U.S. patent application number 10/887020 was filed with the patent office on 2005-03-17 for antifungal proteins.
This patent application is currently assigned to Syngenta Limited. Invention is credited to Broekaert, Willem Frans, De Samblanx, Genoveva Wivina, Rees, Sarah Bronwen.
Application Number | 20050060770 10/887020 |
Document ID | / |
Family ID | 10785360 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050060770 |
Kind Code |
A1 |
Rees, Sarah Bronwen ; et
al. |
March 17, 2005 |
Antifungal proteins
Abstract
Anfifungal proteins which are analogues of the Rs-AFP2 protein
and contain particular mutations in their amino acid sequence are
disclosed. The mutated proteins possess enhanced salt-tolerant
antifungal activity. The proteins are useful for combating fungal
diseases in agricultural, pharmaceutical or preservative
applications.
Inventors: |
Rees, Sarah Bronwen;
(Bracknell, GB) ; Broekaert, Willem Frans;
(Dilbeek, BE) ; De Samblanx, Genoveva Wivina;
(Leuven, BE) |
Correspondence
Address: |
Larry W. Stults, Ph.D.
Syngenta Biotechnology, Inc.
Patent Department
P.O. Box 12257
Research Triangle Park
NC
27709-2257
US
|
Assignee: |
Syngenta Limited
Bracknell
GB
|
Family ID: |
10785360 |
Appl. No.: |
10/887020 |
Filed: |
November 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10887020 |
Nov 18, 2004 |
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10006252 |
Dec 4, 2001 |
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10006252 |
Dec 4, 2001 |
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09077951 |
Mar 11, 1999 |
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6372888 |
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09077951 |
Mar 11, 1999 |
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PCT/GB96/03065 |
Dec 12, 1996 |
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Current U.S.
Class: |
800/279 |
Current CPC
Class: |
A61P 31/10 20180101;
A01N 65/08 20130101; C12N 15/8282 20130101; A61P 31/00 20180101;
C07K 14/415 20130101 |
Class at
Publication: |
800/279 |
International
Class: |
A01H 001/00; C12N
015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 1995 |
GB |
9525474.4 |
Claims
What is claimed is:
1. A transgenic plant comprising a DNA sequence encoding an
antifungal protein having an amino acid sequence which is at least
80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid
sequence of said antifungal protein contains at least one mutation
selected from the group consisting of a basic residue at the
position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a
basic residue at the position corresponding to position 39 in
Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position
corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a
hydrophobic residue other than glycine at the position
corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9).
2. The transgenic plant according to claim 1, wherein said plant is
selected from the group consisting of canola, sunflower, tobacco,
sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum,
tomatoes, mangoes, peaches, apples, pears, strawberries, bananas,
melons, potatoes, carrot, lettuce, cabbage, and onion.
3. The transgenic plant of claim 1 having improved resistance to a
fungal pathogen.
4. The transgenic plant according to claim 3, wherein said fungal
pathogen is selected from the group consisting of Alternaria
brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum,
Nectria haematococca, Phoma betae, and Verticillium dahlias.
5. A transgenic plant comprising a DNA sequence encoding an
antifungal protein having an amino acid sequence selected from the
group consisting of Rs-AFP2 (SEQ ID NO: 9), Rs-AFP1 (SEQ ID NO: 8),
Rs-AFP3 (SEQ ID NO: 10) and Rs-AFP4 (SEQ ID NO: 11), wherein said
amino acid sequence of said antifungal protein contains at least
one mutation selected from the group consisting of a basic residue
at the position corresponding to position 9 in Rs-AFP2 (SEQ ID NO:
9), a basic residue at the position corresponding to position 39 in
Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position
corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a
hydrophobic residue other than glycine at the position
corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9).
6. The transgenic plant according to claim 5, wherein said plant is
selected from the group consisting of canola, sunflower, tobacco,
sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum,
tomatoes, mangoes, peaches, apples, pears, strawberries, bananas,
melons, potatoes, carrot, lettuce, cabbage, and onion.
7. The transgenic plant of claim 5 having improved resistance to a
fungal pathogen.
8. The transgenic plant according to claim 7, wherein said fungal
pathogen is selected from the group consisting of Alternaria
brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum,
Nectria haematococca, Phoma betae, and Verticillium dahliae.
9. A transgenic plant comprising a DNA sequence encoding an
antifungal peptide having at least six amino acid residues
identical to a run of amino acid residues in an antifungal protein
which is at least 80% identical to Rs-AFP2 (SEQ ID NO: 9), wherein
said amino acid sequence of said antifungal protein contains at
least one mutation selected from the group consisting of a basic
residue at the position corresponding to position 9 in Rs-AFP2 (SEQ
ID NO: 9), a basic residue at the position corresponding to
position 39 in Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the
position corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and
a hydrophobic residue other than glycine at the position
corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9), said run of
residues including at least one of the mutated residues.
10. The transgenic plant according to claim 9, wherein said plant
is selected from the group consisting of canola, sunflower,
tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice,
sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries,
bananas, melons, potatoes, carrot, lettuce, cabbage, and onion.
11. The transgenic plant of claim 9 having improved resistance to a
fungal pathogen.
12. The transgenic plant according to claim 11, wherein said fungal
pathogen is selected from the group consisting of Alternaria
brassiccola, Ascochyta pisi, Botrytis cinerea, Fusarium culmorum,
Nectria haematococca, Phoma betae, and Verticillium dahliae.
13. A method of producing a transgenic plant having improved
resistance to a fungal pathogen comprising: a) introducing into a
plant cell a DNA sequence encoding an antifungal protein having an
amino acid sequence which is at least 80% identical to Rs-AFP2 (SEQ
ID NO: 9), wherein said amino acid sequence of said antifungal
protein contains at least one mutation selected from the group
consisting of a basic residue at the position corresponding to
position 9 in Rs-AFP2 (SEQ ID NO: 9), a basic residue at the
position corresponding to position 39 in Rs-AFP2 (SEQ ID NO: 9), a
hydrophobic residue at the position corresponding to position 5 in
Rs-AFP2 (SEQ ID NO: 9) and a hydrophobic residue other than glycine
at the position corresponding to position 16 in Rs-AFP2 (SEQ ID NO:
9); and b) regenerating a transformed plant from said plant cell,
wherein said transformed plant has improved resistance to a fungal
pathogen as compared to an untransformed plant.
14. The method according to claim 13, wherein said plant is
selected from the group consisting of canola, sunflower, tobacco,
sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum,
tomatoes, mangoes, peaches, apples, pears, strawberries, bananas,
melons, potatoes, carrot, lettuce, cabbage, and onion.
15. The method according to claim 13, wherein said fungal pathogen
is selected from the group consisting of Alternaria brassiccola,
Ascochyta pisi, Botrytis cinerea, Fusarium culmorum, Nectria
haematococca, Phoma betae, and Verticillium dahliae.
16. A method of combating a fungal pathogen comprising contacting
said fungal pathogen with the transgenic plant of any one of claims
1, 5, or 9.
17. A method of making a transgenic plant capable of producing an
antifungal peptide, comprising: a) stably integrating into the
genome of a plant cell a DNA sequence encoding an antifungal
protein having an amino acid sequence which is at least 80%
identical to Rs-AFP2 (SEQ ID NO: 9), wherein said amino acid
sequence of said antifungal protein contains at least one mutation
selected from the group consisting of a basic residue at the
position corresponding to position 9 in Rs-AFP2 (SEQ ID NO: 9), a
basic residue at the position corresponding to position 39 in
Rs-AFP2 (SEQ ID NO: 9), a hydrophobic residue at the position
corresponding to position 5 in Rs-AFP2 (SEQ ID NO: 9) and a
hydrophobic residue other than glycine at the position
corresponding to position 16 in Rs-AFP2 (SEQ ID NO: 9); and b)
regenerating stably transformed plants from said transformed plant
cell, wherein said stably transformed plants produce at least one
antifungal peptide.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/077,951, now U.S. Pat. No. 6,372,888, filed Mar. 11, 1999,
which is a national stage application of PCT/GB96/03065, filed Dec.
12, 1996, and published Jun. 19, 1997, as WO 97/21814, which claims
priority of United Kingdom Application Serial No. 9525474.4, filed
Dec. 13, 1995, all of which are incorporated herein by reference in
their entirety
[0002] This invention relates to antifungal proteins, processes for
their manufacture and use, and DNA sequences encoding them.
[0003] In this context, antifungal proteins are defined as proteins
or peptides possessing antifungal activity. Activity includes a
range of antagonistic effects such as partial inhibition or
death.
[0004] A wide range of antifungal proteins with activity against
plant pathogenic fungi have been isolated from certain plant
species. We have previously described a class of antifungal
proteins capable of isolation from radish and other plant species.
These proteins are described in the following publications which
are specifically incorporated herein by reference: International
Patent Application Publication Number WO93/05153 published 18 Mar.
1993; Terras FRG et al., 1992, J Biol Chem, 267:15301-15309; Terras
et al., FEBS Lett, 1993, 316:233-240; Terras et al., 1995, Plant
Cell, 7:573-588. The class includes Rs-AFP1 (antifungal protein 1),
Rs-AFP2, Rs-AFP3 and Rs-AFP4 from Raphanus sativus and homologous
proteins such as Bn-AFP1 and Bn-AFP2 from Brassica napus, Br-AFP1
and Br-AFP2 from Brassica rapa, Sa-AFP1 and Sa-AFP2 from Sinapis
alba, At-AFP1 from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 from
Dahlia merckii, Cb-AMP1 and Cb-AMP2 from Cnicus benedictus, Lc-AFP
from Lathyrus cicera, Ct-AMP1 and Ct-AMP2 from Clitoria ternatea.
The proteins specifically inhibit a range of fungi and may be used
as fungicides for agricultural or pharmaceutical or preservative
purposes. It has been proposed that this class of antifungal
proteins should be named as plant defensins (Terras F. R. G. et al.
1995, Plant Cell 7: 573-588) and these proteins share a similar
motif of conserved cysteines and glycines (Broekaert et al., 1995,
Plant Physiol 108: 1353-1358).
[0005] FIG. 1 shows the amino acid sequences of the protein Rs-AFP2
(SEQ ID NO: 9) and the substantially homologous proteins Rs-AFP1
(SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10), Rs-AFP4 (SEQ ID NO: 11),
Br-AFP1 (SEQ ID NO: 12), Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID
NO: 14), Bn-AFP2 (SEQ ID NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2
(SEQ ID NO: 17) and At-AFP1 (SEQ ID NO: 18) which are small 5 kDa
polypeptides that are highly basic and rich in cysteine. FIG. 1
numbers the positions of the amino acid residues: the dash (-) at
the start of the Rs-AFP3 sequence indicates a gap introduced for
maximum alignment. The sequences shown for Br-AFP1, Br-AFP2,
Bn-AFP1, Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP1 are not complete:
only the N-terminal sequences are shown. The question mark (?) in
the Bn-AFP2 sequence indicates a non-standard amino acid which the
sequencing could not assign and which is thought to be a
post-translational modification on one of the standard amino acid
residues.
[0006] The primary structures of the two antifungal protein
isoforms capable of isolation from radish seeds, Rs-AFP1 (SEQ ID
NO: 8) and Rs-AFP2 (SEQ ID NO: 9), only differ at two positions:
the glutamic acid residue (E) at position 5 in Rs-AFP1 (SEQ ID NO:
8) is a glutamine residue (O) in Rs-AFP2 (SEQ ID NO: 9), and the
asparagine residue (N) at position 27 in Rs-AFP1 (SEQ ID NO: 8) is
substituted by an arginine residue (R) in Rs-AFP2 (SEQ ID NO: 9).
As a result, Rs-AFP2 (SEQ ID NO: 9) has a higher net positive
charge (+2) at physiological pH. Although both Rs-AFPs are 94%
identical at the amino acid sequence level, Rs-AFP2 (SEQ ID NO: 9)
is two- to thirty-fold more active than Rs-AFP1 (SEQ ID NO: 8) on
various fungi and shows an increased salt-tolerence. The proteins
Rs-AFP3 (SEQ ID NO: 10) and Rs-AFP4 (SEQ ID NO: 11) are found in
radish leaves following localized fungal infection. The induced
leaf proteins are homologous to Rs-AFP1 (SEQ ID NO: 8) and Rs-AFP2
(SEQ ID NO: 9) and exert similar antifungal activity in vitro.
[0007] The cDNA encoding Rs-AFP1 (SEQ ID NO: 19) encodes a
preprotein with a signal peptide followed by the mature protein.
The cDNA sequence is shown in FIG. 2. Saccharomyces cerevisiae can
be used as a vector for the production and secretion of Rs-AFP2
(Vilas Alves et al., FEBS Lett, 1994, 348:228-232). Plant-derivable
"wild-type" Rs-AFP2 can be correctly processed and secreted by
yeast when expressed as a N-terminal fusion to the yeast mating
factor .alpha.1 (MF.alpha.1) preprosequence. The Rs-AFP2 protein
does not have adverse effects on yeast even at concentrations as
high as 500 .mu.g/ml.
[0008] We now provide new potent antifungal proteins based on the
structure of the Rs-AFPs and related proteins.
[0009] According to a first aspect the invention provides an
antifungal protein having an amino acid sequence which is
substantially homologous to the Rs-AFP2 sequence (SEQ ID NO: 9)
shown in FIG. 1 and containing at least one mutation selected from
the group consisting of a basic residue at position 9, a basic
residue at position 39, a hydrophobic residue at position 5 and a
hydrophobic residue at position 16.
[0010] According to a preferred embodiment of the first aspect of
the present invention there is provided an antifungal protein
having an amino acid sequence which is substantially homologous to
the Rs-AFP2 sequence (SEQ ID NO: 9) shown in FIG. 1 and containing
at least one mutation selected from the group consisting of an
arginine residue at position 9, an arginine residue at position 39,
a methionine residue at position 5 and a methionine residue at
position 16. An antifungal protein having both a mutation to
arginine at position 9 and a mutation to arginine at position 39
may be particularly active.
[0011] Proteins which are substantially homologous to the Rs-AFP2
protein include the proteins Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ
ID NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12),
Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID
NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and
At-AFP1 (SEQ ID NO: 18) shown in FIG. 1.
[0012] As used herein the term substantially homologous denotes
those proteins which have an amino acid sequence with at least 40%
identity, preferably at least 60% identity and most preferably at
least 80% identity to the Rs-AFP2 sequence (SEQ ID NO: 9).
[0013] The invention further provides an antifungal peptide which
comprises at least six amino acid residues identical to a run of
amino acid residues in an antifungal protein according to the
invention, said run of residues including at least one of the
mutated residues.
[0014] In particular, there are provided the following antifungal
proteins and antifungal peptides derived therefrom:
[0015] a protein having the amino acid sequence of Rs-AFP1 (SEQ ID
NO: 8), Rs-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4
(SEQ ID NO: 11) in which the glycine residue at postion 9 is
replaced by an arginine residue;
[0016] a protein having the amino acid sequence of Rs-AFP1 (SEQ ID
NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which
the valine residue at postion 39 is replaced by an arginine
residue;
[0017] a protein having the amino acid sequence of Rs-AFP4 (SEQ ID
NO: 11) in which the isoleucine residue at postion 39 is replaced
by an arginine residue;
[0018] a protein having the amino acid sequence of Rs-AFP1 (SEQ ID
NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which
the glycine residue at postion 9 is replaced by an arginine residue
and the valine residue at position 39 is replaced by an arginine
residue;
[0019] a protein having the amino acid sequence of Rs-AFP4 (SEQ ID
NO: 11) in which the glycine residue at postion 9 is replaced by an
arginine residue and the isoleucine residue at position 39 is
replaced by an arginine residue;
[0020] a protein having the amino acid sequence of Rs-AFP1 (SEQ ID
NO: 8), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which
the glutamic acid residue at postion 5 is replaced by a methionine
residue;
[0021] a protein having the amino acid sequence of Rs-AFP2 (SEQ ID
NO: 9) in which the glutamine residue at postion 5 is replaced by a
methionine residue;
[0022] a protein having the amino acid sequence of Rs-AFP1 (SEQ ID
NO: 8), RS-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4
(SEQ ID NO: 11) in which the glycine residue at postion 16 is
replaced by a methionine residue.
[0023] Proteins according to the invention include proteins having
one of the following sequences:
1 QKLCERPSRTWSGVCGNNNACKNQCINLEKARHG (SEQ ID NO: 57)
SCNYVFPAHKCICYFPC; QKLCERPSGTWSGVCGNNNACKNQCINLEKARHG (SEQ ID NO:
58) SCNYRFPAHKCICYFPC; QKLCERPSRTWSGVCGNNNACKNQCINLEKARHG (SEQ ID
NO: 59) SCNYRYPAHKCICYFPC; QKLCMRPSGTWSGVCGNNNACKNQCINLEKARHG (SEQ
ID NO: 60) SCNYVFPAHKCICYFPC; QKLCERPSGTWSGVGMNNNACKNQCINLEKARHG
(SEQ ID NO: 61) SCNYVFPAHKCICYFPC;
QKLCQRPSRTWSGVCGNNNACKNQCIRLEKARHG (SEQ ID NO: 62)
SCNYVFPAHKCICYFPC; QKLCQRPSGTWSGVCGNNNACKNQCIRLEKARHG (SEQ ID NO:
63) SCNYREPAHKCICYFPC; QKLCQRPSRTWSGVCGNNNACKNQCIRLEKARHG (SEQ ID
NO: 64) SCNYRFPAHKCICYFPC; QKLCMRPSGTWSGVCGNNNACKNQCIRLEKARHG (SEQ
ID NO: 65) SCNYVFPAHKCICYFPC; QKLCQRPSGTWSGVCMNNNACKNQCIRLEKARHG
(SEQ ID NO: 66) SCNYVFPAHKCICYFPC;
KLCERSSRTWSGVCGNNNACKNQCIRLEGAQHGS (SEQ ID NO: 67)
CNYVFPAHKCICYFPC; KLCERSSGTWSGVCGNNNACKNQCIRLEGAQHGS (SEQ ID NO:
68) CNYRFPAHKCICYFPC; KLCERSSRTWSGVCGNNNACKNQCIRLEGAQHGS (SEQ ID
NO: 69) CNYRFPAHKCICYFPC; KLCMRSSGTWSGVCGNNNACKNQCIRLEGAQHGS (SEQ
ID NO: 70) CNYVFPAHKCICYFPC; KLCERSSGTWSGVCMNNNACKNQCIRLEGAQHGS
(SEQ ID NO: 71) CNYVFPAHKCICYFPC;
QKLCERSSRTWSGVCGNNNACKNQCINLEGARHG (SEQ ID NO: 72)
SCNYIFPYHRCIGYFPC; QKLCERSSGTWSGVCGNNNACKNQCINLEGARHG (SEQ ID NO:
73) SCNYRFPYHRCICYFPC; QKLCERSSRTWSGVCGNNNACKNQCINLEGARHG (SEQ ID
NO: 74) SCNYRFPYHRCICYFPC; QKLCMRSSGTWSGVCGNNNACKNQCINLEGARHG (SEQ
ID NO: 75) SCNYIFPYHRCICYFPC; QKLCERSSGTWSGVCMNNNACKNQCINLEGARHG
(SEQ ID NO: 76) SCNYIFPYHRCICYFPC.
[0024] A cDNA clone encoding the plant-derivable "wild-type"
Rs-AFP2 preprotein was modified by recombinant DNA methods in order
to allow expression in the yeast Saccharomyces cerevisiae. This
peptide was expressed in yeast as a fusion protein carrying at its
N-terminus the prepro sequences derived from the precursor of the
yeast pheromone mating factor .alpha.1. These sequences allow
secretion of the biologically active peptide in a correctly
processed form. The yeast expression system was then used to
express and characterize isoforms of the Rs-AFP2 protein by
introducing deliberate or random changes into the coding region.
These isoforms were subsequently purified and tested for their
antifungal activity.
[0025] The Rs-AFP2 isoform having a mutation at position 5
(glutamine to methionine) (SEQ ID NO: 22) and the Rs-AFP2 isoform
having a mutation at position 16 (glycine to methionine) (SEQ ID
NO: 25) have an enhanced salt-tolerant antifungal activity.
However, two other isoforms were found to possess particularly
advantageous antifungal properties. The Rs-AFP2 isoform having a
mutation at position 9 (glycine to arginine) (SEQ ID NO: 38) and
the Rs-AFP2 isoform having a mutation at position 39 (valine to
arginine) (SEQ ID NO: 43) have a significantly enhanced antifungal
activity. This enhanced activity is prominent in high salt
conditions. An Rs-AFP2 isoform having a mutation at both position 9
(glycine to arginine) and at position 39 (valine to arginine) may
have an even greater salt-tolerance.
[0026] Proteins which maintain their antifungal activity as salt
concentration is increased are particularly suitable for use as
antifungal agents in higher salt conditions. For example, such
proteins are particularly suitable for expression within some
biological organisms including plants. The most abundant divalent
cations in plant tissues are Ca.sup.2+ and Mg.sup.2+. The
concentration of free Ca.sup.2+ in the cytosol is very low (0.1 to
1 .mu.M) (Macklom, 1984, Plant Cell Environ, 7:407-413)), whereas
free Mg.sup.2+ reaches about 1 mM (Hepler and Wyne, 1982, Ann Rev
Plant Physiol, 36:397-439). Free Ca.sup.2+ in plant vacuoles is
about 0.06 to 1 mM and apoplastic free Ca.sup.2+ ranges between
0.02 and 1.3 mM (Harker and Venis, 1991, Plant Cell Environ,
14:525-530). It thus appears that relatively high ionic strength
conditions occur in all cellular compartments. In many cases,
however, fungal infection leads to the disruption of the cells and
contact of the cellular contents with the environment. Therefore it
is difficult to predict the exact ionic conditions under which
antifungal proteins expressed within a plant cell will interact
with invading hyphae. However, proteins whose antifungal activity
is less sensitive to cation concentration are particularly suitable
for expression within plant cells.
[0027] An antifungal protein according to the invention may be
manufactured from its known amino acid sequence by chemical
synthesis using a standard peptide synthesiser, or produced within
a suitable organism (for example, a micro-organism or plant) by
expression of recombinant DNA. The antifungal protein is useful as
a fungicide and may be used for agricultural or pharmaceutical
applications.
[0028] Knowledge of its primary structure enables manufacture of
the antifungal protein, or parts thereof, by chemical synthesis
using a standard peptide synthesiser. It also enables production of
DNA constructs encoding the antifungal protein.
[0029] The invention further provides a DNA sequence encoding an
antifungal protein according to the invention. The DNA sequence may
be predicted from the known amino acid sequence and DNA encoding
the protein may be manufactured using a standard nucleic acid
synthesiser. Alternatively, DNA encoding proteins according to the
invention may be produced by appropriate site-directed mutagenesis
of DNA sequences encoding one of the proteins shown in FIG. 1.
[0030] The DNA sequence encoding the antifungal protein may be
incorporated into a DNA construct or vector in combination with
suitable regulatory sequences (promoter, terminator, transit
peptide etc). The DNA sequence may be placed under the control of a
homologous or heterologous promoter which may be a constitutive or
an inducible promoter (stimulated by, for example, environmental
conditions, presence of a pathogen, presence of a chemical). The
transit peptide may be a homologous or heterologous to the
antifungal protein and will be chosen to ensure secretion to the
desired organelle or to the extracellular space. The transit
peptide is preferably that naturally associated with the antifungal
protein of interest.
[0031] Such a DNA construct may be cloned or transformed into a
biological system which allows expression of the encoded protein or
an active part of the protein. Suitable biological systems include
micro-organisms (for example, bacteria such as Escherichia coli,
Pseudomonas and endophytes such as Clavibacter xyli subsp.
cynodontis (Cxc); yeast; viruses; bacteriophages; etc), cultured
cells (such as insect cells, mammalian cells) and plants. In some
cases, the expressed protein may subsequently be extracted and
isolated for use.
[0032] An antifungal protein according to the invention is useful
for combatting fungal diseases in plants. The invention further
provides a process of combating fungi whereby they are exposed to
an antifungal protein according to the invention.
[0033] For pharmaceutical applications, the antifungal protein may
be used as a fungicide to treat mammalian infections (for example,
to combat yeasts such as Candida).
[0034] An antifungal protein according to the invention may also be
used as a preservative (for example, as a food additive).
[0035] For agricultural applications, the antifungal protein may be
used to improve the disease-resistance or disease-tolerance of
crops either during the life of the plant or for post-harvest crop
protection. Pathogens exposed to the proteins are inhibited. The
antifungal protein may eradicate a pathogen already established on
the plant or may protect the plant from future pathogen attack. The
eradicant effect of the protein is particularly advantageous.
[0036] Exposure of a plant pathogen to an antifungal protein may be
achieved in various ways, for example:
[0037] (a) The isolated protein may be applied to plant parts or to
the soil or other growth medium surrounding the roots of the plants
or to the seed of the plant before it is sown using standard
agricultural techniques (such as spraying).
[0038] The protein may have been extracted from plant tissue or
chemically synthesised or extracted from micro-organisms
genetically modified to express the protein. The protein may be
applied to plants or to the plant growth medium in the form of a
composition comprising the protein in admixture with a solid or
liquid diluent and optionally various adjuvants such as
surface-active agents. Solid compositions may be in the form of
dispersible powders, granules, or grains.
[0039] (b) A composition comprising a micro-organism genetically
modified to express the antifungal protein may be applied to a
plant or the soil in which a plant grows.
[0040] (c) An endophyte genetically modified to express the
antifungal protein may be introduced into the plant tissue (for
example, via a seed treatment process).
[0041] An endophyte is defined as a micro-organism having the
ability to enter into non-pathogenic endosymbiotic relationships
with a plant host. A method of endophyte-enhanced protection of
plants has been described in a series of patent applications by
Crop Genetics International Corporation (for example, International
Application Publication Number WO90/13224, European Patent
Publication Number EP-125468-B1, International Application
Publication Number WO91/10363, International Application
Publication Number WO87/03303). The endophyte may be genetically
modified to produce agricultural chemicals. International Patent
Application Publication Number WO94/16076 (ZENECA Limited)
describes the use of endophytes which have been genetically
modified to express a plant-derived antifungal protein.
[0042] (d) DNA encoding an antifungal protein may be introduced
into the plant genome so that the protein is expressed within the
plant body (the DNA may be cDNA, genomic DNA or DNA manufactured
using a standard nucleic acid synthesiser).
[0043] Plant cells may be transformed with recombinant DNA
constructs according to a variety of known methods (Agrobacterium
Ti plasmids, electroporation, microinjection, microprojectile gun,
etc). The transformed cells may then in suitable cases be
regenerated into whole plants in which the new nuclear material is
stably incorporated into the genome. Both transformed
monocotyledonous and dicotyledonous plants may be obtained in this
way, although the latter are usually more easy to regenerate. Some
of the progeny of these primary transformants will inherit the
recombinant DNA encoding the antifungal protein(s).
[0044] The invention further provides a plant having improved
resistance to a fungal pathogen and containing recombinant DNA
which expresses an antifungal protein according to the invention.
Such a plant may be used as a parent in standard plant breeding
crosses to develop hybrids and lines having improved fungal
resistance.
[0045] Recombinant DNA is DNA, preferably heterologous, which has
been introduced into the plant or its ancestors by transformation.
The recombinant DNA encodes an antifungal protein expressed for
delivery to a site of pathogen attack (such as the leaves). The DNA
may encode an active subunit of an antifungal protein.
[0046] A pathogen may be any fungus growing on, in or near the
plant. In this context, improved resistance is defined as enhanced
tolerance to a fungal pathogen when compared to a wild-type plant.
Resistance may vary from a slight increase in tolerance to the
effects of the pathogen (where the pathogen in partially inhibited)
to total resistance so that the plant is unaffected by the presence
of pathogen (where the pathogen is severely inhibited or killed).
An increased level of resistance against a particular pathogen or
resistance against a wider spectrum of pathogens may both
constitute an improvement in resistance. Transgenic plants (or
plants derived therefrom) showing improved resistance are selected
following plant transformation or subsequent crossing.
[0047] Where the antifungal protein is expressed within a
transgenic plant or its progeny, the fungus is exposed to the
protein at the site of pathogen attack on the plant. In particular,
by use of appropriate gene regulatory sequences, the protein may be
produced in vivo when and where it will be most effective. For
example, the protein may be produced within parts of the plant
where it is not normally expressed in quantity but where disease
resistance is important (such as in the leaves).
[0048] Examples of genetically modified plants which may be
produced include field crops, cereals, fruit and vegetables such
as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize,
wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples,
pears, strawberries, bananas, melons, potatoes, carrot, lettuce,
cabbage, and onion.
[0049] The invention will now be described by way of example only,
with reference to the following drawings wherein:
[0050] FIG. 1 shows the amino acid sequences of the Rs-AFPs and
related proteins. In FIG. 1, the proteins have the following
sequence identifications: Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID
NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12), Br-AFP2
(SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID NO: 15),
Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and At-AFP1 (SEQ
ID NO: 18).
[0051] FIG. 2 shows the nucleotide sequence of the cDNA (SEQ ID NO:
19) encoding Rs-AFP1 (SEQ ID NO: 8).
[0052] FIG. 3 shows the schematic representation of the
construction of the expression vectors pMFprepro/RsAFP2 and
pMFpre/RsAFP2.
[0053] FIG. 4 shows the amino acid sequences of plant-derivable
Rs-AFP2, and a series of yeast-expressed Rs-AFP2 (yRs-AFP2)
isoforms. In FIG. 4, the proteins have the following sequence
identifications:
2 Protein SEQ ID NO: Rs-AFP2 SEQ ID NO: 77 yRs-AFP2 SEQ ID NO: 20
SI.alpha.2 SEQ ID NO: 21 yRs-AFP2/Q5M SEQ ID NO: 22 yRs-AFP2/T10G
SEQ ID NO: 23 yRs-AFP2/W11S SEQ ID NO: 24 yRs-AFP2/G16M SEQ ID NO:
25 yRs-AFP2/A31W SEQ ID NO: 26 yRs-AFP2/Y38G SEQ ID NO: 27
yRs-AFP2/F40M SEQ ID NO: 28 yRs-AFP2/K44Q SEQ ID NO: 29
yRs-AFP2/Y48I SEQ ID NO: 30 yRs-AFP2/T10A SEQ ID NO: 31
yRs-AFP2/H33A SEQ ID NO: 32 yRs-AFP2/Y38A SEQ ID NO: 33
yRs-AFP2/F40A SEQ ID NO: 34 yRs-AFP2/P7- SEQ ID NO: 35
yRs-AFP2/P41- SEQ ID NO: 36 yRs-AFP2/P7R SEQ ID NO: 37 yRs-AFP2/G9R
SEQ ID NO: 38 yRs-AFP2/S12R SEQ ID NO: 39 yRs-AFP2/I26R SEQ ID NO:
40 yRs-AFP2/L28R SEQ ID NO: 41 yRs-AFP2/N37R SEQ ID NO: 42
yRs-AFP2/V39R SEQ ID NO: 43 yRs-AFP2/A42R SEQ ID NO: 44
yRs-AFP2/I46R SEQ ID NO: 45 yRs-AFP2/F49R SEQ ID NO: 46
[0054] FIG. 5 illustrates PCR amplification using the mutagenic
primer OWB41 and the M13 reverse primer. In FIG. 5, the primers
have the following sequence identifications: OWB41-SEQ ID NO: 47;
OWB42-SEQ ID NO: 48; OW1B43-SEQ ID NO: 49; OWB44-SEQ ID NO: 50;
OWB45-SEQ ID NO: 51; OWB77-SEQ ID NO: 52; OWB47-SEQ ID NO: 53;
OWB48-SEQ ID NO: 55; OWB49-SEQ ID NO: 54; OWB50-SEQ ID NO: 56.
[0055] FIG. 6 is a graph of relative specific antifungal activity
(1/IC.sub.50) on F. culmorum of the Rs-AFP isoforms. In FIG. 6, the
peptides have the following sequence identifications: Q5M-SEQ ID
NO: 22; P7R-SEQ ID NO: 37; G9R-SEQ ID NO: 38; TIOG-SEQ ID NO: 23;
S12R-SEQ ID NO: 39; G16M-SEQ ID NO: 25; 126R-SEQ ID NO: 40;
L28R-SEQ ID NO: 41; A31W-SEQ ID NO: 26; N37R-SEQ ID NO: 42;
Y38G-SEQ ID NO: 27; V39R-SEQ ID NO: 43; F40M-SEQ ID NO: 28; P41-SEQ
ID NO: 36; A42R-SEQ ID NO: 44; K44Q-SEQ ID NO: 29; 146R-SEQ ID NO:
45; Y481-SEQ ID NO: 30; F49R-SEQ ID NO: 46.
[0056] FIG. 7 is a graph of the percentage growth inhibition of F.
culmorum caused by Rs-AFP2 isoforms in varying concentrations of
CaCl.sub.2 (panel A) and KCl (panel B).
EXAMPLE 1
[0057] Construction of Expression Vectors for Secretion of Rs-AFP2
by Yeast
[0058] Saccharomyces cerevisiae can be used as a vector for the
production and secretion of Rs-AFP2 as described by Vilas Alves et
al., FEBS Lett, 1994, 348:228-232 using the method described
below.
[0059] Plasmid pFRG1 is a pBluescript IISK derivative containing a
full length cDNA clone encoding Rs-AFP1 (international patent
application publication number WO93/05153). By PCR-mediated
site-directed mutagenesis (Merino et al, 1992, BioTechniques,
12:508-510) two mutations were introduced such that the encoded
protein is the more active isoform Rs-AFP2. A third mutation (CGA
to CGT for Arg.sup.31 of mature Rs-AFP2) was introduced to comply
with the codon usage preference in Saccharomyses cerevisiae
(Bennetzen and Hall, 1982, J. Biol. Chem. 257:3026). The resulting
plasmid was called pBluescript/RsAFP2.
[0060] The vectors pMFpre/RsAFP2 and pMFprepro/RsAFP2 are based on
the yeast/E. coli shuttle vector pTG3828 (Achstetter et al., 1992,
Gene 110:25-31). pTG3828 contains a URA3-d selection marker, the
origin of replication from the yeast 2 .mu. plasmid, the
prokaryotic ColE1 origin of replication and the ampicillin
resistance marker from pBR322. pTG3828 also contains the yeast
phosphoglycerate kinase (PGK) terminator preceded by a polylinker
with multiple unique restriction sites which facilitate insertion
of an expression block.
[0061] The expression blocks in pMFpre/RsAFP2 and pMF prepro/RsAFP2
were derived from the M13 phage derivative M13TG5879 (Reichhart et
al., 1992, Invertebrate Reproduction and Development, 21:15-24)
which contains the promoter of the yeast MF.alpha.1 gene, the
coding region of the MF.alpha.1 pre-sequence with an engineered
NheI site, and the coding region of the MF.alpha.1 pro-sequence
with an engineered HindIII site. The expression cassette of
M13TG5879 was amplified by PCR using the sense primer OWB63:
3 5'TATCAGTCGACGCATGCTATTGATAAGATTTAAA (SEQ ID NO: 1) GG
[0062] (SalI site underlined, SphI site in bold), which introduces
a novel SalI site immediately adjacent to the SphI site at the 5'
end of the MF.alpha.1 promoter, and the M13 reverse primer as an
antisense primer. The resulting PCR product was digested with
SalI-BamHI and subcloned into pBluescriptII SK to yield pVD4.
[0063] Plasmid pBluescript/RsAFP2 was used as a template for the
amplification of the coding sequence of mature Rs-AFP2 in two
separate PCR reactions. In the first PCR reaction the sense primer
OWB61:
4 5'AATAAGCTTGGACAAGAGACAGAAGTTGTGCCAA (SEQ ID NO: 2) AGG
[0064] (HindIII site underlined) was designed such that sixteen
extra nucleotides (coding for the last five amino acids of the
MF.alpha.1 pro-sequence) were added upstream of the coding region
of mature Rs-AFP2. The HindIII site allowed in frame cloning into
the HindIII site in the MF.alpha.1 pro-sequence region of pVD4
Reichhart J M et al., 1991, Invertebrate Reproduction and
Development 21:15-24). The antisense primer OWB64:
5'AAGGATCCCTATTAACAAGGAAAGTAGC (SEQ ID NO: 3) (BamHI site
underlined) introduced a second stop codon and a BamHI site
immediately downstream of the stop codon of the coding region of
Rs-AFP2. In the second PCR reaction, the same antisense primer was
combined with the sense primer OWB62:
5'AATGCTAGCTCAGAAGTTGTGCCAAAGG (SEQ ID NO: 4) (NheI site
underlined) which added seven extra nucleotides (coding for the
last two amino acids of the MF.alpha.1 presequence), including a
NheI site (for in frame cloning into the NheI site in the
MF.alpha.1 pro-sequence region of pVD4) upstream of the coding
region of mature Rs-AFP2. The fragments corresponding to the mature
domain of Rs-AFP2 obtained by PCR amplification in the first or in
the second reaction were digested with HindIII-BamH1 and
NheI-BamHI, respectively, and introduced in the corresponding sites
of pVD4 to yield vectors pVD5 and pVD6, respectively. The resulting
vectors were digested with SalI-BamHI to isolate the expression
blocks, which were then subcloned into SalI-BglII digested pTG3828
to yield the vectors pMFpre-RsAFP2 and pMFprepro/RSAFP2,
respectively.
[0065] FIG. 3 shows the schematic representation of the
construction of the expression vectors pMFprepro/RsAFP2 and
pMFpre/RsAFP2. The different steps in the procedure are (1) PCR
amplification of the coding region of mature RsAFP2 using primers
to add a HindIII site and part of the MF.alpha.1 pro region (5'
site) and a BamHI site (3' site); (2) PCR amplification of the
coding region of mature RsAFP2 using primers to introduce a NheI
and part of the MF.alpha.1 pre region (5' site) and a BamHI (3'
site); (3) subcloning of the PCR product into HindIII-BamHI
digested pVD4; (4) subcloning of the PCR product into NheI-BamHI
digested pVD4; (5) digestion of the resulting plasmids with
SalI/BamHI and subcloning of the inserts in SalI-BglII digested
pTG3828. (Abbreviations in FIG. 3: pre, signal sequence domain of
RsAFP1 cDNA; prea, signal sequence domain of MF.alpha.1 gene;
proc.alpha., propeptide domain of MF.alpha.1 gene; pMF.alpha.1,
promoter domain of MF.alpha.1 gene; tPGK, terminator domain of the
yeast phosphoglycerate kinase gene).
[0066] The plasmids pMFpre-RsAFP2, pMFprepro/RSAFP2 and pTG3828
were transformed in yeast (S. cerevisiae) strain c13-ABYS86
(genotype; .alpha. pral-1, prbl-1, prcl-1, cps1-3, ura3-5, leu2-3,
112, his-) by the lithium acetate method as described by Elble
(1992, BioTechniques, 13:18). Selection of transformants was done
on minimal selective SD medium lacking uracil (Sherman, 1991, Meth.
Enzymol., 194:3-21). Presence of the plasmids in the yeast colonies
was verified by PCR as described by Ward (1990, Nucl. Acids Res.,
18:5319).
EXAMPLE 2
[0067] Purification and Analysis of Yeast-Expressed Rs-AFP2
[0068] Yeast cells transformed with either pTG3828, pMFprepro/Rs
AFP2 or PMFpre/RsAFP2 were grown in selective SD medium until a
saturated culture was obtained. To assess the antifungal activity
of proteins secreted by the yeast cells, the supernatant of the
yeast cultures was filtered (sterile 0.22 .mu.m filter) and
serially diluted in sterile water. Diluted sampels (20 .mu.l) were
incubated in microtiter plate wells with 80 .mu.l of half strength
potato dextrose broth (Difco) containing spores 104 spores/ml) of
Fusarium culmorum.
[0069] Growth of the fungi was monitored by microspectrophotometry
as described by Broekaert et al (1990, FEMS Microbiol Lett,
69:55-60). Homogenates of yeast cells were prepared by spinning
down 1 ml of a saturated yeast culture, suspending the cells in 200
.mu.l of water, vortexing the cells in the presence of 0.2 g of
glass beads (425-600 .mu.m), and clearing the homogenate by
centrifugation (1 min, 10000.times.g). Antifungal activity could
only be detected in the culture medium of yeast cells transformed
with pMFprepro/RsAFP2, which contained about 2 .mu.g/ml of Rs-AFP2
equivalents. The activity of the homogenate of these cells, as well
as that of culture media and cell homogenates of yeast cells
transformed with pMFpre/RsAFP2 or pTG3828 transformed yeasts was
below the detection limit (about 0.2 .mu.g/ml of Rs-AFP2
equivalents). Hence, pMFprepro/RsAFP2 seems to convey significant
expression of Rs-AFP2 in yeast.
[0070] The supernatant of 100 ml of a saturated culture of yeast
transformed with pMFprepro/RsAFP2 (grown on minimal selective SD
medium supplemented with 0.5% w/v of casamino acids) was
centrifuged (4000 rpm, 10 min), and filtered (0.45 .mu.m) to remove
yeast cells and debris. Tris-HCl (pH9) was added to the supernatant
to a final concentration of 50 mM. The sample was loaded at a flow
rate of 2 ml/min on an anion exchange chromatography column
(Q-Sepharose Fast Flow, 20 ml bed volume, Pharmacia), on-line
connected with a disposable reversed-phase C8 silica column (Bond
Elut, 500 mg solid phase, Varian, Harbor City, USA). The antifungal
activity was not retained on the Q-Sepharose matrix but bound to
the C8 silica matrix. The C8 silica column was rinsed with 6 ml of
10% (v/v) acetonitrile in 0.1% (v/v) trifluroacetic acid (TFA) and
subsequently eluted with 4 ml of 30% (v/v) acetonitrile in 0.1%
(v/v) TFA. The latter eluate was dried in a rotating vacuum
concentrator, redissolved in 0.5 ml 15% (v/v) acetonitrile
containing 0.1% (v/v) TFA, and was loaded on a reversed-phase C2/C
18 silica column (Pep-S, 5 .mu.m beads, 0.4.times.25 cm, Pharmacia
connected to a Waters 600 HPLC station pre-equilibriated with 15%
acetonitrile containing 0.1% (v/v) TFA. After loading, the column
was rinsed with the same buffer until the absorbance reached
background level. The column was subsequently eluted with a 15
minute linear gradient from 15% to 50% acetonitrile containing 0.1%
TFA at a flow rate of 1 ml/min. The eluate was monitored for
proteins by on-line measurement of the absorbance at 280 nm. Peak
fractions were collected manually, dried in a rotating vacuum
concentrator to remove the solvents, and redissolved in 200 .mu.l
of distilled water. Twenty .mu.l fractions were tested in a liquid
growth inhibition assay: 20 .mu.l samples were incubated in
microtiter plate wells with 80 p.sup.1 of half strength potato
dextrose broth (Difco) containing 10.sup.4 spores/ml of F.
culmorum; growth of the fungi was monitored by
microspectrophotometry as described by Broekaert et al. (1990, FEMS
Microbiol Lett, 69: 55-60).
[0071] Only the main peak (peak A, elution time 14.7 min) and a
smaller peak (peak B, elution time 15.2 min) coeluted with
antifungal activity. The elution time of peak B was identical to
that of plant-derivable Rs-AFP2 (15.2 min).
[0072] The amino-terminal amino acid sequence obtained by automated
Edman degradation for RPC-purified peak A revealed a sequence of 51
amino acids, all of which being identical to the sequence of
Rs-AFP2. This sequence includes an N-terminal glutamine which is
known to be blocked by cyclisation in plant-derivable Rs-AFP2
(Terras et al., 1992, J Biol Chem, 267:15301-15309). Absence of any
contaminating signals in the amino acid sequence analysis indicated
that the peak A fraction was essentially homogeneous. No sequence
signals could be recorded for RPC-purified peak B material,
probably due to blocking of its N-terminus. This protein fraction
was treated with pyroglutamate aminopeptidase in order to cleave
off the presumed blocked glutamine residue, but also in this case
no amino acid sequence could be determined, whereas the same
treatment successfully deblocked plant-derivable Rs-AFP2. Because
of the uncertain identification of the peak B material and because
of its lower abundancy relative to peak A material, the peak B
material was not further analysed.
[0073] The specific antifungal activity of RPC-purified peak A
material, as well as that of plant-derivable Rs-AFP2, was
determined by measuring the percentage growth inhibition of F.
culmorum caused by serial dilutions of the protein samples. The
IC.sub.50 values (concentration required for 50% growth inhibition)
values derived from dose-response curves, was about 3 .mu.g/ml for
both protein preparataions. Moreover, the type of inhibition caused
by RPC-purified peak A material was identical to that caused by
plant-derivable Rs-AFP2, showing a characteristic morphological
distortion of the fungal hyphae typified by the induction of
multiple branches near the tips.
[0074] These results show that yeast cells transformed with
pMFprepro/Rs-AFP2 produce a protein that has the same biological
activity as plant-derivable Rs-AFP2. Presence of the MF.alpha.1
preprosequence seems to be essential for expression of Rs-AFP2 in
yeast.
EXAMPLE 3
[0075] Production of Rs-AFP2 Isoforms Containing Amino Acid
Mutations
[0076] In order to produce Rs-AFP2 isoforms with single amino acid
substitutions or deletions, mutations were introduced by
PCR-directed mutagenesis in the DNA region coding for the mature
Rs-AFP2 domain.
[0077] FIG. 4 shows the amino acid sequences of plant-derivable
wild-type protein (Rs-AFP2) (SEQ ID NO: 77), yeast-expressed
Rs-AFP2 (yRs-AFP2) (SEQ ID NO: 20), Sorghum bicolor a-amylase
inhibitor 2 (SI.alpha.2) (SEQ ID NO: 21) and four series of
yeast-expressed isoforms of Rs-AFP2 with single amino acid
substitutions or deletions. Z indicates a pyroglutamyl residue.
Amino acids identical to the corresponding residue in Rs-AFP2 are
indicated by dots whereas amino acid deletions relative to the
Rs-AFP2 sequence are represented by a dash.
[0078] The yRS-AFP2 isoforms in Series A (FIG. 4) include a range
of mutations in Rs-AFP2, some of which represent a substitution by
the corresponding amino acid occurring in SI.alpha.2 (Bloch and
Richardson, 1991, FEBS Lett, 279:101-104). SI .alpha.2 is a protein
which is partially homologous to Rs-AFP2 but which (in contrast to
Rs-AFP2) does not exhibit antifungal activity when assayed as
described in Example 2. Series B contains proline deletions. In
Series C, particular amino acids were replaced by a basic residue
(arginine) in order to obtain more basic Rs-AFP2 isoforms.
[0079] The vector for production of yRS-AFP/Q5M, the Rs-AFP2
isoform with an amino acid substitution at position 5 (glutamine to
methionine) was prepared as follows. The vector pVD5 (see Example
1) was used as a template for PCR amplification using the mutagenic
primer OWB41 (SEQ ID NO: 47) and the M13 reverse primer
(5'AGGAAACAGCTATGACCATG) (SEQ ID NO: 5). FIG. 5 illustrates PCR
amplification using the mutagenic primer OWB41 and the M13 reverse
primer. The resulting PCR product was digested with HindIII and Bam
HI and subcloned into the corresponding sites of pVD4 (see Example
1). The resulting vector was digested with SalI and Bam HI and
subcloned into SalI-BglII digested yeast transformation vector
pTG3828 (see Example 1).
[0080] The vectors for the production of Rs-AFP2 isoforms other
than yRs-AFP2/Q5M were constructed as follows. The vector pVD5 was
used as a template for introducing mutations by the two-step PCR
protocol of Merino et al. (1992, BioTechniques, 12:508-510), with
the PCR mutagenic primers being designed according to standard
molecular biology techniques. For example, for Series A and B
isoforms, a first PCR reaction was performed using a mutagenic
primer (either OWB42 (SEQ ID NO: 47), OWB43 (SEQ ID NO: 49), OWB44
(SEQ ID NO: 50), OWB45 (SEQ ID NO: 51), OWB77 (SEQ ID NO: 52),
OWB47 (SEQ ID NO: 53), OWB48 (SEQ ID NO: 55),OWB49 (SEQ ID NO: 54)
or OWB50 (SEQ ID NO: 56): see FIG. 5) and the primer OWB35
(5'GGAATAGCCGATGGAGATCTAGGAAAACAGCTATGACCATG (SEQ ID NO: 6),
nucleotides corresponding to the M13 reverse primer underlined).
The resulting PCR product was used in a second PCR reaction as a
megaprimer and after 5 amplification cycles the primers OWB61 (see
Example 1) and OWB36 (GGAATACCCGATCGAGATCTAGGA) (SEQ ID NO; 7),
corresponding to the first 24 nucleotides of OWB 35) were added.
The PCR product of the second PCR reaction was subcloned in pVD4
and subsequently in pTG3828 as described above. Nucleotide
sequences of all subcloned PCR products were verified by nucleotide
sequencing. The obtained derivatives of pTG3828 were transformed
into yeast and the RsAFP2 isoforms produced by the transformed
yeast strains was purified by reversed-phase chromatography (RPC)
as described in Example 2. All Rs-AFP2 isoforms had the same
electrophoretic mobility as plant-derivable wild-type Rs-AFP2.
[0081] An Rs-AFP2 isoform having a mutation at both position 9
(glycine to arginine) and at position 39 (valine to arginine) may
be readily made in yeast using either the G9R construct or the V39R
construct as the initial PCR template instead of pVD5. The
appropriate mutagenic primer is used for the second amino acid
change.
EXAMPLE 4
[0082] Antifungal Activity of the Rs-AFP2 Isoforms
[0083] In order to assess the effect of single amino acid
substitutions or deletions on the antifungal activity of Rs-AFP2,
yeast-expressed and RPC-purified Rs-AFP2 isoforms (see FIG. 4) were
tested for their specific antifungal activity. The RPC-purified
Rs-AFP2 isoforms were first analysed by SDS-PAGE and the purity of
the preparations was estimated to be at least 50%.
[0084] For each isoform, two independent purifications were carried
out and the antifungal activity was determined in duplicate using
F. culmorum as a test fungus in two different media: a low ionic
strength medium called SMF- (Terras et al, 1992, J Biol Chem,
267:15301-15309) and the same medium supplemented with 1 mM
CaCl.sub.2 and 50 mM KCl called SMF+. The presence of salts in the
test medium, especially salts with divalent cations, reduces the
specific activity of Rs-AFP2. Seed-purified as well as
yeast-expressed wild type Rs-AFP2 served as a control in the
assays.
[0085] Results of preliminary tests are given in Table 1 which
shows the relative specific antifungal activity against F. culmorum
of yeast-expressed wild-type Rs-AFP2 (yRs-AFP2) and the mutant
yRs-AFP2 isoforms. The relative specific activity is expressed as
the specific activity of the mutant divided by the specific
activity of yRs-AFP2 and multiplied by 100. The specific activity
is expressed as the reciprocal of the IC.sub.50 value determined on
F. culmorum after 48 hour of incubation in the presence of the
proteins. The specific activity was measured in medium SMF- and
SMF+.
5 TABLE 1 RELATIVE SPECIFIC ACTIVITY (%) IN MEDIUM PROTEIN SMF-
hc,26 SMF+ yRs-AFP2 (SEQ ID NO: 20) 100 100 SERIES A yRs-AFP2/Q5M
(SEQ ID NO: 22) 100 100 yRs-AFP2/T10G (SEQ ID NO: 23) 30 <16
yRs-AFP2/G16M (SEQ ID NO: 25) 151 114 yRs-AFP2/A31W (SEQ ID NO: 26)
15 <5 yRs-AFP2/Y38G (SEQ ID NO: 27) 30 <4 yRs-AFP2/F40M (SEQ
ID NO: 28) 30 23 yRs-AFP2/K44Q (SEQ ID NO: 29) 100 114
yRs-AFP2/Y48I (SEQ ID NO: 30) 38 114 SERIES B yRs-AFP2/P7- (SEQ ID
NO: 35) 8 17 yRs-AFP2/P41- (SEQ ID NO: 36) 4 <10 SERIES C
yRs-AFP2/P7R (SEQ ID NO: 37) 33 84 yRs-AFP2/G9R (SEQ ID NO: 38) 116
285 yRs-AFP2/S12R (SEQ ID NO: 39) 67 31 yRs-AFP2/I26R (SEQ ID NO:
40) 76 82 yRs-AFP2/L28R (SEQ ID NO: 41) 39 -- yRs-AFP2/N37R (SEQ ID
NO: 42) 100 80 yRs-AFP2/V39R (SEQ ID NO: 43) 74 114 yRs-AFP2/A42R
(SEQ ID NO: 44) 44 26 yRs-AFP2/I46R (SEQ ID NO: 45) 22 --
yRs-AFP2/F49R (SEQ ID NO: 46) 18 22
[0086] It is seen that certain mutations cause a major decrease in
antifungal activity while certain proteins (notably Q5M (SEQ ID NO:
22) and V39R (SEQ ID NO: 43)) maintain their antifungal activity.
However, two mutations cause an increase in antifungal activity.
The isoform with the G16M (SEQ ID NO: 25) mutation shows an
increased activity in low salt (SMF-), although activity is not so
significantly different in high salt (SMF+). The mutant G9R (SEQ ID
NO: 38) is approximately three times more active than yRs-AFP2 (SEQ
ID NO: 20) in high salt (SMF+), although activity is not so
significantly different in low salt (SMF-).
[0087] Table 2 shows results from further comparative tests of the
Rs-AFP2 isoforms in which experiments were carried out in
triplicate. IC.sub.50 values were measured after 72 hours growth in
low salt (SMF-) and high salt (SMF+) media. Deviations are given as
standard error of the mean (sem) based on the triplicate
experiments. In SMF-, the medium without added salts, most of the
derivatives show no decrease or only a minor decrease in antifungal
activity, while in SMF+, the medium with added salts, there is a
significant decrease in antifungal activity for several Rs-AFP2
isoforms. Substitutions that apparently have little effect on the
antifungal activity in SMF-- (low salt medium) include G9R, V39R,
Q5M and G16M. However, in SMF+ (high salt medium) these four
isoforms (in particular G39R and V39R) show an increased antifungal
activity.
6 TABLE 2 SMF- SMF+ PROTEIN IC.sub.50 sem IC.sub.50 sem yRs-AFP2
(SEQ ID NO: 20) 2.7 0.6 8.5 2.7 yRs-AFP2/Q5M (SEQ ID NO: 22) 4.1
0.2 5.4 1.2 yRs-AFP2/T10G (SEQ ID NO: 23) 11.0 4.2 >100
yRs-AFP2/W11S (SEQ ID NO: 24) 16.0 5.7 >100 yRs-AFP2/G16M (SEQ
ID NO: 25) 2.2 0.3 5.0 0.9 yRs-AFP2/A31W (SEQ ID NO: 26) 30.0 5.0
>100 yRs-AFP2/H33A (SEQ ID NO: 32) 32.0 8.7 >100
yRs-AFP2/Y38G (SEQ ID NO: 27) 42.0 17.0 >200 yRs-AFP2/F40M (SEQ
ID NO: 28) 16.0 6.7 54.0 13.0 yRs-AFP2/P41- (SEQ ID NO: 36) 100.0
15.0 >200 yRs-AFP2/K44Q (SEQ ID NO: 29) 3.6 0.4 36.0 9
yRs-AFP2/Y48I (SEQ ID NO: 30) 9.3 1.0 11.0 2.0 yRs-AFP2/P7R (SEQ ID
NO: 37) 6.8 2.4 8.8 1.0 yRs-AFP2/G9R (SEQ ID NO: 38) 3.0 0.5 3.3
0.6 yRs-AFP2/S12R (SEQ ID NO: 39) 3.5 1.0 20.0 6.0 yRs-AFP2/I26R
(SEQ ID NO: 40) 7.2 0.8 9.6 3.7 yRs-AFP2/L28R (SEQ ID NO: 41) 6.4
1.4 >100 yRs-AFP2/N37R (SEQ ID NO: 42) 2.8 0.3 7.0 1.8
yRs-AFP2/V39R (SEQ ID NO: 43) 4.0 0.2 3.2 0.3 yRs-AFP2/A42R (SEQ ID
NO: 44) 4.2 2.5 18.0 5.2 yRs-AFP2/I46R (SEQ ID NO: 45) 12.0 2.4
>40 yRs-AFP2/F49R (SEQ ID NO: 46) 22.0 4.8 23.0 3.0
[0088] FIG. 6 is a graph of relative specific antifungal activity
of the Rs-AFP isoforms as determined on F. culmorum in medium SMF+.
The specific antifungal activity (1/IC.sub.50) of Rs-AFP2 was set
at 100. Bars without indication of standard deviation represent
maximum values; actual values may be even lower. The G9R (SEQ ID
NO: 38) and V39R (SEQ ID NO: 43) isoforms are particularly active,
with the Q5M (SEQ ID NO: 22) and G16M (SEQ ID NO: 25) isoforms also
showing enhanced activity.
EXAMPLE 5
[0089] Rs-AFP2/G9R (SEQ ID NO: 38) and Rs-AFP2/V39R (SEQ ID NO: 43)
Isoforms: Further Tests of Antifungal Activity
[0090] Isoforms Rs-AFP2/G9R and Rs-AFP2/V39R were subjected to
further tests as their antifungal activity in SMF+, in contrast
with that of other isoforms, was approximately two-fold higher than
that of wild-type Rs-AFP2. Their antifungal activity was determined
in SMF with increasing Ca.sup.2+ or K.sup.+ concentration and
compared with that of plant-derivable wild type Rs-AFP2 (isolated
from seed) as well as yeast purified Rs-AFP2. FIG. 7 is a graph of
the percentage growth inhibition of F. culmorum caused by 10
.mu.g/ml of yeast-purified Rs-AFP2 (SEQ ID NO: 20) (open circles),
seed-purified Rs-AFP2 (SEQ ID NO: 77) (closed circles), Rs-AFP2/G9R
(SEQ ID NO: 38) (squares) and Rs-AFP2N39R (SEQ ID NO: 43)
(triangles) in a medium consisting of SMF with varying
concentrations of CaCl.sub.2 (panel A) and KCl (panel B).
[0091] As shown in FIG. 7, the antifungal activity of yRS-AFP2/G9R
and yRS-AFP2/V39R was less reduced by the presence of cations in
the growth medium than the activity of wild-type Rs-AFP2. At a
concentration of 10 .mu.g/ml, both G9R and V39R caused complete
inhibition of the growth of F. culmorum in the presence of 5 mM
CaCl.sub.2 whereas wild type Rs-AFP2 was basically inactive under
the same conditions. At 10 .mu.g/ml, wild-type Rs-AFP2 was fully
active against F. culmorum only when the CaCl.sub.2 concentration
was equal to or lower than 1.25 mM. Similarly, the activity of wild
type Rs-AFP2 was drastically reduced in the presence of 100 mM KCl,
whereas Rs-AFP2 isoforms G9R and V39R were still fully inhibitory
to fungal growth.
[0092] Thus the G9R and V39R Rs-AFP2 isoforms show no increased
activity in the low ionic strength medium, but their activity is
more resistant to the presence of cations in comparison with
wild-type Rs-AFP2. As relatively high ionic strength conditions
occur in all cell compartments, such Rs-AFP2 isoforms displaying a
decreased cation antagonism may be useful for plant transformation
to obtain disease resistant crops. An Rs-AFP2 isoform
yRs-AFP2/G9R/V39R having a mutation at both position 9 (glycine to
arginine) and at position 39 (valine to arginine) would carry a net
+2 positive charge compared to Rs-AFP2 and is expected to show
antifungal activity having an increased salt-tolerence above that
even of the individual isoforms G9R or V39R.
[0093] The antifungal activity of the G9R and V39R Rs-AFP2 isoforms
was also assessed on a set of seven different phytopathogenic fungi
in three media differing in ionic strength: SMF-, SMF plus 1 mM
CaCl.sub.2 and 50 mM KCl, and SMF plus 5 mM CaCl.sub.2 and 50 mM
KCl. The fungi tested were: Alternaria brassicicola, Ascochyta
pisi, Botrytis cinerea, Fusarium culmorum, Nectria haematococca,
Phoma betae and Verticillium dahliae. The results are shown in
Table 3. All IC.sub.50 values were recorded after 72 hours of
growth except for IC.sub.50 values on V. dahliae and F. culmorum
which were determined after 96 hours of growth.
[0094] The data in Table 3 show that the relative strength of the
Rs-AFP2 isoforms may be dependent on the test organism. The
activity of the G9R and V39R isoforms against A. brassicola, A.
pisi and B. cinerea was comparable to the activity of Rs-AFP2,
while Rs-AFP2 appeared to be more active against P. betae. However,
on three fungi (F. culmorum, N. haematococca and V. dahliae) the
isoforms Rs-AFP2/G9R and Rs-AFP2/V39R were more active than Rs-AFP2
itself particularly in the SMF media with added salts. For example,
in the medium SMF plus 1 mM CaCl.sub.2 and 50 mM KCl, Rs-AFP2/G9R
was approximately three-fold more active than Rs-AFP2 against these
three fungi while Rs-AFP2/V39R was approximately two-fold more
active than Rs-AFP2 against F. culmorum and five-fold more active
against N. haematococca and V. dahliae. The three fungi against
which Rs-AFP2NV39R and Rs-AFP2/G9R are more active than Rs-AFP2
belong to the same family of fungi, namely the Nectriaceae.
7 TABLE 3 IC.sub.50 VALUES (.mu.G/ml) SME SMF + 1 mM CaCl.sub.2/50
mM Kcl SMF + 5 mM CaCl.sub.2/50 mM Kcl FUNGUS Rs-AFP2 G9R V39R
Rs-AFP2 G9R V39R Rs-AFP2 G9R V39R A. brassicciola 3.2 2.6 2.5
>50 >50 50 >100 >100 >100 A. pisi 1.9 1.6 2.0 >50
>50 >50 >100 >100 >100 B. cinerea 1.8 1.9 1.6 >50
>50 >50 >100 >100 >100 F. culmorum 2.1 2.2 2.2 4.6
1.5 2.3 22.0 7.2 7.0 N. haemato- 2.0 2.0 2.1 48.0 16.0 9.0 >100
100 62.0 cocca P. betae 0.9 2.0 1.4 14.0 2.50 40.0 27.0 >100
70.0 V. dahliae 1.0 0.5 0.4 11.0 4.0 2.3 50.0 17.0 6.0
[0095]
Sequence CWU 1
1
77 1 36 DNA Artificial Sequence Description of Artificial
Sequenceprimer 1 tatcagtcga cgcatgctat tgataagatt taaagg 36 2 37
DNA Artificial Sequence Description of Artificial Sequenceprimer 2
aataagcttg gacaagagac agaagttgtg ccaaagg 37 3 28 DNA Artificial
Sequence Description of Artificial Sequenceprimer 3 aaggatccct
attaacaagg aaagtagc 28 4 28 DNA Artificial Sequence Description of
Artificial Sequenceprimer 4 aatgctagct cagaagttgt gccaaagg 28 5 20
DNA Artificial Sequence Description of Artificial Sequenceprimer 5
aggaaacagc tatgaccatg 20 6 41 DNA Artificial Sequence Description
of Artificial Sequenceprimer 6 ggaatagccg atggagatct aggaaaacag
ctatgaccat g 41 7 24 DNA Artificial Sequence Description of
Artificial Sequenceprimer 7 ggaatacccg atcgagatct agga 24 8 51 PRT
Raphanus sativus 8 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser
Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile
Asn Leu Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 9 51 PRT
Raphanus sativus 9 Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser
Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile
Arg Leu Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 10 50 PRT
Raphanus sativus 10 Lys Leu Cys Glu Arg Ser Ser Gly Thr Trp Ser Gly
Val Cys Gly Asn 1 5 10 15 Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg
Leu Glu Gly Ala Gln His 20 25 30 Gly Ser Cys Asn Tyr Val Phe Pro
Ala His Lys Cys Ile Cys Tyr Phe 35 40 45 Pro Cys 50 11 51 PRT
Raphanus sativus 11 Gln Lys Leu Cys Glu Arg Ser Ser Gly Thr Trp Ser
Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile
Asn Leu Glu Gly Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Ile Phe
Pro Tyr His Arg Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 12 27 PRT
Brassica rapa 12 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser
Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile
Asn 20 25 13 27 PRT Brassica rapa SITE (11) Xaa is a non-standard
amino acid; thought to be a post-translational modification of a
standard amino acid 13 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Xaa
Ser Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys
Ile Arg 20 25 14 30 PRT Brassica napus 14 Gln Lys Leu Cys Glu Arg
Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala
Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys 20 25 30 15 23 PRT Brassica
napus 15 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val
Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn 20 16 25 PRT Sinapis
alba 16 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys
Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys 20 25 17 26 PRT
Sinapis alba 17 Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly
Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Arg Asn Gln Cys Ile 20 25
18 27 PRT Arabidopsis thaliana 18 Gln Lys Leu Cys Glu Arg Pro Ser
Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Ser Asn Ala Cys Lys
Asn Gln Cys Ile Asn 20 25 19 414 DNA Raphanus sativus 19 gttttattag
tgatcatggc taagtttgcg tccatcatcg cacttctttt tgctgctctt 60
gttctttttg ctgctttcga agcaccaaca atggtggaag cacagaagtt gtgcgaaagg
120 ccaagtggga catggtcagg agtctgtgga aacaataacg catgcaagaa
tcagtgcatt 180 aaccttgaga aagcacgaca tggatcttgc aactatgtct
tcccagctca caagtgtatc 240 tgctactttc cttgttaatt tatcgcaaac
tctttggtga atagttttta tgtaatttac 300 acaaaataag tcagtgtcac
tatccatgag tgattttaag acatgtacca gatatgttat 360 gttggttcgg
ttatacaaat aaagttttat tcaccaaaaa aaaaaaaaaa aaaa 414 20 51 PRT
Raphanus sativus 20 Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser
Gly Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile
Arg Leu Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 21 47 PRT
Sorghum bicolor 21 Arg Val Cys Met Lys Gly Ser Ala Gly Phe Lys Gly
Leu Cys Met Arg 1 5 10 15 Asp Gln Asn Cys Ala Gln Val Cys Leu Gln
Glu Gly Trp Gly Gly Gly 20 25 30 Asn Cys Asp Gly Val Met Arg Gln
Cys Lys Cys Ile Arg Gln Cys 35 40 45 22 51 PRT Raphanus sativus 22
Gln Lys Leu Cys Met Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 23 51 PRT Raphanus sativus 23
Gln Lys Leu Cys Gln Arg Pro Ser Gly Gly Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 24 51 PRT Raphanus sativus 24
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Ser Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 25 51 PRT Raphanus sativus 25
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Met 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 26 51 PRT Raphanus sativus 26
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Trp
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 27 51 PRT Raphanus sativus 27
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Gly Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 28 51 PRT Raphanus sativus 28
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Met Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 29 51 PRT Raphanus sativus 29
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Gln Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 30 51 PRT Raphanus sativus 30
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Pro Pro Ala His Lys Cys
Ile Cys Ile 35 40 45 Phe Pro Cys 50 31 51 PRT Raphanus sativus 31
Gln Lys Leu Cys Gln Arg Pro Ser Gly Ala Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 32 51 PRT Raphanus sativus 32
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 Ala Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 33 51 PRT Raphanus sativus 33
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Ala Val Phe Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 34 51 PRT Raphanus sativus 34
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5
10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala
Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Ala Pro Ala His Lys Cys
Ile Cys Tyr 35 40 45 Phe Pro Cys 50 35 50 PRT Raphanus sativus 35
Gln Lys Leu Cys Gln Arg Ser Gly Thr Trp Ser Gly Val Cys Gly Asn 1 5
10 15 Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg
His 20 25 30 Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile
Cys Tyr Phe 35 40 45 Pro Cys 50 36 50 PRT Raphanus sativus 36 Gln
Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10
15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg
20 25 30 His Gly Ser Cys Asn Tyr Val Pro Ala His Lys Cys Ile Cys
Tyr Phe 35 40 45 Pro Cys 50 37 51 PRT Raphanus sativus 37 Gln Lys
Leu Cys Gln Arg Arg Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 38 51 PRT Raphanus sativus 38 Gln Lys
Leu Cys Gln Arg Pro Ser Arg Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 39 51 PRT Raphanus sativus 39 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Arg Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 40 51 PRT Raphanus sativus 40 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Arg Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 41 51 PRT Raphanus sativus 41 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Arg Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 42 51 PRT Raphanus sativus 42 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Arg Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 43 51 PRT Raphanus sativus 43 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 44 51 PRT Raphanus sativus 44 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Arg His Lys Cys Ile Cys
Tyr 35 40 45 Phe Pro Cys 50 45 51 PRT Raphanus sativus 45 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Arg Cys
Tyr 35 40 45 Phe Pro Cys 50 46 51 PRT Raphanus sativus 46 Gln Lys
Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg 20
25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr 35 40 45 Arg Pro Cys 50 47 43 DNA Artificial Sequence
Description of Artificial Sequenceprimer 47 aataagcttt ggacaagaga
cagaagttgt gcatgaggcc aag 43 48 27 DNA Artificial Sequence
misc_feature (13), (14) & (15) Description of Artificial
Sequenceprimer n= a, t, g, or c 48 ttgtgccaaa ggnnnagtgg gacatgg 27
49 20 DNA Artificial Sequence Description of Artificial
Sequenceprimer 49 ccaagtgggg gttggtcagg 20 50 21 DNA Artificial
Sequence Description of Artificial Sequenceprimer 50 agtgggacat
cctcaggagt c 21 51 23 DNA Artificial Sequence Description of
Artificial Sequenceprimer 51 ggagtctgta tgaacaataa cgc 23 52 20 DNA
Artificial Sequence Description of Artificial Sequenceprimer 52
tcttgcaacg gtgtcttccc 20 53 22 DNA Artificial Sequence Description
of Artificial Sequenceprimer 53 tgcaactatg tcatgccagc ta 22 54 23
DNA Artificial Sequence Description of Artificial Sequenceprimer 54
ttcccagctc accaatgtat ctg 23 55 26 DNA Artificial Sequence
misc_feature (13), (14) & (15) Description of Artificial
Sequenceprimer n= a, t, g, or c 55 aactatgtct tcnnngctca caagtg 26
56 20 DNA Artificial Sequence Description of Artificial
Sequenceprimer 56 tgtatctgca tctttccttg 20 57 51 PRT Raphanus
sativus 57 Gln Lys Leu Cys Glu Arg Pro Ser Arg Thr Trp Ser Gly Val
Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu
Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala
His Lys Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 58 51 PRT Raphanus
sativus 58 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val
Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu
Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Arg Phe Pro Ala
His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 59 51 PRT Raphanus sativus
59 Gln Lys Leu Cys Glu Arg Pro Ser Arg Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 60 51 PRT Raphanus sativus
60 Gln Lys Leu Cys Met Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 61 51 PRT Raphanus sativus
61 Gln Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Met
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 62 51 PRT Raphanus sativus
62 Gln Lys Leu Cys Gln Arg Pro Ser Arg Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 63 51 PRT Raphanus sativus
63 Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 64 51 PRT Raphanus sativus
64 Gln Lys Leu Cys Gln Arg Pro Ser Arg Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 65 51 PRT Raphanus sativus
65 Gln Lys Leu Cys Met Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 66 51 PRT Raphanus sativus
66 Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Met
1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys
Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50 67 50 PRT Raphanus sativus
67 Lys Leu Cys Glu Arg Ser Ser Arg Thr Trp Ser Gly Val Cys Gly Asn
1 5 10 15 Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Gly Ala
Gln His 20 25 30 Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys
Ile Cys Tyr Phe 35 40 45 Pro Cys 50 68 50 PRT Raphanus sativus 68
Lys Leu Cys Glu Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn 1 5
10 15 Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Gly Ala Gln
His 20 25 30 Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys Cys Ile
Cys Tyr Phe 35 40 45 Pro Cys 50 69 50 PRT Raphanus sativus 69 Lys
Leu Cys Glu Arg Ser Ser Arg Thr Trp Ser Gly Val Cys Gly Asn 1 5 10
15 Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Gly Ala Gln His
20 25 30 Gly Ser Cys Asn Tyr Arg Phe Pro Ala His Lys Cys Ile Cys
Tyr Phe 35 40 45 Pro Cys 50 70 50 PRT Raphanus sativus 70 Lys Leu
Cys Met Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn 1 5 10 15
Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Gly Ala Gln His 20
25 30 Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr
Phe 35 40 45 Pro Cys 50 71 50 PRT Raphanus sativus 71 Lys Leu Cys
Glu Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Met Asn 1 5 10 15 Asn
Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Gly Ala Gln His 20 25
30 Gly Ser Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe
35 40 45 Pro Cys 50 72 51 PRT Raphanus sativus 72 Gln Lys Leu Cys
Glu Arg Ser Ser Arg Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Asn
Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg 20 25 30
His Gly Ser Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr 35
40 45 Phe Pro Cys 50 73 51 PRT Raphanus sativus 73 Gln Lys Leu Cys
Glu Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Asn
Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg 20 25 30
His Gly Ser Cys Asn Tyr Arg Phe Pro Tyr His Arg Cys Ile Cys Tyr 35
40 45 Phe Pro Cys 50 74 51 PRT Raphanus sativus 74 Gln Lys Leu Cys
Glu Arg Ser Ser Arg Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Asn
Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg 20 25 30
His Gly Ser Cys Asn Tyr Arg Phe Pro Tyr His Arg Cys Ile Cys Tyr 35
40 45 Phe Pro Cys 50 75 51 PRT Raphanus sativus 75 Gln Lys Leu Cys
Met Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Gly 1 5 10 15 Asn Asn
Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg 20 25 30
His Gly Ser Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr 35
40 45 Phe Pro Cys 50 76 51 PRT Raphanus sativus 76 Gln Lys Leu Cys
Glu Arg Ser Ser Gly Thr Trp Ser Gly Val Cys Met 1 5 10 15 Asn Asn
Asn Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg 20 25 30
His Gly Ser Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr 35
40 45 Phe Pro Cys 50 77 51 PRT Raphanus sativus SITE (1) Xaa is
pyroglutamyl 77 Xaa Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly
Val Cys Gly 1 5 10 15 Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg
Leu Glu Lys Ala Arg 20 25 30 His Gly Ser Cys Asn Tyr Val Phe Pro
Ala His Lys Cys Ile Cys Tyr 35 40 45 Phe Pro Cys 50
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