AU665020B2 - Biocidal proteins - Google Patents

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(51) International Patent Classification 5 (11) International Publication Number: WO 93/04586 AO1N 63/00, 65/00, CO7K 15/10 Al C12N 15/29, A01 5/00, 5/10 (43) International Publication Date: 18 March 1993 (18.03.93) (21) International Application Number: PCT/GB92/01574 (74) Agent: HUSKISSON, Frank, Mackie; Imperial Chemical Industries plc, ICI Group Patent Department, P.O. Box (22) International Filing Date: 28 August 1992 (28.08.92) 6, Bessemer Road, Welwyn Garden City, Herts AL7 IHD (GB).

Priority data: 9118730.2 2 September 1991 (02.09.91) GB (81) Designated States: AU, BB, BG, BR, CA, CS, FI, HU, JP, KP, KR, LK, MG, MN, MW, NO, PL, RO, RU, SD, 2£.EC~ L\VM Te US, European patent (AT, BE, CH, DE, DK, ES, FR, (71) Applicant (for all designated States except US): IMPERIAL GB, GR, IE, IT, LU, MC, NL, SE), OAPI patent (BF, CI ,ICAL INDUSTRIES PLC [GB/GB]; Imperial BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

Chemical House, Millbank, London SWIP 3JF (GB).

(72) Inventors; and Published Inventors/Applicants (for US only): BROEKAERT, Willem, With international search report.

Frans [BE/BE]; Levoldlaan 18, B-1700 Dilbeek (BE).

CAMMUE, Bruno, Philippe, Angelo [BE/BE]; J.B.

Woutersstraat 109A, B-1652 Alsemberg OSBORN, Rupert, William [GB/GB]; 36 Warwick Road, Twickenham, Middlesex TW2 6SW REES, Sarah, Bronwen [GB/GB]; 32 Micheldever Way, Forest Park, Bracknell, U /a \LJ Berkshire RG12 3XX TERRAS, Franky, Raymond, Gerard [BE/BE]; Schaagstraat 96, B-8570 Anze- \A/T O gem VANDERLEYDEN, Jozef [BE/BE]; Kastanjelaan 12, B-3001 Heverlee (BE).

(54) Title: BIOCIDAL PROTEINS (57) Abstract The antimicrobial activity of albumin-type protein is described. Albumin-type proteins include 2S albumins, napins, barley trypsin inhibitor, wheat a-amylase inhibitor and cereal gliadins. The proteins exhibit a range of antifungal Lctivity, and in addition can potentiate the antifungal activity of thionins. The proteins find commercial application as antifungal or antibacterial agents. Compositions containing an albumin-type protein, or an albumin-type protein and a thiunin protein, may be applied externally to a plant, or may be expressed within a plant. Transformed plants will show increased disease resistance.

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WO 93/04586 PCT/GB92/01574 with those deduced from a rarish 2 i alhr,,mn nupa BIOCIDAL PROTEINS This invention relates to biocidal proteins and compositions, and processes for their manufacture and use. In particular, it relates to the antimicrobial activity of seed storage proteins, and to their synergistic activity with other antimicrobial proteins isolated from plants.

In this context, antimicrobial activity includes a range of antagonistic effects (such as partial inhibition or death) on a fungus and/or a bacterium. In particular, the invention relates to antifungal (which may include anti-yeast) activity.

Although plants normally grow on substrates that are extremely rich in fungal organisms, infection remains a rare event. To keep out potential invaders, plants produce a wide array of K antifungal compounds, either in a constitutive or an inducible manner. The best studied of these are phytoalexins, secondary metabolites with a broad antimicrobial activity spectrum that are specifically synthesised upon perception of appropriate defence-related signal molecules. The production of phytoalexins depends on the transcriptional activation of a series of genes encoding enzymes of the phytoalexin biosynthetic i 25 pathway. During the last decade, however, it has become increasingly clear that some plant proteins can play a more direct role in the control of Sphytopathogenic fungi. Several classes of proteins with antifungal properties have now been identified, including 0-1,3-glucanases (Mauch et al, 1988, Plant Physiol, 88, 936-942), ribosome-inactivating proteins (Roberts and Selitrennikoff, 1986, Biosci Rep, 6, 19-29; Leah et al, 1991, J Biol Chem, 266, 1564-1573), chitinases r, 4 1 1 1 1 1-J' 1 WO 93/04586 PCr/GB92/01574 with a linear qradient (40 min) from 0 to WO 93/04586 PCT/GB92/01574 2 (Schlumbaum et al, 1986, Nature, 324, 363-367), chitin-binding lectins (Broekaert et al, 1989, Science, 245, 1100-1102) and permatins (Vigers et al, 1991, Molec Plant-Microbe Interact, 4, 315-323; Woloshuk et al, 1991, Plant Cell, 3, 619-628).

The antifungal activity of thionins has also been reported (Apel et al, 1990, Physiol Plant, 315-321). Thionins are basic cysteine-rich plant proteins that are thought to be involved in host defence (Rodriguez-Palenzuela et al, 1988, Gene, 271-280).

The 2S albumins are, together with the 75 and 11S globulins, the main protein components in seeds of several'different plant families, including the Brassicaceae (Higgins, 1984, Ann Rev Plant Physiol, 191-221). They serve as storage molecules providing nitrogen and sulphur for the germinating seedling. The 2S albumins have been well studied in rapeseed (Brassica napus), where these proteins are termed napins. Napins are composed of two different chains of about 4 kDa (small subunit) and kDa (large subunit), held together by two disulphide bridges. Both subunits originate from K the same 21 kDa precursor polypeptide by post-translational proteolytic processing events (Crouch et al, 1983, J Mol Appl Genet, 2, 273-283; Ericson et al, 1986, J Biol Chem, 261, 14576-14581). Genes or cDNAs encoding napin precursors have been isolated from rapeseed (Scofield and Crouch, 1987, J Biol Chem, 262, 12202-12208; Josefsson et al, 1987, J Biol Chem, 262, 12196-12201), radish (Raphanus sativus) (Raynal et al, 1991, Gene, 99, 77-86) and Arabidopsis thaliana (Krebbers et al, 1988, Plant WO 93/04586 PCT/GB92/01574 16 A- C. 1 -414 Ok- I a WO 93/04586 PCT/GB92/01574 3 Physiol, 87, 859-866). The 2S albumin genes belong to a large family of related genes that are believed to have arisen by triplication of an ancestral protogene (Kreis and Shewry, 1989, Bio Essays, 10, 201-207). Other members of this superfamily include the barley (Hordeum vulgare) trypsin inhibitor, wheat a-amylase inhibitor, and cereal gliadins, all of which are considered as seed storage proteins. Unlike most 2S albumins, however, the aforementioned related proteins exist as uncleaved polypeptides in their mature form.

There has been speculation that seed storage proteins may have secondary functions related to the protection of seeds from damage caused by animals or micro-organisms. However, no such function has ever been attributed to 2S storage I albumins.

We have now shown that 2S albumins and the related proteins (collectively described as "albumin-type proteins") exhibit surprising antifungal activity, and may be used as fungicidal agents. In addition, these proteins exhibit a surprising and unique property in that they can potentiate the antifungal activity of thionins: in some cases up to According to the present invention, we provide an antimicrobial composition comprising an albumin-type protein. The said protein may be a functional polypeptide subunit of an oligomeric protein. The composition may contain more than one protein.

In further aspects, the invention provides a process of combating fungi or bacteria by exposure to such a composition.

I WO 93/04586 P(iT/GB92/01574 The 2S albumins and related proteins show surprising activity: they inhibit the growth of a variety of plant pathogenic fungi. The antifungal properties of 2S albumins from rapeseed and radish and of a trypsin inhibitor from barley have been demonstrated. The antifungal activity of 2S albumins resides mainly in the small subunit of these proteins, and may be antagonised by K+ and Ca2+ at physiological concentrations.

The albumin-type proteins (2S albumins and I related proteins) show a wide range of antifungal activity, and could be used as fungicides by application to plant parts (or surrounding soil) using standard agricultural techniques (such as spraying). Pathogens exposed to the proteins are inhibited. The protein may eradicate a pathogen already established on the plant or the protein may eradicant effect of the antimicrobial proteins is particularly advantageous.

The antimicrobial proteins can be extracted j and purified from plant material, manufactured from their known amino acid sequence by chemical synthesis using a standard peptide synthesiser, or produced within a suitable organism by expression of recombinant DNA. DNA encoding the proteins can be manufactured using a standard nucleic acid synthesiser, or suitable probes (derived from the known sequence) can be used to isolate the actual gene(s) and control sequences from a plant genome.

f This genetic material can then be cloned into a biological system which allows expression of the proteins. Hence the proteins can be produced in a I

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S WO 93/04586 PCr/GB92/01574 1 WO 93/04586 PCT/GB92/01574 and isolated for use. Suitable micro-organisms may include Escherichia coli and Saccharomyces cerevisiae. Suitable cells may include cultured insect cells and cultured mammalian cells.

The antimicrobial proteins may also be used to combat disease by expression of DNA encoding one or more of said proteins within transgenic plant bodies. The fungus or bacterium is thus exposed to i the protein at the site of pathogen attack on the plant. In particular, by use of appropriate inducible gene regulator sequences, the albumins (seed proteins) may be produced in vivo within parts of the plant where they are not normally K expressed in quantity but where disease resistance is important (such as in the leaves).

Plant cells may be transformed with recombinant DNA constructs according to a variety of known methods (Agrobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, etc). The transformed cell_ may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way, although the latter are usually more easy to regenerate. The progeny of these primary transformants will inherit the recombinant DNA encoding the antimicrobial protein(s).

Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, !,l (1 i ii :i SWO 93/04586 PCT/GB92/01574 WO 93/04586 PCT/GB92/01574 6 carrot, lettuce, cabbage, onion.

The invention further provides a plant having improved resistance to a fungal or bacterial pathogen and containing recombinant DNA which expresses an albumin-type protein.

A pathogen may be any fungus or bacterium growing on, in or near the plant. In this context, improved resistance is defined as enhanced tolerance to a fungal or bacterial pathogen when compared to a wild-type plant. Resistance may vary from a slight increase in tolerance to the effects of the pathogen (where the pathogen in partially inhibited) to total resistance so that the plant is unaffected by the presence of pathogen (where the pathogen is severely inhibited or killed). An increased level of resistance against a particular pathogen or resistance against a wider spectrum of pathogens may both constitute an improvement in resistance.

Recombinant DNA is heterologous DNA which has ij been introduced into the plant. The recombinant DNA encodes an antimicrobial protein expressed for delivery to a site of pathogen attack (such as the leaves). The DNA may encode an active subunit of an antimicrobial protein, such as the small subunit (SS) of 2S albumin described in Example The invention further provides an antimicrobial composition comprising an albumin-type protein and a thionin protein. Either or both of said proteins may be a functional polypeptide subunit of an oligomeric protein. The composition may contain more than one albumin-type protein and/or more than one thionin protein.

In further aspects, the invention provides a i 1 l 1-e WO 93/04586 PCT/GB92/01574 7 process of combating fungi or bacteria by exposure to such a composition.

Even more surprising than their own antifungal activity is the synergism exhibited between the albumins and the thionins. The unexpectedly potent antifungal activity of the albumin/thionin mixture can be exploited to combat plant disease. As above, the mixture may be used as a fungicide by application to plant parts (or surrounding soil) using standard agricultural techniques (such as |spraying). Pathogens exposed to the proteins are inhibited. The protein may eradicate a pathogen already established on the plant or the protein may protect the plant from future pathogen attack. The eradicant effect of the antimicrobial proteins is particularly advantageous.

The antimicrobial proteins can be extracted and purified from plant material, manufactured from their known amino acid sequence by chemical synthesis using a standard peptide synthesiser, or produced within a suitable organism by expression of recombinant DNA as previously described.

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The proteins may also be be used to combat 'i disease by expression within transgenic plant bodies as previously described.

The invention further provides a plant having improved resistance to a fungal or bacterial pathogen and containing recombinant DNA which expresses an albumin-type protein and a thionin protein.

A plant with an inherent thionin content may be transformed with DNA encoding one or more albumin proteins, so that a mixture of the two classes of protein is produced in vivo.

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i WO 93/04586 PCT/GB92/01574 8 Alternatively, DNA encoding one or more thionins can be transformed into a plant and expressed within tissue which has an inherent albumin content. A third possibility is to transform both DNA encoding one or more albumins and DNA encoding one or more thionins into a plant, so that both classes of protein are expressed together in the same tissue. The albumin-encoding DNA may be introduced on the same construct as the thionin-encoding DNA, or on a separate construct.

Another possibility is to transform a first plant with DNA encoding an albumin-type protein and to transform a second plant with DNA encoding a thionin protein, and then to cross the first and second plants producing a plant containing recombinant DNA expressing both proteins.

The invention may be illustrated by the following examples and by reference to the drawings, in which: Figure 1 is the chromatogram for the protein fraction extracted from radish seed.

Figure 2 is the chromatogram for the protein fraction extracted from rapeseed seed.

Figure 3 shows the SDS-PAGE analysis of the reduced and unreduced radish 2S albumins.

Figure 4 shows the SDS-PAGE analysis of the reduced and unreduced rapeseed 2S albumins.

Figure 5 shows the reversed-phase chromatogram of the reduced and carboxyamidomethylated Rs-2S5, a radish 2S albumin.

Figure 6 shows the amino acid sequences of the 2S albumins Rs-2S5, pBa3 and napin.

Figure 7 shows the N-terminal amino acid sequences of the trypsin inhibitors from barley and

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WO 93/04586 PCT/GB92/01574 9 wheat.

Figure 8 is a graph showing the degree of Cercospora beticola disease on sugarbeet against rate of 2S albumin application.

Figure 9 is a graph showing the degree of Cercospora beticola disease on sugarbeet against time of 2S albumin application.

Figure 10 shows the nucleotide sequence of Sthe 25 albumin gene pIG8.

Figure 11 shows the nucleotide sequence and derived amino acid sequence of the small subunit of 2S albumin fused to the Mj-AMP2 signal peptide.

Figure 12 shows the construction of the V expression vector pIGl3.

Figure 13 shows the construction of the expression vector Figure 14 shows the construction of the plant transformation vector pIGl9.

Figure 15 shows the construction of the i 20 plant transformation vector EXAMPLE 1 Isolation of 2S albumins from radish and rapeseed seeds.

2S albumins were isolated from radish seeds and rapeseed seeds by a four-step procedure including ammonium sulphate fractionation, heat treatment, anion exchange chromatography and cation exchange chromatography. The detailed methods are I described below.

One kg of radish or rapeseed seeds was ground in a coffee mill and the resulting meal was extracted for 2 hours at 4 0 C with 2 litres of an j r i WO 93/04586 PCT/GB92/01574 ice-cold extraction buffer containing 10 mM NaH 2

PO

4 15 mM Na 2

HPO

4 100 mM KC1, 2 mM EDTA, 2 mM thiourea and 1 mM PMSF. The homogenate was squeezed through cheesecloth and clarified by centrifugation (30 min at 7,000 x Solid ammonium sulphate was added to the supernatant to obtain 30% relative saturation and the precipitate formed after standing overnight at room temperature was removed by centrifugation (30 minutes at 7,000 x The supernatant was adjusted to 70% relative ammonium sulphate saturation and the precipitate formed overnight at room temperature collected by centrifugation (30 min at 7,000 x After redissolving the pellet in 400 ml H 2 0 the solution was heated at 80°C for 15 min. The coagulated insoluble material was removed by centrifugation min at 7000 x g) and the supernatant was I dialyzed extensively against distilled water using dialysis tubing (SpectralPor, Spectrum, USA) with a molecular weight cut off of 1000 Da. After dialysis the solution was adjusted to 50 mM Tris-aCl (pH 9) by addition of the ten-fold concentrated buffer, and subsequently passed over a Q-Sepharose Fast Flow (Pharmacia) column (12 x cm) in equilibrium with 50 mM Tris-HC1, pH 9. The protein fraction passed through the column was dialyzed extensively against H 2 0 and adjusted to mM sodium N-morpholinoethanesulfonic acid (MES)(pH6) by addition of the 10-fold concentrated buffer. About 150 mg protein r this fraction was applied on a S-Sepharose High tarformance (Pharmacia) column (10 x 1.6 cm) previously equilibrated with the sodium MES buffer. The column was eluted at 2.5 ml/min with'a linear lu~xr WO 93/04586 PCr/GB92/01574 11 gradient of 1000 ml from 0 to 500 mM NaCi in 50 mM sodium MES buffer (pH The eluate was monitored for protein by online measurement of the absorbance at 280 nm.

Figure 1 shows results for the radish seed extract and Figure 2 shows results for the rapeseed seed extract. The broad group of non-resolved peaks eluting between 250 and 450 mM NaCl (indicated by Numbers 1 to 5 in Figures 1 and 2) 110 represent different isoforms of the 2S albumins.

The protein fractions corresponding to these peaks are henceforward designated as Rs-2S1, Rs-2S2, Rs-2S3, Rs-2S4 and Rs-2S5, respectively, for the radish 2S 'albumins (Figure 1) and Bn-2Sl, Bn-2S2, Bn-2S3, Bn-2S4 and Bn-2S5 respectively for the rapeseed 2S albumins (Figure 2).

EXAMPLE 2 Characterisation of the isolated 2S albumins.

The isolated isoforms of 2S albumins from radish and rapeseed were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) before and after reduction with dithiothreitol. SDS-PAGE was performed on precast commercial gels (PhastGel High Density from Pharmacia) using a PhastSystem (Pharmacia) electrophoresis apparatus. The sample buffer for analysis of unreduced proteins contained 200 mM Tris-ICl (pH 1% SDS, 1 mM EDTA, 0.005% bromophenol blue, and the sample buffer for analysis of reduced proteins contained a supplement of 1% dithiothreitol (DTT). The unroduced proteins were fixed in 30% ethanol/10% (v/v) Our Ref: 357480 2850n WO 93/04586 PCI/GB92/01574 12 acetic acid and were silver-stained according to Heukeshoven and Dernick (1985, Electrophoresis, 6, 103-112). The reduced proteins were blotted on nitro-cellulose after electrophoresis and the blots were silver-stained according to Kovarik et al (1987, Folia Biologica, 33, 253-257).

Figure 3 shows the SDS-PAGE analysis of the radish 2S albumins in their unreduced (left panel) and reduced (right panel) forms. Lanes 1 show Rs-2S1, lanes 2 show Rs-2S2, lanes 3 show Rs-2S3, lanes 4 show Rs-2S4, lanes 5 show Rs-2S5. Figure 4 shows the SDS-PAGE analysis of the rapeseed 2S albumins in their unreduced (left panel) and reduced (right panel) forms. Lanes 1 show Bn-2S1, lanes 2 show Bn-2S2, lanes 3 show Bn-2S3, lanes 4 I show Bn-2S4, lanes 5 show Bn-2S5. Fifty nanograms of the proteins were loaded on the gels. Lanes M show myoglobin fragments used as molecular weight markers (Pharmacia) with the following sizes: 17 kDa, 14.5 kDa, 8 kDa, 6 kDa, and 2.5 kDa.

As can be seen in Figure 3, the reduced radish 2S albumins migrate as 2 bands of about 9 and 4 kDa, respectively. In their unreduced form the different isoforms yield doublets with an apparent molecular weight af 17 and 18 kDa, respectively.

In Rs-2S1 the 17 kDa band is predominant whereas in the 18 kDa band is the major component. The reduced rapeseed 2S albumins (Figure 4) migrate as 2 bands of about 9 and 4 kDa, respectively, whereas the unreduced 2S albumins migrate as single 17 kDa bands, The presence of the 4 and 9 kDa bands in the SDS-PAGE patterns of reduced radish and rapeseed 2S albumins is consistent with previous observations that these proteins contain a small (4 i 4; ii 4 WO 93/04586 PCT/GB92/01574 13 kDa) subunit and a large (9 kDa) subunit linked together by disulphide bridges (Crouch et al, 1983, J Mol Appi Genet, 2, 273-283; Laroche et al, 1984, Plant Physiol, 74, 487-493).

To confirm the identity of the isolated proteins as 2S albumins, one of the isoforms was subjected to amino acid sequence analysis. Two hundred pg of Rs-2S5 was reduced with dithiothreitol and the cysteines were modified by S-carboxyamidomethylation as described by Creighton (1989, In: Protein structure, a practical approach, ed. Creighton pp. 155-167). The S-carboxyamidomethylated proteins were separated by reversed phase chromatography on a Pep S column (porous silica C2/C18, 25 x 0.4 cm, from Pharmacia). The column was eluted at 1 ml/min with a linear gradient (90 min) from 0 to acetonitrile in 0.1% trifluoracetic acid.

Figure 5 shows the resulting chromatogram: the mixture separated into 2 groups of 3 peaks each.

SDS-PAGE analysis of the different peaks revealed that peaks 1, 2 and 3 correspond to the small subunit (4 kDa) and peaks 4, 5 and 6 to the large subunit (9 kDa). The N-terminal sequence of material from peak 1 (small subunit) and peak 4 (large subunit) was determined by the Edman degradation method on a pulsed liquid-phase sequencer (Applied Biosystems model 477A/120). For the small subunit of Rs-2S5 (peak 1, Figure 5) residues could be identified, whereas for the large subunit of Rs-2S5 (peak 4, Figure 5) 20 amino acids were sequenced.

The results of these analyses are shown in Figure 6, where the obtained sequences are compared 'P :4 i -1 I' I '1 I I' WO 93/04586 PC/GB92/01574 37 0 C under a 5% CO. atmosohere with flfl. rpla4 4 7o -::C1 SWO 93/04586 PCIFGB92/01574 14 with those deduced from a radish 2S albumin cDNA clone, pBA3 (Raynal et al, 1991, Gene, 99, 77-86) and with data obtained by N-terminal sequencing of a rapeseed 2S albumin, napin (Ericson et al, 1986, J. Biol Chem, 261, 14576-14581). Amino acids differing from the corresponding residues in the sequences are indicated in lower case. The obtained partial sequence of the small subunit of is 100% identical to the pBA3 sequence and 87% identical to the napin sequence. The large subunit shows 90% identity with the corresponding pA3 and napin sequences.

EXAMPLE 3 Isolation of the trypsin inhibitors from barley seeds and a-purothionins from wheat seeds.

It is known from the literature that barley (Hordeum vulgare contains a trypsin inhibitor that is highly homologous to 2S albumins from dicotyledonous species. However, in contrast to 2S albumins whose polypeptides are cleaved into a small and large subunit, the barley trypsin inhibitor (hereafter called bti0) consists of a single uncleaved 13 kDa polypeptide (Odani et al, 1983, Biochem J, 213:543-545).

In order to compare the antifungal activity of trypsin inhibitors to 2S albumins, bti0 was isolated by the procedure of Mikola and Suolinna (1969, Eur J Biochem, 9, 555-560). The preparation was further purified by an additional step consisting of reversed-phase chromatography on a Pep S (porous silica C2/C18) column (25 x 0.93 cm) (Pharmacia). Tht column was eluted at 5 ml/min WO 93/04586 PCT/GB92/01574 with a linear gradient (40 min) from 0 to acetonitrile. The identity of the purified protein as bti0 was verified by N-terminal amino acid sequencing. The first 11 N-terminal residues were identical to those previously reported (Odani et al, 1983, J Biol Chem, 258, 7998-8000).

Moreover, by assaying the cation exchange chromatogram for trypsin inhibitor activity, a trypsin inhibitor fraction different from bti0 was discovere. This fraction was further purified by an additional step consisting of reversed-phase chromatography as described above. It yielded two peaks eluting at 33 and 35% acetonitrile, hereafter called btil (barley trfpsin inhibitor 1) and bti2 (barley trypsin inhibitor 2) respectively.

Amino-terminal sequence analysis revealed that both proteins are homologous to WG11, a Bowman-Birk type protease inhibitor from wheat (Odani et al, 1986, J Biochem, 100:975-983).

Bowman-Birk type trypsin inhibitors are known to occur also in barley endosperms (Boisen and Djertoft, 1982, J Sci Food Agric, 33:431-440) and some homology has been found between the Ricinis 2S albumin large subunit and the Bowman-Birk inhibitor from lima bean (Sharif and Li, 1982, J Biol Chem, 257:14753-14759).

Figure 7 shows the N-terminal amino acid sequences of the trypsin inhibitors from barley and from WG11.

a-Purothionin was isolated from wheat (Triticum aestivum) seed flour essentially as described by Redman and Fisher (1969, J Sci Food Agric, 20, 427-432). The preparation was purified by one more step consisting of reversed-phase Si- of 4 L WO 93/04586 PCT/GB92/01574 16 chromatography on a Pep S (porous silica C2/C18) column (25 x 0.93 cm) (Pharmacia). The column was eluted at 5 ml/min with a linear gradient (40 min) from 0 to 40% acetonitrile in 0.1% TFA. The purothionin eluted was two incompletely resolved peaks at 34 and 35% acetonitrile, respectively, which represent the al and a2 isoform (Mak and Jones, 1977, Cereal Chem, 54, 511-523). Both isoforms were pooled, to yield the c-purothionin preparation.

EXAMPLE 4 Antifungal activity assay.

Antifungal activity was measured by microspectrophotometry as previously described (Broekaert, 1990, FEMS Microbiol Lett, 69, 55-60)..

Routinely, tests were performed with 201 of a (filter-sterilised) test solution and 80u1 of a fungal spore suspension (2 x 104 spores/ml). The suspension was prepared either in growth medium A (half strength potato dextrose broth, Difco) or medium B (half strength potato dextrose broth, Difco, supplemented with 1 mM CaCl 2 and 50 mM KC1).

For experiments on the antagonistic effect of cations, a synthetic growth medium was used. The synthetic growth medium consisted of K 2

HPO

4 mM), MgSO 4 (50 pM), CaC1 2 (50 pM), FeSO 4 (5 pM), Cocl 2 (0.1 CuSo 4 (0.1 pM), Na 2 MoO 4 (2 pM),

H

3 B0 3 (0.5 upM), KI (0.1 pM), ZnSO 4 (0.5 MnSO 4 (0.1 pM), glucose (10g/1), asparagine (19/1), methionine (20,mg/l), myo-inositol (2 mg/1), biotin (0.2 mg/1), thiamine-HCl (1 mg/1), and pyridoxine-HCl (0.2 mg/1). Control microcultures i i I' 1 WO 93/,4586 PCT/GB92/01574 17 contained 20 pu of sterile distilled water and pl of the fungal suspension.

Unless otherwise stated incubation was done at 0 C for 48 hours. Percent growth inhibition is defined as 100 times the ratio of the corrected absorbance of the control microculture minus the corrected absorbance of the test microculture over the corrected absorbance at 595 nm of the control microculture. The corrected absorbance values equal the absorbance at 595 nm of the culture measured after 48 hours minus the absorbance at 595 nm measured after 30 minutes.

EXAMPLE Antifungal activity of the radish and rapeseed 2S albumins, barley trypsin inhibitors and a-purothionin.

The antifungal potency of the radish 2S albumins, rapeseed 2S albumins, barley trypsin inhibitors and a-purothionin was assessed by the microspectrophotometric assay described in Example 4. Growth of fungi, collection and harvest of spores was done as previously described (Broekaert et al, 1990, FEMS Microbiol Lett, 69, 55-60). Four different plant pathogenic fungi were used as test organisms: Fusarium culmorum IMI 180420, Alternaria brassicola MUCL 20297, Ascochyta pisi MUCL 30164, and Verticillium dahliae MUCL 19210.

Two different growth media were used in these tests: medium A was half-strength potato dextrose broth, and medium B was medium A supplemented with 1 mM CaC1 2 and 50 mM KC1. The constitution of monovalent and divalent cations of medium B is such 1 WO 93/04586 PCT/GB92/01574 18 that it resembles that inside a plant cell.

Serial dilutions of the different proteins were applied to the fungi, either in medium A or medium B. The concentration required for growth inhibition after 48 hours of incubation

(IC

50 value) was calculated from the dose-response curves. The results of these experiments are summarised in Table 1. The following abbreviations are used for the fungi: Fc, Fusarium culmorum; Ab, Alternaria brassicola; Ap, Ascochyta pisi; Vd, Verticillium dahliae.

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cc 0% NOW- mm-mill 0 TABLE 1 ANTIFUNGAL ACTIVITY OF 2S ALBUMINS FROM RAPESEED AND RADISH, BARLEY TRYPSIN INHIBITORS AND a-PUROTHIONIN Protein IC 50 (pg/mi) Medium A Mediumn B Fc Ab Ap Vd Fc Ab- Ap xR,-2S1 30 20 30 20 >1000 >1000 >1000 >1000 Rs-2S2 30 22 25 20 >1000 >1000 >1000 >1000 Rs-:2S3 35 20 30 2O >1000 >1000 >1000 >1000 Rs-2S4 35 20 25 20 >1000 >1000 >1000 >1000 40 22 30 22 >1000 >1000 >1000 >1000 Bn-2S3 60 45 80 45 >1000 >1000 >1000 >1000 btUo 910 180 >1000 350 >1000 >1000 >1000 >1000 btil 110 55 105 65 >1000 >1000 >1600 >1000 bti2 90 40 90 50 >1000 >1000 >1000 >1000 a-purothie-nin 4 4 8 2 11 11 12 8 I I.

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r i i WO: 93/04586 PCr/GB92/01574 In medium A (low ionic strength), the 2S albumins from rapeseed and radish, the barley trypsin inhibitors btil and bti2 and a-purothionin were active on all four fungi tested. The five isoform fractions of the radish 25 albumins had similar activities. The rapeseed 2S albumin and the barley trypsin inhibitors 1 and 2 had about two to three fold lower activities compared to the radish 2S albumins, whereas the thionins were about five to ten-fold more active relative to the radish 2S albumins.

In medium B (high ionic strength), however, the 2S albumins and the barley trypsin inhibitors were completely inactive at concentrations up to 1 mg/ml. In contrast the a-purothionin remained active in medium B, although its specific activity was decreased by about two- to four-fold in medium B relative to medium A.

EXAMPLE 6 Effect of ions on antifungal activity.

The effect of ions on the antifungal activity of radish 2S albumins was examined in more detail.

The IC 50 values of Rs-2S3 on Fusarium culmorum and Trichoderma hamatum MUCL 29736 were measured in five different media. The reference medium was the synthetic growth medium described in Example 4, which contains a total of 2.5 mM monovalent cations and 0.1 mM divalent cations. The four other media contained 10 mM KC1, 50 mM KC1, 1 mM CaCl 2 or 5 mM CaC1 2 in supplement, respectively.

Results are shown in Table 2.

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A

IaaaxC WO 93/04586 PCT/GB92/01574 21 TABLE 2 VARIATION IN ANTIFUNGAL ACTIVITY OF RS-2S3 IN PRESENCE OF K AND CA 2 Fungus IC 50 (pg/ml) Reference medium supplemented with: 5 2+ 2+ 10mM K 50mM K 1mM Ca 2 5mM Ca 2 F culmorum 12 25 >400 >400 >400 T hamatum' 3 3 12 100 >400 Addition of 50 mM K to the medium decreased the antifungal activity of Rs-2S3 on F culmorum by more than 30-fold, while the activity on T hamatum was reduced by four fold. Also, the presence of Ca2+ at concentrations as low as 1 mM reduced the activity by more than 30-fold on F culmorum and by about 30-fold on T hamatum.

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WO 93/04586 PC/GB!2/01574 22 EXAMPLE 7 Synergism between 2S albumins, barley trypsin inhibitor and a-purothionin.

The synergistic antifungal effect of combinations of 2S albumins and a-purothionin, on the one hand, and barley trypsin inhibitors and a-purothionin, on the other hand, was measured as follows.

To serial dilutions of a-purothionin, a constant subinhibitory concentration of 2S albumins or barley trypsin inhibitors was added. The IC 50 value of the a-purothionin was calculated from dose-response curves for the series with and without addition of 2S albumins or barley trypsin inhibitors. The subinhibitory concentrations of 2S albumins and barley trypsin inhibitors used in the experiment were 10 pg/ml (final concentration) for the tests in medium A and 10, 50 and 250 pg/ml for the tests performed in medium B. The Synergism Factor was calculated as the ratio of the IC 50 value in the control series (no 2S albumins or trypsin inhibitors added) over the IC 50 value in the series supplemented with 2S albumin or barley trypsiL inhibitors. Synergism Factors are rounded to the nearest integer.

The results of these experiments are shown in Table 3. The following abbreviations are used for the fungi: Ab, Alternaria brassicola; Ap, Ascochyta pisi; Fc, Fusarium culmorum; Vd, Verticillium dahliae.

r WO 93/04586 PCT/B920154 TABLE 3 SYNERGISTIC ANTIFUNGAL EFFECT OF COMBINATIONS BETWEEN ALBUMINS/OL-PUROTHIONIN AND TRYPSIN INHIBITORS/cx-PUROTHIONIN Fungus Test IC 50 o of a-purothionin la" pg/mi Protein (Synergism Factor) Medium A Medium B Test protein Test protein -conc. (pg/mi) conc. (pg/mi) 0 10 0 10 50 250 Ab Rs-2S3 4 0.86(5) 11 6(2) 0.45(25) 0.25(44) Bn-2S3 4 0.22(18) 11 2(5) 0.32:34) 0.15(73) bti0 4 0.20(20) 11 2.5(4) 1.2(9) 0.35(31) btil 4 0.8(5) 11 8(1) 2.5(4) ND bti2 4 0.28(14) 11 8(l) 2.5(7 0.2(55) Ap Rs-2S3 8 2.5(3) 12 5.5(2) 1(11) 0.8(14) Bn-2S3 8 2(4) 12 7(2) 3(4) 1.5(8) bti0 8 3(3 12 10(l) 6(2) 1.2(10) btil 8 7(l) 12 11(1) 9(l) 1.4(8) bti2 8 4(2) 12 12(1) 9(1) 1.2(10) Fc Rs-2S3 4 0.45(9) 11 3(4) 0.6(18) Bn-2S3 4 0.25(16) 11 5(2) 1(11) 1-.5(7) bti0 4 0.4(10) 11 10(1 7(2) 0.(12) btil 4 1.5(3) 11 10(1 2.5(4) 1.1(10) bti2 4 0.9(4) 11 10(1 2.5(4) 1.2(9) Vd Rs-2S3 2 0.4(5) 8 1.1(7) 0.4(20) 0.4(20) Bn-25-3 2 0.15(13) 8 0.7(11) 0.22(36) 0.2(40) bti0 2 0.14(14) 8 2.8(3) 0.9(9) 0.4(20) btil 2 0.6(3) 8 3(3) 0.9(9) 1.2(7) bti2 2 0.3(7) 8 1.5(5) 0.4(20) 0.8(10) -I Not determined.

WO 93/04586 PCT/GB92/01574 24 With some exceptions, the synergism factors obtained in medium A were generally about two to three-fold higher than those obtained in medium B.

The highest synergism factor (73) was obtained in medium B for the combination of a-purothionin and Bn-2S3 (at 250 pg/ml) on the fungus Alternaria brassicola. The synergism factors increased drastically when the subinhibitory concentration of 2S albumins in medium B was raised from 10 to pg/ml, but except for A brassicola, no further substantial increase was obtained on going from to 250 pg/ml. With the barley trypsin inhibitor the synergism factors were generally between two and five-fold lower compared to the 2S albumins Rs-2S3 and Bn-2S3.

EXAMPLE 8 Effect of 2S albumins and a-purothionin on bacterial growth.

Antibacterial activity was measured microspectrophotometrically as follows. A bacterial suspension was prepared by inoculating either soft agarose medium C (10 g/1 tryptone, g/l Seaplaque agarose FMC), or soft agarose medium D (10 g/l tryptone, 5 g/1 Seaplaque agarose (FMC), 1 mM CaC12 and 50 mM KC1). Aliquots (80 pl) of the 5 bacterial suspension (10 colony forming units per ml) were added to filter-sterilised samples (20 pl) in flat-bottom 96-well microplates. The absorbance at 595 nm of the culture was measured with the aid of a microplate reader after 30 minutes (blank values) and 24 hours ci incubation at 28 0

C.

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WO 93/04586 PCf/GB92/01374 Percent growth inhibition was calculated as described in Example 4 for the antifungal activity assay.

The following bacterial species were used: Agrobacterium tumefaciens LMG 88, Alcaligenes eutrophus LMG 1195, Azospirillum brasilense ATCC 29145, Bacillus megaterium ATCC 13632, Erwinia carotovora strain 3912, Escherichia coli strain Pseudomonas solanacearum LMG 2293, Sarcina lutea ATCC 9342. The antibacterial effect of Rs-2S3 was assessed by adding serial dilutions of th| protein to bacteria3 suspensions. The highest test concentration was 500 ug/ml (final concentration).

Rs-2S3 only affected the growth of the gram-positive bacterium B megaterium (ICS 0 and the gram-negative bacterium E carotovora (IC 50 250pg/ml). However, when the growth medium C was supplemented with 1 mlX CaCl 2 2 and 50 mM KC1 (medium Rs-2S3 lost its inhibitory activity on these bacteria.

Synergisms in antibacterial activity were assessed for combinations between Rs-2S3 and -purothionin by using the same approach as described in Example 7. A synergistic effect was observed on B megaterium. When tested in growth medium D, synergism factors of 4, 15 and 17 were obtained after addition of Rs-2S3 at subinhibitory concentrations of 10, 50 and 250 ug/ml, respectively, to a serial dilution series of a-purothionin (Table Thus, the thionin-potentiating activity of 2S albumin is not limited to fungi, but is also evident on some bacterial species.

Ii I o b t a n e d a f t r a d d i t on-- -r y ~u I i UIIII~I~1 WO 93/04586 PCT/GB92/01574 TABLE 4 SYNERGISTIC ANTIBACTERIAL EFFECT ON BACILLUS MEGATERIUM OF COMBINATIONS BETWEEN RS-2S3 AND a-PUROTHIONIN Rs-2S3 conc. IC 50 of a-purothionin in pg/ml (pg/ml) (Synergism Factor) Medium C Medium D 0 2 0.6(3) 0.6(4) 0.2(15) 250 0.15(17) EXAMPLE 9 Effect of 2S albumins and a-purothionin on cultured human cells.

Human cell toxicity assays were performed either on umbilical vein endothelial cells (Alessi et al, 1988, Eur J Biochem, 175, 531-540) or skin-mu :2lr fibroblasts (Van Damme et al, 1987, Eur J Immunol, 17, 1-7) cultured in 96-well microplates. The growth medium was replaced by 80 pl of serum-free medium (Optimem 1 for endothelial cells or Eagle's minimal essential medium (EMEM) for fibroblasts, both from GIBCO), to which 20 p1 of a filter-sterilised test solution was added.

The cells were further incubated for 24 hours at 1 6 i' 4 i: :i i a I I II I i WO 93/04586 PCT/GB92/01574 27 37 0 C under a 5% C02 atmosphere with 100% relative humidity. The viability of the cells was assessed microscopically after staining with trypane blue (400 mg/l in phosphate buffered saline, PBS) for minutes. Alternatively, cells were stained with neutral red (56 ml/l in PBS) for 2 hours at 37 0

C.

Neutral red treated cells were washed with PBS, lysed in acidic ethanol (30 mM sodium citrate, pH 4.2, containing 50% ethanol) and .scored for release of the dye by microspectrophotometry at 540 nm.

The 2S albumins were evaluated for their potential toxic effects on mammalian cells. When added at up to 500 ug/ml to either cultured human umbilical vein endothelial cells or human skin-muscle fibroblasts, Rs-2S3 did not affect cell viability after 24 hours of incubation. In contrast, a-purothionin administered at 50 ug/ml and 20 pg/ml decreased the viability of both cell types by more than 90% and 50%, respectively.

Addition of Rs-2S3 at a constant concentration of 250 #g/ml to a serial dilution series of a a-purothionin did not increase the toxic activity of the a-purothionin.

EXAMPLE Antifungal activity of the small and large subunit of 2S albumins.

The small subunit (SS) and the large subunit (LS) of Rs-2S3 were prepared as follows. Rs-2S3 was reduced in 100 mM Tris-HCl, pH 8.4, 100 mM DTE and kept at 45°C for 1 hour. After reduction the preparation was passed over a reversed phase column as described in Example 2. The peaks corresponding to SS and LS, respectively, were pooled and 4 WO 93/04586 PCT/GB92/01574 28 vacuum-dried. The cysteine residues of the SS and LS preparations were reoxidised using an oxido-shuffling system consisting of reduced glutathione and glutathione disulphide as described by Jaenicke and Rudolph (1989, In: Creighton (ed), Protein structure: a practical approach. IRL Press Ltd, Oxford, pp 191-224). After the reoxidation step the oxidised SS and LS were purified by reversed-phase chromatography. Their thiol content was assessed as described by Creighton (1989, In: Creighton Protein structure, a practical approach, pp 155-167) and found to be 9% and 22% of that of the reduced forms for SS and LS, respectively.

The antifungal activity of the reduced and re-oxidised SS and LS was determined on Fusarium culmorum as described in Example 4. The activity of the reduced forms was tested in the presence of subinhibitory concentrations of dithiotreitol (0.25 mM) to prevent spontaneous disulphide formation (Monsalve et al, 1991, Biochem Biophys Acta, 1078, 265-272). The reduced LS had no activity on F culmorum at concentrations below 400 ug/ml, while reduced SS had an IC 50 value of 14 ug/ml. The oxidised SS had an IC 50 value of 32 ug/ml, whereas oxidised LS had no activity below 400 #g/ml, when assayed in medium A. In medium B, the IC 50 values of oxidised SS and LS were higher than 200 pg/ml and higher than 400 pg/ml, respectively.

The synergistic antifuingal effect on F culmorum was assessed for combination between oxidised SS or LS and a-purothionin by using the same approach as described in Example 7. The results of these experiments are shown in Table i" TABLE SYNERGISTIC ANTIFUNGAL EFFECT ON FUSARIUM CULNORUM OF COMBINATIONS BETWEEN OXIDISED SS OR LS AND u-PUROTHIONIN Test protein IC 50 of u-purothionin, ug/ml (Synergism Factor) Medium A Medium B Test protein conc. Test protein conc.

(Pg/mi) (Pg/mi) 0 10 20 0 10 20 50 100 500 Oxidised SS 4 0.12(33) ND 11 5.2(2) ND 0.8(14) ND ND Oxidised LS 4 ND 0.32(13) 11 ND 6(2) ND 0.9(12) 0.7(16) LJ WO 93/04586 PCT/GB92/01574 Table 5 shows that SS as well as LS (though to a much lesser extent) are able to potentiate the activity of a-purothionin. A synergism factor of up to 33 could be obtained when oxidised SS was added at 10 pg/ml to a dilution of a-purothionin and assayed in medium A. In medium B a synergism factor of 2 was measured under these conditions. A synergism factor of 14 was obtained when oxidised SS was added at 50 /g/ml in medium B. Low synergism factors were measured during the same experiments with the oxidised LS.

The antifungal activity and the thionin-potentiating activity of 2S albumins may apparently be attributed primarily to their small subunits. These results suggest that truncated 2S albumin genes encoding only the SS domain could be used for the expression of a novel type of antifungal protein. Since it has been shown that intramolecular disulphide bonds are formed spontaneously in purified reduced SS molecules (Monsalve et al, 1991, Biochem Biophys Acta, 1078, 265-272), the expressed proteins are likely to adapt the configuration necessary for exerting antifungal activity.

EXAMPLE 11 Anti-fungal activity of the 2S albumins against foliar disease: in vivo test Radish 2S albumins were tested against the sugarbeet foliar disease Cercospora beticola (strain K897) to establish whether they exhibited any in vivo anti-fungal activity.

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31 Sugarbeet plants were grown in John Innes potting compost No. 1 or 2 in 4cm diameter mini-pots. 27 day old plants were used for the tests. The protein preparation was formulated immediately prior to use by dissolving in sterile distilled water and diluting to the appropriate concentration. The samples were applied to the plants as a foliar spray to maximum discrete droplet retention. The protein formulations were applied either one day prior to inoculation with the disease (1 day protectant assay) or 48 hours after disease inoculation (2 day eradicant assay).

The pathogen was applied as a foliar spray at a concentration of 50,000 spores/ml. Following inoculation the plants were kept in a humidity chamber for 48 hours and the plants then moved to a 24 0 C growth room. Disease was assessed after a further 12 days.

Results are shown in Figure 8. When applied 2 days after inoculation of the disease eradicant activity the 2S albumins gave good disease control at rates of between 100 uM and 700 uM.

Surprisingly though there was no disease control at rates of up to 700 uM when the protein was applied as a protectant 1 day prior to the disease.

To further investigate this difference the time at which the 2S albumins were applied relative to the disease was varied and the subsequent disease control assessed. The method was essentially as described above and the protein was applied at a single rate of 350 uM. The results are shown in Figure 9. Disease was applied at time zero and the proteins subsequently sprayed on at different times up to 96 hours after inoculation.

i C WO 93/04586 PCT/GB92/01574 32 All the plants were assessed 14 days after inoculation with the disease. The level of disease on plants from each time point was compared to control plants treated in an identical way but sprayed with distilled water. It can be seen that maximum anti-fungal activity is obtained when the protein is applied between 24 and 72 hours after disease inoculation. It is likely that the plants own defence mechanism will also have been induced during this period and the expression of many AFPs including thionins have been shown to reach a maximum within 44 hours of pathogen attack (Bohlmann et al, 1988, EMBO J, 7:1559-1565). It is possible therefore that these in vivo results reflect the synergy demonstrated in vitro example 7 and that the disease control that can be obtained by application of the 2S albumins results from the combination of the protein and a factor which is induced by the pathogen within the plant.

This would account for the inability of the 2S albumins to give any disease control when applied at the same time as the disease or shortly afterwards. The reversal of disease control when the protein is applied 96 hours after inoculation probably reflects the fact that the fungus has become to well established.

EXAMPLE 12 Molecular cloning of the radish 2S albumin cDNA.

From outdoor grown Raphanus sativus plants, seeds at 6 different developmental stages were collected, frozen in liquid nitrogen and stored at S- WO- 9 3/04 8 PC /B 0 54 WO 93/04586 PCT/GB92/01574 93/04586 0 ii a a id i! i-i -1 WO 93/04586 PC/GB92/01574 33 0 C. After homogenisation with a mortar and pestle, total RNA was extracted from 15 g of a mixture of the 6 different developmental stages, using the method of De Vries et al (1988, Plant Molecular Biology Manual, B6, 1-13) with the exception that 6 ml of a 1;2 phenol:RNA extraction buffer mixture and 2 ml of chloroform were used per g of tissue. Poly mRNA was purified by affinity chromatography on oligo(dT)-cellulose as described by Siflow et al (1979, Biochemistry 18, 2725-2731) yielding about 10 ug of poly(A)+ RNA per g of tissue. Double stranded cDNAs were prepared from 1,5 pg of poly(A)+ RNA according to Gubler and Hoffman (1983, Gene 25, 253-269) and ligated to EcoRI/NotI adaptors using the cDNA Synthesis Kit of Pharmacia. The cDNAs were cloned into the lambda ZAPII phage vector (Stratagene) according to the manufacturers instructions. One of the radish 2S albumin cDNAs was cloned by performing PCR (polymerase chain reaction) (under standard conditions Sambrook et al. 1989, Molecular cloning, Cold Spring Harbor Laboratory Press) on 2 pg of total cDNA as template DNA and using the sense oligonucleotide OWB18 (5'TAAGGATCCCATACACGAATGGCGAACAAGCTCTTCCTCG and the antisense oligonucleotide (5'TAATCTAGACTCTCGAGGGTTTCGTTTGG as amplimers.

was designed based on the published sequence of the radish 2S albumin pBA3 cDNA (Raynal et al, 1991, Geine 99, 77-86). As this clone was truncated at its 5' end, OWB18 was designed based on the published sequence of the oilseed rape napin pNAPI cDNA (Ericson et al, 1986, J. Biol. Chem. 261, 14576-14581). OWB18 has the TAAGGATCC ('TAA' ea ^i [(f i" i i it ii L~ I I I I ii- LL~_V ;illui Li-- L WO 93/04586 PCT/GB92/01574 followed by the BamHI recognition site) at its end and OWB20 the TAATCTAGA ('TAA' followed by the XbaI recognition site). The PCR-product was cut with BamHI and XbaI, subcloned into pEMBL18+ pre-digested with the same enzymes and subjected to automated nucleotidt sequencing on a Pharmacia ALF Automated Nucleotide Sequencer. The nucleotide sequence of this clone (hereafter called pIG8) is given in Figure 10 (the signal peptide is underlined with a dashed line, the mature small subunit is underlined with a full line, the mature large subunit is boxed).

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EXAMPLE 13 Molecular cloning of the radish 2S albumin small subunit cDNA and construction of pIG11 To obtain the 2S albumin small subunit cDNA, the same approach was followed as the one outlined in Example 12 though now using the sense oligonucleotide OWB19 and the antisense oligonucleotide OWB21 OWB19 carries the TAATCTAGACTA sequence ('TAA' followed by the Xbal recognition site and 'CTA' which introduces the stop codon 'TAG') at its 5' end and OWB21 carries at its 5' end the AATTGCTAGC sequence ('AATT' followed by the NheI recognition site).

The Mirabilia jalapa antimicrobial protein 2 (Mj- AMP2 Cammue et al, 1992, J. Biol. Chem. 267, 2228-2233) signal peptide was obtained in the same way as described above though using 50 ng of the Mj-AMP2 cDNA clone pMJ9 as template DNA and the M13 WO 93/04586 PCT/GB92/01574 universal primer and the antisense oligonucleotide OWB22 (5'AATTCTAGAATAGCTAGCTTCTATCATGCCTGACATGG 3') as amplimers. OWB22 carries the AATTCTAGAATAGCTAGC sequence ('AAT'-XbaI recognition site-'ATA'-NheI recognition site) at its 5' end. The Mj-AMP2 signal peptide PCR-product was cut with BamHI (occurring in the polylinker of pMJ9) and XbaI and cloned into pEMBL18+ pre-digested with the same enzymes. The additional ATG start-codon upstream of the Mj-AMP2 start-codon was removed by cutting the obtained construct with EcoRV and SmaI followed by blunt ligation. This resulted in the clone Subsequently, the radish 2S albumin small subunit PCR-product was cut with NheI and XbaI and cloned into pIG10 pre-digested with NheI. As NheI and XbaI produce compatible ends, the orientation of the inserted small subunit was checked by digestion of different clones with NheI and BamHI.

One of the clones with the correctly inserted small subunit, piG11, was subjected to automated nucleotide sequence analysis. The nucleotide sequence and the derived amino acid sequence of the pIG11 insert are given in Figure 11. Note that the first amino acid of the small subunit (a proline, see Fig. 10) has been changed into a serine. As the oilseed rape napins have a serine as the first amino acid of their small subunit (Ericson et al, 1983, J. Biol. Chem. 261, 14576-14581) and also exert antifungal activity and display the synergistic effect with the a-purothionin (see Examples 5 and 10), this substitution is not believed to affect nor the antifungal activity nor the synergistic effect of this altered radish 2S

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i I I i i WO 93/04586 PCT/GB92/01574 36 albumin small subunit.

EXAMPLE 14 Construction of the expression vectors pIG13 and The expression vector pIG13 (Figure 12) contains the full coding region of the Rs-2S albumin cDNA flanked at its 5' end by the strong constitutive promoter of the 35S RNA of the cauliflower mosaic virus (Odell et al, 1985, Nature K 313, 810-812) with a duplicated enhancer element to allow for high transcriptional activity (Kay et al, 1987, Science 236, 1299-1302). The coding region of the Rs-2S albumin cDNA is flanked at its 3' end side by the polyadenylation sequence of 35S RNA of the cauliflower mosaic virus (CaMV35S). The I plasmid backbone of this is the phagemid pUC120 (Vieira and Messing Methods Enzymol.

153, 3-11). pIG13 was constructed as follows clone pIG8 which consisted of the Rs-2S albumin cDNA (Figure 10) cloned into the KpnI PstI sites of pEMBL18+ (Boehringer). The 298 bp KpnI PstI fragment was subcloned into the expression vector pFAJ3002 which was pre-digested with KpnI and PstI.

pFAJ3002 is a derivative of the expression vector pFF19 (Timmermans et al. 1990, J. Biotechnol. 14, 333-344) of which the unique EcoRI site is replaced by a HindlII site. The expression vector (Figure 13) contains the hybrid nucleotide sequence coding for the Mj-AMP2 signal peptide followed by the Rs-2S albumin small subunit and was constructed in the same way as pIG13 though starting from pIG11 (Figure 11).

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WO 7~93/04586 PC/GB92/01574 37 EXAMPLE Construction of the plant transformation vector pIGl9 and The expression vector pIG13 was digested with HindIII and the fragment containing the Rs-2S Salbumin cDNA expression cassette was subcloned into the unique HindIII site of pBinl9Ri. pBinl9Ri is a modified version of the plant transformation vector pBinl9 (Bevan 1984, Nucleic Acids Research 12, 8711-8721) wherein the unique EcoRI and HindIII sites are switched and the defective nptII expression cassette (Yenofsky et al, 1990, Proc.

Natl. Acad. Sci. USA 87:3435-3439) is introduced.

The new plant transformation vector is designated pIG19 (Figure 14). The new plant transformation Svector pIG20 was constructed in the same way as pIG19 with the exception that the HindIII fragment of pIG15 (containing the hybrid Mj-AMP2 signal peptide Rs-2S albumin small subunit expression I 20 cassette) was subcloned in pBinl9Ri (Figure I A

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EXAMPLE 16 Plant Transformation.

The disarmed Agrobacterium tumefaciens strain LBA4404 (pAL4404)(Hoekema et al, 1983, Nature 303, 179-180) was transformed with the vectors plGl9 or using the method of de Framond et al, (BioTechnology 1, 262-269).

Tobacco transformation was carried out using leaf discs of Nicotiana tabacum Samsun based on the method of Horsch et al, (1985, Science 227, 1229- 1231) and co-culturing with Agrobacterium strains containing pIG19 or pIG20. Co-cultivation was f IC _-JF I WO93/04586 PCT/GB92/01574 38 carried out under selection pressure of 100 pg/ml kanamycin. Transgenic plants (transformed with pIG19 or pIG20) were regenerated on media containing 100 pg/ml kanamycin. These transgenic plants may be analysed for expression of the newly introduced genes using standard western blotting techniques. Plants capable of constitutive expression of the introduced genes may be selected and self-pollinated to give seed. Fl seedlings of the transgenic plants may be further analysed.

Ultimately, transgenic homozygous (in the p!Gi9 or in the pIG20 trait) plants may be crossed with transgenic thionin-homozygous tobacco plants. The new transgenic plants may be analysed as above.

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Claims (16)

1. An antimicrobial composition including one or more albumin-type proteins and one or more thionin proteins.

2. A composition as clain'rY. n claim 1 in which at least one protein is a functional polypeptide subunit.

3. A composition as claimed in claim 1 or claim 2 in which at least one albumin-type protein is a 2S albumin or a napin.

4. A composition as claimed in claim 3 in which at least one albumin-type protein is the small subunit of 2S albumin. A composition as claimed in any one of claims i to 4 in which at least one albumin-type protein is barley trypsin inhibitor, wheat a-amylase inhibitor, or a cereal gliadin.

6. A composition as claimed in any of claims 1 to 5 which is antifungal.

7. A process of combating fungi or bacteria which includes exposing them to a composition as claimed in any of claims 1 to 6. S, 8. A process as claimed in claim 7 in which the composition is applied externally to a plant.

9. A process as claimed in claim 7 in which the composition is expressed within a plant. A biological system having improved resistance to a bacterial or fungal pathogen, and expressing first and second proteins for delivery to a site of pathogen attack, said first protein being an albumin-type protein and said second protein being a thionin, wherein at least one of said first and second proteins is expressed from recombinant DNA.

11. A biological system as claimed in claim 10 which is a plant.

12. A plant as claimed ii claim 11 where the site of pathogen attack is in a leaf.

13. A lant as claimed in claim 11 or claim 12 in which at least one protein is a, functional polypeptide subunit.

14. A plant as claimed in any one of claims 11 to 13 in which at least one albumin- type protein is a 2S albumin or a napir, A plant as claimed in claim 14 which contains recombinant DNA having substantially the sequence shown in Fige WN :\WNWORfWENDY8WP,,iU4479,r0.Dc1 4 I WO 93/04586 PCT/GB92/01574 13/13

16. A plant as claimed in claim 14 in which at least one albumin-type protein is the small subunit of 2S albumin.

17. A plant as claimed in claim 16 which contains recombinant DNA having sul tantially the sequence shown in Figure 11.

18. A plant as claimed in any one of claims 11 to 17 in which at least one albumin- type protein is barley trypsin inhibitor, wheat a-amylase inhibitor, or a cereal gliadin.

19. Seeds and progeny of a plant as claimed in any one of claims 11 to 18, A composition as claimed in claim 1 substantially as hereinbefore described with reference to any one of the examples.

21. A biological system as claimed in claim 10 substantially as hereinbefore described with reference to any one of the examples. DATED: 13 October, 1995 PHILLIPS ORMONDE FITZPATRICK I l C A ZEC, LU IIT typ prti sbre rpsnihbtr ha aayaeihbtr o eelgidn 18. Sed an rgn fapata liedi n n fcam 1t 8 copostio asclaied n caim1 sbstatialy s hreibefoe dscrbedwit refeenceto ay on of he eampls, 10 2. Abioogial ystm asclamedin lai 10subsantall asherinbfor descibedwit refrene toany ne f th exmple. I I 9 se I i 1 ~i" I DAED:13 Otobr, 195 6 t IS, 1« PH'LIPSORMODE &FITPATRCK I ffr~ Atoney fo: ZNECALIMIED Q ^J i-