[go: up one dir, main page]

WO2008054890A1 - Génération de plantes avec une meilleure résistance aux agents pathogènes - Google Patents

Génération de plantes avec une meilleure résistance aux agents pathogènes Download PDF

Info

Publication number
WO2008054890A1
WO2008054890A1 PCT/US2007/071146 US2007071146W WO2008054890A1 WO 2008054890 A1 WO2008054890 A1 WO 2008054890A1 US 2007071146 W US2007071146 W US 2007071146W WO 2008054890 A1 WO2008054890 A1 WO 2008054890A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
plants
resistance
fur
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2007/071146
Other languages
English (en)
Inventor
Ry D. Wagner
Caroline Bonneau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AGRONOMICS LLC
Original Assignee
AGRONOMICS LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AGRONOMICS LLC filed Critical AGRONOMICS LLC
Publication of WO2008054890A1 publication Critical patent/WO2008054890A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the fungal pathogen Fusarium oxysporum causes severe vascular wilt diseases and major loses in a wide variety of economically important crops (Beckman, 1987, The Nature of Wilt Diseases of Plants).
  • the F. oxysporum pathogen infects plants through the root system, often though wounds.
  • the vascular system of the plant is adversely affected by the organism as it grows due to reduced nutrient and water flower through the plant.
  • the disclosure provides a transgenic plant having increased resistance to a pathogen, such as a fungus (for example,- Fusarium oxysporum) relative to control plants.
  • the transgenic plant has incorporated (e.g., stably incorporated) into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pathogen resistance activity.
  • the nucleotide sequence may be a nucleotide sequence identified in column 3 of Tables 3 and 4, or a complement thereof; a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence identified in column 3 of Tables 3 and 4, or a complement thereof; a nucleotide sequence encoding a polypeptide comprising an amino acid sequence identified in column 4 of Tables 3 and 4; or a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an amino acid sequence identified in column 4 of Tables 3 and 4.
  • the nucleotide sequence is, for instance, operably linked to a promoter that drives expression of a coding sequence in a plant cell.
  • the transgenic plant is selected from the group consisting of rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut, tomato, carrot, lettuce, bean, asparagus, cauliflower, pepper, beetroot, cabbage, eggplant, endive, leek, long cucumber, melon, pea, radish, rootstock, short cucumber (Be ⁇ t alpha), squash, watermelon, white onion, witloof, yellow onion, broccoli, brussel sprout, bunching onion, celery, mache, cucumber, fennel, gourd, pumpkin, sweet corn, and zucchini.
  • the transgenic plants may be produced by introducing into the plant or a cell thereof at least one plant transformation vector comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes an FUR polypeptide identified in column 4 of Tables 3 and 4, or a variant thereof, and growing the transformed plant or cell to produce a transgenic plant, wherein said transgenic plant exhibits increased resistance to at least one pathogen.
  • the FUR polypeptide has at least about 70% sequence identity to an amino acid sequence referred to in column 4 of Tables 3 and 4.
  • the FUR polypeptide has at least about 80% or 90% sequence identity to or has the amino acid sequence referred to in column 4 of Tables 3 and 4.
  • Methods are provided for producing a plant with increased pathogen resistance, including increased fungal resistance, comprising identifying a plant having an altered FUR gene, and generating progeny of the plant, wherein the progeny have increased pathogen resistance, and wherein the FUR gene is one that is identified in column 4 of Tables 3 and 4.
  • Methods are also provided for identifying a plant having increased pathogen resistance, comprising analyzing at least one FUR gene from the plant, and identifying a plant with an altered FUR gene, wherein the plant has increased pathogen resistance.
  • the disclosure further provides plants and plant paits obtained by the methods described herein.
  • nucleic and/or amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NOs: 1 and 2 show the nucleic acid (GI
  • SEQ ID NOs: 3 and 4 show the nucleic acid (GI
  • SEQ ID NOs: 5 and 6 show the nucleic acid (GI
  • SEQ ID NOs: 7 and 8 show the nucleic acid (GI
  • SEQ ID NOs: 9 and 10 show the nucleic acid (GI
  • SEQ ID NOs: 1 1 and 12 show the nucleic acid (GI
  • ⁇ SEQ ID NOs: 13 and 14 show the nucleic acid (GI
  • SEQ ID NOs: 15 and 16 show the nucleic acid (GI)18396346
  • SEQ ID NOs: 17 and 18 show the nucleic acid (GI
  • SEQ ID NOs: 19 and 20 show the nucleic acid (GI
  • SEQ ID NOs: 21 and 22 show the nucleic acid (GI
  • SEQ ID NOs: 25 and 26 show the nucleic acid (GI
  • SEQ ID NOs: 27 and 28 show the nucleic acid (GI
  • SEQ ID NOs: 29 and 30 show the nucleic acid (GI
  • SEQ ID NOs: 31 and 32 show the nucleic acid (GI
  • SEQ ID NOs: 33 and 34 show the nucleic acid (GI
  • SEQ ID NOs: 35 and 36 show the nucleic acid (GI
  • SEQ ID NOs: 41 and 42 show the nucleic acid (GI
  • SEQ ID NOs: 43 and 44 show the nucleic acid (GI
  • SEQ ID NOs: 45 and 46 show the nucleic acid (GI
  • SEQ ID NOs: 47 and 48 show the nucleic acid (GI) 18403251
  • SEQ ID NOs: 49 and 50 show the nucleic acid (GI
  • SEQ ID NOs: 51 and 52 show the nucleic acid (Gl
  • SEQ ID NOs: 53 and 54 show the nucleic acid (GI
  • SEQ ID NOs: 55 and 56 show the nucleic acid (GI
  • SEQ ID NOs: 57 and 58 show the nucleic acid (GI
  • SEQ ID NOs: 59 and 60 show the nucleic acid (GI
  • SEQ ID NOs: 61 and 62 show the nucleic acid (GI
  • SEQ ID NOs: 63 and 64 show the nucleic acid (QIjI 840645 l
  • SEQ ID NOs: 65 and 66 show the nucleic acid (GI
  • SEQ ID NOs: 67 and 68 show the nucleic acid (GI
  • SEQ ID NOs: 69 and 70 show the nucleic acid (GI
  • SEQ ID NOs: 71 and 72 show the nucleic acid (GI
  • SEQ ID NOs: 73 and 74 show the nucleic acid (GI
  • SEQ ID NOs: 75 and 76 show the nucleic acid (GI
  • SEQ ID NOs: 77 and 78 show the nucleic acid (GI)42565568
  • SEQ ID NOs: 79 and 80 show the nucleic acid (GI
  • SEQ ID NOs: 83 and 84 show the nucleic acid (GI
  • SEQ ID NOs: 85 and 86 show the nucleic acid (GI
  • SEQ ID NOs: 87 and 88 show the nucleic acid (GI
  • SEQ ID NOs: 89 and 90 show the nucleic acid (GI
  • SEQ ID NOs: 91 and 92 show the nucleic acid (GI
  • vector or transformation vector refers to a nucleic acid construct designed for transfer between different host cells.
  • expression vector refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell.
  • Many prokaryotic and cukaryotic vectors, including example expression vectors, are commercially available. Selection of appropriate vectors is within the knowledge of those having skill in the art.
  • a “heterologous” nucleic acid construct or sequence has at least a portion of the sequence that is not native to the plant cell in which it is expressed.
  • Heterologous refers to a control sequence (e.g., promoter or enhancer) that docs not function in nature to regulate the same gene the expression of which it is currently regulating.
  • heterologous nucleic acid sequences are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, or the like.
  • a “heterologous” nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native plant.
  • the term “gene” means the segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons) and non-transcribed regulatory sequence.
  • the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules ⁇ e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • complement is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex.
  • recombinant includes reference to a cell o ⁇ vector that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all as a result of deliberate human intervention.
  • the term "gene expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation; accordingly, “expression” may refer to either a polynucleotide or polypeptide sequence, or both. Sometimes, expression of a polynucleotide sequence will not lead to protein translation. "Over-expression” refers to increased expression of a polynucleotide and/or polypeptide sequence relative to its expression in a wild-type (or other reference [e.g., non-transgenic]) plant and may relate to a naturally-occurring or non-naturally occurring sequence.
  • Ectopic expression refers to expression at a time, place, and/or increased level that docs not naturally occur in the non- altered or wild-type plant.
  • Under-expression refers to decreased expression of a polynucleotide and/or polypeptide sequence, generally of an endogenous gene, relative to its expression in a wild-type plant.
  • mi-expression and “altered expression” encompass over-expression, under-expression, and ectopic expression.
  • a nucleic acid sequence into a cell includes, for example, “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a cukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
  • a "plant cell” refers to any cell derived from a plant, including cells from undifferentiated tissue (e.g., callus) as well as plant seeds, pollen, propagules, and embryos.
  • mutant and wild-type refers to the form in which that trait or phenotype is found in the same variety of plant in nature.
  • the term "modified" regarding a plant trait refers to a change in the phenotype of a transgenic plant relative to the similar non-transgenic plant.
  • An "interesting phenotype (trait)" with reference to a transgenic plant refers to an observable or measurable phenotype demonstrated by a Tl and/or subsequent generation plant, which is not displayed by the corresponding non-transgenic (e.g., a genotypically similar plant that has been raised or assayed under similar conditions).
  • An interesting phenotype may represent an improvement in the plant or may provide a means to produce improvements in other plants.
  • An “improvement” is a feature that may enhance the utility of a plant species or variety by providing the plant with a unique and/or novel quality.
  • altered pathogen resistance phenotype or “altered pathogen resistance” refers to a detectable change in the response of a genetically modified plant to pathogenic infection, compared to the similar, but non-modified plant.
  • the phenotype may be apparent in the plant itself (e.g., in growth, viability or particular tissue morphology of the plant) or may be apparent in the ability of the pathogen to proliferate on and/or infect the plant.
  • improved pathogen resistance refers to increased resistance to a pathogen. Methods for measuring pathogen resistance arc well known in the art.
  • pathogen resistance activity or “pathogen resistance” is therefore intended the ability to grow or survive during a pathogenic infection.
  • An "altered fungal resistance phenotype” or “altered fungal resistance” refers to detectable change in the response of a genetically modified plant to fungal infection, compared to the similar, but non-modified plant. The phenotype may be apparent in the plant itself (e.g., in growth, viability or particular tissue morphology of the plant) or may be apparent in the ability of the pathogen to proliferate on and/or infect the plant, or both. As 7 07H46
  • improved fungal resistance refers to increased resistance to a fungal pathogen. Methods for measuring fungal resistance are well known in the art. See, for example: Adam & Somerville, PZa ⁇ /., 1996, 9:341-356, Alan & Earle, MoI. Plant Microbe Interact, 2002, 15:701-708, Castillo-Lluva ef al, J. Cell ScL, 2004, 117:4143-4156, Dufresne et al, MoI. Plant Microbe Interact., 1998, 11 :99-108, Epple et al, Plant Cell,
  • a "mutant" polynucleotide sequence or gene differs from the corresponding wild type polynucleotide sequence or gene either in terms of sequence or expression, where the difference contributes to a modified plant phenotype or trait.
  • the term “mutant” refers to a plant or plant line which has a modified plant phenotype or trait, where the modified phenotype or trait is associated with the modified expression of a wild type polynucleotide sequence or gene.
  • Tl refers to the generation of plants from the seed of TO plants.
  • the Tl generation is the first set of transformed plants that can be selected by application of a selection agent, e.g., an antibiotic or herbicide, for which the transgenic plant contains the corresponding resistance gene.
  • T2 refers to the generation of plants by self-fertilization of the flowers of Tl plants, previously selected as being transgenic.
  • plant part includes any plant organ or tissue, including, without limitation, seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can be obtained from any plant organ or tissue and cultures prepared therefrom.
  • the category of plants which can be used in the methods of the present disclosure is generally as broad as the category of higher 2007/071146
  • plants amenable to transformation techniques including both monocotyledenous and dicotyledenous plants.
  • transgenic plant includes reference to a plant that comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide can be either stably integrated into the genome, or can be extra-chromosomal.
  • the polynucleotide of the present disclosure is stably integrated into the genome such that the polynucleotide is passed on to successive generations.
  • a plant cell, tissue, organ, or plant into which the heterologous polynucleotides have been introduced is considered “transformed,” “transfected,” or “transgenic.”
  • Direct and indirect progeny of transformed plants or plant cells that also contain the heterologous polynucleotide are also considered transgenic.
  • nucleic acid molecule or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid ⁇ e.g. , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • isolated when used to refer to nucleic acid molecules excludes isolated chromosomes.
  • the isolated FUR nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a FUR protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-FUR protein (also referred to herein as a "contaminating protein").
  • Activation tagging in plants refers to a method of generating random mutations by insertion of a heterologous nucleic acid construct comprising regulatory sequences (e.g., an enhancer) into a plant genome.
  • the regulatory sequences can act to enhance transcription of one or more native plant genes; accordingly, activation tagging is a fruitful method for generating gain-of-function mutations that are generally dominant (see, e.g., Hayashi et al., Science, 1992, 258: 1350-1353; Weigel et al, Plant Physiology, 2000, 122:1003-1013).
  • the inserted construct also provides a molecular tag for rapid identification of the native plant gene or sequence the mis-expression of which causes the mutant phenotype.
  • a screen of Arabidopsis activation tagged (ACTTAG) mutants was used to identify the genes [designated FUR# listed in column 1 of TablevS 3 and 4 (below)] which are responsible for an altered pathogen resistance phenotype (specifically, a fungal resistance phenotype).
  • the pSKI015 vector which comprises a T-DNA from the Ti plasmid of Agrobacterium tumefaciens, a viral enhancer element, and a selectable marker gene (Weigel et al, Plant Physiology, 2000, 122:1003- 1013).
  • the enhancer element can cause up-regulation of genes in the vicinity, generally within about 10 kilobase (kb) of the insertion.
  • Tl plants were exposed to the selective agent in order to specifically recover transformed plants that expressed the selectable marker and therefore harbored T-DNA insertions.
  • Tl plants were allowed to grow to maturity, self-fertilize and produce seed.
  • T2 seed was harvested, labeled and stored.
  • ACTTAG lines showing increased resistance to the fungus Fusarium oxysporum sp. conglutinans were identified either in a "forward genetics” or a "reverse genetics” screen.
  • ACTTAG lines that showed increase resistance to F. oxysporum sp. conglulinans were identified by comparing the phenotype of ACTTAG seedlings and of wild-type seedlings after F. oxysporum sp. conglutinans infection.
  • the association of the FUR gene with the pathogen resistance phenotype was discovered by analysis of the genomic DNA sequence flanking the T-DNA insertion in the identified line. Accordingly, FUR genes and/or polypeptides may be employed in the development of genetically modified plants having a modified pathogen ⁇ e.g., fungal) resistance phenotype ("a FUR phenotype").
  • FUR genes may be used in the generation of crops and/or other plant species that ha ⁇ 'e improved resistance to infection by F, oxyspomm sp. conglutinans, other subspecies, isolates or races of F. oxyspomm, other pathogens causing vascular wilt disease and may also be useful in the generation of a plant with improved resistance to fungal, bacterial, and/or other pathogens. Mis-expression of FUR genes may thus reduce the need for fungicides and/or pesticides.
  • the modified pathogen resistance phenotype may further enhance the overall health of the plant.
  • FUR Nucleic Acids and Polypeptides The FUR genes discovered in the "forward genetics" activation tagging screen and
  • “reverse genetics” activation tagging screen are listed in column 1 of Tables 3 and 4, respectively.
  • the Arabidopsis Information Resource (TAIR) identification numbers are provided in column 2.
  • Columns 3-4 provide GenBank identifier numbers (GI#s) for the nucleotide and polypeptide sequences, respectively; each of the referenced published sequences is incorporated herein by reference as of the date on which this application is filed.
  • Column 5 lists biochemical function and/or protein name.
  • Column 6 lists the conserved protein domains.
  • Column 7 provides the GI#s for nucleic acid and polypeptide sequences of orthologous genes from other plant species; each of the referenced published sequences is incorporated herein by reference as of the date on which this application is filed.
  • fragment is intended a portion of the nucleotide sequence encoding a FUR protein or a portion of the amino acid sequence of the FUR protein.
  • a fragment of a nucleotide sequence may encode a biologically active portion of a FUR protein, a biologically active nucleic acid (e.g., an antisense or small inhibitory nucleic acid), or it may be a fragment that can be used as a hybridization probe or PCR primer using methods known in the art.
  • Nucleic acid molecules that are fragments of a FUR nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1150, 1200, 1250, 1300, 1400, 1500, 2000, 2500, 3000 contiguous nucleotides, or up to the number of nucleotides present in a full-length FUR- encoding nucleotide sequence disclosed herein, depending upon the intended use.
  • contiguous nucleotides or amino acids are intended nucleotide or amino acid residues that arc immediately adjacent to one another.
  • a functionally active FUR polypeptide causes an altered pathogen resistance phenotype when mis-expressed in a plant.
  • mis-expression of the functionally active FUR polypeptide causes increased resistance to F. oxysporum sp conglutinans, and/or other pathogens causing vascular wilt disease.
  • a functionally active FUR polypeptide is capable of rescuing defective (including deficient) endogenous FUR activity when expressed in a plant or in plant cells; the rescuing polypeptide may be from the same or from a different species as that with defective activity.
  • a functionally active fragment of a full length FUR polypeptide retains one of more of the biological properties associated with the full-length FUR polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity.
  • binding activity refers to the ability of a protein to bind to another protein, a DNA fragment or some other molecule (Bogdanove, Plant MoI Biol, 2002, 50:981-989, Inohara et al, Annu Rev Biochem., 2005, 74:355-383, Testerink & Munnik, Trends Plant ScL, 2005, 10:368-375).
  • catalytic activity refers to the ability of a protein to catalyze a chemical reaction; sec, e.g., Bhatia et al., CHt Rev Biotechnol., 2002, 22:375-407, Pcdlcy & Martin, Curr Opin Plant Biol., 2005, 8:541-547, Rosahl, Z Naturforsch [C]. 1996, 51 : 123-138, Stone & Walker, Plant Physiol, 1995, 108:451-457.
  • cellular or extra-cellular localizing activity refers to portions of the protein that interact with other components of the cell to localize the protein to a specific subcellular or extra-cellular location.
  • a FUR fragment preferably comprises a FUR domain, such as a C- or N-terminal or catalytic domain, among others, and may comprise at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, or 450 contiguous amino acids of a FUR protein, or up to the total number of amino acids present in a full-length FUR protein disclosed herein.
  • Representative functional domains of FUR genes are listed in column 6 of Table 3 and Table 4 and can be identified using the INTERPRO program (Mulder et al. , 2003 Nucleic Acids Res. 31, 315-318; Mulder el al., 2005 Nucleic Acids Res. 33-.D201-D205).
  • Functionally active variants of full-length FUR polypeptides or fragments thereof include polypeptides with amino acid insertions, deletions, or substitutions that retain one of more of the biological activities associated with the full-length FUR polypeptide.
  • By "retains biological activity” is intended that the variant will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the biological activity of the native protein, such as for instance an anti-fungal activity.
  • variants are generated that change the post-translational processing of a FUR polypeptide. For instance, variants may have altered protein transport or protein localization characteristics or altered protein half-life compared to the native polypeptide.
  • FUR nucleic acid encompasses nucleic acids with the sequence provided in the GenBank entry referenced in column 3 of Table 3 and Table 4. Nucleic acid sequences complementary to the GenBank entry referenced in column 3 of Table 3 and Table 4, as well as functionally active fragments, derivatives, or orthologs thereof may also be used in the methods and compositions disclosed herein.
  • a FUR nucleic acid of this disclosure may be DNA, derived from genomic DNA or cDNA, or RNA.
  • a functionally active FUR nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active FUR polypeptide.
  • genomic DNA that serves as a template for a primary RNA transcript (e.g., an mRNA precursor) that requires processing, such as splicing, before encoding the functionally active FUR polypeptide.
  • a FUR nucleic acid can include other non-coding sequences, which may or may not be transcribed; such sequences include 5' and 3' UTRs, polyadenylation signals and regulatory sequences that control gene expression, among others, as are known in the art. Some polypeptides require processing events, such as proteolytic cleavage, covalent modification, etc., in order to become fully active. Accordingly, functionally active nucleic acids may encode the mature or the pre-processed FUR polypeptide, or an intermediate form.
  • a FUR polynucleotide can also include heterologous coding sequences, for example, sequences that encode a marker included to facilitate the purification of the fused polypeptide, or a transformation marker.
  • a functionally active FUR nucleic acid is capable of being used in the generation of loss-of- function pathogen resistance phenotypes, for instance, via antisensc suppression, co-suppression, etc.
  • a FUR nucleic acid used in the methods of this disclosure may comprise a nucleic acid sequence that encodes or is complementary to a sequence that encodes a FUR polypeptide having at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the polypeptide sequence of the
  • a FUR polypeptide of the disclosure may include a conserved protein domain of the FUR polypeptide, such as one or more protein domain(s) listed in column 6 of Tables 3 and 4.
  • a FUR polypeptide comprises a polypeptide sequence with at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90% or about 95% or more sequence identity to a functionally active fragment of the polypeptide of the GenBank entry referenced in column 4 of Tables 3 and 4.
  • a FUR polypeptide comprises a polypeptide sequence with at least about 50%, about 60 %, about 70%, about 80%, or about 90% identity to the polypeptide sequence of the GenBank entry referenced in column 4 of Tables 3 and 4 over its entire length and comprises a conserved protein domain(s) listed in column 6 of Tables 3 and 4.
  • a FUR nucleic acid sequence used in the methods of the present disclosure comprises a nucleic acid sequence that has at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the nucleic acid sequence of the GenBank entry referenced in column 3 of Tables 3 and 4, or nucleic acid sequences that are complementary to such a FUR sequence or a functionally active fragment thereof.
  • percent (%) sequence identity with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al., J. MoI. Biol., 215:403-410 1990) with search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • “Variants” of FUR-encoding nucleotide sequences include those sequences that encode the FUR proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that have a specific sequence identity as discussed above.
  • conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues.
  • a "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a FUR protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. Amino acid substitutions may be made in non-conserved regions that retain function.
  • Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that selectively hybridize to the nucleic acid sequence of the GenBank entry referenced in column 3 of Tables 3 and 4.
  • the stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are well known (see, e.g., Current Protocol in Molecular Biology, Vol. 1 , Chap. 2,10, John Wiley & Sons, Publishers (1994); Sambrook et ⁇ /., supra).
  • low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37°C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in I x SSC at about 37°C for 1 hour.
  • orthologs of each of the Arabidopsis FUR genes are identified in column 7 of Tables 3 and 4. Methods of identifying the orthologs in other plant species are known in the art. Normally, orthologs in different species retain the same function, due to the presence of one or more protein motifs and/or 3-dimensional structures. In evolution, when a gene duplication event follows speciation, a single gene in one .species, such as Arabidopsis, may correspond to multiple genes (paralogs) in another. As used herein, the term "orthologs" encompasses paralogs. When sequence data is available for a particular plant species, orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.
  • Sequences arc assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen & Bork, Proc. Natl. Acad. ScL U.S.A., 95:5849-5856, 1998; Huynen el al, Genome Research, 10:1204-1210, 2000).
  • Programs for multiple sequence alignment such as CLUSTAL (Thompson JD et al., Nucleic Acids Res. 22:4673-4680, 1994) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.
  • Structural threading or other analysis of protein folding e.g. , using software by ProCeryon, Biosciences, Salzburg, Austria
  • Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available.
  • PCR and screening of cDNA or genomic DNA libraries are common methods for finding related gene sequences and are well known in the art (see, e.g., Sambrook, supra; Dieffenbach and Dveksler (Eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, 1989). For instance, methods for generating a cDNA library from the plant species of interest and probing the library with partially homologous gene probes are described in Sambrook et al, supra. A highly conserved portion of the Arabidopsis FUR coding sequence may be used as a probe.
  • Western blot analysis can determine that a FUR ortholog (e.g., an orthologous protein) is present in a crude extract of a particular plant species.
  • a FUR ortholog e.g., an orthologous protein
  • the sequence encoding the candidate ortholog may be isolated by screening expression libraries representing the particular plant species.
  • Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl 1 , as described in Sambrook, el al., supra. Once the candidate ortholog(s) are identified by any of these means, candidate orthologous sequence are used as bait (the "query") for the reverse BLAST against sequences from Arabidopsis or other species in which FUR nucleic acid and/or polypeptide sequences have been identified.
  • the methods of the disclosure involve incorporating the desired form of the FUR nucleic acid into a plant expression vector for transformation of plant cells, and the FUR polypeptide is expressed in the host plant.
  • FUR nucleic acids and polypeptides may be used in the generation of genetically modified plants having a modified pathogen resistance phenotype; in general, improved resistance phenotypes are of interest.
  • Pathogenic infection may affect seeds, fruits, blossoms, foliage, stems, tubers, roots, etc. Accordingly, resistance may be observed in any part of the plant.
  • altered expression of the FUR gene in a plant is used to generate plants with increased resistance to F. oxysporum sp conglutinans.
  • plants that mis-express FUR may also display altered resistance to other pathogens.
  • fungal pathogens of interest include, but are not limited to, Alternaria brassicicola, Botr ⁇ tis cinerea, Erysiphe cichoracearum, Fusarium oxysporum, Fusarium spp., Plasmodiophora brassica, Rhizoctonla solani, Colletotrichum coccode, Sclerotinia spp., Aspergillus spp,, Penicillium spp., Ustilaga spp., and Tilletia spp., Phytophthora megaspernia f.sp.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Phakopsora pachyrhizi, Pythium aphanidermaturn, Pythium ultimum, Pythium debaryanum, He ' terodera glycines Fusarium solani, Albugo Candida, Alternaria brassicae, Leptosphaeria macula ⁇ s, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternate, Clavibacter michiganensis subsp.
  • Xanthomonas campestris p.v. translucens Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondila f.sp.
  • the bacterial pathogens of interest include, but are not limited to, Agrobacterium tumefaciens, Erwinia tracheiphila, Erwinia stewartii, Xanthomonas phaseoli, Erwinia amylovora, Erwinia carolovora, Pseudomonas syringae, Pelargonium spp, Pseudomonas cichorii, Xanlhomonas fragariae, Pseudomonas morsprunorum, and Xanthomonas campestris.
  • Pathogenic ' infection may affect seeds, fruits, blossoms, foliage, stems, tubers, roots, etc. Accordingly, resistance may be observed in any part of the plant.
  • the methods described herein are generally applicable to all plants, as the FUR gene (or an ortholog, variant or fragment thereof) may be expressed in any type of plant.
  • the disclosure is directed to crops such as maize, soybean, cotton, rice, wheat, barley, tomato, canola, turfgrass, and flax. Other crops include alfalfa, tobacco, and other forage crops.
  • the disclosure may also be directed to fruit- and vegetable-bearing plants including tomato, carrot, lettuce, bean, asparagus, cauliflower, pepper, beetroot, cabbage, eggplant, endive, leek, long cucumber, melon, pea, radish, rootstock, short cucumber (Be ⁇ t alpha), squash, watermelon, white onion, witloof, yellow onion, bunching onion, broccoli, brussel sprout, celery, mache, cucumber, fennel, pumpkin, sweet corn, and zucchini, plants used in the cut flower industry, grain-producing plants, oil-producing plants, and nut-producing plants, among others.
  • fruit- and vegetable-bearing plants including tomato, carrot, lettuce, bean, asparagus, cauliflower, pepper, beetroot, cabbage, eggplant, endive, leek, long cucumber, melon, pea, radish, rootstock, short cucumber (Be ⁇ t alpha), squash, watermelon, white onion, witloof, yellow onion, bunching onion, broccoli, brussel sprout, celery, mach
  • the constructs can be introduced in a variety of forms including, but not limited to, as a strand of DNA, in a plasmid, or in an artificial chromosome.
  • the introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to Agrobaclerium-medi&ted transformation, electroporation, microinjection, microprojectile bombardment calcium-phosphate-DNA co- precipitation or liposome-mediated transformation of a heterologous nucleic acid.
  • the transformation of the plant is preferably permanent, i.e.
  • a heterologous nucleic acid construct comprising a FUR polynucleotide may encode the entire protein or a biologically active portion thereof,
  • the FUR gene expression is under the control of a pathogen-inducible promoter (Rushton et al., The Plant Cell, 14:749-762, 2002). In one embodiment, expression of the FUR gene is under control of regulatory sequences from genes whose expression is associated with the CsVMV promoter.
  • exemplary methods for practicing this aspect of the disclosure include, but are not limited to, antisense suppression (Smith et al., Nature, 334:724-726, 1988; van der Krol et al, Biotechniques, 6:958-976, 1988); co-suppression (Napoli, et al, Plant Cell, 2:279-289, 1990); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse et al, Proc. Natl. Acad. Sci. U.S.A., 95:13959-13964, 1998).
  • Methods for the suppression of endogenous sequences in ahost cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence. Antisense inhibition may use the entire cDNA sequence (Sheehy et al., Proc. Natl. Acad. ScL U.S.A., 85:8805-8809, 1988), a partial cDNA sequence including fragments of 5' coding sequence (Cannon et al, Plant MoI. Biol, 15:39-47, 1990), or 3' non-coding sequences (Ch'ng et al, Proc. Natl Acad. Sci.
  • Cosuppression techniques may use the entire cDNA sequence (Napoli et al, supra; van der Krol et al, The Plant Cell, 2:291-299, 1990), or a partial cDNA sequence (Smith ed/., MoI Gen. Genetics, 224:477-481, 1990). Standard molecular and genetic tests may be performed to further analyze the association between a gene and an observed phenotype. Exemplary techniques are described below.
  • expression profiling is used to simultaneously measure differences or induced changes in the expression of many different genes.
  • Techniques for microarray analysis are well known in the art (see, for example, Schena et al, Science, 270:467-470, 1995; Baldwin et al, Cur. Opin. Plant Biol, 2(2):96-103, 1999; Dangond, Physiol. Genomics, 2:53-58, 2000; van Hal et al, J. Biotechnol, 78:271-280, 2000; Richmond & Somerville, Cur. Opin. Plant Biol, 3:108-116, 2000).
  • Expression profiling of individual tagged lines may be performed. Such analysis can identify other genes that are coordinately regulated as a consequence of the over- expression of the gene of interest, which may help to place an unknown gene in a particular pathway.
  • Gene Product Analysis Analysis of gene products may include recombinant protein expression, antisera production, immuno localization, biochemical assays for catalytic or other activity, analysis of phosphorylation status, and analysis of interaction with other proteins via yeast two- hybrid assays.
  • Pathway analysis may include placing a gene or gene product within a particular biochemical, metabolic or signaling pathway based on its mis-cxprcssion phcnotypc or by sequence homology with related genes.
  • analysis may comprise genetic crosses with wild-type lines and other mutant lines (creating double mutants) to order the gene in a pathway, or determining the effect of a mutation on expression of downstream "reporter" genes in a pathway.
  • the disclosure further provides a method of identifying plants having increased pathogen resistance, in particular, plants that have a mutation in an endogenous FUR gene that confers such resistance.
  • This method comprises analyzing at least one- FUR gene from a population of plants, and identifying a plant with an altered (e.g., mutated) FUR gene.
  • the FUR gene may have a mutation that confers the pathogen resistance, or it may have an altered expression as compared to a wild-type plant.
  • Pathogen-resistant progeny of these plants that are not genetically modified may be generated. Methods for producing and identifying plants with mutations that confer pathogen resistance are known in the art.
  • TILLING for targeting induced local lesions in genomes
  • mutations are induced in the seed of a plant of interest, for example, using EMS treatment.
  • the resulting plants are grown and self-fertilized, and the progeny are used to prepare DNA samples.
  • PCR amplification and sequencing of the FUR gene is used to identify whether a mutated plant has a mutation in the FUR gene.
  • Plants having FUR mutations may then be tested for pathogen resistance, or alternatively, plants may be tested for pathogen resistance, and then PCR amplification and sequencing of the FUR gene is used to determine whether a , plant having increased pathogen resistance has a mutated FUR gene.
  • TILLING can identify mutations that may alter the expression of specific genes or the activity of proteins encoded by these genes (see Colbert et al, 2001, Plant Physiol ⁇ 26:480-484; McCallum et al, 2000, Nature Biotechnology 18:455-457).
  • a candidate gene/Quantitative Trait Loci (QTLs) approach can be used in a marker-assisted breeding program to identify mutations in the FUR gene or ⁇ orthologs of FUR gene that may confer resistance to pathogens (sec Foolad el al, , Theor. Appl Genet, 2002, 104(6-7):945-958; Roman i a/., 2002, Theor. Appl. Genet., 105(l):145- 159; Dekkers and Hospital, 2002, Nat. Rev. Genet., Jan;3(l):22-32).
  • a FUR nucleic acid is used to identify whether a pathogen-resistant plant has a mutation in the endogenous FUR gene or has a particular allele that causes a pathogen resistance phenotype.
  • T2 seed was collected from Tl plants and stored in an indexed collection, and a portion of the T2 seed was accessed for the forward genetic screen.
  • T3 seed was used in the reverse genetic screen.
  • T2 seed was sown in soil and plants were exposed to the herbicide to kill plants lacking the ACTTAG vector.
  • T2 plants were grown to maturity, allowed to self- fertilize and set seed.
  • T3 seed (from the T2 plants) was harvested in bulk for each line, and a portion of the T3 seed was accessed for the reverse genetic screen (see below).
  • the position of the ACTTAG element in the genome in each line was determined using T3 seed by inverse PCR.
  • the PCR product was subjected to sequence analysis and placed on the genome using a basic BLASTN search and/or a search of the' Arabidopsis Information Resource (TAIR) database (available at the arabidopsis.org website). 38,090 lines with recovered flanking sequences were considered in the reverse genetic screen.
  • TAIR Arabidopsis Information Resource
  • the forward genetics screen was conducted as a primary and secondary screen.
  • T2 seed from lines from the Arabidopsis ACTTAG collection and seed from wild-type CoI-O were planted in soil.
  • the seeds were stratified for 2 days at 4°C and then grown in a growth chamber at 23 0 C with 75% relative humidity on a long-day light cycle of 16 hours light and 8 hours dark for 1 week.
  • the plants were sprayed with a solution containing 3xlO 5 spores of F. oxysporum sp. conglutinans.
  • the plants were covered with domes and allowed to grow in growth chambers at 25 0 C with 95% relative humidity for 4 weeks.
  • Each plant was then evaluated for stress caused by the fungus. Any lines with a plant showing no stress were submitted for further analysis.
  • T2 seed from Arabidopsis ACTTAG lines identified in the primary screen and seed from wild-type CoI-O were planted in the same flat. Planting was performed in triplicate for each ACTTAG line identified in the primary screen. These plants were grown, inoculated with F. oxysporum sp. conglutinans spores, and evaluated for stress as described in the primary screen.
  • ACTTAG locus number determination and ACTTAG copy number determination Because ACTTAG lines may have inserts at more than one genetic locus, the number of genetic loci containing the ACTTAG inserts was estimated in each line identified in Example 2. In Tl plants, ACTTAG inserts are present in the hemizygous state (that is, they are present inserted in one of the two copies of the genome of the diploid plant). Because of genetic segregation, in T2 plants each genetic locus containing an ACTTAG insert is present in a 3: 1 ratio; 75% of the T2 plants will have the ACTTAG insert at that locus and 25% will not.
  • Tl plant contains two ACTTAG elements at independently segregating loci
  • the number of T2 plants containing any ACTTAG element will be 87.5% and 12.5% of the plants will not contain an insert. Because each ACTTAG element contains a gene conferring resistance to the herbicide BASTA, the number of genetic loci containing an ACTTAG element can be estimated by determining the percentage of T2 plants that are resistant to BASTA.
  • the proportion T2 plants resistant to the selective agent 50-100 T2 seeds were sown in soil, allowed to germinate, and the number of germinated T2 seedlings was recorded.
  • the T2 seedlings were sprayed with 60 mg/L of the herbicide BASTA 6 times over a period of 2 weeks to kill plants lacking the ACTTAG inserts.
  • the number of BASTA resistant T2 seedlings was determined and the percentage of BASTA resistant plants calculated. Lines that had 60-80% BASTA-resistant T2 seedlings were estimated to carry an ACTTAG insert at a single genetic locus. Lines that had greater than 80% BASTA-resistant T2 seedlings were estimated to carry an ACTTAG insert at more than one genetic locus.
  • each genetic locus can contain more than one insert, the number of ACTTAG elements was estimated in each line identified in Example 2.
  • a TaqMan® polymerase chain reaction (PCR) based method was used using TaqMan® Universal PCR master Mix (Applied Biosystems) and ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Briefly, genomic DNA was isolated from a pool of at least 18 T2 seedlings. Two PCR reactions were carried out simultaneously in a reaction mixture using the DNA of an ACTTAG line as the template.
  • One PCR reaction detects the presence of the BAR gene, which confers resistance to the herbicide glufosinate-ammonium, using the PCR primers specific to the BAR gene.
  • the other PCR reaction detects the presence of the ELF3 gene in Arabidopsis using PCR primers specific to the ELFl gene. The relative amounts of the two PCR products accumulated during the course of the reaction were used to determine the ACTTAG copy number.
  • ACTTAG locus number estimate and ACTTAG copy number estimate for these lines are show in Table 1 below.
  • Phenotype Determination of ACTTAG insertion site in the Arabidopsis genome
  • FURl The right border of the ACTTAG insert is just upstream of nucleotide -54372 of Arabidopsis thaliana DNA chromosome 4, BAC clone F23E13 (>gi
  • FUR2 The left border of the ACTTAG insert is just downstream of nucleotide ⁇ 79693 Arabidopsis thaliana genomic DNA, chromosome 1 , BAC clone:F17L21 (>gi
  • FUR3 The right border of the ACTTAG insert is just upstream of nucleotide ⁇ 64641 Arabidopsis thaliana genomic DNA, chromosome 5, Pl clone:MXC9 (gi
  • FUR4 The right border of the ACTTAG insert is just upstream of nucleotide ⁇ 61146 Arabidopsis thaliana genomic DNA, chromosome 5, BAC clone:F15A17 (>gi
  • FUR5 The right border of the ACTTAG insert is just upstream of nucleotide ⁇ 78585 Arabidopsis thaliana genomic DNA, chromosome 3, Pl clone: MZN24 (>gi
  • SYBR green dye real-time quantitative RT-PCR was performed using primers specific to the genes with sequence IDs presented in column 3 of Table 2 and to a constitutively expressed actin gene (ACT2, positive control).
  • ACT2 constitutively expressed actin gene
  • Genes identii ⁇ ed in the forward and reverse genetic screens were tested to identify whether direct over-expression can confer resistance to fusarium.
  • the plant transformation vector contains a gene encoding a selectable marker driven by the RE4 promoter, to provide resistance to a cytotoxic agent, and serve as a selectable marker. Seed from the transformed plants were plated on agar medium containing the cytotoxic agent. After 10 days, transgenic plants were identified as healthy green plants and transplanted to soil.
  • T2 seed was collected from 20 primary transformants containing each construct, T2 plants were tested for resistance to Fusarium in replicated experiments.
  • approximately 16 T2 seeds from a transgenic event were planted in soil in a 10 row tray.
  • Each tray contained 8 rows seeded with 16 transgenic lines (1 event per row) and 2 rows seeded with wild-type CoI-O seeds; 1 of the rows containing CoI-O was inoculated and served as the negative control, the other was not inoculated and served as the positive control.
  • the seeds were stratified for 2 days at 4 0 C and then grown in a growth chamber at 23 0 C with 75% relative humidity on a long-day light cycle of 16 hours light and 8 hours dark for 1 week.
  • the plants were sprayed with a solution containing 3xlO 5 spores of F. oxysporum sp. conglulinans.
  • the plants were covered with domes and allowed to grow in growth chambers at 25°C with 95% relative humidity for 4 weeks. Each plant was then evaluated for stress caused by the fungus. Resistant plants were identified as plants showing no stress symptoms.
  • the genes in Table 5 showed positiye recapitulation results.
  • At4g36240 FURl-A
  • CsVMV promoter driving expression of At4g36240 was tested by growing T2 plants containing the CsVMV promoter driving expression of At4g36240 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Six of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 6 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table. TABLE 6
  • At4g36250 FURl-B
  • CsVMV promoter driving expression of At4g36250 was tested by growing T2 plants containing the CsVMV promoter driving expression of At4g36250 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Five of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 7 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • Atlg27460 FUR2-D
  • CsVMV promoter driving expression of Atlg27460 was tested by growing T2 plants containing the CsVMV promoter driving expression of Atlg27460 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Six of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 8 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • Fusarium resistance is conferred by over-expression of At5gl 2210
  • the effect of over-expression of At5gl2210 (FUR3-A) on Fusarium resistance was tested by growing T2 plants containing the CsVMV promoter driving expression of At5gl2210 from 20 independent transformation events in two separate experiments as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Five of the transformation events produced more plants than the control that were score resistant to Fusarium in both experiments. Table 9 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • Fusarium resistance is conferred by over-expression of At5gl2230 .
  • At5gl2230 FUR3-C
  • CsVMV promoter driving expression of At5gl2230 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Three of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 10 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At5gl2235 FUR3-D
  • CsVMV promoter driving expression of At5gl2235 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Ten of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 1 1 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At5g03180 FUR4-C
  • the effect of over-expression of At5g03180 (FUR4-C) on Fusarium resistance was tested by growing T2 plants containing the CsVMV promoter driving expression of At5gO3180 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Eleven of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 12 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table. '
  • At5g03190 FUR4-D
  • FUR4-D over-expression of At5g03190
  • Table 13 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At3g22080 FUR5-A
  • the effect of over-expression of At3g22080 (FUR5-A) on Fusarium resistance was tested by growing T2 plants containing the CsVMV promoter driving expression of At3g22080 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Four of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 14 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At3g22090 FUR5-B
  • the effect of over-expression of At3g22090 (FUR5-B) on Fusarium resistance was tested by growing T2 plants containing the CsVMV promoter driving expression of At3g22090 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Eleven of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 15 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At3g22120 FUR5-D
  • CsVMV promoter driving expression of At3g22120 was tested by growing T2 plants containing the CsVMV promoter driving expression of At3g22120 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Six of the transformation events produced more plants than the control that were score resistant to Fusarium. Table 16 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At2g44100 FURl 003
  • CsVMV promoter driving expression of At2g44100 from 20 independent transformation events in two separate experiments as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Five of the transformation events produced more plants than the control that were score resistant to Fusarium in both experiments. Table 17 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At4g35090 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible.
  • Table 18 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At3g45300 FURl 009
  • CsVMY promoter driving expression of At3g45300 from 20 independent transformation events in two separate experiments as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Six of the transformation events produced more plants than the control that were score resistant to Fusarium in both experiments. Table 19 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At3g26100 FURlOl 1
  • CsVMV promoter driving expression of At3g26100 from 20 independent transformation events in two separate experiments as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible. Four of the transformation events produced more plants than the control that were score resistant to Fusarium in both experiments. Table 20 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.
  • At2g36320 from 20 independent transformation events as described above. Each plant was evaluated for symptoms of Fusarium infection and scored as either resistant or susceptible.
  • Table 21 shows the events that produced plants resistant to Fusarium and how many plants were scored resistant. The number of control plants that were scored is also listed in the table.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne des plantes qui présentent un phénotype de résistance aux agents pathogènes modifié (par exemple, une résistance fongique accrue) dû à l'expression modifiée d'un acide nucléique FU. L'invention concerne en outre des procédés permettant de générer des plantes avec un phénotype de résistance aux agents pathogènes modifié.
PCT/US2007/071146 2006-06-13 2007-06-13 Génération de plantes avec une meilleure résistance aux agents pathogènes Ceased WO2008054890A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81363706P 2006-06-13 2006-06-13
US60/813,637 2006-06-13

Publications (1)

Publication Number Publication Date
WO2008054890A1 true WO2008054890A1 (fr) 2008-05-08

Family

ID=38566911

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/071146 Ceased WO2008054890A1 (fr) 2006-06-13 2007-06-13 Génération de plantes avec une meilleure résistance aux agents pathogènes

Country Status (1)

Country Link
WO (1) WO2008054890A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2350291A4 (fr) * 2008-11-03 2012-02-22 Swetree Technologies Ab Matière végétale, plantes, et procédé de production d'une plante dont les propriétés de la lignine sont modifiées
WO2013176548A1 (fr) * 2012-05-25 2013-11-28 Wageningen Universiteit Nouveau gène de résistance pour plantes
WO2019074737A1 (fr) * 2017-10-10 2019-04-18 Dow Agrosciences Llc Molécules d'acide nucléique aldéhyde déshydrogénase (aldh1) qui régulent des pathogènes
CN112250744A (zh) * 2019-07-05 2021-01-22 中国农业大学 蛋白质ZmHEI10在调控玉米产量和抗病性中的应用
CN114480423A (zh) * 2021-06-03 2022-05-13 浙江农林大学 BrMYC3-1基因过表达在提高植物对真菌病原体抗性中的应用
CN116284289A (zh) * 2022-12-02 2023-06-23 中国农业大学 氧化还原感受器apt1在培育抗根腐病植物中的应用
CN120775899A (zh) * 2025-07-10 2025-10-14 西南林业大学 BnGAE1基因在调控甘蓝型油菜菌核病抗性中的应用
CN120775899B (en) * 2025-07-10 2026-01-23 西南林业大学 Application of BnGAE gene in regulation and control of sclerotinia rot resistance of brassica napus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031349A2 (fr) * 2002-09-18 2004-04-15 Mendel Biotechnology, Inc. Polynucleotides et polypeptides chez des plantes
WO2004087923A1 (fr) * 2003-03-31 2004-10-14 Korea Research Institute Of Bioscience And Biotechnology Nouveau facteur de transcription de resistance au stress (capif1 et nbpif1), son gene et vegetal resistant au stress transfecte par ledit gene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031349A2 (fr) * 2002-09-18 2004-04-15 Mendel Biotechnology, Inc. Polynucleotides et polypeptides chez des plantes
WO2004087923A1 (fr) * 2003-03-31 2004-10-14 Korea Research Institute Of Bioscience And Biotechnology Nouveau facteur de transcription de resistance au stress (capif1 et nbpif1), son gene et vegetal resistant au stress transfecte par ledit gene

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
OH SANG-KEUN ET AL: "A plant EPF-type zinc-finger protein, CaPIF1, involved in defence against pathogens", MOLECULAR PLANT PATHOLOGY, vol. 6, no. 3, May 2005 (2005-05-01), pages 269 - 285, XP009091054, ISSN: 1464-6722 *
PARK J M ET AL: "Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco", PLANT CELL, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 13, no. 5, May 2001 (2001-05-01), pages 1035 - 1046, XP002973198, ISSN: 1040-4651 *
REYES JOSE C ET AL: "The GATA family of transcription factors in Arabidopsis and rice", PLANT PHYSIOLOGY (ROCKVILLE), vol. 134, no. 4, April 2004 (2004-04-01), pages 1718 - 1732, XP009091052, ISSN: 0032-0889 *
SANG HEE KIM ET AL: "CAZFP1, Cys2/His2-type zinc-finger transcription factor gene functions as a pathogen-induced early-defense gene in Capsicum annuum", PLANT MOLECULAR BIOLOGY, KLUWER ACADEMIC PUBLISHERS, DO, vol. 55, no. 6, 1 August 2004 (2004-08-01), pages 883 - 904, XP019262547, ISSN: 1573-5028 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2350291A4 (fr) * 2008-11-03 2012-02-22 Swetree Technologies Ab Matière végétale, plantes, et procédé de production d'une plante dont les propriétés de la lignine sont modifiées
WO2013176548A1 (fr) * 2012-05-25 2013-11-28 Wageningen Universiteit Nouveau gène de résistance pour plantes
US9732354B2 (en) 2012-05-25 2017-08-15 Wageningen Universiteit Plant resistance gene
WO2019074737A1 (fr) * 2017-10-10 2019-04-18 Dow Agrosciences Llc Molécules d'acide nucléique aldéhyde déshydrogénase (aldh1) qui régulent des pathogènes
US10913955B2 (en) 2017-10-10 2021-02-09 Dow Agrosciences Llc Aldehyde dehydrogenase (ALDH1) nucleic acid molecules that control pathogens
CN112250744A (zh) * 2019-07-05 2021-01-22 中国农业大学 蛋白质ZmHEI10在调控玉米产量和抗病性中的应用
CN112250744B (zh) * 2019-07-05 2022-04-05 中国农业大学 蛋白质ZmHEI10在调控玉米产量和抗病性中的应用
CN114480423A (zh) * 2021-06-03 2022-05-13 浙江农林大学 BrMYC3-1基因过表达在提高植物对真菌病原体抗性中的应用
CN114480423B (zh) * 2021-06-03 2023-06-23 浙江农林大学 BrMYC3-1基因过表达在提高植物对真菌病原体抗性中的应用
CN116284289A (zh) * 2022-12-02 2023-06-23 中国农业大学 氧化还原感受器apt1在培育抗根腐病植物中的应用
CN120775899A (zh) * 2025-07-10 2025-10-14 西南林业大学 BnGAE1基因在调控甘蓝型油菜菌核病抗性中的应用
CN120775899B (en) * 2025-07-10 2026-01-23 西南林业大学 Application of BnGAE gene in regulation and control of sclerotinia rot resistance of brassica napus

Similar Documents

Publication Publication Date Title
US20090089892A1 (en) Generation of plants with improved pathogen resistance
US20110207608A1 (en) Transcriptional and post-transcription regulation of transcription factor for drought resistance
US8222485B2 (en) Generation of plants with improved pathogen resistance
WO2008054890A1 (fr) Génération de plantes avec une meilleure résistance aux agents pathogènes
US8044262B2 (en) Generation of plants with improved drought tolerance
US7897837B2 (en) Generation of plants with improved pathogen resistance
CN103361325B (zh) 一种与水稻白叶枯抗性相关的蛋白及其编码基因与应用
Wang et al. A pair of GPI‐anchored proteins regulate soybean immunity and disease resistance in coordination with GmLMM1 and GmRALFs
WO2004086853A2 (fr) Generation de plantes a resistance pathogene amelioree

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07863361

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07863361

Country of ref document: EP

Kind code of ref document: A1