Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • 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
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• Loci confening disease resistance have been identified in virtually every plant species examined. Genetic analysis of many plant-pathogen interactions has demonstrated that plants often contain single genes that confer resistance against specific pathogen races containing complementary avirulence (avr) genes (Flor. H.H. (1971 ) Annual Review of Phytophatology 9:275-296). Loss or mutation of the pathogen avr gene or host resistance (R) gene generally results in the loss of host plant resistance (Flor, supra; Kunkel, B.N., et al. (1993). Plant Cell 5:865-875; Salmeron, J.S., et al (1994) Plant Cell 6:51 1-520).
• avr complementary avirulence
• disease R genes have been isolated from diverse species such as tomato, Arabidopsis, tobacco, flax, barley and rice. Interestingly, some of these genes encode related protein motifs, suggesting a common role in signal transduction. For instance, the tomato Pto gene, conferring resistance to Pseudomonas syringae pv. tomato, encodes a serine-threonine protein kinase. suggesting a function for Pto in cellular signaling via protein phosphorylation.
• R genes are also known.
• the largest class of disease R gene products (including RPS2, Rpml, L6, and N ) carry leucine-rich repeats (LRR), cytoplasmic signaling domains and nucleotide binding sites ( ⁇ BS) (Bent, A.F., et al (1994) Science 265: 1856-18592; Staskawicz, B.J., et ⁇ (1995) Science 268:661-667; Whifham, S., et al. (1994) Cell 78: 1101-1 115).
• LRR leucine-rich repeats
• ⁇ BS nucleotide binding sites
• the tomato disease R gene CjV encodes a membrane- anchored extra-cytoplasmic glycoprotein carrying a LRR motif in the presumed extracellular domain (Jones, D.A., et al (1994) Science 266:189-193).
• the broad spectrum R gene mlo encodes a protein anchored by at least six membrane-spanning helices and represents a member of a novel protein family (Buschges. R., et al. (1997) Cell 88:695-705).
• the last class of disease R genes is represented by one gene, the rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae (Xoo) (Song, W., et al. (1995).
• Xa21 encodes a receptor like-kinase protein (RK) with an LRR in the presumed extracellular domain and is proposed to transduce an extracellular signal through activation of the intracellular kinase.
• RK receptor like-kinase protein
• Rice is the most widely-grown crop plant worldwide. Therefore, applied benefits of rice research are likely to have broad applications and impact worldwide.
• Bacterial leaf blight (BB) caused by the gram negative bacteria Xoo is the most destructive bacterial disease of rice in Asia and Africa. In susceptible cultivars, Xoo enters through the hydathodes, multiplies in the epithem and moves to the xylem vessels where the infection becomes systemic. In resistant cultivars, reduced growth of the avirulent race is reflected in lower pathogen populations, delayed movement of the avirulent bacteria in the leaf and reduced lesion development. Host resistance often involves rapid deposition of a lignin-like material followed by a sharp decrease in the rate of bacterial multiplication (Reimers, P.J., et al (1991) Physiological and Molecular Plant Pathology 38:39-55).
• avr genes corresponding to the R genes XaJO, xa5, and Xa7 have been cloned and are highly similar to the Xcv avrBs3 gene family.
• the identification of Xoo avr genes conesponding to specific rice R genes suggests that the avr gene product is directly recognized by the R gene product.
• XalO, xa5 and Xa7 have not yet been cloned, experimental proof of this hypothesis is limited.
• blight R genes have been characterized genetically but only two have been cloned: the Xa21 disease R gene and Xal which encodes a NBS-LRR type R gene (Song, W., et al. (1995). Science 270:661-667; Yoshimura, S; et al. (1998) PNAS, 95(4): 1663-8).
• the corresponding avr genes for Xa21 (named avrXa21) and Xal have not previously been identified.
• Transgenic plants carrying Xa21 show resistance to 29 and susceptibility to three diverse Xoo isolates (Song et al, supra: Wang. G., et al. (1996) Molecular Plant-Microbe Interactions 9:850-855).
• the mechanism by which the Xa21 gene can confer broad spectrum resistance is unknown.
• the Xa21 gene product recognizes a highly conserved Xoo determinant essential for pathogen fitness, reminiscent of the Bs2lavrBs2 interaction.
• the observed stability of the pepper R gene Bs2 in the field is thought to be due to the conservation of the pathogen avrBs2 gene product in Xanthomonas campestris pv. vesicatoria (Kearney, B., et al. (1990) Nature 346:385-386).
• t o Xa21 gene product may recognize unique determinants specific to each race of the pathogen.
• Figure 1 displays a representation of the open reading frames in the approximately 9 kb fragment of clone 10.78 (SEQ ID NO: l).
• This invention relates to avrXa21 polynucleotides and polypeptides, as well as transgenic plants and transgenic prokaryotes comprising Xa21 polynucleofides and polypeptides.
• the present invention provides for isolated nucleic acid constructs comprising at least one open reading frame (ORF) from a polynucleotide at least 70% identical to SEQ ID NO:l.
• ORF open reading frame
• the ORF(s) is at least 90% identical to SEQ ID NO: 1.
• the ORF(s) is identical to SEQ ID NO: 1.
• the present invention provides for a number of open reading frames within SEQ ID NO: 1. including an ORF from position 2407 to 4626, and ORF from position 839 to 2470. and/or an ORF the reverse complement of positions 6454 to 8157.
• An isolated nucleic acid construct comprising any of these ORFs is at least 70% identical to SEQ ID NO: 1, and more preferably, is substantially identical to SEQ ID NO: 1.
• the invention also provides for an isolated polypeptide that is at least 70% identical to a polypeptide encoded by SEQ ID NO: l that is at least 50 amino acids in length.
• the isolated polypeptides can be SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
• the polypeptides are at least 70% identical to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
• the invention provides for transgenic plants and prokaryotes canying a recombinant expression cassette comprising at least one open reading frame from a polynucleotide at least 70% identical to SEQ ID NO: 1.
• the expression cassette also comprises a plant promoter operably to at least one of the open reading frames within SEQ ID NO: 1.
• the plant promoter can be heterologous.
• the plant promoter can also be inducible or constitutive.
• the transgenic plant further comprises a second plant promoter operably linked to an Xa21 nucleic acid (e.g., SEQ ID NO:5). As described above, the second plant promoter can be heterologous, inducible or constitutive.
• the transgenic plants of the invention can include any of a number of open reading frames within SEQ ID NO:l, including an ORF from position 2407 to 4626, an ORF from position 839 to 2470, and/or an ORF the reverse complement of positions 6454 to 8157.
• the transgenic plants comprising any of these ORFs are at least 70% identical to SEQ ID NO: l, or more preferably, are substantially identical to SEQ ID NO:l .
• the present invention also provides for a transgenic prokaryote comprising a recombinant expression cassette comprising a promoter operably linked to at least one ORF from a polynucleotide at least 70% identical to SEQ ID NO: 1.
• the ORF(s) is substantially identical to SEQ ID NO: 1.
• nucleic acid sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
• promoter refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
• a "plant promoter” is a promoter capable of initiating transcription in plant cells.
• plant includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same.
• shoot vegetative organs/structures e.g. leaves, stems and tubers
• roots e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules
• seed including embryo, endosperm, and seed coat
• fruit the mature ovary
• plant tissue e.g. vascular tissue, ground tissue, and the like
• cells e.g. guard cells, egg cells, trichomes
• the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
• a polynucleotide sequence is "heterologous to " ' an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
• a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
• a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. New York, (1989) or Cunent Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette.
• a recombinant expression cassette may comprise polynucleofides combined in such a way that the polynucleofides are extremely unlikely to be found in nature.
• human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide.
• polynucleofides can be manipulated in many ways and are not limited to the examples above.
• a polynucleotide "exogenous to" an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like.
• Such a plant containing the exogenous nucleic acid is refened to here as a Ti (e.g. in Ar ⁇ bidopsis by vacuum infiltration) or R 0 (for plants regenerated from transformed cells in vitro) generation transgenic plant.
• Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
• nucleic acid In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or co-suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived. As explained below, these substantially identical variants are specifically covered by the term nucleic acid.
• polynucleotide sequence is transcribed and translated to produce a functional polypeptide
• codon degeneracy a number of polynucleotide sequences will encode the same polypeptide.
• nucleic acid “polynucleotide” and their equivalents.
• the terms specifically include those full length sequences substantially identical (determined as described below) with an polynucleotide sequence and that encode proteins that retain the function of the polypeptide (e.g., resulting from conservative substitutions of amino acids in the polypeptide).
• AvrXa21 refers to a gene(s) or gene product(s) that confers avirulence to a pathogen when the pathogen infects X ⁇ 21 -containing plants.
• an avirulence gene product such as avrXa21 , specifically confers avirulence to a host plant expressing the conesponding resistance gene product, e.g. Xa21.
• a “host plant” is defined as any plant species that can be infected by a given pathogen or that is capable of producing a resistance response to a given pathogen.
• a “defense response” can be monitored by lack of disease, as a hypersensitive response or by induction of pathogenesis-related proteins or mRNAs encoding pathogenesis proteins.
• the "reverse complement" of a polynucleotide sequence can be determined by reversing the order of the sequence and then generating the complement of the sequence. For example, the reverse complement of ATGC is GCAT.
• Phathogens include, but are not limited to. viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) 1988).
• nucleic acid sequences 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.
• the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum conespondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identit .
• a conservative substitution is given a score between zero and 1.
• the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:1 1-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
• the phrase "substantially identical.” in the context of two nucleic acids or polypeptides. refers to a sequence or subsequence that has at least 25% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 25%> to 100%.
• More prefened embodiments include at least: 25%o, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%. or 99%. compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. This definition also refers to the complement of a test sequence, when the test sequence has substantial identity to a reference sequence.
• BLAST BLAST using standard parameters
• a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, 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.
• Methods of alignment of sequences for comparison are well-known in the art.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988). by computerized implementations of these algorithms (GAP, BESTFIT. FASTA, and
• TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
• PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
• the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
• the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
• This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
• Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
• the final alignment is achieved by a series of progressive, pairwise alignments.
• the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
• HSPs high scoring sequence pairs
• initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 7. Acad. Sci. USA 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.
• Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
• each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
• amino acid sequences As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
• nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid.
• a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
• Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
• stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, highly stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
• T m thermal melting point
• Low stringency conditions are generally selected to be about 15-30 °C below the T m .
• the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50%) of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
• Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
• nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
• genomic DNA or cDNA comprising ANT nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here.
• suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions.
• a positive hybridization is at least twice background.
• Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
• Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
• a further indication that two polynucleofides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.
• the present invention provides the first identification of polynucleotide sequences encoding an avirulence gene determinants specific for Xa21.
• the polynucleofides encode gene products with avrXa21 activity, e.g. gene products that confer cultivar-specific interactions with cultivars expressing Xa21.
• the present invention also provides for avirulence gene products as well as transgenic plants and prokaryotes expressing polynucleotide sequences encoding such gene products.
• the avrXa21 avirulence determinants of the invention are secreted by avirulent Xoo bacterial pathogens and then interact with Xa21 gene products in the plant.
• the polynucleotide sequence carrying the avrXa21 gene determinants described herein is 9 kb long.
• Several of the ORFs in this sequence encode predicted proteins with homology to the type I secretion systems of prokaryotes. (see, e.g., Higgins, C. F. (1992). Annu. Rev. Cel Biol. 8: 67-113). Without wishing to be bound by any theory, it is believed that the components of this secretion system may play a role in secreting the actual avrXa21 determinant.
• the secreted avirulence determinant may also be encoded by the 9 kb polynucleotide.
• the polynucleofides and polypeptides of the invention have many uses.
• the polypeptides of the invention are useful tools as probes or ligands to identify the binding motifs of the corresponding Xa21 R gene product.
• the polypeptides can be used to identify avrXa21 - binding Xa21 variants created by gene shuffling (see, e.g., Ness, J.E., et al. (1999) Nat. Biotechnology 17:893-896; Stemmer, W.P. (1994) Nature 370:389-391 ; Stemmer, W.P.. (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751).
• the polynucleofides and polypeptides of the invention are useful in developing a two-component system to generate resistance response in plants in response to pathogen infection.
• nucleic acids may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a genomic DNA library. To construct 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. Methods for the isolation of Xa21 nucleic acids are taught in, e.g., U.S. Patent Nos. 5,859,339, 5,952,488 and 5,977,434 and in Song, W.Y. et al. (1995) Science 270(5243) 1804-6.
• the genomic library can then be screened using a probe based upon the sequence of a cloned avrXa21 gene disclosed here. Probes may be used to hybridize with genomic DNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide can be used to screen an expression library.
• the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques.
• PCR polymerase chain reaction
• 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 RNA in samples, for nucleic acid sequencing, or for other purposes.
• PCR Protocols A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J.
• 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.
• a cloned gene such as polynucleotides encoding avrXa21
• Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
• Bacterial expression systems for expressing the avrXa21 protein(s) are available in, e.g., E.
• kits for such expression systems are commercially available.
• Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
• the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art. however, some variation in this distance can be accommodated without loss of promoter function.
• the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the avrXa21 encoding nucleic acid in host cells.
• a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding avrXa21and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
• the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
• the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
• the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g.. c-myc.
• Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Ban virus.
• eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
• markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
• high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a avrXa21 encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
• the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
• the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
• the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
• Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of avrXa21 protein, which are then purified using standard techniques (see, e.g., Colley et ⁇ l., J. Biol Chem. 264: 17619- 17622 (1989); Guide to Protein Purification, in Methods in Enzymology. vol. 182 (Deutscher, ed.. 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g.. Morrison. J. Bad. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101 :347-362 (Wu et al, eds. 1983).
• Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing avrXa21.
• the transfected cells are cultured under conditions favoring expression of avrXa21. which is recovered from the culture using standard techniques identified below. 3. Preparation of recombinant plant vectors
• DNA sequence coding for the desired polypeptide for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
• a plant promoter fragment may be employed which will direct expression of the gene 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 V- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.
• Such genes include for example, ACT 11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No.
• the plant promoter may direct expression of nucleic acid in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e. inducible promoters).
• tissue-specific promoters examples include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones.
• the promoter is specifically induced by infection of a pathogen.
• Tissue-specific promoters can be inducible.
• tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue.
• tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
• a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
• tissue-specific promoters can also be used in the invention.
• promoters that direct expression of nucleic acids in leaves, roots or flowers are useful for enhancing resistance to pathogens that infect those organs.
• photosynthetic organ-specific promoters such as the RBCS promoter (Khoudi, et al, Gene 197:343, 1997), can be used.
• Root-specific expression of avrXa21 polynucleotides can be achieved under the control of the root-specific ANR1 promoter (Zhang & Forde, Science, 279:407, 1998).
• Any strong, constitutive promoters such as the CaMV 35S promoter, can be used for the expression of avrXa21 polynucleotides throughout the plant.
• a list of inducible promoters is provided in section 6 below.
• polyadenylation region at the 3'-end of the 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 (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene that 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 chlorosulfuron or
• DNA constructs of the invention 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 and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
• Microinjection 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).
• 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.
• Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et ⁇ l. Science 233:496-498 (1984), and Fraley et ⁇ l. Proc. N ⁇ tl. Ac ⁇ d. Sci. USA 80:4803 (1983) and Gene Transfer to Plants.
• 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 phenotype such as increased seed mass.
• Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, 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 nucleic acids of the invention can be used to confer desired traits on essentially any plant.
• the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Mains, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum. S
• 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.
• polypeptides of the invention Either naturally occuning or recombinant polypeptides can be purified for use in functional assays. Naturally occuning polypeptides can be purified, e.g., from bacteria such as Xoo. Recombinant polypeptides can be purified from any suitable expression system.
• Polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4.673,641 ; Ausubel et al, supra; and Sambrook et al, supra).
• polypeptides having established molecular adhesion properties can be reversible fused to the polypeptides.
• the polypeptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally the polypeptides could be purified using immunoaffinity columns. 6. Induction of non-specific plant disease resistance to pathogens
• the polynucleotides of the invention can be expressed in plants in coordination with Xa21 to create a two component system that triggers the plants' resistance reaction in response to specific stimuli.
• This system acts to limit a pathogen by inducing a hypersensitive response (HR) and/or other defense-related mechanisms via the stimulation of the Xa21 -specific resistance reaction when the protein interacts with avrXa21.
• HR hypersensitive response
• the approach involves the expression of avrXa21 in a plant that also expresses Xa21.
• both components When at least one of the two components are expressed under the control of an inducible promoter, both components will only be present in a plant cell when the promoter is active. Thus, in some embodiments, most or all of the cells will express only one of the components when not confronted with a pathogen infection.
• any cells containing both avrXa21 and Xa21 will induce a resistance response, including preferably, an HR.
• the resistance response will result in systemic acquired resistance (SAR) (see, e.g., Ross, A.F. Virology 14: 340-358 (1961); Ryals, et al . Plant Cell 8: 1809-1819 (1996)), thus preventing pathogen infection of plant cells that do not express both components.
• SAR systemic acquired resistance
• Either component or both can be expressed from an inducible promoter.
• Xa21 can be expressed from its native promoter or a constitutive promoter and avrXa21 can be expressed from an inducible promoter.
• a rice cultivar that carrys Xa21, such as IRBB21 is transformed with avrXa21 operably linked to an inducible promoter.
• both gene products can be expressed from inducible promoters.
• Preferred inducible promoters include those promoters that are specifically induced upon infection by a virulent pathogen.
• Selected promoters useful in the invention are discussed in PCT application WO 99/43824, and include promoters from: a. lipoxygenases (e.g., Peng et al, (1994) J. Biol Chem. 269:3755- 3761), b. peroxidases (e.g., Chittoor et al. (1997) Molec. Plant-Microbe Interact. 10:861-871), c. hydroxymethylglutaryl-CoA reductase, d. phenylalanine ammonia lyase, e.
• glutathione-S — transferase f. chitinases (e.g.. Zhu et al. (1991) Mol Gen. Genet. 226:289-296), and g. genes involved in the plant respiratory burst (e.g.. Groom et al. (1996) Plant J. 10(3):515-522).
• Example 1 This example shows the identification a virulent Xoo strain with a high conjugation frequency.
• strain DY8903 had tri-parental conjugation frequencies of 10 "5 and 10 "6 , respectively, using the pUFR027 (DeFeyter, R., et al. (1990) Gene 88:65- 72) vector and the vector with a 1 1 kb insert. These frequencies are sufficient for large- scale conjugation experiments.
• avrXa7, avrXalO and avrBs3 do not confer avirulence to DY89031 when inoculated into Xa21 plants. Therefore, we reasoned that another gene must confer Xa21 -specific avirulence.
• This example shows the construction of a cosmid library capable of high frequency conjugation.
• Complementation experiments also require a library that can be conjugated into the virulent recipient strain with high frequency. Since Xoo strain PXO99 is virulent on rice cultivars that lack Xa21 and is avirulent on rice cultivars that have an active copy of Xa21, it was hypothesized that strain PXO99 carried an avirulence gene capable of conferring avirulence on Xoo strains. Thus, a library of Xoo PXO99 genomic DNA was constructed in pUFR027 (DeFeyter. R., et al. (1990) Gene 88:65-72). Xoo genomic DNA from PXO99 was isolated by established procedures and partially digested with Sau3Al.
• the partially digested DNA was then size fractionated in a 0.7% agarose gel. Fragments between 7 to 15 kb were recovered, ligated to pUFR027 at the compatible BamHl restriction sites and amplified in E. coli.
• the Xoo library contains 10 4 clones with an average insert size of 8.8 kb.
• This example shows the identification of a candidate clone carrying the avrXa21 avirulence determinants.
• the Xoo library was conjugated into the normally virulent Xoo strain DY89031 via triparental mating on solid media.
• the recipient Xoo strain was grown on PSA (Tsuchiya, K... Mew, T. W., et al, (1982) Phytopathology 72:43-46) plates (without antibiotics) for 3 days previous to the mating.
• the donor and helper E. coli strains were grown on Luria agar (LA) (Miller, J. H. 1972. EXPERIMENTS IN MOLECULAR GENETICS. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.)plates with kanamycin (50 ug/ml) one day before the mating.
• the cells were concentrated by centrifugation at 13,000 rpm in a centrifuge for 2 minutes and re-suspended on 40 ⁇ l of NB.
• the concentrated mixture was then spot plated on PSA plates (12 spots/plate) and allowed to dry in flow hood for approximately 30 minutes and then incubated at 30° C for 48 hours.
• the bacteria were then plated on PSA plates with Kanamycin (25 ⁇ g/ml) + Cephalexin (20 ⁇ g/ ⁇ l) that were previously air-dried for two days. After approximately one hour of drying in the flow hood, the plates were incubated at 30° C for 4-5 days until singles conjugant colonies appeared.
• Transconjugants were single colony purified by growing them on PSA + Kanamycin (25 ⁇ g/ ⁇ l) + Cephalexin (20 ⁇ g/ ⁇ l). The clones were subsequently inoculated onto Xa21 -containing rice lines and assayed for lesion length according to the protocols discussed in Song, W., et al. (1995) Science 270:661- 667. Two thousand clones were inoculated. One of the clones. 10.78, consistently displayed a strong avirulent reaction and therefore was selected for further experiments.
• Example 4 This example shows further characterization of the Xa21 -specific avirulence of DY89031(10.78).
• DY89031(10.78) was inoculated on a variety of rice cultivars to measure its virulence relative to other Xoo strains. Results from those experiments showed that DY89031(10.78) developed lesion lengths comparable to the avirulent donor strain PXO99. Strains DY89031(10.78) and PXO99 were virulent on the Xa21 -lacking rice cultivar IR24, but were avirulent on the Xa21 -containing rice cultivar IRBB21. Recipient strain DY89031 as well as DY89031 carrying a random member of the conjugation library were equally virulent on both rice cultivars.
• DY89031(10.78) like strain PXO99, was virulent on IR24 but was avirulent on IRBB21. Further experiments revealed that DY89031(10.78) grew 100 fold less than the wild type DY89031 , and to the same levels as the avirulent strain PX099A, on IRRBB21 rice plants. Therefore, clone 10.78 was chosen as a likely to contain the avrXa21 avirulence determinants(s).
• This example shows sequence analysis of a clone carrying the avrXa21 avirulence determinants.
• avrXa21 gene product is a molecule secreted through a Type I secretion system (also known as the ABC export system).
• Bacterial ABC export systems for protein secretion require the ABC transporter and two accessory proteins: a membrane fusion protein and an outer membrane proteins (Fath, M. J., et al (1993) Microbiol. Rev. 57:995-1017).
• ABC transporters are membrane transporters comprising both eukaryotic and prokaryotic proteins for the export or import of a wide range of substrates such as peptides, sugars, proteins and antibiotics (Fath. et al, supra; Higgins, C. F. (1992). Annu. Rev. Cel Biol. 8: 67-113).
• the 9 kb clone encodes two predicted proteins with high homology to two of the components of an ABC export system, e.g., a membrane fusion protein and the ABC exporter itself.
• these two proteins are present in the same operon, along with the secreted protein itself (Duong F, et al. (1992) Gene 121(l):47-54; Fath, et al, supra; Letoffe, S.,et al. (1993) J. Bacteriology 175(22): 7321-7328).
• the gene coding for the outer membrane protein is normally not present in the same operon (Wandersman, C, and C. Delepelaire (1990) Proc. Natl. Acad. Sci. USA 87:317-322). Since other metalloproteases in bacteria are secreted by ABC transporters
• avrXa21 is a colicin V functional homologue.
• the E. coli Colicin V a 103 amino acid bacteriocin, is secreted by an ABC transporter.
• the protein has with no homology to any other toxin or bacteriocin in the database (Gilson, L, et al. (1990) EMBO J. 9(12):3875-3884).
• This bacteriocin has an unusual amino acid composition of 17%> glycine, 14%o Alanine and 10% serine and a double- glycine-type leader peptide on its N-terminus (Van Belkun, M. J., et al. (1997). Molecular Microbiology 23(6): 1293-1301). It is believed that this glycine, alanine and serine composition is an important characteristic for a small secreted peptide.
• the double- glycine-type leader peptide is believed to direct the secretion of Colicin V.
• ORFs contained in the 9 kb which encode predicted proteins similar to Colicin V. These predicted proteins are small and have a glycine, alanine and serine composition similar to colicin V. Some of these predicted proteins also contain double-glycine-leader type peptides. Therefore, it is possible that one of them may be the secreted avrXa21 protein.
• the cvaA homologue encodes avrXa21.
• either cvaA is non functional in the virulent strains, preventing secretion of a Xa21 ligand, or part of the cvaA gene is cleaved off and exported.
• the N terminal region of the Xoo cvaA is longer than the cvaA from Bordetella pertussis and E. coli, and may be cleaved and exported.
• the secreted avirulence determinant may not be encoded on the 9 kb fragment and/or may not be a protein.
• at least one ORF is required for proper expression of the avrXa21 -specific avirulence phenotype.
• Example 6 This example shows marker exchange mutagenesis disrupting ORFs of
• ORF(s) confers Xa21 -specific avirulence
• marker exchange mutagenesis was employed.
• Four ORFs (the CvaA homologue, the CvaB homologue, the ACE homologue and an ORF downstream of the CvaB homologue were knocked out by partial deletion and/or insertion of a kanamycin resistance gene cassette.
• a 2.9kb Hindlll/EcoRI fragment from clone 10.78 was subcloned into pUC18. Two internal Notl cutting sites were used to delete part of the gene and to insert the kanamycin resistance gene.
• ACE homologue For the ACE homologue, a 3.6kb BamHI/Kpnl fragment was subcloned into pBluescript SK. The internal RsrII site was used for the insertion of kan .
• the four mutant constructs were transformed into X. o. oryzae strain PXO99 by electroporation after the orientation of the inserted kan R gene was confirmed.
• the transformed cells were plated onto PS agar plates containing kanamycin. After three days of culture, 100-120 colonies for each construct were replica-transferred onto PS agar plates containing kanamycin or ampicillin to select recombinants with double crossover events. Out of 100-120 colonies transferred, 7-10 clones were confirmed to have the replacement of the wild type genes with an insertionally-mutated gene.
• PXO113, PXO112 and PXO99 are Philippine races 4, 5 and 6 of Xoo and avirulent on IRBB21.
• C21202, C21203 and C21303 are virulent Philippine strains of Xoo and DY89031, CK89021 and JW8901 lare virulent Korean strains of Xoo. No polymorphism between the avirulent and virulent Philippine strains was observed, though there was a polymorphism between Philippine and Korean strains.
• SEQ ID NO:2 cvaB ABC transporter
• SEQ ID NO:3 ABC membrane fusion a gcgctggacgggctgcgccaggcgttctacggcgaccatgctg
• G F G F A D V G V L W R ⁇ G Y 7797 gacatgccgccggcgcaactggccagcgaaaccgaccgcctgtgg D M P P A Q L A S E T D R L
• G G L L P A H L M G N M Q Q 7617 gacrggagcaatctgtgggatctgctgcagccctaccccggggcc

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