Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/08—Magnoliopsida [dicotyledons]
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/40—Liliopsida [monocotyledons]
  • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

Definitions

• This invention relates to biocidal proteins and compositions, and processes for their manufacture and use.
• it relates to the antimicrobial activity of seed storage proteins, and to their synergistic activity with other antimicrobial proteins isolated from plants.
• antimicrobial activity includes a range of antagonistic effects (such as partial inhibition or death) on a fungus and/or a bacterium.
• the invention relates to antifungal (which may include anti-yeast) activity.
• plants normally grow on substrates that are extremely rich in fungal organisms, infection remains a rare event.
• plants produce a wide array of 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 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 phytopathogenic fungi.
• thionins are basic cysteine-rich plant proteins that are thought to be involved in host defence (Rodriguez-Palenzuela et al, 1988, Gene, 70, 271-280).
• the 2S albumins are, together with the 7S and IIS globulins, the main protein components in seeds of several' different plant families, including the Brassicaceae (Higgins, 1984, Ann Rev Plant Physiol, 35, 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 9.5 kDa (large subunit), held together by two disulphide bridges. Both subunits originate from the same 21 kDa precursor polypeptide by post-translational proteolytic processing events
• seed storage proteins may have secondary functions related to the protection of seeds from damage caused by animals or micro-organisms.
• no such function has ever been attributed to 2S storage albumins.
• albumin-type proteins exhibit surprising antifungal activity, and may be used as fungicidal agents.
• these proteins exhibit a surprising and unique property in that they can potentiate the antifungal activity of thionins: in some cases up to 70-fold.
• 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.
• the invention provides a process of combating fungi or bacteria by exposure to such a composition.
• 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 Ca + at physiological concentrations.
• the albumin-type proteins (2S albumins and 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 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 reco binant 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( ⁇ ) and control sequences from a plant genome. This genetic material can then be cloned into a biological system which allows expression of the proteins.
• the proteins can be produced in a suitable micro-organism or cultured cell, extracted 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 the protein at the site of pathogen attack on the plant.
• the albumins seed proteins
• the albumins may be produced i ⁇ vivo within parts of the plant where they are not normally 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 plasmid ⁇ , electroporation, microinjection, microprojectile gun, etc).
• the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed 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).
• genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes. carrot, lettuce, cabbage, 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.
• 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 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 10.
• the invention further provides an antimicrobial composition
• 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.
• the invention provides a process of combating fungi or bacteria by exposure to such a composition.
• the synergism exhibited between the albumins and the thionins 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.
• 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.
• the proteins may also be be used to combat 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 ⁇ n vivo.
• 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.
• 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 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
• Figure 10 shows the nucleotide sequence of the 2S 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 expression vector pIGl3.
• Figure 13 shows the construction of the expression vector pIGl ⁇ .
• Figure 14 shows the construction of the plant transformation vector pIG19.
• Figure 15 shows the construction of the plant transformation vector pIG20.
• 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 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°C with 2 litres of an ice-cold extraction buffer containing 10 mM NaH 2 P0 4 , 15 mM Na 2 HP0 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 g). 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 g) .
• 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 g). After redissolving the pellet in 400 ml H-0 the solution was heated at 80°C for 15 min. The coagulated insoluble material was removed by centrifugation (30 min at 7000 x g) and the supernatant was dialyzed extensively against distilled water using dialysis tubing (SpectralPor, Spectrum, USA) with a molecular weight cut off of 1000 Da.
• 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-2S1, Bn-2S2, Bn-2S3, Bn-2S4 and Bn-2S5 respectively for the rapeseed 2S albumins ( Figure 2).
• 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-HCl (pH 8.3), 1% (w/v) SDS, 1 M EDTA, 0.005% bromophenol blue, and the sample buffer for analysis of reduced proteins contained a supplement of 1% (w/v) dithiothreitol (DTT).
• the unreduced proteins were fixed in 30% (v/v) ethanol/10% (v/v) 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 show Bn-2S4, lanes 5 show Bn-2S5.
• 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.
• the reduced radish 2S albumins migrate as 2 bands of about 9 and 4 kDa, respectively.
• the different isoforms yield doublets with an apparent molecular weight of 17 and 18 kDa, respectively.
• the column was eluted at 1 ml/min with a linear gradient (90 min) from 0 to 45% 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) 30 residues could be identified, whereas for the large subunit of Rs-2S5 (peak 4, Figure 5) 20 amino acids were sequenced.
• barley Hordeum vulgare L.
• barley trypsin inhibitor that is highly homologous to 2S albumins from dicotyledonous species.
• the barley trypsin inhibitor hereafter called btiO
• btiO barley trypsin inhibitor
• btiO 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). The column was eluted at 5 ml/min with a linear gradient (40 min) from 0 to 40% acetonitrile. The identity of the purified protein as btiO was verified by N-terminal amino acid sequencing. The first 11 N-terminal residues were identical to those previously reported (Odani e_t al, 1983, J Biol Chem, 258, 7998-8000).
• bti-1 barley trypsin inhibitor 1
• bti2 barley trypsin inhibitor 2
• Figure 7 shows the N-terminal amino acid sequences of the trypsin inhibitors from barley and from WG11. ⁇ -Purothionin was isolated from wheat
• the purothionin eluted was two incompletely resolved peaks at 34 and 35% acetonitrile, respectively, which represent the ⁇ l and ⁇ 2 isofor (Mak and Jones, 1977, Cereal Chem, 54, 511-523). Both isoforms were pooled, to yield the o-purothionin preparation.
• Antifungal activity assay was measured by microspectrophoto etry as previously described
• the synthetic growth medium consisted of K-HPO ⁇ (2.5 mM), MgS0 4 (50 ,vM), CaCl 2 (50 //M) , FeS0 4 (5 ⁇ VL) , C ⁇ Cl 2 (0.1 ⁇ K) , CuS ⁇ 4 (0.1 ⁇ K ) , a 2 Mo0 4 (2 ,vM) , H 3 B0 3 (0.5 ⁇ ) , KI (0.1 /M) , ZnS0 4 (0.5 /M), MnS0 4 (0.1 ⁇ ) , glucose (lOg/l), asparagine (lg/1), methionine (20 mg/1), myo-inositol (2 mg/1), biotin (0.2 mg/1), thiamine-HCl (1 mg/1), and pyridoxine-HCl (0.2 mg/1). Control microculture
• 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.
• the antifungal potency of the radish 2S albumins, rapeseed 2S albumins, barley trypsin inhibitors and ⁇ -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.
• medium A was half-strength potato dextrose broth
• medium B was medium A supplemented with 1 mM CaCl 2 and 50 mM KCl.
• the constitution of monovalent and divalent cations of medium B is such that it resembles that inside a plant cell.
• medium B high ionic strength
• the 2S albumins and the barley trypsin inhibitors were completely inactive at concentrations up to 1 mg/ml.
• the ⁇ -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.
• the effect of ions on the antifungal activity of radish 2S albumins was examined in more detail.
• the IC CQ 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 KCl, 50 mM KCl, 1 mM CaCl 2 or 5 mM CaCl 2 in supplement, respectively. Results are shown in Table 2. TABLE 2 VARIATION IN ANTIFUNGAL ACTIVITY OF RS-2S3 IN PRESENCE OF K + AND CA 2+
• EXAMPLE 7 Synergism between 2S albumins, barley trypsin inhibitor and ⁇ -purothionin.
• the Synergism Factor was calculated as the ratio of the C 50 value in the control series (no 2S albumins or trypsin inhibitors added) over the IC 5Q value in the series supplemented with 2S albumin or barley trypsin inhibitors. Synergism Factors are rounded to the nearest integer.
• 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 ⁇ -purothionin and Bn-2S3 (at 250 ⁇ q/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 50 ,vg/ml, but except for A brassicola, no further substantial increase was obtained on going from 50 to 250 ⁇ q/ml.
• 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 ⁇ -purothionin on bacterial growth.
• a bacterial suspension was prepared by inoculating either soft agarose medium C (10 g/1 tryptone, 5 g/1 Seaplaque agarose FMC), or soft agarose medium D (10 g/1 tryptone, 5 g/1 Seaplaque agarose (FMC),
• the following bacterial species were used: Agrobacterium tumefaciens LMG 188, Alcaligenes eutrophus LMG 1195, Azospirillum brasilense ATCC 29145, Bacillus megaterium ATCC 13632, Erwinia carotovora strain 3912, Escherichia coli strain HB101, Pseudomonas solanacearum LMG 2293, Sarcina lutea ATCC 9342.
• RS-2S3 was assessed by adding serial dilutions of the protein to bacterial suspensions. The highest test concentration was 500 ⁇ q/ml (final concentration) .
• Rs-2S3 only affected the growth of the gram-positive bacterium B megaterium (IC ⁇ 0 « 10/g/ml) and the gram-negative bacterium E carotovora (IC 50 - 250,vg/ml). However, when the growth medium C was supplemented with 1 mM CaCl- and 50 mM KCl (medium D), 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.
• synergism factors of 4, 15 and 17 were obtained after addition of Rs-2S3 at subinhibitory concentrations of 10, 50 and 250 ⁇ q/ml , respectively, to a serial dilution series of ⁇ -purothionin (Table 4).
• the thionin-potentiating activity of 2S albumin is not limited to fungi, but is also evident on some bacterial species.
• EXAMPLE 9 Effect of 2S albumins and ⁇ -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-muscle fibroblasts (Van Damme et al, 1987, Eur J Immunol, 17, 1-7) cultured in 96-well microplates. The growth medium was replaced by 80 ⁇ l of serum-free medium (Optimem 1 for endothelial cells or Eagle's minimal essential medium (EMEM) for fibroblasts, both from GIBCO), to which 20 ,vl of a filter-sterilised test solution was added. The cells were further incubated for 24 hours at 37°C under a 5% C0 2 atmosphere with 100% relative humidity.
• EMEM Eagle's minimal essential medium
• the viability of the cells was assessed microscopically after staining with trypane blue (400 mg/1 in phosphate buffered saline, PBS) for 10 minutes. Alternatively, cells were stained with neutral red (56 ml/1 in PBS) for 2 hours at 37°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.
• RS-2S3 When added at up to 500 ,vg/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, ⁇ -purothionin administered at 50 ⁇ q/ml and 20 ⁇ q/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 ⁇ -purothionin did not increase the toxic activity of the - ⁇ -purothionin.
• 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 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.
• Test protein cone Test protein cone.
• Table 5 shows that SS as well as LS (though to a much lesser extent) are able to potentiate the activity of ⁇ -purothionin.
• a synergism factor of up to 33 could be obtained when oxidised SS was added at 10 ⁇ q/ml to a dilution of ⁇ -purothionin and assayed in medium A.
• 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 ⁇ q/ml in medium B.
• Low synergism factors were measured during the same experiments with the oxidised LS.
• EXAMPLE 11 Anti—fungal activity of the 2S albumins against foliar disease: ii 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. 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°C growth room. Disease was assessed after a further 12 days.
• Results are shown in Figure 8.
• the 2S albumins gave good disease control at rates of between 100 uM and 700 uM.
• Double stranded cDNAs were prepared from 1,5 ⁇ q of poly(A) + RNA according to Gubler and Hoffman (1983, Gene 25, 263-269) 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.
• OWB20 was designed based on the published sequence of the radish 2S albumin pBA3 cDNA (Raynal et al, 1991, Gene 99, 77-86).
• OWB18 was designed based on the published sequence of the oilseed rape napin pNAPl cDNA (Ericson e_t al, 1986, J. Biol. Chem. 261, 14576-14581). OWB18 has the TAAGGATCC ('TAA' followed by the BamHI recognition site) at its 5' end and OWB20 the TAATCTAGA ('TAA' followed by the Xbal recognition site).
• the PCR-product was cut with BamHI and Xbal, subcloned into pEMBL18+ pre-digested with the same enzymes and subjected to automated nucleotide 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).
• 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 ( 'AAT r followed by the Nhel recognition site).
• the Mirabilis jalapa antimicrobial protein 2 (Mj- AMP2 ; Cammue et al, 1992, J. Biol. Chem.
• the MJ-AMP2 signal peptide PCR-product was cut with BamHI (occurring in the polylinker of pMJ9) and Xbal and cloned into pEMBLl8+ 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 Smal followed by blunt ligation. This resulted in the clone pIGlO.
• the radish 2S albumin small subunit PCR-product was cut with NheI and Xbal and cloned into pIGlO pre-digested with NheI.
• NheI and Xbal produce compatible ends
• the orientation of the inserted small subunit was checked by digestion of different clones with Nhel and BamHI.
• One of the clones with the correctly inserted small subunit, pIGll was subjected to automated nucleotide sequence analysis.
• the nucleotide sequence and the derived amino acid sequence of the pIGll 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.
• 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 ⁇ -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 albumin small subunit.
• EXAMPLE 14 Construction of the expression vectors pIG13 and plGlS.
• the expression vector pIGl3 ( 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 e_t al ⁇ , 1985, Nature 313, 810-812) with a duplicated enhancer element to allow for high transcriptional activity (Kay e_t 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).
• pIG13 was constructed as follows : clone pIG8 which consisted of the Rs-2S albumin cDNA ( Figure 10) cloned into the Kpnl / PstI sites of pEMBLl8+ (Boehringer) . The 298 bp Kpnl / PstI fragment was subcloned into the expression vector pFAJ3002 which was pre-digested with Kpnl and PstI. pFAJ3002 is a derivative of the expression vector pFFl9 (Timmermans et al.
• the expression vector pIG15 ( Figure 13) contains the hybrid nucleotide sequence coding for the MJ-AMP2 signal peptide followed by the Rs-25 albumin small subunit and was constructed in the same way as pIG13 though starting from pIGll ( Figure 11) .
• EXAMPLE 15 Construction of the plant transformation vector pIG19 and pIG20.
• pBinl9Ri is a modified version of the plant transformation vector pBinl9 (Bevan 1984, Nucleic Acids Research 12, 8711-8721) wherein the unique EcoRI and Hindlli sites are switched and the defective nptll expression cassette (Yenofsky et a_l, 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 vector pIG20 was constructed in the same way as pIG19 with the exception that the Hindlli fragment of pIG15 (containing the hybrid MJ-AMP2 signal peptide / Rs-2S albumin small subunit expression cassette) was subcloned in pBinl9Ri ( Figure 15).
• LBA4404 (pAL4404) (Hoekema et. al, 1983, Nature 303, 179-180) was transformed with the vectors pIGl9 or pIG20 using the method of de Framond e_t 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 carried out under selection pressure of 100 ⁇ q/ml kanamycin.
• Transgenic plants (transformed with pIG19 or pIG20) were regenerated on media containing 100 ⁇ q/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.
• transgenic homozygous plants in the pIGl9 or in the plG20 trait
• transgenic thionin-homozygous tobacco plants may be crossed with transgenic thionin-homozygous tobacco plants.
• the new transgenic plants may be analysed as above.

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