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WO1990007001A1 - Surexpression de la chitinase dans des plantes transgeniques - Google Patents

Surexpression de la chitinase dans des plantes transgeniques Download PDF

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Publication number
WO1990007001A1
WO1990007001A1 PCT/US1989/005501 US8905501W WO9007001A1 WO 1990007001 A1 WO1990007001 A1 WO 1990007001A1 US 8905501 W US8905501 W US 8905501W WO 9007001 A1 WO9007001 A1 WO 9007001A1
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Prior art keywords
plant
chitinase
recombinant dna
dna construct
plants
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PCT/US1989/005501
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English (en)
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Karen Elizabeth Broglie
Richard Martin Broglie
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E.I. Du Pont De Nemours And Company
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Priority to KR1019900701797A priority Critical patent/KR910700346A/ko
Publication of WO1990007001A1 publication Critical patent/WO1990007001A1/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • 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)
    • C12Y302/01014—Chitinase (3.2.1.14)

Definitions

  • This invention relates to the preparation of novel recombinant DNA constructs used to introduce and ⁇ verexpress chitinase polypeptide(s) in plants to confer resistance to plant pathogenic fungi, and to such transgenic plants and their seeds.
  • ⁇ -1,3-glucanase have been proposed to function in defense by causing extensive degradation of pathogen cell walls. Lysis of pathogenic fungal hyphae has been observed in vivo in plants infected with a number of vascular wilt pathogens. G. F. Pegg and J. C. Vessey (Physiol. Plant Path. 3:207-222, 1973) suggested that lysis of Verticillium albo-atrum
  • bacterial chitinase obtained from a commercial source, on plant parasitic nematodes. Their results indicate that this enzyme is toxic to certain nematodes, in particular, Tylenchorhynchus dubius. Their data indicate that this toxicity is greater in aqueous solution than in soil.
  • Endo-type chitinase activities have been observed in many species of higher plants including bean, pea, soybean, tomato, sunflower, melon, cotton, corn, wheat, barley and tobacco (Boiler, T.,
  • Plant chitinases have been purified from wheat germ, tomato, bean and pea. The enzymes isolated from these sources have been shown to correspond to basic proteins of approximately 30 kilodaltons
  • Chitinase cDNA and genomic clones have been isolated from bean, tomato, tobacco and potato. As discussed in Broglie et al. (1986) Proc. Natl. Acad. Sci. USA 83:6820-6824, an endochitinase from bean was found to be encoded by a 1.2 kilobase messenger RNA comprised of a short, 33 base untranslated region followed by a 984 nucleotide open reading frame and 115 nucleotides of 3' untranslated RNA.
  • the protein coding region specifies a 328 amino acid polypeptide which consists of a 27 amino acid residue signal peptide and the 301 residues of the mature chitinase polypeptide chain. The amino terminal signal sequence presumably
  • chitinase cDNA and genomic clones provide the opportunity to manipulate the expression of this protein and to evaluate the effect of this genetic modification on the fungal resistance of the derived plants.
  • the involvement of chitinase in the defense of the plant against chitin-containing fungal pathogens is based upon the following indirect evidence and upon data generated from model, in vitro systems.
  • chitin is known to be a ubiquitous component of the cell walls of most fungi except the oomycetes (Wessels, J.G.H. and Sietsma, J.H. (1981) in "Plant Carbohydrates II" (Tanner, W.
  • purified bean chitinase has been found to inhibit the vegetative growth of the non-pathogenic test fungus, Trichoderma viride (Schlumbaum, A., Mauch, F., Vogeli, U. and Boiler, T. (1986) Nature 324:365-367).
  • Trichoderma viride Scholadium viride
  • fungus was grown on solid, agar-containing medium and purified bean chitinase or a protein extract from ethylene-treated bean was introduced into wells in the agar plate. Zones of growth inhibition were found to develop around wells containing purified chitinase or the bean protein extract. This effect was attributed to
  • fungal-infected pea pods inhibited the growth of 15 of the 18 fungi tested. These extracts were shown to contain high levels of chitinase and ( ⁇ -1,3-glucanase activity. Eight fungi were tested for growth
  • Trichoderma viride was susceptible to the action of chitinase alone and only Fusarium solani f .sp.
  • Trichoderma viride is not a plant pathogen; indeed, Trichoderma species are known to be parasites of other fungi and as such have been utilized as effective biocontrol agents to inhibit the growth of plant pathogenic fungi (Chet, I. (1987) in "Innovative Approaches To Plant Disease Control” (Chet, I., ed.) pp. 137-160). While Mauch et al.
  • Fusarium solani f .sp. pisi is sensitive to the presence of chitinase and glucanase during growth on agar plates, this fungus is nevertheless a
  • the presence or absence of sensitivity to the two hydrolytic enzymes in the plate assay may have little bearing on the phytopathogenic properties of the fungus.
  • Growth on nutritive agar media is distinguished from infection of plant tissue by the striking lack of specialized infection structures in the former case.
  • Mendgen et al. (Mendgen, K. Freytag, S., Lange, M and Bretschneider, K. (1986) J. Cellular Biochem. (Suppl.) 10C: 25) determined that in the rust fungi, different infection structures display different surface carbohydrate patterns. In the germ tube that recognizes the host cuticle, chitin is mainly found. In contrast, the structures of the rust fungi in the leaf (substomatal vesicles and infection hyphae) contain mainly ⁇ -1,3-glucans on their surface.
  • RNA from infected plants is infected with an incompatible ( ⁇ ) and a compatible (Y) race of Colletotrichum lindemuthianum and the RNA from infected plants analyzed on Northern blots, a difference is observed in the appearance of transcripts for phytoalexin biosynthesis in the two interactions.
  • phenylalanine ammonia lyase and chalcone synthase mRNAs accumulate rapidly and early in infection, being localized mainly at the site of fungal infection.
  • appearance of the RNAs is delayed and more widespread than in the incompatible interaction (Bell, J.N., Ryder, T.B., Wingate, V.P.M., Bailey, J.A.
  • the promoter region containing the DNA sequence elements for inducible expression, has been removed from an endochitinase gene from Phaseolus vulgaris and replaced with a promoter fragment of the cauliflower mosaic virus (CaMV) 35S transcript in order to promote high level, constitutive expression and to eliminate the time necessary for induction of chitinase activity in response to pathogen attack.
  • CaMV 35S promoter fragment controls the
  • a bean chitinase gene which encodes a polypeptide consisting of a 26 amino acid residue signal peptide and 301 amino acids of the mature chitinase polypeptide.
  • Transgenic plants of the present invention containing this modified chitinase gene have been shown to display increased resistance to infection by the foliar pathogen, Botrytis cinerea and by the soil-borne pathogen, Rhizoctonia solani.
  • this approach is applicable mainly to soil borne pathogens and is dependent on the stability of the enzyme in the rhizosphere.
  • the inhibitory eff-ects of a chitinase produced by rhizobacteria would not be selective against pathogenic fungi but would also be inhibitory to non-pathogenic fungi that inhabit the rhizosphere and are beneficial to the growth and development, of the plant.
  • the coding region of Serratia chitinase includes a 23 residue amino terminal extension which serves as a signal for the secretion of the protein into the extracellular milieu of this gram negative bacterium.
  • the signal peptide is at least partially cleaved to yield a protein form which co-migrates with purified Serratia marcescens chitinase (Taylor, J. L. et al., Mol. Gen. Genet. (1987) 210: 572-577; Jones, J. D. G. et al., Mol. Gen. Genet. (1988)
  • lamB is translocated across the vesicle membrane.
  • translocation machinery of the microsomal membrane is able to recognize the bacterial signal sequence, it is unable to recognize the stop-transfer signals required for membrane integration (Watanabe, M.
  • chitinase In bean, chitinase is known to be synthesized as a precursor protein containing an amino terminal peptide extension (Broglie, K.E., Gaynor, J.J. and Broglie, R.M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:6820-6824). This signal sequence presumably functions in determining the vacuolar localization of the mature bean enzyme (Boiler, T. and Vogeli, U.
  • the signal sequence of the bean chitinase polypeptide was obtained by comparing the amino acid sequence deduced from the nucleotide sequence of a chitinase cDNA clone with the N-terminal sequence of the purified protein. This analysis indicates that the bean chitinase encoded by clone ⁇ CH18 contains a 27 residue signal peptide (Broglie, K.E., Gaynor, J.J. and Broglie, R.M. (1986) Proc. Natl. Acad. Sci.
  • the coding sequence for bean chitinase (specified by the chitinase gene of bean genomic clone ⁇ CH 5B) is preceeded by its cognate 26 amino acid residue signal peptide.
  • the bean chitinase precursor protein is found to be efficiently processed to the mature form of the enzyme.
  • Immunoblots of soluble protein isolated from these plants show the presence of a protein band, immunoreactive with anti-chitinase IgG and identical in size with purified bean chitinase. Efficient recognition and cleavage of the bean signal peptide in the heterologous plant background is indicative of its translocation to the central vacuole of the plant cell.
  • the chitin in the insect integument is highly cross-linked and
  • the present invention utilizes a genetically engineered chitinase gene consisting of a high level promoter, a signal sequence and a protein coding sequence, which functions in plants to provide protection against chitin-containing pathogens.
  • transgenic plants which contain a chimeric chitinase gene in which the inducible, regulatory region (promoter) of a natural chitinase gene was replaced with a viral DNA fragment in order to promote high level expression and to eliminate the need for induction of chitinase activity in response to pathogen attack.
  • the present invention also contains a DNA sequence which encodes a short signal peptide which is required to direct the mature chitinase enzyme to the central vacuole of the cell.
  • This invention discloses a novel DNA construct which when introduced into plants, confers resistance to plant pathogenic fungi.
  • Such transgenic plants incorporate a high level promoter and a coding sequence for a plant chitinase polypeptide under the control of the high level promoter.
  • one aspect of the present invention is a recombinant DNA construct capable of transforming a plant comprising the following DNA fragments: (a) a high level promoter operably linked to (b) a coding sequence for a plant chitinase gene or effective sequence thereof, wherein said high level promoter causes the overexpression of the chitinase
  • polypeptide transport thereby conferring resistance to plant pathogenic fungi.
  • An advantage of this invention is that unlike genes from other sources, the plant genes may contain one or more signal sequences which facilitate transport of said
  • chitinase polypeptide to a plant cell vacuole.
  • Preferred high level promoters are derived from the genome of a plant virus, a plant, or from the T-DNA region of Agrobacterium tumafaciens. More preferred high level promoters include the 35S and 19S
  • Agrobacterium the promoter of the RUBP carboxylase small subunit gene, and the promoter from the
  • chlorophyll A/B binding protein genes Most of the chlorophyll A/B binding protein genes.
  • coding sequences for a signal peptide include a plant signal peptide, a chitinase signal peptide, and a synthetic signal peptide whose DNA sequence encodes a peptide which allows efficient transport of a protein to a plant vacuole. More preferred, by virtue of activity or ease of preparation, is the DNA sequence coding for the bean chitinase signal peptide.
  • Preferred coding sequences for chitinase polypeptides include those derived from plants, while those more preferred would be the bean chitinase polypeptide. The most
  • preferred recombinant DNA construct includes a high activity promoter from the 35S constituent of the cauliflower mosaic virus, a coding sequence for a plant signal sequence from a bean chitinase
  • chitinase enzyme from a bean chitinase structural gene.
  • Another aspect of the invention involves a plant containing a recombinant DNA construct
  • Preferred monocotyledonous plants include corn, alfalfa, oat, millet, wheat, rice, barley, and sorghum, while preferred
  • dicotyledonous plants include soybean, tobacco, petunia, cotton, sugarbeet, sunflower, carrot, celery, flax, canola, cabbage, cucumber, pepper, tomato, potato, oilseed rape, bean, strawberry, grape, and lettuce.
  • tobacco, tomato, canola and rice plants transformed with a recombinant DNA construct incorporating the high activity promoter of the 35S RNA transcript of the cauliflower mosaic virus, the plant signal sequence of the bean
  • chitinase signal peptide and the coding sequence of the bean chitinase structural gene.
  • Figure 1 is a restriction map of bean genomic clone lambda CH5B and the 4.7 kb Hindlll-EcoRI fragment containing a bean chitinase gene.
  • the arrows depict the sequencing strategy employed to obtain the nucleotide sequence of this fragment.
  • the bold line shows the open reading frame encoding the chitinase polypeptide.
  • the following symbols are used to represent restriction enzyme sites in the genomic clone: B, BamHl; E, EcoRI; H, Hindlll.
  • Figure 2 is the nucleotide sequence of the 4.7 kb Hindlll-EcoRI fragment containing the bean
  • the 981 bp open reading frame encodes the chitinase precursor polypeptide which consists of a 301 amino acid mature enzyme (amino acid residues 27-301) and a 26 amino acid signal peptide (amino acid residues 1-26).
  • the open reading frame is preceded by approximately 2 kb of 5'
  • flanking DNA is followed by approximately 1.7 kb of 3* flanking DNA.
  • the deduced amino acid sequence is shown below the corresponding triplet codons .
  • FIG. 3 is a summary of the steps involved in the construction of pK35CHN.
  • pBR322 refers to plasmid DNA sequences donated by the vector, pBR322.
  • the following symbols are used to represent restriction enzyme cleavage sites: B, BamHl : C, Clal : E, EcoRI : H, HindllI; S, Sall.
  • Figure 4 describes the immunodetection of bean chitinase in protein extracts from transgenic tobacco plants. Antibodies raised against gel-purified bean chitinase were used to detect the presence of the bean protein in transgenic tobacco plants.
  • Antigen-antibody complexes were visualized using alkaline phosphatase conjugated goat anti-rabbit IgG and an alkaline phosphatase specific histochemical reaction. Lanes contain the following protein extracts: Lanes 1-8, protein extract from 8
  • Rhizoctonia solani infection on root fresh weight of transgenic tobacco plants containing a chimeric bean chitinase gene (plants # 230, 238, 329, and 373).
  • Plant #548 contains a kanamycin resistance gene and serves as a control in this study. Data points are the mean root fresh weight of 10 plants determined two weeks after inoculation. Figure 5A and Figure 5B represent two different experiments.
  • Figure 6 describes the survival of transgenic tobacco plants containing the chimeric chitinase gene (#373) in soil infected with the plant pathogen
  • Rhizoctonia solani compared to control tobacco plants lacking the modified gene (#548) and grown under identical conditions. Seedlings were transplanted into soil infested with R. solani and allowed to grow for an additional 16 days. Disease progression was monitored by scoring seedling survival at intervals following infection.
  • Figure 7 describes the partial resistance of transgenic tobacco plants containing the modified chitinase gene to infection by the foliar pathogen Botrytis cinerea. Plants were inoculated with a suspension of conidia and the number and size of the lesions determined after development of disease symptoms. Plant #548 lacks the chimeric gene and served as a control in this experiment; plants #230, #329 and #238 all contained the chimeric gene and showed a reduction in lesion size following infection.
  • Figure 8 is an outline of the binary
  • transformation vector pMChAD The chimeric chitinase gene is inserted into the vector as a Kpn I
  • the vector contains the right (RB) and the left (LB) borders of the T-DNA of A ⁇ robacterium
  • the vector also contains a chimeric marker gene consisting of the nopaline synthase promoter fused to the bacterial Npt II gene
  • This vector also contains a sulfonylurea herbicide
  • Figure 9 describes the irnmunodetection of bean chitinase in protein extracts of transgenic tomato plants. Lanes contain the following protein
  • Lanes 2-4 protein extracts from transgenic tomato plants containing the chimeric chitinase gene in the binary vector pMChAD; Lane 5, transgenic tomato plants lacking the chimeric chitinase gene.
  • Figure 10 describes the irnmunodetection of bean chitinase in protein extracts of transgenic oil seed rape. Lanes contain the following protein extracts: Lanes 1 and 8, purified bean chitinase; Lanes 2 and 7, transgenic tobacco plants containing the chimeric chitinase gene; Lane 9, wild type (WT) untransformed Brassica napus; Lanes 4-6, 3 individual transformed B. napus plants.
  • Figures 11A and 11B describe the increased survival rate and delay in symptom appearance
  • Figure 12 describes the irnmunodetection of bean chitinase in protein extracts of transgenic rice cells. Lanes contain the following protein
  • Lane 1 purified bean chitinase
  • Lanes 2-6 5 individual kan R rice callus samples
  • the arrow indicates the bean chitinase polypeptide in transgenic rice cells.
  • the present invention describes a genetically engineered nucleic acid fragment which, when
  • This novel DNA fragment consists of (a) a promoter region which specifies high level expression fused to the coding region of a plant chitinase gene, and (b) a coding sequence for a plant chitinase gene or effective sequence thereof, wherein said high level promoter causes the overexpression of the chitinase polypeptide transport thereby
  • the chitinase enzyme catalyzes the hydrolysis of chitin (Boiler, T., et al. (1983) 157:22), a ⁇ -1,4-linked N-acetyl glucosamine polymer and an important
  • promoter region refers to a sequence of DNA, usually upstream (5') of the coding sequence, which controls the expression of a coding region of a gene.
  • a promoter region can include a recognition site(s) for the binding of RNA polymerase and/or other factors required for correct transcription initiation.
  • the promoter region may also contain DNA sequences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions.
  • a "promoter fragment” constitutes a DNA sequence consisting of a promoter region.
  • regulatory sequence refers to a nucleotide sequence located upstream (5'), within, and/or downstream (3') to a DNA sequence for a selected gene product whose transcription and
  • An “enhancer” is a DNA sequence which can operate in an orientation- and location-independent manner to stimulate the activity of a promoter.
  • a transcriptional “stimulator” or “activator” is a DNA sequence which operates in an
  • tissue-specific promoters as referred to herein are those that direct gene expression only in specific tissues such as roots, leaves and stems.
  • expression is intended to mean the translation to gene product from a gene coding for the sequence of the gene product.
  • a DNA chain coding for a gene product is first transcribed into a complementary RNA which is called a messenger RNA and then, the thus transcribed RNA is translated into the above-mentioned gene product in conjunction with the protein synthesis apparatus of the cell. Expression which is constitutive producing multiple copies of mRNA and large quantities of the specified gene product continuously throughout the life cycle of the plant.
  • “Overexpression” refers to the production of a gene product in transgenic plants that exceeds levels of production in normal plants, including but not limited to constitutive or induced expression.
  • Nucleic acid refers to a large molecule which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar,
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA.
  • homologous to refers to the
  • substantially homologous refers to nucleic acid molecules which require less stringent conditions of hybridization than those for homologous sequences, and coding DNA sequence which may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter an amino acid, but not affect the functional properties of the protein encoded by the DNA sequence.
  • Effectivee sequence of a DNA sequence coding for a protein, refers to a truncated version of the DNA sequence which encodes a peptide which is at least partially functional with respect to the utility of the
  • gene refers to a segment of DNA that is involved in producing a polypeptide chain; including regulatory regions preceding and following the coding region as well as intervening sequences between individual coding segments.
  • coding region or “coding sequence” refers to a region of a gene or a DNA sequence that codes for a specific protein.
  • plant chitinase gene refers to a segment of plant DNA which codes for an enzyme with chitinolytic
  • the term "recombinant DNA construct” refers to a DNA fragment, linear or circular, in which a number of nucleotide sequences have been joined into a unique and novel construction, capable of being introduced into a plant cell, and containing a promoter fragment and DNA sequence coding for a selected gene product.
  • operably linked refers to the chemical fusion of two DNA fragments in a proper orientation and reading frame to be transcribed into functional RNA.
  • the "translational start codon” refers to a unit of three nucleotides (codon) in a DNA sequence that specifies the initiation of the structural gene of protein sequence.
  • a “signal sequence” refers to a peptide
  • the signal peptide is cleaved from the remainder of the
  • polypeptide precursor to provide an active or mature protein.
  • secretion means the transfer of a polypeptide molecule into the
  • Transferring refers to methods to transfer DNA into cells including, but not limited to, microinjection, microprojectile bombardment, permeabilizing the cell membrane with various physical (e.g.,
  • protoplast refers to a plant cell without a cell wall or extracellular matrix.
  • Total DNA was isolated from etiolated bean leaves. Tissue was frozen in liquid nitrogen, ground to a fine powder and then transferred to a buffer consisting of 10 mM Tris-HCl, pH 7.6, 0.35 M NaCl, ImM EDTA, 7 M urea, 2% sarkosyl and 5% phenol (2 ml per gram tissue). After stirring at room temperature for 10 minutes, the sample was centrifuged to remove insoluble material and the supernatant was extracted with a 3:1 mixture of phenol: chloroform until a clear interface was evident.
  • the DNA sample was dialyzed against 2 changes of 4 liters 10 mM Tris-HCl, pH 8.0, 10 mM EDTA and 10 mM NaCl at 4°C for 4 hours. To the dialyzed material, 1 gram of CsCl was added per ml solution and ethidium bromide was added to 0.125 mg/ml. The DNA samples were centrifuged to
  • the ethanol precipitate was centrifuged, washed with 80% ethanol and dissolved in TE buffer, pH 7.8 at 0.12 mg/ml.
  • ⁇ EMBL 4 vector DNA was prepared essentially as described in T. Maniatis, E. F. Fritsch and J.
  • the DNA was centrifuged, washed with 80% ethanol and dissolved in TE buffer, pH 8.0 at a concentration of 150 ⁇ g/ml. MgCl 2 was added to
  • Tris-HCl, pH 8.0, 1 M NaCl and 5 mM EDTA Tris-HCl, pH 8.0, 1 M NaCl and 5 mM EDTA.
  • the sample was centrifuged in an SW 27 rotor at 26,000 rpm for 24 hours at 15°C Following centrifugation, 0.5 ml fractions were collected. A 15 ⁇ l aliquot of every third fraction was heated at 68°C to disrupt the cohesive arms and then subjected to electrophoresis on a 0.5% agarose gel. Fractions containing the left and right arms but lacking uncut DNA or stuffer fragment were pooled, dialyzed extensively against TE buffer, pH 8.0 and concentrated by ethanol
  • the precipitated DNA was centrifuged, washed with 80% ethanol and dissolved in TE buffer, pH 8.0 at 0.24 mg/ml.
  • ligase/ml After a 16 hour incubation at 15°C, the ligation mixture was packaged into viable phage particles using the Packagene system available through the Promega Corporation (2800 S. Fish
  • the DNA of these purified clones was digested with the restriction enzymes EcoRI, BamHl,HindIII and KpnI.
  • the derived restriction maps indicate that the genomic clones comprise three different bean chitinase genes.
  • the DNA fragments harboring the chitinase genes were identified by hybridization of Southern blots of restricted phage DNA to nick-translated pCH18 insert DNA.
  • genomic clone ⁇ CH5B was subcloned into a plasmid vector to allow determination of the nucleotide sequence of this chitinase gene.
  • E. coli strain JM 101 After a 16 hour incubation at 12.5°C, 10 ⁇ l of the ligation mixture was used to transform E. coli strain JM 101. Transformants were selected on Luria-Bertani (LB) (Table IV) media containing 100 ⁇ g/ml ampicillin. Plasmid DNA was isolated from 1.5 ml cultures of individual transformants, essentially as described in Maniatis et al, pg 368. The mini prep DNA was digested with the restriction enzymes EcoRI and Bglll to determine the orientation of the inserted fragment in pEMBL 8+.
  • LB Luria-Bertani
  • Plasmid DNA was isolated from 1.5 ml cultures of individual transformants, essentially as described in Maniatis et al, pg 368. The mini prep DNA was digested with the restriction enzymes EcoRI and Bglll to determine the orientation of the inserted fragment in pEMBL 8+.
  • Plasmid DNA was isolated from transformants containing the 4.68 kb genomic fragment in both orientations in the vector, pEMBL 8+ (designated pCH34 and pCH35). pCH34 and pCH35 DNA was then purified by two cycles of CsCl/ethidium bromide density gradient centrifugation. A nest of ordered deletions was created across the insert sequence using a modification of the procedure of Barnes, W. M., Bevan, M. and Son, P. H. (1983) Methods in
  • Deletions were created by digesting 10 ⁇ g of each DNA sample with 50 units BamHI in 100 ⁇ l 25 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA at 37°C for 45 minutes. The ends were repaired by the addition of 2 ⁇ l 0.1 M DTT, 10 ⁇ l 0.5 mM dGTP, dATP, dTTP, dCTP and 10 units Klenow
  • Figure 1 shows a restriction map of the
  • Figure 1 also shows a restriction map of the 4.7 kb Hindlll-EcoRI fragment which contains a bean
  • chitinase gene and hybridizes to the chitinase cDNA clone, pCH18.
  • the arrows in the figure represent the sequencing strategy used to obtain the complete nucleotide sequence of this DNA fragment.
  • polypeptide is shown in Figure 2.
  • the polypeptide is encoded by a single uninterrupted open reading frame consisting of 981 base pairs. This region is surrounded by 2.03 kb of 5' flanking DNA and 1.67 kb of 3' flanking DNA.
  • pCH35 ⁇ 6 contains 600 base pairs (bp) of 5' flanking DNA, a 981 bp open reading frame consisting of the mature chitinase polypeptide and a 26 amino acid residue signal peptide and 1670 bp of 3' flanking DNA.
  • pCH35 ⁇ 6 plasmid DNA was first linearized by digestion with the restriction enzyme
  • Linearized pCH35 ⁇ 6 DNA was then incubated at 0.1 mg/ml in 25 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 12 mM MgCl 2 , 12 mM CaCl 2 , 1 mM EDTA and 250 ⁇ g/ml BSA containing 0.05 units nuclease Bal 31/ ⁇ g DNA.
  • the DNA was repaired in an end filling reaction consisting of 0.1 mg/ml DNA in 50 mM Tris-HCl, pH 7.2, 10 mM MgSO 4 , 0.1 mM DTT, 80 ⁇ M dGTP, 80 ⁇ M dATP, 80 ⁇ M dCTP, 80 um dTTP, 50 ⁇ g/ml BSA and 0.8 units Klenow/ ⁇ g DNA. After 30 minutes at room temperature, the reaction was terminated by heating to 70°C for 5 minutes. 2 ⁇ g of the
  • blunt-ended DNA was ligated to 0.75 ⁇ g phosphorylated Hindlll linkers in 42 mM Tris-HCl, pH 7.5, 8 mM
  • the DNA was collected by centrifugation, washed with 80% ethanol, and dissolved in medium salt buffer.
  • the DNA was digested in a total volume of 50 ⁇ l buffer with 80 units HindIII at 37°C After 4 hours, the salt concentration was increased to 100 mM NaCl, 20 units of the restriction enzyme Bglll were added, and incubation resumed at 37°C for 2
  • the sample was concentrated by ethanol precipitation and the precipitate dissolved in 12 ⁇ l TE buffer, pH 8.0. 3 ⁇ l of gel loading buffer (25% Ficoll, 0.25% bromophenol blue and 0.25% xylene cyanol) was added and the sample run on a 0.8% low melting point agarose gel in TAE buffer. After electrophoresis, the gel was stained in 1 ⁇ g/ml ethidium bromide, destained in H 2 O and visualized under long wave UV light.
  • gel loading buffer (25% Ficoll, 0.25% bromophenol blue and 0.25% xylene cyanol
  • Hindlll-Bglll fragment was excised from the gel, the agarose melted at 68°C, and the DNA ligated to 0.48 ⁇ g of HindIII-BamHl digested pEMBL 8+ in 170 ⁇ l ligation buffer
  • Transformants were analyzed by nucleotide sequence analysis in order to define the 5' end point of the DNA fragment containing the chitinase coding and 3' untranslated region. Single colonies were inoculated into 1.5 ml LB broth containing 100 ⁇ g/ml ampicillin and 1.7 ⁇ 10 8 IR1 phage/ml. After 16 hours at 37°C, single stranded DNA was isolated from the liquid cultures using the procedure of Dente et al. (Nucleic Acids Res. (1983) 11: 1645-1655). Single stranded DNA from selected transformants was sequenced by the dideoxy chain termination procedure of Sanger et al. (Proc. Natl. Acad. Sci.
  • 695 is identical to 641 except that the 5' endpoint of the chitinase fragment is found at +23. Since it was not initially known whether the amount of 5' untranslated DNA would influence expression of bean chitinase through an effect on the stability of the mRNA, both 641 (which contains 21 bp of 5'
  • Plasmid DNA was isolated from 10 ml liquid cultures of clones 641 and 695 using a scaled down version of the alkaline-SDS lysis procedure of
  • pK35CAT 35S-chitinase constructs, pK35CHN641 and pK35CHN695, is termed pK35CAT.
  • pK35CAT has been deposited with the American Type Culture Collection under the terms of the Budapest Treaty and has the deposit
  • pKNK contains in pBR322, a neomycin phosphotransferase II (NPT II) promoter fragment, a nopaline synthase (NOS). promoter fragment, the coding region of neomycin phosphotransferase II and the polyadenylation signals of the nopaline synthase gene.
  • NPT II neomycin phosphotransferase II
  • NOS nopaline synthase
  • This segment was derived from a Hindll-Bglll fragment by conversion of the HindIII site to a Clal site through linker addition.
  • the NPT II promoter fragment is followed by a 296 bp nopaline synthase promoter fragment (corresponding to
  • nucleotides -263 to +33 (Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. and Goodman, H. J. (1982) J. Appl. Genet. 1:561-574). This was obtained by the creation of a PstI site at the ATG initiation codon and subcloning of the Sau3A-PstI fragment behind the NPT II segment. The NOS promoter is followed by a 998 bp HindIII-BamHl sequence containing the NPT II coding region. The NPT II coding region was obtained from the transposon Tn5 (Beck, E., Ludwig, G.,
  • NPT II structural region is then followed by a 702 bp BamHl-Clal fragment corresponding to the 3' end of the nopaline synthase gene (nucleotides 848 to 1550) (Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. and Goodman, H.J. (1982) J. Mol. Appl. Genet. 1:561-574).
  • the remainder of pKNK consists of pBR322 sequences from 29 to 4361.
  • pK35CAT is a pBR322 based construct which contains a chimeric gene consisting of the 35S promoter of cauliflower mosaic virus, the protein coding region of chloramphenicol acetyl transferase (CAT) and the polyadenylation signals of the nopaline synthase gene.
  • the 35S promoter fragment of pK35CAT was obtained from a 1.15 kb Bglll segment of the CaMV genome (corresponding to sequences -941 to +208 relative to the 35S transcription start site) cloned in the plasmid vector pUC13 (Odell, J.T., Nagy, F. and Chua, N-H., (1985) Nature 313:
  • This plasmid was linearized with the restriction enzyme Sall and the 3' end of the
  • the 35S promoter fragment was isolated as an EcoRI-Hindlll fragment and substituted for the
  • the chloramphenicol acetyl transferase coding region of pK35CAT was obtained as a 975 bp Sau3A fragment from pBR325.
  • the 5' protruding ends were filled in by reaction with the Klenow fragment of DNA polymerase I and the blunt-ended fragment ligated into a similarly blunt-ended Sall site of pGEM2.
  • a selected clone, pGCAT9 contains the insert oriented such that the Hindlll and BamHl sites of the
  • polylinker are located 5' and 3' respectively to the CAT coding region.
  • the CAT coding region was
  • pK35K The resultant construct, termed pK35CAT, also contains the NOS 3' end fragment which remains
  • the 975 bp CAT coding sequence was excised by
  • p35CHN641 or p35CHN695 depending upon the source of the chitinase coding, 3' end fragment.
  • each plasmid was digested with a two fold excess of EcoRI in 25 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA containing 5 units calf intestinal alkaline phosphatase for 1.5 hours at 37°C After this time, 1/10 volume 1 M Tris-HCl, pH 8.8 and 5 units more alkaline phosphatase were added and the samples incubated at 55°C for 30 minutes.
  • the treatments were quenched by the addition of EDTA to 10 mM, followed by heating to 70°C for 5 minutes.
  • the digested DNAs were purified by phenol/chloroform extraction, precipitated with ethanol and dissolved in sterile H 2 O at 0.2 ⁇ g/ ⁇ l. A 0.4 ⁇ g aliquot of each vector was combined with 0.1 ⁇ g of a DNA
  • the 3.5 kb drug resistance marker consists of a bacterial NPTI and a chimeric NOS:NPTII :OCS gene.
  • the NPTI gene confers kanamycin resistance in E. coli and A. tumefaciens while the NOS :NPTII :OCS gene confers kanamycin resistance to plant cells.
  • E. coli strains HB101 carrying the plasmids pK35CHN641 and pK35CHN695 were deposited
  • the construct of the present invention contains the coding region and 3' end of a bean chitinase gene fused to a DNA fragment bearing cauliflower mosaic virus 35S promoter DNA sequences
  • Other constitutive promoters which function in plants (e.g. nopaline synthase promoter (Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. and Goodman, H. M. (1982) J. Mol. Appl. Genet.
  • octopine synthase enhancer e.g. the octopine synthase enhancer (Ellis, J.G., Llewellyn, D.J., Dennis, E.S. and Peacock, W.J. (1987) EMBO J.
  • the first intron of the maize Adhl gene may provide reference
  • the 35S transcription stimulator Kay, R., Chan, A., Daly, M.
  • Tissue or developmentally specific promoters may also be employed.
  • promoters such as those derived from ribulose bisphosphate carboxylase small subunit (rbcS) genes (Morelli, G., Nagy, F., Fraley, R.T., Rogers, S.G. and Chua, N-H. (1985) Nature 315:200-204; Dean, C, van den Elzen, P., Tamaki, S., Black, M., Dunsmuir, P. and Bedbrook, J. (1987) Mol. Gen. Genet. 206:465-474), of the ribulose bisphosphate carboxylase small subunit (rbcS) genes (Morelli, G., Nagy, F., Fraley, R.T., Rogers, S.G. and Chua, N-H. (1985) Nature 315:200-204; Dean, C, van den Elzen, P., Tamaki, S., Black, M., Dunsmuir, P. and Bedbrook
  • chlorophyll a/b binding (Cab) protein genes Jones, J.D.G., Dunsmuir, P. and Bedbrook, J. (1985) EMBO J. 10:2411-2418; Simpson, J., Timko, M.P., Cashmore, A.R., Schell, J., VanMontagu, M. and
  • Herrera-Estrella, L. (1985) EMBO J. 4: 2723-2729) would optimize production of chitinase in leaf tissue to specifically combat foliar pathogens.
  • promoter sequences derived from root or stem-specific (Goldberg, R.B. Science 240:1460-1467) genes would provide preferential expression in these tissues and may thus provide protection against root and stem rot pathogens. Promoters obtained from developmentally regulated genes (Goldberg, R.B. (1988) Science
  • Mol. Gen. Genet. 203:15-20; Chen, Z.-L., Pan, N.-S. and Beachy, R.N. (1988) EMBO J. 7: 297-302) may allow timing of the expression of the chimeric chitinase gene to coincide with developmental stages of the plant which are particularly susceptible to attack by fungal pathogens. As discussed above, if the desired tissue or developmentally specific promoter proves to be of insufficient strength, one may combine this element with a transcriptional activator or
  • untranslated leader segments can influence gene expression by regulation of mRNA translation.
  • Kozak (1988, Mol. Cell. Biol. 8:2737-2744) has discussed the importance of the lack of secondary structure in and the length of the 5' leader.
  • Lutke et al. (1987) have proposed an optimal context for ATG initiation codons in plant mRNAs (Lutke, H.A., Chow, K.C, Michel, F.S., Moss, K.A., Kern, H.F. and Scheele, G. (1987) EMBO J. 6:43-48).
  • the nucleotide sequence of the 5' untranslated segment may also influence translational efficiency.
  • the 5* untranslated region of several plant virus RNAs have been found to increase the expression of the reporter RNA to which they are linked (Gallie, D.R., Sleat, D.E., Watts, J.W., Turner, P.C and Wilson, T.M.A. (1987) Nucleic Acids Res. 15:3257-3273; Gallie, D.R., Sleat, D.E., Watts, J.W., Turner, P.C. and Wilson, T.M.A. (1987) Nucleic Acids Res. 15:8693-8711; Jobling, S.A. and Gehrke, L. (1987) Nature 325:622-625).
  • a 5' leader sequence which stimulates translation of the chimeric chitinase gene may be inserted between the promoter region and the DNA segment which encodes the chitinase polypeptide.
  • Termination signals may be used in place of the cognate chitinase 3' end. Alternate 3' untranslated sequences may contribute to increased stability of the mRNA thus facilitating strong expression of the chitinase polypeptide in
  • the target pathogens may be those which invade specific tissues (foliar vs. root/stem rot fungi) or specific stages in the development of the plant (young seedling, mature plant, flowering stage, etc.).
  • chitinase coding region in the construct of the present invention is derived from an endochitinase gene from common bean (Phaseolus vulgaris), other structural sequences encoding functionally equivalent chitinase enzymes may also be used.
  • cDNA clones complementary to endochitinase mRNAs have been isolated and characterized from tobacco (Shinshi, H., Mohnen, D. and Meins, F. (1987) Proc. Natl. Acad. Sci. USA 84:89-93) and potato
  • oligonucleotides prepared from strongly conserved regions of the chitinase polypeptide (based upon comparison of the presently available amino acid sequences from bean, tomato, tobacco and potato), one skilled in the art can isolate corresponding
  • the coding region of bean chitinase is preceded by its cognate 26 amino acid residue signal peptide.
  • the location of this segment in the bean chitinase 5B gene is indicated in Figure 2 and its primary sequence given below:
  • 35S-chitinase gene of the present invention are found (Callis, J. Fromm, M. and Walbot, V. (1987) Genes and Development, 1:1183-1200) to correctly and efficiently process the bean chitinase precursor to the mature form of the enzyme. Efficient recognition and cleavage of the bean signal peptide in the heterologous plant background is indicative of its translocation to the central vacuole of the plant cell.
  • the bean lectin In the case of the bean lectin,
  • the chimeric gene of the present invention can be used in transformation experiments to obtain plants exhibiting increased resistance to plant pathogenic fungi.
  • Nucleic acids can generally be introduced into plant protoplasts, with or without the aid of electroporatio ⁇ , polyethylene glycol or other processes known to alter membrane
  • Nucleic acid constructs can also be introduced into plants using vectors comprising part of the Ti- or Ri- plasmids, a plant virus or an autonomously replicating sequence. Nucleic acid constructs can also be introduced into plants
  • DNA-coated microprojectiles into various plant parts.
  • One preferred means of introducing a nucleic acid fragment into plant cells involves the use of
  • Agrobacterium tumefaciens containing the nucleic acid fragment between T-DNA borders either on a disarmed Ti-plasmid (that is, a Ti-plasmid from which the genes for tumorigenicity have been deleted) or in a binary vector in trans to a disarmed Ti-plasmid.
  • the agrobacterium can be used to transform plants by inoculation of tissue explants, such as stems or leaf discs, or by co-cultivation with plant protoplasts.
  • Another preferred means of introducing the present nucleic acid fragment comprises direct introduction of the fragment or a vector containing the construct into plant protoplasts or cells.
  • the nucleic acid construct of the invention can be used to transform protoplasts or cell cultures from a wide range of higher plant species to form plant tissue cultures of the present invention.
  • These species include the dicotyledonous plants tobacco, petunia, cotton, sugarbeet, potato, tomato, sunflower, soybean, Brassica species and poplars; and the monocotyledonous plants corn, wheat, rice, yam, Lolium multiflorum and Asparagus officinalis. It is expected that all protoplast-derived plant cell lines can be stably transformed with the fragments of the invention.
  • nucleic acid fragments of the invention can also be introduced into plant cells with subsequent formation of transformed plants of the present invention. Transformation of whole plants is
  • Transformed plants can be monocotyledonous and dicotyledonous plants.
  • the transformed plants are selected from the group consisting of tobacco, petunia, cotton, sugarbeet, canola, potato, tomato, sunflower, carrot, celery, flax, alfalfa, lettuce, cabbage, cucumber, pepper, bean, soybean, Brassica species, poplars, clover, sugarcane, barley, oats, rice and millet; see "Handbook of Plant Cell Culture” Vols. 1-4, Evans, D. A. et al., Sharp, et al., and Ammirato et al., respectively, MacMillan, N. Y. (1983,84,86).
  • the range of crop species in which foreign genes can be introduced is expected to increase rapidly as tissue culture and transformation methods improve and as selectable markers become available.
  • the cointegrate Ti plasmids containing chimeric chitinase genes were introduced into tobacco by leaf disc transformation. All manipulations of sterile media and plant materials were done in a laminar flow hood, under suitable containment. Plant growth and plant cell cultures were carried out at 27°C
  • the leaf disks were transferred to fresh CN medium containing 500 mg/l cefotaxime and 100 mg/1
  • Cefotaxime was kept as a frozen 100 mg/ml stock solution and added aseptically (filter
  • Leaf disks were incubated under the growth conditions described above for 3 weeks and then transferred to fresh media of the same composition for an additional 1-2 weeks.
  • kanamycin were excised with a sterile scalpel and planted in root induction medium (A) (Table II) containing 100 mg/1 kanamycin. Root formation on selective and non-selective media was recorded within 3 weeks . Within 2 weeks of planting, small leaves were removed from excised shoots to determine levels of resistance to kanamycin in a callus induction assay on selective media. To induce callus formation, small leaves were excised and cut into several sections with a scalpel and plated on callus
  • induction medium (B) (Table II) containing 50 mg/l kanamycin. Callus growth on selective and
  • non-selective media was recorded within 3 weeks.
  • kanamycin resistant transgenic tobacco plants were selected and analyzed further for expression of the bean chitinase polypeptide.
  • 3-4 leaves 500-1000 mg fresh wt were excised from tobacco seedlings and homogenized in a small amount of buffer containing 50 mM HEPES, pH 6.8, 5%
  • phenylmethylsulfonyl fluoride 5 mM benzamidine, 1 mM ⁇ -amino caproic acid (to inhibit proteases).
  • the homogenized tissue was filtered through two layers of cheesecloth and the filtrate centrifuged at 20,000 rpm x 30 minutes to remove membranes. Soluble proteins were precipitated by the addition 1/10 volume of 100% trichloroacetic acid (TCA) . After incubation on ice for 30 minutes, the precipitated protein was collected by centrifugation in a
  • dithiothreitol An equal volume of a 5% SDS, 30% sucrose, 0.1% bromophenol blue solution was then added and the sample heated to 100°C for 2 minutes. The solubilized protein was then subjected to
  • chitinase is synthesized by a co-translational mechanism on membrane bound ribosomes as a precursor polypeptide of ⁇ 32 kd.
  • the precursor polypeptide is processed to a mature size of 30 kd during the course of its transport to the vacuole.
  • the identification of a 30 kd bean polypeptide in individual transgenic tobacco plants is evidence that (1) the signal peptide is cleaved in the heterologous system, (2) the enzyme has been transported to the vacuole, and (3) the bean chitinase polypeptide is expressed constitutively.
  • Transgenic tobacco plants expressing the bean chitinase polypeptide were found to contain a
  • Rhizoctonia solani is an endemic chitinous soil fungus which infects many plant species, including corn and soybeans, and produces severe stem and root rotting symptoms.
  • Rhizoctonia rarely kills the plants it infects, seed planted in heavily infested fields have problems with standability and early season growth. This disease is especially severe on oilseed rape grown in Canada. Infection by Rhizoctonia generally results in stunting and an overall reduction in seed yields. Rhizoctonia is a very adaptable organism which can survive in dry soils, wet soils, warm temperatures and cold temperatures. It is a very common soil inhabitant and feeds not only on live plants but also on crop residue.
  • Rhizoctonia infection has largely been attributed to poor seed quality, herbicide damage and low fertility and not to the presence of the fungus (Kirby, W. (1987) Seed Trade News, p. 28-30).
  • Herbicides tend to disrupt the growing point leading to increased absorption of water causing the roots and stems to crack. Breakage of the external tissues in these areas makes it easier for the pathogen to gain entry into the plant.
  • girdling root-rot are important diseases of young seedlings.
  • partial to nearly complete loss of plant stands and an 80-100% infection of established stands have been reported (Davidson, J.G.N. (1977) in "Rapeseed Production on the Peace River region of Alberta” NGR-77-7. Agric. Can. Res. Stn., Beaverlodge, Alberta. 37 pp.).
  • the estimated average yield loss due to root rot was 36 and 23%, respectively.
  • Rhizoctonia solani The organism most frequently associated with the root rot complex of canola is Rhizoctonia solani; it is the only organism isolated from diseased canola plants that is capable of inducing symptoms on artificially inoculated seedlings that are similar to the symptoms observed on seedlings damped-off in the field (Gugel, R.K. et al. (1987) Can. J. Plant Path. 9:119-128).
  • transgenic plants have been obtained that are resistant to
  • Rhizoctonia solani Resistance is due to the presence of a modified chitinase gene of the present invention which allows over-expression, of a bean chitinase enzyme.
  • oilseed rape plants containing the modified
  • modified chitinase gene The increased survival rate of the transgenic plants is dependent upon the concentration of R. solani inoculum applied to the soil.
  • concentration of R. solani inoculum applied to the soil In a quantitative assay, in which 12-14 day-old transgenic tobacco plants are transplanted into soil infested with increasing amounts of
  • transgenic tobacco plants show an average 10% reduction in root fresh weight. Three of the five plants tested showed only a 4% reduction in root mass when compared to uninoculated plants. These results, consistant with the results of survival tests, demonstrate that transgenic plants exhibit an increased resistance to infection by R. solani when production of chitinase is
  • the resistant phenotype is further manifested by a delay in progression of the disease with time.
  • Rhizoctonia is a soil-borne pathogen which produces severe root and stem rotting disease
  • the utility of the present invention is not limited to R. solani. Essentially all fungi, except the oomycetes, contain chitin in their cell walls and are potential targets. Transgenic tobacco plants of the present invention also exhibit increased
  • Botrytis cinerea a sclerotinaceous ascomycete, commonly referred to as grey mold. This pathogen is responsible for significant post-harvest deterioration of fruits and vegetables, especially strawberries and grapes.
  • Transgenic plants which are inoculated with conidia of B . cinerea were found to exhibit a reduction in the number and size of the lesions produced on young leaves.
  • Three of the five transgenic plants tested, #329, #230 and #238 exhibited an average 30%, 23% and 60% reduction, respectively, in lesion size when compared to control tobacco plants inoculated under the same conditions.
  • Two additional transformants which showed no reduction in fungal damage were found to contain 2- to 4-fold lower levels of the bean chitinase polypeptide in their leaves when compared to other transformants.
  • the CaMV 35S promoter is a constitutive promoter, the absolute levels and tissue-specificity of genes expressed under the control of this promoter can also be influenced by the environment surrounding the chromosomal insertion site. As stated previously, it may be possible to use alternative leaf specific promoters, such as the rbcS or Cab promoters, to enhance the levels of chitinase in leaves of
  • Transformation of tomato plants with the chimeric gene of the present invention may be used to provide protection against such tomato pathogens as Alternaria. Botrytis, Colletotrichum.
  • Rhizoctonia Sclerotium. Selerotinia, and Fusarium. Additionally, the stable introduction of the
  • chimeric gene into rice may be of commercial value against the causal agent of rice sheath blight
  • Rhizoctonia oryzae In oilseed rape, the potential targets of commercial value include white mold
  • Resistance to these pathogens may be enhanced further by choosing the appropriate promoter, transcription stimulator, and termination signals fused to the coding region of a higher plant chitinase gene in order to create transgenic plants with optimum resistance to either a broad range of fungal pathogens or to specific fungal pathogens, whether foliar or root/stem pathogens.
  • the plasmid pCH35 ⁇ 6 provided a convenient starting point in the construction of pK35CHN.
  • ⁇ CH35 ⁇ 6 consists of a deleted chitinase gene
  • Plasmid DNA was isolated from E. coli JM 101 cells harboring pCH35 ⁇ 6 according to the procedure of Birnboim and Doly
  • the DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and the concentration determined by measuring the absorbance at 260 nm assuming an extinction coefficient of 20 cm ⁇ /mg.
  • 50 ⁇ g of linearized pCH35 ⁇ 6 DNA was incubated at 30°C with 2.5 units of nuclease Bal 31 in 500 ⁇ l of buffer containing 20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 12 mM MgCl 2 , 12 mM CaCl 2 , 1 mM EDTA and 250 ⁇ g/ml bovine serum albumin (BSA) .
  • 50 ⁇ l aliquots of the digestion mixture were removed at 3, 6, 8, 10, 11, 12, 13, 14, 15 and 16 minutes and the Bal 31 digestion quenched by the addition of EGTA to 20 mM final concentration.
  • Hindlll 80 units in 50 ⁇ l medium salt buffer at 37°C for 4 hours. After this period of time, the salt concentration was increased to 100 mM NaCl and 20 units of Bglll were added. The reaction was incubated at 37°C for an additional 2 hours. The digested DNA was concentrated by ethanol precipitation and subjected to electrophoresis on a 0.8% low melting point agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.2).
  • Hindlll-Bglll chitinase fragment was excised from the gel and the agarose melted at 68°C
  • the DNA was ligated to 0.48 ⁇ g of Hindlll + BamHl digested pEMBL8+ in 170 ⁇ l 50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP containing 3 units T4 DNA ligase. After incubation at 12.5°C for 16 hours, 10 ⁇ l of the ligation mix was used to transform E. coli strain JM 101.
  • Transformants were selected by plating on LB agar containing 100 ⁇ g/ml ampicillin.
  • the samples were centrifuged for 10 minutes at 4°C, resuspended in 120 ⁇ l of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.3 M sodium acetate and 0.0025% bromophenol blue, and extracted with 100 ⁇ l phenol.
  • the aqueous phase was extracted twice with 1.4 ml ether and the single stranded DNA precipitated by the addition of 300 ⁇ l of ethanol. After 1 hour incubation at -70°C, the DNA was collected by centrifugation, washed with 80% ethanol and dissolved in 25 ⁇ l TE, pH 8.0.
  • Plasmid DNA was isolated from clones 641 and 695 by a mini-prep version of the alkaline-SDS lysis procedure of Birnboim and Doly (Nucleic Acids Res. 7: 1513). Cultures were grown in 10 ml LB broth containing 100 ⁇ g/ml ampicillin at 37°C overnight. The cells were harvested, resuspended in 400 ⁇ l 25 mM Tris-HCl , pH 8 . 0 , 10 mM EDTA , 50 mM glucose and 5 mg/ml lysozyme and incubated at room temperature for 5 minutes.
  • Plasmid DNA was precipitated at room temperature for 2 minutes by the addition of 2.5 volumes of absolute ethanol. The precipitated DNA was collected by centrifugation, washed with 80% ethanol and dissolved in 200 ⁇ l TE buffer containing 20 ⁇ g/ml ribonuclease.
  • One-fifth volume of gel loading buffer (25% Ficoll, 0.25% bromophenol blue and 0.25% xylene cyanol) was added to each sample and 15 ⁇ l of the 641 and 695 digests were run on a 0.75% low melting agarose gel in TAE buffer.
  • P35CHN641 and p35CHN695 plasmid DNA was isolated by the alkaline-SDS lysis procedure and purified by CsCl/ethidium bromide density gradient centrifugation. 10 ⁇ g of each plasmid was digested with 20 units EcoRI in 50 ⁇ l 25 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA containing 5 units calf intestinal alkaline
  • Tris-HCl, pH 8.8 and 5 units more phosphatase were added and the samples incubated at 55°C for 30 minutes. After this time, EDTA was added to a final concentration of 10 mM and the samples heated to 70°C for 5 minutes.
  • the digested DNAs were purified by phenol/chloroform and ether extraction, followed by precipitation with ethanol. A 0.4 ⁇ g aliquot of each vector was combined with 0.1 ⁇ g of an EcoRI fragment bearing an NPTI and a chimeric
  • E. coli strains HB101 carrying the plasmids pK35CHN641 and ⁇ K35CHN695 were deposited
  • the deposit identification numbers are ATCC67811 and 67812, respectively.
  • Example 1 The recombinant DNA construct described in Example 1 was transformed into tobacco by
  • Agrobacterium tumefaciens infection of tobacco leaf discs Primary transformants were analyzed to demonstrate constitutive expression of the bean chitinase polypeptide in tobacco. Progeny of the transformants were also analyzed to demonstrate resistance to fungal infection and inheritance of the inserted DNA construct. Standard aseptic techniques for the manipulation of sterile media and axenic plant/ bacterial cultures were followed, including the use of a laminar flow hood for all transfers. The plasmids pK35CHN641 and pK35CHN695 were introduced into Agrobacterium tumefaciens
  • strain GV 3850 (Zambryski, P., Joos, H., Genetello, C, Leemans, J., Van Montagu, M. and Schell, J.
  • the cells were harvested by centrifugation at 6000 rpm for 10 minutes in an SS-34 rotor at 4°C
  • the pellets were resuspended in 100 ⁇ l 0.15 M NaCl, 0.1 M EDTA and 25 ⁇ l of a fresh solution of lysozyme (2mg/ml) was added.
  • the samples were incubated at 37°C for 30 minutes and then transferred to a dry ice/ethanol bath. After thawing, 125 ⁇ l 0.1 M Tris-HCl, pH 9.0, 0.1 M NaCl, 1% SDS was added and the samples mixed gently by inversion. They were then extracted once with phenol, once with chloroform and the DNA
  • nitrocellolose filter according to the method of Southern, E. (J. Mol. Biol. [1975] 98:503).
  • the DNA blots were then probed with nick translated EcoRI insert of pCH18 in order to verify the integrity of the chimeric chitinase gene.
  • Young leaves, not fully expanded and 4-6 inches in length were harvested from 4-6 week old tobacco plants (Nicotiana tabacum cv xanthi). The leaves were surface sterilized for 30 minutes by submerging them in approximately 500 mis of a 10% Clorox, 0.1% SDS solution and then rinsed three times with sterile distilled water. Leaf disks, 6 mm in diameter, were prepared from whole leaves using a sterile paper punch.
  • Leaf disks were inoculated by submerging them for several minutes in 20 mis of a 1:10 dilution of an overnight culture of Agrobacterium. The culture was started by inoculating 5 mis of YEB broth
  • the leaf discs were placed in petri dishes containing CN agar medium (Table II) and sealed with parafilm.
  • the petri dishes were incubated under mixed fluorescent and "Gro and Sho” plant lights (General Electric) for 3 days in a culture room maintained at approximately 25°C
  • the leaf discs were transferred to fresh CN medium
  • Cefotaxime was kept as a frozen 100 mg/ml stock solution and added aseptically (filter sterilized through a 0.45 ⁇ m filter) to the media after autoclaving. A fresh kanamycin stock (50 mg/ml) was made for each use and was filter
  • polyacrylamide gels consisting of either a 7.5-15% gradient of acrylamide or 12% acrylamide prepared as described by
  • Electrophoretic Transfer System (or
  • FIG. 4 shows the results of this experiment for 8 transformants containing the chimeric chitinase gene. These independent transformed lines represent plants containing either plasmid pK35CHN641 or pK35CHN695. No differences were observed in the level of chitinase expression among these plants other than those attributable to the effect of different chromosome insertion sites. To demonstrate that constitutive expression of the bean chitinase
  • Chitinase enzyme activity was determined using a radiometric assay which utilized regenerated
  • radioactive chitin as a substrate
  • the reaction mixture consisted of enzyme extract (25 ⁇ g protein), 1 mg [ 3 H] chitin, 0.3 mmol sodium azide, 20 mM sodium phosphate (pH 6.5) in a final volume of 0.25 ml.
  • the reaction was stopped after 90 min at 37°C by the addition of 0.25 ml 1M trichloroacetic acid. After centrifugation (1,000g for 5 min), the radioactivity of 0.3 ml of the supernatant was determined by liquid scintilation counting.
  • Table IV The results of this analysis, shown in Table IV,
  • transgenic tobacco plants containing the modified chitinase gene exhibit approximately 1.5-2.5-fold increases in the level of chitinase enzyme activity.
  • Bacto-Yeast Extract 5.0 g
  • chitinase gene were chosen for analysis: #230, #235, #238, #329 and #373.
  • Inoculum for these experiments was prepared by growing R. solani on a sand/cereal medium consisting of 500 ml quartz sand, 40 ml cream of wheat, 40 ml corn meal and 75 ml water.
  • the medium was prepared by placing the corn meal and cream of wheat in a metal mixing bowl with 500 ml quartz sand and mixing thoroughly. The medium was then poured into a wide mouth jar, covered with a glass petri dish top, and autoclaved for 2.5 hours. Upon removing from the autoclave, the media was shaken to loosen and break up the sand /cereal in order to prevent hardening.
  • This medium is suitable for growing a number of soil organisms including
  • Rhizoctonia Selerotinia. Fusarium, and Thielaviopsis.
  • transformants #548, #230, #235, #238, #329, and #373 were surface sterilized and germinated on SG medium containing 100 mg/L kanamycin, as described above, to select for plants containing the transferred
  • kanamycin-containing medium were transferred to soil and allowed to grow to maturity in the greenhouse. As above, bags were placed on individual flowers to permit self-fertilization. Seeds of several plants derived from individual transformants were collected and subjected to segregation analysis by germinating seed on SG medium containing 100 mg/L kanamycin. R1 plants which were originally heterozygous would produce progeny which segregated with a ratio of 3 resistant:1 sensitive. On the other hand, R1 plants which were homozygous would yield 100% kanamycin resistant progeny after self-fertilization. Using this procedure, homozygous seed stock of each transformant were identified for further analysis.
  • Transgenic tobacco plants of the present invention were analyzed for resistance to the foliar pathogen Botrytis cinerea.
  • five independently isolated transgenic tobacco plants (#329, #235, #238, #230, #373) containing the
  • modified chitinase gene of the invention and control plants (#548) lacking the modified chitinase gene, were tested for resistance to the fungal pathogen
  • B. cinerea isolate used in this experiment is a Benlate resistant isolate. This isolate was used because it grows faster and sporulates more profusely than Benylate sensitive isolates, however, any publicly available virulent strain of B. cinerea (such as are available from the ATCC) would be useful for this purpose.
  • B. cinerea was grown on Potato Dextrose Yeast Agar (PDYA) containing 20 mg/L benomyl (99.5% a.i.) PDYA was prepared by melting 39 grams potato dextrose agar (Difco Laboratories, Detroit, Michigan) and 5 grams yeast extract (Difco
  • the modified chitinase gene of Example 1 is carried as a Kpn I fragment on the binary vector pMChAD in Agrobacterium tumefaciens strain LBA4404. This vector was used to introduce the modified chitinase gene into tomato plants by infection of cotyledon explants.
  • An outline of the features of pMChAD are shown in Figure 8 and are described below.
  • pMChAD was assembled from the parent binary vector ⁇ ZS97.
  • the plasmid pZS97 contains a left border fragment of the octopine Ti plasmid, pTiA6 and a right border fragment derived from pTiAch5 (van den Elzen, P. et al. (1985) Plant Molec. Biol. 5:149).
  • the border fragments delimit the segment of DNA which becomes stably incorporated into the host plant genome during the process of
  • Agrobacterium-mediated transformation Between the left and right border fragments is positioned the polylinker sequence of pUC18 and a chimeric marker gene (NOS/NPTII/OCS) which specifies kanamycin resistance in plant cells.
  • the amp r segment provides ampicillin resistance to bacteria harboring this plasmid and the ori segment is required for
  • Plasmid 11:206 are essential for replication and stable maintenance, respectively, of pZS97 and its derivatives in Agrobacterium tumefaciens.
  • the plasmid pMChAD also contains a tobacco acetolactate synthase (ALS) gene.
  • ALS tobacco acetolactate synthase
  • This gene consists of the upstream and termination sequences of the SurB allele and the coding region of the SurA allele containing a proline to alanine mutation at amino acid 197 and a tryptophan to leucine mutation at amino acid 591. These mutations in ALS confer resistance to sulfonylurea herbicides when introduced into plants.
  • pMChAD contains both a
  • any number of Agrobacterium based Ti-plasmid vectors would allow efficient transfer and identification of plants containing the modified chitinase gene of the present invention.
  • Seeds of tomato (Lycopersicon esculentum var. Bonnie Best ) were surface sterilized for 30 minutes in a 10% Clorox, 0.1% SDS solution and rinsed 3 times with sterile deionized water.
  • the seeds were planted in Magenta boxes (Magenta Corp.) containing 100 ml of OMS agar medium (Table VIII) and germinated under mixed fluorescent and "Gro and Sho” plant lights (General Electric) in a culture room maintained at approximately 25°C. Cotyledons from 10-15 day old seedlings were used for the Agrobacterium
  • carbenicillin was inoculated into a flask containing 30 ml of Min A broth and grown for 2 days at 28°C in a New Brunswick shaker incubator.
  • the bacterial culture was diluted with sterile Min A broth to an OD 650 of 0.1 and allowed to grow to an OD 650 of 0.2 under the same growth conditions. This culture was then used undiluted for the transformation experiment.
  • CTM agar plates (Table VIII) containing the cotyledon explants were flooded with 5 ml of the bacterial suspension for approximately 5 minutes before removal of the solution. The plates were then secured with Time Tape (Shamrock Scientific Specialty Co.) on two sides of the dish and incubated for two days under mixed fluorescent and "Gro and Sho” plant lights at approximately 25°C for two days.
  • the cotyledon explants were transferred to fresh CTM medium containing 500 mg/liter cefotaxime and 50 mg/liter kanamycin, respectively, and incubated under the same culture conditions for approximately 3 weeks. After this period of time, the cotyledons were transferred to fresh CTM medium containing the same selective agents as above but with 1/10 the zeatin concentration.
  • Plant 75 seeds per crystallizing dish, and place in the dark at 25°C for 5 days. DAY 5
  • Acetosyringone is kept as a 100 mM stock in DMSO for a maximum of three weeks.
  • concentration is about 10% cells per ml.
  • B. napus forms roots very inefficiently in culture
  • normalized shoots were planted directly into potting mix without attempting to root in vitro .
  • the shoot was excised near the agar surface, the cut surface dipped in Rootone, and the shoot planted in water-saturated Metro-mix in an 8 inch pot.
  • the pot was covered with a plastic bag until the plant was clearly growing.
  • Three transgenic B. napus plants were obtained using this procedure and were grown in the greenhouse. These plants were analyzed for expression of the chimeric chitinase gene by
  • MS Minimal Organic Medium (MS salts, 100 mg/L i-inositol, 0.4 mg/L thiamine)
  • MSV-1A (Shoot Maintenance Medium)
  • MS Minimal Organic Medium 100 mg/L i-inositol
  • Vitamins Myo-Inositol 100x 100 mg/l 10000 mg
  • Nicotinic Acid 100 mg/100 ml
  • Thiamine Hydrochloride 1000 mg/100 ml
  • Pyrid ⁇ xine Hydrochloride 100 mg/100 ml
  • Nicotinic Acid 500 mg/100 ml
  • Nicotinic Acid (Shelf) 10 mg/100 ml
  • transgenic canola plants of Example 6 are shown to be resistant to infection by Rhizoctonia solani.
  • the resistant phenotype of these transgenic plants is characterized by a delay in the appearance of disease and a reduction in disease severity.
  • Seeds were surface sterilized as outlined in the transformation procedure of Example 6. The seed were then placed on MSV-1A medium (TABLE X) containing 10 ppb chlorsulfuron in Magenta boxes. Approximately 30-40 seeds were used and divided between two Magenta boxes. Plants were allowed to germinate and grow for approximately two weeks with a 16 hr. photoperiod at 25°C. Seedlings which displayed elongated hypocotyls (6-12 cm), expanded cotyledons, true leaf formation, and well developed root systems were scored as
  • chitinase gene modified according to Example 1 for constitutive expression in plants, 16 of the pooled Rl seed were germinated in soil and grown in a growth chamber for 14 days at 20°C with a photoperiod of 16 hr. day: 8 hr. night. The seedlings were
  • R. solani inoculum prepared as described in Example 3. This level of inoculum was determined empirically to result in the survival of approximately 50% of the transplanted seedlings when wild type B. napus cv. Westar was used. In contrast to tobacco, canola is extremely sensitive to infection by R. solani and lower levels of inoculum were required in these experiments. The extent of disease was monitored by recording the number of surviving plants at various time intervals following infection. The results of two independent experiments are shown in Figure 11 and indicate that transgenic canola plants containing the modified chitinase gene exhibit increased
  • transgenic plants which did not survive fungal infection lacked the modified chitinase gene as a result of genetic segregation.
  • the availability of homozygous lines of these and other transgenic canola plants containing the modified chitinase gene of the present invention should result in even higher levels of resistance and allow more quantitative evaluations of the resistant phenotype.
  • transgenic plants exhibit increased survival rates and a delay in disease development when grown in infested soil. This is likely to be of important practical value by enabling canola seedlings to survive the critical period during stand
  • suspension cultures were used to introduce the chimeric chitinase gene, carried on the plasmid ⁇ K35CHN, into rice. Suspension cultures were
  • degrading enzymes cellulase and macerozyme Four mis of the enzyme mixture consisting of 2% (wt/vol) cellulase "Onozuka” RS and 0.5% (wt/vol) Macerozyme (both from Vakult Homsh, Nishinomiya, Japan) in 13% mannitol pH 5.6, were used per gram of cells. The mixture was incubated on a rotary shaker (30 rpm) at 25°C for 16-18 hours. Released protoplasts were filtered through a 60 ⁇ m nylon mesh, transferred to 50 ml Pyrex test tubes and washed twice by
  • Protoplasts were resuspended at 10°/ml in N6 medium containing 17% sucrose and 2mg/liter 2,4-D, pH 5.8. An equal volume of molten 2.5% (wt/vol)
  • the agarose blocks were cut into 1 ⁇ 1 cm segments which were placed in the 3.5 cm diameter wells of a 3 ⁇ 2 well cluster dish (Gibco).
  • Protoplast division was supported by immersing the slabs in culture medium containing 0.1 g of
  • the agarose slabs were transferred to fresh culture medium without nurse cells after two weeks. Clusters of 20 or more cells were visible after 3 weeks, at which stage 100 ⁇ g/ml kanamycin sulfate was included in the culture medium.
  • the agarose slabs were transferred onto the surface of agarose-solified N6 medium containing 2mg/liter 2,4-D and 8% (wt/vol) sucrose with 100ug/ml kanamycin sulfate. After 10 weeks, the most vigorous colonies were individually transferred to fresh agarose-solidified medium. 92 individual kanamycin tolerant calli were recovered from 5 ⁇ 10 6
  • rice cells transformed with pK35CHN also contain an additional immunoreactive polypeptide which co-migrates with purified bean chitinase. This indicates that the precursor form, encoded by the chimeric chitinase gene is processed correctly in monocot cells and suggests that it is localized in the plant vacuole. The lower molecular weight bands present in this experiment are likely due to
  • regeneration occurs through somatic embryogenesis of the protoplast-derived calli. Therefore, one skilled in the art could obtain rice plants containing the recombinant DNA construct of the present invention through protoplast transformation using cell cultures capable of regeneration.
  • pH Adjusted to 5.8 with NaOH (if too acid) or HCl (if too basic).
  • Callus culture medium 80 g/l sucrose and 0.4% seaplaque low melting point agarose

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Abstract

Préparation de constructions nouvelles d'ADN recombinant et leur utilisation dans la transformation de plantes pour obtenir une surexpression de la chitinase et par là une résistance accrue à divers champignons pathogéniques de plantes.
PCT/US1989/005501 1988-12-16 1989-12-13 Surexpression de la chitinase dans des plantes transgeniques WO1990007001A1 (fr)

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EP0460753A2 (fr) * 1990-06-07 1991-12-11 Mogen International N.V. Nouvelles préparations antifungiques, procédé pour leur production, procédé d'obtention des plantes ayant une sensibilité réduite aux champignons
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EP1009805A1 (fr) * 1997-07-27 2000-06-21 Yissum Research Development Company Of The Hebrew University Of Jerusalem PLANTES TRANSGENIQUES A MORPHOLOGIE MODIFIEE ET GENE, PROMOTEUR ET PROTEINE DE L'ENDO-1,4-$g(b)-GLUCANASE ISOLES A PARTIR D'ARABIDOPSIS THALIANA
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