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WO1997049822A1 - Gene conferring disease resistance in plants and uses thereof - Google Patents

Gene conferring disease resistance in plants and uses thereof Download PDF

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Publication number
WO1997049822A1
WO1997049822A1 PCT/EP1997/001218 EP9701218W WO9749822A1 WO 1997049822 A1 WO1997049822 A1 WO 1997049822A1 EP 9701218 W EP9701218 W EP 9701218W WO 9749822 A1 WO9749822 A1 WO 9749822A1
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WIPO (PCT)
Prior art keywords
plant
gene
plants
nim1
nimi
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PCT/EP1997/001218
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French (fr)
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John Andrew Ryals
Terrence Patrick Delaney
Leslie Bethards Friedrich
Kristianna Weymann
Jay Earl Johnson
Kay Ann Lawton
Daniel Murray Ellis
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Novartis Ag
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Priority to AU20261/97A priority Critical patent/AU719639B2/en
Priority to IL12706697A priority patent/IL127066A0/en
Priority to JP10502146A priority patent/JP2000512502A/en
Priority to EP97908210A priority patent/EP0923648A1/en
Priority to BR9709925A priority patent/BR9709925A/en
Priority to PL97330599A priority patent/PL187851B1/en
Priority to UA98126736A priority patent/UA73713C2/en
Publication of WO1997049822A1 publication Critical patent/WO1997049822A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to disease resistance in plants and to identifying and breeding disease resistance into plants. More particularly, the present invention relates to the identification, isolation and characterization of a gene involved in broa ⁇ spectrum disease resistance in plants.
  • Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe.
  • an initial inoculation by a necrotizing pathogen can immunize the plant to subsequent infection.
  • This acquired disease resistance was first documented in 1901 and is thought to play an important role in the preservation of plants in nature.
  • Particularly well characterized examples of plant immunity are the phenomenon o ⁇ systemic acquired resistance (SAR) and induced resistance in plants such as tobacco, Arabiaopsis and cucumber.
  • SAR systemic acquired resistance
  • inoculation by a necrotizing pathogen results in systemic protection against subsequent infections by that pathogen as well as a number of other agronomically important bacterial, fungal and viral pathogens
  • Systemic acquired resistance can also be triggered by chemical immunization compounds, certain chemicals that induce the immunity response in plants.
  • Such compounds can be of natural origin, such as salicylic acid (SA), or can be synthetic chemicals, such as 2,6-dichloroisonicotinic acid (INA) and benzo(1 ,2,3)thiadiazole-7- carbothioic acid S-methyl ester (BTH).
  • SA salicylic acid
  • INA 2,6-dichloroisonicotinic acid
  • BTH benzo(1 ,2,3)thiadiazole-7- carbothioic acid S-methyl ester
  • the SAR genes are induced following infection by a pathogen. Some of these genes have a role in providing systemic acquired resistance to the plant. These plant proteins are induced in large amounts in response to infection by various pathogens, including viruses, bacteria and fungi. PR proteins were first discovered in tobacco plants (Nicotiana tabacum) reacting hypersensitively to infection with tobacco mosaic virus (TMV). Subsequently, PR proteins have been found in many plant species (see Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254: Van Loon (1985) Plant Mol. Biol. 4: 111-116; and Uknes et al. (1992) Plant Cell 4: 645-656.) Such proteins are believed to be a common defensive systemic response of plants to infection by pathogens.
  • TMV tobacco mosaic virus
  • Pathogenesis-related proteins include but are not limited to SAR8.2a and SAR8.2b proteins, the acidic and basic forms of tobacco PR-la, PR-lb, and PR-lc; PR-1', PR-2, PR-2', PR-2", PR-N, PR-O, PR-O ⁇ PR-4, PR-P, PR-Q, PR-S, and PR-R major proteins; cucumber peroxidases; basic cucumber peroxidase; the chitinase which is a basic counterpart of PR-P or PR-Q; the beta- 1 ,3-glucanase (glucan endo- 1 ,3-beta-glucosidase, EC 3.2.1.39) which is a basic counterpart of PR-2, PR-N or PR-O; and the pathogen-inducible chitinase from cucumber.
  • PR proteins are disclosed, for example, in Uknes et al. (1992) The Plant Cell 4- 645-656 and the references cited therein.
  • SAR or SAR-hke genes are expressed in all plant species exhibiting systemic acquired resistance. Expression of such genes can be determined by probing with known SAR DNA sequences. For example, see Lawton et al (1992) Proceedings of the Second European Federation of Plant Pathology (1983), In: Mechanisms of Defense Responses in Plants, B. F ⁇ tig and M. Legrand (eds), Kluwer Academic Publishers, Dordrecht, pp. 410- 420; Uknes et al. (1992) The Plant Cell 4: 645-656; and Ward et al. (1991) The Plant Cell 3: 1085-1094 Methods for hybridization and cloning are well known in the art. See, for example, Molecular Cloning. A Laboratory Manual. 2nd Edition, Vol. 1 -3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press (1989) and the references cited therein.
  • SAR or SAR-hke genes can be found by other methods such as protein screening, +/- screening, etc. See, for example, Liang and Pardee (1992) Science 257: 967-971 ; and St. John and Davis (1979) CeJ 16: 443
  • mutants contain a mutant gene, which gene in its wildtype form controls SAR gene expression and SAR itself.
  • the present invention recognizes that the mutant gene confers broad spectrum disease susceptibility to mutant plants and renders them noninducible to pathogens and chemical inducers.
  • the present invention concerns the identification, isolation and characterization of the wildtype (NIM 1) gene, a gene which allows activation in a plant of SAR and SAR gene expression in response to biological and chemical inducers.
  • NIM 1 wildtype gene
  • a mutant gene has been identified in Mutagenized Arabidopsis plants. These plants have been found to be defective in their normal response to pathogen infection in that they do not express genes associated with systemic acquired resistance (SAR) nor are they capable of exhibiting SAR. These mutants contain a defective gene which has been labelled niml (for noninducible immunity).
  • the present invention also concerns the use of the cloned NIM1 gene and variants thereof to create transgenic plants that have broad spectrum disease resistance and to the transgenic plants produced thereby.
  • the invention further concerns the use of the cloned NIMl gene and variants thereof in a screening method for identifying compounds capable of inducing broad spectrum disease resistance in plants.
  • Figure 1 shows the effect of chemical inducers on the induction of PR gene expression in wild-type and nimi plants.
  • Figure 2 depicts PR-1 gene expression in pathogen-infected Ws-O and nimi plants over the course of 6 days from the initiation of infection.
  • Figure 3 shows the levels of SA accumulation in Ws-O and nimi plants infected with P. syringae.
  • Figure 4 shows the genetic map of the NIM1 region as determined by AFLP and SSLP analysis.
  • Figure 5 depicts a physical map of the NIM1 region as determined by YAC clone analysis.
  • Figure 6 shows a physical map of an extended P1/BAC contig.
  • Figure 7 shows a physical map setting forth the positions of P1 and BAC clones with respect to the flanking AFLP markers and YACs.
  • Figure 8 shows a physical map of a further extended P1/BAC contig containing the NIM1 gene.
  • Figure 9 shows an integrated genetic and physical fine map of the NIM region.
  • Figure 10 shows an integrated map of the NIM1 region.
  • Figure 11 shows an integrated map of the NIM1 region including the new AFLP markers.
  • Figure 12 is a schematic representation of recombinants D169 and C105.
  • Figure 13 is a global map of the chromosomal region centered on NIM1 with recombinants indicated, including, BACs, YACs and Cosmids in NIM1 region.
  • Figure 14 provides the sequence of the 9.9kb region of clone BAC-04 containing the NIM1 gene.
  • Figure 15 shows the nucleic acid sequence of the NIM1 gene and the amino acid sequence of the NIM1 gene product, including changes in the various alleles.
  • Figure 16 shows the expression of NIM1 induced by INA, BTH, SA and pathogen in wild type and mutant alleles of nimi.
  • Figure 17 shows the expression of PR-1 in nimi mutants and wild-type plants.
  • Figure 18 shows disease resistance in various nimi mutants.
  • Figure 19 is an amino acid sequence comparison of Expressed Sequence Tag regions of the NIM1 protein and cDNA protein products of 4 rice gene sequences (see SEQ ID NO: 3).
  • AFLP Amplified Fragment Length Polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syri ⁇ gae
  • NIM1 the wildtype gene, conferring disease resistance to the plant nim: mutant allele of NIM 1, conferring disease susceptibility to the plant nimi: mutant plant line
  • SSLP Simple Sequence Length Polymorphism
  • Ws-O Arabidopsis ecotype Wassilewskija
  • the NIM1 gene has been cloned by mapping and walking techniques which indicate that the gene is contained in a -105 Kb region. (See Figure 13 and Table 16). This region is delineated by the L84.6b marker on the left and the L84.T2 marker on the right. Only three overlapping cosmids made from wild-type DNA from the 105 Kb region complement the nimi mutant phenotype ( Figure 13 and Table 16). These three cosmids only overlap in a 9.9 Kb region defined by the left end of cosmid clone D7 and the right end of cosmid D5 as pictured in Figure 13. Many other cosmids made to other areas of the 105 Kb region do not complement the nimi phenotype ( Figure 13 and Table 16).
  • a near full length cDNA clone to the NIM1 gene indicates the appropriate intron-exon borders and defines the amino acid sequence of the gene product.
  • Only the NIM1 gene region within the 9.9 Kb complementing region has sequence changes in various nimi mutant alleles (Table 18). Three other potential gene regions showed no sequence changes that are assosciated with the nimi phenotype.
  • the sequence changes found in the NIM1 gene region are consistent with altered function or loss of function of the gene product.
  • the severity of the change to the NIM1 gene region in a particular mutant allele is roughly correlated with the observed physiological severity of that nimi allele.
  • the present invention relates to an isolated gene fragment, the NIM 1 gene, which is a key component of the systemic acquired resistance (SAR) pathway in plants.
  • the NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression.
  • the location of the NIM gene is determined by molecular biological analysis of the genome of mutant plants known to carry the mutant nimi gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR.
  • Nimi mutants are useful as "universal disease susceptible” (UDS) plants by virtue of their being susceptible to many strains and pathotypes of pathogens of the host plant and also to pathogens which do not normally infect the host plant, but which infect other hosts. They can be generated by treating seeds or other biological material with mutagenic agents and then selecting progeny plants for the UDS phenotype by treating progeny plants with known chemical inducers (e.g. INA) of the systemic acquired response and then infecting the plants with a known pathogen.
  • UDS universal disease susceptible
  • Noninducible mutants develop severe disease symptoms under these circumstances, whereas non-mutants are induced by the chemical compound to systemic acquired resistance, nim mutants can be equally selected from mutant populations generated by chemical and irradiation mutagenesis, as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis.
  • nim plant phenotype is used as a tool to identify an isolated gene fragment which allows expression of broad spectrum disease resistance in plants.
  • Comprised by the present invention is an isolated DNA molecule comprising a mutant gene of the NIM1 gene which is a nimi gene.
  • a mutant gene of the NIM1 gene which is a nimi gene.
  • the resistance trait in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding. John Wiley & Sons, NY (1981 ); Crop Breeding. Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of Plant Breeding.
  • a further object of the invention is a chimeric gene comprising a promotor active in plant operably linked to a heterologuous DNA molecule encoding the aminoacid sequence of a NIM 1 gene product and variants thereof according to the invention.
  • the components of the expression cassette may be modified to increase expression.
  • truncated sequences, nucleotide substitutions or other modifications may be employed. Plant cells transformed with such modified expression systems, then, would exhibit overexpression or constitutive expression of SAR genes necessary for activation of SAR.
  • the DNA molecule or gene fragment conferring disease resistance to plants by allowing induction of SAR gene expression can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems IgtH , IgtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems.
  • the DNA sequences can be cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory
  • a further object of the invention a recombinant vector comprising the chimeric gene according to the invention.
  • a promoter In order to obtain efficient expression of the gene or gene fragment of the present invention, a promoter must be present in the expression vector.
  • RNA polymerase normally binds to the promoter and initiates transcription of a gene. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used. Suitable promoters include ubiquitin, nos promoter, the small subunit ribulose bisphosphate carboxylase gene promoter, the small subunit chlorophyll A/B binding polypeptide promoter, the 35S promoter of cauliflower mosaic virus, and promoters isolated from plant genes.
  • Plant tissues suitable for transformation include leaf tissues, root tissues, meristems, and protoplasts.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells.
  • This technique is disclosed in U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792 all to Sanford et al.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
  • the isolated gene fragment of the present invention can be utilized to confer disease resistance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots.
  • the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • the expression system of the present invention can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the gene imparts disease resistance to the intact transgenic plants. As set forth above, the expression system can be modified so that the disease resistance gene is continuously or constitutively expressed.
  • the present system can be utilized in any plant which can be transformed and regenerated. Such methods for transformation and regeneration are well known in the art. As well as the above cited references, see also, An, G., Watson, B.D., and Chiang, C.C. Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81 :301 -305, 1986; Fry, J., Barnason, A., and Horsch, R.B
  • Transgenic Soybean Glycine Max Plants from Electroporated Protoplasts PLANT PHYSIOL 99:81-88, 1992; Ha, S.B., Wu, F.S., and Thorne, T. K. Transgenic turf- type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. PI. Cell Rep. 11 :601 -604, 1992; Blechl, A.E. Genetic Transformation The New Tool for Wheat Improvement 78th Annual Meeting Keynote Address.
  • Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts. J BIOTECHNOL 32: 1 -10, 1994; Borkowska, M , Kleczkowski, K , Klos, B., Jakubiec, J., and Wielgat, B. Transformation of Diploid Potato with an Agrobacterium Tumefaciens Binary Vector System .1. Methodological Approach ACTA PHYSIOL PLANT 16:225-230, 1994, Brar, G.S., Cohen, B A., Vick, C.L., and Johnson, G.W. Recovery of Transgenic Peanut (Arachis Hypogaea L) Plants from Elite Cultivars Utilizing Accell(R) Technology.
  • nimi host plants may also be susceptible to pathogens outside of the host-range into which they normally fall, these plants also have significant utility in the molecular, genetic, and biological study of host-pathogen interactions.
  • the UDS phenotype of nimi plants also renders them of utility for fungicide screening, nimi mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host.
  • the advantage lies in the UDS phenoytpe of the mutant, which circumvents the problems encountered due to hosts being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes.
  • Pathogens of the invention include but are not limited to viruses or viroids, e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses; fungi, e.g. Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabacr, insects such as aphids, e.g.
  • viruses or viroids e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses
  • fungi e.g. Phythophthora parasitica and Peronospora tabacina
  • bacteria e.g. Pseudomonas syringae and Pseudomonas tabacr
  • the methods of the invention are useful against a number of disease organisms of maize including but not limited to downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis; rusts such as Puccinia sorphi, Puccinia polysora and Physopella zeae; other fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exseroh
  • SEQ ID NO:3 - AA sequence of wild-type NIM protein encoded by eds of seq id no:2.
  • Plasmid BAC-04 was deposited with ATCC on May 8, 1996 as ATCC 97543.
  • Plasmid P1-18 was deposited with ATCC on June 13, 1996 as ATCC 97606.
  • Cosmid D7 was deposited with ATCC on September 25, 1996 as ATC 97736.
  • NIM1 clones by map-based cloning. High resolution genetic mapping and physical mapping of NIM1 in Arabidopsis.
  • Nimi mutants were isolated from two Arabidopsis ecotype Ws-O plant populations, as described by Delaney et al., (1995) PNAS 92, 6602-6606.
  • One mutant population was in the form of an M2 library derived from ethyl methane sulfonate (EMS) mutagenized seeds (purchased from Lehle, Round Rock, TX) and the other was in the form of a T-DNA population derived from seed obtained from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH).
  • EMS ethyl methane sulfonate
  • Plants from the mutant populations were grown at high density in large trays in commercial planting mix. When the plants were 2 weeks of age, the trays were sprayed with 0.25 mg/ml INA. Four days later, the plants were sprayed with a spore suspension of Peronospora parasitica, isolate EmWa (EmWa), at 5x10 4 to 1x10 5 spores/ml. This fungus is normally virulent on the Arabidopsis Ws-O ecotype, unless resistance is first induced in these plants with INA or a similar compound.
  • nim mutant plants were isolated from the flat, placed under low humidity conditions and allowed to set seed. Plants derived from this seed were screened in an identical manner for susceptibility to the fungus EmWa, again after pretreatment with INA. The progeny plants that showed infection symptoms were defined as nim mutants. Six nim lines were thus identified. One line (nimi) was isolated from the T-DNA population and five from the EMS population. 2. Scoring Plant Reactions to INA and Other Chemical Inducers of Disease Resistance. i. Phenotypic analysis of nimi.
  • Salicylic acid (SA) and benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) are two chemicals that, like (NA, induce broad spectrum disease resistance, termed Systemic Acquired Resistance (SAR), in wildtype plants. Since INA did not induce resistance in the nimi plants, these plants were also evaluated for their disease resistance response following pretreatment with SA and BTH (as partly described in Delaney et al, 1995, PNAS 92, 6602-6606).
  • Nimi was also evaluated for its disease response to infection with 2 incompatible P. parasitica isolates, Wela and Noco (i.e. these fungal strains do not cause disease on wildtype Ws-O plants), nimi plants were sprayed with conidial suspensions of 5-10x10 4 spores/ml of Wela or Noco and incubated under high humidity for 7 days. Unlike wildtype plants, nimi plants developed disease symptoms in response to both Wela and Noco infection. The symptoms were necrotic flecking and trailing, with some sporulation. Following lactophenol blue staining, fungal hyphae were easily observed in the leaves of nimi plants. Thus, the nimi plants are susceptible to normally incompatible P. parasitica isolates. This result shows that the nimi plants are not only defective in chemically induced disease resistance, but are also defective in natural resistance to microorganisms that are normally not pathogenic.
  • SA, INA and BTH induce SAR and expression of the SAR genes, which include the Pathogenesis Related genes PR-1 , PR-2 and PR-5 in Arabidopsis. Since these compounds did not induce disease resistance in nimi (as described in example 1.2 above), this mutant line was analyzed for SAR gene expression following SA, INA or BTH treatment.
  • Nimi plants were tested for their ability to accumulate SA following pathogen infection.
  • Pseudomonas syringae tomato strain DC 3000, carrying the avrRpt2 gene was injected into leaves of 4-week-old nimi plants. The leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 92, 6602-6606. This analysis showed that the nimi plants accumulated high levels of SA in infected leaves, as shown in Figure 3. Uninfected leaves also accumulated SA, but not to the same levels as the infected leaves, similar to what has been observed in wild-type Arabidopsis. This indicated that the nim mutation maps downstream of the SA marker in the signal transduction pathway.
  • Nimi plants were backcrossed to wild-type Ws-O plants, and F1 progeny were tested for resistance to EmWa after INA pretreatment, as described in Example 1.1 above. None of the INA-pretreated F1 plants had infection symptoms, whereas the nimi control plants did show infection. Hence, the nimi mutation was determined to be recessive.
  • the F2 population from the Ws-O x nimi cross was also assayed for its disease resistance after INA pretreatment. Of this population, approximately 1/4 (32/130 plants) showed disease symptoms after EmWa treatment of INA pre-sprayed plants and 3/4 (98/130 plants) showed no disease.
  • markers had to be identified that were genetically closely linked to the mutation. This was accomplished in 2 steps. First, the nimi plants were crossed to a different Arabidopsis genotype, Landsberg erecta (Ler), and F2 plants from this cross which had a nimi phenotype (i.e. plants that are homozygous nim/nim at the NIM locus) were identified. From these, plants that had a Ler genotype at a nearby DNA marker were identified by molecular analysis. These plants, by virtue of the identification criterion, are recombinant between the marker and the NIM locus. The frequency of recombinants defines the genetic distance between the marker and the NIM locus.
  • nimi phenotype i.e. plants that are homozygous nim/nim at the NIM locus
  • the second prerequisite for map based cloning is that markers are identified that are genetically very close to the NIM locus, i.e. markers that identify very few recombinants. If genetic markers are identified that are very close, than these can be used to isolate genomic DNA clones that are close to the NIM locus.
  • the NIM locus can then be cloned by walking, if not already present on the cloned DNA. Walking can be initiated from both sides of the gene. It relies on obtaining overlapping clones that are successively closer to the gene of interest. When a single DNA marker is obtained from a walk initiated from, for instance, the North end and it identifies no recombinants between this marker and the gene of interest, it must be very close to the gene.
  • the clone from which the marker was obtained must have crossed the gene.
  • the gene of interest is cloned. It must be located between this marker and the last North-end marker that identifies the least number of recombinants from the North end.
  • a large number of recombinants are generated by genetic crossing.
  • recombinants that are close to the NIM gene are identified with the use of molecular markers.
  • Many markers have been described in the literature and several methods exist to develop additional markers. Our approach has relied on a number of marker systems, including SSLPs and AFLPs (see below).
  • nimi was crossed to Ler to make a mapping population. F2 plants from this cross were grown and leaves harvested for future DNA extractions. Next, the F2 plants were scored for the nimi phenotype, as described in example 1 .1 above. Also, F3 populations derived from individual F2 plants were grown and scored for the nim phenotype. DNA was extracted from the stored tissue of nimi phenotype F2 and F3 plants by the CTab method, as described (Rogers and Bendich, 1988, Plant Molecular Biology Manual, A6, 1-10). This DNA was used for mapping the NIM gene, as described below.
  • SSLP Simple Sequence Length Polymorphism
  • primer set primer sequence (5' to 3')
  • primerset ATHGENEA (1) (Table 1 ) was used to amplify genomic DNA in the presence of 6-carboxyrhodamine-labelled UTP (dUTP-RH O, obtained from ABI), yielding rhodamine-labelled PCR fragments.
  • the PCR reactions were analyzed on a DNA Sequencer, which detects DNA fragments with single nucleotide resolution.
  • the specific reagents were: 1xPCR buffer, 2 mM MgCI2, dNTPs each 200 mM, 2 mM dUTP-R1 10, ATHGENEA (1 ) primers at 0.75 mM, 10 ng DNA and 0.75 units Taq polymerase in a 20 ml reaction volume.
  • Amplification conditions were: 3 minutes 94°C followed by 35 cycles of 15 seconds at 94°C, 15 seconds at 55°C and 30 seconds at 72°C. These samples were analyzed on an ABI 377 DNA Sequencer, capable of detecting fluorescently labeled DNA fragments with single nucleotide (nt.) resolution. This allowed for genotyping the plant samples: a 205-nucleotide DNA fragment was obtained from Ler DNA and a 21 1 -nucleotide band from Ws-O DNA.
  • DNA fragments differing by 6 nucleotides in length could be easily distinguished, allowing for easy genotyping of samples as homozygous Ws-O, homozygous Ler and heterozygous Ws-O/Ler at the ATHGENEA locus.
  • Primer set ATHGENEA (2) was made based on the published sequence of ATHGENEA (Simoens et al., 1988, Gene 67, 1-11). This primerset amplified a DNA fragment of 139 bp from Ler DNA and a 145-bp band from Ws-O DNA. Amplification reaction conditions for primerset ATHGENEA (2) were identical to those described for primerset ATHGENEA (1), above.
  • This primer was labeled with FAM-6 and used in a PCR amplification reaction with the following reagents (Perkin Elmer): 1xXL buffer, 1 mM MgCI2, dNTPs each at 200 mM, primers each at 0.50 mM (forward primer FAM-6 labeled), 10 ng genomic DNA and 0.5 units XL polymerase in a 20-ml reaction volume.
  • the cycling conditions were: 3 minutes at 94°C, followed by 35 cycles of 15 seconds at 94°C, 15 seconds at 59°C and 30 seconds at 72°C.
  • Primer set ngal 11 (2) (listed in Table 1 , and a derivative of primer set nga111 (1 )) was used under different conditions: IxPCR buffer, 1.5 mM MgCI2, dNTPs each 200 mM, primers at 1 mM, 10 ng DNA and 1 unit Taq polymerase in a 20 ml reaction volume. Both reactions were amplified by incubation at 94°C for 1 minute, followed by 40 cycles of 15 seconds at 94°C, 15 seconds at 55°C and 30 seconds at 72°C.
  • NIM1 locus was determined to be between these 2 markers, with ATHGENEA located North of the NIM1 gene and ngal 1 1 located South of the NIM1 gene. This placed the NIM1 gene approximately 10 cM north of nga111 , near position 85 on chromosome 1 (Lister and Dean, 1993, Plant J. 4, 745-750; Bell and Ecker, 1994, Genomics 19, 137-144).
  • iii Amplified Fragment Length Polymorphism markers.
  • AFLP Amplified Fragment Length Polymorphism
  • the use of the AFLP technology in mapping relies on selective amplification of a set of DNA bands in 2 genetically distinct samples. Finding that any of the obtained bands are different between the 2 genotypes identifies those bands as markers for that genotype. If the marker cosegregates at high frequency with the gene (mutation) of interest, then the marker is close to the genetic locus.
  • DNA fragments are generated by digesting the DNA with restriction enzymes, followed by ligation of adapters to the ends.
  • primers consisting of a sequence complementary to the adapters plus a 3' extension (typically 0-3 nucleotides) are used to amplify only those DNA fragments with ends that are complementary to these primers. If a single nucleotide extension is used, then theoretically, each primer will "fit" on approximately 1/4 of all fragments, with 1/16 of all fragments having a primer fit on both ends. Thus, a limited set of DNA fragments is amplified with these primers. By further radiolabelling one primer, an even smaller subset of visible bands can be obtained. AFLP analysis.
  • DNA samples 50 ng DNA was digested with the appropriate enzymes (usually EcoRI and Msel; see below) and adapters (listed in table 2 below) were ligated to the restriction fragments (usually EcoRI and Msel).
  • the sequences of the primers and the YAC, P1 and BAC clones are described in detail below.
  • the templates were used for amplification reactions (approximately 0.5 ng DNA per reaction), using primers that were complementary to the adapters, with short 3' extensions (2 or 3 nucleotides; primer sequences are listed below). Since one of the primers is radioactively labelled (usually the EcoRI primer), only a subset of the amplified fragments is visible upon autoradiographic analysis of the gel used to separate the bands.
  • Amplification conditions for cloned DNA were as follows: 36 cycles of 30 sec. at 94°C (denaturation), 30 sec annealing and 1 min extension at 72°C. The annealing temperature in the first cycle was 65°C and was reduced by 0.7°C in each cycle for the next 12 cycles and then kept at 56°C.
  • genomic DNA of Arabidopsis plants the amplification was performed in 2 steps: in the first step (preamplification), the DNA was amplified with primers that have a single nucleotide extension (neither primer was labeled). Reaction conditions for this amplification reaction were: 20 cycles of 30 sec.
  • the first amplification reaction was diluted 10 fold and reamplified 36 cycles with primers containing the full-size extensions (using one labeled primer) under the following conditions: 30 sec. at 94°C (denaturation), 30 sec annealing and 1 min extension at 72°C.
  • the annealing temperature in the first cycle was 65°C and was reduced by 0.7°C in each cycle for the next 12 cycles and then kept at 56°C.
  • the final reaction products were separated on a polyacrylamide gel and the gel was exposed to film, allowing visualization of the radiolabeled PCR bands. When this procedure was applied to DNA from 2 genotypes simultaneously, AFLP bands were identified that were diagnostic for one genotype or the other. Such bands are called informative AFLP bands, or AFLP markers. Table 2 shows Adapters used in the AFLP analysis.
  • AFLP markers and fine mapping of the NIM1 locus.
  • the primers used for the AFLP screening were:
  • N indicates that this part was variable (A, C, G or T)
  • W indicates A or T
  • X indicates a C.
  • All 8 possible primers were used for both the EcoRl- and Msel-primer. This gave a total of 64 (8 x 8) primer combinations (PCs) that were used to amplify DNA from the recombinant inbred line and the parental genotypes, Ler and Col, as described above.
  • the amplification reactions were run on a denaturing polyacrylamide gel to separate AFLP fragments by size and the gel was exposed to film. The film was inspected for bands that were present in only one genotype, i.e. inspected for AFLP markers.
  • the AFLP markers i.e., DNA fragments that are polymorphic between both parents of the recombinant inbred lines, were used for constructing a genetic map of the recombinant inbred line population.
  • Example 1.5i describes the mapping of the NIM 1 gene on Arabidopsis chromosome 1 , at approximately position 85. Those AFLP markers that had been mapped (using the recombinant inbred line) between positions 81 and 88 of Arabidopsis chromosome 1 were chosen for analyzing recombinant plants for the presence of said AFLP markers and thus for mapping the NIM1 gene more precisely.
  • AFLP markers from this region were identified as being informative; they were polymorphic between both parents of the nimlxLer cross.
  • Six AFLP markers were Ler-specific, i.e. these AFLP markers were absent in Ws (and in Col as well).
  • One AFLP marker was Ws- specific, i.e. a Col-specific AFLP marker (absent in Ler) was also present in Ws.
  • Ws-specific AFLP marker abent in Ler
  • These AFLP markers are: L81.1 , L81.2, W83.1 , L84, L85, L87 and L88 (an L-marker is specific for ecotype Ler and a W-marker is specific for both the ecotypes Col and Ws; the number indicates the map position).
  • AFLP markers were used to analyze recombinant plants from the nimlxLer cross (see below).
  • AFLP marker C86 a recombinant, inbred- line-derived marker specific for Col
  • Table 3 lists the primer sequences that were used to obtain these AFLP markers. Table 3 shows primer combinations of AFLP markers derived from recombinant inbred line population.
  • EcoRI- refers to the sequence 5'-GACTGCGTACCAATTC-3' and "Msel-” refers to the sequence 5'-GATGAGTCCTGAGTAA-3'.
  • a detailed genetic map of the region was constructed using the AFLP markers described above by typing the recombinants. A total of 337 recombinant plants were available out of 1144 F2 nimi plants. These recombinants were first screened with the North-flanking AFLP markers L81.2 and ATHGENEA and the South-flanking markers L88 and nga1 1 1 . Forty-eight plants were homozygous nimllnimi and heterozygous at ATHGENEA and L81.2, and 21 plants were homozygous nimllnimi and heterozygous at ngal 11 and L88.
  • AFLP markers in the NIM region, including 4 AFLP markers that were derived from the recombinant inbred line mapping population, (W83.1 , L84, L85 and L87) and 5 AFLP markers derived from analysis of YAC clones (W83.3/W84.1 , W84.2, W85.1 , W86.1 and L86, see below).
  • NIM1 The genetic map of NIM1, based on this analysis, is depicted in Figure 4. As seen, 27 recombinants were found between marker W84.2 and NIM1 and 14 recombinants were found between W85.1 and NIML Marker L85 is linked closely to NIM1, but this marker could not be mapped on the YAC, BAC or P1 clones (see below) and was, therefore, not useable for identification of the NIM1 gene.
  • the CIC library an Arabidopsis ecotype Columbia YAC library (Bouchez et al, 1995, 6th Int. Conf on Arabidopsis Research, Madison, Wl), was screened for YAC clones in the NIM region. This library has about 2.5 nuclear genome equivalents and has an average insert size of 450 kb.
  • the YAC library was screened with two AFLP markers: W83.1 and C86. W83.1 is the most closely linked recombinant, inbred-line-derived AFLP marker north of NIM1, and C86 is a recombinant, inbred-line-derived AFLP marker specific for Col (absent in Ler and Ws).
  • This Col AFLP marker has been used instead of the closely linked Ler AFLP markers ( Figure 4), because the latter AFLP markers detected only ecotype Landsberg erecta and hence cannot be used for screening the Columbia YAC library.
  • the YAC library was screened in two steps. Firstly, the cells of the YAC clones of each plate of the twelve 96-well microtiter plates were pooled (a plate pool) and used for DNA isolation as described by Ross et al (1991 , Nucleic Acids Res. 19, 6053). The pools were screened with both AFLP markers. Subsequently, from each positive plate pool, the DNA samples of each row (a pool of 8 clones) and of each column (a pool of 12 clones) were screened with the AFLP marker for which the plate pool was positive. In this way, the individual positive YAC clones could be identified.
  • YAC 12F04 and YAC 12H07 were isolated using the North AFLP marker W83.1 , and YAC 10G07 and YAC 7E03 using the south AFLP marker C86 (for the nomenclature of the YAC clones the CIC numbering is used).
  • the YACs were "fingerprinted" by AFLP, giving YAC-specific AFLP fragments. Fingerprints of the YACs were compared and used to estimate overlaps between the YACs (see also Tables 5 and 6). Based on the AFLP fingerprints, clone 7E03 is essentially covered by clone 10G07 (see also table 5) and clone 12H07 is likewise essentially covered by clone 12F04 (see also table 6).
  • a screening for additional YAC-derived AFLP markers was performed on DNA samples of the following: DNA of the isolated YAC clones (4 YACs were identified, as described above), the yeast strain without a YAC, and the three Arabidopsis ecotypes Col, Ler and Ws. In this way, the fragments specific for the YAC clones (absent in the yeast strain and present in Col) could be tested for polymorphism in Ler and Ws (the parents of the recombinant plants identified in Example 1.5 below). All identified polymorphic fragments would thus be additional AFLP markers. In the first AFLP screening the enzyme combination (EC) EcoRI/Msel was used.
  • Table 5 shows a number of shared and unique AFLP fragments detected in YACs 10G07 and 7E03 and the number of informative AFLP markers among these fragments in Ws and Ler genotypes.
  • a second screening for AFLP markers was performed assaying all four identified YAC clones (see below) and using the enzyme combination Pstl/Msel.
  • the primers used are:
  • a total of 144 (12 x 12) primer combinations was screened on all four isolated YAC clones, 12F04, 12H07, 10G07 and 7E03; the yeast strain without a YAC; and the three Arabidopsis ecotypes Col, Ler and Ws.
  • Table 6 lists the number of shared and unique AFLP fragments detected in YACs and the number of informative AFLP markers among these fragments in Ws and Ler genotypes.
  • Table 7 lists the primer sequences used to obtain these AFLP markers. These 5 additional AFLP markers raised the total number of AFLP markers to 12 in the region from L81.1 to L88 (see figure 4 and below).
  • Table 7 shows primer combinations of AFLP markers derived from YAC clones.
  • "EcoRI-” refers to the sequence 5'-GACTGCGTACCAATTC-3'
  • Mel- refers to the sequence 5'-GATGAGTCCTGAGTAA-3' and "Pstl-” refers to the sequence 5'-GACTGCGTACATGCAG-3'.
  • the libraries used for gap closure were an Arabidopsis ecotype Columbia P1 library described by Liu et al (The Plant J. 7. 351-358, 1995) and an ecotype Columbia BAC library described by Choi et al (http/genome-www.stanford.edu/Arabidopsis/ww/Vol2/ choi.html).
  • the P1 -library consists of about 10,000 clones with an average insert size of 80 kb and the BAC library consists of about 4000 clones with an average insert size of 100 kb. In theory these libraries represent about 10 nuclear genome equivalents (assuming a haploid genome size for Arabidopsis of 120 Mb).
  • flanking markers Ws84.2 and Ws85.1 were used to screen pools of P1 clones using a similar strategy as previously described for screening of the YAC library (see Example 1.5i). P1 clones having the marker fragments were selected and "plasmid" DNA was isolated. The various P1 clone DNAs were fingerprinted using the ECs EcoRI/Msel and Hindlll/Msel and primers without selective nucleotides. A physical map was constructed, i.e. a map giving the size and overlaps of the clones, by comparing the AFLP fingerprints. The number of AFLP fragments that are unique and the number of AFLP fragments that are common between clones indicate the extent of the overlaps.
  • the map is displayed in Figure 6.
  • the AFLP fingerprinting revealed that two sets of non-overlapping P1-contigs had been constructed each containing one of the flanking markers: P1-1 and P1-2 containing marker Ws84.2; P1 -3 and P1 -4 containing marker W85.1 . Consequently, the gap between the flanking markers was not closed ( Figure 6).
  • the positions of the P1 contigs with respect to the YAC contig was determined by AFLP fingerprinting of the YACs and P1 clones with a number of YAC-specific PCs described above.
  • P1 clones P1 -1 and P1 -2 appeared to overlap completely with YAC CIC12F04, but only partially with YAC CIC12H07. Therefore, the latter P1 clones could be positioned on the YAC contig CIC12H07/12F04 ( Figure 6).
  • P1 clones P1 -3 and P1 -4 overlapped completely with both YACs CIC7E03 and CIC10G07 and it appeared that AFLP marker W86.1 , like W85.1 , was mapped to this P1 contig ( Figure 6).
  • L85 was used to identify corresponding P1 and BAC clones.
  • L85 is an ecotype-Landsberg-specific marker and, therefore, colony hybridization of radioactively labelled L85 DNA to P1 and BAC filters was employed. Not a single P1 or BAC clone hybridizing to L85 was identified. This supported our earlier findings that the L85 sequence is lacking in the Arabidopsis ecotype Columbia genome and is, therefore, the most likely explanation of why no corresponding clones were identified.
  • CIC12F04 not present in CIC 12H07 were used to identify P1 clones by AFLP screening of pools of the library.
  • YAC AFLP fragments from YAC 10G07 and overlapping with P1-4 were used to identify P1 clones by AFLP screening of pools of the P1 library.
  • Table 8 represents the various AFLP PCs used to screen the P1 library. The top half of the table shows PCs specific for the North YACs and the bottom half shows the PCs specific for the South YACs. Also indicated are the YACs and P1 clones wherein the AFLP fragments were detected. Table 8
  • P1/BAC contig of about 250 kb was obtained covering the South end of YAC CIC12F04 (not extending from this YAC) and containing marker W84.2.
  • a P1 contig of about 150 kb containing markers W85.1 and W86.1 was obtained; this contig is completely contained within YAC CIC7E03.
  • Table 9 is an overview of the various walking steps showing the hybridization probe used to screen the P1 and BAC libraries and the selected clones hybridizing to the probes and extending in the South direction.
  • Step l PI -7 BAC-02
  • Step 2 BAC-02 Pl-16, BAC-03
  • Step 4 Pl-18 Pl-21 , Pl-20, BAC-04
  • Step 5 BAC-04 Pl-22, Pl-23, Pl-24, BAC-06, BAC-05
  • FIG. 8 A physical map of the various clones resulting from this walking effort is depicted in Figure 8.
  • a total distance of about 600 kb was covered starting from the initial walking point marker W84.2.
  • the South end of the contig presented in Figure 8 appeared to contain the NIM1 gene (see next section).
  • the contig extends more than 300 kb South from YAC CIC12F04 and appeared not to overlap with YACs CIC10G07 and CIC7E03, indicating that the NIM1 gene is in the gap between the flanking YAC contigs and that this gap is at least 300 kb.
  • the P1 and BAC clones of the contig extension were characterized by AFLP fingerprinting using the ECs EcoRI/Msel and Hindlll/Msel. This defined quite accurately the extent of the overlaps between the various P1 and BAC clones and, in addition, generated a number of AFLP fragments specific for these clones.
  • AFLP primers without selective nucleotides are used in fingerprinting of purified plasmid DNA of the P1 or BAC clones. Selective nucleotides will be necessary, however, to be able to use these P1 or BAC-specific AFLP fragments for detection in Arabidopsis.
  • AFLP primers having the appropriate selective bases can be designed to amplify the P1- or BAC-specific AFLP fragment in
  • AFLP fragments originate from the ecotype Columbia (Col) and, therefore, it also should be determined if the Columbia AFLP markers are informative in the NIM1 recombinants which are derived from a cross of the ecotypes Landsberg erecta (Ler) and a nimi mutant of the ecotype Wassilewskija ( ⁇ Ns-nim).
  • AFLP fragments there are 4 types of AFLP fragments, two of which are useful markers as indicated in Table 10 below:
  • Table 10 is an overview of the types of AFLP markers found. (+) or (-) indicates presence or absence of the AFLP fragment. Table 10
  • fingerprinting of the P1 and BAC clones generated 30 to 40 EcoRI/Msel AFLP fragments and 60 to 80 Hindlll/Msel AFLP fragments for each individual clone. The end sequences of individual fragments were determined by standard sequencing techniques. Next, specific AFLP primers sets with selective extensions of 3 nucleotides for both the EcoRI or Hindlll primer and the Msel primer were tested on the following panel of DNAs:
  • YAC clone CIC12F04 (only for AFLP fragments from P1-7) 2c. YAC clone CIC10G07
  • BAC-01/P1 -7 Six clones were selected for sequence analyses of their EcoRI/Msel and Hindlll/Msel AFLP fragments: BAC-01/P1 -7, P1-17/P1 -18, BAC-04/BAC-06.
  • the AFLP fragments from clone P1-7 were all detected in YAC CIC12F04, indicating that this clone is completely contained within this YAC. None of the P1 /BAC-specific AFLP fragments was detected in YAC clone CIC10G07, indicating that the P1/BAC contig does not bridge the gap between the two flanking YAC contigs.
  • AFLP markers selected for analysis of the nim recombinants are depicted in Table 11.
  • Table 11 is an overview of the selected AFLP markers from the AFLP PCs specific for the various P1 and BAC clones.
  • a "WL" marker is a marker originating from the same PC and displaying two AFLP markers, a Ws and a Ler marker, which appeared to be completely linked in repulsion phase upon analysis of the NIM recombinants.
  • Table 11 is an overview of the selected AFLP markers from the AFLP PCs specific for the various P1 and BAC clones.
  • a "WL" marker is a marker originating from the same PC and displaying two AFLP markers, a Ws and a Ler marker, which appeared to be completely linked in repulsion phase upon analysis of the NIM recombinants.
  • the AFLP markers were all analyzed on a selected set of the recombinants. The results obtained are summarized in Tables 12a, 12b and 12c.
  • AFLP markers Ler84.8, Ler84.9a, Ler84.9b and Ler84.9c appeared to map at the South side of NIML Recombinants were found that were phenotypically nimi (homozygous, genotype ⁇ Ns-nim1DNs-nim1) and heterozygous for these AFLP markers (the Ler-specific AFLP marker was detected, genotype is Ws- ⁇ /m7/Ler).
  • AFLP marker Ler84.8 appeared to be closest to NIMI: only a single recombinant (C-105) was scored as heterozygous ⁇ Ns-nimKLer and homozygous ⁇ Ns-nim1/ ⁇ Ns-nim1.
  • AFLP markers Ler84.7 and Ler84.6c appeared to completely cosegregate with NIMI: all recombinants had an identical NIM1 and AFLP marker genotype. North of NIM1, marker L84.6b appeared to be closest to NIM1: three nimi phenotype recombinant plants, C-074, D-169 and E-103 (Table 12c), were found to be heterozygous Ws-n/m7/Ler at this marker. With the aid of the cosmid contig generated from P1-18, BAC-04 and BAC-06, AFLP markers Ler84.6b and Ler84.8 were mapped in P1-18 and BAC-04, respectively, and found to have a physical distance of approximately 110 kb.
  • nimi to be located on a DNA segment estimated to be 110 kb in length. From this analysis it has been determined that the NIM1 gene is contained in clone BAC-04 or P1 -18. Clones BAC-04 and P1 -18 have been deposited with ATCC and given deposit numbers ATCC 97543 and ATCC 97606, respectively.
  • NIMI Pstl/Msel, Xbal/Msel, BstYI/Msel and Taql/Msel.
  • Pstl/Msel and Xbal/Msel AFLP fragments were generated on clone P1-18 and BAC-04 and the selective sequences necessary for detection in Arabidopsis were determined. Similarly, the AFLP fragments and selective sequences were determined for BstYI/Msel and Taql/Msel; however, in this case the procedure was performed using cosmid DNAs: A11 , C7, E1 and E8 for BstYI/Msel
  • Table 13 shows the AFLP markers used for genetic and physical fine mapping of NIML "BstYI(T)" indicates that the restriction site and corresponding primer was either AGATCT or GGATCT.
  • the new AFLP markers were genetically mapped by AFLP analysis of the closest North and South recombinants.
  • the closest North (recombinant D169) and South (recombinant C105) recombination points were mapped (see Table 15).
  • the AFLP analysis showed that recombinant D169 had a recombination South of marker L84.Y1 , but North of marker W84.Y2.
  • the recombination point in recombinant C105 mapped between markers L84.T2 and L84.8. Using the available set of recombinants this allowed further delineation of the chromosomal interval containing NIMI; the distance between the flanking recombination points appeared to be 60-90 kb ( Figure 12). 56/1
  • nimi plants For complementation of the nimi plant phenotype, transformation of nimi plants is required with a wildtype NIM1 gene. This can be accomplished by transforming these plants with a cosmid containing the gene. For this purpose, a cosmid contig of the NIM1 region is constructed. Since Arabidopsis is transformed using Agrobacterium, the cosmid vector used is a binary vector.
  • DNA was isolated from BAC-04, BAC-06 and P1 -18, and used to make a partial digest using restriction enzyme Sau3AI.
  • the 20-25 kb fragments were isolated using a sucrose gradient, pooled, and filled in with dATP and dGTP.
  • the binary vector (04541 ) was cleaved with Xhol and filled in with dCTP and dTTP. The fragments were next ligated into the vector. The ligation mix was packaged and transduced into E.coli.
  • This cosmid library was screened with the BAC-04, BAC-06 and P1 -18 clones and positive clones isolated. These cosmids were next AFLP fingerprinted and arranged into a contig of overlapping clones spanning the NIM1 region. The insert sizes of the cosmids were determined, and limited restriction mapping was performed. The results are shown in Figure 10.
  • Cosmids that are generated from clones that span the NIM1 region are moved into Agrobacterium by triparental mating. These cosmids are then used to transform nimi Arabidopsis by vacuum infiltration (Mindrinos et al., 1994, CeJ 78, 1089- 1099) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection are transferred to soil and tested for the nim phenotype or their progeny are tested for the nim phenotype. Transformed plants that no longer have the nim phenotype identify cosmid(s) that contain a functional NIM1 gene.
  • nimi Arabidopsis plants containing a PR1-luciferase (PR1 -lux) transgene are used as bombardment recipient material. These plants are generated by transforming Columbia ecotype plants with a PR1-lux construct by vacuum infiltration, followed by kanamycin selection of the harvested seed, as described above. Transformed plants that express luciferase activity after induction with INA are selfed and homozygous plants are generated. These are crossed to nimi plants. In the transient assay, progeny plants from this cross that are homozygous for nimi and for PR1 -lux are used for identification of DNA clones that can complement the nimi phenotype.
  • PR1 -lux PR1-luciferase
  • the plants are first treated with INA, as described in example 1.1 above. Two days later these plants are harvested, surface sterilized and plated on GM agar medium. The leaf tissue is then bombarded with cosmid, P1 or BAC clones (or subclones) from the NIM1 region and after one day, the luciferase activity of the leaves is measured. Clones that induce luciferase activity contain the NIM1 gene.
  • nimi plants are treated with INA (as described in example 1.1 above) and 2 days later bombarded with cloned DNA (cosmid, P1 , BAC and/or YAC clones or subclones) from the NIM1 locus region and a reporter plasmid.
  • the reporter plasmid contains the luciferase gene, driven by the Arabidopsis PR1 promoter (PR1-lux).
  • PR1-lux the Arabidopsis PR1 promoter
  • INA does not activate the PR1 promoter (as described in example 1.2 above) and thus can not induce luciferase activity from the reporter plasmid.
  • INA does induce the PR1 promoter, as evidenced by an induction of luciferase activity.
  • DNA clones (cosmids, P1 or BAC clones or subclones) that induce luciferase activity that is significantly above background levels contain the NIM1 gene.
  • nimi phenotype plants have mutations in the NIM1 gene, it is conceivable that in some lines the gene is altered in such a manner that there is no mRNA transcribed, or an aberrant mRNA (size) is produced. To test for this, RNA blot analysis is performed on the nimi lines.
  • RNA is isolated from Ws and Ler plants of these lines, (after water or INA or BTH treatment) and used to prepare northern blots. These blots are hybridized with DNA fragments isolated from clones of the DNA contig of the NIM1 locus DNA fragments that identify nimi lines with aberrant RNA expression (aberrant in size or concentration), likely identify (part of) the NIM1 gene.
  • the DNA fragment and surrounding DNA is sequenced and used to isolate a cDNA (by library screen or by reverse transcription-PCR), which is also sequenced.
  • the clone from which the fragment was isolated or the isolated cDNA is used to show complementation of the nimi phenotype in stable and transient expression systems.
  • Genomic clones that may contain the NIM1 gene are sequenced using methods known in the art. These include BAC-04, P1-18 and the cosmids from the NIM1 region. For instance, the cosmids are digested with restriction enzymes and fragments that are derived from the insert are cloned into a general purpose vector, such as pUC18 or Bluescnpt. The larger P1 and BAC clones are randomly sheared and fragments cloned into a general purpose vector. The fragments in these vectors are sequenced by conventional methods (e.g. by "primer walking" or generation of deletions of inserts). The obtained sequences are assembled into a contiguous sequence.
  • the sequence of the insert of a complementing clone contains the NIM1 gene.
  • the approximate start and end of the NIM1 gene are deduced based on the DNA sequence, sequence motifs such as TATA boxes, the open reading frames present in the sequence, codon usage, the cosmid complementation data, the relative location of the AFLP markers and additional relevant data that is gathered (see Example 4, below). 2. cDNA sequencing.
  • the cosm ⁇ d(s) or larger clones that contain the NIM1 gene are used to isolate cDNAs. This is accomplished by using the clones (or DNA fragments) as probes in a screen of a cDNA library of wildtype Arabidopsis plants.
  • the cDNAs that are isolated are sequenced as described for cosmid sequencing and used in complementation tests.
  • full length cDNAs are cloned into a suitable plant expression vector, behind a constitutive promoter. These constructs are used in the transient assays as described above.
  • the cDNAs are cloned into a binary expression vector, allowing for expression in plant tissues and for Agrobacterium- mediated plant transformation, as described in Example 2 above.
  • a cDNA that contains the NIM1 gene is sequenced.
  • the genes from Ws-O and nimi plants are isolated and sequenced.
  • the genes are obtained from a cosmid of cDNA library, using a fragment of the isolated NIM1 gene as a probe Alternatively, the genes or cDNAs are isolated by PCR, using ⁇ ///W ⁇ -gene-spec ⁇ f ⁇ c primers and genomic DNA or cDNA as template. Likewise, the nimi alleles from other nimi lines (see Example 1.1 above) are isolated and sequenced in a similar manner.
  • Example 4 Description of the NIM1 gene and deduced protein sequence
  • the DNA sequence of the NIM1 gene or cDNA is determined as described in Example 3 above. This sequence is analyzed with the use of DNA analysis programs, such as can be found in the Genetics Computer Group (GCG) package, in the Sequencer or Staden packages, or any similar DNA analysis program package. Specifically, the start and end of the gene are determined, based on open reading frame analysis, the presence of stop and potential start codons, the presence of potential promoter motifs (such as the TATA box), the presence of polyadenylation signals and the like. Also, the predicted amino acid is deduced from the open reading frame. Both the DNA and protein sequence are used to search databases for sequences with homologies, such as transcription factors, enzymes or motifs of such genes or proteins.
  • GCG Genetics Computer Group
  • Example 5 Isolation of NIM1 homologs
  • the Arabidopsis NIM1 gene may be used as a probe in the low stringency hybridization screening of a genomic or cDNA library in order to isolate NIM1 homologs from other plant species. Alternatively, this is accomplished by PCR amplification, using primers designed based on the Arabidopsis NIM1 gene sequence and using genomic DNA or cDNA as template.
  • the NIM1 gene may be isolated from corn, wheat, rice, barley.rape seed, sugarbeet, potato, tomato, bean, cucumber, grape, tobacco and other crops of interest and sequenced. With a set of sequences from NIM 1 gene homologs in hand, new primers can be designed from conserved portions of the gene, in order to isolate NIM1 homologs from more distantly related plant species by PCR amplification.
  • Example 6 Complementation of the nim1-1 gene with genomic fragments. 1. Construction of a cosmid contig.
  • a cosmid contig of the NIM1 region was constructed using CsCI-purified DNA from BAC04, BAC06 and P1 -18.
  • the DNAs of the three clones were mixed in equimolar quantities and were partially digested with the restriction enzyme Sau3A.
  • the 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP.
  • Plasmid pCLD04541 was used as T-DNA cosmid vector. This plasmid contains a broad host range pRK290-based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection.
  • the vector was cleaved with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector . The ligation mix was packaged and transduced into E. coli strain XL1-blue MR (Stratagene). Resulting transformants were screened by hybridization with the BAC04, BAC06 and P1-18 clones and positive clones isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using the ECs EcoRI/Msel and Hindlll/Msel. The resulting AFLP finge ⁇ rint patterns were analyzed to determine the order of the cosmid clones.
  • a set of 15 semi-overlapping cosmids was selected spanning the nim region ( Figure 13).
  • the cosmid DNAs were also restricted with EcoRI, Pstl, BssHII and SgrAI. This allowed for the estimation of the cosmid insert sizes and the verification of the overlaps between the various cosmids as determined by AFLP fingerprinting.
  • Cosmids generated from clones spanning the NIM1 region were moved into Agrobacterium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013). These cosmids were then used to transform a kanamycin- sensitive nim1-1 Arabidopsis line using vacuum infiltration (Mindrinos et al., 1994, Cell 78, 1089-1099). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive.
  • Seedlings that survive the selection were transferred to soil approximately two weeks after plating and tested for the nimi phenotype as described below. Transformed plants that no longer have the nimi phenotype identify cosm ⁇ d(s) that contain a functional NIM1 gene.
  • Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300 ⁇ m INA was applied as a fine mist to completely cover the plants using a chromister. After two days, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate EmWa) and grown under high humidity conditions in a growing chamber with 19°C day/17° night temperatures and 8h l ⁇ ght/16h dark cycles. Eight to ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. Ws and nimi plants were treated in the same way to serve as controls for each experiment.
  • Table 16 shows complementation of the nimi phenotype by cosmid clones.
  • BAC04 DNA 25 ug, obtained from KeyGene was the source of DNA used for sequence analysis. This BAC was shown to be the clone completely encompassing the region that complemented the nim i mutants. DNA was randomly sheared using an approach from Cold Spring Harbor. Briefly, BAC DNA was sheared in a nebulizer to an average molecular weight of about 2 kb. Ends of the sheared fragments were repaired using a two-step protocol with dNTPS, T4 DNA polymerase and Klenow fragment (Boehringer).
  • pBRKanF4 is a derivative of pBRKanFI , which was obtained from Kolavi Bhat at Vanderbilt University (Bhat, K.S., Gene 134(1 ), 83-87 (1993)).
  • E. coli strain DH5a was transformed with the ligation mix, and the transformation mix was plated onto plates containing kanamycin and X-gal.
  • Plasmids were sequenced using Dye Terminator chemistry (Applied BioSystems PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, P/N 402078) and primers designed to sequence both strands of the plasmids. Data was collected on ABI 377 DNA sequencers. Approximately 75% of these reactions yielded useful sequence information. Sequences were edited and were assembled into contigs using Sequencher 3.0 (Gene Codes Corporation), Staden gap4 (Roger Staden, e-mail address rs@mrc-lmb.cam.ac.uk), and PHRED (Phil Green, e-mail address phg@u. washington.edu).
  • the largest contig (approximately 76 kb) covered the complementing region to an average depth of 7 independent calls/base.
  • a region of approximately 9.9 kb defined by the overlap of cosmids E1 and D7 was identified by complementation analysis to contain the nimi region.
  • Primers which flanked the insertion site of the vector and specific to the cosmid backbone were designed using Oligo 5.0 Primer Analysis Software (National Biosciences, Inc.).
  • DNA was isolated from cosmids D7 and E1 using a modification of the ammonium acetate method (Traynor, P.L., 1990. BioTechniques 9(6): 676.) This DNA was directly sequenced using Dye Terminator chemistry above. The sequence obtained allowed determination of the endpoints of the complementing region.
  • a truncated version of the BamHI-EcoRV fragment was also constructed, resulting in a construct which contains none of the "Gene 3" region (Fig. 13).
  • the following approach was necessary due the presence of Hindlll sites in the Bam-Spe region of the DNA.
  • the BamHI-EcoRV construct was completely digested with Spel, then was split into two separate reactions for double digestion. One aliquot was digested with BamHl, the other Hindlll.
  • a BamHI-Spel fragment of 2816 bp and a Hindlll-Spel fragment of 1588 bp were isolated from agarose gels (QiaQuick Gel extraction kit) and were ligated to BamHI-Hindlll- digested pSGCGOL DH5a was transformed with the ligation mix. Resulting colonies were screened for the correct insert by digestion with Hindlll following preparation of DNA using Wizard Magic MiniPreps (Promega). A clone containing the correct construct was electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.
  • Wild type denotes the wildtype Ws-O strain.
  • the 9.9 kb region containing the NIM1 region was analyzed for the presence of open reading frames in all six frames using Sequencher 3.0 and the GCG package.
  • Four regions containing large ORF's were identified as possible genes (Gene regions 1 -4). These four regions were PCR amplified from DNA of the wild-type parent and six different nimi allelic variants. Primers for these amplifications were selected using Oligo 5.0 (National Biosciences, Inc.) and were synthesized by Integrated DNA Technologies, Inc. PCR products were separated on 1.0% agarose gels and were purified using the QIAquick Gel Extraction Kit. The purified genomic PCR products were directly sequenced using the primers used for the initial amplification and with additional primers designed to sequence across any regions not covered by the initial primers. Average coverage for these gene regions was approximately 3.5 reads/base.
  • NIM1 gene was cloned and that it lies within Gene Region 2, since there are amino acid changes or alterations of sequence within the open reading frame of Gene Region 2 in all 6 nimi alleles. At the same time, at least one of the nimi alleles shows no changes in the open reading frames within Gene Regions 1 , 3 and 4 Therefore, the only gene within the 9.9 kb region that could be NIM1 is the Gene Region 2, the NIM1 gene.
  • the Ws section of Table 18 indicates the changes in the Ws ecotype of Arabidopsis relative to the Columbia ecotype of Arabidopsis.
  • Figures 13, 14, 15 and all others wherein sequence is shown relate to the Columbia ecotype of Arabidopsis, which contains the wild type gene in the experiments that were conducted.
  • the changes are listed as amino acid changes within the gene 2 or NIM1 region and are listed as changes in base pairs in the other regions.
  • Figure 13 shows 4 different panels that describe the cloning of the NIM1 gene and describe the entire 9.9 kb region.
  • Figure 14 is the sequence of the entire 9.9 kb region in the same orientation as described in Figure 13.
  • Figure 15 is the sequence of the specific NIM1 gene region which is gene region 2 indicated in Figure 13; the sequence of Figure 15 contains the NIM1 gene.
  • Figure 15 shows the ammo acid sequence in single letter code and shows the full length cDNA and RACE product that was obtained in capital letters in the DNA sequence. Some of the allele mutations that were found are shown above the DNA sequence and the particular mm1 allele that had that change is indicated.
  • coli strain DH5a Colonies containing the insert were selected, DNA was isolated, and confirmation was made by digestion with Hindlll. The Bam- EcoRV fragment was then engineered into a binary vector (pSGCGOI) for transformation into Arabidopsis.
  • RNA samples isolated from water-, SA-, BTH- and INA-treated Ws and nimi lines as previously described (Delaney et al, 1995, PNAS 92, 6602-6606). These blots were hybridized with PCR products generated from the four gene regions identified in the 9.9 kb NIM1 gene region. Only the gene region containing the NIM1 gene (Gene Region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcribed gene ( Figure 16 and Table 18).
  • Table 18 shows nimi allele sequence variation.
  • Fig. 13 contains the functional NIM1 gene by doing additional complementation experiments.
  • a BamHI/Hindlll genomic DNA fragment containing gene region 2 was isolated from cosmid D7 and was cloned into the binary vector pSGCGOI containing the gene for kanamycin resistance (Fig. 13; Steve Goff, personal communication).
  • the resulting plasmid was transformed into the Agrobacterium strain GV3101 and positive colonies were selected on kanamycin. PCR was used to verify that the selected colony contained the plasmid.
  • Kanamycin-sensitive nim1-1 plants were infiltrated with this bacteria as prevously described.
  • the resulting seed was harvested and planted on GM agar containing 50 ⁇ g/ml kanamycin.
  • Plants surviving selection were transferred to soil and tested for complementation. Transformed plants and control Ws and nimi plants were sprayed with 300 ⁇ m INA. Two days later, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate EmWa) and grown as previously described. Ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. All of the
  • the resulting RACE products were sequenced and found to include the additional bases indicated in Figure 15.
  • the transcribed region present in both cDNA clones and detected in RACE is shown as capital letters in Figure 15. Changes in the alleles are shown above the DNA strand. Capitals indicate the presence of the sequence in a cDNA clone or detected after RACE PCR.
  • Example 9 Characterization of the NIM1 gene The multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5:151 -153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994).
  • NIM1 protein is homologous in amino acid sequence to 4 different rice cDNA protein products.
  • the homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 19.
  • the NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.
  • Mutant plants were treated with chemical inducers and then assayed for PR gene expression and disease resistance.
  • Wild-type seeds and seeds for each of the nimi alleles were sown onto MetroMix 300 growing media, covered with a transparent plastic dome and placed at 4° C in the dark for 3 days. After 3 days of 4° C treatment the plants were moved to a phytotron for 2 weeks. At approximately 2 weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H 2 0, 5mM SA, 300 uM BTH or 300 uM INA. Chemicals were applied as a fine mist to completely cover the seedlings using a chromister. Water control plants were returned to the growing phytotron while the chemically treated plants were held in a separate but identical phytotron. After 3 days plants were divided into 2 groups. One group was harvested for RNA extraction and analysis. The second group was inoculated with P. parasitica. 3. Peronospora parasitica inoculation and analysis.
  • P. parasitica isolate 'EmWa' is a P.p. isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of causing disease on a particular host.
  • the P. parasitica isolate 'NoCo' is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogens are recognized by the potential host, eliciting a host response that prevents disease development.
  • water and chemically treated plants were inoculated with the compatible 'EmWa' isolate.
  • 'NoCo' inoculation was conducted on water treated plants only. Following inoculation plants were covered with a clear plastic dome to maintain high humidity required for successful P. parasitica infection and placed in a growing chamber with 19° C day/17° C night temperatures and 8h l ⁇ ght/16h dark cycles.
  • Figure 18 shows the disease assessment of the various nimi alleles following P. parasitica inoculation. The most distinguishing timepoints are 5 and 6 days post- inoculation. At 5 days post-inoculation n ⁇ m1-4 shows -80% infection under all inducing chemical treatments performed, clearly indicating that this allele/genotype has the most severe disease susceptibility. At 6 days post-inoculation, nimi, -2, -3, -4 and -6 show significant disease incidence under all inducing chemical treatments. However, n ⁇ m1-5 shows less infection than Ws wild-type under all treatments at day 6. Therefore, n ⁇ m1-5 is the most disease resistant of the various nimi alleles.
  • PR-1 gene expression indicates that nim1-4 is the least responsive to all of the inducing chemicals tested ( Figure 17), while nim1-5 shows elevated levels of PR-1 expression in the absence of inducers. These PR-1 gene expression results are consistent with the disease assessment performed with P. parasitica ( Figure 18) and indicate that nimi alleles can cause resistance or susceptibility.
  • NIM1 gene expression Figure 17
  • NIM1 mRNA was present in the untreated control samples. Following treatment with SA, INA, BTH or infection with a compatible pathogen the NIM1 mRNA accumulated to higher levels. Differences in NIM1 message (mRNA) abundance were observed in the nimi alleles compared to wildtype. The abundance of NIM1 mRNA in untreated mutant plants was lower than observed in the wildtype with the exception of niml- 2 and -5 where the amounts were similar. The nim1-1, -3 and -4 had low levels of NIM1 message while the nim1-6 had very low accumulation of NIM1 mRNA.
  • NIM1 mRNA Increases in NIM1 mRNA following SA, INA or BTH were observed in nim1-1, -2, -3 but not nim1-5 or -6. However, this increase was less than observed in wildtype plants. Following pathogen infection additional bands hybridizing to the NIM1 cDNA probe were observed in both wildtype and mutants and the NIM1 mRNA level was elevated relative to untreated controls, except in nim 1-6.
  • Figure 18 shows the disease resistance assessment via infection rating of the various nimi alleles as well as the NahG plants at various times after innoculation with Peronospora parasitica.
  • WsWT indicates the Ws wild type parent line in which the nimi alleles are found.
  • the various nimi alleles are indicated in the table and the NahG plant is indicated also.
  • the NahG plant has been previously published. (Delaney et al. Science 266, pp. 1247-1250 (1994)).
  • the NahG Arabidopsis is also described in WO 95/19443.
  • the NahG gene is a gene from Pseudomonas putida that converts salicylic acid to catechol, thereby eliminating the accumulation of salicylic acid, a necessary signal transduction component for SAR in plants.
  • NahG Arabidopsis plants do not display normal SAR. In addition, they show much greater susceptibility in general to pathogens. Therefore, the NahG plants serve as a kind of universal susceptibility control.
  • the NahG plants still respond to the chemical inducers INA and BTH; this is shown in the bottom two panels of the Figure 17.
  • the NahG plants show very good response to both INA and BTH and look very similar to the mm 1-5 allele.
  • the disease resistance induced in the NahG plants begins to fade, and there is a profound difference between INA and BTH in that the INA-induced resistance fades much faster and more severely than the resistance induced in the NahG plants by BTH.
  • INA and BTH induced very good resistance in Ws to EmWa, and the n ⁇ m1-1, n ⁇ m1-2 and other nimi alleles show virtually no response to SA or INA in regard to disease resistance.
  • Figure 18 lists the percent of plants that are showing sporulation after infection with the EmWa race of P. parasitica, and each of the bar graphs indicates the number of days after infection that the disease resistance was rated.
  • FIG. 17 shows that the wild type plant shows very good response to salicylate, INA, BTH and also to pathogen infection, as manifested by enhanced PR1 gene expression.
  • the nim1-1 allele shows only very limited response to all the chemical inducers including pathogen.
  • the pathogen induction is at least several fold lower in the nim1-1 allele than it is in the wild type.
  • the nim1-2, nim1-3 and nim1-6 alleles show response similar to the nim1-1 allele to the various treatments.
  • the nim1-4 allele shows virtually no expression in response to any of the inducers used. Basically, background level is all that is observed.
  • the nim 1-5 allele shows a very high background level relative to controls with water and that background level is maintained in all the treatments; however, there is limited or no induction by the chemical inducers.
  • the NahG plants serve as a good control, showing that they are unable to induce PR-1 in the presence of SA; on the other hand, INA and BTH both induce very strong high level expression of PR-1.
  • the effect of pathogen infection is similar to that of SA; there is no expression of PR-1 in the EmWa-treated NahG plants.
  • RNA samples produced in the induction studies were also probed with a NIM1 gene using a full-length cDNA clone as probed.
  • INA induces the NIM1 gene in the wild type Ws allele.
  • the nim1-1 mutation allele shows a lower basal level expression of the NIM1 gene, and it is not inducible by INA. This is similar to what is observed in the nim 1-3 allele and the nim1-6 allele.
  • the /7/m7-_? allele shows approximately normal levels in the untreated sample and shows similar induction to that of the wild type sample, as does the nim1-4 allele.
  • the nim1-5 allele seems to show higher basal level expression of the NIM1 gene and much stronger expression when induced by chemical inducers.
  • the induction of NIM1 by chemical inducers of resistance and other inducers is consistent with its role in pathogen defense and is also further evidence that we have obtained the right gene in the 9.9 kb region.
  • MOLECULE TYPE DNA (genomic)
  • AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360
  • CTGCATTTCA CTCATCTAAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480
  • TTTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA 900
  • AAAATTAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT 7380
  • AAAAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520
  • ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820
  • GAAGTATCCA GAGATATTGT TGGTTCGATG GAGATTTAGG TTGACAAACC
  • AAGACTCGTA 8880 GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAATTTT CCGATTTCGG ACCCCCGAAG 8940
  • TGT GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT 3546 Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He 215 220 225
  • GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866
  • AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226

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Abstract

The invention concerns the location and characterization of a gene (designated NIM1) which is a key component of the SAR pathway and which in connection with chemical and biological inducers enables induction of SAR gene expression and broad spectrum disease resistance to plants. The invention further concerns plants transformed with the NIM1 gene as well as methods employing the gene to create the transgenic plants and employing the gene in a screening assay for compounds capable of inducing broad spectrum disease resistance in plants.

Description

GENE CONFERRING DISEASE RESISTANCE IN PLANTS AND USES THEREOF
The present invention relates to disease resistance in plants and to identifying and breeding disease resistance into plants. More particularly, the present invention relates to the identification, isolation and characterization of a gene involved in broaα spectrum disease resistance in plants.
Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe.
However, most plants have their own innate mechanisms of defense against pathogenic organisms Natural variation for resistance to plant pathogens nas been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens.
In many plant species an initial inoculation by a necrotizing pathogen can immunize the plant to subsequent infection. This acquired disease resistance was first documented in 1901 and is thought to play an important role in the preservation of plants in nature. Particularly well characterized examples of plant immunity are the phenomenon oτ systemic acquired resistance (SAR) and induced resistance in plants such as tobacco, Arabiaopsis and cucumber. In these systems, inoculation by a necrotizing pathogen results in systemic protection against subsequent infections by that pathogen as well as a number of other agronomically important bacterial, fungal and viral pathogens Systemic acquired resistance can also be triggered by chemical immunization compounds, certain chemicals that induce the immunity response in plants. Such compounds can be of natural origin, such as salicylic acid (SA), or can be synthetic chemicals, such as 2,6-dichloroisonicotinic acid (INA) and benzo(1 ,2,3)thiadiazole-7- carbothioic acid S-methyl ester (BTH). Treatment with a pathogen or an immunization compound induces the expression of at least nine sets of genes in tobacco, the best characterized species. Different numbers and types of genes can be expressed in other plants. The level of induction for SAR-related genes induced by immunization compounds is as high as 10,000-fold over background. In particular, SAR is characterized by the expression of SAR genes, including the pathogenesis-related (PR) genes.
The SAR genes are induced following infection by a pathogen. Some of these genes have a role in providing systemic acquired resistance to the plant. These plant proteins are induced in large amounts in response to infection by various pathogens, including viruses, bacteria and fungi. PR proteins were first discovered in tobacco plants (Nicotiana tabacum) reacting hypersensitively to infection with tobacco mosaic virus (TMV). Subsequently, PR proteins have been found in many plant species (see Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254: Van Loon (1985) Plant Mol. Biol. 4: 111-116; and Uknes et al. (1992) Plant Cell 4: 645-656.) Such proteins are believed to be a common defensive systemic response of plants to infection by pathogens.
Pathogenesis-related proteins include but are not limited to SAR8.2a and SAR8.2b proteins, the acidic and basic forms of tobacco PR-la, PR-lb, and PR-lc; PR-1', PR-2, PR-2', PR-2", PR-N, PR-O, PR-O\ PR-4, PR-P, PR-Q, PR-S, and PR-R major proteins; cucumber peroxidases; basic cucumber peroxidase; the chitinase which is a basic counterpart of PR-P or PR-Q; the beta- 1 ,3-glucanase (glucan endo- 1 ,3-beta-glucosidase, EC 3.2.1.39) which is a basic counterpart of PR-2, PR-N or PR-O; and the pathogen-inducible chitinase from cucumber. Such PR proteins are disclosed, for example, in Uknes et al. (1992) The Plant Cell 4- 645-656 and the references cited therein.
SAR or SAR-hke genes are expressed in all plant species exhibiting systemic acquired resistance. Expression of such genes can be determined by probing with known SAR DNA sequences. For example, see Lawton et al (1992) Proceedings of the Second European Federation of Plant Pathology (1983), In: Mechanisms of Defence Responses in Plants, B. Fπtig and M. Legrand (eds), Kluwer Academic Publishers, Dordrecht, pp. 410- 420; Uknes et al. (1992) The Plant Cell 4: 645-656; and Ward et al. (1991) The Plant Cell 3: 1085-1094 Methods for hybridization and cloning are well known in the art. See, for example, Molecular Cloning. A Laboratory Manual. 2nd Edition, Vol. 1 -3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press (1989) and the references cited therein.
Alternatively such SAR or SAR-hke genes can be found by other methods such as protein screening, +/- screening, etc. See, for example, Liang and Pardee (1992) Science 257: 967-971 ; and St. John and Davis (1979) CeJ 16: 443
Despite much research and the use of sophisticated and intensive crop-protection measures, including genetic transformation of plants, losses due to disease remain in the billions of dollars annually. Disease resistance genes have previously been cloned but transgenic plants transformed with these genes would typically be resistant only to a subset of strains of a particular pathogen species. Despite efforts to clone genes involved in SAR, a gene controlling broad spectrum disease resistance has not been isolated and characterized.
Several lines of evidence indicate that endogenously produced SA is involved in the signal transduction pathway coupling the perception of pathogen infection with the onset of SAR Mutants which retain the ability to accumulate SA in response to pathogen yet have lost the ability to induce SAR genes or resistance after application of SA or INA have been described by Delaney, et al., Proc. Natl. Acad. Sci. 92: 6602-6606 (1995) and in WO94/16077 the whole of which is incorporated herein by reference.
It has now been discovered that these mutants contain a mutant gene, which gene in its wildtype form controls SAR gene expression and SAR itself. The present invention recognizes that the mutant gene confers broad spectrum disease susceptibility to mutant plants and renders them noninducible to pathogens and chemical inducers.
The present invention concerns the identification, isolation and characterization of the wildtype (NIM 1) gene, a gene which allows activation in a plant of SAR and SAR gene expression in response to biological and chemical inducers.
A mutant gene has been identified in Mutagenized Arabidopsis plants. These plants have been found to be defective in their normal response to pathogen infection in that they do not express genes associated with systemic acquired resistance (SAR) nor are they capable of exhibiting SAR. These mutants contain a defective gene which has been labelled niml (for noninducible immunity).
The present invention also concerns the use of the cloned NIM1 gene and variants thereof to create transgenic plants that have broad spectrum disease resistance and to the transgenic plants produced thereby. The invention further concerns the use of the cloned NIMl gene and variants thereof in a screening method for identifying compounds capable of inducing broad spectrum disease resistance in plants. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the effect of chemical inducers on the induction of PR gene expression in wild-type and nimi plants.
Figure 2 depicts PR-1 gene expression in pathogen-infected Ws-O and nimi plants over the course of 6 days from the initiation of infection.
Figure 3 shows the levels of SA accumulation in Ws-O and nimi plants infected with P. syringae.
Figure 4 shows the genetic map of the NIM1 region as determined by AFLP and SSLP analysis.
Figure 5 depicts a physical map of the NIM1 region as determined by YAC clone analysis.
Figure 6 shows a physical map of an extended P1/BAC contig.
Figure 7 shows a physical map setting forth the positions of P1 and BAC clones with respect to the flanking AFLP markers and YACs.
Figure 8 shows a physical map of a further extended P1/BAC contig containing the NIM1 gene.
Figure 9 shows an integrated genetic and physical fine map of the NIM region.
Figure 10 shows an integrated map of the NIM1 region.
Figure 11 shows an integrated map of the NIM1 region including the new AFLP markers.
Figure 12 is a schematic representation of recombinants D169 and C105.
Figure 13 is a global map of the chromosomal region centered on NIM1 with recombinants indicated, including, BACs, YACs and Cosmids in NIM1 region.
Figure 14 provides the sequence of the 9.9kb region of clone BAC-04 containing the NIM1 gene. Figure 15 shows the nucleic acid sequence of the NIM1 gene and the amino acid sequence of the NIM1 gene product, including changes in the various alleles.
Figure 16 shows the expression of NIM1 induced by INA, BTH, SA and pathogen in wild type and mutant alleles of nimi.
Figure 17 shows the expression of PR-1 in nimi mutants and wild-type plants.
Figure 18 shows disease resistance in various nimi mutants.
Figure 19 is an amino acid sequence comparison of Expressed Sequence Tag regions of the NIM1 protein and cDNA protein products of 4 rice gene sequences (see SEQ ID NO: 3).
DEFINITIONS
AA: Amino Acid
AFLP: Amplified Fragment Length Polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syriπgae
BAC: Bacterial Artificial Chromosome
BTH: benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester
Col: Arabidopsis ecotype Columbia
ECs: Enzyme combinations
INA: 2,6-dichloroisonicotinic acid
Ler: Arabidopsis ecotype Landsberg erecta
NIM1: the wildtype gene, conferring disease resistance to the plant nim: mutant allele of NIM 1, conferring disease susceptibility to the plant nimi: mutant plant line
ORF: open reading frame
PCs: Primer combinations SA: salicylic acid
SAR: Systemic Acquired Resistance
SSLP: Simple Sequence Length Polymorphism
Ws-O: Arabidopsis ecotype Wassilewskija
YAC: Yeast Artificial Chromosome
The NIM1 gene has been cloned by mapping and walking techniques which indicate that the gene is contained in a -105 Kb region. (See Figure 13 and Table 16). This region is delineated by the L84.6b marker on the left and the L84.T2 marker on the right. Only three overlapping cosmids made from wild-type DNA from the 105 Kb region complement the nimi mutant phenotype (Figure 13 and Table 16). These three cosmids only overlap in a 9.9 Kb region defined by the left end of cosmid clone D7 and the right end of cosmid D5 as pictured in Figure 13. Many other cosmids made to other areas of the 105 Kb region do not complement the nimi phenotype (Figure 13 and Table 16). A near full length cDNA clone to the NIM1 gene indicates the appropriate intron-exon borders and defines the amino acid sequence of the gene product. Only the NIM1 gene region within the 9.9 Kb complementing region has sequence changes in various nimi mutant alleles (Table 18). Three other potential gene regions showed no sequence changes that are assosciated with the nimi phenotype. The sequence changes found in the NIM1 gene region are consistent with altered function or loss of function of the gene product. The severity of the change to the NIM1 gene region in a particular mutant allele is roughly correlated with the observed physiological severity of that nimi allele. Only the NIM1 gene region had detectable RNA (transcription) and this RNA showed abundant changes consistent with the physiological role of NIM1 in pathogenesis (Table 18 and Figure 16). The present invention relates to an isolated gene fragment, the NIM 1 gene, which is a key component of the systemic acquired resistance (SAR) pathway in plants. The NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression.
The location of the NIM gene is determined by molecular biological analysis of the genome of mutant plants known to carry the mutant nimi gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR.
Nimi mutants are useful as "universal disease susceptible" (UDS) plants by virtue of their being susceptible to many strains and pathotypes of pathogens of the host plant and also to pathogens which do not normally infect the host plant, but which infect other hosts. They can be generated by treating seeds or other biological material with mutagenic agents and then selecting progeny plants for the UDS phenotype by treating progeny plants with known chemical inducers (e.g. INA) of the systemic acquired response and then infecting the plants with a known pathogen. Noninducible mutants develop severe disease symptoms under these circumstances, whereas non-mutants are induced by the chemical compound to systemic acquired resistance, nim mutants can be equally selected from mutant populations generated by chemical and irradiation mutagenesis, as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis.
Techniques for generating mutant plant lines are well known in the art. The nim plant phenotype is used as a tool to identify an isolated gene fragment which allows expression of broad spectrum disease resistance in plants.
Comprised by the present invention is an isolated DNA molecule comprising a mutant gene of the NIM1 gene which is a nimi gene. Following the use of a nimi mutant or plant to isolate the wild-type NIM1 gene necessary for constitutive expression of SAR genes, the resistance trait, in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding. John Wiley & Sons, NY (1981 ); Crop Breeding. Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of Plant Breeding. Second Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests. Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding. Walter de Gruyter and Co., Berlin 1986).
A further object of the invention is a chimeric gene comprising a promotor active in plant operably linked to a heterologuous DNA molecule encoding the aminoacid sequence of a NIM 1 gene product and variants thereof according to the invention.
Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants is generally described in the art. Generally, for the introduction of foreign DNA into plants, Ti plasmid vectors have been utilized for the delivery of foreign DNA. Also utilized for such delivery have been direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. Such methods had been published in the art. See, for example, Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) MoL Gen. Genet. 228: 104-112; Guerche et al., (1987) Plant Science 52: 11 1-116; Neuhause et al., (1987) Theor. Appl. Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265: Horsch et al., (1985) Science 227: 1229-1231 ; DeBlock et al., (1989) Plant Physiology 91 : 694-701 ; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). See also US. patent application Serial Nos. 08/438,666 filed May 10, 1995, and WO 93/07278, both of which are incorporated herein by reference in their entirety. It is understood that the method of transformation will depend upon the plant cell to be transformed.
It is further recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Plant cells transformed with such modified expression systems, then, would exhibit overexpression or constitutive expression of SAR genes necessary for activation of SAR.
The DNA molecule or gene fragment conferring disease resistance to plants by allowing induction of SAR gene expression can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems IgtH , IgtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems. The DNA sequences can be cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory, Cold Spring Harbor, New York (1982).
A further object of the invention a recombinant vector comprising the chimeric gene according to the invention. In order to obtain efficient expression of the gene or gene fragment of the present invention, a promoter must be present in the expression vector. RNA polymerase normally binds to the promoter and initiates transcription of a gene. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used. Suitable promoters include ubiquitin, nos promoter, the small subunit ribulose bisphosphate carboxylase gene promoter, the small subunit chlorophyll A/B binding polypeptide promoter, the 35S promoter of cauliflower mosaic virus, and promoters isolated from plant genes. See C.E. Vallejos, et al. , "Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the RRNA (45S), the major chlorophyll A/βBinding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes," Genetics 112: 93-105 (1986), which discloses the small subunit materials. The nos promoter and the 35S promoter of cauliflower mosaic virus are well known in the art.
Once the disease resistance gene of the present invention has been cloned into an expression system, it is ready to be transformed into a plant cell. Plant tissues suitable for transformation include leaf tissues, root tissues, meristems, and protoplasts.
Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
The isolated gene fragment of the present invention can be utilized to confer disease resistance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots. Although the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
The expression system of the present invention can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the gene imparts disease resistance to the intact transgenic plants. As set forth above, the expression system can be modified so that the disease resistance gene is continuously or constitutively expressed.
Transformation
The present system can be utilized in any plant which can be transformed and regenerated. Such methods for transformation and regeneration are well known in the art. As well as the above cited references, see also, An, G., Watson, B.D., and Chiang, C.C. Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81 :301 -305, 1986; Fry, J., Barnason, A., and Horsch, R.B
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(Solanum tuberosum) using Agrobacterium tumefaciens. Theor.appl. genet. 76:767-774,
1988; Deblock, M., Brouwer, D.D., and Tenning, P Transformation of Brassica napus and
Brassica oleracea using Agrobacterium tumefaciens and the Expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91 :694-701 , 1989; Banbault, T.J., Skene,
K.G.M., Cain, P. A., and Scott, N.S Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase. Pl.Cell Rep. 41 :1045-1049, 1990; Hinchee, M.A W., Newell,
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As nimi host plants may also be susceptible to pathogens outside of the host-range into which they normally fall, these plants also have significant utility in the molecular, genetic, and biological study of host-pathogen interactions. Furthermore, the UDS phenotype of nimi plants also renders them of utility for fungicide screening, nimi mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host. The advantage lies in the UDS phenoytpe of the mutant, which circumvents the problems encountered due to hosts being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes.
Pathogens of the invention include but are not limited to viruses or viroids, e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses; fungi, e.g. Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabacr, insects such as aphids, e.g. Myzus persicae; and lepidoptera, e.g., Heliothus spp.; and nematodes, e.g., Meloidogyne incognita. The methods of the invention are useful against a number of disease organisms of maize including but not limited to downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis; rusts such as Puccinia sorphi, Puccinia polysora and Physopella zeae; other fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae and Bipolaris maydis; and bacteria such as Erwinia stewartii.
DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO:1 - 9919-bp genomic sequence from Figure 14.
SEQ ID NO:2 - 5655-bp genomic sequence from Figure 15.
SEQ ID NO:3 - AA sequence of wild-type NIM protein encoded by eds of seq id no:2.
SEQ ID NO:4 - Rice-1 AA sequence 33-155 from Figure 19.
SEQ ID NO:5 - Rice-1 AA sequence 215-328 from Figure 19.
SEQ ID N0:6 - Rice-2 AA sequence 33-155 from Figure 19.
SEQ ID N0:7 - Rice-2 AA sequence 208-288 from Figure 19. SEQ ID NO:8 - Rice-3 AA sequence 33-155 from Figure 19. SEQ ID NO:9 - Rice-3 AA sequence 208-288 from Figure 19. SEQ ID NO: 10 - Rice-4 AA sequence 33-155 from Figure 19. SEQ ID NO:11 - Rice-4 AA sequence 215-271 from Figure 19.
DEPOSITS
The following vector molecules have been deposited with American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852 U.S.A. on the dates indicated below: Plasmid BAC-04 was deposited with ATCC on May 8, 1996 as ATCC 97543.
Plasmid P1-18 was deposited with ATCC on June 13, 1996 as ATCC 97606.
Cosmid D7 was deposited with ATCC on September 25, 1996 as ATC 97736.
EXAMPLES
Example 1
Identifying NIM1 clones by map-based cloning. High resolution genetic mapping and physical mapping of NIM1 in Arabidopsis.
1. Plant Material and Isolation of nimi Mutants.
Nimi mutants were isolated from two Arabidopsis ecotype Ws-O plant populations, as described by Delaney et al., (1995) PNAS 92, 6602-6606. One mutant population was in the form of an M2 library derived from ethyl methane sulfonate (EMS) mutagenized seeds (purchased from Lehle, Round Rock, TX) and the other was in the form of a T-DNA population derived from seed obtained from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH). The basis of the screen for noninducible immunity (nimi) mutants was to screen mutagenized plant populations for plants in which resistance to a virulent pathogen could not be induced by INA (2,6 dichloro isonicotinic acid; Metraux, et al., 1991. In: Advances in Molecular Genetics of Plant-Microbe Interactions. Vol 1, 432-439. Hennecke and Verma, eds.; Kessmann et al. 1993 In: Mode of action of agrochemicals. Y Honma, ed.; Vernooij et al, 1995, Molec. PI. Microbe Interaction 8, 228-234).
Plants from the mutant populations were grown at high density in large trays in commercial planting mix. When the plants were 2 weeks of age, the trays were sprayed with 0.25 mg/ml INA. Four days later, the plants were sprayed with a spore suspension of Peronospora parasitica, isolate EmWa (EmWa), at 5x104 to 1x105 spores/ml. This fungus is normally virulent on the Arabidopsis Ws-O ecotype, unless resistance is first induced in these plants with INA or a similar compound.
Following incubation in a high humidity environment, plants with visible disease symptoms were identified, typically 7 days after the infection. These plants did not show resistance to the fungus, despite the application of the resistance-inducing chemical and were thus potential nim (non-immunity) mutant plants. From 360,000 plants, 75 potential nim mutants were identified.
These potential mutant plants were isolated from the flat, placed under low humidity conditions and allowed to set seed. Plants derived from this seed were screened in an identical manner for susceptibility to the fungus EmWa, again after pretreatment with INA. The progeny plants that showed infection symptoms were defined as nim mutants. Six nim lines were thus identified. One line (nimi) was isolated from the T-DNA population and five from the EMS population. 2. Scoring Plant Reactions to INA and Other Chemical Inducers of Disease Resistance. i. Phenotypic analysis of nimi.
Salicylic acid (SA) and benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) are two chemicals that, like (NA, induce broad spectrum disease resistance, termed Systemic Acquired Resistance (SAR), in wildtype plants. Since INA did not induce resistance in the nimi plants, these plants were also evaluated for their disease resistance response following pretreatment with SA and BTH (as partly described in Delaney et al, 1995, PNAS 92, 6602-6606).
Plants were sprayed with 1 , 5, or 15 mM SA or 0.25 mg/ml BTH and challenge inoculated with EmWa 5 days later (as described in example 1 above). Both SA and BTH failed to protect nimi plants from fungal infection, as evidenced by the presence of disease symptoms and fungal growth on these plants. Thus, the nimi plants were not responsive to any of the SAR-inducing chemicals, implying that the mutation was downstream of the entry point(s) for these chemicals in the resistance induction pathway.
Nimi was also evaluated for its disease response to infection with 2 incompatible P. parasitica isolates, Wela and Noco (i.e. these fungal strains do not cause disease on wildtype Ws-O plants), nimi plants were sprayed with conidial suspensions of 5-10x104 spores/ml of Wela or Noco and incubated under high humidity for 7 days. Unlike wildtype plants, nimi plants developed disease symptoms in response to both Wela and Noco infection. The symptoms were necrotic flecking and trailing, with some sporulation. Following lactophenol blue staining, fungal hyphae were easily observed in the leaves of nimi plants. Thus, the nimi plants are susceptible to normally incompatible P. parasitica isolates. This result shows that the nimi plants are not only defective in chemically induced disease resistance, but are also defective in natural resistance to microorganisms that are normally not pathogenic.
ii. Biochemical analysis of nimi.
SA, INA and BTH induce SAR and expression of the SAR genes, which include the Pathogenesis Related genes PR-1 , PR-2 and PR-5 in Arabidopsis. Since these compounds did not induce disease resistance in nimi (as described in example 1.2 above), this mutant line was analyzed for SAR gene expression following SA, INA or BTH treatment.
After treatment of nimi plants with SA, INA or BTH, plant tissue was harvested and analyzed for accumulation of RNA from the PR-1 , PR-2 and PR-5 genes. To this end, total RNA was isolated from the treated tissues and electrophoresed on an agarose gel. Triplicate gel blots were prepared and each was hybridized with a probe for one of these 3 SAR genes as described in Delaney et al, 1995, PNAS 92, 6602-6606. In contrast to the case in wildtype plants, the chemicals did not induce RNA accumulation from any of these 3 SAR genes in nimi plants, as shown in figure 1. Taken together, the results indicate that the chemicals induce neither SAR nor SAR gene expression in nimi plants.
Since the chemicals did not induce SAR, or SAR gene expression in nimi plants, it was of interest to investigate whether pathogen infection could induce SAR gene expression in these plants, as it does in wildtype plants. Ws-O and nimi plants were sprayed with EmWa spores as described and tissue collected for RNA analysis at several timepoints. Pathogen infection (EmWa) of wildtype Ws-O plants induced PR-1 gene expression within 4 days after infection, as shown in figure 2. In nimi plants, however, PR- 1 gene expression is not induced until 6 days after infection and the level is reduced relative to the wildtype at that time. Thus, following pathogen infection, PR-1 gene expression in nimi plants is delayed and reduced relative to the wildtype. Infection of wildtype plants with pathogens that cause a necrotic reaction leads to accumulation of SA in the infected tissues. It has been shown that this endogenous SA is required for signal transduction in the SAR pathway, i.e., breakdown of the endogenous SA leads to a decrease in disease resistance. This defines SA accumulation as a marker in the SAR pathway (Gaffney et al, 1993, Science 261., 754-756).
Nimi plants were tested for their ability to accumulate SA following pathogen infection. Pseudomonas syringae tomato strain DC 3000, carrying the avrRpt2 gene, was injected into leaves of 4-week-old nimi plants. The leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 92, 6602-6606. This analysis showed that the nimi plants accumulated high levels of SA in infected leaves, as shown in Figure 3. Uninfected leaves also accumulated SA, but not to the same levels as the infected leaves, similar to what has been observed in wild-type Arabidopsis. This indicated that the nim mutation maps downstream of the SA marker in the signal transduction pathway. This was anticipated, as INA and BTH (inactive in nimi plants) are known to stimulate a component in the SAR pathway downstream of SA (Vernooij et al., 1995, Molec. PL Microbe Interaction 8, 228-234; Friedrich, et al., 1996, The Plant Journal 9, in press; and Lawton, et al., 1996, The Plant Journal 9, in press). In addition, as described in Example 1.2, exogenously applied SA did not protect nimi from EmWa infection.
3. Genetic analysis of nimi.
Nimi plants were backcrossed to wild-type Ws-O plants, and F1 progeny were tested for resistance to EmWa after INA pretreatment, as described in Example 1.1 above. None of the INA-pretreated F1 plants had infection symptoms, whereas the nimi control plants did show infection. Hence, the nimi mutation was determined to be recessive. The F2 population from the Ws-O x nimi cross was also assayed for its disease resistance after INA pretreatment. Of this population, approximately 1/4 (32/130 plants) showed disease symptoms after EmWa treatment of INA pre-sprayed plants and 3/4 (98/130 plants) showed no disease. These results indicate that the nim mutation identifies a single genetic locus and corroborates the F1 data that show the recessive nature of the mutation.
4. Identification of markers in and genetic mapping of the NIM locus.
For conventional map-based cloning of the NIM gene, markers had to be identified that were genetically closely linked to the mutation. This was accomplished in 2 steps. First, the nimi plants were crossed to a different Arabidopsis genotype, Landsberg erecta (Ler), and F2 plants from this cross which had a nimi phenotype (i.e. plants that are homozygous nim/nim at the NIM locus) were identified. From these, plants that had a Ler genotype at a nearby DNA marker were identified by molecular analysis. These plants, by virtue of the identification criterion, are recombinant between the marker and the NIM locus. The frequency of recombinants defines the genetic distance between the marker and the NIM locus.
The second prerequisite for map based cloning is that markers are identified that are genetically very close to the NIM locus, i.e. markers that identify very few recombinants. If genetic markers are identified that are very close, than these can be used to isolate genomic DNA clones that are close to the NIM locus. The NIM locus can then be cloned by walking, if not already present on the cloned DNA. Walking can be initiated from both sides of the gene. It relies on obtaining overlapping clones that are successively closer to the gene of interest. When a single DNA marker is obtained from a walk initiated from, for instance, the North end and it identifies no recombinants between this marker and the gene of interest, it must be very close to the gene. However, if the marker does identify recombinant(s) from the South end, the clone from which the marker was obtained must have crossed the gene. By definition then, the gene of interest is cloned. It must be located between this marker and the last North-end marker that identifies the least number of recombinants from the North end.
In a first step, a large number of recombinants are generated by genetic crossing. In a second step, recombinants that are close to the NIM gene are identified with the use of molecular markers. Many markers have been described in the literature and several methods exist to develop additional markers. Our approach has relied on a number of marker systems, including SSLPs and AFLPs (see below).
i. Genetic crosses.
In order to map the chromosomal position of the NIM gene relative to the SSLP and AFLP markers, nimi was crossed to Ler to make a mapping population. F2 plants from this cross were grown and leaves harvested for future DNA extractions. Next, the F2 plants were scored for the nimi phenotype, as described in example 1 .1 above. Also, F3 populations derived from individual F2 plants were grown and scored for the nim phenotype. DNA was extracted from the stored tissue of nimi phenotype F2 and F3 plants by the CTab method, as described (Rogers and Bendich, 1988, Plant Molecular Biology Manual, A6, 1-10). This DNA was used for mapping the NIM gene, as described below.
ii. Simple Sequence Length Polymorphism markers.
The Simple Sequence Length Polymorphism (SSLP) markers ATHGENEA and nga111 have been described (Bell and Ecker, 1994, Genomics 19, 137-144). Primers used for detection of these SSLPs are listed in Table 1. Table 1. SSLP primer sequences.
primer set primer sequence (5' to 3')
ATHGENEA (1) ACC ATG CAT AGC TTA AAC
TTC TTG
ACA TAA CCA CAA ATA GGG
GTG C
ATHGENEA (2) ACC ATG CAT AGC TTA AAC
TTC TTG
CCA AAT GTC AAA ATA CTC
GTC nga111 (1 ) CTC CAG TTG GAA GCT AAA
GGG
TGT TTT TTA GGA CAA ATG
GCG nga11 1 (2) CTC CAG TTG GAA GCT AAA G
TGT TTT TTA GGA CAA ATG G
Genetic mapping of the NIM gene relative to marker ATHGENEA.
Using the ATHGENEA (1) primers for PCR amplification of Ler genomic DNA, a 205- basepair (bp) band was expected, whereas with Ws-O genomic DNA a band of 211 bp was expected (Bell and Ecker, 1994, Genomics 19, 137-144). The amplification products proved to be difficult to separate on conventional agarose gels. Hence, two alternative methods were developed for separation and detection of these PCR fragments.
In a first method, primerset ATHGENEA (1) (Table 1 ) was used to amplify genomic DNA in the presence of 6-carboxyrhodamine-labelled UTP (dUTP-RH O, obtained from ABI), yielding rhodamine-labelled PCR fragments. The PCR reactions were analyzed on a DNA Sequencer, which detects DNA fragments with single nucleotide resolution. The specific reagents were: 1xPCR buffer, 2 mM MgCI2, dNTPs each 200 mM, 2 mM dUTP-R1 10, ATHGENEA (1 ) primers at 0.75 mM, 10 ng DNA and 0.75 units Taq polymerase in a 20 ml reaction volume. Amplification conditions were: 3 minutes 94°C followed by 35 cycles of 15 seconds at 94°C, 15 seconds at 55°C and 30 seconds at 72°C. These samples were analyzed on an ABI 377 DNA Sequencer, capable of detecting fluorescently labeled DNA fragments with single nucleotide (nt.) resolution. This allowed for genotyping the plant samples: a 205-nucleotide DNA fragment was obtained from Ler DNA and a 21 1 -nucleotide band from Ws-O DNA. Thus, DNA fragments differing by 6 nucleotides in length could be easily distinguished, allowing for easy genotyping of samples as homozygous Ws-O, homozygous Ler and heterozygous Ws-O/Ler at the ATHGENEA locus.
In order to increase the throughput of this system, a multiplexing scheme was used. Some DNA samples were PCR amplified as described above with primer set ATHGENEA (1) , whereas other samples were analyzed with primerset ATHGENEA (2) (listed in table 2), in each case in the presence of 6-carboxyrhodamine-labeled dUTP. Primer set ATHGENEA (2) was made based on the published sequence of ATHGENEA (Simoens et al., 1988, Gene 67, 1-11). This primerset amplified a DNA fragment of 139 bp from Ler DNA and a 145-bp band from Ws-O DNA. Amplification reaction conditions for primerset ATHGENEA (2) were identical to those described for primerset ATHGENEA (1), above.
Single reactions using primer set ATHGENEA (1) and single reactions using primer set ATHGENEA (2) were mixed together before electrophoresis on the ABI 377 DNA Sequencer. This multiplexing approach allowed for genotyping 2 samples in a singe lane of the Sequencer, one at positions 145/139 nt. and one at positions 211/205 nt. on the Sequencer. In the second method, PCR fragments were labelled by using a primer labelled with the fluorescent dye FAM-6 (6-carboxyfluorescein) (Integrated DNA Technologies, Inc.). The forward ATHGENEA primers of the ATHGENEA (1) and (2) primer sets are identical in sequence (see Table 1 ). This primer was labeled with FAM-6 and used in a PCR amplification reaction with the following reagents (Perkin Elmer): 1xXL buffer, 1 mM MgCI2, dNTPs each at 200 mM, primers each at 0.50 mM (forward primer FAM-6 labeled), 10 ng genomic DNA and 0.5 units XL polymerase in a 20-ml reaction volume. The cycling conditions were: 3 minutes at 94°C, followed by 35 cycles of 15 seconds at 94°C, 15 seconds at 59°C and 30 seconds at 72°C. Again, single reactions using primer set ATHGENEA (1 ) and single reactions using primer set ATHGENEA (2) were mixed together before electrophoresis on the ABI 377 DNA Sequencher. This multiplexing approach allowed for genotyping 2 samples in a single lane of the Sequencer, one at positions 145/139 nt. and one at positions 211/205 nt.
All F2 and F3 samples from nimi phenotype plants were scored for their genotype at the ATHGENEA locus as described above. All samples that were heterozygous at this locus identified plants that were recombinant between the NIM1 locus and the ATHGENEA locus. In a population of 1 144 F2 nimi phenotype plants and F3 nimi phenotype populations that were scored in this way, 98 were heterozygous at the ATHGENEA locus, giving an estimate of the genetic distance between this SSLP locus and the NIM1 locus of 4.3 cM. This established that the NIM1 locus was on chromosome 1 , near the ATHGENEA marker.
Genetic mapping of the NIM 1 gene relative to marker ngal 11. Two primer sets for SSLP marker ngal 1 1 (described in Bell and Ecker, 1994, Genomics 19, 137-144) were used to amplify genomic DNA of F2 and F3 nimi phenotype plants and control Ws-O and Ler plants. Primer set nga111 (1) (described in Bell and Ecker, 1994, Genomics 19, 137-144 and listed in Table 1) was used under the following conditions: IxPCR buffer, 2 mM MgCI2, dNTPs each at 200 mM, primers at 0.75 mM, 10 ng DNA and 0.75 unit Taq polymerase in a 20 ml reaction volume. Primer set ngal 11 (2) (listed in Table 1 , and a derivative of primer set nga111 (1 )) was used under different conditions: IxPCR buffer, 1.5 mM MgCI2, dNTPs each 200 mM, primers at 1 mM, 10 ng DNA and 1 unit Taq polymerase in a 20 ml reaction volume. Both reactions were amplified by incubation at 94°C for 1 minute, followed by 40 cycles of 15 seconds at 94°C, 15 seconds at 55°C and 30 seconds at 72°C.
The samples were analyzed on 3-5% agarose gels. The band obtained from amplification of Ws-O DNA with either primer set was 146 bp, whereas amplifying Ler DNA resulted in a 162-bp band. Plant samples that were heterozygous at the ngal 1 1 locus identified plants that were recombinant between this SSLP marker and the NIM locus. Among 1 144 F2 nimi phenotype plants and F3 nimi phenotype populations, 239 were identified as heterozygous for the ngal 1 1 marker, giving an estimate for the genetic distance between the SSLP marker and the NIM locus of 10.4 cM. This corroborated that the NIM1 locus was on chromosome 1. Since few nimi phenotype plants existed that were heterozygous at both ATHGENEA and ngal 1 1 , the NIM1 locus was determined to be between these 2 markers, with ATHGENEA located North of the NIM1 gene and ngal 1 1 located South of the NIM1 gene. This placed the NIM1 gene approximately 10 cM north of nga111 , near position 85 on chromosome 1 (Lister and Dean, 1993, Plant J. 4, 745-750; Bell and Ecker, 1994, Genomics 19, 137-144). iii. Amplified Fragment Length Polymorphism markers.
For map-based cloning of the NIM1 gene, it is necessary to identify molecular markers that are successively closer to this gene. For this purpose, Amplified Fragment Length Polymorphism (AFLP) markers were generated by using the selective restriction fragment amplification method described by Zabeau and Vos (1993, European Patent Application EP 0534858) and Vos et al. (1995, Nucleic Acid Research 23, 4407-4414).
Outline of the AFLP Technology.
The use of the AFLP technology in mapping relies on selective amplification of a set of DNA bands in 2 genetically distinct samples. Finding that any of the obtained bands are different between the 2 genotypes identifies those bands as markers for that genotype. If the marker cosegregates at high frequency with the gene (mutation) of interest, then the marker is close to the genetic locus.
Selective amplification of a small set of DNA fragments in a complex DNA sample is achieved in a 2-step process. First, DNA fragments are generated by digesting the DNA with restriction enzymes, followed by ligation of adapters to the ends. Second, primers consisting of a sequence complementary to the adapters plus a 3' extension (typically 0-3 nucleotides) are used to amplify only those DNA fragments with ends that are complementary to these primers. If a single nucleotide extension is used, then theoretically, each primer will "fit" on approximately 1/4 of all fragments, with 1/16 of all fragments having a primer fit on both ends. Thus, a limited set of DNA fragments is amplified with these primers. By further radiolabelling one primer, an even smaller subset of visible bands can be obtained. AFLP analysis.
For AFLP analysis of DNA samples, 50 ng DNA was digested with the appropriate enzymes (usually EcoRI and Msel; see below) and adapters (listed in table 2 below) were ligated to the restriction fragments (usually EcoRI and Msel). The sequences of the primers and the YAC, P1 and BAC clones are described in detail below. The templates were used for amplification reactions (approximately 0.5 ng DNA per reaction), using primers that were complementary to the adapters, with short 3' extensions (2 or 3 nucleotides; primer sequences are listed below). Since one of the primers is radioactively labelled (usually the EcoRI primer), only a subset of the amplified fragments is visible upon autoradiographic analysis of the gel used to separate the bands.
Amplification conditions for cloned DNA (YAC, P1 , cosmid) were as follows: 36 cycles of 30 sec. at 94°C (denaturation), 30 sec annealing and 1 min extension at 72°C. The annealing temperature in the first cycle was 65°C and was reduced by 0.7°C in each cycle for the next 12 cycles and then kept at 56°C. For genomic DNA of Arabidopsis plants, the amplification was performed in 2 steps: in the first step (preamplification), the DNA was amplified with primers that have a single nucleotide extension (neither primer was labeled). Reaction conditions for this amplification reaction were: 20 cycles of 30 sec. denaturation (94°C), 1 min annealing (56°C) and 1 min extension (72°C). In the second step, the first amplification reaction was diluted 10 fold and reamplified 36 cycles with primers containing the full-size extensions (using one labeled primer) under the following conditions: 30 sec. at 94°C (denaturation), 30 sec annealing and 1 min extension at 72°C. The annealing temperature in the first cycle was 65°C and was reduced by 0.7°C in each cycle for the next 12 cycles and then kept at 56°C. The final reaction products were separated on a polyacrylamide gel and the gel was exposed to film, allowing visualization of the radiolabeled PCR bands. When this procedure was applied to DNA from 2 genotypes simultaneously, AFLP bands were identified that were diagnostic for one genotype or the other. Such bands are called informative AFLP bands, or AFLP markers. Table 2 shows Adapters used in the AFLP analysis.
Table 2
Figure imgf000034_0001
Generation of AFLP markers and fine mapping of the NIM1 locus. A population of recombinant inbred lines derived from a cross between the Arabidopsis ecotypes Landsberg erecta (Ler) and Columbia (Col) (Lister and Dean, 1993, Plant J. 4, 745-750) was used for AFLP marker screening. The primers used for the AFLP screening were:
EcoRI-primers: 5'-GACTGCGTACCAATTCWN-3' MseI-primers: 5'-GATGAGTCCTGAGTAAXWN-3'
An "N" in the primers indicates that this part was variable (A, C, G or T), a "W" indicates A or T, and an "X" indicates a C. All 8 possible primers were used for both the EcoRl- and Msel-primer. This gave a total of 64 (8 x 8) primer combinations (PCs) that were used to amplify DNA from the recombinant inbred line and the parental genotypes, Ler and Col, as described above. The amplification reactions were run on a denaturing polyacrylamide gel to separate AFLP fragments by size and the gel was exposed to film. The film was inspected for bands that were present in only one genotype, i.e. inspected for AFLP markers.
The AFLP markers, i.e., DNA fragments that are polymorphic between both parents of the recombinant inbred lines, were used for constructing a genetic map of the recombinant inbred line population. Example 1.5i below describes the mapping of the NIM 1 gene on Arabidopsis chromosome 1 , at approximately position 85. Those AFLP markers that had been mapped (using the recombinant inbred line) between positions 81 and 88 of Arabidopsis chromosome 1 were chosen for analyzing recombinant plants for the presence of said AFLP markers and thus for mapping the NIM1 gene more precisely. Seven AFLP markers from this region were identified as being informative; they were polymorphic between both parents of the nimlxLer cross. Six AFLP markers were Ler-specific, i.e. these AFLP markers were absent in Ws (and in Col as well). One AFLP marker was Ws- specific, i.e. a Col-specific AFLP marker (absent in Ler) was also present in Ws. These AFLP markers are: L81.1 , L81.2, W83.1 , L84, L85, L87 and L88 (an L-marker is specific for ecotype Ler and a W-marker is specific for both the ecotypes Col and Ws; the number indicates the map position). These AFLP markers were used to analyze recombinant plants from the nimlxLer cross (see below). In addition, AFLP marker C86 (a recombinant, inbred- line-derived marker specific for Col) was used in isolating DNA clones (see below). Table 3 lists the primer sequences that were used to obtain these AFLP markers. Table 3 shows primer combinations of AFLP markers derived from recombinant inbred line population.
"EcoRI-" refers to the sequence 5'-GACTGCGTACCAATTC-3' and "Msel-" refers to the sequence 5'-GATGAGTCCTGAGTAA-3'.
Table 3
AFLP marker Corresp< anding primer combinations
L81.1 EcoRI-CA Msel-CCG
L81.2 EcoRI-AA Msel -CAA
W83.1 EcoRI-CA Msel-CTC
L84 EcoRI-AAT Msel-CAA
L85 EcoRI-CA Msel-CCT
L87 EcoRI-CA Msel-CTT
L88 EcoRI-AG Msel-CTA
C86 EcoRI-AG Msel-CCT
A detailed genetic map of the region was constructed using the AFLP markers described above by typing the recombinants. A total of 337 recombinant plants were available out of 1144 F2 nimi plants. These recombinants were first screened with the North-flanking AFLP markers L81.2 and ATHGENEA and the South-flanking markers L88 and nga1 1 1 . Forty-eight plants were homozygous nimllnimi and heterozygous at ATHGENEA and L81.2, and 21 plants were homozygous nimllnimi and heterozygous at ngal 11 and L88. These recombinant plants were further analyzed with 9 AFLP markers in the NIM region, including 4 AFLP markers that were derived from the recombinant inbred line mapping population, (W83.1 , L84, L85 and L87) and 5 AFLP markers derived from analysis of YAC clones (W83.3/W84.1 , W84.2, W85.1 , W86.1 and L86, see below).
The genetic map of NIM1, based on this analysis, is depicted in Figure 4. As seen, 27 recombinants were found between marker W84.2 and NIM1 and 14 recombinants were found between W85.1 and NIML Marker L85 is linked closely to NIM1, but this marker could not be mapped on the YAC, BAC or P1 clones (see below) and was, therefore, not useable for identification of the NIM1 gene.
5. Physical mapping of the NIM1 region. i. Isolation of YAC clones using AFLP markers closely linked to NIML
The CIC library, an Arabidopsis ecotype Columbia YAC library (Bouchez et al, 1995, 6th Int. Conf on Arabidopsis Research, Madison, Wl), was screened for YAC clones in the NIM region. This library has about 2.5 nuclear genome equivalents and has an average insert size of 450 kb. The YAC library was screened with two AFLP markers: W83.1 and C86. W83.1 is the most closely linked recombinant, inbred-line-derived AFLP marker north of NIM1, and C86 is a recombinant, inbred-line-derived AFLP marker specific for Col (absent in Ler and Ws). C86 mapped south of the NIM1 gene on the map of the recombinant inbred line population. This Col AFLP marker has been used instead of the closely linked Ler AFLP markers (Figure 4), because the latter AFLP markers detected only ecotype Landsberg erecta and hence cannot be used for screening the Columbia YAC library.
The YAC library was screened in two steps. Firstly, the cells of the YAC clones of each plate of the twelve 96-well microtiter plates were pooled (a plate pool) and used for DNA isolation as described by Ross et al (1991 , Nucleic Acids Res. 19, 6053). The pools were screened with both AFLP markers. Subsequently, from each positive plate pool, the DNA samples of each row (a pool of 8 clones) and of each column (a pool of 12 clones) were screened with the AFLP marker for which the plate pool was positive. In this way, the individual positive YAC clones could be identified. The screening yielded a total of 4 YAC clones: YAC 12F04 and YAC 12H07 were isolated using the North AFLP marker W83.1 , and YAC 10G07 and YAC 7E03 using the south AFLP marker C86 (for the nomenclature of the YAC clones the CIC numbering is used). The YACs were "fingerprinted" by AFLP, giving YAC-specific AFLP fragments. Fingerprints of the YACs were compared and used to estimate overlaps between the YACs (see also Tables 5 and 6). Based on the AFLP fingerprints, clone 7E03 is essentially covered by clone 10G07 (see also table 5) and clone 12H07 is likewise essentially covered by clone 12F04 (see also table 6).
ii. Generation of AFLP markers from YAC clones.
Since the AFLP markers described above were genetically relatively far from the NIM1 gene (see figure 3), additional AFLP markers were developed in an effort to find markers that were closer to the NIM gene.
A screening for additional YAC-derived AFLP markers was performed on DNA samples of the following: DNA of the isolated YAC clones (4 YACs were identified, as described above), the yeast strain without a YAC, and the three Arabidopsis ecotypes Col, Ler and Ws. In this way, the fragments specific for the YAC clones (absent in the yeast strain and present in Col) could be tested for polymorphism in Ler and Ws (the parents of the recombinant plants identified in Example 1.5 below). All identified polymorphic fragments would thus be additional AFLP markers. In the first AFLP screening the enzyme combination (EC) EcoRI/Msel was used. In this screening two YAC clones, 10G07 and 7E03 (detected with AFLP marker C86, see below), the yeast strain without a YAC and the three Arabidopsis ecotypes Col, Ler and Ws were assayed. The primer combinations with the selective extensions used can be divided into three groups and are depicted in Table 4. A total of 256 (64 + 96 + 96) primer combinations were screened.
In Table 4 below the primer sequences used in the AFLP screening of two YAC clones, 10G07 and 7E03, the yeast strain without a YAC, and the three Arabidopsis ecotypes Col, Ler and Ws are shown. Three groups of primer combinations have been used. An "N" in the primers indicates that this part was variable (A, C, G or T), an "S" indicates C or G, a "W" indicates A or T, and a "Y" indicates C or T.
Table 4
EcoRI-primers:
5'-GACTGCGTACCAATTCGW-3'
5'-GACTGCGTACCAATTCTS-3'
Msel-primers:
5'-GATGAGTCCTGAGTAAAAS-3'
5'-GATGAGTCCTGAGTAAASA-3'
5'-GATGAGTCCTGAGTAAATN-3'
5'-GATGAGTCCTGAGTAACAN-3'
5'-GATGAGTCCTGAGTAACTN-3'
EcoRI-primers:
5'-GACTGCGTACCAATTCAN-3'
5'-GACTGCGTACCAATTCCW-3'
5'-GACTGCGTACCAATTCTW-3'
Msel-primers:
5'-GATGAGTCCTGAGTAAAAS-3'
5'-GATGAGTCCTGAGTAAASA-3'
5'-GATGAGTCCTGAGTAAGAY-3'
5'-GATGAGTCCTGAGTAAGTW-3'
5'-GATGAGTCCTGAGTAATCG-3'
5'-GATGAGTCCTGAGTAATCT-3'
5'-GATGAGTCCTGAGTAATGW-3' EcoRI-primers:
5'-GACTGCGTACCAATTCGW-3'
S'-GACTGCGTACCAATTCTN-S'
Msel-primers:
5'-GATGAGTCCTGAGTAAGAW-3'
5'-GATGAGTCCTGAGTAAGCW-3'
5'-GATGAGTCCTGAGTAAGTW-3'
5'-GATGAGTCCTGAGTAATAN-3'
5'-GATGAGTCCTGAGTAATCW-3'
5'-GATGAGTCCTGAGTAATGW-3'
5,-GATGAGTCCTGAGTAATTS-3'
In total, 83 Col-specific fragments were generated, of which 62 were shared by both YAC clones. Three fragments were AFLP markers polymorphic between Ws and Ler, of which two were Ws AFLP markers (a Col fragment also present in Ws and absent in Ler) and one was a Ler AFLP marker (a Col fragment also present in Ler and absent in Ws). These results are presented in Table 5 below.
Table 5 shows a number of shared and unique AFLP fragments detected in YACs 10G07 and 7E03 and the number of informative AFLP markers among these fragments in Ws and Ler genotypes.
Table 5
AFLP fragment! > in YAC clones AFLP marker
10G07 7E03 Ws Ler
shared 62 62 2 1 unique 21 0 0 0
This AFLP analysis thus yielded 3 new AFLP markers (see Figure 4 and below). Their positions relative to each other and relative to the recombinant, inbred-line-derived AFLP markers were determined by analysis of the recombinants with these AFLP markers.
A second screening for AFLP markers was performed assaying all four identified YAC clones (see below) and using the enzyme combination Pstl/Msel. The primers used are:
Pstl-primers:
5'-GACTGCGTACATGCAGAN-3'
5'-GACTGCGTACATGCAGCW-3'
5'-GACTGCGTACATGCAGGW-3'
5'-GACTGCGTACATGCAGTN-3'
Msel-primers:
5'-GATGAGTCCTGAGTAAAN-3'
5'-GATGAGTCCTGAGTAACW-3'
5'-GATGAGTCCTGAGTAAGW-3'
5'-GATGAGTCCTGAGTAATN-3'
An "N" in the primers indicates that this part was variable (A, C, G or T) and a "W" in the primers indicates that this was A or T. A total of 144 (12 x 12) primer combinations was screened on all four isolated YAC clones, 12F04, 12H07, 10G07 and 7E03; the yeast strain without a YAC; and the three Arabidopsis ecotypes Col, Ler and Ws. In total, 219 AFLP fragments were generated, of which 144 were present in YAC clones 12F04 and 12H07 (72 were unique for clone 12F04 and 72 were shared between both YACs) and of which 75 were present in YAC clones 10G07 and 7E03 (33 were unique for clone 10G07 and 42 were shared between the 2 YACs). Three fragments derived from the first set of YAC clones were polymorphic (Ws AFLP markers). These results are presented in Table 6 below.
Table 6 lists the number of shared and unique AFLP fragments detected in YACs and the number of informative AFLP markers among these fragments in Ws and Ler genotypes.
Table 6
number of AFLP fragments in YAC clones AFLP markers
12F04 12H07 10G07 7E03 Ws Ler
shared 72 72 0 0 1 0 unique 72 0 0 0 2 0 shared 0 0 42 42 0 0 unique 0 0 33 0 0 0
The results indicate that YAC clone 12H07 is part of the larger YAC clone 12F04, and that YAC clone 7E03 is a part of the larger YAC clone 10G07. These data indicate that the larger YAC clones, 12F04 and 10G07, do not overlap. These data do not allow the positioning of the NIM1 gene on any of these YAC clones. The whole screening, involving 400 primer combinations producing 302 AFLP fragments in the NIM region, yielded 5 useful AFLP markers, of which 4 were Ws-specific and one Ler-specific. These 5 additional AFLP markers have been mapped by analysis of recombinant plants (see figure 4 and below) and are denominated W84.1 (a.k.a. W83.3), W84.2, W85.1 , W86.1 and L86.
Table 7 lists the primer sequences used to obtain these AFLP markers. These 5 additional AFLP markers raised the total number of AFLP markers to 12 in the region from L81.1 to L88 (see figure 4 and below).
Table 7 shows primer combinations of AFLP markers derived from YAC clones. "EcoRI-" refers to the sequence 5'-GACTGCGTACCAATTC-3',
"Msel-" refers to the sequence 5'-GATGAGTCCTGAGTAA-3' and "Pstl-" refers to the sequence 5'-GACTGCGTACATGCAG-3'.
Table 7
AFLP marker Primer combination with selective extensions
W84.1 Pstl-AT Msel-TT
W84.2 Pstl-AA Msel-TT
W85.1 EcoRI-CT Msel-GTA
W86.1 EcoRI-GT Msel-CTT
L86 EcoRI-GT Msel-CTT
This information was used to construct a physical map of the region, as shown in Figure 5, with approximate positions of the YAC clones, relative to the genetic map. The map showed that the region containing the NIM1 locus, between markers W83.1 and W85.1 , is partly covered by 3 YAC clones: 12F04 and 10G07/7E03. iii. Construction of a P1/BAC contig containing the NIM1 gene.
In the previous sections it was described how AFLP markers linked to the NIM1 region were isolated and how YACs corresponding to these markers were identified and mapped. The results obtained while localizing the NIM1 gene to a chromosome fragment, did not allow the definition of a specific DNA segment containing the NIM1 gene: the flanking AFLP markers were mapped to different YACs that did not overlap. It was, therefore, not possible to determine the precise physical position of the NIM1 gene; it could be located on either of the two YACs or in the gap between the YACs. An alternative approach was selected to close the physical gap between the flanking markers: a P1 and BAC library were employed to bridge the gap between the flanking AFLP markers.
The libraries used for gap closure were an Arabidopsis ecotype Columbia P1 library described by Liu et al (The Plant J. 7. 351-358, 1995) and an ecotype Columbia BAC library described by Choi et al (http/genome-www.stanford.edu/Arabidopsis/ww/Vol2/ choi.html). The P1 -library consists of about 10,000 clones with an average insert size of 80 kb and the BAC library consists of about 4000 clones with an average insert size of 100 kb. In theory these libraries represent about 10 nuclear genome equivalents (assuming a haploid genome size for Arabidopsis of 120 Mb).
iv. Identification of P1 clones corresponding to the flanking markers.
The flanking markers Ws84.2 and Ws85.1 were used to screen pools of P1 clones using a similar strategy as previously described for screening of the YAC library (see Example 1.5i). P1 clones having the marker fragments were selected and "plasmid" DNA was isolated. The various P1 clone DNAs were fingerprinted using the ECs EcoRI/Msel and Hindlll/Msel and primers without selective nucleotides. A physical map was constructed, i.e. a map giving the size and overlaps of the clones, by comparing the AFLP fingerprints. The number of AFLP fragments that are unique and the number of AFLP fragments that are common between clones indicate the extent of the overlaps. The map is displayed in Figure 6. The AFLP fingerprinting revealed that two sets of non-overlapping P1-contigs had been constructed each containing one of the flanking markers: P1-1 and P1-2 containing marker Ws84.2; P1 -3 and P1 -4 containing marker W85.1 . Consequently, the gap between the flanking markers was not closed (Figure 6).
The positions of the P1 contigs with respect to the YAC contig was determined by AFLP fingerprinting of the YACs and P1 clones with a number of YAC-specific PCs described above. P1 clones P1 -1 and P1 -2 appeared to overlap completely with YAC CIC12F04, but only partially with YAC CIC12H07. Therefore, the latter P1 clones could be positioned on the YAC contig CIC12H07/12F04 (Figure 6). P1 clones P1 -3 and P1 -4 overlapped completely with both YACs CIC7E03 and CIC10G07 and it appeared that AFLP marker W86.1 , like W85.1 , was mapped to this P1 contig (Figure 6).
Next, marker L85 was used to identify corresponding P1 and BAC clones. L85 is an ecotype-Landsberg-specific marker and, therefore, colony hybridization of radioactively labelled L85 DNA to P1 and BAC filters was employed. Not a single P1 or BAC clone hybridizing to L85 was identified. This supported our earlier findings that the L85 sequence is lacking in the Arabidopsis ecotype Columbia genome and is, therefore, the most likely explanation of why no corresponding clones were identified.
v. Extending the NIM 7-flanking P1 Contigs.
Various approaches were employed to extend from the flanking P1 contigs:
YAC AFLP fragments specific to the South end of YAC CIC12F04 (unique to
CIC12F04, not present in CIC 12H07) were used to identify P1 clones by AFLP screening of pools of the library.
1. YAC AFLP fragments from YAC 10G07 and overlapping with P1-4 were used to identify P1 clones by AFLP screening of pools of the P1 library.
2. EcoRI restriction fragments from P1 clone P1 -6 (resulting from the AFLP- based P1 library screening of step 1) were used as hybridization probes on filters of the BAC library.
Various P1 and BAC clones resulted from this screening and all were AFLP- fingerprinted with the ECs EcoRI/Msel and Hindlll/Msel using primers without selective nucleotides. A new map was constructed as described above and is depicted in Figure 7. Table 8 shows the various AFLP PCs having AFLP fragments mapped to flanking YACs and used to screen the P1 -library for corresponding P1 clones.
Table 8 represents the various AFLP PCs used to screen the P1 library. The top half of the table shows PCs specific for the North YACs and the bottom half shows the PCs specific for the South YACs. Also indicated are the YACs and P1 clones wherein the AFLP fragments were detected. Table 8
AFLP PCs CIC YACs PI -clones Comments
Pstl-AA Msel-TT 12F04 and l2H07 P1-1. P1-2 Marker Ws84.2
Pstl-AT Msel-GT 12F04-specifϊc P1-1. P1-2
Pstl-CA Msel-AA 12F04-specifιc Pl-6
Pstl-AC Msel-TG 12F04-specifιc Pl-7
Pstl-AG Msel-TG 12F04-specifιc Pl-7
Pstl-CT Msel-GT 12F04-specifιc Pl-7
EcoRI-CT Msel-GTA 10G07 and 7E03 Pl-3, Pl-4 Marker Ws85.1
EcoRI-GT Msel-CTT 10G07 and 7E03 Pl-4 Marker Ws86.1
EcoRI-AA Msel-GT 10G07 and 7E03 Pl-4, Pl-9
EcoRI-AT Msel-GA 10G07 and 7E03 P1-4. P1-9
EcoRI-GG Msel-CT 10G07 and 7E03 P1-4. P1-9
P1/BAC contig of about 250 kb was obtained covering the South end of YAC CIC12F04 (not extending from this YAC) and containing marker W84.2. A P1 contig of about 150 kb containing markers W85.1 and W86.1 was obtained; this contig is completely contained within YAC CIC7E03.
Construction of a P1/BAC contig covering the NIM1 gene AFLP marker analysis on the recombinants with markers from the South end of the North P1/BAC contig (WL84.4 and WL84.5, see below and table 11 ) showed that the previous "walking" steps were unsuccessful in the construction of a contig containing the NIM1 gene (see next section). Therefore, the existing North P1/BAC contig was extended South with the purpose of "walking" across the NIM1 gene, which would enable the definition and isolation of a specific DNA segment containing the NIM1 gene. A hybridization-based approach was followed in which P1 or BAC clones located at the South end of the North P1/BAC contig were used to identify clones positioned closer to NIM1 (South bound). New clones resulting from the walking steps were mapped with respect to the existing contigs using AFLP fingerprinting with the ECs EcoRI/Msel and Hindlll/Msel as described above. A total of 5 subsequent walking steps appeared to be necessary to "cross" the NIM1 gene. Table 9 shows the clones obtained in the various walking steps.
Table 9 is an overview of the various walking steps showing the hybridization probe used to screen the P1 and BAC libraries and the selected clones hybridizing to the probes and extending in the South direction.
Table 9
Probe New clones extending South
Step l : PI -7 BAC-02
Step 2: BAC-02 Pl-16, BAC-03
Step 3: BAC-03 Pl-17, Pl-18
Step 4: Pl-18 Pl-21 , Pl-20, BAC-04
Step 5: BAC-04 Pl-22, Pl-23, Pl-24, BAC-06, BAC-05
A physical map of the various clones resulting from this walking effort is depicted in Figure 8. A total distance of about 600 kb was covered starting from the initial walking point marker W84.2. The South end of the contig presented in Figure 8 appeared to contain the NIM1 gene (see next section). The contig extends more than 300 kb South from YAC CIC12F04 and appeared not to overlap with YACs CIC10G07 and CIC7E03, indicating that the NIM1 gene is in the gap between the flanking YAC contigs and that this gap is at least 300 kb. vi. Construction of an Integrated Genetic and Physical Map of the NIM1 Region.
In the previous sections it was described how AFLP markers linked to the NIM1 region were isolated, how YACs corresponding to the flanking markers were identified, and how a P1/BAC contig was constructed extending about 550 kb South from the closest North flanking AFLP marker W84.2. This section describes the generation of new AFLP markers from the P1/BAC contig, the physical mapping of these markers on this contig and the genetic mapping of these markers with the available recombinants.
1. Generation of New AFLP Markers from the P1/BAC Contig
As described in the previous section, the P1 and BAC clones of the contig extension were characterized by AFLP fingerprinting using the ECs EcoRI/Msel and Hindlll/Msel. This defined quite accurately the extent of the overlaps between the various P1 and BAC clones and, in addition, generated a number of AFLP fragments specific for these clones. AFLP primers without selective nucleotides are used in fingerprinting of purified plasmid DNA of the P1 or BAC clones. Selective nucleotides will be necessary, however, to be able to use these P1 or BAC-specific AFLP fragments for detection in Arabidopsis. By determining the end sequences of the amplified restriction fragments, AFLP primers having the appropriate selective bases can be designed to amplify the P1- or BAC-specific AFLP fragment in
Arabidopsis. All AFLP fragments originate from the ecotype Columbia (Col) and, therefore, it also should be determined if the Columbia AFLP markers are informative in the NIM1 recombinants which are derived from a cross of the ecotypes Landsberg erecta (Ler) and a nimi mutant of the ecotype Wassilewskija (\Ns-nim). In principle, there are 4 types of AFLP fragments, two of which are useful markers as indicated in Table 10 below:
Table 10 is an overview of the types of AFLP markers found. (+) or (-) indicates presence or absence of the AFLP fragment. Table 10
Col Ler Ws-n/m marker-type
+ + + not informative
+ + - Ler marker
+ - + Ws marker
+ - - not informative
In general, fingerprinting of the P1 and BAC clones generated 30 to 40 EcoRI/Msel AFLP fragments and 60 to 80 Hindlll/Msel AFLP fragments for each individual clone. The end sequences of individual fragments were determined by standard sequencing techniques. Next, specific AFLP primers sets with selective extensions of 3 nucleotides for both the EcoRI or Hindlll primer and the Msel primer were tested on the following panel of DNAs:
1. P1/BAC clone from which the AFLP marker was derived
2a. Yeast
2b. YAC clone CIC12F04 (only for AFLP fragments from P1-7) 2c. YAC clone CIC10G07
3a. Col, origin of the P1 and BAC libraries
3b. Ler, parent 1 of the nim recombinants
3c. Ws-nim, parent 2 of the nim recombinants
Six clones were selected for sequence analyses of their EcoRI/Msel and Hindlll/Msel AFLP fragments: BAC-01/P1 -7, P1-17/P1 -18, BAC-04/BAC-06. The AFLP fragments from clone P1-7 were all detected in YAC CIC12F04, indicating that this clone is completely contained within this YAC. None of the P1 /BAC-specific AFLP fragments was detected in YAC clone CIC10G07, indicating that the P1/BAC contig does not bridge the gap between the two flanking YAC contigs. AFLP markers selected for analysis of the nim recombinants are depicted in Table 11.
Table 11 is an overview of the selected AFLP markers from the AFLP PCs specific for the various P1 and BAC clones. A "WL" marker is a marker originating from the same PC and displaying two AFLP markers, a Ws and a Ler marker, which appeared to be completely linked in repulsion phase upon analysis of the NIM recombinants. Table 11
Origin Marker name AFLP primers combination
Pl-7 WL84.4 EcoRI-AGC Msel-ACT
Pl -7 WL84.5 Hindlll-CTC Msel-TTC
P1-17/P1- 18 Ler84.6a Hindlll-CGT Msel-ATT
P1-17/P1-18 Ler84.6b Hindm-ATT Msel-CAT
Pl-18 Ler84.6c Hindlll-TCT Msel-TAT
Pl -18 Ler84.7 EcoRI-AAA Msel-AGA
BAC-04/06 Ler84.8 EcoRI-TTC Msel-AGT
BAC-06 Ler84.9a EcoRI-AAA Msel-TGT
BAC-06 Ler84.9b EcoRI-ATC Msel-TCC
BAC-06 Ler84.9c EcoRI-ATG Msel-GTA
2. Physical Mapping of the New AFLP Markers.
The AFLP markers described above were physically mapped by detecting their presence in the various P1 and BAC clones. The results are presented in Figures 9-11.
3. Genetic Mapping of the New AFLP Markers.
The AFLP markers were all analyzed on a selected set of the recombinants. The results obtained are summarized in Tables 12a, 12b and 12c.
Table 12a
NIM RECOMBINANTS NORTH OF WL84.4&5
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
51/1
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
The AFLP markers Ler84.8, Ler84.9a, Ler84.9b and Ler84.9c appeared to map at the South side of NIML Recombinants were found that were phenotypically nimi (homozygous, genotype \Ns-nim1DNs-nim1) and heterozygous for these AFLP markers (the Ler-specific AFLP marker was detected, genotype is Ws-π/m7/Ler). AFLP marker Ler84.8 appeared to be closest to NIMI: only a single recombinant (C-105) was scored as heterozygous \Ns-nimKLer and homozygous \Ns-nim1/\Ns-nim1. AFLP markers Ler84.7 and Ler84.6c appeared to completely cosegregate with NIMI: all recombinants had an identical NIM1 and AFLP marker genotype. North of NIM1, marker L84.6b appeared to be closest to NIM1: three nimi phenotype recombinant plants, C-074, D-169 and E-103 (Table 12c), were found to be heterozygous Ws-n/m7/Ler at this marker. With the aid of the cosmid contig generated from P1-18, BAC-04 and BAC-06, AFLP markers Ler84.6b and Ler84.8 were mapped in P1-18 and BAC-04, respectively, and found to have a physical distance of approximately 110 kb. This defines nimi to be located on a DNA segment estimated to be 110 kb in length. From this analysis it has been determined that the NIM1 gene is contained in clone BAC-04 or P1 -18. Clones BAC-04 and P1 -18 have been deposited with ATCC and given deposit numbers ATCC 97543 and ATCC 97606, respectively.
vii. Genetic and Physical Fine Mapping of the NIM1 Gene.
The previous section described how a DNA segment containing the NIM gene was delineated by physical mapping of the flanking AFLP markers (Ler84.6b and Ler84.8) on the P1/BAC contig. The flanking markers appeared to map on two overlapping clones, P1 - 18 and BAC-04. This section describes how additional BAC-04-specific and P1 -18-specific AFLP markers were generated to increase the resolution of the genetic and physical map in the region containing the NIM1 gene. viii. Generation of Additional AFLP Markers from the Cosmid Array.
Four ECs were selected to generate additional AFLP markers for fine mapping of
NIMI: Pstl/Msel, Xbal/Msel, BstYI/Msel and Taql/Msel. Pstl/Msel and Xbal/Msel AFLP fragments were generated on clone P1-18 and BAC-04 and the selective sequences necessary for detection in Arabidopsis were determined. Similarly, the AFLP fragments and selective sequences were determined for BstYI/Msel and Taql/Msel; however, in this case the procedure was performed using cosmid DNAs: A11 , C7, E1 and E8 for BstYI/Msel
(complete NIM1 region) and D7, E8 and E6 for Taql/Msel (South side of NIM1 region).
Informative AFLP markers selected for further genetic and physical mapping are shown in
Table 13. Additional adapters used in this work are shown in Table 14.
Table 13 shows the AFLP markers used for genetic and physical fine mapping of NIML "BstYI(T)" indicates that the restriction site and corresponding primer was either AGATCT or GGATCT.
Table 13
Marker EC/PC
Ler84.Yl BstYI(T)-GCT Msel-AAC
Ws84.Y2 BstYI(T)-TCT Msel-GCA
Ler84.Y3 BstYI(T)-AAG Msel-TAT
Ler84.Y4 BstYI(T)-GTT Msel-AGA
Ws84.Tl Taql-TAC Msel-GGA
Ler84.T2 Taql-TTG Msel-GGA Table 14 shows same Additional adapters used for identifying new AFLP markers.
Table 14
BstYI: 5'-CTCGTAGACTGCGTACC-3'
3'-CATCTGACGCATGGCTAG-5'
TaqI: 5'-CTCGTAGACTGCGTACC-3'
3'-CATCTGACGCATGGGC-5'
ix. Physical mapping of new AFLP markers to the cosmid contig. The markers indicated above were physically mapped on the cosmid array by determining their presence in the various cosmid clones (Figure 11).
1. Genetic Mapping of New AFLP Markers.
The new AFLP markers were genetically mapped by AFLP analysis of the closest North and South recombinants. The closest North (recombinant D169) and South (recombinant C105) recombination points were mapped (see Table 15). The AFLP analysis showed that recombinant D169 had a recombination South of marker L84.Y1 , but North of marker W84.Y2. The recombination point in recombinant C105 mapped between markers L84.T2 and L84.8. Using the available set of recombinants this allowed further delineation of the chromosomal interval containing NIMI; the distance between the flanking recombination points appeared to be 60-90 kb (Figure 12). 56/1
Figure imgf000062_0001
I
Figure imgf000063_0001
Figure imgf000064_0001
2. Construction of a Cosmid Contig.
For complementation of the nimi plant phenotype, transformation of nimi plants is required with a wildtype NIM1 gene. This can be accomplished by transforming these plants with a cosmid containing the gene. For this purpose, a cosmid contig of the NIM1 region is constructed. Since Arabidopsis is transformed using Agrobacterium, the cosmid vector used is a binary vector.
DNA was isolated from BAC-04, BAC-06 and P1 -18, and used to make a partial digest using restriction enzyme Sau3AI. The 20-25 kb fragments were isolated using a sucrose gradient, pooled, and filled in with dATP and dGTP. The binary vector (04541 ) was cleaved with Xhol and filled in with dCTP and dTTP. The fragments were next ligated into the vector. The ligation mix was packaged and transduced into E.coli.
This cosmid library was screened with the BAC-04, BAC-06 and P1 -18 clones and positive clones isolated. These cosmids were next AFLP fingerprinted and arranged into a contig of overlapping clones spanning the NIM1 region. The insert sizes of the cosmids were determined, and limited restriction mapping was performed. The results are shown in Figure 10.
Example 2 Identification of a Clone Containing the NIM1 gene.
1. Complementation Via Stable Transformation
Cosmids that are generated from clones that span the NIM1 region (described above) are moved into Agrobacterium by triparental mating. These cosmids are then used to transform nimi Arabidopsis by vacuum infiltration (Mindrinos et al., 1994, CeJ 78, 1089- 1099) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection are transferred to soil and tested for the nim phenotype or their progeny are tested for the nim phenotype. Transformed plants that no longer have the nim phenotype identify cosmid(s) that contain a functional NIM1 gene.
2. Complementation in a Transient Expression System.
The ability of DNA clones to complement the nimi mutation is tested in 2 transient expression systems.
In the first system, nimi Arabidopsis plants containing a PR1-luciferase (PR1 -lux) transgene are used as bombardment recipient material. These plants are generated by transforming Columbia ecotype plants with a PR1-lux construct by vacuum infiltration, followed by kanamycin selection of the harvested seed, as described above. Transformed plants that express luciferase activity after induction with INA are selfed and homozygous plants are generated. These are crossed to nimi plants. In the transient assay, progeny plants from this cross that are homozygous for nimi and for PR1 -lux are used for identification of DNA clones that can complement the nimi phenotype. To this end, the plants are first treated with INA, as described in example 1.1 above. Two days later these plants are harvested, surface sterilized and plated on GM agar medium. The leaf tissue is then bombarded with cosmid, P1 or BAC clones (or subclones) from the NIM1 region and after one day, the luciferase activity of the leaves is measured. Clones that induce luciferase activity contain the NIM1 gene.
In a second system, nimi plants are treated with INA (as described in example 1.1 above) and 2 days later bombarded with cloned DNA (cosmid, P1 , BAC and/or YAC clones or subclones) from the NIM1 locus region and a reporter plasmid. The reporter plasmid contains the luciferase gene, driven by the Arabidopsis PR1 promoter (PR1-lux). In nimi plants, INA does not activate the PR1 promoter (as described in example 1.2 above) and thus can not induce luciferase activity from the reporter plasmid. However, when a cotransformed DNA clone contains the complementing NIM1 gene, INA does induce the PR1 promoter, as evidenced by an induction of luciferase activity. One day after the cobombardment, the luciferase activity of the whole plant is measured DNA clones (cosmids, P1 or BAC clones or subclones) that induce luciferase activity that is significantly above background levels contain the NIM1 gene.
3. Changes in transcripts in nimi phenotype lines.
Since nimi phenotype plants have mutations in the NIM1 gene, it is conceivable that in some lines the gene is altered in such a manner that there is no mRNA transcribed, or an aberrant mRNA (size) is produced. To test for this, RNA blot analysis is performed on the nimi lines.
RNA is isolated from Ws and Ler plants of these lines, (after water or INA or BTH treatment) and used to prepare northern blots. These blots are hybridized with DNA fragments isolated from clones of the DNA contig of the NIM1 locus DNA fragments that identify nimi lines with aberrant RNA expression (aberrant in size or concentration), likely identify (part of) the NIM1 gene. The DNA fragment and surrounding DNA is sequenced and used to isolate a cDNA (by library screen or by reverse transcription-PCR), which is also sequenced. The clone from which the fragment was isolated or the isolated cDNA is used to show complementation of the nimi phenotype in stable and transient expression systems.
Example 3 Determination of the DNA sequence of the NIM1 gene.
1. Genomic sequencing.
Genomic clones that may contain the NIM1 gene are sequenced using methods known in the art. These include BAC-04, P1-18 and the cosmids from the NIM1 region. For instance, the cosmids are digested with restriction enzymes and fragments that are derived from the insert are cloned into a general purpose vector, such as pUC18 or Bluescnpt. The larger P1 and BAC clones are randomly sheared and fragments cloned into a general purpose vector. The fragments in these vectors are sequenced by conventional methods (e.g. by "primer walking" or generation of deletions of inserts). The obtained sequences are assembled into a contiguous sequence.
The sequence of the insert of a complementing clone contains the NIM1 gene. The approximate start and end of the NIM1 gene are deduced based on the DNA sequence, sequence motifs such as TATA boxes, the open reading frames present in the sequence, codon usage, the cosmid complementation data, the relative location of the AFLP markers and additional relevant data that is gathered (see Example 4, below). 2. cDNA sequencing.
The cosmιd(s) or larger clones that contain the NIM1 gene (as described in Example 2 above), are used to isolate cDNAs. This is accomplished by using the clones (or DNA fragments) as probes in a screen of a cDNA library of wildtype Arabidopsis plants. The cDNAs that are isolated are sequenced as described for cosmid sequencing and used in complementation tests. To this end, full length cDNAs are cloned into a suitable plant expression vector, behind a constitutive promoter. These constructs are used in the transient assays as described above. Alternatively, the cDNAs are cloned into a binary expression vector, allowing for expression in plant tissues and for Agrobacterium- mediated plant transformation, as described in Example 2 above. A cDNA that contains the NIM1 gene (as determined by complementation, isolation with a closely linked AFLP marker, isolation with a cosmid fragment, or by other deduction) is sequenced.
The genes from Ws-O and nimi plants are isolated and sequenced. The genes are obtained from a cosmid of cDNA library, using a fragment of the isolated NIM1 gene as a probe Alternatively, the genes or cDNAs are isolated by PCR, using Λ///Wϊ-gene-specιfιc primers and genomic DNA or cDNA as template. Likewise, the nimi alleles from other nimi lines (see Example 1.1 above) are isolated and sequenced in a similar manner.
Example 4 Description of the NIM1 gene and deduced protein sequence The DNA sequence of the NIM1 gene or cDNA is determined as described in Example 3 above. This sequence is analyzed with the use of DNA analysis programs, such as can be found in the Genetics Computer Group (GCG) package, in the Sequencer or Staden packages, or any similar DNA analysis program package. Specifically, the start and end of the gene are determined, based on open reading frame analysis, the presence of stop and potential start codons, the presence of potential promoter motifs (such as the TATA box), the presence of polyadenylation signals and the like. Also, the predicted amino acid is deduced from the open reading frame. Both the DNA and protein sequence are used to search databases for sequences with homologies, such as transcription factors, enzymes or motifs of such genes or proteins.
Example 5 Isolation of NIM1 homologs The Arabidopsis NIM1 gene may be used as a probe in the low stringency hybridization screening of a genomic or cDNA library in order to isolate NIM1 homologs from other plant species. Alternatively, this is accomplished by PCR amplification, using primers designed based on the Arabidopsis NIM1 gene sequence and using genomic DNA or cDNA as template. The NIM1 gene may be isolated from corn, wheat, rice, barley.rape seed, sugarbeet, potato, tomato, bean, cucumber, grape, tobacco and other crops of interest and sequenced. With a set of sequences from NIM 1 gene homologs in hand, new primers can be designed from conserved portions of the gene, in order to isolate NIM1 homologs from more distantly related plant species by PCR amplification.
Example 6 Complementation of the nim1-1 gene with genomic fragments. 1. Construction of a cosmid contig.
A cosmid contig of the NIM1 region was constructed using CsCI-purified DNA from BAC04, BAC06 and P1 -18. The DNAs of the three clones were mixed in equimolar quantities and were partially digested with the restriction enzyme Sau3A. The 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP. Plasmid pCLD04541 was used as T-DNA cosmid vector. This plasmid contains a broad host range pRK290-based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection. The vector was cleaved with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector . The ligation mix was packaged and transduced into E. coli strain XL1-blue MR (Stratagene). Resulting transformants were screened by hybridization with the BAC04, BAC06 and P1-18 clones and positive clones isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using the ECs EcoRI/Msel and Hindlll/Msel. The resulting AFLP fingeφrint patterns were analyzed to determine the order of the cosmid clones. A set of 15 semi-overlapping cosmids was selected spanning the nim region (Figure 13). The cosmid DNAs were also restricted with EcoRI, Pstl, BssHII and SgrAI. This allowed for the estimation of the cosmid insert sizes and the verification of the overlaps between the various cosmids as determined by AFLP fingerprinting.
2. Identification of a clone containing the NIM gene.
Cosmids generated from clones spanning the NIM1 region were moved into Agrobacterium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013). These cosmids were then used to transform a kanamycin- sensitive nim1-1 Arabidopsis line using vacuum infiltration (Mindrinos et al., 1994, Cell 78, 1089-1099). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection were transferred to soil approximately two weeks after plating and tested for the nimi phenotype as described below. Transformed plants that no longer have the nimi phenotype identify cosmιd(s) that contain a functional NIM1 gene.
3. Testing for the nimi phenotype of transformants.
Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300μm INA was applied as a fine mist to completely cover the plants using a chromister. After two days, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate EmWa) and grown under high humidity conditions in a growing chamber with 19°C day/17° night temperatures and 8h lιght/16h dark cycles. Eight to ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. Ws and nimi plants were treated in the same way to serve as controls for each experiment.
Total RNA was extracted from the collected tissue using a LiCI/phenol extraction buffer (Verwoerd, et al. NAR 17:2362). RNA samples were run on a formaldehyde agarose gel and blotted to GeneScreen Plus (DuPont) membranes. Blots were hybridized with a 32P-labeled PR-1 cDNA probe. The resulting blots were exposed to film to determine which transformants were able to induce PR-1 expression after INA treatment. The results are summarized in Table 16.
Table 16 shows complementation of the nimi phenotype by cosmid clones. Table 16
Figure imgf000073_0001
NA-not applicable
Example 7 Sequencing of the 9.9 Kb NIM1 gene region. BAC04 DNA (25 ug, obtained from KeyGene) was the source of DNA used for sequence analysis. This BAC was shown to be the clone completely encompassing the region that complemented the nim i mutants. DNA was randomly sheared using an approach from Cold Spring Harbor. Briefly, BAC DNA was sheared in a nebulizer to an average molecular weight of about 2 kb. Ends of the sheared fragments were repaired using a two-step protocol with dNTPS, T4 DNA polymerase and Klenow fragment (Boehringer). The end-repaired DNA was run on a 1% low-melt agarose gel and the region between 1.3 kb and 2.0 kb was cut from the gel. DNA was isolated from the gel fragment by a freeze-thaw approach. DNA was then mixed with EcoRV-digested pBRKanF4 and was ligated overnight at 4°C. pBRKanF4 is a derivative of pBRKanFI , which was obtained from Kolavi Bhat at Vanderbilt University (Bhat, K.S., Gene 134(1 ), 83-87 (1993)). E. coli strain DH5a was transformed with the ligation mix, and the transformation mix was plated onto plates containing kanamycin and X-gal. 1600 white or light blue KanR colonies were selected for plasmid isolation. Individual colonies were picked into 96-well deep well plates (Polyfiltronics, #U508) containing 1.5 ml of TB + Kan (50 ug/ml). Plates were covered and were placed on a rotating platform shaker at 37°C for 16 hrs. Plasmid DNA was isolated using the Wizard Plus 9600 Miniprep system (Promega, #A7000) according to manufacturer's recommendations.
Plasmids were sequenced using Dye Terminator chemistry (Applied BioSystems PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, P/N 402078) and primers designed to sequence both strands of the plasmids. Data was collected on ABI 377 DNA sequencers. Approximately 75% of these reactions yielded useful sequence information. Sequences were edited and were assembled into contigs using Sequencher 3.0 (Gene Codes Corporation), Staden gap4 (Roger Staden, e-mail address rs@mrc-lmb.cam.ac.uk), and PHRED (Phil Green, e-mail address phg@u. washington.edu). The largest contig (approximately 76 kb) covered the complementing region to an average depth of 7 independent calls/base. A region of approximately 9.9 kb defined by the overlap of cosmids E1 and D7 was identified by complementation analysis to contain the nimi region. Primers which flanked the insertion site of the vector and specific to the cosmid backbone were designed using Oligo 5.0 Primer Analysis Software (National Biosciences, Inc.). DNA was isolated from cosmids D7 and E1 using a modification of the ammonium acetate method (Traynor, P.L., 1990. BioTechniques 9(6): 676.) This DNA was directly sequenced using Dye Terminator chemistry above. The sequence obtained allowed determination of the endpoints of the complementing region.
A truncated version of the BamHI-EcoRV fragment was also constructed, resulting in a construct which contains none of the "Gene 3" region (Fig. 13). The following approach was necessary due the presence of Hindlll sites in the Bam-Spe region of the DNA. The BamHI-EcoRV construct was completely digested with Spel, then was split into two separate reactions for double digestion. One aliquot was digested with BamHl, the other Hindlll. A BamHI-Spel fragment of 2816 bp and a Hindlll-Spel fragment of 1588 bp were isolated from agarose gels (QiaQuick Gel extraction kit) and were ligated to BamHI-Hindlll- digested pSGCGOL DH5a was transformed with the ligation mix. Resulting colonies were screened for the correct insert by digestion with Hindlll following preparation of DNA using Wizard Magic MiniPreps (Promega). A clone containing the correct construct was electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.
Example 8 Identification of the NIM J gene region by allele sequencing.
Table 17. Genetic segregation of non-inducible immunity mutants.
Phenotype Mutant Generation Female MaleWildtype ntml
nιml-1" FI wildtype mm/ -I 24 0
F2 98 32
ntml -2 FI nιmI-2 Wildtype 3
nιml-3 FI nιml-3 Wildtype 3
nιtnl-4 FI nιmI-4 Wildtype 3
nimi -5 FI nimi -5 Wildtype 0 35
nιml-6 FI niml-6 Wildtype 3
nιml-2 FI nιml-2 nιml-l 0 15
nimi -3 FI ntml -3 nimi -I 0 10
nimi -4 FI mml-4 ntml -I 0 15 nιml-5 FI nιml-5 nιml-1 0 14
F2 9 85
nιml-6 FI ntml -6 nιml-1 0 12
a Data from Delaney et al . ( 1995 ) PNAS 92,6602-6606. b Wild type denotes the wildtype Ws-O strain.
1. Genetic Analyses
To determine dominance of the various mutants that displayed the nimi phenotype, pollen from wildtype plants was transferred to the stigmata of nιm1-1, -2, -3, -4, -5, -6. If the mutation is dominant, then the nimi phenotype will be observed in the resulting F1 plants. If the mutation is recessive, then the resulting F1 plants will exhibit a wildtype phenotype
The data presented in Table 17 show that when nιm1-1, -2, -3, -4 and -6 are crossed with the wildtype, the resulting F1 exhibit the wildtype phenotype. Thus, these mutations are recessive. In contrast, the nιm 1-5 X wildtype F1 progeny all exhibit the nimi phenotype, indicating that this is a dominant mutation. Following INA treatment, no P parasitica sporulation was observed on wildtype plants, while the F1 plants supported growth and some sporulation of P. parasitica. However, the nimi phenotype in these F1 plants was less severe then observed when mm 1-5 was homozygous.
To determine allelism, pollen from the kanamycin-resistant nιm1-1 mutant plants was transferred to the stigmata of nιm1-2, -3, -4, -5, -6. Seeds resulting from the cross were plated onto Murashige-Skoog B5 plates containing kanamycin at 25 ug/ml to verify the hybrid origin of the seed. Kanamycin resistant (F1 ) plants were transferred to soil and assayed for the nimi phenotype Because the F1 progeny of the cross of the nιm1-5 mutant with the Ws wildtype displayed a nimi phenotype, analysis of nιm1-5 X nim1-1 F2 was also carried out.
As shown in Table 17, all of the resulting F1 plants exhibited the nιm1-1 phenotype. Thus, the mutation in the nιm1-2, -3, -4, -5, -6 was not complemented by the nιm1-1; these plants all fall within the same complementation group and are therefore allelic. Analysis of F2 progeny from the nιm1-5 X nim1-1 cross also displayed the nimi phenotype, confirming that mm 1-5 is a nimi allele.
2. Sequence Analysis and Subcloning of the NIM1 Region
The 9.9 kb region containing the NIM1 region was analyzed for the presence of open reading frames in all six frames using Sequencher 3.0 and the GCG package. Four regions containing large ORF's were identified as possible genes (Gene regions 1 -4). These four regions were PCR amplified from DNA of the wild-type parent and six different nimi allelic variants. Primers for these amplifications were selected using Oligo 5.0 (National Biosciences, Inc.) and were synthesized by Integrated DNA Technologies, Inc. PCR products were separated on 1.0% agarose gels and were purified using the QIAquick Gel Extraction Kit. The purified genomic PCR products were directly sequenced using the primers used for the initial amplification and with additional primers designed to sequence across any regions not covered by the initial primers. Average coverage for these gene regions was approximately 3.5 reads/base.
Sequences were edited and were assembled using Sequencher 3.0. Base changes specific to various nimi alleles were identified only in the region designated Gene Region 2.
The positions listed in Table 18 relate to Figure 14 and relate to the top strand of the
9.9 kb region featured in Figure 13. The open reading frames from the gene regions described in Figure 13 as 1 , 2, 3 and 4 were sequenced and the changes in the different nimi alleles are shown in the Table. The changes that are described are on the top strand, 5φ to 3Φ, as it would relate to Figure 13.
It is apparent that the NIM1 gene was cloned and that it lies within Gene Region 2, since there are amino acid changes or alterations of sequence within the open reading frame of Gene Region 2 in all 6 nimi alleles. At the same time, at least one of the nimi alleles shows no changes in the open reading frames within Gene Regions 1 , 3 and 4 Therefore, the only gene within the 9.9 kb region that could be NIM1 is the Gene Region 2, the NIM1 gene.
The Ws section of Table 18 indicates the changes in the Ws ecotype of Arabidopsis relative to the Columbia ecotype of Arabidopsis. Figures 13, 14, 15 and all others wherein sequence is shown relate to the Columbia ecotype of Arabidopsis, which contains the wild type gene in the experiments that were conducted. The changes are listed as amino acid changes within the gene 2 or NIM1 region and are listed as changes in base pairs in the other regions.
Figure 13 shows 4 different panels that describe the cloning of the NIM1 gene and describe the entire 9.9 kb region. Figure 14 is the sequence of the entire 9.9 kb region in the same orientation as described in Figure 13. Figure 15 is the sequence of the specific NIM1 gene region which is gene region 2 indicated in Figure 13; the sequence of Figure 15 contains the NIM1 gene. Figure 15 shows the ammo acid sequence in single letter code and shows the full length cDNA and RACE product that was obtained in capital letters in the DNA sequence. Some of the allele mutations that were found are shown above the DNA sequence and the particular mm1 allele that had that change is indicated.
Sequence analysis of the region and sequencing of various nimi alleles (see below) allowed identification of a cosmid region that contains the nimi gene. This region is delineated by a BamHI -EcoRV restriction fragment of -5.3 kb Cosmid DNA from D7 and plasmid DNA from pBlueScriptll(pBSII)were digested with Bam HI and with EcoRV (NEB). The 5.3 kb fragment from D7 was isolated from agarose gels and was purified using the QIAquick gel extraction kit (# 28796, Qiagen). The fragment was ligated overnight to the Bam-EcoRV-digested pBSII and the ligation mixture was transformed into E. coli strain DH5a. Colonies containing the insert were selected, DNA was isolated, and confirmation was made by digestion with Hindlll. The Bam- EcoRV fragment was then engineered into a binary vector (pSGCGOI) for transformation into Arabidopsis.
3. Northern analysis of the four gene regions.
Identical Northern blots were made from RNA samples isolated from water-, SA-, BTH- and INA-treated Ws and nimi lines as previously described (Delaney et al, 1995, PNAS 92, 6602-6606). These blots were hybridized with PCR products generated from the four gene regions identified in the 9.9 kb NIM1 gene region. Only the gene region containing the NIM1 gene (Gene Region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcribed gene (Figure 16 and Table 18).
Table 18 shows nimi allele sequence variation.
Table 18
Figure imgf000081_0001
Figure imgf000082_0001
Positions listed in the table relate to Figure 14 containing the 9.9Kb sequence. All alleles nim1-1 to nim1-6 are WS strain. Columbia-0 represents the wild type
We also demonstrated that the gene region 2 (Fig. 13) contains the functional NIM1 gene by doing additional complementation experiments. A BamHI/Hindlll genomic DNA fragment containing gene region 2 was isolated from cosmid D7 and was cloned into the binary vector pSGCGOI containing the gene for kanamycin resistance (Fig. 13; Steve Goff, personal communication). The resulting plasmid was transformed into the Agrobacterium strain GV3101 and positive colonies were selected on kanamycin. PCR was used to verify that the selected colony contained the plasmid. Kanamycin-sensitive nim1-1 plants were infiltrated with this bacteria as prevously described. The resulting seed was harvested and planted on GM agar containing 50μg/ml kanamycin. Plants surviving selection were transferred to soil and tested for complementation. Transformed plants and control Ws and nimi plants were sprayed with 300μm INA. Two days later, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate EmWa) and grown as previously described. Ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. All of the
15 transformed plants, as well as the Ws controls, were negative for fungal growth following
INA treatment, while the n/mϊcontrols were positive for fungal growth. RNA was extracted and analyzed as described above for these transformants and controls. Ws controls and all
15 transformants showed PR-1 gene induction following INA treatment, while the nimi controls did not show PR-1 induction by INA. 4. Isolation of a NIM1 cDNA.
An Arabidopsis cDNA library made in the IYES expression vector (Elledge et al, 1991 , PNAS 88, 1731 -1735) was plated and plaque lifts were performed. Filters were hybridized with a 32P-labeled PCR product generated from the gene region containing nim- 1. 14 positives were identified from a screen of approximately 150,000 plaques. Each plaque was purified and plasmid DNA was recovered. cDNA inserts were digested out of the vector using EcoRI, agarose-gel-purified and sequenced. Sequence obtained from the longest cDNA is indicated in Figure 15. To confirm that we had obtained the 5c end of the cDNA, a Gibco BRL 5' RACE kit was used following manufacturer's instructions. The resulting RACE products were sequenced and found to include the additional bases indicated in Figure 15. The transcribed region present in both cDNA clones and detected in RACE is shown as capital letters in Figure 15. Changes in the alleles are shown above the DNA strand. Capitals indicate the presence of the sequence in a cDNA clone or detected after RACE PCR.
Example 9 Characterization of the NIM1 gene The multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5:151 -153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994).
It has been determined that certain regions of the NIM1 protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 19. The NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.
Example 10 Phenotypic characterization of the various nimi alleles
1. Analysis of chemical responsiveness in nimi alleles.
We analyzed the differences among the various nimi alleles in terms of chemical induction of PR gene expression and Peronospora parasitica resistance (see Figures 17 and 18).
Mutant plants were treated with chemical inducers and then assayed for PR gene expression and disease resistance.
2. Plant growth and chemical application.
Wild-type seeds and seeds for each of the nimi alleles (nim1-1, -2, -3, -4, -5, -6) were sown onto MetroMix 300 growing media, covered with a transparent plastic dome and placed at 4° C in the dark for 3 days. After 3 days of 4° C treatment the plants were moved to a phytotron for 2 weeks. At approximately 2 weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H20, 5mM SA, 300 uM BTH or 300 uM INA. Chemicals were applied as a fine mist to completely cover the seedlings using a chromister. Water control plants were returned to the growing phytotron while the chemically treated plants were held in a separate but identical phytotron. After 3 days plants were divided into 2 groups. One group was harvested for RNA extraction and analysis. The second group was inoculated with P. parasitica. 3. Peronospora parasitica inoculation and analysis.
P. parasitica isolate 'EmWa' is a P.p. isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of causing disease on a particular host. The P. parasitica isolate 'NoCo' is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogens are recognized by the potential host, eliciting a host response that prevents disease development. At 3 days post-chemical application water and chemically treated plants were inoculated with the compatible 'EmWa' isolate. 'NoCo' inoculation was conducted on water treated plants only. Following inoculation plants were covered with a clear plastic dome to maintain high humidity required for successful P. parasitica infection and placed in a growing chamber with 19° C day/17° C night temperatures and 8h lιght/16h dark cycles.
At various timepoints after inoculation plants were analyzed microscopically to assess symptom development. Under magnification sporulation of the fungus can be observed at very early stages of disease development. The percentage of plants/pot showing sporulation at 5d, 6d, 7d, 11d and 14d after inoculation was determined and the density of sporulation was also recorded.
Figure 18 shows the disease assessment of the various nimi alleles following P. parasitica inoculation. The most distinguishing timepoints are 5 and 6 days post- inoculation. At 5 days post-inoculation nιm1-4 shows -80% infection under all inducing chemical treatments performed, clearly indicating that this allele/genotype has the most severe disease susceptibility. At 6 days post-inoculation, nimi, -2, -3, -4 and -6 show significant disease incidence under all inducing chemical treatments. However, nιm1-5 shows less infection than Ws wild-type under all treatments at day 6. Therefore, nιm1-5 is the most disease resistant of the various nimi alleles. nιm1-2 appears intermediate with respect to disease susceptibility after BTH but not the other inducing treatments PR-1 gene expression indicates that nim1-4 is the least responsive to all of the inducing chemicals tested (Figure 17), while nim1-5 shows elevated levels of PR-1 expression in the absence of inducers. These PR-1 gene expression results are consistent with the disease assessment performed with P. parasitica (Figure 18) and indicate that nimi alleles can cause resistance or susceptibility.
The samples obtained above were used to analyze NIM1 gene expression (Figure 17). In wildtype plants NIM1 mRNA was present in the untreated control samples. Following treatment with SA, INA, BTH or infection with a compatible pathogen the NIM1 mRNA accumulated to higher levels. Differences in NIM1 message (mRNA) abundance were observed in the nimi alleles compared to wildtype. The abundance of NIM1 mRNA in untreated mutant plants was lower than observed in the wildtype with the exception of niml- 2 and -5 where the amounts were similar. The nim1-1, -3 and -4 had low levels of NIM1 message while the nim1-6 had very low accumulation of NIM1 mRNA. Increases in NIM1 mRNA following SA, INA or BTH were observed in nim1-1, -2, -3 but not nim1-5 or -6. However, this increase was less than observed in wildtype plants. Following pathogen infection additional bands hybridizing to the NIM1 cDNA probe were observed in both wildtype and mutants and the NIM1 mRNA level was elevated relative to untreated controls, except in nim 1-6.
Figure 18 shows the disease resistance assessment via infection rating of the various nimi alleles as well as the NahG plants at various times after innoculation with Peronospora parasitica. WsWT indicates the Ws wild type parent line in which the nimi alleles are found. The various nimi alleles are indicated in the table and the NahG plant is indicated also. The NahG plant has been previously published. (Delaney et al. Science 266, pp. 1247-1250 (1994)). The NahG Arabidopsis is also described in WO 95/19443. The NahG gene is a gene from Pseudomonas putida that converts salicylic acid to catechol, thereby eliminating the accumulation of salicylic acid, a necessary signal transduction component for SAR in plants. Thus, NahG Arabidopsis plants do not display normal SAR. In addition, they show much greater susceptibility in general to pathogens. Therefore, the NahG plants serve as a kind of universal susceptibility control. In addition, the NahG plants still respond to the chemical inducers INA and BTH; this is shown in the bottom two panels of the Figure 17.
From Figure 18 it can be seen that the nιm1-4 and nιm1-6 alleles seem to be the most severe; this is most easily observable at the earlier time points, described earlier in the results section herein, and from the results set forth in the EmWa BTH panel, the lowest panel, in the Figure. In addition, the nim 1 -5 allele shows the greatest response to both INA and BTH and therefore it is the weakest nimi allele.
The NahG plants show very good response to both INA and BTH and look very similar to the mm 1-5 allele. However, at late time points, Day 11 in the Figure, the disease resistance induced in the NahG plants begins to fade, and there is a profound difference between INA and BTH in that the INA-induced resistance fades much faster and more severely than the resistance induced in the NahG plants by BTH. Also seen in these experiments is that INA and BTH induced very good resistance in Ws to EmWa, and the nιm1-1, nιm1-2 and other nimi alleles show virtually no response to SA or INA in regard to disease resistance.
Figure 18 lists the percent of plants that are showing sporulation after infection with the EmWa race of P. parasitica, and each of the bar graphs indicates the number of days after infection that the disease resistance was rated.
Northern analysis analyzing the expression of the SAR gene PR1 was also performed on the same samples, as shown in Figure 17. Figure 17 shows that the wild type plant shows very good response to salicylate, INA, BTH and also to pathogen infection, as manifested by enhanced PR1 gene expression. The nim1-1 allele, on the other hand, shows only very limited response to all the chemical inducers including pathogen.
The pathogen induction is at least several fold lower in the nim1-1 allele than it is in the wild type. The nim1-2, nim1-3 and nim1-6 alleles show response similar to the nim1-1 allele to the various treatments. However, the nim1-4 allele shows virtually no expression in response to any of the inducers used. Basically, background level is all that is observed. The nim 1-5 allele shows a very high background level relative to controls with water and that background level is maintained in all the treatments; however, there is limited or no induction by the chemical inducers.
The NahG plants serve as a good control, showing that they are unable to induce PR-1 in the presence of SA; on the other hand, INA and BTH both induce very strong high level expression of PR-1. The effect of pathogen infection is similar to that of SA; there is no expression of PR-1 in the EmWa-treated NahG plants.
These same RNA samples produced in the induction studies were also probed with a NIM1 gene using a full-length cDNA clone as probed. In Figure 16 it can be seen that INA induces the NIM1 gene in the wild type Ws allele. However, the nim1-1 mutation allele shows a lower basal level expression of the NIM1 gene, and it is not inducible by INA. This is similar to what is observed in the nim 1-3 allele and the nim1-6 allele. The /7/m7-_? allele shows approximately normal levels in the untreated sample and shows similar induction to that of the wild type sample, as does the nim1-4 allele. The nim1-5 allele seems to show higher basal level expression of the NIM1 gene and much stronger expression when induced by chemical inducers. The induction of NIM1 by chemical inducers of resistance and other inducers is consistent with its role in pathogen defense and is also further evidence that we have obtained the right gene in the 9.9 kb region.
82
SEQUENCE LISTING
(1) GENERAL INFORMATION:
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(ii) TITLE OF INVENTION: GENE CONFERRING DISEASE RESISTANCE IN PLANTS AND USES THEREOF
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(2) INFORMATION FOR SEQ ID Nθ:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9919 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
TGATCATGAA TTGCGTGTAG GGTTGTGTTT TAAAGATAGG GATGAGCTGA AGAAGGCGGT 60
GGACTGGTGT TCCATTAGAG GGCAGCAAAA GTGTGTAGTA CAAGAGATTG AGAAGGACGA 120
GTATACGTTT AAATGCATCA GATGGAAATG CAATTGGTCG CGTCGGGCAG ATTGAATAGA 180
AGAACATGGA CTTGTTAAGA TAACTAAGTG TAGTTGGTCC ACATACTTGT TGTTCTATTA 240
AGCCGGAAAA CTTCAACTTG TAATTTGCAG CAGAAGAGAT TGAGTGTCTG ATCAGGGTAC 300
AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360
GGCAGCAGAA CAGGAAGTAA TCAAAGATCA GAGTTTCAGA GTATTGCCTA AACTAATTGG 420
CTGCATTTCA CTCATCTAAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480
TGAATTTGCA TCCTTCGGTG GCGCGTTTTG GGCGTTTCCA CAGTCCATTG AAGGGTTTCA 540
ACACTGTAGA CCTCTGATCA TAGTGGATTC AAAAGACTTG AACGGCAAGT ACCCTATGAA 600
ATTGATGATT TCCTCAGGAC TCGACGCTGA TGATTGCTTT TTCCCGCTTG CCTTTCCGCT 660
TACCAAAGAA GTGTCCACTG ATAGTTGGCG TTGGTTTCTC ACTAATATCA GAGAGAAGGT 720
AACACAAAGG AAAGACGTTT GCCTCGTCTC CAGTCCTCAC CCGGACATAG TTGCTGTTAT 780
TAACGAACCC GGATCACTGT GGCAAGAACC TTGGGTCTAT CACAGGTTCT GTCTGGATTG 840
TTTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA 900
GGCTGGATCC ACAAGTCAGA AGGAAGAATT TGATTCCTAC ATAAAGGACA TCAAAAAGAA 960
GGACTCAGAA GCTCGGAAAT GGTTAGCCCA ATTCCCTCAA AATCAGTGGG CTCTGGCTCA 1020
TGACCAGTGG TCGGAGATAT GGAGTCATGA CGATAGAAAC AGAAGATTTG AGGGCAATTT 1080
GTGAAAGCTT TCAGTCTCTT GGTCTATCAG TGACAGCGAA CGCACCTGCA CATGTGGGAA 1140 GTTTCAATCG AAGAAGTTTC CATGTATGCA CCCAGAAATG GTGCAAAGGA TTGTTAACTT 1200
GTGTCATTCA CAAATGTTGG ATGCAATGGA GCTGACTAGG AGAATGCACC TTACACGCCC 1260
ACTCAGTGTT CTCTTATCTC TAGACCTGAA ACTAACTTGC TGTGTAATTC GAGTTACAAA 1320
AGGTTAAAGG AAGAATTAGG AAGATACATA TAACATGAAT GTTGCCAGAA GTTCAGGGAA 1380
CTTGAATATT CTTTTGGTTC TTGGTGGAAA ATATCCAACA GATGAACAAT TTGACATTAT 1440
TTCACACTTT GATTCTAGCA ACTCTGTAAC ACCATCATGG GTTATTGTTG ATGTACATAA 1500
ATATATATTA CAAATCTGTA TACCATTGGT TCAAATTGTT ACAACATTTG TTTGAAGCAC 1560
ACCTGCAGCA ATAATACACA GGATGCAAAA CGAAGAGCGA AACTATATGA CGCCAACGAT 1620
AGACATAAAC AGTTACAGTC ATCATGAAAA CAGAATTATA TGGTACAGCA AAAATTACAC 1680
TAAGAGGCAA GAGTCTCACC GACGACGATG AGAGAGTTTA CGGTTAGACC TCTTTCCACC 1740
GGTTGATTTC GATGTGGAAG AAGTCGAATC TGTCAGGGAC GAATTTCCTA ATTCCAAATT 1800
GTCCTCACTA AAGGCCTTCT TTAGTGTCTC TTGTATTTCC ATGTACCTTT GCTTCTTTTG 1860
TAGTCGTTTC TCAGCAGTGT CGTCTTCTCC GCAAGCCAGT TGAGTCAAGT CCTCACAGTT 1920
CATAATCTGG TCGAGCACTG CCGAACAGCG CGGGAAGAAT CGTTTCCCGA GTTCCACTGA 1980
TGATAAAAAA AACAAGGTCA GACAGCAAGT AACAAAACCA TGTTTAAAGA TCATTTAGTT 2040
TTGTTTTTTG TGATAAGGAG TCCGATGAAG TGGGTGAGAA TCCATACCGG TTTTAGAAAG 2100
CGCTTTTAGT CTACTTTGAT GCTCTTCTAG GATTCTGAAA GGTGCTATCT TTACACCCGG 2160
TGATGTTCTC TTCGTACCAG TGAGACGGTC AGGCTCGAGG CTAGTCACTA TGAACTCACA 2220
TGTTCCCTTC ATTTCGGCGA TCTCCATTGC AGCTTGTGCT TCCGTTGGAA AAAGACGTTG 2280
AGCAAGTGCA ACTAAACAGT GGACGACACA AAGAATAGTT ATCATTAGTT CACTCAGTTT 2340
CCTAATAGAG AGGACATAAA TTTAATTCAA ACATATAAGA AATAAGACTT GATAGATACC 2400
TCTATTTTCA AGATCGAGCA GCGTCATCTT CAATTCATCG GCCGCCACTG CAAAAGAGGG 2460 AGGAACATCT CTAGGAATTT GTTCTCGTTT GTCTTCTTGC TCTAGTATTT CTACACATAG 2520
TCGGCCTTTG AGAGAATGCT TGCATTGCTC CGGGATATTA TTACATTCAA CCGCCATAGT 2580
GGCTTGTTTT GCGATCATGA GTGCGGTTCT ACCTTCCAAA GTTGCTTCTG ATGCACTTGC 2640
ACCTTTTTCC AATAGAGATA GTATCAATTG TGGCTCCTTC CGCATCGCAG CAACATGAAG 2700
CACCGTATAT CCCCTCGGAT TCCTATGGTT GACATCGGCA AGATCAAGTT TTAAAAGATC 2760
TGTTGCGGTC TTCACATTGC AATATGCAAC AGCGAAATGA AGAGCACACG CATCATCTAG 2820
ATTGGTGTGA TCCTCTTTCA AAAGCAACTT GACTAACTCA ATATCATCCG AGTCAAGTGC 2880
CTTATGTACA TTCGAGACAT GTTTCTTTAC TTTAGGTACC TCCAAACCAA GCTCTTTACG 2940
TCTATCAATT ATCTCTTTAA CAAGCTCTTC CGGCAATGAC TTTTCAAGAC TAACCATATC 3000
TACATTAGAC TTGACAATAA TCTCTTTACA TCTATCCAAT AGCTTCATAC AAGCTTTACC 3060
ACATATATTA GCAAGCTTGA GTATAACCAA TGTGTCCTCT ATAACAACTT TGTCTACAAC 3120
GTCCAATAAG TGCCTCTGAA ATACAAATAC AAGTACTCAA GTAAGAACAT ATTCATGAAT 3180
GTGTAACCAT AGCTTAATGC AGATGGTGTT TTACCTGATA GAGAGTAATT AATTCAGGGA 3240
TCTTGAAGAT GAAAGCCAAA TAGAGAACCT CCAACATGAA ATCCACCGCC GGCCGGCAAG 3300
CCACGTGGCA GCAATTCTCG TCTGCGCATT CAGAAACTCC TTTAGGCGGC GGTCTCACTC 3360
TGCTGCTGTA AACATAAGCC AAAACAGTCA CAACCGAATC GAAACCGACT TCGTAATCCT 3420
TGGCAATCTC CTTAAGCTCG AGCTTCACGG CGGCGGTGTT GTTGGAGTCT TTCTCCTTCT 3480
TAGCGGCGGC TAAAGCGCTC TTGAAGAAAG AGCTTCTCGC TGACAAAACG CACCGGTGGA 3540
AAGAAACTTC CCGGCCGTCG GAGAGAACAA GCTTAGCGTC GCTGTAGAAA TCATCCGGCG 3600
AGTCAAAGAC GGATTCGAAG CTGTTGGAGA GCAATTGCAG AGCAGATACA TCAGGTCCGG 3660
TGAGTACTTG TTCGGCGGCC AGATAAACAA TAGAGGAGTC GGTGTTATCG GTAGCGACGA 3720 AACTAGTGCT GCTGATTTCA TAAGAATCGG CGAATCCATC AATGGTGGTG TCCATCAACA 3780
GGTTCCGATG AATTGAAATT CACAAATTAA AGAGATCTCT GCTAATCAAC GAAGAGACCT 3840
TATCAACTGG ATTTGGTTAA AGATCGAAGA TAACCATTGA CGAGCAGAGC CAAGTCAAGT 3900
CAACGAGAGT GGTGGTGAGA TATGAAGAAG CATCCTCGTC CCACGGTTTA CATTTCACCA 3960
AAACCGGTAA ATTTCCAGGA AAGGAATCTT TGTCAGAGAT CTTTTTTAAA AAGATATAAC 4020
AGGAAGCTAA ACCGGTTCGG GTTATAAATG TTAGTATTTA TACCGGAGAC ATTTTGTGTT 4080
GCTAATTTTT GTATATGAGA AGTTCAATCC GGTTCGGTAA GCCCCTGAAC CAAACTAGAT 4140
TTGGAGATGA TATAAATATA TAAAATTTAT TTTTCATCCG GTTCGTTATT TTCATATAAA 4200
TATATAAATA TTATTTTTTA AATTTAAGAA TTAGATTTAC ATGTGAAAGT TACATTTCTG 4260
TTTATTTTCT TTGAAGTAAA ATGATAAAGG GAACGTATAT TAAGTTTCAT GCTTTATTCA 4320
CATAAGTTTT GTAATGTATA TTATATTTTT CGTTTATTGA AAAAGTAATT TTCAGTGTTC 4380
AGCATGTTTA CACTATAATT AAATCAAGTC GAATATTTCC TGGAACTATT CTCCTTGTTC 4440
TATAGCAAAT GAAAACGCTC TTCACAACAA AATCATTATA GATATAGGAA TAAATTACAT 4500
TAAAAACATG AAAGTCATAA TGAATATATT TTTTTAATTA GGATTTGATT TAAAAACAAT 4560
TATTGTATAC ATATAAAAGA CTTCTTTAGT TATTTGCCTT CAACTTCTCG TTCTGAATCA 4620
TGCGATAAAT CAGCTTTTTC AATAACTACG ACGTAAAAGC AAATTCATAA CACGTCTAAA 4680
CAAATTTGGC TCATCCTTCA CTTGATTGGT GTTTTCCGGA CTCGATGTTG CTGGAAACTG 4740
AGAAGAAGAA GGAATCTGCA TAATCACCTC TTGGTTCCTC ACCGGTAGAC TCATTTTGTT 4800
GGATCGAAAA CGATCGAGAT CAGAAAATGA AAAGATAGGT TAAAGATGCC TATGAATACA 4860
ACAACGTAAG ATTATGTTGA ATAAACAGAG TACTTTATAT AGGAGTTATA ATAAGGTAAA 4920
TAAATTATTG CTTTCCGCGT TTTTTACTTT TGTATTTCTT AAATGATAAG TTAAATTAGG 4980
ATAAGATTTG TATGATTTTA AGTAAATTTA CAATAACTCT CTATAACTCA ATAGCATCAC 5040 ATATTTAATT AATTTTACTA ATTATCTTTT GAACAATTTT ATGAAATAGT TTTCTTTTAA 5100
TTAATTTTTT AAAATGATAT ATTATAAAAT TTAATTGAAT CAATCTGATA TAATTTTTTT 5160
ATCTTCTACC ATCTATTATA GTTGATAAAT ATTGTGATAA ACTTTAGATA AACACCCAAT 5220
TGCCAAATAT TTAATAAATT TTGTGTACCA TGCGTTTTTT TTGGAGAATA TATATACGTG 5280
GACAGCATAC CGTACATATA TTGTATAAAA GCTTATAAAA CATAGATACG GGTTATATTG 5340
GTAAGCTATA AATATATGTA AACAATAGTA AGATATTACG TGTTGTGTCT AAATATGTGT 5400
TGCTTTAGAT ATTATGTATA TCTAATATAT TAAAATATCT TTTATTAACT AATATATTAT 5460
TTAAGAGAGA AAATTGGGAC ACTATTTTCT ATACAGTAAC TGTTTTCAAC TATAAACAGG 5520
AACCCTTGAT ATAATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATG 5580
TTTGGTATTA TTACATAAAC CTAAGAAACA AAATTCAATA TTCCTTTTTA CCTTATAAAA 5640
AACAATTAAA CATCACTAGA TATATTTATG CCCCACAATG AGCGAGCCAA TTGAGACTTG 5700
AGACTTGAGA TCCTTGTCAA CTACGTTTGC ATTTGTCGGC CCATTTTTTT TATTTTTTTT 5760
TTAAAGTGTC GGCCCGTTGC TTCTTCCGTT CAGATCAACC CTCTCGTAAT CAGAACAAAA 5820
CGGAAAACAA ACGAAAGAAC AATCAGATCC CTCTTTTTTT GCATAAACTA AATTCAACTT 5880
CTCTGCGTTT ATGTTGTAGA GGCAACCACG ATCACTACTA CGAAACAATA CAACGTCGTT 5940
GCTTGGAGTC CACGTAATCA AATCTACTCC AATGCTTTTA ATATCTTTCA CTTTAACCCA 6000
CGACTTTTCA AAACTGCTCT TTAAAACCCA TAACTCGTGA ACATCTTCTT GATCTTTGTT 6060
TGTCCACTGA CGAATAGCAC CTAGCTTCCC TTCGTATCTG ACTAATCCTG AGAAAACATC 6120
AGAGTTCGGA GTATGGAAGA AGGACCAAGT TTCGGTTTTG AGACAAAACC GGATCACATT 6180
GTTGTTCCGT GATATCCAAT GCAAGAACCC CGAAACTTGT ATCGGGTTGG AAAAAATTAA 6240
TCTGTCTGTT TTTGGTAGAC GCAAATTTTC TAATCTCTTC CAGGTAAACG AATCAGAATC 6300 GAAAACTTCG CACATAAAAG TTCTGTGATT CAAATGGTAG ATACCCCGAG ACATACACAT 6360
ACGCCGAGAC TGCGAAAGCC TTTGTATTTT ATACCGGAAA GGGTTCAATC CGATTACCGC 6420
TAAACCCAAT GACATATCCC AACCCTTCAC TTCTGGCTTT GGTATGACCT GATACTGTTT 6480
AGTGGTTGGT TTGAAGACTA TGTATCCACG TGATGGTTTT GTATACTTAA CACAAAGCAA 6540
TATCCCATGA CTTGCATCAC AAGCTTCGAT CTTTATCATT CCGGGTGGCA GAAAGTCGAT 6600
GGAGACTCCA TTGTTTTGTA AATCACTCCT CTCATGGACA AAACTGGTTC GAAGTTCGTG 6660
TCCTTTTACT ATGTAGTGTT GTATGAAGTA TCCCGAAATA CGATTGGTTC TAAGGAGATT 6720
AAGATTGACA AACCATGACT CGTAGCTTCT CTTGTTGCAC TCTTTATTCA GGAGCCTGAA 6780
TTTTCCGATT TTTGACGCCG GAAGATAAGA AAGAAATTCT TGGATCATGT CTTGATTTAT 6840
CACCGGAGAA CTCATGATCC TGTCGGGAAT AAAGAGATGA GCACGATCAC TGAATGAGAA 6900
ATGAAAAAAT CAGGATCGGT AGAGAACAAC TTATGATGAA TAAAGTGTTT ATATATCCTT 6960
TCTTTGTTTA AGGAAAGTAT CAAAATTTGC CTTTTTCTTC GCTAGTCCTA AAACAAACAA 7020
ATTAACCAAA AGATAAAATC TTTCATGATT AATGTTACTT GTGATACCTT AAGCCAAAAC 7080
TTTATCTTTA GACTTTTAAC CAAATCTACA GTAATTTAAT TGCTAGACTT AGGAAACAAC 7140
TTTTTTTTTT ACCCAACAAT CTTTGGATTT TAATTGTTTT TTTTTCTACT AATAGATTAA 7200
CAACTCATTA TATAATAATG TTTCTATCAT AATTGACAAT TCTTTCTTTT TAATAAACAT 7260
CCAGCTTGTA TAATAATCCA CAAGTCAATT TCACCATTTT GGCCAATTTA TTTTCTTATA 7320
AAAATTAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT 7380
AATTTCCATT TTCCATAGAT TTATCTTTCT TTTTATTTCC TTAGTTATCT TAGTACTTTC 7440
TTAGTTTCCT TAGTAATTTT AAATTTTAAG ATAATATATT GAAATTAAAA GAAGAAAAAA 7500
AACTCTAGTT ATACTTTTGT TAAATGTTTC ATCACACTAA CTAATAATTT TTTTTAGTTA 7560
AATTACAATA TATAAACACT GAAGAAAGTT TTTGGCCCAC ACTTTTTTGG GATCAATTAG 7620 TACTATAGTT AGGGGAAGAT TCTGATTTAA AGGATACCAA AAATGACTAG TTAGGACATG 7680
AATGAAAACT TATAATCTCA ATAACATACA TACGTGTTAC TGAACAATAG TAACATCTTA 7740
CGTGTTTTGT CCATATATTT GTTGCTTATA AATATATTCA TATAACAATG TTTGCATTAA 7800
GCTTTTAAGA AGCACAAAAC CATATAACAA AATTAAATAT TCCTATCCCT ACCAAAAAAA 7860
AAAATTAAAT ATTCCTACAG CCTTGTTGAT TATTTTATGC CCTACGTTGA GCCTTGTTGA 7920
CTAGTTTGCA TTTGTCGGTC CATTTCTTCT TCCGTCCAGA TCAACCCTCT CGTAATCAGA 7980
ACAAAAGGGG AAACAAACGT AAGAGGCAAA ATCCTTGTTT GTATGAACTA AGTTTAACTT 8040
CTCTGTGTTT AAGTTGTAGA GGCAAACATG ATCCCAACTA GAAAGCATTA CGACGTCGTT 8100
GCTTGGTATC CACGTAATAT GCTCTACTCC AATGCTTTCA ATATCTTTCA CTTTTTCCCA 8160
CGACTTTTCA AAACTGCTCT TTAAAACCCA TAATCTGTGA ACATCTTCTT GATTGTTGTT 8220
TATCCAGTGA CGAATAACAC CTAGCTTCCC TTCGTAGCTG ACTAACTCTG GGAATAAACC 8280
AACGTTTGGA GTATGTAAGA AAGACCAAGT TTCGGTTTTG GGACATAACC GGATCACATT 8340
GTGGTTCCAT GATCTCCAAT GCAAGAACCC TGAAGCTTGT ACCGGGTTTG AAAGAATTAG 8400
ACCGTCTGTT CTCGGTAGAC GCAAATTTTT TAATCTCTTC CACATAAACG AATCGGAATC 8460
AAAAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520
AAATATTTTA TACCGGAAAG GCTGCAATCC GGTTACCGTT AGACCTAATG ACTTATCACA 8580
ACTCCTCACT TTTGGGTTTG GTATGATCTG ATACTGTTTT GTTGTTGGTT TGCAGACTAT 8640
GTATTCCGGT ATTGGTCTTG TATCATTATA ACAAAGCAAT ATCCCATGAC GTGCATCACA 8700
AGCTTTGATC TTTACCTCTC CTTGTGGCAG AAAATCGATG GAGACTCCTT TGTTATCCAA 8760
ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820
GAAGTATCCA GAGATATTGT TGGTTCGATG GAGATTTAGG TTGACAAACC AAGACTCGTA 8880 GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAATTTT CCGATTTCGG ACCCCCGAAG 8940
ATAAGAAAGA ACCTCTTGGA TCGTGTCCTG ATTTATCACC GGAGAACTCA TGATCTTATT 9000
GGAAAAAAGA AAGAAAGAGA TGAGCACGAT CAGTGAATGA GATATATAGA AATCAGGATT 9060
GGTAGAGAAC CGACGATGAT GAATATACAA GTGTTTATAA GTATCACAAA TTGCCTTTTT 9120
CTTCGCTAGT CCCAAAACAA GCAAATTAAC CAAAGATAAA ATCTTCATTA ATGTTTTCCT 9180
TTTTCTTCGC CAGTCCCAGA TAAAAATATA TATAAAATAT TTCATTAGGT TACTTGTAGT 9240
ACCTTGAGCC CAAAGTTTCT CTTTTGACTT TTAACCAAAT TAACAGTAAT TTAATAGCTA 9300
GACTTAGAAA ACAACATTTT GTATATATAT TCTTTGACAT CAAAATTCAA CAATCTTTGG 9360
GTTTCTATAG TGTTTTTTTT CTTATTCTAA TAGATTACCA CTCATTATAT CATATACAAA 9420
GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480
ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540
CTTTCTTAGT TTAGTACTTT AAATTTTCAT ATATATAATT TATTAAAATT AAAAGTAAAA 9600
ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA AGAGCATTAA GTAATAAATA 9660
TTTTAGCTTT ATGAAAAAAA ATATCAAATC ACTGAAGACA TTTGTTGGCC TATACTCTAT 9720
TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA TATGATATCT CTCTAATTCT 9780
GGCGAAACGA ATATTCTGAT TCTAAAGATA GTAAAAATGA ATTTTGATGA AGGGAATACT 9840
ATTTCACACA CCTAGAAAGA GTAAGGTAGA AACCTTTTTT TTTTTGGTCA GATTCTTGTA 9900
TCAAGAAGTT CTCATCGAT 9919
(2) INFORMATION FOR SEQ ID NO:2 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5555 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (genomic)
(ill) HYPOTHETICAL: NO
(IV) ANTI-SENSE: NO
(IX) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 2787..3347
(D) OTHER INFORMATION: /product= "1st exon of NIMl"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION. 3427..4162
(D) OTHER INFORMATION: /product= "2nd exon of NIMl"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 4271..4474
(D) OTHER INFORMATION: /product= "3rd exon of NIMl"
(IX) FEATURE.
(A) NAME/KEY: exon
(B) LOCATION: 4586. -4866
(D) OTHER INFORMATION: /product= "4th exon of NIMl"
(IX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(2787..3347, 3427..4162, 4271. 4474, 4586..4866)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC 60
ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC CAAAGCCAGA AGTGAAGGGT 120
TGGGATATGT CATTGGGTTT AGCGGTAATC GGATTGAACC CTTTCCGGTA TAAAATACAA 180
AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA TCTACCATTT GAATCACAGA 240 ACTTTTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT GGAAGAGATT AGAAAATTTG 300
CGTCTACCAA AAACAGACAG ATTAATTTTT TCCAACCCGA TACAAGTTTC GGGGTTCTTG 360
CATTGGATAT CACGGAACAA CAATGTGATC CGGTTTTGTC TCAAAACCGA AACTTGGTCC 420
TTCTTCCATA CTCCGAACTC TGATGTTTTC TCAGGATTAG TCAGATACGA AGGGAAGCTA 480
GGTGCTATTC GTCAGTGGAC AAACAAAGAT CAAGAAGATG TTCACGAGTT ATGGGTTTTA 540
AAGAGCAGTT TTGAAAAGTC GTGGGTTAAA GTGAAAGATA TTAAAAGCAT TGGAGTAGAT 600
TTGATTACGT GGACTCCAAG CAACGACGTT GTATTGTTTC GTAGTAGTGA TCGTGGTTGC 660
CTCTACAACA TAAACGCAGA GAAGTTGAAT TTAGTTTATG CAAAAAAAGA GGGATCTGAT 720
TGTTCTTTCG TTTGTTTTCC GTTTTGTTCT GATTACGAGA GGGTTGATCT GAACGGAAGA 780
AGCAACGGGC CGACACTTTA AAAAAAAAAT AAAAAAAATG GGCCGACAAA TGCAAACGTA 840
GTTGACAAGG ATCTCAAGTC TCAAGTCTCA ATTGGCTCGC TCATTGTGGG GCATAAATAT 900
ATCTAGTGAT GTTTAATTGT TTTTTATAAG GTAAAAAGGA ATATTGAATT TTGTTTCTTA 960
GGTTTATGTA ATAATACCAA ACATTGTTTT ATGAATATTT AATCTGATTT TTTGGCTAGT 1020
TATTTTATTA TATCAAGGGT TCCTGTTTAT AGTTGAAAAC AGTTACTGTA TAGAAAATAG 1080
TGTCCCAATT TTCTCTCTTA AATAATATAT TAGTTAATAA AAGATATTTT AATATATTAG 1140
ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200
TTACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260
ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320
AAATTTATTA AATATTTGGC AATTGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380
CTATAATAGA TGGTAGAAGA TAAAAAAATT ATΛTCAGATT GATTCAATTA AATTTTATAA 1440
TATATCATTT TAAAAAATTA ATTAAAAGAA AACTATTTCA TAAAATTGTT CAAAAGATAA 1500 TTAGTAAAAT TAATTAAATA TGTGATGCTA TTGAGTTATA GAGAGTTATT GTAAATTTAC 1560
TTAAAATCAT ACAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AAAAGTAAAA 1620
AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTTTA 1680
TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740
GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA AGAGGTGATT 1800
ATGCAGATTC CTTCTTCTTC TCAGTTTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860
GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTACG TCGTAGTTAT 1920
TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980
GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040
ATTATGACTT TCATGTTTTT AATGTAATTT ATTCCTATAT CTATAATGAT TTTGTTGTGA 2100
AGAGCGTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAAATATT CGACTTGATT 2160
TAATTATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220
ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCCTTTATCA 2280
TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCTTAAAT 2340
TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400
ATATATTTAT ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460
TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACATTTATAA 2520
CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580
TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640
ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700
TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760
GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC 2813 Met Asp Thr Thr lie Asp Gly Phe Ala
1 5
GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861 Asp Ser Tyr Glu lie Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr 10 15 20 25
GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909 Asp Ser Ser lie Val Tyr Leu Ala Ala Glu Gin Val Leu Thr Gly Pro 30 35 40
GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957 Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser Phe Glu Ser Val Phe 45 50 55
GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005 Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp 60 65 70
GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053 Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser 75 80 85
TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101 Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn 90 95 100 105
AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT GCC AAG GAT TAC 3149 Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu lie Ala Lys Asp Tyr 110 115 120
GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC 3197 Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser 125 130 135
AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245 Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu 140 145 150
AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG 3293 Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu 155 160 165 GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC 3341 Val Leu Tyr Leu Ala Phe He Phe Lys He Pro Glu Leu He Thr Leu 170 175 180 185
TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397 Tyr Gin
TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTG GAC GTT GTA GAC 3450
Arg His Leu Leu Asp Val Val Asp 190 195
AAA GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA 3498 Lys Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He 200 205 210
TGT GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT 3546 Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He 215 220 225
GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594 Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu 230 235 240
GAG CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG 3642 Glu Leu Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu 245 250 255
GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC 3690 Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp 260 265 270 275
TCG GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC 3738 Ser Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr 280 285 290
AAT CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT 3786 Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn 295 300 305
GTG AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC 3834 Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn 310 315 320 CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG 3882 His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg 325 330 335
AAG GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA 3930 Lys Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala 340 345 350 355
TCA GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978 Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He Ala Lys Gin 360 365 370
GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026 Ala Thr Met Ala Val Glu Cys Asn Asn He Pro Glu Gin Cys Lys His 375 380 385
TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074 Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin Glu Asp Lys 390 395 400
CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122 Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala 405 410 415
GAT GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA G 4162
Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430
GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA 4222
AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT 4278
Val Ala Leu 435
GCT CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326 Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu He Ala 440 445 450
GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374 Glu Met Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp 455 460 465 CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422 Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro 470 475 480
TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470 Phe Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys 485 490 495
ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524
Thr
500
TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584
G TG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC 4629 Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu 505 510 515
GAC CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA 4677 Asp Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu 520 525 530
GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725 Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu 535 540 545
ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773 He Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu 550 555 560
GGA AAT TCG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC 4821 Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr 565 570 575
GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866
Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 580 585 590
GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 4926
TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 4986
ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 5046 ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA 5106
GAATCAAAGT GTGAAATAAT GTCAAATTGT TCATCTGTTG GATATTTTCC ACCAAGAACC 5166
AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226
TTCTTCCTTT AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286
AAGAGAACAC TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346
ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406
TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGCGTTCGC TGTCACTGAT AGACCAAGAG 5466
ACTGAAAGCT TTCACAAATT GCCCTCAAAT CTTCTGTTTC TATCGTCATG ACTCCATATC 5526
TCCGACCACT GGTCATGAGC CAGAGCCCAC TGATTTTGAG GGAATTGGGC TAACCATTTC 5586
CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG AATCAAATTC TTCCTTCTGA 5646
CTTGTGGAT 5655
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 594 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:3:
Met Asp Thr Thr He Asp Gly Phe Ala Asp Ser Tyr Glu He Ser Ser 1 5 10 15
Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser He Val Tyr Leu 20 25 30
Ala Ala Glu Gin Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu 35 40 45 Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr 50 55 60
Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80
Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala 85 90 95
Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu 100 105 110
Glu Leu Lys Glu He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125
Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro 130 135 140
Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160
Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He 165 170 175
Phe Lys He Pro Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp 180 185 190
Val Val Asp Lys Val Val He Glu Asp Thr Leu Val He Leu Lys Leu 195 200 205
Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220
Glu He He Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240
Leu Pro Glu Glu Leu Val Lys Glu He He Asp Arg Arg Lys Glu Leu 245 250 255
Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270 Ala Leu Asp Ser Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285
Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala 290 295 300
Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320
Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335
Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly 340 345 350
Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He 355 360 365
Ala Lys Gin Ala Thr Met Ala Val Glu Cys Asn Asn He Pro Glu Gin 370 375 380
Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin 385 390 395 400
Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala 405 410 415
Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430
Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met 435 440 445
Glu He Ala Glu Met Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu 450 455 460
Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480
He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala 485 490 495
Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510
Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525
Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540
Tyr Met Glu He Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn 545 550 555 560
Leu Glu Leu Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser 565 570 575
Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg 580 585 590
Arg
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:4:
He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15
Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30
Val His Tyr Ala Val Gin His Cys Asn 35 40 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 15
Asp Met Val Ser Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg 20 25 30
Thr Xaa Asp Gly Val Thr 35
(2) INFORMATION FOR SEQ ID NO: 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 :
He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30
Val His Tyr Ala Val Gin His Cys Asn 35 40
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1 5 10 15
Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15
Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30
Val His Tyr Ala Val Gin His Cys Asn 35 40
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val
1 5 10 15
Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15
Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30
Val His Tyr Ala Val Gin His Cys Asn 35 40
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 15
Asp Met Val
8
Figure imgf000114_0001
Figure imgf000115_0001
105/3
Figure imgf000116_0002
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule \3bts)
Figure imgf000116_0001
Figure imgf000117_0001
105/5
Figure imgf000118_0002
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule \3bιs)
Figure imgf000118_0001
Foπn PCT/RO/I34 (July 1992)
Figure imgf000119_0001

Claims

What Is Claimed Is:
1. An isolated DNA molecule comprising a NIM 1 gene.
2. An isolated DNA molecule according to claim 1 , comprising the nucleotide sequence set forth in SEQ ID NO:2.
3. An isolated DNA molecule of about 9.9kb which encodes the NIM1 gene product.
4. An isolated DNA molecule according to claim 1 , comprising the nucleotide sequence set forth in SEQ ID NO: 1.
5. An isolated DNA molecule of claim 1 , encoding the aminoacid sequence of the NIM1 gene product set forth in SEQ ID NO:2.
6. An isolated DNA molecule comprising a mutant gene of NIM1 of claim 1 , which is a nimi gene.
7. Clone BAC-04, ATCC Deposit No. 97543.
8. A chimeric gene comprising a promotor active in plant operably linked to a heterologuous DNA molecule encoding the aminoacid sequence of a NIM1 gene product.
9. A chimeric gene comprising a promotor active in plant operably linked to the heterologuous DNA fragment according to claim 3.
10. A chimeric gene comprising a promotor active in plant operably linked to a heterologuous DNA molecule encoding the aminoacid sequence set forth in SEQ ID NO:2.
11. A chimeric gene comprising a promotor active in plant operably linked to a heterologuous DNA molecule encoding the aminoacid sequence of a nimi gene product.
12. A recombinant vector comprising the chimeric gene of anyone of claims 8 to 11.
13. A recombinant vector according to claim 12, wherein said vector is capable of being stably transformed into a host cell.
14. A recombinant vector according to claim 12, wherein said vector is capable of being stably transformed into a plant, plant seeds, plant tissue or plant cell. 26. Use of an isolated DNA molecule according to claim 1 and variants thereof in a screening method for identifying compounds capable of inducing broad spectrum disease resistance in plants.
27. Use of a plant phenotyp according to claim 17 to identify an isolated gene fragment which allows expression of broad spectrum of disease resistance in plants.
28. Use of an isolated DNA molecule according to anyone of claims 1 to 5 to confer disease resistance to plant cells, plants and the progeny thereof.
29. Use of an isolated DNA molecule according to claim 6 to confer universal disease susceptibility to plant cells, plants and the progeny thereof.
30. Use of resistant plants and the progeny thereof according to claim 20 to incorporate the resistant trait into plant lines through breeding.
15. A plant expression cassette comprising a chimeric gene of anyone of claims 8 to 11.
16. A plant expression cassette comprising a chimeric gene of claim 8 to 10.
17. A plant expression cassette comprising a chimeric gene of claim 11.
18. A plant expression cassette according to claim 15 to 17 which expresses the chimeric gene continously or constitutively.
19. A plant, plant cells and the progeny thereof comprising the chimeric gene of anyone of claims 8 to 11.
20. A plant, plant cells and the progeny thereof comprising the chimeric gene of anyone of claims 8 to 10, which have a broad spectrum of disease resistance.
21. A plant, plant cells and the progeny thereof comprising the chimeric gene of claim 11.
22. A plant, plant cells and the progeny thereof of claim 19, wherein said plant is selected form the group consisting of gymnosperms, monocots, and dicots.
23. A plant, plant cells and the progeny thereof of claim 19, wherein said plant is a crop plant.
24. A plant, plant cells and the progeny thereof of claim 23, wherein said plant is selected form the group consisting of rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
25. Use of an isolated DNA molecule according to claim 1 , gene to confer disease resistance in plants.
PCT/EP1997/001218 1996-06-21 1997-03-10 Gene conferring disease resistance in plants and uses thereof WO1997049822A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU20261/97A AU719639B2 (en) 1996-06-21 1997-03-10 Gene conferring disease resistance in plants and uses thereof
IL12706697A IL127066A0 (en) 1996-06-21 1997-03-10 Gene conferring disease resistance in plants and uses thereof
JP10502146A JP2000512502A (en) 1996-06-21 1997-03-10 Genes that confer disease resistance on plants and their use
EP97908210A EP0923648A1 (en) 1996-06-21 1997-03-10 Gene conferring disease resistance in plants and uses thereof
BR9709925A BR9709925A (en) 1996-06-21 1997-03-10 Gene conferring disease resistance in plants and their uses
PL97330599A PL187851B1 (en) 1996-06-21 1997-03-10 Gene giving immunity from diseases among plants and application thereof
UA98126736A UA73713C2 (en) 1996-12-27 1997-10-03 Nim1 gene imparting plants a persistence to diseases and use thereof

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US2027296P 1996-06-21 1996-06-21
US2488396P 1996-08-30 1996-08-30
US3317796P 1996-12-13 1996-12-13
US60/033,177 1996-12-13
US77355996A 1996-12-27 1996-12-27
US3502297P 1997-01-10 1997-01-10
US08/773,559 1997-01-10
US60/024,883 1997-01-10
US60/035,022 1997-01-10
US60/020,272 1997-01-10

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BR (1) BR9709925A (en)
CA (1) CA2258576A1 (en)
HU (1) HUP9901749A3 (en)
IL (1) IL127066A0 (en)
PL (1) PL187851B1 (en)
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WO1999014350A1 (en) * 1997-09-15 1999-03-25 Institute Of Molecular Agrobiology Rank1, an ankyrin-repeat containing peptide from rice associated with disease resistance
EP1013767A1 (en) * 1998-12-22 2000-06-28 American Cyanamid Company Method of screening for agrochemicals
EP1038965A1 (en) * 1999-03-23 2000-09-27 American Cyanamid Company Method of screening for chemical compounds capable of inducing ERS in plants
WO2000028036A3 (en) * 1998-11-05 2000-11-09 Du Pont Disease resistance factors
WO2000070069A1 (en) * 1999-05-13 2000-11-23 Monsanto Technology Llc Acquired resistance genes in plants
WO2000071748A3 (en) * 1999-05-21 2001-05-31 American Cyanamid Co Ers-genes, method of screening for chemical compounds capable of inducing ers in plants
EP1019436A4 (en) * 1996-08-09 2002-09-18 Gen Hospital Corp ACQUIRED RESISTANCE TO NPR GENES AND THEIR USE
WO2001066755A3 (en) * 2000-03-06 2002-10-10 Syngenta Participations Ag Monocotyledonous plant genes and uses thereof
WO2003000898A1 (en) * 2001-06-22 2003-01-03 Syngenta Participations Ag Plant genes involved in defense against pathogens
US6504084B1 (en) 1999-04-23 2003-01-07 Pioneer Hi-Bred International, Inc. Maize NPR1 polynucleotides and methods of use
US6706952B1 (en) 1999-12-15 2004-03-16 Syngenta Participations Ag Arabidopsis gene encoding a protein involved in the regulation of SAR gene expression in plants
US7199286B2 (en) 1999-12-15 2007-04-03 Syngenta Participations Ag Plant-derived novel pathogen and SAR-induction chemical induced promoters, and fragments thereof

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Cited By (24)

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EP1019436A4 (en) * 1996-08-09 2002-09-18 Gen Hospital Corp ACQUIRED RESISTANCE TO NPR GENES AND THEIR USE
FR2757875A1 (en) * 1996-12-13 1998-07-03 Ciba Geigy Ag METHODS OF USING THE NIM1 GENE TO PROVIDE PLANT RESISTANCE TO VEGETABLES
NL1007779C2 (en) * 1996-12-13 1998-07-22 Ciba Geigy Ag Methods of using the NIM1 gene to confer disease resistance to plants.
WO1998026082A1 (en) * 1996-12-13 1998-06-18 Novartis Ag Methods of using the nim1 gene to confer disease resistance in plants
WO1999014350A1 (en) * 1997-09-15 1999-03-25 Institute Of Molecular Agrobiology Rank1, an ankyrin-repeat containing peptide from rice associated with disease resistance
US6512163B1 (en) 1997-09-15 2003-01-28 Institute Of Molecular Agrobiology RANK1, an ankyrin-repeat containing peptide from rice associated with disease resistance
WO2000028036A3 (en) * 1998-11-05 2000-11-09 Du Pont Disease resistance factors
EP1013767A1 (en) * 1998-12-22 2000-06-28 American Cyanamid Company Method of screening for agrochemicals
WO2000037651A3 (en) * 1998-12-22 2000-09-14 American Cyanamid Co Method of screening for agrochemicals
EP1038965A1 (en) * 1999-03-23 2000-09-27 American Cyanamid Company Method of screening for chemical compounds capable of inducing ERS in plants
US6504084B1 (en) 1999-04-23 2003-01-07 Pioneer Hi-Bred International, Inc. Maize NPR1 polynucleotides and methods of use
US6713665B2 (en) 1999-04-23 2004-03-30 Pioneer Hi-Bred International, Inc. Maize NPR1 promoter and methods of use in plant gene expression
US7030293B2 (en) 1999-05-13 2006-04-18 Monsanto Technology Llc Methods of inducing resistance in plants to fungal pathogens by transformation with acquired genes
US6506962B1 (en) 1999-05-13 2003-01-14 Monsanto Technology Llc Acquired resistance genes in plants
WO2000070069A1 (en) * 1999-05-13 2000-11-23 Monsanto Technology Llc Acquired resistance genes in plants
US7148398B2 (en) 1999-05-13 2006-12-12 Monsanto Technology Llc Acquired resistance genes in plants
US7294757B2 (en) 1999-05-13 2007-11-13 Monsanto Technology Llc Acquired resistance genes in plants
US7459605B2 (en) 1999-05-13 2008-12-02 Monsanto Technology Llc Acquired resistance genes in plants
US7465849B2 (en) 1999-05-13 2008-12-16 Monsanto Technology Llc Acquired resistance genes in plants
WO2000071748A3 (en) * 1999-05-21 2001-05-31 American Cyanamid Co Ers-genes, method of screening for chemical compounds capable of inducing ers in plants
US6706952B1 (en) 1999-12-15 2004-03-16 Syngenta Participations Ag Arabidopsis gene encoding a protein involved in the regulation of SAR gene expression in plants
US7199286B2 (en) 1999-12-15 2007-04-03 Syngenta Participations Ag Plant-derived novel pathogen and SAR-induction chemical induced promoters, and fragments thereof
WO2001066755A3 (en) * 2000-03-06 2002-10-10 Syngenta Participations Ag Monocotyledonous plant genes and uses thereof
WO2003000898A1 (en) * 2001-06-22 2003-01-03 Syngenta Participations Ag Plant genes involved in defense against pathogens

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BR9709925A (en) 1999-08-10
PL330599A1 (en) 1999-05-24
AU719639B2 (en) 2000-05-11
HUP9901749A2 (en) 1999-09-28
KR20000022203A (en) 2000-04-25
IL127066A0 (en) 1999-09-22
HUP9901749A3 (en) 2001-11-28
CN1228813A (en) 1999-09-15
PL187851B1 (en) 2004-10-29
CA2258576A1 (en) 1997-12-31
JP2000512502A (en) 2000-09-26
AU2026197A (en) 1998-01-14
EP0923648A1 (en) 1999-06-23
TR199802660T2 (en) 1999-04-21

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