treating, controlling, combating,
reducing, or inhibiting damage to a plant susceptible to damage
from a species of fungus selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumanomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp., comprising providing to the locus of said
susceptible plant an antifungal effective amount of the peptide of
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/277,157, filed Feb. 15, 2019, which is a
divisional of the U.S. patent application Ser. No. 14/888,011,
filed Oct. 29, 2015, which is the United States National Phase
entry of International Application No. PCT/US2014/035786, filed
Apr. 29, 2014, which claims the benefit of priority of U.S.
Provisional Application Ser. No. 61/817,415, filed Apr. 30, 2013,
the contents of which are herein incorporated by reference in their
entireties.
[0002] The material contained in the text file identified as
"47004-181214_ST25.txt" (created Feb. 15, 2019; 27114 bytes) is
hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to small antifungal proteins
and peptides derived from Medicago truncatula (Barrel Medic, Barrel
Medick, or Barrel Clover), i.e., MtDef5 Defensins, and methods for
controlling pathogenic fungi susceptible to the antifungal activity
of these molecules. The antifungal proteins and peptides can be
applied directly to a plant as a component of an antifungal
composition, applied to a plant in the form of microorganisms that
produce the proteins or peptides, or plants themselves can be
genetically modified to produce the proteins or peptides. The
present invention also relates to DNA constructs, microorganisms,
and plants transformed with the DNA constructs, and compositions
useful in controlling pathogenic plant and other fungi.
Description of Related Art
[0004] Protection of agriculturally important crops from pathogenic
fungi is crucial in improving crop yields. Fungal infections are a
particular problem in damp climates, and may become a major concern
during crop storage, where such infections can result in spoilage
and contamination of food or feed products with fungal toxins.
Unfortunately, modern growing methods and harvesting and storage
systems can promote plant pathogen infections.
[0005] Control of plant pathogens is further complicated by the
need to simultaneously control multiple fungi of distinct genera.
For example, fungi such as Alternaria; Aspergillus; Ascochyta;
Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Fusarium;
Gaeumannomyces; Helminthosporium; Macrophomina; Nectria;
Peronospora; Phakopsora; Phoma; Phymatotrichum; Phytophthora;
Plasmopara; Podosphaera; Puccinia; Pythium; Pyrenophora;
Pyricularia; Rhizoctonia; Scerotium; Sclerotinia; Septoria;
Thielaviopsis; Uncinula; Venturia; and Verticillium species are all
recognized plant pathogens. Consequently, resistant crop plant
varieties or fungicides that control only a limited subset of
fungal pathogens may fail to deliver adequate protection under
conditions where multiple pathogens are present. It is further
anticipated that plant pathogenic fungi may become resistant to
existing fungicides and transgenic and non-transgenic crop
varieties, necessitating the introduction of fungal control agents
with distinct modes of action to combat resistant fungi.
[0006] One approach to inhibiting plant pathogenic fungal activity
has been to identify and isolate peptides, polypeptides, and
proteins exhibiting antifungal activity against plant pathogenic
fungi (Bowles, 1990; Brears et al., 1994). The antifungal peptides,
polypeptides, and proteins that include chitinases, cysteine-rich
chitin-binding proteins, .beta.-1,3-glucanases, permatins
(including zeamatins), thionins, ribosome-inactivating proteins,
and non-specific lipid transfer proteins are believed to play
important roles in plant defense against fungal infection. The use
of these protein products to control plant pathogens in transgenic
plants has been reported, for example, in European Patent
Application 0 392 225.
[0007] Another group of peptides known as defensins have been shown
to inhibit plant pathogens. Defensins are small cysteine-rich
peptides of 45-54 amino acids that constitute an important
component of the innate immunity of plants (Thomma et al., 2002;
Lay and Anderson, 2005). Widely distributed in plants, defensins
vary greatly in their amino acid composition. However, they all
have a compact shape that is stabilized by either four or five
intramolecular disulfide bonds. Plant defensins have been
extensively studied for their role in plant defense. Some plant
defensins inhibit the growth of a broad range of fungi at
micromolar concentrations (Broekaert et al., 1995; Broekaert et
al., 1997; da Silva Conceicao and Broekaert, 1999) and, when
expressed in transgenic plants, confer strong resistance to fungal
pathogens (da Silva Conceicao and Broekaert, 1999; Thomma et al.,
2002; Lay and Anderson, 2005). Two small cysteine-rich proteins
isolated from radish seed, Rs-AFP1 and Rs-AFP2, inhibited the
growth of many pathogenic fungi when the pure protein was added to
an in vitro antifungal assay medium (U.S. Pat. No. 5,538,525).
Transgenic tobacco plants containing the gene encoding Rs-AFP2
protein were found to be more resistant to attack by fungi than
non-transformed plants.
[0008] Antifungal defensin proteins have also been identified in
Alfalfa (Medicago sativa) and shown to inhibit plant pathogens such
as Fusarium and Verticillium in both in vitro tests and in
transgenic plants (U.S. Pat. No. 6,916,970). Under low salt in
vitro assay conditions, the Alfalfa defensin AlfAFP1 inhibited
Fusarium culmorum growth by 50% at 1 .mu.g/ml and Verticillium
dahliae growth by 50% at 4 .mu.g/ml (i.e., IC.sub.50 values of 1
.mu.g/ml and 4 .mu.g/ml, respectively). Expression of the AlfAFP1
protein in transgenic potato plants was also shown to confer
resistance to Verticillium dahliae in both greenhouse and field
tests (Gao et al, 2000). Mode-of-action analyses have also shown
that AlfAFP1 (which is alternatively referred to as MsDef1, for
Medicago sativa Defensin 1) induces hyper-branching of F.
graminearum and can block L-type calcium channels (Spelbrink et al,
2004).
[0009] Other defensin genes have also been identified in the legume
Medicago truncatula (Hanks et al, 2005). The cloned MtDef2 protein
has been demonstrated through in vitro experiments to have little
or no antifungal activity (Spelbrink et al, 2004). Analysis of the
sequence database search identified 10 tentative consensus
sequences (10 unique defensin-encoding genes represented by
multiple ESTs) and six singletons (i.e., six unique defensin genes
represented by a single EST) with homology to known Medicago
defensin genes. One of the tentative consensus sequences was
identified as TC85327 and shown to be expressed in both
mock-treated and mycorrhizal fungus-infected Medicago truncatula
roots. There was no demonstration that proteins encoded by any of
the TC85327 Medicago truncatula sequences possessed anti-fungal
activity in this study (Hanks et al, 2005).
[0010] Although defensin proteins such as AlfAFP1 (MsDef1) and
Rs-AFP2 have been used to obtain transgenic plants that are
resistant to fungal infections, other proteins that provide for
increased levels of resistance are needed. In particular, proteins
with increased specific activities against fungal pathogens would
be particularly useful in improving the levels of fungal resistance
obtained in transgenic plants. Furthermore, proteins that inhibit
fungal pathogens via distinct modes of action would also be useful
in combating fungal pathogens that have become resistant to
defensin proteins such as AlfAFP1 (MsDef1) and Rs-AFP2.
[0011] The present invention meets this need by providing a family
of novel small antifungal proteins and peptides from Medicago
truncatula, MtDef5.1-5.6 (SEQ ID NOs:1, 3-9, 16-22, and 49-64).
SUMMARY OF THE INVENTION
[0012] Accordingly, among its various aspects, the present
invention provides the following. [0013] 1. An isolated, purified
antifungal protein or peptide, comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64. [0014] 2. The isolated,
purified antifungal protein or peptide of 1, comprising a plant
apoplast, vacuolar, or endoplasmic reticulum targeting amino acid
sequence at its N-terminus. [0015] 3. The isolated, purified
antifungal protein or peptide of 2, wherein said targeting sequence
comprises an amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in shown in SEQ ID
NOs:30-35 and 42-48. [0016] 4. An isolated, purified nucleotide
sequence encoding said isolated, purified antifungal protein or
peptide of any one of 1-3. [0017] 5. The isolated, purified
nucleotide sequence of 4, codon-optimized for expression in a plant
of interest. [0018] 6. The isolated, purified nucleotide sequence
of 4 or 5, wherein said plant of interest is a food crop plant.
[0019] 7. The isolated, purified nucleotide sequence of 6, wherein
said food crop plant is selected from the group consisting of
soybean, wheat, maize, sugarcane, rice, and potato. [0020] 8. A
transgenic plant, cells of which contain an antifungal protein or
peptide comprising an amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64. [0021] 9. The transgenic plant of 8, wherein said
antifungal protein or peptide further comprises a plant apoplast,
vacuolar, or endoplasmic reticulum targeting amino acid sequence at
its N-terminus. [0022] 10. The transgenic plant of 8 or 9, wherein
said antifungal protein or peptide is present in said cells in an
antifungal effective amount. [0023] 11. The transgenic plant of any
one of 8-10, wherein said cells are root cells. [0024] 12. The
transgenic plant of any one of 8-11, wherein said antifungal
protein or peptide inhibits damage to said plant caused by a
species of fungus selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumannomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp. [0025] 13. The transgenic plant of any one of
8-12, the genome of which further comprises: [0026] DNA encoding a
plant defensin selected from the group consisting of MsDef1,
MtDef2, MtDef4, NaD1, Rs-AFP1, Rs-AFP2, KP4, and KP6, wherein said
DNA is expressed and produces an anti-fungal effective amount of
said defensin, and/or [0027] DNA encoding a Bacillus thuringiensis
endotoxin, wherein said DNA is expressed and produces an
anti-insect effective amount of said Bacillus thuringiensis
endotoxin, and/or [0028] DNA encoding a protein that confers
herbicide resistance to said plant, wherein said DNA is expressed
and produces an anti-herbicide effective amount of said protein
that confers herbicide resistance. [0029] 14. The transgenic plant
of any one of 8-13, produced by a method comprising: [0030] a)
inserting into the genome of a plant cell a recombinant,
double-stranded DNA molecule comprising, operably linked for
expression: [0031] (i) a promoter sequence that functions in plant
cells to cause the transcription of an adjacent coding sequence to
RNA; [0032] (ii) optionally, an intron; [0033] (iii) a coding
sequence encoding an antifungal protein or peptide comprising an
amino acid sequence having at least 90% sequence identity to an
amino acid sequence selected from the group consisting of amino
acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a
plant apoplast, vacuolar, or endoplasmic reticulum targeting amino
acid sequence at its N-terminus; [0034] (iv) a 3' non-translated
sequence that functions in plant cells to cause transcriptional
termination and the addition of polyadenylate nucleotides to the 3'
end of said transcribed RNA; [0035] b) obtaining a transformed
plant cell; and [0036] c) regenerating from said transformed plant
cell a genetically transformed plant, cells of which express said
antifungal protein or peptide. [0037] 15. The transgenic plant of
14, wherein said antifungal protein or peptide is expressed in an
antifungal effective amount in cells of said transformed plant.
[0038] 16. The transgenic plant of 14 or 15, wherein said coding
sequence comprises a nucleotide sequence having a sequence identity
to a nucleotide sequence selected from the group consisting of
nucleotide sequences shown in SEQ ID NOs:10-15, 23-29, and 65-78
sufficient to enable said coding sequence to encode said antifungal
protein or peptide, or a codon-optimized version of said coding
sequence to optimize expression thereof in said plant. [0039] 17.
The transgenic plant of any one of 14-16, wherein said promoter is
a root-specific promoter. [0040] 18. The transgenic plant of 17,
wherein said root-specific promoter is selected from the group
consisting of RB7, RD2, ROOT1, ROOT2, ROOT3, ROOT4, ROOT5, ROOT6,
ROOT7, and ROOT5. [0041] 19. The transgenic plant of any one of
8-18, which is selected from the group consisting of maize,
soybean, wheat, sugarcane, rice, and potato. [0042] 20. A part of
said transgenic plant of any one of 8-19. [0043] 21. The part of
20, which is selected from the group consisting of a protoplast, a
cell, a tissue, an organ, a cutting, and an explant. [0044] 22. The
part of 21, which is selected from the group consisting of an
inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a
style, an ovary, an ovule, an embryo, a receptacle, a seed, a
fruit, a stamen, a filament, an anther, a male or female
gametophyte, a pollen grain, a meristem, a terminal bud, an
axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a
tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in
culture, a tissue of said plant in culture, an organ of said plant
in culture, and a callus. [0045] 23. Progeny of said transgenic
plant of any one of 8-19. [0046] 24. Seed of said transgenic plant
of any one of 8-19. [0047] 25. A transgenic plant, cells of which
comprise a nucleotide coding sequence encoding an antifungal
protein or peptide comprising an amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of amino acid sequences shown in SEQ ID NOs:1,
3-9, 16-22, and 49-64. [0048] 26. The transgenic plant of 25,
wherein said nucleotide coding sequence further encodes a plant
apoplast, vacuolar, or endoplasmic reticulum targeting amino acid
sequence at the N-terminus of said antifungal protein or peptide.
[0049] 27. The transgenic plant of 25 or 26, wherein said
antifungal protein or peptide is expressed in an antifungal
effective amount in said cells. [0050] 28. The transgenic plant of
any one of 25-27, wherein said cells are root cells. [0051] 29. The
transgenic plant of any one of 25-28, produced by a method
comprising: [0052] a) inserting into the genome of a plant cell a
recombinant, double-stranded DNA molecule comprising, operably
linked for expression: [0053] (i) a promoter that functions in
plant cells to cause transcription of an adjacent coding sequence
to RNA; [0054] (ii) optionally, an intron; [0055] (iii) a coding
sequence encoding an antifungal protein or peptide comprising an
amino acid sequence having at least 90% sequence identity to an
amino acid sequence selected from the group consisting of amino
acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a
plant apoplast, vacuolar, or endoplasmic reticulum targeting amino
acid sequence at its N-terminus; and [0056] (iv) a 3'
non-translated region that functions in plant cells to cause
transcriptional termination and the addition of polyadenylate
nucleotides to the 3' end of said transcribed RNA; [0057] b)
obtaining a transformed plant cell; and [0058] c) regenerating from
said transformed plant cell a genetically transformed plant, cells
of which express said antifungal protein or peptide. [0059] 30. The
transgenic plant of 29, wherein said antifungal protein or peptide
is expressed in an antifungal effective amount in cells of said
plant. [0060] 31. The transgenic plant of 29 or 30, wherein said
coding sequence comprises a nucleotide sequence having a sequence
identity to a nucleotide sequence selected from the group
consisting of nucleotide sequences shown in SEQ ID NOs:10-15,
23-29, and 65-78 sufficient to enable said coding sequence to
encode said antifungal protein or peptide, or a codon-optimized
version of said coding sequence to optimize expression thereof in
said plant. [0061] 32. The transgenic plant of any one of 29-31,
wherein said promoter is a root-specific promoter. [0062] 33. The
transgenic plant of 32, wherein said root-specific promoter is
selected from the group consisting of RB7, RD2, ROOT1, ROOT2,
ROOT3, ROOT4, ROOT5, ROOT6, ROOT7, and ROOT5. [0063] 34. The
transgenic plant of any one of 29-33, the genome of which further
comprises: [0064] DNA encoding a plant defensin selected from the
group consisting of MsDef1, MtDef2, MtDef4, NaD1, Rs-AFP1, Rs-AFP2,
KP4, and KP6, wherein said DNA is expressed and produces an
anti-fungal effective amount of said defensin, and/or [0065] DNA
encoding a Bacillus thuringiensis endotoxin, wherein said DNA is
expressed and produces an anti-insect effective amount of said
Bacillus thuringiensis endotoxin, and/or [0066] DNA encoding a
protein that confers herbicide resistance to said plant, wherein
said DNA is expressed and produces an anti-herbicide effective
amount of said protein that confers herbicide resistance. [0067]
35. The transgenic plant of any one of 29-34, which is selected
from the group consisting of maize, soybean, wheat, sugarcane,
rice, and potato. [0068] 36. A plant normally susceptible to damage
from a species of fungus selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumannomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp., cells of which contain a coding sequence encoding
a antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64. [0069] 37. The plant of 36,
wherein said antifungal protein or peptide further comprises a
plant apoplast, vacuolar, or endoplasmic reticulum targeting amino
acid sequence at its N-terminus. [0070] 38. The plant of 36 or 37,
wherein said antifungal protein or peptide is present in said cells
in an antifungal effective amount. [0071] 39. The plant of any one
of 36-38, wherein said cells are root cells. [0072] 40. The plant
of any one of 36-39, which is selected from the group consisting of
maize, soybean, wheat, sugarcane, rice, and potato.
[0073] 41. A method of preventing, treating, controlling,
combating, reducing, or inhibiting damage to a plant susceptible to
damage from a species of fungus selected from the group consisting
of an Alternaria sp., an Ascochyta sp., an Aspergillus sp., a
Botrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia
sp., an Erysiphe sp., a Fusarium sp., Gaeumannomyces sp.,
Helminthosporium sp., Macrophomina sp., a Nectria sp., a
Peronospora sp., a Phakopsora sp., a Phoma sp., a Phymatotrichum
sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a
Podosphaera sp., a Pyrenophora sp., a Pyricularia sp., a Pythium
sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a
Septoria sp., a Thielaviopsis sp., an Uncinula sp., a Venturia sp.,
and a Verticillium sp., comprising providing to the locus of said
susceptible plant an antifungal effective amount of a antifungal
protein or peptide comprising an amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of amino acid sequences shown in SEQ ID NOs:1,
3-9, 16-22, and 49-64, and any combination thereof [0074] 42. The
method of 41, wherein said antifungal protein or peptide is
provided to said susceptible plant locus by expressing DNA encoding
said antifungal protein or peptide within cells of said susceptible
plant. [0075] 43. The method of 42, wherein said DNA encoding said
antifungal protein or peptide comprises a nucleotide sequence
having a sequence identity to a nucleotide sequence selected from
the group consisting of nucleotide sequences shown in SEQ ID
NOs:10-15, 23-29, and 65-78 sufficient to enable said coding
sequence to encode said antifungal protein or peptide, or a
codon-optimized version of said coding sequence to optimize
expression thereof in said plant. [0076] 44. The method of 43,
wherein said DNA further comprises a nucleotide sequence encoding a
plant apoplast, vacuolar, or endoplasmic reticulum targeting amino
acid sequence at the N-terminus of said antifungal protein or
peptide. [0077] 45. The method of any one of 41-44, wherein said
cells are root cells. [0078] 46. The method of 41, wherein said
antifungal protein or peptide is provided to said susceptible plant
locus by plant colonizing microorganisms that produce said
antifungal protein or peptide. [0079] 47. The method of 41, wherein
said antifungal protein or peptide is provided to said susceptible
plant locus by applying a composition comprising plant colonizing
microorganisms that produce said antifungal protein or peptide, or
by applying said antifungal protein or peptide itself thereto.
[0080] 48. The method of 47, wherein said composition comprises an
agriculturally acceptable diluent, excipient, or carrier. [0081]
49. The method of any one of 41-48, wherein said susceptible plant
is selected from the group consisting of maize, soybean, wheat,
sugarcane, rice, and potato. [0082] 50. A method of preventing,
treating, controlling, combating, reducing, or inhibiting damage to
a plant susceptible to damage from a species of fungus selected
from the group consisting of an Alternaria sp., an Ascochyta sp.,
an Aspergillus sp., a Botrytis sp.; a Cercospora sp., a
Colletotrichum sp., a Diplodia sp., an Erysiphe sp., a Fusarium
sp., Gaeumannomyces sp., Helminthosporium sp., Macrophomina sp., a
Nectria sp., a Peronospora sp., a Phakopsora sp., a Phoma sp., a
Phymatotrichum sp., a Phytophthora sp., a Plasmopara sp., a
Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia
sp., a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a
Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula
sp., a Venturia sp., and a Verticillium sp., comprising expressing
DNA comprising a nucleotide sequence encoding a protein or peptide
comprising an amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64, and any combination thereof, in cells of said
susceptible plant at a level sufficient to inhibit said fungal
damage. [0083] 51. The method of 50, wherein said antifungal
protein or peptide is targeted to apoplasts, vacuoles, or the
endoplasmic reticulum of cells of said susceptible plant. [0084]
52. The method of 50 or 51, wherein said antifungal protein or
peptide is encoded by a nucleotide sequence having a sequence
identity to a nucleotide sequence selected from the group
consisting of nucleotide sequences shown in SEQ ID NOs:10-15,
23-29, and 65-78 sufficient to enable said nucleotide sequence to
encode said antifungal protein or peptide, or a codon-optimized
version of said nucleotide sequence to optimize expression thereof
in said plant. [0085] 53. The method of any one of 50-52, wherein
said cells are root cells. [0086] 54. The method of any one of
50-53, wherein said susceptible plant is selected from the group
consisting of maize, soybean, wheat, sugarcane, rice, and potato.
[0087] 55. A method of inhibiting damage to a plant susceptible to
damage from a species of fungus selected from the group consisting
of an Alternaria sp., an Ascochyta sp., an Aspergillus sp., a
Botrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia
sp., an Erysiphe sp., a Fusarium sp., Gaeumannomyces sp.,
Helminthosporium sp., Macrophomina sp., a Nectria sp., a
Peronospora sp., a Phakopsora sp., a Phoma sp., a Phymatotrichum
sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a
Podosphaera sp., a Pyrenophora sp., a Pyricularia sp., a Pythium
sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a
Septoria sp., a Thielaviopsis sp., an Uncinula sp., a Venturia sp.,
and a Verticillium sp., comprising: [0088] a) inserting into the
genome of a plant cell a recombinant, double stranded DNA molecule
comprising, operably linked for expression: [0089] (i) a promoter
that functions in plant cells to cause the transcription of an
adjacent coding sequence to RNA; [0090] (ii) optionally, an intron;
[0091] (iii) a coding sequence comprising a nucleotide sequence
encoding an antifungal protein or peptide comprising an amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of amino acid sequences
shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64; and [0092] (iv) a 3'
nontranslated region that functions in said plant cells to cause
transcriptional termination and the addition of polyadenylate
nucleotides to the 3' end of said transcribed RNA; [0093] b)
obtaining a transformed plant cell; and [0094] c) regenerating from
said transformed plant cell a genetically transformed plant, cells
of which express said antifungal protein or peptide. [0095] 56. The
method of 55, wherein said antifungal protein or peptide is
expressed in an antifungal amount in cells of said transformed
plant. [0096] 57. The method of 55 or 56, wherein said antifungal
protein or peptide is targeted to apoplasts, vacuoles, or the
endoplasmic reticulum of cells of said transformed plant. [0097]
58. The method of any one of 55-57, wherein said nucleotide
sequence encoding said antifungal protein or peptide is a
nucleotide sequence having a sequence identity to a nucleotide
sequence selected from the group consisting of nucleotide sequences
shown in SEQ ID NOs:10-15, 23-29, and 65-78 sufficient to enable
said nucleotide sequence to encode said antifungal protein or
peptide, or a codon-optimized version of said nucleotide sequence
to optimize expression thereof in said plant. [0098] 59. The method
of any one of 55-58, wherein said promoter is a root-specific
promoter. [0099] 60. The method of 59, wherein said root-specific
promoter is selected from the group consisting of RB7, RD2, ROOT1,
ROOT2, ROOT3, ROOT4, ROOT5, ROOT6, ROOT7, and ROOT8. [0100] 61. The
method of any one of 55-60, wherein said susceptible plant is
selected from the group consisting of maize, soybean, wheat,
sugarcane, rice, and potato. [0101] 62. A method of controlling,
combating, or inhibiting a species of fungus selected from the
group consisting of an Alternaria sp., an Ascochyta sp., an
Aspergillus sp., a Botrytis sp.; a Cercospora sp., a Colletotrichum
sp., a Diplodia sp., an Erysiphe sp., a Fusarium sp.,
Gaeumannomyces sp., Helminthosporium sp., Macrophomina sp., a
Nectria sp., a Peronospora sp., a Phakopsora sp., a Phoma sp., a
Phymatotrichum sp., a Phytophthora sp., a Plasmopara sp., a
Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia
sp., a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a
Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula
sp., a Venturia sp., and a Verticillium sp., comprising contacting
said fungal species with a composition comprising an antifungal
effective amount of an antifungal protein or peptide comprising an
amino acid sequence having at least 90% sequence identity to an
amino acid sequence selected from the group consisting of amino
acid sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and
any combination thereof [0102] 63. The method of 62, wherein said
composition comprises said antifungal protein or peptide, and an
agriculturally acceptable carrier, diluent, or excipient. [0103]
64. The method of 62, wherein said composition comprises
microorganisms expressing said antifungal protein or peptide.
[0104] 65. A method of preventing, treating, controlling,
combating, reducing, or inhibiting damage to a plant caused by a
fungus, comprising: [0105] a) inserting into the genome of a plant
cell a recombinant, double stranded DNA molecule comprising,
operably linked for expression: [0106] (i) a promoter that
functions in plant cells to cause the transcription of an adjacent
coding sequence to RNA; [0107] (ii) optionally, an intron; [0108]
(iii) a coding sequence comprising a nucleotide sequence encoding
an antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64; and [0109] (iv) a 3'
nontranslated region that functions in said plant cells to cause
transcriptional termination and the addition of polyadenylate
nucleotides to the 3' end of said transcribed RNA; [0110] b)
obtaining a transformed plant cell; and [0111] c) regenerating from
said transformed plant cell a genetically transformed plant, cells
of which express said antifungal protein or peptide. [0112] 66. The
method of 65, wherein said antifungal protein or peptide is
expressed in an antifungal effective amount in cells of said
transformed plant. [0113] 67. The method of 65 or 66, wherein said
coding sequence further comprises a nucleotide sequence encoding a
plant apoplast, vacuolar, or endoplasmic reticulum targeting amino
acid sequence at the N-terminus of said antifungal protein or
peptide. [0114] 68. The method of any one of 65-67, wherein said
nucleotide sequence encoding said antifungal protein or peptide
comprises a nucleotide sequence having a sequence identity to a
nucleotide sequence selected from the group consisting of
nucleotide sequences shown in SEQ ID NOs:10-15, 23-29, and 65-78
sufficient to enable said coding sequence to encode said antifungal
protein or peptide, or a codon-optimized version of said coding
sequence to optimize expression thereof in said plant. [0115] 69.
The method of any one of 65-68, wherein said promoter is a
root-specific promoter. [0116] 70. The method of 69, wherein said
root-specific promoter is selected from the group consisting of
RB7, RD2, ROOT1, ROOT2, ROOT3, ROOT4, ROOT5, ROOT6, ROOT7, and
ROOT8. [0117] 71. The method of any one of 65-70, wherein said
fungus is a species selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumannomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp. 72. The method of any one of 65-71, wherein said
plant is selected from the group consisting of maize, soybean,
wheat, sugarcane, rice, and potato. 73. A method of preventing,
treating, controlling, combating, reducing, or inhibiting damage to
a plant caused by a fungus, comprising:
[0118] transforming a plant with a DNA molecule encoding an
antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a plant apoplast,
vacuolar, or endoplasmic reticulum targeting amino acid sequence at
the N-terminus of said antifungal protein or peptide to produce a
transformed plant, wherein cells of said transformed plant produce
said antifungal protein or peptide, and wherein said transformed
plant exhibits reduced fungal damage as compared to the fungal
damage of an otherwise identical, untransformed control plant that
does not produce said antifungal protein or peptide when both
plants are contacted with similar amounts of said fungus and are
grown under the same conditions.
[0119] 74. The method of 73, wherein said cells are root cells. 75.
The method of 73 or 74, wherein said fungus is a species selected
from the group consisting of an Alternaria sp., an Ascochyta sp.,
an Aspergillus sp., a Botrytis sp.; a Cercospora sp., a
Colletotrichum sp., a Diplodia sp., an Erysiphe sp., a Fusarium
sp., Gaeumannomyces sp., Helminthosporium sp., Macrophomina sp., a
Nectria sp., a Peronospora sp., a Phakopsora sp., a Phoma sp., a
Phymatotrichum sp., a Phytophthora sp., a Plasmopara sp., a
Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia
sp., a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a
Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula
sp., a Venturia sp., and a Verticillium sp. [0120] 76. The method
of any one of 73-75, wherein said plant is selected from the group
consisting of maize, soybean, wheat, sugarcane, rice, and potato.
[0121] 77. A method of reducing or inhibiting fungal contamination
of soil, comprising cultivating in said soil transgenic plants
expressing an antifungal protein or peptide comprising an amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of amino acid
sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64 in cells of
roots of said transgenic plants. [0122] 78. The method of 77,
wherein said root cells produce said antifungal protein or peptide
in an antifungal effective amount. [0123] 79. The method of 77 or
78, wherein said antifungal protein or peptide is targeted to
apoplasts, vacuoles, or the endoplasmic reticulum of said root
cells. [0124] 80. The method of any one of 77-79, wherein said
wherein said fungus is a species selected from the group consisting
of an Alternaria sp., an Ascochyta sp., an Aspergillus sp., a
Botrytis sp., a Cercospora sp., a Colletotrichum sp., a Diplodia
sp., an Erysiphe sp., a Fusarium sp., Gaeumannomyces sp.,
Helminthosporium sp., Macrophomina sp., a Nectria sp., a
Peronospora sp., a Phakopsora sp., a Phoma sp., a Phymatotrichum
sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a
Podosphaera sp., a Pyrenophora sp., a Pyricularia sp., a Pythium
sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a
Septoria sp., a Thielaviopsis sp., an Uncinula sp., a Venturia sp.,
and a Verticillium sp. [0125] 81. The method of any one of 77-80,
wherein said transgenic plants are transgenic food crop plants.
[0126] 82. The method of 81, wherein said transgenic food crop
plants are selected from the group consisting of maize, soybean,
wheat, sugarcane, rice, and potato. [0127] 83. A recombinant,
double-stranded DNA molecule comprising, operatively linked for
expression: [0128] a) a promoter that functions in plant cells to
cause transcription of an adjacent coding sequence to RNA; [0129]
b) optionally, an intron; [0130] c) a nucleotide sequence encoding
an antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a plant apoplast,
vacuolar, or endoplasmic reticulum targeting amino acid sequence at
the N-terminus of said antifungal protein or peptide; and [0131] d)
a 3' non-translated sequence that functions in plant cells to cause
transcriptional termination and the addition of polyadenylate
nucleotides to the 3' end of said transcribed RNA. [0132] 84. The
recombinant, double-stranded DNA molecule of 83, wherein said
promoter is a root-specific promoter. [0133] 85. The recombinant,
double-stranded DNA molecule of 84, wherein said root-specific
promoter is selected from the group consisting of RB7, RD2, ROOT1,
ROOT2, ROOT3, ROOT4, ROOT5, ROOT6, ROOT7, and ROOT8. [0134] 86. The
recombinant, double-stranded DNA molecule of any one of 83-85,
which is codon-optimized for expression in a plant of interest.
[0135] 87. The recombinant, double-stranded DNA molecule of 86,
wherein said plant of interest is selected from the group
consisting of maize, soybean, wheat, sugarcane, rice, and potato.
[0136] 88. An expression construct, comprising a recombinant,
double-stranded DNA molecule comprising, operably linked for
expression: [0137] a) a promoter that functions in plant cells to
cause transcription of an adjacent coding sequence to RNA; [0138]
b) optionally, an intron; [0139] c) a nucleotide sequence encoding
an antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a plant apoplast,
vacuolar, or endoplasmic reticulum targeting amino acid sequence at
the N-terminus of said antifungal protein or peptide; and [0140] d)
a 3' non-translated sequence that functions in plant cells to cause
transcriptional termination and the addition of polyadenylate
nucleotides to the 3' end of said transcribed RNA. [0141] 89. The
expression construct of 88, wherein said promoter is a
root-specific promoter. [0142] 90. The expression construct of 89,
wherein said root-specific promoter is selected from the group
consisting of RB7, RD2, ROOT1, ROOT2, ROOT3, ROOT4, ROOT5, ROOT6,
ROOT7, and ROOT8. [0143] 91. The expression construct of any one of
89-90, wherein said recombinant, double-stranded DNA molecule is
codon-optimized for expression in a plant of interest. [0144] 92.
The expression construct of 91, wherein said plant of interest is
selected from the group consisting of maize, soybean, wheat,
sugarcane, rice, and potato. [0145] 93. A plant transformation
vector, comprising said recombinant, double-stranded DNA molecule
of any one of 83-87, or the expression construct of any one of
88-92, and a selectable or scoreable marker for selection of
transformed plant cells. [0146] 94. An antifungal composition,
comprising an antifungal protein or peptide comprising an amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of amino acid
sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and any
combination thereof [0147] 95. The antifungal composition of 94,
wherein said antifungal protein or peptide, or combination thereof,
is present in an antifungal effective amount. [0148] 96. The
antifungal composition of 94 or 95, further comprising an
agriculturally or pharmaceutically acceptable carrier, diluent, or
excipient. [0149] 97. The antifungal composition of any one of
94-96, wherein said antifungal protein or peptide, or combination
thereof, is present in a concentration in the range of from about
0.1 microgram per milliliter to about 500 milligrams per
milliliter. [0150] 98. The antifungal composition of any one of
94-96, wherein said wherein said antifungal protein or peptide, or
combination thereof, is present in a concentration in the range of
from about 5 micrograms per milliliter to about 250 milligrams per
milliliter. [0151] 99. The antifungal composition of any one of
94-98, having a pH in the range of from about 3 to about 9. [0152]
100. The antifungal composition of any one of 94-99, formulated
with one or more additives selected from the group consisting of an
inert material, a surfactant, and a solvent. [0153] 101. The
antifungal composition of any one of 94-100, formulated in a
mixture of one or more other active agents selected from the group
consisting of a pesticidally active substance, a fertilizer, an
insecticide, an attractant, a sterilizing agent, an acaricide, a
nematocide, a herbicide, and a growth regulator. [0154] 102. The
antifungal composition of 101, wherein said pesticidally active
substance is selected from the group consisting of a fungal
antibiotic and a chemical fungicide. [0155] 103. The antifungal
composition of 102, wherein said fungal antibiotic or chemical
fungicide is selected from the group consisting of a polyoxine, a
nikkomycine, a carboxyamide, an aromatic carbohydrate, a carboxine,
a morpholine, a sterol biosynthesis inhibitor, and an
organophosphate. [0156] 104. Use of said antifungal composition of
any one of 94-103 to inhibit the growth of a susceptible fungal
species. [0157] 105. The use of 104, wherein said susceptible
fungal species is selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp., a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumannomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp. [0158] 106. The antifungal composition of any one
of 94-103 for use in inhibiting the growth of a susceptible fungal
species. [0159] 107. The use of 106, wherein said susceptible
fungal species is selected from the group consisting of an
Alternaria sp., an Ascochyta sp., an Aspergillus sp., a Botrytis
sp., a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an
Erysiphe sp., a Fusarium sp., Gaeumannomyces sp., Helminthosporium
sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a
Phakopsora sp., a Phoma sp., a Phymatotrichum sp., a Phytophthora
sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a
Pyrenophora sp., a Pyricularia sp., a Pythium sp., a Rhizoctonia
sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a
Thielaviopsis sp., an Uncinula sp., a Venturia sp., and a
Verticillium sp. [0160] 108. The antifungal composition of any one
of 94-103, provided to a plant locus by plant colonizing
microorganisms producing said protein, peptide, or combination
thereof, or by a composition comprising said plant colonizing
microorganisms. [0161] 109. The antifungal composition of 94 or 95,
wherein said protein, peptide, or combination thereof is expressed
from DNA encoding said protein, peptide, or combination thereof
within cells of a transgenic plant. [0162] 110. A method of
controlling, combating, or inhibiting a susceptible fungus,
comprising contacting said susceptible fungus with a transgenic
plant, cells of which comprise and express a nucleotide sequence
encoding an antifungal protein or peptide comprising an amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of amino acid sequences
shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and a plant apoplast,
vacuolar, or endoplasmic reticulum targeting amino acid sequence at
the N-terminus of said antifungal protein or peptide. [0163] 111.
The method of 110, wherein said antifungal protein or peptide is
expressed in cells of roots of said transgenic plant. [0164] 112.
The method of 111, wherein said antifungal protein or peptide is
expressed by said root cells in an antifungal effective amount.
[0165] 113. The method of any one of 110-112, wherein said
susceptible fungus is a species is selected from the group
consisting of an Alternaria sp., an Ascochyta sp., an Aspergillus
sp., a Botrytis sp., a Cercospora sp., a Colletotrichum sp., a
Diplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumannomyces sp.,
Helminthosporium sp., Macrophomina sp., a Nectria sp., a
Peronospora sp., a Phakopsora sp., a Phoma sp., a Phymatotrichum
sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a
Podosphaera sp., a Pyrenophora sp., a Pyricularia sp., a Pythium
sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a
Septoria sp., a Thielaviopsis sp., an Uncinula sp., a Venturia sp.,
and a Verticillium sp. [0166] 114. The method of any one of
110-113, wherein said transgenic plant is selected from the group
consisting of maize, soybean, wheat, sugarcane, rice, and potato.
[0167] 115. An antifungal protein or peptide comprising an amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of amino acid
sequences shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, for use in
human therapy. [0168] 116. An antifungal protein or peptide
comprising an amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64, for use in treating a susceptible fungal
infection. [0169] 117. Use of an antifungal protein or peptide
comprising an amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64, in human therapy. [0170] 118. Use of an
antifungal protein or peptide comprising an amino acid sequence
having at least 90% sequence identity to an amino acid sequence
selected from the group consisting of amino acid sequences shown in
SEQ ID NOs:1, 3-9, 16-22, and 49-64, in treating a susceptible
fungal infection. [0171] 119. Use of an antifungal protein or
peptide comprising an amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64, for the manufacture of a medicament to treat a
susceptible fungal infection. [0172] 120. A method of treating a
susceptible fungal infection in a patient in need thereof,
comprising administering to said patient an antifungal effective
amount of an antifungal protein or peptide comprising an amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of amino acid sequences
shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, and any combination
thereof [0173] 121. The method of 120, wherein said administering
is performed topically.
[0174] Further scope of the applicability of the present invention
will become apparent from the detailed description and drawing(s)
provided below. However, it should be understood that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration only
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0175] The above and other aspects, features, and advantages of the
present invention will be better understood from the following
detailed descriptions taken in conjunction with the accompanying
drawing(s), all of which are given by way of illustration only, and
are not limitative of the present invention, in which:
[0176] FIG. 1 shows transformation vector AKK/FMV/Def5 used to make
MtDef5-expressing soybean lines. "LB"=T-DNA left border; "T-DNA
RB"=T-DNA right border; "tNOS"=Nopaline synthase terminator
sequence; "FMV"=Figwort mosaic virus 35S; "SU intron"=Super
ubiquitin intron; "BAR"=Bialophos resistance gene; "NOS"=Nopaline
synthase promoter; "Ori"=Origin of replication; "TraF"=Transfer F;
"Tet(R)"=tetracycline resistance gene; "Kan(R)"=Kanamycin
resistance gene; "TrfA"=T-DNA replication factor, and "Def5"
represents a MtDef5 protein or peptide coding sequence.
[0177] FIG. 2 shows transformation vector pZP212-Def5 to be used
for making MtDef5-expressing wheat lines. "LB"=T-DNA left border;
"T-DNA RB"=T-DNA right border; nptII=neomycin phosphotransferase
II; 35S=CaMV35S promoter; Ubi and Ubi intron=Maize Ubi1A promoter
and intron; TEV=tobacco etch virus leader; Def5=a MtDef5 protein or
peptide coding sequence; ter=CaMV 35S termination signal.
[0178] FIG. 3 shows the quantitative assessment of the in vitro
antifungal activity of the chemically synthesized GMA-5C peptide
GACHRQGFGFACFCYKKC (SEQ ID NO:58) of MtDef5.1a at 24 h after
incubation of Fusarium graminearum PH-1 conidia with the peptide.
Values are means of thee replications. Error bars indicate standard
deviations. The in vitro antifungal activity of the peptide was
determined as previously described (Ramamoorthy et al. (2007)
Cellular Microbiology 9:1491-1506).
DETAILED DESCRIPTION OF THE INVENTION
[0179] The following detailed description of the invention is
provided to aid those skilled in the art in practicing the present
invention. Even so, the following detailed description should not
be construed to unduly limit the present invention, as
modifications and variations in the embodiments herein discussed
may be made by those of ordinary skill in the art without departing
from the spirit or scope of the present inventive discovery.
[0180] The contents of each of the references discussed in this
specification, including the references cited therein, are herein
incorporated by reference in their entirety.
[0181] Any feature, or combination of features, described herein
is(are) included within the scope of the present disclosure,
provided that the features included in any such combination are not
mutually inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. Additional advantages and aspects of the present disclosure
are apparent in the following detailed description and claims.
[0182] The amino acid and nucleotide sequences of the MtDef5
proteins and peptides, as well as those of the other elements
useful in the constructs, methods, and organisms of the present
invention, can be found at the end of the specification. All the
amino acid and nucleotide sequences encompassed by the present
invention include sequences consisting of, consisting essentially
of, or comprising those specifically disclosed. As would be
appreciated by one of ordinary skill in the art, as a result of the
degeneracy of the genetic code, there are many nucleotide sequences
that encode the protein and peptide molecules disclosed herein.
Some of these polynucleotides may bear minimal homology to the
nucleotide sequence of any native coding sequence.
[0183] In addition, polynucleotides that vary due to differences in
codon usage are specifically contemplated by the present invention,
and include those that are optimized for expression in monocots,
dicots, yeasts, or bacteria. Nakamura et al. (2000) Nucl. Acids
Res. 28(1):292 discusses the incorporation of preferred codons to
enhance the expression of polynucleotides in various organisms.
Codon usage in various monocot or dicot genes has been disclosed in
Kawabe and Miyashita (2003) "Patterns of codon usage bias in three
dicot and four monocot plant species", Genes Genet. Syst.
78:343-352, and in Murray et al. (1989) "Codon Usage in Plant
Genes" NAR 17:477-498. Methods for optimizing codon usage in plants
are also disclosed in U.S. Pat. Nos. 5,500,365; 5,689,052;
5,500,365; and 5,689,052.
[0184] MtDef5 protein- and peptide-encoding nucleotide sequences,
and promoter nucleotide sequences used to drive their expression,
can be genomic or non-genomic nucleotide sequences. Genomic and
non-genomic nucleotide sequences encoding MtDef5 proteins and
peptides, and promoters, include, for example, naturally-occurring
mRNA, synthetically produced mRNA, naturally-occurring nucleotide
sequences encoding MtDef5 proteins and peptides, and promoters, or
synthetically produced nucleotide sequences encoding MtDef5
proteins and peptides, and promoters. Synthetic nucleotide
sequences can be produced by means well known in the art,
including, for example, by chemical or enzymatic synthesis of
oligonucleotides, and include, for example, cDNA, codon-optimized
sequences for efficient expression in different transgenic plants
reflecting the pattern of codon usage in such plants, variants
containing conservative (or non-conservative) amino acid
substitutions that do not adversely affect their normal activity,
PCR-amplified nucleotide sequences, etc.
Definitions
[0185] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention pertains. Any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention in place of the methods and materials described
herein.
[0186] For the purposes of the present invention, the following
terms are defined below.
[0187] The term "food crop plant" to which the methods and
compositions disclosed herein can be applied refers to plants that
are either directly edible, or which produce edible products, and
that are customarily used to feed humans either directly, or
indirectly through animals. Non-limiting examples of such plants
include: [0188] 1. Cereal crops: wheat, rice, maize (corn), barley,
oats, sorghum, rye, and millet; [0189] 2. Protein crops: peanuts,
chickpeas, lentils, kidney beans, soybeans, lima beans; [0190] 3.
Roots and tubers: potatoes, sweet potatoes, and cassavas; [0191] 4.
Oil crops: corn, soybeans, canola (rapeseed), wheat, peanuts, palm,
coconuts, safflower, cottonseed, sunflower, flax, olive, and
safflower; [0192] 5. Sugar crops: sugar cane and sugar beets;
[0193] 6. Fruit crops: bananas, oranges, apples, pears, breadfruit,
pineapples, and cherries; [0194] 7. Vegetable crops and tubers:
tomatoes, lettuce, carrots, melons, asparagus, etc. [0195] 8. Nuts:
cashews, peanuts, walnuts, pistachio nuts, almonds; [0196] 9.
Forage and turf grasses; [0197] 10. Forage legumes: alfalfa,
clover; [0198] 11. Drug crops: coffee, cocoa, kola nut, poppy;
[0199] 12. Spice and flavoring crops: vanilla, sage, thyme, anise,
saffron, menthol, peppermint, spearmint, coriander The terms
"biofuels crops" or "energy crops" to which the present methods and
compositions can also be applied include the oil crops listed in
item 4, above, and further include plants such as sugarcane, castor
bean, Camelina, switchgrass, Miscanthus, and Jatropha.
[0200] The singular terms "a", "an", and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. Hence, comprising A or B means
including A, or B, or A and B.
[0201] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight, length, or
the like, that varies by as much as .+-.30, 25, 20, 15, 10, 9, 8,
7, 6, 5, 4, 3, 2, or 1% compared to a reference quantity, level,
value, number, frequency, percentage, dimension, size, amount,
weight, length, or the like.
[0202] The endpoints of all ranges disclosed herein directed to the
same component or property are inclusive and independently
combinable (e.g., ranges of "up to about 25 wt. %, or, more
specifically, about 5 wt. % to about 20 wt. %," is inclusive of the
endpoints and all intermediate values of the ranges of "about 5 wt.
% to about 25 wt. %," etc.).
[0203] The term "comprising" as used in a claim herein is
open-ended, and means that the claim must have all the features
specifically recited therein, but that there is no bar on
additional features that are not recited being present as well. The
term "comprising" leaves the claim open for the inclusion of
unspecified ingredients even in major amounts. The term "consisting
essentially of" in a claim means that the invention necessarily
includes the listed ingredients, and is open to unlisted
ingredients that do not materially affect the basic and novel
properties of the invention. A "consisting essentially of" claim
occupies a middle ground between closed claims that are written in
a closed "consisting of" format and fully open claims that are
drafted in a "comprising` format". These terms can be used
interchangeably herein if, and when, this may become necessary.
[0204] Furthermore, the use of the term "including", as well as
other related forms, such as "includes" and "included", is not
limiting.
[0205] The term "heterologous" refers to a nucleic acid fragment or
protein that is foreign to its surroundings. In the context of a
nucleic acid fragment, this is typically accomplished by
introducing such fragment, derived from one source, into a
different host. Heterologous nucleic acid fragments, such as coding
sequences that have been inserted into a host organism, are not
normally found in the genetic complement of the host organism. As
used herein, the term "heterologous" also refers to a nucleic acid
fragment derived from the same organism, but which is located in a
different, e.g., non-native, location within the genome of this
organism. Thus, the organism can have more than the usual number of
copy(ies) of such fragment located in its(their) normal position
within the genome and in addition, in the case of plant cells,
within different genomes within a cell, for example in the nuclear
genome and within a plastid or mitochondrial genome as well. A
nucleic acid fragment that is heterologous with respect to an
organism into which it has been inserted or transferred is
sometimes referred to as a "transgene."
[0206] A "heterologous" MtDef5 protein- or peptide-encoding
nucleotide sequence can be one or more additional copies of an
endogenous MtDef5 protein- or peptide-encoding nucleotide sequence,
or a nucleotide sequence from another plant or other source.
Furthermore, these can be genomic or non-genomic nucleotide
sequences. Non-genomic nucleotide sequences encoding MtDef5
proteins or peptides include, for example, naturally occurring
mRNA; synthetic mRNA produced, for example, by enzymatic synthesis
or chemical oligonucleotide synthesis; synthetically produced
MtDef5 protein- or peptide-encoding DNA sequences including, for
example, those made by chemical oligonucleotide synthesis or
enzymatic synthesis, including, for example, cDNA, codon-optimized
sequences for efficient expression in different transgenic plants
plants-reflecting the pattern of codon usage in such plants,
nucleotide sequences that differ from naturally occurring genomic
sequences due to the degeneracy of the genetic code but that still
encode MtDef5 proteins or peptides disclosed herein, nucleotide
sequences encoding MtDef5 proteins or peptides comprising
conservative (or non-conservative) amino acid substitutions that do
not adversely affect their normal antifungal activity,
PCR-amplified nucleotide sequences, and other non-genomic forms of
nucleotide sequences familiar to those of ordinary skill in the
art.
[0207] A "transgenic" organism, such as a transgenic plant, is a
host organism that has been genetically engineered to contain one
or more heterologous nucleic acid fragments, including nucleotide
coding sequences, expression cassettes, vectors, etc. Introduction
of heterologous nucleic acids into a host cell to create a
transgenic cell is not limited to any particular mode of delivery,
and includes, for example, microinjection, adsorption,
electroporation, particle gun bombardment, whiskers-mediated
transformation, liposome-mediated delivery, Agrobacterium-mediated
transfer, the use of viral and retroviral vectors, etc., as is well
known to those skilled in the art.
[0208] The term "genome" can collectively refer to the totality of
different genomes within plant cells, i.e., nuclear genome, plastid
(e.g., chloroplast genome), and mitochondrial genome, or separately
to the each of these individual genomes when specifically
indicated. As used herein, the term "genome" refers to the nuclear
genome unless indicated otherwise. The "genome" for expression of
the MtDef5 proteins or peptides employed in the present recombinant
methods and plants is the nuclear genome. However, expression in a
plastid genome, e.g., a chloroplast genome, or targeting of a
MtDef5 protein or peptide to a plastid genome such as a chloroplast
via the use of a plastid targeting sequence, is also encompassed by
the present disclosure.
[0209] The term "control plant" refers to a plant without
introduced trait-improving recombinant DNA. A control plant is used
as a standard against which to measure and compare trait
improvement in a transgenic plant comprising such trait-improving
recombinant DNA. One suitable type of control plant is a
non-transgenic plant of the parental line that was used to generate
a transgenic plant, i.e., an otherwise identical wild-type plant.
Another type of suitable control plant is a transgenic plant that
comprises recombinant DNA without the specific trait-producing DNA,
e.g., simply an empty vector.
[0210] The phrases "antifungal protein" or "antifungal peptide" as
used herein refer to proteins and peptides that exhibit any one or
more of the following characteristics: inhibiting or retarding the
growth of fungal cells; killing fungal cells; disrupting or
retarding stages of the fungal life cycle, such as spore
germination, sporulation, or mating; and/or disrupting fungal cell
infection, penetration, or spread within a plant. The net effect is
thus to limit, decrease, or eliminate fungal pathogenesis and/or
damage to a plant.
[0211] The phrase "biological functional equivalents" and the like
as used herein refer to peptides, polypeptides, and proteins that
contain a sequence or structural feature(s) similar or identical to
that of an MtDef5 protein or peptide of the present invention, and
which exhibit the same or similar, e.g., about .+-.30%, antifungal
activity of an MtDef5 protein or peptide of the present invention.
Biological functional equivalents also include peptides,
polypeptides, and proteins that react with (i.e., specifically
bind) to monoclonal and/or polyclonal antibodies raised against an
MtDef5 protein or peptide as disclosed herein, and that exhibit the
same or similar, e.g., about .+-.30%, antifungal activity as an
MtDef5 protein or peptide of the present invention.
[0212] The phrases "combating fungal damage", "combating or
controlling fungal damage", "controlling fungal damage", or the
like as used herein refer to reduction in damage to a crop plant or
crop plant product due to infection by a fungal pathogen. More
generally, these phrases refer to reduction in the adverse effects
caused by the presence of an undesired fungus in the crop plant.
Adverse effects of fungal growth are understood to include any type
of plant tissue damage or necrosis, any type of plant yield
reduction, any reduction in the value of the crop plant product,
and/or production of undesirable fungal metabolites or fungal
growth by-products including, but not limited to, mycotoxins.
[0213] The phrase "DNA construct" as used herein refers to any DNA
molecule in which two or more ordinarily distinct DNA sequences
have been covalently linked. Examples of DNA constructs include,
but are not limited to, plasmids, cosmids, viruses, BACs (bacterial
artificial chromosome), YACs (yeast artificial chromosome), plant
minichromosomes, autonomously replicating sequences, phage, or
linear or circular single-stranded or double-stranded DNA
sequences, derived from any source, that are capable of genomic
integration or autonomous replication. DNA constructs can be
assembled by a variety of methods including, but not limited to,
recombinant DNA techniques, DNA synthesis techniques, PCR
(Polymerase Chain Reaction) techniques, or any combination of
techniques.
[0214] The phrases "a plant pathogenic fungus inhibitory amount" or
"antifungal effective amount", or the like, as used herein in the
context of a transgenic plant or microorganism expressing a MtDef5
protein or peptide, or an agricultural or pharmaceutical
composition containing a MtDef5 protein, peptide, or any
combination thereof used for antifungal purposes, as the case may
be, refers to an amount of an MtDef5 protein and/or peptide that
results in any measurable decrease in fungal growth in the
transgenic plant and/or any measurable decrease in the adverse
effects caused by fungal growth in the transgenic plant, or that
results in any measurable decrease in fungal growth and/or any
measurable decrease in the adverse effects caused by fungal growth
in the particular application in which it is employed,
respectively. The latter includes, for example, human and
veterinary therapeutic applications. An antifungal effective amount
of a MtDef5 protein or peptide, or any combination thereof, is an
amount or dose that, upon single or multiple dose administration to
a patient or subject, provides the desired prevention or
treatment.
[0215] In the context of a transgenic plant, a plant pathogenic
fungus inhibitory amount (antifungal effective amount) of a MtDef5
protein or peptide, is at least about 0.05 PPM, at least about 0.5
PPM, at least about 1.0 PPM, or at least about 2.0 PPM, where PPM
are "parts per million" of MtDef5 protein or peptide present in
fresh weight plant tissue, where 1 microgram of MtDef5 protein or
peptide per 1 gram of fresh weight plant tissue represents a
concentration of 1 PPM.
[0216] The phrase "a heterologous promoter", as used herein in the
context of a DNA construct, refers to either: i) a promoter that is
derived from a source distinct from the operably linked structural
coding sequence or ii) a promoter derived the same source as the
operably linked structural gene, where the promoter's sequence is
modified from its original form.
[0217] The term "homolog" as used herein refers to a gene related
to a second gene by identity of either the DNA sequences or the
encoded protein sequences. Genes that are homologs can be genes
separated by the event of speciation (see "ortholog"). Genes that
are homologs may also be genes separated by the event of genetic
duplication (see "paralog"). Homologs can be from the same or a
different organism and may perform the same biological function in
either the same or a different organism.
[0218] The phrase "MtDef5 protein or peptide" as used herein refers
to: i) proteins or peptides with at least about 70% sequence
identity to a mature MtDef5 protein sequence, or peptide sequence,
disclosed herein and ii) proteins with at least about 70% sequence
identity to MtDef5 proprotein sequences comprising an MtDef5 signal
peptide and a mature MtDef5 protein. Mature MtDef5 protein
sequences include, but are not limited to, a MtDef5.1a mature
protein sequence (SEQ ID NO:16), a MtDef5.1b mature protein
sequence (SEQ ID NO:17), a MtDef5.2 mature protein sequence (SEQ ID
NO:18), aMtDef5.3 mature protein sequence (SEQ ID NO:19), a
MtDef5.4 mature protein sequence (SEQ ID NO:20), a MtDef5.5 mature
protein sequence (SEQ ID NO:21), and a MtDef5.6 mature protein
sequence (SEQ ID NO:22), as well as coding sequences therefor as
exemplified by SEQ ID NOs:23-29, respectively; proteins that have
at least about 70% sequence identity to these sequences; and
biological functional equivalents of these sequences. MtDef5
proprotein sequences include, but are not limited to, a MtDef5.1a
proprotein sequence (SEQ ID NO:1), a MtDef5.1a-5.1b proprotein
sequence (SEQ ID NO:4), a MtDef5.2 proprotein sequence (SEQ ID
NO:5), aMtDef5.3 proprotein sequence (SEQ ID NO:6), a MtDef5.4
proprotein sequence (SEQ ID NO:7), a MtDef5.5 proprotein sequence
(SEQ ID NO:8), and a MtDef5.6 proprotein sequence (SEQ ID NO:9), as
well as coding sequences therefor as exemplified by SEQ ID
NOs:10-15, respectively; proteins that have at least about 70%
sequence identity to these sequences; and biological functional
equivalents of these sequences.
[0219] It is interesting to note that mature MtDef5.1a protein (SEQ
ID NO:16) and mature MtDef5.1b protein (SEQ ID NO:17) appear to be
expressed as a dimer as components of MtDef5.1a-MtDef5.1b
proprotein (SEQ ID NO:4), wherein the peptide having the amino acid
sequence shown in SEQ ID NO:2, i.e., APKKVEP, acts as a
"linker".
[0220] The phrase "inhibiting growth of a plant pathogenic fungus"
as used herein refers to methods that result in any measurable
decrease in fungal growth, where fungal growth includes, but is not
limited to, any measurable decrease in the numbers and/or extent of
fungal cells, spores, conidia, or mycelia. As used herein,
"inhibiting growth of a plant pathogenic fungus" is also understood
to include any measurable decrease in the adverse effects cause by
fungal growth in a plant. Adverse effects of fungal growth in a
plant include any type of plant tissue damage or necrosis, any type
of plant yield reduction, any reduction in the value of the crop
plant product, and/or production of undesirable fungal metabolites
or fungal growth by-products including, but not limited to,
mycotoxins.
[0221] The term "orthologs" as used herein refers to two or more
homologous genes in different species that evolved from a common
ancestral gene by speciation. Orthologs may have the same
biological function in different species.
[0222] The phrase "operably linked" as used herein refers to the
joining of nucleic acid sequences such that one sequence can
provide a required function to a linked sequence. In the context of
a promoter, "operably linked" means that the promoter is connected
to a sequence of interest such that the transcription of that
sequence of interest is controlled and regulated by that promoter.
When the sequence of interest encodes a protein and when expression
of that protein is desired, "operably linked" means that the
promoter is linked to the sequence in such a way that the resulting
transcript will be efficiently translated. If the linkage of the
promoter to the coding sequence is a transcriptional fusion and
expression of the encoded protein is desired, the linkage is made
so that the first translational initiation codon in the resulting
transcript is the initiation codon of the coding sequence.
Alternatively, if the linkage of the promoter to the coding
sequence is a translational fusion and expression of the encoded
protein is desired, the linkage is made so that the first
translational initiation codon contained in the 5' untranslated
sequence associated with the promoter and the coding sequence is
linked such that the resulting translation product is in frame with
the translational open reading frame that encodes the desired
protein. Nucleic acid sequences that can be operably linked
include, but are not limited to, sequences that provide gene
expression functions (e.g., gene expression elements such as
promoters, 5' untranslated regions, introns, protein coding
regions, 3' untranslated regions, polyadenylation sites, and/or
transcriptional terminators), sequences that provide DNA transfer
and/or integration functions (e.g., T-DNA border sequences, site
specific recombinase recognition sites, integrase recognition
sites), sequences that provide for selective functions (e.g.,
antibiotic resistance markers, biosynthetic genes), sequences that
provide scoreable marker functions (e.g., reporter genes),
sequences that facilitate in vitro or in vivo manipulations of the
sequences (e.g., polylinker sequences, site specific recombination
sequences) and sequences that provide replication functions (e.g.,
bacterial origins of replication, autonomous replication sequences,
centromeric sequences).
[0223] The phrase "percent identity" as used herein refers to the
number of elements (i.e., amino acids or nucleotides) in a sequence
that are identical within a defined length of two optimally aligned
DNA, RNA, or protein segments. To calculate the "percent identity",
the number of identical elements is divided by the total number of
elements in the defined length of the aligned segments and
multiplied by 100. When percentage of identity is used in reference
to proteins it is understood that certain amino acid residues may
not be identical but are nonetheless conservative amino acid
substitutions that reflect substitutions of amino acid residues
with similar chemical properties (e.g., acidic or basic,
hydrophobic, hydrophilic, hydrogen bond donor or acceptor
residues). Such substitutions may not change the functional
properties of the molecule. Consequently, the percent identity of
protein sequences can be increased to account for conservative
amino acid substitutions.
[0224] The term "regeneration" as used herein refers to any method
of obtaining a whole plant from any one of, for example, a seed, a
protoplast, a plant cell, a group of plant cells, plant callus
tissue, or an excised piece of a plant.
[0225] The terms "susceptible fungus (or fungi)", susceptible
fungal infection, and the like refer to fungi that infect plants,
or human or animal patients or subjects, or fungal infections
thereof, that are responsive to, and detrimentally affected by, the
growth inhibiting or growth retarding effect, and/or
pathogenesis-retarding effect, of the MtDef5 molecules disclosed
herein. Such susceptible fungi can be killed by these molecules, or
stages of their life cycle, such as spore germination, sporulation,
or mating; infection, penetration, or spread within a plant or
mammal, are disrupted or retarded by the MtDef5 molecules. The net
effect of the Def5 molecules on such fungi and fungal infections is
to prevent, limit, decrease, treat, inhibit, or completely
eliminate fungal pathogenesis and/or damage to a plant, human, or
animal in contact with such fungus. Susceptible fungi can be
identified by any of the assay methods disclosed herein.
[0226] The term "transformation" as used herein refers to a process
of introducing an exogenous DNA sequence (e.g., a vector, a
recombinant DNA molecule) into a cell or protoplast in which that
exogenous DNA is incorporated into a chromosome or is capable of
autonomous replication.
[0227] The phrase "transgenic plant" refers to a plant or progeny
thereof derived from a transformed plant cell, protoplast, or other
transformed plant tissue wherein the plant DNA contains an
introduced exogenous DNA molecule not originally present in a
corresponding native, non-transgenic plant of the same species.
[0228] The term "treating" (or "treat" or "treatment") means
slowing, interrupting, arresting, controlling, stopping, reducing,
or reversing the progression or severity of a symptom, disorder,
condition, or disease caused by a plant or other fungal pathogen,
and can include a total elimination of all fungal disease-related
symptoms, conditions, or disorders of affected plants or human or
veterinary subjects.
[0229] The term "vector" as used herein refers to a DNA or RNA
molecule capable of replication in a host cell and/or to which
another DNA or RNA segment can be operatively linked so as to bring
about replication of the attached segment. A plasmid is an
exemplary vector.
[0230] The present invention provides isolated DNA constructs
useful for expressing the MtDef5 antifungal proteins and peptides
disclosed herein. The isolated DNA constructs comprise a
heterologous promoter, optional intron, a sequence that encodes a
MtDef5 protein or peptide with at least about 85% sequence identity
to any one of SEQ ID NOs:1, 3-9, 16-22, and 49-64), and a
polyadenylation sequence, wherein the promoter, optional intron,
the sequence encoding a signal peptide, the sequence encoding a
mature MtDef5 protein or peptide, and the polyadenylation sequence
are operably linked for expression.
[0231] Isolated DNA constructs of the invention can further
comprise a sequence that encodes a signal peptide that is operably
linked to the sequence that encodes the MtDef5 protein. The signal
peptides used in DNA constructs of the invention are selected from
the group consisting of yeast signal peptides, monocot plant signal
peptides, dicot plant signal peptides, and synthetic signal
peptides. Yeast signal peptides can be selected from the group
consisting of an .alpha.-factor signal peptide, an invertase signal
peptide, and a PHO1 signal peptide. Dicot plant signal peptides can
be selected from the group consisting of a MtDef1.1, a MsDef1.6, a
MtDef2.1, a MtDef4, a MtDef5, and a tobacco PR1b signal peptide.
Monocot plant signal peptides can be selected from the group
consisting of a cysteine endoproteinase signal peptide and an
.alpha.-amylase signal peptide. The MtDef5 signal peptides can be
selected from the group consisting of SEQ ID NOs:30-35 signal
peptide sequences.
[0232] A variety of DNA sequences encoding the MtDef5 proteins and
peptides can be used in the DNA constructs of the instant
invention. In general, a sequence that encodes a MtDef5 protein or
peptide with at least about 85% sequence identity to any one of SEQ
ID NOs:1, 3-9, 16-22, and 49-64 can be used. The encoded MtDef5
protein or peptide sequence of the DNA construct includes, but is
not limited to, sequences that encode mature MtDef5 proteins. The
encoded MtDef5 protein and peptide sequences of the DNA construct
also include nucleic acid sequences that encode MtDef5 proproteins
comprising a MtDef5 signal peptide and a mature MtDef5 protein.
MtDef5.1a-5.6 proprotein sequences are shown in SEQ ID NOs:1 and
4-9. Proteins having at least 70% sequence identity to these
sequences, and biological functional equivalents of these
sequences, are encompassed by the present invention. The MtDef5
proteins and peptides encoded by the DNA construct can thus
comprise the amino acid sequences shown in SEQ ID NOs:1, 3-9,
16-22, and 49-64. The mature MtDef5 proteins encoded by the DNA
construct can also comprise the amino acid sequences shown in SEQ
ID NOs:16-22.
[0233] In certain embodiments of the invention, the isolated DNA
construct uses promoter and polyadenylation sequences that provide
for expression of operably linked sequences when introduced into a
transgenic plant. Sequences encoding MtDef5 proteins and peptides
that are at least about 85% identical to any of SEQ ID NOs:1, 3-9,
16-22, and 49-64 are operably linked to the promoter and
polyadenylation sequences that provide for expression in transgenic
plants. The promoter that provides for expression in plants can be
selected from the group consisting of a constitutive promoter, a
tissue specific promoter, a stress induced promoter, a temporal
promoter, and a fungal infection induced promoter. Constitutive
promoters are selected from the group consisting of a CaMV35S
promoter, a FMV35S promoter, a maize ubiquitin promoter, and a rice
actin promoter. Polyadenylation sequences can be selected from the
group consisting of a CaMV35S, a NOS, a rice lactate dehydrogenase,
and a wheat Hsp17 polyadenylation sequence.
[0234] In other embodiments of the invention, the isolated DNA
construct can further comprise an intron sequence that provides for
expression of operably linked sequences when introduced into the
nuclear genome of a plant and when the intron sequence is operably
linked to the promoter, the sequence that encodes a signal peptide,
the sequence that encodes a mature MtDef5 polypeptide, and the
polyadenylation sequence. This intron sequence can be selected from
the group comprising a rice actin intron, a maize hsp70 intron, a
maize small subunit RUBISCO intron, a maize ubiquitin intron, a
maizeAdh1 intron, a rice phenylalanine ammonia lyase intron, a
sucrose synthase intron, a CAT-1 intron, a pKANNIBAL intron, the
PIV2 intron and a Super Ubiquitin intron.
[0235] The DNA construct that provides for expression of a mature
MtDef5 protein in plants can comprise a polynucleotide containing a
maize ubiquitin promoter and intron, a synthetic MtDef5 gene
encoding both an MtDef5 signal peptide and a mature MtDef5 protein
and polyadenylation signal. Another DNA construct that provides for
expression of a mature MtDef5 protein in plants can comprise a FMV
promoter, a super ubiquitin intron, a genomic MtDef5 sequence
encoding a signal peptide, an intron, and a mature MtDef5 protein,
and a tNOS terminator. DNA constructs encompassed by the present
invention include those that provide for MtDef5 protein or peptide
expression in plants further comprising sequences encoding vacuolar
targeting peptides or endoplasmic reticulum retention peptides that
are operably linked to the MtDef5 protein or peptide. A DNA
construct that provides for expression of a mature MtDef5 protein
in plants can comprise a FMV promoter, a super ubiquitin intron, a
genomic MtDef5 sequence encoding a signal peptide, an intron, and a
mature MtDef5 protein that is operably linked to a vacuolar or
endoplasmic reticulum targeting peptide, and a tNOS terminator
[0236] The DNA constructs of the invention can further comprise a
sequence encoding a selectable marker. This selectable marker is
typically used to select transgenic plants containing the DNA
construct. The selectable marker can be selected from the group
consisting of a neomycin phosphotransferase protein, a
phosphinothricin acetyltransferase protein, a glyphosate resistant
5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein, a
hygromycin phosphotransferase protein, a dihydropteroate synthase
protein, a sulfonylurea insensitive acetolactate synthase protein,
an atrazine insensitive Q protein, a nitrilase protein capable of
degrading bromoxynil, a dehalogenase protein capable of degrading
dalapon, a 2,4-dichloro-phenoxyacetate monoxygenase protein, a
methotrexate insensitive dihydrofolate reductase protein, and an
aminoethylcysteine insensitive octopine synthase protein.
[0237] The DNA constructs of the invention can further comprise a
sequence encoding a scoreable marker. This scoreable marker is
typically used to identify transgenic plants containing the DNA
construct. The scoreable marker can be selected from the group
consisting of a beta-glucuronidase protein, a green fluorescent
protein, a yellow fluorescent protein, a beta-galactosidase
protein, a luciferase protein derived from a luc gene, a luciferase
protein derived from a lux gene, a sialidase protein, streptomycin
phosphotransferase protein, a nopaline synthase protein, an
octopine synthase protein, and a chloramphenicol acetyl transferase
protein.
[0238] Transgenic plants comprising the aforementioned DNA
constructs that express MtDef5 proteins and peptides in such plants
are also provided by this invention. The transgenic plants can be
food crop plants or biofuel crop plants, and either monocots or
dicots. Transgenic monocot plants of the invention can be selected
from the group consisting of barley, corn, flax, oat, rice, rye,
sorghum, turf grass, sugarcane, and wheat. Transgenic dicot plants
of the invention can be selected from the group consisting of
alfalfa, Arabidopsis, barrel medic, banana, broccoli, bean,
cabbage, canola, carrot, cassava, cauliflower, celery, citrus,
cotton, a cucurbit, eucalyptus, garlic, grape, onion, lettuce, pea,
peanut, pepper, potato, poplar, pine, sunflower, safflower,
soybean, strawberry, sugar beet, sweet potato, tobacco, and
tomato.
[0239] In other embodiments of the invention, the DNA construct
uses a promoter and a polyadenylation sequence that provide for
expression of operably linked sequences when introduced into a
yeast cell. This promoter can be selected from the group consisting
of an AOX1 promoter, an AOX2 promoter, a PHO promoter, a MOX
promoter, a DAS promoter, an ADH promoter, a GAPDH promoter, and a
LAC4 promoter. This polyadenylation sequence can selected from the
group consisting of an AOX1, an AOX2, a CYC 1, a p40, a p76, a MOX,
a LAC4, and an actin polyadenylation sequence. The DNA construct
can comprise a polynucleotide encoding an operably linked AOX1
promoter, yeast .alpha.-factor signal sequence, mature MtDef5
defensin sequence, and an AOX1 polyadenylation sequence.
Alternatively, the DNA construct can comprise a polynucleotide
encoding an operably linked AOX1 promoter, yeast .alpha.-factor
signal sequence, mature MtDef5defensin sequence, and an AOX1
polyadenylation sequence.
[0240] DNA constructs for expression of mature MtDef5 proteins or
peptides in yeast can further comprise a selectable or scoreable
marker gene. The selectable marker gene can be selected from the
group consisting of genes encoding a ADE protein, a HIS5 protein, a
HIS4 protein, a LEU2 protein, a URA3 protein, a ARG4 protein, a
TRP1 protein, a LYS2 protein, a protein conferring resistance to a
bleomycin or phleomycin antibiotic, a protein conferring resistance
to chloramphenicol, a protein conferring resistance to G418 or
geneticin, a protein conferring resistance to hygromycin, a protein
conferring resistance to methotrexate, an a ARO4-OFP protein, and a
FZF1-4 protein.
[0241] The scoreable marker gene can be selected from the group
consisting of genes encoding a beta-glucuronidase protein, a green
fluorescent protein, a yellow fluorescent protein, a
beta-galactosidase protein, a luciferase protein derived from a luc
gene, a luciferase protein derived from a lux gene, a sialidase
protein, streptomycin phosphotransferase protein, a nopaline
synthase protein, an octopine synthase protein, and a
chloramphenicol acetyl transferase protein.
[0242] Yeast cells comprising the aforementioned DNA constructs
that comprise a promoter and a polyadenylation sequence that
provide for expression of operably linked sequences encoding MtDef5
proteins and peptides in yeast are also provided by this invention.
The yeast cells can be selected from the group consisting of
Candida, Kluveromyces, Hansuela, Pichia, Saccharomyces,
Schizosaccharomyces, and Yarrowia.
[0243] The present invention further provides for methods of
obtaining transgenic plants capable of inhibiting growth of a plant
pathogenic fungus. These methods comprise the steps of: introducing
the DNA construct that provides for expression of a MtDef5 protein
or peptide in a plant, plant cell, or plant tissue, and obtaining a
transgenic plant comprising the DNA construct that expresses a
plant pathogenic fungus inhibitory amount of a MtDef5 protein or
peptide. To practice this method, the DNA construct can be
introduced into the plant by a method selected, for example, from
the group consisting of particle bombardment, DNA transfection, DNA
electroporation, and T-DNA mediated transformation.
[0244] The DNA construct used in certain embodiments of this method
can further comprise a selectable marker gene. When the DNA
construct further comprises a selectable marker gene, a transgenic
plant of the invention is obtained by growing the plant, plant
cell, or plant tissue under conditions requiring expression of the
selectable marker gene for plant growth. This selectable marker
gene can be selected from the group consisting of genes encoding a
neomycin phosphotransferase protein, a phosphinothricin
acetyltransferase protein, a glyphosate resistant
5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein, a
hygromycin phosphotransferase protein, a dihydropteroate synthase
protein, a sulfonylurea insensitive acetolactate synthase protein,
an atrazine insensitive Q protein, a nitrilase protein capable of
degrading bromoxynil, a dehalogenase protein capable of degrading
dalapon, a 2,4-dichlorophenoxyacetate monoxygenase protein, a
methotrexate insensitive dihydrofolate reductase protein, and an
aminoethylcysteine insensitive octopine synthase protein.
[0245] These methods of obtaining transgenic plants can also employ
DNA constructs that further comprise a scoreable marker gene that
functions in plants. In this case, expression of the scoreable
marker gene is assayed to obtain a transgenic plant cell or a
regenerated transgenic plant. Scoreable marker genes can be
selected from the group consisting of genes encoding a
beta-glucuronidase protein, a green fluorescent protein, a yellow
fluorescent protein, a beta-galactosidase protein, a luciferase
protein derived from a luc gene, a luciferase protein derived from
a lux gene, a sialidase protein, streptomycin phosphotransferase
protein, a nopaline synthase protein, an octopine synthase protein,
and a chloramphenicol acetyl transferase protein.
[0246] In this method, a transgenic plant that expresses a plant
pathogenic fungus inhibitory amount of the mature MtDef5 protein or
peptide can be obtained by assaying expression of a MtDef5 encoding
transgene in the transgenic plant. Expression of the MtDef5
encoding transgene can be assayed by a method selected from, for
example, the group consisting of an immunoassay, an enzyme-linked
immunoassay, an assay based on detection by RNA hybridization, and
an assay based on detection by a reverse-transcriptase polymerase
chain reaction. Alternatively, the expression of the MtDef5
encoding transgene is assayed by exposing the transgenic plant to a
plant pathogenic fungus and determining if growth of the plant
pathogenic fungus is inhibited. A plant pathogenic fungus
inhibitory amount of mature MtDef5 protein is at least about 0.05
PPM, about 0.5 PPM, about 1.0 PPM, or about 2.0 PPM, where PPM is
"parts per million" of MtDef5 protein present in fresh weight plant
tissue. Typically, microgram amounts of MtDef5 protein are present
per gram fresh weight of transgenic plant tissue. In preferred
embodiments, the plant pathogenic fungus inhibitory amount of
MtDef5 protein is at least about 0.5 PPM. In more preferred
embodiments, the plant pathogenic fungus inhibitory amount of
MtDef5 is at least about 1.0 PPM. In the most preferred
embodiments, the plant pathogenic fungus inhibitory amount of
MtDef5 is at least about 2.0 PPM.
[0247] This method provides for inhibition of the growth of a
variety of MtDef5 molecule-susceptible plant pathogenic fungi.
Plant pathogenic fungi inhibited by the method can be selected from
the group consisting of an Alternaria sp., an Ascochyta sp., an
Aspergillus sp., a Botrytis sp.; a Cercospora sp., a Colletotrichum
sp., a Diplodia sp., an Erysiphe sp., a Fusarium sp.,
Gaeumannomyces sp., Helminthosporium sp., Macrophomina sp., a
Nectria sp., a Peronospora sp., a Phakopsora sp., a Phoma sp., a
Phymatotrichum sp., a Phytophthora sp., a Plasmopara sp., a
Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia
sp., a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a
Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula
sp., a Venturia sp., and a Verticillium sp.
[0248] The present invention also provides for transgenic plants
capable of inhibiting the growth of a plant pathogenic fungus that
are produced by the methods described herein. Transgenic plants
produced by a process comprising the steps of introducing a DNA
construct of the invention into a plant, a plant cell, or a plant
tissue and obtaining a transgenic plant comprising the DNA
construct that expresses a plant pathogenic fungus inhibitory
amount of a MtDef5 protein or peptice are also thus contemplated by
this invention. A plant pathogenic fungus inhibitory amount of
mature MtDef5 protein or peptide is at least about 0.05 PPM, 0.5
PPM, 1.0 PPM, or 2.0 PPM, where PPM is "parts per million" of
MtDef5 protein present in fresh weight plant tissue.
[0249] The instant invention further provides for methods of
producing a MtDef5 protein or peptide that has at least about 85%
sequence identity to the amino acid sequences shown in SEQ ID
NOs:1, 3-9, 16-22, and 49-64. The methods for producing the mature
MtDef5 protein or peptide comprise the steps of culturing a yeast
cell comprising a DNA construct that provides for expression of a
MtDef5 protein or peptide in yeast under conditions wherein the
yeast cell expresses a MtDef5 protein or peptide, and isolating the
MtDef5 protein or peptide from the culture of the preceding step.
The MtDef5 protein or peptide can be isolated in the second step
from the cell culture medium. Alternatively, the MtDef5 protein or
peptide can be isolated from the yeast cells. In certain
embodiments of this method, the MtDef5 protein is a mature MtDef5
protein. The yeast cell used in this method can be a Pichia cell
that comprises a DNA construct containing an AOX1 promoter that is
operably linked to a sequence encoding a signal peptide and a
sequence encoding a mature MtDef5 protein, and the conditions that
provide for expression of the mature MtDef5 protein would comprise
culturing the Pichia cell in the presence of methanol.
[0250] The present invention also provides for an antibody that
recognizes a MtDef5 protein or peptide having at least about 85%
sequence identity to any one of sequences SEQ ID NOs:1, 3-9, 16-22,
and 49-64. Kits for specifically detecting a MtDef5 protein or
peptide with at least about 85% sequence identity to any one of SEQ
ID NOs:1, 3-9, 16-22, and 49-64, comprising the antibody that
recognizes a MtDef5 protein or peptide with at least about 85%
sequence identity to any one of SEQ ID NOs:1, 3-9, 16-22, and 49-64
and a reagent for detecting the antibody, are also provided by this
invention.
[0251] Certain isolated nucleotide sequences are also provided by
this invention. The isolated nucleotide sequences of the invention
comprise a MtDef5 coding sequence selected from the group
consisting of SEQ ID NOs:10-15, 23-29, and 65-78, and a MtDef5 or
other signal peptide coding sequence selected from the group
consisting of SEQ ID NOs:30-35 and 42-48. Oligo-nucleotides derived
from the above sequences are further contemplated. Such
oligonucleotides can be used to identify transgenic plants
containing the noted nucleotide sequences, or to identify material
obtained from these transgenic plants. Methods for using these
oligonucleotides to identify the transgenic plant materials and
kits for performing these methods are also contemplated.
[0252] Various isolated, purified MtDef5 proprotein, mature, and
peptide sequences are also provided by this invention as variously
shown in SEQ ID NOs:1, 3-9, 16-22, and 49-64, as well as coding
sequences therefor as shown in SEQ ID NOs:10-15, 23-29, and 65-78,
respectively, as noted above.
DNA Constructs Comprising Plant Expression Cassettes
[0253] The construction of expression cassettes for use in
monocotyledonous or dicotyledonous plants is well established.
Expression cassettes are DNA constructs wherein various promoter,
coding, and polyadenylation sequences are operably linked. In
general, expression cassettes typically comprise a promoter that is
operably linked to a sequence of interest, which is operably linked
to a polyadenylation or terminator region. In certain instances
including, but not limited to, the expression of transgenes in
monocot plants, it may also be useful to include an intron
sequence. When an intron sequence is included it is typically
placed in the 5' untranslated leader region of the transgene. In
certain instances, it may also be useful to incorporate specific 5'
untranslated sequences in a transgene to enhance transcript
stability or to promote efficient translation of the
transcript.
Promoters
[0254] A variety of promoters can be used in the practice of this
invention. One broad class of useful promoters are referred to as
"constitutive" promoters in that they are active in most plant
organs throughout plant development. For example, the promoter can
be a viral promoter such as a CaMV35S or FMV35S promoter. The
CaMV35S and FMV35S promoters are active in a variety of transformed
plant tissues and most plant organs (e.g., callus, leaf, seed and
root). Enhanced or duplicate versions of the CaMV35S and FMV35S
promoters are particularly useful in the practice of this invention
(U.S. Pat. No. 5,378,619, incorporated herein by reference in its
entirety). Other useful promoters include the nopaline synthase
(NOS) and octopine synthase (OCS) promoters (which are carried on
tumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic
virus (CaMV) 19S promoters, a maize ubiquitin promoter, the rice
Act1 promoter, and the Figwort Mosaic Virus (FMV) 35S promoter
(see, e.g., U.S. Pat. No. 5,463,175, incorporated herein by
reference in its entirety). It is understood that this group of
exemplary promoters is non-limiting and that one skilled in the art
could employ other promoters that are not explicitly cited here in
the practice of this invention.
[0255] Promoters that are active in certain plant tissues (i.e.,
tissue specific promoters) can also be used to drive expression of
MtDef5 proteins and peptides. Expression of MtDef5 proteins and
peptides in the tissue that is typically infected by a fungal
pathogen is anticipated to be particularly useful. Thus, expression
in reproductive tissues, seeds, roots, stems, or leaves can be
particularly useful in combating infection of those tissues by
certain fungal pathogens in certain crops. Examples of useful
tissue-specific, developmentally regulated promoters include but
are not limited to the .beta.-conglycinin 7S promoter (Doyle et
al., 1986), seed-specific promoters (Lam and Chua, 1991), and
promoters associated with napin, phaseolin, zein, soybean trypsin
inhibitor, ACP, stearoyl-ACP desaturase, or oleosin genes. Examples
of root specific promoters include but are not limited to the RB7
and RD2 promoters described in U.S. Pat. Nos. 5,459,252 and
5,837,876, respectively.
[0256] Another class of useful promoters are promoters that are
induced by various environmental stimuli. Promoters that are
induced by environmental stimuli include, but are not limited to,
promoters induced by heat (e.g., heat shock promoters such as
Hsp70), promoters induced by light (e.g., the light-inducible
promoter from the small subunit of ribulose 1,5-bisphosphate
carboxylase, ssRUBISCO, a very abundant plant polypeptide),
promoters induced by cold (e.g., COR promoters), promoters induced
by oxidative stress (e.g., catalase promoters), promoters induced
by drought (e.g., the wheat Em and rice rab16A promoters), and
promoters induced by multiple environmental signals (e.g., rd29A
promoters, Glutathione-S-transferase (GST) promoters).
[0257] Promoters that are induced by fungal infections in plants
can also be used to drive expression of MtDef5 proteins and
peptides. Useful promoters induced by fungal infections include
those promoters associated with genes involved in phenylpropanoid
metabolism (e.g., phenylalanine ammonia lyase, chalcone synthase
promoters), genes that modify plant cell walls (e.g.,
hydroxyproline-rich glycoprotein, glycine-rich protein, and
peroxidase promoters), genes encoding enzymes that degrade fungal
cell walls (e.g., chitinase or glucanase promoters), genes encoding
thaumatin-like protein promoters, or genes encoding proteins of
unknown function that display significant induction upon fungal
infection. Maize and flax promoters, designated as Mis1 and Fis1,
respectively, are also induced by fungal infections in plants and
can be used (U.S. Patent Application 20020115849).
[0258] Depending on the fungus to which protection is sought, the
present MtDef5 proteins and peptides can be expressed in any tissue
or organ in the plant where the fungus attacks. In the case of
Fusarium for example, a preferred site for expression is in the
roots. In the case of those fungi that infect by entering external
plant surfaces, accumulation of the MtDef5 proteins and peptides in
the apoplast is preferred, and these molecules can be expressed in
roots, stems, leaves, etc., by the use of tissue-specific
promoters.
[0259] Promoters active at particular developmental stages in the
plant life cycle can also be used to optimize resistance to fungal
infection and/or damage when it is most needed.
Introns
[0260] An intron can also be included in the DNA expression
construct, especially in instances when the sequence of interest is
to be expressed in monocot plants. For monocot plant use, introns
such as the maize hsp70 intron (U.S. Pat. No. 5,424,412;
incorporated by reference herein in its entirety), the maize
ubiquitin intron, the Adh intron 1 (Callis et al., 1987), the
sucrose synthase intron (Vasil et al., 1989) or the rice Act1
intron (McElroy et al., 1990) can be used. Dicot plant introns that
are useful include introns such as the CAT-1 intron (Cazzonnelli
and Velten, 2003), the pKANNIBAL intron (Wesley et al., 2001;
Collier et al., 2005), the PIV2 intron (Mankin et al., 1997) and
the "Super Ubiquitin" intron (U.S. Pat. No. 6,596,925, incorporated
herein by reference in its entirety; Collier et al., 2005) that
have been operably integrated into transgenes. It is understood
that this group of exemplary introns is non-limiting and that one
skilled in the art could employ other introns that are not
explicitly cited here in the practice of this invention.
[0261] Certain embodiments of this invention comprise a sequence
encoding a signal peptide that facilitates secretion of the mature
MtDef5 proteins or peptides from plant cells. Portions of the
MtDef5.1a-5.6 cDNAs (SEQ ID NOs:1 and 4-9) contain sequences (SEQ
ID NOs:30-35) that encode MtDef5 signal peptides that can be used
for secreting MtDef5 proteins or peptides from plant or other
cells.
[0262] MtDef5 signal peptide-encoding sequences can be used in the
DNA constructs of the invention in a variety of ways. These MtDef5
signal sequences can be the MtDef5 signal sequences that are
associated with a given MtDef5 proprotein coding sequence in a
given MtDef5 cDNA or genomic clone. Alternatively, the MtDef5
signal peptide can be operably linked to a distinct mature MtDef5
protein (or MtDef5 peptide) encoding sequence (i.e., MtDef5 signal
peptide and mature protein (or peptide) encoding sequences derived
from distinct genomic or cDNA clones can be operably linked). In
the DNA constructs, nucleotide sequences encoding any MtDef5
proprotein comprising both a MtDef5 signal peptide and a mature
MtDef5 protein can also be used instead of two distinct signal
peptide and mature MtDef5 protein encoding sequences. MtDef5
proproteins encoded by these sequences include, but are not limited
to, MtDef5 proprotein sequences including SEQ ID NOs:1 and 4-9,
proteins that have at least about 70% sequence identity to these
sequences, and the biological functional equivalents of these
sequences. It is anticipated that the MtDef5 signal peptides can be
used to secrete mature MtDef5 proteins and peptices from either
monocot or dicot plant cells. Synthetic nucleotide sequences that
encode the MtDef5 signal peptide sequences can also be used. Such
synthetic sequences can be deduced from the MtDef5 signal peptide
sequences disclosed herein through application of the genetic code.
Table 1 provides a list of MtDef5 and other signal peptides that
can be used to secrete mature MtDef5 or other proteins from
cells.
TABLE-US-00001 TABLE 1 MtDef5 and MtDef4 Signal Peptide Sequences
SEQ ID NO: Amino Acid Sequence Source SEQ ID
MTSSASKFYTIFIFVCLAFLFISTSEVEA MtDef5.1a- NO: 30 5.1b SEQ ID
MASSSPKLFTIFLFLILVVLLFSTSEVQA MtDef5.2 NO: 31 SEQ ID
MTSSATKFYTIFVFVCLALLLISICEVEA MtDef5.3 NO: 32 SEQ ID
MASSTLKFNTIFLFLSLALLLFFTLEVQG MtDef5.4 NO: 33 SEQ ID
MASSALKYYTFFLFFILALILLPTLEVQG MtDef5.5 NO: 34 SEQ ID MVCTEVQA
MtDef5.6 NO: 35 SEQ ID MARSVPLVSTIFVFLLLLVATGPSMVAEA MtDef4 NO: 48
signal peptide consensus SEQ ID MARSVPLVSTIFVFLLLLVATGPSMVAEA
MtDef4.1 NO: 42 (H33R) SEQ ID MARSVPLVSTIFVFFLLIVATEMGPSMVAA
MtDef4.2 NO: 43 SEQ ID MARSVPLVSTIFVFFLLLVATEMGPIMVAEA MtDef4.3 NO:
44 SEQ ID MARSVFLVSTIFVFLLVLVATGPSMVAEA AL385796* NO: 45 SEQ ID
MARSVSLVFTIFVFLLLVVATGPSMVAEA AW573770* NO: 46 SEQ ID
MARSVPLVSTIFVFLLLLVATGPSMVGEA BE999096* NO: 47 *GenBank Accession
Number (including signal and mature peptide sequences)
[0263] Alternatively, signal peptide sequences derived from other
Medicago defensin proteins (Hanks et al, 2005) can be used.
Examples of such other Medicago defensin protein signal peptides
include, but are not limited to, signal peptides of MtDef1.1,
MsDef1.6, and MtDef2.1. Another example of a useful signal peptide
encoding sequence that can be used in monocot plants is the signal
peptide derived from a barley cysteine endoproteinase gene (Koehler
and Ho, 1990). Another example of a useful signal peptide encoding
sequence that can be used in dicot plants is the tobacco PR1b
signal peptide. This group of signal peptides is meant to be
exemplary and non-limiting, and one skilled in the art could employ
other signal peptides in the practice of the present invention that
are not explicitly cited here.
[0264] In the present invention, a sequence encoding a mature
MtDef5 protein or peptide is typically linked to the signal peptide
encoding sequence. A variety of DNA sequences encoding a variety of
mature MtDef5 proteins and peptides can be used in practicing this
invention. The DNA sequence can encode mature MtDef5 proteins and
peptides that include, but are not limited to, the amino acid
sequences shown in SEQ ID NOs:16-22 and 49-64, and biological
functional equivalents of any of the foregoing amino acid
sequences. Biological functional equivalents of an MtDef5
proprotein, mature protein, or peptide also include, but are not
limited to, MtDef5 proproteins, mature proteins, and peptides with
at least about 85% sequence identity to any of SEQ ID NOs:1, 3-9,
16-22, and 49-64. In certain embodiments of the invention, a mature
MtDef5 protein- or peptide-encoding sequence can be physically
derived or obtained from either genomic DNA or cDNA obtained from
Medicago truncatula plant tissue. Such methods for obtaining
similar defensin genes from Medicago truncatula have been described
(Hanks et al, 2005). The native or endogenous MtDef5-encoding
nucleotide sequence is derived from a dicotyledonous plant in which
it is ordinarily expressed under the control of the endogenous
MtDef5 promoter sequence. Consequently, it is expected that the
endogenous or naturally occurring MtDef5-encoding nucleotide
sequence can be expressed in plants. In general, nucleic acids that
encode MtDef5 proteins and peptides can be obtained from MtDef5
consensus nucleotide sequences, from synthetic MtDef5 genes derived
by "back-translation" of MtDef5 polypeptide sequences, from genomic
clones, from deduced coding sequences derived from genomic clones,
from cDNA or EST sequences, and from any of the foregoing sequences
that have been subjected to mutagenesis. Examples of nucleic acids
that contain mature MtDef5 protein-encoding nucleotide sequences
include, but are not limited to, SEQ ID NOs:10-15 and 23-29.
Nucleotide sequences encoding various MtDef5 proteins and peptides
can also be obtained from genomic sequences by removing deduced
intron sequences to obtain the deduced MtDef5 coding sequences for
MtDef5 proteins and peptides. Removal of the intron sequences from
MtDef5 genomic clones can be effected by in vitro mutagenesis
techniques. Alternatively, the intron sequences can be removed in
silico (in a computer file) and the resultant deduced coding
sequence synthesized by standard DNA synthesis techniques. It
should also be noted that the closely related plant Medicago sativa
may be a source of MsDef4 ESTs that encode MsDef4 proteins that are
either identical to, or otherwise biologically equivalent to, the
MtDef5 proteins ands and peptides of the present invention.
Nucleotide sequences encoding the MtDef5 proteins and peptides of
this invention can thus also be derived from other Medicago sp.
such as Medicago sativa, and can be used in this invention.
Portions of the aforementioned nucleotide sequences containing the
sequences that encode mature MtDef5 proteins and peptides can be
operably linked to either the native MtDef5 signal peptide sequence
to which they are ordinarily linked or to another signal peptide
via standard recombinant DNA techniques, or by DNA synthesis
methods.
[0265] In other embodiments of the invention, the MtDef5-encoding
nucleotide sequence can be synthesized de novo from an MtDef5
protein or peptide sequence disclosed herein. The sequence of the
MtDef5-encoding nucleotide sequence can be deduced from the MtDef5
protein or peptide sequence through use of the genetic code.
Computer programs such as "BackTranslate" (GCG.TM. Package,
Acclerys, Inc. San Diego, Calif.) can be used to convert a protein
or peptide sequence to the corresponding nucleotide sequence that
encodes the protein or peptide.
[0266] Furthermore, the synthetic MtDef5-encoding nucleotide
sequence can be designed so that it will be optimally expressed in
plants. U.S. Pat. No. 5,500,365 describes a method for synthesizing
plant genes to optimize the expression level of the protein encoded
by the synthesized gene. This method relates to the modification of
the structural gene sequences of the exogenous transgene, to make
them more "plant-like" and therefore more efficiently transcribed,
processed, translated, and expressed by the plant. Features of
genes that are expressed well in plants include use of codons that
are commonly used by the plant host and elimination of sequences
that can cause undesired intron splicing or polyadenylation in the
coding region of a gene transcript. A similar method for obtaining
enhanced expression of transgenes in monocotyledonous plants is
disclosed in U.S. Pat. No. 5,689,052. Furthermore, the synthetic
design methods disclosed in U.S. Pat. Nos. 5,500,365 and 5,689,052
could also be used to synthesize a signal peptide encoding sequence
that is optimized for expression in plants in general or monocot
plants in particular.
[0267] In other embodiments of the invention, sequences encoding
peptides that provide for the localization of an MtDef5 in
subcellular organelles can be operably linked to the sequences that
encode the MtDef5 protein or peptide. MtDef5 proteins and peptides
that are operably linked to a signal peptide are expected to enter
the secretion pathway and can be retained by organelles such as the
endoplasmic reticulum (ER) or targeted to the vacuole by operably
linking the appropriate retention or targeting peptides to the
C-terminus of the MtDef5 protein or peptide. Examples of vacuolar
targeting peptides include, but are not limited to, a CTPP vacuolar
targeting signal from the barley lectin gene. Examples of ER
targeting peptides include, but are not limited to, a peptide
comprising a KDEL amino acid sequence.
[0268] Localization of MtDef5 proteins and peptides in either the
endoplasmic reticulum or the vacuole can provide for desirable
properties such as increased expression in transgenic plants and/or
increased efficacy in inhibiting fungal growth in transgenic
plants.
[0269] As noted above, the sequence of interest can also be
operably linked to a 3' non-translated region containing a
polyadenylation signal. This polyadenylation signal provides for
the addition of a polyadenylate sequence to the 3' end of the RNA.
The Agrobacterium tumor-inducing (Ti) plasmid nopaline synthase
(NOS) gene 3' and the pea ssRUBISCO E9 gene 3' un-translated
regions contain polyadenylate signals and represent non-limiting
examples of such 3' untranslated regions that can be used in the
practice of this invention. It is understood that this group of
exemplary polyadenylation regions is non-limiting and that one
skilled in the art could employ other polyadenylation regions that
are not explicitly cited here in the practice of this
invention.
[0270] The DNA constructs that comprise the plant expression
cassettes described above are typically maintained in various
vectors. Vectors contain sequences that provide for the replication
of the vector and covalently linked sequences in a host cell. For
example, bacterial vectors will contain origins of replication that
permit replication of the vector in one or more bacterial hosts.
Agrobacterium-mediated plant transformation vectors typically
comprise sequences that permit replication in both E. coli and
Agrobacterium as well as one or more "border" sequences positioned
so as to permit integration of the expression cassette into the
plant chromosome. Such Agrobacterium vectors can be adapted for use
in either Agrobacterium tumefaciens or Agrobacterium rhizogenes.
Selectable markers encoding genes that confer resistance to
antibiotics are also typically included in the vectors to provide
for their maintenance in bacterial hosts.
Methods for Obtaining Antifungal Plants
[0271] Methods of obtaining a transgenic plant capable of
inhibiting growth of a plant pathogenic fungus are also provided by
this invention. First, expression vectors suitable for expression
of the MtDef5 protein or peptide in various dicot and monocot
plants are introduced into a plant, a plant cell, a protplast, or a
plant tissue using transformation techniques as described herein.
Next, a transgenic plant containing or comprising the MtDef5
expression vector is obtained by regenerating that transgenic plant
from the plant, plant cell, protoplast, or plant tissue that
received the expression vector. The final step is to obtain a
transgenic plant that expresses a plant pathogenic fungus
inhibitory amount of the mature MtDef5 protein or peptide, where a
"plant pathogenic fungus inhibitory amount" is a level of MtDef5
protein or peptide sufficient to provide any measurable decrease in
fungal growth in the transgenic plant and/or any measurable
decrease in the adverse effects caused by fungal growth in the
transgenic plant.
[0272] Any of the MtDef5 expression vectors can be introduced into
the chromosomes of a host plant via methods such as
Agrobacterium-mediated transformation, Rhizobium-mediated
transformation, Sinorhizobium-mediated transformation,
particle-mediated transformation, DNA transfection, DNA
electroporation, or "whiskers"-mediated transformation. The
aforementioned methods of introducing transgenes are well known to
those skilled in the art and are described in U.S. Patent
Application No. 20050289673 (Agrobacterium-mediated transformation
of corn), U.S. Pat. No. 7,002,058 (Agrobacterium-mediated
transformation of soybean), U.S. Pat. No. 6,365,807 (particle
mediated transformation of rice), and U.S. Pat. No. 5,004,863
(Agrobacterium-mediated transformation of cotton), each of which
are incorporated herein by reference in their entirety. Methods of
using bacteria such as Rhizobium or Sinorhizobium to transform
plants are described in Broothaerts, et al., Nature. 2005,10;
433(7026):629-33. It is further understood that the MtDef5
expression vector can comprise cis-acting site-specific
recombination sites recognized by site-specific recombinases,
including Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. Methods of
integrating DNA molecules at specific locations in the genomes of
transgenic plants through use of site-specific recombinases can
then be used (U.S. Pat. No. 7,102,055). Those skilled in the art
will further appreciate that any of these gene transfer techniques
can be used to introduce the expression vector into the chromosome
of a plant cell, a protoplast, a plant tissue, or a plant.
[0273] Methods of introducing plant minichromosomes comprising
plant centromeres that provide for the maintenance of the
recombinant minichromosome in a transgenic plant can also be used
in practicing this invention (U.S. Pat. No. 6,972,197). In these
embodiments of the invention, the transgenic plants harbor the
minichromosomes as extrachromosomal elements that are not
integrated into the chromosomes of the host plant.
[0274] Transgenic plants are typically obtained by linking the gene
of interest (in this case an MtDef5-encoding nucleotide sequence)
to a selectable marker gene, introducing the linked transgenes into
a plant cell, a protoplast, a plant tissue, or a plant by any one
of the methods described above, and regenerating or otherwise
recovering the transgenic plant under conditions requiring
expression of the selectable marker gene for plant growth. The
selectable marker gene can be a gene encoding a neomycin
phosphotransferase protein, a phosphinothricin acetyltransferase
protein, a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate
synthase (EPSPS) protein, a hygromycin phosphotransferase protein,
a dihydropteroate synthase protein, a sulfonylurea insensitive
acetolactate synthase protein, an atrazine insensitive Q protein, a
nitrilase protein capable of degrading bromoxynil, a dehalogenase
protein capable of degrading dalapon, a 2,4-dichlorophenoxyacetate
monoxygenase protein, a methotrexate insensitive dihydrofolate
reductase protein, or an aminoethylcysteine insensitive octopine
synthase protein. The corresponding selective agents used in
conjunction with each gene can be: neomycin (for neomycin
phosphotransferase protein selection), phosphinotricin (for
phosphinothricin acetyltransferase protein selection), glyphosate
(for glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate
synthase (EPSPS) protein selection), hygromycin (for hygromycin
phosphotransferase protein selection), sulfadiazine (for a
dihydropteroate synthase protein selection), chlorsulfuron (for a
sulfonylurea insensitive acetolactate synthase protein selection),
atrazine (for an atrazine insensitive Q protein selection),
bromoxinyl (for a nitrilase protein selection), dalapon (for a
dehalogenase protein selection), 2,4-dichlorophenoxyacetic acid
(for a 2,4-dichlorophenoxyacetate monoxygenase protein selection),
methotrexate (for a methotrexate insensitive dihydrofolate
reductase protein selection), or aminoethylcysteine (for an
aminoethylcysteine insensitive octopine synthase protein
selection).
[0275] Transgenic plants can also be obtained by linking a gene of
interest (in this case an MtDef5-encoding nucleotide sequence) to a
scoreable marker gene, introducing the linked transgenes into a
plant cell by any one of the methods described above, and
regenerating the transgenic plants from transformed plant cells
that test positive for expression of the scoreable marker gene. The
scoreable marker gene can be a gene encoding a beta-glucuronidase
protein, a green fluorescent protein, a yellow fluorescent protein,
a beta-galactosidase protein, a luciferase protein derived from a
luc gene, a luciferase protein derived from a lux gene, a sialidase
protein, streptomycin phosphotransferase protein, a nopaline
synthase protein, an octopine synthase protein, or a
chloramphenicol acetyl transferase protein.
[0276] When the expression vector is introduced into a plant cell
or plant tissue, the transformed cells or tissues are typically
regenerated into whole plants by culturing these cells or tissues
under conditions that promote the formation of a whole plant (i.e.,
the process of regenerating leaves, stems, roots, and, in certain
plants, reproductive tissues). The development or regeneration of
transgenic plants from either single plant protoplasts or various
explants is well known in the art (Horsch, R. B. et al. 1985). This
regeneration and growth process typically includes the steps of
selection of transformed cells and culturing selected cells under
conditions that will yield rooted plantlets. The resulting
transgenic rooted shoots are thereafter planted in an appropriate
plant growth medium such as soil. Alternatively, transgenes can
also be introduced into isolated plant shoot meristems and plants
regenerated without going through callus stage tissue culture (U.S.
Pat. No. 7,002,058). When the transgene is introduced directly into
a plant, or more specifically into the meristematic tissue of a
plant, seed can be harvested from the plant and selected or scored
for presence of the transgene. In the case of transgenic plant
species that reproduce sexually, seeds can be collected from plants
that have been "selfed" (self-pollinated) or out-crossed (i.e.,
used as a pollen donor or recipient) to establish and maintain the
transgenic plant line. Transgenic plants that do not sexually
reproduce can be vegetatively propagated to establish and maintain
the transgenic plant line. As used herein, "transgenic plant line"
refers to transgenic plants derived from a transformation event
where the transgene has inserted into one or more locations in the
plant genome. In a related aspect, the present invention also
encompasses a seed produced by the transformed plant, a progeny
from such seed, and a seed produced by the progeny of the original
transgenic plant, produced in accordance with the above process.
Such progeny and seeds will have an MtDef5 protein- or
peptide-encoding transgene stably incorporated into their genome,
and such progeny plants will inherit the traits afforded by the
introduction of a stable transgene in Mendelian fashion. All such
transgenic plants having incorporated into their genome transgenic
DNA segments encoding one or more MtDef5 proteins or peptide are
aspects of this invention. It is further recognized that transgenic
plants containing the DNA constructs described herein, and
materials derived therefrom, may be identified through use of PCR
or other methods that can specifically detect the sequences in the
DNA constructs.
Identification of Transgenic Plants and Quantitation of MtDef5
Expression
[0277] Once a transgenic plant is regenerated or recovered, a
variety of methods can be used to identify or obtain a transgenic
plant that expresses a plant pathogenic fungus inhibitory amount of
MtDef5. One general set of methods is to perform assays that
measure the amount of MtDef5 that is produced. For example, various
antibody-based detection methods employing antibodies that
recognize MtDef5 can be used to quantitate the amount of MtDef5
produced. Examples of such antibody-based assays include, but are
not limited to, ELISAs, RIAs, or other methods wherein an
MtDef5-recognizing antibody is detectably labelled with an enzyme,
an isotope, a fluorophore, a lanthanide, and the like. By using
purified or isolated MtDef5 protein or peptide as a reference
standard in such assays (i.e., providing known amounts of MtDef5),
the amount of MtDef5 present in the plant tissue in a mole per gram
of plant material or mass per gram of plant material can be
determined. The MtDef5 protein or peptide will typically be
expressed in the transgenic plant at the level of "parts per
million" or "PPM", where microgram levels of MtDef5 are present in
gram amounts of fresh weight plant tissue. In this case, 1
microgram of MtDef5 per 1 gram of fresh weight plant tissue would
represent a MtDef5 concentration of 1 PPM. A plant pathogenic
fungus inhibitory amount of MtDef5 protein or peptide is at least
about 0.05 PPM (i.e., 0.05 .mu.g MtDef5 protein or peptide per gram
fresh weight plant tissue). In preferred embodiments, a plant
pathogenic fungus inhibitory amount of MtDef5 is at least about 0.5
PPM. In more preferred embodiments, the amount of MtDef5 is at
least about 1.0 PPM. In the most preferred embodiments, the amount
of MtDef5 protein or peptide is at least about 2.0 PPM.
[0278] Alternatively, the amount of MtDef5-encoding mRNA produced
by the transgenic plant can be determined to identify plants that
express plant pathogenic fungus inhibitory amounts of MtDef5.
Techniques for relating the amount of protein produced to the
amount of RNA produced are well known to those skilled in the art
and include methods such as constructing a standard curve that
relates specific RNA levels (i.e., MtDef5 mRNA) to levels of the
MtDef5 protein or peptide (determined by immunologic or other
methods). Methods of quantitating MtDef5 mRNA typically involve
specific hybridization of a polynucleotide to either the MtDef5
mRNA or to a cDNA (complementary DNA) or PCR product derived from
the MtDef5 RNA. Such polynucleotide probes can be derived from
either the sense and/or antisense strand nucleotide sequences of
the MtDef5-encoding transgene. Hybridization of a polynucleotide
probe to the MtDef5 mRNA or cDNA can be detected by methods
including, but not limited to, use of probes labelled with an
isotope, a fluorophore, a lanthanide, or a hapten such as biotin or
digoxigenin. Hybridization of the labelled probe can be detected
when the MtDef5 RNA is in solution or immobilized on a solid
support such as a membrane. When quantitating MtDef5 RNA by use of
a quantitative reverse-transcriptase Polymerase Chain Reaction
(qRT-PCR), the MtDef5-derived PCR product can be detected by use of
any of the aforementioned labelled polynucleotide probes, by use of
an intercalating dye such as ethidium bromide or SYBR green, or use
of a hybridization probe containing a fluorophore and a quencher
such that emission from the fluorophore is only detected when the
fluorophore is released by the 5' nuclease activity of the
polymerase used in the PCR reaction (i.e., a TaqMan.TM. reaction;
Applied Biosystems, Foster City, Calif.) or when the fluorophore
and quencher are displaced by polymerase mediated synthesis of the
complementary strand (i.e., Scorpion.TM. or Molecular Beacon.TM.
probes). Various methods for conducting qRT-PCR analysis to
quantitate mRNA levels are well characterized (Bustin, S. A.;
2002). Fluorescent probes that are activated by the action of
enzymes that recognize mismatched nucleic acid complexes (i.e.,
Invader.TM., Third Wave Technologies, Madison, Wis.) can also be
used to quantitate RNA. Those skilled in the art will also
understand that RNA quantitation techniques such as Quantitative
Nucleic Acid Sequence Based Amplification (Q-NASBA.TM.) can be used
to quantitate MtDef5-encoding mRNA and identify expressing
plants.
[0279] Transgenic plants that express plant pathogenic fungus
inhibitory amounts of MtDef5 proteins or peptides can also be
identified by directly assaying such plants for inhibition of the
growth of a plant pathogenic fungus. Such assays can be used either
independently or in conjunction with MtDef5 expression assays to
identify the resistant transgenic plants. Infection of certain
plants with certain plant pathogen fungi can result in distinctive
effects on plant growth that are readily observed. Consequently,
one can distinguish MtDef5-expressing transgenic plants by simply
challenging such plants transformed with MtDef5-encoding transgenes
with pathogenic plant fungi and observing reduction of the symptoms
normally associated with such infections. Such observations are
facilitated by co-infecting otherwise identical, non-transgenic
control plants that do not contain an MtDef5 encoding transgene
with the same type and dose of plant pathogenic fungi used to
infect the transgenic plants that contain an MtDef5-encoding
transgene. Identification of transgenic plants that control or
combat fungal infection can be based on observation of decreased
disease symptoms, measurement of the decreased fungal growth in the
infected plant (e.g., by determining the numbers of colony forming
units per gram of infected tissue) and/or by measurement of the
amount of mycotoxins present in infected plant tissue. The use of
fungal disease severity assays and colony formation assays in
conjunction with expression assays to identify transgenic
MsDef1-expressing potato plants that are resistant to Verticillium
dahliae has been described (U.S. Pat. No. 6,916,970 and Gao et al,
2000). It is similarly anticipated that a variety of
MtDef5-expressing transgenic plants that combat or control fungal
pathogens can be identified by scoring transgenic plants for
resistance to fungal pathogens that infect those plants. Examples
of MtDef5 transgene-conferred fungal resistance that can be assayed
by observing reductions in disease symptoms or reductions in fungal
growth include, but are not limited to, resistance of transgenic
corn to Fusarium verticillioides, Fusarium moniliforme,
Stenocarpella maydis, and/or Cercospora zeae-maydis; resistance of
transgenic wheat to head blight (Fusarium graminearum), powdery
mildew (Erysiphe graminis f. sp. tritici), or leaf rust (Puccinia
recondita f. sp. tritici); resistance of transgenic cotton to
Fusarium oxysporum; resistance of transgenic rice to Magnaporthe
grisea and Rhizoctonia solani, and resistance of transgenic soybean
to Asian Soybean rust (Phakopsora pachyrhizi), Phytophthora Root
Rot (Phytophthora sp.), White Mold (Sclerotinia sp.), Sudden Death
Syndrome (Fusarium solani) and/or Brown Stem Rot (Phialophora
gregata).
[0280] Transgenic plants that express plant pathogenic fungus
inhibitory amounts of MtDef5 can also be identified by measuring
decreases in the adverse effects cause by fungal growth in such
plants. Such decreases can be ascertained by comparing the extent
of the adverse effect in an MtDef5-expressing transgenic plant
relative to an otherwise identical, non-transgenic control plant
that does not express MtDef5. Adverse effects of fungal growth in a
plant that can be measured include any type of plant tissue damage
or necrosis, any type of plant yield reduction, any reduction in
the value of the crop plant product, and/or production of
undesirable fungal metabolites or fungal growth by-products
including, but not limited to, mycotoxins. Mycotoxins comprise a
number of toxic molecules produced by fungal species, including but
not limited to polyketides (including aflatoxins,
demethylsterigmatocystin, O-methylsterigmatocystin, etc.),
fumonisins, alperisins (e.g., A.sub.1, A.sub.2, B.sub.1, B.sub.2),
sphingofungins (A, B, C and D), trichothecenes, fumifungins, and
the like. Methods of quantitating mycotoxin levels are widely
documented. Moreover, commercial kits for measurement of the
mycotoxins such as aflatoxin, fumonisin, deoxynivalenol, and
zearalenone are also available (VICAM, Watertown, Mass., USA).
Target Plants/Plants of Interest
[0281] A wide variety of plants can be transformed with
MtDef5-expressing vectors to obtain transgenic plants that combat
or control fungal infections, or that resist such infections.
Plants of interest include both food crop plants and biofuels or
energy crop plants, as listed above. Transgenic monocot plants
obtainable by the expression vectors and methods described herein
include but are not limited to barley, corn, flax, oat, rice, rye,
sorghum, turf grass, sugarcane, and wheat. Transgenic dicot plants
obtainable by the expression vectors and methods described herein
include but are not limited to alfalfa, Arabidopsis, barrel medic,
banana, broccoli, bean, cabbage, canola, carrot, cassava,
cauliflower, celery, citrus, cotton, cucurbits, eucalyptus, garlic,
grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine,
sunflower, safflower, soybean, strawberry, sugar beet, sweet
potato, tobacco, and tomato.
Stacked Genes: Multiple Resistances
[0282] Simultaneous co-expression of multiple antifungal and/or
other anti-pathogen proteins in plants is advantageous in that it
exploits more than one mode of control of plant pathogens. This
may, where two or more antifungal proteins are expressed, minimize
the possibility of developing resistant fungal species, broaden the
scope of resistance, and potentially result in a synergistic
antifungal effect, thereby enhancing the level of resistance.
[0283] Other proteins conferring certain advantages that can be
co-expressed with the DNAs encoding the MtDef5 molecules of the
present invention include: (1) DNAs encoding enzymes such as
glucose oxidase (which converts glucose to gluconic acid,
concomitantly producing hydrogen peroxide which confers broad
spectrum resistance to plant pathogens); pyruvate oxidase; oxalate
oxidase; cholesterol oxidase; amino acid oxidases; and other
oxidases that use molecular oxygen as a primary or secondary
substrates to produce peroxides, including hydrogen peroxide; (2)
pathogenesis-related proteins such as SAR8.2a and SAR8.2b proteins;
the acidic and basic forms of tobacco PR-1a, PR-1b, PR-1c, PR-1',
PR-2, PR-3, PR-4, PR-5, PR-N, PR-O, PR-O', PR-P, PR-Q, PR-S, and
PR-R proteins; chitinases such as tobacco basic chitinase and
cucumber chitinase/lysozyme; peroxidases such as cucumber basic
peroxidase; glucanases such as tobacco basic glucanase;
osmotin-like proteins; (3) viral capsid proteins and replicases of
plant viruses; (4) plant R-genes (resistance genes) and homologs
thereof, including but not limited to Arabidopsis RPS2 (Bent et
al., 1994), Arabidopsis RPM1 (Grant et al., 1995), tobacco N-gene
and N'-gene, tomato Cf-9, flax L6, and rice Xa21; (5) pathogen Avr
genes, such as Cladosporium fulvum Avr9, that can be expressed
using pathogen- or chemical-inducible promoters; (6) genes that are
involved in the biosynthesis of salicylic acid, such as benzoic
acid 2-hydroxylase; and (7) other defensin proteins with antifungal
modes-of-action distinct from the mode-of-action of MtDef5
including, but not limited to, MsDef1, MtDef2, NaD1, Rs-AFP1 and
Rs-AFP2. Other antifungal proteins that can be co-expressed in
transgenic plants with the present MtDef5 defensins to confer
resistance to fungal pathogen infections include the KP4 and KP6
proteins.
[0284] Co-expression of insect and herbicide resistance genes in
transgenic plants expressing MtDef5 proteins and peptides confers
even further agricultural benefits. The genomes of such transgenic
plants can therefore further comprise:
[0285] DNA encoding a plant defensin selected, for example, from
the group consisting of MsDef1, MtDef2, MtDef4, NaD1, Rs-AFP1,
Rs-AFP2, KP4, and KP6, wherein such DNA is expressed and produces
an anti-fungal effective amount of the defensin, and/or
[0286] DNA encoding a Bacillus thuringiensis endotoxin, wherein
such DNA is expressed and produces an anti-insect effective amount
of the Bacillus thuringiensis endotoxin, and/or
[0287] DNA encoding a protein that confers herbicide resistance to
such plants, wherein such DNA is expressed and produces an
anti-herbicide effective amount of the protein that confers
herbicide resistance.
Yeast Expression Vectors and Transformation Systems
[0288] Expression of MtDef5 proteins and peptides in yeast is
specifically contemplated herein. The construction of expression
vectors for production of heterologous proteins in various yeast
genera is well established. In general, such expression vectors
typically comprise a promoter that is operably linked to a sequence
of interest which is operably linked to a polyadenylation or
terminator region. Examples of yeast genera that have been used to
successfully express heterologous genes include Candida,
Kluveromyces, Hansuela, Pichia, Saccharomyces, Schizosaccharomyces,
and Yarrowia. A general description of expression vectors and
transformation systems for Saccharomyces is found in Kingsman et al
(1985). Expression vectors and transformation systems useful for
yeasts other than Saccharomyces are described in Reiser et al
(1990).
[0289] In general, the promoter and polyadenylation region are
selected based on their operability in the desired yeast host. For
example, the AOX1 or AOX2 promoters of Pichia can be used in
conjunction with the AOX1, AOX2, p40, or p76 polyadenylation
sequences of Pichia to express a heterologous protein such as an
MtDef5 protein or peptide. Both the AOX1 and AOX2 promoters are
particularly useful in Pichia as both promoters provide for
abundant expression of the linked heterologous gene when induced by
addition of methanol to the growth medium. The use of these Pichia
promoters and polyadenylation sequences is described in U.S. Pat.
No. 4,855,231, which is expressly incorporated herein by reference
in its entirety.
[0290] Similarly, the Hansuela MOX, DHAS, or FMDH promoters can be
used to express heterologous proteins such as MtDef5 in Hansuela.
The MOX, DHAS, or FMDH promoters are particularly useful in
Hansuela as these promoters provide for abundant expression of the
linked heterologous gene when induced by addition of methanol to
the growth medium. The use of the MOX and DHAS promoters in
Hansuela is described in U.S. Pat. No. 5,741,672, while the use of
the FMDH promoter in Hansuela is described in U.S. Pat. No.
5,389,525, each of which is expressly incorporated herein by
reference in its entirety.
[0291] For Kluveromyces, a Lactase promoter and polyadenylation
sequence can be used to express heterologous genes such as MtDef5.
Expression of heterologous genes that are operably linked to the
Lactase promoter and polyadenylation sequence is achieved by
growing Kluveromyces in the presence of galactose. The use of the
Lactase promoter and polyadenylation sequences in Kluveromyces is
described in U.S. Pat. No. 6,602,682, which is expressly
incorporated herein by reference in its entirety.
[0292] Yeast expression vectors that provide for secretion of
heterologous proteins such as MtDef5 into the growth medium by
transformed yeast are also contemplated. Secretion of the mature
MtDef5 protein or peptide is typically achieved by operable linkage
of a signal peptide sequence or a signal peptide and propeptide
sequence to the mature MtDef5 protein- or peptide-encoding
sequence. Examples of useful signal peptides for secretion of
heterologous proteins in yeast include but are not limited to an
.alpha.-factor signal peptide, an invertase signal peptide, and a
PHO1 signal peptide, all of which are derived from yeast. The
.alpha.-factor signal peptide is typically derived from
Saccharomyces, Kluveromyces, or Candida, while the PHO1 signal
peptide is derived from Pichia.
[0293] A particularly useful signal peptide sequence or signal
peptide and propeptide sequence for secretion of proteins in yeast
is derived from the S. cerevisiae .alpha.-factor, and is described
in U.S. Pat. Nos. 4,546,082, 4,588,684, 4,870,008, and 5,602,034.
The S. cerevisiae .alpha.-factor signal peptide and propeptide
sequence consist of amino acids 1-83 of the primary, unprocessed
translation product of the S. cerevisiae alpha mating factor gene
(GenBank Accession Number: P01149). In certain embodiments, the
signal peptide sequence of the alpha-mating factor comprising amino
acids 1 to about 19 to 23 of the alpha-mating factor proprotein can
be directly linked to the N-terminus of the mature MtDef5 protein
to provide for secretion of mature MtDef5 protein. In this case,
the signal peptide is cleaved from the mature MtDef5 protein in the
course of the secretion process. Alternatively, the signal peptide
and propeptide of the alpha mating factor can be operably linked to
the mature MtDef5 encoding sequence via a spacer sequence. This
spacer sequence can comprise a variety of sequences that provide
for proteolytic processing of the leader sequence and gene of
interest. In the native S. cerevisiae alpha mating factor gene the
spacer sequence corresponds to amino acid residues 84-89 and is
represented by the sequence Lys84-Arg85-Glu86-Ala87-Glu88-Ala 89
(SEQ ID NO:79). The sequence Lys-Arg corresponds to a KEX2 protease
recognition site while the Glu-Ala-Glu-Ala (SEQ ID NO:80) sequence
corresponds to a duplicated dipeptidylaminopeptidase or STE13
recognition site. In certain embodiments, a DNA fragment encoding
the 89 amino acid S. cerevisiae alpha factor signal, propeptide
coding region, and entire native spacer coding region (i.e., the
N-terminal 89 amino acid residues of the alpha mating factor
precursor protein containing both the Lys-Arg KEX2 protease
cleavage site at residues 84 and 85 as well as the Glu-Ala-Glu-Ala
(SEQ ID NO:80) dipeptidylaminopeptidase or STE13 recognition site
at residues 86-89) is operably linked to the sequence encoding the
mature MtDef5 protein. When the N-terminal 89 amino acids of the
alpha mating factor precursor protein are fused to the N-terminus
of a heterologous protein such as MtDef5, the propeptide sequence
is typically dissociated from the heterologous protein via the
cleavage by endogenous yeast proteases at either the KEX2 or STE13
recognition sites. In other embodiments, a DNA fragment encoding
the smaller 85 amino acid Saccharomyces cerevisiae alpha factor
signal peptide, propeptide, and KEX2 spacer element (i.e., the
N-terminal 85 amino acid residues of the alpha mating factor
precursor protein containing just the Lys-Arg KEX2 protease
cleavage site at residues 84 and 85) is operably linked to the
sequence encoding the mature MtDef5 protein. When the N-terminal 85
amino acids of the alpha mating factor precursor protein are fused
to the N-terminus of a heterologous protein such as MtDef5, the
propeptide sequence is typically dissociated from the heterologous
protein via cleavage by endogenous yeast proteases at the KEX2
recognition site. The MtDef5 protein can thus be expressed without
the glu-ala repeats.
[0294] To obtain transformed yeast that express MtDef5 proteins and
peptides, the yeast MtDef5 expression cassettes (e.g., yeast
promoter, yeast signal peptide encoding sequence, mature MtDef5
protein sequence, and polyadenylation sequence) are typically
combined with other sequences that provide for selection of
transformed yeast. Examples of useful selectable marker genes
include, but are not limited to, genes encoding a ADE protein, a
HIS5 protein, a HIS4 protein, a LEU2 protein, a URA3 protein, ARG4
protein, a TRP1 protein, a LYS2 protein, a protein conferring
resistance to a bleomycin or phleomycin antibiotic, a protein
conferring resistance to chloramphenicol, a protein conferring
resistance to G418 or geneticin, a protein conferring resistance to
hygromycin, a protein conferring resistance to methotrexate, an a
ARO4-OFP protein, and a FZF1-4 protein.
[0295] DNA molecules comprising the yeast MtDef5 expression
cassettes and selectable marker genes are introduced into yeast
cells by techniques such as transfection into yeast spheroplasts or
electroporation. In certain embodiments of the invention, the DNA
molecules comprising the yeast MtDef5 expression cassettes and
selectable marker genes are introduced as linear DNA fragments that
are integrated into the genome of the transformed yeast host cell.
Integration may occur either at random sites in the yeast host cell
genome or at specific sites in the yeast host cell genome.
Integration at specific sites in the yeast host cell genome is
typically accomplished by homologous recombination between
sequences contained in the expression vector and sequences in the
yeast host cell genome. Homologous recombination is typically
accomplished by linearizing the expression vector within the
homologous sequence (for example, within the AOX1 promoter sequence
of a Pichia expression vector when integrating the expression
vector into the endogenous AOX1 gene in the Pichia host cell). In
other embodiments of the invention, the yeast expression cassettes
can also comprise additional sequences such as autonomous
replication sequences (ARS) that provide for the replication of DNA
containing the expression cassette as an extrachromosomal
(non-integrated) element. Such extra-chromosomal elements are
typically maintained in yeast cells by continuous selection for the
presence of the linked selectable marker gene. Yeast artificial
chromosomes (YACs) containing sequences that provide for
replication and mitotic transmission are another type of vector
that can be used to maintain the DNA construct in a yeast host.
Methods of Producing MtDef5 Protein in Yeast
[0296] Yeast cells transformed with the yeast MtDef5 expression
cassettes can be used to produce MtDef5 proteins and peptides.
These MtDef5 molecules can be used directly as antifungal agents,
to produce antifungal compositions that can be applied to plants,
as immunogens to raise antibodies that recognize the MtDef5
proteins or peptides, or as reference standards in kits for
measuring concentrations of MtDef5 proteins and peptides in various
samples. The transformed yeast cells expressing MtDef5 antifungal
molecules can also be applied to plants to combat/control
pathogenic fungal infections. The methods of producing MtDef5
proteins and peptides typically first comprise the step of
culturing yeast cells transformed with MtDef5 expression cassettes
under conditions wherein the yeast cells express a mature MtDef5
molecule. In general, the conditions where the yeast cells express
the mature MtDef5 molecules are conditions that allow for or
specifically induce expression of the yeast promoter that is
operably linked to the MtDef5 coding sequence in the yeast
expression cassette. When the yeast is Pichia and the
signal-peptide/MtDef5 gene is under the control of an AOX1 or AOX2
promoter, addition of methanol to the growth medium will provide
for expression of mature MtDef5 protein. Similarly, when the yeast
is Hansuela and the signal-peptide/MtDef5 gene is under the control
of a MOX, DHAS, or FMDH promoter, addition of methanol to the
growth medium will provide for expression of mature MtDef5 protein.
Alternatively, when the yeast is Kluveromyces and the
signal-peptide/MtDef5 gene is under the control of a Lactase
promoter, addition of galactose to the growth medium will provide
for expression of mature MtDef5 protein.
[0297] Once the transformed yeast culture has been incubated under
culture conditions that provide for expression of mature MtDef5
protein or peptide for a sufficient period of time, the mature
MtDef5 molecule can be isolated from the culture. A sufficient
period of time can be determined by periodically harvesting
portions or aliquots of the culture and assaying for the presence
of MtDef5 protein or peptide. Analytical assays such as SDS-PAGE
with protein staining, Western blot analysis, or any
immunodetection method (e.g., such as an ELISA) can be used to
monitor MtDef5 production. For example, incubation in the presence
of methanol for between 1 to 8 days is sufficient to provide for
expression of mature MtDef5 protein from the AOX1 promoter in
Pichia.
[0298] Isolation of the MtDef5 protein or peptide from the culture
can be partial or complete. For MtDef5 expression vectors where a
yeast signal peptide is operably linked to the sequence encoding
the mature MtDef5 protein, the mature MtDef5 protein can be
recovered from the yeast cell culture medium. Yeast cell culture
medium that contains the mature MtDef5 protein can be separated
from the yeast cells by centrifugation or filtration, thus
effecting isolation of mature MtDef5 protein. Yeast cell culture
medium that contains the mature MtDef5 protein can be further
processed by any combination of dialysis and/or concentration
techniques (e.g., precipitation, lyophilization, filtration) to
produce a composition containing the MtDef5 protein. Production of
MtDef5 protein can also comprise additional purification steps that
result in either a partially or completely pure preparation of the
MtDef5 protein. To effect such purification, filtration
size-exclusion membranes can be used. Alternatively, various types
of chromatographic techniques such as size exclusion
chromatography, ion-exchange chromatography, or affinity
chromatography can be used to produce a partially or completely
pure preparation of the MtDef5 protein.
[0299] Combinations of various isolation techniques can also be
employed to produce the mature MtDef5 protein. For example, the
cell culture medium can be separated from the cells by
centrifugation and dialyzed or adjusted. A preferred buffer for
dialysis or adjustment is a 25 mM sodium acetate buffer at about
pH4.5-pH6.0. This dialysate is then subjected to ion-exchange
chromatography. For example, a cation-exchange resin such as
CM-Sephadex C-25 equilibrated with a 25 mM sodium acetate buffer at
about pH6.0 can be used. MtDef5 protein bound to the cation
exchange resin is washed and then eluted. For example, the
aforementioned column is washed with 25 mM sodium acetate buffer at
about pH6.0 and subsequently eluted in 1M NaCl, 50 mM Tris, pH7.6.
Fractions containing the defensin protein are identified by an
assay or by UV absorbance and then concentrated by a size-cut-off
filtration membrane. The concentrated MtDef5 protein is then
dialyzed to obtain an essentially pure MtDef5 protein in a
desirable buffer. Desirable buffers include, but are not limited
to, buffers such as 10 mM Tris, pH 7.6.
Peptides, Polypeptides, and Proteins Containing Conservative Amino
Acid Changes in MtDef5 Sequences
[0300] Peptides, polypeptides, and proteins biologically
functionally equivalent to MtDef5 proproteins, mature proteins, and
peptides include, but are not limited to, amino acid sequences
containing conservative amino acid substitutions in MtDef5
sequences SEQ ID NOs:1, 3-9, 16-22, and 49-64 disclosed herein. In
such amino acid sequences, one or more amino acids in the sequence
is (are) substituted with another amino acid(s), the charge and
polarity of which is similar to that of the native amino acid,
i.e., a conservative amino acid substitution, resulting in a silent
change.
[0301] Substitutes for an amino acid within the MtDef5 sequence can
be selected from other members of the class to which the naturally
occurring amino acid belongs. Amino acids can be divided into the
following four groups: (1) acidic amino acids; (2) basic amino
acids; (3) neutral polar amino acids; and (4) neutral non-polar
amino acids. Representative amino acids within these various groups
include, but are not limited to: (1) acidic (negatively charged)
amino acids such as aspartic acid and glutamic acid; (2) basic
(positively charged) amino acids such as arginine, histidine, and
lysine; (3) neutral polar amino acids such as glycine, serine,
threonine, cysteine, cystine, tyrosine, asparagine, and glutamine;
(4) neutral nonpolar (hydrophobic) amino acids such as alanine,
leucine, isoleucine, valine, proline, phenylalanine, tryptophan,
and methionine.
[0302] Conservative amino acid changes within MtDef5 sequences can
be made by substituting one amino acid within one of these groups
with another amino acid within the same group. Biologically
functional equivalents of MtDef5 can have 10 or fewer conservative
amino acid changes, more preferably seven or fewer conservative
amino acid changes, and most preferably five, four, three, two, or
one conservative amino acid changes. The encoding nucleotide
sequence (e.g., gene, plasmid DNA, cDNA, or synthetic DNA) will
thus have corresponding base substitutions, permitting it to encode
biologically functional equivalent forms of MtDef5 molecules.
[0303] The biologically functional equivalent peptides,
polypeptides, and proteins contemplated herein should possess about
70% or greater sequence identity, preferably about 85% or greater
sequence identity, more preferably about 90% to about 95% sequence
identity, e.g., about 90%, about 91%, about 92%, about 93%, about
94%, or about 95% sequence identity, and even more preferably
greater than about 96%, 97%, or 98% sequence identity, or about 99%
sequence identity to the sequence of, or corresponding moiety
within, the particular MtDef5 protein or peptide sequence of
interest. Such biologically functional equivalent peptides,
polypeptides, and proteins preferably exhibit about .+-.30% of the
antifungal activity of the corresponding MtDef5 molecule disclosed
herein, or even greater than about +30% antifungal activity,
determined by any of the antifungal activity assay methods
disclosed herein.
[0304] Coding sequences for such biologically functional equivalent
peptides, polypeptides, and protein should comprise a nucleotide
sequence having a sequence identity to a nucleotide sequence
selected from the group consisting of nucleotide sequences shown in
SEQ ID NOs:10-15, 23-29, and 65-78 sufficient to enable the coding
sequence to encode the biologically functional equivalent
antifungal peptide, polypeptide, or protein, or a codon-optimized
version of such coding sequence to optimize expression thereof in a
plant.
Methods for Determining Sequence Identity
[0305] Methods of alignment of sequences for comparison are
well-known in the art. Optimal alignment of sequences for
comparison can be conducted by the local homology algorithm of
Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443
(1970); by the search for similarity method of Pearson and Lipman,
Proc. Nat. Acad. Sci. 85: 2444 (1988); by computerized
implementations of these algorithms, including, but not limited to:
CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
Calif.; and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG.RTM.
Wisconsin Package.TM. from Accelrys, Inc., San Diego, Calif.
[0306] Gene identities can be determined by conducting BLAST (Basic
Local Alignment Search Tool; Altschul, S. F., et al., (1993) J.
Mol. Biol. 215:403-410; see also information available from NCBI
(National Center for Biotechnology Information, U.S. National
Library of Medicine, 8600 Rockville Pike, Bethesda, Md. 20894))
searches under default parameters for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences are analyzed for similarity to all
publicly available DNA sequences contained in the "nr" database
using the BLASTN program. The DNA sequences are translated in all
reading frames and compared for similarity to all publicly
available protein sequences contained in the "nr" database using
the BLASTX program (Gish, W. and States, D. J. Nature Genetics
3:266-272 (1993)) provided by the NCBI. In some cases, the
sequencing data from two or more clones containing overlapping
segments of DNA are used to construct contiguous DNA sequences.
[0307] Sequence alignments and percent identity calculations can be
performed using software such as GAP, BestFit, PileUp or Pretty,
available as part of the GCG.RTM. Wisconsin Package.TM. from
Accelrys, Inc., San Diego, Calif. Default parameters for pairwise
alignments of polynucleotide sequences using GAP and BestFit are
Gap Creation Penalty=50, Gap Extension Penalty=3; nwsgapdna.cmp is
the scoring matrix. Default parameters for pairwise alignments for
polypeptide sequences using GAP and BestFit are Gap Creation
Penalty=8, Gap Extension Penalty=2; BLOSUM62 is the scoring matrix.
There is no penalty for gaps at ends of polynucleotide or
polypeptide alignments.
[0308] Default parameters for polynucleotide sequence comparison
using PileUp and Pretty are: Gap Creation Penalty=5, Gap Extension
Penalty=1. Default parameters for polypeptide sequence comparison
using PileUp or Pretty are Gap Creation Penalty=8, Gap Extension
Penalty=2; BLOSUM62 is the scoring matrix.
[0309] Sequence alignments can also be accomplished with the
Megalign program of the LASERGENE bioinformatics computing suite
(DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences
can be performed using the Clustal method of alignment (Higgins and
Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise
alignments using the Clustal method are KTUPLE 1, GAP PENALTY=3,
WINDOW=5 and DIAGONALS SAVED=5.
[0310] Other pairwise comparison tools are also available and known
to those of skill in the art.
[0311] As indicated, modifications and changes can be made in the
structure of the MtDef5 proteins and peptides of the present
invention, and DNA segments that encode them, and still obtain a
functional molecule that encodes a peptide, polypeptide, or protein
with desirable antifungal characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second-generation
molecule. In particular embodiments of the invention, mutated
MtDef5 proteins or peptides are contemplated to be useful for
increasing the antifungal activity of the these molecules, and
consequently increasing the antifungal activity and/or expression
of the recombinant transgene in a plant cell. The amino acid
changes can be achieved by changing the codons of the DNA sequence,
according to the codons given in Table 2.
TABLE-US-00002 TABLE 2 Amino Acid Amino Acids Codes Codons Alanine
Ala (A) GCA GCC GCG GCU Cysteine Cys (C) UGC UGU Aspartic acid Asp
(D) GAC GAU Glutamic acid Glu (E) GAA GAG Phenylalanine Phe (F) UUC
UUU Glycine Gly (G) GGA GGC GGG GGU Histidine His (H) CAC CAU
Isoleucine Ile (I) AUA AUC AUU Lysine Lys (K) AAA AAG Leucine Leu
(L) UUA UUG CUA CUC CUG CUU Methionine Met (M) AUG Asparagine Asn
(N) AAC AAU Proline Pro (P) CCA CCC CCG CCU Glutamine Gln (Q) CAA
CAG Arginine Arg (R) AGA AGG CGA CGC CGG CGU Serine Ser (S) AGC AGU
UCA UCC UCG UCU Threonine Thr (T) ACA ACC ACG ACU Valine Val (V)
GUA GUC GUG GUU Tryptophan Trp (W) UGG Tyrosine Tyr (Y) UAC UAU
[0312] For example, certain amino acids can be substituted for
other amino acids in a protein structure without appreciable loss
of biochemical or biological activity. Since it is the interactive
capacity and nature of a protein that defines that protein's
biological functional activity, certain amino acid sequence
substitutions can be made in a protein sequence, and, of course,
its underlying DNA coding sequence, and nevertheless obtain a
protein with like properties. It is thus contemplated by the
inventors that various changes can be made in the MtDef5 protein
sequences of the invention, or corresponding DNA sequences that
encode them, without appreciable loss of their biological utility
or activity.
[0313] Betts and Russell ((2003), "Amino Acid Properties and
Consequences of Substitutions", Bioinformatics for Geneticists,
Michael R. Barnes and Ian C. Gray, Eds., John Wiley & Sons,
Ltd, Chapter 14, pp. 289-316) review the nature of mutations and
the properties of amino acids in a variety of different protein
contexts with the purpose of aiding in anticipating and
interpreting the effect that a particular amino acid change will
have on protein structure and function. The authors point out that
features of proteins relevant to considering amino acid mutations
include cellular environments, three-dimensional structure, and
evolution, as well as the classifications of amino acids based on
evolutionary, chemical, and structural principles, and the role for
amino acids of different classes in protein structure and function
in different contexts. The authors note that classification of
amino acids into categories such as those shown in FIG. 14.3 of
their review, which involves common physico-chemical properties,
size, affinity for water (polar and non-polar; negative or positive
charge), aromaticity and aliphaticity, hydrogen-bonding ability,
propensity for sharply turning regions, etc., makes it clear that
reliance on simple classifications can be misleading, and suggests
that alternative amino acids could be engineered into a protein at
each position. Criteria for interpreting how a particular mutation
might affect protein structure and function are summarized in
section 14.7 of this review, and include first inquiring about the
protein, and then about the particular amino acid substitution
contemplated.
[0314] As a starting point in making amino acid changes, the
hydropathic index of amino acids can be considered. The importance
of the hydropathic amino acid index in conferring interactive
biologic function on a protein is generally understood in the art
(Kyte and Doolittle, 1982, incorporated herein by reference). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like.
[0315] Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982). These are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0316] It is known in the art that certain amino acids can be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within +2 is preferred, those which
are within +1 are particularly preferred, and those within +0.5 are
even more particularly preferred.
[0317] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein.
[0318] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); glutamate
(+3.0.+0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+0.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
Non-Conservative Substitutions in MtDef5 Sequences
[0319] It is further recognized that non-conservative substitutions
in MtDef5 sequences can be made to obtain MtDef5 proteins and
peptides that are the functional biological equivalents of the
MtDef5 molecules disclosed herein. In these instances, the
non-conservative substitutions can simply be tested for its effect
on inhibition of fungal growth to identify non-conservative
substitutions that provide for functional biological equivalents of
a given MtDef5 protein or peptide.
Fragments and Variants of MtDef5 Proteins and Peptides
[0320] The antifungal defensins of the present invention include
MtDef5 proproteins, mature MtDef5 proteins, and MtDef5 peptides.
Fragments and variants of these sequences possessing the same,
similar, or even greater antifungal activity compared to that of
these MtDef5 molecules are also encompassed by the present
invention. Thus, contiguous sequences of at least 8 or more amino
acids in a mature MtDef5 protein exhibiting antifungal activity are
encompassed by this invention. Fragments or variants of MtDef5
molecules with antifungal activity that are encompassed by this
invention can also comprise amino acid substitutions, deletions,
insertions or additions in an MtDef5 sequence.
[0321] Fragments of mature MtDef5 proteins, which can be truncated
forms, wherein one or more amino acids are deleted from the
N-terminal end, C-terminal end, the middle of the protein, or
combinations thereof, and which possess antifungal activity, are
also encompassed by this invention. These fragments can be
naturally occurring or synthetic mutants of MtDef5 molecules, and
retain the antifungal activity of MtDef5, preferably about .+-.30%
of the antifungal activity of the corresponding MtDef5 protein or
peptide disclosed herein, or even greater than about +30%
antifungal activity, determined by any of the presently disclosed
antifungal activity assay methods.
[0322] Variants of MtDef5 proteins include forms wherein one or
more amino acids has/have been inserted into the natural sequence.
These variants can also be naturally occurring or synthetic mutants
of MtDef5, and should retain about .+-.30% of the antifungal
activity of the corresponding MtDef5 protein disclosed herein, or
even greater than about +30% antifungal activity, determined by any
of the presently disclosed antifungal activity assay methods.
[0323] Combinations of the foregoing, i.e., forms of the antifungal
MtDef5 defensin containing both amino acid deletions and additions,
are also encompassed by the present invention. Amino acid
substitutions can also be present therein as well.
[0324] The fragments and variants of MtDef5 proteins encompassed by
the present invention should preferably possess about 70-75% or
greater sequence identity, preferably about 85% or greater sequence
identity, more preferably about 90% to 95% sequence identity, and
even more preferably greater than about 96%, 97%, 98%, or 99%
sequence identity to the sequence of, or corresponding moiety
within, the MtDef5 sequence. Such biologically functional
equivalent peptides, polypeptides, and proteins preferably exhibit
about .+-.30% of the antifungal activity of the corresponding
MtDef5 molecule disclosed herein, or even greater than about +30%
antifungal activity, determined by any of the presently disclosed
antifungal activity assay methods.
Use of MtDef5 Defensin Structure Function Relationships to Design
MtDef5 Variants
[0325] The MtDef5 proteins are members of the Defensin gene family
and are thus anticipated to possess certain structural and
biochemical properties shared by Defensins. In particular, the
MtDef5 proteins are anticipated to possess a cysteine-stabilized
a/(3 motif, composed of three antiparallel .beta.-strands and one
.alpha.-helix, that are typically observed in Defensin proteins
(Almeida et al, J. Mol. Biol. (2002) 315, 749-757; Thomma et al,
Planta (2002) 216: 193-202). Without being limited by theory, the
structural homology between MtDef5 and other defensins can be used
to identify variants that possess similar or even increased
antifungal activity.
[0326] Alternatively, the conserved structural features of the
MtDef5 defensins can also be used to engineer variant MtDef5
derivatives with other desirable properties. For example, the 8
canonical cysteine residues of MtDef5 that typically form disulfide
linkages in Defensin proteins would typically be conserved or
maintained in any MtDef5 variants. The predicted pairing of
disulfide bonds in MtDef5 is between cysteine residues 3 and 50, 14
and 35, 20 and 44, and 24 and 46. Thus, Cys-pair 1 is predicted to
be formed by a Cys3-Cys50 disulfide bond, Cys-pair 2 is predicted
to be formed by a Cys14-Cys35 disulfide bond, Cys-pair 3 is
predicted to be formed by a Cys20-Cys44 disulfide bond, and
Cys-pair 4 is predicted to be formed by a Cys24-Cys46 disulfide
bond. While not being limited by theory, it is believed that MtDef5
cysteine variants that lack one or more disulfide linkages may be
desirable for use in transgenic plants that are ultimately used as
animal feed or as food for human consumption as such variants are
predicted to be more readily digested by animal or humans that
consume the transgenic plant products. MtDef5 variant proteins that
have shorter half-lives in the digestive tracts of animals or
humans are in theory anticipated to have less potential to become
food allergens while retaining their antifungal activity. It would
thus be desirable to design MtDef5 defensin derivatives that have
fewer disulfide bonds, yet retain antifungal activity.
Other Biologically Functional Equivalent Forms of MtDef5 Proteins
and Peptides
[0327] Other biologically functional equivalent forms of MtDef5
proteins and peptides useful in the present invention include
conjugates of these molecules, or biologically functional
equivalents thereof as described above, with other peptides,
polypeptides, or proteins, forming fusion products therewith
exhibiting the same, similar, or greater antifungal activity as
compared with that of the corresponding MtDef5 molecule.
[0328] As noted above in the discussion of stacked genes,
simultaneous co-expression of multiple antifungal and/or other
anti-pathogen proteins in plants is advantageous in that it
exploits more than one mode of control of plant pathogens. This
may, where two or more antifungal proteins are expressed, minimize
the possibility of developing resistant fungal species, broaden the
scope of resistance, and potentially result in a synergistic
antifungal effect, thereby enhancing the level of resistance.
Site-Specific Mutagenesis
[0329] Site-specific mutagenesis is a technique useful in the
preparation of individual peptides, or biologically functional
equivalent peptides, polypeptides, or proteins, through specific
mutagenesis of the DNA encoding the molecule. The technique further
provides a ready ability to prepare and test sequence variants,
incorporating, for example, one or more of the foregoing
considerations, by introducing one or more nucleotide sequence
changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences that encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0330] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
that exists in both a single-stranded and double-stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage vectors are readily commercially
available, and their use is generally well known to those skilled
in the art. Double-stranded plasmids are also routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage. Commercially
available kits for performing mutagenesis are also available and
can be used. Exemplary kits include the QuikChange.RTM. sited
directed mutagenesis kits (Stratagene, La Jolla, Calif., USA).
[0331] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double-stranded vector that includes
within its sequence a DNA sequence that encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected that include recombinant vectors bearing the mutated
sequence arrangement.
[0332] The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis is
provided as a means of producing potentially useful species and is
not meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them can be
obtained. For example, recombinant vectors encoding the desired
peptide sequence can be treated with mutagenic agents, such as
hydroxylamine, to obtain sequence variants.
MtDef5 Antibody Compositions and Methods of Making Antibodies
[0333] In particular embodiments, the inventors contemplate the use
of antibodies, either monoclonal or polyclonal, that bind to the
MtDef5 proteins and peptides disclosed herein. Means for preparing
and characterizing antibodies are well known in the art (see, for
example, Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1999, and U.S. Pat. No. 4,196,265, both incorporated
herein by reference).
MtDef5 Protein Screening and Detection Kits
[0334] The present invention contemplates immunodetection methods
and kits for screening samples suspected of containing MtDef5
proteins or peptides, or MtDef5-related peptides, polypeptides, or
proteins, or cells producing such molecules. A kit can contain one
or more antibodies of the present invention, and can also contain
reagent(s) for detecting an interaction between a sample and an
antibody of the present invention. The provided reagent(s) can be
radio-, fluorescently- or enzymatically-labeled. The kit can
contain a known radiolabeled agent capable of binding or
interacting with a nucleic acid or antibody of the present
invention. Detection of immunocomplex formation can be achieved,
for example, via the application of ELISA, RIA, immunoblot (e.g.,
dot blot), indirect immunofluorescence techniques, and the
like.
MtDef5 Agricultural and Pharmaceutical Antifungal Compositions
[0335] The present invention encompasses agricultural and
pharmaceutical antifungal compositions comprising either an
antifungal plant, or antifungal human or veterinary, pathogenic
fungus inhibitory amount ("antifungal effective amount") of one or
more the present isolated, purified antifungal MtDef5 proteins or
peptides, or biologically functional equivalents thereof, of the
present invention. Such compositions can comprise one, or any
combination of, MtDef5 proteins or peptides disclosed herein, and
an agriculturally or pharmaceutically/veterinarily acceptable
carrier, diluent, or excipient. As indicated below, other
components relevant in agricultural and therapeutic contexts can be
included in such compositions as well. The antifungal compositions
can be used for inhibiting the growth of, or killing, MtDef5
protein- or peptide-susceptible pathogenic fungi associated with
plant, or human or animal, fungal infections. Such antifungal
compositions can be formulated for topical administration, and
applied topically to either plants, the plant environment
(including soil), or humans or animals.
Agricultural Compositions Comprising MtDef5 Proteins and Peptides,
and Protein- and Peptide-Producing Microorganisms
[0336] Agricultural compositions comprising any of the present
MtDef5 molecules alone, or in any combination, can be formulated as
described in, for example, Winnacker-Kuchler (1986) Chemical
Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; van
Falkenberg (1972-1973) Pesticide Formulations, Second Edition,
Marcel Dekker, N.Y.; and K. Martens (1979) Spray Drying Handbook,
Third Edition, G. Goodwin, Ltd., London. Necessary formulation
aids, such as carriers, inert materials, surfactants, solvents, and
other additives are also well known in the art, and are described,
for example, in Watkins, Handbook of Insecticide Dust Diluents and
Carriers, Second Edition, Darland Books, Caldwell, N.J., and
Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition,
Volume 7, Hanser Verlag, Munich. Using these formulations, it is
also possible to prepare mixtures of the present MtDef5 proteins
and peptides with other pesticidally active substances,
fertilizers, and/or growth regulators, etc., in the form of
finished formulations or tank mixes.
[0337] Whether alone or in combination with other active agents,
the present antifungal MtDef5 proteins and peptides can be applied
at a concentration in the range of from about 0.1 ml to about 100
mg ml, preferably between about 5 .mu.g ml and about 5 mg ml, at a
pH in the range of from about 3.0 to about 9.0. Such compositions
can be buffered using, for example, phosphate buffers between about
1 mM and 1 M, preferably between about 10 mM and 100 mM, more
preferably between about 15 mM and 50 mM. In the case of low buffer
concentrations, it is desirable to add a salt to increase the ionic
strength, preferably NaCl in the range of from about 1 mM to about
1 M, more preferably about 10 mM to about 100 mM.
[0338] Numerous conventional fungal antibiotics and chemical
fungicides with which the present MtDef5 proteins and peptides can
be combined are known in the art, and are described in Worthington
and Walker (1983) The Pesticide Manual, Seventh Edition, British
Crop Protection Council. These include, for example, polyoxines,
nikkomycines, carboxy amides, aromatic carbohydrates, carboxines,
morpholines, inhibitors of sterol biosynthesis, and
organophosphorus compounds. Other active ingredients which can be
formulated in combination with the present antifungal proteins and
peptides include, for example, insecticides, attractants,
sterilizing agents, acancides, nematocides, and herbicides. U.S.
Pat. No. 5,421,839 contains a comprehensive summary of the many
active agents with which substances such as the present antifungal
MtDef5 proteins and peptides can be formulated.
[0339] MtDef5 proteins and peptides and biologically functional
equivalents thereof are expected to be useful in controlling a wide
variety of susceptible fungi in plants, exemplified by those in the
following genera and species: Alternaria (Alternaria brassicola;
Alternaria solani); Ascochyta (Ascochyta pisi); Aspergillus
(Aspergillus flavus; Aspergillus fumigatus); Botrytis (Botrytis
cinerea); Cercospora (Cercospora kikuchii; Cercospora zeae-maydis);
Colletotrichum (Colletotrichum lindemuthianum); Diplodia (Diplodia
maydis); Erysiphe (Erysiphe graminis f. sp. graminis; Erysiphe
graminis f. sp. hordei); Fusarium (Fusarium nivale; Fusarium
oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium
solani; Fusarium moniliforme; Fusarium roseum); Gaeumannomyces
(Gaeumannomyces graminis f.sp. tritici); Helminthosporium
(Helminthosporium turcicum; Helminthosporium carbonum;
Helminthosporium maydis); Macrophomina (Macrophomina phaseolina;
Maganaporthe grisea); Nectria (Nectria heamatococca); Peronospora
(Peronospora manshurica; Peronospora tabacina); Phakopsora
(Phakopsora pachyrhizi); Phoma (Phoma betae); Phymatotrichum
(Phymatotrichum omnivorum); Phytophthora (Phytophthora cinnamomi;
Phytophthora cactorum; Phytophthora phaseoli; Phytophthora
parasitica; Phytophthora citrophthora; Phytophthora megasperma
f.sp. sojae; Phytophthora infestans); Plasmopara (Plasmopara
viticola); Podosphaera (Podosphaera leucotricha); Puccinia
(Puccinia sorghi; Puccinia striiformis; Puccinia graminis f.sp.
tritici; Puccinia asparagi; Puccinia recondita; Puccinia
arachidis); Pythium (Pythium aphanidermatum); Pyrenophora
(Pyrenophora tritici-repentens); Pyricularia (Pyricularia oryzae);
Pythium (Pythium ultimum); Rhizoctonia (Rhizoctonia solani;
Rhizoctonia cerealis); Scerotium (Scerotium rolfsii); Sclerotinia
(Sclerotinia sclerotiorum); Septoria (Septoria lycopersici;
Septoria glycines; Septoria nodorum; Septoria tritici);
Thielaviopsis (Thielaviopsis basicola); Uncinula (Uncinula
necator); Venturia (Venturia inaequalis); Verticillium
(Verticillium dahliae; Verticillium alboatrum).
[0340] Agriculturally useful antifungal compositions encompassed
herein also include those in the form of host cells, such as
bacterial and fungal cells, capable of the producing the MtDef5
proteins and peptides, and which can colonize plants, including
roots, shoots, leaves, or other parts of plants.
[0341] The term "plant-colonizing microorganism" is used herein to
refer to a microorganism that is capable of colonizing the "plant
environment", and which can express the present MtDef5 antifungal
proteins and peptides in the "plant environment". A plant
colonizing micro-organism is one that can exist in symbiotic or
non-detrimental relationship with a plant in the plant
environment.
[0342] U.S. Pat. No. 5,229,112 discloses a variety of
plant-colonizing microorganisms that can be engineered to express
antifungal proteins, and methods of use thereof, applicable to the
MtDef5 antifungal proteins and peptides disclosed herein.
Plant-colonizing microorganisms expressing the presently disclosed
MtDef5 antifungal proteins and peptides useful in inhibiting fungal
infection and damage in plants according to the present invention
include, for example, bacteria selected from the group consisting
of genera selected from spore forming organisms of the family
Bacillaceae, for example Bacillus species such as Bacillus
thuringiensis, Bacillus israelensis, and Bacillus subtilis;
Pseudomonas; Arthrobacter; Azospyrillum; Clavibacter; Escherichia;
Agrobacterium, for example A. radiobacter; Rhizobium; Erwinia;
Azotobacter; Azospirillum; Klebsiella; Alcaligenes; Rhizobacterium;
and Flavobacterium; and yeasts selected from the group consisting
of Saccharomyces cerevisiae; Pichia pastoris; and Pichia
methanolica.
[0343] The term "plant environment" includes the surface of a
plant, e.g., leaf, stem, buds, stalk, floral parts, or root
surface, the interior of the plant and its cells, and to the
"rhizosphere", i.e., the soil which surrounds and which is
influenced by the roots of the plant. When it is desired to apply
the present MtDef5 molecules to the rhizosphere,
rhizosphere-colonizing bacteria from the genus Pseudomonas are
particularly useful, especially the fluorescent pseudomonads, e.g.,
Pseudomonas fluorescens, which is especially competitive in the
plant rhizosphere and in colonizing the surface of the plant roots
in large numbers. Examples of suitable phylloplane (leaf)
colonizing bacteria are P. putida, P. syringae, and Erwinia
species.
[0344] The antifungal plant-colonizing microorganisms of the
invention can be applied directly to the plant environment, e.g.,
to the surface of leaves, buds, roots, shoots, floral parts, seeds,
etc., or to the soil. When used as a seed coating, the
plant-colonizing microorganisms of the invention are applied to the
plant seed prior to planting. Generally, small amounts of the
antifungally active microorganism will be required to treat such
seeds.
[0345] The determination of an antifungal effective amount of
plant-colonizing microorganisms useful in the methods of the
present invention required for a particular plant is within the
skill of the art, and will depend on such factors as the plant
species, the fungal pathogen, method of planting, and the soil
type, (e.g., pH, organic matter content, moisture content).
[0346] Theoretically, a single plant-colonizing microorganism of
the invention containing DNA encoding the MtDef5 antifungal
proteins and peptides disclosed herein is sufficient to control
fungal pathogens because it can grow into a colony of clones of
sufficient number to express antifungal amounts of the Defensin.
However, in practice, due to varying environmental factors which
may affect the survival and propagation of the microorganism, a
sufficient number of plant colonizing microorganisms should be
provided in the plant environment (e.g., roots or foliage) to
assure survival and/or proliferation. For example, application of
10.sup.3 to 10.sup.10 bacteria or yeasts per seed may be sufficient
to insure colonization on the surface of the roots by the
microorganism. It is preferred to dose the plant environment with
enough bacteria or other plant-colonizing microorganism to maintain
a population that expresses 50 to 250 nanograms of Defensin. For
example, 10.sup.5 to 10.sup.8 bacteria per square centimeter of
plant surface may be adequate to control fungal infection. At least
0.5 nanograms, preferably 1 to 100 nanograms, of anti-fungal active
protein or peptides may be sufficient to control fungal damage to
plants.
[0347] Compositions containing the antifungally active
plant-associated microorganisms of the invention can be prepared by
formulating the biologically active microorganism with adjuvants,
diluents, carriers, etc., to provide compositions in the form of
finely-divided particulate solids, granules, pellets, wettable
powders, dusts, aqueous suspensions, dispersions, or emulsions.
Illustrative of suitable carrier vehicles are: solvents, e.g.,
water or organic solvents, and finely divided solids, e.g., kaolin,
chalk, calcium carbonate, talc, silicates, and gypsum.
[0348] The present invention also encompasses the use of the
antifungal plant-colonizing microorganisms in the methods and
compositions of the invention in encapsulated form, e.g., the
plant-colonizing microorganisms can be encapsulated within shell
walls of polymer, gelatin, lipid, and the like. Other formulation
aids such as, for example, emulsifiers, dispersants, surfactants,
wetting agents, anti-foam agents, and anti-freeze agents, can be
incorporated into the antifungal compositions, especially if such
compositions will be stored for any period of time prior to
use.
[0349] In addition to the antifungally active plant-colonizing
microorganisms, the compositions of the invention can additionally
contain other known biologically active agents, such as, for
example, a fungicide, herbicide, or insecticide. Also, two or more
antifungally active plant-colonizing microorganisms can be
combined.
[0350] The application of antifungal compositions containing the
genetically engineered plant-colonizing microorganisms of the
invention as the active agent can be carried out by conventional
techniques utilizing, for example, spreaders, power dusters, boom
and hand sprayers, spry dusters, and granular applicators.
[0351] The compositions of the invention are applied in an
antifungal effective amount, which will vary depending on such
factors as, for example, the specific fungal pathogen to be
controlled, the specific plant (and plant part or soil) to be
treated, and the method of applying the antifungally active
compositions.
MtDef5 Antifungal Protein- and Peptide-Containing Pharmaceutical
Compositions
[0352] The present invention not only encompasses transgenic plants
expressing MtDef5 proteins and peptides and transformed
microorganisms that can be applied to the loci of plants, but also
pharmaceutical compositions that can be used for inhibiting the
growth of, or killing, susceptible pathogenic fungi that infect
humans or animals, i.e., treating such fungal infections by
administering to a patient or other subject in need thereof an
antifungal effective amount of MtDef5 proteins, peptides, or
biologically functional equivalents thereof.
[0353] Such pharmaceutical compositions comprising MtDef5 proteins
and peptides, and biologically functional equivalents thereof, can
be formulated by conventional methods such as those described in
Remington: The Science and Practice of Pharmacy (2005), 21.sup.st
Edition, University of the Sciences in Philadelphia, Lippincott
Williams & Wilkins. Such compositions can contain MtDef5
proteins and peptides, and various combinations thereof, at
concentrations in the range of from about 0.1 .mu.g ml to about 100
mg ml, preferably between about 5 .mu.g ml and about 5 mg ml, at a
pH in the range of from about 3.0 to about 9.0. Such compositions
can be buffered using, for example, phosphate buffers between about
1 mM and 1 M, preferably between about 10 mM and 100 mM, more
preferably between about 15 mM and 50 mM. In the case of low buffer
concentrations, it is desirable to add a salt to increase the ionic
strength, preferably NaCl in the range of from about 1 mM to about
1 M, more preferably about 10 mM to about 100 mM.
[0354] The MtDef5 proteins and peptides can be formulated alone, in
any combination with one another, and either of these can
additionally be formulated in combination with other conventional
antifungal therapeutic compounds such as, by way of non-limiting
example, polyene antifungals; imidazole, triazole, and thiazole
antifungals; allylamines; and echinocandins that are routinely used
in human and veterinary medicine.
[0355] Administration of the present MtDef5 molecules can be
accomplished via a variety of conventional routes, with topical
application being preferred.
[0356] The following examples describe various aspects of the
present invention, and are merely intended to be illustrative
rather than limiting of the compounds, compositions, and methods
useful therein.
Example 1
Construction of Soybean Transformation Vector AKK/FMV/MtDef5
[0357] As shown in FIG. 1, a MtDef5 gene or cDNA with its own
signal peptide is cloned as a Nco I-Xba I fragment between the
Figwort mosaic virus 35S promoter (Sanger et al. (1990) Plant
Molecular Biology 14:433-443) and nopaline synthase polyadenylation
signal (Gleave (1992) Plant Molecular Biology 20:1203-1207) in the
soybean expression vector AKK1472 (Hammes et al. (2005) Molecular
Plant Microbe Interactions 18:1247-1257). The AKK1472 vector
containing a MtDef5 chimeric gene or cDNA and bar gene conferring
Basta.RTM. resistance as a selectable marker gene (Thompson et al.
(1985) EMBO Journal 6:2519-2523) is transferred to Agrobacterium
tumefaciens strain EHA105 for soybean transformation (Clemente et
al. (2000) Crop Sci. 40:797-803; Zhang et al. (1999) Plant Cell,
Tiss. Organ Cult. 56:37-46).
Example 2
Soybean Transformation and Regeneration of Transgenic Plants
[0358] The transformation protocol used in this example to create
transgenic soybean lines using Agrobacterium has been previously
described (Clemente et al. (2000) Crop Sci. 40:797-803; Zhang et
al. (1999) Plant Cell, Tiss. Organ Cult. 56:37-46).
[0359] The exterior of the seeds (in this case the soybean variety
called "Jack") are first sterilized using commercial grade
Clorox.RTM. (5% aqueous sodium hypochlorite, NaClO) overnight. The
sterilized seeds are then allowed to germinate in germination
medium (GM; Gamborg's B5 medium (Gamborg et al. (1968) Experimental
Cell Research 50:151) supplemented with 2% sucrose, pH 5.8,
solidified with 0.8% agar) for 5 days at 24.degree. C. (18/6) light
regime). The A. tumefaciens transformed with the vector of Example
1 are collected via low speed centrifugation and suspended in
co-cultivation medium to a final OD.sub.650 of 0.6 to 1.0.
Co-cultivation medium is 1/10th Gamborg's B5 medium supplemented
with 1.67 mg/l 6-benzylaminopurine (BAP), 0.25 mg/l gibberellic
acid (GA3), 3% sucrose, 200 .mu.M acetosyringone, 20 mM
2-(N-morpholino)-ethanesulfonic acid (MES), pH 5.4.
[0360] The following protocol has been previously described
(Clemente et al. (2000) supra; Zhang et al. (1999) supra).
[0361] Agrobacterium inoculum is placed in a petri plate with the
prepared explants (from wounded, germinating seed) for 30 min.,
with occasional agitation. The explants are then placed on
co-cultivation plates (Petri dishes containing 0.76 g Gamborg Basal
Salt Mixture, 7.4 g MES, 60 g sucrose, pH adjusted to 5.4 using 1 M
KOH, and 5 g/L agarose dissolved in warm media), adaxial side down.
The plates are wrapped with parafilm and placed at 24.degree. C.,
18/6 light regime for 3 days. Following the co-cultivation period,
the explants are briefly washed in liquid shoot initiation medium
(3.08 g of Gamborgs B5 salts, 30 g Sucrose, 0.56 g MES, adjusted to
pH to 5.6 using 1 M KOH) supplemented with 0.25 mg/l GA3. After the
first two weeks, the hypocotyl region is cut flush to the
developing node, and incubated for two weeks in the absence of
glufosinate. The tissue is then transferred to fresh shoot
initiation medium every two weeks, for a total of approximately 10
weeks with 3 mg/l glufosinate. The tissue is oriented so that the
freshly cut surface is imbedded in the medium, with the
differentiating region flush to the surface. At the end of the
shoot initiation period, only the differentiating explants are
used. The cotyledons are removed from the explants, and a fresh cut
is made at the base of the developing node (horizontally), the
tissue is transferred to shoot elongation medium, and is cultured
at 24.degree. C. with a 18/6 light regime. Shoot elongation medium
is composed of MS salts/Gamborg's vitamins supplemented with 1 mg/l
zeatin riboside, 0.1 mg/l indole acetic acid (IAA), 0.5 mg/l GA3,
3% sucrose, 100 mg/l pyroglutamic acid, 50 mg/l asparagine, 3 mM
MES (pH 5.6), solidified with 0.8% purified agar. Since the bar
gene is used as a marker, 3 mg/l glufosinate is added. The tissue
is transferred to fresh shoot elongation medium every two weeks. At
each transfer, a fresh horizontal slice is made at the base of the
tissue. Elongated shoots (greater than 3 cm) are rooted on rooting
medium without further selection. Rooting medium is composed of
4.33 g of Murashige & Skoog Basal Salt Mixture, 20 g sucrose,
0.56 g MES. The pH is adjusted to 5.6 using 1 M KOH and 3 g
Phytagel per liter are added. The solution is autoclaved (20 min.)
and when cooled, 1 ml Gamborg B5 vitamins (1000.times.), 1 ml
L-asparagine monohydrate (50 mg/ml stock), and 1 ml L-Pyroglutamic
acid (100 mg/ml stock) are added.
[0362] The plants are then grown and selected for using PCR to
detect the presence of the MtDef5 gene or cDNA. The expression of
the MtDef5 gene or cDNA at the RNA level is determined using the
qRT-PCR assay, and expression of the MtDef5 protein is determined
by sandwich ELISA using a MtDef5 polyclonal antibody. Homozygotes
are eventually selected using quantitative PCR using the Promega
GoTaq.RTM. qPCR master mix (Promega Corporation, Madison, Wis.) on
an AB StepOne Plus-Real time PCR system (Applied Biosystems,
Carlsbad, Calif.) per the manufacturer's instructions.
[0363] Transgenic plants that are homozygous for the MtDef5 DNA
construct insertion will be obtained, identified, and subsequently
tested for resistance to Fusarium head blight as described in Chen
et al. (1999) Theor. Appl. Genet. 99:755-760; leaf rust and stripe
rust as described in Cheng et al. (2010) Theor. Appl. Genet.
121:195-204); and stem rust as described in Nirmala et al. (2011)
PNAS 108:14676-14681.
Example 3
Construction of Wheat Transformation Vector pZP212/Maize Ubi1A
Promoter/MtDef5/35S 3'
[0364] For expression of MtDef5 in wheat, a MtDef5 gene or cDNA
will be synthesized based on monocot preferred codons such that the
amino acid sequence of the MtDef5 signal peptide and mature protein
remain unchanged. The synthetic MtDef5 gene or cDNA will be
obtained from the GenScript Corporation (Piscataway, N.J., USA).
The synthetic MtDef5 gene will be placed between the maize
ubiquitin (Ubi1) promoter/intron and CaMV 35S polyadenylation
signal sequence and cloned between the T-DNA borders of the binary
plant expression vector pZP212 (Hajdukiewicz et al., 1994) as shown
in FIG. 2.
[0365] The pZP212 vector containing the synthetic MtDef5 gene or
cDNA and a neomycin phosphotransferase selectable marker gene
(nptII) will be introduced into Bobwhite and/or XC9 wheat. The
transgenic wheat will be obtained as described below in Example
4.
Example 4
Wheat Transformation and Regeneration of Transgenic Plants
[0366] The protocol used for wheat transformation was previously
described by Cheng et al. (1997) Plant Physiology 115:971-980.
[0367] For these transformations, Triticum aestivum cv Bobwhite, is
used. Immature caryopses are collected from plants 14 days after
anthesis. Immature embryos are dissected aseptically and cultured
on a semisolid or liquid CM4 medium (Zhou et al. (1995) Plant Cell
Replication 15:159-163) with 100 mg L-ascorbic acid (CM4C). The MS
salts (Murashige and Skoog (1962) Physiology Plant. 15:473-497) in
the CM4 medium are adjusted to full strength (the original amounts)
or one-tenth-strength (Fry et al. (1987) Plant Cell Reports 6:
321-325). The immature embryos are cultured for 3 to 4 hours.
Embryogenic calli are prepared by culturing the immature embryos on
CM4C medium for 10 to 25 days. The callus pieces derived from
immature embryos are inoculated with A. tumefaciens using the
embryogenic callus sectors. A. tumefaciens C58 (ABI) harboring the
vector described in Example 3 (FIG. 2) is prepared as described
above in Example 3 for soybean transformation. The A. tumefaciens
is grown to a cell density of A600 of 1 to 2 for inoculation. The
immature embryos and embryogenic calli maintained on the CM4C
medium are transferred into an A. tumefaciens cell suspension in
Petri dishes. The inoculation is conducted at 23 to 25.degree. C.
for 3 hours in the dark. After inoculation, the A. tumefaciens
cells are removed and the explants are placed on semisolid medium
(Gelrite) with liquid CM4C with full-strength MS salts and
supplemented with 10 g/L glucose and 200 .mu.M acetosyringone. The
co-cultivation is performed at 24 to 26.degree. C. in the dark for
2 or 3 days. After co-culture, the infected immature embryos and
calli are cultured on the solid CM4C medium with 250 mg/L
carbenicillin for 2 to 5 days without selection. A. tumefaciens
infected explants are then transferred to CM4C medium supplemented
with 3 mg/l glufosinate and 250 mg/L carbenicillin for callus
induction. Two weeks later, the explants are transferred to the
first regeneration medium, MMS0.2C (consisting of MS salts and
vitamins, 1.95 g/L MES, 0.2 mg/L 2,4-dichloro-phenoxyacetic acid,
100 mg/L ascorbic acid, and 40 g/L maltose, solidified by 2 g/L
gelrite supplemented with 3 mg/l glufosinate and 250 mg/L
carbenicillin). At transfer to the regeneration medium, each piece
of callus derived from one immature embryo or one piece of
inoculated callus is divided into several small pieces
(approximately 2 mm). In another 2 weeks, young shoots and viable
callus tissues are transferred to the second regeneration medium,
MMSOC, which contains the same components as MMS0.2C, with all
antibiotics included. When the shoots develop into about 3 cm or
longer plantlets, they are transferred to larger culture vessels
containing the regeneration medium for further growth and
selection. Leaf samples are taken from some of the plantlets for
PCR testing at this stage. Plants that are highly glufosinate
resistant are transferred to soil. All of the plants derived from
the same embryo or piece of callus are considered to be clones of a
given event.
[0368] The plants are then grown and selected for using PCR to
detect the presence of the MtDef5 gene or cDNA. The expression of
the MtDef5 gene or cDNA at the RNA level is determined using the
qRT-PCR assay, and expression of the MtDef5 protein is determined
by sandwich ELISA using a MtDef5 polyclonal antibody. Homozygotes
are eventually selected using quantitative PCR using the Promega
GoTaq.RTM. qPCR master mix (Promega Corporation, Madison, Wis.) on
an AB StepOne Plus-Real time PCR system (Applied Biosystems,
Carlsbad, Calif.) per the manufacturer's instructions.
[0369] Transgenic plants that are homozygous for the MtDef5 DNA
construct insertion will be obtained, identified, and subsequently
tested for resistance to Fusarium head blight as described in Chen
et al. (1999) Theor. Appl. Genet. 99:755-760; leaf rust and stripe
rust as described in Cheng et al. (2010) Theor. Appl. Genet.
121:195-204); and stem rust as described in Nirmala et al. (2011)
PNAS 108:14676-14681.
Example 5
In Vitro Antifungal Activity of Chemically Synthesized MtDef5.1a
Gamma Core Peptide with C-Terminal Extension (GMA-5C)
(GACHRQGFGFACFCYKKC (SEQ ID NO:58))
[0370] In order to determine if a small peptide derived from the
full-length MtDef5 protein exhibits antifungal activity, a peptide
consisting of the C-terminal 18 amino acids is obtained from
Genemed, Inc., Texas, and tested for its antifungal activity
against Fusarium graminearum PH-1. This peptide contains the
gamma-core motif and the last 6 amino acids of the mature MtDef5
protein.
[0371] The antifungal activity of the gamma-core motif of MtDef5.1a
is assessed using the chemically synthesized GMA-5C peptide having
the amino acid sequence GACHRQGFGFACFCYKKC (SEQ ID NO:58).
Quantitative assessment of the in vitro antifungal activity of the
chemically synthesized GMA-5C peptide is assessed at 24 h after
incubation of Fusarium graminearum PH-1 conidia with the peptide as
described in Ramamoorthy et al. (2007) Cellular Microbiology
9:1491-1506. The results are shown in FIG. 3, where the values
reported are means of thee replications. Error bars indicate
standard deviations.
[0372] The data demonstrate that the IC.sub.50 (concentration
required for 50% inhibition of fungal growth) of this peptide is
about 6-12 .mu.M, while the IC.sub.100 (concentration required for
100% inhibition of fungal growth) is about 2404.
[0373] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
TABLE-US-00003 Amino Acid and Nucleotide Sequence Information
Medicago truncatula MtDef5.1a-MtDef5.6 defensin proprotein amino
acid sequences, comprising a MtDef5 signal peptide (8-29 amino
acids, underlined) and a mature MtDef5 defensin protein
(approximately 50 amino acids), with the .gamma.-core motif of each
MtDef5 defensin shown in bold, italic, 10 point type MtDef5.1a: SEQ
ID NO: 1
MTSSASKFYTIFIFVCLAFLFISTSEVEAKLCQKRSTTWSGPCLNTGNCKRQCINVEHATF
FCYKKC MtDef5.1a - MtDef5.1b "linker sequence": SEQ ID NO: 2
APKKVEP MtDef5.1b: SEQ ID NO: 3 KLCERRSKTWSGPCLISGNCKRQCINVEHATS
FCKKKC MtDef5.1a-"Linker"-MtDef5.1b: SEQ ID NO: 4
MTSSASKFYTIFIFVCLAFLFISTSEVEAKLCQKRSTTWSGPCLNTGNCKRQCINVEHATF
FCYKKC APKKVEPKLCERRSKTWSGPCLISGNCKRQCINVEHATS FCKKKC MtDef5.2: SEQ
ID NO: 5
MASSSPKLFTIFLFLILVVLLFSTSEVQAKLCRGRSKLWSGPCINSKCKRQCINVERAVS FCDFKC
MtDef5.3: SEQ ID NO: 6
MTSSATKFYTIFVFVCLALLLISICEVEAKVCQKRSKTWSGPCLNTGNCKRQCVDVENATF
FCYKKC MtDef5.4: SEQ ID NO: 7
MASSTLKFNTIFLFLSLALLLFFTLEVQGNICKRKSTTWSGPCLNTGNCKNQCINVEHATF
FCYFNC MtDef5.5: SEQ ID NO: 8
MASSALKYYTFFLFFILALILLPTLEVQGNTCQRKSKTWSGPCLNTANCKNQCISKEPPATF
FCYFNC MtDef5.6: SEQ ID NO: 9
MVCTEVQAKLCRGRSKLWSGPCINSKCKRQCINVERAVS FCDFKC Medicago truncatula
MtDef5.1a-MtDef5.6 proprotein cDNA coding sequences, comprising a
MtDef5 signal peptide (8-29 amino acids) coding sequence
(underlined), and a mature MtDef5 protein (approximately 50 amino
acids) coding sequence, with the .gamma.-core motif coding sequence
shown in bold type MtDef5.1a and MtDef5.1b cDNA (SEQ ID NO: 10).
The nucleotide sequence shown in bold, italics, and underlined
encodes "linker" peptide amino acid sequence APKKVEP:
ATGACTTCCTCTGCTAGTAAATTCTATACCATCTTCATTTTTGTCTGCCTTGCCTTTCTCTTTATTTC
CACATCTGAGGTGGAAGCAAAACTTTGTCAAAAGCGAAGTACAACATGGTCAGGACCTTGTCTTAACA
CAGGAAACTGCAAAAGACAATGCATTAATGTGGAGCATGCTACTTTTGGTGCTTGTCATCGTCAAGGC
TTTGGTTTTGCTTGCTTCTGCTACAAAAAATGT AAACTTTGTGAAAG
GCGAAGCAAAACATGGTCAGGACCTTGTCTTATCTCAGGAAATTGTAAAAGACAGTGCATCAATGTTG
AGCATGCAACTTCTGGTGCTTGTCACCGTCAAGGCATTGGTTTTGCTTGCTTCTGCAAGAAAAAATGT
TGA MtDef5.2 cDNA (SEQ ID NO: 11):
ATGGCTTCCTCTTCTCCTAAATTGTTTACCATCTTTCTGTTTCTCATCCTTGTCGTGCTCCTTTTCTC
AACTTCGGAGGTGCAAGCAAAACTTTGTAGAGGGAGAAGCAAACTTTGGTCAGGGCCTTGTATTAACT
CAAAATGCAAAAGACAATGCATCAACGTGGAGCGCGCAGTTAGCGGGGGTTGTCACCTTGATAACACT
GGAGTTTTTTGTTTCTGCGACTTCAAATGCTGA MtDef5.3 cDNA (SEQ ID NO: 12):
ATGACTTCCTCTGCTACTAAATTTTACACCATCTTTGTTTTTGTCTGCCTTGCCCTTCTCCTTATTTC
CATATGTGAGGTGGAAGCAAAAGTGTGTCAAAAACGAAGTAAAACGTGGTCAGGACCTTGTCTTAACA
CAGGAAACTGTAAAAGACAATGCGTTGATGTGGAGAATGCAACCTTCGGTGCTTGTCACCGTCAAGGC
TATGGTTTTGCTTGCTTCTGCTACAAAAAGTGTTGA MtDef5.4 cDNA (SEQ ID NO: 13):
ATGGCTTCATCTACTCTTAAATTCAACACTATCTTTCTGTTTCTCAGCCTTGCACTTCTCCTGTTCTT
CACATTGGAGGTACAAGGAAATATTTGTAAAAGGAAAAGCACAACATGGTCAGGGCCATGTTTAAACA
CGGGAAACTGTAAAAATCAGTGCATCAATGTGGAACATGCTACTTTTGGGGCATGCCACCAAGATGGA
TTTGGATTTGCTTGCTTCTGCTACTTCAATTGCTGA MtDef5.5 cDNA (SEQ ID NO: 14):
ATGGCTTCCTCTGCTCTTAAATACTACACTTTCTTTCTGTTTTTCATCCTTGCACTTATCCTGTTACC
CACATTGGAGGTACAAGGAAATACTTGTCAAAGGAAAAGCAAAACATGGTCAGGGCCATGTTTAAACA
CGGCAAACTGTAAAAATCAGTGCATCAGTAAGGAACCACCGGCAACATTTGGGGCATGTCACCGTGAT
GGCATTGGATTTGCTTGCTTCTGTTACTTCAACTGCTAA MtDef5.6 cDNA (SEQ ID
NO:15):
ATGGTGTGTACAGAGGTGCAAGCAAAACTTTGTAGAGGGAGAAGCAAACTTTGGTCAGGGCCTTGTAT
TAACTCAAAATGCAAAAGACAATGCATCAACGTGGAGCGCGCAGTTAGCGGGGGTTGTCACCTTGATA
ACACTGGAGTTTTTTGTTTCTGCGACTTCAAATGCTGA Mature Medicago truncatula
MtDef5.1a-MtDef5.6 Defensin Protein Amino Acid Sequences MtDef5.1a:
SEQ ID NO: 16 KLCQKRSTTWSGPCLNIGNCKRQCINVEHATFGACHRQGFGFACFCYKKC
MtDef5.1b: SEQ ID NO: 17
KLCERRSKTWSGPCLISGNCKRQCINVEHATSGACHRQGIGFACFCKKKC MtDef5.2: SEQ ID
NO: 18 KLCRGRSKLWSGPCINSKCKRQCINVERAVSGGCHLDNIGVFCFCDFKC MtDef5.3:
SEQ ID NO: 19 KVCQKRSKTWSGPCLNIGNCKRQCVDVENATFGACHRQGYGFACFCYKKC
MtDef5.4: SEQ ID NO: 20
NICKRKSTTWSGPCLNIGNCKNQCINVEHATFGACHQDGFGFACFCYFNC MtDef5.5: SEQ ID
NO: 21 NTCQRKSKTWSGPCLNTANCKNQCISKEPPATFGACHRDGIGFACFCYFNC
MtDef5.6: SEQ ID NO: 22
KLCRGRSKLWSGPCINSKCKRQCINVERAVSGGCHLDNIGVFCFCDFKC Mature Medicago
truncatula MtDef5.1a-MtDef5.6 Defensin Protein cDNA Coding
Sequences MtDef5.1a: SEQ ID NO: 23
AAACTTTGTCAAAAGCGAAGTACAACATGGTCAGGACCTTGTCTTAACACAGGAAACTGCAAAAGACA
ATGCATTAATGTGGAGCATGCTACTTTTGGTGCTTGTCATCGTCAAGGCTTTGGTTTTGCTTGCTTCT
GCTACAAAAAATGT MtDef5.1b: SEQ ID NO: 24
AAACTTTGTGAAAGGCGAAGCAAAACATGGTCAGGACCTTGTCTTATCTCAGGAAATTGTAAAAGACA
GTGCATCAATGTTGAGCATGCAACTTCTGGTGCTTGTCACCGTCAAGGCATTGGTTTTGCTTGCTTCT
GCAAGAAAAAATGT MtDef5.2: SEQ ID NO: 25
AAACTTTGTAGAGGGAGAAGCAAACTTTGGTCAGGGCCTTGTATTAACTCAAAATGCAAAAGACAATG
CATCAACGTGGAGCGCGCAGTTAGCGGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGTTTCTGCG
ACTTCAAATGC MtDef5.3: SEQ ID NO: 26
AAAGTGTGTCAAAAACGAAGTAAAACGTGGTCAGGACCTTGTCTTAACACAGGAAACTGTAAAAGACA
ATGCGTTGATGTGGAGAATGCAACCTTCGGTGCTTGTCACCGTCAAGGCTATGGTTTTGCTTGCTTCT
GCTACAAAAAGTGT MtDef5.4: SEQ ID NO: 27
AATATTTGTAAAAGGAAAAGCACAACATGGTCAGGGCCATGTTTAAACACGGGAAACTGTAAAAATCA
GTGCATCAATGTGGAACATGCTACTTTTGGGGCATGCCACCAAGATGGATTTGGATTTGCTTGCTTCT
GCTACTTCAATTGC MtDef5.5: SEQ ID NO: 28
AATACTTGTCAAAGGAAAAGCAAAACATGGTCAGGGCCATGTTTAAACACGGCAAACTGTAAAAATCA
GTGCATCAGTAAGGAACCACCGGCAACATTTGGGGCATGTCACCGTGATGGCATTGGATTTGCTTGCT
TCTGTTACTTCAACTGC MtDef5.6: SEQ ID NO: 29
AAACTTTGTAGAGGGAGAAGCAAACTTTGGTCAGGGCCTTGTATTAACTCAAAATGCAAAAGACAATG
CATCAACGTGGAGCGCGCAGTTAGCGGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGTTTCTGCG
ACTTCAAATGC MtDef5.1a - 5.6 Signal Peptide Amino Acid Sequences
MtDef5.1a - 5.1b Signal Peptide Sequence:
MTSSASKFYTIFIFVCLAFLFISTSEVEA (SEQ ID NO: 30) MtDef5.2 Signal
Peptide Sequence: MASSSPKLFTIFLFLILVVLLFSTSEVQA (SEQ ID NO: 31)
MtDef5.3 Signal Peptide Sequence: MTSSATKFYTIFVFVCLALLLISICEVEA
(SEQ ID NO: 32) MtDef5.4 Signal Peptide Sequence:
MASSTLKFNTIFLFLSLALLLFFTLEVQG (SEQ ID NO: 33) MtDef5.5 Signal
Peptide Sequence: MASSALKYYTFFLFFILALILLPTLEVQG (SEQ ID NO: 34)
MtDef5.6 Putative Signal Peptide Sequence: MVCTEVQA (SEQ ID NO: 35)
MtDef5.1a - 5.6 Signal Peptide cDNA Coding Sequences MtDef5.1a-5.1b
Signal Peptide cDNA Coding Sequence:
ATGACTTCCTCTGCTAGTAAATTCTATACCATCTTCATTTTTGTCTGCCTTGCCTTTCTCTTTATTTC
CACATCTGAGGTGGAAGCA (SEQ ID NO: 36) MtDef5.2 Signal Peptide cDNA
Coding Sequence:
ATGGCTTCCTCTTCTCCTAAATTGTTTACCATCTTTCTGTTTCTCATCCTTGTCGTGCTCCTTTTCTC
AACTTCGGAGGTGCAAGCA (SEQ ID NO: 37) MtDef5.3 Signal Peptide cDNA
Coding Sequence:
ATGACTTCCTCTGCTACTAAATTTTACACCATCTTTGTTTTTGTCTGCCTTGCCCTTCTCCTTATTTC
CATATGTGAGGTGGAAGCA (SEQ ID NO: 38) MtDef5.4 Signal Peptide cDNA
Coding Sequence:
ATGGCTTCATCTACTCTTAAATTCAACACTATCTTTCTGTTTCTCAGCCTTGCACTTCTCCTGTTCTT
CACATTGGAGGTACAAGGA (SEQ ID NO: 39) MtDef5.5 Signal Peptide cDNA
Coding Sequence:
ATGGCTTCCTCTGCTCTTAAATACTACACTTTCTTTCTGTTTTTCATCCTTGCACTTATCCTGTTACC
CACATTGGAGGTACAAGGA (SEQ ID NO: 40) MtDef5.6 Signal Peptide cDNA
Coding Sequence: ATGGTGTGTACAGAGGTGCAAGCA (SEQ ID NO: 41) MtDef4.1
- 4.3 and Other Signal Peptide Amino Acid Sequences MtDef4.1(H33R)
Signal Peptide Sequence: MARSVPLVSTIFVFLLLLVATGPSMVAEA (SEQ ID NO:
42) MtDef4.2 Signal Peptide Sequence:
MARSVPLVSTIFVFFLLIVATEMGPSMVAA (SEQ ID NO: 43) MtDef4.3 Signal
Peptide Sequence: MARSVPLVSTIFVFFLLLVATEMGPIMVAEA (SEQ ID NO: 44)
AL385796 Signal Peptide Sequence: MARSVFLVSTIFVFLLVLVATGPSMVAEA
(SEQ ID NO: 45) AW573770 Signal Peptide Sequence:
MARSVSLVFTIFVFLLLVVATGPSMVAEA (SEQ ID NO: 46) BE999096 Signal
Peptide Sequence: MARSVPLVSTIFVFLLLLVATGPSMVGEA (SEQ ID NO: 47)
MtDef4 Signal Peptide Consensus Sequence:
MARSVPLVSTIFVFLLLLVATGPSMVAEA (SEQ ID NO: 48) MsDef1, MtDef4, and
MtDef5.1a-5.6 .gamma.-Core Motif (GXCX.sub.3-9C) (SEQ ID NO: 81)
Peptide Amino Acid Sequences MsDef1: GRCRDDFRC (SEQ ID NO: 49)
MtDef4: GRCRGFRRRC (SEQ ID NO: 50) MtDef5.1a: GACHRQGFGFAC (SEQ ID
NO: 51) MtDef5.1b: GACHRQGIGFAC (SEQ ID NO: 52) MtDef5.2:
GGCHLDNTGVFC (SEQ ID NO: 53) MtDef5.3: GACHRQGYGFAC (SEQ ID NO: 54)
MtDef5.4: GACHQDGFGFAC (SEQ ID NO: 55) MtDef5.5: GACHRDGIGFAC (SEQ
ID NO: 56) MtDef5.6: GGCHLDNTGVFC (SEQ ID NO: 57) MtDef5.1a-5.6
.gamma.-Core Peptides With C-Terminal Extensions (GMA-5C)
MtDef5.1a: GACHRQGFGFACFCYKKC (SEQ ID NO: 58) MtDef5.1b:
GACHRQGIGFACFCKKKC (SEQ ID NO: 59) MtDef5.2: GGCHLDNTGVFCFCDFKC
(SEQ ID NO: 60) MtDef5.3: GACHRQGYGFACFCYKKC (SEQ ID NO: 61)
MtDef5.4: GACHQDGFGFACFCYFNC (SEQ ID NO: 62) MtDef5.5:
GACHRDGIGFACFCYFNC (SEQ ID NO: 63) MtDef5.6: GGCHLDNTGVFCFCDFKC
(SEQ ID NO: 64) MtDef5.1a-5.6 .gamma.-Core Motif (GXCX.sub.3-9C)
Peptide Amino Acid Sequence cDNA Coding
Sequences MtDef5.1a: GGTGCTTGTCATCGTCAAGGCTTTGGTTTTGCTTGC (SEQ ID
NO: 65) MtDef5.1b: GGTGCTTGTCACCGTCAAGGCATTGGTTTTGCTTGC (SEQ ID NO:
66) MtDef5.2: GGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGT (SEQ ID NO: 67)
MtDef5.3: GGTGCTTGTCACCGTCAAGGCTATGGTTTTGCTTGC (SEQ ID NO: 68)
MtDef5.4: GGGGCATGCCACCAAGATGGATTTGGATTTGCTTGC (SEQ ID NO: 69)
MtDef5.5: GGGGCATGTCACCGTGATGGCATTGGATTTGCTTGC (SEQ ID NO: 70)
MtDef5.6: GGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGT (SEQ ID NO: 71)
MtDef5.1a-5.6 .gamma. Core Peptide With C-Terminal Extensions
(GMA-5C) cDNA Coding Sequences MtDef5.1a:
GGTGCTTGTCATCGTCAAGGCTTTGGTTTTGCTTGCTTCTGCTACAAAAAATGT (SEQ ID NO:
72) MtDef5.1b:
GGTGCTTGTCACCGTCAAGGCATTGGTTTTGCTTGCTTCTGCAAGAAAAAATGT (SEQ ID NO:
73) MtDef5.2:
GGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGTTTCTGCGACTTCAAATGC (SEQ ID NO:
74) MtDef5.3:
GGTGCTTGTCACCGTCAAGGCTATGGTTTTGCTTGCTTCTGCTACAAAAAGTGT (SEQ ID NO:
75) MtDef5.4:
GGGGCATGCCACCAAGATGGATTTGGATTTGCTTGCTTCTGCTACTTCAATTGC (SEQ ID NO:
76) MtDef5.5:
GGGGCATGTCACCGTGATGGCATTGGATTTGCTTGCTTCTGTTACTTCAACTGC (SEQ ID NO:
77) MtDef5.6:
GGGGGTTGTCACCTTGATAACACTGGAGTTTTTTGTTTCTGCGACTTCAAATGC (SEQ ID NO:
78)
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Sequence CWU 1
1
81179PRTMedicago truncatula 1Met Thr Ser Ser Ala Ser Lys Phe Tyr
Thr Ile Phe Ile Phe Val Cys1 5 10 15Leu Ala Phe Leu Phe Ile Ser Thr
Ser Glu Val Glu Ala Lys Leu Cys 20 25 30Gln Lys Arg Ser Thr Thr Trp
Ser Gly Pro Cys Leu Asn Thr Gly Asn 35 40 45Cys Lys Arg Gln Cys Ile
Asn Val Glu His Ala Thr Phe Gly Ala Cys 50 55 60His Arg Gln Gly Phe
Gly Phe Ala Cys Phe Cys Tyr Lys Lys Cys65 70 7527PRTMedicago
truncatula 2Ala Pro Lys Lys Val Glu Pro1 5350PRTMedicago truncatula
3Lys Leu Cys Glu Arg Arg Ser Lys Thr Trp Ser Gly Pro Cys Leu Ile1 5
10 15Ser Gly Asn Cys Lys Arg Gln Cys Ile Asn Val Glu His Ala Thr
Ser 20 25 30Gly Ala Cys His Arg Gln Gly Ile Gly Phe Ala Cys Phe Cys
Lys Lys 35 40 45Lys Cys 504136PRTMedicago truncatula 4Met Thr Ser
Ser Ala Ser Lys Phe Tyr Thr Ile Phe Ile Phe Val Cys1 5 10 15Leu Ala
Phe Leu Phe Ile Ser Thr Ser Glu Val Glu Ala Lys Leu Cys 20 25 30Gln
Lys Arg Ser Thr Thr Trp Ser Gly Pro Cys Leu Asn Thr Gly Asn 35 40
45Cys Lys Arg Gln Cys Ile Asn Val Glu His Ala Thr Phe Gly Ala Cys
50 55 60His Arg Gln Gly Phe Gly Phe Ala Cys Phe Cys Tyr Lys Lys Cys
Ala65 70 75 80Pro Lys Lys Val Glu Pro Lys Leu Cys Glu Arg Arg Ser
Lys Thr Trp 85 90 95Ser Gly Pro Cys Leu Ile Ser Gly Asn Cys Lys Arg
Gln Cys Ile Asn 100 105 110Val Glu His Ala Thr Ser Gly Ala Cys His
Arg Gln Gly Ile Gly Phe 115 120 125Ala Cys Phe Cys Lys Lys Lys Cys
130 135578PRTMedicago truncatula 5Met Ala Ser Ser Ser Pro Lys Leu
Phe Thr Ile Phe Leu Phe Leu Ile1 5 10 15Leu Val Val Leu Leu Phe Ser
Thr Ser Glu Val Gln Ala Lys Leu Cys 20 25 30Arg Gly Arg Ser Lys Leu
Trp Ser Gly Pro Cys Ile Asn Ser Lys Cys 35 40 45Lys Arg Gln Cys Ile
Asn Val Glu Arg Ala Val Ser Gly Gly Cys His 50 55 60Leu Asp Asn Thr
Gly Val Phe Cys Phe Cys Asp Phe Lys Cys65 70 75679PRTMedicago
truncatula 6Met Thr Ser Ser Ala Thr Lys Phe Tyr Thr Ile Phe Val Phe
Val Cys1 5 10 15Leu Ala Leu Leu Leu Ile Ser Ile Cys Glu Val Glu Ala
Lys Val Cys 20 25 30Gln Lys Arg Ser Lys Thr Trp Ser Gly Pro Cys Leu
Asn Thr Gly Asn 35 40 45Cys Lys Arg Gln Cys Val Asp Val Glu Asn Ala
Thr Phe Gly Ala Cys 50 55 60His Arg Gln Gly Tyr Gly Phe Ala Cys Phe
Cys Tyr Lys Lys Cys65 70 75779PRTMedicago truncatula 7Met Ala Ser
Ser Thr Leu Lys Phe Asn Thr Ile Phe Leu Phe Leu Ser1 5 10 15Leu Ala
Leu Leu Leu Phe Phe Thr Leu Glu Val Gln Gly Asn Ile Cys 20 25 30Lys
Arg Lys Ser Thr Thr Trp Ser Gly Pro Cys Leu Asn Thr Gly Asn 35 40
45Cys Lys Asn Gln Cys Ile Asn Val Glu His Ala Thr Phe Gly Ala Cys
50 55 60His Gln Asp Gly Phe Gly Phe Ala Cys Phe Cys Tyr Phe Asn
Cys65 70 75880PRTMedicago truncatula 8Met Ala Ser Ser Ala Leu Lys
Tyr Tyr Thr Phe Phe Leu Phe Phe Ile1 5 10 15Leu Ala Leu Ile Leu Leu
Pro Thr Leu Glu Val Gln Gly Asn Thr Cys 20 25 30Gln Arg Lys Ser Lys
Thr Trp Ser Gly Pro Cys Leu Asn Thr Ala Asn 35 40 45Cys Lys Asn Gln
Cys Ile Ser Lys Glu Pro Pro Ala Thr Phe Gly Ala 50 55 60Cys His Arg
Asp Gly Ile Gly Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70 75
80957PRTMedicago truncatula 9Met Val Cys Thr Glu Val Gln Ala Lys
Leu Cys Arg Gly Arg Ser Lys1 5 10 15Leu Trp Ser Gly Pro Cys Ile Asn
Ser Lys Cys Lys Arg Gln Cys Ile 20 25 30Asn Val Glu Arg Ala Val Ser
Gly Gly Cys His Leu Asp Asn Thr Gly 35 40 45Val Phe Cys Phe Cys Asp
Phe Lys Cys 50 5510411DNAMedicago truncatula 10atgacttcct
ctgctagtaa attctatacc atcttcattt ttgtctgcct tgcctttctc 60tttatttcca
catctgaggt ggaagcaaaa ctttgtcaaa agcgaagtac aacatggtca
120ggaccttgtc ttaacacagg aaactgcaaa agacaatgca ttaatgtgga
gcatgctact 180tttggtgctt gtcatcgtca aggctttggt tttgcttgct
tctgctacaa aaaatgtgct 240ccaaagaagg tggaacctaa actttgtgaa
aggcgaagca aaacatggtc aggaccttgt 300cttatctcag gaaattgtaa
aagacagtgc atcaatgttg agcatgcaac ttctggtgct 360tgtcaccgtc
aaggcattgg ttttgcttgc ttctgcaaga aaaaatgttg a 41111237DNAMedicago
truncatula 11atggcttcct cttctcctaa attgtttacc atctttctgt ttctcatcct
tgtcgtgctc 60cttttctcaa cttcggaggt gcaagcaaaa ctttgtagag ggagaagcaa
actttggtca 120gggccttgta ttaactcaaa atgcaaaaga caatgcatca
acgtggagcg cgcagttagc 180gggggttgtc accttgataa cactggagtt
ttttgtttct gcgacttcaa atgctga 23712240DNAMedicago truncatula
12atgacttcct ctgctactaa attttacacc atctttgttt ttgtctgcct tgcccttctc
60cttatttcca tatgtgaggt ggaagcaaaa gtgtgtcaaa aacgaagtaa aacgtggtca
120ggaccttgtc ttaacacagg aaactgtaaa agacaatgcg ttgatgtgga
gaatgcaacc 180ttcggtgctt gtcaccgtca aggctatggt tttgcttgct
tctgctacaa aaagtgttga 24013240DNAMedicago truncatula 13atggcttcat
ctactcttaa attcaacact atctttctgt ttctcagcct tgcacttctc 60ctgttcttca
cattggaggt acaaggaaat atttgtaaaa ggaaaagcac aacatggtca
120gggccatgtt taaacacggg aaactgtaaa aatcagtgca tcaatgtgga
acatgctact 180tttggggcat gccaccaaga tggatttgga tttgcttgct
tctgctactt caattgctga 24014243DNAMedicago truncatula 14atggcttcct
ctgctcttaa atactacact ttctttctgt ttttcatcct tgcacttatc 60ctgttaccca
cattggaggt acaaggaaat acttgtcaaa ggaaaagcaa aacatggtca
120gggccatgtt taaacacggc aaactgtaaa aatcagtgca tcagtaagga
accaccggca 180acatttgggg catgtcaccg tgatggcatt ggatttgctt
gcttctgtta cttcaactgc 240taa 24315174DNAMedicago truncatula
15atggtgtgta cagaggtgca agcaaaactt tgtagaggga gaagcaaact ttggtcaggg
60ccttgtatta actcaaaatg caaaagacaa tgcatcaacg tggagcgcgc agttagcggg
120ggttgtcacc ttgataacac tggagttttt tgtttctgcg acttcaaatg ctga
1741650PRTMedicago truncatula 16Lys Leu Cys Gln Lys Arg Ser Thr Thr
Trp Ser Gly Pro Cys Leu Asn1 5 10 15Thr Gly Asn Cys Lys Arg Gln Cys
Ile Asn Val Glu His Ala Thr Phe 20 25 30Gly Ala Cys His Arg Gln Gly
Phe Gly Phe Ala Cys Phe Cys Tyr Lys 35 40 45Lys Cys
501750PRTMedicago truncatula 17Lys Leu Cys Glu Arg Arg Ser Lys Thr
Trp Ser Gly Pro Cys Leu Ile1 5 10 15Ser Gly Asn Cys Lys Arg Gln Cys
Ile Asn Val Glu His Ala Thr Ser 20 25 30Gly Ala Cys His Arg Gln Gly
Ile Gly Phe Ala Cys Phe Cys Lys Lys 35 40 45Lys Cys
501849PRTMedicago truncatula 18Lys Leu Cys Arg Gly Arg Ser Lys Leu
Trp Ser Gly Pro Cys Ile Asn1 5 10 15Ser Lys Cys Lys Arg Gln Cys Ile
Asn Val Glu Arg Ala Val Ser Gly 20 25 30Gly Cys His Leu Asp Asn Thr
Gly Val Phe Cys Phe Cys Asp Phe Lys 35 40 45Cys1950PRTMedicago
truncatula 19Lys Val Cys Gln Lys Arg Ser Lys Thr Trp Ser Gly Pro
Cys Leu Asn1 5 10 15Thr Gly Asn Cys Lys Arg Gln Cys Val Asp Val Glu
Asn Ala Thr Phe 20 25 30Gly Ala Cys His Arg Gln Gly Tyr Gly Phe Ala
Cys Phe Cys Tyr Lys 35 40 45Lys Cys 502050PRTMedicago truncatula
20Asn Ile Cys Lys Arg Lys Ser Thr Thr Trp Ser Gly Pro Cys Leu Asn1
5 10 15Thr Gly Asn Cys Lys Asn Gln Cys Ile Asn Val Glu His Ala Thr
Phe 20 25 30Gly Ala Cys His Gln Asp Gly Phe Gly Phe Ala Cys Phe Cys
Tyr Phe 35 40 45Asn Cys 502151PRTMedicago truncatula 21Asn Thr Cys
Gln Arg Lys Ser Lys Thr Trp Ser Gly Pro Cys Leu Asn1 5 10 15Thr Ala
Asn Cys Lys Asn Gln Cys Ile Ser Lys Glu Pro Pro Ala Thr 20 25 30Phe
Gly Ala Cys His Arg Asp Gly Ile Gly Phe Ala Cys Phe Cys Tyr 35 40
45Phe Asn Cys 502249PRTMedicago truncatula 22Lys Leu Cys Arg Gly
Arg Ser Lys Leu Trp Ser Gly Pro Cys Ile Asn1 5 10 15Ser Lys Cys Lys
Arg Gln Cys Ile Asn Val Glu Arg Ala Val Ser Gly 20 25 30Gly Cys His
Leu Asp Asn Thr Gly Val Phe Cys Phe Cys Asp Phe Lys 35 40
45Cys23150DNAMedicago truncatula 23aaactttgtc aaaagcgaag tacaacatgg
tcaggacctt gtcttaacac aggaaactgc 60aaaagacaat gcattaatgt ggagcatgct
acttttggtg cttgtcatcg tcaaggcttt 120ggttttgctt gcttctgcta
caaaaaatgt 15024150DNAMedicago truncatula 24aaactttgtg aaaggcgaag
caaaacatgg tcaggacctt gtcttatctc aggaaattgt 60aaaagacagt gcatcaatgt
tgagcatgca acttctggtg cttgtcaccg tcaaggcatt 120ggttttgctt
gcttctgcaa gaaaaaatgt 15025147DNAMedicago truncatula 25aaactttgta
gagggagaag caaactttgg tcagggcctt gtattaactc aaaatgcaaa 60agacaatgca
tcaacgtgga gcgcgcagtt agcgggggtt gtcaccttga taacactgga
120gttttttgtt tctgcgactt caaatgc 14726150DNAMedicago truncatula
26aaagtgtgtc aaaaacgaag taaaacgtgg tcaggacctt gtcttaacac aggaaactgt
60aaaagacaat gcgttgatgt ggagaatgca accttcggtg cttgtcaccg tcaaggctat
120ggttttgctt gcttctgcta caaaaagtgt 15027150DNAMedicago truncatula
27aatatttgta aaaggaaaag cacaacatgg tcagggccat gtttaaacac gggaaactgt
60aaaaatcagt gcatcaatgt ggaacatgct acttttgggg catgccacca agatggattt
120ggatttgctt gcttctgcta cttcaattgc 15028153DNAMedicago truncatula
28aatacttgtc aaaggaaaag caaaacatgg tcagggccat gtttaaacac ggcaaactgt
60aaaaatcagt gcatcagtaa ggaaccaccg gcaacatttg gggcatgtca ccgtgatggc
120attggatttg cttgcttctg ttacttcaac tgc 15329147DNAMedicago
truncatula 29aaactttgta gagggagaag caaactttgg tcagggcctt gtattaactc
aaaatgcaaa 60agacaatgca tcaacgtgga gcgcgcagtt agcgggggtt gtcaccttga
taacactgga 120gttttttgtt tctgcgactt caaatgc 1473029PRTMedicago
truncatula 30Met Thr Ser Ser Ala Ser Lys Phe Tyr Thr Ile Phe Ile
Phe Val Cys1 5 10 15Leu Ala Phe Leu Phe Ile Ser Thr Ser Glu Val Glu
Ala 20 253129PRTMedicago truncatula 31Met Ala Ser Ser Ser Pro Lys
Leu Phe Thr Ile Phe Leu Phe Leu Ile1 5 10 15Leu Val Val Leu Leu Phe
Ser Thr Ser Glu Val Gln Ala 20 253229PRTMedicago truncatula 32Met
Thr Ser Ser Ala Thr Lys Phe Tyr Thr Ile Phe Val Phe Val Cys1 5 10
15Leu Ala Leu Leu Leu Ile Ser Ile Cys Glu Val Glu Ala 20
253329PRTMedicago truncatula 33Met Ala Ser Ser Thr Leu Lys Phe Asn
Thr Ile Phe Leu Phe Leu Ser1 5 10 15Leu Ala Leu Leu Leu Phe Phe Thr
Leu Glu Val Gln Gly 20 253429PRTMedicago truncatula 34Met Ala Ser
Ser Ala Leu Lys Tyr Tyr Thr Phe Phe Leu Phe Phe Ile1 5 10 15Leu Ala
Leu Ile Leu Leu Pro Thr Leu Glu Val Gln Gly 20 25358PRTMedicago
truncatula 35Met Val Cys Thr Glu Val Gln Ala1 53687DNAMedicago
truncatula 36atgacttcct ctgctagtaa attctatacc atcttcattt ttgtctgcct
tgcctttctc 60tttatttcca catctgaggt ggaagca 873787DNAMedicago
truncatula 37atggcttcct cttctcctaa attgtttacc atctttctgt ttctcatcct
tgtcgtgctc 60cttttctcaa cttcggaggt gcaagca 873887DNAMedicago
truncatula 38atgacttcct ctgctactaa attttacacc atctttgttt ttgtctgcct
tgcccttctc 60cttatttcca tatgtgaggt ggaagca 873987DNAMedicago
truncatula 39atggcttcat ctactcttaa attcaacact atctttctgt ttctcagcct
tgcacttctc 60ctgttcttca cattggaggt acaagga 874087DNAMedicago
truncatula 40atggcttcct ctgctcttaa atactacact ttctttctgt ttttcatcct
tgcacttatc 60ctgttaccca cattggaggt acaagga 874124DNAMedicago
truncatula 41atggtgtgta cagaggtgca agca 244229PRTMedicago
truncatula 42Met Ala Arg Ser Val Pro Leu Val Ser Thr Ile Phe Val
Phe Leu Leu1 5 10 15Leu Leu Val Ala Thr Gly Pro Ser Met Val Ala Glu
Ala 20 254330PRTMedicago truncatula 43Met Ala Arg Ser Val Pro Leu
Val Ser Thr Ile Phe Val Phe Phe Leu1 5 10 15Leu Ile Val Ala Thr Glu
Met Gly Pro Ser Met Val Ala Ala 20 25 304431PRTMedicago truncatula
44Met Ala Arg Ser Val Pro Leu Val Ser Thr Ile Phe Val Phe Phe Leu1
5 10 15Leu Leu Val Ala Thr Glu Met Gly Pro Ile Met Val Ala Glu Ala
20 25 304529PRTMedicago truncatula 45Met Ala Arg Ser Val Phe Leu
Val Ser Thr Ile Phe Val Phe Leu Leu1 5 10 15Val Leu Val Ala Thr Gly
Pro Ser Met Val Ala Glu Ala 20 254629PRTMedicago truncatula 46Met
Ala Arg Ser Val Ser Leu Val Phe Thr Ile Phe Val Phe Leu Leu1 5 10
15Leu Val Val Ala Thr Gly Pro Ser Met Val Ala Glu Ala 20
254729PRTMedicago truncatula 47Met Ala Arg Ser Val Pro Leu Val Ser
Thr Ile Phe Val Phe Leu Leu1 5 10 15Leu Leu Val Ala Thr Gly Pro Ser
Met Val Gly Glu Ala 20 254829PRTMedicago truncatula 48Met Ala Arg
Ser Val Pro Leu Val Ser Thr Ile Phe Val Phe Leu Leu1 5 10 15Leu Leu
Val Ala Thr Gly Pro Ser Met Val Ala Glu Ala 20 25499PRTMedicago
sativa 49Gly Arg Cys Arg Asp Asp Phe Arg Cys1 55010PRTMedicago
truncatula 50Gly Arg Cys Arg Gly Phe Arg Arg Arg Cys1 5
105112PRTMedicago truncatula 51Gly Ala Cys His Arg Gln Gly Phe Gly
Phe Ala Cys1 5 105212PRTMedicago truncatula 52Gly Ala Cys His Arg
Gln Gly Ile Gly Phe Ala Cys1 5 105312PRTMedicago truncatula 53Gly
Gly Cys His Leu Asp Asn Thr Gly Val Phe Cys1 5 105412PRTMedicago
truncatula 54Gly Ala Cys His Arg Gln Gly Tyr Gly Phe Ala Cys1 5
105512PRTMedicago truncatula 55Gly Ala Cys His Gln Asp Gly Phe Gly
Phe Ala Cys1 5 105612PRTMedicago truncatula 56Gly Ala Cys His Arg
Asp Gly Ile Gly Phe Ala Cys1 5 105712PRTMedicago truncatula 57Gly
Gly Cys His Leu Asp Asn Thr Gly Val Phe Cys1 5 105818PRTMedicago
truncatula 58Gly Ala Cys His Arg Gln Gly Phe Gly Phe Ala Cys Phe
Cys Tyr Lys1 5 10 15Lys Cys5918PRTMedicago truncatula 59Gly Ala Cys
His Arg Gln Gly Ile Gly Phe Ala Cys Phe Cys Lys Lys1 5 10 15Lys
Cys6018PRTMedicago truncatula 60Gly Gly Cys His Leu Asp Asn Thr Gly
Val Phe Cys Phe Cys Asp Phe1 5 10 15Lys Cys6118PRTMedicago
truncatula 61Gly Ala Cys His Arg Gln Gly Tyr Gly Phe Ala Cys Phe
Cys Tyr Lys1 5 10 15Lys Cys6218PRTMedicago truncatula 62Gly Ala Cys
His Gln Asp Gly Phe Gly Phe Ala Cys Phe Cys Tyr Phe1 5 10 15Asn
Cys6318PRTMedicago truncatula 63Gly Ala Cys His Arg Asp Gly Ile Gly
Phe Ala Cys Phe Cys Tyr Phe1 5 10 15Asn Cys6418PRTMedicago
truncatula 64Gly Gly Cys His Leu Asp Asn Thr Gly Val Phe Cys Phe
Cys Asp Phe1 5 10 15Lys Cys6536DNAMedicago truncatula 65ggtgcttgtc
atcgtcaagg ctttggtttt gcttgc 366636DNAMedicago truncatula
66ggtgcttgtc accgtcaagg cattggtttt gcttgc 366736DNAMedicago
truncatula 67gggggttgtc accttgataa cactggagtt ttttgt
366836DNAMedicago truncatula 68ggtgcttgtc accgtcaagg ctatggtttt
gcttgc 366936DNAMedicago truncatula 69ggggcatgcc accaagatgg
atttggattt gcttgc 367036DNAMedicago truncatula 70ggggcatgtc
accgtgatgg cattggattt gcttgc 367136DNAMedicago truncatula
71gggggttgtc accttgataa cactggagtt ttttgt 367254DNAMedicago
truncatula 72ggtgcttgtc atcgtcaagg ctttggtttt gcttgcttct gctacaaaaa
atgt 547354DNAMedicago truncatula 73ggtgcttgtc accgtcaagg
cattggtttt gcttgcttct gcaagaaaaa atgt 547454DNAMedicago truncatula
74gggggttgtc accttgataa cactggagtt ttttgtttct gcgacttcaa atgc
547554DNAMedicago truncatula 75ggtgcttgtc accgtcaagg ctatggtttt
gcttgcttct gctacaaaaa gtgt 547654DNAMedicago truncatula
76ggggcatgcc accaagatgg atttggattt gcttgcttct gctacttcaa ttgc
547754DNAMedicago truncatula 77ggggcatgtc accgtgatgg cattggattt
gcttgcttct gttacttcaa ctgc 547854DNAMedicago truncatula
78gggggttgtc accttgataa cactggagtt ttttgtttct gcgacttcaa atgc
54796PRTSaccharomyces cerevisiae 79Lys Arg Glu Ala Glu Ala1
5804PRTSaccharomyces cerevisiae 80Glu Ala Glu
Ala18113PRTSaccharomyces cerevisiaeMISC_FEATURE(2)..(2)Xaa is any
amino acidMISC_FEATURE(4)..(4)Xaa is any amino
acidMISC_FEATURE(5)..(5)Xaa is any amino
acidMISC_FEATURE(6)..(6)Xaa is any amino
acidMISC_FEATURE(7)..(7)Xaa is any amino acid and (7) may or may
not be presentMISC_FEATURE(8)..(8)Xaa is any amino acid and (8) may
or may not be presentMISC_FEATURE(9)..(9)Xaa is any amino acid and
(9) may or may not be presentMISC_FEATURE(10)..(10)Xaa is any amino
acid and (10) may or may not be presentMISC_FEATURE(11)..(11)Xaa is
any amino acid and (11) may or may not be
presentMISC_FEATURE(12)..(12)Xaa is any amino acid and (12) may or
may not be present 81Gly Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Cys1 5 10
* * * * *