Classifications

• • 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

Definitions

• the present invention relates generally to plant molecular biology.
• it relates to nucleic acids and methods for conferring disease resistance in plants.
• Loci conferring disease resistance have been identified in many plant species. Genetic analysis of many plant-pathogen interactions has demonstrated that plants contain loci that confer resistance against specific races of a pathogen containing a complementary avirulence gene. Molecular characterization of these genes should provide means for conferring disease resistance to a wide variety of crop plants.
• Those plant resistance genes that have been characterized at the molecular level fall into four classes.
• the Pto gene confers resistance against Pseudomonas syringae that express the avrPto avirulence gene (Martin et al. Science 262:1432 (1993)).
• the predicted Pto gene encodes a serine threonine protein kinase.
• the tomato Cf-9 gene confers resistance to races of the fungus Cladosporium fulvum that carry the avirulence gene Avr9 (Jones et al. Science 266:789- 793 (1994).
• the tomato Cf-9 gene encodes a putatitive extracellular LRR protein.
• the RPS2 gene of Arabidopsis thaliana confers resistance to P. syringae that express the avrRpt2 avirulence gene (Bent et al. Science 265: 1856-1860 (1994)).
• RPs2 encodes a protein with an LRR motif and a P-loop motif.
• Bacterial blight disease caused by Xanthomonas spp. infects virtually all crop plants and leads to extensive crop losses worldwide.
• Bacterial blight disease of rice (Oryza sativa), caused by Xanthomonas oryzae pv. oryzae (Xoo), is an important disease of this crop. Races of Xoo that induce resistant or susceptible reactions on rice cultivars with distinct resistance (Xa) genes have been identified.
• One source of resistance (Xa21) had been identified in the wild species Oryza longistaminata (Khush et al. in Proceedings of the International Workshop on Bacterial Blight of Rice. (International Rice Research Institute, 1989) and Ikeda et al. Jpn J.
• Xa21 is a dominant resistance locus that confers resistance to all known isolates of Xoo and is the only characterized Xa gene that carries resistance to Xoo race 6. Genetic and physical analysis of the Xa21 locus has identified a number of tightly linked markers on chromosome 11 (Ronald et al. Mol. Gen. Genet. 236: 113-120 (1992)). The molecular mechanisms by which the Xa21 locus confers resistance to this pathogen were not identified, however.
• the present invention provides isolated nucleic acid constructs comprising an RRK polynucleotide sequence, which hybridizes to SEQ. ID. No. 1 or to SEQ. ID. No. 3 under stringent conditions.
• Exemplary RRK polynucleotide sequences are Xa21 sequences which encode an Xa21 polypeptide as shown in SEQ. ID. No. 4.
• the RRK polynucleotides encode a protein having a leucine rich repeat motif and/or a cytoplasmic protein kinase domain.
• the nucleic acid constructs of the invention may further comprise a promoter operably linked to the RRK polynucleotide sequence.
• the promoter may be a tissue-specific promoter or a constitutive promoter.
• the invention also provides nucleic acid constructs comprising a promoter sequence from an RRK gene linked to a heterologous polynucleotide sequence.
• heterologous polynucleotide sequences include structural genes which confer pathogen resistance on plants.
• the invention further provides transgenic plants comprising a recombinant expression cassette comprising a promoter from an RRK gene operably linked to a polynucleotide sequence as well as transgenic plants comprising a recombinant expression cassette comprising a plant promoter operably linked to an RRK polynucleotide sequence.
• transgenic plants comprising a recombinant expression cassette comprising a promoter from an RRK gene operably linked to a polynucleotide sequence
• transgenic plants comprising a recombinant expression cassette comprising a plant promoter operably linked to an RRK polynucleotide sequence.
• rice and tomato plants may be conveniently used.
• the invention further provides methods of enhancing resistance to
• the methods comprise introducing into the plant a recombinant expression cassette comprising a plant promoter operably linked to an RRK
• polynucleotide sequence The methods may be conveniently carried out with rice or tomato plants.
• plant includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
• plant organs e.g., leaves, stems, roots, etc.
• the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and
• heterologous sequence is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form.
• a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
• RRK gene is member of a new class of disease resistance genes which encode RRK polypeptides comprising an extracellular LRR domain, a
• an LRR domain is a region of a repeated unit of about 24 residues as shown in Figure 1 and found in Cf-9 and RLK5).
• a nucleic acid probe from an Xa21 gene detected polymorphisms that segregated with the blast (Pyricularia oryzae) resistance gene (Pi7) in 58 recombinant inbred lines of rice.
• the same probe also detected polymorphism in nearly isogenic lines carrying xa5 and Xa10 resistance genes.
• members of this class of disease resistance genes can be identified by their ability to be amplified by degenerate PCR primers which correpsond to the LRR and kinase domains.
• primers have been used to isolate homologous genes in tomato.
• Exemplary primers for this purpose are tcaagcaacaatttgtcaggnca a/g at a/c/t cc (for the LRR domain sequence GQIP) and taacagcacattgcttgatttnan g/a tcncg g/a tg (the kinase domain sequence HCDIK).
• Xa21 polynucleotide sequence is a subsequence or full length polynucleotide sequence of an Xa21 gene, such as the rice Xa21 gene, which, when present in a transgenic plant confers resistance to Xanthomonas spp. (e.g. , X. oryzae) on the plant.
• exemplary polynucleotides of the invention include the coding region of SEQ. ID. No. 3.
• An Xa21 polynucleotide is typically at least about 3100 nucleotides to about 6500 nucleotides in length, usually from about 4000 to about 4500 nucleotides.
• Xa21 polypeptide is a gene product of an Xa21 polynucleotide sequence, which has the activity of Xa21, i.e., the ability to confer resistance to
• Xanthomonas spp. Xa21 polypeptides are characterized by the presence of an extracellular domain comprising a region of leucine rich repeats
• Exemplary Xa21 polypeptides of the invention include SEQ. ID. No. 4.
• transgenes In the expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be "substantially identical" to a sequence of the gene from which it was derived. As explained below, these variants are specifically covered by this term.
• polynucleotide sequence specifically includes those full length sequences substantially identical (determined as described below) with an RRK gene sequence and that encode proteins that retain the function of the RRK protein.
• the above term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode proteins capable of conferring resistance to Xanthomonas or other plant diseases and pests on a transgenic plant comprising the sequence.
• Two polynucleotides or polypeptides are said to be "identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
• complementary to is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
• Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity.
• the segment used for purposes of comparison may be at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFTT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
• Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 60% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described above (preferably BESTFIT) using standard parameters.
• BESTFIT the programs described above
• One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
• Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95%.
• substantially similar share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
• Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
• a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
• a group of amino acids having amide-containing side chains is asparagine and glutamine
• a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
• a group of amino acids having basic side chains is lysine, arginine, and histidine
• a group of amino acids having sulfur-containing side chains is cysteine and methionine.
• Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine
• nucleotide sequences are substantially identical if two molecules hybridize to each other under appropriate conditions.
• Appropriate conditions can be high or low stringency and will be different in different circumstances.
• stringent conditions are selected to be about 5°C to about 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• stringent wash conditions are those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60°C.
• nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
• high stringency wash conditions will include at least one wash in 0.1X SSC at 65°C.
• Nucleic acids of the invention can be identified from a cDNA or genomic library prepared according to standard procedures and the nucleic acids disclosed here (e.g. , SEQ. ID. Nos. 1 or 3) used as a probe.
• Low stringency hybridization conditions will typically include at least one wash using 2X SSC at 65°C. The washes are preferrably followed by a subsequent wash using IX SSC at 65°C.
• a homolog of a particular RRK gene is a second gene (either in the same species or in a different species) which encodes a protein having an amino acid sequence having at least 25% identity or 45% similiarity to (determined as described above) to a polypeptide sequence in the first gene product. It is believed that, in general, homologs share a common evolutionary past.
• Figure 1 shows a comparison of leucine rich repeat proteins.
• Figure 2A-F show partial restriction maps of BAC and cosmid clones containing regions that hybridized to Xa21-specific probes.
• Figure 3 shows a restriction map of pB822, the most active copy.
• Figure 4 shows the results of assays measuring Xanthomonas resistance in transgenic plants comprising the Xa21 gene from the pB822 clone.
• Figure 5 shows the map position of TRK1.
• Figure 6 shows the map position of TRL1.
• This invention relates to plant RRK genes, such as the Xa21 genes of rice.
• Nucleic acid sequences from RRK genes, in particular Xa21 genes, can be used to confer resistance to Xanthomonas and other pathogens in plants.
• the invention has use in conferring resistance in all higher plants susceptible to pathogen infection.
• the invention thus has use over a broad range of types of plants, including species from the genera Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum,
• Heterocallis Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Zea, Avena, Hordeum, Secale, Triticum, and, Sorghum.
• Example section below which describes the isolation and characterization of Xa21 genes in rice, is exemplary of a general approach for isolating Xa21 genes and other RRK genes.
• the isolated genes can then be used to construct recombinant vectors for transferring RRK gene expression to transgenic plants.
• DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA
• oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library.
• genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
• cDNA library mRNA is isolated from the desired organ, such as leaf and a cDNA library which contains the RRK gene transcript is prepared from the mRNA.
• cDNA may be prepared from mRNA extracted from other tissues in which RRK genes or homologs are expressed.
• the cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned RRK gene such as rice Xa21 genes disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.
• the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the RRK and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
• PCR polymerase chain reaction
• PCR Protocols A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990), incorporated herein by reference.
• Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al. , Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al. , J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
• Isolated sequences prepared as described herein can then be used to provide RRK gene expression and therefore Xanthomonas resistance in desired plants.
• nucleic acid encoding a functional RRK protein need not have a sequence identical to the exemplified gene disclosed here.
• polypeptides encoded by the RRK genes like other proteins, have different domains which perform different functions.
• the RRK gene sequences need not be full length, so long as the desired functional domain of the protein is expressed.
• the proteins of the invention comprise an extracellular leucine rich repeat domain, as well as an intracellular kinase domain.
• Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art.
• the chains can vary from the naturally occurring sequence at the primary structure level by amino acid
• Modification can also include swapping domains from the proteins of the invention with related domains from other pest resistance genes.
• the extra cellular domain (including the leucine rich repeat region) of the proteins of the invention can be replaced by that of the tomato Cf-9 gene and thus provide resistance to fungal pathogens of rice.
• a DNA sequence coding for the desired RRK polypeptide will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
• An expression cassette will typically comprise the RRK polynucleotide operably linked to transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the RRK gene in the intended tissues of the
• a plant promoter fragment may be employed which will direct expression of the RRK in all tissues of a regenerated plant.
• Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
• constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.
• the plant promoter may direct expression of the RRK gene in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as "inducible" promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.
• promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
• the operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
• the endogenous promoters from the RRK genes of the invention can be used to direct expression of the genes. These promoters can also be used to direct expression of heterologous structural genes. Thus, the promoters can be used in recombinant expression cassettes to drive expression of genes conferring resistance to any number of pathogens, including fungi, bacteria, and the like.
• promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site.
• TATAAT TATA box consensus sequence
• promoter element with a series of adenines surrounding the trinucleotide G (or T) N G.
• a polyadenylation region at the 3'-end of the RRK coding region should be included.
• the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
• the vector comprising the sequences from an RRK gene will typically comprise a marker gene which confers a selectable phenotype on plant cells.
• the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
• DNA constructs may be introduced into the genome of the desired plant host by a variety of conventional techniques.
• the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment and microinjection of plant cell protoplasts or embryogenic callus, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
• the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
• Transformation techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984).
• Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987). Using a number of approaches, cereal species such as rye (de la Pena et al. , Nature 325:274-276 (1987)), corn (Rhodes et al. , Science 240:204-207 (1988)), and rice (Shimamoto et al. , Nature 338:274-276 (1989) by electroporation; Li et al. Plant Cell Rep. 12:250-255 (1993) by ballistic techniques) can be transformed.
• Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
• Agrobacterium is useful primarily in dicots, certain monocots can be transformed by
• Agrobacterium transformation of rice is described by Hiei et al, Plant J. 6:271-282 (1994).
• Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired RRK-controlled phenotype.
• Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the RRK nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al. , Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.
• Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
• the methods of the present invention are particularly useful for
• the RRK polypeptides may be expressed at times or in quantities which are not characteristic of natural plants.
• the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
• the effect of the modification of RRK gene expression can be measured by detection of increases or decreases in mRNA levels using, for instance, Northern blots.
• the phenotypic effects of gene expression can be detected by measuring lesion length as in plants. Suitable assays for determining resistance are described below.
• Plant genes may also be isolated using map-based cloning methods. This strategy consists of identifying DNA markers that are tightly linked to the gene or genes of interest.
• map-based cloning and physical analysis of large chromosomal regions is the availability of libraries containing large inserts of genomic DNA.
• Shizuya, H., et al. Proc. Natl. Acad. Sci. 89, 8794-8797 (1992)
• BAC bacterial artificial chromosome
• This system utilizes an F-factor-based vector and is capable of maintaining human genomic DNA fragments of >300 kb. DNA can be cloned with high efficiency, manipulated easily and stably maintained in E. coli. The following is a description of the use of this technique to isolate genes of the invention.
• IRRI International Rice Research Institute
• IR-BB21 carrying Xa-21 was used as the plant material.
• the plants were grown in the greenhouse for 3-5 weeks.
• Leaf tissue was harvested and washed with distilled water before grinding.
• High molecular weight DNA was extracted from rice tissue essentially as described by Hatano, S., et al., Plant Sciences, 83, 55-64, (1992) and Zhang, H.B., et al., Plant J. 7:175-184 (1994), with the following modifications: approximately 20 grams of leaf tissue was ground into powder using a cold mortar and pestle in liquid nitrogen.
• the powder was suspended by stirring in 200 ml cold nuclei-extraction (NE) buffer (1 MM spermidine, 1 mM spermine, 10 mM Na 2 EDTA, 10 mm Trizma base, 80 mM KCI, 0.5% Triton-X 100 and 0.4 M sucrose, pH 9.4).
• NE cold nuclei-extraction
• the mixture was filtered through two layers of cheesecloth into a GSA bottle and centrifuged at 1200 g at 4°C for 20 min. The supernatant was poured off and the nuclear pellet (pale green) was resuspended in 50 ml cold NE Buffer.
• the resuspended pellet was then filtered through an 80-micron sieve into a 50 ml tube to remove green tissue debris and then centrifuged at 1000 g for 10 min. The pellet was resuspended and centrifuged as above without passing through the 80-micron sieves.
• the nuclear pellet (about 5 ⁇ 10 8 nuclei/ml) was resuspended in 2.5 ml of SCE buffer (1M sorbitol, 0.1M NaCitrate, 60 mM EDTA, pH 7.0) and embedded in 2.5 ml 1 % low-melting-point (LMP) agarose (Ultrapure).
• 80 ⁇ l plugs were incubated in 25 ml ESP solution (0.5M EDTA, pH 9.3, 1 % sodium laurel sarcosine, 5 mg/ml proteinase K, Boehringer Mannheim) at 50°C for two days with one change of the buffer. Each plug contained about 5 ⁇ g DNA.
• ESP solution 0.5M EDTA, pH 9.3, 1 % sodium laurel sarcosine, 5 mg/ml proteinase K, Boehringer Mannheim
• Agarose plugs were dialyzed twice against TE (10 mm Tris-HCI and 1 mM EDTA, pH 8.0) plus 1 mm PMSF (phenylmethyl sulphonyl fluoride) at 50°C for one h, and then equilibrated with Hindin buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 and 1 mM dithiothreitol, pH 7.9) twice at room temperature for one hr. Plugs were melted at 65°C for 15 min and kept at 37°C for 5 min before partial digestion. Five to seven units of HindIII (NEB, USA) per plug were added to the DNA solution and incubated at 37°C for 30 min.
• TE 10 mm Tris-HCI and 1 mM EDTA, pH 8.0
• PMSF phenylmethyl sulphonyl fluoride
• the reaction was stopped by addition of 1/10 volume of 0.5 M EDTA, pH 8.0.
• Partially digested DNA was immediately loaded into a 0.8% LMP agarose gel with a pipette tip cut off to an inside diameter of 2 mm and separated by PFGE (CHEF DR II system, BioRad, USA).
• PFGE CHEF DR II system, BioRad, USA.
• Two different PFGE methods were used for the library construction. Firstly, the gel was subjected to electrophoresis at 15OV, using an 8 s initial and 8 s final switch time for 16 h at 14°C.
• the unresolved DNA ( ⁇ 200 kb) was focused into a thin band.
• the gel was subject to electrophoresis at 150V, ramped switching time from 60 to 90 s for 16 h at 14°C.
• the gel containing the partially digested DNA was cut and soaked in TE while the marker lanes of the gel were stained with ethidium bromide.
• the agarose slice containing fragments larger than 200 kb (the first PFGE method) or agarose slice containing 250-350 kb (the second method) was excised from the gel.
• the agarose slice was equilibrated in TE for 2 h at 4°C, placed in a 1.5 ml tube, melted at 65°C for 10 min, digested with Gelase (Epicentre, USA) (one unit of enzyme per 100 mg agarose) and incubated at 45°C for one hr.
• the DNA solution was directly used for the ligation reaction.
• the vector, pBeloBAC II was provided by Drs. H. Shizuya and M.
• This vector contains the lacz gene inserted into the vector pBAC108L. Shizuya, et al. (1992). A single colony was inoculated into 5 ml LB media containing 12.5 ⁇ g/ml chloramphenicol and grown at 37°C for 4-5 h before adding to 6 liters of LB media. The inoculum was grown for about 16 h at 37°C to an OD 600nm 1.3-1.5. The plasmid was isolated using Qiagen's plasmid maxi isolation kit (Qiagen, USA). Vector DNA was further purified by cesium chloride/ethidium bromide equilibrium centrifugation at 45,000 RPM for 60 h.
• the rotor was decelerated to 35,000 RPM for one hr. to allow the gradient to relax, using a fixed anger rotor 70.1 (Beckman, USA).
• the plasmid was digested with HindIII to completion and assayed by gel electrophoresis.
• Vector ends were dephosphorylated with HK phosphatase (Epicenter, USA) at 30°C for one hr., using 1 unit of the enzyme per 1 ⁇ g of vector ]DNA.
• the HK phosphatase was inactivated by heating at 65°C for 30 min.
• the ligation was carried out in a 100 ⁇ l volume in which about 40 ng of the
• size-selected rice DNA (about 85 ⁇ l) was ligated to 10 ng of HindIII-digested vector (1 ⁇ l) molar ratio of about 10 to 1 in vector excess) with 400 units of T4 DNA ligase (NEB, USA) at 16°C overnight. Before transformation, the litigation was dialyzed against TE in an ULTRAFREE-MC filter tube (Millipore, USA) at 4°C overnight.
• Transformation of competent E. coli D ⁇ 10B cells was carried out by electroporation using a Cell-Porator (GIBCO-BRL, USA) at the following settings: voltage: 400; charge rate: fast; voltage booster resistance: 4,000; capacitance: 330 ⁇ ; impedance: low. Thirteen ⁇ l of competent cells were mixed with 0.5-1.0 ⁇ l of ligation solution for each electroporation.
• cells were transferred to 1 ml SOC solution (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 MM NaCl, 2.5 mM KCI, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM Glucose, pH 7.0) and incubated at 37°C with gentle shaking (90-95 RPM,) for 45 min.
• SOC solution 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 MM NaCl, 2.5 mM KCI, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM Glucose, pH 7.0
• the cells were spread on LB plates containing chloramphenicol (12.5 ⁇ g/ml), X-gal (40 ⁇ g/ml) and IPTG
• the BAC clones were transferred to 384-well microtiter plates (Genetix, UK) containing 60 ⁇ l of LB freezing buffer (36 mM K 2 HPO 4 , 13.2 mM KH 2 PO 4 , 1.7 mM Citrate, 0.4 MM MgSO 4 , 6.8 mM (NH 4 ) 2 SO 4 , 4.4% v/v Glycerol, 12.5 ⁇ g/ml chloramphenicol, LB) and incubated at 37oC for 24 h. Since more than 95% of the colonies were still white on the second screen, only one screen was used in the subsequent experiments, and white colonies were directly picked to 384-well microtiter plates. The library was replicated in duplicate and stored in two different -80°C freezers.
• the BAC clones in each 384-well microtiter plate were replicated onto a Hybond N + filter (Amersham, USA).
• the filter was put into a plastic box containing LB/agar with 12.5 ⁇ g/ml chloramphenicol and the box was kept at 37°C overnight until the colonies were about 2-3 mm in diameter.
• Treatment of the filters was as described. Nizetic, D., et al., Nucl. Acids Res. 19, 182 (1990); Hoheisel, J.D., et al, Cell, 73, 109-120 (1993). Hybridization and washing conditions were the same as described in Hoheisel, et al. (1993). Probes were labeled using random primer extension. Feinberg, A.P. and Vogelstein, B., Anal. Biochem. 132, 6-13 (1983); Addendum 137, 266-267 (1984).
• the BAC library described above of consists of 11,000 clones.
• the library was constructed using two different approaches. A first half of the library having 7269 BAC clones was made with one size selection using a compression zone method as described in Ramsay, M. and Wicking, C., Protocols in Human Molecular Genetics, 197-221 (1991). A second half of the library having 3731 clones was made using double size-selection of partially digested DNA. Double size-selection failed, however, to increase the average DNA insert size. Apparently, there were small DNA molecules still present in the size-selected DNA solution (only 250-350 kb DNA isolated).
• High molecular weight DNA used to construct the BAC library was isolated from purified rice nuclei. Most of the chloroplasts and mitochondria were removed by low speed centrifugation ( ⁇ 1000 g). The low frequency of chloroplast or mitochondrial clones found in the inventive BAC library ( ⁇ O.3%) reduces the possibility of organellar/nuclear DNA co-ligation.
• the BAC library was used to construct a contiguous set of clones (contig) spanning the Xa21 locus.
• RAPD818 in Ronald, et al. were used to screen the BAC library.
• RG103 is found in 8 copies in the Xa21-containing line and hybridizes with 8 genomic HindIII DNA fragments in this line. All of these fragments are genetically and physically linked to the Xa21 disease resistance locus.
• pTA818 hybridizes with 2 DNA fragments and at least one of these fragments is linked to the Xa21 locus.
• the pTA818 sequences and four (out of eight) of the RG103 hybridizing sequences are over represented in this portion of the library.
• the other four RG103 hybridizing sequences are under represented.
• the DNA insert sizes of these clones ranged from 40 to 140 kb.
• the rice line, 1188 carrying the Xa-21 locus was used as the plant material for isolation of HMW DNA.
• 120 g 4-6 weeks old leaf tissue was harvested and ground into fine powder using a cold mortar and pestle in liquid nitrogen.
• the powder was then suspended by stirring in 800 ml cold H buffer [4 mM spermidine, 1 mM spermine, 10 mM EDTA, 10 mM Tris-HCl, 80 mM KCl, 0.5 M sucrose, 1mM PMSF (phenylmethyl sulphony fluoride, add just before use), 0.5% (v/v) Triton-X 100, 1/1000 (v/v) ⁇ -mercaptoethanol (add just before use), pH 9.5].
• 800 ml cold H buffer [4 mM spermidine, 1 mM spermine, 10 mM EDTA, 10 mM Tris-HCl, 80 mM KCl, 0.5 M sucrose, 1mM PMSF (pheny
• the mixture was filtered through an 80-micron sieve into GSA bottles and the pellet resuspended in 400 ml H buffer and filtered again. The two filtrate volumes were combined and centrifuged at 3500 rpm for 10 min at 4°C. The pellet was resuspended in 300 ml washing buffer (same as H buffer except PMSF and ⁇ -mercaptoethanol) and centrifuged at 3500 rpm for 10 min at 4°C. The pellet was washed two additional times until the color of the pellet was pale green.
• the pellet was resuspended in 40 ml washing solution and the nuclei were lysed by adding an equal volume of lysis buffer ( 2% Na laurel sarcosine, 100 mM Tris-HCl, 0.5 M EDTA, pH 9.5) containing 2 mg/ml proteinase K (Boehringer Mannheim). Proteins were removed by incubation at 50°C for 5 hr and then extraction of the solution (by gentle inversion) with an equal volume of phenol-chloroform-isoamyl alcohol (24:24:1) for 30 min at room temperature. The HMW DNA was precipitated by gently layering 1/10 vol. of 3M sodium acetate (pH 5.5), 2 vol. of ethanol and inverting several times.
• lysis buffer 2% Na laurel sarcosine, 100 mM Tris-HCl, 0.5 M EDTA, pH 9.5
• proteinase K Boehringer Mannheim
• the DNA was removed from the ethanol using wide-mouth pipette tips, washed with 70% ethanol, dried and dissolved into 1 ml of TE (10 mM Tris-HCl, 1mM EDTA, pH8.0) at 4°C overnight without shaking. Normally, 250 ug HMW DNA can be isolated from 120 g leaves.
• the partially digested DNA was fractionated on a sucrose density gradient of 5 to 40% by centrifuge in an SW27 rotor at 26,000 rpm at 20°C for 13 hr. 0.8 ml fractions (20 total) were collected by carefully placing a capillary tube at the bottom of the centrifuge tube and pumping out the gradient at a very slow speed. 20 ul of each samples was assayed on a 0.3% agarose gel at 2 V/cm gel length for 36 hr. DNA fractions with approximately 35-50 kb were pooled together. After diluting the sucrose with an equal vol. of H2O, the DNA was precipitated with 2 vol. ethanol. The partial fill in reaction was achieved using standard protocols.
• the cosmid vector, pHC80 was kindly provided by Dr. Scot Hulbert.
• Vector and insert DNA were ligated in a 2 to 1 molar ratio, at a final concentration of 0.8 ug/ul.
• the ligation reaction was carried out with 600 units of T4 DNA ligase (NEB, USA) at 16°C for overnight.
• the ligated DNA was in vitro packaged with GigapackII packaging extract (Stratagene, USA) and transfected into competent cell, E.coli NM554, according to the Stratagene manual.
• 61440 cosmid colonies (more than five genome equivalents) in 160 384-well microtiter plates were transferred onto Hybond N+ filters (Amersham, USA) in two type densities.
• the cosmid clones were replicated in low density (1536 colonies/11.5x15 cm filter) using manual replicators (Genetix, U.K.) and grown on LB/agar with 100 ug/ml ampicillin for overnight. Forty filters were made to cover the whole cosmid library.
• the cosmid clones were replicated in high density arrays using a Beckman BiomekTM robotic workstation and grown using the same method as above. Using 3 ⁇ 3 arrays, 3456 colonies were transferred onto an 8.5 ⁇ 12cm filter.
• a reference cosmid colony (containing the RG103 marker) was plated in the first position of each 3 ⁇ 3 grid. The remaining eight offset position were plated with colonies from eight microtiter plates of the cosmid library. In this case, 20 filters in size of 8.5 ⁇ 12 cm each can cover the whole library.
• the RG103 probe was mixed with the unique probe in a ratio of 1:4 to produce the reference pattern.
• Bacteria on the filters were lysed and fixed using the steaming water bath procedure with the following modification: colonies were placed face up on top of two pieces of 3 MM Whatman soaked in lysis solution (0.5 M NaOH, 1.5 M NaCl) for 4 min at room temperature, the plastic boxes containing the filters were incubated in a steaming water bath at 85 °C for 6 min and then the filters were transferred to 3 MM Whatman soaked in neutralization buffer (1 M Tris-HCl (pH7.4), 1.5 M NaCl) for 4 min. Proteins and cell debris were removed by submergence in 50 ml proteinase K solution (50 mM
• Tris-HCl pH8.5
• 50 mM EDTA pH8.0
• 100 mM NaCl 1 % (w/v)
• Hybridization was performed according to standard procedures as follows: filters were subjected to prehybridization solution (7% SDS, 0.5 M Na 2 PO 4 (pH 7.2), 1 mM EDTA, 100 ug/ml ssDNA) at 65°C for 2 hr to overnight. Probes were labeled using the random primer extension procedure and hybridization was performed at 65 °C with shaking overnight. The filters were washed briefly in (40 mM Na 2 PO 4 (pH 7.2), 0.1 % SDS) at room temperature and the filters were incubated in the same solution at 65 °C for 20 min with gentle shaking. B. RESULTS
• RG103 Three Xa21-linked markers (RG103, RAPD 248 and RAPD 818) were used to screen the cosmid library. Genomic Southern analysis showed that the copy numbers of these three markers in resistant lines are 8, 1 and 2 respectively (unpublished results). Six positive cosmid clones hybridizing with the RG103 marker were identified and confirmed by further Southern analysis. However, no positive clones were identified to contain RAPD248 and RAPD818.
• Figures 2A-2E are partial restriction maps of the cosmid clones.
• Figure 2F is a partial restriction map of the BAC clone.
• SEQ. ID. No. 1 It includes the promoter region, the predicted intron and a partial 3' sequence.
• SEQ. ID. No. 2 shows the predicted amino acid sequence. The predicted intron has been spliced out.
• the predicted amino acid sequence has revealed two features of the protein which indicate it is encoded by a member of the new class of plant disease resistance genes referred to here as RRK genes.
• the extracellular domain of the proteins encoded by these genes comprise a block of about 23 tandem leucine-rich repeats (LRR) with an average length of 24 amino acids.
• LRR motif has been implicated in protein-protein interactions and ligand binding in a variety of proteins.
• the extracellular domain also comprises a region between the LRRs and the signal peptide which contain a motif, SWNTS, which is conserved among a number of proteins, including, Cf-9, PGIP, and RLK5.
• the protein comprises a region with high sequence identity to receptor-like protein kinases (RLPKs) such as RLK5 and TMK1 (Walker et al. Plant J. 3:451 (1993); Chang et al. Plant Cell 4: 1263 (1992); Valon et al. Plant Molec. Biol. 23:415 (1993)) as well as the tomato resistance gene product, Pto (Martin et al. Science 262: 1432 (1993).
• the signal domain, the extracellular domain (including the LRR region), the transmembrane domain and the cytoplasmic kinase domain are identified in SEQ. ID. No. 2.
• Figure 3 is a restriction map of a second clone, pB822, which was used to construct the plasmid used in the transformation experiments described in Example 3, below.
• the Xa21 gene in this clone has also been sequenced (SEQ. ID. No. 3).
• the predicted amino acid sequence revealed the same motifs identified in SEQ. ID. No. 2.
• the protein kinase domain carries 11 subdomains containing 15 conserved residues diagnostic of protein kinases and is flanked by a 31 aa juxtamembrane domain (aa 677-707) and a C terminus domain.
• the presumed intron is located between the two highly conserved residues P and E (aa 892 and aa 893) in the putative catalytic domain.
• KID kinase interacting domain
• Arabidopsis KID is functionally analogous to the SH2 domain of animal proteins.
• this region act as a high affinity and specific binding site for proteins containing KID. Modification of the amino acid sequence of this region of Xa21 can thus be used to alter affinity for the KID protein and thus control intracellular signalling in response to ligand binding of the LRR domain.
• the Xa21 gene described above was used to transform rice plants to demonstrate that the genes could confer Xanthomonas resistance to susceptible plants.
• the gene was introduced into susceptible rice strains using a variation of the methods of Li et al , Plant Cell Rep. 12:250-255 (1993). Briefly, co-transformation was carried out using the hygromycin construct pMON410 (from Monsanto) and a bluescript vector containing the sequences of interest.
• the Kpn fragment of pB822 was cloned into the pTA818 vector, which is derived from Invitrogen vector per 1000 and contains the 1kb fragment RAPD818 (Ronald et al , supra). The resulting plasmid is referred to as pC822.
• the plants were selected on hygromycin (30mg/L) and then screened for resistance to Xoo race 6.
• Standard methods were used to test Xanthomonas resistance in the transformants.
• the assays were carried out according to the methods of Kaufman et al Plant Disease Rep. 57:537-541 (1973). Briefly, Xoo race 6 was grown on PSA plates for 3 days. The bacteria were scraped up, resuspended in water and the OD adjusted to 10 9 colony forming units per ml. Scissors were dipped in the suspension and leaves from the transformed plants (4 months post bombardment) were cut 5 cm from the tip.
• Plants were scored for the presence of lesions 11 days post inoculation.
• Figure 4 shows lesion length data from experiments using an expression vector comprising the gene from the pC822 clone.
• Individuals derived from independent transformants 106, -9, -22, -11, -17, -1, -12, -4, 16, and -29 carry the pC822 construct and showed increased resistance as compared to susceptible untransformed controls (IR24), as well as rice plants transformed with the vector (1-15).
• Xa21 sequence can be used to isolate RRK genes from other plant species using a degenerate primer or low stringency hybridization approach.
• This example describes the isolation and characterization of two RRK genes from tomato using a degenerate primer approach.
• PCR polymerase Chain Reaction
• the forward primers were taken from motifs conserved in the LRR region of Xa21 and several other plant proteins (e.g. , Cf-9, RLK5, and PG1P).
• the reverse primers were taken from motifs conserved in the Xa21 kinase domain and other plant serine-threonine kinase domains( e.g., RLK5, Pto, and Fen (Martin et al. Plant Cell 6: 1543-1552 (1994)).
• the degenerate primers used for amplification of PCR products were as follows:
• the PCR conditions were as follows (20 micro liter reaction): First cycle:
• the annealing temperature dropped down 1C every cycle. After the 20 cycles are finished, the reaction was incubated for 10 min at 72C .
• the PCR products were cloned and used as probes to a tomato cDNA library.
• the library was constructed from tomato cDNA thatwas primed with oligo DT primers, ligated to EcoRI adaptors and cloned into a lambda GT11 vector.
• TRKl Tomato Receptor Kinase 1
• TRKl is shown in SEQ. ID. No. 12.
• This clone is present as one or two copies in the tomato genome and onecopy maps to the short arm of chromosome 1 in the proximity of a resistance gene to
• the second clone TRLl (Tomato Receptor Like 1) is a 496bp
• TRLl maps within a few cM of men (see, Figure 6) a mutation on chromosome 3 that causes spotty necrosis on plants, typical of a defense phenotype.
• TRK1 and TRL1 genes that were isolated are important components of plant signal transduction pathways leading to a defense response. These genes are useful to engineer disease resistance in tomato and other plant species.

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