U.S. patent application number 11/674765 was filed with the patent office on 2007-07-12 for crop plant cystatin proteinase inhibitors and methods of use.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Daniel J. Altier, Zhongmeng Bao, Guihua Lu, Pedro A. Navarro Acevedo, Vincent J.H. Sewalt, Carl R. Simmons, Nasser Yalpani.
Application Number | 20070162999 11/674765 |
Document ID | / |
Family ID | 34393092 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070162999 |
Kind Code |
A1 |
Altier; Daniel J. ; et
al. |
July 12, 2007 |
Crop Plant Cystatin Proteinase Inhibitors and Methods of Use
Abstract
Methods and compositions for modulating development and defense
responses are provided. Nucleotide sequences encoding maize,
soybean, wheat and rice cystatin proteins are provided. The
sequences can be used in expression cassettes for modulating
development, developmental pathways, and defense responses.
Transformed plants, plant cells, tissues, and seed are also
provided.
Inventors: |
Altier; Daniel J.; (Granger,
IA) ; Bao; Zhongmeng; (Urbandale, IA) ; Lu;
Guihua; (Johnston, IA) ; Navarro Acevedo; Pedro
A.; (Ankeny, IA) ; Sewalt; Vincent J.H.; (West
Des Moines, IA) ; Simmons; Carl R.; (Des Moines,
IA) ; Yalpani; Nasser; (Johnston, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE
P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
34393092 |
Appl. No.: |
11/674765 |
Filed: |
February 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10947979 |
Sep 23, 2004 |
7205453 |
|
|
11674765 |
Feb 14, 2007 |
|
|
|
60505948 |
Sep 25, 2003 |
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Current U.S.
Class: |
800/279 ;
435/419; 435/468; 536/23.2; 800/285 |
Current CPC
Class: |
C12N 15/8279 20130101;
C07K 14/8139 20130101 |
Class at
Publication: |
800/279 ;
800/285; 435/468; 435/419; 536/023.2 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
1. An isolated polynucleotide comprising a nucleic acid sequence
selected from the group consisting of: (a) a nucleic acid sequence
set forth in SEQ ID NO: 5; (b) a nucleotide sequence that encodes a
polypeptide having the amino acid sequence set forth in SEQ ID NO:
6; (c) a nucleic acid sequence having at least 95% sequence
identity over the entire length of SEQ ID NO: 5 as determined by
the GAP algorithm under default parameters, wherein said nucleic
acid sequence encodes a polypeptide with cysteine proteinase
inhibitor activity; (d) a nucleic acid sequence that encodes a
polypeptide with cysteine proteinase inhibitor activity, wherein
said polypeptide has at least 95% sequence identity to SEQ ID NO:
6; and (e) a nucleic acid sequence that comprises the full length
complement of any one of (a) to (d).
2. The isolated polynucleotide of claim 1, wherein said
polynucleotide is optimized for expression in a plant.
3. A DNA construct comprising the isolated polynucleotide of claim
1, wherein said polynucleotide is operably linked to a promoter
that drives expression in a host cell.
4. The DNA construct of claim 3, wherein said polynucleotide is
operably linked in an antisense orientation to said promoter.
5. An expression cassette comprising the DNA construct of claim
3.
6. A host cell having stably incorporated into its genome at least
one DNA construct of claim 3.
7. The host cell of claim 6, wherein said host cell is a plant
cell.
8. A transgenic plant having stably incorporated into its genome
the DNA construct of claim 3.
9. The transgenic plant according to claim 8, wherein said plant is
a monocot.
10. The transgenic plant according to claim 8, wherein said plant
is a dicot.
11. The transgenic plant according to claim 8, wherein said plant
is selected from the group consisting of: corn, soybean, wheat,
rice, alfalfa, barley, millet, sunflower, sorghum, canola and
cotton.
12. Transformed seed of the transgenic plant of claim 8, wherein
said transformed seed comprises the DNA construct of claim 3.
13. A method for enhancing the disease resistance of a plant
comprising: (a) introducing into a plant cell at least one DNA
construct comprising a polynucleotide operably linked to a promoter
that drives expression of a cysteine proteinase inhibitor
polypeptide in plant cells, wherein said polynucleotide comprises a
nucleotide sequence selected from the group consisting of: (i) a
nucleic acid sequence set forth in SEQ ID NO: 5; (ii) a nucleic
acid sequence that encodes a polypeptide having the amino acid
sequence set forth in SEQ ID NO: 6; (iii) a nucleic acid sequence
having at least 95% sequence identity over the entire length of SEQ
ID NO: 5 as determined by the GAP algorithm under default
parameters, wherein said nucleic acid sequence encodes a
polypeptide with cysteine proteinase inhibitor activity; (iv) a
nucleic acid sequence that encodes a polypeptide with cysteine
proteinase inhibitor activity, wherein said polypeptide has at
least 95% sequence identity to SEQ ID NO: 6; and (v) a nucleotide
sequence that comprises the full length complement of any one of
(i) through (iv). (b) growing the plant cell under plant growing
conditions to produce a regenerated plant; and (c) inducing
expression of said polynucleotide for a time sufficient to enhance
the disease resistance of said plant.
14. The method of claim 13, wherein said promoter is selected from
the group consisting of: (a) a strong constitutive promoter; (b) a
tissue-specific promoter; (c) a temporally-defined promoter; and
(d) an inducible promoter.
15. The method of claim 13, wherein said plant expresses a
polypeptide having pesticidal activity against fungal
pathogens.
16. The method of claim 15, wherein said fungus is Fusarium
ssp.
17. The method of claim 13, wherein said plant expresses a
polypeptide having pesticidal activity against insects.
18. The method of claim 13, wherein said plant expresses a
polypeptide having pesticidal activity against nematodes.
19. The method of claim 18, wherein said nematode is a Soybean Cyst
Nematode.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Application No. 60/505,948, filed Sep. 25, 2003, and
U.S. Utility application Ser. No. 10/947,979, filed Sep. 23, 2004,
which are hereby incorporated in their entirety by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of the genetic
manipulation of plants, particularly the modulation of gene
activity and development in plants resulting in improvements in
agronomic traits.
BACKGROUND OF THE INVENTION
[0003] Agronomic traits, such as disease resistance, nutritional
quality, senescence and cell proliferation, have been subject to
improvement attempts by various methods in the past. Often,
improvements are attempted through plant breeding methods, which
are often very expensive and of uncertain success. More recently,
genetic modifications, such as those creating transgenic plants,
have been used in attempts to reach these trait improvement goals.
These approaches are meeting with varied success. No one strategy
or gene has proven to be a panacea, although some show promise.
Successful broad improvement of crop disease resistance, among
other traits, will require multiple strategies. The addition of
novel genes and methods is especially of value in the area of
disease resistance, where pathogens are continually evolving and no
single-gene method will have sustained success for long. Thus
multiple genes and strategies for genetic improvement of agronomic
traits are sought. This invention provides novel genes and methods
of use through which agronomic traits can be improved.
SUMMARY OF THE INVENTION
[0004] Compositions and methods relating to disease resistance and
other plant agronomic traits are provided. Particularly, the
nucleotide and amino acid sequences for cystatin homologs from
maize, soybean, wheat and rice are provided. The nucleotide
sequences of the invention encode proteinase inhibitors of the
cystatin cysteine proteinase inhibitor class.
[0005] The cystatin genes of the present invention may find use in
enhancing agronomic traits of plants, including a wide variety of
crop plants. The compositions and methods of the invention can be
used to manipulate the plant pathogen defense system, the control
of senescence, the control of cell proliferation and cell death,
and the nutritional quality of plant seeds intended for human and
animal consumption. The methods involve stably transforming a plant
with a nucleotide sequence capable of modulating the production of
one or more cystatins in the plant, operably linked with a promoter
capable of driving expression of a gene in a plant cell.
[0006] Specifically, the present invention is directed to an
isolated polynucleotide set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75,
isolated polynucleotides encoding the amino acid sequences set
forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, and 76, isolated polypeptides
having the sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76,
variant polynucleotide and amino acid sequences, DNA constructs
comprising the sequences of the present invention, and host cells
having incorporated such DNA constructs. Transformed plants, plant
cells, and seeds, as well as methods for making such plants, plant
cells, and seeds are additionally provided. It is recognized that a
variety of promoters will be useful in the invention, the choice of
which will depend in part upon the desired level of expression of
the disclosed genes. It is recognized that the levels of expression
can be controlled to modulate the levels of expression in the plant
cell.
[0007] Further embodiments of the invention include methods of
enhancing disease resistance of plants; methods of modulating the
timing of plant maturation; methods of reducing cell death in plant
tissue culture preparations; and methods of modulating protein
digestibility and energy availability in plant products; wherein
the preceding methods use plants transformed with the nucleotide
sequences of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows the chemical reaction inhibited by the
cystatins of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Units, prefixes, and symbols are denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation and amino acid
sequences are written left to right in amino to carboxy
orientation, respectively. Numeric ranges recited within the
specification are inclusive of the numbers defining the range and
include each integer within the defined range. Amino acids may be
referred to herein by either their commonly known three letter
symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission. Nucleotides, likewise, may be
referred to by their commonly accepted single-letter codes. Unless
otherwise provided for, software, electrical, and electronics terms
as used herein are as defined in The New IEEE Standard Dictionary
of Electrical and Electronics Terms (5.sup.th edition, 1993). The
terms below are more fully defined by reference to the
specification as a whole.
[0010] "Amplified" means the construction of multiple copies of a
nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Diagnostic Molecular
Microbiology: Principles and Applications, D. H. Persing et al.,
Ed., American Society for Microbiology, Washington, D.C. (1993).
The product of amplification is termed an amplicon.
[0011] As used herein, "antisense orientation" includes reference
to a duplex polynucleotide sequence that is operably linked to a
promoter in an orientation where the antisense strand is
transcribed. The antisense strand is sufficiently complementary to
an endogenous transcription product such that translation of the
endogenous transcription product is often inhibited.
[0012] "Encoding" or "encoded", with respect to a specified nucleic
acid, means comprising the information for translation into the
specified protein. A nucleic acid encoding a protein may comprise
non-translated sequences (e.g., introns) within translated regions
of the nucleic acid, or may lack such intervening non-translated
sequences (e.g., as in cDNA). The information by which a protein is
encoded is specified by the use of codons. Typically, the amino
acid sequence is encoded by the nucleic acid using the "universal"
genetic code. However, variants of the universal code, such as are
present in some plant, animal, and fungal mitochondria, the
bacterium Mycoplasma capricolum, or the ciliate Macronucleus, may
be used when the nucleic acid is expressed therein.
[0013] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledons or
dicotyledons as these preferences have been shown to differ (Murray
et al. Nucl. Acids Res. 17: 477-498 (1989)). Thus, the maize
preferred codon for a particular amino acid may be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants is listed in Table 4 of Murray et al., supra.
[0014] As used herein "full-length sequence" in reference to a
specified polynucleotide or its encoded protein means having the
entire amino acid sequence of, a native (non-synthetic),
endogenous, biologically active form of the specified protein.
Methods to determine whether a sequence is full-length are well
known in the art including such exemplary techniques as northern or
western blots, primer extension, S1 protection, and ribonuclease
protection. See, e.g., Plant Molecular Biology: A Laboratory
Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to
known full-length homologous (orthologous and/or paralogous)
sequences can also be used to identify full-length sequences of the
present invention. Additionally, consensus sequences typically
present at the 5' and 3' untranslated regions of mRNA aid in the
identification of a polynucleotide as full-length. For example, the
consensus sequence ANNNNAUGG, where the underlined codon represents
the N-terminal methionine, aids in determining whether the
polynucleotide has a complete 5' end. Consensus sequences at the 3'
end, such as polyadenylation sequences, aid in determining whether
the polynucleotide has a complete 3' end.
[0015] As used herein, "heterologous," in reference to a nucleic
acid, is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous nucleotide sequence can be from a species different
from that from which the nucleotide sequence was derived, or, if
from the same species, the promoter is not naturally found operably
linked to the nucleotide sequence. A heterologous protein may
originate from a foreign species, or, if from the same species, is
substantially modified from its original form by deliberate human
intervention.
[0016] "Host cell" means a cell which contains a vector and
supports the replication and/or expression of the vector. Host
cells may be prokaryotic cells such as E. coli, or eukaryotic cells
such as yeast, insect, amphibian, or mammalian cells, excluding
human cells. Preferably, host cells are monocotyledonous or
dicotyledonous plant cells. A particularly preferred
monocotyledonous host cell is a maize host cell.
[0017] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0018] The term "isolated" refers to material, such as a nucleic
acid or a protein, which is: (1) substantially or essentially free
from components that normally accompany or interact with it as it
is found in its naturally occurring environment. The isolated
material optionally comprises material not found with it in its
natural environment; or (2) if the material is in its natural
environment, the material has been synthetically (non-naturally)
altered by deliberate human intervention to a composition and/or
placed at a location in the cell (e.g., genome or subcellular
organelle) not native to a material found in that environment. The
alteration to yield the synthetic material can be performed on the
material within or removed from its natural state. For example, a
naturally occurring nucleic acid becomes an isolated nucleic acid
if it is altered, or if it is transcribed from DNA which has been
altered, by means of human intervention performed within the cell
from which it originates. See, e.g., Compounds and Methods for Site
Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No.
5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic
Cells; Zarling et al., PCT/US93/03868. Likewise, a naturally
occurring nucleic acid (e.g., a promoter) becomes isolated if it is
introduced by non-naturally occurring means to a locus of the
genome not native to that nucleic acid. Nucleic acids which are
"isolated" as defined herein, are also referred to as
"heterologous" nucleic acids.
[0019] As used herein, "nucleic acid" and "polynucleotide" are used
interchangeably and include reference to a deoxyribonucleotide or
ribonucleotide polymer, or chimeras thereof, in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides. A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including among other things, simple and complex
cells.
[0020] "Nucleic acid library" means a collection of isolated DNA or
RNA molecules which comprise and substantially represent the entire
transcribed fraction of a genome of a specified organism or of a
tissue from that organism. Construction of exemplary nucleic acid
libraries, such as genomic and cDNA libraries, is taught in
standard molecular biology references such as Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol.
152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et
al., Molecular Cloning--A Laboratory Manual, 2nd ed., Vol. 1-3
(1989) (hereinafter Sambrook); and Current Protocols in Molecular
Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc. (1994) (hereinafter Ausubel).
[0021] As used herein, "operably linked" includes reference to a
functional linkage between a promoter and a second sequence,
wherein the promoter sequence initiates and mediates transcription
of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences
being linked are contiguous and, where necessary to join two
protein coding regions, contiguous and in the same reading
frame.
[0022] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. Plant cell, as used herein
includes, without limitation, seeds, suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen, and microspores. The classes of
plants which can be used in the methods of the invention include
both monocotyledonous and dicotyledonous plants. A particularly
preferred plant is Zea mays.
[0023] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The essential nature of
such analogues of naturally occurring amino acids is that, when
incorporated into a protein, that protein is specifically reactive
to antibodies elicited to the same protein but consisting entirely
of naturally occurring amino acids. The terms "polypeptide",
"peptide" and "protein" are also inclusive of modifications
including, but not limited to, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation. Further, this invention
contemplates the use of both the methionine-containing and the
methionine-less amino terminal variants of the protein of the
invention.
[0024] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells whether or not its origin
is from a plant cell. Exemplary plant promoters include, but are
not limited to, those that are obtained from plants, plant viruses,
and bacteria which comprise genes expressed in plant cells such as
Agrobacterium or Rhizobium. Examples of promoters under
developmental control include promoters that preferentially
initiate transcription in certain tissues, such as leaves, roots,
or seeds. Such promoters are referred to as "tissue preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue specific". A "cell type" specific promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" or "repressible" promoter is a promoter which is under
environmental control. Examples of environmental conditions that
may effect transcription by inducible promoters include anaerobic
conditions or the presence of light. Tissue specific, tissue
preferred, cell type specific, and inducible promoters constitute
the class of "non-constitutive" promoters. A "constitutive"
promoter is a promoter which is active under most environmental
conditions.
[0025] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all as a result of deliberate human intervention. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention.
[0026] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements which permit
transcription of a particular nucleic acid in a host cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed, and a promoter.
[0027] The terms "residue," "amino acid residue," and "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass non-natural analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0028] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 80% sequence identity, 90% sequence identity, 95% or 100%
sequence identity (i.e., complementary) with each other.
[0029] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will selectively hybridize to its target sequence, to a detectably
greater degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences can be identified which are 100% complementary to the
probe (homologous probing). Alternatively, stringency conditions
can be adjusted to allow some mismatching in sequences so that
lower degrees of similarity are detected (heterologous probing).
Generally, a probe is less than about 1000 nucleotides in length,
and optionally less than 500 nucleotides in length.
[0030] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of a
destabilizing agent such as formamide. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.
for at least 4 hours, more preferably up to 12 hours or longer, and
a final wash in 0.1.times.SSC at 60 to 65.degree. C. for 30
minutes.
[0031] Specificity is typically a function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m (thermal melting point) can be approximated from the
equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):
T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (%
form)-500/L; where M is the molarity of monovalent cations, % GC is
the percentage of guanosine and cytosine nucleotides in the DNA, %
form is the percentage of formamide in the hybridization solution,
and L is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. T.sub.m is reduced by about 1.degree. C. for each 1% of
mismatching; thus, T.sub.m, hybridization and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity.
For example, if sequences with >90% identity are sought, the
T.sub.m can be decreased 10.degree. C.
[0032] Generally, stringent conditions are selected to be about
5.degree. C. lower than the T.sub.m for the specific sequence and
its complement at a defined ionic strength and pH. However,
severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3, or 4.degree. C. lower than the T.sub.m; moderately
stringent conditions can utilize a hybridization and/or wash at 6,
7, 8, 9, or 10.degree. C. lower than the T.sub.m; low stringency
conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 15, or 20.degree. C. lower than the T.sub.m. Using the
equation, hybridization and wash compositions, and desired T.sub.m,
those of ordinary skill will understand that variations in the
stringency of hybridization and/or wash solutions are inherently
described. If the desired degree of mismatching results in a
T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree.
C. (formamide solution) it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York (1993); and
Ausubel.
[0033] As used herein, "transgenic plant" includes reference to a
plant which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of a
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0034] As used herein, "vector" includes reference to a nucleic
acid used in the introduction of a polynucleotide of the present
invention into a host cell. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0035] The following terms are used to describe the sequence
relationships between a polynucleotide/polypeptide of the present
invention with a reference polynucleotide/polypeptide: (a)
"reference sequence", (b) "comparison window", (c) "sequence
identity", and (d) "percentage of sequence identity".
[0036] (a) As used herein, a "reference sequence" is a defined
sequence used as a basis for sequence comparison with a
polynucleotide/polypeptide of the present invention. A reference
sequence may be a subset of, or the entirety of a specified
sequence; for example, as a segment of a full-length cDNA or gene
sequence, or the complete cDNA or gene sequence.
[0037] (b) As used herein, a "comparison window" includes reference
to a contiguous and specified segment of a
polynucleotide/polypeptide sequence, wherein the
polynucleotide/polypeptide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide/polypeptide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. Generally, the comparison window is at least 20
contiguous nucleotide or amino acid residues in length, and
optionally can be 30, 40, 50, 100, or longer. Those of skill in the
art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide/polypeptide
sequence, a gap penalty is typically introduced and is subtracted
from the number of matches.
[0038] Methods of alignment of sequences for comparison are
well-known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Optimal alignment of sequences for comparison may be
conducted by the local alignment algorithm of Smith and Waterman,
Adv. Appl. Math. 2: 482 (1981); by the global alignment algorithm
of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the local
alignment method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:
2444 (1988), by the algorithm of Karlin and Altschul (1990) Proc
Natl Acad Sci USA 87: 2264, modified as in Karlin and Altschul
(1993) Proc Natl Acad Sci USA 90: 5873-5877.; by computerized
implementations of these algorithms, including, but not limited to:
CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package.RTM., Genetics Computer Group (GCG.RTM.),
(Accelrys, Inc., San Diego, Calif.). The CLUSTAL program is well
described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins
and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids
Research 16: 10881-90 (1988); Huang, et al., Computer Applications
in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods
in Molecular Biology 24: 307-331 (1994).
[0039] The BLAST family of programs which can be used for database
similarity searches includes: BLASTN for nucleotide query sequences
against nucleotide database sequences; BLASTX for nucleotide query
sequences against protein database sequences; BLASTP for protein
query sequences against protein database sequences; TBLASTN for
protein query sequences against nucleotide database sequences; and
TBLASTX for nucleotide query sequences against nucleotide database
sequences. See, Current Protocols in Molecular Biology, Chapter 19,
Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,
New York (1995).
[0040] Software for performing BLAST analyses is publicly
available, e.g., through the National Center for Biotechnology
Information website, located on the world wide web at the address
ncbi.nlm.nih.gov, preceded by the www prefix. This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold. These initial
neighborhood word hits act as seeds for initiating searches to find
longer HSPs containing them. The word hits are then extended in
both directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are calculated
using, for nucleotide sequences, the parameters M (reward score for
a pair of matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0041] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5877, 1993). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance.
[0042] BLAST searches assume that proteins can be modeled as random
sequences. However, many real proteins comprise regions of
nonrandom sequences which may be homopolymeric tracts, short-period
repeats, or regions enriched in one or more amino acids. Such
low-complexity regions may be aligned between unrelated proteins
even though other regions of the protein are entirely dissimilar. A
number of low-complexity filter programs can be employed to reduce
such low-complexity alignments. For example, the SEG (Wooten and
Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie and
States, Comput. Chem., 17:191-201, 1993) low-complexity filters can
be employed alone or in combination.
[0043] GAP can also be used to compare a polynucleotide or
polypeptide of the present invention with a reference sequence. GAP
uses the algorithm of Needleman and Wunsch (J. Mol. Biol.
48:443-453, 1970) to find the alignment of two complete sequences
that maximizes the number of matches and minimizes the number of
gaps. GAP considers all possible alignments and gap positions and
creates the alignment with the largest number of matched bases and
the fewest gaps. It allows for the provision of a gap creation
penalty and a gap extension penalty in units of matched bases. GAP
must make a profit of gap creation penalty number of matches for
each gap it inserts. If a gap extension penalty greater than zero
is chosen, GAP must, in addition, make a profit for each gap
inserted of the length of the gap times the gap extension penalty.
Default gap creation penalty values and gap extension penalty
values in Version 10 of the Wisconsin Genetics Software Package for
protein sequences are 8 and 2, respectively. For nucleotide
sequences the default gap creation penalty is 50 while the default
gap extension penalty is 3. The gap creation and gap extension
penalties can be expressed as an integer selected from the group of
integers consisting of from 0 to 200. Thus, for example, the gap
creation and gap extension penalties can each independently be: 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 65 or
greater.
[0044] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0045] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters (Altschul et al.,
Nucleic Acids Res. 25:3389-3402, 1997; Altschul et al., J. Mol.
Bio. 215: 403-410, 1990) or to the value obtained using the GAP
program using default parameters (see the Wisconsin Genetics
Software Package, (Accelrys, Inc., San Diego, Calif.)).
[0046] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences which are the same
when aligned for maximum correspondence over a specified comparison
window. When the percentage of sequence identity is used in
reference to proteins it is recognized that residue positions which
are not identical often differ by conservative amino acid
substitutions, where amino acid residues are substituted for other
amino acid residues with similar chemical properties (e.g. charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences which differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988)
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0047] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0048] (e) As used herein, "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will
recognize that these values can be appropriately adjusted to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, more preferably
at least 70%, 80%, 90%, and most preferably at least 95%.
[0049] The present invention provides, inter alia, compositions and
methods for modulating the total level of proteins of the present
invention and/or altering their ratios in a plant. "Modulation" is
intended to mean an increase or decrease in a particular character,
quality, substance, or response.
[0050] The compositions comprise the nucleotide and amino acid
sequence for 38 homologs of cysteine proteinases from maize, wheat,
rice, and soybean, as presented in Table 1. These plant cystatin
genes are characterized by their cysteine proteinase inhibitory
activity. "Plant cystatin genes" is intended to mean genes that are
structurally related to plant cystatins, also known as plant
cysteine proteinases. As is well known in the art, "proteinases"
are also called "proteases" and "peptidases" interchangeably. Thus,
"cystatin-like" activity is intended to include the activity of
peptides that inhibit the activity of cysteine proteinases in
plants. In addition, at least some of these peptides also retain
antifungal and/or antibacterial activity. The genes of the present
invention are called cystatins after a structural classification of
proteins (SCOP) classification system. TABLE-US-00001 TABLE 1 The
Cystatin Genes of the Present Invention Full Length Full Length
Nucleotide Peptide Nucleotide Corresponding Peptide Sequence
Sequence Plant Gene Name SEQ ID NO: SEQ ID NO: Length (nt) Length
(aa) Maize Zm-Cys1 1 2 786 135 Maize Zm-Cys3 3 4 915 134 Maize
Zm-Cys4 5 6 915 134 Maize Zm-Cys5 7 8 1102 245 Maize Zm-Cys6 9 10
944 176 Maize Zm-Cys7 11 12 688 116 Maize Zm-Cys8 13 14 622 110
Maize Zm-Cys9 15 16 802 157 Maize Zm-Cys10 17 18 871 174 Maize
Zm-Cys11 19 20 716 174 Maize Zm-Cys12 21 22 1102 245 Maize Zm-Cys13
23 24 761 127 Maize Zm-Cys14 25 26 749 150 Soybean Gm-Cys1 27 28
1140 245 Soybean Gm-Cys2 29 30 552 97 Soybean Gm-Cys3 31 32 484 103
Soybean Gm-Cys4 33 34 814 92 Soybean Gm-Cys5 35 36 504 112 Soybean
Gm-Cys6 37 38 708 104 Soybean Gm-Cys7 39 40 505 97 Soybean Gm-Cys8
41 42 573 142 Soybean Gm-Cys9 43 44 473 114 Rice Os-Cys1 45 46 797
102 Rice Os-Cys2 47 48 1091 250 Rice Os-Cys3 49 50 744 108 Rice
Os-Cys4 51 52 919 184 Rice Os-Cys5 53 54 798 151 Rice Os-Cys6 55 56
780 123 Wheat Ta-Cys1 57 58 626 142 Wheat Ta-Cys2 59 60 609 125
Wheat Ta-Cys3 61 62 557 128 Wheat Ta-Cys4 63 64 608 107 Wheat
Ta-Cys6 65 66 622 107 Wheat Ta-Cys8 67 68 750 152 Wheat Ta-Cys9 69
70 801 152 Wheat Ta-Cys10 71 72 1149 243 Wheat Ta-Cys11 73 74 959
180 Wheat Ta-Cys13 75 76 518 127
[0051] Cystatins are a group of proteins which inhibit the activity
of cysteine proteinases. The cystatins identified in vertebrates,
insects, and plants have been classified into four groups, all
belonging to a cystatin superfamily. Groups 1 through 3 are
primarily vertebrate cystatin molecules, while group 4 comprises
all the known plant cystatins. Group 1 cystatins are referred to as
the stefins, single chain proteins with molecular weights of about
11 kDa, which contain no disulfide bonds or carbohydrates. The
second group is referred to as the cystatins, and comprise single
chain proteins of about 13 kDa, with two disulfide bonds located
toward the carboxyl terminus. The kininogens, group 3, are the
largest of the cystatins. They are characterized by having three
Type 2-like domains, bound carbohydrates, and an additional
polypeptide (kinin) unrelated to the cystatin segments.
[0052] The cysteine proteinase inhibitors of plant origin have been
grouped into a fourth cystatin family, the "phytocystatins," or
"plant cystatins" based on their sequence similarity and absence of
disulfide bonds or cysteine residues. The phytocystatins are single
polypeptide chains with molecular weights ranging from 10 to 16
kDa. Many share several reported conserved sequence motifs,
including glycine residue(s) in the vicinity of the N-terminal
region, a Gln-Xaa-Val-Xaa-Gly (SEQ ID NO: 77) motif in the first
hairpin loop, and a Pro-Trp in the second hairpin loop. In
addition, many also share a longer conserved sequence at a part of
the N-terminal .alpha.-1 helix identified as Leu-Ala-Arg-[Phe or
Tyr]-Ala-[Val or Ile]-Xaa-Xaa-Xaa-Asn (SEQ ID NO: 78) (Margis et
al. (1998) Arch Biochem Biophys 359(1): 24-30). After an
examination of 32 members of the plant cystatin family, Margis et
al. (supra) indicate this conserved region of the N-terminal
.alpha.-1 helix can be rewritten as [Leu or Val or Ile]-[Ala or Gly
or Thr]-[Arg or Lys or Glu]-[Phe or Tyr]-[Ala or Ser]-[Val or
Ile]-Xaa-[Glu or Asp or Gln or Val]-[His or Tyr or Phe or Gln]-Asn
(SEQ ID NO: 79).
[0053] Analysis of the 38 plant cystatins (see Table 62--multiple
sequence alignment) of the present invention when compared with
those analyzed by Margis et al. (supra) shows this N-terminal
domain analysis to be generally consistent across the plant
cystatin group. The sequences examined by Margis et al. were
primarily dicot sequences, only 5 of 32 sequences were monocot
species. The present analysis (Table 62) shows some trends that are
more particular to monocot species. In particular, Table 62 shows
that the fourth residue of the N-terminal .alpha.-1 helix, shown by
Margis et al. to be [Phe or Tyr], should be expanded to be [Phe or
Tyr or Trp]. This is supported by the fact that all eight of the
sequences of the instant invention not having a phenylalanine or
tyrosine at position 4 had a tryptophan residue instead, and is
further supported by the chemical similarity of tryptophan to both
phenylalanine and tyrosine, which are often referred to as the
aromatics group of amino acids. Similarly, in the first hairpin
loop, while the Margis et al. analysis showed the consensus
sequence of the final six amino acids to be Thr-Met-Tyr-Tyr-Ile-Thr
(SEQ ID NO: 80), the present analysis showed this consensus to be
Thr-Leu-Tyr-Tyr-Leu-Thr (SEQ ID NO: 81), in which quite often the
Tyr-Tyr was replaced with His-His. The replacement of the
methionine residue with a leucine and the isoleucine residue with a
leucine is not surprising in view of the fact that methionine,
leucine, isoleucine and valine are all in the same family and are
considered to be conservative substitutes for each other.
[0054] Table 2 shows the sequences of the first and second hairpin
loops and their locations in the cystatin sequences of the present
invention. In particular, the highly conserved nature of the QXVXG
(SEQ ID NO: 77) motif within the first hairpin loop is evident.
Furthermore, the second hairpin loop, while less highly conserved
than the first, generally presents a tryptophan residue at the end
of the motif. TABLE-US-00002 TABLE 2 First Hairpin Loop (FHL) and
Second Hairpin Loop (SHL) Motifs of Plant Cystatin Genes FHL FHL
SHL SHL Full Motif Start Motif Start Protein SEQ Amino SEQ Amino
SEQ Gene ID Acid ID Acid ID NO: Name FHL Motif NO: Position SHL
Motif NO: Position 28 gm-cys1 QVVAGTLHHLT 82 93 EAKVWVKPW 120 116
30 gm-cys2 QVVSGTLYTIT 83 49 EAKVWEKSW 121 72 32 gm-cys3
QVVSGTLYYIT 84 49 ETKVLEKPW 122 72 34 gm-cys4 QVVEGFIYYIT 85 40
ETKVWVRSW 123 63 36 gm-cys5 QVVSGTNYRLV 86 68 EAIVWEKPW 124 91 38
gm-cys6 QVVSGMKYYLK 87 54 TSVVVVKPW 125 77 40 gm-cys7 QVVSGTLYTIT
88 49 EAKVWEKAW 126 72 42 gm-cys8 QVVSGMKYYLK 89 80 NSVVVVKPW 127
103 44 gm-cys9 QVVAGLNYRLS 90 70 QAIVYEKAW 128 90 46 os-cys1
QVVAGTLYYFT 91 53 EAKVWEKPW 129 76 48 os-cys2 QVVAGTLHHLT 92 93
EAKVWVKPW 130 116 50 os-cys3 QVVGGFMHYLT 93 59 EAKVWERAW 131 83 52
os-cys4 QVVTGTLHDLM 94 90 SAKVWVKPW 132 113 54 os-cys5 QVVSDVAYYLK
95 96 DAVVVVKAW 133 127 56 os-cys6 QVVSGMNYRLV 96 76 VAVVYEQSW 134
100 58 ta-cys1 QTVAGTMHYIT 97 93 EAKVWEKPW 135 116 72 ta-cys10
QTVAGTVHHLT 98 86 EAKVWVKPW 136 109 74 ta-cys11 QVVAGTLHDLM 99 85
KAKVWVKPW 137 108 76 ta-cys13 QVVAGTMYYLT 100 78 EAKVWEKPW 138 101
60 ta-cys2 QTVAGTMHYIT 101 76 EAKVWEKPW 139 99 62 ta-cys3
QLVSGMNYELI 102 83 KAEVYEQTW 140 107 64 ta-cys4 QVVAGCMHYFT 103 63
EAKVWEKAW 141 86 66 ta-cys6 QVVAGCMHYFT 104 63 EAKVWEKAW 142 86 68
ta-cys8 QVVSGIKYYLR 105 98 DAVVVVKPW 143 129 70 ta-cys9 QVVSGIKYYLR
106 98 DAVVVVKPW 144 129 2 ZmCys1 QVVAGTMYYLT 107 86 EAKVWEKPW 145
109 18 ZmCys10 QVVTGTLHDLI 108 77 RAKVWVKSW 146 100 20 ZmCys11
QVVAGTNYKLN 109 131 QAVVFDPLP 147 152 22 ZmCys12 QVVAGTLHHLT 110 87
EAKVWVKPW 148 110 24 ZmCys13 QIVAGKNYRLR 111 83 RAVVYEQLT 149 107
26 ZmCys14 QVVSGLKYYLR 112 99 DAVVVVKPW 150 127 4 ZmCys3
QVVAGTMYYLT 113 85 EAKVWEKPW 151 108 6 ZmCys4 QVVAGTMYYLT 114 85
EAKVWEKPW 152 108 8 ZmCys5 QVVAGTLHHLT 115 87 EAKVWVKPW 153 110 10
ZmCys6 QVVTGTLHDLI 116 80 RAKVWVKPW 154 103 12 ZmCys7 QVVSGMNYKLV
117 71 GAFVYEQSW 155 95 14 ZmCys8 QVVAGTLHHFT 118 59 EAKVWEKAW 156
84 16 ZmCys9 QVVSGMNYRLY 119 81 VAVVYEQVW 157 105
[0055] Table 3 shows the conserved region of the N-terminal
alpha-1-helix in each of the sequences of the present invention.
TABLE-US-00003 TABLE 3 N-terminal Alpha-1 Helix Motif Full Motif
Protein Motif Start SEQ SEQ Amino ID Gene Motif ID Acid NO: Name
Sequence NO: Position 28 gm-cys1 LARFAVDEHN 158 66 30 gm-cys2
LARFAVEEHN 159 22 32 gm-cys3 LARFAVDEHN 160 22 34 gm-cys4
LARFAVEEQN 161 13 36 gm-cys5 IANYALSEYD 162 41 38 gm-cys6
LGRFAVEEYN 163 20 40 gm-cys7 LARFAVEEHN 164 22 42 gm-cys8
LGRFAVEEYN 165 42 44 gm-cys9 IANFAVTEYD 166 43 46 os-cys1
LARFAVTEHN 167 26 48 os-cys2 LARFAVDEHN 168 66 50 os-cys3
LARFAVAEHN 169 32 52 os-cys4 AARFAVAEYN 170 63 54 os-cys5
LGRFAVAEHN 171 57 56 os-cys6 LGGWAVERHA 172 49 58 ta-cys1
LARFAVSEHN 173 66 72 ta-cys10 LARFAVDEHN 174 59 74 ta-cys11
AARFAVAEHN 175 58 76 ta-cys13 LARFAVDEHN 176 51 60 ta-cys2
LARFAVSEHN 177 49 62 ta-cys3 LGRWAVLEFG 178 56 64 ta-cys4
LARFAVAEHN 179 36 66 ta-cys6 LARFAVAEHN 180 36 68 ta-cys8
LGRYSVEEHN 181 61 70 ta-cys9 LGRYSVEEHN 182 61 2 ZmCys1 LARFAVNEHN
183 59 18 ZmCys10 AARFAVAHYN 184 50 20 ZmCys11 VGEWAVKEHN 185 104
22 ZmCys12 LGRFAVDEHN 186 60 24 ZmCys13 IGRWAVSEHI 187 56 26
ZmCys14 LGRFSVAEYN 188 66 4 ZmCys3 LARFAVDEHN 189 58 6 ZmCys4
LARFAVDEHN 190 58 8 ZmCys5 LGRFAVDEHN 191 60 10 ZmCys6 AARFAVAYHN
192 53 12 ZmCys7 LGGWAVTEHV 193 44 14 ZmCys8 LARFAVAEHN 194 32 16
ZmCys9 LGGWALGQAK 195 35
[0056] The protein sequences of the present invention were analyzed
for percent identities and similarities using the GAP algorithm.
These analyses were performed by species, such that the maize
sequences were compared to the other maize sequences, and so on for
each of soybean, rice, and wheat. It is evident from the tables
that follow that the sequences of the present invention, although
they are all cystatins, can vary markedly at the protein level
while still retaining cystatin activity. In Tables 4 through 11,
sequence similarities and identities among crop plant sequence
groups are presented. Those SEQ ID NOs: for which activity data are
provided in the instant application are shown in bold faced type
(see Examples). TABLE-US-00004 TABLE 4 GAP Analysis: Maize Amino
Acid Sequence Percent Identities SEQ ID NO: 3 5 7 9 11 13 15 18 20
22 24 26 1 90.3 90.3 44.2 38.7 36.2 56.0 33.0 37.3 34.1 44.3 35.2
33.3 3 100 45.0 38.2 39.1 56.0 33.9 37.3 33.3 45.0 37.1 33.3 5 45.0
38.2 39.1 56.0 33.9 37.3 33.3 45.0 37.1 33.3 7 38.9 33.9 50.5 28.7
36.9 32.3 100 29.5 37.6 9 36.8 44.9 25.5 88.4 32.5 39.0 24.6 38.1
11 29.0 57.4 33.9 37.5 33.9 53.9 38.8 13 25.2 43.5 32.3 50.5 34.7
36.9 15 28.0 35.2 28.7 43.0 36.7 18 29.9 36.9 22.5 34.9 20 32.3
44.3 29.6 22 29.5 37.6 24 37.1
[0057] TABLE-US-00005 TABLE 5 GAP Analysis: Maize Amino Acid
Sequence Percent Similarities SEQ ID NO: 3 5 7 9 11 13 15 18 20 22
24 26 1 92.5 92.5 53.4 48.1 44.2 69.2 40.3 46.8 40.0 53.4 44.0 43.2
3 100 54.2 46.9 47.8 70.1 42.7 45.2 41.7 54.2 48.4 43.4 5 54.2 46.9
47.8 70.1 42.7 45.2 41.7 54.2 48.4 43.4 7 46.5 40.0 57.0 35.3 44.6
35.4 100 40.2 44.8 9 44.7 49.5 34.6 91.3 37.5 46.5 38.5 45.2 11
38.0 61.1 42.6 45.5 40.0 64.3 46.6 13 32.7 47.2 38.4 57.0 46.5 45.6
15 36.7 43.4 35.3 49.6 42.2 18 36.8 44.6 33.3 41.5 20 35.4 54.9
35.2 22 40.2 44.8 24 48.4
[0058] TABLE-US-00006 TABLE 6 GAP Analysis: Glycine max Amino Acid
Sequence Percent Identities SEQ ID NO: 30 32 34 36 38 40 42 44 28
67.0 55.3 45.7 33.9 41.2 65.0 37.8 27.7 30 69.1 61.4 41.3 42.2 94.9
38.0 30.8 32 67.4 39.1 34.0 69.1 33.0 32.2 34 34.5 37.8 60.2 35.9
32.9 36 34.4 41.3 32.1 59.6 38 42.2 82.7 31.8 40 38.0 31.9 42
28.4
[0059] TABLE-US-00007 TABLE 7 GAP Analysis: Glycine max Amino Acid
Sequence Percent Similarities SEQ ID NO: 30 32 34 36 38 40 42 44 28
77.3 63.1 59.8 44.6 55.7 75.3 48.8 38.4 30 78.4 68.2 48.9 51.1 94.9
47.8 42.9 32 72.8 48.9 47.4 78.4 42.7 41.1 34 44.0 47.8 67.0 44.6
40.2 36 42.2 48.9 39.3 67.0 38 51.1 85.6 42.0 40 47.8 44.0 42
36.7
[0060] TABLE-US-00008 TABLE 8 GAP Analysis: Oryza sativa Amino Acid
Sequence Percent Identities SEQ ID NO: 48 50 52 54 56 46 54.9 60.0
40.2 34.7 31.3 48 48.1 37.0 40.2 34.7 50 38.3 34.9 36.9 52 31.3
25.0 54 42.3
[0061] TABLE-US-00009 TABLE 9 GAP Analysis: Oryza sativa Amino Acid
Sequence Percent Similarities SEQ ID NO: 48 50 52 54 56 46 62.7
71.0 49.0 44.9 40.4 48 57.5 42.0 46.5 41.3 50 46.7 44.3 40.8 52
36.7 31.7 54 48.0
[0062] TABLE-US-00010 TABLE 10 GAP Analysis: Wheat Amino Acid
Sequence Percent Identities SEQ ID NO: 60 62 64 66 68 70 72 74 76
58 92.8 28.6 61.7 61.7 29.9 31.3 46.7 38.8 63.8 60 33.3 63.6 63.6
31.4 33.1 48.7 38.0 62.6 62 33.6 33.6 29.7 29.7 30.3 24.4 35.0 64
98.1 34.0 34.9 47.6 43.0 63.2 66 33.0 34.0 47.6 42.1 63.2 68 91.5
38.8 43.4 37.2 70 35.7 40.0 33.1 72 38.3 47.9 74 39.5
[0063] TABLE-US-00011 TABLE 11 GAP Analysis: Wheat Amino Acid
Sequence Percent Similarities SEQ ID NO: 60 62 64 66 68 70 72 74 76
58 93.6 34.1 66.4 65.4 41.8 41.8 54.1 47.8 70.9 60 39.3 67.3 66.4
43.0 43.8 57.1 47.1 68.3 62 40.2 40.2 37.5 39.1 41.0 34.4 44.2 64
98.1 42.5 43.4 57.1 51.4 70.8 66 41.5 42.5 57.1 50.5 70.8 68 94.1
46.5 50.0 47.1 70 44.2 47.7 46.0 72 43.4 57.0 74 51.6
[0064] The phytocystatins play a role in a wide range of plant
physiological processes, including plant defense mechanisms. Plant
cystatins have been found in various tissues in numerous plant
species, including, but not limited to, crop plants such as rice
(Abe et al. (1987) J Biol Chem 262: 16793-16797; Kondo et al.
(1990) J Biol Chem 265: 15832-15837), tomato (Wu et al. (2000) Comp
Biochem Phys C 127: 209-220), maize (Yamada et al. (2000) Plant
Cell Physiol 42(7): 710-716), sunflower (Doi-Kawano et al. (1998) J
Biochem 124: 11-916), soybean (Misaka et al. (1996) Eur J Biochem
240: 609-614) and potato (Rowan et al. (1990) FEBS Lett 269:
328-330; Waldron et al. (1993) Plant Mol Biol 23: 801-812).
Information on their amino acid sequences has been obtained through
either protein or cDNA sequencing.
[0065] Cystatins play a role in many plant physiological functions,
including defense, more specifically plant defense against
pathogens. A range of functions performed by plant cystatins are
responsible for enhancing plant defense against different
pathogens. While not wishing to be bound by any one mechanism of
action, the sequences and related genes of the present invention
encode proteins with antimicrobial and antifungal activity. These
proteins may inhibit the proteinases of the pathogen, so as to
thwart their utilization of the plant tissue. In addition,
cystatins which are expressed around disease-induced lesions may
control symptom development, as in a hypersensitive response (HR),
by controlling the proteinase-mediated cell death mechanism.
[0066] Compositions of the present invention include the sequences
for maize, soybean, rice and wheat nucleotide sequences which have
been identified as cystatins that are involved in plant defense
response and development. In particular, the present invention
provides for isolated nucleic acid molecules comprising nucleotide
sequences encoding the amino acid sequences shown in SEQ ID NOs: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, and 76. Further provided are polypeptides having an amino
acid sequence encoded by a nucleic acid molecule described herein,
for example those nucleotide sequences set forth in SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, and 75.
[0067] The nucleotide sequences of the invention are maize,
soybean, rice and wheat sequences comprising plant cysteine
proteinases. The claimed sequences are members of the plant
cystatin class of genes and polypeptides. These plant cystatins are
identified herein as "Zm-Cys", "Ta-Cys", "Gm-Cys" and "Os-Cys" for
cystatins originating from Zea mays, Triticum aestivum, Glycine max
and Oryza sativa, respectively, and are numbered for easy reference
(e.g. Zm-Cys10 or Gm-Cys8). These sequences represent a diverse and
conserved supergene family in plants.
[0068] The compositions of the invention can be used in a variety
of methods whereby the protein products can be expressed in crop
plants to function as antimicrobial proteins. Such expression
results in the alteration or modulation of the level, tissue, or
timing of expression to achieve enhanced disease or stress
resistance. The compositions of the invention may be expressed in
the same species from which the particular cystatin originates, or
alternatively, can be expressed in any plant of interest. In this
manner, the coding sequence for the cystatin can be used in
combination with a promoter that is introduced into a crop plant.
In one embodiment, a high-level expressing constitutive promoter
may be utilized and would result in high levels of expression of
the cystatin. In other embodiments, the coding sequence may be
operably linked to a tissue-specific promoter to direct the
expression to a plant tissue known to be susceptible to a pathogen.
Likewise, manipulation of the timing of expression may be utilized.
For example, by judicious choice of promoter, expression can be
enhanced early in plant growth to prime the plant to be responsive
to pathogen attack. Likewise, pathogen inducible promoters can be
used wherein expression of the cystatin is turned on in the
presence of the pathogen.
[0069] The cystatin genes of the present invention additionally
find use in enhancing the plant pathogen defense system. The
compositions and methods of the invention can be used for enhancing
resistance to plant pathogens including fungal pathogens, plant
viruses, and the like. The method involves stably transforming a
plant with a nucleotide sequence capable of modulating the plant
pathogen defense system operably linked with a promoter capable of
driving expression of a gene in a plant cell. "Enhancing
resistance" means that the plant's tolerance to pathogens is
increased. That is, the cystatin may slow or prevent pathogen
infection and spread.
[0070] In specific embodiments, methods for increasing pathogen
resistance in a plant comprise stably transforming a plant with a
DNA construct comprising an anti-pathogenic nucleotide sequence of
the invention operably linked to a promoter that drives expression
in a plant. Such methods find use in agriculture, particularly in
limiting the impact of plant pathogens on crop plants. While the
choice of promoter will depend on the desired timing and location
of expression of the anti-pathogenic nucleotide sequences,
preferred promoters include constitutive and pathogen-inducible
promoters.
[0071] Additionally, the compositions can be used in formulations
used for their disease resistance activities. The proteins of the
invention can be formulated with an acceptable carrier into a
pesticidal composition(s) that is for example, a suspension, a
solution, an emulsion, a dusting powder, a dispersible granule, a
wettable powder, an emulsifiable concentrate, an aerosol, an
impregnated granule, an adjuvant, a coatable paste, or an
encapsulation in, for example, polymer substances.
[0072] Transformed plants, plant cells, plant tissues and seeds
thereof are additionally provided.
[0073] It is recognized that the present invention is not dependent
upon a particular mechanism of defense. Rather, the genes and
methods of the invention work to increase resistance of the plant
to pathogens independent of how that resistance is increased or
achieved.
[0074] It is understood in the art that plant DNA viruses and
fungal pathogens remodel the control of the host replication and
gene expression machinery to accomplish their own replication and
effective infection. The present invention may be useful in
preventing such corruption of the cell.
[0075] The cystatin sequences find use in disrupting cellular
function of plant pathogens or insect pests as well as altering the
defense mechanisms of a host plant to enhance resistance to disease
or insect pests. While the invention is not bound by any particular
mechanism of action to enhance disease resistance, the gene
products, probably proteins or polypeptides, function to inhibit or
prevent diseases in a plant.
[0076] The methods of the invention can be used with other methods
available in the art for enhancing disease resistance in plants.
For example, any one of a variety of second nucleotide sequences
may be utilized, embodiments of the invention encompass those
second nucleotide sequences that, when expressed in a plant, help
to increase the resistance of a plant to pathogens. It is
recognized that such second nucleotide sequences may be used in
either the sense or antisense orientation depending on the desired
outcome. Other plant defense proteins include those described in
PCT patent publications WO 99/43823 and WO 99/43821, both of which
are herein incorporated by reference.
[0077] Plant senescence is an important trait affecting life cycle
duration or maturity, seed dry down, the disease resistance
profile, and `stay green`, which in turn affect yield, stalk
strength, appearance, and nutritional value (silage quality). All
of these factors, which relate to cell death processes, are
considered in maize breeding efforts. Plant proteinases have been
implicated in these processes and thus comprise an area of active
research.
[0078] In particular, cysteine proteinases are induced upon plant
organ senescence, such as in tomato leaves (Drake et al. (1996)
Plant Mol Biol 30(4): 755-767), sweet potato (Chen et al. (2002)
Plant and Cell Phys 43(9): 984-991), and day-lily flowers
(Valpuesta et al. (1995) Plant Mol Biol 28(3): 575-582).
Furthermore, the maize cysteine proteinase Seel ("Senescence
enhanced") has been linked to the stay green phenotype in maize
(Griffiths et al. (1997) Plant Mol Biol 34: 815-821). Cysteine
proteinase inhibitors have also been shown to delay flower
senescence (Eason et al. (2002) Functional Plant Biol 29(9):
1055-1064). While the biology is undoubtedly complex in senescence,
to the extent that the cysteine proteinases are involved in
senescence related processes, then their cognate proteinase
inhibitors, here cystatins, are involved in the control of the
timing and onset of senescence as well. Modulation of cystatins can
provide agronomic advantages by promoting or delaying senescence
and other developmental signals.
[0079] In a plant breeding effort, the position of the starting
breeding material relative to the desired outcome, will dictate
what direction one will want to push a trait. For example, one may
want increase or decrease maturity time; generally decrease. To
increase senescence one may want to suppress cystatin expression,
and to decrease senescence one may want to increase cystatin
expression. Methods for increasing or decreasing cystatin
expression are outlined elsewhere in this specification. However,
tissue-targeted or developmental-targeted expression may be
desirable to reach these ends. The proteinase promoters, such as
those from Seel, can be useful in conjunction with forward or
antisense constructs of the proteinase inhibitor gene in question,
to coordinately augment or cancel, respectively, the
death-promoting capacity of the cysteine proteinase.
[0080] The proteinase inhibitor genes herein are useful for
controlling the senescence of special crop plant tissues. For
certain crops particular tissue or organs are desired to senesce.
This includes controlled dropping of cotton leaves to facilitate
cotton boll harvesting. Sometimes organs are desired not to
senesce, as in the petioles of fruit; premature fruit drop can
cause loss of yield. For maize, delay of senescence of the
pedicel/hilum region of kernels may be desirable to allow for
prolonged kernel fill or delayed maturation of seed, with higher
yield and/or higher digestibility as possible outcomes.
[0081] Over-expression or transgenic expression of proteinase
inhibitors provides effective control of both cyst and root knot
nematodes. The primary mechanism by which cystatins confer nematode
resistance is most likely associated with disruption of nematode
development. Currently over-expression of proteinase inhibitors
(PIs) offers the most advanced approach for nematode control.
Furthermore, transgenic expression of PIs provides effective
control of both cyst and root knot nematodes. Recent research has
shown the value of using proteinase inhibitors in controlling
certain species of nematodes.
[0082] Oryzacystatin-I (Oc-I) is a cysteine proteinase inhibitor
from rice seeds (Abe et al. (1987) Supra), while Oc-1D86 is a
modified form of Oc-1 which has shown stronger inhibitory activity
(Urwin et al. (1995) Plant J 8: 121-131). When expressed in tomato
hairy roots both Oc-1 and Oc-ID86 had a detrimental effect on the
growth and development of potato cyst nematode G. pallida (Id.).
Similarly, when expressed in transgenic Arabidopsis thaliana
Oc-ID86 had a profound effect on the size and fecundity of females
of both the beet-cyst nematode Heterodera schachtii and the
root-knot nematode Meloidogyne incognita as well as reniform
nematode Rotylenchulus reniformis (Urwin et al. (1997) Plant J 12:
455-461; Urwin et al. (1998) Planta 204: 472-479; Urwin et al.
(2000) Mol Breeding 6: 257-264). Compositions of the instant
invention indicate that cystatins can also confer resistance to
soybean cyst nematode (SCN) in soybean and other crops, by
inhibiting nematode growth and development.
[0083] The recovery of viable transgenic plants from crop plants,
in particular for monocot cereal crop plants such as maize, rice
and wheat, is still a laborious and expensive process. This can be
a particular problem when transformation-recalcitrant varieties,
often those with desirable breeding characteristics, perform poorly
in the transgenic production transformation process. Methods are
consequently sought to identify new methods that will improve the
transformation and recovery of viable plants.
[0084] One of the chief problems is cell death in tissue culture.
This is caused not only by the general poor viability of some lines
in culture, but also by the fact that in order to select for the
transformants, often antibiotics are added that kill the
non-transformed cells. Amidst this cell death the positively
transformed lines are also killed. Recalcitrance to death, or the
signals of death, and as well positive cell growth, are thus
desirable features.
[0085] To the extent that these proteinase inhibitors can retard
cell death by suppressing proteinase inhibitor activity, they can
be used to help transformed cells survive. Cysteine proteinases are
known to be induced in the plant HR response, and transgenic
ectopically expressed cystatins can counteract this response
(Pechan et al. (2000) Plant Cell 12(7): 1031-1040; Solomon et al.
(1999) Plant Cell 11 (3): 431-443). The transformed cells receive a
copy of one or more of these proteinase inhibitor coding region(s)
driven by an appropriate promoter. Promoter choices are discussed
elsewhere in this application, however, this embodiment can benefit
from the use of a constitutive promoter, or by a transiently
expressed promoter targeted to the cell culture phase or induced by
plant hormones used in culture. Constitutive expression may help
disease resistance generally, and as such, constitutive promoters,
for example the ubiquitin promoter, can be useful beyond cell
culture. Of course a variety of promoters would be effective. The
resulting transformed cells would be more viable. This would effect
a cleaner separation of the dying non-transformed cells and allow
for cleaner and more rapid growth of the transformed line. Plant
transformation techniques would be improved as a result.
[0086] It should also be recognized that plants can be wounded
abiotically, as by drought stress, wind stress (which includes
damage by wind-blown soil particles), and chemical and nutrient
stress. Such stresses can precipitate cell death that can reduce
plant yield. To the extent that these proteinase inhibitors may
retard cell death by thwarting proteinase inhibitor activity, they
can retard the symptom development of necrosis resulting from these
stresses when driven by a death-induced promoter.
[0087] Second, the proteinase inhibitor genes can have application
in the development and implementation of herbicide resistance
mechanisms in crop plants. Ectopic expression of the proteinase
inhibitors, as in leaves, can result in a retardation of cell death
following the application of herbicides. This would be subject to
the kind of herbicide used and its mode of action, but it is an
area of utility for these genes. Herbicides and herbicide
resistance systems are often used as selectable markers in plant
transformation experiments. Thus, in a way similar to the herbicide
resistance application, these proteinase inhibitor genes can be
used as selectable markers--only cells expressing the proteinase
inhibitor genes (ectopically) would grow or stay alive in the face
of an antibiotic/herbicide medium. This application of course bears
direct overlap with the examples given above for improving plant
transformation.
[0088] Cell death can also be a mechanism of male infertility.
Consequently similar methods, probably with anther- or tapetum- or
pollen-preferred expression, could be a means of enhancing or
controlling male fertility. For example, expressing cystatins can
suppress cell death and thus suppress sterility, rendering the
plants male fertile. This could be used in a conditional situation,
where the plants would be sterile until induced to be fertile.
[0089] Many proteinase inhibitors, including some of the present
invention, are expressed in seeds. The chief biological role of
seed expression of proteinase inhibitors is to inhibit, or
otherwise control, proteinase activities in the seeds. This is
especially important during seed development/maturation, in order
to regulate protein processing by proteinases. Cereal cysteine
proteinases play a chief role in the digestion of seed storage
proteins, especially during germination (Gruis et al. (2002) Plant
Cell 14(11):2863-2882; Debarros & Larkins (1994) Plant Sci
99(2) 189-197; Koehler & Ho (1990) Plant Physiol 94(1):251-258;
Poulle & Jones (1988) Plant Physiol 88(4): 1454-1460).
Regulating the activity of cysteine proteinases in seeds prevents
undesirable loss of seed proteins, including storage proteins, and
also prevents premature germination (Corre et al (2002) Plant Mol
Biol 50(4-5):687-698). Furthermore, regulating the processing of
proteins can serve as an anti-nutritional/protective agent against
microbes, insects, and herbivores. However, crop plant seeds, such
as maize caryopses, are mostly intended for animal consumption as
feed grain, and some also for human food consumption. As such, the
proteinase inhibitors from the seed can inhibit digestive
proteinases in the gastrointestinal tract of livestock and humans.
This can change the site and extent of digestion of protein and
other grain components within, as well as elicit hyper-secretion of
pancreatic enzymes. The impact on overall nutritional status may
either be positive or negative.
[0090] Lowering the digestibility of grain proteins lowers the
effectiveness of the grain for weight gain for monogastric animals
and humans. The reduced protein digestibility will also reduce
access of starch-degrading enzymes to starch granules (which are
encompassed by a protein matrix), thereby reducing digestible
energy content in addition to digestible protein. Moreover, various
proteinase inhibitors induce the release of pancreatic
cholecystokinin, a known satiety factor resulting in lower feed or
food intake (Elsaesser et al. (1990) Cell Tissue Res 262(1):
143-148; Garlicki et al. (1990) Am J Physiol 258: E40-E45; Schwartz
et al. (1994) Diabetes Care 17(4): 255-262; Choi et al. (2000)
Domest Anim Endocrin 19(3): 159-175). For livestock, this is
clearly a negative factor, as well as for undernourished humans in
developing countries; reduced caloric value and reduced food intake
may be very positive, however, for overweight people.
[0091] Lowering fermentative proteolysis to reduce the formation of
non-protein-nitrogen (NPN) is beneficial, however, for ruminant
livestock (dairy and beef cattle, sheep and goats), especially if
the protein is of high Biological Value (i.e., of balanced amino
acid composition, containing especially lysine, tryptophan,
threonine, and methionine). First, silage made of various forages,
such as ryegrass and alfalfa, is subject to excessive proteolysis
during the ensiling process. Total protein losses can amount to 50%
and the dairy cow poorly utilizes the resulting NPN. Proteinase
inhibitors can be employed to reduce these proteolytic losses.
Second, lowering the proteolysis in the rumen is beneficial to
allow otherwise easily digestible high-protein concentrates (such
as fat-extracted soybean & canola meals) and high-protein
forages (such as alfalfa) to bypass rumen fermentation. Rapid and
extensive ruminal breakdown of protein leads to decreased protein
efficiency because 1) the rumen microbes do not use the degraded
protein as fast as it is broken down, leading to excessive
formation of ammonia, much of which will be excreted in the urine
as urea, and 2) the microbial protein that is re-synthesized from
ammonia is generally of lower biological value than soybean or
canola protein.
[0092] Consequently, to the extent that these cystatin proteinase
inhibitors can alter digestion characteristics of the grain, it
would be desirable to reduce the level of their expression to
increase protein digestibility and energy availability for
monogastric livestock and humans. On the other hand, given the
reasoning above, it would be desirable to increase the level of
cystatin expression to reduce proteolysis in silage and generate
rumen by-pass protein for ruminant livestock, and to produce diet
foods by reducing the caloric value of cereals and/or inducing
satiety.
[0093] It is this over-expression that would be the best mode of
using these cystatin genes, which would be achieved by
over-expression of one or more of the cystatin genes. The various
advantages and disadvantages of using different promoters to drive
such over-expression is well known by those skilled in the art.
However, by way of example, a constitutive promoter could drive the
expression, but a more ideal promoter would target tissues, such as
the grain. For silage production, a high-level vegetative promoter
would be desirable. Over-expression of one or several of the
cystatins would be technically more easy to achieve than
suppression of most of these cystatins, especially given their
sequence diversity.
[0094] The different proteinase inhibitor genes have somewhat
different expression profiles. Based upon the maize EST library
distributions, Zm-Cys5, Zm-Cys6, Zm-Cys9, Zm-Cys10, Zm-Cys13, and
Zm-Cys14 are abundant in, or specific to, maize endosperm or other
kernel tissues. Generally, the best mode for this invention (in
maize or cereals) is to express the sense version of the proteinase
inhibitor genes under the control of a seed-preferred, especially
endosperm-preferred, especially R3-R5-preferred promoter, or, in
the case of alfalfa, a constitutive promoter. Thus when the sense
transcript is produced, it will result in either over-expression or
silencing of the targeted proteinase inhibitor gene. There are
other methods for suppression of gene expression that may be
applied. This strategy could be applied to one or several of the
proteinase inhibitor genes in the same crop plant.
[0095] Sequences of the invention, as discussed in more detail
below, encompass coding sequences, antisense sequences, and
fragments and variants thereof. Expression of the sequences of the
invention can be used to modulate or regulate the expression of
corresponding cystatin proteins. The invention encompasses isolated
or substantially purified nucleic acid or protein compositions. An
"isolated" or "purified" nucleic acid molecule or protein, or
biologically active portion thereof, is substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Preferably, an "isolated"
nucleic acid is free of sequences (preferably protein encoding
sequences) that naturally flank the nucleic acid (i.e., sequences
located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the organism from which the nucleic acid is derived. For
example, in various embodiments, the isolated nucleic acid molecule
can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb,
or 0.1 kb of nucleotide sequences that naturally flank the nucleic
acid molecule in genomic DNA of the cell from which the nucleic
acid is derived. A protein that is substantially free of cellular
material includes preparations of protein having less than about
30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When
the protein of the invention or biologically active portion thereof
is recombinantly produced, preferably culture medium represents
less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-protein-of-interest chemicals.
[0096] Fragments and variants of the disclosed nucleotide sequences
and proteins encoded thereby are also encompassed by the present
invention. "Fragment" means a portion of the nucleotide sequence or
a portion of the amino acid sequence and hence protein encoded
thereby. Fragments of a nucleotide sequence may encode protein
fragments that retain the biological activity of the native protein
and hence have cystatin-like activity and thereby affect
development, developmental pathways, and defense responses.
Alternatively, fragments of a nucleotide sequence that are useful
as hybridization probes generally do not encode fragment proteins
retaining biological activity. Thus, fragments of a nucleotide
sequence may range from at least about 20 nucleotides, about 50
nucleotides, about 100 nucleotides, and up to the full-length
nucleotide sequence encoding the proteins of the invention.
[0097] A fragment of a cystatin nucleotide sequence that encodes a
biologically active portion of a cystatin protein of the invention
will encode at least 15, 25, 30, 50, 100, 150, 200, or 250
contiguous amino acids, or up to the total number of amino acids
present in a full-length protein of the invention (for example,
135, 134, 134, 245, 176, 116, 110 or 157 amino acids for SEQ ID
NO:2, 4, 6, 8, 10, 12, 14, or 16, respectively). Fragments of a
cystatin nucleotide sequence that are useful as hybridization
probes for PCR primers generally need not encode a biologically
active portion of a cystatin protein.
[0098] Thus, a fragment of a cystatin nucleotide sequence may
encode a biologically active portion of a cystatin protein, or it
may be a fragment that can be used as a hybridization probe or PCR
primer using methods disclosed below. A biologically active portion
of a cystatin protein can be prepared by isolating a portion of one
of the cystatin nucleotide sequences of the invention, expressing
the encoded portion of the cystatin protein (e.g., by recombinant
expression in vitro), and assessing the activity of the encoded
portion of the cystatin protein. Nucleic acid molecules that are
fragments of a cystatin nucleotide sequence comprise at least 16,
20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, or 800 nucleotides, or up to the number of nucleotides
present in a full-length cystatin nucleotide sequence disclosed
herein (for example, 408, 405, 405, 738, 531, 351, 333, or 474
nucleotides for SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15,
respectively).
[0099] "Variants" is intended to mean substantially similar
sequences. For nucleotide sequences, conservative variants include
those sequences that, because of the degeneracy of the genetic
code, encode the amino acid sequence of one of the cystatin
polypeptides of the invention. Naturally occurring allelic variants
such as these can be identified with the use of well-known
molecular biology techniques, as, for example, with polymerase
chain reaction (PCR) and hybridization techniques as outlined
below. Variant nucleotide sequences also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode a
cystatin protein of the invention. Generally, variants of a
particular nucleotide sequence of the invention will have at least
about 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to that particular nucleotide sequence as determined by
sequence alignment programs described elsewhere herein using
default parameters.
[0100] These variant nucleotide sequences can also be evaluated by
comparison of the percent sequence identity shared by the
polypeptides they encode. For example, isolated nucleic acids which
encode a polypeptide with a given percent sequence identity to the
polypeptide of SEQ ID NO: 2, 4, 6, 8 and 10 are disclosed. Identity
can be calculated using, for example, the BLAST, CLUSTALW, or GAP
algorithms under default conditions. The percentage of identity to
a reference sequence is at least 50% and, rounded upwards to the
nearest integer, can be expressed as an integer selected from the
group of integers consisting of from 50 to 99. Thus, for example,
the percentage of identity to a reference sequence can be at least
60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
[0101] A "variant" protein is intended to mean a protein derived
from the native protein by deletion (so-called truncation) or
addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native protein; deletion or addition of one
or more amino acids at one or more sites in the native protein; or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
cystatin-like activity as described herein. Such variants may
result from, for example, genetic polymorphism or from human
manipulation. Biologically active variants of a native cystatin
protein of the invention will have at least about 40%, 50%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to the amino acid sequence of
the native protein as determined by sequence alignment programs
described elsewhere herein using default parameters. A biologically
active variant of a protein of the invention may differ from that
protein by as few as 1-15 amino acid residues, as few as 1-10, such
as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0102] The polypeptides of the invention may be altered in various
ways including amino acid substitutions, deletions, truncations,
and insertions. Novel proteins having properties of interest may be
created by combining elements and fragments of proteins of the
present invention as well as other proteins. Methods for such
manipulations are generally known in the art. For example, amino
acid sequence variants of the cystatin proteins can be prepared by
mutations in the DNA. Methods for mutagenesis and nucleotide
sequence alterations are well known in the art. See, for example,
Kunkel (1985) Proc Nat Acad Sci USA 82:488-492; Kunkel et al.
(1987) Method Enzymol 154:367-382; U.S. Pat. No. 4,873,192; Walker
and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan
Publishing Company, New York) and the references cited therein.
Guidance as to appropriate amino acid substitutions that do not
affect biological activity of the protein of interest are well
known in the art and may be found in the model of Dayhoff et al.
(1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res.
Found., Washington, D.C.), herein incorporated by reference.
Conservative substitutions, such as exchanging one amino acid with
another having similar properties, may be preferred. Table 12,
below, shows potential amino acid substitution groups which are
considered to be highly conserved. TABLE-US-00012 TABLE 12
Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine
(T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)
Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine
(L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y)
Tryptophan (W)
[0103] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the invention encompass both
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
developmental activity, or defense response activity. Obviously,
the mutations that will be made in the DNA encoding the variant
must not place the sequence out of reading frame and preferably
will not create complementary regions that could produce secondary
mRNA structures. See, EP Patent Application Publication No.
0075444.
[0104] In nature, some polypeptides are produced as complex
precursors which, in addition to targeting labels such as the
signal peptides discussed elsewhere in this application, also
contain other fragments of peptides which are removed (processed)
at some point during protein maturation, resulting in a mature form
of the polypeptide that is different from the primary translation
product (aside from the removal of the signal peptide). "Mature
protein" refers to a post-translationally processed polypeptide;
i.e., one from which any pre- or propeptides present in the primary
translation product have been removed. "Precursor protein" or
"prepropeptide" or "preproprotein" all refer to the primary product
of translation of mRNA; i.e., with pre- and propeptides still
present. Pre- and propeptides may include, but are not limited to,
intracellular localization signals. "Pre" in this nomenclature
generally refers to the signal peptide. The form of the translation
product with only the signal peptide removed but not further
processing yet is called a "propeptide" or "proprotein". The
fragments or segments to be removed may themselves also be referred
to as "propeptides." A proprotein or propeptide thus has had the
signal peptide removed, but contains propeptides (here referring to
propeptide segments) and the portions that will make up the mature
protein. The skilled artisan is able to determine, depending on the
species in which the proteins are being expressed and the desired
intracellular location, if higher expression levels might be
obtained by using a gene construct encoding just the mature form of
the protein, the mature form with a signal peptide, or the
proprotein (i.e., a form including propeptides) with a signal
peptide. For optimal expression in plants or fungi, the pre- and
propeptide sequences may be needed. The propeptide segments may
play a role in aiding correct peptide folding.
[0105] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect can be evaluated by routine
screening assays. That is, the activity can be evaluated by
cystatin activity assays. Additionally, differences in the
expression of specific genes between uninfected and infected plants
can be determined using gene expression profiling.
[0106] Variant nucleotide sequences and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different cystatin coding sequences can be manipulated to create a
new cystatin protein possessing the desired properties. In this
manner, libraries of recombinant polynucleotides are generated from
a population of related sequence polynucleotides comprising
sequence regions that have substantial sequence identity and can be
homologously recombined in vitro or in vivo. For example, using
this approach, sequence motifs encoding a domain of interest may be
shuffled between the cystatin genes and partial sequences of the
invention and other known cystatin genes to obtain a new gene
coding for a protein with an improved property of interest, such as
an increased K.sub.m in the case of an enzyme. Such shuffling of
domains may also be used to assemble novel proteins having novel
properties. Strategies for such DNA shuffling are known in the art.
See, for example, Stemmer (1994) Proc Natl Acad Sci USA 91:
10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al.
(1997) Nature Biotech 15: 436-438; Moore et al. (1997) J Mol Biol
272: 336-347; Zhang et al. (1997) Proc Natl Acad Sci USA 94:
4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Pat.
Nos. 5,605,793 and 5,837,458.
[0107] The nucleotide sequences of the invention can be used to
isolate corresponding sequences from other organisms, particularly
other plants, more particularly other monocots. In this manner,
methods such as PCR, hybridization, and the like can be used to
identify such sequences based on their sequence homology to the
sequences set forth herein. Sequences isolated based on their
sequence identity to the entire cystatin sequences set forth herein
or to fragments thereof are encompassed by the present invention.
Such sequences include sequences that are orthologs of the
disclosed sequences. "Orthologs" means genes derived from a common
ancestral gene and which are found in different species as a result
of speciation. Genes found in different species are considered
orthologs when their nucleotide sequences and/or their encoded
protein sequences share substantial identity as defined elsewhere
herein. Functions of orthologs are often highly conserved among
species.
[0108] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any plant of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in, for example, Sambrook. See
also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods
and Applications (Academic Press, New York); Innis and Gelfand,
eds. (1995) PCR Strategies (Academic Press, New York); and Innis
and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New
York). Known methods of PCR include, but are not limited to,
methods using paired primers, nested primers, single specific
primers, degenerate primers, gene-specific primers, vector-specific
primers, partially-mismatched primers, and the like.
[0109] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the cystatin sequences of the invention.
Methods for preparation of probes for hybridization and for
construction of cDNA and genomic libraries are generally known in
the art and are disclosed in Sambrook.
[0110] For example, an entire cystatin sequence disclosed herein,
or one or more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding cystatin sequences and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique among
cystatin sequences and are preferably at least about 10 nucleotides
in length, and most preferably at least about 20 nucleotides in
length. Such probes may be used to amplify corresponding sequences
from a chosen organism by PCR. This technique may be used to
isolate additional coding sequences from a desired organism or as a
diagnostic assay to determine the presence of coding sequences in
an organism. Hybridization techniques include hybridization
screening of plated DNA libraries (either plaques or colonies; see,
for example, Sambrook.
[0111] Thus, isolated sequences that encode for a cystatin
polypeptide and which hybridize under stringent conditions to the
cystatin sequences disclosed herein, or to fragments thereof, are
encompassed by the present invention.
[0112] Biological activity of the cystatin polypeptides (i.e.,
influencing the plant defense response and various developmental
pathways, including, for example, influencing cell division) can be
assayed by any method known in the art. Biological activity of the
polypeptides of the present invention can be assayed by any method
known in the art. For example, most published cystatin activity
assays are based on inhibition of papain-mediated substrate
hydrolysis. A variety of synthetic papain substrates are known in
the art and can be used for this purpose, such as
N-benzoyl-asparaginyl-p-nitroanilide (Schlereth et al. (2001)
Planta 212: 718-727), .alpha.-N-benzoyl-L-arginine-p-nitroanilide
(Masoud et al. (1993) Plant Mol Biol 21: 655-663),
N-Cbz-Phe-Arg-7-amido-4-methylcoumarin (Urwin et al. (1998) Supra),
and Z-Phe-Arg-7-(4-methylcoumarylamide) (Barrett & Kirschke
(1981) Method Enzymol 80: 535-561), all of which are herein
incorporated by reference. Furthermore, papain could be substituted
by a cysteine proteinase that is more relevant to the biological
system studied (e.g., a Fusarium cysteine proteinase). Assays to
detect cystatin-like activity include, for example, assessing
antifungal and/or antimicrobial activity (Soares-Costa et al.
(2002) Biochem Biophys Res Comm 296: 1194-1199; Duvick et al.
(1992) J Biol Chem 267(26): 18814-18820; Pernas-Monica et al.
(1999) Mol Plant Microbe In 12 (7): 624-627; Blankenvoorde-Michiel
et al. (1998) Biol Chem 379(11): 1371-1375, all of which are herein
incorporated by reference).
[0113] Assays that measure antipathogenic activity are commonly
known in the art, as are methods to quantitate disease resistance
in plants following pathogen infection. See, for example, U.S. Pat.
No. 5,614,395, herein incorporated by reference. Such techniques
include, measuring over time, the average lesion diameter, the
pathogen biomass, and the overall percentage of decayed plant
tissues. For example, a plant either expressing an antipathogenic
polypeptide or having an antipathogenic composition applied to its
surface shows a decrease in tissue necrosis (i.e., lesion diameter)
or a decrease in plant death following pathogen challenge when
compared to a control plant that was not exposed to the
antipathogenic composition. Alternatively, antipathogenic activity
can be measured by a decrease in pathogen biomass. For example, a
plant expressing an antipathogenic polypeptide or exposed to an
antipathogenic composition is challenged with a pathogen of
interest. Over time, tissue samples from the pathogen-inoculated
tissues are obtained and RNA is extracted. The percent of a
specific pathogen RNA transcript relative to the level of a plant
specific transcript allows the level of pathogen biomass to be
determined. See, for example, Thomma et al. (1998) Plant Biology
95:15107-15111, herein incorporated by reference.
[0114] Furthermore, in vitro antipathogenic assays include, for
example, the addition of varying concentrations of the
antipathogenic composition to paper disks and placing the disks on
agar containing a suspension of the pathogen of interest. Following
incubation, clear inhibition zones develop around the discs that
contain an effective concentration of the antipathogenic
polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892, herein
incorporated by reference). Additionally, microspectrophotometrical
analysis can be used to measure the in vitro antipathogenic
properties of a composition (Hu et al. (1997) Plant Mol. Biol.
34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,
both of which are herein incorporated by reference).
[0115] Compositions and methods for controlling pathogenic agents
are provided. The anti-pathogenic compositions comprise maize,
soybean, rice and wheat cystatin nucleotide and amino acid
sequences. Particularly, the nucleic acid and amino acid sequences
and fragments and variants thereof set forth herein. Accordingly,
the compositions and methods are also useful in protecting plants
against fungal pathogens, viruses, nematodes, insects and the
like.
[0116] "Plant pathogen" or "plant pest" is intended to mean any
microorganism that can cause harm to a plant, such as by inhibiting
or slowing the growth of a plant, by damaging the tissues of a
plant, by weakening the immune system of a plant or the resistance
of a plant to abiotic stresses, and/or by causing the premature
death of the plant, etc. Plant pathogens and plant pests include
microbes such as fungi, viruses, bacteria, and nematodes.
[0117] "Disease resistance" or "pathogen resistance" is intended to
mean that the organisms avoid the disease symptoms which are the
outcome of organism-pathogen interactions. That is, pathogens are
prevented from causing diseases and the associated disease
symptoms, or alternatively, the disease symptoms caused by the
pathogen is minimized or lessened. The methods of the invention can
be utilized to protect plants from disease, particularly those
diseases that are caused by plant pathogens. "Anti-pathogenic
compositions" is intended to mean that the compositions of the
invention are capable of suppressing, controlling, and/or killing
the invading pathogenic organism. An antipathogenic composition of
the invention will reduce the disease symptoms resulting from
pathogen challenge by at least about 5% to about 50%, at least
about 10% to about 60%, at least about 30% to about 70%, at least
about 40% to about 80%, or at least about 50% to about 90% or
greater. Hence, the methods of the invention can be utilized to
protect plants from disease, particularly those diseases that are
caused by plant pathogens.
[0118] An "antimicrobial agent," a "pesticidal agent," a
"cystatin," and/or a "fungicidal agent" will act similarly to
suppress, control, and/or kill the invading pathogen. A defensive
agent will possess defensive activity. "Defensive activity" means
an antipathogenic, antimicrobial, or antifungal activity.
[0119] "Antipathogenic compositions" is intended to mean that the
compositions of the invention have activity against pathogens;
including fungi, microorganisms, viruses, and nematodes, and thus
are capable of suppressing, controlling, and/or killing the
invading pathogenic organism. An antipathogenic composition of the
invention will reduce the disease symptoms resulting from plant
pathogen challenge by at least about 5% to about 50%, at least
about 10% to about 60%, at least about 30% to about 70%, at least
about 40% to about 80%, or at least about 50% to about 90% or
greater. Hence, the methods of the invention can be utilized to
protect organisms, particularly plants, from disease, particularly
those diseases that are caused by invading pathogens.
[0120] Pathogens of the invention include, but are not limited to,
viruses or viroids, bacteria, insects, nematodes, fungi, and the
like. Viruses include any plant virus, for example, tobacco or
cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf
mosaic virus, etc. Specific fungal and viral pathogens for the
major crops include, but are not limited to: Soybeans: Phytophthora
megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia
solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe
phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum
var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora
sojina, Peronospora manshurica, Colletotrichum dematium
(Colletotichum truncatum), Corynespora cassiicola, Septoria
glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas
syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli,
Microsphaera diffusa, Fusarium semitectum, Phialophora gregata,
Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus,
Tobacco Streak virus, Phakopsora pachyrhizi, Pythium
aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted
wilt virus, Heterodera glycines, Fusarium solani; Canola: Albugo
candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia
solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola,
Pythium ultimum, Peronospora parasitica, Fusarium roseum,
Alternaria alternata; Alfalfa: Clavibater michiganese subsp.
insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens,
Pythium debaryanum, Pythium aphanidermatum, Phytophthora
megasperma, Peronospora trifoliorum, Phoma medicaginis var.
medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis,
Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v.
alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium
alfalfae; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis
agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas
syringae p.v. syringae, Alternaria alternata, Cladosporium
herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium
culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium
gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp.
tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp.
tritici, Puccinia striiformis, Pyrenophora tritici-repentis,
Septoria nodorum, Septoria tritici, Septoria avenae,
Pseudocercosporella herpotrichoides, Rhizoctonia solani,
Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium
aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris
sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil
Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle
Streak Virus, American Wheat Striate Virus, Claviceps purpurea,
Tilletia tritici, Tilletia laevis, Tilletia indica, Pythium
gramicola, High Plains Virus, European wheat striate virus;
Sunflower: Broomrape, Plasmophora halstedii, Sclerotinia
sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis
helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis
cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe
cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus
stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia
carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora
cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.
subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella
zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis),
Pythium irregulare, Pythium debaryanum, Pythium graminicola,
Pythium splendens, Pythium ultimum, Pythium aphanidermatum,
Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus
heterostrophus), Helminthosporium carbonum I, II & III
(Cochliobolus carbonum), Exserohilum turcicum I, II & III,
Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta
maydis, Kabatiella-maydis, Cercospora sorghi, Ustilago maydis,
Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina,
Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum,
Curvularia lunata, Curvularia inaequalis, Curvularia pallescens,
Clavibacter michiganense subsp. nebraskense, Trichoderma viride,
Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus,
Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae,
Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt
spiroplasma, Diplodia macrospora, Sclerophthora macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinensis,
Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca
reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium
acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize
Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize
Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum
turcicum, Colletotrichum graminicola (Glomerella graminicola),
Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina,
Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v.
holcicola, Pseudomonas andropogonis, Puccinia purpurea,
Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme,
Alternaria alternata, Bipolaris sorghicola, Helminthosporium
sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae
(Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora
sorghicola, Phyllachara sacchari, Sporisorium reilianum
(Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium
sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B,
Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
Sclerophthona macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium
graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium
graminicola; Rice: rice brownspot fungus (Cochliobolus miyabeanus),
rice blast fungus--Magnaporthe grisea (Pyricularia grisea),
Magnaporthe salvinii (Sclerotium oryzae), Xanthomomas oryzae pv.
oryzae, Xanthomomas oryzae pv. oryzicola, Rhizoctonia spp.
(including but not limited to Rhizoctonia solani, Rhizoctonia
oryzae and Rhizoctonia oryzae-sativae), Pseudomonas spp. (including
but not limited to Pseudomonas plantarii, Pseudomonas avenae,
Pseudomonas glumae, Pseudomonas fuscovaginae, Pseudomonas
alboprecipitans, Pseudomonas syringae pv. panici, Pseudomonas
syringae pv. syringae, Pseudomonas syringae pv. oryzae and
Pseudomonas syringae pv. aptata), Erwinia spp. (including but not
limited to Erwinia herbicola, Erwinia amylovaora, Erwinia
chrysanthemi and Erwinia carotovora), Achyla spp. (including but
not limited to Achyla conspicua and Achyla klebsiana), Pythium spp.
(including but not limited to Pythium dissotocum, Pythium
irregulare, Pythium arrhenomanes, Pythium myriotylum, Pythium
catenulatum, Pythium graminicola and Pythium spinosum), Saprolegnia
spp., Dictyuchus spp., Pythiogeton spp., Phytophthora spp.,
Alternaria padwickii, Cochliobolus miyabeanus, Curvularia spp.
(including but not limited to Curvularia lunata, Curvularia
affinis, Curvularia clavata, Curvularia eragrostidis, Curvularia
fallax, Curvularia geniculata, Curvularia inaequalis, Curvularia
intermedia, Curvularia oryzae, Curvularia oryzae-sativae,
Curvularia pallescens, Curvularia senegalensis, Curvularia
tuberculata, Curvularia uncinata and Curvularia verruculosa),
Sarocladium oryzae, Gerlachia oryzae, Fusarium spp. (including but
not limited Fusarium graminearum, Fusarium nivale and to different
pathovars of Fusarium monoliforme, including pvs. fujikuroi and
zeae), Sclerotium rolfsii, Phoma exigua, Mucor fragilis,
Trichoderma viride, Rhizopus spp., Cercospora oryzae, Entyloma
oryzae, Dreschlera gigantean, Sclerophthora macrospora,
Mycovellosiella oryzae, Phomopsis oryzae-sativae, Puccinia
graminis, Uromyces coronatus, Cylindrocladium scoparium,
Gaeumannomyces graminis pv. graminis, Myrothecium verrucaria,
Pyrenochaeta oryzae, Ceratobasidium oryzae-sativae, Microdochium
oryzae (Rhynchosporium oryzae), Cercospora janseana, Thanatephorus
cucumeris, Ustilaginoidea virens, Neovossia spp. (including but not
limited to Neovossia horrida), Tilletia spp., Balansia
oryzae-sativae, Phoma spp. (including but not limited to Phoma
sorghina, Phoma insidiosa, Phoma glumarum, Phoma glumicola and
Phoma oryzina), Nigrospora spp. (including but not limited to
Nigrospora oryzae, Nigrospora sphaerica, Nigrospora panici and
Nigrospora padwickii), Epiococcum nigrum, Phyllostica spp., Wolkia
decolorans, Monascus purpureus, Aspergillus spp., Penicillium spp.,
Absidia spp., Mucor spp., Chaetomium spp., Dematium spp., Monilia
spp., Streptomyces spp., Syncephalastrum spp., Verticillium spp.,
Nematospora coryli, Nakataea sigmoidea, Cladosporium spp.,
Bipolaris spp., Coniothyrium spp., Diplodia oryzae, Exserophilum
rostratum, Helococera oryzae, Melanomma glumarum, Metashaeria spp.,
Mycosphaerella spp., Oidium spp., Pestalotia spp., Phaeoseptoria
spp., Sphaeropsis spp., Trematosphaerella spp., rice black-streaked
dwarf virus, rice dwarf virus, rice gall dwarf virus, barley yellow
dwarf virus, rice grassy stunt virus, rice hoja blanca virus, rice
necrosis mosaic virus, rice ragged stunt virus, rice stripe virus,
rice stripe necrosis virus, rice transitory yellowing virus, rice
tungro bacilliform virus, rice tungro spherical virus, rice yellow
mottle virus, rice tarsonemid mite virus, Echinochloa hoja blanca
virus, Echinochloa ragged stunt virus, rice bunchy stunt virus,
rice giallume virus, orange leaf mycoplasma-like organism, yellow
dwarf mycoplasma-like organism, Aphelenchoides besseyi, Ditylenchus
angustus, Hirschmanniella spp., Criconemella spp., Meloidogyne
spp., Heterodera spp., Pratylenchus spp., Hoplolaimus indicus.
[0121] Nematodes include plant-parasitic nematodes such as
root-knot, cyst, and lesion nematodes, including Heterodera and
Globodera spp. such as Globodera rostochiensis and Globodera
pailida (potato cyst nematodes); Heterodera glycines (soybean cyst
nematode); Heterodera schachtii (beet cyst nematode); and
Heterodera avenae (cereal cyst nematode).
[0122] Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Coleoptera and Lepidoptera. Insect pests of the invention for the
major crops include, but are not limited to: Maize: Ostrinia
nubilalis, European corn borer; Agrotis ipsilon, black cutworm;
Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall
armyworm; Diatraea grandiosella, southwestern corn borer;
Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea
saccharalis, surgarcane borer; Diabrotica virgifera, western corn
rootworm; Diabrotica longicornis barberi, northern corn rootworm;
Diabrotica undecimpunctata howardi, southern corn rootworm;
Melanotus spp., wireworms; Cyclocephala borealis, northern masked
chafer (white grub); Cyclocephala immaculata, southern masked
chafer (white grub); Popillia japonica, Japanese beetle;
Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize
billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis
maidiradicis, corn root aphid; Blissus leucopterus leucopterus,
chinch bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus sanguinipes, migratory grasshopper; Hylemya platura,
seedcorn maggot; Agromyza parvicornis, corn blot leafminer;
Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief
ant; Tetranychus urticae, two-spotted spider mite; Sorghum: Chilo
partellus, sorghum borer; Spodoptera frugiperda, fall armyworm;
Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser
cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga
crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,
wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema
pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug;
Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow
sugarcane aphid; Blissus leucopterus leucopterus, chinch bug;
Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus,
carmine spider mite; Tetranychus urticae, twospotted spider mite;
Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda,
fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer;
Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus,
lesser cornstalk borer; Oulema melanopus, cereal leaf beetle;
Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata
howardi, southern corn rootworm; Russian wheat aphid; Schizaphis
graminum, greenbug; Macrosiphum avenae, English grain aphid;
Melanoplus femurrubrum, redlegged grasshopper; Melanoplus
differentialis, differential grasshopper; Melanoplus sanguinipes,
migratory grasshopper; Mayetiola destructor, Hessian fly;
Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem
maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca,
tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae,
wheat curl mite; Sunflower: Suleima helianthana, sunflower bud
moth; Homoeosoma electellum, sunflower moth; zygogramma
exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton:
Heliothis virescens, cotton budworm; Helicoverpa zea, cotton
bollworm; Spodoptera exigua, beet armyworm; Pectinophora
gossypiella, pink bollworm; Anthonomus grandis grandis, boll
weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus,
cotton fleahopper; Trialeurodes abutilonea, banded-winged whitefly;
Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum,
redlegged grasshopper; Melanoplus differentialis, differential
grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca,
tobacco thrips; Tetranychus cinnabarinus, carmine spider mite;
Tetranychus urticae, two-spotted spider mite; Rice: Diatraea
saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm;
Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis;
Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae,
rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus
leucopterus leucopterus, chinch bug; Acrosternum hilare, green
stink bug; Soybean: Pseudoplusia includens, soybean looper;
Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra,
green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis
ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis
virescens, cotton budworm; Helicoverpa zea, cotton bollworm;
Epilachna varivestis, Mexican bean beetle; Myzus persicae, green
peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus differentialis, differential grasshopper; Hylemya
platura, seedcorn maggot; Sericothrips variabilis, soybean thrips;
Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry
spider mite; Tetranychus urticae, two-spotted spider mite; Barley:
Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm; Schizaphis graminum, greenbug; Blissus leucopterus
leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Euschistus servus, brown stink bug; Delia platura, seedcorn maggot;
Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat
mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha
armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root
maggots.
[0123] The nucleic acid sequences of the present invention can be
expressed in a host cell such as bacteria, yeast, insect,
mammalian, or preferably plant cells. It is expected that those of
skill in the art are knowledgeable in the numerous expression
systems available for expression of a nucleic acid encoding a
protein of the present invention. No attempt to describe in detail
the various methods known for the expression of proteins in
prokaryotes or eukaryotes will be made.
[0124] The cystatin sequences of the invention are provided in
expression cassettes or DNA constructs for expression in the plant
of interest. The cassette will include 5' and 3' regulatory
sequences operably linked to a cystatin sequence of the invention.
The cassette may additionally contain at least one additional gene
to be cotransformed into the organism. Alternatively, the
additional gene(s) can be provided on multiple expression
cassettes.
[0125] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the cystatin sequence to be
under the transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0126] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional initiation region (i.e., a
promoter), translational initiation region, a polynucleotide of the
invention, a translational termination region and, optionally, a
transcriptional termination region functional in the host organism.
The regulatory regions (i.e., promoters, transcriptional regulatory
regions, and translational termination regions) and/or the
polynucleotide of the invention may be native/analogous to the host
cell or to each other. Alternatively, the regulatory regions and/or
the polynucleotide of the invention may be heterologous to the host
cell or to each other. As used herein, "heterologous" in reference
to a sequence is a sequence that originates from a foreign species,
or, if from the same species, is substantially modified from its
native form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous polynucleotide is from a species different from the
species from which the polynucleotide was derived, or, if from the
same/analogous species, one or both are substantially modified from
their original form and/or genomic locus, or the promoter is not
the native promoter for the operably linked polynucleotide.
[0127] While it may be preferable to express the sequences using
heterologous promoters, the native promoter sequences may be used.
Such constructs would change expression levels of cystatin in the
host cell (i.e., plant or plant cell). Thus, the phenotype of the
host cell (i.e., plant or plant cell) is altered.
[0128] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, or may be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also Guerineau et al.
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903;
and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0129] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed plant. That is, the genes
can be synthesized using plant-preferred codons for improved
expression. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0130] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0131] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for
example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)
Virology 154:9-20); and human immunoglobulin heavy-chain binding
protein (BiP), (Macejak et al. (1991) Nature 353:90-94);
untranslated leader from the coat protein mRNA of alfalfa mosaic
virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);
tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in
Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256);
and maize chlorotic mottle virus leader (MCMV) (Lommel et al.
(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987)
Plant Physiol. 84:965-968. Other methods known to enhance
transcription can also be utilized.
[0132] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0133] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells.
Selectable marker genes are utilized for the selection of
transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate, glyphosate, ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See
generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;
Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595;
Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993)
Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al. (1988) Nature 334:721-724; and WO Publication No. 02/36782.
Such disclosures are herein incorporated by reference.
[0134] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention.
[0135] A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. That is, the nucleic acids can be combined with
constitutive, tissue-preferred, or other promoters for expression
in the host cell of interest. Such constitutive promoters include,
for example, the core promoter of the Rsyn7 (WO 99/48338 and U.S.
Pat. No. 6,072,050); the core CaMV 35S promoter (Odell et al.
(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant
Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.
Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.
18:675-689); PEMU (Last et al. (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. Pat. No. 5,659,026), and the like. Other
constitutive promoters include, for example, those disclosed in
U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; and 5,608,142.
[0136] Generally, it will be beneficial to express the gene from an
inducible promoter, particularly from a pathogen-inducible
promoter. Such promoters include those from pathogenesis-related
proteins (PR proteins), which are induced following infection by a
pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,
chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J.
Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656;
and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also, U.S.
application Ser. No. 09/257,583 and WO 99/43819, herein
incorporated by reference.
[0137] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant. Path. 41:189-200).
[0138] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the invention. Such wound-inducible promoters
include potato proteinase inhibitor (pin 11) gene (Ryan (1990) Ann.
Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology
14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2
(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin
(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.
(1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS
Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J.
6(2):141-150); and the like, herein incorporated by reference.
[0139] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize ln2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0140] Tissue-preferred promoters can be utilized to target
enhanced cystatin expression within a particular plant tissue.
Tissue-preferred promoters include those disclosed in Yamamoto et
al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant
Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet.
254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;
Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et
al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996)
Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.
20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590;
and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such
promoters can be modified, if necessary, for weak expression.
[0141] Leaf-specific promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0142] Just as expression of an antipathogenic polypeptide of the
invention may be targeted to specific plant tissues or cell types
through the use of appropriate promoters, it may also be targeted
to different locations within the cell through the use of targeting
information or "targeting labels". Unlike the promoter, which acts
at the transcriptional level, such targeting information is part of
the initial translation product. Depending on the mode of infection
of the pathogen or the metabolic function of the tissue or cell
type, the location of the protein in different compartments of the
cell may make it more efficacious against a given pathogen or make
it interfere less with the functions of the cell. For example, one
may produce a protein preceded by a signal peptide, which directs
the translation product into the endoplasmic reticulum, by
including in the construct (i.e. expression cassette) sequences
encoding a signal peptide (such sequences may also be called the
"signal sequence"). The signal sequence used could be, for example,
one associated with the gene encoding the polypeptide, or it may be
taken from another gene.
[0143] There are many signal peptides described in the literature,
and they are largely interchangeable (Raikhel N, Chrispeels M J
(2000) Protein sorting and vesicle traffic. In B Buchanan, W
Gruissem, R Jones, eds, Biochemistry and Molecular Biology of
Plants. American Society of Plant Physiologists, Rockville, Md., pp
160-201, herein incorporated by reference). The addition of a
signal peptide will result in the translation product entering the
endoplasmic reticulum (in the process of which the signal peptide
itself is removed from the polypeptide), but the final
intracellular location of the protein depends on other factors,
which may be manipulated to result in localization most appropriate
for the pathogen and cell type. The default pathway, that is, the
pathway taken by the polypeptide if no other targeting labels are
included, results in secretion of the polypeptide across the cell
membrane (Raikhel and Chrispeels, supra) into the apoplast. The
apoplast is the region outside the plasma membrane system and
includes cell walls, intercellular spaces, and the xylem vessels
that form a continuous, permeable system through which water and
solutes may move.
[0144] The method of transformation/transfection is not critical to
the instant invention; various methods of transformation or
transfection are currently available. As newer methods are
available to transform crops or other host cells they may be
directly applied. Accordingly, a wide variety of methods have been
developed to insert a DNA sequence into the genome of a host cell
to obtain the transcription and/or translation of the sequence to
effect phenotypic changes in the organism. Thus, any method, which
provides for effective transformation/transfection may be
employed.
[0145] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606), Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055 and Zhao et al., U.S.
Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984)
EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for
example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.
(1995) "Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg and Phillips
(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology
6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926
(soybean); Finer and McMullen (1991) In vitro Cell Dev. Biol.
27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize);
Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos.
5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.
91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839
(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)
311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New
York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell
Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.
84:560-566 (whisker-mediated transformation); D'Halluin et al.
(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993)
Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals
of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature
Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all
of which are herein incorporated by reference.
[0146] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
constitutive expression of the desired phenotypic characteristic is
stably maintained and inherited and then seeds harvested to ensure
constitutive expression of the desired phenotypic characteristic.
One of skill will recognize that after the recombinant expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0147] In vegetatively propagated crops, mature transgenic plants
can be propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants. Selection of
desirable transgenics is made and new varieties are obtained and
propagated vegetatively for commercial use. In seed propagated
crops, mature transgenic plants can be self-crossed to produce a
homozygous inbred plant. The inbred plant produces seed containing
the newly introduced heterologous nucleic acid. These seeds can be
grown to produce plans that would produce the selected
phenotype.
[0148] Parts obtained from the regenerated plant, such as flowers,
seeds, leaves, branches, fruit, and the like are included in the
invention, provided that these parts comprise cells comprising the
isolated nucleic acid of the present invention. Progeny and
variants, and mutants of the regenerated plants are also included
within the scope of the invention, provided that these parts
comprise the introduced nucleic acid sequences.
[0149] A preferred embodiment is a transgenic plant that is
homozygous for the added heterologous nucleic acid; i.e., a
transgenic plant that contains two added nucleic acid sequences,
one gene at the same locus on each chromosome of a chromosome pair.
A homozygous transgenic plant can be obtained by sexually mating
(selfing) a heterozygous transgenic plant that contains a single
added heterologous nucleic acid, germinating some of the seed
produced and analyzing the resulting plants produced for altered
expression of a polynucleotide of the present invention relative to
a control plant (i.e., native, non-transgenic). Backcrossing to a
parental plant and out-crossing with a non-transgenic plant are
also contemplated.
[0150] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),
particularly those Brassica species useful as sources of seed oil,
alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale
cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana)), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine
max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot
esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),
pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa
(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium
guajava), mango (Mangifera indica), olive (Olea europaea), papaya
(Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0151] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum. Conifers that may be employed in
practicing the present invention include, for example, pines such
as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),
ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta),
and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga
menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea
glauca); redwood (Sequoia sempervirens); true firs such as silver
fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars
such as Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). Preferably, plants of the present
invention are crop plants (for example, corn, alfalfa, sunflower,
Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,
millet, tobacco, etc.), more preferably corn and soybean plants,
yet more preferably corn plants.
[0152] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli, however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding sequences,
include such commonly used promoters as the beta lactamase
(penicillinase) and lactose (lac) promoter systems (Chang et al.
(1977) Nature 198:1056), the tryptophan (trp) promoter system
(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda
derived P L promoter and N-gene ribosome binding site (Shimatake et
al. (1981) Nature 292:128). Examples of selection markers for E.
coli include, for example, genes specifying resistance to
ampicillin, tetracycline, or chloramphenicol.
[0153] The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of plasmid
or phage origin. Appropriate bacterial cells are infected with
phage vector particles or transfected with naked phage vector DNA.
If a plasmid vector is used, the bacterial cells are transfected
with the plasmid vector DNA. Expression systems for expressing a
protein of the present invention are available using Bacillus sp.
and Salmonella (Palva et al. (1983) Gene 22:229-235 and Mosbach et
al. (1983) Nature 302:543-545).
[0154] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, a polynucleotide of
the present invention can be expressed in these eukaryotic systems.
In some embodiments, transformed/transfected plant cells, as
discussed infra, are employed as expression systems for production
of the proteins of the instant invention. Such antimicrobial
proteins can be used for any application including coating surfaces
to target microbes. In this manner, target microbes include human
pathogens or microorganisms.
[0155] Synthesis of heterologous nucleotide sequences in yeast is
well known. Sherman, F., et al. (1982) Methods in Yeast Genetics,
Cold Spring Harbor Laboratory is a well recognized work describing
the various methods available to produce a protein in yeast. Two
widely utilized yeasts for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like as
desired.
[0156] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates. The monitoring of the
purification process can be accomplished by using Western blot
techniques, radioimmunoassay, or other standard immunoassay
techniques.
[0157] The sequences of the present invention can also be ligated
to various expression vectors for use in transfecting cell cultures
of, for instance, mammalian, insect, or plant origin. Illustrative
cell cultures useful for the production of the peptides are
mammalian cells. A number of suitable host cell lines capable of
expressing intact proteins have been developed in the art, and
include the HEK293, BHK21, and CHO cell lines. Expression vectors
for these cells can include expression control sequences, such as
an origin of replication, a promoter (e.g. the CMV promoter, a HSV
tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen et al. (1986) Immunol. Rev. 89:49), and necessary processing
information sites, such as ribosome binding sites, RNA splice
sites, polyadenylation sites (e.g., an SV40 large T Ag poly A
addition site), and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection.
[0158] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth and Drosophila cell lines such as a
Schneider cell line (See, Schneider, J. Embryol. Exp. Morphol.
27:353-365 (1987)).
[0159] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al. (1983) J. Virol. 45:773-781).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors. See, Saveria-Campo, M., (1985)
Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA
Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,
Arlington, Va. pp. 213-238.
[0160] Animal and lower eukaryotic (e.g., yeast) host cells are
competent or rendered competent for transfection by various means.
There are several well-known methods of introducing DNA into animal
cells. These include: calcium phosphate precipitation, fusion of
the recipient cells with bacterial protoplasts containing the DNA,
treatment of the recipient cells with liposomes containing the DNA,
DEAE dextrin, electroporation, biolistics, and micro-injection of
the DNA directly into the cells. The transfected cells are cultured
by means well known in the art. See, Kuchler, R. J. (1997)
Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson and Ross, Inc.
[0161] It is recognized that with these nucleotide sequences,
antisense constructions, complementary to at least a portion of the
messenger RNA (mRNA) for the cystatin sequences can be constructed.
Antisense nucleotides are constructed to hybridize with the
corresponding mRNA. Modifications of the antisense sequences may be
made as long as the sequences hybridize to and interfere with
expression of the corresponding mRNA. In this manner, antisense
constructions having 70%, preferably 80%, more preferably 85%
sequence identity to the corresponding antisensed sequences may be
used. Furthermore, portions of the antisense nucleotides may be
used to disrupt the expression of the target gene. Generally,
sequences of at least 50 nucleotides, 100 nucleotides, 200
nucleotides, or greater may be used.
[0162] The nucleotide sequences of the present invention may also
be used in the sense orientation to suppress the expression of
endogenous genes in plants. Methods for suppressing gene expression
in plants using nucleotide sequences in the sense orientation are
known in the art. The methods generally involve transforming plants
with a DNA construct comprising a promoter that drives expression
in a plant operably linked to at least a portion of a nucleotide
sequence that corresponds to the transcript of the endogenous gene.
Typically, such a nucleotide sequence has substantial sequence
identity to the sequence of the transcript of the endogenous gene,
preferably greater than about 65% sequence identity, more
preferably greater than about 85% sequence identity, most
preferably greater than about 95% sequence identity. See, U.S. Pat.
Nos. 5,283,184 and 5,034,323; herein incorporated by reference.
[0163] The present invention further provides a method for
modulating (i.e., increasing or decreasing) the concentration or
composition of the polypeptides of the present invention in a plant
or part thereof. Increasing or decreasing the concentration and/or
the composition of polypeptides in a plant can affect modulation.
For example, increasing the ratio of polypeptides of the invention
to native polypeptides can affect modulation. The method comprises:
introducing a polynucleotide of the present invention into a plant
cell with a recombinant expression cassette as described above to
obtain a transformed plant cell, culturing the transformed plant
cell under appropriate growing conditions, and inducing or
repressing expression of a polynucleotide of the present invention
in the plant for a time sufficient to modulate the concentration
and/or the composition of polypeptides in the plant or plant
part.
[0164] In some embodiments, the content and/or composition of
polypeptides of the present invention in a plant may be modulated
by altering, in vivo or in vitro, the promoter of the nucleotide
sequence to up- or down-regulate expression. For instance, an
isolated nucleic acid comprising a promoter sequence is transfected
into a plant cell. Subsequently, a plant cell comprising the
promoter operably linked to a polynucleotide of the present
invention is selected for by means known to those of skill in the
art such as, but not limited to, Southern blot, DNA sequencing, or
PCR analysis using primers specific to the promoter and to the gene
and detecting amplicons produced therefrom. A plant or plant part
altered or modified by the foregoing embodiments is grown under
plant forming conditions for a time sufficient to modulate the
concentration and/or composition of polypeptides of the present
invention in the plant. Plant forming conditions are well known in
the art and discussed briefly, supra.
[0165] In general, concentration or composition is increased or
decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
or 90% relative to a native control plant, plant part, or cell
lacking the aforementioned recombinant expression cassette.
Modulation in the present invention may occur during and/or
subsequent to growth of the plant to the desired stage of
development. Modulating nucleic acid expression temporally and/or
in particular tissues can be controlled by employing the
appropriate promoter operably linked to a polynucleotide of the
present invention in, for example, sense or antisense orientation
as discussed in greater detail, supra. Induction of expression of a
polynucleotide of the present invention can also be controlled by
exogenous administration of an effective amount of inducing
compound. Inducible promoters and inducing compounds, which
activate expression from these promoters, are well known in the
art. In preferred embodiments, the polypeptides of the present
invention are modulated in monocots, particularly maize.
[0166] In certain embodiments the nucleic acid sequences of the
present invention can be stacked with any combination of
polynucleotide sequences of interest in order to create plants with
a desired phenotype. For example, the polynucleotides of the
present invention may be stacked with any other polynucleotides of
the present invention, such as any combination of the maize,
soybean, rice and wheat cystatin sequences presented (SEQ ID NOS:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, and 75), or with other genes implicated in disease
resistance pathways, especially those that have antimicrobial
activity like cystatins, such as: (a) other proteinase inhibitors,
such as trypsin proteinase inhibitors (Chen et al. (1999) Appl
Environ Microb 65(3): 1320-1324.), subtilisin/chymotrypsin
inhibitors (Cordero et al. (1994) Plant J 6(2): 141-150),
trypsin/alpha-amylase inhibitors (Wen et al. (1992) Plant Mol.
Biol. 18(4): 813-814), and Bowman-Birk proteinase inhibitors
(Prakash et al. (1996) J Mol Evol 1996; 42 (5): 560-569); (b) small
cysteine-rich antimicrobial proteins, such as defensins (Thomma et
al. (2002) Planta 261(2): 193-202) or gamma-thionins (Nitti et al.
(1995) Eur J Biochem 228(2): 250-6), kistrin-like cysteine-rich
proteins (Segura, et al. (1999) Mol Plant Microbe Interact 12:
16-23), cyclotides (Craik et al. (1999) J Mol Biol 294(5):1327-36),
and basal layer antifungal peptides (Hueros et al. (1995) Plant
Cell 7: 747-757); (c) antimicrobial enzymes, such as
endo-1,3-beta-glucanases, chitinases (Simmons (1994) CRC Cr Rev
Plant Sci 13: 325-387), RNAses (Hugot et al. (2002) Mol Plant
Microbe Interact 15(3): 243-250); (d) other pathogenesis-related
proteins, such as PR-1 homologs (Tornero et al. (1997) Mol Plant
Microbe Interact 10(5): 624-34), PR-10/ocatin/major latex protein
homologs (McGee et al. (2001) Mol Plant Microbe Interact 14(7):
877-86), PR-5/thaumatin/osmotin homologs (Barre et al. (2000)
Planta. 211 (6):791-9); (e) and other antimicrobial proteins such
as lipid transfer proteins (Park et al. (2002) Plant Mol. Biol.
48(3): 243-54), puroindolines (Krishnamurthy et al. (2001). Mol
Plant Microbe Interact 14: 1255-1260), alpha- and beta-thionins
(Rodriguez-Palenzuela et al. (1988) Gene 70: 271-281; Van
Campenhout et al. (1998) Theor Appl Genet 96: 80-86), maize basic
proteins (Duvick et al. (1992) J Biol Chem 267(26): 18814-18820),
and small histidine-glycine rich proteins (Park et al. (2000) Plant
Mol Biol 44: 187-197) and the like, the disclosures of which are
herein incorporated by reference. It is understood that other genes
and their products that are not themselves antimicrobial, but which
contribute to the disease response by a number mechanisms, could
also be employed in conjunction, among them LRR-proteins
(Mondragon-Palomino et al. (2002) Genome Res (9): 1305-15) and
other R gene analogues, transcription factors such as WRKY (Eulgem
et al. (2000) Trends Plant Sci (5): 199-206), multidrug
transporters (Diener et al. (2001) Plant Cell 13(7): 1625-38),
phenylpropanoid pathway enzymes (Dixon et al. (1996) Gene
179(1):61-71), and polygalacturonase inhibitor proteins (Berger et
al. (2000). Phys Mol Plant Pathol. 57 (1): 5-14). Such genes could
come from maize or non-maize sources, including non-plant
sources.
[0167] The combinations generated can also include multiple copies
of any one of the polynucleotides of interest. The polynucleotides
of the present invention can also be stacked with any other gene or
combination of genes to produce plants with a variety of desired
trait combinations including but not limited to traits desirable
for animal feed such as high oil genes (e.g., U.S. Pat. No.
6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat.
Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409)); barley high
lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and
WO 98/20122); and high methionine proteins (Pedersen et al. (1986)
J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and
Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased
digestibility (e.g., modified storage proteins (U.S. application
Ser. No. 10/053,410, filed Nov. 7, 2001)); and thioredoxins (U.S.
application Ser. No. 10/005,429, filed Dec. 3, 2001), the
disclosures of which are herein incorporated by reference.
[0168] The polynucleotides of the present invention can also be
stacked with traits desirable for insect, disease or herbicide
resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat.
Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser
et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant
Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No.
5,792,931); avirulence and disease resistance genes (Jones et al.
(1994) Science 266:789; Martin et al. (1993) Science 262:1432;
Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS)
mutants that lead to herbicide resistance such as the S4 and/or Hra
mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene and GAT gene)); and traits desirable for
processing or process products such as high oil (U.S. Pat. No.
6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S.
Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)); and
polymers or bioplastics (U.S. Pat. No. 5,602,321);
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170:5837-5847), which facilitate expression of
polyhydroxyalkanoates (PHAs)), the disclosures of which are herein
incorporated by reference. One could also combine the
polynucleotides of the present invention with polynucleotides
providing agronomic traits such as male sterility (see U.S. Pat.
No. 5,583,210), stalk strength, flowering time, or transformation
technology traits such as cell cycle regulation or gene targeting
(see, WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of
which are herein incorporated by reference.
[0169] These stacked combinations can be created by any method
including, but not limited to, polynucleotide sequences of interest
can be combined at any time and in any order. For example, a
transgenic plant comprising one or more desired traits can be used
as the target to introduce further traits by subsequent
transformation. The traits can be introduced simultaneously in a
co-transformation protocol with the polynucleotides of interest
provided by any combination of transformation cassettes. For
example, if two sequences will be introduced, the two sequences can
be contained in separate transformation cassettes (trans) or
contained on the same transformation cassette (cis). Expression of
the sequences can be driven by the same promoter or by different
promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of the
polynucleotide of interest. This may be combined with any
combination of other suppression cassettes or overexpression
cassettes to generate the desired combination of traits in the
plant.
[0170] The present invention provides a method of genotyping a
plant comprising a polynucleotide of the present invention.
Optionally, the plant is a monocot, such as maize or sorghum.
Genotyping provides a means of distinguishing homologs of a
chromosome pair and can be used to differentiate segregants in a
plant population. Molecular marker methods can be used for
phylogenetic studies, characterizing genetic relationships among
crop varieties, identifying crosses or somatic hybrids, localizing
chromosomal segments affecting monogenic traits, map based cloning,
and the study of quantitative inheritance. See, e.g., Plant
Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,
Springer-Verlag, Berlin (1997). For molecular marker methods, see
generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter
2) in: Genome Mapping in plants (Ed., Andrew H. Paterson) by
Academic Press/R.G. Lands Company, Austin, Tex., pp. 7-21.
[0171] The particular method of genotyping in the present invention
may employ any number of molecular marker analytic techniques such
as, but not limited to, restriction fragment length polymorphisms
(RFLPs). RFLPs are the product of allelic differences between DNA
restriction fragments resulting from nucleotide sequence
variability. As is well known to those of skill in the art, RFLPs
are typically detected by extraction of genomic DNA and digestion
with a restriction enzyme. Generally, the resulting fragments are
separated according to size and hybridized with a probe; single
copy probes are preferred. Restriction fragments from homologous
chromosomes are revealed. Differences in fragment size among
alleles represent an RFLP. Thus, the present invention further
provides a means to follow segregation of a gene or nucleic acid of
the present invention as well as chromosomal sequences genetically
linked to these genes or nucleic acids using such techniques as
RFLP analysis. Linked chromosomal sequences are within 50
centiMorgans (cM), often within 40 or 30 cM, preferably within 20
or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the
present invention.
[0172] In the present invention, the nucleic acid probes employed
for molecular marker mapping of plant nuclear genomes hybridize,
under selective hybridization conditions, to a gene encoding a
polynucleotide of the present invention. In preferred embodiments,
the probes are selected from polynucleotides of the present
invention. Typically, these probes are cDNA probes or restriction
enzyme treated (e.g., PST I) genomic clones. The length of the
probes is typically at least 15 bases in length, more preferably at
least 20, 25, 30, 35, 40, or 50 bases in length. Generally,
however, the probes are less than about 1 kilobase in length.
Preferably, the probes are single copy probes that hybridize to a
unique locus in a haploid chromosome compliment. Some exemplary
restriction enzymes employed in RFLP mapping are EcoRI, EcoRV, and
SstI. As used herein the term "restriction enzyme" includes
reference to a composition that recognizes and, alone or in
conjunction with another composition, cleaves at a specific
nucleotide sequence.
[0173] The method of detecting an RFLP comprises the steps of (a)
digesting genomic DNA of a plant with a restriction enzyme; (b)
hybridizing a nucleic acid probe, under selective hybridization
conditions, to a sequence of a polynucleotide of the present
invention of the genomic DNA; (c) detecting therefrom a RFLP. Other
methods of differentiating polymorphic (allelic) variants of
polynucleotides of the present invention can be had by utilizing
molecular marker techniques well known to those of skill in the art
including such techniques as: 1) single stranded conformation
analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE);
3) RNase protection assays; 4) allele-specific oligonucleotides
(ASOs); 5) the use of proteins which recognize nucleotide
mismatches, such as the E. coli mutS protein; and 6)
allele-specific PCR. Other approaches based on the detection of
mismatches between the two complementary DNA strands include
clamped denaturing gel electrophoresis (CDGE); heteroduplex
analysis (HA); and chemical mismatch cleavage (CMC). Thus, the
present invention further provides a method of genotyping
comprising the steps of contacting, under stringent hybridization
conditions, a sample suspected of comprising a polynucleotide of
the present invention with a nucleic acid probe. Generally, the
sample is a plant sample, preferably, a sample suspected of
comprising a maize polynucleotide of the present invention (e.g.,
gene, mRNA). The nucleic acid probe selectively hybridizes, under
stringent conditions, to a subsequence of a polynucleotide of the
present invention comprising a polymorphic marker. Selective
hybridization of the nucleic acid probe to the polymorphic marker
nucleic acid sequence yields a hybridization complex. Detection of
the hybridization complex indicates the presence of that
polymorphic marker in the sample. In preferred embodiments, the
nucleic acid probe comprises a polynucleotide of the present
invention.
[0174] The use of the term "nucleotide constructs" herein is not
intended to limit the present invention to nucleotide constructs
comprising DNA. Those of ordinary skill in the art will recognize
that nucleotide constructs, particularly polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides may also be employed in
the methods disclosed herein. Thus, the nucleotide constructs of
the present invention encompass all nucleotide constructs that can
be employed in the methods of the present invention for
transforming plants including, but not limited to, those comprised
of deoxyribonucleotides, ribonucleotides, and combinations thereof.
Such deoxyribonucleotides and ribonucleotides include both
naturally occurring molecules and synthetic analogues. The
nucleotide constructs of the invention also encompass all forms of
nucleotide constructs including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0175] Furthermore, it is recognized that the methods of the
invention may employ a nucleotide construct that is capable of
directing, in a transformed plant, the expression of at least one
protein, or at least one RNA, such as, for example, an antisense
RNA that is complementary to at least a portion of an mRNA.
Typically such a nucleotide construct is comprised of a coding
sequence for a protein or an RNA operably linked to 5' and 3'
transcriptional regulatory regions. Alternatively, it is also
recognized that the methods of the invention may employ a
nucleotide construct that is not capable of directing, in a
transformed plant, the expression of a protein or an RNA.
[0176] In addition, it is recognized that methods of the present
invention do not depend on the incorporation of the entire
nucleotide construct into the genome, only that the plant or cell
thereof is altered as a result of the introduction of the
nucleotide construct into a cell. In one embodiment of the
invention, the genome may be altered following the introduction of
the nucleotide construct into a cell. For example, the nucleotide
construct, or any part thereof, may incorporate into the genome of
the plant. Alterations to the genome of the present invention
include, but are not limited to, additions, deletions, and
substitutions of nucleotides in the genome. While the methods of
the present invention do not depend on additions, deletions, or
substitutions of any particular number of nucleotides, it is
recognized that such additions, deletions, or substitutions
comprise at least one nucleotide.
[0177] The nucleotide constructs of the invention also encompass
nucleotide constructs that may be employed in methods for altering
or mutating a genomic nucleotide sequence in an organism,
including, but not limited to, chimeric vectors, chimeric
mutational vectors, chimeric repair vectors, mixed-duplex
oligonucleotides, self-complementary chimeric oligonucleotides, and
recombinogenic oligonucleobases. Such nucleotide constructs and
methods of use, such as, for example, chimeraplasty, are known in
the art. Chimeraplasty involves the use of such nucleotide
constructs to introduce site-specific changes into the sequence of
genomic DNA within an organism. See, U.S. Pat. Nos. 5,565,350;
5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; all of
which are herein incorporated by reference. See also, WO 98/49350,
WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778; herein incorporated by reference.
[0178] The methods of the invention can be used with other methods
available in the art for enhancing disease resistance in plants.
Similarly, the antimicrobial compositions described herein may be
used alone or in combination with other nucleotide sequences,
polypeptides, or agents to protect against plant diseases and
pathogens. Although any one of a variety of second nucleotide
sequences may be utilized, specific embodiments of the invention
encompass those second nucleotide sequences that, when expressed in
a plant, help to increase the resistance of a plant to
pathogens.
[0179] Proteins, peptides, and lysozymes that naturally occur in
insects (Jaynes et al. (1987) Bioassays 6:263-270), plants
(Broekaert et al. (1997) Critical Reviews in Plant Sciences
16:297-323), animals (Vunnam et al. (1997) J. Peptide Res.
49:59-66), and humans (Mitra and Zang (1994) Plant Physiol.
106:977-981; Nakajima et al. (1997) Plant Cell Reports 16:674-679)
are also a potential source of plant disease resistance (Ko, K.
(2000) www.scisoc.org/feature/BioTechnology/antimicrobial.html).
Examples of such plant resistance-conferring sequences include
those encoding sunflower rhoGTPase-Activating Protein (rhoGAP),
lipoxygenase (LOX), Alcohol Dehydrogenase (ADH), and
Sclerotinia-Inducible Protein-1 (SCIP-1) described in U.S.
application Ser. No. 09/714,767, herein incorporated by reference.
These nucleotide sequences enhance plant disease resistance through
the modulation of development, developmental pathways, and the
plant pathogen defense system. Other plant defense proteins include
those described in WO 99/43823 and WO 99/43821, all of which are
herein incorporated by reference. It is recognized that such second
nucleotide sequences may be used in either the sense or antisense
orientation depending on the desired outcome.
[0180] In another embodiment, the cystatins comprise isolated
polypeptides of the invention. The cystatins of the invention find
use in the decontamination of plant pathogens during the processing
of grain for animal or human food consumption; during the
processing of feedstuffs, and during the processing of plant
material for silage. In this embodiment, the cystatins of the
invention, are presented to grain, plant material for silage, or a
contaminated food crop, or during an appropriate stage of the
processing procedure, in amounts effective for anti-microbial
activity. The compositions can be applied to the environment of a
plant pathogen by, for example, spraying, atomizing, dusting,
scattering, coating or pouring, introducing into or on the soil,
introducing into irrigation water, by seed treatment, or dusting at
the time when the plant pathogen has begun to appear or before the
appearance of pests as a protective measure. It is recognized that
any means to bring the defensive agent polypeptides in contact with
the plant pathogen can be used in the practice of the
invention.
[0181] Additionally, the compositions can be used in formulations
used for their antimicrobial activities. Methods are provided for
controlling plant pathogens comprising applying a decontaminating
amount of a polypeptide or composition of the invention to the
environment of the plant pathogen. The polypeptides of the
invention can be formulated with an acceptable carrier into a
composition(s) that is, for example, a suspension, a solution, an
emulsion, a dusting powder, a dispersible granule, a wettable
powder, an emulsifiable concentrate, an aerosol, an impregnated
granule, an adjuvant, a coatable paste, and also encapsulations in,
for example, polymer substances.
[0182] Such compositions disclosed above may be obtained by the
addition of a surface-active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV
protectant, a buffer, a flow agent or fertilizers, micronutrient
donors or other preparations that influence plant growth. One or
more agrochemicals including, but not limited to, herbicides,
insecticides, fungicides, bacteriocides, nematocides,
molluscicides, acaracides, plant growth regulators, harvest aids,
and fertilizers, can be combined with carriers, surfactants, or
adjuvants customarily employed in the art of formulation or other
components to facilitate product handling and application for
particular target pathogens. Suitable carriers and adjuvants can be
solid or liquid and correspond to the substances ordinarily
employed in formulation technology, e.g., natural or regenerated
mineral substances, solvents, dispersants, wetting agents,
tackifiers, binders, or fertilizers. The active ingredients of the
present invention are normally applied in the form of compositions
and can be applied to the crop area or plant to be treated,
simultaneously or in succession, with other compounds. In some
embodiments, methods of applying an active ingredient of the
present invention or an agrochemical composition of the present
invention (which contains at least one of the proteins of the
present invention) are foliar application, seed coating, and soil
application.
[0183] Suitable surface-active agents include, but are not limited
to, anionic compounds such as a carboxylate of, for example, a
metal; a carboxylate of a long chain fatty acid; an
N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty
alcohol ethoxylates or salts of such esters; fatty alcohol sulfates
such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium
cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated
alkylphenol sulfates; lignin sulfonates; petroleum sulfonates;
alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower
alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of
sulfonated phenol-formaldehyde condensates; more complex sulfonates
such as the amide sulfonates, e.g., the sulfonated condensation
product of oleic acid and N-methyl taurine; or the dialkyl
sulfosuccinates, e.g., the sodium sulfonate or dioctyl succinate.
Non-ionic agents include condensation products of fatty acid
esters, fatty alcohols, fatty acid amides or fatty-alkyl- or
alkenyl-substituted phenols with ethylene oxide, fatty esters of
polyhydric alcohol ethers, e.g., sorbitan fatty acid esters,
condensation products of such esters with ethylene oxide, e.g.
polyoxyethylene sorbitar fatty acid esters, block copolymers of
ethylene oxide and propylene oxide, acetylenic glycols such as
2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic
glycols. Examples of a cationic surface-active agent include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate, or oleate; or oxygen-containing amine such as an amine
oxide of polyoxyethylene alkylamine; an amide-linked amine prepared
by the condensation of a carboxylic acid with a di- or polyamine;
or a quaternary ammonium salt.
[0184] Examples of inert materials include, but are not limited to,
inorganic minerals such as kaolin, phyllosilicates, carbonates,
sulfates, phosphates, or botanical materials such as cork, powdered
corncobs, peanut hulls, rice hulls, and walnut shells.
[0185] The compositions of the present invention can be in a
suitable form for direct application or as concentrate of primary
composition, which requires dilution with a suitable quantity of
water or other diluent before application. The decontaminating
concentration will vary depending upon the nature of the particular
formulation, specifically, whether it is a concentrate or to be
used directly.
[0186] In a further embodiment, the compositions, as well as the
polypeptides of the present invention can be treated prior to
formulation to prolong their activity when applied to the
environment of a plant pathogen as long as the pretreatment is not
deleterious to the activity. Such treatment can be by chemical
and/or physical means as long as the treatment does not
deleteriously affect the properties of the composition(s). Examples
of chemical reagents include, but are not limited to, halogenating
agents; aldehydes such as formaldehyde and glutaraldehyde;
anti-infectives, such as zephiran chloride; alcohols, such as
isopropanol and ethanol; and histological fixatives, such as
Bouin's fixative and Helly's fixative (see, for example, Humason
(1967) Animal Tissue Techniques (W.H. Freeman and Co.)).
[0187] In an embodiment of the invention, the compositions of the
invention comprise a microbe having stably integrated the
nucleotide sequence of a defensive agent. The resulting microbes
can be processed and used as a microbial spray. Any suitable
microorganism can be used for this purpose. See, for example,
Gaertner et al. (1993) in Advanced Engineered Pesticides, Kim
(Ed.). In one embodiment, the nucleotide sequences of the invention
are introduced into microorganisms that multiply on plants
(epiphytes) to deliver the cystatins to potential target crops.
Epiphytes can be, for example, gram-positive or gram-negative
bacteria.
[0188] It is further recognized that whole, i.e., unlysed, cells of
the transformed microorganism can be treated with reagents that
prolong the activity of the polypeptide produced in the
microorganism when the microorganism is applied to the environment
of a target plant. A secretion signal sequence may be used in
combination with the gene of interest such that the resulting
enzyme is secreted outside the microorganism for presentation to
the target plant.
[0189] In this manner, a gene encoding a defensive agent of the
invention may be introduced via a suitable vector into a microbial
host, and said transformed host applied to the environment, plants,
or animals. Microorganism hosts that are known to occupy the
"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or
rhizoplane) of one or more crops of interest may be selected for
transformation. These microorganisms are selected so as to be
capable of successfully competing in the particular environment
with the wild-type microorganisms, to provide for stable
maintenance and expression of the gene expressing the detoxifying
polypeptide, and for improved protection of the enzymes of the
invention from environmental degradation and inactivation.
[0190] Such microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms, such as bacteria, e.g.,
Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly
yeast, e.g., Saccharomyces, Pichia, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, Aureobasidium, and Gliocladium. Of
particular interest are such phytosphere bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,
Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,
Clavibacter xyli, and Azotobacter vinlandii; and phytosphere yeast
species such as Rhodotorula rubra, R. glutinis, R. marina, R.
aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,
Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces
rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium
pullulans.
[0191] Illustrative prokaryotes, both Gram-negative and -positive,
include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as
Rhizobium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and
Acetobacter; Azotobacteraceae; and Nitrobacteraceae. Among
eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which
includes yeast, such as Saccharomyces and Schizosaccharomyces; and
Basidiomycetes yeast, such as Rhodotorula, Aureobasidium,
Sporobolomyces, and the like.
[0192] In an embodiment of the invention, the cystatins of the
invention may be used as a pharmaceutical compound for treatment of
fungal and microbial pathogens in humans and other animals.
Diseases and disorders caused by fungal and microbial pathogens
include but are not limited to fungal meningoencephalitis,
superficial fungal infections, ringworm, Athlete's foot,
histoplasmosis, candidiasis, thrush, coccidioidoma, pulmonary
cryptococcus, trichosporonosis, piedra, tinea nigra, fungal
keratitis, onychomycosis, tinea capitis, chromomycosis,
aspergillosis, endobronchial pulmonary aspergillosis, mucormycosis,
chromoblastomycosis, dermatophytosis, tinea, fusariosis,
pityriasis, mycetoma, pseudallescheriasis, and sporotrichosis.
[0193] The compositions of the invention may be used as
pharmaceutical compounds to provide treatment for diseases and
disorders associated with, but not limited to, the following fungal
pathogens: Histoplasma capsulatum, Candida spp. (C. albicans, C.
tropicalis, C. parapsilosis, C. guilliermondii, C. glabratal
Torulopsis glabrata, C. krusei, C. lusitaniae), Aspergillus
fumigatus, A. flavus, A. niger, Rhizopus spp., Rhizomucor spp.,
Cunninghamella spp., Apophysomyces spp., Saksenaee spp., Mucor
spp., and Absidia spp. Efficacy of the compositions of the
invention as anti-fungal treatments may be determined through
anti-fungal assays known to one of skill in the art.
[0194] The cystatins may be administered to a patient through
numerous means. Systemic administration can also be by transmucosal
or transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art, and include, for example, for
transmucosal administration, detergents, bile salts, and fusidic
acid derivatives. Transmucosal administration can be accomplished
through the use of nasal sprays or suppositories. For transdermal
administration, the active compounds are formulated into ointments,
salves, gels, or creams as generally known in the art. The
compounds can also be prepared in the form of suppositories (e.g.,
with conventional suppository bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.
[0195] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0196] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated with each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. Depending on the type and severity of the
disease, about 1 .mu.g/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to about 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this therapy is easily monitored by
conventional techniques and assays. An exemplary dosing regimen is
disclosed in WO 94/04188. The specification for the dosage unit
forms of the invention are dictated by and directly dependent on
the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an active compound for the
treatment of individuals.
[0197] "Treatment" is herein defined as the application or
administration of a therapeutic agent to a patient, or application
or administration of a therapeutic agent to an isolated tissue or
cell line from a patient, who has a disease, a symptom of disease
or a predisposition toward a disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve or
affect the disease, the symptoms of disease or the predisposition
toward disease. A "therapeutic agent" includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0198] The cystatins of the invention can be used for any
application including coating surfaces to target microbes. In this
manner, target microbes include human pathogens or microorganisms.
Surfaces that might be coated with the cystatins of the invention
include carpets and sterile medical facilities. Polymer bound
polypeptides of the invention may be used to coat surfaces. Methods
for incorporating compositions with anti-microbial properties into
polymers are known in the art. See U.S. Pat. No. 5,847,047 herein
incorporated by reference.
[0199] An isolated polypeptide of the invention can be used as an
immunogen to generate antibodies that bind cystatins using standard
techniques for polyclonal and monoclonal antibody preparation. The
full-length cystatins can be used or, alternatively, the invention
provides antigenic peptide fragments of cystatins for use as
immunogens. The antigenic peptide of a defensive agent comprises at
least 8, preferably 10, 15, 20, or 30 amino acid residues of the
amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76, and
encompasses an epitope of a cystatin such that an antibody raised
against the peptide forms a specific immune complex with the
anti-microbial polypeptides. Preferred epitopes encompassed by the
antigenic peptide are regions of cystatins that are located on the
surface of the protein, e.g., hydrophilic regions.
[0200] Accordingly, another aspect of the invention pertains to
anti-cystatin polyclonal and monoclonal antibodies that bind a
cystatin. Polyclonal cystatin-like antibodies can be prepared by
immunizing a suitable subject (e.g., rabbit, goat, mouse, or other
mammal) with an defensive agent-like immunogen. The anti-cystatin
antibody titer in the immunized subject can be monitored over time
by standard techniques, such as with an enzyme linked immunosorbent
assay (ELISA) using immobilized anti-microbial polypeptides. At an
appropriate time after immunization, e.g., when the anti-defensive
agent antibody titers are highest, antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
by standard techniques, such as the hybridoma technique originally
described by Kohler and Milstein (1975) Nature 256:495-497, the
human B cell hybridoma technique (Kozbor et al. (1983) Immunol.
Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) in
Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell
(Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or trioma
techniques. The technology for producing hybridomas is well known
(see generally Coligan et al., eds. (1994) Current Protocols in
Immunology (John Wiley & Sons, Inc., New York, N.Y.); Galfre et
al. (1977) Nature 266:55052; Kenneth (1980) in Monoclonal
Antibodies: A New Dimension In Biological Analyses (Plenum
Publishing Corp., NY; and Lerner (1981) Yale J. Biol. Med.,
54:387-402).
[0201] Alternatively to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-cystatin-like antibody can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with a cystatin to thereby isolate immunoglobulin library members
that bind the defensive agent. Kits for generating and screening
phage display libraries are commercially available (e.g., the
Pharmacia Recombinant Phage Antibody System, Catalog No.
27-9400-01; and the Stratagene SurfZAP.TM. Phage Display Kit,
Catalog No. 240612). Additionally, examples of methods and reagents
particularly amenable for use in generating and screening an
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO
92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and
90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.
The antibodies can be used to identify homologs of the cystatins of
the invention.
[0202] The following examples are offered by way of illustration
and not by way of limitation.
[0203] The following stock solutions and media were used for
transformation and regeneration of soybean roots:
Stock Solutions (Per Liter):
[0204] 10.times.B-5 Majors: 25.00 g KNO.sub.3, 1.34 g
(NH.sub.4).sub.2 SO.sub.4, 2.50 g MgSO.sub.4.7H.sub.2O, 1.50 g
CaCl.sub.2.2H.sub.2O, 1.31 g NaH.sub.2PO.sub.4 (anhydrous). [0205]
100.times.B-5 Minors: 1.00 g MnSO.sub.4.H.sub.2O, 0.30 g
H.sub.3BO.sub.3, 0.20 g ZnSO.sub.4.7H.sub.2O, 0.075 g KI. [0206]
100.times.B-5 Vitamins with Thiamine: 10.00 g myo-Inositol, 1.00 g
Thiamine*HCl, 0.10 g Nicotinic acid, 0.10 g Pyridoxine HCl. [0207]
100.times. Iron Mix: 3.73 g. Na.sub.2EDTA, 2.78 g
FeSO.sub.4.7H.sub.2O. Media (per Liter): [0208] Minimal A medium:
10.5 g K.sub.2HPO.sub.4, 4.5 g KH.sub.2PO.sub.4, 1.0 g
(NH.sub.4).sub.2SO.sub.4, 0.5 g
(Na).sub.2C.sub.6H.sub.5O.sub.7.2H.sub.2O, 246.5 mg
MgSO.sub.4.7H.sub.2O, 2 g sucrose, 15 g agar. [0209] 0 B-5 medium:
0.6 g MES [2-(N-Morpholino) ethane-sulfonic acid (Sigma, M5287), 20
g sucrose, 10 mL 100.times.B-5 minors, 100 mL 10.times.B-5 majors,
10 mL 100.times.B-5 vitamins with Thiamine, 10 mL 100.times.Iron
mix. [0210] MXB medium: Murashige and Skoog Basal nutrient salts
(M5524, Sigma), 10 mL 100.times.B-5 Vitamins with thiamine, 30 g
sucrose, 3 g gelrite, pH 5.7. [0211] SOC medium: 20 g
bactotryptone, 5 g yeast extract (Difco), 2 mL 5 M NaCl, 2.5 mL 1 M
KCl, 10 mL 1 M MgCl.sub.2, 10 mL 2 M glucose, 10 mL 1 M
MgSO.sub.4.
EXAMPLE 1
Transformation and Regeneration of Transgenic Plants
[0212] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the cystatin sequences of the
present invention operably linked to a ubiquitin promoter and the
selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),
which confers resistance to the herbicide Bialaphos. Alternatively,
the selectable marker gene is provided on a separate plasmid.
Transformation is performed as follows. Media recipes follow
below.
Preparation of Target Tissue
[0213] The ears are husked and surface sterilized in 30% Clorox
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the 2.5
cm target zone in preparation for bombardment.
Preparation of DNA
[0214] This plasmid DNA plus plasmid DNA containing a PAT
selectable marker is precipitated onto 1.1 .mu.m (average diameter)
tungsten pellets using a CaCl.sub.2 precipitation procedure as
follows:
[0215] 100 .mu.L prepared tungsten particles in water
[0216] 10 .mu.L (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0217] 100 .mu.L 2.5 M CaCl.sub.2
[0218] 10 .mu.L 0.1 M spermidine
[0219] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 .mu.L 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.L spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
Particle Gun Treatment
[0220] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
Subsequent Treatment
[0221] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/L Bialaphos, and subcultured every 2 weeks. After approximately
10 weeks of selection, selection-resistant callus clones are
transferred to 288J medium to initiate plant regeneration.
Following somatic embryo maturation (2-4 weeks), well-developed
somatic embryos are transferred to medium for germination and
transferred to the lighted culture room. Approximately 7-10 days
later, developing plantlets are transferred to 272V hormone-free
medium in tubes for 7-10 days until plantlets are well established.
Plants are then transferred to inserts in flats (equivalent to
2.5'' pot) containing potting soil and grown for 1 week in a growth
chamber, subsequently grown an additional 1-2 weeks in the
greenhouse, then transferred to classic 600 pots (1.6 gallon) and
grown to maturity. Plants are monitored and scored for and altered
level of expression of the cystatin sequence of the invention.
Alternatively, the cysteine proteinase activity can be assayed.
Bombardment and Culture Media
[0222] Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts
(SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000X SIGMA-1511),
0.5 mg/L thiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88
g/L L-proline (brought to volume with D-1 H.sub.20 following
adjustment to pH 5.8 with KOH); 2.0 g/L Gelrite (added after
bringing to volume with D-I H.sub.20); and 8.5 mg/L silver nitrate
(added after sterilizing the medium and cooling to room
temperature). Selection medium (560R) comprises 4.0 g/L N6 basal
salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000X
SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L
2,4-D (brought to volume with D-I H.sub.20 following adjustment to
pH 5.8 with KOH); 3.0 g/L Gelrite (added after bringing to volume
with D-I H.sub.20); and 0.85 mg/L silver nitrate and 3.0 mg/L
bialaphos (both added after sterilizing the medium and cooling to
room temperature).
[0223] Plant regeneration medium (288J) comprises 4.3 g/L MS salts
(GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and
0.40 g/L glycine brought to volume with polished D-I H.sub.20)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/L
myo-inositol, 0.5 mg/L zeatin, 60 g/L sucrose, and 1.0 mL/L of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.20
after adjusting to pH 5.6); 3.0 g/L Gelrite (added after bringing
to volume with D-I H.sub.20); and 1.0 mg/L indoleacetic acid and
3.0 mg/L bialaphos (added after sterilizing the medium and cooling
to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/L MS
salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock solution (0.100
g/L nicotinic acid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL,
and 0.40 g/L glycine brought to volume with polished D-I H.sub.20),
0.1 g/L myo-inositol, and 40.0 g/L sucrose (brought to volume with
polished D-I H.sub.20 after adjusting pH to 5.6); and 6 g/L
bacto-agar (added after bringing to volume with polished D-I
H.sub.20), sterilized and cooled to 60.degree. C.
EXAMPLE 2
Agrobacterium-Mediated Transformation
[0224] For Agrobacterium-mediated transformation of maize with a
cystatin nucleotide sequence of the invention, operably linked to a
ubiquitin promoter, preferably the method of Zhao is employed (U.S.
Pat. No. 5,981,840, and PCT patent publication WO98/32326; the
contents of which are hereby incorporated by reference). Briefly,
immature embryos are isolated from maize and the embryos contacted
with a suspension of Agrobacterium, where the bacteria are capable
of transferring the DNA construct containing the cystatin
nucleotide sequence to at least one cell of at least one of the
immature embryos (step 1: the infection step). In this step the
immature embryos are preferably immersed in an Agrobacterium
suspension for the initiation of inoculation. The embryos are
co-cultured for a time with the Agrobacterium (step 2: the
co-cultivation step). Preferably the immature embryos are cultured
on solid medium following the infection step. Following this
co-cultivation period an optional "resting" step is contemplated.
In this resting step, the embryos are incubated in the presence of
at least one antibiotic known to inhibit the growth of
Agrobacterium without the addition of a selective agent for plant
transformants (step 3: resting step). Preferably the immature
embryos are cultured on solid medium with antibiotic, but without a
selecting agent, for elimination of Agrobacterium and for a resting
phase for the infected cells. Next, inoculated embryos are cultured
on medium containing a selective agent and growing transformed
callus is recovered (step 4: the selection step). Preferably, the
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step), and preferably calli grown on selective medium are cultured
on solid medium to regenerate the plants.
EXAMPLE 3
Identification of the Gene from a Computer Homology Search
[0225] Gene identities were determined by conducting BLAST (Basic
Local Alignment Search Tool; Altschul, S. F., et al. (1993) J. Mol.
Biol. 215:403-410) 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).
[0226] The cDNA sequences of the present invention were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm. The DNA sequences
were translated in all reading frames and compared for similarity
to all publicly available protein sequences contained in the "nr"
database using the BLASTX algorithm (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 were used to construct contiguous DNA
sequences.
[0227] Sequence alignments and percent identity calculations were
performed using GAP, as well as the Megalign program of the
LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignments of the sequences were 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.
EXAMPLE 4
Soybean Embryo Transformation
[0228] Soybean embryos are bombarded with a plasmid containing the
cystatin sequence operably linked to a ubiquitin promoter as
follows. To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface-sterilized, immature seeds of the soybean
cultivar A2872, are cultured in the light or dark at 26.degree. C.
on an appropriate agar medium for six to ten weeks. Somatic embryos
producing secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos that multiplied as early, globular-staged embryos,
the suspensions are maintained as described below.
[0229] Soybean embryogenic suspension cultures are maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0230] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0231] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene composed of the 35S promoter
from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The expression cassette
comprising the cystatin sequence operably linked to the ubiquitin
promoter can be isolated as a restriction fragment. This fragment
can then be inserted into a unique restriction site of the vector
carrying the marker gene.
[0232] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0233] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi,
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0234] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days
post-bombardment with fresh media containing 50 mg/mL hygromycin.
This selective media can be refreshed weekly. Seven to eight weeks
post-bombardment, green, transformed tissue may be observed growing
from untransformed, necrotic embryogenic clusters. Isolated green
tissue is removed and inoculated into individual flasks to generate
new, clonally propagated, transformed embryogenic suspension
cultures. Each new line may be treated as an independent
transformation event. These suspensions can then be subcultured and
maintained as clusters of immature embryos or regenerated into
whole plants by maturation and germination of individual somatic
embryos.
EXAMPLE 5
Sunflower Meristem Tissue Transformation
[0235] Sunflower meristem tissues are transformed with an
expression cassette containing the cystatin sequence operably
linked to a ubiquitin promoter as follows (see also European Patent
Number EP 0 486233, herein incorporated by reference, and
Malone-Schoneberg et al. (1994) Plant Science 103:199-207). Mature
sunflower seed (Helianthus annuus L.) are dehulled using a single
wheat-head thresher. Seeds are surface sterilized for 30 minutes in
a 20% Clorox bleach solution with the addition of two drops of
Tween 20 per 50 mL of solution. The seeds are rinsed twice with
sterile distilled water.
[0236] Split embryonic axis explants are prepared by a modification
of procedures described by Schrammeijer et al. (Schrammeijer et al.
(1990) Plant Cell Rep. 9: 55-60). Seeds are imbibed in distilled
water for 60 minutes following the surface sterilization procedure.
The cotyledons of each seed are then broken off, producing a clean
fracture at the plane of the embryonic axis. Following excision of
the root tip, the explants are bisected longitudinally between the
primordial leaves. The two halves are placed, cut surface up, on
GBA medium consisting of Murashige and Skoog mineral elements
(Murashige et al. (1962) Physiol. Plant., 15: 473-497), Shepard's
vitamin additions (Shepard (1980) in Emergent Techniques for the
Genetic Improvement of Crops (University of Minnesota Press, St.
Paul, Minn.), 40 mg/L adenine sulfate, 30 g/L sucrose, 0.5 mg/L
6-benzyl-aminopurine (BAP), 0.25 mg/L indole-3-acetic acid (IAA),
0.1 mg/L gibberellic acid (GA3), pH 5.6, and 8 g/L Phytagar.
[0237] The explants are subjected to microprojectile bombardment
prior to Agrobacterium treatment (Bidney et al. (1992) Plant Mol.
Biol. 18: 301-313). Thirty to forty explants are placed in a circle
at the center of a 60.times.20 mm plate for this treatment.
Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 mL of sterile TE buffer (10 mM Tris HCl, 1 mM
EDTA, pH 8.0) and 1.5 mL aliquots are used per bombardment. Each
plate is bombarded twice through a 150 mm nytex screen placed 2 cm
above the samples in a PDS 1000.RTM. particle acceleration
device.
[0238] Disarmed Agrobacterium tumefaciens strain EHA105 is used in
all transformation experiments. A binary plasmid vector comprising
the expression cassette described above is introduced into
Agrobacterium strain EHA105 via freeze-thawing as described by
Holsters et al. (1978) Mol. Gen. Genet. 163:181-187. This plasmid
further comprises a kanamycin selectable marker gene (i.e, nptII).
Bacteria for plant transformation experiments are grown overnight
(28.degree. C. and 100 RPM continuous agitation) in liquid YEP
medium (10 g/L yeast extract, 10 g/L Bactopeptone, and 5 g/L NaCl,
pH 7.0) with the appropriate antibiotics required for bacterial
strain and binary plasmid maintenance. The suspension is used when
it reaches an OD.sub.600 of about 0.4 to 0.8. The Agrobacterium
cells are pelleted and resuspended at a final OD.sub.600 of 0.5 in
an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 g/L
NH.sub.4Cl, and 0.3 g/L MgSO.sub.4.
[0239] Freshly bombarded explants are placed in an Agrobacterium
suspension, mixed, and left undisturbed for 30 minutes. The
explants are then transferred to GBA medium and co-cultivated, cut
surface down, at 26.degree. C. and 18-hour days. After three days
of co-cultivation, the explants are transferred to 374B (GBA medium
lacking growth regulators and a reduced sucrose level of 1%)
supplemented with 250 mg/L cefotaxime and 50 mg/L kanamycin
sulfate. The explants are cultured for two to five weeks on
selection and then transferred to fresh 374B medium lacking
kanamycin for one to two weeks of continued development. Explants
with differentiating, antibiotic-resistant areas of growth that
have not produced shoots suitable for excision are transferred to
GBA medium containing 250 mg/L cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green,
kanamycin-resistant shoots are assayed for the presence of NPTII by
ELISA and for the presence of transgene expression by assaying for
the activity of the cystatin sequences.
[0240] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid
6440 in vitro-grown sunflower seedling rootstock. Surface
sterilized seeds are germinated in 48-0 medium (half-strength
Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and
grown under conditions described for explant culture. The upper
portion of the seedling is removed, a 1 cm vertical slice is made
in the hypocotyl, and the transformed shoot inserted into the cut.
The entire area is wrapped with parafilm to secure the shoot.
Grafted plants can be transferred to soil following one week of in
vitro culture. Grafts in soil are maintained under high humidity
conditions followed by a slow acclimatization to the greenhouse
environment. Transformed sectors of T.sub.0 plants (parental
generation) maturing in the greenhouse are identified by NPTII
ELISA and/or by the analysis of the activity of the cystatin
sequences in the leaf extracts while transgenic seeds harvested
from NPTII-positive T.sub.0 plants are identified by the analysis
of the activity the cystatin sequences in small portions of dry
seed cotyledon.
[0241] An alternative sunflower transformation protocol allows the
recovery of transgenic progeny without the use of chemical
selection pressure. Seeds are dehulled and surface-sterilized for
20 minutes in a 20% Clorox bleach solution with the addition of two
to three drops of Tween 20 per 100 mL of solution, then rinsed
three times with distilled water. Sterilized seeds are imbibed in
the dark at 26.degree. C. for 20 hours on filter paper moistened
with water. The cotyledons and root radical are removed, and the
meristem explants are cultured on 374E (GBA medium consisting of MS
salts, Shepard vitamins, 40 mg/L adenine sulfate, 3% sucrose, 0.5
mg/L 6-BAP, 0.25 mg/L IAA, 0.1 mg/L GA, and 0.8% Phytagar at pH
5.6) for 24 hours under the dark. The primary leaves are removed to
expose the apical meristem, around 40 explants are placed with the
apical dome facing upward in a 2 cm circle in the center of 374M
(GBA medium with 1.2% Phytagar), and then cultured on the medium
for 24 hours in the dark.
[0242] Approximately 18.8 mg of 1.8 .mu.m tungsten particles are
resuspended in 150 .mu.L absolute ethanol. After sonication, 8
.mu.l of it is dropped on the center of the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture
discs in the first shelf at 26 mm of Hg helium gun vacuum.
[0243] The plasmid of interest is introduced into Agrobacterium
tumefaciens strain EHA105 via freeze thawing as described
previously. The pellet of overnight-grown bacteria at 28.degree. C.
in a liquid YEP medium (10 g/L yeast extract, 10 g/L Bactopeptone,
and 5 g/L NaCl, pH 7.0) in the presence of 50 .mu.g/L kanamycin is
resuspended in an inoculation medium (12.5 mM 2-mM 2-(N-morpholino)
ethanesulfonic acid, MES, 1 g/L NH.sub.4Cl and 0.3 g/L MgSO.sub.4
at pH 5.7) to reach a final concentration of 4.0 at OD 600.
Particle-bombarded explants are transferred to GBA medium (374E),
and a droplet of bacteria suspension is placed directly onto the
top of the meristem. The explants are co-cultivated on the medium
for 4 days, after which the explants are transferred to 374C medium
(GBA with 1% sucrose and no BAP, IAA, GA3 and supplemented with 250
.mu.g/mL cefotaxime). The plantlets are cultured on the medium for
about two weeks under 16-hour day and 26.degree. C. incubation
conditions.
[0244] Explants (around 2 cm long) from two weeks of culture in
374C medium are screened for cystatin activity using assays known
in the art. After positive (i.e., for cystatin expression) explants
are identified, those shoots that fail to exhibit cystatin activity
are discarded, and every positive explant is subdivided into nodal
explants. One nodal explant contains at least one potential node.
The nodal segments are cultured on GBA medium for three to four
days to promote the formation of auxiliary buds from each node.
Then they are transferred to 374C medium and allowed to develop for
an additional four weeks. Developing buds are separated and
cultured for an additional four weeks on 374C medium. Pooled leaf
samples from each newly recovered shoot are screened again by the
appropriate protein activity assay. At this time, the positive
shoots recovered from a single node will generally have been
enriched in the transgenic sector detected in the initial assay
prior to nodal culture.
[0245] Recovered shoots positive for defense-inducible expression
are grafted to Pioneer hybrid 6440 in vitro-grown sunflower
seedling rootstock. The rootstocks are prepared in the following
manner. Seeds are dehulled and surface-sterilized for 20 minutes in
a 20% Clorox bleach solution with the addition of two to three
drops of Tween 20 per 100 mL of solution, and are rinsed three
times with distilled water. The sterilized seeds are germinated on
the filter moistened with water for three days, then they are
transferred into 48 medium (half-strength MS salt, 0.5% sucrose,
0.3% gelrite pH 5.0) and grown at 26.degree. C. under the dark for
three days, then incubated at 16-hour-day culture conditions. The
upper portion of selected seedling is removed, a vertical slice is
made in each hypocotyl, and a transformed shoot is inserted into a
V-cut. The cut area is wrapped with parafilm. After one week of
culture on the medium, grafted plants are transferred to soil. In
the first two weeks, they are maintained under high humidity
conditions to acclimatize to a greenhouse environment.
EXAMPLE 6
Transformation of BL21 Star Cells and Cystatin Expression and
Purification
Strain Transformation
[0246] One .mu.L samples of mini-prep DNA were incubated on ice
with 20 .mu.L of BL21 Star cells (Invitrogen) for 30 minutes. The
DNA/cell mixtures were then heated at 42.degree. C. for 45 sec,
then iced for 2 min. 200 .mu.L of SOC were added to each tube, and
these mixtures were then incubated at 37.degree. C. for 1 hour. 50
.mu.L samples were spread out on LB broth plates containing 100
mg/L carbenicillin and 0.1 M MgSO.sub.4 to incubate overnight at
37.degree. C.
Protein Expression
[0247] Bacteria samples in duplicate were selected from the plates,
and these were incubated overnight in 2.0 mL of 2xYT media
containing 100 .mu.g/mL carbenicillin. The incubations took place
in a 48 well, pyramid bottom plate (Innovative Microplate) at
37.degree. C. on a shaker (225 rpm).
[0248] The next day, the OD readings for the wells were obtained
after diluting 10 .mu.L bacterial samples with 90 .mu.L of water in
a standard 96-well, flat bottomed microtiter plate, with the
absorbance read at 600 nm. Based on these values, the cultures were
diluted with fresh 2xYT (+carb.) to the two target induction
OD.sub.600 values of 0.1 and 0.6, to a final volume of 2 mL. Each
target OD value was diluted in quadruplicate to account for two
isopropyl-beta-D-thiogalactopyranoside (IPTG) induction values each
and two incubation temperatures each. When the plates were ready,
the wells were induced with IPTG at concentrations of either 0.05
mM or 1.0 mM. The plates were then incubated overnight on shakers
(225 rpm) at temperatures of either 16.degree. C. or 30.degree. C.
After approximately 20 hours of incubation, the OD values for all
the wells were obtained as described supra. The cells were then
harvested by centrifuging the plates at 1700 rpm for 10 minutes.
The supernatants were discarded and the pelleted cells frozen at
-20.degree. C. until purification could take place.
Protein Purification
[0249] The cells were allowed to thaw for several minutes, then 200
.mu.L of B-Peril protein extraction reagent (Pierce) containing 0.2
units of benzonase (Novagen) were added to each well, and the
solution was pipetted repeatedly to resuspend the pellet. The cells
were incubated for 10 min in the B-Peril solution.
[0250] 25-30 .mu.m MBPP 800 .mu.L purification filter plates
(Whatman) were prepared by adding 200 .mu.L of 50% slurry of
glutathione Sepharose 4B resin (Amersham) for GST-tagged proteins,
or TALON resin (Clontech) for HIS-tagged proteins into each well.
The plates were centrifuged briefly to remove excess buffer. The
lysates were transferred directly from the growth plate to the
plates with the resins, and the solutions were pipetted up and down
several times to mix them well. The plates were then centrifuged at
760 rpm for 5 min, and the flow-through was discarded. The resins
with the bound proteins were washed twice with 200 .mu.L of buffer
A (50 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol), and each time
the plates were centrifuged at 760 rpm for 5 min and the wash was
discarded. Standard 96 well plates were placed underneath the
Whatman filter plates, and the proteins were eluted by adding and
mixing 100 .mu.L of buffer A containing either 20 mM reduced
glutathione (Sigma) for GST tags, or 500 mM imidazole (Sigma) for
HIS tags. The plates were centrifuged at 1000 rpm for 5 min, and
the purified proteins were collected in the standard 96 well plates
placed underneath.
[0251] Bradford reactions were performed to determine the protein
concentrations for all the samples. These values were used to
establish normalization plates for the cystatin assays where equal
volumes contained equal amounts of protein. However, when the
protein concentration was too low for a given sample, 20 .mu.L were
used directly in the cystatin assay to come as close as possible to
the target amount of 6 .mu.g of protein.
EXAMPLE 7
Anti-Fungal and Anti-Bacterial Assays
[0252] The anti-fungal and anti-bacterial activity of the cystatin
polypeptides of the invention can be tested using a variety of
assays.
[0253] BL21 cells were transformed with cystatin genes using the
Lambda CE6 Induction Kit (Stratagene) according to the
manufacturer's protocol. The induced BL21 cells were grown and
harvested, and then resuspended in the binding buffer from a
His-Bind Kit (Novagen). The suspended cells were sonicated to
release the expressed protein from the cells. The crude protein
extract was denatured by dissolving it into a 6 M urea solution for
1 hour on ice in order to allow the His-tag to efficiently bind to
the resin. The denatured cystatins were then purified by His-Bind
Resin Chromatography according to the manufacturer's protocol,
except that 6M Urea was added to the binding, washing and elution
buffers.
[0254] The purified proteins were renatured by dialysis using
1.times.PBS buffer with gradually decreased concentration of urea
(5M, 4M, 3M, 1.5M, and 0.75M). The dialyses were performed with
each concentration of urea for one hour and then with 1.times.PBS
buffer without urea overnight at 4.degree. C. Bradford reactions
were performed to determine the protein concentrations for all the
samples. The isolated polypeptide was then tested for activity.
[0255] Anti-Fungal Assays:
[0256] Fusarium verticillioides (strain M033) was grown on
1/2.times. potato dextrose agar plates: (For each liter, 12 grams
Difco Potato Dextrose Broth (#0549-17-9) and 15 grams agar were
suspended in dH.sub.2O, final volume was raised to 1 liter and
autoclaved at 121.degree. C. for 20 minutes. Plates were poured in
sterile hood.) Spores were collected from 10 day old to 3 week old
culture plates of Fusarium verticillioides by rinsing a portion of
the plate with potato dextrose broth (24 grams Difco Potato
Dextrose Broth (#0549-17-9) per liter+0.08% tween-20). The
collected spore solution was then vortexed and placed on ice. The
spores were counted with a hemocytometer and used within 2 hours.
4.times. assay medium with spores was prepared by diluting the
collected spores with potato dextrose broth+0.08% tween-20 to
16,000 spores per milliliter.
[0257] The two purified cystatins used in this assay were supplied
in PBS buffer, which was prepared by diluting a 10.times.
commercial stock buffer (10.times. phosphate buffered saline [137
mM NaCl, 2.7 mM KCl, 10 mM phosphate pH 7.3-7.5] EM Science Catalog
#6505) with water. The protein concentration of each cystatin was
measured using the Pierce BCA protein assay (BCA Protein Assay
Reagent, Pierce catalog #23225; Smith, P. K., et al. (1985).
Measurement of protein using bicinchoninic acid (Anal. Biochem.
150, 76-85.) calibrated against BSA. The purified GmCys-2 (SEQ ID
NO: 30) protein was supplied at 5.7 mg/mL and the ZmCys-4 (SEQ ID
NO: 6) protein was supplied at 3.2 mg/mL.
[0258] For an assay, 25 .mu.L of the 4.times. assay medium with
spores was diluted to 1.times. with 75 .mu.L test sample/water.
Assays were conducted in 96 well microtiter plates.
[0259] Plates were covered and incubated at 280 in the dark for
24-48 h. Growth was evaluated visually using an inverted
microscope, and a scale of 0-4 was used to rate the effect of added
peptide
[0260] Antifungal activity was rated as follows:
[0261] 0=no observable inhibition relative to water control
[0262] 1=slight inhibition
[0263] 2=strong inhibition, contained growth (fuzzy balls)
[0264] 3=strong inhibition, very little branching
[0265] 4=complete inhibition of germination
[0266] PBS buffer was tested at an equivalent dilution and no
effect was observed (score=0) for all control samples.
TABLE-US-00013 TABLE 13 Anti-Fusarium activity of maize cystatin
Zm-Cys4 and soybean cystatin Gm-Cys2. ZmCys-4 GmCys-2 (SEQ ID NO:
30) (SEQ ID NO: 6) Score Concentration Score Concentration 48
(.mu.g/mL) 26 hours (.mu.g/mL) 24 hours hours 400 3.5 570 4 3.5 200
3.5 280 4 0 100 3.5 140 3 0 50 3 71 2 0 25 2 35 1 0 12.5 1 18 0 0 6
1 9 0 0 3 1 4 0 0
The results shown in Table 13, above, are a clear indication that
both ZmCys-4 (SEQ ID NO: 6) and GmCys-2 (SEQ ID NO: 30) have a
significant anti-fungal activity towards F. verticillioides.
[0267] Anti-bacterial Assays. Cultures are grown to midlog phase
(E. coli in LB broth and C. nebraskense in NBY) and are then
harvested by centrifugation (2000.times.g for 10 min). Cells are
washed with 10 mM sodium phosphate buffer, pH 5.8 (C. nebraskense)
or pH 6.5 (E. coli) by centrifugation and then colony forming units
are estimated spectrophotometrically at 600 nm with previously
established colony forming unit-optical density relationships used
as a reference.
[0268] Assays for bactericidal activity are performed by incubating
10.sup.5 bacterial colony forming units in 90 .mu.L with 10 mL of
peptide (or water for control). After 60 min at 37.degree. C. (E.
coli) or 25.degree. C. (C. nebraskense), four serial, 10-fold
dilutions are made in sterile phosphate buffer. Aliquots of 100
.mu.L are plated on LB or NBY plates, using 1 or 2 plates/dilution.
Resulting colonies are counted, and the effect of the peptide is
expressed as percent of initial colony count (Selsted et al. (1984)
Infect. Immun. 45:150-154).
[0269] Assays for bacteriostatic activity are performed by
incubating 10.sup.5 bacteria with MBP-1 in 200 .mu.L of dilute
medium (1 part NBY broth to 4 parts 10 mM sodium phosphate, pH 5.8)
in microtiter plate wells. Plates are covered, sealed, and
incubated at 28.degree. C. Growth is monitored
spectrophotometrically at 600 nm. After 41 h controls will have
grown sufficiently (optical density >0.20) to measure effect of
peptide as percent of control.
EXAMPLE 8
Proteinase Inhibition Assays
Cystatin Assay
[0270] The measurement of inhibition activity of cystatins toward
papain was based on the method by Kouzuma et al. (1996) (J.
Biochem. 119: 1106-1113) (see FIG. 1). The assay was performed in a
96 well plate. Twenty .mu.L of a cystatin solution containing
either 6 .mu.g or 0.06 .mu.g of cystatin protein were incubated
with 20 .mu.L of papain solution (0.1 mg/mL papain (Calbiochem)
stock in 100 mM phosphate buffer, pH 6.5, 0.3 M KCl, 0.1 mM EDTA,
and 1 mM freshly added DTT) at 37.degree. C. for 15 min. 200 .mu.L
of filtered substrate solution (504 .mu.M
L-pyroglutamyl-L-phenylalanyl-L-leucine p-nitroanilide
(Pyr-Phe-Leu-pNA; Sigma, P3169) with 10% DMSO (Sigma) in the same
buffer as above) were then added to the reaction mix and the plate
incubated at 37.degree. C. for one hour. Formation of
p-nitroaniline was measured by measuring absorbance at 420 nm.
Further 37.degree. C. incubation took place with 420 nm readings
being taken at desired time intervals. The reaction was stopped by
the addition of 30 .mu.L of 30% acetic acid without significantly
affecting the absorbance values. Cysteine proteinase inhibitory
activity was indicated by smaller absorbance values for the 6 .mu.g
protein wells versus the 0.06 .mu.g wells for each cystatin.
Negative controls consisted of buffer without cystatin protein.
Positive controls consisted of previously tested cystatins.
TABLE-US-00014 TABLE 14 Impact of N-terminal tag and induction
conditions on cystatin activity. Cystatin activity Gene SEQ ID NO:
GST-tagged His-tagged Conditions for highest activity GmCys2 29
low/medium Low GST: 16 C. His: 25 C., OD 0.8 GmCys4 33 not not
detected detected GmCys5 35 low/medium not detected GST: 30 C.,
0.05 mM IPTG, OD 0.1 GmCys7 39 low not detected GST: 30 C., 0.05 mM
IPTG, OD 0.1 GmCys9 43 high High GST: 30 C., 0.05 mM IPTG, OD 0.1
His: 16 C., 1 mM IPTG, OD 0.6 OsCys4 51 not not detected detected
OsCys6 55 high High GST: 30 C., 1 mM IPTG, OD 0.6 His: 16 C., 0.05
mM IPTG, OD 0.6 TaCys8 67 low/medium low/medium GST: 30 C.
(transient) (transient) His: 30 C. ZmCys4 5 high High GST: 25 C.
HIS: 30 C. ZmCys6 9 not not detected detected ZmCys7 11 high Low
GST: 30 C. His: 30 C., OD 0.6 ZmCys8 13 not not detected detected
ZmCys10 17 not not detected detected ZmCys11 19 not very low His:
16 C., 0.05 mM IPTG, OD detected 0.1 ZmCys12 21 low Low GST: 30 C.,
0.05 mM IPTG, OD 0.1 His: 30 C., 1 mM IPTG, OD 0.6 ZmCys13 23 high
High GST: 30 C. His: 16 C., OD 0.6 ZmCys14 25 low/medium low/medium
GST: 30 C. His: 30 C.
[0271] Table 14, above, is a summary of the cystatin activity
detected in the tested genes of the present invention. Detailed
data for the assay results on each of the above cystatins is
presented in tables 15 through 60.
[0272] Some of the identified cystatins did not show any proteinase
inhibitor activity in the conditions of the assay as conducted
above. There can be numerous reasons for these results. As is well
known in the art, certain microorganisms perform better than others
for expression of a given protein. Often it is difficult to predict
which one will work best. The cystatins of the present invention
have been expressed in BL21 Star cells (Invitrogen). Other
bacterial lines or expression systems may be more effective for a
selection of the genes. Furthermore, the cystatin proteins were
expressed with a N-terminal His- or GST-tag to facilitate cystatin
purification from the bacterial culture. It is possible that the
BL21 bacteria were unable to express sufficient amounts of
correctly folded protein or, that for unknown reasons, recovery of
certain cystatin proteins from the bacterial culture was low. It is
also possible that correct folding and cystatin activity are
impacted by the presence of the GST or His tag. See results given
in Table 14. A C-terminal tag may be more effective for certain
cystatins. A preferred method for expression of these proteins in
plants would be to express the cystatins without a tag. Untagged
cystatins can be expected to have improved activity compared to
tagged cystatins. It is also possible that certain cystatins have a
pH optimum that is distinct from pH 6.5 at which papain inhibition
was measured. Another possibility is that while certain cystatins
appear to be inactive as inhibitors of papain the expressed
proteins inhibit other proteinases. TABLE-US-00015 TABLE 15
Cystatin Activity Results GST fusion tagged protein, first
replicate GmCys4 (SEQ ID NO: 34) Cystatin Assay 9/26 First
Replicate Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C.
(mM) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng
.DELTA. BL21 16 0.05 0.1 2.404 0.082 0.281 0.206 -0.08 0.556 0.537
-0.02 16 1.0 0.1 2.312 0.048 0.258 0.207 -0.05 0.552 0.553 0.00 16
0.05 0.6 3.654 0.079 0.276 0.205 -0.07 0.550 0.541 -0.01 16 1.0 0.6
3.747 0.039 0.260 0.209 -0.05 0.557 0.552 -0.01 30 0.05 0.1 5.419
0.194 0.303 0.247 -0.06 0.545 0.552 0.01 30 1.0 0.1 6.305 0.161
0.293 0.240 -0.05 0.548 0.556 0.01 30 0.05 0.6 6.259 0.179 0.305
0.241 -0.06 0.551 0.554 0.00 30 1.0 0.6 6.259 0.150 0.302 0.245
-0.06 0.552 0.555 0.00 BL21 Star 16 0.05 0.1 0.882 0.021 0.229
0.204 -0.03 0.562 0.552 -0.01 16 1.0 0.1 0.882 0.026 0.237 0.206
-0.03 0.551 0.550 0.00 16 0.05 0.6 1.712 0.064 0.262 0.214 -0.05
0.547 0.552 0.01 16 1.0 0.6 1.389 0.046 0.255 0.219 -0.04 0.549
0.555 0.01 30 0.05 0.1 4.025 0.096 0.242 0.226 -0.02 0.544 0.554
0.01 30 1.0 0.1 4.303 0.154 0.269 0.221 -0.05 0.542 0.556 0.01 30
0.05 0.6 4.675 0.154 0.261 0.224 -0.04 0.551 0.547 0.00 30 1.0 0.6
5.047 0.110 0.202 0.224 0.02 0.547 0.550 0.00
[0273] TABLE-US-00016 TABLE 16 Cystatin Activity Results GST fusion
tagged protein, second replicate GmCys4 (SEQ ID NO: 34) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.543 0.095 0.292
0.246 -0.05 0.549 0.564 0.01 16 1.0 0.1 2.404 0.077 0.302 0.250
-0.05 0.554 0.564 0.01 16 0.05 0.6 3.191 0.064 0.288 0.230 -0.06
0.551 0.556 0.01 16 1.0 0.6 3.283 0.066 0.313 0.224 -0.09 0.549
0.549 0.00 30 0.05 0.1 5.233 0.119 0.296 0.247 -0.05 0.549 0.552
0.00 30 1.0 0.1 5.746 0.165 0.295 0.251 -0.04 0.543 0.552 0.01 30
0.05 0.6 6.119 0.182 0.311 0.259 -0.05 0.557 0.553 0.00 30 1.0 0.6
6.445 0.152 0.303 0.251 -0.05 0.557 0.552 -0.01 BL21 Star 16 0.05
0.1 0.928 0.023 0.254 0.222 -0.03 0.568 0.551 -0.02 16 1.0 0.1
0.882 0.019 0.245 0.228 -0.02 0.561 0.554 -0.01 16 0.05 0.6 1.758
0.064 0.285 0.221 -0.06 0.546 0.545 0.00 16 1.0 0.6 1.620 0.05
0.283 0.226 -0.06 0.544 0.549 0.01 30 0.05 0.1 4.350 0.125 0.259
0.234 -0.03 0.545 0.550 0.01 30 1.0 0.1 4.396 0.152 0.272 0.235
-0.04 0.543 0.556 0.01 30 0.05 0.6 5.419 0.1 0.278 0.242 -0.04
0.546 0.544 0.00 30 1.0 0.6 5.419 0.137 0.273 0.240 -0.03 0.525
0.538 0.01
[0274] TABLE-US-00017 TABLE 17 Cystatin Activity Results His fusion
tagged protein, first replicate GmCys4 (SEQ ID NO: 34) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.404 0.233 0.254 0.205
-0.05 0.543 0.554 0.01 16 1.0 0.1 1.896 0.228 0.247 0.202 -0.05
0.536 0.549 0.01 16 0.05 0.6 3.422 0.347 0.273 0.197 -0.08 0.535
0.544 0.01 16 1.0 0.6 2.543 0.302 0.273 0.202 -0.07 0.542 0.543
0.00 30 0.05 0.1 6.212 0.457 0.270 0.229 -0.04 0.541 0.552 0.01 30
1.0 0.1 5.187 0.410 0.267 0.223 -0.04 0.542 0.548 0.01 30 0.05 0.6
5.839 0.570 0.275 0.225 -0.05 0.536 0.550 0.01 30 1.0 0.6 5.559
0.463 0.266 0.215 -0.05 0.533 0.547 0.01 BL21 Star 16 0.05 0.1
1.389 0.084 0.271 0.206 -0.07 0.543 0.547 0.00 16 1.0 0.1 1.389
0.079 0.271 0.218 -0.05 0.542 0.544 0.00 16 0.05 0.6 2.867 0.302
0.273 0.206 -0.07 0.538 0.540 0.00 16 1.0 0.6 2.219 0.246 0.280
0.200 -0.08 0.534 0.540 0.01 30 0.05 0.1 5.001 0.435 0.269 0.226
-0.04 0.527 0.552 0.03 30 1.0 0.1 5.187 0.456 0.267 0.229 -0.04
0.521 0.549 0.03 30 0.05 0.6 4.629 0.412 0.282 0.219 -0.06 0.545
0.546 0.00 30 1.0 0.6 4.861 0.456 0.275 0.216 -0.06 0.535 0.544
0.01
[0275] TABLE-US-00018 TABLE 18 Cystatin Activity Results His fusion
tagged protein, second replicate GmCys4 (SEQ ID NO: 34) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.219 0.255 0.289
0.223 -0.07 0.539 0.547 0.01 16 1.0 0.1 1.989 0.261 0.291 0.225
-0.07 0.543 0.553 0.01 16 0.05 0.6 2.867 0.3 0.298 0.221 -0.08
0.538 0.547 0.01 16 1.0 0.6 2.635 0.282 0.284 0.213 -0.07 0.536
0.540 0.00 30 0.05 0.1 5.746 0.528 0.282 0.232 -0.05 0.539 0.544
0.01 30 1.0 0.1 4.954 0.312 0.274 0.231 -0.04 0.543 0.551 0.01 30
0.05 0.6 5.932 0.534 0.282 0.243 -0.04 0.531 0.548 0.02 30 1.0 0.6
5.606 0.44 0.278 0.239 -0.04 0.537 0.542 0.01 BL21 Star 16 0.05 0.1
1.297 0.086 0.294 0.226 -0.07 0.546 0.544 0.00 16 1.0 0.1 1.389
0.084 0.291 0.223 -0.07 0.544 0.549 0.01 16 0.05 0.6 2.728 0.016
0.221 0.215 -0.01 0.550 0.543 -0.01 16 1.0 0.6 2.497 0.212 0.289
0.214 -0.08 0.538 0.537 0.00 30 0.05 0.1 5.094 0.471 0.277 0.209
-0.07 0.540 0.541 0.00 30 1.0 0.1 5.140 0.515 0.281 0.235 -0.05
0.533 0.546 0.01 30 0.05 0.6 4.303 0.463 0.280 0.239 -0.04 0.528
0.546 0.02 30 1.0 0.6 4.629 0.377 0.286 0.231 -0.06 0.530 0.529
0.00
[0276] TABLE-US-00019 TABLE 19 Cystatin Activity Results GST fusion
tagged protein, first replicate GmCys2 (SEQ ID NO: 30) Cystatin
Assay 6/12 Cell Temp IPTG Bradford Overnight Type .degree. C. (Mm)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
4.559 0.089 0.270 0.232 0.233 -0.038 16 0.5 0.8 6.894 0.129 0.258
0.230 0.224 -0.031 16 0.05 0.1 7.694 0.150 0.275 0.229 0.240 -0.040
16 0.05 0.8 11.171 0.143 0.263 0.229 0.220 -0.038 25 0.5 0.1 3.140
0.000 0.237 0.206 0.201 -0.034 25 0.5 0.8 3.186 0.004 0.233 0.211
0.197 -0.029 25 0.05 0.1 4.285 0.062 0.245 0.216 0.214 -0.030 25
0.05 0.8 4.461 0.067 0.244 0.211 0.206 -0.036 BL21 Star 16 0.5 0.1
1.906 0.134 0.258 0.222 0.223 -0.036 16 0.5 0.8 5.131 0.171 0.216
0.224 0.219 0.006 16 0.05 0.1 1.726 0.024 0.241 0.223 0.221 -0.019
16 0.05 0.8 4.415 0.159 0.201 0.219 0.218 0.017 25 0.5 0.1 3.547
0.212 0.210 0.195 0.193 -0.016 25 0.5 0.8 4.248 0.276 0.209 0.188
0.194 -0.018 25 0.05 0.1 3.241 0.259 0.215 0.199 0.195 -0.019 25
0.05 0.8 3.714 0.524 0.204 0.194 0.189 -0.012
[0277] TABLE-US-00020 TABLE 20 Cystatin Activity Results GST fusion
tagged protein, second replicate GmCys2 (SEQ ID NO: 30) Cystatin
Assay 7/24 Cell Temp IPTG Bradford Overnight Type .degree. C. (Mm)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
5.331 0.058 0.243 0.200 0.186 -0.049 16 0.5 0.8 7.142 0.081 0.240
0.200 0.198 -0.041 16 0.05 0.1 7.769 0.082 0.239 0.200 0.193 -0.043
16 0.05 0.8 10.354 2.948 0.254 0.201 0.203 -0.052 25 0.5 0.1 2.894
0.000 0.253 0.204 0.191 -0.055 25 0.5 0.8 3.010 0.009 0.243 0.196
0.185 -0.052 25 0.05 0.1 3.686 0.048 0.250 0.208 0.211 -0.040 25
0.05 0.8 4.294 0.052 0.244 0.197 0.198 -0.046 BL21 Star 16 0.5 0.1
3.269 0.135 0.226 0.181 0.186 -0.042 16 0.5 0.8 6.576 0.119 0.211
0.190 0.191 -0.020 16 0.05 0.1 2.899 0.155 0.215 0.188 0.185 -0.028
16 0.05 0.8 5.401 0.097 0.178 0.186 0.187 0.008 25 0.5 0.1 3.862
0.542 0.232 0.185 0.183 -0.048 25 0.5 0.8 3.802 0.246 0.221 0.186
0.180 -0.038 25 0.05 0.1 3.492 0.302 0.231 0.186 0.190 -0.043 25
0.05 0.8 3.329 0.669 0.215 0.192 0.195 -0.022
[0278] TABLE-US-00021 TABLE 21 Cystatin Activity Results His fusion
tagged protein, first replicate GmCys2 (SEQ ID NO: 30) Cystatin
Assay 6/12 Cell Temp IPTG Bradford Overnight Type .degree. C. (Mm)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
5.312 0.351 0.256 0.227 0.225 -0.030 16 0.5 0.8 7.076 0.456 0.297
0.295 0.285 -0.007 16 0.05 0.1 7.324 0.410 0.260 0.224 0.228 -0.033
16 0.05 0.8 10.935 0.578 0.294 0.290 0.296 -0.001 25 0.5 0.1 3.241
0.280 0.235 0.198 0.210 -0.031 25 0.5 0.8 3.468 0.203 0.258 0.208
0.216 -0.046 25 0.05 0.1 4.498 0.300 0.238 0.244 0.215 -0.008 25
0.05 0.8 4.215 0.345 0.259 0.227 0.222 -0.035 BL21 Star 16 0.5 0.1
5.760 0.342 0.263 0.220 0.225 -0.041 16 0.5 0.8 7.058 0.456 0.283
0.294 0.290 0.009 16 0.05 0.1 4.698 0.295 0.259 0.224 0.219 -0.037
16 0.05 0.8 5.690 0.453 0.285 0.288 0.294 0.005 25 0.5 0.1 4.016
0.337 0.215 0.192 0.194 -0.022 25 0.5 0.8 3.714 0.353 0.243 0.216
0.210 -0.030 25 0.05 0.1 3.492 0.383 0.224 0.199 0.197 -0.026 25
0.05 0.8 2.941 0.410 0.249 0.216 0.212 -0.035
[0279] TABLE-US-00022 TABLE 22 Cystatin Activity Results His fusion
tagged protein, second replicate GmCys2 (SEQ ID NO: 30) Cystatin
Assay 7/24 Cell Temp IPTG Bradford Overnight Type .degree. C. (Mm)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
5.131 0.245 0.234 0.188 0.206 -0.037 16 0.5 0.8 6.049 0.421 0.254
0.205 0.201 -0.051 16 0.05 0.1 7.268 0.306 0.244 0.200 0.203 -0.042
16 0.05 0.8 8.599 0.559 0.245 0.213 0.538 0.130 25 0.5 0.1 2.728
0.204 0.234 0.197 0.241 -0.015 25 0.5 0.8 2.880 0.290 0.266 0.219
0.206 -0.053 25 0.05 0.1 3.473 0.300 0.223 0.240 0.217 0.006 25
0.05 0.8 3.825 0.352 0.251 0.207 0.202 -0.046 BL21 Star 16 0.5 0.1
4.991 0.302 0.258 0.184 0.222 -0.055 16 0.5 0.8 6.553 0.434 0.245
0.208 0.194 -0.044 16 0.05 0.1 3.927 0.144 0.220 0.191 0.188 -0.030
16 0.05 0.8 5.014 0.458 0.245 0.201 0.203 -0.043 25 0.5 0.1 3.556
0.259 0.229 0.203 0.208 -0.024 25 0.5 0.8 3.427 0.219 0.247 0.213
0.207 -0.038 25 0.05 0.1 3.093 0.298 0.225 0.210 0.220 -0.010 25
0.05 0.8 3.019 0.259 0.271 0.189 0.214 -0.069
[0280] TABLE-US-00023 TABLE 23 Cystatin Activity Results GST fusion
tagged protein, first replicate OsCys4 (SEQ ID NO: 52) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.312 0.073 0.275 0.203
-0.07 0.554 0.536 -0.02 16 1.0 0.1 2.035 0.046 0.264 0.211 -0.05
0.553 0.550 0.00 16 0.05 0.6 3.839 0.073 0.282 0.204 -0.08 0.559
0.539 -0.02 16 1.0 0.6 3.515 0.059 0.271 0.187 -0.08 0.549 0.540
-0.01 30 0.05 0.1 6.352 0.171 0.304 0.242 -0.06 0.543 0.546 0.00 30
1.0 0.1 5.746 0.156 0.296 0.235 -0.06 0.540 0.548 0.01 30 0.05 0.6
5.932 0.100 0.292 0.235 -0.06 0.550 0.552 0.00 30 1.0 0.6 6.305
0.110 0.285 0.233 -0.05 0.542 0.554 0.01 BL21 Star 16 0.05 0.1
1.067 0.019 0.232 0.194 -0.04 0.566 0.552 -0.01 16 1.0 0.1 1.021
0.017 0.239 0.195 -0.04 0.558 0.547 -0.01 16 0.05 0.6 1.896 0.070
0.263 0.198 -0.07 0.541 0.546 0.01 16 1.0 0.6 1.989 0.075 0.269
0.191 -0.08 0.542 0.545 0.00 30 0.05 0.1 4.489 0.182 0.275 0.224
-0.05 0.539 0.545 0.01 30 1.0 0.1 4.954 0.123 0.205 0.220 0.02
0.539 0.551 0.01 30 0.05 0.6 4.582 0.158 0.265 0.217 -0.05 0.536
0.549 0.01 30 1.0 0.6 5.094 0.150 0.232 0.219 -0.01 0.552 0.545
-0.01
[0281] TABLE-US-00024 TABLE 24 Cystatin Activity Results GST fusion
tagged protein, second replicate OsCys4 (SEQ ID NO: 52) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.035 0.075 0.291
0.214 -0.08 0.552 0.540 -0.01 16 1.0 0.1 2.173 0.062 0.297 0.210
-0.09 0.554 0.543 -0.01 16 0.05 0.6 3.654 0.098 0.297 0.222 -0.08
0.551 0.545 -0.01 16 1.0 0.6 3.144 0.068 0.303 0.228 -0.08 0.548
0.557 0.01 30 0.05 0.1 6.072 0.228 0.296 0.244 -0.05 0.542 0.545
0.00 30 1.0 0.1 6.025 0.154 0.285 0.240 -0.05 0.535 0.551 0.02 30
0.05 0.6 6.679 0.085 0.280 0.241 -0.04 0.549 0.547 0.00 30 1.0 0.6
6.119 0.182 0.294 0.245 -0.05 0.542 0.554 0.01 BL21 Star 16 0.05
0.1 0.975 0.023 0.252 0.220 -0.03 0.562 0.549 -0.01 16 1.0 0.1
0.975 0.025 0.262 0.230 -0.03 0.559 0.554 -0.01 16 0.05 0.6 1.804
0.062 0.294 0.225 -0.07 0.548 0.558 0.01 16 1.0 0.6 1.850 0.077
0.295 0.230 -0.07 0.544 0.562 0.02 30 0.05 0.1 4.350 0.217 0.256
0.238 -0.02 0.530 0.550 0.02 30 1.0 0.1 4.954 0.282 0.266 0.233
-0.03 0.535 0.546 0.01 30 0.05 0.6 5.513 0.266 0.243 0.238 -0.01
0.537 0.553 0.02 30 1.0 0.6 5.419 0.163 0.275 0.236 -0.04 0.541
0.548 0.01
[0282] TABLE-US-00025 TABLE 25 Cystatin Activity Results His fusion
tagged protein, first replicate OsCys4 (SEQ ID NO: 52) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.219 0.194 0.257 0.197
-0.06 0.542 0.540 0.00 16 1.0 0.1 1.942 0.212 0.263 0.193 -0.07
0.538 0.544 0.01 16 0.05 0.6 3.283 0.304 0.275 0.186 -0.09 0.533
0.542 0.01 16 1.0 0.6 2.728 0.313 0.262 0.186 -0.08 0.537 0.542
0.01 30 0.05 0.1 6.072 0.459 0.279 0.218 -0.06 0.535 0.543 0.01 30
1.0 0.1 5.466 0.391 0.278 0.210 -0.07 0.532 0.542 0.01 30 0.05 0.6
5.979 0.448 0.273 0.219 -0.05 0.530 0.542 0.01 30 1.0 0.6 5.559
0.438 0.266 0.228 -0.04 0.522 0.558 0.04 BL21 Star 16 0.05 0.1
1.389 0.095 0.272 0.198 -0.07 0.543 0.547 0.00 16 1.0 0.1 1.343
0.100 0.281 0.215 -0.07 0.541 0.543 0.00 16 0.05 0.6 3.329 0.295
0.260 0.190 -0.07 0.534 0.536 0.00 16 1.0 0.6 2.820 0.270 0.265
0.196 -0.07 0.534 0.541 0.01 30 0.05 0.1 5.326 0.433 0.279 0.200
-0.08 0.534 0.534 0.00 30 1.0 0.1 6.072 0.459 0.278 0.203 -0.08
0.528 0.537 0.01 30 0.05 0.6 5.280 0.415 0.260 0.216 -0.04 0.533
0.542 0.01 30 1.0 0.6 4.954 0.423 0.271 0.218 -0.05 0.539 0.538
0.00
[0283] TABLE-US-00026 TABLE 26 Cystatin Activity Results His fusion
tagged protein, second replicate OsCys4 (SEQ ID NO: 52) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.989 0.217 0.291
0.224 -0.07 0.544 0.552 0.01 16 1.0 0.1 2.035 0.237 0.284 0.219
-0.07 0.543 0.548 0.01 16 0.05 0.6 2.820 0.304 0.297 0.222 -0.08
0.545 0.549 0.00 16 1.0 0.6 2.867 0.288 0.279 0.225 -0.05 0.543
0.547 0.00 30 0.05 0.1 5.606 0.463 0.277 0.232 -0.05 0.531 0.542
0.01 30 1.0 0.1 5.280 0.412 0.269 0.231 -0.04 0.529 0.545 0.02 30
0.05 0.6 5.885 0.505 0.276 0.231 -0.05 0.537 0.547 0.01 30 1.0 0.6
5.466 0.49 0.275 0.231 -0.04 0.538 0.549 0.01 BL21 Star 16 0.05 0.1
1.435 0.1 0.291 0.221 -0.07 0.541 0.548 0.01 16 1.0 0.1 1.389 0.088
0.289 0.207 -0.08 0.539 0.536 0.00 16 0.05 0.6 2.682 0.205 0.279
0.228 -0.05 0.543 0.561 0.02 16 1.0 0.6 2.312 0.156 0.297 0.229
-0.07 0.539 0.550 0.01 30 0.05 0.1 5.140 0.517 0.267 0.237 -0.03
0.521 0.544 0.02 30 1.0 0.1 5.326 0.509 0.274 0.236 -0.04 0.523
0.540 0.02 30 0.05 0.6 5.792 0.469 0.223 0.235 0.01 0.546 0.547
0.00 30 1.0 0.6 5.140 0.45 0.276 0.223 -0.05 0.529 0.534 0.01
[0284] TABLE-US-00027 TABLE 27 Cystatin Activity Results GST fusion
tagged protein, first replicate ZmCys4 (SEQ ID NO: 6) Cystatin
Assay 6/12 Cell Temp IPTG Bradford Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
5.298 0.061 0.272 0.238 0.236 -0.035 16 0.5 0.8 6.749 0.097 0.229
0.231 0.223 -0.002 16 0.05 0.1 8.848 0.093 0.277 0.246 0.244 -0.032
16 0.05 0.8 12.435 0.137 0.270 0.247 0.233 -0.030 25 0.5 0.1 3.038
0.830 0.234 0.206 0.207 -0.027 25 0.5 0.8 3.237 0.145 0.222 0.206
0.189 -0.025 25 0.05 0.1 5.452 0.188 0.240 0.226 0.221 -0.017 25
0.05 0.8 4.577 0.241 0.226 0.208 0.195 -0.025 BL21 Star 16 0.5 0.1
1.550 0.124 0.233 0.231 0.229 -0.003 16 0.5 0.8 3.552 0.364 0.188
0.237 0.222 0.042 16 0.05 0.1 1.906 0.088 0.215 0.218 0.212 0.000
16 0.05 0.8 2.991 0.588 0.223 0.227 0.222 0.002 25 0.5 0.1 3.575
0.112 0.184 0.198 0.196 0.013 25 0.5 0.8 3.830 0.354 0.169 0.200
0.197 0.029 25 0.05 0.1 3.459 0.150 0.141 0.235 0.202 0.077 25 0.05
0.8 3.839 0.353 0.116 0.209 0.190 0.084
[0285] TABLE-US-00028 TABLE 28 Cystatin Activity Results GST fusion
tagged protein, second replicate ZmCys4 (SEQ ID NO: 6) Cystatin
Assay 7/24 Cell Temp IPTG Bradford Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 600 ng 60 ng 6 ng .DELTA. BL21 16 0.5 0.1
5.243 0.195 0.198 0.202 0.200 0.003 16 0.5 0.8 7.029 0.112 0.194
0.187 0.321 0.060 16 0.05 0.1 8.322 0.153 0.247 0.217 0.204 -0.037
16 0.05 0.8 11.639 0.170 0.244 0.252 0.224 -0.006 25 0.5 0.1 2.982
0.094 0.239 0.195 0.222 -0.031 25 0.5 0.8 2.968 0.082 0.234 0.195
0.199 -0.036 25 0.05 0.1 4.601 0.272 0.240 0.211 0.203 -0.033 25
0.05 0.8 4.294 0.167 0.236 0.210 0.201 -0.030 BL21 Star 16 0.5 0.1
1.744 0.082 0.162 0.198 0.200 0.037 16 0.5 0.8 4.275 0.319 0.127
0.205 0.206 0.078 16 0.05 0.1 2.002 0.154 0.165 0.214 0.191 0.038
16 0.05 0.8 4.164 0.414 0.124 0.200 0.218 0.085 25 0.5 0.1 3.167
0.812 0.216 0.192 0.190 -0.025 25 0.5 0.8 3.965 0.237 0.092 0.208
0.191 0.107 25 0.05 0.1 3.140 0.725 0.116 0.203 0.194 0.082 25 0.05
0.8 3.478 0.273 0.097 0.191 0.194 0.095
[0286] TABLE-US-00029 TABLE 29 Cystatin Activity Results His fusion
tagged protein, first replicate ZmCys4 (SEQ ID NO: 6) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.850 0.226 0.274 0.193
-0.08 0.540 0.537 0.00 16 1.0 0.1 1.804 0.211 0.268 0.192 -0.08
0.539 0.534 -0.01 16 0.05 0.6 2.913 0.338 0.270 0.185 -0.09 0.540
0.538 0.00 16 1.0 0.6 2.358 0.325 0.260 0.198 -0.06 0.536 0.532
0.00 30 0.05 0.1 5.326 0.490 0.295 0.248 -0.05 0.524 0.526 0.00 30
1.0 0.1 5.419 0.478 0.291 0.258 -0.03 0.518 0.538 0.02 30 0.05 0.6
5.513 0.455 0.303 0.268 -0.04 0.529 0.537 0.01 30 1.0 0.6 5.140
0.490 0.303 0.273 -0.03 0.536 0.538 0.00 BL21 Star 16 0.05 0.1
1.297 0.045 0.082 0.194 0.11 0.402 0.535 0.13 16 1.0 0.1 1.343
0.051 0.047 0.203 0.16 0.144 0.529 0.39 16 0.05 0.6 2.358 0.213
0.045 0.205 0.16 0.123 0.539 0.42 16 1.0 0.6 2.450 0.215 0.046
0.199 0.15 0.115 0.535 0.42 30 0.05 0.1 5.094 0.467 0.053 0.265
0.21 0.120 0.538 0.42 30 1.0 0.1 5.140 0.533 0.048 0.261 0.21 0.123
0.531 0.41 30 0.05 0.6 4.768 0.400 0.265 0.278 0.01 0.545 0.535
-0.01 30 1.0 0.6 4.768 0.488 0.052 0.274 0.22 0.119 0.531 0.41
[0287] TABLE-US-00030 TABLE 30 Cystatin Activity Results His fusion
tagged protein, second replicate ZmCys4 (SEQ ID NO: 6) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.758 0.425 0.318
0.247 -0.07 0.536 0.545 0.01 16 1.0 0.1 1.712 0.453 0.313 0.233
-0.08 0.537 0.539 0.00 16 0.05 0.6 2.312 0.414 0.328 0.260 -0.07
0.539 0.540 0.00 16 1.0 0.6 2.219 0.348 0.325 0.258 -0.07 0.543
0.546 0.00 30 0.05 0.1 5.699 0.216 0.290 0.267 -0.02 0.561 0.569
0.01 30 1.0 0.1 5.699 0.222 0.292 0.263 -0.03 0.559 0.566 0.01 30
0.05 0.6 5.466 0.293 0.297 -- -- 0.521 -- -- 30 1.0 0.6 5.979 0.316
0.294 -- -- 0.530 -- -- BL21 Star 16 0.05 0.1 1.297 0.427 0.265
0.234 -0.03 0.549 0.547 0.00 16 1.0 0.1 1.251 0.446 0.235 0.232
0.00 0.536 0.536 0.00 16 0.05 0.6 2.404 0.427 0.079 0.233 0.15
0.305 0.541 0.24 16 1.0 0.6 2.589 0.453 0.063 0.222 0.16 0.243
0.542 0.30 30 0.05 0.1 4.629 0.083 0.095 0.260 0.17 0.371 0.557
0.19 30 1.0 0.1 4.118 0.109 0.102 0.269 0.17 0.426 0.558 0.13 30
0.05 0.6 4.582 0.175 0.067 -- -- 0.150 -- -- 30 1.0 0.6 5.280 0.354
0.068 -- -- 0.272 -- --
[0288] TABLE-US-00031 TABLE 31 Cystatin Activity Results GST fusion
tagged protein, first replicate ZmCys6 (SEQ ID NO: 10) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.358 0.093 0.293 0.211
-0.08 0.553 0.544 -0.01 16 1.0 0.1 2.219 0.060 0.286 0.221 -0.07
0.552 0.561 0.01 16 0.05 0.6 3.237 0.103 0.292 0.220 -0.07 0.549
0.554 0.01 16 1.0 0.6 2.589 0.072 0.291 0.223 -0.07 0.558 0.557
0.00 30 0.05 0.1 5.606 0.142 0.298 0.247 -0.05 0.552 0.549 0.00 30
1.0 0.1 6.165 0.197 0.289 0.248 -0.04 0.539 0.554 0.02 30 0.05 0.6
5.746 0.166 0.296 0.256 -0.04 0.547 0.552 0.01 30 1.0 0.6 6.445
0.170 0.305 0.262 -0.04 0.549 0.558 0.01 BL21 Star 16 0.05 0.1
1.113 0.027 0.252 0.200 -0.05 0.559 0.545 -0.01 16 1.0 0.1 0.975
0.037 0.268 0.210 -0.06 0.549 0.549 0.00 16 0.05 0.6 1.758 0.128
0.266 0.213 -0.05 0.543 0.543 0.00 16 1.0 0.6 1.758 0.170 0.279
0.214 -0.07 0.540 0.544 0.00 30 0.05 0.1 4.396 0.632 0.266 0.226
-0.04 0.537 0.552 0.02 30 1.0 0.1 4.257 0.516 0.269 0.231 -0.04
0.536 0.550 0.01 30 0.05 0.6 4.954 0.676 0.274 0.241 -0.03 0.544
0.551 0.01 30 1.0 0.6 4.536 0.710 0.268 0.237 -0.03 0.542 0.553
0.01
[0289] TABLE-US-00032 TABLE 32 Cystatin Activity Results GST fusion
tagged protein, second replicate ZmCys6 (SEQ ID NO: 10) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.896 0.068 0.252
0.213 -0.04 0.548 0.548 0.00 16 1.0 0.1 1.896 0.072 0.271 0.222
-0.05 0.555 0.550 -0.01 16 0.05 0.6 2.635 0.116 0.291 0.212 -0.08
0.549 0.545 0.00 16 1.0 0.6 2.266 0.076 0.291 0.232 -0.06 0.550
0.549 0.00 30 0.05 0.1 5.932 0.174 0.335 0.274 -0.06 0.540 0.542
0.00 30 1.0 0.1 6.212 0.178 0.350 0.307 -0.04 0.544 0.546 0.00 30
0.05 0.6 5.746 0.145 0.337 0.283 -0.05 0.553 0.540 -0.01 30 1.0 0.6
6.259 0.178 0.366 0.302 -0.06 0.547 0.542 -0.01 BL21 Star 16 0.05
0.1 0.836 0.024 0.233 0.207 -0.03 0.566 0.550 -0.02 16 1.0 0.1
0.882 0.025 0.232 0.210 -0.02 0.565 0.551 -0.01 16 0.05 0.6 1.389
0.153 0.265 0.201 -0.06 0.543 0.535 -0.01 16 1.0 0.6 1.389 0.159
0.282 0.205 -0.08 0.528 0.538 0.01 30 0.05 0.1 5.001 0.657 0.355
0.312 -0.04 0.545 0.543 0.00 30 1.0 0.1 4.768 0.594 0.357 0.311
-0.05 0.539 0.542 0.00 30 0.05 0.6 4.489 0.63 0.375 0.297 -0.08
0.539 0.532 -0.01 30 1.0 0.6 4.582 0.68 0.384 0.231 -0.15 0.547
0.511 -0.04
[0290] TABLE-US-00033 TABLE 33 Cystatin Activity Results His fusion
tagged protein, first replicate ZmCys6 (SEQ ID NO: 10) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.942 0.292 0.282 0.202
-0.08 0.546 0.549 0.00 16 1.0 0.1 2.035 0.238 0.266 0.199 -0.07
0.537 0.542 0.01 16 0.05 0.6 2.959 0.338 0.274 0.213 -0.06 0.542
0.552 0.01 16 1.0 0.6 2.913 0.300 0.284 0.197 -0.09 0.544 0.538
-0.01 30 0.05 0.1 5.373 0.416 0.219 0.233 0.01 0.540 0.546 0.01 30
1.0 0.1 4.954 0.452 0.263 0.231 -0.03 0.533 0.548 0.02 30 0.05 0.6
5.419 0.448 0.275 0.245 -0.03 0.544 0.553 0.01 30 1.0 0.6 5.373
0.459 0.275 0.236 -0.04 0.542 0.553 0.01 BL21 Star 16 0.05 0.1
1.297 0.093 0.290 0.210 -0.08 0.542 0.543 0.00 16 1.0 0.1 1.343
0.091 0.287 0.218 -0.07 0.542 0.552 0.01 16 0.05 0.6 2.266 0.284
0.264 0.206 -0.06 0.536 0.542 0.01 16 1.0 0.6 2.266 0.253 0.274
0.220 -0.05 0.532 0.545 0.01 30 0.05 0.1 4.675 0.463 0.214 0.228
0.01 0.548 0.550 0.00 30 1.0 0.1 5.094 0.490 0.252 0.226 -0.03
0.526 0.539 0.01 30 0.05 0.6 4.814 0.402 0.263 0.230 -0.03 0.538
0.546 0.01 30 1.0 0.6 4.861 0.452 0.266 0.226 -0.04 0.540 0.545
0.01
[0291] TABLE-US-00034 TABLE 34 Cystatin Activity Results His fusion
tagged protein, second replicate ZmCys6 (SEQ ID NO: 10) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.758 0.267 0.270
0.199 -0.07 0.539 0.541 0.00 16 1.0 0.1 1.666 0.25 0.271 0.196
-0.08 0.544 0.541 0.00 16 0.05 0.6 2.635 0.306 0.277 0.187 -0.09
0.539 0.532 -0.01 16 1.0 0.6 2.497 0.286 0.276 0.190 -0.09 0.532
0.528 0.00 30 0.05 0.1 5.979 0.448 0.340 0.299 -0.04 0.541 0.546
0.01 30 1.0 0.1 5.979 0.414 0.349 0.303 -0.05 0.536 0.542 0.01 30
0.05 0.6 6.072 0.436 0.356 0.296 -0.06 0.540 0.538 0.00 30 1.0 0.6
6.072 0.431 0.366 0.303 -0.06 0.533 0.536 0.00 BL21 Star 16 0.05
0.1 1.113 0.207 0.258 0.206 -0.05 0.552 0.540 -0.01 16 1.0 0.1
1.159 0.132 0.267 0.220 -0.05 0.543 0.546 0.00 16 0.05 0.6 2.219
0.257 0.253 0.205 -0.05 0.526 0.533 0.01 16 1.0 0.6 2.081 0.186
0.284 0.219 -0.07 0.531 0.534 0.00 30 0.05 0.1 5.885 0.452 0.362
0.324 -0.04 0.535 0.538 0.00 30 1.0 0.1 6.259 0.471 0.341 0.284
-0.06 0.530 0.533 0.00 30 0.05 0.6 5.140 0.509 0.379 0.305 -0.07
0.529 0.534 0.01 30 1.0 0.6 5.652 0.476 0.345 0.269 -0.08 0.528
0.524 0.00
[0292] TABLE-US-00035 TABLE 35 Cystatin Activity Results GST fusion
tagged protein, first replicate ZmCys7 (SEQ ID NO: 12) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 2.127 0.078 0.295 0.245
-0.05 0.550 0.565 0.01 16 1.0 0.1 2.127 0.072 0.291 0.224 -0.07
0.557 0.555 0.00 16 0.05 0.6 3.191 0.224 0.291 0.221 -0.07 0.544
0.543 0.00 16 1.0 0.6 2.959 0.261 0.281 0.225 -0.06 0.544 0.547
0.00 30 0.05 0.1 5.606 0.353 0.314 0.261 -0.05 0.559 0.551 -0.01 30
1.0 0.1 6.025 0.370 0.320 0.266 -0.05 0.554 0.553 0.00 30 0.05 0.6
5.979 0.313 0.335 0.262 -0.07 0.533 0.532 0.00 30 1.0 0.6 6.445
0.277 0.328 0.268 -0.06 0.529 0.537 0.01 BL21 Star 16 0.05 0.1
1.067 0.093 0.218 0.202 -0.02 0.552 0.538 -0.01 16 1.0 0.1 0.975
0.097 0.173 0.209 0.04 0.525 0.540 0.02 16 0.05 0.6 1.896 0.375
0.042 0.207 0.17 0.068 0.537 0.47 16 1.0 0.6 1.758 0.373 0.040
0.208 0.17 0.065 0.542 0.48 30 0.05 0.1 4.025 0.600 0.047 0.224
0.18 0.069 0.539 0.47 30 1.0 0.1 4.536 0.621 0.045 0.219 0.17 0.064
0.532 0.47 30 0.05 0.6 4.164 0.594 0.042 0.252 0.21 0.058 0.535
0.48 30 1.0 0.6 4.350 0.602 0.042 0.259 0.22 0.057 0.545 0.49
[0293] TABLE-US-00036 TABLE 36 Cystatin Activity Results GST fusion
tagged protein, second replicate ZmCys7 (SEQ ID NO: 12) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.942 0.19 0.308
0.251 -0.06 0.548 0.536 -0.01 16 1.0 0.1 1.804 0.196 0.292 0.240
-0.05 0.538 0.539 0.00 16 0.05 0.6 2.774 0.167 0.321 0.248 -0.07
0.544 0.536 -0.01 16 1.0 0.6 2.404 0.152 0.310 0.245 -0.07 0.531
0.544 0.01 30 0.05 0.1 6.072 0.054 0.327 0.262 -0.07 0.534 0.556
0.02 30 1.0 0.1 6.539 0.051 0.319 0.266 -0.05 0.529 0.562 0.03 30
0.05 0.6 5.932 0.075 0.329 0.273 -0.06 0.567 0.561 -0.01 30 1.0 0.6
6.585 0.086 0.320 0.276 -0.04 0.556 0.572 0.02 BL21 Star 16 0.05
0.1 0.928 0.713 0.235 0.210 -0.03 0.551 0.532 -0.02 16 1.0 0.1
0.928 0.425 0.228 0.225 0.00 0.553 0.547 -0.01 16 0.05 0.6 1.666
0.785 0.149 0.227 0.08 0.489 0.543 0.05 16 1.0 0.6 1.527 0.421
0.056 0.204 0.15 0.140 0.529 0.39 30 0.05 0.1 4.257 0.019 0.043
0.244 0.20 0.061 0.545 0.48 30 1.0 0.1 4.443 0.021 0.041 0.251 0.21
0.065 0.556 0.49 30 0.05 0.6 4.489 0.077 0.044 0.246 0.20 0.060
0.562 0.50 30 1.0 0.6 4.350 0.105 0.045 0.250 0.21 0.062 0.550
0.49
[0294] TABLE-US-00037 TABLE 37 Cystatin Activity Results His fusion
tagged protein, first replicate ZmCys7 (SEQ ID NO: 12) Cystatin
Assay 9/26 First Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.989 0.275 0.287 0.227
-0.06 0.542 0.555 0.01 16 1.0 0.1 1.850 0.244 0.282 0.194 -0.09
0.545 0.529 -0.02 16 0.05 0.6 2.404 0.311 0.323 0.206 -0.12 0.536
0.541 0.01 16 1.0 0.6 2.266 0.280 0.271 0.187 -0.08 0.531 0.531
0.00 30 0.05 0.1 5.652 0.396 0.279 0.248 -0.03 0.544 0.549 0.01 30
1.0 0.1 5.326 0.461 0.265 0.236 -0.03 0.539 0.539 0.00 30 0.05 0.6
5.280 0.486 0.301 0.268 -0.03 0.519 0.537 0.02 30 1.0 0.6 5.187
0.448 0.304 0.262 -0.04 0.523 0.534 0.01 BL21 Star 16 0.05 0.1
1.159 0.122 0.147 0.211 0.06 0.503 0.539 0.04 16 1.0 0.1 1.251
0.112 0.220 0.211 -0.01 0.540 0.535 -0.01 16 0.05 0.6 2.219 0.263
0.043 0.215 0.17 0.076 0.541 0.47 16 1.0 0.6 2.266 0.358 0.058
0.203 0.15 0.148 0.520 0.37 30 0.05 0.1 5.187 0.554 0.175 0.236
0.06 0.517 0.543 0.03 30 1.0 0.1 5.094 0.537 0.212 0.230 0.02 0.521
0.535 0.01 30 0.05 0.6 5.187 0.020 0.196 0.271 0.08 0.504 0.534
0.03 30 1.0 0.6 4.768 0.520 0.234 0.265 0.03 0.506 0.535 0.03
[0295] TABLE-US-00038 TABLE 38 Cystatin Activity Results His fusion
tagged protein, second replicate ZmCys7 (SEQ ID NO: 12) Cystatin
Assay 9/26 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 16 0.05 0.1 1.804 0.482 0.286
0.222 -0.06 0.529 0.529 0.00 16 1.0 0.1 1.804 0.453 0.295 0.212
-0.08 0.532 0.527 -0.01 16 0.05 0.6 2.266 0.463 0.291 0.220 -0.07
0.532 0.533 0.00 16 1.0 0.6 2.358 0.491 0.288 0.222 -0.07 0.527
0.537 0.01 30 0.05 0.1 5.606 0.237 0.298 0.263 -0.04 0.530 0.562
0.03 30 1.0 0.1 5.094 0.224 0.290 0.255 -0.04 0.530 0.540 0.01 30
0.05 0.6 5.513 0.32 0.301 0.264 -0.04 0.558 0.565 0.01 30 1.0 0.6
5.513 0.337 0.295 0.266 -0.03 0.554 0.534 -0.02 BL21 Star 16 0.05
0.1 1.021 0.47 0.214 0.227 0.01 0.549 0.540 -0.01 16 1.0 0.1 1.113
0.485 0.238 0.224 -0.01 0.544 0.540 0.00 16 0.05 0.6 2.127 0.431
0.051 0.220 0.17 0.110 0.539 0.43 16 1.0 0.6 1.989 0.395 0.088
0.219 0.13 0.325 0.535 0.21 30 0.05 0.1 5.280 0.036 0.173 0.258
0.09 0.504 0.548 0.04 30 1.0 0.1 5.140 0.049 0.230 0.262 0.03 0.515
0.553 0.04 30 0.05 0.6 5.373 0.164 0.193 0.265 0.07 0.535 0.552
0.02 30 1.0 0.6 5.187 0.203 0.208 0.268 0.06 0.533 0.549 0.02
[0296] TABLE-US-00039 TABLE 39 Cystatin Activity Results GST fusion
tagged protein, first and second replicates OsCys6 (SEQ ID NO: 56)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.758 -0.018
0.651 0.482 -0.169 1.235 1.4 0.165 16 1.0 0.1 1.067 -0.025 0.638
0.49 -0.148 1.286 1.382 0.096 16 0.05 0.6 2.45 0.058 0.701 0.477
-0.224 1.222 1.333 0.111 16 1.0 0.6 1.758 0.015 0.657 0.427 -0.230
1.239 1.103 -0.136 30 0.05 0.1 5.094 0.313 0.057 0.494 0.437 0.14
1.037 0.897 30 1.0 0.1 3.052 0.196 0.051 0.489 0.438 0.126 1.204
1.078 30 0.05 0.6 2.682 0.276 0.06 0.457 0.397 0.117 1.002 0.885 30
1.0 0.6 2.589 0.191 0.053 0.486 0.433 0.12 1.402 1.282 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.573 -0.016
0.501 0.38 -0.121 1.231 1.36 0.129 16 1.0 0.1 1.021 -0.025 0.447
0.361 -0.086 1.269 1.176 -0.093 16 0.05 0.6 2.035 0.076 0.525 0.406
-0.119 1.386 1.689 0.303 16 1.0 0.6 1.85 -0.034 0.477 0.367 -0.110
1.456 1.325 -0.131 30 0.05 0.1 3.237 0.287 0.061 0.453 0.392 0.129
1.198 1.069 30 1.0 0.1 3.283 0.142 0.077 0.426 0.349 0.288 1.39
1.102 30 0.05 0.6 3.607 0.268 0.06 0.462 0.402 0.116 1.242 1.126 30
1.0 0.6 3.283 -0.004 0.415 0.385 -0.030 1.114 1.188 0.074
[0297] TABLE-US-00040 TABLE 40 Cystatin Activity Results His fusion
tagged protein, first and second replicates OsCys6 (SEQ ID NO: 56)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.113 0.004
0.429 0.389 -0.040 1.107 1.284 0.177 16 1.0 0.1 1.251 0.024 0.297
0.38 0.083 1.291 1.32 0.029 16 0.05 0.6 2.173 0.265 0.056 0.389
0.333 0.111 1.148 1.037 16 1.0 0.6 2.358 -0.034 0.359 0.378 0.019
1.18 1.293 0.113 30 0.05 0.1 4.675 0.462 0.567 0.453 -0.114 1.123
1.294 0.171 30 1.0 0.1 4.629 0.429 0.599 0.459 -0.140 1.346 1.45
0.104 30 0.05 0.6 6.305 0.374 0.585 0.455 -0.130 1.321 1.282 -0.039
30 1.0 0.6 3.932 0.368 0.559 0.447 -0.112 1.484 1.359 -0.125
Cystatin Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1
hour Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng
60 ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.021
-0.01 0.479 0.427 -0.052 1.252 1.597 0.345 16 1.0 0.1 1.159 -0.013
0.405 0.368 -0.037 1.187 1.311 0.124 16 0.05 0.6 1.896 0.21 0.056
0.424 0.368 0.116 1.326 1.210 16 1.0 0.6 2.035 0.216 0.07 0.384
0.314 0.126 1.392 1.266 30 0.05 0.1 3.654 0.394 0.609 0.499 -0.110
1.22 1.291 0.071 30 1.0 0.1 3.607 0.286 0.599 0.503 -0.096 1.314
1.409 0.095 30 0.05 0.6 3.468 0.292 0.599 0.51 -0.089 1.693 1.392
-0.301 30 1.0 0.6 3.329 0.347 0.522 0.495 -0.027 1.321 1.721
0.400
[0298] TABLE-US-00041 TABLE 41 Cystatin Activity Results GST fusion
tagged protein, first and second replicates GmCys5 (SEQ ID NO: 36)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.758
-0.013 0.654 0.423 -0.231 1.163 1.368 0.205 16 1.0 0.1 1.343 -0.016
0.608 0.468 -0.140 1.241 1.252 0.011 16 0.05 0.6 1.896 0.04 0.662
0.454 -0.208 1.244 1.304 0.060 16 1.0 0.6 1.804 0.085 0.656 0.438
-0.218 1.176 1.309 0.133 30 0.05 0.1 2.959 0.174 0.288 0.494 0.206
1.195 1.382 0.187 30 1.0 0.1 2.867 0.134 0.359 0.491 0.132 0.985
1.17 0.185 30 0.05 0.6 3.422 0.147 0.299 0.492 0.193 1.309 1.434
0.125 30 1.0 0.6 3.747 0.119 0.362 0.446 0.084 1.126 0.993 -0.133
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.021
0.004 0.463 0.347 -0.116 1.185 1.256 0.071 16 1.0 0.1 1.389 -0.002
0.496 0.342 -0.154 1.48 1.099 -0.381 16 0.05 0.6 2.127 0.036 0.479
0.351 -0.128 1.17 1.417 0.247 16 1.0 0.6 2.081 0.044 0.486 0.35
-0.136 1.067 1.248 0.181 30 0.05 0.1 3.237 0.187 0.114 0.425 0.311
0.731 1.534 0.803 30 1.0 0.1 3.052 0.162 0.126 0.38 0.254 0.717
1.194 0.477 30 0.05 0.6 3.515 0.113 0.206 0.428 0.222 1.214 1.437
0.223 30 1.0 0.6 2.635 0.091 0.182 0.392 0.210 1.063 1.279
0.216
[0299] TABLE-US-00042 TABLE 42 Cystatin Activity Results His fusion
tagged protein, first and second replicates GmCys5 (SEQ ID NO: 36)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.251 -0.006
0.451 0.362 -0.089 1.166 1.283 0.117 16 1.0 0.1 2.219 -0.015 0.442
0.356 -0.086 1.19 1.143 -0.047 16 0.05 0.6 2.404 0.218 0.266 0.374
0.108 1.118 1.124 0.006 16 1.0 0.6 2.543 0.193 0.238 0.357 0.119
1.336 1.344 0.008 30 0.05 0.1 3.561 0.345 0.586 0.431 -0.155 1.355
1.374 0.019 30 1.0 0.1 5.932 0.38 0.569 0.435 -0.134 1.339 1.308
-0.031 30 0.05 0.6 4.303 0.386 0.576 0.435 -0.141 1.29 1.521 0.231
30 1.0 0.6 4.303 0.372 0.555 0.388 -0.167 1.06 1.048 -0.012
Cystatin Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1
hour Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng
60 ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.113
-0.021 0.465 0.364 -0.101 1.273 1.347 0.074 16 1.0 0.1 1.021 -0.023
0.467 0.359 -0.108 1.232 1.176 -0.056 16 0.05 0.6 2.312 0.165 0.306
0.38 0.074 1.323 1.369 0.046 16 1.0 0.6 2.173 0.206 0.202 0.388
0.186 0.973 1.686 0.713 30 0.05 0.1 3.468 0.284 0.581 0.478 -0.103
1.223 1.173 -0.050 30 1.0 0.1 3.376 0.286 0.593 0.422 -0.171 1.541
1.766 0.225 30 0.05 0.6 3.098 0.276 0.61 0.49 -0.120 1.265 1.205
-0.060 30 1.0 0.6 2.867 0.255 0.594 0.445 -0.149 1.784 1.802
0.018
[0300] TABLE-US-00043 TABLE 43 Cystatin Activity Results GST fusion
tagged protein, first and second replicates GmCys7 (SEQ ID NO: 40)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.435 -0.013
0.609 0.452 -0.157 1.179 1.263 0.084 16 1.0 0.1 1.113 -0.02 0.562
0.433 -0.129 1.155 1.184 0.029 16 0.05 0.6 1.573 0.067 0.556 0.361
-0.195 1.187 1.191 0.004 16 1.0 0.6 1.573 0.053 0.52 0.376 -0.144
1.171 1.231 0.060 30 0.05 0.1 3.515 0.274 0.246 0.501 0.255 1.164
1.217 0.053 30 1.0 0.1 2.867 0.17 0.336 0.45 0.114 1.295 1.061
-0.234 30 0.05 0.6 3.422 0.225 0.216 0.461 0.245 1.05 1.126 0.076
30 1.0 0.6 2.82 0.179 0.258 0.446 0.188 1.156 1.069 -0.087 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 0.975 -0.019
0.525 0.404 -0.121 1.204 1.393 0.189 16 1.0 0.1 1.389 -0.034 0.492
0.366 -0.126 1.343 1.333 -0.010 16 0.05 0.6 1.666 0.054 0.512 0.402
-0.110 1.298 1.29 -0.008 16 1.0 0.6 1.573 0.024 0.486 0.374 -0.112
1.333 1.285 -0.048 30 0.05 0.1 3.654 0.216 0.212 0.482 0.270 1.115
1.348 0.233 30 1.0 0.1 3.098 0.104 0.3 0.475 0.175 1.222 1.297
0.075 30 0.05 0.6 3.191 0.245 0.236 0.445 0.209 1.244 1.262 0.018
30 1.0 0.6 2.82 0.145 0.322 0.462 0.140 1.185 1.326 0.141
[0301] TABLE-US-00044 TABLE 44 Cystatin Activity Results His fusion
tagged protein, first and second replicates GmCys7 (SEQ ID NO: 40)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.113 0
0.486 0.403 -0.083 1.134 1.185 0.051 16 1.0 0.1 1.159 -0.017 0.423
0.374 -0.049 1.051 1.178 0.127 16 0.05 0.6 2.266 0.12 0.387 0.397
0.010 1.075 1.144 0.069 16 1.0 0.6 2.404 0.094 0.289 0.367 0.078
1.858 1.2 -0.658 30 0.05 0.1 3.886 0.429 0.475 0.515 0.040 1.277
1.771 0.494 30 1.0 0.1 3.515 0.411 0.471 0.479 0.008 1.318 1.317
-0.001 30 0.05 0.6 3.747 0.405 0.478 0.456 -0.022 1.499 1.364
-0.135 30 1.0 0.6 4.21 0.386 0.443 0.479 0.036 1.475 1.615 0.140
Cystatin Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1
hour Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng
60 ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 0.975 0
0.571 0.473 -0.098 1.236 1.18 -0.056 16 1.0 0.1 1.159 -0.011 0.54
0.438 -0.102 1.259 1.11 -0.149 16 0.05 0.6 1.942 0.087 0.453 0.429
-0.024 1.035 1.078 0.043 16 1.0 0.6 1.942 0.067 0.345 0.446 0.101
1.033 1.178 0.145 30 0.05 0.1 3.283 0.322 0.641 0.539 -0.102 1.249
1.331 0.082 30 1.0 0.1 3.376 0.308 0.622 0.54 -0.082 1.345 1.453
0.108 30 0.05 0.6 3.329 0.285 0.579 0.552 -0.027 1.263 1.36 0.097
30 1.0 0.6 3.329 0.302 0.605 0.503 -0.102 1.551 1.294 -0.257
[0302] TABLE-US-00045 TABLE 45 Cystatin Activity Results GST fusion
tagged protein, first and second replicates GmCys9 (SEQ ID NO: 44)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.021 -0.016
0.591 0.434 -0.157 1.131 1.066 -0.065 16 1.0 0.1 1.067 -0.018 0.586
0.45 -0.136 1.069 1.173 0.104 16 0.05 0.6 3.237 0.056 0.541 0.454
-0.087 1.178 1.196 0.018 16 1.0 0.6 2.127 0.053 0.543 0.452 -0.091
1.111 1.256 0.145 30 0.05 0.1 3.144 0.187 0.066 0.501 0.435 0.194
1.236 1.042 30 1.0 0.1 3.191 -0.002 0.44 0.471 0.031 1.267 1.162
-0.105 30 0.05 0.6 3.607 0.153 0.09 0.554 0.464 0.371 1.364 0.993
30 1.0 0.6 2.728 0.094 0.217 0.481 0.264 1.007 1.203 0.196 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.343 -0.007
0.477 0.371 -0.106 1.416 1.062 -0.354 16 1.0 0.1 1.113 -0.013 0.444
0.362 -0.082 1.286 1.167 -0.119 16 0.05 0.6 1.989 0.033 0.501 0.405
-0.096 1.036 1.251 0.215 16 1.0 0.6 2.035 0.011 0.471 0.403 -0.068
1.222 1.511 0.289 30 0.05 0.1 3.747 0.238 0.063 0.455 0.392 0.145
1.119 0.974 30 1.0 0.1 5.094 0.134 0.123 0.458 0.335 0.725 1.233
0.508 30 0.05 0.6 3.376 0.151 0.131 0.496 0.365 0.73 1.314 0.584 30
1.0 0.6 3.005 0.085 0.236 0.456 0.220 1.048 1.32 0.272
[0303] TABLE-US-00046 TABLE 46 Cystatin Activity Results His fusion
tagged protein, first and second replicates GmCys9 (SEQ ID NO: 44)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.113 0.007
0.476 0.402 -0.074 1.148 1.388 0.240 16 1.0 0.1 1.113 0 0.295 0.393
0.098 1.246 1.421 0.175 16 0.05 0.6 2.45 0.206 0.056 0.405 0.349
0.137 1.084 0.947 16 1.0 0.6 2.312 0.201 0.059 0.404 0.345 0.114
1.286 1.172 30 0.05 0.1 5.047 0.417 0.497 0.469 -0.028 1.369 1.199
-0.170 30 1.0 0.1 3.839 0.392 0.558 0.474 -0.084 1.263 1.429 0.166
30 0.05 0.6 4.443 0.382 0.423 0.489 0.066 1.115 1.221 0.106 30 1.0
0.6 3.839 0.321 0.432 0.496 0.064 0.937 1.26 0.323 Cystatin Assay
10/22 Second Replicate Cell Temp IPTG Bradford 1 hour Overnight
Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA.
6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.297 0.014 0.613 0.495
-0.118 1.303 1.166 -0.137 16 1.0 0.1 1.067 0.012 0.381 0.513 0.132
1.081 1.422 0.341 16 0.05 0.6 1.942 0.236 0.08 0.477 0.397 0.33
1.115 0.785 16 1.0 0.6 2.081 0.259 0.054 0.474 0.420 0.114 1.228
1.114 30 0.05 0.1 3.561 0.397 0.292 0.53 0.238 1.077 1.269 0.192 30
1.0 0.1 3.376 0.304 0.48 0.501 0.021 1.195 1.305 0.110 30 0.05 0.6
3.144 0.257 0.364 0.511 0.147 1.372 1.222 -0.150 30 1.0 0.6 3.191
0.332 0.383 0.491 0.108 1.291 1.293 0.002
[0304] TABLE-US-00047 TABLE 47 Cystatin Activity Results GST fusion
tagged protein, first and second replicates TaCys8 (SEQ ID NO: 68)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.481 -0.007
0.608 0.392 -0.216 1.323 1.516 0.193 16 1.0 0.1 1.021 -0.007 0.587
0.428 -0.159 1.272 1.198 -0.074 16 0.05 0.6 2.173 0.076 0.546 0.417
-0.129 0.95 1.197 0.247 16 1.0 0.6 1.896 0.04 0.537 0.433 -0.104
1.338 1.188 -0.150 30 0.05 0.1 3.005 0.208 0.121 0.471 0.350 0.879
1.15 0.271 30 1.0 0.1 2.774 0.166 0.191 0.444 0.253 1.025 1.156
0.131 30 0.05 0.6 4.536 0.183 0.134 0.46 0.326 0.985 1.058 0.073 30
1.0 0.6 2.913 0.159 0.142 0.448 0.306 0.838 1.037 0.199 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.343 -0.002
0.45 0.34 -0.110 1.227 1.191 -0.036 16 1.0 0.1 1.62 0.002 0.398
0.357 -0.041 1.092 1.304 0.212 16 0.05 0.6 2.035 0.031 0.4 0.352
-0.048 1.118 1.364 0.246 16 1.0 0.6 2.081 0.024 0.336 0.358 0.022
1.091 1.19 0.099 30 0.05 0.1 3.515 0.272 0.095 0.406 0.311 0.753
1.103 0.350 30 1.0 0.1 2.774 0.164 0.116 0.392 0.276 0.916 1.159
0.243 30 0.05 0.6 3.468 0.191 0.117 0.433 0.316 0.818 1.184 0.366
30 1.0 0.6 2.728 0.189 0.111 0.416 0.305 0.76 1.099 0.339
[0305] TABLE-US-00048 TABLE 48 Cystatin Activity Results His fusion
tagged protein, first and second replicates TaCys8 (SEQ ID NO: 68)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.389 -0.006
0.484 0.387 -0.097 1.28 1.614 0.334 16 1.0 0.1 1.113 -0.008 0.417
0.354 -0.063 1.163 1.451 0.288 16 0.05 0.6 2.266 0.158 0.38 0.414
0.034 1.21 1.428 0.218 16 1.0 0.6 2.404 0.152 0.357 0.406 0.049
1.307 1.227 -0.080 30 0.05 0.1 3.839 0.517 0.093 0.438 0.345 0.796
1.514 0.718 30 1.0 0.1 3.839 0.53 0.097 0.416 0.319 0.78 1.245
0.465 30 0.05 0.6 4.814 0.462 0.099 0.455 0.356 0.678 1.101 0.423
30 1.0 0.6 3.839 0.415 0.092 0.422 0.330 0.676 0.99 0.314 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.205 0
0.636 0.498 -0.138 1.461 1.677 0.216 16 1.0 0.1 0.975 0 0.63 0.494
-0.136 1.463 1.354 -0.109 16 0.05 0.6 2.127 0.197 0.339 0.455 0.116
0.98 1.102 0.122 16 1.0 0.6 2.035 0.22 0.261 0.447 0.186 1.035
1.056 0.021 30 0.05 0.1 3.839 0.277 0.386 0.503 0.117 1.165 1.225
0.060 30 1.0 0.1 3.191 0.306 0.425 0.498 0.073 1.169 1.406 0.237 30
0.05 0.6 3.7 0.297 0.223 0.479 0.256 1.097 1.226 0.129 30 1.0 0.6
3.191 0.304 0.216 0.488 0.272 1.062 1.071 0.009
[0306] TABLE-US-00049 TABLE 49 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys8 (SEQ ID NO: 14)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.666 -0.009
0.606 0.425 -0.181 1.435 1.288 -0.147 16 1.0 0.1 0.882 -0.011 0.581
0.437 -0.144 1.283 1.369 0.086 16 0.05 0.6 1.758 0.047 0.526 0.351
-0.175 1.027 1.126 0.099 16 1.0 0.6 1.758 0.067 0.541 0.368 -0.173
1.168 1.156 -0.012 30 0.05 0.1 2.589 0.213 0.484 0.464 -0.020 0.914
1.219 0.305 30 1.0 0.1 2.635 0.174 0.508 0.444 -0.064 0.951 1.116
0.165 30 0.05 0.6 2.173 0.189 0.463 0.416 -0.047 1.069 0.968 -0.101
30 1.0 0.6 2.173 0.138 0.523 0.434 -0.089 1.349 1.404 0.055
Cystatin Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1
hour Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng
60 ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.159
-0.017 0.49 0.348 -0.142 1.326 1.308 -0.018 16 1.0 0.1 1.251 -0.013
0.495 0.37 -0.125 1.411 1.256 -0.155 16 0.05 0.6 1.297 0.052 0.512
0.364 -0.148 1.306 1.447 0.141 16 1.0 0.6 1.297 0.062 0.51 0.369
-0.141 1.365 1.387 0.022 30 0.05 0.1 2.682 0.173 0.469 0.426 -0.043
1.212 1.153 -0.059 30 1.0 0.1 2.543 0.149 0.544 0.447 -0.097 1.349
1.282 -0.067 30 0.05 0.6 2.543 0.165 0.481 0.426 -0.055 1.146 1.359
0.213 30 1.0 0.6 2.173 0.118 0.541 0.422 -0.119 1.57 1.603
0.033
[0307] TABLE-US-00050 TABLE 50 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys8 (SEQ ID NO: 14)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.527 -0.013
0.464 0.382 -0.082 1.168 1.229 0.061 16 1.0 0.1 1.343 -0.021 0.413
0.368 -0.045 1.028 1.08 0.052 16 0.05 0.6 2.543 0.099 0.487 0.368
-0.119 1.117 1.147 0.030 16 1.0 0.6 2.358 0.064 0.486 0.366 -0.120
1.311 1.355 0.044 30 0.05 0.1 4.025 0.403 0.557 0.464 -0.093 1.346
1.576 0.230 30 1.0 0.1 5.28 0.446 0.548 0.445 -0.103 1.337 1.448
0.111 30 0.05 0.6 4.071 0.353 0.517 0.466 -0.051 1.199 1.389 0.190
30 1.0 0.6 3.376 0.308 0.491 0.438 -0.053 1.062 1.665 0.603
Cystatin Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1
hour Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng
60 ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 0.975
-0.013 0.522 0.436 -0.086 1.283 1.163 -0.120 16 1.0 0.1 0.975
-0.013 0.53 0.421 -0.109 1.314 1.256 -0.058 16 0.05 0.6 1.942 0.161
0.527 0.427 -0.100 1.148 1.055 -0.093 16 1.0 0.6 1.804 0.095 0.505
0.417 -0.088 1.071 1.075 0.004 30 0.05 0.1 3.144 0.257 0.646 0.554
-0.092 1.176 1.318 0.142 30 1.0 0.1 3.515 0.332 0.675 0.509 -0.166
1.541 1.5 -0.041 30 0.05 0.6 2.774 0.214 0.661 0.505 -0.156 1.396
1.296 -0.100 30 1.0 0.6 2.543 0.234 0.678 0.505 -0.173 1.532 1.418
-0.114
[0308] TABLE-US-00051 TABLE 51 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys10 (SEQ ID NO: 18)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 2.127
-0.027 0.581 0.463 -0.118 1.184 1.334 0.150 16 1.0 0.1 1.481 -0.011
0.632 0.478 -0.154 1.324 1.385 0.061 16 0.05 0.6 2.127 0.056 0.612
0.457 -0.155 1.252 1.243 -0.009 16 1.0 0.6 2.219 0.047 0.668 0.464
-0.204 1.208 1.353 0.145 30 0.05 0.1 4.35 0.1 0.622 0.482 -0.140
1.158 1.187 0.029 30 1.0 0.1 4.443 0.106 0.629 0.483 -0.146 1.122
1.151 0.029 30 0.05 0.6 3.7 0.072 0.592 0.466 -0.126 1.23 1.146
-0.084 30 1.0 0.6 2.635 0.068 0.617 0.477 -0.140 1.29 1.268 -0.022
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.021
-0.018 0.446 0.341 -0.105 1.12 1.218 0.098 16 1.0 0.1 1.113 -0.009
0.473 0.352 -0.121 1.217 1.172 -0.045 16 0.05 0.6 2.266 0.009 0.459
0.334 -0.125 1.494 1.18 -0.314 16 1.0 0.6 2.127 0.038 0.486 0.358
-0.128 1.229 1.161 -0.068 30 0.05 0.1 2.728 0.074 0.547 0.412
-0.135 1.193 1.136 -0.057 30 1.0 0.1 2.959 0.083 0.512 0.403 -0.109
1.094 1.178 0.084 30 0.05 0.6 2.173 0.047 0.528 0.424 -0.104 1.122
1.315 0.193 30 1.0 0.6 2.358 0.045 0.564 0.425 -0.139 1.594 1.58
-0.014
[0309] TABLE-US-00052 TABLE 52 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys10 (SEQ ID NO: 18)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.62
0.002 0.481 0.357 -0.124 1.291 1.21 -0.081 16 1.0 0.1 1.113 0.011
0.468 0.336 -0.132 1.161 1.278 0.117 16 0.05 0.6 2.497 0.214 0.499
0.365 -0.134 1.192 1.136 -0.056 16 1.0 0.6 2.266 0.214 0.495 0.36
-0.135 1.175 1.211 0.036 30 0.05 0.1 4.722 0.446 0.56 0.415 -0.145
1.233 1.295 0.062 30 1.0 0.1 3.654 0.339 0.58 0.387 -0.193 1.2
1.276 0.076 30 0.05 0.6 5.001 0.339 0.543 0.418 -0.125 1.14 1.254
0.114 30 1.0 0.6 4.118 0.38 0.576 0.39 -0.186 1.291 1.29 -0.001
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.021
-0.008 0.491 0.356 -0.135 1.137 1.392 0.255 16 1.0 0.1 1.021 -0.004
0.434 0.353 -0.081 1.163 1.474 0.311 16 0.05 0.6 1.896 0.287 0.54
0.386 -0.154 1.48 1.423 -0.057 16 1.0 0.6 2.173 0.227 0.51 0.362
-0.148 1.242 1.362 0.120 30 0.05 0.1 3.468 0.31 0.597 0.472 -0.125
1.308 1.232 -0.076 30 1.0 0.1 3.329 0.325 0.559 0.469 -0.090 1.042
1.169 0.127 30 0.05 0.6 2.959 0.272 0.632 0.485 -0.147 1.597 1.161
-0.436 30 1.0 0.6 3.098 0.308 0.577 0.484 -0.093 1.119 1.107
-0.012
[0310] TABLE-US-00053 TABLE 53 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys11 (SEQ ID NO: 20)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.942
-0.016 0.598 0.443 -0.155 1.414 1.123 -0.291 16 1.0 0.1 2.081
-0.042 0.581 0.456 -0.125 1.247 1.244 -0.003 16 0.05 0.6 3.283
0.053 0.603 0.427 -0.176 1.136 1.193 0.057 16 1.0 0.6 2.589 0.091
0.621 0.45 -0.171 1.125 1.282 0.157 30 0.05 0.1 3.7 0.104 0.554
0.434 -0.120 1.192 1.171 -0.021 30 1.0 0.1 4.025 0.085 0.557 0.454
-0.103 1.001 1.202 0.201 30 0.05 0.6 3.098 0.074 0.485 0.446 -0.039
0.964 1.082 0.118 30 1.0 0.6 2.913 0.089 0.529 0.46 -0.069 1.013
1.1 0.087 Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05
0.1 1.067 -0.013 0.438 0.335 -0.103 1.093 1.296 0.203 16 1.0 0.1
1.067 0.011 0.455 0.338 -0.117 1.167 1.015 -0.152 16 0.05 0.6 2.404
0.082 0.461 0.331 -0.130 0.946 1.235 0.289 16 1.0 0.6 2.404 0.013
0.46 0.358 -0.102 1.106 1.1 -0.006 30 0.05 0.1 3.237 0.079 0.509
0.401 -0.108 1.149 1.099 -0.050 30 1.0 0.1 3.793 0.07 0.506 0.404
-0.102 1.082 1.01 -0.072 30 0.05 0.6 2.867 0.068 0.5 0.44 -0.060
1.264 1.201 -0.063 30 1.0 0.6 3.747 0.072 0.433 0.442 0.009 1.293
1.236 -0.057
[0311] TABLE-US-00054 TABLE 54 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys11 (SEQ ID NO: 20)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.712
0.024 0.467 0.372 -0.095 1.176 1.526 0.350 16 1.0 0.1 1.159 0.404
0.456 0.364 -0.092 1.088 1.235 0.147 16 0.05 0.6 2.682 0.265 0.492
0.431 -0.061 1.122 1.418 0.296 16 1.0 0.6 2.312 0.319 0.479 0.454
-0.025 1.04 1.543 0.503 30 0.05 0.1 4.768 0.433 0.514 0.439 -0.075
1.308 1.335 0.027 30 1.0 0.1 3.468 0.356 0.527 0.435 -0.092 1.453
1.511 0.058 30 0.05 0.6 3.839 0.339 0.525 0.461 -0.064 1.228 1.229
0.001 30 1.0 0.6 3.283 0.272 0.523 0.454 -0.069 1.337 1.172 -0.165
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.021
0.032 0.518 0.48 -0.038 1.326 1.467 0.141 16 1.0 0.1 1.067 0.026
0.64 0.491 -0.149 1.298 1.255 -0.043 16 0.05 0.6 1.989 0.344 0.519
0.431 -0.088 1.041 1.007 -0.034 16 1.0 0.6 1.989 0.336 0.656 0.507
-0.149 1.222 1.288 0.066 30 0.05 0.1 3.237 0.314 0.599 0.493 -0.106
1.147 1.117 -0.030 30 1.0 0.1 2.913 0.318 0.646 0.501 -0.145 1.453
1.682 0.229 30 0.05 0.6 3.237 0.275 0.599 0.49 -0.109 1.262 1.523
0.261 30 1.0 0.6 3.561 0.269 0.621 0.483 -0.138 1.33 1.337
0.007
[0312] TABLE-US-00055 TABLE 55 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys12 (SEQ ID NO: 22)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 0.882
-0.02 0.552 0.428 -0.124 1.307 1.093 -0.214 16 1.0 0.1 1.021 -0.007
0.561 0.434 -0.127 1.609 1.369 -0.240 16 0.05 0.6 1.573 0.04 0.505
0.359 -0.146 1.18 1.41 0.230 16 1.0 0.6 1.942 0.071 0.491 0.355
-0.136 1.171 1.108 -0.063 30 0.05 0.1 4.814 0.198 0.165 0.455 0.290
0.881 1.121 0.240 30 1.0 0.1 3.793 0.136 0.435 0.458 0.023 1.069
1.252 0.183 30 0.05 0.6 2.173 0.234 0.257 0.415 0.158 0.994 1.083
0.089 30 1.0 0.6 3.283 0.213 0.211 0.405 0.194 0.934 1.406 0.472
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.067
-0.013 0.511 0.375 -0.136 1.372 1.221 -0.151 16 1.0 0.1 0.882
-0.015 0.491 0.347 -0.144 1.234 1.209 -0.025 16 0.05 0.6 1.527
0.041 0.477 0.367 -0.110 1.309 1.418 0.109 16 1.0 0.6 2.404 0.037
0.468 0.348 -0.120 1.246 1.229 -0.017 30 0.05 0.1 2.635 0.165 0.219
0.436 0.217 1.26 1.424 0.164 30 1.0 0.1 3.144 0.132 0.417 0.462
0.045 1.773 1.297 -0.476 30 0.05 0.6 3.654 0.12 0.322 0.414 0.092
1.559 1.443 -0.116 30 1.0 0.6 3.607 0.106 0.389 0.437 0.048 1.27
1.249 -0.021
[0313] TABLE-US-00056 TABLE 56 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys12 (SEQ ID NO: 22)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.297
-0.025 0.508 0.364 -0.144 1.289 1.036 -0.253 16 1.0 0.1 1.067
-0.015 0.538 0.375 -0.163 1.589 1.006 -0.583 16 0.05 0.6 2.035
0.186 0.49 0.365 -0.125 1.376 1.348 -0.028 16 1.0 0.6 2.219 0.154
0.47 0.377 -0.093 1.276 1.171 -0.105 30 0.05 0.1 3.932 0.425 0.423
0.47 0.047 1.345 1.489 0.144 30 1.0 0.1 3.561 0.411 0.491 0.432
-0.059 1.141 1.016 -0.125 30 0.05 0.6 3.839 0.513 0.321 0.458 0.137
1.676 1.602 -0.074 30 1.0 0.6 3.747 0.38 0.396 0.456 0.060 1.118
1.771 0.653 Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05
0.1 0.882 0.01 0.533 0.418 -0.115 1.266 1.19 -0.076 16 1.0 0.1
1.021 0.01 0.553 0.435 -0.118 1.328 1.209 -0.119 16 0.05 0.6 1.666
0.324 0.33 0.442 0.112 1.202 1.172 -0.030 16 1.0 0.6 1.85 0.273
0.344 0.438 0.094 1.1 1.188 0.088 30 0.05 0.1 2.497 0.299 0.136
0.493 0.357 1.092 1.511 0.419 30 1.0 0.1 2.728 0.359 0.263 0.482
0.219 1.251 1.224 -0.027 30 0.05 0.6 3.376 0.365 0.2 0.518 0.318
1.117 1.177 0.060 30 1.0 0.6 2.589 0.336 0.15 0.453 0.303 0.962 1.2
0.238
[0314] TABLE-US-00057 TABLE 57 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys13 (SEQ ID NO: 24)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 2.82 0
0.701 0.5 -0.201 1.364 1.272 -0.092 16 1.0 0.1 1.62 -0.011 0.66
0.512 -0.148 1.244 1.55 0.306 16 0.05 0.6 3.191 0.071 0.672 0.478
-0.194 1.303 1.18 -0.123 16 1.0 0.6 3.005 0.047 0.654 0.489 -0.165
1.286 1.261 -0.025 30 0.05 0.1 4.025 0.387 0.056 0.473 0.417 0.135
1.148 1.013 30 1.0 0.1 2.82 0.257 0.055 0.486 0.431 0.158 1.344
1.186 30 0.05 0.6 4.35 0.347 0.053 0.472 0.419 0.126 1.727 1.601 30
1.0 0.6 2.45 0.291 0.051 0.432 0.381 0.133 0.99 0.857 Cystatin
Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.527 0.011
0.473 0.36 -0.113 1.096 1.082 -0.014 16 1.0 0.1 1.343 0.015 0.622
0.47 -0.152 1.004 1.124 0.120 16 0.05 0.6 2.219 0.089 0.478 0.365
-0.113 1.07 1.225 0.155 16 1.0 0.6 2.312 0.091 0.641 0.492 -0.149
1.158 1.236 0.078 30 0.05 0.1 3.283 0.357 0.053 0.371 0.318 0.132
0.989 0.857 30 1.0 0.1 3.932 0.249 0.056 0.406 0.350 0.142 1.265
1.123 30 0.05 0.6 2.82 0.349 0.05 0.397 0.347 0.113 1.207 1.094 30
1.0 0.6 4.443 0.323 0.054 0.42 0.366 0.121 1.324 1.203
[0315] TABLE-US-00058 TABLE 58 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys13 (SEQ ID NO: 24)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.343
0.019 0.417 0.386 -0.031 1.141 1.314 0.173 16 1.0 0.1 1.804 0.022
0.429 0.396 -0.033 1.205 1.318 0.113 16 0.05 0.6 2.82 0.276 0.066
0.409 0.343 0.232 1.148 0.916 16 1.0 0.6 3.005 0.231 0.068 0.4
0.332 0.239 1.155 0.916 30 0.05 0.1 5.14 0.411 0.459 0.412 -0.047
1.052 1.256 0.204 30 1.0 0.1 3.561 0.376 0.463 0.44 -0.023 1.205
1.383 0.178 30 0.05 0.6 5.233 0.362 0.129 0.448 0.319 0.917 1.397
0.480 30 1.0 0.6 3.422 0.358 0.132 0.433 0.301 0.895 1.308 0.413
Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1 1.666
0.004 0.504 0.448 -0.056 1.094 1.401 0.307 16 1.0 0.1 1.205 -0.013
0.424 0.385 -0.039 1.182 1.215 0.033 16 0.05 0.6 2.543 0.291 0.064
0.475 0.411 0.241 1.301 1.060 16 1.0 0.6 2.404 0.178 0.059 0.409
0.350 0.26 1.592 1.332 30 0.05 0.1 3.376 0.368 0.446 0.45 0.004
1.052 1.036 -0.016 30 1.0 0.1 4.396 0.394 0.457 0.415 -0.042 0.915
0.932 0.017 30 0.05 0.6 4.164 0.364 0.174 0.474 0.300 0.8 1.355
0.555 30 1.0 0.6 4.396 0.315 0.181 0.464 0.283 1.06 1.272 0.212
[0316] TABLE-US-00059 TABLE 59 Cystatin Activity Results GST fusion
tagged protein, first and second replicates ZmCys14 (SEQ ID NO: 26)
Cell Temp IPTG Bradford 1 hour Overnight Type .degree. C. (mM)
OD600 Final OD (ug/uL) 6000 ng 60 ng .DELTA. 6000 ng 60 ng .DELTA.
Cystatin Assay 10/22 First Replicate BL21 Star 16 0.05 0.1 1.113
-0.018 0.604 0.477 -0.127 1.298 1.255 -0.043 16 1.0 0.1 1.343
-0.016 0.643 0.493 -0.150 1.39 1.257 -0.133 16 0.05 0.6 2.45 0.056
0.476 0.456 -0.020 1.185 1.314 0.129 16 1.0 0.6 3.052 0.051 0.488
0.436 -0.052 1.025 1.012 -0.013 30 0.05 0.1 3.654 0.245 0.082 0.459
0.377 0.836 1.257 0.421 30 1.0 0.1 3.283 0.204 0.08 0.484 0.404
0.813 1.27 0.457 30 0.05 0.6 5.326 0.264 0.062 0.436 0.374 0.592
1.122 0.530 30 1.0 0.6 3.747 0.251 0.064 0.416 0.352 0.538 0.923
0.385 Cystatin Assay 10/22 Second Replicate BL21 Star 16 0.05 0.1
1.989 0.002 0.426 0.348 -0.078 1.043 1.017 -0.026 16 1.0 0.1 1.113
-0.002 0.587 0.46 -0.127 1.215 1.019 -0.196 16 0.05 0.6 1.942 0.053
0.377 0.354 -0.023 1.092 0.95 -0.142 16 1.0 0.6 2.913 0.022 0.526
0.488 -0.038 0.943 1.159 0.216 30 0.05 0.1 3.144 0.289 0.061 0.374
0.313 0.532 0.949 0.417 30 1.0 0.1 3.329 0.221 0.07 0.414 0.344
0.647 1.23 0.583 30 0.05 0.6 4.768 0.274 0.059 0.43 0.371 0.512
1.246 0.734 30 1.0 0.6 2.959 0.221 0.085 0.455 0.370 0.716 1.224
0.508
[0317] TABLE-US-00060 TABLE 60 Cystatin Activity Results His fusion
tagged protein, first and second replicates ZmCys14 (SEQ ID NO: 26)
Cystatin Assay 10/22 First Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (Mm) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.251 0.032
0.372 0.408 0.036 1.206 1.292 0.086 16 1.0 0.1 1.251 0.017 0.41
0.396 -0.014 1.249 1.293 0.044 16 0.05 0.6 2.497 0.265 0.13 0.539
0.409 0.764 1.31 0.546 16 1.0 0.6 3.191 0.235 0.093 0.477 0.384
0.705 1.498 0.793 30 0.05 0.1 3.515 0.481 0.065 0.453 0.388 0.602
1.365 0.763 30 1.0 0.1 3.932 0.481 0.078 0.465 0.387 0.672 1.29
0.618 30 0.05 0.6 5.047 0.394 0.069 0.454 0.385 0.504 1.465 0.961
30 1.0 0.6 5.513 0.472 0.062 0.449 0.387 0.517 1.373 0.856 Cystatin
Assay 10/22 Second Replicate Cell Temp IPTG Bradford 1 hour
Overnight Type .degree. C. (mM) OD600 Final OD (ug/uL) 6000 ng 60
ng .DELTA. 6000 ng 60 ng .DELTA. BL21 Star 16 0.05 0.1 1.021 0.022
0.518 0.541 0.023 1.22 1.407 0.187 16 1.0 0.1 1.021 0.02 0.489
0.589 0.100 1.147 1.308 0.161 16 0.05 0.6 2.035 0.306 0.092 0.499
0.407 0.785 1.188 0.403 16 1.0 0.6 2.081 0.273 0.081 0.475 0.394
0.636 1.037 0.401 30 0.05 0.1 3.607 0.324 0.17 0.489 0.319 0.881
1.484 0.603 30 1.0 0.1 3.561 0.359 0.181 0.496 0.315 0.941 1.592
0.651 30 0.05 0.6 3.515 0.365 0.065 0.462 0.397 0.566 1.169 0.603
30 1.0 0.6 3.237 0.355 0.076 0.485 0.409 0.64 1.223 0.583
EXAMPLE 9
Microinjection Assay for Anti-Nematodal Activity of Cystatins
[0318] Two of the cystatin genes of the present invention, ZmCys4
(SEQ ID NO: 5) and GmCys2 (SEQ ID NO: 29), were expressed in, and
their encoded proteins purified from E. coli as set forth in
Example 7. A control expression vector was also prepared which
contained no cystatin gene. The purified cystatins and control were
injected into sugar beet nematode-induced syncytia in Arabidopsis
roots one week after inoculation, as per the methods outlined in
Bockenhoff and Grundler ((1994), Parasitology. 109: 249-255),
hereby incorporated by reference. Fluorescent dye was used to
monitor the growth of the nematodes following injection, as per
Bockenhoff and Grundler (1994). The anti-nematodal activity of the
cystatins was measured by comparing the nematode growth and
development 10 days following injection, and comparing it with the
control. Results of the experiment are presented in Table 61,
below. TABLE-US-00061 TABLE 61 Effect of Two Cystatins on Nematode
Development Protein Injected Lethality (%) Control 30 Zm-Cys4 (SEQ
ID NO: 6) 50 Gm-Cys2 (SEQ ID NO: 30) 58
[0319] These results show that both Zm-Cys4 and Gm-Cys2 had
significant inhibitory effects on the growth and development of
sugar beet nematode juveniles in Arabidopsis. Sugar beet nematode
is a genetically close relative of soybean cyst nematode. It has
been reported that there is a high cysteine proteinase activity in
SCN intestines (Lilley, C. J. et al. (1996) 113: 415-424). These
data indicate that both Zm-Cys4 and Gm-Cys2 confer resistance to
nematodes by inhibiting SCN growth and development in roots.
EXAMPLE 10
Use of C. elegans as a Model to Analyze Cystatin Anti-Nematodal
Activity
[0320] C. elegans populations are cultured on NGM agar carrying a
lawn of E. coli OP50 cells as described by Wood ((1988) The
nematode C. elegans, Cold Spring Harbor, N.Y. Cold Spring Harbor
Laboratory Press). After populations are maintained for five days,
agar plugs are removed to fresh plates. Cystatins are added to the
medium to a final concentration of 2.5 mg/L just prior to pouring.
In order to study the effect of the cystatins on egg laying,
hermaphrodite nematodes are taken from their normal growth media
and transferred individually to fresh plates containing the
cystatin(s) to be used for testing. Egg laying is carefully
monitored.
[0321] Half of the eggs laid on each plate are removed to fresh
plates containing media not supplemented with the cystatin(s) being
tested. Development of the hatched larvae is monitored.
[0322] Alternately, groups of larvae hatched on normal media can be
transferred to plates containing the cystatin(s) to be tested at
time points corresponding to larval stages L1, L2, L3 and L4. The
larvae for each stage should be removed, respectively, 6, 12, 24,
and 30 hours post hatching. Development of the various larval
stages on the supplemented media is monitored.
[0323] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications, patents and
patent applications are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
[0324] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. TABLE-US-00062 TABLE 62 Multiple Alignment of All Cystatin
Sequences Plurality: 2.00 Threshold: 4 AveWeight 1.00 AveMatch 2.78
AvMisMatch -2.25 Symbol comparison table: blosum62.cmp CompCheck:
1102 GapWeight: 8 GapLengthWeight: 2 Pileup MSF: 314 Type: P May
13, 2003 15:11 Check: 6646 . . . // 1 50 gm-cys6
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out.
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.
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(SEQ ID NO:38) gm-cys8
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out.
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(SEQ ID NO:42) ta-cys8
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out.
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.
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.about..about..about..about..about..about..about.MAR VIGASGACAL
(SEQ ID NO:68) ta-cys9
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about.MAR LVGAAGACAL
(SEQ ID NO:70) ZmCys14
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about.MAR ...ALGACVL
(SEQ ID NO:26) os-cys5
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about.M
(SEQ ID NO:54) ZmCys3
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about.MRK (SEQ ID NO:4)
ZmCys4
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about.MRK (SEQ ID NO:6)
ta-cys13
.about..about..about..about..about..about..about..about..about..a-
bout.
.about..about..about..about..about..about..about..about..about..abou-
t.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:76) ZmCys1
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about.MRK (SEQ ID NO:2)
os-cys1
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:46) ta-cys1
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about.MEMWKYRVV (SEQ ID NO:58) ta-cys2
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:60) ta-cys4
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:64) ta-cys6
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:66) os-cys3
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:50) ZmCys8
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:14) ZmCys12
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
MRVAAT...R (SEQ ID NO:22) ZmCys5
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
MRVAAT...R (SEQ ID NO:8) os-cys2
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
MRVAATTRPA (SEQ ID NO:48) ta-cys10
.about..about..about..about..about..about..about..about..about..a-
bout.
.about..about..about..about..about..about..about..about..about..abou-
t.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
MRVAATRPAS (SEQ ID NO:72) gm-cys2
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out.
.about..about..about..about..about..about..about..about..about..about-
.
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.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:30) gm-cys7
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:40) gm-cys1
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
MRALTSSSST (SEQ ID NO:28) gm-cys3
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:32) gm-cys4
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:34) ZmCys10
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:18) ZmCys6
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:10) os-cys4
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about.M
LRRRGFCCCS (SEQ ID NO:52) ta-cys11
.about..about..about..about..about..about..about..about..about..a-
bout.
.about..about..about..about..about..about..about..about..about..abou-
t.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about.MVRRCGCS (SEQ ID NO:74) gm-cys5
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:36) gm-cys9
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:44) ZmCys7
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:12) os-cys6
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:56) ZmCys9
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
(SEQ ID NO:16) ZmCys13
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about.M
(SEQ ID NO:24) ta-cys3
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about.M
(SEQ ID NO:62) ZmCys11 MAFLSTNALM SVPITAAAAP RHRRSLVVVR AAAVKSNEHL
QEEQASVADG (SEQ ID NO:20) 51 100 gm-cys6
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about.MVGG KTEVP.DVRT gm-cys8
.about..about..about..about..about..about..about..about..about..ab-
out. .about..about..about..about.MAVALT ILVTLLSVLS SASCARMVGG
KTEIP.EVRK ta-cys8 LVVLLVACA. ASAARTE... .PGAA.RQLW ED..GRKVGG
RTEVR.DVES ta-cys9 LVILLMACA. ASAARSE... .PGAA.RQLW DD..GRKVGG
RTEVT.DVEG ZmCys14 LAVLLGALAP AAAARAHDDQ GSGAGIRQPS GEYRGRKVGA
RTEVR.DVEG os-cys5 ATSPMLFLVS LLLVLVAAAT GDEASPSNAA APAAPVLVGG
RTEIR.DVGS ZmCys3 HRIVSLVAAL LILLAL.AVS STRNAQEDSM ADNTGTLAGG
IKDVP.GNEN ZmCys4 HRIVSLVAAL LILLAL.AVS STRNAQEDSM ADNTGTLAGG
IKDVP.GNEN ta-cys13 .about..about..about..about.SLVAAL LILLAL.AVS
STRNAQEDSM ADNTGTLAGG IKDVP.GNEN ZmCys1 HRIVSLVAAL LVLLALAAVS
STRSAQKESV ADNAGMLAGG IKDVP.ANEN os-cys1
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
. .about..about..about..about..about..about..about..about..about.M
SSDGGPVLGG VEPV..GNEN ta-cys1 GSVAALLLLL AIVVPFTQTQ TQSARDKAAM
AEDAGPLVGG ISDSPMGQEN ta-cys2
.about..about..about..about..about..about..about..about.LL
AIVVPFTQTR TQSARDKAAM AEDAGPLVGG IKDSPMGQEN ta-cys4
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
. .about.MAEAAQGGG LRGRGALLGG VQDAPAGREN ta-cys6
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
. .about.MAEAAQGGG LRGRGVLLGG VQDAPAGREN os-cys3
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
. .about.MAEEAQ... .QPRGVKVGG IHDAPAGREN ZmCys8
.about..about..about..about..about..about..about..about..about..abo-
ut.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about.MAEVHN ERPVG.MVGD VRDAPVGREN ZmCys12
AAAAAHPPSA FLLLLLLLGC ASL.AIGGA. .AMAGHVLGG VKENP.AAAN ZmCys5
AAAAAHPPSA FLLLLLLLGC ASL.AIGGA. .AMAGHVLGG VKENP.AAAN os-cys2
SSSAAAPLPL FLLLAVAAAA AALFLVGSAS LAMAGHVLGG AHDAP.SAAN ta-cys10
SAPVA..LLA ALALLFLVGS ASL.AIG... .AMASHVLGG KSENP.DAAN gm-cys2
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about.MAALGG NRDVT.GSQN gm-cys7
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about.MAALGG NRDVA.GSQN gm-cys1 FIPKRYSFFF
FLSILFALRS SSGGCSEYHH HHAPMATIGG LRDSQ.GSQN gm-cys3
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about.MAALGG FTDIT.GAQN gm-cys4
.about..about..about..about..about..about..about..about..about..ab-
out.
.about..about..about..about..about..about..about..about..about..about-
.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about..about..about..about..about..about..about..about.
.about..about..about.MP.TLGA ZmCys10 .about..about..about.MMPRRAL
LFAAVLLAAS A.AAVSGFHL GGDESGLVRG VL.AALRER. ZmCys6
.about..about.MTMPRRAL LFAAVLLAAS A.AAVSGFHL AGDESGLVRG VL.TAVRERA
os-cys4 GAPAAAAAAL LLLAV..AAA A.PRAAGFHL GGDESVLVRG ML.AAIR.RE
ta-cys11 GAMLLA.... LSLAVLLAAS AVPGAAGFHL GGDESGLVRG ML.AAVRER.
gm-cys5
.about..about..about..about..about..about..about..about..about..ab-
out. .about..about.MRHHCL.L LVSLVLVSYA AR.SESALGG WS..PIKDVN
gm-cys9
.about..about..about..about..about..about..about..about..about..ab-
out. .about..about.MKQKCLVV LVFVVLLACA VGWDEGTPGG WN..PIKNIN ZmCys7
.about..about..about..about..about..about..about..about.MS
ARALLLTTAT LLLLVAAAR. ..AGQPLAGG WS..PIRNVS os-cys6
.about..about..about.MARIPLL LALLLAVSAA AAAQVGGNR. ..GHGPLVGG
WS..PITDVG ZmCys9 .about..about..about.MATHRHC LPLLLLVAAA
LAAVPARAAL GGGRGPLLGG WN..PIPDVS ZmCys13 AMTMTLGSML IAAAAVVGLC
SVAPAASARE EPLQPQIVGG WK..PIKNVN ta-cys3 RTSSFLLIIV VAFLYAIGSP
AIGCGERMGN QLWNTAIENG WE..PIGNIN ZmCys11 ARGRRRAMVL LAATAAVTGS
SVAICRSARA AGV.TTLSGQ YV..KIENVK 101 150 gm-cys6 NREVQELGRF
AVEEYNRGLK Q.WKN....N GSEQLNFSEV VEAQQQVVSG gm-cys8 NRQVQELGRF
AVEEYNLGLK L.LKNNNVDN GREQLNFSAV VEAQQQVVSG ta-cys8 DREVQELGRY
SVEEHNRRRE EGCEGGGGVC GR..LEFARV VSAQRQVVSG ta-cys9 DREVQELGRY
SVEEHNRRRE EGCEGGGGVC GR..LEFARV VSAQRQVVSG ZmCys14 DGEVQELGRF
SVAEYNRQLR EG..GGGG.. GR..LEFGRV VAAQRQVVSG os-cys5 NKAVQSLGRF
AVAEHNRRLR HGGSGGPADP VPVKLAFARV VEAQKQVVSD ZmCys3 DLHLQELARF
AVDEHN...K KA........ .NALLGFEKL VKAKTQVVAG ZmCys4 DLHLQELARF
AVDEHN...K KA........ .NALLGFEKL VKAKTQVVAG ta-cys13 DLHLQELARF
AVDEHN...K KA........ .NALLGFEKL VKAKTQVVAG ZmCys1 DLQLQELARF
AVNEHN...Q KA........ .NALLGFEKL VKAKTQVVAG os-cys1 DLHLVDLARF
AVTEHN...K KA........ .NSLLEFEKL VSVKQQVVAG ta-cys1 DLDVIALARF
AVSEHN.. N KA........ .NALLEFENV VKVKKQTVAG ta-cys2 DLDVIALARF
AVSEHN.. N KA........ .NALLEFENV VKLKKQTVAG ta-cys4 DLETIELARF
AVAEHN...I KA........ .NALLEFERL VKVRQQVVAG ta-cys6 DLATIELARF
AVAEHN...I KA........ .NALLEFERL VKVRQQVVAG os-cys3 DLTTVELARF
AVAEHN...S KA........ .NAMLELERV VKVRQQVVGG ZmCys8 DLEAIELARF
AVAEHN...S KT........ .NAMLEFERL VKVRHQVVAG ZmCys12 SAESDGLGRF
AVDEHN...R RE........ .NALLEFVRV VEAKEQVVAG ZmCys5 SAESDGLGRF
AVDEHN...R RE........ .NALLEFVRV VEAKEQVVAG os-cys2 SVETDALARF
AVDEHN...K RE........ .NALLEFVRV VEAKEQVVAG ta-cys10 SLETDGLARF
AVDEHN...K RE........ .NALLEFVRV VEAKEQTVAG gm-cys2 SVEIDALARF
AVEEHN...K KQ........ .NALLEFEKV VIAKQQVVSG gm-cys7 SLEIDGLARF
AVEEHN...K KQ........ .NALLEFEKV VSAKQQVVSG gm-cys1 SVQTEALARF
AVDEHN...K KQ........ .NSLLEFSRV VRTQEQVVAG gm-cys3 SIDIENLARF
AVDEHN...K KE........ .NAVLEFVRV ISAKKQVVSG gm-cys4 GGEIDHLARF
AVEEQN...K RE........ .NANLEFVGV IRAKQQVVEG ZmCys10 .AEAEDAARF
AVAHYN...K NQ........ .GAALEFTRV LKSKRQVVTG ZmCys6 EAEAEDAARF
AVAYHN...R NQ........ .GAALEFTRV LKSKRQVVTG os-cys4 QAEAEDAARF
AVAEYN...K NQ........ .GAELEFARI VKAKRQVVTG ta-cys11 .AXAXDAARF
XVAEHN...R XQ........ .GSALEFTRV VNAKXQVVAG gm-cys5 DSHVAEIANY
ALSEYD...K RS........ .GAKLTLVKV VKGETQVVSG gm-cys9 DPHVTEIANF
AVTEYD...K QS........ .GEKLKLVKV TKGDLQVVAG ZmCys7 DPHIQELGGW
AVTEHV...R RA........ .NDGLRFGEV TGGEEQVVSG os-cys6 DPHIQELGGW
AVERHA...S LS........ .SDGLRFRRV TSGEQQVVSG ZmCys9 DSHIQELGGW
ALGQA.KHQK LA........ .ADGLRFRRV VRGEQQVVSG ZmCys13 DPHVQEIGRW
AVSEHI...K TA........ .NDGLGFGRV VSGEEQIVAG ta-cys3 DQHIQELGRW
AVLEFGKHVN CV........ ....LKFNKV VSGRQQLVSG ZmCys11 DPYVQGVGEW
AVKEHN...R QT........ .GESLQFAEV VSGMEQVVAG 151 200 gm-cys6
MKYYLKISAT HKG....... .VHKMFTSVV VVKPWLHS.. KQLLHFAPAA gm-cys8
MKYYLKISAT HNG....... .VHEMFNSVV VVKPWLHS.. KQLLHFAPAS ta-cys8
IKYYLRVAAA EENGAGSNVV SDGRVFDAVV VVKPWLQS.. RALVRFAPAD ta-cys9
IKYYLRVAAA EEGGAGSNGV TDGRVFDAVV VVKPWLQS.. RALIRFAPAD ZmCys14
LKYYLRVVAV EEGGAGNGG. ..ERVFDAVV VVKPWLDS.. RTLLTFAPAA os-cys5
VAYYLKVAAS ARDPRGGAAA GGDRVFDAVV VVKAWLKS.. KELVSFTPAS ZmCys3
TMYYLTIEVK D..GEVK... ...KLYEAKV WEKPWEN..F KELQEFKPVE ZmCys4
TMYYLTIEVK D..GEVK... ...KLYEAKV WEKPWEN..F KELQEFKPVE ta-cys13
TMYYLTIEVK D..GEVK... ...KLYEAKV WEKPWEN..F KELQEFKPVE ZmCys1
TMYYLTIEVK D..GEVN... ...KLYEAKV WEKPWEN..F KQLQEFKPVE os-cys1
TLYYFTIEVK E..GDAK... ...KLYEAKV WEKPWMD..F KELQEFKPVD ta-cys1
TMHYITIRVT E..GGAK... ...KLYEAKV WEKPWEN..F KKLEEFKLVE ta-cys2
TMHYITIRVT E..GGAK... ...KLYEAKV WEKPWEN..F KQLQEFKPVE ta-cys4
CMHYFTIEVK E.GGA.K... ...KLYEAKV WEKAWEN..F KQLQDFKPAA ta-cys6
CMHYFTIEVK E.GGA.K... ...KLYEAKV WEKAWEN..F KQLQDFKPAA os-cys3
FMHYLTVEVK EPGGA.N... ...KLYEAKV WERAWEN..F KQLQDFKPLD ZmCys8
TLHHFTVEVK EAGGGEK... ...KLYEAKV WEKAWEN..F KQLQSFELVG ZmCys12
TLHHLTLEAV F..AGRK... ...KLYEAKV WVKPWLD..F KELQEFSHKG ZmCys5
TLHHLTLEAV F..AGRK... ...KLYEAKV WVKPWLD..F KELQEFSHKG os-cys2
TLHHLTLEAL F..AGRK... ...KVYEAKV WVKPWLD..F KELQEFRNTG ta-cys10
TVHHLTLEAL F..AGRK... ...KLYEAKV WVKPWLD..F KELQEFRHTG gm-cys2
TLYTITLEAK D..GGQK... ...KVYEAKV WEKSWLN..F KEVQEFKLVG gm-cys7
TLYTITLEAK D..GGQK... ...KVYEAKV WEKAWLN..F KEVQEFKLVG gm-cys1
TLHHLTLEAI F..AGEK... ...KLYEAKV WVKPWLN..F KELQEFKPAG gm-cys3
TLYYITLEAN D..GVTK... ...KVYETKV LEKPWLN..I KEVQEFKPIT gm-cys4
FIYYITLEAK D..GETK... ...NVYETKV WVRSWLN..S KEVLEFKPIS ZmCys10
TLHDLILEAA D..AGKK... ...SVYRAKV WVKSWED..F KSVVEFRLVG ZmCys6
TLHDLILEAA D..AGKK... ...SLYRAKV WVKPWED..F KSVVEFRLAG os-cys4
TLHDLMLEVV D..SGKK... ...SLYSAKV WVKPWLD..F KAVVEFRHVG ta-cys11
TLHDLMVEVV D..SGXK... ....ICTTQS LGEAWQN..F XAVVEFRHAG gm-cys5
TNYRLVLKAK D.GSATA... ...S.YEAIV WEKPWL..HF MNLTSFKPLH gm-cys9
LNYRLSLTAS D.SN...... ...N.YQAIV YEKAWAREHY RNLTSFTPLH ZmCys7
MNYKLVLDAT DADGKVA... ...A.YGAFV YEQSWTNT.. RELVSFAPAS os-cys6
MNYRLVVSAS DPAGATA... ...S.YVAVV YEQSWTNT.. RQLTSFKPAA ZmCys9
MNYRLYVDAA DPAGRTV... ...P.YVAVV YEQVWTAP.. ...ASSPPST ZmCys13
KNYRLRIQAT KVGGQKA... ...M.YRAVV YEQL.TNT.. RQLLSFDPAN ta-cys3
MNYELIIEAS DIGGKED... ...K.YKAEV YEQTWTHK.. RQLLSFAKVK ZmCys11
TNYKLNLATK DP...TS... ...S.YQAVV FDPLPNSSKN RQLMSFKSI.about. 201
250 gm-cys6 PSSKDF.about..about..about..about.
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gm-cys8 SSTTTTNNNM HPIVRKDN.about..about.
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ta-cys8 AK.about..about..about..about..about..about..about..about.
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ta-cys9 AK.about..about..about..about..about..about..about..about.
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ZmCys14 AK.about..about..about..about..about..about..about..about.
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os-cys5 STK.about..about..about..about..about..about..about.
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ZmCys3 EGASA.about..about..about..about..about.
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ZmCys4 EGASA.about..about..about..about..about.
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ta-cys13 EGASA.about..about..about..about..about.
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ZmCys1 EGASA.about..about..about..about..about.
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os-cys1 ASANA.about..about..about..about..about.
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ta-cys1 DVPSA.about..about..about..about..about.
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ta-cys2 DAAIA.about..about..about..about..about.
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ta-cys4
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out.
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.
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ta-cys6
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out.
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.
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os-cys3 DATA.about..about..about..about..about..about.
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ZmCys8 DAAVA.about..about..about..about..about.
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ZmCys12 DATAFTNADL GAKQGGHEPG WREVPVEDPV VKDAAHHAVK SIQERSNSLF
ZmCys5 DATAFTNADL GAKQGGHEPG WREVPVEDPV VKDAAHHAVK SIQERSNSLF
os-cys2 DATTFTNADL GAKKGGHEPG WRDVPVHDPV VKDAADHAVK SIQQRSNSLF
ta-cys10
D.about..about..about..about..about..about..about..about..about.
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gm-cys2 DAPA.about..about..about..about..about..about.
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gm-cys7 DAPA.about..about..about..about..about..about.
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gm-cys1 DVPSFTSADL GVKKDGHQPG WQSVPTHDPQ VQDAANHAIK TIQQRSNSLV
gm-cys3 VAVNPLSVTV
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gm-cys4 INPLSVSV.about..about.
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ZmCys10 DSESEPEPSV ASDVSSGQAI AKLSLEADIV QEEARLHTIE NDGLSGDFTS
ZmCys6 DSESEPEPSV ASDEGSGQGV AKLSLEADII HEEAHLHTIE NDGLSSDFAS
os-cys4 DSQS..QSAT AADDNAGQDT AD.....PTV ASRNDLHNTE NNKVSVVLST
ta-cys11 TXSXLPLLYG RXGKLPQASL KXHAXRXQHX NTXSVTHLSR KHXVWCXYIX
gm-cys5
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out.
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.
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gm-cys9
A.about..about..about..about..about..about..about..about..about.
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ZmCys7
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ut.
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os-cys6 AH.about..about..about..about..about..about..about..about.
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ZmCys9 RCPAPTETIR TRVGRSDVLR QLRVELILLL
FLLL.about..about..about..about..about..about.
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ZmCys13
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out.
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.
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ta-cys3
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out.
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.
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ZmCys11
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out.
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251 300 gm-cys6
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out.
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gm-cys8
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out.
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ta-cys8
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out.
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ta-cys9
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out.
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ZmCys14
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out.
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.
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os-cys5
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out.
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ZmCys3
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ut.
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ZmCys4
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ut.
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ta-cys13
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bout.
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t.
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ZmCys1
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ut.
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os-cys1
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out.
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.
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ta-cys1
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out.
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.
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ta-cys2
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out.
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.
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ta-cys4
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out.
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.
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ta-cys6
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out.
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.
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os-cys3
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out.
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.
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ZmCys8
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ut.
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ZmCys12 PYELLEILRA HAQVVEDFAK FDILMKLKRG SKEEKIKAEV HKSLEGAFVL
ZmCys5 PYELLEILRA HAQVVEDFAK FDILMKLKRG SKEEKIKAEV HKSLEGAFVL
os-cys2 PYELLEIVRA KAEVVEDFAK FDILMKLKRG NKEEKFKAEV HKNLEGAFVL
ta-cys10
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bout.
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t.
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gm-cys2
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out.
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.
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gm-cys7
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out.
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.
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gm-cys1 PYELHEVADA KAEVIDDFAK FNLLLKVKRG QKEEKFKVEV HKNNQGGFHL
gm-cys3
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out.
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.
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gm-cys4
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out.
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.
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ZmCys10 SSS.about..about..about..about..about..about..about.
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ZmCys6 SA.about..about..about..about..about..about..about..about.
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References