WO2014098088A1

WO2014098088A1 - Antifungal agent

Info

Publication number: WO2014098088A1
Application number: PCT/JP2013/083783
Authority: WO
Inventors: 森山　裕充; 俊一浦山
Original assignee: 国立大学法人東京農工大学
Priority date: 2012-12-19
Filing date: 2013-12-17
Publication date: 2014-06-26
Also published as: JPWO2014098088A1

Abstract

The purpose of the present invention is to provide a polypeptide having strong antibacterial activity. Furthermore, the purpose of the present invention is to provide a nucleic acid that encodes said polypeptide. In addition, the purpose of the present invention is to provide an antifungal composition containing the polypeptide, nucleic acid, or the like. The present invention provides: a peptide that has the ability to inhibit the growth of fungi, and comprises the amino acid sequence of SEQ ID NO:2 that includes at least from isoleucine at position 16 to glutamine at position 435; and functional analogs of said peptide.

Description

Antifungal

The present invention relates to a polypeptide useful for controlling the growth of pathogenic or phytopathogenic fungi and a nucleic acid encoding the same.

Fungi cause various diseases. For example, these diseases include circulatory organs, respiratory organs, digestive organs, urinary tract or meninges, carini pneumonia, candidiasis, cryptococcosis, aspergillus (filamentous mycosis), mucormycosis (zygomycosis) and Includes deep mycosis such as penicillosis. In particular, since carini pneumonia can be fatal to a patient whose biological defense ability is reduced due to AIDS infection or the like, the treatment and prevention of the infection is an important medical target. Other effective treatments for deep skin mycosis such as sporotricosis, chromomycosis (mycomycosis), mycoma (mycetoma) and superficial mycosis such as ringworm, cutaneous candidiasis, oral candidiasis and There remains a need to provide preventive measures. That is, since both fungi and humans are the same eukaryotes, many of the cellular mechanisms of both are common. Therefore, it is considered relatively difficult to find compounds that exhibit selective cytotoxic activity only on fungal cells.

There is no time to enumerate plant diseases caused by fungi. For example, rice blast disease, rice sesame leaf blight, coat blight, wheat leafy mildew, powdery mildew; citrus sunspot and scab; apple monilia, brown spot and red blight; pear Ring-rot and body blight; peach leaf blight, ash scab and black scab; grape black rot and late rot; cucurbit vine blight and vine split; tomato leaf mold; eggplant Brown rot; Brassicaceae white rust and vitiligo; Onion gray rot; Strawberry snake eye disease; Potato summer plague; Soybean stem blight and purpura; And plant diseases such as Helmintosporium leaf blight, but these are just a few of the fungal plant diseases known so far. That is, fungal infection is one of the most important risk factors for plant survival, and therefore selective and effective control of phytopathogenic bacteria continues to be a fundamental requirement in plant cultivation.

In recent years, “killer factors” have attracted attention as compounds that selectively inhibit fungal growth. In certain yeasts, there are killer strains that kill other strains and sensitive strains that are killed by these strains, and the proteins responsible for this phenomenon are called killer factors. Non-Patent Document 1 discloses the use of “HM-1”, which is a killer factor of Hansenula yeast, as an antifungal agent. HM-1 is thought to bind to the mannoprotein or sugar chain structure of the cell wall and inhibit glucan synthase. The same document also uses the “K1 killer factor” of the genus Saccharomyces , the killer factor of Kluyveromyces lactis , and the “SMKT” killer factor of the genus Pichia as an antibacterial agent. The prospect of doing it. Further, the technology for applying the killer factor “HM-1” to an antifungal agent is also described in

Patent Documents

1 and 2. Further, Patent Document 3 describes the application of the killer factor of Kluyveromyces lactis. Some of these killer factors are known to be encoded by cytoplasmic nucleic acid molecules, ie virus-like elements (VLE), that are replicated and transcribed independently of the nucleus by the enzyme they encode. (Non-Patent Document 2).

In connection with the selective control of plant pathogens using nucleic acid derived from a virus, Patent Document 4, dsRNA of the coding strand of chestnut blight fungus (Cryphonectria parasitica) in hypo virus is a type of mycovirus infects (Hypovirus) Has been able to reduce the phytopathogenicity of the fungus by transfecting to a natural phytopathogenic strain. And it is supposed that the disease caused by a highly toxic phytopathogenic strain can be reduced by infecting the plant with such a transfection phytopathogenic strain simultaneously.

Recently, the present inventors have found a novel mycovirus that is endogenously present in a specific rice blast strain. This mycovirus (hereinafter sometimes referred to as “MoCV1”) has four types of double-stranded RNAs of 2.8 to 3.6 kb that differ from any of the known mycovirus RNA base sequences. It was. Moreover, the number of lesions could be remarkably reduced by inoculating rice plants with conidia of Magnaporthe oryzae infected with MoCV1 together with conidia of highly toxic rice blast fungi (Patent Document 5). .

Furthermore, the present inventors have reported that Saccharomyces cerevisiae overexpressing ORF4 encoding the P70 protein of the four main proteins of MoCV1 is morphologically abnormal, such as cell hypertrophy and intracellular granulation, and poor colonization. (Non-Patent Document 3). In addition, it has been shown that MoCV1 can exist extracellularly unlike the mycovirus reported so far. As for the P70 protein, the full-length protein (P84) is expressed at the initial stage of culture, but it has been confirmed that the P70 protein is processed into the P70 protein as the culture is prolonged (Non-patent Document 3). .

However, none of the prior literatures disclose or suggest the function of the partial peptide of the protein encoded by ORF4 of MoCV1.

JP-A-10-75789 International Publication No. WO2007 / 023782 Pamphlet JP 2007-228937 A US Pat. No. 5,665,581 International Publication No. WO2009 / 093409 Pamphlet

An object of the present invention is to provide a polypeptide having strong antibacterial activity. Another object of the present invention is to provide a nucleic acid encoding the polypeptide. Furthermore, this invention aims at providing the antifungal composition containing the said polypeptide or nucleic acid.

It was found that the partial polypeptide of the ORF4 gene translation product of MoCV1 (SEQ ID NO: 2) still retains fungal growth inhibitory activity. More surprisingly, such partial polypeptides showed higher antifungal activity than the full-length ORF4 gene translation product. Therefore, the first aspect of the present invention is
[1] A polypeptide having the ability to inhibit fungal growth, which has the following amino acid sequence:
1) a partial sequence of the amino acid sequence represented by SEQ ID NO: 2 and comprising at least the 16th position of SEQ ID NO: 2—isoleucine to 435 position—glutamine;
2) Amino acid sequence showing at least 60% homology with respect to 1) above, or 3) Deletion, substitution, insertion or addition of one or several amino acids to the amino acid sequence of 1) above An amino acid sequence having,
The polypeptide consisting of:
It is.

A typical partial polypeptide of 1) above is an amino acid sequence obtained by truncating positions 1 to 15 of SEQ ID NO: 2, as shown in Examples below; The amino acid sequence is truncated. Accordingly, a preferred embodiment of the present invention is
[2] The polypeptide of [1] above, wherein the amino acid sequence of 1) above is the sequence from SEQ ID NO: 2 from 16-isoleucine to 812-serine, and [3] the amino acid sequence of 1) above The polypeptide according to [1] above, which is a sequence from SEQ ID NO: 2 from position 16-isoleucine to position 435-glutamine,
It is.

The polypeptide of the present invention can be suitably produced by overexpressing the nucleic acid encoding it in an appropriate host cell. Therefore, the second aspect of the present invention is
[4] A nucleic acid encoding the polypeptide according to any one of [1] to [3] above,
[5] A vector in which the nucleic acid according to [4] is operably linked, and [6] a host cell transformed with the vector according to [5],
It is.

The polypeptide and nucleic acid of the present invention can be suitably used as an active ingredient of an antifungal composition. In particular, it is advantageous to use these compositions for the treatment of infections by fungi and the control of plant diseases by phytopathogenic fungi. Accordingly, a further aspect of the present invention is
[7] An antifungal composition comprising the polypeptide according to any one of [1] to [3] above, the nucleic acid according to [4] above, or the vector according to [5] above,
[8] The composition according to [7] above, which is a pharmaceutical composition containing a pharmaceutically acceptable carrier, and [9] the agricultural composition according to [7] above, which includes an agriculturally acceptable carrier. Composition,
It is. In other words, this aspect of the present invention is the polypeptide according to any one of the above [1] to [3], which is used for inhibiting fungal growth. Or it is a nucleic acid as described in said [4] used in order to inhibit the growth of fungi. Furthermore, the vector according to the above [5], which is used for inhibiting fungal growth.

Furthermore, the present invention contemplates a method of inhibiting harmful fungal growth in a subject that may be an animal or a plant. That is, another aspect of the present invention is
[10] A method for treating an infection caused by a fungus, wherein the polypeptide according to any one of [1] to [3] above is used for a patient in need of such treatment, Administration of the nucleic acid according to [5] above, and [11] a method for controlling plant diseases caused by fungi, wherein the plant individual needs such treatment. On the other hand, applying the polypeptide according to any one of [1] to [3], the nucleic acid according to [4], the vector according to [5] or the host cell according to [6]. Including the method,
It is.

Since the polypeptide of the present invention exhibits a remarkable fungal growth inhibitory activity, the polypeptide and the nucleic acid encoding it are useful as active ingredients of antifungal agents. In addition, since the minimum length of the polypeptide of the present invention is about half of the original ORF4 gene translation product, such a minimum polypeptide contains the sequence of the cytotoxic activity domain in the P70 protein encoded by the ORF4 gene. It is thought that it more accurately reflects the structure. Therefore, the polypeptide of the present invention can be used in studies for elucidating the mechanism of fungal growth inhibition by MoCV1 virus. Further, such a minimal polypeptide can be used as a lead substance for a further highly active polypeptide.

FIG. 1 shows the nucleotide sequence of MoCV1-dsRNA4 cDNA (SEQ ID NO: 1). FIG. 2 shows the amino acid sequence of the ORF4 gene translation product of MoCV1 virus (SEQ ID NO: 2). FIG. 3 shows a map of plasmid pRS426. FIG. 4 shows a map of the expression plasmid pRST426. FIG. 5 shows the procedure for constructing the expression plasmid pRST426. FIG. 6 shows the results of growth tests (changes in pH, turbidity, dissolved oxygen concentration, glucose concentration, and viable cell number over time) of yeast into which full-

length ORFs

4, 1B, and 24 were introduced. As a negative control, only an empty vector was introduced. In all graphs, ■ represents a negative control. Δ represents the full length ORF4. * Represents Reference Example (24). O represents Example 1 (1B). FIG. 7 shows the results of cell morphology observation and colony morphology of a yeast into which full-

length ORFs

4, 1B and 24 have been introduced. In the figure, “pRST426” is a negative control strain into which an empty vector was introduced. “PRST426-ORF4” is a transformant expressing full-length ORF4. “PRST426-1B” is a transformant expressing the partial ORF4 (position 1-15 truncated) of the present invention. “PRST426-24” is a transformant that expresses a region other than the partial peptide of the present invention. The white bar in the micrograph of the cell morphology corresponds to 10 μm. FIG. 8 shows the result of Western blotting analysis using a protein extracted from yeast introduced with full-length ORF4, 1B and 24 and using an anti-ORF4p antibody. All samples are insoluble fractions. The numbers at the top indicate the culture time. The gel used is an 8% gel. From the 24 region sample, a signal is seen at a size of approximately 28 kDa. FIG. 9 shows the results of a growth test (change in pH, turbidity, and number of viable cells over time) of yeast into which full-

length ORFs

4, 1B, and 13 were introduced. In all the graphs, Δ represents the full length ORF4. O represents Example 1 (1B). ◇ represents Example 2 (13).

Polypeptide The polypeptide of the present invention is a partial polypeptide containing a partial sequence of the ORC4 gene translation product of the MoCV1 virus. As used herein, the term “partial sequence” means that in any case the sequence containing the sequence does not match the sequence of the full-length ORF4 gene translation product. Similarly, the term “partial polypeptide” means that in any event the polypeptide is not identical in amino acid sequence to the full-length ORF4 gene translation product. Typically, the polypeptides of the invention are shorter than the full-length ORF4 gene translation product.

As mentioned above, it was a surprising discovery that such a partial polypeptide exhibited higher antibacterial activity than the original full-length ORF4 gene translation product.

Specifically, as shown in Examples below, the partial polypeptide obtained by truncating amino acid residues from the 1st position to the 15th position of the ORF4 gene translation product expresses the polypeptide more than the full-length ORF4 gene translation product. The increase in viable cell count and turbidity as well as the decrease in pH and glucose concentration were further suppressed. Similarly, a partial polypeptide obtained by truncating all amino acid residues from the 1st to 15th positions and the 436th and subsequent positions of the ORF4 gene translation product also expresses the polypeptide more than the full-length ORF4 gene translation product. The increase in viable cell count and turbidity and the decrease in pH were further suppressed in the culture. Therefore, these results mean that the activity of inhibiting fungal growth is increased by deleting the amino acid on the N-terminal side of the ORF4 gene translation product. And it may be suitable that such deletion of amino acids includes at least deletions from the 1st position to the 15th position of the ORF4 gene translation product.

On the other hand, as described later (Reference Example 2), it is also clear that the partial polypeptide consisting of amino acid residues from position 436-glycine to position 693-cysteine of the ORF4 gene translation product significantly promotes fungal growth rather. (The inventors have disclosed and claimed this invention in a Japanese patent application having the same priority claim and filing date as the present application.) The coexistence of a part that inhibits the growth of host cells and a part that promotes growth on a single polypeptide chain was a more surprising discovery, but from the viewpoint of the survival of the virus species, Failure to completely kill the host may be a reasonable strategy for MoCV1 virus. In any case, it is considered that the C-terminal side of the ORF4 gene translation product is not important for the activity of inhibiting fungal growth.

Therefore, the minimum unit of the polypeptide of the present invention consists of the following sequence from ORF4 gene translation product from position 16-isoleucine to position 435-glutamine.

The DNA sequence corresponding to the ORF4 gene of MoCV1 virus (that is, since the MoCV1 virus is a dsRNA virus, the gene is originally RNA) and the amino acid sequence of the full-length translation product thereof are disclosed in Patent Document 5, respectively. It is disclosed as SEQ ID NO: 3 and SEQ ID NO: 7 in the same document. Patent Document 5 discloses that the dsRNA fragment of La France disease virus has homology with the ORF4 gene of MoCV1 virus. Accordingly, a person skilled in the art obtains a polypeptide having high amino acid sequence homology with the partial polypeptide of the present invention containing at least the 16th-isoleucine to 435-glutamine sequences of the ORF4 gene translation product, from which a fungus is obtained. Those having the ability to inhibit the growth of can be easily identified. Such polypeptides are also within the scope of the present invention. The homology of the amino acid sequence may be 60% or more, 70% or more, or 80% or more. In particular, polypeptides showing 90% or more amino acid sequence homology are preferred. In this specification, the homology of amino acid sequences is determined by searching using the default parameters (matrix = Blosum62; gap existence cost = 11, gap extension cost = 1) on the Internet site http: // www. ncbi. n / m. nih. Defined as the percentage of positives shown by the BLASTP algorithm, which can be implemented with gov / egi-gin / BLAST.

Further, the present invention has a deletion, substitution, insertion or addition of one or several amino acids with respect to the partial polypeptide containing at least the 16th-isoleucine to 435-glutamine sequences of the ORF4 gene translation product of the present invention. Polypeptides can also be used in the present invention. For example, substitution of leucine for valine, lysine for arginine, and glutamine for asparagine may not change the function of the polypeptide. Accordingly, it is within the scope of the present invention that such polypeptides have the ability to inhibit fungal growth.

Nucleic acid The polypeptide of the present invention can be easily obtained by overexpressing a nucleic acid encoding the amino acid sequence in an appropriate host. Such overexpression can also take place in situ, ie in the cells of the individual to be treated or protected. A nucleic acid that can be used for such purposes will have at least the nucleotide sequence from position 207-adenine to position 1466-guanine of SEQ ID NO: 1 herein. The portion is a natural type sequence encoding the amino acid sequence from position 16-isoleucine to position 435-glutamine of the ORF4 gene translation product. However, as long as the nucleic acid of the present invention is translated into the above-described polypeptide of the present invention, it may have all mutations that can occur in the natural world, and artificially introduced mutations and modifications. For example, it is known that there are extra codons in various codons that code for specific amino acids. Therefore, in the present invention, alternative codons that are finally translated into the same amino acid may be used. That is, since the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, so that the amino acid sequence can be encoded with any set of similar DNA oligonucleotides. Only the only member of the set is identical to the gene sequence of the native enzyme, but even mismatched DNA oligonucleotides hybridize under appropriate stringency conditions (eg 3 × SSC, 68 ° C., 2 × SSC, 0.1 Can be hybridized to the native sequence by washing with% SDS and 68 ° C.), and the DNA encoding the native sequence can be identified and isolated, and such genes can also be used in the present invention. In particular, most organisms are known to preferentially use a specific subset of codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.). Performing “codon optimization” may also be useful in the present invention.

Since the polypeptide of the present invention is derived from a dsRNA virus protein, the nucleic acid of the present invention may be not only DNA but also RNA. Furthermore, the nucleic acid of the present invention may be modified DNA and modified RNA. That is, the nucleic acid of the present invention may be labeled with a fluorescent substance such as Cy3 or Cy5, a chemiluminescent substance, or the like, depending on the application. Alternatively, the nucleic acid of the present invention may be prepared as a stable DNA derivative such as phosphorothioate or methylphosphonate, or a stable RNA derivative such as 2'-O-alkyl RNA. All of these nucleic acids are also included in the nucleic acids of the present invention.

Vectors However, nucleic acids of the present invention, by being introduced into the host cell as an "expression cassette" Those skilled in the art will understand that to achieve a more stable high-level polypeptide production of the present invention . As used herein, “expression cassette” means a nucleotide comprising a nucleic acid to be expressed or a nucleic acid sequence that regulates transcription and translation operably linked to a gene to be expressed. Typically, the expression cassette of the present invention comprises a promoter sequence 5 ′ upstream from the coding sequence, a terminator sequence 3 ′ downstream, and optionally further normal regulatory elements, operably linked, such as In some cases, the nucleic acid to be expressed or the gene to be expressed is “operably” introduced into the host.

A promoter is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis, whether it is a structural promoter or a regulated promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is also preferably used in the present invention. For example, when the host cell is a fungal cell, TDH3, ADH1, ADC1, MFa, AC, P-60, CYC1, GAPDH, amylase gene and trpC promoters can be used, and TDH3 promoter is preferred. When the host is an animal cell, examples include SV40 gene promoter, adenovirus major late gene promoter, thymidine kinase gene promoter, metallothionein gene promoter, immunoglobulin gene promoter, and the like. When the host is a plant cell, 35S transcript derived from CaMV, maize ubiquitin, nopaline synthase (NOS) gene, octopine (OCT) synthesis gene promoter and the like can be mentioned. For prokaryotic host cells, the lac system, trp system, TAC or TRC system, lambda phage major operator and promoter region, fd coat protein control region, glycolytic enzymes (eg, 3-phosphoglycerates) Kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamate decarboxylase A, a promoter for serine hydroxymethyltransferase, and the like can be used. In addition to promoter and terminator sequences, examples of other regulatory elements that may be mentioned include selection markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are described, for example, in “Gene Expression Technology: Methods in Enzymology 185”, Academic Press (1990).

The expression cassette described above is inserted into a host cell after being incorporated into a vector comprising, for example, a plasmid, virus, phage, transposon, IS element, fasmid, cosmid, or linear or circular DNA. These vectors may be autonomously replicated in the host cell or may be replicated by chromosome. Suitable vectors for fungal host cells include pRS426 (available from ATCC), pAUR101 and pAUR316 (available from Takara BIO Inc.), pYepSec1, pMFa, pJRY88, pYES2 and “Gene transfer system: fungi ", Applied Molecular Genetics of Fungi, J. F. Peberdy et al. Eds. , P. 1-28, Cambridge University Press. Suitable vectors for animal host cells include pCDM8 and pMT2PC, as well as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, and the like. Suitable vectors for plant host cells include binary vector plasmids such as pBE2113Not, pBI2113Not, pBI2113, pBI101, pBI121, pGA482, pGAH, pBIG, and pGreen, and intermediate vector plasmids such as pLGV23Neo, pNCAT, and pMON200. Can be mentioned. Suitable plasmids for prokaryotic host cells are, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKK30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG90, pU2R2 pIN-III113-B1, λgt11 or pBdCI; Neisseria gonorrhoeae pUB110, pC194 or pBD214; Corynebacterium pSA77 or pAJ667. Other usable plasmids are described in “Cloning Vectors”, Elsevier, 1985. The expression cassette can be introduced into a vector by a conventional method including excision, cloning, and ligation with an appropriate restriction enzyme.

As a technique that can be applied when the vector having the expression cassette of the present invention is constructed as described above and then introduced into a host microorganism, for example, coprecipitation, protoplast fusion, electroporation, retrovirus transfection, etc. Conventional cloning and transfection methods are used. Examples thereof are “Current Protocols in Molecular Biology (Current Protocols in Molecular Biology)”, F.M. Ausubel et al., Publ. Included in Wiley Interscience, New York, 1997, or Sambrook et al., “Molecular Cloning: Laboratory Manual”, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, ColdNbSrH, 1987.

Pharmaceutical Composition The polypeptide of the present invention can be produced by incubating the host cell transformed with the vector of the present invention as described above under the optimal culture conditions for the cell. Thereafter, the polypeptide of the present invention can be purified from the culture by combining centrifugation, salting out, pH precipitation, dialysis, and various types of chromatography. For example, a His tag may be added to the end of the polypeptide of the present invention, and it may be purified by affinity chromatography. Therefore, the active ingredient of the pharmaceutical composition of the present invention can be the polypeptide of the present invention. In addition, the polypeptide of the present invention can be produced by directly transforming fungal cells whose growth should be inhibited with the nucleic acid of the present invention operably linked in a fungal vector, so that the fungus itself can produce the polypeptide. Also good. Such production will have fatal consequences for the transformed fungus. Furthermore, the polypeptide of the present invention can be produced in situ by transforming cells of an individual to be treated or protected with the vector of the present invention. Therefore, the active ingredient of the pharmaceutical composition of the present invention may be the nucleic acid of the present invention or the vector of the present invention.

Accordingly, the pharmaceutical composition of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to an effective amount of the polypeptide, nucleic acid or vector of the present invention.

Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, citric acid Fragrances such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspensions such as methylcellulose and polyvinylpyrrolide, dispersants such as surfactants, water And diluents such as physiological saline, base wax and the like, but are not limited thereto.

When the active ingredient is a nucleic acid or a vector, the pharmaceutical composition of the present invention can further contain a nucleic acid introduction reagent. As the reagent for introducing nucleic acid, cationic lipids such as atelocollagen, ribosome, nanoparticle, lipofectin, lipfectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDAB, HDAB, polybrene, or polyethyleneimine are used. I can do it. That is, by including the nucleic acid or vector of the present invention in atelocollagen or the like, the nucleic acid or vector of the present invention can be efficiently delivered to the cell in which the polypeptide of the present invention is to be produced, and efficiently incorporated into the cell. Can do.

The pharmaceutical composition of the present invention can be administered to mammals orally or parenterally, but is preferably administered parenterally. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. The preparation can be enclosed in a container such as an ampoule or a vial by unit dose or multiple doses. In addition, the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried together and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use. As another preparation suitable for parenteral administration, a spray etc. can be mentioned.

As the content of the active ingredient in the pharmaceutical composition of the present invention, for example, about 0.1 to 100% by weight of the whole pharmaceutical composition is exemplified. The dosage of such a pharmaceutical composition varies depending on the administration method, the type and size of infection, and the situation of the subject of administration (sex, age, weight, etc.). The amount of polypeptide may be about 0.1 to 100 mg / kg administered once to several times a day. When the active ingredient is a nucleic acid or a vector, the amount of the nucleic acid or vector is about 1 to 10 nmol / kg administered about 1 to 10 times.

As fungi for which the pharmaceutical composition of the present invention is effective, Aspergillus genus such as Aspergillus fumigatus and Aspergillus flavus; Pneumocystis carinii; Kumonskabi genus; Coccidioides spp .; Blastomyces spp .; Paracoccidioides spp. Cryptococcus genus such as Formans; Pseudolesia genus; Sporotrix genus; Black fungus; Trichophyton genus; Microsporum genus; Epidermophyton genus; A genus; Honsenkaea spp; Fusarium; Paecilomyces spp; Trichosporon such Trichosporon Kutaneumu; Hiarohora genus; and may be exemplified Cladosporium and the like. Diseases caused by these pathogens include fungiemia, respiratory mycosis, gastrointestinal mycosis, uromycosis, fungal meningitis, sporotrichosis, chromomycosis, mycosis ( Mysetoma), normal disease type ringworm, deep ringworm, refractory ringworm, onychomycosis, folding screen, cutaneous candidiasis, oral candidiasis and the like.

As in the case of the agrochemical composition pharmaceutical composition, the active ingredient of the agrochemical composition of the present invention can be the polypeptide of the present invention. Further, by directly transforming a fungal cell whose growth should be inhibited with the nucleic acid of the present invention operably linked in a fungal vector, the fungus itself can produce the polypeptide of the present invention, and May inhibit the growth of the fungus. Further, in Patent Document 5, the number of lesions can be remarkably reduced by inoculating rice with conidia of rice blast fungus ( Magnaporthe oryzae ) infected with MoCV1 together with conidia of highly toxic rice blast fungus. It has been shown. Therefore, a fungus whose growth should be inhibited is transformed with the nucleic acid or vector of the present invention to produce an attenuated strain of the fungus, and a live cell of the attenuated strain thus obtained, preferably a conidia Can be applied to plants to control plant diseases caused by wild-type highly toxic strains. Alternatively, the polypeptide of the present invention can be produced in situ by transforming cells of a plant individual to be protected with the vector of the present invention. Therefore, the active ingredient of the agrochemical composition of the present invention may be the nucleic acid of the present invention or the vector of the present invention, or the host cell of the present invention (for example, conidia derived from attenuated phytopathogenic fungi).

Therefore, the agrochemical composition of the present invention contains any carrier, for example, an agriculturally acceptable carrier, in addition to an effective amount of the polypeptide, nucleic acid or vector of the present invention, or the host cell (conidia etc.) of the present invention. Can be included.

Agriculturally acceptable solid carriers include, for example, animal and vegetable powders such as starch, activated carbon, soybean flour, wheat flour, wood flour, fish flour, milk powder, talc, kaolin, bentonite, zeolite, diatomaceous earth, white carbon, clay, alumina And mineral powders such as calcium carbonate, potassium chloride and ammonium sulfate. Examples of liquid carriers include water, alcohols such as isopropyl alcohol and ethylene glycol, ketones such as cyclohexanone and methyl ethyl ketone, ethers such as propylene glycol monomethyl ether and diethylene glycol mono-n-butyl ether, and aliphatic carbonization such as kerosene and light oil. Hydrogens, xylenes, trimethylbenzenes, tetramethylbenzenes, aromatic hydrocarbons such as methylnaphthalene, solvent naphtha, amides such as N-methyl-2-pyrrolidone, esters such as glycerol esters of fatty acids, soybean oil, rapeseed And vegetable oils such as oil.

Moreover, it is preferable that the agrochemical composition of the present invention contains additional components such as a binder, a thickener, and a sticking agent. Examples include starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylstarch, pullulan, sodium alginate, ammonium alginate, propylene glycol alginate, guar gum, locust Examples include bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene / propylene block polymer, sodium polyacrylate, polyvinyl pyrrolidone and the like. Furthermore, as already explained regarding the pharmaceutical composition, also in the agrochemical composition of the present invention, the nucleic acid or vector of the present invention contains atelocollagen, ribosome, nanoparticle, lipofectin, lipfectamine, DOGS (transfectam), DOPE, DOTAP. , DDAB, DHDAB, HDAB, polybrene, or a nucleic acid introduction reagent comprising a cationic lipid such as polyethyleneimine.

The dosage form of the agrochemical composition of the present invention is, for example, an emulsion, suspension, powder, granule, tablet, wettable powder, aqueous solvent, liquid, flowable, granulated wettable powder, aerosol, paste, oil. Or an emulsion. As content of the active ingredient in the agrochemical composition of this invention, about 0.1 to 100 weight% of the whole agrochemical composition is illustrated, for example. The application amount of the composition includes the concentration and type of the active ingredient, the form of the preparation, the target disease or crop type, the degree of damage caused by the disease, the application location, the application method, the application timing, the drugs or fertilizers to be mixed and used together, etc. What is necessary is just to select suitably according to conditions, such as usage-amount and a kind.

For example, in the case of an agrochemical composition containing the polypeptide of the present invention, the polypeptide of the present invention may be applied at a rate of about 0.01 to 2.0 kg / ha. Application can be repeated as desired, for example, at 8-30 day intervals. In the case of an agrochemical composition containing the nucleic acid or vector of the present invention, the amount of the nucleic acid or vector applied is about 1 mol / kg to 200 μmol / ha about 1 to 10 times.

In the case of an agrochemical composition containing the host cell (conidia etc.) of the present invention, for example, a conidia-containing solution adjusted to 1 × 10 ³ to 1 × 10 ¹⁰ cells / mL is about 10 to 1,000 L / ha. It may be applied at a rate of.

Examples of plant diseases that can be controlled by the agrochemical composition of the present invention include rice blast (causal fungus " Magnaporthe oryzae "), rice sesame leaf blight (causal fungus " Cochliobolus miyabeanus "), and blight (causal fungus " Thanatephorus "). cucumeris "), stupid seedlings (causal fungus" Gibberella fujikuroi "), seedling blight (causal fungi" Fusarium fungus "," Rhizopus fungus "," Pythium fungus "," Trichoderma viride "fungus, rice fungus) Claviceps virens "), red rot of wheat (causal organisms" Gibberella zeae "," Fusarium avenaceum "," Fusarium culmorum "," Monographella nivale "), snow mold disease (causing bacteria" Pythium bacteria "," Typhula bacteria "," Monographella nivalis "," Myriosclerotinia borealis "), loose smut (bacteria causing" Ustilago nuda "), fishy smell smut (causing bacteria "Tilletia controversa"), Memonbyo (bacteria causing "Pseudocercosporella herpotrichoides"), leaf blight (bacteria causing "Septoria tritici"), glume blotch (bacteria causing "Phaeosphaeria nodorum"), powdery mildew (causing bacteria "Blumeria graminis "), sunspot disease of citrus (causative bacteria" Diaporthe citri "), small Black spot disease (causing bacteria "Diaporthe medusa, Alternaria citri"), scab (bacteria causing "Elsinoe fawcettii"), brown rot (bacteria causing "Phytophthora citrophthra"), green mold disease (causing bacteria "Penicillium digitatum"), Blue mold disease (causal fungus " Penicillium italicum "), apple Monilia disease (cause fungus " Monilinia mali "), black star disease (cause fungus " Venturia inaequalis "), spotted leaf disease (causing fungus " Alternaria mali " (black spot disease), black spot disease ( Alternaria mali ) Causative bacteria " Mycosphaerella pomi "), soot spot (causal bacteria " Gloodes pomigena "), soot spot disease (causal bacteria " Z ygophiala jamaicensis "), early blight (bacteria causing" Botryosphaeria berengeriana "), brown spot disease (causing bacteria" Diplocarpon mali "), gymnosporangium (bacteria causing" Gymnosporangium yamadae "), canker (bacteria causing" Valsa ceratosperma ") , pear scab (bacteria causing "Venturia nashicola"), gymnosporangium (bacteria causing "gymnosporangium asiaticum"), early blight (bacteria causing "Botryosphaeria berengeriana"), blight (bacteria causing "Phomopsis fukushii"), peach of leaf curl disease (causing bacteria "Taphrina deformans"), Haihoshibyo (bacteria causing "Monil nia fructicola, Monilinia fructigena "), scab (bacteria causing" Cladosporium c arpophilum "), Homopushisu rot (bacteria causing" Phomopsis sp. "), Haiboshi disease of cherry (bacteria causing" Monilinia fructicola "," Monilinia fructigena "), Yohatekinkakubyo (bacteria causing" Monilinia kusanoi "), plum of scab (bacteria causing" Cladosporium carpophilum "), grapes of sphaceloma disease (causing bacteria "Elsinoe ampelina"), ripe rot (bacteria causing "Colletotrichum acutatum", "Glomerella cingulata"), brown spot disease (causing bacteria "Pseudocercospora vitis"), Fusarium disease (causing bacteria "Phomopsis viticola "), oyster leaf spot disease (causal bacterium" Cercospora kaki "), lobster leaf disease (causal bacterium" Mycosphaer ") ella nawae "), tea of the wheel plaque disease (causing bacteria" Pestalotiopsis longiseta "," Pestalotiopsis theae "), Kasshokuen Hoshibyo (bacteria causing" Pseudocercospora ocellata "," Cercospora chaae "), has disease (causing bacteria" Exobasidium vexans "), the network has disease (causing bacteria" Exobasidium reticulatum "), vine blight of cucurbits (bacteria causing" Mycosphaerella melonis "), Fusarium disease (causing bacteria" Fusarium oxysporum "), scab (bacteria causing" Cladosporium cucumerinum "), brown spot disease (causing bacteria" Corynespora cassiicola "), tomato Leaf blight (bacteria causing "Fulvia fulva"), early blight (bacteria causing "Alternaria solani"), eggplant褐紋disease (causing bacteria "Phomopsis vexans"), Susukabi disease (causing bacteria "Mycovellosiella nattrassii"), cruciferous vegetables of white rust disease (causing bacteria "Albugo macrospora"), white spot disease (causing bacteria "Cercosporella brassicae", "P seudocercosporella capsellae"), onion gray rot (bacteria causing "Botrytis allii"), strawberry bull's-eye Disease (causal fungus " Mycosphaerella fragariae "), potato summer plague (cause fungus " Alternaria solani "), soybean Plague of the stalk (bacteria causing "Phytophthora sojae"), purpura (bacteria causing "Cercospora kikuchii"), stems plague of adzuki bean (bacteria causing "Phytophthora vignae"), peanut brown spot disease (causing bacteria "Mycosphaerella arachidis"), Brown spot disease of sugar beet (cause fungus " Cercospora beticola "), leaf rot (cause " Thanatephorus cucumeris "), buckwheat leaf blight of cabbage (cause fungus " Curvularia fungus"), dollar spot disease (cause fungus " Sclerotinia homoma ") ), Helmintosporium leaf blight (causative " Cochliobolus "), rose scab (causing " Diplocarpon ros ") ae "), chrysanthemum rust (causative fungus" Puccinia horiana "), various crop downy mildews (causal fungus" Peronospora fungus "," Pseudoperonospora fungus "," Plasmopara fungus "," Bremia fungus "), plague Causative fungus " Phytophthora "), powdery mildew (causing fungus " Erysiphe fungus", " Blumeria fungus", " Sphaerotheca fungus", " Podophaerea fungus", " Phyllactinia fungus", " Uncinula fungus", " Uncinula " Rust (causal bacteria " Puccinia ", " Uromyces ", " Physopella "), black spot (causal " Alternaria "), gray mold (causal bacteria " Botrytis ci " nerea "), Sclerotinia rot (bacteria causing" Sclerotinia sclerotiorum "), Shiromon rot (bacteria causing" Rosellinia necatrix "), Murasakimon rot (bacteria causing" Helicobasidium mompa "), Southern blight (bacteria causing" Sclerotium rolfsii "), and various other soil-borne diseases (bacteria causing" Fusarium fungus "," Rhizoctonia bacteria "," Pythium bacteria "," Aphanomyces bacteria "," Phoma fungus "," Verticillium fungus "includes" such as Plasmodiophora brassicae ") .

Other uses As described above, the minimum length of the polypeptide of the present invention is about half of the original ORF4 gene translation product. Therefore, such a minimal polypeptide is considered to more accurately reflect the sequence and structure of the cytotoxic activity domain in the P70 protein encoded by the ORF4 gene. Therefore, the polypeptide of the present invention can be used for studies for elucidating the mechanism of fungal growth inhibition by MoCV1 virus. Further, such a minimal polypeptide can be used as a lead substance for a further highly active polypeptide. For such applications, any of the polypeptides, nucleic acids, and vectors of the present invention can be used as reagents for research. The reagent comprises the polypeptide, nucleic acid or vector of the present invention, for example, a preservative such as sodium benzoate or sodium bisulfite, a stabilizer such as citric acid or sodium citrate, and a dispersant such as a surfactant. It can be obtained by dissolving in a buffer solution having an appropriate pH.

Those skilled in the art given the above explanation can fully practice the present invention. In the following, examples are given for the purpose of further explanation and therefore the invention is not limited to these examples. In the present specification, unless otherwise specified, nucleotide sequences are described in the 5 'to 3' direction, and amino acid sequences are described in the N-terminal to C-terminal direction.

Reference Example 1: Preparation of yeast introduced with full-length ORF4 coding sequence A transformant producing a protein encoded by an open reading frame in the ORF4 gene of MoCV1 was prepared by the following procedure.

<1-a> plasmid pRS426 purchased from the construction of the expression vector ATCC (Figure 3) based on, S. Plasmid pRST426 having a multi-cloning site sandwiched between the promoter region and terminator region of C. cerevisiae TDH3 gene was prepared (FIG. 4). For details, see S.A. cerevisiae W303-1A [LAO] strain was extracted, and the TDH3 gene promoter region (Primer1: forward2 and reverse2) and terminator region (Primer3: forward and Primer 4: reverse) were amplified by PCR. Each was cloned into a commercially available pUC19 plasmid. The PCR conditions were in accordance with the manual for KOD-plus- (trade name, manufactured by TOYOBO). Next, the promoter region was excised from the plasmid containing the promoter region with BlnI1 / BamHI and inserted into the plasmid containing the terminator region cleaved with the same restriction enzymes. The gene expression cassette portion was cut out from this plasmid with NotI / XhoI and inserted into the pRS426 plasmid cut with the same restriction enzymes (FIG. 5).

On the other hand, ORF4 (Primer A: forward and Primer B: reverse) was amplified by PCR using MoCV1-dsRNA4 cDNA (SEQ ID NO: 1) as a template. PCR conditions followed the manual of KOD-plus- (trade name, manufactured by TOYOBO). The PCR amplification product was cloned into the pUC19 plasmid, and the inserted fragment was excised from the plasmid with EcoRI / HpaI. The fragment was inserted into pRST426 cut with the same restriction enzyme to construct an expression vector for full-length ORF4.

<1-b> transformed S. yeast C. cerevisiae W303-1A (Mata ura3 leu2 his3 trp1 ade2 LA) was cultured at 30 ° C., collected by centrifugation, suspended in distilled water, and washed again by centrifugation. SolA (0.1 M Lithium acetate, 10 mM Tris-HCl pH 7.8, 1 mM EDTA) was added to the precipitate, suspended, and centrifuged. SolA was again added to the precipitate to suspend it to obtain a yeast solution. After incubation at 30 ° C. for 50 minutes, heat-treated ssDNA (1 mg / ml, salmon palm) 10 μl, yeast solution 50 μl, the above full-length ORF4 expression vector 10 μl, SolA 20 μl, 50% polyethylene glycerol 750 μl are added in this order. And incubated at 30 ° C. for 30 minutes and 42 ° C. for 15 minutes. After centrifugation, distilled water was added to the precipitate, and the mixture was spread on an SC agar plate medium (a dropout medium was selected according to the selection marker of the shuttle vector) and cultured at 30 ° C.

Example 1: Preparation of yeast in which coding sequence from ORF4 position 16-isoleucine to position 812-serine was introduced In the multi-cloning site of expression vector pRST426 prepared in the above <1-a>, position 16 of ORF4 -A coding sequence from isoleucine to position 812-serine (hereinafter, the sequence and its translation product are abbreviated as "1B") was inserted. Specifically, 1B (PrimerC: forward and PrimerB: reverse) was amplified by PCR using MoCV1-dsRNA4 cDNA (SEQ ID NO: 1) as a template. PCR conditions followed the manual of KOD-plus- (trade name, manufactured by TOYOBO). The PCR amplification product was cloned into the pUC19 plasmid, and the inserted fragment was excised from the plasmid with EcoRI / HpaI. The fragment was inserted into pRST426 cut with the same restriction enzyme to construct a 1B expression vector.

By the same procedure as in the above <1-b>, S. cerevisiae W303-1A (Mata ura3 leu2 his3 trp1 ade2 LA) strain was transformed.

Example 2: Preparation of yeast into which coding sequence from ORF4 position 16-isoleucine to position 435-glutamine was introduced At the multi-cloning site of expression vector pRST426 prepared in the above <1-a>, position 16 of ORF4 A coding sequence from isoleucine to position 435 to glutamine (hereinafter, the sequence and its translation product are abbreviated as “13”) was inserted. Specifically, 13 (PrimerC: forward and Primer D: reverse) was amplified by PCR using MoCV1-dsRNA4 cDNA (SEQ ID NO: 1) as a template. PCR conditions followed the manual of KOD-plus- (trade name, manufactured by TOYOBO). The PCR amplification product was cloned into the pUC19 plasmid, and the inserted fragment was excised from the plasmid with EcoRI / SmaI. The fragment was inserted into pRST426 cut with the same restriction enzymes to construct 13 expression vectors.

Reference Example 2: Preparation of yeast in which coding sequence from ORF4 position 436-glycine to 693-cysteine was introduced In the multi-cloning site of expression vector pRST426 prepared in <1-a> above, position 436 of ORF4 -A coding sequence from glycine to position 693-cysteine (hereinafter, the sequence and its translation product are abbreviated as "24") was inserted. Specifically, 24 (Primer E: forward and Primer F: reverse) was amplified by PCR using MoCV1-dsRNA4 cDNA (SEQ ID NO: 1) as a template. PCR conditions followed the manual of KOD-plus- (trade name, manufactured by TOYOBO). The PCR amplification product was cloned into the pUC19 plasmid, and the inserted fragment was excised from the plasmid with EcoRI / SmaI. The fragment was inserted into pRST426 cut with the same restriction enzymes to construct 24 expression vectors.

In the same procedure as in the above <1-b>, S. cerevisiae W303-1A (Mata ura3 leu2 his3 trp1 ade2 LA) strain was transformed.

Example 3 Growth Test of Yeasts Introduced with Full-length ORF4, 1B, 13 and 24 The growth of each transformed yeast was tested by jar fermenter culture.

<3-a> Comparison of full length ORF4, 1B and 24 Using a microbial culture apparatus (BM2-02NP3, manufactured by ABLE), yeast introduced with the title sequence was cultured and its growth was tested. As a negative control, yeast introduced with pRST426 was used. The medium used was SC-ura liquid medium. Specifically, yeast cultured on an agar medium at 30 ° C. for 3 days was inoculated into 5 ml of liquid medium (test tube), and shake-cultured at 28 ° C. and 145 rpm for 16 hours (pre-culture). This culture solution was added to 50 ml of a liquid medium (200 ml Kolben) so that OD600 = 0.1, and cultured at 28 ° C. and 140 rpm for 12 hours (preculture). This culture solution was inoculated into a microorganism culture apparatus containing 1 L of a liquid medium so that OD600 = 0.03, and cultured under conditions of a temperature of 35 ° C., an air flow rate of 1.5 L / min, and stirring of 100 rpm (main culture). . The dissolved oxygen concentration was measured with a DO sensor (12 mm diameter sealed type L = 220 mm) manufactured by ABLE.

The above culture solution was sampled over time. Specifically, a syringe was connected to the sampling port of the microorganism culture apparatus with a silicon tube, 10 ml of the culture solution was taken out with the syringe, and transferred to a sterile centrifuge tube. A part of the culture solution sample thus obtained was diluted with sterilized water and then spread on an agar plate. After incubating these plates at 28 ° C. for 3 days, the number of colonies formed on the plates was counted to determine the number of viable cells. Further, the absorbance of another part of the culture solution sample was measured with a spectrophotometer (UV-1800, manufactured by Shimadzu Corporation). Next, the sample after measuring the absorbance was centrifuged to separate into a supernatant and cells. The glucose concentration in the supernatant was measured using a biosensor (BF-5, BF-30AS, glucose sensor: ED05-0003, manufactured by Oji Scientific Instruments). Moreover, protein was extracted from the microbial cells, and it confirmed that the target protein was produced by the western blotting using the anti- ORF4p antibody (FIG. 8). Furthermore, cell morphology was observed for some of the cells using a microscope (an inverted research microscope, IX71 Olympus) (magnification 1,000 times, differential interference).

As a test result of the culture solution sample, remarkable growth failure was observed in the 1B-introduced strain. On the other hand, an increase in growth rate was observed in the 24 introduced strain (FIG. 6). In the 24 introduced strains, the increase in turbidity and the increase in CFU were stagnant at around 26 hours of culture, but abnormal aggregation of bacterial cells was confirmed as described below, which was considered to be the effect.

Also, as a result of observation of cell morphology, it was confirmed that cells were enlarged and granulated inside the ORF4 and 1B-introduced strains. On the other hand, in the 24 introduced strain, cell enlargement was not observed so much, but many cells gathered to form a huge cell aggregate (FIG. 7). In addition, the colonies on the plate were remarkably small in size with the 1B-introduced strain, and conversely, with the 24-introduced strain, large uneven colonies were seen (FIG. 7).

<3-b> Comparison of full-length ORF4, 1B and 13 The growth test (pH, turbidity and viable cell number) of <3-a> was carried out on yeasts into which full-length ORF4, 1B and 13 were introduced. As a result, the 1B and 13 introduced strains exhibited growth inhibitory activity superior to the full-length ORF4 in any of the items of pH decrease, turbidity increase and viable cell number increase. In addition, the decrease in pH and the rate of increase in turbidity were the slowest in the 1B-introduced strain. However, the number of viable cells was suppressed as much as the 1B-introduced strain even in the 13-introduced strain, and always showed a low value (FIG. 9).

Since the polypeptide of the present invention exhibits remarkable fungal growth inhibitory activity, it can be used in the pharmaceutical manufacturing industry, agricultural chemical manufacturing industry, agriculture and the like. Further, since it can be used as a research reagent, it can be useful in the chemical manufacturing industry.

Claims

A polypeptide having the ability to inhibit fungal growth, comprising the following amino acid sequence:
1) a partial sequence of the amino acid sequence represented by SEQ ID NO: 2 and comprising at least the 16th position of SEQ ID NO: 2—isoleucine to 435 position—glutamine;
2) Amino acid sequence showing at least 60% homology with respect to 1) above, or 3) Deletion, substitution, insertion or addition of one or several amino acids to the amino acid sequence of 1) above An amino acid sequence having,
The polypeptide comprising:
The polypeptide according to claim 1, wherein the amino acid sequence of 1) above is the sequence from SEQ ID NO: 2 from position 16-isoleucine to position 812-serine.
2. The polypeptide according to claim 1, wherein the amino acid sequence of 1) is the sequence from SEQ ID NO: 2 from position 16-isoleucine to position 435-glutamine.
A nucleic acid encoding the polypeptide according to any one of claims 1 to 3.
A vector to which the nucleic acid according to claim 4 is operably linked.
A host cell transformed with the vector according to claim 5.
An antifungal composition comprising the polypeptide according to any one of claims 1 to 3, the nucleic acid according to claim 4, or the vector according to claim 5.
8. The composition of claim 7, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
8. The composition of claim 7, which is an agrochemical composition comprising an agriculturally acceptable carrier.
A method of treating an infection caused by a fungus, wherein the polypeptide according to any one of claims 1 to 3, the nucleic acid according to claim 4 or the claim for a patient in need of such treatment. 6. The method comprising administering the vector according to item 5.
A method for controlling plant diseases caused by fungi, wherein the polypeptide according to any one of claims 1 to 3, the nucleic acid according to claim 4, for a plant individual in need of such treatment, 7. The method comprising applying a vector according to claim 5 or a host cell according to claim 6.