WO1998049331A1 - Antifungal composition containing beta-(1,6)-glucanase, and hosts incorporating same - Google Patents
Antifungal composition containing beta-(1,6)-glucanase, and hosts incorporating same Download PDFInfo
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- WO1998049331A1 WO1998049331A1 PCT/EP1998/002580 EP9802580W WO9849331A1 WO 1998049331 A1 WO1998049331 A1 WO 1998049331A1 EP 9802580 W EP9802580 W EP 9802580W WO 9849331 A1 WO9849331 A1 WO 9849331A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01075—Glucan endo-1,6-beta-glucosidase (3.2.1.75)
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- 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
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
Definitions
- the present invention relates to a novel glucanase protein, a combination of antifungal proteins and plants transformed with the glucanase or the combination, as well as methods of combatting fungal pathogens by causing said fungal pathogens to be contacted with said protein or proteins.
- the invention further relates to transformed plants which show reduced susceptibility to fungal pathogens.
- a protein with antifungal activity isolated from TMV-induced tobacco leaves, which is capable of causing lysis of germinating spores and hyphal tips of PhyCophthora mfestans and whicn causes the hyphae to grow at a reduced rate, was disclosed in 091/18984 Al and in Woloshuk et al . (Plant cell 3., 619-628, 1991).
- This protein, an osmotin has an apparent molecular weight of about 24 kDa and was named AP24. Comparison of its complete ammo acid sequence, as deduced from the nucleic acid sequence of the AP24 gene, with proteins known from databases revealed that the protein was an osmotm-like protein.
- WO 9b/Ji534 describes the ⁇ - (1 , 6) -glucanase (or pustulanase) from Tri choderma harzianum as an agent which catalyzes the cleavage of ⁇ - ( 1, 6 ) -linkages in ⁇ -glucan, a major component of the cell walls of fungal cells. It has also been found to act cooperativally with cellulases and chitmases against fungal plant pathogens such as Botrytis, Phytophthora and Gibberella fu iko ⁇ (de la C ruz, J . Bacteriol. 177 (7) . 1864-1871, 1995).
- the present invention provides for a new protein with ⁇ -
- the present invention further provides an antifungal composition comprising a ⁇ - (1, 6) -glucanase and optionally an osmotin
- the present invention further provides a method for making plants pathogen resistant by transforming them with recombinant DNA comprising a sequence coding for an osmotin and a sequence coding for a ⁇ - (1, 6) -glucanase .
- the osmotin is AP24, preferably having the amino acid sequence of SEQ ID NO: 2, and preferably encoded by the nucleotide sequence of SEQ ID N0:1.
- the ⁇ - (1, 6 ) -glucanase is of fungal origin, for instance from Tri choder a harzianum, or from edible fungi such as mushroom or shu-take.
- the invention also provides a recombinant DNA sequence according to the invention further comprising a transcriptional initiation region and, optionally, a transcriptional termination region, so linked to said open reading frames as to enable the DNA to be transcribed in a living host cell when present therein, thereby producing RNAs which comprise said open reading frames.
- a preferred chimeric DNA sequence according to the invention is one, wherein the RNAs comprising said open reading frames are capable of being translated into protein in said host cell, when present therein, thereby producing said proteins.
- the invention also embraces a chimeric DNA sequence comprising a DNA sequence according to the invention, which may be selected from replicons, such as bacterial cloning plasmids and vectors, such as a bacterial expression vector, a (non-mtegrative) plant viral vector, a Ti-plasmid vector of Agrobacterium, such as a binary vector, and the like, as well as a host cell comprising a replicon or vector according to the invention, and which s capable of maintaining said replicon once present therein.
- replicons such as bacterial cloning plasmids and vectors, such as a bacterial expression vector, a (non-mtegrative) plant viral vector, a Ti-plasmid vector of Agrobacterium, such as a binary vector, and the like
- a host cell comprising a replicon or vector according to the invention, and which s capable of maintaining said replicon once present therein.
- the invention further provides a host cell stably incorporating in its genome a chimeric DNA sequence according to the invention, such as a plant cell, as well as multicellular hosts comprising such cells, or essentially consisting of such cells, such as plants.
- a host cell stably incorporating in its genome a chimeric DNA sequence according to the invention, such as a plant cell, as well as multicellular hosts comprising such cells, or essentially consisting of such cells, such as plants.
- a method for producing a protein with ⁇ - ( 1 , 6) -glucanase activity characterised in that a host cell according to the invention is grown under conditions allowing the said protein to be produced by said host cell, optionally followed by the step of recovering the protein from the host cells.
- Another part of the invention is directed to the targeting of the osmotin and/or the ⁇ - (1 , 6 ) -glucanase to the extracellular space .
- the invention also provides a method for obtaining plants with reduced susceptibility to fungi, comprising the steps of
- a chimeric DNA sequence comprising open reading frames capable of encoding an osmotin and a ⁇ - (1 , 6) -glucanase, said open reading frames being operatively linked to one or more transcriptional and translat onal regions and, optionally, a transcriptional termination region, allowing the said proteins to be produced in a plant cell that is susceptible to infection by said fungus, and a chimeric DNA sequence capable of encoding a plant selectable marker allowing selection of transformed ancestor cells when said selectable marker is present therein, and
- step (b) regenerating said ancestor cells into plants under conditions favouring ancestor cells which have the said selectable marker, and (c) identifying a plant which produces the proteins, thereby reducing the susceptibility of said plant to infection.
- step (a) is performed using an Agrobacterium tumefaciens strain capable of T-DNA transfer to plant cells and which harbours the said chimeric DNA cloned into binary vector pMOG ⁇ OO; another preferred method is when step (b) is performed in the presence of an antibiotic favouring cells which have a neomycm phosphotransferase or a cyanamid hydratase gene.
- Figure 1 Purification of ⁇ l-6 glucanase by cation exchange chromatography. Three peaks of ⁇ l-6 glucanase activity can be seen, indicated by the numbers 1, 2, and 3 respectively. The chromatogram of one representative run is shown Other runs showed simmilar, if not identical, protein patterns and distribution of ⁇ l-6 glucanase activity. Protein is measured at A280. ⁇ l-6 glucanase activity is measured as described and is depicted as the absorbance at 510 nanometre .
- Figure 2 Purification of ⁇ l-6 glucanase (pool no. 1, pooled fractions 4 and 5 from the cation exchange chromatography experiment; figure 1) by gel filtration chromatography. ⁇ l-6 glucanase activity elutes from the column at fractions 24-26. Protein is measured at A280 and is depicted milli-absorption units, ⁇ l-6 glucanase activity is measured as described and is depicted as the absorbance at 510 nanometre.
- FIG 4. Purification of ⁇ l-6 glucanase (pool no. 1, pooled fractions 25 and 26 from the gel filtration chromatography experiment; figure 2) by chromatophocusmg chromatography on a Mono P exchanger column, ⁇ l-6 glucanase activity elutes from the column at fractions 11-12, corresponding to a pH of about 6.7. Protein is measured at A280 and is depicted in milli-absorption units.
- Figure 5. SDS-PAGE analysis of the ⁇ l-6 glucanase activity containing fractions of pool no. 1 after chromatophocusmg chromatography (figure 4).
- Figure 6 Purification of ⁇ l-6 glucanase (pool no. 2, pooled fractions 10, 11, and 12 from the cation exchange chromatography experiment; figure 1) by gel filtration chromatography. ⁇ l-6 glucanase activity elutes from the column at fractions 15-17. Protein is measured at A280 and is depicted in milli-absorption units, ⁇ l-6 glucanase activity is measured as described and is depicted as the absorbance at 510 nanometre.
- FIG. 7 SDS-PAGE analysis of the protein fractions of pool no. 2 after gel filtration chromatograpny (figure 6) .
- the arrow points towards the presumed ⁇ l-6 glucanase bands.
- Figure 8 Purification of ⁇ l-6 glucanase (pool no. 2, fraction 16 from the gel filtration chromatography experiment; figure 6) by chromatofocusmg chromatography on a Mono P exchanger column, ⁇ l-6 glucanase activity elutes from the column at fractions 3-5, corresponding to the flow-through of the column, meaning that the isoelectric point is above pH 6.8. Protein is measured at A280 and is depicted in milli-absorption units.
- FIG. 9 SDS-PAGE analysis of the ⁇ l-6 glucanase activity containing fractions of pool no. 2 after chromatophocusmg chromatography (figure 8) .
- the arrow points towards the ⁇ l-6 glucanase band.
- Figure 10 Purification of ⁇ l-6 glucanase (pool no. 3, pooled fractions 15, 16, and 17 from the cation exchange chromatography experiment; figure 1) by gel filtration chromatography. ⁇ l-6 glucanase activity elutes from the column at fractions 15-18. Protein is measured at A280 and is depicted in milli-absorption units ⁇ l-6 glucanase activity is measured as described and is depicted as the absorbance at 510 nanometre.
- Figure 11 Double reciprocal plot showing ⁇ l-6 glucanase activity as a function of pustulan concentration. Fraction number 4 from the chromatophocusmg column from pool no. 2 (figure 8) was used as source of ⁇ l-6 glucanase activity, ⁇ l-6 glucanase activity is measured as described and is depicted as the absorbance at 510 nanometre .
- Figure 12 Effect of AP24 and ⁇ l-6 glucanase on m vitro growth of Fusar ⁇ um oxysporu .
- the fungus was grown in a microtiter plate assay as described, in the presence of 5 ⁇ g/ml AP24 plus 5 ⁇ g/ml ⁇ l-6 glucanase ( ⁇ l-6/AP24) or the absence of these proteins (Ref ) .
- Fungal growth was followed time by reading the absorbance at 620 nanometres in a microtiter plate reader.
- Figure 14 Effect of AP24 and ⁇ l-6 glucanase on in vitro growth of Pemcillium rogueforti . Same assay as depicted m fig. 12.
- the ⁇ - ( 1 , 6) -glucanase protein according to the present invention may be obtained by isolating it from any suitable edible fungus source material containing it.
- Edible fungi m this respect means fungi which are commonly eaten.
- a particularly suitable source comprises fruit bodies and or mycelium of mushrooms (Agaricus spp . ) , oyster mushrooms ( Pleurotus ostreatus) , shn-take (Le ⁇ tmus edodes) or of other commonly eaten fungi such as Volvariella spp. , Tricholoma matsutake, Tuber uncmatum, Tuber melanosporum, Tuber spp .
- proteins according to the invention may be obtained by cloning DNA comprising an open reading frame capable of encoding said protein, or the precursor thereof, linking said open reading frame to a transcriptional, and optionally a translational initiation and transcriptional termination region, inserting said DNA into a suitable host cell and allowing said host cell to produce sa d protein Subsequently, the protein may be recovered from said host cells, preferably after secretion of the protein mto the culture medium by said host cells.
- said host cells may be used directly in a process of combating fungal pathogens according to the invention as a pesticidal acceptable composition.
- Host cells suitable for use in a process of obtaining a protein according to the invention may be selected from prokaryotic microbial hosts, such as bacteria e . g. Agrobacterium, Bacillus, Cyanobacteria, E. coli , Pseudomonas , and the like, as well as eukaryotic hosts including yeasts, e.g. Saccharomyces cerevisiae, fungi, e . g. T ⁇ choderma and plant cells, including protoplasts.
- the word protein means a sequence of ammo acids connected trough peptide bonds.
- Mutems of the protein of the invention are proteins that are obtained from the proteins depicted m the sequence listing by replacing, adding and/or deleting one or more ammo acids, while still retaining their glucanase activity.
- Such mutems can readily be made by protein engineering m vivo, e . g . by changing the open reading frame capable of encoding tne protein such that the ammo acid sequence is thereby affected. As long as the changes in the ammo acid sequences do not altogether abolish the glucanase activity such mutems are embraced in the present invention.
- the present invention provides a chimeric DNA sequence which comprises an open reading frame capable of encoding the protein according to the invention or a combination of osmotin and ⁇ -(l,6)- glucanase.
- the expression chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found m nature.
- chimeric DNA shall mean to comprise DNA comprising the said open reading frame in a non- natural location of the host genome, notwithstanding the fact that said host genome normally contains a copy of the sa d open reading frame in its natural chromosomal location.
- the said open reading frame may be incorporated in the host genome wherein it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector.
- Chimeric DNA shall not be limited to DNA molecules which are replicable in a host, but shall also mean to comprise DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor sequences, physically linked to the open reading frame according to the invention.
- the open reading frame may or may not be linked to its natural upstream and downstream regulatory elements .
- the open reading frame may be derived from a genomic library. In this latter it may contain one or more introns separating the exons making up the open reading frame that encodes a protein according to the invention
- the open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention.
- Open reading frames according to the invention also comprise those in which one or more nitrons have been artificially removed or added Each of these variants is embraced by the present invention.
- the open reading frame coding for the osmotin preferably comprises a nucleotide sequence that codes for a protein as is depicted in SEQ ID NO : 2. Most preferably the nucleotide sequence comprises the nucleotide sequence of the open reading frame depicted n SEQ ID NO:l.
- the open reading frame coding for the ⁇ - (1 , 6) -glucanase can either be composed of the sequence coding for the protein which can be derived from Trichoderma harzianum, or it can be derived from an edible fungus or a plant.
- a chimeric DNA according to the invention will usually be provided with regulatory elements enabling it to be recognised by the biochemical machinery of the host and allowing for the open reading frame to be transcribed and/or translated in the host. It will usually comprise a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ⁇ bosome recognition and attachment.
- an expression cassette usually comprises in addition a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur.
- the codon usage may be adapted to accepted codon usage of the host of choice.
- a host cell In order for the open reading frame to be maintained m a host cell it will usually be provided in the form of a replicon comprising said open reading frame according to the invention linked to DNA which is recognised and replicated by the chosen host cell. Accordingly the selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary skilled person the art.
- a special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame according to the invention to said plant cell. Replicons with such capability are herein referred to as vectors.
- Ti-plasmid vector which, when present in a suitable host, such as Agro-bacterium tumefaciens , is capable of transferring part of itself, the so-called T-region, to a plant cell.
- a suitable host such as Agro-bacterium tumefaciens
- Different types of Ti-plasmid vectors vide : EP 0 116 718 BI
- a particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 BI and US 4,940,838).
- suitable vectors which may be used to introduce DNA according to the invention mto a plant host, may be selected from the viral vectors, e.g. non-mtegrative plant viral vectors, such as derivable from the double stranded plant viruses (e.g. CaMV) and single stranded viruses, gemmi viruses and the like.
- the use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines.
- host cells incorporating a chimeric DNA sequence according to the invention in their genome shall mean to comprise cells, as well as multicellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA mto their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis.
- plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric DNA mto their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion.
- chimeric DNA capable of being expressed in substantially a constitutive fashion, that is, m substantially most cell types of the plant and substantially without serious temporal and/or developmental restrictions, is now routine.
- promoters obtainable from the cauliflower mosaic virus, notably the 35S RNA and 19S RNA transcript promoters and the so-called T-DNA promoters of Agrobacterium tumefaciens , in particular to be mentioned are the nopaline synthase promoter, octopme synthase promoter (as disclosed m EP 0 122 791 BI) and the mannopme synthase promoter.
- plant promoters may be used, which may be substantially constitutive, such as the rice actin gene promoter, or e.g. organ- specific, such as the root-specific promoter.
- pathogen-mducible promoters may be used such as the PRP1 promoter (also named gstl promoter) obtainable from potato (Martini N. et al . (1993), Mol. Gen. Genet. 261, 179-186).
- PRP1 promoter also named gstl promoter
- the choice of the promoter is not essential, although it must be said that constitutive high-level promoters are slightly preferred.
- enhancers may considerably enhance the expression level of the DNA under its regime ⁇ vide for instance: Kay R. et al . (1987), Science 236 , 1299-1302: the duplication of the sequence between -343 and - 90 of the CaMV 35S promoter increases the activity of that promoter) .
- high-level promoters are the light-mducible ⁇ bulose bisphosphate carboxylase small subunit (rbcSSU) promoter and the chlorophyll a/b binding protein (Cab) promoter.
- hybrid promoters which comprise elements of different promoter regions physically linked.
- CaMV enhanced mannop e synthase promoter US Patent 5,106,739, which comprises elements of the mannopme synthase promoter linked to the CaMV enhancer.
- transcriptional terminator region As regards the necessity of a transcriptional terminator region, it is generally believed that such a region enhances the reliability as well as the efficiency of transcription m plant cells. Use thereof is therefore strongly preferred in the context of the present invention.
- Naturally mtracellularly occurring proteins may be caused to be targeted to the apoplast by removal of the C-terminal propeptide, e.g. by modifying the open reading frame at its 3' end such that protein is caused to be C-termmally truncated.
- a certain number of ammo acids of the C-termmal part of the protein was found to be responsible for targeting of the protein to the vacuole (e.g. vide W091/18984 Al) .
- By introducing a translational stopcodon in the open reading frame the truncated protein is caused to be targeted to the apoplast.
- plants for the purpose of this description shall include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants, be they for feed, food or industrial processing purposes; included are plants used for any agricultural or horticultural purpose including forestry and flower culture, as well as home gardening or indoor gardening, or other decorative purposes.
- any transformation method may be used to introduce chimeric DNA according to the invention mto a suitable ancestor cell.
- Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al . , 1982, Nature 296. 72- 74; Negrutiu I. et al , June 1987, Plant Mol. Biol. 8., 363-373), electroporation of protoplasts (Shillito R.D. et al . , 1985
- a preferred method according to the invention comprises Agro acte ⁇ u_ ⁇ -med ⁇ ated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,940,838).
- monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material.
- Transgenic maize plants have been obtained by introducing the Strepfcomyces hygroscopicus bar-gene , which encodes phosph othricm acetyltransferase (an enzyme which inactivates the herbicide phosphmothricm) , into embryogenic cells of a maize suspension culture by micropro ⁇ ectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2., 603-618) .
- Monocotyledonous plants including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains ( vide WO 94/00977; EP 0 159 418 BI; Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. ____, 426-434).
- Agrobacterium strains vide WO 94/00977; EP 0 159 418 BI; Gould J, Michael D, Hasegawa O, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. ____, 426-434.
- DNA e.g a T-DNA on a binary plasmid
- a number of modified genes physically coupled to a second selectable marker gene.
- the advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus .
- the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci .
- the actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids) but is not critical with respect to the described invention.
- the means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Shoots may be induced directly, or indirectly from callus via organogenesis or embryogenesis and subsequently rooted. Next to the selectable marker, the culture media will generally contain various ammo acids and hormones, such as auxin and cytokinms . It is also advantageous to add glutamic acid and prolme to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype and on the history of the culture. If these three variables are controlled regeneration is usually reproducable and repeatable.
- the traits conferred by them can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
- a marker gene linked to the plant expressible gene according to the invention to be transferred to a plant cell.
- the choice of a suitable marker gene in plant transformation is well within the scope of the average skilled worker; some examples of routinely used marker genes are the neomycm phosphotransferase genes conferring resistance to kanamycm (EP-B 131 623), the glutathion-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides (EP-A 256 223), glutamme synthetase conferring upon overexpression resistance to glutamme synthetase inhibitors such as phosphmothricm (WO 87/05327), the acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphmothricm (EP-A 275 957), the gene encoding a 5-enolsh ⁇ k
- WO 91/02071 the cyanamid hydratase gene and the like.
- the actual choice of the marker is not crucial as long as it is functional (i.e. selective) m combination with the plant cells of choice.
- the marker gene and the gene of interest do not have to be linked, since co-transformation of unlinked genes (U.S. Patent 4,399,216) is also an efficient process m plant transformation.
- Preferred plant material for transformation especially for dicotyledonous crops are leaf-discs which can be readily transformed and have good regenerative capability (Horsch R.B. et al . , (1985) Science 222, 1229-1231).
- Another embodiment of the present invention is an antifungal composition
- a ⁇ - (1 , 6) -glucanase and an osmotin act synergistically to yield an antifungal effect in vi tro .
- Such antifungal effects are very useful in food applications. Especially low-fat spreads, cheese and other dairy products, tea-based beverages, fruit- and tomato-based products are vulnerable food products in th s respect.
- Combinations of antifungal compounds which act synergistically are preferable to combat fungi in food applications because less preservative substance is needed.
- the composition according to the invention may contain auxiliary ingredients which are usual for fungi combatting compositions and which may include solid diluents, solvents, stabilizers and pH-regulators .
- the composition may be in the form of a powder, a paste or a liquid, depending on the envisaged way of application.
- the composition is particularly suitable as a preservative for combatting fungi in food, but it can be used as well for preventing or combatting undesired fungal growth on other products, such as cosmetic products, where the fungal growth inhibiting composition is not only useful to keep the surface of the cosmetic product (e.g. soap) free of fungi, but also for an advantageous effect on the skin which is treated with such product, e.g. a shampoo being applied to a scalp with a fungal affliction.
- the cosmetic product e.g. soap
- Both the antifungal composition according to the invention as the plant transformed with the gene coding for the protein of the invention may contain additional antifungal components.
- proteins that may be used in combination with the proteins according to the invention include, but are not limited to, ⁇ - ( 1, 3 ) -glucanases and chitmases which are obtainable from barley (Swegle M. et al . , 1989, Plant Mol. Biol. 12 . , 403-412; Balance G.M. et al . , 1976, Can. J. Plant Sci. 5£, 459-466 ; Ho: P.B. et al . , 1988, FEBS Lett. 210 . , 67-71; Ho: P-B. et al . , 1989, Plant Mol. Biol. 12, 31-42 1989), bean (Boiler T. et al . , 1983,
- plants already containing chimeric DNA capable of encoding antifungal proteins may form a suitable genetic background for introducing chimeric DNA according to the invention, for instance in order to enhance resistance levels, or broaden the resistance.
- the cloning of other genes corresponding to proteins that can suitably be used in combination with DNA, and the obtention of transgenic plants, capable of relatively over-expressing same, as well as the assessment of their effect on pathogen resistance m planta is now within the scope of the ordinary skilled person in the art.
- transgenic plants capable of expressing, or relatively over-expressing, proteins according to the invention is a preferred method for counteracting the damages caused by pathogens such as fungi, as will be clear from the above description
- Plants, or parts thereof, which relatively over-express the protein combination according to the invention, including plant varieties, w th improved resistance against diseases, especially diseases caused by fungi may be grown in the field, m the greenhouse, or at home or elsewhere.
- Plants or edible parts thereof may be used for animal feed or human consumption, or may be processed for food, feed or other purposes in any form of agriculture or industry.
- Agriculture shall mean to include horticulture, arboriculture, flower culture, and the like.
- Plants for the purpose of this invention shall mean multicellular organisms capable of photosynthesis, and subject to some form of fungal disease. They shall at least include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants.
- plants which relatively over-express a protein shall mean plants which contain cells expressing a transgene- encoded protein which is either not naturally present in said plant, or f it is present by virtue of an endogenous gene encoding an identical protein, not m the same quantity, or not in the same cells, compartments of cells, tissues or organs of the plant. It is known for instance that proteins which normally accumulate mtracellularly may be targeted to the apoplastic space. The following state of the art may be taken mto consideration, especially as illustrating the general level of skill in the art to which this invention pertains.
- a first evaluation may include the level of expression of the newly introduced genes, the level of fungal resistance of the transformed plants, stable he ⁇ tability of the desired properties, field trials and the like.
- the transformed plants can be crossbred with other varieties, for instance varieties of higher commercial value or varieties in which other desired characteristics have already been introduced, or used for the creation of hybrid seeds, or be subject to another round of transformation and the like.
- EXPERIMENTAL PART Standard methods for the isolation, manipulation and amplification of DNA, as well as suitable vectors for replication of recombinant DNA, suitable bacterium strains, selection markers, media and the like are described for instance m Maniatis et al . , molecular cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press; DNA Cloning: Volumes I and II (D.N. Glover ed. 1985); and in: From Genes To Clones (E.-L. Wmnacker ed. 1987).
- ⁇ (l,6) glucanase activity was determined by measuring the amount of reducing sugars released from pustulan at 37°C.
- Pustulan was dissolved in hot buffer (50mM Kac, pH 5.0) and, after cooling, remained solution without precipitation.
- the standard assay i (0.5ml) contained the enzyme preparation, 1.3 mg of pustulan and 50mM Kac, pH 5.0.
- ⁇ (l,3) glucanase activity was assayed with lammarm as the reaction substrate (1.25 mg of lammarm in 0.5ml 50mM NaAc , pH 5.5) .
- Novozym 234 17 Mushrooms do contain ⁇ - (1 , 6) -glucanase activity, its specific activity being about one order of magnitude lower compared to the Trichoderma derived enzyme. In protein fractions of sunflower and TMV- infected tobacco no activity could be detected.
- the newly determined specific activity for the Agaricus bisporus extract is substantial lower than that depicted in table 1.
- the extract used for the data of Table 2 is a different extract, and we do not know the reason for this discrepancy.
- the crude protein extract (homogenate) was then further purified by ammonium sulfate precipitation, the ⁇ - ( 1, 6) -glucanase activity was precipitated at 80% saturation.
- this extract was mixed with finely dispersed pustulan particles, incubated for 20 minutes at room temperature, and the mixture centrifuged for 10 minutes at maximum speed in a microfuge centrifuge. Then the pellets were washed twice in 70 mM Potassium phosphate buffer pH 6.0 containing 1 M NaCl.
- the crude fungi homogenates contain, besides the ⁇ - (1 , 6) -glucanase activity (see above), also ⁇ - (1 , 3 ) -glucanase activity and chitmase activity.
- Specific activities for ⁇ - (1, 3) -glucanase are 9.1 and 30.2 nkat/mg for Oyster mushrooms and Shu Take respectively.
- the activities for chitmase are 13.8 and 53.5 OD(550nm) umts/mg respectively.
- the purified proteins (Mono-P fractions) contained no detectable activity for ⁇ - ( 1, 3 ) -glucanase, however, a small amount of chitmase activity was still present.
- the supernatant was filtered over a paper filter and applied to a phenyl-sepharose 6FastFlow High sub column (1.6x15cm) pre- equilibrated with 2M ammonium sulphate in 50mM potassium-acetate buffer, pH 5.0 at a flow rate of 10 ml/mm.
- the column was washed with at least 10 column volumes of buffer A after which bound protein was eluted with a negative linear gradient ending at 50mM potassium acetate buffer pH 5.0. This was followed by a positive gradient starting with 50mM potassium acetate buffer and ended with the same acetate buffer containing 50% ethylene glycol (flow rate 5 ml/mm. ) .
- Fractions of 10 ml were collected and assayed for ⁇ - (1, 6) -glucanase activity.
- Fractions containing ⁇ - (1 , 6 ) -glucanase activity were pooled and concentrated m an Amicon ultra-filtration device with a molecular weight cut-off point of 10 kDa.
- the concentrated protein solution was diluted 10 times with 25mM sodium acetate buffer pH 4.0 and subjected to cation exchange chromatography on a small Resource S column (Pharmacia) Unbound protein was washed off with 2 column volumes of 25mM sodium acetate buffer pH 4.0. Retained proteins were eluted with a linear gradient ending at 250 mM NaCl in 25mM sodium acetate pH 4.0 after 40 column volumes. Fractions of 2 ml were collected and and assayed for ⁇ - (1 , 6) -glucanase activity Three protein peaks containing ⁇ -(l,6)- glucanase activity were found (see fig. 1) .
- the pools were subjected to gelf ltration chromatography (Superdex 75, 10/30, Pharmacia), with 200mM NaCl in 50mM potassium acetate, pH 5 0 as the running buffer.
- the sample volume was 200 ⁇ l; flow rate 0.5 ml/mm, fractions of 0.5 ml were collected.
- the antifungal activity elutes from the gel-filtration column (fig. 2) at about 12kDa, based on standard marker proteins.
- ⁇ - (1 , 6) -glucanase activity was found in the flow-through of the column, indicating an isoelectric point of 6.8 or higher (fig. 8).
- SDS-PAGE of the ⁇ - ( 1 , 6 ) -glucanase containing fractions (fig. 9) showed two bands. Comparison of this gel (fig. 9) with the SDS-PAGE patterns after gel-filtration chromatography (fig. 7) and the respective ⁇ - ( 1 , 6 ) -glucanase activities, showed that ⁇ - ( 1 , 6) -glucanase corresponds to a protein band with an apparent molecular weight of about 50kDa. This band is indicated with an arrow in fig. 7 and fig. 9. These fractions did not contain ⁇ (l,3) glucanase activity.
- the antifungal activity elutes from the gel-filtration column at the same position as for pool no .2 , 88kDa (fig. 10) .
- Example 3 In vitro antifungal assay on fungi.
- the antifungal activity of proteins is measured m a microtiter plate assay.
- Each well of a 96-well microtiter dish contained 75 ⁇ l of potato dextrose agar containing 450 spores.
- the spore solution is added to PDA (0.75% agar, pH 4.9 at 39°C) just prior to filling of the microtiterplate wells.
- PDA 0.75% agar, pH 4.9 at 39°C
- 75 ⁇ l filter sterilized (0.22 ⁇ filter) protein solution is added.
- the tests are carried out in microtiter plates at 20°C.
- the fungus is monitored m a microtiter plate reader at 620nm.
- AP24 or ⁇ - (1-6) -glucanase from Trichoderma harzianum are added seperately at a final concentration of 5 ⁇ g/ml or are added in combination (5 ⁇ g/ml of each).
- AP24 was purified according to W091/18984
- ⁇ -(l- 6) -glucanase was purified from a Pichia pasto ⁇ s expression system according to Lora, J.M. et al , Mol. Gen. Genet., 247, 639-645,
- GGT AAA CCA CCA AAC ACC TTG GCT GAA TAC GCT TTG GAC CAA TTC AGT 336 Gly Lys Pro Pro Asn Thr Leu Ala Glu Tyr Ala Leu Asp Gin Phe Ser 100 105 110
Abstract
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Priority Applications (4)
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EP98925515A EP1027445A1 (en) | 1997-04-29 | 1998-04-27 | Antifungal composition containing beta-(1,6)-glucanase, and hosts incorporating same |
JP54661998A JP2001523962A (en) | 1997-04-29 | 1998-04-27 | Antifungal composition containing β- (1,6) -glucanase and host incorporating the same |
AU77608/98A AU743529B2 (en) | 1997-04-29 | 1998-04-27 | Antifungal composition containing beta-(1,6)-glucanase, and hosts incorporating same |
CA002288074A CA2288074A1 (en) | 1997-04-29 | 1998-04-27 | Antifungal composition containing beta-(1,6)-glucanase, and hosts incorporating same |
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JP (1) | JP2001523962A (en) |
AR (1) | AR015112A1 (en) |
AU (1) | AU743529B2 (en) |
CA (1) | CA2288074A1 (en) |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002059333A2 (en) * | 2001-01-23 | 2002-08-01 | Ziyu Dai | Gene promoters isolated from potato and use thereof |
JP2003516753A (en) * | 1999-12-16 | 2003-05-20 | モンサント テクノロジー エルエルシー | DNA constructs for the expression of heterologous polypeptides in plants |
EP1363487A2 (en) * | 2001-01-29 | 2003-11-26 | Cargill, Incorporated | Fungal resistant transgenic plants |
US6900343B1 (en) | 1998-11-25 | 2005-05-31 | Basell Polyolefine Gmbh | Method for the purification of metallocenes |
CN104974225A (en) * | 2015-07-22 | 2015-10-14 | 中国农业大学 | Matsutake polypeptide and matsutake extract, and application thereof |
WO2020128991A1 (en) * | 2018-12-21 | 2020-06-25 | Covertis | Biologically active substance, method for manufacturing same and use thereof as an agent for protecting a biological tissue |
Families Citing this family (1)
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JP2004189737A (en) * | 2002-11-28 | 2004-07-08 | Keiichiro Hiyama | Antifungal agent |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4748021A (en) * | 1983-07-28 | 1988-05-31 | Yissum Research And Development Company Of The Hebrew University Of Jerusalem | Antifungal compositions containing trichoderma active against fusarium |
WO1991018984A2 (en) * | 1990-06-07 | 1991-12-12 | Mogen International N.V. | New antifungal preparations, process for making such preparations, process for obtaining plants with decreased susceptibility to fungi |
WO1994008010A1 (en) * | 1992-09-28 | 1994-04-14 | Monsanto Company | Method of controlling plant pathogenic fungi |
WO1994024271A1 (en) * | 1993-04-21 | 1994-10-27 | Cornell Research Foundation, Inc. | Nagase and glucosidase isolated from trichoderma harzianum and antifungal synergistic combinations of fungal cell wall degrading enzymes |
WO1995002319A1 (en) * | 1993-07-16 | 1995-01-26 | The Salk Institute For Biological Studies | Transgenic plants containing multiple disease resistance genes |
WO1995031534A1 (en) * | 1994-05-11 | 1995-11-23 | Novo Nordisk A/S | AN ENZYME WITH β-(1-6)-ENDOGLUCANASE ACTIVITY |
-
1998
- 1998-04-27 AU AU77608/98A patent/AU743529B2/en not_active Ceased
- 1998-04-27 JP JP54661998A patent/JP2001523962A/en active Pending
- 1998-04-27 CA CA002288074A patent/CA2288074A1/en not_active Abandoned
- 1998-04-27 EP EP98925515A patent/EP1027445A1/en not_active Withdrawn
- 1998-04-27 WO PCT/EP1998/002580 patent/WO1998049331A1/en not_active Application Discontinuation
- 1998-04-28 ZA ZA983554A patent/ZA983554B/en unknown
- 1998-04-28 AR ARP980101956A patent/AR015112A1/en not_active Application Discontinuation
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4748021A (en) * | 1983-07-28 | 1988-05-31 | Yissum Research And Development Company Of The Hebrew University Of Jerusalem | Antifungal compositions containing trichoderma active against fusarium |
WO1991018984A2 (en) * | 1990-06-07 | 1991-12-12 | Mogen International N.V. | New antifungal preparations, process for making such preparations, process for obtaining plants with decreased susceptibility to fungi |
WO1994008010A1 (en) * | 1992-09-28 | 1994-04-14 | Monsanto Company | Method of controlling plant pathogenic fungi |
WO1994024271A1 (en) * | 1993-04-21 | 1994-10-27 | Cornell Research Foundation, Inc. | Nagase and glucosidase isolated from trichoderma harzianum and antifungal synergistic combinations of fungal cell wall degrading enzymes |
WO1995002319A1 (en) * | 1993-07-16 | 1995-01-26 | The Salk Institute For Biological Studies | Transgenic plants containing multiple disease resistance genes |
WO1995031534A1 (en) * | 1994-05-11 | 1995-11-23 | Novo Nordisk A/S | AN ENZYME WITH β-(1-6)-ENDOGLUCANASE ACTIVITY |
Non-Patent Citations (2)
Title |
---|
MULENGA, D.K., ET AL.: "isolation and characterization of a unique endo-beta-1,6-glucanase from yeast Saccharomycopsis fibuligera NCYC 451", MICROBIOS, vol. 80, no. 324, 1994, pages 143 - 154, XP002076682 * |
PANDEY M ET AL: "Expression and characterization of Pichia etchellsii beta -glucosidase in Escherichia coli", GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, vol. 190, no. 1, 29 April 1997 (1997-04-29), pages 45-51, XP004064383 * |
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JP2003516753A (en) * | 1999-12-16 | 2003-05-20 | モンサント テクノロジー エルエルシー | DNA constructs for the expression of heterologous polypeptides in plants |
US8822666B2 (en) | 1999-12-16 | 2014-09-02 | Monsanto Technology Llc | Hybrid caulimovirus promoters and constructs thereof |
WO2002059333A3 (en) * | 2001-01-23 | 2004-02-12 | Ziyu Dai | Gene promoters isolated from potato and use thereof |
WO2002059333A2 (en) * | 2001-01-23 | 2002-08-01 | Ziyu Dai | Gene promoters isolated from potato and use thereof |
US7141723B2 (en) | 2001-01-29 | 2006-11-28 | Cargill, Incorporated | Transgenic plants resistant to Sclerotinia and Phoma lingam |
EP1363487A2 (en) * | 2001-01-29 | 2003-11-26 | Cargill, Incorporated | Fungal resistant transgenic plants |
EP1363487A4 (en) * | 2001-01-29 | 2005-08-17 | Cargill Inc | Fungal resistant transgenic plants |
CN104974225A (en) * | 2015-07-22 | 2015-10-14 | 中国农业大学 | Matsutake polypeptide and matsutake extract, and application thereof |
CN104974225B (en) * | 2015-07-22 | 2018-04-27 | 中国农业大学 | Matsutake polypeptide, Fructificatio Tricholomatis Matsutakis extract and their application |
WO2020128991A1 (en) * | 2018-12-21 | 2020-06-25 | Covertis | Biologically active substance, method for manufacturing same and use thereof as an agent for protecting a biological tissue |
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CN113490419A (en) * | 2018-12-21 | 2021-10-08 | 利摩日大学 | Bioactive substances, method for producing same, and use thereof as biological tissue protective agents |
CN113490419B (en) * | 2018-12-21 | 2023-06-23 | 利摩日大学 | Bioactive substance, method for producing same, and use of same as biological tissue protective agent |
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EP1027445A1 (en) | 2000-08-16 |
AR015112A1 (en) | 2001-04-18 |
ZA983554B (en) | 1998-10-29 |
CA2288074A1 (en) | 1998-11-05 |
AU7760898A (en) | 1998-11-24 |
JP2001523962A (en) | 2001-11-27 |
AU743529B2 (en) | 2002-01-31 |
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