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WO1999043823A1 - Genes activant le systeme de defense de plantes contre les elements pathogenes - Google Patents

Genes activant le systeme de defense de plantes contre les elements pathogenes Download PDF

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Publication number
WO1999043823A1
WO1999043823A1 PCT/US1999/003336 US9903336W WO9943823A1 WO 1999043823 A1 WO1999043823 A1 WO 1999043823A1 US 9903336 W US9903336 W US 9903336W WO 9943823 A1 WO9943823 A1 WO 9943823A1
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plant
gene
promoter
avimlence
avrrxv
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PCT/US1999/003336
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English (en)
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Steven P. Briggs
Carl R. Simmons
John T. Tossberg
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Pioneer Hi-Bred International, Inc.
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Priority to AU26832/99A priority Critical patent/AU2683299A/en
Publication of WO1999043823A1 publication Critical patent/WO1999043823A1/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
    • C12N15/63—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

Definitions

  • the invention relates to the genetic manipulation of plants, particularly to transforming plants with genes that enhance disease resistance.
  • Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants.
  • Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
  • a host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism.
  • plants can activate an array of biochemical responses. Generally, the plant responds by inducing several local responses in the cells immediately surrounding the infection site. The most common resistance response observed in both nonhost and race- specific interactions is termed the "hypersensitive response" (HR). In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.
  • HR hypersensitive response
  • the hypersensitive response in many plant-pathogen interactions results from the expression of a resistance (R) gene in the plant and a corresponding avirulence (avr) gene in the pathogen. This interaction is associated with the rapid, localized cell death of the hypersensitive response.
  • R genes that respond to specific bacterial, fungal, or viral pathogens have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins.
  • the resistance gene in the plant and the avirulence gene in the pathogen often conform to a gene-for-gene relationship. That is, resistance to a pathogen is only observed when the pathogen carries a specific avirulence gene and the plant carries a corresponding or complementing resistance gene.
  • ⁇ vrR gene-for-gene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying ⁇ vrR gene mediated resistance has been postulated.
  • a simple model which has been proposed is that pathogen avr genes directly or indirectly generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R genes.
  • expressing an avirulence gene does not stop the pathogen from being virulent on hosts that lack the corresponding R gene.
  • a single plant can have many R genes, and a pathogen can have many avr genes.
  • the phytopathogenic fungi play the dominant role. Phytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. All of the approximately 300,000 species of flowering plants are attacked by pathogenic fungi.
  • a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range.
  • Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change.
  • the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose.
  • the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
  • compositions and methods for creating or enhancing resistance to plant pests involve stably transforming a plant with an avirulence gene operably linked with a promoter capable of driving expression of a gene in a plant cell.
  • the avirulence gene product is capable of interacting with a complementing resistance gene in the plant.
  • a plant can be stably transformed with both an avirulence gene and with the complementing resistance gene, both of which are operably linked with an appropriate promoter.
  • promoters will be useful in the invention the choice of which will depend in part upon the desired level of expression of the avirulence and/or the resistance genes in the plant, or alternatively, in the plant organ in which expression is desired. It is recognized that the levels of expression can be controlled to induce the disease resistance pathway resulting in levels of immunity in the plant or to induce cell death.
  • the methods of the invention find use in controlling plant pests, including fungal pathogens, viruses, nematodes, insects, and the like.
  • Fig. 1 schematically illustrates the plasmid construct comprising the ubiquitin promoter and CRC fusion protein gene.
  • Fig. 2 schematically illustrates the plasmid construct comprising the ubiquitin promoter and AvrRxv gene.
  • Fig. 3 schematically illustrates the plasmid construct comprising the PRtns promoter and AvrRxv gene.
  • the invention is drawn to methods for creating or enhancing resistance in a plant to plant pests. Accordingly, the methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.
  • disease resistance is intended that the plants avoid the disease symptoms which are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms.
  • the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.
  • Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like.
  • Viruses include tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
  • Specific fungal and viral pathogens for the major crops include:
  • Soybeans Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora par asitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum,
  • nebraskense Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv.
  • Peronosclerospora philippinensis Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum. Pythium arrhenomanes, Pythium graminicola, etc.
  • Nematodes include parasitic nematodes such as root knot, cyst and lesion nematodes, etc.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
  • Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp.
  • Sphenophorus maidis maize billbug; Rhopalosiphum maidis; corn leaf aphid; Siphaflava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hyper a punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid
  • the methods of the invention rely upon the gene-for-gene interaction between avirulence and resistance genes.
  • robust defense responses to invading pathogens often conform to a gene-for-gene relationship. Resistance to a pathogen is observed when the pathogen carries a specific avirulence (avr) gene and the plant carries a corresponding resistance (R) gene. Therefore, the pathogen carries a specific avirulence (avr) gene and the plant carries a corresponding resistance (R) gene. Therefore, the
  • corresponding or complementing is intended that the R gene is capable of recognizing and interacting with the particular avr gene product to invoke the hypersensitive response, or alternatively, to induce a level of immunity in the plant to minimize, reduce, and/or avoid pathogen infection.
  • the method relies upon the presence of a resistance (R) gene in the plant which is able to complement the avimlence gene. That is, for the HR to occur the R gene must recognize the gene product of the avimlence gene. Accordingly, a plant is transformed with an avimlence gene. Where the plant contains the complementing or corresponding R gene, a disease resistance reaction occurs. Where the plant does not contain a complementing R gene, methods are available for crossing into or transforming the plant with the appropriate R gene. Dominance of the appropriate R gene facilitates introduction of the gene by breeding methods. Then, expression of the two genes involves a hypersensitive reaction that includes cell death. The programmed cell death process in plant disease responses has definite characteristics such as DNA degradation. Additionally, it is involved in response to receptor-type R or resistance gene interactions. See, for example, Ryerson and Heath (1996) Plant Cell 5:393-402 and Dangl et al. (1996) Plant Cell 5:1793-1807.
  • promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. When the genes are expressed at levels to cause cell death, an inducible promoter can be used to drive the expression of either the avr or both the avr and R genes. Where the R gene is present in the plant or is crossed into the plant through breeding methods, the avr gene can be expressed utilizing an inducible promoter.
  • the inducible promoter generally needs to be tightly regulated to prevent unnecessary cell death yet be expressed in the presence of a pathogen to prevent infection and disease symptoms.
  • an inducible promoter particularly from a pathogen-inducible promoter.
  • promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta- 1,3 -glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Net/?. J Plant Pathol. 89:245-254; Uknes et al. (1992) The Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See, also application serial numbers 60/076,100 and 60/079,648 entitled “Inducible Maize Promoters", filed February 26, 1998, and March 27, 1998, respectively, and herein incorporated by reference.
  • PR proteins pathogenesis-related proteins
  • promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325- 331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Molecular and General Genetics 2:93-98; and Yang, Y (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al.
  • a wound inducible promoter may be used in the constructions of the invention.
  • wound inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan, C, Annu Rev Phytopath 28:425-449; Duan et al.
  • weak promoters will be used. It is recognized that weak inducible promoters may also be used. Likewise, either a weak constitutive or a weak tissue specific promoter may be used. Such weak promoters cause activation of the plant defense system short of hypersensitive cell death. Thus, there is an activation of the plant defense system at levels sufficient to protect from pathogen invasion. In this state, there is at least a partial activation of the plant defense system wherein the plant produces increased levels of antipathogenic factors such as PR proteins, i.e., PR1, chitinases, ⁇ -glucanases, etc.; secondary metabolites; phytoalexins; reactive oxygen species; and the like.
  • PR proteins i.e., PR1, chitinases, ⁇ -glucanases, etc.
  • weak promoter is intended either a promoter that drives expression of a coding sequence at a low level.
  • low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts.
  • weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.
  • Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 (copending application serial number 08/661,601), the core 35S CaMV promoter, and the like.
  • Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142. See also, application serial number 60/076,075 entitled “Constitution Maize Promoters” filed February 26, 1998 and herein incorporated by reference.
  • Tissue specific promoters include Yamamoto et al. (1997) Plant J. 112(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol Gen Genet. 254(3):337-343; Russell et al. (1997)
  • avimlence genes are known in the art and can be used in the invention.
  • the avimlence gene will be chosen based upon the presence of a corresponding or complementing R gene in the plant to be transformed. Alternatively, where no corresponding R gene is present in the plant, it will be necessary to use an avimlence gene wherein the complementing or corresponding R gene is available to be cotransformed or crossed into the plant.
  • the plant to be transformed with the avimlence gene will be tested for the presence of a complementing resistance gene.
  • the resistance gene may be introduced by recombinant methods or alternatively by breeding. For particular avimlence genes, see Puri et al. (1997) Mol Plant Microbe
  • Kanazin et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93(21):11746-11750: Hammond-Kosack et al. (1996) Plant Cell 8(10):1773-1791 ; and Buschges et al.
  • the avimlence gene can be introduced into the plant in a transgenic experiment.
  • the avimlence gene is introduced into the plant, along with a reporter gene. Constitutive promoiers will be used to drive both the avirulence gene and the reporter gene or genes. If
  • Reporter genes are available in the art. Reporter genes should ideally exhibit low background activity and should not have any detrimental effects on metabolism. The reporter gene products will have moderate stability in vivo, so that down-regulation of gene expression as well as gene activity can be detected. Finally, the reporter gene should be able to be assayed by a quantitative, sensitive, simple to perform and inexpensive system.
  • GUS Beta-glucuronidase
  • This gene is encoded by the uidA locus of E coli.
  • GUS enzyme activity can be assayed easily and sensitively in plants.
  • the expression of GUS gene fusions can be quantified by fluorometric assay, and histochemical analysis can be used to localize gene activity in transgenic tissues.
  • Luciferase (DeWet et al. (1987) Mol. Cell. Biol, 7:725-737). Luciferase catalyzes the oxidation of D(-)-luciferin in the presence of ATP to generate oxyluciferin and yellow-green light.
  • Anthocyanins (Goff et al. (1990) EMBO J. 9:2517-2522).
  • Anthocyanin is a reporter system that does not require the application of external substrates for its detection.
  • the anthocyanin system utilizes the CI, Bz and R genes, which code for trans-acting factors that regulate the anthocyanin biosynthetic pathway in maize seeds.
  • the introduction of these regulatory genes under the control of constitutive promoters includes cell-autonomous pigmentation in non-seed tissues.
  • Green fluorescent protein (GFP) from the jellyfish Aequorea victoria
  • GFP emits bright green light when excited with UV or blue light. GFP fluorescence does not require a substrate or cofactor, is stable, and can be monitored non-invasively in living cells.
  • the avimlence and/or R genes of the invention can be introduced into any plant.
  • the genes to be introduced will be used in expression cassettes for expression in any plant of interest.
  • Such expression cassettes will comprise a transcriptional initiation region linked to the gene encoding the R or avr gene of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest are constitutive promoters (See, for example, U.S. Patent Nos.
  • the transcriptional cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell
  • the genes of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
  • the additional gene(s) can be provided on another expression cassette.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant preferred codons for improved expression.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Vims); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macej ak and Samow ( 1991 ) Nature
  • picornavirus leaders for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch
  • RNA 4 alfalfa mosaic vims
  • TMV tobacco mosaic vims leader
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g. transitions and transversions may be involved.
  • the genes of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium mediated transformation (Townsend et al. U.S. Patent No. 5,563,055), direct gene transfer (Paszkowski et al.
  • Patent No. 5,240,855 Buising et al. U.S. Patent Nos. 5,322,783 and 5,324, 646 (maize); Klein et al. (1988) Plant Physiol. 91:440-444(maize); Fromm ei al. (1990) Biotechnology 8:833-839 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London) 311 :763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.
  • the cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
  • the methods of the present invention provide an improvement over the previous approach of matching avirulence and resistance genes via traditional breeding methods.
  • breeders of many crops initiated breeding programs with the expectation that the resulting control of plant diseases would be permanent.
  • Durable disease resistance based on the utilization of one or more single dominant R genes has been achieved in some cases. More frequently, however, the rapid evolution of matching pathotypes virulent on previously resistant cultivars has forced breeders into a repetitive cycle of cultivar replacement demanding the continual introgression of new resistance specialties.
  • the present method relies upon an inducible promoter which is turned on in the presence of the pathogen and is not necessarily dependent upon the recognition of a ligand or protein produced by the pathogen.
  • weak constitutive promoters are likely to induce a level of immunity in the plant. Accordingly, there may be no increased selection pressure against the matching avr allele in the pathogen population. Therefore, single mutational events at the corresponding avr locus may not result in a new virulent pathotype.
  • the methods of the invention can be used with other methods available in the art for enhancing disease resistance in plants.
  • a transient gene expression assay as modified from Nelson and Bushnell (Transgenic Res. (1997) 6:233-244), was used to evaluate the ability of an introduced avimlence gene, whose expression product would induce expression of an unknown resident resistance gene in a host plant cell, to confer a hypersensitive response within the host cell.
  • a particle bombardment system was used to simultaneously introduce a constmct comprising a reporter gene driven by a constitutive promoter and a constmct comprising an avimlence gene with its promoter into maize cells for the purposes of studying physiological processes, foremost amongst them the plant defense response.
  • the first constmct comprised a ubiquitin promoter driving the expression of the reporter CRC fusion protein gene, which when expressed causes cells to turn red due to anthocyanin production.
  • Other reporter
  • the second constmct comprised the AvrRxv gene driven by the constitutive ubiquitin promoter.
  • This gene the nucleotide sequence of which is published (Whalen et al. (1993) Mol. Plant Microb. Interact. 6:616-627; Accession No. L20423), is from Xanthomonas campestris pv vesicatoria, a pathogen of tomato.
  • Expression of the AvrRxv gene product causes interaction of that gene product with a resident maize resistance gene, termed Rxv, if such a gene is present.
  • Plasmid pi 1416 (Fig. 1), comprising the ubiquitin promoter (ubi) and the CRC fusion protein gene (ubi::CRC fusion), the expression of which yields the anthocyanin- producing, or red cell, phenotype
  • plasmid pi 0249 (Fig. 2), comprising the ubiquitin promoter and the A rR gene (ubi: AvrRxv), the expression of which yields the AvrRxv product.
  • Plasmid p7770 (not shown), comprising an empty ubiquitin promoter constmct (ubi::pinll terminator), was used as a control to balance promoter site molarity; and plasmid p7731 (not shown), an inert DNA
  • Embryos were transformed by the tungsten particle biolistic method (Tomes et al. (1995) supra; Koziel et al. (1993) Bio/Technology 11 : 194-200) using a high pressure particle delivery system (Biolistic Particle Delivery System Model PDS-100 by DuPont). Forty-five embryos, arranged in 5 plates, each with 9 embryos, were subjected to bombardment with the ubi::CRC fusion constmct alone (Treatment A) or to cobombardment with the ubi::CRC fusion constmct and the ubi: A rR v constmct (Treatment B). Following bombardment, embryos were stored in the dark for 36 hours at 23°C.
  • CRC fusion gene Expression of the CRC fusion gene was quantified by visual means 16 to 48 hours, more usually 36 hours, following bombardment.
  • Cells expressing the CRC fusion protein gene are red in color. The number of red cells on all 9 embryos within each plate was counted and an average number of spots calculated for the 45 embryos bombarded with the ubi::CRC fusion constmct (Treatment A) and the 45 embryos bombarded with the ubi::CRC fusion and ubi: : Avr Rxv constmct (Treatment B).
  • Activation of the defense system using the maize PR1 protein as a marker, was verified with an antibody Western blot for the PR1 class of pathogenesis-related proteins. Forty-eight hours after bombardment, 18 embryos for each treatment were pooled and their protein extracted and mn on SDS- PAGE, electroblotted onto 0.2 micron PVDV membrane, and probed with antibodies raised against tobacco PR1 protein.
  • a rRxv gene sequences in maize tissue causes elevated expression of defense-related markers, such as PR1 protein.
  • the elevated PR1 protein expression was observed in two types of bombarded tissues, immature embryos and leaves, indicating the likely widespread developmental expression of
  • Tissues are ME, mature embryo, IE, immature embryo, and LF, leaf
  • This system basically involves primer extension using a primer site on the 5' end of the cDNA.
  • promoters are cloned and linked to the AvrRxv coding regions for testing in both transient assays and stable transformed plants.
  • the first such promoter to have been cloned next to the AvrRxv gene is that of the PRms gene.
  • This is a maize gene that is induced by Fusarium moniliforme treatment. It also has some developmental expression apart from pathogen treatment.
  • the PRms gene is related to the PR1 class of pathogenesis- related proteins, a class first identified and characterized in tobacco.
  • the PRms expression pattern has been published (see, for example, Cordero et al. (1992) Physiological and Molecular Plant Pathology 41:189-200; Casacuberta et al. (1992) Molecular and General Genetics 234:97-104; and Murillo et al. (1997) 77ze Plant Cell 9:145-156), and the PRms promoter has been sequenced (Raventos et al. (1995) Plant! 7(1):147-155; Accession No. X78337).
  • a plasmid constmct comprising the PRms promoter: AvrRxv coding region has been tested in the transient assay system described in Example 1 using mature embryo scutellum of the maize inbred line in Example 1. It was observed that the PRms: AvrRxv construct did not cause a suppression of the CRC anthocyanin-producing reporter system. This appears to indicate that the basal or background expression of the AvrR v gene with the PRms promoter does not produce enough AvrRxv product to elicit the vrRxv-Rxv defense response. This being the case, finding an inducible promoter to drive A rRxv expression may not require an especially low basal (i.e., not pathogen induced) level of expression to avoid triggering the A vrRxv-Rxv defense response.
  • pathogen inducible promoters such as the maize PR1 promoter
  • Other pathogen inducible promoters are isolated, characterized, and linked to the AvrRxv coding region for testing similar to that described for the PRms promoter.
  • the transient system is used to identify promoters that have low-level background expression before proceeding with production of stable transformants.
  • Example 3 Use of a Constitutive Promoter and the AyrRxy Gene to Enhance the Pathogen Defense Response System in Leaf Tissue
  • the ⁇ vrR gene or other avimlent gene may be used to develop a pathogen defense response system in particular plant tissues.
  • the AvrRxv coding region is driven by a constitutive promoter.
  • the ubi: AvrR v constmct was cobombarded with CRC into maize leaf tissue. Protocols for the transformations was as described above.
  • the leaf tissue was isolated from L6- L7 plants grown in the greenhouse for approximately 3 weeks.
  • the tender nearly white leaves wrapped at the center of the whorl were isolated, unfurled and laid on the agar bombing medium. After bombardment the tissue was incubated for 48 hours in the dark and red spots were counted.
  • the results demonstrated a suppression of the CRC expression relative to controls.
  • a 2-3 fold suppression of the CRC expression was achieved by the following: A 2-3 fold suppression
  • Example 4 AyrRxy Defense-Inducing System in Stable Transformed Maize Tissue.
  • Transgenic maize callus/cell lines expressing the avrRxv gene under the direction of the ERE (estrogen response element) promoter constmct were regenerated.
  • the ERE promoter constmct is an estrogen inducible gene expression system. The results indicate that induced avrRxv expression causes the activation of maize pathogen defense systems.
  • ERE-avrRxv callus treated with estradiol were subjected to mRNA profiling. A set of induced gene expression changes were identified. These induced genes are largely genes whose expression is known or suspected to be involved in pathogen defense.
  • the purpose of the experiment was to profile gene expression changes associated with the induced expression of avrRxv, and to determine whether these gene expression changes are consistent with activation of the plant (cells) defense system.
  • the callus line used for this experiment previously showed elevated expression of PR1, chitinase, and cationic peroxidases following induction with estradiol. Additionally, the line showed browning and accumulation of phenolics.
  • Transgenic maize callus transformed with the ERE- avrRxv constmct was used. This callus is from line "197" and was the same used in earlier experiments described above. Similar GS3 callus transformed with a constmct containing only the selectable marker gene, but not the ERE- avrRxv chimeric gene, was used as control. The callus from these two backgrounds was divided into two samples. These samples were plated on fresh agar selection medium. For the induction with estradiol, one plate for each genotype was treated with estradiol in an aqueous/ethanolic solution. The other plate for each genotype served as a non-induced control and received aqueous/ethanolic solution without estradiol. This application was time 0.
  • Hybridization, image detection, data normalization, and algorithmic analysis were conducted. Relative fold change in hybridizing intensity was compared between estradiol-treated versus control samples for both the ERE-avrRxv genotype and the GS3 genotype.
  • the results indicate those genes (cDNAs) that have changed (here induced) expression of at least two-fold in the ERE-avrRxv callus treated with estradiol relative to the GS3 callus treated with estradiol represent a set of genes that is defense related. In fact, nearly all, if not all, of the genes are known or suspected to be defense related.
  • the purpose of this experiment was two-fold. The first is to show that the avrRxv gene mRNA expression is indeed induced in ERE-avrRxv callus treated with estradiol. The second is to obtain a time course of its induced and to relate that to the rate of induction of defense-related factors.
  • results The results indicated that induction of the AvrRxv transcript by treatment with estradiol occurs in as little as 1 hour, is strongly induced in 4 hrs., and continues until 48 hrs. Without estradiol treatment very little AvrRxv transcript is made.
  • the purpose of this experiment was to determine whether the callus purported to be transgenic with the ERE-avrRxv constmct does indeed contain that constmct as revealed by Southern blots and PCR tests.

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Abstract

L'invention porte sur des compositions et procédés renforçant la résistance de plantes aux maladies. Le procédé consiste à transformer une plante à l'aide d'un gène d'avirulence ou d'un gène d'avirulence associé au gène de résistance complémentaire. On utilise un promoteur activable par un agent pathogène ou un promoteur constitutif faible pour obtenir le niveau désiré de résistance aux maladies dans la plante. L'invention porte également sur des plantes transformées, des cellules de plantes, des tissus et des semences présentant une résistance renforcée aux maladies.
PCT/US1999/003336 1998-02-26 1999-02-17 Genes activant le systeme de defense de plantes contre les elements pathogenes WO1999043823A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000863A2 (fr) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
AU2008221617B2 (en) * 2008-03-19 2010-09-23 Jose A. Fernandez-Pol Tandem repeat DNA constructs producing proteins that attack plant pathogenic viruses, fungi and bacteria by disrupting transcription factors essential for replication thereof in plants
WO2012030759A1 (fr) 2010-09-01 2012-03-08 Pioneer Hi-Bred International, Inc. Peptides de ciblage de vacuole et leurs procédés d'utilisation
DE102012003848A1 (de) 2012-02-29 2013-08-29 Kws Saat Ag Pathogenresistente transgene Pflanze

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015585A1 (fr) * 1990-04-02 1991-10-17 Rijkslandbouwuniversiteit Wageningen Procede de protection des plantes contre les pathogenes
WO1995028423A1 (fr) * 1994-04-13 1995-10-26 The General Hospital Corporation Famille, amorces et sondes de genes rps et methodes de detection associees
WO1995031564A2 (fr) * 1994-05-11 1995-11-23 John Innes Centre Innovations Limited Procede d'introduction d'une resistance aux agents pathogenes chez les vegetaux
WO1996022375A2 (fr) * 1995-01-17 1996-07-25 The Regents Of The University Of California Procedures et materiaux destines a conferer a des plantes la resistance a des maladies
WO1996035790A1 (fr) * 1995-05-11 1996-11-14 John Innes Centre Innovations Limited Genes conferant a une plante une resistance aux elements pathogenes, et leurs utilisations
WO1997047756A1 (fr) * 1996-06-11 1997-12-18 Pioneer Hi-Bred International, Inc. Promoteur de coeur de plante synthetique et element regulateur en amont

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015585A1 (fr) * 1990-04-02 1991-10-17 Rijkslandbouwuniversiteit Wageningen Procede de protection des plantes contre les pathogenes
WO1995028423A1 (fr) * 1994-04-13 1995-10-26 The General Hospital Corporation Famille, amorces et sondes de genes rps et methodes de detection associees
WO1995031564A2 (fr) * 1994-05-11 1995-11-23 John Innes Centre Innovations Limited Procede d'introduction d'une resistance aux agents pathogenes chez les vegetaux
WO1996022375A2 (fr) * 1995-01-17 1996-07-25 The Regents Of The University Of California Procedures et materiaux destines a conferer a des plantes la resistance a des maladies
WO1996035790A1 (fr) * 1995-05-11 1996-11-14 John Innes Centre Innovations Limited Genes conferant a une plante une resistance aux elements pathogenes, et leurs utilisations
WO1997047756A1 (fr) * 1996-06-11 1997-12-18 Pioneer Hi-Bred International, Inc. Promoteur de coeur de plante synthetique et element regulateur en amont

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HONÉE G. ET AL.: "Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants", PLANT MOLECULAR BIOLOGY, vol. 29, no. 5, 1995, pages 909 - 920, XP002106959 *
PARKER J E ET AL: "Molecular intimacy between proteins specifying plant-pathogen recognition", TIBS TRENDS IN BIOCHEMICAL SCIENCES, vol. 22, no. 8, 1 August 1997 (1997-08-01), pages 291-296, XP004085814 *
WHALEN, M. C. ET AL: "Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998", MOL. PLANT-MICROBE INTERACT. (1993), 6(5), 616-27 CODEN: MPMIEL;ISSN: 0894-0282, XP002106960 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000863A2 (fr) 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
EP2270187A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270186A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270165A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270185A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270188A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2270184A2 (fr) 2001-06-22 2011-01-05 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
EP2333070A2 (fr) 2001-06-22 2011-06-15 Pioneer Hi-Bred International, Inc. Polynucléotides de défensine et procédés d'utilisation
AU2008221617B2 (en) * 2008-03-19 2010-09-23 Jose A. Fernandez-Pol Tandem repeat DNA constructs producing proteins that attack plant pathogenic viruses, fungi and bacteria by disrupting transcription factors essential for replication thereof in plants
WO2012030759A1 (fr) 2010-09-01 2012-03-08 Pioneer Hi-Bred International, Inc. Peptides de ciblage de vacuole et leurs procédés d'utilisation
DE102012003848A1 (de) 2012-02-29 2013-08-29 Kws Saat Ag Pathogenresistente transgene Pflanze
WO2013127379A1 (fr) 2012-02-29 2013-09-06 Kws Saat Ag Plante transgénique résistante aux pathogènes

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