The present invention relates to polypeptides that are effective against fungi,
especially against fungi causing plant diseases, and against fungi colonizing
agricultural products. The invention further relates to processes for preparing such
polypeptides, and to nucleic acids coding for such polypeptides. In addition, the
5 invention relates to processes and preparations for treating plants using the
polypeptides according to the invention, and to the use of the nucleic acids
according to the invention for producing crops that are protected against damage
from fungi.
Infections of humans by fungi present significant problems, above all for
10 immunodeficient patients, because only a few reliable antimycotically active drugs
have been known to date. Those employed most frequently include azole derivatives,
such as clotrimazole or ketoconazole, which are less toxic as compared to
other compounds (e.g., amphotericine B), and therefore often represent the only
possibility for the therapy of fungal infections.
15 In addition to infections in humans and animals, fungi may also cause significant
damage to plants. Yield losses because of infections of crops by fungi are among
the greatest problems of modern agriculture. More than 8000 fungal species that
may cause a plant disease are known. For example, Phytophthora species cause
the rotting of potato plants, so that 20% of the potato harvest are lost per year
2 0 worldwide. Of particular importance are Fusarium species, which infest cereal
plants, such as wheat, corn and barley. For example, Fusarium graminearum
causes diseases in these plants that are manifested as eye spots on the stalks, leaf
necrosis or damaged roots, as well as cob rot in corn. The damage caused by
Fusarium graminearum in wheat cultivation in the U.S.A. in the period from 1992
25 to 2001 amounted to more than 2.6 billion U.S. dollars (Schisler et al. Biological
control of Fusarium head blight of wheat and deoxynivalenol levels in grain via
use of microbial antagonists; Adv Exp Med Biol. 2002; 504: 53-69). In addition
to the high yield and quality losses in infested cereals, the fungus also causes a
significant contamination of the cereal with toxic metabolites (mycotoxins),
3 0 which when ingested cause severe poisoning (toxicoses) with significant health
problems. This in turn results in massive economical damage in the field of
animal production. To date, about 100 toxins produced by Fusarium spp. have
- 3 -
been identified, of which deoxynivalenol and nivalenol from the group of
Trichothecenes are the most important mycotoxins in cereal cultivation. The
effect of these potent inhibitors of protein synthesis includes skin toxicity and
adverse affection of the nervous and digestive systems of higher animals. These
5 substances are thus an important danger for both humans and farm animals.
Based on the quantitative proportion, dithiocarbamates and thiuram disulfides
are the most important fungicides employed in agriculture, followed by azoles.
The application of dithiocarbamates and thiuram disulfides is regarded with
skepticism, in particular because of their water-polluting potential and their little
10 specific mechanisms of action (Bayerisches Landesamt fur Wasserwirtschaft,
Leaflet No. 4.5/14, as of July 25, 2005). The problems in the use of azoles, such
as propiconazole or triticonazole, in plant protection mainly reside in the fact
that chemically related substances with the same mechanism of action (inhibition
of fungal-specific lanosterol-14a-demethylase) are also employed in
15 medicine. Thus, there is a danger that the effectiveness of the medically
employed azole antimycotics is reduced through selection for resistance in
potentially human-pathogenic fungi in the environment (Report of the German
Federal Institute for Health-Related Consumer Protection and Veterinary Medicine
of June 7, 2001). Although the formation of resistance against azoles
2 0 proceeds relatively slowly and is therefore considered a moderate risk from the
point of view of plant protection, it is considered that a resistance-free situation
cannot be restored even today. Nevertheless, azoles are considered an indispensable
element of integrated plant cultivation today.
In addition to the infestation of growing cultures, fungi also represent an
2 5 important problem in the storage and stocking of seeds, cereals, fruits and other
agricultural products. Colonization with fungi not only leads to immediate losses
when products have to be discarded, but also carries the risk of severe poisoning
if foods and feeds contaminated by mycotoxins are ingested. However, the use
of fungicides in stock protection, which is possible in principle and desirable from
3 0 an economical point of view, is highly restricted. In plant protection, usually
enough time elapses between the application of pesticides and the harvest, so
that the active substances can be degraded. In contrast, for stored crops, their
- 4 -
sale or consumption is to be expected any time. Thus, many highly effective
pesticides cannot be applied in stock protection for hygienic or toxicological
reasons. Other preparations can be employed only in reduced concentrations,
observing the allowable maximum levels. This results in a reduction of the
5 success of control and in the necessity of repeating the measure, which calls the
purpose of the treatment into question from an economical point of view. For
example, currently no fungicides are approved for use in stock protection in
Germany and Austria. Thus, no satisfactory solution exists for the control of
mycotoxicogenic fungi in stocked products.
10 In order to improve the situation, mainly in the field of plant protection, various
alternatives to synthetic low molecular weight fungicides are researched into and in
part already employed. One possibility is the controlled inoculation of the crop
cultures with non-virulent soil fungi, which compete with fungal pests, killing them
or inhibiting the growth thereof by mechanisms that are in part incompletely
15 researched. Mycoparasitism, antibiosis, release of siderophores, competition for
habitat and nutrients, induction of systemic resistance mechanisms, and promotion
of plant growth are discussed in this connection. To date, a good performance
could be achieved mainly in the treatment of cocoa cultures to control the witches'
broom disease caused by Moniliophthora (syn. Crinipellis) perniciosa in South
20 America. A preparation formulated on the basis of Trichoderma stromaticum was
approved in Brazil in February 2012. Other preparations based on symbiotic or
endophytic Trichoderma species (T. martiale, T. ovalisporum) are under development
for use against black rot of cocoa, which is caused by Phytophthoras
palmivora, and against Moniliophthora roreri, the pathogen causing Monilia fruit rot
2 5 of cocoa.
Another possible strategy for controlling harmful fungi, which has not yet entered
the medical or agricultural practice, is the application of so-called antimicrobial
peptides. These include gene-encoded oligopeptides that typically consist of 15 to
45 proteinogenic amino acids linked together in a linear chain (Zasloff, M. Antimi-
30 crobial peptides of multicellular organisms. Nature 2002, 415: 389-95; Borman,
H.G. Antibacterial peptides: basic facts and emerging concepts. J Intern Med.
2003, 254: 197-215). Antimicrobial peptides have been found in the large
- 5 -
majority of polycellular organisms, and are also an important component of the
immune system in mammals including humans. Antimicrobial peptides are of
utmost importance to the immune defense of organisms like insects and other
arthropods, which do not have an adaptive immune system. Most of the known
5 . antimicrobial peptides have an amphipathic structure, wherein the hydrophilic
moiety bears a positive charge. This enables peptides to interact with the
typically negatively charged membrane of bacteria, and to form pore-like
structures after embedding into the membrane. Since the membranes of
eukaryotes usually bear no or only a weak negative charge, the activity of most
10 antimicrobial peptides is primarily antibacterial, the effectiveness often being
more pronounced against either Gram-positive or Gram-negative bacteria. A few
antimicrobial peptides, such as the peptides cecropin, sarcotoxin and stomoxyn,
which are characterized by an alpha-helical structure, show a broad range of
activities and are also active, to various extents, against fungi, protozoans and
15 tumor cells. However, a tendency to non-specific interactions of such antimicrobial
peptides with biological membranes often also results in a hemolytic activity
and thus toxicity for mammals. Further limitations to the practical application of
many antimicrobial peptides are due to the reduction of the activity thereof by
higher salt concentrations, often even by the physiological NaCI concentration of
20 150 mM (Marr et al. Antibacterial peptides for therapeutic, use: obstacles and
realistic outlook. Curr Opin Pharmacol. 2006, 6: 468-72). This is accounted for
by the neutralization of the electrostatic interaction with the target membrane.
For some antimicrobial peptides, a predominantly or exclusively fungicidal
activity has been described. These antimicrobial peptides include gallerimycin,
25 heliomicin, termicin, Alo3, drosomycin, PsDl and NaDl (Lobo et al. Antifungal
Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to
the cell cycle. Biochemistry. 2007, 46: ,987-96; van der Weerden et al. The plant
defensin, NaDl, enters the cytoplasm of Fusarium oxysporum hyphae. J Biol
Chem. 2008, 283: 14445-52). A compact three-dimensional structure stabilized
3 0 by three or four disulfide bridges is characteristic of such antimicrobial peptides.
Antimicrobial peptides having such structural features are referred to as
defensins independently of their range of activities and of the organisms by
which they are produced. Alo3 is a special case because it is the only known
defensin that exclusively consists of three beta strands without the alpha-helical
- 6 -
portions found in other defensins. The recognition of fungal membranes takes
place independently of charges, at least in some cases by specific binding to
neutral glucosylceramides (Aerts et al. The mode of antifungal action of plant,
insect and human defensins. Cell Mol Life Sci. 2008, 65: 2069-79). Activity
5 against both fungi and, above all, Gram-positive bacteria has further been
described for metchnikowin (Levashina et al. Metchnikowin, a novel immuneinducible
proline-rich peptide from Drosophila with antibacterial and antifungal
properties. Eur J Biochem. 1995, 233: 694-700). This proline-rich peptide, which
consists of 26 amino acid residues, differs from typical antimicrobial peptides by
10 lacking a membrane-lysing activity. Shorter proline-rich antimicrobial peptides
with activity predominantly against Gram-negative bacteria penetrate biological
membranes without disrupting them and inhibit the chaperone protein DnaK in
the interior of the cell (Kragol et al. The antibacterial peptide pyrrhocoricin
inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein
15 folding. Biochemistry. 2001, 40: 3016-26). Whether this mechanism of action
also holds for metchnikowin is unclear.
The application of fungicidally active peptides in medicine and cosmetics appears to
be reasonable, above all, by application to the skin or mucosae, for example, in
the form of powders, creams, gels, lotions, shampoos, sprays, mouth washes or
2 0 toothpastes. In addition, there is a possibility of oral application or injection, for
example, in the case of a life-threatening acute infection. External application is
also possible in the field of plant protection and stock protection, for example, by
spraying or applying powdery formulations. The preparation of active peptides can
be effected synthetically, primarily by solid-phase synthesis, or by the heterological
25 expression of corresponding nucleic acids in different host organisms. Since the
preparation of oligo- and polypeptides in a pure form is complicated, alternative
application forms are sought, mainly for application in plant protection. One
method that is well established at least in the field of research consists in the
production of genetically engineered plants into which nucleic acids coding for the
3 0 active peptides to be produced were introduced. Because the constant production
of such active substances by the plant is undesirable in some cases, it may be
advantageous to introduce the nucleic acids in such a way that the expression
- 7 -
thereof is under the control of promoters that are activated directly by the fungal
infestation, or by the stress reactions by the plant associated therewith.
Nucleic acids coding for the defensins heliomicin and drosomycin were introduced
into tobacco plants and could mediate a small, but statistically significant re-
5 sistance against Cercospora nicotianae (Banzet et al. Expression of insect
cysteine-rich antifungal peptides in transgenic tobacco enhances resistance to a
fungal disease. Plant Sci. 2002, 162: 995-1006). Similarly, resistance against
powdery mildew Erysiphe cichoracearum and Sclerotinia minor could be achieved
by introducing nucleic acids coding for gallerimycin into tobacco plants (Langen
10 et al. Transgenic expression of gallerimycin, a novel antifungal insect defensin
from the greater wax moth Galleria mellonella, confers resistance to pathogenic
fungi in tobacco. Biol Chem. 2006, 387: 549-57). In addition to these defensins
that are specifically active against fungi, genetically engineered plants producing
antimicrobial peptides with a broader range of activities were also prepared. For
15 example, the expression in rice of a foreign gene coding for cecropin A resulted
in increased resistance against the rice blast pathogen Magnaporthe grisea (Coca
et al. Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred
by expression of a cecropin A gene in transgenic rice. Planta 2006, 223: 392-
406). Increased resistance against Fusarium solani was achieved by the heterol-
20 ogous expression in potato and tobacco plants of a gene coding for an artificial
hybrid peptide consisting of sequence elements of cecropin and of melittin,
which occurs in bee venom (Osusky et al. Transgenic plants expressing cationic
peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nat
Biotechnol. 2000, 18: 1162-6; Yevtushenko et al. Pathogen-induced expression
2 5 of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance
in transgenic tobacco. J Exp Bot. 2005, 56: 1685-95). In barley, increased
resistance against Fusarium graminearum and Blumeria graminis was observed
upon introduction of a foreign gene for metchnikowin (Rahnamaeian et al. Insect
peptide metchnikowin confers on barley a selective capacity for resistance to
3 0 fungal ascomycetes pathogens. J Exp Bot. 2009, 60: 4105-14).
Because of these data, it is to be considered as an established fact that antimicrobial
fungi are suitable in principle for controlling fungal infections in plants and in
- 8 -
stock protection. However, only a few of these peptides having a specific activity
against fungi in general and, in particular, against plant-pathogenic fungi are
currently known. The use of peptides having a broad range of activities bears the
risk that the growth of useful bacteria, for example, those associated with the
5 plants as symbionts, is impeded. In addition, the presence of such peptides in
products intended for human or animal ingestion may lead to interference with the
natural intestinal flora and thus adversely affect health. Further problems arise
from the fact that most of the known antimicrobial peptides bear a positive net
charge at physiological pH values. The resulting non-specific interactions with
10 different negatively charged biological polymers may lead to toxic effects that are
difficult to predict. Thus, there is a risk of histamine release by mast cells upon
contact with strongly cationic substances (Vaara, M. New approaches in peptide
antibiotics. Curr Opin Pharmacol. 2009, 9: 571-6). In addition, many of the
known antimicrobial peptides have an amphipathic structure, which involves a
15 tendency to embedding in biological membranes of mammal cells, which is
measurable as a hemolytic activity, so that there is further toxic potential.
EP 0 798 381 A2 discloses a polypeptide from flies of the order Diptera and the use
thereof for preparing transgenic plants having an increased resistance against
Fusarium. The entry in Database EMBL (online) of March 1, 2011, EBI Accession
2 0 No. JG 4D7441 discloses a polynucleotide from Lucilia cuprina. No functions
associated with this polynucleotide are disclosed.
Surprisingly, it has been found that a group of polypeptides have fungicidal
activity. The polypeptides according to the invention are characterized by being
identical with at least 12 contiguous amino acid residues of SEQ ID No. 2, especial-
2 5 ly having at least 65% sequence homology with amino acid sequence SEQ ID No.
2.
Typically, exposure to the polypeptides according to the invention at a concentration
of 1 to 1000 uM, especially 2 uM, leads to a half-maximum inhibition of the
spore germination of Fusarium graminearum.
- 9 -
In one embodiment, the polypeptide according to the invention is identical with
SEQ ID No. 1 orSEQID No. 2.
In another embodiment of the invention, the polypeptide according to the invention
is effective against the growth of organisms of the genera Fusarium and/or
5 Phytophthora.
The polypeptide according to the invention may be derivatized, especially at the N
terminal, C terminal and/or in the peptide chain. The derivatization of the N
terminal may include partial or complete alkylation, acylation or another N
modification. The derivatization of the C terminal may include amidation, esterifi-
10 cation or another modification of the terminal carboxy group. The derivatization of
the polypeptide chain may include, in particular, a modification to improve the
properties of the polypeptide according to the invention, for example, PEGylation,
HESylation or the like. If desired, the polypeptide according to the invention may
also be coupled to structural units having binding affinity for cellular structures of
15 the organism against which the polypeptide according to the invention is to be
employed. Suitable derivatizations for achieving modified properties of polypeptides
are known to the skilled person. Derivatives of the polypeptide according to
the invention having the mentioned activities are also claimed according to the
invention.
20 In particular, the present invention relates to a polypeptide with the following
sequence:
Zx-QHGYGAGGHGQQGYGSQHSSHAPQGGHVVREQGFSGHVHEQQAGHHHEAGHHEQAGHHEQ
SGQQVHGQGHGYK-Z2,
where
25 Zi is the N-terminal end of the polypeptide, or a derivative of the terminal
amino group of the polypeptide, or a chain of up to ten arbitrary amino acids;
Z2 is the C-terminal end of the polypeptide, or a derivative of the terminal
carboxy group of the polypeptide, or a chain of-up to ten arbitrary amino ac-
30 ids.
- 10 -
In particular, Z2 may have an amino acid sequence of SHGY-Z3, wherein Z3 is the
C-terminal end of the polypeptide, or a derivative of the terminal carboxy group of
the polypeptide, or a chain of up to six arbitrary amino acids.
In another embodiment of the invention, the polypeptide according to the inven-
5 tion may have D-amino acids or a D-retro-inverso peptide structure in part or
completely in the peptide chain.
The invention also relates to the use of a polynucleotide coding for the polypeptide
according to the invention for producing the polypeptide according to the invention,
or for controlling fungi, wherein said polynucleotide has a sequence that
10 hybridizes under conditions of stringency using 0.2xSSC at 42 °C for washing with
an oligo- or polynucleotide probe selected from the group:
the complementary strand to nucleotide residues 1 to 231 from SEQ ID No.
3;
the complementary strand to nucleotide residues 1 to 102 from SEQ ID No.
15 3;
the complementary strand to nucleotide residues 103 to 331 from SEQ ID
No. 3;
the complementary strand to nucleotide residues 18 to 251 from SEQ ID No.
4;
20 the complementary strand to nucleotide residues 18 to 119 from SEQ ID No.
4;
the complementary strand to nucleotide residues 120 to 251 from SEQ ID
No. 4.
The present invention also relates to the use of the polypeptide according to the
2 5 invention or of a nucleic acid according to the invention coding for such a polypeptide
for controlling fungi, especially fungi causing plant diseases; organisms of the
genera Fusarium and/or Phytophthora; organisms of the class
Peronosporomycetes.
- 1 1 -
Further, the present invention relates to a process for producing the polypeptide
according to the invention.
The present invention also relates to a cell, except for wild type cells of Lucilia
sericata, containing a nucleic acid for the expression of a polypeptide according to
5 the invention, especially a polypeptide of SEQ ID Nos. 1 or 2.
The invention also relates to a transgenic crop containing a cell according to the
invention.
Figure 1 shows the result of the analysis of the protein LserFCPl-77, which was
prepared in a completely synthetic way, by reverse-phase chromatography and
10 mass spectrometry.
Figure 2 shows the result of an SDS-PAGE analysis after different periods of time
from the induction with IPTG of E. coli cells that were genetically engineered for
the production of a fusion polypeptide consisting of thioredoxin, a hexahistidine
sequence, a factor Xa recognition sequence, and LserFCPl-77.
15 Figure 3 shows the result of an immobilized metal ion affinity chromatography for
the purification of a fusion polypeptide consisting of thioredoxin, a hexahistidine
sequence, a factor Xa recognition sequence, and LserFCPl-77, and the result of
the related SDS PAGE analysis.
Figure 4 shows the result of a Mono-Q chromatography for the separation of the
2 0 cleavage products obtained after treatment with factor Xa of a fusion polypeptide
consisting of thioredoxin, a hexahistidine sequence, a factor Xa recognition
sequence, and LserFCPl-77, and the result of the related SDS PAGE analysis.
Figure 5 shows the result of the analysis of the polypeptide LserFCPl-77, which
was prepared in a recombinant form, by reverse phase chromatography and mass
25 spectrometry.
- 12 -
Figure 6 shows the result of a Mono-Q chromatography for the separation of the
cleavage products obtained after treatment with enterokinase of a fusion polypeptide
consisting of thioredoxin, a hexahistidine sequence, an enterokinase recognition
sequence, and LserFCPl-77, and the result of the mass-spectrometric analysis
5 of the obtained polypeptide LserFCPl-73.
Figure 7 shows the result of tests on the polypeptide LserFCPl-77 for inhibition of
the spore germination of Fusarium graminearum at the stated concentrations as
compared to a water control.
Figure 8 shows the dose-effect relationship for the inhibition of the spore germina-
10 tion of Fusarium graminearum by LserFCPl-77.
Figure 9 shows the result of tests on the polypeptide LserFCPl-73 for inhibition of
the spore germination of Fusarium graminearum at the stated concentrations as
compared to a water control.
Figure 10 shows the result of tests on the polypeptide LserFCPl-77 for inhibition of
15 the spore germination of Phytophthora parasitica at the stated concentrations as
compared to a water control.
According to the invention, the term "fungicidal activity" is understood to mean the
killing of fungi, the inhibition of fungal growth, and the prevention of the germination
of fungal spores. These effects can be completely or partially pronounced.
2 0 According to the invention, the term "control of fungi" is understood to mean the
killing of fungi, or the inhibition of the growth of existing fungi as well as the
prevention of fungal colonization, and the prevention of the germination of fungal
spores.
According to the invention, "fungi" is also understood to mean those organisms
2 5 that are colloquially referred to as fungi (or molds), even though this does not
correctly reflect the scientific evidence relating to phylogenesis. In particular,
"fungi" according to the invention is intended to include representatives of the
- 13 -
class Peronosporomycetes (former designations Oomycota or Oomycetes),
including the genus Phytophthora.
The polypeptides according to the invention are suitable for controlling fungi that
cause health-related or economical damage.
5 In a preferred embodiment of the invention, the polypeptides according to the
invention are employed for controlling fungi that cause plant diseases or damage in
the storage of agricultural products.
In another preferred embodiment of the invention, the polypeptides according to
the invention are employed for controlling fungi of the genera Phytophthora and
10 Fusarium.
The polypeptides according to the invention can be used in a pure form or in the
form of different formulations for controlling fungi. For external application, the
polypeptides can be diluted to form a liquid solution or suspension containing from
0.01 to 30 mg/ml of the respective polypeptide, or mixed with an extender solid
15 for application as a dust or powder. Methods for adapting common methods for
application to particular crops and pathogens are known in the literature (Methods
for Evaluating Pesticides for Control of Plant Pathogens, K.D. Hickey, Ed., The
American Phytopathological Society, 1986). Methods for application include the
singular or periodically performed aqueous and non-aqueous spraying of plants or
2 0 plant parts, seed coating, and the incorporation in spraying systems. Auxiliary
additives that may be added to the formulation include stabilizers, agents for
improving the dissolving performance, and wetting agents, and also agents that
allow microencapsulation.
For a particularly effective control of fungi, the polypeptides according to the
2 5 invention can be employed in combination with other fungicidally active substances.
Also, combination with bactericidal, antiviral, nematocidal, insecticidal and
other active substances common in plant protection is possible. Combination with
fertilizers, plant hormones and growth regulators is also possible. One possibility of
controlling fungi by means of the polypeptides according to the invention is to
- 14 -
genetically engineer plants by introducing polynucleotide sequences coding for
such polypeptides into their genetic material by a process known as transformation.
Methods for preparing such genetically engineered plants according to the
invention are known to the skilled person (Methods for Plant Molecular Biology, A.
5 Weissbach, H. Weissbach, Eds., Saunders College Publishing/Harcourt Brace,
June 1988; Methods in plant molecular biology and biotechnology, B.R. Glick,
J.E. Thompson, CRC Press, Boca Raton, FL, 1993). In an advantageous variant
of the invention, artificial gene constructs of suitably modified vectors are
incorporated into plants, plant parts or plant cells by bombardment with DNA-
10 coated microparticles, by the so-called floral dip method, or by Agrobacteriummediated
transformation. Other possible methods include, for example, microinjection,
chemical permeabilization, electroporation, and protoplast fusion
with DNA-containing units, such as cells, minicells, protoplasts, or liposomes. In
another advantageous variant of the invention, a gene coding for a polypeptide
15 according to the invention is prepared synthetically, in which the nucleotide
triplets, which code for one amino acid each, are adapted to the preferred codon
usage of the genetically engineered plant. Further, it is possible to place the
foreign gene to be transferred under the control of a promoter that is activatable
by injury and auxine activity according to a known method, whereby an en-
20 hanced expression after fungal colonization is achieved (Rahnamaeian. Insect
peptide metchnikowin confers on barley a selective capacity for resistance to
fungal ascomycetes pathogens. J Exp Bot. 2009, 60: 4105-14). In an advantageous
embodiment of the invention, corn and/or potato plants are genetically
engineered to be able to produce the polypeptides according to the invention.
2 5 A polypeptide corresponding to SEQ ID No. 1 or No. 2 can be used for controlling
fungi. In addition, it is also possible to employ polypeptides or oligopeptides
resulting from truncation of the sequence or the addition of further amino acid
residues.
Further, it is possible to employ variants of the polypeptide that have additional
3 0 sequence elements having been added, for example, in order to achieve a higher
yield in the preparation in a recombinant form, or a facilitated purification.
- 15 -"
In some cases, it may be advantageous to employ oligopeptides consisting of at
least twelve contiguous amino acid residues of SEQ ID No. 1. In addition, it may be
advantageous to remove individual amino acid residues, or replace them by the
residues of different amino acids. The insertion of additional amino acid residues is
5 also possible. Such changes are also possible when proceeding from truncated or
extended variants of the polypeptide. In particular, it may be advantageous to
replace individual amino acid residues by those having similar physico-chemical
properties. Thus, mainly residues of the amino acids are mutually interchangeable
within the following groups:
io Arginine and lysine;
glutamic acid and aspartic acid;
glutamine, asparagine and threonine;
glycine, alanine and proline; ,
leucine, isoleucine and valine;
15 tyrosine, phenylalanine and tryptophan;
serine and threonine.
The identification of polypeptides that are derived from SEQ ID No. 1 can be
effected, for example, by the screening of genomic and/or cDNA gene libraries
using known methods, which are described, for example, in Sambrook and
20 Russell 2001 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NJ. The gene libraries can be prepared, for example, using
bacteria or bacteriophages as receptor organisms. Also, the gene libraries could
be in the form of sequence data electronically stored on suitable media, especially
if such data were produced by means of the techniques known as Next
25 Generation Sequencing without molecular cloning of nucleic acid molecules. As
probes for the screening, there can be employed, in particular, nucleotide
sequences corresponding to the complementary strand of SEQ ID No. 3 or SEQ
ID No. 4, or of fragments of these sequences. The sequences of these probes
may vary within the scope of the degeneracy of the genetic code, and may also
3 0 contain nucleosides such as inosine, which do not naturally occur in proteinencoding
nucleic acids, in order to enhance the capability of hydrogen bonding.
- 16 -
The screening may also be effected using antibodies raised against polypeptides
corresponding to SEQ ID No. 1, SEQ ID No. 2, or fragments of these sequences.
Because of their comparatively small size, the polypeptides according to the
invention can be prepared by methods of chemical peptide synthesis (Example 1).
5 This may involve the use of known solid-phase methods, for example, according to
B. Merrifield. The synthesis may be effected manually or with the aid of an
automated peptide synthesizer using Fmoc or Boc protective group strategy. It is
possible to synthesize the polypeptides in smaller fragments, which are subsequently
linked together.
10 The preparation of the polypeptides according to the invention may also be
effected by the heterologous expression of suitable nucleic acid constructs in
different receptor organisms and receptor cells. The required methods of genetic
engineering are described in Sambrook and Russell 2001 Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, NJ. Prokaryotic
15 systems (for example, Escherichia coli or Pseudomonas fluorescens) or eukaryotic
systems (for example, insect cells, plant cells or mammal cells) may be
employed.
In a preferred embodiment of the invention, the preparation is effected in E. coli as
a fusion polypeptide with thioredoxin and a hexahistidine sequence, wherein a
2 0 specific protease recognition sequence is provided for the cleavage of the polypeptide
according to the invention (Examples 2, 3 and 4).
The polypeptides according to the invention obtained by heterologous expression
can be purified from cell lysates or supernatants of the genetically engineered
organisms or cells by a number of known methods. Particularly suitable methods
25 include immobilized metal ion affinity chromatography, anion-exchange chromatography,
and reverse-phase chromatography (Example 3).
Example 1:
Synthetic preparation of the polypeptide LserFCPl-77
- 17 -
The polypeptide having the amino acid sequence according to SEQ ID No. 1 was
prepared completely by solid-phase synthesis on a polymeric support resin. An
analysis of the product by reverse-phase chromatography (column: Alltech Alltima
C18 4.6 x 250 mm, Fischer Scientific) with an ascending methanol gradient in
5 water yielded an essentially homogeneous peak, so that a purity of at least 80%
could be assumed (Figure 1). During the further analysis by electrospray
ionization mass spectrometry (ESI-IMS), a dominant molecule ion was observed
at m/z 746.50, which corresponds to the eleven times protonated target
molecule (Figure 1).
10 Example 2:
Construction of plasmids for the recombinant preparation of LserFCPl-77 and
LserFCPl-73 in Escherichia coli
For the heterologous expression in E. coli, a synthetic gene was prepared that
codes for the sequence of the polypeptide LserFCPl-77 (SEQ ID No. 1). The
15 codon usage was adapted to that of the receptor organism E coli K12. The gene
synthesis was performed by the company Eurofins MWG Operon (Anzingerstr.
7a, 85560 Ebersberg, Germany) on a contract basis. Further sequences enabling
the insertion into the vector pASK-IBA33plus were added to the coding sequence
at the 5' and 3' terminals. Thus, the complete synthetically prepared polynucleo-
2 0 tide had the sequence as shown in SEQ ID No. 4. The synthetic gene was
inserted into the vector pASK-IBA33plus in an oriented way by using two Bsal
restriction endonuclease cleaving sites. The thus obtained construct was used for
the transformation of E. coli cells of the strains TOP10 and BL21.
In further experiments proceeding from the construct based on pASK-IBA33plus,
25 the synthetic gene was recloned into the vector pET-32a(+). The recloning was
performed to obtain a plasmid coding for a fusion polypeptide consisting of
thioredoxin, a hexahistidine sequence, a protease recognition sequence, and the
polypeptide LserFCPl-77. Two variants of this plasmid were prepared; in one, the
encoded recognition sequence was specific for enterokinase, and in the other, for
30 coagulation factor Xa. Accordingly, the plasmids were designated as pET-32-FCPEK
and pET-32-FCP-Xa.
- 18 -
For the construction of these plasmids, the synthetic gene was amplified by PCR,
wherein the additional sequences required for the insertion into the new vector
were added with the primers. pET-32-FCP-EK was constructed using the forward
primer 1 consisting of a Kpnl restriction site, a linking sequence, a sequence
5 coding for the enterokinase recognition sequence, and a specific sequence coding
for the first amino acid residues of the polypeptide LserFCPl-77. pET-32-FCP-Xa
was constructed using the forward primer 2 consisting of a Kpnl restriction site,
a linking sequence, a sequence coding for the factor Xa recognition sequence,
and a specific sequence coding for the first amino acid residues of the polypeptide
10 LserFCPl-77. For both constructs, the reverse primer 3 consisting of an EcoRI
restriction site, a stop codon and a specific sequence coding for the last amino
acid residues of the polypeptide LserFCPl-77 was used.
Primer 1 (forward):
5'-tgaggtaccgacgacgacgacaagcagcatggctatggagcgg-3'
15 Primer 2 (forward):
5•-tgaggtaccggtggtggctccggtattgagggtegccageatggctatggagcgg-3•
Primer 3 (reverse):
5'-tcagaattettaatacccgtgegatttgtag-3'
The PCR products were inserted through the Kpnl and EcoRI restriction sites into
20 the vector pET-32a(+), and the constructs obtained were used for the transformation
of E. coli cells of the strain BL21(DE3) (Novagen/Merck). The identification
of transformed cells was effected by selection on ampicillin-containing
nutrient medium. In its genomic DNA, the E. coli strain employed contains a
lysogenic lambda phage on which a gene coding forT7 RNA polymerase is under
25 the control of a lacUV5 promoter. The expression of the T7 RNA polymerase
gene and thus the expression of the foreign gene being under the control of a T7-
promoter on the expression plasmid is induced by adding isopropyl-beta-Dthiogalactopyranoside
(IPTG). The formation of the corresponding fusion
polypeptide at different times after the induction was detected by SDS PAGE
30 followed by Coomassie blue staining (Figure 2).
- 19 -
Example 3:
Production of the recombinant polypeptide LserFCPl-77 in Escherichia coli
E. coli cells of the strain BL21(DE3) (Novagen/Merck) were transformed with the
plasmid pET-32-FCP-Xa constructed as described in Example 2, and cultured in
5 six one-liter Erlenmeyer flasks with baffles, each of which contained 400 ml of
LB medium supplemented with 300 mg/l ampicillin, at 37 °C with shaking at
250 rpm. After an absorption value of 0.4 at 600 nm as observed by turbidity
measurement had been achieved, the induction was effected by adding 1 mM
IPTG. After the culture had been continued for 3 hours, the bacterial cells were
10 harvested by centrifugation (10,000 x g, lOmin, 4°C). The pellet was resuspended
in 200 ml of buffer A (100 mM NaCI, 30 mM Tris, pH 7.5), and the cells
were lysed by shear forces in a high-pressure homogenizer (Microfluidizer
M110PS, Microfluidics, 30 Ossipee Road,. Newton, MA 02464 U.S.A.). After *
centrifugation (70,000 x g, 30 min, 4°C), the supernatant was filtered through a
15 0.22 mm membrane. The cell lysate was charged at a flow rate of 4 ml/min onto
an immobilized metal ion affinity chromatographic column loaded with Co2+ ions
(16 x 100 mm, TALON Superflow Resin, Clontech Laboratories, 1290 Terra Bella
Avenue, Mountain View, CA 94043, U.S.A.). The column had previously been
equilibrated with buffer A. After the sample had been charged, the column was
2 0 washed with buffer A until the detection at 280 nm yielded a constant value. The
elution was effected with a step gradient from 30 mM to 100 mM imidazole in
buffer A. The main quantity of the fusion polypeptide was eluted with the 100
mM imidazole step. An analysis by SDS PAGE followed by Coomassie blue
staining yielded a purity of more than 90% (Figure 3).
2 5 The fusion polypeptide was rebuffered by gel permeation chromatography
(HiPrep Desalting Column 26/10, GE Healthcare, Buckinghamshire, UK) in
10 mM NaCI, 10 mM Tris, pH 7.5. Ten micrograms of the fusion polypeptide was
incubated with 500 units of factor Xa (Merck) in 10 ml of cleavage buffer
(100 mM; Tris-HCI, 20 mM NaCI, pH 7.5) for 16 h at room temperature. The
3 0 reaction product was charged at a flow rate of 1 ml/min onto a strong ionexchange
column (MonoQ 5/50 GL, GE Healthcare) equilibrated with 10 mM Tris-
HCI, pH 8, and the column was washed with the same buffer. The elution was
- 20 -
effected with a gradient from 0 to 300 mM NaCI over 20 minutes. An analysis by
SDS PAGE showed that the polypeptide LserFCPl-77, which was obtained as a
cleaving product and eluted at 11 min, was completely separated from the
remaining polypeptide components (Figure 4). The corresponding peak in the
5 chromatogram was visible only by detection by UV absorption, but not by
fluorescence measurement. This can be explained by the absence of tryptophan
residues in the amino acid sequence of the polypeptide LserFCPl-77. In an SDS
PAGE analysis, LserFCPl-77 exhibited an apparent molecular mass of 30 kDa
instead of the expected 8.2 kDa. This atypical electrophoretic behavior is
10 possibly accounted for by ineffective binding of SDS molecules to the polypeptide.
The identity of the polypeptide was checked by reverse-phase chromatography
(column: BioBasic 8, 2 x 150 mm, Dionex; gradient: 10-80% acetonitrile
in water with 0.1% formic acid) with direct injection into an ESI mass spectrometer
(amaZon ETD, Bruker Daltonik, Fahrenheitstr. 4, D-28359 Bremen). The
15 predominant molecular ion observed at m/z 684 corresponded to the twelve
times protonated target molecule (Figure 5).
Example 4:
Production of the recombinant polypeptide LserFCPl-73 in Escherichia coli
E. coli cells of the strain BL21(DE3) (Novagen/Merck) were transformed with the
2 0 plasmid pET-32-FCP-EK constructed as described in Example 2. The cuituring of
the cells and the processing of the cell lysate were performed by analogy with
the operations described in Example 3, except that 0.1 units of enterokinase
(Novagen) were employed for cleaving the fusion polypeptide. In this case too, it
was possible to separate the target molecule by anion-exchange chromatog-
25 raphy (Figure 6). However/ the mass-spectrometric analysis (micrOTOF I I,
Bruker Daltonik, Fahrenheitstr. 4, D-28359 Bremen) showed that the expected
polypeptide LserFCPl-77 had not been formed (Figure 6). The data showed
clearly that the polypeptide that had formed was LserFCPl-73 having a molecular
mass of 7755 Da. This is to be explained by the fact that the last four amino
3 0 acid residues of the LserFCPl-77 sequence had been cleaved off, apparently by a
non-characteristic activity of the enterokinase.
- 21 -
Example 5:
Determination of the activity of LserFCPl-77 against Fusarium graminearum
Fusarium graminearum (strain IFA 65)
Source of supply: Interuniversitary Department for Agricultural Biotechnology of
5 Tulln, Austria
Reference: Steiner B, Kurz H, Lemmens M, Buerstmayr H. Differential gene
expression of related wheat lines with contrasting levels of head blight resistance
after Fusarium graminearum inoculation. Theor Appl Genet. 2009 Feb; 118(4):
753-64.
10 Fusarium graminearum was cultured on SNA-agar plates at room temperature to
sporulation. The spores were swept off the plate with water, adjusted to a
density of 20,000 spores/ml, and stored at 4 °C until further use. For performing
the tests, 0.05 ml each of the spore suspension was combined with 0.05 ml each
of differently concentrated LserFCPl-77 solutions in the wells of a 96-well
15 microtitration plate. After incubation for 24 h at room temperature, the spores
were examined microscopically for germination using objective lenses with 4 fold
and 10 fold magnifications. The result was documented photographically (Figure
7). The proportion of germinated and non-germinated spores was counted, and
the data obtained were used for the graphic representation of the dose-effect
20 relationship (Figure 8). The thus established concentration in which a halfmaximum
inhibition occurred was at 1.6 uM. In addition, the length of the
hyphae formed by the germinated spores was observed as a semiquantitative
measure of the inhibition (Figure 7).
Example 6:
25 Determination of the activity of LserFCPl-73 against Fusarium graminearum
The effectiveness of LserFCPl-73 at a concentration of 100 uM was determined
as compared to a water control by means of the test system described in
Example 5. A complete inhibition of spore germination by LserFCPl-73 was
observed (Figure 9).
- 22 -
Example 7:
Determination of the activity of LserFCPl-77 against Phytophthora parasitica
. Phytophthora parasitica (isolate 329)
Source of supply: INRA (institut national de la recherche agronomique), France
5 Reference: Keller H, Pamboukdjian N, Ponchet M, Poupet A, Delon R, Verrier JL,
Roby D, Ricci P. Pathogen-induced elicitin production in transgenic tobacco
generates a hypersensitive response and nonspecific disease resistance. Plant
Cell. 1999 Feb; 11(2): 223-35.
Phytophthora parasitica was cultured on Rye B agar plates for 8 days at 25 °C.
io The sporangia were washed off with water, and the sporangia suspension
obtained was incubated for 4 h at 4 °C to induce the formation of zoospores.
After 1:50 dilution in RPMI 1640 medium, the spore density was determined and
adjusted to 20,000 spores/ml. The further performance of the test was effected
by using the methods described in Example 5. The result was also documented
15 photographically (Figure 10).
Sequence Listings
The amino acids were abbreviated according to the IUPAC nomenclature as
follows: alanine A, arginine R, asparagine N, aspartic acid E, cysteine C, glutamic
acid D, glutamine Q, glycine G, histidine H, isoleucine I, leucine L, lysine K,
20 methionine M, phenylalanine F, proline P, serine S, threonine T, tryptophan W,
tyrosine Y, valine V
SEQ ID NO: 1- LserFCPl-77
QHGYGAGGHGQQGYGSQHSSHAPQGGHWREQGFSGHVHEQQAGHHHEAGHHEQAGHHEQSG
QQVHGQGHGYKSHGY
25 SEQ ID NO: 2 - LserFCPl-73
QHGYGAGGHGQQGYGSQHSSHAPQGGHWREQGFSGHVHEQQAGHHHEAGHHEQAGHHEQSG
QQVHGQGHGYK
SEQ ID NO: 3 — L sericata cDNA coding for LserFCPl-77
5'-CAACACGGCTATGGTGCCGGTGGCCATGGCCAACAAGGCTATGGTAGCCAACATAGCAG
3 0 TCATGCTCCCCAAGGTGGACATGTTGTCCGTGAACAAGGTTTTAGTGGTCATGTTCATGAAC
- 23 -
AACAGGCTGGGCATCATCATGAAGCTGGCCATCATGAGCAAGCTGGTCATCATGAACAATCT
GGTCAACAAGTTCATGGTCAAGGTCATGGCTATAAAAGTCATGGTTAT-3'
SEQ ID NO: 4 — synthetic gene with. E. coli adapted codon usage coding for
LserFCPl-77

We claim:
1. A polypeptide comprising an amino acid sequence being identical with at
least 12 contiguous amino acid residues of SEQ ID No. 2.
2. The polypeptide according to claim 1, comprising an amino acid sequence
having at least 65% sequence homology with amino acid sequence SEQ ID
No. 2.
3. The polypeptide according to claim 1 or 2, which leads to a half-maximum
inhibition of the spore germination of Fusarium graminearum at a concentration
of 1 to 1000 uM, especially 2 uM.
4. The polypeptide according to any of claims 1 to 3, comprising an amino acid
sequence being identical with SEQ ID No. 1 or SEQ ID No. 2.
5. The polypeptide according to at least one of claims 1 to 4, which is effective
against the growth of organisms of the genera Fusarium and/or Phytophthora.
6. The polypeptide according to at least one of claims 1 to 5, wherein the N
terminal is derivatized by partial or complete alkylation, or by acylation.
7. The polypeptide according to at least one of claims 1 to 6, wherein the C
terminal is derivatized by amidation or esterification.
8. The polypeptide according to at least one of claims 1 to 7, wherein the
peptide chain is derivatized by PEGylation or HESylation.
9. The polypeptide according to at least one of claims 1 to 8, having the
following sequence
Zi-QHGYGAGGHGQQGYGSQHSSHAPQGGHWREQGFSGHVHEQQAGHHHEAGHHEQA
GHHEQSGQQVHGQGHGYK- Z2,
- 25 -
where
Zi is the N-terminal end of the polypeptide, or a derivative of the terminal
amino group of the polypeptide, or a chain of up to ten arbitrary amino acids;
Z2 is the C-terminal end of the polypeptide, or a derivative of the terminal
carboxy group of the polypeptide, or a chain of up to ten arbitrary amino acids.
10. The polypeptide according to at least one of claims 1 to 9, wherein Z2 has an
amino acid sequence of SHGY-Z3, wherein Z3 is the C-terminal end of the
polypeptide, or a derivative of the terminal carboxy group of the polypeptide,
or a chain of up to six arbitrary amino acids.
11. The polypeptide.according to at least one of claims 1 to 10, wherein the
polypeptide has D-amino acids in part or completely, or has a D-retroinverso
peptide structure.
12. A polynucleotide coding for the polypeptide according to claim 1 or 11.
13. Use of the polypeptide according to at least one of claims 1 to 11 or of a
polynucleotide coding for such a polypeptide and having a sequence that
hybridizes under conditions of stringency using 0.2xSSC at 42 °C for washing
with an oligo- or polynucleotide probe selected from the group:
the complementary strand to nucleotide residues 1 to 231 from SEQ ID No.
3 ;
the complementary strand to nucleotide residues 1 to 102 from SEQ ID No.
3 ;
the complementary strand to nucleotide residues 103 to 331 from SEQ ID
No. 3;
the complementary strand to nucleotide residues 18 to 251 from SEQ ID No.
4 ;
the complementary strand to nucleotide residues 18 to 119 from SEQ ID No.
4;
- 26 -
the complementary strand to nucleotide residues 120 to 251 from'SEQ ID
No. 4;
for controlling fungi, especially fungi causing plant diseases; organisms of
the genera Fusarium and/or Phytophthora; organisms of the class
Peronosporomycetes.
14. A process for preparing a polypeptide according to at least one of claims 1
. to 11.
15. A cell, except for wild type cells of Lucilia sericata, wherein said cells contain
a nucleic acid for the expression of a polypeptide according to either of
claims 1, 2 or with the SEQ ID Nos. 1 or 2.
16. A transgenic crop containing a cell according to claim 15.