EP0667905A1 - Proteines antifongiques de liaison de la chitine et adn les codant - Google Patents

Proteines antifongiques de liaison de la chitine et adn les codant

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Publication number
EP0667905A1
EP0667905A1 EP93921929A EP93921929A EP0667905A1 EP 0667905 A1 EP0667905 A1 EP 0667905A1 EP 93921929 A EP93921929 A EP 93921929A EP 93921929 A EP93921929 A EP 93921929A EP 0667905 A1 EP0667905 A1 EP 0667905A1
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EP
European Patent Office
Prior art keywords
plant
antifungal
cbp
gene
chitin binding
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EP93921929A
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German (de)
English (en)
Inventor
Leo Sjoerd Melchers
Marianne Beatrix Sela-Buurlage
Alexandra Aleida Bres-Vloemans
Anne Silene Ponstein
Marion Apotheker
Bernardus Johannes Clemens Cornelissen
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Syngenta Mogen BV
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Mogen International NV
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Priority to EP93921929A priority Critical patent/EP0667905A1/fr
Priority claimed from PCT/EP1993/002790 external-priority patent/WO1994008009A1/fr
Publication of EP0667905A1 publication Critical patent/EP0667905A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to antifungal chitin binding proteins, methods for isolating such proteins and recombinant polynucleotides encoding therefor, as well as plants which have been transformed to contain said recombinant DNA and parts of such plants.
  • chitin binding proteins comprises proteins of various nature such as chitinases, occurring inter alia in bean (Boiler T. et al, 1983, Planta 157, 22-31), wheat (Molano J. et al., 1979, J. Biol. Chem. Z5 . 4901-4907), tobacco (Shinsi H. et al., 1987, Proc. Natl. Acad. Sci. USA 84. 89-93), poplar” (Parsons, T.J. et al, 1989, P.N.A.S. 8j5, 7895-7899), and potato (Laflamme D.
  • chitin binding proteins share a similar amino acid sequence which is called the hevein domain because its approximate 50% homology with a small chitin binding protein found in the latex of the rubber tree Hevea brasiliensis (Waiserono K. et aJL. , 1975, Proceedings of the International Rubber Conference, Kuala Lumpur, Malaysia, 518-531) .
  • Some chitin binding proteins have been reported to possess antifungal activity in in vitro assays.
  • Bean intracellular chitinase is capable of inhibiting the growth of Trichoderma viride at a concentration of at least 2 ⁇ g/ml (Schlumbaum A. et al.
  • th s chitinase belongs to the so-called class-I chitinases (see: Lawton K. et al. , 1992, Plant Mol. Biol. ⁇ , 735-743). Initially, the chitin binding lectins have been reported to possess antifungal activity in in vitro assays (e.g. Mirelman D. et aJL., 1975, Nature 256. 414-416) , but this effect appeared attributable to contaminating chitinases (Schlumbaum et al, 1986, supra) .
  • Fusariu oxysporum (Van Parijs et al.. , Planta 183, 258-264). Its activity was reported to be stronger than tobacco chitinases and somewhat less than that of nettle lectin UDA. Nonetheless, the required concentrations by far exceed physiologically feasible concentrations; the IC 50 ranges from 90 ⁇ g/ml for Trichoderma hamatum to 1250 ⁇ g/ml for Fusarium oxysporum. Its molecular weight as estimated by SDS-PAGE was 14 kDa, but using gel filtration its size was determined at 9 - 10 kDa. Several authors speculated about a possible correlation between the small size of both chitin binding proteins and antifungal activity.
  • the present invention provides a new class of antifungal chitin binding proteins, which are characterized in that they have low chitinase activity, a molecular weight of at least 15 kDa, and a strong synergistic antifungal effect in combination with 1,3- ⁇ -glucanases; the antifungal effect of these proteins is not markedly decreased by divalent cations.
  • Preferred antifungal chitin binding proteins are those which have an estimated molecular weight of about 20 kDa using SDS- PAGE and are obtainable from tobacco.
  • the invention also comprises an antifungal composition comprising an antifungal amount of an antifungal CBP according to the invention.
  • Preferred according to th invention is a composition which further comprises g__... anase, more preferably an intracellular plant ⁇ -l,3-glucanase, yet more preferably from tobacco.
  • Another aspect of the invention is a substantially pure polynucleotide sequence encoding an antifungal CBP according to the invention.
  • a preferred embodiment is the polynucleotide sequences represented by SEQIDNO: 7 and 9, as well as DNA sequences which hybridize therewith.
  • Yet another aspect of the invention is a chimeric plant expressible gene encoding an antifungal CBP according to the invention.
  • the plant expressible antifungal CBP gene comprises in sequence:
  • the invention also includes plasmids suitable for cloning in a microorganism which plasmid harbours a DNA sequence encoding an antifungal CBP according to the invention. Also included are plasmids harbouring a said DNA sequence and which are suitable for the transformation of plant material. Other embodiments of the invention are microorganims , including Agrobacterium strains, containing a said plasmid or plasmids.
  • the invention provides a method for obtaining a plant host which contains a chimeric plant expressible antifungal CBP comprising the steps of: (1) introducing into a recipient cell of said plant host a chimeric plant expressible antifungal chitin binding protein gene and a selectable marker gene that is functional in said plant host,
  • step (2) (2) generating a plant from a recipient cell obtained from step (1) under conditions that allow for selection for the presence of the selectable marker gene.
  • the invention also provides a recombinant plant DNA genome which contains a chimeric plant expressible gene encoding an antifungal CBP according to the invention. More preferred recombinant plant DNA genomes according to the invention are those which further comprise a chimeric plant expressible 1,3- ⁇ -glucanase gene, such that both genes are expressed and the proteins they encode are produced.
  • the invention further provides plant cells, including protoplasts, which have a recombinant plant DNA genome according to the invention, as well as plants or parts of plants, such as a bulbs, flowers, fruits, leaves, pollen, roots or root cultures, seeds, stalks, tubers (including microtubers) and the like, containing a cell harbouring a recombinant DNA genome according to the invention.
  • plants or parts thereof which substantially consist of cells having a recombinant plant DNA genome according to the invention.
  • plants which harbour a recombinant plant DNA genome according to the invention which as a result exhibit reduced susceptibility to fungal infection.
  • the invention further provides a method for breeding a plant variety which has reduced susceptibility to fungi, characterized in that at least one of the parental lines has a recombinant DNA genome according to the invention.
  • the invention also *-provides a method for reducing the damage to agricultural crop plants as a result of fungal infection characterized in that a plant is used which exhibits reduced susceptility to fungal infection.
  • the invention also provides a method for the isolation of an antifungal CBP from plant material comprising the steps of:
  • FIGURES Figure 1: Binary vector pMOG685, containing in addition to the plant expressible NPTII marker gene a plant expressible fungal chitin binding protein gene.
  • Figure 2 Binary vector pMOG686, containing in addition to the plant expressible NPTII marker gene a plant expressible, C-terminally modified (*) , chitin binding protein gene; the modified CBP is targeted to the extracellular space.
  • Figure 3 Binary vector pMOG687 containing in addition to the plant expressible NPTII marker gene an unmodified plant expressible CBP gene and a C-terminally modified (*) plant expressible glucanase gene; the glucanase encoded by this gene is targeted to the extracellular space.
  • Figure 4 Binary vector pM0G688 containing in addition to the plant expressible NPTII marker gene a plant expressible, C-terminally modified (*) , CBP gene and a plant expressible, C-terminally modified (*) , glucanase gene; both the glucanase and the CBP encoded by these genes are targeted to the extracellular space.
  • Glu- I intracellular glucanase
  • class-I chitinases in a microtiter plate assay
  • the new CBPs are characterized by a molecular weight in the range of 15 to 25 kDa of the mature protein, a a drastic synergistic antifungal effect in combination with ⁇ -1,3- glucanase and low chitinase activity (not more than 10%, more particularly not more than 5% of the class-I chitinases from tobacco as determined with the tritiated chitin method according to Molano et al . , 1977, supra) .
  • a composition containing 5 ⁇ g/ml i abacco CBP and 0.5 ⁇ g/ml intracellular ⁇ - 1,3-glucanase from tobacco almost completely inhibited the growth of Fusarium solani and Trichoderma viride.
  • a plant chimeric plant expressible gene construct containing a said CBP encoding DNA sequence under the control of the high-level CaMV 35S promoter with double enhancer and the alfalfa mosaic virus (A1MV) untranslated leader was introduced into tobacco and tomato plants and plants producing CBP either intracellularly (unmodified construct) or extracellulary (C-terminal vauolar targeting signal deleted construct) were assayed for fungal resistance using Rhizoctonia solani or Fusarium oxysporu f. sp. Lycopersici as test fungi.
  • any plant species that is subject to some form of fungal attack may be provided with one or more plant expressible gene constructs, which when expressed overproduce CBP and/or glucanase in said plant in order to decrease the rate of infectivity and/or the effects of such attack.
  • the invention can even be practiced in plant species that are presently not amenable for transformation, as the amenability of such species is just a matter of time and because transformation as such is of no relevance for the principles underlying the invention.
  • 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 a plant expressible gene according to the invention into a plant species of choice.
  • useful methods are 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 Bio/Technol. 3., 1099-1102) , microinjection into plant material (Crossway A. et al., 1986, Mol. Gen. Genet. 2J02, 179-185), (DNA or RNA- coated) particle bombardment of various plant material (Klein T.M. et al. , 1987, Nature 327. 70), infection with viruses and the like.
  • use is made of Agrobacterium-mediated DNA transfer.
  • use is made of the so-called binary vector technology as disclosed in EP-A 120 516 and U.S. Patent 4,940,838).
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant expressible genes co- transferred with the plant expressible gene according to the invention, whereafter the transformed material is regenerated into a whole plant.
  • monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or cell groupings.
  • preferred methods for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation (Shima oto, et al. 1989, Nature 338. 274-276).
  • Transgenic maize plants have been obtained by introducing the Streptomvces hvgroscopicus bar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin) , into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon- Kam , 1990, Plant Cell, 2., 603-618) .
  • the introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, 1989, Plant Mol. Biol. 13, 21-30) .
  • Monocotyledonous plants including commercially important crops such as corn are also amenable to DNA transfer by Agrobacterium strains (Gould J, Michael D, Hasegawa 0, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. 95, 426- 434).
  • a DNA sequence is generally linked to a regulatory sequence, which should at least comprise a trancriptional initiation site; such a promoter is occasionally referred to in the art as a 'minimal promoter'.
  • Regulatory sequences may include additional elements such as enhancers to promote transcription.
  • Enhancers may increase expression in a constitutive fashion or in a tissue-specific or developmentally, or environmentally regulated fashion.
  • Preferred according to the invention are constitutive high-level promoters, such as the CaMV 19S promoter and the CaMV 35S promoter, or the promoters derivable from the T-DNA of Ti-plasmids from Agrobacterium. This promoter may be flanked by so-called enhancer sequences to further enhance expression levels.
  • the invention also embraces the use of hybrid promoters, i.e. promoters that comprise elements derived from regulatory elements of different genes.
  • Plant expressible genes generally comprise a so-called terminator sequence including a polyadenylation signal. Suitable terminators may be selected from homologous or heterologous genes, the choice is not critical to the invention.
  • 'gene' as used here is meant to comprise cDNAs as well as transcribed regions of genomic clones, either of which may be synthetic or partially synthetic.
  • Plant expressible gene' shall mean a DNA sequence which is operably linked to a regulatory sequence required for transcription in a plant cell and which yields RNA upon transcription which can be translated into protein.
  • a gene is 'plant expressible' if it is expressed at least in one tis'sue in one particular phase of the life cycle of the plant.
  • a gene is understood to be plant expressible even if it is not expressed 'of its own motion' but must be triggered or induced by an external stimulus, such as pathogen attack.
  • a chimeric plant expressible gene according to the invention is a plant expressible gene which at least combines two sequences that are not naturally associated.
  • chimeric plant expressible genes may comprise genes which comrise combinations of functional regions of a eukaryotic gene such as enhancers, transcription initiation regions, coding regions, non-translated leaders, signal sequences, vacuolar targeting sequences, terminator sequences, introns, exons, and the like, or parts thereof.
  • Preferred according to the invention are chimeric plant expressible genes which comprise a gene encoding an antifungal CBP according to the invention linked to a promoter not naturally associated therewith.
  • a very effective site of action of hydrolytic enzymes in the protection of transformed plants against a range of plant pathogenic fungi is believed to be the apoplastic space.
  • plants are transformed with a recombinant DNA construct comprising a gene encoding a plant expressible gene according to the invention which exerts its action in the apoplastic space of the plant, either naturally or by virtue of genetic modification.
  • Naturally intracellular genes may be modified such that the C-terminal amino acids involved in vacuolar targeting are not present (e.g. by introducing a translational stopcodon in the coding region of the gene, or otherwise) , resulting in apoplast-targeting of the vacuolar protein produced in that plant.
  • a first evaluation may include the level of expression of the newly introduced genes, the level of fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.
  • the transformed plants can be cross-bred 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.
  • synergizing proteins that may be used in combination with antifur " CBPs according to the invention include, but are not limited to, ⁇ -l,3-glucanases and chitinases 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. 56, 459-466 ; Hoj P.B. et al. , 1988, FEBS Lett. 230, 67-71; Hoj P.B. et al. , 1989, Plant Mol. Biol. 13, 31-42 1989), bean (Boiler T.
  • transgenic plants capable of constitutively expressing more than one chimeric gene
  • a number of alternatives are available, which are encompassed by the present invention, including the following:
  • B Cross-pollination of transgenic plants which are already capable of expressing one or more chimeric genes coupled to a gene encoding a selection marker, with pollen from a trans ⁇ genic plant which contains one or more gene constructions coupled to another selection marker. Afterwards the seed, which is obtained by this crossing, is selected on the basis of the presence of the two markers. The plants obtained from the selected seeds can afterwards be used for further crossing.
  • C The use of a number of various recombinant poly- nucleotides, e.g. plasmids, each having one or more chimeric genes and one other selection m ⁇ rker. If the frequency of cotransformation is high, then selection on the basis of only one marker is sufficient.
  • Plants, or parts thereof of commercial interest, with improved resistance against phytopathogenic fungi can be grown in the field or in greenhouses, and subsequently be used for animal feed, direct consumption by humans, for prolonged storage, used in food- or other industrial processing, and the like.
  • the advantages of the plants, or parts thereof, according to the invention are the decreased need for fungicide treatment, thus lowering costs of material, labour, and environmental pollution, or prolonged shelf-life of products (e.g. fruit, seed, and the like) of such plants.
  • MOG101 A helper plasmid conferring the Agrobacterium tumefaciens virulence functions derived from the octcpine Ti-plasmid pTiB6 was constructed, MOG101.
  • MOG101 is a Agrobacterium tumefaciens strain carrying a non-oncogenic Ti-plasmid from which the entire T-region was substituted by a bacterial Spectinomycin resistance marker from transposon Tn 1831 (Hooykaas et al.. 1980 Plasmid 4., 64-75) .
  • the Ti-plasmid pTiB6 contains two adjacent T-regions, TL (T-left) and TR (T-right) .
  • Plasmid pMOG621 is a pBR322 derivative, which contains the 2 Ti-plasmid fragments that are located to the left and right, outside the T-regions ( Figure 2).
  • the 2 fragments shown in dark
  • Plasmid 4., 64-75 carrying the spectinomycin resistance marker ( Figure 2) .
  • Transconjugants were selected for resistance to Rifampicin (20 mg/1) and spectinomycin (250 mg/1) .
  • EXAMPLE 1 Leaves of 7 to 8 weeks old Samsun NN tobacco plants were inoculated with tobacco mosaic virus (TMV) . Seven days after inoculation 400 grams leaves were harvested and homogenized at 4 ⁇ C in 500 ml 0,5 M NaOAc pH5.2, 15 mM 2-mercapto-ethanol, and 4 gram active carbon, using a Waring blendor. The homogenate was filtered over four layers of cheese cloth and subsequently the filtrate was centrifuged for 15 minutes at 3,000g.
  • TMV tobacco mosaic virus
  • the supernatant was centrifugated for 50 minutes at 20,000g and desalted by passage through a Sephadex G25 column (medium course; Pharmacia), length 60 cm, diameter 11,5 cm, and equilibrated in 40mM NaOAc pH 5.2.
  • the desalted protein solution was stored overnight at 4*C and subsequently centrifugated during 45 minutes at 20,000g.
  • the supernatant was passed through a S-sepharose (Fast Flow, Pharmacia) column, length 5 cm, diameter 5 cm, which was equilibrated with 40 mM NaOAc pH 5.2.
  • the column was washed with the above mentioned buffer (flow rate 400 to 500 ml/hr) until the OD 280 dropped to zero.
  • the unbound proteins were collected.
  • the bound proteins were eluted using an increasing linear NaCl gradient (o to 300 mM) in 500 ml of the above mentioned buffer, and a flow rate of 3 ml per minute; fractions of approximately 5 ml were collected. All fractions were assayed for chitinase activity.
  • Chitinase activity was assayed radiometrically with tritiated chitin as substrate (Molano et a 1977, Anal. Biochem. .83., 648-656) - The specific activity of the final product was approximately 1.2 x 10° cpm/mg. Before use the tritiated chitin was washed three times. To 100 ⁇ l 10 mM potassium phosphate buffer pH t.4 with 0.02% sodium azide, 50 ⁇ l tritiated chitin (approximately 150,000 counts per minute, cpm) and 50 ⁇ l protein solution was added. The mixture was incubated while shaking for 30 minutes at 37 ⁇ C.
  • the reaction was stopped by adding 600 ⁇ l 10% trichloro acetic acid. After centrifugation to pellet the chitin (10 minutes in a microfuge) , 500 ⁇ l supernatant was filtered over glasswool and pipetted into a scintillation vial. 5 ml scintillation fluid was added and the radioactivity released (expressed as counts per minute) was taken as a measure for chitinase activity.
  • the fractions containing chitinase activity were pooled and concentrated by ultrafiltration through an Amicon membrane (cut off lOkDa) .
  • the concentrated fraction was brought to 20 mM NaHC0 3 and the pH was adjusted to 8.3 with NaOH. Subsequently, the fraction was adsorbed to 50 ml regenerated chitin (Molano et al. , 1977) equilibrated in 20 mM NaHC0 3 .
  • the chitin matrix was washed with 100 ml 20 mM NaHC0 3 and subsequently with 100 ml 20 mM NaOAc pH 5.2. Bound proteins were eluted with approximately 150 ml 20 mM HAc (pH 3,5).
  • the protein containing fractions were dialyzed against 0.2 M NaCl, 50 mM K 2 HP0 4 /KH 2 P0 4 , pH 7,0 and subjected to gelfiltration chromatography on a Superdex 75 column (HR 10/30; Pharmacia) at a flow rate of 0,5 ml per minute. Fractions of approximately 0,5 ml were collected. Each fraction was analyzed by electrophoresis (Laemmli, Nature 227. 680-685) using a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS) , using molecular weight markers of (18-97 kD) as reference. A separate portion of each fraction was tested for chitinase activity.
  • SDS sodium dodecyl sulphate
  • Fractions containing a 32 kD proteins could be identified as the two isoforms of class I tobacco intracellular chitinase (Shinshi et al. , 1990) .
  • CBP Chitin Binding protein
  • CBP The antifungal activity of CBP was assessed in a microtiter plate assay using the fungi Trichoderma viride and Fusarium solani.
  • PDA potato dextrose agar
  • Fungal spores were suspended in water and 400-600 spores in 50 ⁇ l were added to the wells. Spores were pregerminated 6 to 16 hours at 25"C.
  • 100 ⁇ l filter sterilized (0.22 ⁇ m filter) protein solution in 50 mM K 2 HP0 4 /KH 2 P0 4 ,pH 6.0 was added.
  • Microtiter dishes were wrapped with Parafilm and incubated at room temperature.
  • the fungus was monitored microscopically for effects of the added protein. After 2-3 days the mycelium of the growing fungus in the wells was stained with lactophenol cotton blue and the extent of growth was estimated. With 2 ⁇ _ viride addition of 1 ⁇ g purified CBP per well resulted in lysis of the hyphal tips of the fungus. Moreover, an inhibition of growth could be observed. One to ten ⁇ g of CBP was not sufficient to lyse hyphal tips of F ⁇ _ solani or to inhibit the growth of the fungus. However, microscopically a clear swelling of tips was observed.
  • Lysis is indicated by a percentage with respect to untreated control.
  • CBP does not cause lysis of Fusarium solani f but it has a strong growth inhibitory effect at 5 ⁇ g. Chitinase as such has no lytic effect on Fusarium solani at 0.5 ⁇ g, but in combination with 1 ⁇ g CBP it has a strong growth inhibitory effect.
  • CBP has a synergistic antifungal effect in combination with glucanases as well as with class-I chitinases.
  • results in table 2 indicate a synergistic antifungal effect of CBP and intracellular glucanase and at least an additive effect of CBP and class-I chitinase on Alternaria radicina.
  • CBP has a growth inhibitory effect on Alternaria radicina. albeit rather weak.
  • Further in vitro antifungal activities have been determined with Alternaria porri as test fungus. Up to 10 ⁇ g CBP had no detectable effect against A. porri. whereas 5 ⁇ g CBP in combination with 0.3 ⁇ g intracellular ⁇ -1,3-glucanase from tobacco has a moderate growth inhibitory effect on a_j.
  • V 8 protease cuts proteins at glutamic acid residues.
  • the digestion products were run over a 12,5% polyacrylamide gel containing 0,05% SDS (Laemmli, supra) and electroblotted onto a PVDF membrane as described by Matsudaira et aJL. (1987, J. Biol. Chem. 262. 10035-10038) .
  • the protein band migrating as a polypeptide 3-4 kDa smaller than the undigested material was cut out of the gel and sequenced using Edman degradation on an Applied Biosystems 477A protein sequencer according to the protocol provided by the manufacturer. The following sequence was obtained:
  • NCS N-c_hlorosuccini ⁇ nide/urea
  • the polypeptide migrating on the gel as a 9-11 kDa protein was cut out and sequenced using Edman degradation on an Applied Biosystems 477A protein sequencer according to the protocol provided by the manufacturer. The following sequence was obtained: (W) T A F (Y) G P V G P (P/R) G R D S (SEQIDNO: 1) .
  • the amino acid sequence is given using the one-letter code.
  • Amino acid 1 (W) was not determined, but since NCS cuts proteins at tryptophan residues, it has been placed at that position. Amino acids 5 (Y) and 11 (P/R) could not be determined unambiguously.
  • the primary structure of the tobacco extracellular proteins PR-4a and PR- 4b (Linthorst et al. 1991, Mol. Plant-Microbe Interact. 4_. 586-592) and the tomato extracellular protein P2 (Linthorst et al. , supra) show homology with sequence 1 as well.
  • the tobacco PR-4 proteins and the tomato P2 protein are serologically related (Joosten e_t al. , 1990, Plant Physiol. £4, 585-591) .
  • Antisera raised against either the PR-4 proteins or P2 cross-react with CBP.
  • the tobacco PR-4 proteins do not exert a fungal growth inhibiting activity.
  • SEQIDNO: 4 is deduced from the partial amino acid sequence (SEQIDNO: 1) as determined from CBP (see EXAMPLE 1) .
  • the CBP cDNA-fragment was cloned as a EcoRI fragment into the EcoRI linearized vector pBluescript (pBS) plasmid to yield clone pMOG684.
  • the nucleotide sequence of the EcoRI-fragment of clone pMOG684 was determined using the double strand DNA sequencing method (Chen J. & Seeburg P.H., 1985, DNA 4., 165-170) and showed that a partial CBP cDNA clone was isolated.
  • the CBP gene was cloned as a BamHI fragment into the BamHI linearized vector pMOG180 (described in EP-A 460 753 Al) .
  • the expression construct obtained contains on a EcoRI-Hindlll fragment the cauliflower mosaic virus (CaMV) 35S promoter in front of the CBP gene which in its turn is followed by the nopaline synthase (nos) transcription terminator.
  • the expression construct was cloned into the EcoRl-site of the binary vector pMOG23 (deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands, No.
  • the resulting plasmid pMOG687 contains the following expression constructs, the CBP gene, the modified basic ⁇ -l,3-glucanase gene and the NPTII gene localized between the left and right T-DNA border sequences.
  • these binary vectors were mobilized indepentdently from E.coli DH5 ⁇ to Agrobacterium tumefaciens strain MOG101.
  • the transconjugants MOG101(pMOG685) and MOG101(pMOG687) were isolated from these matings on selection medium containing 40 mg/1 rifampicin, 250 mg/1 spectinomycin, and 100 mg/1 kanamycin.
  • CBP Wild-type CBP is found intracellularly, most likely in vacuoles of plant cells.
  • a translational stop-codon is introduced into wild-type CBP cDNA as present in pMOG685, between codon 13 and 14 as numbered from the C-terminal end of the protein encoded by the cDNA.
  • PCR technique a stop-codon is created by the insertion of a Thy idine (T) residue between nucleotide 619 and 620 with respect to the sequence presented in SEQIDNO: 13 in the sequence protocol.
  • T Thy idine
  • the mutated cDNA encodes a CBP lacking the 13 C- terminal amino acids of the primary translation product of the wild-type CBP mRNA.
  • the binary vector thus obtained was called pMOG686 (modified CBP) and the corresponding Agrobacterium transconjugant MOG101(pMOG686) .
  • the CBP encoded by pMOG686 is indeed targeted extracellularly.
  • a pM0G686 derived binary plasmid was constructed which contains in addition to the modified CBP gene a modified tobacco basic ⁇ - 1,3-glucanase gene encoding an extracellularly targeted protein (described in more detail in EP-A 440 304 Al) .
  • the binary vector thus obtained was called pMOG688 and the corresponding Agrobacterium transconjugant MOG101(pMOG688) .
  • EXAMPLE 4 Transformation of plants The transformation of tomato (Lycopersicon esculentum cv. Moneymaker) with Agrobacterium strains MOG101 (pMOG685) , MOG101 (pMOG686) , MOG101 (pMOG687) and MOG101(pMOG688) was performed essentially according to the procedure described by McCor ick et al. (1986, Plant Cell Rep. 5, 81-84). For the transformation of tobacco use is made of the leaf-disc dip method (1985, Horsch et al., Science 227., 1229-1231). Leaf- discs were cocultivated with Agrobacterium strains
  • MOG101(pMOG685) , MOG101(pM0G686) , MOG101(pMQG687) or MOG101(pMOG688) were regenerated into whole plants and analyzed for expression of the newly introduced genes.
  • the transgenic shoots were regenerated into whole plants and analyzed for expression of the newly introduced genes.
  • For this analysis use was made of the so-called Western blotting technique, using antibodies raised against either pathogenesis-related protein PR-4 (detection of CBP) or the tobacco basic ⁇ -1,3-glucanase protein.
  • the Northern blotting technique was performed using the CBP cDNA and the basic ⁇ -l,3-glucanase gene as a probe.
  • EXAMPLE 5 Analysis of pM0G685- and pMOG686-transgenic tobacco plants for targeting of the transgene product.
  • the following experiment was carried out. Leaves of FI plants from pMOG685-transgenic plant lines and from pMOG6864-transgenic plant lines, and leaves of non-transgenic plants were used for the extraction of total proteins and of extracellular proteins.
  • the extracellular fluid (EF) of these plants was collected according to the procedure described by De Wit and Spikman (1982, Physiol. Plant Pathol. 2J), 1-11).
  • minus EF fraction For the analysis of proteins in the total extracts (Total) , in the EF and in extracts of leaves from which the EF was removed (- EF) , use was made of the Western blotting technique, using CBP specific antibodies, or antibodies raised against tomato P2 protein, or antibodies raised against tobacco PR-4. The results shown in Table 3 indicate that with the pM0G686- transgenic plants CBP is indeed targeted extracellularly.
  • EXAMPLE 6 Analysis of fungal resistance in transgenic tobacco plants With the aid of Agrobacterium tumefaciens different transgenic tobacco plants were obtained that express the chimeric gene constructs delivered from the binary plasmids pMOG685, pMOG686, pMOG687 and pMOG688. Transgenic tobacco plants displaying good expression of the transgenes were analyzed for resistance to Rhizoctonia solani.
  • Rhizoctonia solani causes disease symptoms on roots (root-rot) and stems (stem-canker) of a wide range of plant species, including tobacco. Infection of tobacco produces necrosis that reduces the ability of plants to collect and transport nutrients, which results in significant reductions in growth and biomass. Plant growth is correlated with the degree of fungal infection (1992, Logemann et al- , Bio/technology, 10; 305- 308) . To assess the fungal resistance of transgenic tobacco plants were tested essentially according to the procedure described by Jach et al. (1992, Biopractice 1; 33-40) the following experiment was performed.
  • the pMOG685- and pMOG686-transgenic plants show a slightly enhanced resistance against R.solani as the growth of these plants is less retarded compared to the control plants. However, a better protection was observed in pM0G687- and pM0G688-transgenic plants, which illustrates the synergistic effect of CBP and basic ⁇ -1,3-glucanase against R.solani. ../.
  • EXAMPLE 7 Analysis of fungal resistance in transgenic tomato plants
  • the fungus Fusarium oxysporum f.SP. Ivcopersici is a pathogen of the tomato plant causing complete wilting of the leaves, affection of the stem and eventually death of the plant.
  • lycopersici fysio 1 (10 6 spores/ml) and were continued to grow in soil at 18 e C. The disease symptoms are scored 21 days after infection of the plant. On a scale of 0 to 9 the plants are classified according to the infection grade of. the vascular tissue and also the degree of wilting of the plant. In Table 5 estimations are given of the results. Table 5. Fusarium oxysporum f.sp. Ivcopersici assay on transgenic tomato plants expressing either CBP or simultaneously CBP and basic ⁇ -1,3- glucanase.
  • MDIECUIE TYPE CENA to iriRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORIGINAL SOURCE
  • GAATTOGGCA CGAGG GGA AAG CTA ACT ACT CTT TTG CIT GIT CTG ATC CTC 51
  • Gly Lys Leu Ser Bit Leu Leu Leu Val Leu lie Leu 1 5 10 TAT TTC ATA GCC GCA GGT GCC AAC GCA CAG CAG TGC GGA AGG CAA AGG 99 Tyr Phe lie Ala Ala Gly Ala Asn Ala Gin Gin Cys Gly Arg Gin Arg 15 20 25
  • MOLECULE TYPE protein

Abstract

Nouvelle catégorie de protéines antifongiques de liaison de la chitine (CBP antifongiques) à très faible activité de chitinase (10 % au moins par rapport à celle des chitinases de classe I du tabac). Des séquences d'ADN sensiblement pures codant les CBP antifongiques et des gènes de CBP antifongiques pouvant être exprimés par des plantes sont également décrits, et permettent d'obtenir des plantes transgéniques produisant des CBP antifongiques. Les plantes exprimant un gène de CBP antifongiques, éventuellement en combinaison avec un gène de glucanase et pouvant être exprimé par une plante, présentent une sensibilité réduite aux champignons.
EP93921929A 1992-10-05 1993-10-05 Proteines antifongiques de liaison de la chitine et adn les codant Withdrawn EP0667905A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP92203071 1992-10-05
EP92203071 1992-10-05
EP93201370 1993-05-13
EP93203071 1993-05-13
PCT/EP1993/002790 WO1994008009A1 (fr) 1992-10-05 1993-10-05 Proteines antifongiques de liaison de la chitine et adn les codant
EP93921929A EP0667905A1 (fr) 1992-10-05 1993-10-05 Proteines antifongiques de liaison de la chitine et adn les codant

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EP0667905A1 true EP0667905A1 (fr) 1995-08-23

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9408009A1 *

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