WO2000035947A1

WO2000035947A1 - Organic compounds

Info

Publication number: WO2000035947A1
Application number: PCT/CN1998/000307
Authority: WO
Inventors: Zhangliang Chen; Qing Xu
Original assignee: The National Laboratory Of Protein Engineering And Plant Genetic Engineering, College Of Life Science, Peking University
Priority date: 1998-12-15
Filing date: 1998-12-15
Publication date: 2000-06-22
Also published as: CN1327451A

Abstract

The present invention is in the area of plant biotechnology. It particularly relates to novel antifungal proteins obtainable from the medicinal herb Gastrodial, but especially Gastrodia elata and the use thereof in combating fungal pathogens.

Description

Organic Compounds

The present invention is in the area of plant biotechnology. It particularly relates to novel antifungal proteins and the use thereof in combating fungal pathogens.

The present invention relates in particular to the following subject matter. An antifungal protein which is

-insensitive to high temperatures in that it is capable of retaining about 75% of its antifungal activity after 30 minutes at 60*C; -rich in Asp, G!y and Leu but does contain essentially no Pro; •is incapable of agglutinating trypsiπ-treatεd rabbit erγthrocytβs; -is capable of binding to chitin, immobilized mannose and N-acεtylglucosamine in SO mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate. The antifungal protein according to the invention, wherein the Asp, Gly and Leu content is about 22%, about 10% and about 9.5%, respβctiveiy. The antifungal protein according to invention, which exhibits an inhibitory activity against Trichoderma viride, Valsa ambiens, Rhizαctonia soiaπi, Gibbenslla zea, Ganoderma lυάdum and Botrytis cinereal, but preferably no inhibitory activity against, Piπcularia oryzae, Helminthospαriυm turcieυm and Fusarium oxysporum The antifungal protein according to the invention, which comprises at the N-terminus the a ino acid sequence given in SEQ ID NO: 1

A DNA comprising a nucleotide sequence encoding for a protein according to the invention.

A host organism, preferably a a microorganism or a plant, comprising stably integrated into its genome a DNA according to the invention.

A plant and the progeny thereof, but preferably a plant selected from the group consisting of maize, sugar beet, cotton, rice, wheat, barley, sorghum, tomato, melon, pepper and Brassica, comprising stably integrated into its genome a DNA comprising a nucieotide sequence encoding for a protein according to the invention. A fungicidal composition comprising a protein or a host organism according the inventions together with a suitable carrier. A method of protecting a plant against a fungal pathogen comprising directly or indirectly applying to said plant a protein or a fungicidal composition according to the invention

A method of protecting a plant against a fungal pathogen, wherein the protein is indirectly applied to the plant by expressing said protein within said plant.

Definitions:

Expression: refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only. preferably expressed: as used herein means that the extent of expression of the DNA molecule from the nuclear genome and from the genome of a cellular organelle having circular DNA is sufficiently different so that plants having stably transformed circular DNA molecules are readily selected for.

Gene: refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

Heterolooous: "heterologous" as used herein means "of different natural or of synthetⁱc origin". For example, if a host cell is transformed with a nucleic acid sequence that does not occur in the untransformed host cell, that nucleic acid sequence is said to be heterologous with respect to the host cell. The transforming nucleic acid may comprise a heterologous promoter, heterologous coding sequence, or heterologous termination sequence.

Alternatively, the transforming nucleic acid may be completely heterologous or may comprise any possible combination of heterologous and endogenous nucleⁱc acⁱd sequences.

Marker gene: a gene encoding a selectable or screenable trait.

Ooerablv linked to/associated v/ith: a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence. Plant: refers to any plant, particularly to seed plants.

-Plant cell: a structural and physiological unit of the plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.

Plant material: refers to ieaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, plastids, mitochondria, cuttings, cell or tissue cultures, or any other part or product of a plant.

Promoter: a DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.

Protoplast: isolated plant cell where the cell wall has been totally or partially removed.

Recombinant DNA molecule: a combination of DNA sequences that are joined together using recombinant DNA technology.

Recombinant DNA technology: procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Screeπable marker αene: a gene v/hose expression does not confer a selective advantage to a transformed cell, but whose expression makes the transformed cell phenotypically distinct from unlransformed cells.

Selectable marker oene: a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells. The selective advantage possessed by the transformed cells, compared to non- transformed cells, may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source. Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant ceil gives the cell both, a negative and a positive selective advantage.

Transformation: Introduction of a nucleic acid into a cell. In particular, the stable integration of a DNA molecule into the genome of an organism of interest.

In the present invention an antifungal protein is provided that exhibits a broad spectrum of inhibitory activity against phytopathogenic fungi and thus can be widely used to combat fungal pathogens, but especially fungal plant pathogens. An antifungal protein as described and claimed herein can, for example, be found in and isolated from the medicinal herb Gastrodial, but especially Gastrodia elata. Gastrodia elata Bl. f. flavida S. Chow is in Gastrodia R. Br. of Gastrodinae, Gastrodieae, Epidendroideaβ, Orchidaceaβ. The cαrrπ of G. elata has been widely used as a tranquilizer and anodyne with no side effects in Chinese medicinal science for about two thousand years. The morphology of G. elata has also been well studied. Tne orchid, G. elata leads a parasitic life on the fungus Anvillaria mellea. There is no chlorophyll in this plant. The hyphae of Ar illaria mellea can infect the nutritive corm of G. elata, but are arrested and digested in the cortical cells. The released nutrients are mainly transported into the terminal corm for its growth. It has been noticed that the developing terminal corm is never infected by the fungus.

The antifungal protein according to the invention is distinguished over know proteins essentially by one or more of the following unique characteristics.

-thermal stability, that is insensitivity to low and high temperatures;

-high content of Asx, Gly and Leu but essentially no Pro;

-incapability of agglutinating trypsin-treated rabbit erythrocytes;

-capability of binding to chitin, immobilized mannose and N-acetylglucosamine;

-inhibitory activity against saprophytic fungi such as, for example, Tπchoderma viήde abd Valsa ambiens as well as some facultative parasite fungi such as, for example,

Rhizoctonia solani, Glbbβrella zea (Gibberella zeae F34, G. zeae F11 , G. zeae JF10,

G. zeae H28), Gaπodetτna lucidum and Botrytis ciner^βa; essentially no inhibitory activity against Piήcularia oryzae, Helminthosporium tυrvieum and Fusariυm oxysporum (Fusarium oxyspαtvm f. sp. vasinfectum). In a preferred embodiment of the present invention the anitfungal protein is in a size range of 8 to 15 kDa, but preferably has a size of about 10 kD. The size of a protein can be approximated by methods known to those skilled in the art, such as, for example, by means of SDS-PAGE in Tris-tricine buffer and gel filtration in 50 mM sodium acetate buffer (pH 5.0) on Superose 12 ™ column on FPLC system.

The preferred antifungal protein according to the invention is further characterized by a a pi of 8.45 (determined by IEF gel) and has preferably no sugar covalently bound to it. As mentioned herein previously, the antifungal protein according to the invention ⁱs capable of binding to chitin_, immobilized mannose and N-acetylglucosamine. This characteπstⁱc property of the antifungal protein according to the invention can be easily tested by a skⁱlled artisan employing methods well known in the art. For example, in a specirfic embodiment of the invention it can be shown that an exemplary antifungal protein of the invention in 50 mM sodium acetate buffer (pH 5.0) with 2 ammonium sulfate can be adsorbed by the mannose-Sepharose 6B column and be eluted with the same buffer with low concentration of ammonium sulfate (0.2 M). The addition of 1 mannose to the loading sample slightly reduces the binding efficiency of the protein, but 1 mannose in 50 M sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate is inefficient for elutiπg the antifungal protein. Said exemplary protein according to the invention can also bind to N-acetylglucosamine- Sepharose 6B and chitin and be eluted in the same condition as the mannose-Sepharose

6B.

The antifungal acitivity of the protein according to the invention can be easily determined using_, for example, an assay as descirbed in the examples involving 7. viride as an indicator organism. In a preferred embodiment of the invention, the antifungal protein exhibits a growth inhibitory activity against saprophytic fungi such as, for example,

Trichoderma viride abd Valsa ambiens as well as some facultative parasite fungi such as^, for example, Rhizoctonia solani_, Gibberella zea (Gibberella zeae F34. G. zeae F11 , G. zeae

JF10. G. zeae H28), Gaπoderma lucidum and Botrytis cinerea, but has essentially no activity against. Piricularia oryzae_, Helminthosporium turcieυm and Fusarium oxysporum

(Fusarium oxysporum f. sp. vasinfectum).

The antifungal protein according to the invention can inhibit the growth^, but especially the hypha_l growth of saprophytic fungi like T. viride, Valsa ambiens and some facultative parasite fungi such as. for example, Rhizoctonia solani, Gibberella zeae F34, G. zeae FH ,

G. zeae JF10, G. zeae H28, but has no effect against some other facultative parasitic fungi such as, for example. Piricularia oryzeae, Helminthosporium turcieum and Fusarium oxysporum f. sp. vasinfectum. Potentially the saprophytic fungi are more significantly inhibited by the protein according to the invention than the facultative specⁱes.

The antifungal protein according to the invention further has an inhibitory effect on spore germination, which can be demonstrated, for example, in a spore germinating test.

A futher characteristic of the antifungal protein according to the invention is its incapability of agglutinating human erythrocytes B type or trypsin-treated rabbit erythrocytes. This specific protperty can be easily tested in a hemagglutiπation assay as described herein ⁱn the examples. The antifungal protein according to the invention is further characterized by its thermal stability. The thermal stability of the antifungal protein can be measured, for example, by its inhibitory activity against T. viride.

In a preferred embodiment of the invention the antifungal protein fully maintains its antifungal activity when it is preserved at 4 °C for one month in 50 mM sodium acetate buffer (pH 5.0). When the protein is incubated at 20 -C and 37 °C for 30 min in the same buffer, there is no activity lost compared with the untreated sample.

The antifungal protein according to the invention is also insensitive to high temperatures. In particular, after high temperature treatments for 30 min, an exemplary antifungal protein according to the present invention still possesses inhibitory activity: between 70% and 80%, but especially about 75 % activity is retained when treated at 60 °C and between 45% and 65%. but especially about 55 % activity is retained when treated at 95 °C depending on the

PH.

The highest stability is obtained at a pH of between about pH 5 and pH 6, as can be demonstrated, for example, in a pH gradient test in disodium hydrogen phosphate-citric acid buffer, as further described in the examples.

The antifungal protein according to the present invention is further characterized by its amino acid content. It is rich in Asx, Gly and Leu, but does not contain Pro.

In a specific embodiment of the invention the antifungal protein has a protein content as shown in table II.

The antifungal protein can be further characterized by its N-terminal sequence, which is shown in SE_Q ID NO: 1 Also comprised within the invention is a protein which is characterized by a N-terminal sequence of SEQ ID NO:1 which has the N-terminal residue Ser truncated and begins with Asp. while the remainder residues of the two polypeptides are identical.

It is a further objective of the present invention to provide a DNA comprising a nucleof.de sequence encoding the antifungal protein according to the invention. Such a DNA can be obtained by methods well known in the art. For example, a cDNA can be obtained by anchored PCR. In a first step, mRNA is prepared from a suitable starting material such as, for example, the corm of aGastrodia elata plant. The cDNA is then synthesized with a cDNA synthesis primer and Reverse Transcriptase. Suitable Adaptors are then linked to the cDNA with DNA ligase. The cDNA is amplified by Adaptor primers and degenerate primers derⁱved from the N-terminal amino acid sequence of the antifungal protein in forward and reverse direction ,o generate the 6'- and 3' part, o. .he cDNA. The PCR products are then inserted into a suitable vecor such as. for example, the pGEM-T vector (Promega) and sequenced. At a suitable restriction enzyme digestion site, the tv»c parts are linked together to obtam the full cDNA.

The coding sequence of the novel antifungal proteins of the present invents can be operably fused to a variety of promoters for expression in a host organism such as a microorganism or a plant including constitutive, inducible, temporally regulated, developmental^ regulated, chemically regulated, tissue-preferred and tissue-specⁱfⁱc promoters to prepare recombinant DNA molecules, i.e.. chimeric genes. Preferred constitutive promoters for expression in plants include the CaMV 35S and 19S promoters (Fraley et a,.. U.S. Patent No. 5.352,605. issued October 4. 1994). An additionally preferred promoter is derived from any one of several of the actin genes^, which are known to be expressed in most cell types. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expressⁱon of the novel antifungal gene and are particularly suitable for use in monocotyledonous hosts.

Yet another preferred constitutive promoter is derived from ubiquitⁱn, wh.ch ⁱs another gene product known to accumulate in many cell types. The ubiquitin promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - B.net et al. Plant Science 79: 87-94 (1991), maize - Christensen et al. Plant Molec Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed ,n the patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expressⁱon of the novel antifungal gene in transgenic plants, especially monocotyledons.

Tissue-specific or tissue-preferential promoters useful for the expression of the novel antifungal gene in plants, particularly maize, are those which direct expression ,n root, pⁱth leaf or pollen. Such promoters, e.g. those isolated from PEPC or frpA, are disclosed ,n U~. Patent No. 5,625,136, or MTL, disclosed in U.S. Patent No. 5,466,785. Both U.S. patents are herein incorporated by reference in their entirety. Chemically inducible promoters useful for directing the expression of the novel antifungal gene in plants are d.sclosed ⁱn U.S. Patent No. 5.614,395. herein incorporated by reference in its entirety. in addition to promoters, a variety of transcriptional terminators are also avaⁱlab e to, us_* in chimeric gene construction using the novel antifungal gene of the present ⁱnvention. Transcriptional terminators are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml termⁱnator, the nopaline synthase terminator, the pea rbcS E9 terminator and others known in the art. These can be used in both monocotyledons and dicotyledons.

Numerous sequences have also been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the novel antifungal gene of this invention to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly ⁱn monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Callis et el.. Genes Develop. 1 : 1183-1200 (1987)). intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "fl-sequ^βnce"). Maⁱze Chiorotic Mottle Virus (MCMV)_, and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et el. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Bio!. 15; 65-79 (1990)).

It may futher be advantagous to co-introduce a marker gene into the plant to be transformed with the DNA comprising the novel antifungal gene. Examples of marker genes are described below. For certain target species, different antibiotic or herbic.de selectⁱon markers may be preferred. Selection markers used routinely in transformation mclude the npfll gene which confers resistance to kanamycin, paromomyciπ, geneticin and related antibiotics (Vieira and Messing, 1982; Bevan et al., 1983) the bacterial aadA gene (Goldschmidt-Clermont. 1991), encoding aminoglycoside 3'-adenylyltransferase and conferring resistance to streptomycin or spectinomycin, the hph gene whⁱch confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann. 1984), and the gene, which confers resistance to methotrexate (Bourouis and Jarry, 1983). Other markers to be used include a phosphinothricin acetyltransferase gene, which confers res.stance to the herbicide phosphinothricin (White et al., 1990; Spencer et al. 1990). a mutant EPSP synthase gene encoding glyphosate resistance (Hinchee et al., ¹988), a mutant acetolactate synthase (ALS) gene which confers imidazolione or su.fonylurea resⁱstance (Lee et al., 1988), a mutant psbA gene conf erring resistance to atrazinε (Smeda et al., 1993), or a mutant protoporphyrinogeπ oxidase gene as described in EP 0769 059. Selection markers resulting in positive selection, such as a p'πosphomannose isomerase gene, as described in patent application WO 93/05153, are also used. Identification of transformed cells may also be accomplished through expression of screenable marker genes such as genes coding for chlorampheπicol acetyl transferase (CAT), β-glucuronidase (GUS), luciferase, and green fluorescent protein (GFP) or any other protein that confers a phenotypically distinct trait to the transformed cell. The recombinant DNA comprising the nucleotide sequence encoding the antifungal protein according to the invention and, optionally, a marker gene sequence can be introduced into the plant cell in a number of well known ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al.. 1986, BioTechniques 4: 320-334), electroporation (Riggs and Bates, 1986, Proc. Natl. Acad. Sci. USA 83: 5602-5606), Agrobacfeπ^'um-mediated transformation (Hinchee et l., 1988, Bio/Technology 6: 915- 922; EP 0 853 675), direct gene transfer (Paszkowski et al., 1984, EMBO J. 3: 2717- 2722). and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, US patent 4 945 050 and McCabe et al., 1988, Bio/Technology 6: 923-926). The cells to be transformed may be differentiated leaf cells, embryogenic cells, or any other type of cell.

In the direct transformation of protoplasts, the uptake of exogenous genetic material into a protoplast may be enhanced by use of a chemical agent or electric field. The exogenous material may then be integrated into the nuclear genome. The early work is conducted in the dicot tobacco where it is shown that the foreign DNA is incorporated and transmitted to progeny plants (Paszkowski et al., 1984, EMBO J. 3: 2717-2722; Potrykus et al.. 1985. Mol. Gen. Genet.199: 169-177). Monocot protoplasts have also been transformed by this procedure in, for example, Triticum monococcum, Lolium multiflorum (Italian rye grass), maize, and Black Mexican sweet corn. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435. as well as in EP 0 846 771. For maize transformation also see Koziel et a!., 1993 (3ic.Technoiogy JJ.: 194-200). Transformed of rice can be undertaken by direct gene transfer techniques ufflizing protoplasts or pamcle bombardment. Protoplast-mediated transformation has been described for Japc/c-.types and .nd.ca-types (Zhang et a,.. 1988. Plan, C.J Rep.7. 379-3841 Shimamoto et al.. 1989. Nature 32 : 274-276; Datta e. al.. 1990. Biotechnology ft 736-740). Both types are a,so routinely transformable us.ng paraoe bombardment (Christou et al., 1991 , BioTechnology 2: 9o7-962). Patent application EP 0 332 581 describes techniques for the generate, transformation and regeneration c, Pooideae protoplasts. These techniques ailo the transformation of all Pooideae plants including Dae ,* and wheat. Furthermore, wheat transformation has been described in patent appϋcation EP 0 674 715 by and Weeks et al.. 1993 (Plant Physiol. 102: 1077-1084).

The present invention thus further relates to transformed host organisms ⁱnCud.ng microorganisms and piants expressing the antifungal protein according to the invention and to the use of those organsims to combat pathogenic funga, pests.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook et al. (1989) Molecular Clonⁱng and by Ausub_θl et al. (1994) Current Protocols in Molecular Biology.

Example 1 : Biological materials

The fresh terminal corm of Gastrodia elata Bl. f. flavida S. Chow is purchased from the plantation at Fengcheng in Liaoning Province, and stored at - 70 -C. The tested fung, Trichoderma viride, Ganoderma lucidum, Piricularia oryzeae, Helmimthosporium turc^teum, Fusarium oxysporium 1. vasinfectum and Botrytis cinerea are purchased from Insftute of Microbiology, Academia Sinica. Rhizoctonia solani is from institute of Plant Protectⁱon, Chinese Academy of Agricultural Sciences, and Valsa ambiens and Gibberella zeae are from the inventor's laboratory. They are cultured at 28 °C on the PDA medium accord.ng to China catalogue of cultures (China committee for culture collections of microorganⁱsms, China catalogue of cultures, China Machine Press. Beijing, (1992) 249).

Example 2: Extraction and purification of GAFP-1

The terminal corms are washed and their outer halves are cut off and collected, then homogenized in a blender in 2 volumes (v/w) of 20 mM sodium phosphate buffer (pH 6.0 with 0.2 M sodium chloride at 4 -O. The homogenate is filtered through four layers of cheesecloth and centrifuged a. 6,000 x g for 20 min. The supernatant is adjusted to 45 % saturation with solid ammonium su-fate! and set still for 8 h a, 4 -O. After centrifuge-en (10,000 X g. 20 min). the supernatant Is adjusted to 80 % saturation of ammonium sulfa.e and kept a, 4 -C overnight. The precipitation is collected by centrilugation (10.000 X g. min) and dissolved in 50 mM sodium acetate buffer (pH 5.0). After centrifugation at 10 000 x _g for 15 min. the insoluble par. is discarded. The supernatant is then desalled on Sephadex G-25 medium column (G26/40). The first peak is collected and applied to DEAE- cellulose co,umn (36 mm x 20 cm,. The unadsorbed portion is collected and "<-*«<" « 70 ml stirred cell (Sigma) with PTGC membrane (NM L - 10.0 kDa) at 4 C. ^ concentrated solution is loaded on Sephadex G-50 fine column (C 6 70). The second pea. is collected and introduced to CM-Sepharose Fas, Flow coiumn (C26/20), then the column

- n - is eluted with a 0 - 0.3 M sodium chloride linear gradient. The main peak is collected and adjusted to 2 M solid ammonium sulfate with 4 M stock solution. This sample is applied to a Phenyl-Superose™ column (HR 10/10) on a FPLC system (Pharmacia), and then eluted with 2.0 - 0 M sodium ammonium sulfate linear gradient in 50 mM sodium acetate buffer (pH 5.0). The main peak is collected and purified GAFP-1 is obtained. The purified GAFP-1 sample is desalted for its amino acid composition and sequence analysis with distilled water on column PD-10 Sephadex G-25 M (Pharmacia) according to its usage manual. The purity of GAFP-1 is evaluated by Tris-tricine SDS-PAGE (Schagger and Jagow, 1987). The amount of protein is determined either by UV absorption at 280 nm using a DU-65 Spectrophotometer (Beckman), or by the method of Lowry et al. (1951) with bovine serum albumin as standard.

The purification procedure of GAFP-1 is illustrated in table I. GAFP-1 can not bind to a Mono S column on a FPLC system in 50 mM sodium acetate buffer (pH 5.0), but it is well adsorbed by the CM-Sepharose Fast Flow and Phenyl-Superose™ in this buffer.

About 140 mg of purified GAFP-1 can be obtained from 1.0 kg of fresh corm after hydrophobic interaction chromatography on a Phenyl-Superose™ column. The purity of GAFP-1 is monitored on SDS-PAGE with the Tris - tricine buffer and checked on IEF with polyacrylamide gel. One band is detected in both cases.

Example 3: Test for antifungal activity

The inhibitory activity of GAFP-1 against fungi is tested on 9 cm Petri dishes. When the fungus inoculum on the PDA medium grows to a diameter of 3 cm at 28 °C, 5μl solutⁱon with different concentrations of GAFP-1 and its corresponding buffer as control are added on the medium surface 0.5 cm away from hyphae. Twelve strains of fungi are tested. Tests are performed in triplicates and the averages of the width of inhibition zone are used in table III. For testing the inhibitory effect on fungal spore germination, the spores are collected after T. viride is cultivated on PDA medium plate for three days and filtered with three layers of cheesecloth. Then they are suspended in the PDA liquid medium at a concentration of 3 - 5 x 10 spores per ml. GAFP-1 is added to a final concentration of 0.1 mg _■ l-' and its corresponding buffer is used as control. The test is carried out in a 96-well plate and observed under the inverted microsco^pe.

The inhibitory activity of GAFP-1 at a concentration of 0.36 mg ^• ml ^"' on the hypha^l extension of 12 kinds of phytopathogenic fungi strains is tested in vitro. As shown in table IV, GAFP-1 can inhibit the hyphal growth of saprophytic fungi like T. viride, Valsa ambiens and some facultative parasite fungi: Rhizoctonia solani, Gibberella zeae F34, G. zeae F11 , G. zeae JF10, G. zeae H28, but had no effect against some other facultative parasitic fungi such as Piricularia oryzeae, Helminthosporium turcieum and Fusarium oxysporum f. sp. vasinfectum. Potentially the saprophytic fungi are more significantly inhibited by GAFP-1 than the facultative species.

On the spore germinating test. 0.1 mg • ml '' of GAFP-1 can inhibit about 30 % spores from germinating, and the average hyphal length of those germinated is about two times shorter than that of the buffer control. Under the phase contrast microscope, no significant abnormal morphogenesis of the hyphae is observed.

Example 4: Characterization of GAFP-1

The molecular mass of GAFP-1 is determined by both SDS-PAGE with Tris-tricine buffer and gel Filtration. Tris-tricine SDS-PAGE is performed by using a discontinuous system according to Schagger and Jagow (1987) on a 10 % spacer gel and a 16.5 % resolving gel with a Mini-PROTEIN II Electrophoresis Cell (Bio-Rad). Gel Filtration is carried out using a Superose-12™ column (HR10/30) on a FPLC system. Isoelectric point is determined by electric focusing on 5 % polyacrylamide gel with a Model 111 IEF Cell (Bio-Rad). The manπose-binding capacity is confirmed by performing affinity chromatography on the column (HR 5/2) of mannose - Sepharose 6B prepared according to Vretblad (1976). Purified GAFP-1 in 50 mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate is loaded onto the column and eluted by the same buffer with 0.2 M ammonium sulfate. Sugar content is determined by the pheπol-H₂SO_< method Dubois et al. (1956). By means of SDS-PAGE in Tris-tricine buffer and gel filtration in 50 mM sodium acetate buffer (pH 5.0) on Superose 12 ™ column on FPLC system. Both of them showed that the molecular mass of GAFP-1 is of 10 kDa. So we can deduce it is a monomer. GAFP-1 has a pi of 8.45 determined by IEF gel. No sugar covalently bound to GAFP-1 can be detected by the pheπol-H₂SO₄ method.

GAFP-1 in 50 mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate can be adsorbed by the mannose-Sepharose 6B column and be eluted with the same buffer with low concentration of ammonium sulfate (0.2 M). The add-on of 1 mannose to the loading sample slightly reduced the GAFP-1 binding efficiency, but 1 M mannose in 50 mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate is inefficient for eluting GAFP-1.

GAFP-1 can also bind to N-acetylglucosamine-Sepharose 6B and chitin and be eluted in the same condition as the mannose-Sepharose 6B.

Using T. viride as indicator, the least inhibitory concentration of GAFP-1 obtained from Phenyl-Superose™ in the last purification step is 0.09 μg in 5 μJ buffer. Under such a concentration, GAFP-1 made a just visible crescent shape zone of inhibition of T. viride hyphae on a PDA medium plate, which we define as one anti-fungal unit. The least inhibitory concentration of the eluted sample from the mannose-Sepharose 6B column is the same as that of sample purified by ion exchange chromatography on a CM-Sepharose Fast Flow column (table I). Both of them are 0.11 μg in 5 μl buffer.

Example 5: Hemagglutination assays

The rabbit erythrocytes are treated with trypsin as described by Lis and Sharon (1972).

GAFP-1 in 0.9 % NaCI is introduced into 1 % suspension of trypsin-treated rabbit erythrocyte and human erythrocytes B-type. Agglutination is monitored under a light microscope after 1 h at room temperature.

Neither human erythrocytes B type nor trypsin-treated rabbit erythrocytes can be agglutinated by GAFP-1 at a concentration of 60 μg • ml^'1.

Example 6: Stability assays

For testing the thermal-stability, newly purified GAFP-1 is used and kept at 4 ^CC. Samples of the same concentration are incubated at different temperatures in thermostatic bath in 50 mM sodium acetate buffer (pH 5.0) for 30 min. Each treated sample is then diluted into series (1 , 2, 3, and 4folds...) to test their inhibitory activity. For testing the pH stability of GAFP-1 , samples of the same concentration are treated in 50 mM disodium hydrogen phosphate-citric acid buffer at different pH at 4 ^eC for 2 h, then adjusted to pH 5.0 for testing the remaining inhibitory activity. The follov/ing equation is used to calculate the remaining inhibitory activity after treatment: Remaining inhibitory activity = the highest dilution multiple of the treated sample X 100 . the highest dilution multiple of the untreated sample More than five independent duplicates tor each test are carried out to ensure the accuracy of the results.

The stability of GAFP-1 is measured by its inhibitory activity against T. vtnde. GAFP-1 thermal-stability is quite high as shown in figure 3. No loss of activity is detected when ⁱt ⁱs preserved at 4 -C for one month in 50 mM sodium acetate buffer (pH 5.0). When GAFP-1 ,s incubated at 20 -C and 37 °C for 30 min in the same buffer, there is no activity lost compared with the untreated sample. After high temperature treatments for 30 min, GAFP-1 still possessed inhibitory activity: about 75 % activity is retained when treated at 60 -C and

55 % activity when treated at 95 °C.

The pH gradient test shows that GAFP-1 has the highest stability at pH 5 and pH 6 in disodium hydrogen phosphate-citric acid buffer.

Example 7: Amino acid composition and N-terminal sequence analysis One tenth milligram desalted GAFP-1 is hydrolyzed under N₂ gas with 0.4 ml 5.7 N of constant-boiling HC. at 110 -C for 24 h, and analyzed on a Beckman 121MB Amino Acⁱd Analyzer To determine the number of cysteine residues. 0.1 mg GAFP-1 is treated wⁱth 0.2 ml performic acid (88 % formic acid / 30 % hydrogen peroxide. 9/1. vΛ ) at 4 ^<C for 4 h and lyophilized before HCI hydrolysis. The number of tryptophane residues is calculated by comparing to tyrosine, measuring the optic density of GAFP-1 in 6 M guanidine HCI at 280 nm and 288 nm Robyt and White (1987). GAFP-1 N-termina. sequence is analyzed on an ABI 491 Protein Sequencer combined with ABI 610 Protein Sequencing Software. The result of the amino acid composition analysis is shown in table II. It reveals that GAFP- 1 is rich in Asx, G.y and Leu. It does not contain Pro. There are four cysteine and three tryptophane residues (table II).

Table 1 Eva/uato of lΛe purifealron process of GAFP-1. One inhibitory activity unit is defined as the amount of GAFP-1 in 5 μl o, solution obtained by serial dilution, that can cause a minimal detectable crescent shaped zone of inhibition o, Tπcπodem.a vrnde on PDA medium plates. Steps Total Total Specific Yield Purification protein inhibitory activity {%) ^fold

(mg) activity (units • mg") (units)

Corm extract 16,500 4,000,000 240 100 1

Ammonium sulfate 2,000 3,120,000 1 ,600 78.0 6.7

precipitation

Sephadex G-25 880 2,800,000 3,180 70.0 13.3

DEAE-Cellulose 750 2,640,000 3.520 66.0 14.7

Ultrafiltration 680 2,400,000 3,529 60.0 14.7

1 ,900,000 9,048 47.5 37.7

Sephadex G-50 210

CM-Sepharose 180 1 ,700,000 9,444 42.5 39.4

Phenyi Superose ™ 140 1 ,500,000 10,714 37.5 44.6

Table U. A ino add compos../ , ot GAFP-1. The numbers of Trp and Cys are determined as described in ,he section o, Methods. By ,he addi.iona, corredmg method, there are four C ys resiαu eS In one IIIUIC--I- ^«■

Amino acid mol % Residues molecule

Aspartic acid 22.1 20

Threonine 4.4 4

Serine 7.4 7

Glutamic acid 6.0 6

Proline 0 0

Glycine 10.0 9 Alanine 7.5 7

Valine 4.7 4

Methionine 1.3 1

Isoleucine 5.2 5

Leucine 9.4 9

Tyrosine 4.5 4

Phenylalanine 1.9 2

Histidine 1.7 2

Lysine 7.4 7

Arginine 5.5 5

3

Tryptophane

Cysteine 1.08(4.0) 1(4) Total 99.99 96(99)

, _m nf GAFP-1 Sodium acetate buffer (50 mM, _PH 5.0)

Table III. Anti-fungal spectrum of GAFP 1. _ ^ _mi -¹ «f GAFP-1 is used for testing each fungus^{, T} containing 0.36 mg ^• ml ot ι**rr _indicates no inhibition is avera₉es o, the width o, inhibition zone from triples of es ^■ « «*» ^ _

_Λ c m mm ++• 10 - 15 mm, +++: 15 - 20 mm, +-++. 20 observed. +: 5 - 10 mm, ++^• ' υ than 25 mm.

Anti-fungal ability

Tested fungi

++++

Trichodarma viride

+++++ Valsa ambiens

+++ Rhizoctonia solani

+++ Gibberella zeae F34

+++

Gibberella zeae F11

+-r

Gibberella zeae JF10

++

Gibberella zeae H28

++

Ganoderma lucidum

+T

Botrytis cinerea .

Piricularia oryzeae _

Helminthosporium tυrcieum Fusarium oxysporium f. sp. vasinfectum

References

Bevan et l. (1983) Nature 304:184-187

Blochlinger and Diggelmann (1984) Mol. Cell. Biol. 4: 2929-2931

Bourouis and Jarry (1983) EMBO J. 2: 1099-1104

GoldschmidC-Clermont (1991) Nucl. Acids Res. 19: 4083-4089

Hinchee et al. (1988) Bio/Technology 6: 915-922

Lee et al. (1988) EMBO J. 7: 1241 -1248

Smeda et al. (1993) Plant Physio!. 103: 911-917

Spencer et al. (1990) Thβor. Appl. Genet. Z9: 625-631

Vieira and Messing (1982) Gene 19: 259-268

White et al. (1990) Nucl. Acids Res. 18: 1062

Ausubel et al., eds (1994) New York: John Wiley and Sons

Dubois et al. (1956) Anal. Chem. 28: 350-356

Lis and Sharon (1972) Meths. Enz. 28: 360-368

Lowry et al. (1951 ) J. Biol. Chem. 193: 265-275

Robyt and White (1987 Biochemica. techniques-theory and practice. Brooks/cole, Monte ,

California _

Sambrook et al. (1989) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press

Schagger and Jagow (1987) Anal. Biochem. 166: 368-379 Vretblad (1976) Biochim. Biophys. Acta. 434: 169-176

Patent Literature EP 0 769 059 WO 93/05163

R_riaf π rin-icπ of the -mi rr— ^;" *^ho ^gπsnce listing:

SEQ ID NO:1 N-terminal GAFP-1 amino acid sequence SEQUENCE LISTING

(1) GENERAL ----FORMATION:

(i) A??LIC?.NT:

(A) NAME: NOVART S AG

₍B₎ STREET: Scbrfarzwaidallee 215

(C) CITY: Easel

(E) COUNTRY: Switzεrla-nά

(F) POSTAL CODE (ZIP) : 4058

(G) TELEPHONE: +41 61 324 11 11 (HJ TELEFAX: + 41 61 32275 32

(ii) TITLE OF j-NVΞOTION: Orgar.c Ccr-pounds

(iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORX:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: ISM P compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Pace-n.tln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SΞQUrKCE CHARACTERISTICS:

(A) LΞN3TH: 24 s-ri.no acids (3) TYPE: aπino acid

(C) STRA DE3NE5S : ^'single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) KYPOTKΞTICAL: NO (iv) ANTI-SENSE: NO

(v) FRGMENT? TYPE: N-tentάnal

(xi) SE_QUENCE DESCRIPTION: SEQ ID NO: 1:

Ser Aso Arg Leu Asa Ser Gly Kis Gin Leu Asp T-r Gly Gly Ser Leu 1 ^* 5 10 ¹⁵

Ala Gin Gly Gly Tyr Leu Phe He 20

Claims

What we claim is:

1. An antifungal protein which is

-insensitive to high temperatures in that it is capable of retaining about 75% of its antifungal activity after 30 minutes at 60°C;

-rich in Asp, Gly and Leu but does contain essentially no Pro;

-is incapable of agglutinating trypsin-treated rabbit erythrocytes;

-is capable of binding to chitin, immobilized mannose and N-acetylglucosamine in 50 M sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate.

2. The antifungal protein according to claim 1 , wherein the Asp, Gly and Leu content is about 22%, about 10% and about 9.5%, respectively.

3. The antifungal protein according to claim 1 , which exhibits an inhibitory activity against Trichoderma viride, Valsa ambiens, Rhizoctonia solani, Gibberella zea, Ganoderma lucidum and Botrytis cinβrea.

4. The antifungal protein according to claim 4, which does not exhibit an inhibitory activity against, Piricularia oryzae, Helminthosporium turcieum and Fusarium oxysporum

5. The antifungal protein according to claim 2, which comprises the at the N-terminus the amino acid sequence given in SEQ ID NO: 1

6. A DNA comprising a nucleotide sequence encoding for a protein according to any one of claims 1 to 5.

7. A host organism comprising stably integrated into its genome a DNA according to claim 6.

8. A host organism according to claim 8 wherein said organism is a microorganism or a plant.

9. A plant and the progeny thereof comprising stably integrated into its genome a DNA according to claim 6.

10. A plant according to claim 9 selected from the group consisting of maize, sugar beet, cotton, rice, wheat, barley, sorghum, tomato, melon, pepper and Brassica.

11. A fungicidal composition comprising a protein according to any one of claims 1 to 5 together with a suitable carrier.

12. A fungicidal composition comprising a host organism according to claim 8.

13. A method of protecting a plant against a fungal pathogen comprising directly or indirectly applying to said plant a protein according to any one of claims 1 to 5 or a fungicidal composition according to claims 11 or 12.

14. A method according to claim 13, wherein the protein is indirectly applied to the plant by expressing said protein within said plant.