WO2004099417A1 - Sequences genetiques de genes d'avirulence de pathogenes vegetaux et utilisations de celles-ci - Google Patents

Sequences genetiques de genes d'avirulence de pathogenes vegetaux et utilisations de celles-ci Download PDF

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WO2004099417A1
WO2004099417A1 PCT/AU2004/000602 AU2004000602W WO2004099417A1 WO 2004099417 A1 WO2004099417 A1 WO 2004099417A1 AU 2004000602 W AU2004000602 W AU 2004000602W WO 2004099417 A1 WO2004099417 A1 WO 2004099417A1
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plant
rust
gene
avirulence
nucleic acid
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Peter Norman Dodds
Gregory James Lawrence
Michael Anthony Ayliffe
Jeffrey Graham Ellis
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Commonwealth Scientific And Industrial Research Organisation
Grains Research And Development Corporation
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Publication of WO2004099417A1 publication Critical patent/WO2004099417A1/fr

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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

  • the present invention relates generally to genetic sequences, and more particularly to genetic sequences of avirulence genes of plant pathogens such as rust.
  • the present invention also extends to uses of these genetic sequences to induce disease resistance in plants.
  • the present invention further provides for transgenic plants carrying the subject genetic sequences enabling the generation of disease resistant plants.
  • the present invention is particularly useful for developing disease resistance, particularly rust resistance, in crop or cereal plants.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
  • nucleotide sequence information prepared using the program Patentln Version 3.1. Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210>2, etc). The length, type of sequence (DNA) and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as ⁇ 400>1).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents Thymidine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymidine, S represents Guanine or Cytosine, W represents Adenine or Thymidine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymidine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • A represents Adenine
  • C represents Cytosine
  • G Guanine
  • T Thymidine
  • Y a pyrimidine residue
  • R represents a purine residue
  • M represents Adenine or Cytosine
  • rust resistance is very important for protection of crop plants from disease, and genes conditioning resistance to plant diseases have been investigated extensively in agriculture. Of particular economic importance are genes controlling resistance to rust and mildew. Rust is an especially significant problem amongst broad acre crops such as wheat, barley and cereal grains and is caused by infection with a class of fungi known as the Basidiomycetes. Although rust resistance genes are a potentially invaluable genetic resource in agriculture, the molecular basis of major gene resistance to rusts and many other fungi is still not well known, with only a very few exceptions.
  • rust resistance genes control specific recognition of the products of rust avirulence genes. This may occur by a direct protein-protein interaction between the products of the resistance gene and the avirulence gene, or it may be indirect.
  • the expressed avirulence protein may act as an enzyme in the formation of a product that is recognized by the resistance gene product.
  • plant breeders deploy resistance genes that match the avirulence genes present in the local strains ofthe pathogens.
  • the pathogen populations are dynamic and frequently new pathogenic strains arise by any number of means such as by mutation, recombination or accidental or natural introduction of new pathogenic strains.
  • the existing disease resistant varieties then become susceptible to the new pathogenic strains.
  • Plant breeders are then forced to develop new disease resistant varieties. At present, breeders generally use resistance genes that exist in any one plant species or its relatives as their pool of new resistance genes for that species. Many instances of genetic disease resistance in plants are characterized by a "gene-for-gene” interaction, in which a plant resistance (R) gene provides resistance to only those pathogen strains carrying a corresponding avirulence (Avr) gene. This relationship was first elucidated in the flax-flax rust system (Flor 1971), but has been observed in many other disease systems. A simple model to explain the "gene-for-gene” observation is that resistance genes encode receptors that recognise the direct or indirect products of pathogen avirulence genes (van der Biezen and Jones, 1998).
  • the avirulence genes Avr2, Avr4 and Avr9 were isolated from Cladosporium fulvum and shown to encode small secreted peptides of approximately 23 amino acid residues. These products are recognized by the products of the tomato resistance genes Cf-2, Cf-4 and Cf-9 respectively (van den Ackerveken et al., 1992; Joosten et al., 1994; Luderer et al., 2002).
  • the race-specific elicitor NIP1 a small protein of 60 residues secreted by the barley pathogen Rhynchosporium secalis, is encoded by the nip I gene of the fungus.
  • the NIP1 protein elicits defense responses in barley cultivars carrying the resistance gene Rrsl (Rohe et al., 1995).
  • the rice blast avirulence gene AVR-Pita was isolated from the pathogenic fungus Magnaporthe grisea.
  • the protein product of this gene has features typical of metalloproteases, in particular neutral zinc metalloproteases (Orbach et al., 2000).
  • the AVR-Pita avirulence product interacts directly with the corresponding gene product encoded by the resistance gene, Pi-ta from rice (Jia et al., 2000). None of these avirulence genes are from rust fungi.
  • the avirulence gene products are generally thought to be secreted from the fungus and act in the apoplast, outside ofthe plant cell membrane.
  • the cloning of the avirulence gene sequences of the present invention from rust fungi provides the means of generating transgenic plants with de novo, increased or otherwise enhanced rust resistance.
  • these genes induce defence responses if expressed in flax or tobacco along with the corresponding resistance genes.
  • the present invention extends to the use of these avirulence (Avr) and corresponding resistance (R) genes in combination to induce disease resistance responses in plants.
  • the present invention also permits the screening through genetic means to identify similar avirulence genes in other plant pathogens for use in developing or enhancing rust resistance in commercially and economically important plant species.
  • the application of knowledge of the molecular basis behind Avr and R gene action, and the specificity thereof offers the potential of a new source of genes produced by a variety of recombinant techniques.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding an avirulence product of a plant rust fungus.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding an avirulence product of a plant rust fungus which is capable of being recognised by a disease resistance gene product in a plant, particularly a disease resistance gene product in a crop or cereal plant.
  • the invention also provides an isolated nucleic acid molecule comprising a sequence of nucleotides selected from:
  • nucleotide sequence having at least 45% identity overall to a sequence selected from the group consisting ofthe coding regions ofthe 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-1, 2F2-J, 2F2-K and 2F2-L sequences;
  • nucleotide sequence capable of hybridising under at least low stringency conditions to at least about 20 contiguous nucleotides complementary to a sequence selected from the group consisting ofthe 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-1, 2F2-J, 2F2-K and 2F2-L sequences; (iv) a nucleotide sequence encoding a 2F2 protein; and
  • the present invention provides a gene construct comprising a nucleic acid molecule as described above, for example, an expression gene construct produced for expression ofthe avirulence product of a plant rust fungus in a bacterial, insect, yeast, plant, fungal or animal cell.
  • a further aspect ofthe invention contemplates an isolated cell comprising a non-endogenous nucleic acid molecule as described above, preferably wherein said nucleic acid molecule is present in said cell in an expressible format.
  • a further aspect ofthe invention contemplates a transformed plant comprising a nucleic acid molecule as described above introduced into its genome in an expressible format, particularly a plant which has increased disease resistance compared to an isogenic non- transformed plant.
  • the nucleic acid molecule may be co-expressed with a corresponding disease resistance gene in the plant.
  • This aspect ofthe invention clearly extends to any plant cells, tissues, organs or other plant parts, plant seeds, or progeny plants, cells, tissues, organs or other parts, that are derived from the primary transformed plant.
  • the present invention also provides a method of identifying a nucleic acid sequence which encodes an avirulence product of a plant rust fungus, which comprises
  • the method may further comprise the step of isolating the hybridised or amplified nucleic acid sequence.
  • This invention further contemplates a method of inducing a disease resistance response in a plant, which comprises the step of transforming the plant, or a cell, tissue, organ or other part thereof, with a nucleic acid molecule as described above to obtain expression of an avirulence product of a plant rust fungus in the plant.
  • the nucleic acid molecule may be co- expressed with a corresponding disease resistance gene in the plant.
  • the corresponding disease resistance gene may be either endogenous or non-endogenous in the plant.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding an avirulence product of a plant rust fungus.
  • the plant rust fungus is a pathogen of flax.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding an avirulence product of a plant rust fungus which is capable of being recognised by a disease resistance gene product in a plant, particularly a disease resistance gene product in a crop or cereal plant.
  • the avirulence product is capable of being recognised by a product of one or more of the disease resistance genes L, M, N or P which control host resistance to rust in flax, including the L5, L6, L7, L2 or L10 alleles.
  • flax includes any species of Linum, particularly cultivated flax, L. usitatisimum.
  • the avirulence product encoded by the isolated nucleic acid molecule preferably functions predominantly in the cytoplasm of a plant cell. Alternatively, it may function predominantly in the apoplast , i.e. be predominantly secreted from the cell or be located partly or entirely outside of the plant cell membrane.
  • the avirulence product encoded by the isolated nucleic acid molecule preferably comprises a signal sequence which functions for secretion of the protein, however the signal sequence may be removed to provide a truncated avirulence product designed for expression in the cytoplasm.
  • the present invention also provides an isolated nucleic acid molecule comprising a sequence of nucleotides selected from:
  • nucleotide sequence selected from the group consisting ofthe coding regions ofthe 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L sequences;
  • nucleotide sequence capable of hybridising under at least low stringency conditions to at least about 20 contiguous nucleotides complementary to a sequence selected from the group consisting of the 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G,
  • an isolated nucleic acid molecule as described above is at times hereinafter referred to as "an avirulence gene” according to the invention.
  • An “avirulence gene” more generally refers to a gene which, when present in a cell of a pathogen, functions to decrease the virulence or pathogenicity or ability to proliferate of the pathogen cell in or on at least one host cell or host plant or a part thereof, relative to the absence ofthe avirulence gene from the cell ofthe pathogen.
  • An “avirulence product” is the RNA and/or protein product encoded by an avirulence gene, particularly a protein product encoded by the gene. The gene would ordinarily, but not necessarily, be expressed to produce a protein product by transcription and translation of the gene.
  • the cell of the pathogen is commonly a fungal pathogen and particularly a rust fungus. It is thought that the avirulence gene may directly or indirectly produce a product that triggers a defence response in the host cell, particularly in the presence of a corresponding resistance gene in the host cell. However, the avirulence gene may also function heterologously in a host cell such as a plant cell to increase the level of resistance to the pathogen cell, even in the absence of a specific corresponding resistance gene.
  • avirulence genes include “avirulence-like genes” which are genes that encode proteins that are homologous to known avirulence proteins, but where the resistance gene product that corresponds to or interacts with the "avirulence-like protein” that is the product of the "avirulence-like gene” has not been identified. It will be readily understood that an avirulence gene may function to decrease the virulence ofthe cell ofthe pathogen in or on one host but not necessarily on all hosts or in all circumstances.
  • nucleotide sequences encoding an avirulence product of the flax rust fungus in accordance with the present invention correspond with the genomic sequences of SEQ ID NOs: 1, 2 and 3 as follows:
  • 2F2-A nucleotides 12084-12116, 12202-12268 and 12484-12984 of SEQ ID NO: 1
  • 2F2-B nucleotides 19427-19460, 19546-19612 and 19828-20328 of SEQ ID NO: 1
  • 2F2-C nucleotides 9256-9558, 9644-9710 and 9926-12426 of SEQ ID NO: 2
  • 2F2-D nucleotides 3600-3632, 3718-3784 and 4000-4500 of SEQ ID NO: 3.
  • the nucleotide sequence of the 2F2-A cDNA clone is set out in Figure 5, together with the predicted amino acid sequence ofthe 2F2-A transcript.
  • Figure 6 A is an amino acid sequence alignment of the predicted products of the flax rust 2F2-A, 2F2-B, 2F2-C and 2F2-D avirulence genes.
  • Figure 10 is a cDNA alignment ofthe flax rust 2F2-A, 2F2-B, 2F2-C and 2F2-D avirulence genes.
  • the 2F2-A to 2F2-D sequences are cDNA sequences (no introns), and include primer sequences at each end.
  • the coding sequence is from 104 to 553.
  • Sequences 2F2-E and 2F2- F are RT-PCR (i.e. cDNA) sequences.
  • the 5' UTR (82b ⁇ ) is underlined and the coding sequence is from 83 to 532.
  • Sequences 2F2-G to 2F2-L were amplified from genomic DNA and therefore include the intron sequences (bp 13-97 and 165-379; underlined in sequence 2F2-G).
  • the coding sequence is from 383-832.
  • avirulence product is used to refer to both the full-length amino acid product encoded by an avirulence gene, as well as to fragments thereof which have avirulence activity. This term also encompasses truncated avirulence products, particularly truncated products in which the signal peptide sequence (see Figure 6A) is removed.
  • the term "2F2 protein” includes products comprising an amino acid sequence selected from the sequences , 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L above, as well as products having at least 45% identity overall to such sequences.
  • the percentage identity is at least 50% or 60% more preferably at least about 70% or about 80%, and even more preferably at least about 90%.
  • the percentage identity overall of a nucleotide sequence to the 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L sequences is at least 50%, more preferably at least about 60%, or at least about 70%. Even more preferably, the percentage identity is at least about 80%, or at least about 90%.
  • a “low” stringency wash is defined herein as being in approximately 1 x SSC, 0.1-0.5% (w/v) SDS at 37-45°C for 2-3 hours.
  • alternative conditions of stringency may be employed such as "medium” stringent conditions which are considered herein to be a wash in about 1 x SSC, 0.25-0.5% (w/v) SDS at > 45°C for 2-3 hours or "high” stringent conditions as disclosed by Sambrook et al. (1989).
  • nucleic acid molecules comprising sequences encoding an avirulence product of the flax rust fungus
  • the present invention also extends in particular to nucleic acid molecules comprising sequences encoding avirulence products of other rust fungi of plants as set out, by way of example, in Table 2 (see also: Diseases of Field Crops, Dickson JG; Mc-Graw-Hill Book Company, New York, 1956. The Cereal Rusts, Volume II. Diseases, distribution, epidemiology and control. Edited by Roelfs AP and Bushnell WR. Academic Press, Orlando, US, 1985).
  • nucleic acid molecules comprising sequences encoding avirulence products of other rust fungi of plants can be identified by the genetic screening method described herein, using appropriate gene libraries and hybridisation probes or primers derived from the flax rust avirulence gene sequences or genetic markers linked thereto as disclosed hereinafter.
  • a gene construct comprising a nucleic acid molecule as described above, for example, an expression gene construct produced for expression of the avirulence product of a plant rust fungus in a bacterial, insect, yeast, plant, fungal or animal cell.
  • avirulence genes are derived from a naturally occurring avirulence gene by standard recombinant techniques. Generally, an avirulence gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.
  • Nucleotide insertional derivatives of the avirulence gene of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides.
  • Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more nucleotides from the sequence.
  • Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place.
  • substitutions may be "silent" in that the substitution does not change the amino acid defined by the codon.
  • substituents are designed to alter one amino acid for another similar acting amino acid. Typical substitutions are those made in accordance with the following: Suitable residues for amino acid substitutions
  • avirulence gene or a complementary sequence thereto, in a cell, requires said gene to be placed in operable connection with a promoter sequence.
  • the choice of promoter for the present purpose may vary depending upon the level of expression required and/or the tissue, organ and species in which expression is to occur.
  • Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence.
  • a promoter is usually, but not necessarily, positioned upstream, or at the 5 '-end, of the nucleic acid molecule it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting (i.e., the gene from which the promoter is derived).
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting (i.e., the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in gene constructs of the present invention include promoters derived from the genes of viruses, yeast, moulds, bacteria, insects, birds, mammals and plants, preferably those capable of functioning in isolated yeast or plant cells.
  • the promoter may regulate expression constitutively, or differentially, with respect to the tissue in which expression occurs. Alternatively, expression may be differential with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or temperature, or the presence of a pathogen.
  • the nature of the promoter depends on the desired outcome. For certain applications, it is preferable to express the avirulence gene of the invention specifically, in particular tissues of a plant, such as, for example, to avoid any pleiotropic effects that may be associated with expressing said gene throughout the plant. As will be known to the skilled artisan, tissue-specific or cell- specific promoter sequences may be required for such applications.
  • the promoter is an inducible promoter that is preferentially expressed in the presence of a plant pathogen on the plant, for example only in the presence of plant disease.
  • the inducible promoter may need to be tightly regulated to prevent unnecessary cell death and yet be expressed in the presence of the pathogen. A low level of expression in the absence of the pathogen may be acceptable, indeed may even pre-induce resistance in the plant.
  • the pathogen-inducible promoter may be any of those known in the art, for example the promoters from rust-induced genes such as the promoters of fisl from flax (Roberts et al 1995) and its maize and barley homologues (misl and bisl, Ayliffe et al 2002).
  • pathogen-inducible promoters include those from: the tobacco gene TobRB7 (Opperman et al 1994), which is induced during nematode infection; the tomato gene CEVI-1 (Mayda et al 2000) which is induced during infection by ToMV or citrus exocortis viroid; the potato prpl-1 gene (Martini et al 1993), which is induced during infection by Phytophthora infestans, Globodera spp (nematode and Glomus spp (mycorrhizae).
  • pathogen-inducible promoters are generally those from the Pathogenesis Related (PR) genes which are highly induced during incompatible interactions or in systemic acquired resistance (SAR), but are often also induced more weakly during compatible infections.
  • PR Pathogenesis Related
  • Some examples of these promoters which have been characterized include those from: the PRB-lb gene of tobacco (Eyal et al 1993) which is induced by various pathogens; AtPRBl, from Arabidopsis (Santamaria et al 2001) induced by ethylene and jasmonate signalling; bean chitinase (Roby et al 1990), which is induced during various fungal infections in transgenic tobacco; bean chalcone synthase (Stermer et al 1990) which is induced during Pseudomonas syringae infection in transgenic tobacco; a tobacco sesquiterpene cyclase gene (Yin et al 1997) induced by microbial pathogens; and tobacco H
  • PR class genes are induced in various infections/defense responses (Somssich and Hahlbrook, 1998).
  • genome-wide expression studies are now identifying new pathogen responsive genes (Wan et al., 2002) and the promoters of some of these genes may prove useful for controlling expression of rust-derived Avr genes.
  • the promoter may be expressed locally at or near the site of pathogen infection. Alternatively, the promoter may be wound inducible.
  • the promoter may be a weak promoter or modified to alter, particularly weaken, the expression level. One particular way to modify a promoter is to delete 5' portions such as enhancer elements.
  • promoters which are generally useful for expression in plants include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, the meristem-specific promoter (meril), napin seed-specific promoter, actin promoter sequence, sub-clover stunt virus promoters (International Patent Application No. PCT/AU95/00552), and the like.
  • cellular promoters for so-called housekeeping genes are useful. Promoters derived from genomic gene sequences described herein are particularly contemplated for regulating expression of avirulence genes, or complementary sequences thereto, in plants.
  • Inducible promoters such as, for example, a heat shock-inducible promoter, heavy metal- inducible promoter (e.g. metallotheinin gene promoter), ethanol-inducible promoter, or stress-inducible promoter, may also be used to regulate expression of the introduced nucleic acid ofthe invention under specific environmental conditions.
  • a heat shock-inducible promoter such as, for example, a heat shock-inducible promoter, heavy metal- inducible promoter (e.g. metallotheinin gene promoter), ethanol-inducible promoter, or stress-inducible promoter
  • heavy metal- inducible promoter e.g. metallotheinin gene promoter
  • ethanol-inducible promoter e.g. ethanol-inducible promoter
  • stress-inducible promoter e.g. stress-inducible promoter
  • the promoter is selected from the group consisting of: GAL1, GAL10, CYC1, CUP1, PGK1, ADH2, PH05, PRB1, GUT1, SP013, ADH1, CMV, SV40, LACZ, T3, SP6, T5, and 77 promoter sequences.
  • the gene construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant dominant- negative polypeptide gene product or alternatively, a co-suppression molecule, a ribozyme, gene silencing or antisense molecule.
  • terminal refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription.
  • Eukaryotic terminators are 3 '-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of poly(A) sequences to the 3 '-end of a primary transcript.
  • Terminators active in cells derived from viruses, yeast, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
  • terminators particularly suitable for use in the gene constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator ofthe Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences, subclover stunt virus (SCSN) gene sequence terminators (International Patent Application No. PCT/AU95/00552), and the terminator of the Flaveria bidentis malic enzyme gene meA3 (International Patent Application No. PCT/AU95/00552).
  • NOS nopaline synthase
  • CaMV Cauliflower mosaic virus
  • SCSN subclover stunt virus
  • the gene constructs of the invention may further include an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (e.g. plasmid or cosmid molecule) in said cell.
  • an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (e.g. plasmid or cosmid molecule) in said cell.
  • Preferred origins of replication for use in bacterial cells include, but are not limited to, the fl- ori and colEl origins of replication.
  • the 2-micron origin of replication may be used in gene constructs for use in yeast cells.
  • the gene construct may further comprise a selectable marker gene or genes that are functional in a cell into which said gene construct is introduced.
  • selectable marker gene includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct ofthe invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampicillin resistance (Amp r ), tetracycline resistance gene (Tc r ), bacterial kanamycin resistance gene (Kan r ), phosphinothricm resistance gene, neomycin phosphotransferase gene (npt ⁇ l), hygromycin resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.
  • the gene construct is a binary gene construct, more preferably a binary gene construct comprising a selectable marker gene selected from the group consisting of: bar, npt ⁇ l and spectinomycin resistance genes.
  • selectable marker genes selected from the group consisting of: bar, npt ⁇ l and spectinomycin resistance genes.
  • the binary construct comprises the Streptomyces hygroscopicus bar gene, placed operably in connection with the CaMN 35S promoter sequence. Still more preferably, the binary construct comprises the Streptomyces hygroscopicus bar gene, placed operably in connection with the CaMV 35S promoter sequence and upstream ofthe terminator sequence ofthe octopine synthase (ocs) gene.
  • ocs octopine synthase
  • a further aspect ofthe invention contemplates an isolated cell comprising a non-endogenous nucleic acid molecule as described above, preferably wherein said nucleic acid molecule is present in said cell in an expressible format.
  • cell shall be taken to include an isolated cell, or a cell contained within organised tissue, a plant organ, or whole plant.
  • the cell is a bacterial cell, such as, for example, E.coli ox A. tumefaciens, or cell of a plant, which may be monocotyledonous or dicotyledonous, such as flax or other species of Linum, or a grain crop or cereal plant such as wheat, barley, maize, rye, lupin or rice, or wild varieties of such plants.
  • the cell is an Agrobacterium tumefaciens strain carrying a disarmed Ti plasmid, such as, for example, the Agrobacterium tumefaciens strain designated AGL1.
  • the isolated nucleic acid of the present invention may be introduced to any cell and maintained or replicated therein, for the purposes of generating probes or primers, or to produce recombinant avirulence product, or a peptide derivative thereof. Accordingly, the present invention is not limited by the nature ofthe cell.
  • the present invention also extends to any plant material which comprises a gene construct according to any of the foregoing embodiments or expresses a sense, antisense, ribozyme, PTGS or co-suppression molecule, and to any cell, tissue, organ, plantlet or whole plant derived from said material.
  • a further aspect ofthe invention contemplates a transformed plant comprising a nucleic acid molecule as described above introduced into its genome in an expressible format, particularly a plant which has increased disease resistance compared to an isogenic non- transformed plant.
  • the nucleic acid molecule may be co-expressed with a corresponding disease resistance gene in the plant.
  • This aspect ofthe invention clearly extends to any plant cells, tissues, organs or other plant parts, plant seeds, or progeny plants, cells, tissues, organs or other parts, that are derived from the primary transformed plant.
  • endogenous refers to the normal complement of a stated integer which occurs in an organism in its natural setting or native context (i.e. in the absence of any human intervention, in particular any genetic manipulation).
  • non-endogenous shall be taken to indicate that the stated integer is derived from a source which is different to the plant material, plant cell, tissue, organ, plantlet or whole plant into which it has been introduced.
  • non-endogenous shall also be taken to include a situation where genetic material from a particular species is introduced, in any form, into an organism belonging to the same species as an addition to the normal complement of genetic material of that organism.
  • the plant material, plant cell, tissue, organ, plantlet or whole plant comprises or is derived from a crop or cereal plant as described herein, or a tissue, cell or organ culture of any of said plants or the seeds of any of said plants.
  • the present invention extends to the progeny and clonal derivatives of a plant according to any one ofthe embodiments described herein.
  • transformed plants are generally produced by introducing a gene construct, or vector, into a plant cell, by transformation or transfection means.
  • the isolated nucleic acid molecule ofthe invention especially the avirulence gene of the invention, or a gene construct comprising same, is introduced into a cell using any known method for the transfection or transformation of a plant cell.
  • a cell is transformed by the gene construct ofthe invention, a whole plant may be regenerated from a single transformed cell, using methods known to those skilled in the art.
  • transfect is meant that the avirulence gene or an antisense molecule, co-suppression molecule, PTGS, or ribozyme comprising sequences derived from the avirulence gene, is introduced into a cell without integration into the cell's genome.
  • a gene construct comprising said gene, said molecule, or said ribozyme, placed operably under the control of a suitable promoter sequence, can be used.
  • transform is meant the avirulence gene or an antisense molecule, co-suppression molecule, PTGS, or ribozyme comprising sequences derived from the avirulence gene, is introduced into a cell and integrated into the genome of the cell.
  • a gene construct comprising said gene, said molecule, or said ribozyme, placed operably under the control of a suitable promoter sequence, can be used.
  • Means for introducing recombinant DNA into plant cells or tissue include, but are not limited to, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, electroporation, microinjection of DNA, microparticle bombardment of tissue explants or cells, vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated transfer from Agrobacterium to the plant tissue.
  • transformed plants can be produced by the method of in planta transformation method using Agrobacterium tumefaciens, wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed.
  • Agrobacterium tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed.
  • A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed.
  • Those skilled in the art will be aware that the selection of tissue for use in such a procedure may vary, however it is preferable generally to use plant material at the zygote formation stage for in plant
  • microparticle is propelled into a cell to produce a transformed cell.
  • Any suitable biolistic cell transformation methodology and apparatus can be used in performing the present invention.
  • Stomp et al. U.S. Patent No. 5,122,466) or Sanford and Wolf (U.S. Patent No. 4,945,050) discloses exemplary apparatus and procedures.
  • the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • Exemplary microparticles suitable for use in such systems include 1 to 5 micron gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • a whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • existing meristematic tissue e.g., apical meristem, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
  • organogenesis means a process by which shoots and roots are developed sequentially from a meristematic centre.
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or Tl) transformed plant may be selfed to give homozygous second generation (or T2) transformant and the T2 plants further propagated through classical breeding techniques.
  • the avirulence gene of the present invention may be used to induce a disease resistance response in plants by co-expression with the corresponding resistance genes.
  • the avirulence gene of the invention may be used by itself to trigger a generalized defence response in a host plant in the absence of an added resistance gene, relying on one or more endogenous resistance genes in the plant which have some ability to recognize the avirulence product.
  • Such responses are similar to the systemic acquired resistance (SAR) response and are well known to those skilled in the art.
  • the present invention provides a method of inducing a disease resistance response in a plant, which comprises the step of transforming the plant, or a cell, organ or other part thereof, with a nucleic acid molecule as described above to obtain expression of an avirulence product of a plant rust fungus in the plant.
  • the nucleic acid molecule may be co-expressed with a corresponding disease resistance gene in the plant.
  • the expression "inducing a disease resistance response” is used to include de novo, increased or otherwise enhanced disease resistance in a plant.
  • the corresponding disease resistance gene may be either endogenous or non-endogenous in the plant.
  • the transformed plant co-expresses a flax rust avirulence gene with a corresponding flax resistance gene so as to induce a rust resistance response in the plant.
  • the transformed plant is preferably flax or other species of Linum, or a grain crop or cereal plant such as wheat, barley, maize, rye, lupin or rice, or wild varieties of such plant.
  • This aspect of the invention also extends to any plant cells, tissues, organs or other parts, or progeny plants, cells, tissues, organs or other parts, that are derived from the primary transformed plant.
  • the present invention provides a method of screening to identify avirulence genes in plant pathogens other than the flax rust fungus (Melampsora lini), by homology based methods using a probe or primer derived from the avirulence genes of the flax rust fungus disclosed herein, or alternatively a probe or primer derived from the highly conserved flax rust sec 14 homolog as a genetic marker for linked avirulence loci.
  • the present invention clearly encompasses within its scope, isolated nucleic acid molecules from plant pathogens, particularly rust fungi, other than flax rust fungus as specifically described herein, that encode avirulence products of those other plant pathogens, for example, nucleic acid molecules encoding avirulence products of other rust fungus diseases of plants as set out in Table 2.
  • the present invention provides a method of identifying a nucleic acid sequence which encodes an avirulence product of a plant rust fungus, which comprises
  • probe or primer comprises at least about 20 contiguous nucleotides of (a) a nucleotide sequence encoding an avirulence product of a plant rust fungus, particularly flax rust fungus, or a degenerate or complementary nucleotide sequence thereto, or (b) a nucleotide sequence which is genetically linked to a gene encoding an avirulence product of a plant rust fungus.
  • the method may further comprise the step of isolating the hybridised or amplified nucleic acid sequence.
  • the probe or primer comprises at least about 20 contiguous nucleotides of a nucleotide sequence selected from the group consisting ofthe 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L sequences.
  • the probe or primer may be based on the nucleotide sequence of the highly conserved sec 14 homolog, particularly a flax rust sec 14 homolog.
  • the plant pathogen sequence being identified may be present in a gene library, such as, for example, a cDNA or genomic gene library.
  • the library may be any library, such as, for example, a BAG library, YAC library, cosmid library, bacteriophage library, genomic gene library, or a cDNA library. Methods for the production, maintenance, and screening of such libraries with nucleic acid probes or primers, are well known to those skilled in the art.
  • the sequences of the library are usually in a recombinant form, such as, for example, a cDNA contained in a virus vector, bacteriophage vector, yeast vector, baculovirus vector, or bacterial vector. Furthermore, such vectors are generally maintained in appropriate cellular contents of virus hosts.
  • cDNA or genomic DNA may be contacted, under at least low stringency hybridisation conditions or equivalent, with a hybridisation-effective amount of a probe or primer derived from a nucleotide sequence selected from the group consisting of 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L sequences, or a complementary sequence thereto, or alternatively, with a probe or primer comprising a nucleotide sequence derived from a flax rust sec 14 homolog, and the hybridisation detected using a detection means.
  • a probe or primer derived from a nucleotide sequence selected from the group consisting of 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L
  • the detection means is a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotinylated molecule) covalently linked to the isolated nucleic acid molecule of the invention.
  • an identifiable signal e.g. a radioisotope such as 32 P or 35 S or a biotinylated molecule
  • Conventional nucleic acid hybridisation reactions are encompassed by the use of such detection means.
  • the detection means is any known format of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • degenerate pools of nucleic acid "primer molecules" of about 20-50 nucleotides in length are designed based upon any one or more of the nucleotide sequences disclosed herein, or a complementary sequence thereto.
  • related sequences i.e.
  • the "template molecule" are hybridised to two of said primer molecules, such that a first primer hybridises to a region on one strand ofthe double- stranded template molecule and a second primer hybridises to the other strand of said template, wherein the first and second primers are not hybridised within the same or overlapping regions of the template molecule and wherein each primer is positioned in a 5'- to 3'- orientation relative to the position at which the other primer is hybridised on the opposite strand.
  • Specific nucleic acid molecule copies of the template molecule are amplified enzymatically, in a polymerase chain reaction (PCR), a technique that is well known to persons skilled in the art.
  • PCR polymerase chain reaction
  • screening method of the present invention extends to use of probes or primers of only about 20 nucleotides in length, those skilled in the art will recognise that the specificity of hybridisation increases using longer probes, or primers, to detect genes in standard hybridisation and PCR protocols.
  • nucleotide sequences for probes or primers according to this embodiment of the invention will hybridise to at least about 30 contiguous nucleotides, more preferably at least about 50 contiguous nucleotides, even more preferably at least about 100 contiguous nucleotides, and still even more preferably at least about 500 contiguous nucleotides, derived from the 2F2-A, 2F2-B, 2F2-C, 2F2-D, 2F2-E, 2F2-F, 2F2-G, 2F2-H, 2F2-I, 2F2-J, 2F2-K and 2F2-L sequences or a degenerate or complementary sequence thereto, or a flax rust sec 14 homolog sequence.
  • Figure 1 A rust mapping family in which sixteen avirulence genes segregate at ten loci. The rusts C and H were crossed together to create the FI hybrid CH5 which was then selfed to generate an F2 family of 81 individuals (Lawrence et al., 1981).
  • the avirulence genes (A-Ll etc) that segregate in the F2 family are listed against the parent from which they were inherited. Where multiple avirulence specificities co- segregate at a single locus they are listed together (eg A-L5/L6/L7).
  • the A-N avirulence gene is present in both parents and it is not known which parent is the source of the allele that segregates in the F2 family.
  • the A-P and A-P1/P2/P3 specificities segregate as alternative alleles of a single locus.
  • Figure 2A Differential expression screen by microarray hybridisation.
  • Figure 2B A list of cDNA clones (identified by their positions on the 96 well stock plate arrays) that were selected for further analysis based on a relative expression ratio of about threefold difference between the two probes.
  • the IU2F2 entry which is derived from the A-L5/L6/L7 locus is shaded.
  • FIG. 3 Schematic diagram of the genomic DNA regions sequenced from the A- L5/A-L6/A-L7 avirulence locus in flax rust. Two sequence contigs of 26.5 and 17.5 kbp come from rust strain H, which is avirulent on flax containing the -L5, L6 or L7 resistance genes. The relative positions of lambda clones used for sequence analysis are indicated. A 11.5 kbp sequence contig from the virulent allele was derived by PCR-amplified DNA fragments as described in the text.
  • Kluyveromyces lactis Kl; P24859
  • C. glabrata Cg; P53989
  • Yarrowia lipolytica Yl; P45816
  • Schizosaccharamyces pombe Sp; NP_593003
  • FIG. 5 Nucleotide and predicted amino acid sequence of the 2F2-A transcript. The positions of two introns in the corresponding genomic sequence are indicated by numbered arrowheads. The underlined region is the sequence that was present in the IU2F2 cDNA clone. The positions of the 10-1.15 and 10-1.16 oligonucleotide primers (arrows) and the BamRl and Xba restriction sites (shaded boxes) used for preparation ofthe expression constructs are also shown.
  • Figure 6A Amino acid sequence alignment of the predicted products of the flax rust 2F2-A, -B, -C and -D avirulence genes. Only those amino acids that differ from the consensus (upper line) are shown, with identical residues indicated by a ".”. The position ofthe predicted signal peptide is also shown.
  • FIG. 7 Transient expression of avirulence genes in flax.
  • Agrobacterium cultures containing T-DNA vectors with 35-S-driven avirulence gene constructs were infiltrated into leaves of 4 week old flax plants.
  • the flax line Bison contains the L9 resistance gene, while the near isogenic lines B6xL5, B12xL6 and B6xL7 contain the L5, L6 or L7 resistance genes which have been backcrossed into Bison for 6, 12 or 6 generations respectively. Plant cell death was assayed 11 days post-infiltration.
  • An empty vector produced no response on any of the flax lines.
  • FIG. 8 Transient co-expression assays with the L6 resistance gene and 2F2-A rust avirulence gene. Co-infiltration of these two constructs into the rust susceptible cultivar, Hoshangabad, resulted in cell death of the infiltrated tissue, but either gene alone did not cause obvious cell death in Hoshangabad.
  • FIG. 9 Transient expression of avirulence genes in tobacco. Agrobacterium cultures containing T-DNA vectors with 35S-driven avirulence gene constructs or the L6 resistance gene from flax driven by its own promoter were infiltrated into leaves of tobacco plants. Plant cell death was assayed 11 days post-infiltration. Both 2F2-A and 2F2-C induced cell death when expressed in conjunction with the L6 resistance gene, but neither produced a response in the absence of L6. Expression of L6 alone also did not induce any response.
  • Figure 10 A cDNA alignment of the 2F2-A, 2F2-B, 2F2-C and 2F2-D sequences.
  • 2F2-A and 2F2-B sequences are from rust H (see SEQ ID NO: 1)
  • the 2F2-C sequence is from rust H (see SEQ ID NO: 2)
  • the 2F2-D sequence is from rust C (see SEQ ID NO: 3).
  • the start (nt 104) and stop (nt 554) codons are indicated in bold.
  • FIG. 11 Flax rust avirulence gene homologs occur in wheat leaf rust (Puccinia recondita). Probes from 2F2-A (left panel) or Sec 14 (right panel) were hybridised to DNA gel blots of genomic DNA isolated from rust-infected wheat plants digested with either Hindll or Xbal restriction enzymes. Both probes hybridise to similar fragments indicating that homologs of both genes are present in leaf rust, and are located on a small contiguous DNA segment.
  • FIG. 12 The 2F2 gene variants (A to L) present at each allele in various rust strains are shown. Rust strains 228 and are homozygous for avirulence on L6, L6 and L7 (A/A), while rust C is homozygous for virulence (a a) on these resistance genes and rust strains CH5 and 271 are heterozygous (A/a). Rust 339 is avirulent on L5, L6 and L7, but its genotype is not known and the AvrL567 genes in this strain have not been assigned to alleles. Rust strain WA was isolated from a native Australian L. marginale population.
  • FIG. 13 Alignment of polymorphic amino acids in the AvrL567 homologs.
  • the consensus amino acid at each position is shown above the sequences, and amino acids identical to the consensus are indicated by dots. Numbers above the alignment identify the amino acid position relative to the first methionine. The final columns indicate whether a necrotic response (+) was observed when these proteins were expressed in flax lines containing L5 L6 or L7. A ++ indicates a very strong necrotic response, while +/- indicates a weak response.
  • FIG. 14 RNA gel blot analysis of infiltrated flax leaves. RNA extracted from flax leaves six days after infiltration with Agrobacterium strains containing the 35S-2F2- A, B, etc expression constructs (lanes A to L) was separated on an agarose gel and hybridized to a 32 P-labeled 2F2-derived DNA probe.
  • RNA samples (5 ⁇ g) from uninfected flax leaves (F), rust CH5-infected flax leaves 5-8 days post-infection (5d-8d) and in vitro germinated CH5 rust spores (Sp) were separated on a 1.5% agarose gel, transferred to nylon membranes and hybridised with probes for the 2F2, Sec 14, and tubulin genes of flax rust.
  • the infected leaf samples contained largely flax RNA with an increasing proportion of rust RNA as the fungal biomass increased during infection.
  • the 2F2 probe was also hybridised to RNA from leaves infected with rust strain CH5-F2-89 (homozygous for the avirulence allele) or CH5-F2-112 (homozygous for the virulence allele).
  • RNA extracted from 50 mg of haustoria purified from flax leaves 6 days after infection with rust CH5 (Haus) was run alongside 2.5 ⁇ g RNA from rust CH5 infected leaves
  • RNA filters were stained with methylene blue to detect rRNA loading (top panel). Given the low amount of haustorial RNA, the 2F2 avirulence gene signal in haustoria reflected a considerable enrichment compared to the infected leaf RNA.
  • RNA from in vitro germinated rust spores (Sp) and infected flax leaves after 1-5 days (ld-5d) was reversed transcribed and amplified by PCR using primers specific for the 2F2, Secl4 or tubulin genes.
  • RT-PCR products were separated on a 2% low melting point agarose gel and visualised by ethidium bromide staining and UN irradiation.
  • Tissues were ground in liquid nitrogen in 1-2 gram batches and placed in 4 ml Urea buffer, which contains 7 M urea, 1% (w/v) sodium dodecylsulfate (SDS), 100 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (EDTA), and 100 mM ⁇ - mercaptoethanol (Sigma). These samples were extracted once with 4 ml of PCI, which is a 25:24:1 v/v mixture of phenol (Sigma) : chloroform (BDH chemicals) : isoamyl alcohol (BDH chemicals).
  • PCI which is a 25:24:1 v/v mixture of phenol (Sigma) : chloroform (BDH chemicals) : isoamyl alcohol (BDH chemicals).
  • nucleic acids were precipitated by the addition of 0.1 volumes of 3 M ⁇ a-acetate (pH5.5, BDH chemicals) and 1.0 volume cold ethanol and incubation at -20°C for 30 minutes. Nucleic acids were collected by centrifugation at 8000g, resuspended in 1.0 ml water and transferred to a 1.5 ml centrifuge tube.
  • PolyA RNA was isolated from total RNA using the PolyAtract kit (Promega, Madison WI) according to the manufacturer's instructions. Five to 10 ⁇ g of polyA RNA was isolated from 1.0 mg total RNA for each sample, precipitated with 0.1 volumes 3 M Na-acetate and 2.0 volumes ethanol as described above, and re-dissolved in water to a final concentration of 250-300 ⁇ g/ml.
  • Double stranded cDNA was synthesized using reagents from the Superscript Choice cDNA synthesis kit (Gibco BRL, Rockville MD). Two ⁇ g polyA RNA was mixed with 10 pmol of oligonucleotide primer Prl6 (5'-TTTTGTACAAGCT-3' (SEQ ID NO: 28), prepared on an Applied Biosystems 391 DNA synthesizer) in a volume of ll ⁇ l, denatured at 70°C for 10 min and placed on ice.
  • Prl6 5'-TTTTGTACAAGCT-3' (SEQ ID NO: 28)
  • coli DNA polymerase I (4 ⁇ l, 40 units), RNaseH (l ⁇ l, 2 units), 3 ⁇ l dNTPs (10 mM) and 92 ⁇ l water. This mixture was incubated at 16°C for 2 hours and then T4 DNA polymerase (2 ⁇ l, 10 units) was added followed by a further 5 minutes at 16°C before transfer to ice and addition of 5 ⁇ l 0.5 M EDTA. After extraction with 150 ⁇ l PCI, the double stranded cDNA was precipitated by the addition of 70 ⁇ l NH 4 -acetate (7.5 M, BDH chemicals) and 500 ⁇ l ethanol, collected by immediate centrifugation at 18000g for 20 minutes at room temperature. After washing in 70% ethanol and air drying for 10 minutes at room temperature, the cDNA was re-dissolved in 50 ⁇ l water. Preparation of driver and tester cDNAs.
  • driver and tester cDNA samples were prepared by restriction digestion ofthe double stranded cDNA.
  • the cDNAs (44 ⁇ l) were digested with the four-base blunt-end restriction enzyme Rsal (20 units, MBI Fermentas), then extracted with PCI and precipitated as described above and resuspended in 6.5 ⁇ l water. This sample constituted the driver cDNA.
  • Adaptor 1 upper strand 5'- GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3' (SEQ ID NO: 29); lower strand 5'-CCCGTCCA-3' (SEQ ID NO: 30); or Adaptor 2: (upper strand 5'- TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3' (SEQ ID NO: 31), lower strand 5'-ACCGCCCTCCG-3' (SEQ ID NO: 32). l ⁇ l of the driver cDNA was 'diluted with 5 ⁇ l water.
  • HEPES (Sigma), 2 M NaCl, 0.8 mM EDTA, pH 8.0) and 1.5 ⁇ l of tester cDNA with either adaptor 1 or adaptor 2. This was overlaid with mineral oil, denatured at 98°C for 1.5 minutes and incubated at 68°C for 9.5 hours in a Hybaid PCRExpress thermocycler. A third tube containing l ⁇ l driver cDNA, 2 ⁇ l water, l ⁇ l 4 x buffer and mineral oil was incubated at 98°C for 2 minutes and then cooled to 68°C. The contents of all three tubes were then mixed and incubated for a further 16 hours at 68°C to allow complete re-hybridisation of the cDNA samples. The renatured cDNA samples were diluted with lOO ⁇ l dilution buffer (20 mM HEPES, 50 mM NaCl, 0.2 mM EDTA, pH 8.3).
  • PCR cycling conditions were: 75°C 5 minutes, 94°C 30 seconds, followed by 30 cycles of 94°C 20 seconds, 66°C 30 seconds and 72°C 1.5 minutes.
  • Products ofthe first PCR round were diluted 1 in 10 in water and then l ⁇ l was re-amplified as above but using the primers PN1 : 5'- TCGAGCGGCCGCTCGGGCAGGT-3' (SEQ ID NO: 35) and PN2: 5'- AGGGCGTGGTGCGGAGGGCGGT-3' (SEQ ID NO: 36) and the following cycling conditions: 15 cycles of 94°C 20 seconds, 66°C 30 seconds and 72°C 1.5 minutes.
  • This reaction product constituted the subtracted cDNA samples.
  • the selectively amplified cDNAs were cloned using the pGEMT-Easy PCR cloning kit (Promega).
  • a 3 ⁇ l sample of the second round PCR reaction was mixed with 5 ⁇ l 2 x ligase buffer, 50 ng pGEMT-Easy plasmid and 2 units T4 DNA ligase and incubated at room temperature for 1-2 hours.
  • the ligated cDNA/plasmid solutions were precipitated with Na- acetate/ethanol, resuspended in lO ⁇ l water and then l ⁇ l was transformed into E.
  • Electrocompetent cells were prepared from a 500ml liquid 2xYT culture (16 g/1 Tryptone, Merck, 10 g/1 yeast extract, DIFCO, 5 g/1 NaCl) grown to OD600 of 0.3-0.5. The culture was chilled on ice and centrifuged at 5000 rpm for 15 minutes at 4°C, resuspended in 500 ml sterile water, spun again and resuspended again in 250 ml water.
  • the cell were resuspended in 10 ml 10% (v/v) sterile glycerol, spun again and resuspended in 1.5 ml 10% glycerol and dispensed into 40 ⁇ l aliquots stored at -70°C.
  • 500 ⁇ l liquid LB medium (10 g/1 Tryptone, 5 g/1 yeast extract, 10 g/1 NaCl) was added and 10-50 ⁇ l aliquots were spread on LB agar plates (LB plus 15g/l Agar, DIFCO) containing lOO ⁇ g/ml ampicillin (Sigma) to select for transformed cells.
  • 50 ⁇ l X-Gal BioVectra, 2% w/v in Dimethylformamide, BDH chemicals
  • 40 ⁇ l IPTG (2% w/v, Progen) was spread on the LB plates.
  • the cDNA inserts in the 1056 selected subtracted cDNA clones were amplified by PCR using the PN1 and PN2 primers.
  • the PCR conditions were: 50 ⁇ l volume, l ⁇ l E. coli culture as template, 10 pmol each primer, 0.1 mM dNTPs (Amersham), 2 M MgCl 2 , 0.05% w/v Tween-20 (Sigma), 50mM KC1, 1 unit Taq polymerase; cycle parameters: 94°C 2 minutes, followed by 30 cycles of 94°C 20 seconds, 68°C 30 seconds and 72°C 1 minute.
  • the H-C or C-H subtracted cDNA samples (described above) were labeled with 32 P-dCTP as follows. Five ⁇ l of the subtracted cDNA was first digested with Rsal to remove the PN1 and PN2 ends. Then 6 ⁇ l of random 9mer oligonucleotides (0.1 mg/ml) and water was added to a final volume of 18 ⁇ l and the DNA denatured by placing in a boiling water bath for 2 minutes.
  • 5 x buffer 1.0 M H ⁇ P ⁇ S, 0.25 M Tris, 25 mM DTT (Sigma), 25 mM MgCl 2 , 0.1 mg/ml Bovine serum albumin (BSA, Sigma) and 1.0 mM each dATP, dGTP and dTTP (Amersham), l ⁇ l Klenow (2 units; MBI Fermentas ) and 5 ⁇ l ⁇ - 32 P-dCTP (>1 mCurie/ml; Amersham) were added and the mixture incubated at 37°C for 1-2 hours.
  • 5 x buffer 1.0 M H ⁇ P ⁇ S, 0.25 M Tris, 25 mM DTT (Sigma), 25 mM MgCl 2 , 0.1 mg/ml Bovine serum albumin (BSA, Sigma) and 1.0 mM each dATP, dGTP and dTTP (Amersham), l ⁇ l Klenow (2 units; MBI Fermentas ) and 5 ⁇ l ⁇ - 32
  • filters were washed at 68°C twice for 15 minutes in 1 x SSC, 0.1% SDS and twice in 0.2 x SSC, 0.1% SDS. Washed filters were exposed to X-ray film (Fuji) at -70°C with an intensifying screen for 24 hours.
  • PolyA RNA 250 ng or total RNA (25 ⁇ g) was mixed with 1 ⁇ g of an oligodT23 primer with an A/C/G degenerate 3' nucleotide in a 23 ⁇ l volume and denatured at 70°C for 10 minutes then transferred to ice.
  • First strand cDNA was synthesized using the Superscriptll system by adding 8 ⁇ l 1 st strand buffer, 4 ⁇ l 0.1 M DTT, 2 ⁇ l 10 M dNTPs, 2 ⁇ l water and l ⁇ l superscriptll RT (200 units) and incubating at 42°C for 1 hour. Then 2 units of RNase H (4 units) was added followed by a 30 minute incubation at 37°C.
  • TE 160 ⁇ l TE was added and the cDNA purified and concentrated using a Microcon YM-30 filter (Millipore) by spinning at 13000rpm for 5 minutes, adding a further 200 ⁇ l TE, spinning again and adjusting the final volume to 8 ⁇ l by the addition of TE.
  • 5.5 ⁇ l water, 2 ⁇ l 10 x Klenow buffer (USB), l ⁇ l random 6mer oligonucleotides (USB) and the mixture was denatured at 95°C for 3 minutes and re- annealed at room temperature for 5 minutes.
  • the labeled probes were resuspended in 20 ⁇ l hybridisation mix - 5 x SSC, 0.1% SDS, 25% formamide (BDH chemicals), 1.5 mg/ml ssDNA (Sigma) - and denatured for 5 minutes at 95°C.
  • Slides were prehybridised in 5 x SSC, 0.1% SDS, 25% formamide, 10 mg/ml BSA at 42°C for 1 hour, rinsed in water and then spun dry at lOOOrpm for 2 minutes. The probe solution was then added and a cover slip placed over the printed area and the slides were sealed inside a hybridisation chamber and incubated in a 42°C water bath for 16 hours.
  • the slides were washed in the following solutions: a brief rinse in 2 x SSC, 0.1%SDS to remove the cover slips; 10 minutes at 42°C in 2 x SSC, 0.1%SDS; 10 minutes at room temperature in 0.1 x SSC, 0.1% SDS; 4 washes of 2 minutes each at room temperature in 0.1 x SSC.
  • the slides were then spun dry (2 minutes, 1000 rpm) and read using a fluorescence laser scanner. The scanned images were analysed using Genepix software
  • Plasmids were isolated from 1.5 ml E. coli cultures, grown in LB medium, as follows. After 16 hours growth at 37°C, cells were collected by centrifugation at 18000g and resuspended in lOO ⁇ l GT ⁇ (50 mM glucose, 10 mM Tris, 1 mM ⁇ DTA, pH 8.0) and lysed by the addition of 200 ⁇ l 0.2 N NaOH, 1% SDS (room temperature 5 minutes). Cell debris was precipitated by adding 150 ⁇ l 5 M K-acetate followed by incubation on ice for 10 minutes and 10 minutes spin at 18000g, 4°CrPlasmid DNA was precipitated from the supernatant by the addition of 350 ⁇ l isopropanol and collected by centrifugation.
  • the DNA was resuspended in 50 ⁇ l water and then further purified by precipitation with 12 ⁇ l 5 M NaCl and 60 ⁇ l 13% (w/v) polyethylene glycol (P ⁇ G8000, Sigma) on ice for 1 hour. After collection by centrifugation, washing in 70% ethanol and drying under vacuum, the purified plasmid DNA was resuspended in 50 ⁇ l water.
  • Samples (l ⁇ l) were sequenced using the ml 3 forward: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO: 37); or reverse 5'-GGAAACAGCTATGACCATG-3' (SEQ ID NO: 38) primers with the ABI BigDye version 3.0 system and analyzed on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Sequences were analyzed using Sequencher software (Gene Codes Corporation, Ann Arbor, MI).
  • DNA gel blot analysis Five micrograms of DNA prepared from uninfected flax plants or flax leaves infected with rusts C or H was digested with 20 units of the restriction enzyme H dIII (MBI Fermentas) in 20 ⁇ l volumes of lxR+ buffer (MBI Fermentas) at 37°C for 5 hours, separated by electrophoresis on 1% agarose (Progen) gels in SEB buffer (40 mM Tris, 1 mM EDTA, 40 mM Na-acetate p ⁇ 7.8) and transferred to ⁇ ybond N+ nylon membranes (Amersham, Buckinghamshire, UK) in 0.4 N NaO ⁇ blotting solution.
  • H dIII MBI Fermentas
  • SEB buffer 40 mM Tris, 1 mM EDTA, 40 mM Na-acetate p ⁇ 7.8
  • the cDNA inserts were amplified by PCR with the PN1/PN2 primers as described above and purified using the QiaQuick PCR purification kit (QIAGEN). These fragments were labeled with ⁇ 32 P-dCTP and hybridised to nylon filters as described above (except that hybridisation was at 65°C and the final wash was in 1 x SCC, 0.1% SDS at 65°C). Filters were exposed to either Kodak BiomaxMS or Fuji X-ray film at -70°C with appropriate intensifying screens.
  • Genomic DNA (5 ⁇ g) from rust C or rust H infected tissue was digested with the restriction enzymes Hin ⁇ lll, BamE ⁇ , EcoRI, EcoKV, Pst ⁇ , or Xbal (MBI Fermentas) as described above with the appropriate buffers recommended for each enzyme. Digested DNA fragments were separated by agarose gel electrophoresis and then blotted onto Hybond nylon filters and hybridised to probes as described above.
  • Genomic library of flax rust and isolation of clones This library was prepared from DNA isolated from germinated spores of rust CH5, digested with the enzyme Sau3Al and size selected by passage through a stepped sucrose gradient containing 10-50% sucrose. DNA fragments of 15-20 kbp in size were ligated into the BamH ⁇ site of a lambda EMBL3 cloning vector and packaged into lambda particles using a Packagene lambda packaging extract (Promega). A primary titre of 2x10 5 plaque forming units (pfu) was obtained and the library was then amplified and stored at -80°C.
  • a total of 250,000 pfu was plated onto ten 135 mm LB agar plates using agarose overlays. For each plate, 25000pfu were used to infect 400 ⁇ l E. coli K803 cells (grown overnight in LB medium with 2% w/v maltose and 10 mM MgSO and then centrifuged at 5000rpm for 10 minutes and re-suspended in an equal volume of 10 mM MgSO ). After growth at 37°C for 16 hours, plaques were lifted onto Hybond N+ filters (Amersham) and screened by hybridisation with a 32 P-dCTP -labelled probes as described above.
  • Hybridising clones were extracted into 1ml ⁇ dilution buffer (10 mM Tris; 10 mM MgSO 4 ) and screened again at low density on 100 mm LB agar plates with agarose overlays to isolate pure lambda clones. High titre samples were obtained by elution of phage from confluently lysed 100 mm plates in 3ml ⁇ dilution buffer. Then 3x10 6 pfu was used to infect 400 ⁇ l K803 cells (37°C, 15 minutes) 15ml LB was added followed by incubation at 37°C shaking (200 rpm) for 6-9 hours for DNA preparation.
  • Lysed cultures were treated with RNaseA (lOmg, Sigma) and DNasel (2mg, Sigma) at 37°C for 15min and then precipitated by adding 1 volume of 20% (w/v) PEG8000, 2 M NaCl and incubating on ice for 1 hour. Phage particles were recovered by centrifugation at 10,000g for 10 minutes at 4°C and resuspended in 0.5ml TE (10 mM Tris- HCl, 1 mM EDTA, pH 8.0). Particles were disrupted by adding 5 ml of 10% (w/v) SDS and incubating at 65°C for 5-10 minutes and then proteins were removed by PCI extraction.
  • DNA was precipitated with Na-acetate/ethanol and resuspended in lOO ⁇ l TE.
  • Ten microlitre samples were digested with the restriction enzymes H dlll, BamW., EcoRI, Xbal or Pstl and then precipitated with Na-acetate/ethanol and re-suspended in lO ⁇ l water. Fragments were ligated into plasmid vectors using standard conditions.
  • the Perkin Elmer/ Applied Biosystems GeneAmp XL PCR kit was used (Roche Molecular Systems Inc., Branchburg, New Jersey) to generate long PCR products with modification of the reaction mix to give more reproducible amplification.
  • For the bottom mix we combined 6 ⁇ l 3.3 x bufferll, 8 ⁇ l Mg-acetate (25 mM), 4 ⁇ l dNTPs (10 mM each), 2 ⁇ l each primer (lO ⁇ M) and 3 ⁇ l water.
  • An Ampliwax PCR gem wax bead (Roche Molecular Systems) was placed over this mix and the tube heated to 70°C for 2 minutes to melt the bead.
  • Amplified fragments of about 700bp and 800bp were obtained for the 10-1.41/10-1.39 (BamHI) and 10-1.6/10-1.39 ( ⁇ coRV nested) reactions and these were cloned into pG ⁇ MT- ⁇ asy and sequenced as described above.
  • Agrobacterium strains were inoculated in 50 ml LB liquid medium supplemented with the above antibiotics and grown at 28°C for 16-24 hours.
  • Three loci contain multiple avirulence specificities (A-L5/L6/L7, A-M1/M4 and A-P/P1/P2/P3) and these may represent complex loci with duplicated genes.
  • a third subtracted library contained transcripts present in leaves infected by rust H, but absent in uninfected leaves, and was expected to consist largely of unselected rust H-derived transcripts. The procedure for constructing and screening these libraries is detailed above.
  • Driver and tester cDNAs were prepared and hybridised as described above.
  • Libraries enriched for cDNAs more abundant in the tester than driver samples were prepared by a two-step PCR amplification of the renatured cDNA mixtures using the Advantage cDNA polymerase kit (CLONTECH) as described above.
  • CLONTECH Advantage cDNA polymerase kit
  • the selectively amplified cDNAs were cloned using the pGEMT-Easy PCR cloning kit (Promega). Colonies containing recombinant plasmids were identified by blue/white screening on X-Gal medium.
  • the driver cDNA was derived from flax leaves infected with rust C and the tester was from flax leaves infected with rust H. This library should be enriched for cDNAs that are more highly expressed during infection by rust H than rust C. 480 clones from this library were selected and arrayed into five 96 well plates (designated HC4-8).
  • the driver cDNA was derived from uninfected flax leaves and the tester was from flax leaves infected with rust H. This library should be enriched for cDNAs that are present in leaves infected by rust H, but not in uninfected leaves and therefore should represent unselected rust H derived cDNAs.
  • Figure 2 shows a plot of the transformed signal intensities for the two cDNA probes for each ofthe cDNA spots on the microarray and a list of the clones showing greater than about threefold difference in signal intensity between the two probes. These were selected for further analysis.
  • a total of 130 putatively differentially expressed cDNA clones were identified in the differential hybridisation screens. Plasmid DNAs were isolated as described above, and the nucleotide sequences determined. Some of these 130 clones were identical and a total of 74 unique sequences of between 40 and 500 nucleotides were identified. BLAST searches (Altschul et al., 1997) against the NCBI sequence databases detected matches to known genes for some of the cDNA clones, but most showed no matches to genes in the public databases. Nine cDNAs of less than 80bp were not analysed further, while the remainder were tested by DNA gel blot analysis to determine whether they were derived from flax or rust genes.
  • IU2F2 identifies an RFLP that co segregates with the A-L6 locus
  • the 23 cDNAs from flax rust were used as probes to detect restriction fragment length polymorphisms (RFLPs) between rust strains C and H. Where restriction fragment length polymorphisms (RFLPs) were detected between rusts C and H, the same probe/enzyme combination was used to score the segregation of these RFLP markers among the individual progeny of the CH5F2 family. For this analysis, genomic DNA isolated from in vitro germinated rust spores rather than infected plant tissue was used.
  • this culture contained a mixture of two plasmids with different cDNA inserts, which was apparent from the presence of two fragments of ⁇ 400bp and ⁇ 200bp in the PCR amplified probe. These were isolated separately by retransformation of E. coli with the plasmid mixture isolated from this culture and selection of transformed colonies containing different cDNA inserts. Probes derived from the two inserts were then prepared as described above and hybridised to the rust genomic DNA blots as described above. The larger cDNA probe of ⁇ 400bp failed to hybridise to rust genomic DNA, while the smaller probe detected the same RFLPs as the original mixed probe, indicating that this cDNA was closely linked to the Avirulence locus.
  • This cDNA clone is henceforth referred to as the IU2F2 cDNA. It contained a 187 bp sequence that appeared to consist of a 5' untranslated region of 105 bp followed by an open reading frame encoding 27 amino acids that resembled a hydrophobic secretion signal (see Figure 5).
  • genomic DNA clones ofIU2F2 locus Since the IU2F2 cDNA represented a candidate for encoding one or more ofthe A-L5, A-L6 or A-L7 avirulence functions, we isolated larger genomic DNA clones from this region from a lambda vector genomic library prepared from the FI hybrid rust CH5 (Ayliffe et al, 2001). Hybridisation under stringent conditions identified 17 genomic clones. Digested lambda DNA from each clone was analysed by gel blotting and hybridisation to the IU2F2 probe as described above. Comparison of the restriction fragment and hybridisation patterns of the lambda clones was used to group the clones into several independent classes with different inserted DNA fragments.
  • the lambda clones 6-1, 8-1 and 10-1 were initially chosen for analysis by sequencing sub-cloned restriction fragments in plasmid vectors. Fragments were inserted into pBluescriptSK+ plasmids (either ampicillin or kanamycin resistance versions) after treatment with calf intestinal alkaline phosphatase (1 unit, MBI Fermentas) to remove the 3' phosphate groups and prevent self-ligation. Plasmid DNA was prepared from isolated colonies as described above and analysed by restriction digest (as above) to identify clones containing different fragments.
  • sequence of the corresponding region from the virulent allele was determined from PCR amplified genomic DNA fragments from the F2 individual CH5F2-112, which is homozygous for the virulent allele ofthe A-L5/L6/L7 locus and also the linked 1U2F2 allele.
  • the primer combinations 10-1.8/10-1.16, 10-1.27/10-1.16, 10-1.15/10-1.9, 10-1.4/10-1.41, 10-1.4/10-1.37 were used to generate overlapping PCR fragments of 4.5kbp, 3.1kbp, 3.3kbp, 3.7kbp, 4.5kbp respectively. These were purified using the QIAquick PCR purification kit (QIAGEN) and sequenced directly using internal primers as described above to generate a complete sequence contig of approximately 10.5 kbp. An additional 500bp region at the 3' end of this contig was identified by inverse PCR and sequenced. The inverse PCR clones contained sequence from the 5' ends of the predicted
  • the genomic sequences reveal that the IU2F2 cDNA occurs in a multigene complex.
  • the allelic version associated with the A-L5/L6/L7 avirulence haplotype in flax rust contains three related copies of the cDNA (2F2-A,-B,-C), while the alternative version associated with the virulence allele contains a single copy (2F2-D).
  • Analysis of the cloned genomic DNA sequences suggested that one other duplicated gene family was present in this region ofthe flax rust genome in addition to the IU2F2 homologs.
  • the remaining 15 clones represented two novel sequences, 2F2-E (6 clones) and 2F2-F (9 clones) apparently derived from the alternative allele present in rust H but not inherited in CH5. All 21 sequences lacked the two predicted introns and contained the predicted 450bp open reading frame and the predicted stop codon. Thus this sequence represents the full coding sequence of these genes.
  • the predicted 150 amino acid product of the 2F2 genes includes a predicted 23aa cleavable secretion signal peptide that is completely conserved between all the gene copies (detected by the PSORT analysis program, http://psort.nibb.ac.jp/), suggesting that a mature protein of 127aa is secreted from flax rust.
  • the primers 10-1.15/10-1.16 were used to amplify the genomic DNA versions from the subcloned lambda vectors by PCR.
  • the PCR fragments were purified, cloned into pGEMT-Easy and sequenced to ensure no errors were introduced.
  • the Bam QJXbal fragments ( Figure 5) from the PCR clones were inserted into the corresponding sites of pGEMT-2F2-A to generate pGEMT2F2-B, pGEMT-2F2-C and pGEMT-2F2-D. These genes were also inserted into the EcoRI site ofthe 35S/Nos binary vector.
  • the truncated versions of the putative avirulence genes, which initiate translation at methionine-24, the first amino acid of the predicted mature proteins after signal peptide cleavage were made using the primers 10-1.48/10-1.16 (for 2F2-A and -B) or 10-1.53/10- 1.16 (for 2F2-C and -D) and the coding sequences amplified using the pGEMT2F2-A, -B, - C, and -D plasmids as templates. These primers change an alternative upstream out-of-frame ATG codon to TTG to ensure that translation initiates at the desired ATG codon.
  • PCR products were cloned in pGEMT-Easy and sequenced as described above to identify error- free clones, then the EcoRI fragments were inserted into the 35S/Nos binary vector as described above. All gene expression constructs were fully sequenced to confirm their integrity.
  • Binary plasmids were transferred from E. coli into Agrobacterium tumefaciens strain GV3101 by a triparental mating with the helper ⁇ . coli strain RK2013. Cultures of the Agrobacterium transconjugants were infiltrated into flax leaves by pressing the syringe opening against the underside of the leaf and applying a gentle pressure until the leaf was visibly flooded. The six lowest leaves of four week old plants were injected with the Agrobacterium suspensions. Leaves were observed in the following days to detect the induction of cell death/HR-like phenotypes in the presence of the appropriate resistance genes.
  • Hybridisation was performed at a low stringency, with a 55°C hybridisation step and filters were washed in lxSCC 0.1%SDS at 45°C.
  • the Secl4 probe hybridised strongly to two bands in a H dIII digest and three in the Xba ⁇ digest ( Figure 11).
  • the 2F2-A probes hybridised to the same bands as the Sec 14 probe, plus 1 or 2 additional bands.
  • Genomic DNA was isolated from leaf rust infected wheat by grinding 2g heavily infected leaf tissue (10 days after rust inoculation) in liquid nitrogen and then adding 7 mL extraction buffer (100 mM Tris-HCl, 50 mM EDTA, 0.5 M NaCl, 1.25% [w/v] SDS, 10 mM ⁇ - mercaptoethanol, pH 8.0) and incubating at 65°C for 10 minutes. Then 2 ml 5 M K-acetate was added followed by a 20 minute incubation on ice and a 15 minute spin at 4000 rpm to remove cell debris.
  • extraction buffer 100 mM Tris-HCl, 50 mM EDTA, 0.5 M NaCl, 1.25% [w/v] SDS, 10 mM ⁇ - mercaptoethanol, pH 8.0
  • the supernatant was then filtered through a kimwipe tissue and DNA precipitated by the addition of 1 volume of isopropanol.
  • the DNA was spooled from the interphase between the two liquids, placed into 1 ml of 70% ethanol and centrifuged at 14000 rpm in a microfuge.
  • the DNA pellet was resuspended in 0.4 ml of T5E (50 mM Tris- HCl, 10 mM EDTA, pH 8.0) with 20 ⁇ g RNaseA (NEB) and incubated at 37°C for 15 minutes to remove RNA.
  • the DNA solution was extracted once with PCI and then 0.5 volumes of 10 M NH -acetate was added and the solution placed on ice for 10 minutes.
  • a precipitate was removed by a 10 minute spin at 4°C 14000 rpm, and DNA was then precipitated from the supernatant by adding 1 volume of isopropanol. After rinsing with 70% ethanol the DNA pellet was resuspended in 100 ⁇ l TE.
  • Homologs of the 2F2 avirulence genes are isolated from rust fungi other than flax rust fungi by using DNA fragments of the sequences described here as probes to screen a DNA library prepared from a fungus species by standard techniques. These libraries are of genomic DNA fragments in a lambda phage vector such as is described in Example 1, or are genomic or cDNA libraries prepared in a variety of other vector systems such as plasmid, cosmid, YAC or BAC vectors. The DNA probes are labelled using 32P or other labelling reagent and hybridised at high or low stringency to individual DNA clones using the methods as described in Example 1 to identify clones containing the homologous sequences.
  • primers based on the DNA sequences described herein may be used to amplify related sequences from genomic DNA or cDNA of another rust fungus.
  • the DNA sequences are mapped using standard techniques such as RFLP or PCR based markers in a family segregating for known avirulence phenotypes such as the wheat stem rust family described by Zambinoet al (2000).
  • the avirulence gene homologs are expressed in host plants using a transient transformation system such as the Agrobacterium infiltration method described herein or alternatively a particle bombardment technique such as described by Shirasu et al (1999) or expressed stably in transformed plant.
  • the Avr gene homolog is also expressed stably in transformed plants generated by standard techniques and crossed to lines containing potential corresponding R genes, or the R genes may be expressed transiently in the transgenic line as described in Example 1 .
  • Rust strain 339 contained three 2F2 gene variants (A, B and L). These data are shown schematically in Figure 12.
  • Homologous genes were also amplified from the rust strain WA, which was isolated from a different host species, the native Australian flax, Linum marginale.
  • Two 2F2 variants (C and J) were identified in this isolate, one of which was identical to the 2F2-C gene identified from the genomic DNA library in Example 4.
  • the 12 predicted 2F2 amino acid sequences varied in 35 of the 150 amino acid positions ( Figure 13).
  • the translation products were all of 150 amino acids with a predicted 23-amino acid signal peptide, resulting in 127-amino acid mature polypeptides.
  • T-DNA vectors for use in Agrobacterium were generated that encoded the 2F2-E, F, G, H, I, J, K and L avirulence gene proteins, without the predicted signal peptides, each driven by the 35S promoter. These were tested by Agrobacterium-mediated introduction of the genes into the leaves of flax plants, for transient expression in flax lines carrying the L5, L6 or L7 resistance genes as described above. When tested in the flax transient assay system, all ofthe genes isolated from known avirulence alleles gave positive necrotic responses (hypersensitive response) with L5, L6 or L7, while all of those genes found at virulence alleles gave no response with these resistance genes (Figure 13).
  • Progeny of these seven T 0 plants crossed to L6 showed a range of weaker phenotypes from seedlings arrested after cotyledon emergence (score 2, one To plant) to dwarf seedlings (4 To plants with score 3, one To plant with score 4) and one To plant gave only wildtype progeny (score 5).
  • This weaker phenotype in the L6 background compared to L5 was consistently observed for each of the individual T 0 plants, with seedling phenotype scores 2-3 points higher in the crosses to L6 than in the crosses to L5. No effects were observed when the 2F2- B plants were crossed to 17 or Lx (Table 3).
  • the 2F2-C gene showed no phenotype in crosses to L5, but induced a strong phenotype (score 0) when crossed to L6 and a weaker phenotype in crosses to Lx (score 2-4).
  • DNA gel blot hybridisation using the 2F2 derived probe on genomic DNA from individual progeny of several crosses (as described above) showed that the 2F2 transgenes co-segregated with the abnormal growth phenotypes.
  • constructs encoding the 2F2-A, -B and -C full-length proteins showed weaker responses than the truncated versions, but with a similar relative specificity (Table 3). For instance, nine independent full-length 2F2-A To plants gave rise to progeny with a range of severe to mild stunted phenotypes when crossed to L5 or L6, a very mild phenotype when crossed to Lx and only wild-type progeny when crossed to L7. This weaker interaction was not due to lower transgene expression, since we detected equivalent transcript levels in transgenic plants with the full length and truncated constructs.
  • Tobacco plants stably transformed with the 35S2F2-C gene construct were generated and assayed by infiltration with an Agrobacterium strain containing the flax L6 resistance gene. This treatment triggered a rapid cell death response, indicating that the specific HR induction was induced in stably transformed plants expressing the flax rust avirulence gene.
  • Flax seeds were surface sterilised by immersion in a 70% [v/v] ethanol for 5 minutes followed by a 2% [w/v] solution of Zephirin in 10% ethanol [v/v] for 5 minutes and then washed in sterile distilled water. Seeds were germinated on solid MS media (containing 0.8% agar). Agrobacterium strains were grown overnight in LB medium plus appropriate antibiotics, centrifuged at 2500g and resuspended in liquid MS medium as described above.
  • leaves of W38 tobacco maintained in sterile tissue culture on solid MS medium were sliced into ⁇ lcm 2 pieces and floated on Agrobacterium suspensions as above then transferred to MS plates for two days. Explants were then transferred to tobacco shooting medium (as for flax but with 0.5 ⁇ g/mL NAA) until shoots appeared. Shoots were transferred to rooting medium (as for flax but with 0.05 5 ⁇ g/mL NAA) and then to soil after roots appeared.
  • AvrL567 genes are expressed in haustoria
  • Rust infection involved germination of the rust spore, growth of the germ tube on the leaf surface, and then appresorium formation over a stoma to allow penetration ofthe leaf. Inside the leaf a sub-stomatal vesicle formed and growth of infection hyphae leads to differentiation of a haustorial mother cell that extended a haustorium into a leaf mesophyll cell. In rust- infected flax, the first haustoria appear about 12 hours after infection and by 24 hours haustoria have formed at almost all penetration sites (Kobayashi et al, 1994).
  • Haustoria are the primary site of contact between rust pathogens and host mesophyll cells and in L6- mediated resistance, hypersensitive cell death was first induced in cells containing developing haustoria by 24 hours post infection (Kobayashi et al, 1994), suggesting that avirulence genes were likely to be expressed in these structures.
  • Affinity chromatography with a sepharose ConA column was used to purify haustoria from flax leaves infected with rust CH5. No contaminating plant cells or fungal mycelia were observed by microscopy in the purified haustorial samples, although some chloroplasts were present.
  • the 2F2 avirulence gene transcript was highly abundant in RNA isolated from the purified haustoria ( Figure 15 A).
  • a cDNA library prepared from the purified flax rust haustorial RNA was screened with a 2F2 probe, which identified 4, 2 and 13 cDNA clones derived from 2F2-A, -B and -D respectively, indicating that each of the three genes was expressed in haustoria.
  • the observed expression pattern of the 2F2 avirulence genes was consistent with the timing and location of HR induction in resistant L6 plants.
  • RNA was prepared from infected plant tissue or purified haustoria using the QIAGEN Plant RNeasy kit.
  • total RNA (lO ⁇ g) was denatured at 65°C for 10 minutes in 50% formamide, 5% formaldehyde, IX MOPS buffer (200 mM MOPS ⁇ 3-[N-Morpholino]propanesulfonic acid; Sigma ⁇ , 50 mM sodium acetate, 10 mM EDTA, pH7.0) and then separated on 1.5% agarose genes containing 6% formaldehyde in IX MOPS buffer.
  • RNA was blotted onto nylon membranes in 20X SSC and hybridised to 32 P -labeled DNA probes as described above.
  • RNA was reverse transcribed using Superscript Reverse Transcriptase (Gibco BRL) with an oligo-dT 25 primer and then amplified by Taq polymerase with the following thermal profile: 94°C 2 minutes; 38 cycles 94°C 20 seconds, 55°C 30 seconds, 72°C 1 minute; 72°C 5 minutes.
  • 2F2 transcripts were amplified with the primers 10-1.15 and 10-1.16, Seel 4 transcripts with 10- 1.4 and 10-1.9, and flax rust tubulin transcripts with tublO: 5'- AAACACTAAATCAAACATGAGGG-3' (SEQ ID NO: 41), and tub 12 5'- ACAAAGAACCAAAAGGACCCGA-3' (SEQ ID NO: 42).
  • Each primer set spanned one or more introns to distinguish cDNA and genomic DNA derived products.
  • Haustoria were isolated from flax leaves 7 days after inoculation with rust strain CH5 by affinity chromatography essentially as described by Hahn and Mendgen (1992).
  • An affinity column was prepared by covalently attaching Concanavalin-A (Pharmacia Biotech AB, Uppsala, Sweden) to CNBR-activated Sepharaose 6MB (Pharmacia Biotech AB).
  • CNBr-Sepharose 6MB was swelled in ImM HC1 for 15 min, then washed on a glass filter with 200ml ImM HC1.
  • the gel was then washed quickly with 30ml coupling buffer (0.1 M NaHCO 3 , 0.5M NaCl, pH 8.6).
  • the gel was incubated with 10-20mg Concanavalin-A (in coupling buffer; ratio gel to buffer approx. 1 :1) overnight at 4°C under gentle shaking.
  • infected leaf material Thirty grams of infected leaf material were placed in 180 mis of homogenisation medium (0.3M sorbitol, 20mM MOPS, pH 7.2, 0.1% (w/v) BSA, 0.2 % (v/v) 2-mercaptoethanol, 0.2 % (w/v) PEG 6000).
  • the leaves were homogenised in a Waring Blender at maximum speed for 30 seconds.
  • the homogenate was filtered through a 100 ⁇ m nylon mesh, then through a 11 ⁇ m nylon mesh.
  • the filtrate was divided into 6 30 ml centrifuge tubes and centrifuged in a HB-6 rotor for 5 minutes at 6500 rpm.
  • the pellets were each resuspended in 1 ml suspension medium (0.3M sorbitol, lOmM MOPS, pH 7.2, 0.2% (w/v) BSA, ImM KC1, ImM MgCl 2 , ImM CaCl 2 ).
  • the resuspended pellet suspension was loaded onto the column, which contained 5 mis of gel in volume, equilibrated with suspension medium. The loading was carried out in two consecutive rounds, across three columns, each loading containing 1 ml of suspension. Each loading of suspension was allowed to incubate on the column for 15 minutes. After the second incubation the suspension was washed through the column with 10-15 mis of suspension buffer.
  • the haustoria were released by adding 5 mis of suspension buffer to the column and agitating the beads using a blunt ended 1ml pipette tip by pipetting up and down. The supernatant containing the haustoria were removed after the beads had settled. The haustoria were pelleted by centrifugation for 3 min at 9000 g. The pellet was then quickly frozen in liquid nitrogen and stored at -80°C.
  • Coffee Rust (orange or leaf rust) Hemileia vastatrix Berk. & Br. Rust (powdery or grey rust) Hemileia coffeicola Mauble. & Rog.
  • Flax Rust Melampsora lini (Ehrenb.) Desmaz.
  • Saccharomyces cerevisiae SEC 14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108, 1271-1281. 7. Diatchenko, L., Lau, Y-F.C, Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B.,
  • Suppression subtractive hybridisation a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025-
  • the L6 gene for flax resistance is related to the Arabidopsis bacterial resistance gene Rps2 and the tobacco viral resistance gene N. Plant Cell 7, 1195-1206.
  • a phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form. J. Cell Biol. 125, 113-127
  • Cladosporium fulvum overcomes Cf-2-mediated resistance by producing truncated AVR2 elicitor proteins. Molecular Microbiol. 45, 875-884.
  • Arabidopsis RIN4 is a target of the type III effector AvrRpt2 and modulates RPS2- mediated resistance. Cell 112, 379-389.
  • Root knot nematode- directed expression of a plant root-specific gene Science 263 :221 -223.
  • telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 12, 2019-2032.

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Abstract

L'invention concerne une molécule isolée d'acide nucléique comprenant une séquence de nucléotides codant une séquence de nucléotides ou complémentaire de celle-ci, laquelle code un produit d'avirulence d'une rouille végétale, et l'utilisation de cette molécule dans la transformation d'un végétal permettant d'induire une réponse de résistance aux maladies chez le végétal, éventuellement au moyen d'un gène de résistance aux maladies correspondant dans le végétal. L'invention concerne également un procédé d'identification d'une séquence d'acide nucléique codant un produit d'avirulence d'une rouille végétale.
PCT/AU2004/000602 2003-05-07 2004-05-07 Sequences genetiques de genes d'avirulence de pathogenes vegetaux et utilisations de celles-ci WO2004099417A1 (fr)

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US8148604B2 (en) 2004-10-21 2012-04-03 Venganza Inc. Methods and materials for conferring resistance to pests and pathogens of plants
CN110551674A (zh) * 2019-09-10 2019-12-10 中国农业大学 一种玉米多堆柄锈菌的接种扩繁方法
CN111763654A (zh) * 2020-06-11 2020-10-13 南京林业大学 一种杨树落叶松杨珊锈菌单孢扩繁方法

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CN110951914B (zh) * 2020-02-18 2022-11-25 黑龙江八一农垦大学 豇豆单胞锈菌的巢式pcr检测引物及检测方法

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AU698060B2 (en) * 1994-04-21 1998-10-22 Commonwealth Scientific And Industrial Research Organisation Genetic sequences conferring disease resistance in plants and uses therefor
US20020004944A1 (en) * 2000-03-21 2002-01-10 Baker Barbara J. Non-pathogen induced systemic acquired resistance (SAR) in plants
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8148604B2 (en) 2004-10-21 2012-04-03 Venganza Inc. Methods and materials for conferring resistance to pests and pathogens of plants
US8461416B2 (en) 2004-10-21 2013-06-11 Venganza, Inc. Methods and materials for conferring resistance to pests and pathogens of plants
US8581039B2 (en) 2004-10-21 2013-11-12 Venganza, Inc. Methods and materials for conferring resistance to pests and pathogens of plants
US9121034B2 (en) 2004-10-21 2015-09-01 Venganza Inc Methods and materials for conferring resistance to pests and pathogens of corn
CN110551674A (zh) * 2019-09-10 2019-12-10 中国农业大学 一种玉米多堆柄锈菌的接种扩繁方法
CN110551674B (zh) * 2019-09-10 2021-07-06 中国农业大学 一种玉米多堆柄锈菌的接种扩繁方法
CN111763654A (zh) * 2020-06-11 2020-10-13 南京林业大学 一种杨树落叶松杨珊锈菌单孢扩繁方法

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