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WO2007120820A2 - Plant disease resistance genes and proteins - Google Patents

Plant disease resistance genes and proteins Download PDF

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Publication number
WO2007120820A2
WO2007120820A2 PCT/US2007/009124 US2007009124W WO2007120820A2 WO 2007120820 A2 WO2007120820 A2 WO 2007120820A2 US 2007009124 W US2007009124 W US 2007009124W WO 2007120820 A2 WO2007120820 A2 WO 2007120820A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
plant
percentage
conserved domain
Prior art date
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PCT/US2007/009124
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French (fr)
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WO2007120820A3 (en
Inventor
T. Lynne Reuber
Karen S. Century
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Mendel Biotechnology, Inc.
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Publication date
Application filed by Mendel Biotechnology, Inc. filed Critical Mendel Biotechnology, Inc.
Publication of WO2007120820A2 publication Critical patent/WO2007120820A2/en
Priority to US12/077,535 priority Critical patent/US8030546B2/en
Priority to US12/157,329 priority patent/US7956242B2/en
Priority to US12/169,527 priority patent/US7960612B2/en
Publication of WO2007120820A3 publication Critical patent/WO2007120820A3/en
Priority to US13/244,288 priority patent/US20120137382A1/en
Priority to US14/480,473 priority patent/US20150135360A1/en
Priority to US15/347,676 priority patent/US10597667B2/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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

Definitions

  • the claimed invention in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Corporation as a result of activities undertaken within the scope of a joint research agreement in effect on or before the date the claimed invention was made.
  • the present invention relates to plant genomics and more specifically pertains to polynucleotides that encode polypeptides that confer disease resistance in plants.
  • Plant pathogens cause enormous world-wide annual losses in yield. Using plant biotechnology to engineer disease resistant crops has the potential to make a significant economic impact on agriculture and forestry industries in two ways: reducing the monetary and environmental expense of fungicide application and reducing both pre-harvest and post-harvest crop losses that occur now despite the use of costly disease management practices.
  • the use of genetic engineering technologies to enhance the natural ability of plants to resist pathogen attack holds great potential for enhancing yields while reducing chemical use.
  • genetic engineering of disease resistance may be a critical component of a long-term environmentally sound and economically feasible strategy for increasing global food production. Plant pathogens fall into two major classes: biotrophs and necrotrophs (reviewed in Oliver and Ipcho (2004) MoI. Plant Pathol.
  • Biotrophic pathogens obtain energy by parasitizing living plant tissue, while necrotrophs obtain energy from dead plant tissue.
  • biotrophs include the powdery mildews, rusts, and downy mildews; these pathogens can only grow in association with living plant tissue, and parasitize plants through intracellular feeding structures called haustoria.
  • necrotrophs include Sclerotinia sclerotiorum (white mold), Bottytis cinerea (grey mold), and Cochliobolus heterostrophus (Southern corn leaf blight).
  • the general pathogenic strategy of necrotrophs is to kill plant tissue through toxins and lytic enzymes, and live off the released nutrients.
  • Pathologists also recognize a third class of pathogens, called hemibiotrophs: these pathogens have an initial biotrophic stage, followed by a necrotrophic stage once a parasitic association with plant cells has been established.
  • biotrophic pathogens Infection by biotrophic pathogens often induces defense responses mediated by the plant hormone salicylic acid, while attack by a necrotrophic pathogen often induces defense responses mediated by coordinated action of the hormones ethylene and jasmonate.
  • Genetically engineered traits such as defense response, may be controlled through a number of regulatory processes.
  • One important way to manipulate control of cellular processes is through transcription factors, proteins that influence the expression of a particular gene or sets of genes.
  • transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism.
  • Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits.
  • Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with new and/or improved commercially valuable properties such as pathogen resistance.
  • the present invention pertains to transgenic plants that have greater resistance to a pathogen than a control plant.
  • the transgenic plants of the present invention have each been transformed with at least one a transcription gene sequence encoding an AP2, Myb, HLH, or WRKY family polypeptide.
  • the invention also encompasses polynucleotide and polypeptide sequences that are closely-related to the AP2, MYB HLH 5 or WRKY family polynucleotide and polypeptides.
  • polypeptide sequences may be compared to any of the sequences in the Sequence Listing, and in particular to the sequences, henceforth referred to as reference sequences, that have been shown to confer increased disease resistance in plants, including G207 (SEQ ID NO: 2), G1750 (SEQ ID NO: 50), G440 (SEQ ID NO: 52), G 1274 (SEQ ID NO: 84), G591 (SEQ ID NO: 128), G233 (SEQ ID NO: 146), G4 (SEQ ID NO: 160), G869 (SEQ ID NO: 181), or G237 (SEQ ID NO: 188).
  • Polypeptide sequences that are closely-related to sequences in the Sequence Listing may be identified by having similar conserved domains and at least one similar function of conferring a transcriptional regulatory activity of the reference sequence.
  • the transcriptional regulatory activity may confer greater disease resistance as compared to a control plant.
  • conserved domain sequences are optimally aligned using a BLOSUM62 matrix, a gap existence penalty of 1 1 , and a gap extension penalty of 1 , similar conserved domains may be identified by virtue of having a minimum percentage identity or similarity.
  • polypeptides of the invention have conserved domains that have: a percentage identity of at least 67%, 69%, 70%, 7 1%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%, 83%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2; a percentage identity of at least 61%, 62%, 65%, 69%, 70%, 71%, 72%, 74%, 77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81%, 82%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 93%, or 96%,
  • any of the polypeptides of the invention is overexpressed in a transgenic plant (in other words its expression levels are greater than the expression level of the same sequence in a control plant), the polypeptide regulates expression of other plant genes involved in the defense response, and thus increases pathogen resistance in the plant.
  • Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
  • CD-ROMs Copy 1 and Copy 2 are read-only memory computer-readable compact discs.
  • Each CD-ROM contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named
  • Figure 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Soltis et al. (1997) Ann. Missouri Bot. Gard. 84: 1-49). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales.
  • Figure 1 was adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333.
  • Figure 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including cladcs containing tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl. Acad. Set. USA 97: 9121 -912; and Chase et al. ( ⁇ 993) Ann. Missouri Bot. Gard. 80: 528-580.
  • the present invention relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased resistance to pathogens and/or disease, and/or increased yield with respect to a control plant (for example, a genetically unaltered or non- transgenic plant such as a wild-type plant of the same species, or a transgenic plant line that comprises an empty expression vector).
  • a control plant for example, a genetically unaltered or non- transgenic plant such as a wild-type plant of the same species, or a transgenic plant line that comprises an empty expression vector.
  • nucleic acid molecule refers to an oligonucleotide, polynucleotide or any fragment thereof.
  • Polynucleotide is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides.
  • a polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof.
  • a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, or the like.
  • the polynucleotide can be single-stranded or double-stranded DNA or RNA.
  • the polynucleotide optionally comprises modified bases or a modified backbone.
  • the polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like.
  • the polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the polynucleotide can comprise a sequence in either sense or antisense orientations.
  • "Oligonucleotide" is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded.
  • Gene refers to the partial or complete coding sequence of a gene, its complement, and its 5' or 3' untranslated regions.
  • a gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide.
  • a gene may be isolated, partially isolated, or found with an organism's genome.
  • a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.
  • genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. (1976) Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th ed., Springer Verlag, Berlin).
  • a gene generally includes regions preceding ("leaders”; upstream) and following ("trailers”; downstream) the coding region.
  • a gene may also include intervening, non-coding sequences, referred to as "introns", located between individual coding segments, referred to as "exons". Most genes have an associated promoter region, a regulatory sequence 5 1 of the transcription initiation codon (there are some genes that do not have an identifiable promoter).
  • the function of a gene may also be regulated by enhancers, operators, and other regulatory elements.
  • a "recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity.
  • the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
  • isolated polynucleotide is a polynucleotide, whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not.
  • an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.
  • polypeptide is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues.
  • a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof.
  • the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a DNA-binding domain; or the like.
  • the polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
  • Protein refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
  • a “Portion”, as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies.
  • a “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide.
  • a “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art.
  • the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
  • “Homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.
  • Identity refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison.
  • percent identity refers to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
  • Sequence similarity refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences.
  • a degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences.
  • a degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.
  • “Alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. conserveed domains such as those of Tables 1 -9 and a suitable method such as, for example, the BLOSUM62 matrix, may be used to identify conserved domains and relatedness within these domains.
  • An alignment may also be generated by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, CA) or CLUSTALX (Thompson et al. (1997) Nucl. Acids Res. 24: 4876-4882).
  • optical alignment refers to an alignment (including the introduction of gaps in the sequences as necessary) thai results in the highest similarity score (Holman (2004) "Protein similarity score: a simplified version of the BLAST score as a superior alternative to percent identity for claiming genuses of related protein sequences"; 21 Santa Ciara Computer & High Tech. L.J. pp. 55-99). Similarity scores may be determined, for example, using BLAST analysis.
  • a “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences.
  • a “Myb” domain” such as is found in a polypeptide member of the MYB-(R 1)R2R3 transcription factor family, is an example of a conserved domain. With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is preferably at least nine base pairs (bp) in length.
  • a conserved domain with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology, such as the percentage identities or percentage similarities listed in Tables 1-9, to a conserved domain of a polypeptide of the invention. Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological activity to the present transcription factor sequences, thus being members of a clade of transcription factor polypeptides, are encompassed by the invention.
  • a fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular transcription factor class, family, or sub-family.
  • the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site.
  • a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide can be "outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
  • conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000a) Science 290, 2105-21 10; and Riechmann (2000b) Curr. Opin. Plant Biol. 3, 423-434).
  • conserved domains of the plant transcription factors see, for example, Marchler-Bauer et al. (2003) Nucleic Acids Res. 31 : 383- 387; or Magnani et al. (2004) Plant Cell 16: 2265-2277, may be determined.
  • the conserved domains for many of the transcription factor sequences of the invention are listed in Tables 1 -9. Also, the polypeptides of Tables 1-9 have conserved domains specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Marchler-Bauer et al. (2003) supra) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure. "Complementary" refers to the natural hydrogen bonding by base pairing between purines and pyrimidines.
  • sequence A-C-G-T ( 1 — > 3 1 ) forms hydrogen bonds with its complements A-C-G-T (5' — > ⁇ 3 1 ) or A-C-G-U (5' — * 3').
  • Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or "completely complementary " if all of the nucleotides bond.
  • the degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization and amplification reactions.
  • “Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.
  • highly stringent or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs.
  • Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313: 402-404, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual.
  • stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see the section "Identifying
  • nucleic acid sequences from a variety of sources can be isolated on the basis of their ability to hybridize with known transcription factor sequences.
  • nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein.
  • Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, encoded transcription factors having 38% or greater identity with the conserved domain of disclosed transcription factors.
  • orthologs and paralogs are evolutionarily related genes that have similar sequences and functions.
  • Orthologs are structurally related genes in different species that are derived by a speciation event.
  • Paralogs are structurally related genes within a single species that are derived by a duplication event.
  • equivalog describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) World Wide Web
  • variable refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.
  • polynucleotide variants differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide.
  • Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
  • a variant of a transcription factor nucleic acid listed in the Sequence Listing that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code.
  • polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.
  • allelic variant or “polynucleotide allelic variant” refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be "silent" or may encode polypeptides having altered amino acid sequence.
  • Allelic variant and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.
  • "Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism.
  • “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.
  • polynucleotide variants may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences.
  • Polypeptide variants may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.
  • a polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the transcription factor is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
  • a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Similar minor variations may also include amino acid deletions or insertions, or both.
  • Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see USPN 5,840,544).
  • “Fragment" with respect to a polynucleotide refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic.
  • Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation.
  • a "polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein.
  • Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing.
  • Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a transcription factor.
  • Exemplary fragments also include fragments that comprise a conserved domain of a transcription factor.
  • Exemplary fragments include fragments that comprise an conserved domain of a transcription factor, for example, amino acid residues 6 to 106 of G207 (SEQ ID NO: 215, or the Myb domain of SEQ ID NO: 2), 115 to 177 of G1750 (SEQ ID NO: 239, or the AP2 domain of SEQ ID NO: 50), 122 to 184 of G440 (SEQ ID NO: 240, or the AP2 domain of SEQ ID NO: 52), amino acid residues 1 10 to 166 of Gl 274 (SEQ ID NO: 256, or the WRKY domain of SEQ ID NO: 84), amino acid residues 149 to 206 of G591 (SEQ ID NO: 278, or the HLH domain of SEQ ID NO: 128), amino acid residues 13 to 115 of G233 (SEQ ID NO: 287, or the Myb domain of SEQ ID NO: 146), amino acid residues
  • Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential.
  • the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide.
  • a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds Io a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription.
  • Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.
  • the invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.
  • “Derivative” refers to the chemical modification of a nucleic acid molecule or amino acid sequence. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process that retains or enhances biological activity or lifespan of the molecule or sequence.
  • plant includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and ceils (for example, guard cells, egg cells, and the like), and progeny of same.
  • shoot vegetative organs/structures for example, leaves, stems and tubers
  • roots for example, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules)
  • seed including embryo, endosperm, and seed coat
  • fruit the mature ovary
  • plant tissue for example, vascular tissue, ground tissue, and the like
  • ceils for example, guard cells, egg cells, and the like
  • the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, ps ⁇ lophytes, lycophytes, bryophytes, and multicellular algae (see for example, Figure 1, adapted from Daly et al. (2001) supra, Figure 2, adapted from Ku et al. (2000) supra; and see also Tudge (2000) in The Variety of Life, Oxford University Press, New York, NY pp. 547-606.
  • control plant refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant.
  • a control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated.
  • a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested.
  • a suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
  • a "transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar.
  • the genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty.
  • the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
  • a transgenic plant may contain an expression vector or cassette.
  • the expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the controlled expression of polypeptide.
  • the expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
  • a plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • Wild type or wild-type, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a transcription factor expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
  • a “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring resistance to pathogens or tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as extent of disease, hyperosmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
  • Trait modification refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant.
  • the trait modification can be evaluated quantitatively.
  • the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild-type plants.
  • the plants When two or more plants have “similar morphologies”, “substantially similar morphologies”, “a morphology that is substantially similar”, or are “morphologically similar”, the plants have comparable forms or appearances, including analogous features such as overall dimensions, height, width, mass, root mass, shape, glossiness, color, stem diameter, leaf size, leaf dimension, leaf density, internode distance, branching, root branching, number and form of inflorescences, and other macroscopic characteristics, and the individual plants are not readily distinguishable based on morphological characteristics alone.
  • Modulates refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.
  • transcript profile refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state.
  • transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor.
  • the transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.
  • the term "knockout” refers to a plant or plant cell having a disruption in at least one transcription factor gene in the plant or cell, where the disruption results in a reduced expression or activity of the transcription factor encoded by that gene compared to a control cell.
  • the knockout can be the result of, for example, genomic disruptions, including transposons, tilling, and homologous recombination, antisense constructs, sense constructs, RNA silencing constructs, or RNA interference.
  • a T-DNA insertion within a transcription factor gene is an example of a genotypic alteration that may abolish expression of that transcription factor gene.
  • Ectopic expression or altered expression in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species.
  • the pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species.
  • the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant.
  • the term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression.
  • the resulting expression pattern can be transient or stable, constitutive or inducible.
  • the term "ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
  • overexpression refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also under the control of an inducible or tissue specific promoter. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter used. Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors.
  • a strong promoter e.g., the cauliflower mosaic virus 35S transcription initiation region
  • Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the transcription factor in the plant, cell or tissue.
  • transcription regulating region refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors of the present invention possess an conserved domain.
  • the transcription factors of the invention also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more biotic stress resistance genes in a plant when the transcription factor binds to the regulating region.
  • a transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes.
  • transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding site or DNA-binding site motif (see, for example, Riechmann et al. (2000a) supra).
  • the plant transcription factors of the present invention belong to the AP2, Myb, or HLH transcription factor families.
  • the transcription factors encoded by the present sequences are involved in cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to osmotic stresses.
  • the sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.
  • sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant.
  • the sequences of the invention may also include fragments of the present amino acid sequences.
  • amino acid sequence is recited to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • the polynucleotides and polypeptides of the invention have a variety of additional uses.
  • the polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like.
  • the polynucleotide can comprise a sequence in either sense or antisense orientations.
  • transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins.
  • Examples include Peng et al. (1997) Genes Development 1 1 : 3194-3205, and Peng et al. ( ⁇ 999) Nature 400: 256-261.
  • Peng et al. 1997) Genes Development 1 1 : 3194-3205
  • Peng et al. ⁇ 999
  • Many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or a very similar phenotypic response. See, for example, Fu et al. (2001) Plant Cell 13: 1791-1802; Nandi et al.
  • Gilmour et al. (1998) Plant /. 16: 433-442 teach an Arabidopsis AP2 transcription factor, CBFl, which, when overexpressed in transgenic plants, increases plant freezing tolerance.
  • CBFl Arabidopsis AP2 transcription factor
  • Jaglo et al. (2001) Plant Physiol. 127: 910-917 further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato.
  • Transcription factors mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced transcription factor. It is well appreciated in the art that the effect of a transcription factor on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (e.g., by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor.
  • the PAP2 gene and other genes in the Myb family have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000) Plant Cell 12: 65-79; and Borevitz et al. (2000) Plant Cell 12: 2383-2393).
  • global transcript profiles have been used successfully as diagnostic tools for specific cellular states (e.g., cancerous vs. non-cancerous; Bhattacharjee et al. (2001) Proc. Natl. Acad. Sci. USA 98: 13790-13795; and Xu et al. (2001) Proc. Natl. Acad. Sci. USA 98: 15089-15094). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.
  • the present invention provides, among other things, transcription factors (TFs), and transcription factor homolog polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided in the Sequence Listing. Also provided are plants comprising novel transcription factors or variants of the transcription factors, where the plants have greater resistance to a biotrophic pathogen, a neurotrophic pathogen, or both classes of pathogens, than a control plant.
  • the invention also includes methods for increasing a plant's resistance to disease, including disease caused by biotrophic pathogens, necrotrophic pathogens, or both.
  • Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.
  • Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences, were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5' and 3' ends. The full-length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5' and 3' ends. Exemplary sequences are provided in the Sequence Listing.
  • polynucleotides of the invention were also ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be used to change expression levels of a genes, polynucleotides, and/or proteins of plants or plant cells.
  • the present invention relates to compositions and methods for modifying a plant's traits.
  • the compositions include plants comprising polynucleotides that encode novel plant transcription factor polypeptides first identified in Arabidopsis thaliana, a plant used experimentally as a model plant species.
  • the methods include using the polynucleotides and their encoded polypeptides to modify a trait in a transgenic plant, such as the resistance of a plant to a biotic stress, including a plant pathogen.
  • the data presented herein represent the results obtained in experiments with transcription factor polynucleotides and polypeptides that may be expressed in plants for the purpose of reducing reduced quality or yield losses that arise from biotic stress.
  • Arabidopsis sequences including G207 (SEQ ID NO: 2), G 1750 (SEQ ID NO: 50), G440 (SEQ ID NO: 52), G1274 (SEQ ID NO: 84), G591 (SEQ ID NO: 128), G233 (SEQ ID NO: 146), G4 (SEQ ID NO: 160), G869 (SEQ ID NO: 181), or G237 (SEQ ID NO: 188) have been shown to confer increased disease resistance in plants, as compared to the resistance of control plants, when the sequences are overexpressed.
  • Polypeptide sequences that are closely-related to the reference sequences may be identified by having at least one function of each of the reference sequence to which it is compared, and having descended from a common ancestral sequence, and/or having similar conserved domains.
  • the present invention provides for polynucleotide and polypeptide sequences that function by conferring at least one transcriptional regulatory activity of the reference sequence.
  • the transcriptional regulatory activity will generally confer greater disease resistance to a plant overexpressing a reference or related sequence, as compared to a control plant.
  • Closely-related polypeptide sequences with related conserved domains may be identified by BLAST and phylogenetic analysis, as noted below.
  • BLAST and phylogenetic analysis When the reference sequence conserved domain and the conserved domains of putatively related sequences are optimally aligned using a BLOSUM62 matrix, a gap existence penalty of 1 1, and a gap extension penalty of 1, similar conserved domains may be identified by virtue of having a minimum percentage identity or similarity.
  • polypeptides of the invention have conserved domains that have:
  • polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2;
  • polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50;
  • polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52;
  • G207 corresponds to AtMYBR2 and AtMYB77 (Kirik et al. (1998) Plant MoI Biol. 37:, 819-827; Stracke et al. (2001) Curr. Opin. Plant Biol. 4: 447-456). Tn earlier disease studies, a G207 knockout was found to be more susceptible to Botrytis.
  • Table 1 shows a number of G207 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G207 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G207 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G207, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G207 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • At Arabidopsis thaliana
  • Gm Glycine max
  • Os Oryza sativa
  • Zm Zea mays.
  • G 1750 The G 1750 clade, and related sequences
  • G 1750 is a member of the ERF-B5 class of AP2 domain transcription factors. It is a putative ortholog of Tsi 1 from tobacco, which produces enhanced tolerance to multiple pathogens when overexpressed (Park et al. (2001) Plant Cell 13: 1035-1046; Shin et al. (2002) MoI. Plant Microbe Interact. 15: 983-989). It is also related to Pti6, which is implicated in Pto-dependent disease resistance in tomato (Zhou et al. (1997) EMBOJ. 16: 3207-3218). Plants overexpressing G1750 under a constitutive promoter were found to exhibit reduced size and poor fertility; therefore, dexamethasone-inducible G 1750 lines were also produced.
  • G440 is also a member of the ERF-B5 class of AP2 domain transcription factors. Plants overexpressing G440 under a constitutive promoter were found to exhibit reduced size and poor fertility; therefore, dexamethasone-inducible G440 lines were also produced. .
  • Table 2 shows a number of G 1750 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier"
  • Column 2 the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of ⁇ Q Arabidopsis G 1750 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G 1750 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of Gl 750, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G 1750 conserved domain (in Tables 2 and 3, the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • At Arabidopsis thaliana
  • Gm Glycine max
  • Le Lycopersicon esculentum
  • Nt Nicotiana tabacum
  • Zm Zea mays
  • sequences in Table 2 may be compared against the conserved domain of G440 as the reference sequence rather than the similar domain of G 1750 (i.e., in Table 3, G440 is listed first, and subsequent sequences are compared to the conserved domain of G440).
  • the clade as judged by sequences identified to data as clade member is bounded by sequences having 100% identity and similarity to the reference conserved domain sequence of G440 to 65% identity and 79% similarity.
  • Table 3 conserveed AP2 domains of G440 and closely-related sequences
  • At Arabidopsis thaliana
  • Gm Glycine max
  • Le Lycopersicon esculentum
  • Nt Nicotiana tabacum
  • Zm Zea mays
  • G 1274 polynucleotide SEQ ID NO: 83 from Arabidopsis encodes a member of the WRKY family of transcription factors (SEQ FD NO: 84). G1274 corresponds to AtWRKYS 1 (At5g64810), a gene for which there is currently no published information .
  • WRKY transcription factors WRXY genes appear to have originated in primitive eukaryotes such as Giardia lamblia, Dictyostelium discoideum, and the green alga Chlamydomonas reinhardtii, and have since greatly expanded in higher plants (Zhang and Wang (2005) BMC Evol. Biol. 5: 1). In Arabidopsis alone, there are more than 70 members of the WRKY superfamily. The defining feature of the family is the ⁇ 57 amino acid DNA binding domain that contains a conserved WRKYGQK heptapeptide motif. Additionally, all WRKY proteins have a novel zinc-finger motif contained within the DNA binding domain.
  • Group I members have two WRKY domains, while Group II members contain only one. Members of the Group II family can be further split into five distinct subgroups (Ila-e) based on conserved structural motifs.
  • Group III members have only one WRKY domain, but contain a zinc finger domain that is distinct from Group II members. The majority of WRKY proteins are Group II members, including G 1274 and the related genes being studied here.
  • WRKY genes An additional common feature found among WRKY genes is the existence of a conserved intron found within the region encoding the C-terminal WRKY domain of group I members or the single WRKY domain of group I I/I 11 members. In G1274, this intron occurs between the sequence encoding amino acids Rl 30 and N 131.
  • the founding members of the WRKY family are SPFl from sweet potato (Ishiguro and Nakamura, (1994) MoI. Gen. Genet. 244: 563-571), ABF 1/2 from oat (Rushton et al. (1995) Plant MoI. Biol. 29: 691-702), PcWRKYl,2,3 from parsley (Rushton et al. (1996) EMBO J. 15: 5690- 5700) and ZAPl from Arabidopsis (de Pater et al. (1996) Nucleic Acids Res. 24: 4624-4631).
  • the two WRKY domains of Group I members appear functionally distinct, and it is the C- terminal sequence that appears to mediate sequence-specific DNA binding.
  • the function of the N- terminal domain is unclear, but may contribute to the binding process, or provide an interface for protein-protein interactions.
  • the single WRKY domain in Group II members appears more like the C-terminal domain of Group I members, and likely performs the similar function of DNA binding.
  • Structural features of G 1274 The primary amino acid sequence for the predicted G 1274 protein is presented in the Sequence Listing as SEQ ID NO: 84, and the conserved domain of G 1274 (SEQ ID NO: 256) is shown in Table 4. Discoveries made in earlier genomics programs. G 1274 expression in wild-type plants was detected in leaf, root and flower tissue.
  • G 1274 was also enhanced slightly by hyperosmotic and cold stress treatments, and by auxin or ABA application. Additionally, the gene appears induced by Erysiphe infection and salicylic acid treatment, consistent with the known role of WRKY family members in defense responses.
  • the closely related gene G 1275 (SEQ ID NO: 85) is strongly repressed in wild-type plants during soil drought, and remains significantly down- regulated compared to well-watered plants even after rewatering.
  • G 1274 overexpression studies were more tolerant to low nitrogen conditions and were less sensitive to chilling than wild-type plants.
  • G1274 overexpress ' ing seedlings were also positive in a C:N sensing screen, indicating that G1274 may alter the plant's ability to modulate carbon and/or nitrogen uptake and utilization and improve germination and/or growth in low nitrogen conditions.
  • G 1274 overexpression also produced alterations in inflorescence and leaf morphology. Approximately 20% of overexpressors were slightly small and developed short inflorescences that had reduced internode elongation. Overall, these plants were bushier and more compact in stature than wild-type plants. In T2 populations, rosettes of some 35S::G1274 plants were distinctly broad with greater biomass than wild-type.
  • 35S::G1274 plants also out-performed wild-type plants in a soil drought assay; the overexpressors were more tolerant to water deprivation, and also recovered better from water deprivation, than the control plants.
  • Overexpression of G 1275 (SEQ ID NO: 85, AtWRKY50), a gene closely related to G 1274 and also being studied here, had a more severe effect on morphology than G 1274.
  • 35S::G1275 plants were small, with reduced apical dominance and stunted inflorescences. While the plants were fertile, seed yield was low and these plants were not tested in physiological assays.
  • G 1275 was primarily expressed in rosettes and siliques, and had lower but detectable expression in shoots, roots, flowers and embryos.
  • Table 4 shows a number of G 1274 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved WRXY domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the ⁇ rabidopsis G1274 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G 1274 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G 1274, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G 1274 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • At Arabidopsis thaliana; Sb- Sorghum bicolor; Ca - Capsicum anrtuum; Gm — Glycine max; Hv- Hordeum vulgare; Le — Lycopersicon esculentum; Ls — Latuca saliva; Os — Oi ⁇ za sativa; St - Solarium tuberosum; Zm — Zea mays
  • G591 the G591 clade, and related sequences G591 corresponds to Atb ⁇ L ⁇ 059.
  • the only functional information that is published about this gene comes from a post-transcriptional silencing study, in which partial silencing of G591 resulted in strong stunting (Brummell et al. (2003) Plant J. 33: 793-800).
  • G591 has one close paralog, G793 (AtbHLH007), about which no functional information is known.
  • Table 5 shows a number of G591 clade polypeptides of the invention and includes the SEQ
  • Column 1 the species from which the sequence was derived and the*'GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved HLH domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G591 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G591 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G591, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G591 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • At Arabidopsis thaliana
  • Gm Glycine max
  • Os Oryza sativa
  • Zm Zea mays
  • G233 corresponds to AtMYBl 5.
  • G233 is homologous to NtMYBl, which is involved in SA signal transduction (Yang and Klessig (1996) Proc. Natl. Acad. Sci. USA 93, 14972-14977) and NtMYB2 (LBMl), which is involved in phenylpropanoid regulation (Sugimoto el al. (2000) Plant Cell 12, 2511-2528).
  • NtMYBl which is involved in SA signal transduction
  • LBMl NtMYB2
  • Table 6 shows a number of G233 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G233 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G233 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G233, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G233 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • SEQ ID NO: 146 was predicted from the complete coding sequence of SEQ ID NO: 145, whereas SEQ ID NO: 148 is predicted from the nucleotide sequence (SEQ ID NO: 147) actually used in the expression vector to create transgenic plants.
  • SEQ ID NO: 148 is truncated, lacking ten residues found in SEQ ID NO: 146 at its C-terminus, and in their place SEQ ID NO: 148 has three spurious residues at its C-terminus.
  • G4 is a putative ortholog of CaPFl of hot pepper, which produces resistance to Pseudomonas syringae and freezing tolerance when overexpressed (Yi et al. (2004) Plant Physiol. 136: 2862-2874). G4 is a member of the ERF-B2 subfamily of AP2/ERF genes. A related ERFB-2 gene from hot pepper, CaERFLPl , also produces enhanced tolerance to P. syringae and salt stress, supporting a potential role for ERF B-2 genes in disease resistance (Lee et al. (2004) Plant MoI. Biol. 55: 61-81 ).
  • Table 7 shows a number of G4 clade polypeptides (or predicted polypeptides) of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G4 sequence (Column 6), the similarity in percentage terms to the conserved domain of the Arabidopsis G4 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G4, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G4 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • G869 is a member of the ERF-B6 subfamily of AP2/ERF proteins. G869 overexpressing lines showed reduced growth of E. orontii in an earlier functional genomic study. Since G869 overexpressing lines were small and stunted, dexamethasone-inducible lines were also produced for analysis.
  • Table 8 shows a number of G869 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G869 sequence (Column 6), the similarity in percentage terms to the conserved domain of the Arabidopsis G869 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G869, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G869 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • G237 The G237 clade, and related sequences G237 corresponds to AtMYB 18 (Kranz et al. (1998) Plant J. 16, 263-276) and to LAFl, a transcriptional activator involved in phytochrome A signaling (Ballesteros et al. (2001) Genes Dev. 15: 2613-2625). G237 overexpressing lines were found to be small with developmental abnormalities. Therefore, dexamethasone-inducible G237 lines were also characterized in pathogen assays.
  • Table 9 shows a number of G237 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G237 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G237 sequence (Column 7).
  • the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G237, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G237 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
  • Homologous sequences as described above can comprise orthologous or paralogous sequences.
  • Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences.
  • General methods for identifying orthologs and paralogs including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.
  • Eisen Eisen (1998) Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions.
  • gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs.
  • a paralog is therefore a similar gene formed by duplication within the same species.
  • Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. MoL Evol. 25: 351-360).
  • a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122- 132), and a group of very similar AP2 domain transcription factors from
  • Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433- 442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 543)
  • orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined.
  • Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence. It is often found that orthologous groups of genes identified by the methods above share particular signature sequences that are not found in other homologs, and that these signature sequences can be used to predict other functional orthologous sequences (e.g. Panslita (2005) Plant MoI Biol. 59: 485-500). Distinct Arabidopsis transcription factors, including G482 (found in US Patent Application
  • transcription factors that are phylogenetically related to the transcription factors of the invention have similar functions and have conserved domains that share at least 61% amino acid sequence identity or 79% amino acid sequence similarity.
  • sequences of the invention will typically share at least about
  • nucleotide sequence identity preferably at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed full-length sequences, or to a listed sequence but excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains.
  • the degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.
  • Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.).
  • the MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988) Gene 73: 237-244).
  • the cluslal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • Other alignment algorithms or programs may be used, including FASTA, BLAST 5 or ENTRJEZ, FASTA and BLAST, and which may be used to calculate percent similarity.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1 , e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see USPN 6,262,333).
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • sequence identity refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter "off (see, for example, internet website at http://www.ncbi.nlm.nih.gov/).
  • the blastp program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Hen ikoff ( 1989) Proc. Natl. Acad. ScI USA 89:10915).
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Hen ikoff ( 1989) Proc. Natl. Acad. ScI USA 89:10915.
  • the percentage similarity between two polypeptide sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method (see, for example, Hein (1990) Methods Enzymol. 183: 626-645) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see Hillman et al., US Patent " No. 6, 168,920).
  • the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence.
  • a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.
  • polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions.
  • Methods that search for primary sequence patterns with secondary structure gap penalties Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990) J. MoI. Biol. 215: 403-410; Altschul (1993) J. MoI. Evol.
  • a further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related transcription factors. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions.
  • Fowler and Thomashow (2002) Plant Cell 14: 1675-1690 have shown that three paralogous AP2 family genes (CBFl, CBF2 and CBF3), each of which is induced upon cold treatment, and each of which can condition improved freezing tolerance, have highly similar transcript profiles.
  • a transcription factor Once a transcription factor has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function.
  • methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and AP2, Myb or HLH domains.
  • Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined.
  • Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat
  • Orthologs and paralogs of presently disclosed transcription factors may be cloned using compositions provided by the present invention according to methods well known in the art.
  • cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present transcription factors.
  • Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present transcription factor sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue.
  • Transcription factor-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed transcription factor gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • the cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
  • the invention encompasses isolated nucleotide sequences that are phylogenetically and structurally similar to the sequences listed in the Sequence Listing, and can function in a plant by conferring a transcriptional regulatory activity of a reference sequence in the Sequence Listing and increasing resistance to pathogens when ectopically expressed in a plant.
  • a significant number of these sequences are phylogenetically and sequentially related to each other have been shown to increase a plant's resistance to pathogens, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of transcription factors would also perform similar functions when ectopically expressed.
  • Table 10 lists sequences within the UniGene database determined to be closely-related to transcription factor sequences of the present invention (reference sequences G207, G 1750, G440, G1274, G591, G233, G4, G869 or G237, SEQ ID NOs: 2, 50, 52, 84, 128, 146, 160, 181 or 188, respectively).
  • the column headings include the transcription factors listed by: the Reference Arabidopsis sequence used to identify each clade (Column 1); the UniGene identifier of closely- related homologs found by BLAST analysis (Column 2); the species from which the homologs to the clade reference transcription factors were derived (Column 3); the SEQ ID NO: of the homolog, where provided (Column 4); the smallest sum probability relationship (probability or p-value) of the homologous sequence to the Arabidopsis reference sequence for that row, determined by tBLASTx analysis (Column 5); and the percentage identity of the homolog to the Arabidopsis reference sequence for that row, determined by tBLASTx analysis (Column 6).
  • Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions.
  • Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like.
  • the stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-51 1).
  • full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods.
  • the cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
  • T m The melting temperature
  • L is the length of the duplex formed
  • [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution
  • % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C is required to reduce the melting temperature for each 1% mismatch.
  • Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young (1985) supra).
  • one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), poly vinyl-pyrrol idone, ficoll and Denhardt's solution.
  • Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time.
  • conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
  • Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms.
  • the stringency can be adjusted either during the hybridization step or in the post-hybridization washes.
  • Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at T m -5° C to T m -20° C, moderate stringency at T m -20° C to T n ,- 35° C and low stringency at T m -35° C to T m -50° C for duplex >150 base pairs.
  • Hybridization may be performed at low to moderate stringency (25-50° C below T m ), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T m -25° C for DNA-DNA duplex and T m -15° C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
  • High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences.
  • An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C and about 70° C.
  • high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C.
  • Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.
  • Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate.
  • Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C with formamide present.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS) and ionic strength.
  • SDS sodium dodecyl sulfate
  • ionic strength e.g., sodium dodecyl sulfate (SDS) and ionic strength
  • washing steps that follow hybridization may also vary in stringency; the post- hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium citrate.
  • hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example: 6X SSC at 65° C;
  • wash steps of even greater stringency including about 0.2x SSC, 0.1% SDS at 65° C and washing twice, each wash step being about 30 minutes, or about 0.1 x SSC, 0.1% SDS at 65° C and washing twice for 30 minutes.
  • the temperature for the wash solutions will ordinarily be at least about 25° C, and for greater stringency at least about 42° C.
  • Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C to about 5° C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C to about 9° C.
  • wash steps may be performed at a lower temperature, e.g., 50 0 C.
  • An example of a low stringency wash step employs a solution and conditions of at least 25°
  • Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5- 10x higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15x or more, is obtained.
  • a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2x or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide.
  • the particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like.
  • Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987) supra, pages 399-407, supra; and Kimmel (1987) supra).
  • full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods.
  • the cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
  • a transcription factor that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the First trait.
  • Two-component dex-inducible lines In order to test transcription factors under the regulatory control of a dex-inducible promoter (the two component strategy), a kanamycin resistant 35S::LexA-GAL4-TA driver line was established.
  • Component 1 promoter driver lines (Promoter::LexA/GAL4). Initially, a large number of 35S::LexA-GAL4-TA independent driver lines containing construct pMEN262 (also known as P5486, SEQ ID NO: 203) were generated. Primary transformants were selected on kanamycin plates and screened for GFP (green fluorescent protein) fluorescence at the seedling stage. Any lines that showed constitutive GFP activity were discarded. At 10 days, lines that showed no GFP activity were then transferred onto MS agar plates containing 5 ⁇ M dexamethasone. Lines that showed strong GFP activation at two to three days following the dexamethasone treatments were marked for follow-up in the T2 generation.
  • promoter driver lines Promoter driver lines (Promoter::LexA/GAL4). Initially, a large number of 35S::LexA-GAL4-TA independent driver lines containing construct pMEN262 (also known as P5486, SEQ ID NO: 203)
  • line 65 Following similar experiments in the T2 generation, a single line, line 65, was selected for future studies. Line 65 lacked any obvious background expression and all plants showed strong GFP fluorescence following dexamethasone application. A homozygous population for line 65 was then obtained and re-checked to ensure that it still exhibited induction following dexamethasone application. 35S::LexA-GAL4-TA line 65 was also crossed to an opLexA::GUS line to demonstrate that it could drive activation of targets arranged in trans.
  • Component 2 TF construct (opLexA::TF). Having established a driver line, the transcription factors of the invention could be expressed by super-transforming or crossing in a second construct carrying the transcription factor polynucleotide of interest cloned behind a LexA operator site
  • opLexA::TF the second construct carried a sulfonamide selectable marker and was contained within vector backbone pMEN53.
  • the opLexA::TF constructs prepared and used to supertransform plants were opLexA::G 1750 (construct P3963), opLexA::G440 (construct P3963), and opLexA::G869 (construct P9105).
  • Transformation of Arabidopsis was performed by an ⁇ grobacterium-mediated protocol based on the method of Bechtold and Pelletier (1998) Methods MoI. Biol. 82: 259-266. Unless otherwise specified, all experimental work was done using the Columbia ecotype.
  • Plant preparation Arabidopsis seeds were sown on mesh covered pots. The seedlings were thinned so that 6-10 evenly spaced plants remained on each pot 10 days after planting. The primary bolts were cut off a week before transformation to break apical dominance and encourage auxiliary shoots to form. Transformation was typically performed at 4-5 weeks after sowing.
  • Bacterial culture preparation Bacterial culture preparation. Agrobacterium stocks were inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics and grown until saturation. On the morning of transformation, the saturated cultures were centrifuged and bacterial pellets were re-suspended in Infiltration Media (0.5X MS, IX B5 Vitamins, 5% sucrose, I mg/ml benzylaminopurine riboside, 200 ⁇ l/L Silwet L77) until an A600 reading of 0.8 is reached.
  • Infiltration Media 0.5X MS, IX B5 Vitamins, 5% sucrose, I mg/ml benzylaminopurine riboside, 200 ⁇ l/L Silwet L77
  • Transformation and seed harvest The Agrobacterium solution was poured into dipping containers. All flower buds and rosette leaves of the plants were immersed in this solution for 30 seconds. The plants were laid on their side and wrapped to keep the humidity high. The plants were kept this way overnight at 4 0 C and then the pots were turned upright, unwrapped, and moved to the growth racks.
  • the plants were maintained on the growth rack under 24-hour light until seeds were ready to be harvested. Seeds were harvested when 80% of the siliques of the transformed plants are ripe (approximately 5 weeks after the initial transformation). This seed was deemed TO seed, since it was obtained from the TO generation, and was later plated on selection plates (either kanamycin or sulfonamide). Resistant plants that were identified on such selection plates comprised the Tl generation.
  • Example III Plant Growth and Morphological Analysis
  • Plant Growth and Morphological analysis was performed to determine whether changes in transcription factor levels affect plant growth and development. This was primarily carried out on the Tl generation, when at least 10-20 independent lines were examined. However, in cases where a phenotype required confirmation or detailed characterization, plants from subsequent generations were also analyzed. Primary transformants were selected on MS medium with 0.3% sucrose and 50 mg/1 kanamycin. T2 and later generation plants were selected in the same manner, except that kanamycin was used at 35 mg/1. In cases where lines carry a sulfonamide marker (as in all lines generated by super-transformation), seeds were selected on MS medium with 0.3% sucrose and 1.5 mg/1 sulfonamide. KO lines were usually germinated on plates without a selection.
  • Seeds were cold- treated (stratified) on plates for 3 days in the dark (in order to increase germination efficiency) prior to transfer to growth cabinets. Initially, plates were incubated at 22°C under a light intensity of approximately 100 microEinsteins ( ⁇ E) for 7 days. At this stage, transformants were green, possessed the first two true leaves, and were easily distinguished from bleached kanamycin or sulfonamide-susceptible seedlings. Resistant seedlings were then transferred onto soil (Sunshine potting mix). Following transfer to soil, trays of seedlings were covered with plastic lids for 2-3 days to maintain humidity while they became established. Plants were grown on soil under fluorescent light at an intensity of 70-95 ⁇ E and a temperature of 18-23°C.
  • Plate-based assays were used to assess resistance to three neurotrophic pathogens. All lines were screened with a Sclerotinia sclerotiorum plate-based assay. Most lines were also subjected to Botrytis cinerea plate assays. A Fusarium oxysporum plate assay was used for selected sequences that gave an altered response to Fusarium in previous studies. Typically, eight lines were subjected to plate assays.
  • seed for all experiments were surface sterilized in the following manner: (1 ) 5 minute incubation with mixing in 70 % ethanol; (2) 20 minute incubation with mixing in 30% bleach, 0.01% Triton X-100; (3) five rinses with sterile water. Seeds were resuspended in 0.1% sterile agarose and stratified at 4 0 C for 2-4 days. Sterile seeds were sown on starter plates (15 mm deep) containing the following medium:
  • Sclerotinia inoculum preparation A Sclerolinia liquid culture was started three days prior to plant inoculation by cutting a small agar plug (1/4 sq. inch) from a 14- to 21-day old Sclerotinia plate (on Potato Dextrose Agar; PDA) and placing it into 100 ml of half-strength Potato Dextrose Broth (PDB). The culture was allowed to grown in the PDB at room temperature under 24-hour light for three days. On the day of seedling inoculation, the hyphal ball was retrieved from the medium, weighed, and ground in a blender with water (50 ml/gm tissue). After grinding, the mycelial suspension was filtered through two layers of cheesecloth and the resulting suspension was diluted 1:5 in water. Plants were inoculated by spraying to run-off with the mycelial suspension using a Preval aerosol sprayer.
  • Botrytis inoculum preparation Botrytis inoculum was prepared on the day of inoculation. Spores from a 14- to 21-day old plate were resuspended in a solution of 0.05% glucose, 0.03M KH 2 PO 4 to a final concentration of 10 4 spores/ml. Seedlings were inoculated with a Preval aerosol sprayer, as with Sclerotinia inoculation. Fusarium inoculum preparation. Fusarium inoculum was started one day prior to plant inoculation. Spores from a 14- to 21-day old plate were resuspended in 100 ml of Czapek-Dox medium (Sigma) and grown overnight under constant light. The suspension was shaken several times throughout the day until the time of inoculation. Seedlings were inoculated with a suspension of 10 6 spores/ml, using a Preval aerosol sprayer. Data Interpretation
  • each line was given one of the following overall scores: (++) Substantially enhanced resistance compared to controls. The phenotype was very consistent across all plates for a given line. (+) Enhanced resistance compared to controls. The response was consistent but was only moderately above the normal levels of variability observed for that assay,
  • Each project i.e., lines from transformation with a particular construct was tested in a soil- based assay for resistance to the biotrophic pathogen powdery mildew ⁇ Erysiphe cichoracearum). Typically, eight lines per project were subjected to the Er ⁇ siphe assay. Unless otherwise stated, all experiments were performed with the Arabidopsis thaliana ecotype Columbia (Col-0). Erysiphe assays were usually performed on non-selected segregating T2 populations (in order to avoid the extra stress of selection). Kanamycin or sulfonamide selection was used for the Sclerotima assay to minimize variability and improve statistics on the results.
  • Control plants for assays on lines containing direct promoter-fusion constructs were wild-type plants or CoI-O plants transformed an empty transformation vector (pMEN65).
  • Controls for 2- component lines were the background promoter-driver lines (i.e. promoter::LexA-GAL4TA lines), into which the super-transformations were initially performed.
  • Ervsiphe soil assay Er ⁇ siphe inocuia were propagated on apad4 mutant line in the CoI-O background, which is highly susceptible to Erysiphe (Reuber et al. (1998) Plant J. 16: 473-485). Inocuia were maintained by using a small paintbrush to dust conidia from a 2-3 week old culture onto new plants (generally three weeks old). In addition, the inocuia were passaged through squash plants once a month to ensure the purity of the isolate; the isolate used causes disease on both squash and Arabidopsis, while most isolates are host-specific.
  • seedlings were grown on plates for one week under 24-hour light in a germination chamber, then transplanted to soil and grown in a walk-in growth chamber under a 12-hour light/12-hour dark regimen, 70% humidity.
  • Each line was transplanted to two 13 cm square pots, nine plants per pot.
  • three control plants were transplanted to each pot for direct comparison with the test line.
  • plants were inoculated using settling towers, as described by Reuber et al. (1998) supra. Generally, three to four heavily infested leaves were used per pot for the disease assay. Level of fungal growth was evaluated eight to eleven days after inoculation.
  • G 1750 (Arabidopsis SEO ID NO: 49 and 50 ⁇ P 1034 (SEQ ID NO: 202) contained a 35S::G 1750 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G 1750.
  • P5486 (SEQ ID NO: 203) and P3963 (SEQ ID NO: 204) comprised a two-component system.
  • P5486 encoded a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker.
  • a transgenic line carrying this construct was established and supertransformed with P3963 (carrying a sulfonamide marker) which contained an opLexA::G1750 cD " NA fragment.
  • a large number of 35S::G 1750 lines were generated. Constitutive expression of G 1750 caused severe stunting, and approximately half of the plants died before reaching maturity.
  • 35S::G1750 lines were small, dark-green and vitrified early in development. Very few of the lines were fertile, and thus disease assays were performed on lines that had weak phenotypes.
  • this transcription factor was also tested under the control of a dex-inducible promoter.
  • Eight two- component dex-inducible Gl 750 lines were tested for Sclerotinia and Botrytis resistance. Two lines (325 and 326) showed enhanced resistance to Botrytis when grown on dex-containing plates; line 326 also showed enhanced Sclerotinia resistance. Conversely, line 322 displayed enhanced susceptibility to Botrytis.
  • Table 1 Response of G 1750 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
  • G 1750 may be useful for engineering disease resistance, particularly to biotrophic pathogens.
  • the results from the dex-inducible lines indicate that morphological off- types can be separated from positive disease phenotypes.
  • G440 (Arabidopsis SEO ID NO: 51 and 52V P258 (SEQ ID NO: 205) contained a 35S::G440 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G440.
  • P5486 (SEQ ID NO: 203) and P5265 (SEQ ID NO: 206) comprised a two-component system.
  • P5486 encodes a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker.
  • a transgenic line carrying this construct was established and supertransformed with P5265 (carrying a sulfonamide marker) which contained an opLexA::G440 cDNA fragment.
  • this transcription factor was also tested under the control of a dex-inducible promoter.
  • Eight two-component dex- inducible G440 lines were tested for Sclerotinia and Botrytis resistance. Three of these lines showed enhanced susceptibility to Botrytis when grown on dex-containing plates, while one line was more resistant. An additional line showed mildly increased resistance to Sclerotinia.
  • G440 has potential use for engineering resistance to biotrophic pathogens, such as powdery mildews and rusts.
  • G207 (Arahidopsis SEQ ID NO: 1 and 2)
  • P800 (SEQ ID NO: 207) contained a 35S::G207 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a genomic clone of G207.
  • G207 overexpressing lines were found to have increased sensitivity to glucose in a germination assay, indicating a potential role for G207 in sugar signaling.
  • G207 has previously been reported to be regulated during late embryonic development (Kirik et al. (1998) Plant MoI. Biol. 37:, 819-827), induced in secondary xylem formation (Ko et al. (2004) Plant Physiol. 135: 1069-1083), and repressed by K+ deprivation, which activates reactive oxygen species (Shin and Schachtman (2004) Proc. Natl. Acad. Sci. USA 101 : 8827-8832).
  • 35S::G207 lines were tested for Erysiphe resistance in a soil assay. Constitutive overexpression of G207 caused enhanced resistance to Erysiphe when compared to wild-type plants, clearly evident in five of the transgenic lines. No significant difference compared to wild- type was observed in these lines in response to Botrytis and Sclerotinia. One line showed slightly enhanced susceptibility to Scleroiinia, otherwise the 35S::G207 lines appeared wild-type in response to the two pathogens.
  • G207 represents an excellent candidate for engineering resistance to biotrophic pathogens, such as powdery mildews and rusts. No significant negative side effects of G207 overexpression were observed, decreasing the need for extensive expression pattern optimization in engineered plants.
  • Pl 6 contained a 35S::G233 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G233.
  • Twenty new 35S::G233 lines were generated. These lines showed no consistent differences in morphology from control plants. Eight lines were tested for Botrytis, Sclerotinia, and Erysiphe resistance. One line showed enhanced resistance to all three pathogens, one line showed enhanced resistance to Erysiphe and Botrytis, and three lines were resistant to one pathogen (one each for Botrytis, Sclerotinia, and Erysiphe).
  • G233 shows clear potential for engineering plants for resistance to multiple pathogens, including both biotrophs and necrotrophs. Overexpression of G233 does not result in obvious negative side effects on morphology, decreasing the need for extensive expression pattern optimization in engineered plants.
  • P77 (SEQ ID NO: 209) contained a 35S::G591 direct promoter fusion and carries a kanamycin resistance marker.
  • the construct contains a cDNA clone of G591.
  • G591 has potential for engineering broad- spectrum disease resistance.
  • the resistance to Erysiphe, a biotroph is clear in multiple 35S::G591 lines.
  • the response to the necrotrophs was more variable, although the fact that several lines showed resistance to Botrytis and/or Sclerotinia is promising.
  • Overexpression of G591 does not result in obvious negative side effects on morphology, decreasing the need for extensive expression pattern optimization in engineered plants.
  • G4 (Arabidopsis SEQ ID NO: 159 and 160)
  • Pl 63 (SEQ ID NO: 210) contained a 35S::G4 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G4.
  • G4 might be used to increase resistance to Botrytis or related pathogens.
  • the morphological effects suggest that G4 might also be used to regulate growth rate, biomass, and flowering time.
  • G237 (Arabidopsis SEQ ID NO: 187 and 188)
  • P17 (SEQ ID NO: 21 1) contained a 35S::G237 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a genomic clone of G237.
  • P384 (SEQ ID NO: 212) contained a 35S::G869 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G869.
  • P5486 (SEQ ID NO: 203) and P9105 (SEQ ID NO: 213) comprised a two-component system.
  • P5486 encodes a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker.
  • a transgenic line carrying this construct was established and supertransformed with P9105 (carrying a sulfonamide marker) that contained an opLexA::G869 cDNA fragment.
  • Experimental observations Twenty 35S::G869 lines were generated. The majority of the Tl plants showed dwarfing to various degrees. Lines that were dwarfed were also relatively late developing, spindly and had decreased fertility.
  • Two-component lines containing a dex-inducible promoter were also isolated. Most of these plants were normal in the absence of dexamethasone, except that about half of the lines flowered early.
  • 35S::G869 lines were tested for Erysiphe resistance in a soil assay. Three of these lines showed moderately enhanced resistance. These 35S::G869 lines were tested by Sclerotinia plate assay. Three of these lines showed enhanced susceptibility to Sclerotinia. Note that constitutive overexpression of G869 causes growth retardation in transgenic lines. The apparent enhanced susceptibility of these lines may be an artifact of the assay, since smaller plants, in general, succumb faster than larger plants in the Sclerotinia plate assay.
  • G869 causes a reduction in plant size
  • this gene was also tested under the control of the dex-inducible promoter.
  • Eight two-component dex-inducible G869 lines were tested for Sclerotinia and Botrytis resistance on dex-containing plates. Three lines showed slightly enhanced resistance to Botrytis when tested on dex plates. These results suggest that G869 expression can provide some protective effect against necrotrophic pathogens, and that the increased susceptibility to Sclerotinia observed in the 35S::G869 lines may be an artifact of the assay.
  • G869 may be useful for engineering plants with enhanced disease resistance.
  • P15038 (SEQ ID NO: 214) contained a 35S::G 1274 direct promoter fusion and carried a kanamycin resistance marker.
  • the construct contained a cDNA clone of G 1274.
  • G 1274 has previously been shown by us to confer tolerance to water deprivation, low nitrogen conditions, and cold.
  • three G 1274 lines were tested in a soil assay for resistance to Erysiphe cichor ace arum, and in plate assays for Sclerotinia sclerotiorum and Botrytis cinerea. Two lines showed moderate to strong resistance to Erysiphe. One line showed slightly enhanced susceptibility to Sclerotinia, but overall there was no significant difference from control plants in response to the two necrotrophic pathogens.
  • G 1274 might be used to increase resistance to biotrophic pathogens such as powdery mildew or rusts.
  • Transcription factor polynucleotide sequences listed in the Sequence Listing or Tbale 10, or that encode polypeptides listed in the Sequence Listing or Table 10, recombined into, for example, one of the expression vectors of the invention, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield and/or quality.
  • the expression vector may contain a constitutive, tissue-specific or inducible promoter operably linked to the transcription factor polynucleotide.
  • the cloning vector may be introduced into a variety of plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation.
  • microprojectile-mediated transformation in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al. (1987) Part. ScL Technol. 5:27-37; Christou et al. (1992) Plant. J. 2: 275-281 ; Sanford (1993) Methods Enzymol. 217: 483-509; Klein et al. (1987) Nature 327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991 ; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21 , 1994).
  • sonication methods see, for example, Zhang ct al. ( 1991 ) Bio/Technology 9:
  • Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986) In Tomato Biotechnology: Alan R. Liss, Inc., 169-178, and in U.S. Patent 6,613,962, the latter method described in brief here.
  • Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 ⁇ M ⁇ -naphthalene acetic acid and 4.4 ⁇ M 6-benzylaminopurine.
  • the explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the invention for 5-10 minutes, blotted dry on sterile filter paper and cqcultured for 48 hours on the original feeder layer plates. Culture conditions are as described above.
  • Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Patent 5,563,055 (Townsend et al., issued October 8, 1996), described in brief here.
  • soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.
  • Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the invention are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified.
  • Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Patent 5,563,055).
  • the explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.
  • Example VIII Transformation of monocots to produce increased disease resistance Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may be transformed with the present polynucleotide sequences, including monocot or dicot-derived polynucleotide sequences such as those presented in the present Tables or Sequence Listing, or which encode the polypeptides found in the present Tables of Sequence Listing, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV 35S or CORl 5 promoters, or with tissue-specific or inducible promoters.
  • a vector such as pGA643 and containing a kanamycin-resistance marker
  • pMEN020 may be modified to replace the Nptll coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin.
  • the Kpnl and BgIII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.
  • the cloning vector may be introduced into a variety of cereal plants by means well known in the art including direct DNA transfer or Agrobacterium transformation.
  • the latter approach may be accomplished by a variety of means, including, for example, that of U.S. Patent No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector.
  • the sample tissues are immersed in a suspension of 3x10 "9 cells of Agrobacterium containing the cloning vector for 3-10 minutes.
  • the callus material is cultured on solid medium at 25° C in the dark for several days.
  • the calli grown on this medium are transferred to Regeneration medium. Transfers are continued every 2-3 weeks (2 or 3 times) until shoots develop. Shoots are then transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.
  • the transformed plants are then analyzed for the presence of the NPTlI gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, CO).
  • embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al. (1997) supra; Vasil (1994) supra).
  • a 188XB73 genotype is the preferred genotype (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra).
  • the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990) supra).
  • Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) supra; Gordon- Kamm et al. (1990) supra).
  • Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a transcription factor polypeptide or the invention and related genes that are capable of inducing disease resistance.
  • mature plants overexpressing a transcription factor of the invention may be challenged by a pathogen.
  • control plants for example, wild type
  • transgenic plants may be shown to have greater resistance to the particular pathogen.
  • the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild- type monocot plant, or another transformed monocot plant from a different transgenic line of plants.
  • transcription factor polypeptides of the invention can be identified and shown to confer greater yield and greater disease resistance in dicots or monocots, including resistance to multiple pathogens.
  • Example X Sequences that Confer Significant Improvements to non-Arabidopsis species
  • the function of specific transcription factors of the invention have been analyzed using BLAST and phylogenetic analysis, and they or their closely related homologs may be further characterized and incorporated into crop plants.
  • the ectopic overexpression of these sequences may be regulated using constitutive, inducible, or tissue specific regulatory elements. Genes that have been examined and have been shown to increase disease resistance encode transcription factor polypeptides found in the Sequence Listing or the present Tables.
  • polynucleotide and polypeptide sequences closely related to polynucleotide and polypeptide sequences found in the Sequence Listing can also confer alteration of traits in a similar manner to the reference sequences G207, G1750, G440, G1274, G591, G233, G4, G867, or G237, when transformed into a any of a considerable variety of plants of different species, and including dicots and monocots.
  • Closely related sequences that have similar function include, for example, those sequences that are closely, phylogenetically related to the sequences of the invention by virtue of being within the same clade and having descended from a common ancestral sequence.
  • the closely-related polynucleotide and polypeptide sequences derived from monocots may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy genes) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived.
  • the sequences of the transcription factors of the invention may be overexpressed under the regulatory control of constitutive, tissue specific or inducible promoters.
  • transcription factors may confer disease resistance when they are overexpressed under the regulatory control of non- constitutive promoters or a transactivation domain fused to the clade member, without having a significant adverse impact on plant morphology and/or development.
  • the lines that display useful traits may be selected for further study or commercial development.
  • Monocotyledonous plants including rice, corn, wheat, rye, sorghum, barley and others, may be transformed with a plasm id containing a transcription factor polynucleotide.
  • the transcription factor gene sequence may include dicot or monocot-derived sequences such as those presented herein. These transcription factor genes may be cloned into an expression vector containing a kanamycin-resistance marker, and then expressed constitutively or in a tissue-specific or inducible manner.
  • the cloning vector may be introduced into monocots by, for example, means described in the previous Example, including direct DNA transfer or Agrobacterium tumefaciens-med ' iated transformation.
  • the transformed plants are then analyzed for the presence of the NPTIl gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Pri ⁇ ne Inc. (Boulder, CO).
  • Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a transcription factor polypeptide of the invention that is capable of conferring increased disease resistance, or increased size or yield, in the transformed plants.
  • mature plants expressing a monocot- derived equivalog gene may be challenged using methods described in the above Examples.
  • wild type plants and the transgenic plants By comparing wild type plants and the transgenic plants, the latter are shown be more resistant to disease as compared to wild-type or non-transformed control plants, or controls plants transformed with an empty vector, similarly treated.

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Abstract

Transcription factor polynucleotides and polypeptides incorporated into expression vectors have been introduced into plants and were ectopically expressed. Transgenic plants transformed with many of these expression vectors have been shown to be more resistant to disease, and in some cases, to more than one type of pathogen.

Description

PLANT DISEASE RESISTANCE GENES AND PROTEINS
JOINT RESEARCH AGREEMENT
The claimed invention, in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Corporation as a result of activities undertaken within the scope of a joint research agreement in effect on or before the date the claimed invention was made.
FIELD OF THE INVENTION
The present invention relates to plant genomics and more specifically pertains to polynucleotides that encode polypeptides that confer disease resistance in plants.
BACKGROUND OF THE INVENTION
Plant pathogens cause enormous world-wide annual losses in yield. Using plant biotechnology to engineer disease resistant crops has the potential to make a significant economic impact on agriculture and forestry industries in two ways: reducing the monetary and environmental expense of fungicide application and reducing both pre-harvest and post-harvest crop losses that occur now despite the use of costly disease management practices. Importantly, the use of genetic engineering technologies to enhance the natural ability of plants to resist pathogen attack holds great potential for enhancing yields while reducing chemical use. Moreover, genetic engineering of disease resistance may be a critical component of a long-term environmentally sound and economically feasible strategy for increasing global food production. Plant pathogens fall into two major classes: biotrophs and necrotrophs (reviewed in Oliver and Ipcho (2004) MoI. Plant Pathol. 5, 347-352). Biotrophic pathogens obtain energy by parasitizing living plant tissue, while necrotrophs obtain energy from dead plant tissue. Examples of biotrophs include the powdery mildews, rusts, and downy mildews; these pathogens can only grow in association with living plant tissue, and parasitize plants through intracellular feeding structures called haustoria. Examples of necrotrophs include Sclerotinia sclerotiorum (white mold), Bottytis cinerea (grey mold), and Cochliobolus heterostrophus (Southern corn leaf blight). The general pathogenic strategy of necrotrophs is to kill plant tissue through toxins and lytic enzymes, and live off the released nutrients. Pathologists also recognize a third class of pathogens, called hemibiotrophs: these pathogens have an initial biotrophic stage, followed by a necrotrophic stage once a parasitic association with plant cells has been established. In general, different defense responses have been found to be induced in plants in response to attack by a biotrophic or necrotrophic pathogen. Infection by biotrophic pathogens often induces defense responses mediated by the plant hormone salicylic acid, while attack by a necrotrophic pathogen often induces defense responses mediated by coordinated action of the hormones ethylene and jasmonate.
Genetically engineered traits, such as defense response, may be controlled through a number of regulatory processes. One important way to manipulate control of cellular processes is through transcription factors, proteins that influence the expression of a particular gene or sets of genes. Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with new and/or improved commercially valuable properties such as pathogen resistance. We have identified polynucleotides encoding transcription factors, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for a disease resistance to biotrophic and necrotrophic pathogens. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.
SUMMARY OF THE INVENTION
The present invention pertains to transgenic plants that have greater resistance to a pathogen than a control plant. The transgenic plants of the present invention have each been transformed with at least one a transcription gene sequence encoding an AP2, Myb, HLH, or WRKY family polypeptide. The invention also encompasses polynucleotide and polypeptide sequences that are closely-related to the AP2, MYB HLH5 or WRKY family polynucleotide and polypeptides. The polypeptide sequences may be compared to any of the sequences in the Sequence Listing, and in particular to the sequences, henceforth referred to as reference sequences, that have been shown to confer increased disease resistance in plants, including G207 (SEQ ID NO: 2), G1750 (SEQ ID NO: 50), G440 (SEQ ID NO: 52), G 1274 (SEQ ID NO: 84), G591 (SEQ ID NO: 128), G233 (SEQ ID NO: 146), G4 (SEQ ID NO: 160), G869 (SEQ ID NO: 181), or G237 (SEQ ID NO: 188).
Polypeptide sequences that are closely-related to sequences in the Sequence Listing may be identified by having similar conserved domains and at least one similar function of conferring a transcriptional regulatory activity of the reference sequence. The transcriptional regulatory activity may confer greater disease resistance as compared to a control plant. When the conserved domain sequences are optimally aligned using a BLOSUM62 matrix, a gap existence penalty of 1 1 , and a gap extension penalty of 1 , similar conserved domains may be identified by virtue of having a minimum percentage identity or similarity. For example, polypeptides of the invention have conserved domains that have: a percentage identity of at least 67%, 69%, 70%, 7 1%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%, 83%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2; a percentage identity of at least 61%, 62%, 65%, 69%, 70%, 71%, 72%, 74%, 77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81%, 82%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 93%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50; a percentage identity of at least 65%, 67%, 69%, 72%, 73%, 74%, 75%, 79%, 83%, 85%, or 88%, or 100%, or a percentage similarity of at least 79%, 82%, 83%, 86%, 87%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52; a percentage identity of at least 75%, 77%, 78%, 80%, 82%, or 84%, or 100%, or a percentage similarity of at least 85%, 87%, 89%, 91%, or 92%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 256-277, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 84; a percentage identity of at least 74%, 75%, 91%, or 98%, or 100%, or a percentage similarity of at least 91%, 93%, 94%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 278-286, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 128; a percentage identity of at least 82%, 84%, 58%, 86%, or 100%, or a percentage similarity of at least 93%, 94%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 287-292, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NOs: 146; a percentage identity of at least 82%, 83%, 85%, 86%, 87%, 88%, or 91%, or 100%, or a percentage similarity of at least 86%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 293-308, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 160; a percentage identity of at least 60%, 68%, 72%, or 100%, or a percentage similarity of at least 68%, 73%, 83%, 91%, or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 309-314, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 181 ; or a percentage identity of at least 70%, 71 %, 72%, 73%, 75%, 77%, 85%, or 100%, or a percentage similarity of at least 84%, 85%, 87%, 88%, 89%, 92% or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 315-327, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 188. When any of the polypeptides of the invention is overexpressed in a transgenic plant (in other words its expression levels are greater than the expression level of the same sequence in a control plant), the polypeptide regulates expression of other plant genes involved in the defense response, and thus increases pathogen resistance in the plant.
Methods for making and using these transgenic plants that have greater resistance to a pathogen than a control plant are also encompassed by the present invention. Brief Description of the Sequence Listing and Drawings
The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
CD-ROMs Copy 1 and Copy 2, and the CRF copy of the Sequence Listing under CFR Section 1.821(e), are read-only memory computer-readable compact discs. Each CD-ROM contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named
"MBI0077P.ST25.txt", the electronic file of the Sequence Listing contained on each of these CD- ROMs was created on April 12, 2007, and the electronic file of the Sequence Listing is 666 kilobytes in size. The copies of the Sequence Listing on the CD-ROM discs are hereby incorporated by reference in their entirety.
Figure 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Soltis et al. (1997) Ann. Missouri Bot. Gard. 84: 1-49). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. Figure 1 was adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333. Figure 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including cladcs containing tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl. Acad. Set. USA 97: 9121 -912; and Chase et al. (\993) Ann. Missouri Bot. Gard. 80: 528-580.
DETAILED DESCRIPTION
The present invention relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased resistance to pathogens and/or disease, and/or increased yield with respect to a control plant (for example, a genetically unaltered or non- transgenic plant such as a wild-type plant of the same species, or a transgenic plant line that comprises an empty expression vector). Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses.
While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of "incorporation by reference" is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.
As used herein and in the appended claims, the singular forms "a", "an", and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "a stress" is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth. DEFINITIONS "Nucleic acid molecule" refers to an oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). "Polynucleotide" is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single-stranded or double-stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. "Oligonucleotide" is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded. "Gene" or "gene sequence" refers to the partial or complete coding sequence of a gene, its complement, and its 5' or 3' untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.
Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. (1976) Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th ed., Springer Verlag, Berlin). A gene generally includes regions preceding ("leaders"; upstream) and following ("trailers"; downstream) the coding region. A gene may also include intervening, non-coding sequences, referred to as "introns", located between individual coding segments, referred to as "exons". Most genes have an associated promoter region, a regulatory sequence 51 of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.
A "recombinant polynucleotide" is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
An "isolated polynucleotide" is a polynucleotide, whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.
A "polypeptide" is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a DNA-binding domain; or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues. "Protein" refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
"Portion", as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies. A "recombinant polypeptide" is a polypeptide produced by translation of a recombinant polynucleotide. A "synthetic polypeptide" is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An "isolated polypeptide," whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 1 10%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein. "Homology" refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.
"Identity" or "similarity" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
"Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.
"Alignment" refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Conserved domains such as those of Tables 1 -9 and a suitable method such as, for example, the BLOSUM62 matrix, may be used to identify conserved domains and relatedness within these domains. An alignment may also be generated by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, CA) or CLUSTALX (Thompson et al. (1997) Nucl. Acids Res. 24: 4876-4882).
The term "optimal alignment" or "optimally aligned" refers to an alignment (including the introduction of gaps in the sequences as necessary) thai results in the highest similarity score (Holman (2004) "Protein similarity score: a simplified version of the BLAST score as a superior alternative to percent identity for claiming genuses of related protein sequences"; 21 Santa Ciara Computer & High Tech. L.J. pp. 55-99). Similarity scores may be determined, for example, using BLAST analysis.
A "conserved domain" or "conserved region" as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. A "Myb" domain", such as is found in a polypeptide member of the MYB-(R 1)R2R3 transcription factor family, is an example of a conserved domain. With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is preferably at least nine base pairs (bp) in length. A conserved domain with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology, such as the percentage identities or percentage similarities listed in Tables 1-9, to a conserved domain of a polypeptide of the invention. Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological activity to the present transcription factor sequences, thus being members of a clade of transcription factor polypeptides, are encompassed by the invention. A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular transcription factor class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be "outside a conserved domain" if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000a) Science 290, 2105-21 10; and Riechmann (2000b) Curr. Opin. Plant Biol. 3, 423-434). Thus, by using alignment methods well known in the art, the conserved domains of the plant transcription factors (see, for example, Marchler-Bauer et al. (2003) Nucleic Acids Res. 31 : 383- 387; or Magnani et al. (2004) Plant Cell 16: 2265-2277), may be determined.
The conserved domains for many of the transcription factor sequences of the invention are listed in Tables 1 -9. Also, the polypeptides of Tables 1-9 have conserved domains specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Marchler-Bauer et al. (2003) supra) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure. "Complementary" refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (51 — > 31) forms hydrogen bonds with its complements A-C-G-T (5' — > 31) or A-C-G-U (5' — * 3'). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or "completely complementary " if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization and amplification reactions. "Fully complementary" refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.
The terms "highly stringent" or "highly stringent condition" refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313: 402-404, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and by Haymes et al. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL Press, Washington, D. C, which references are incorporated herein by reference.
In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see the section "Identifying
Polynucleotides or Nucleic Acids by Hybridization", below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity.
Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known transcription factor sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, encoded transcription factors having 38% or greater identity with the conserved domain of disclosed transcription factors.
The terms "paralog" and "ortholog" are defined below in the section entitled "Orthologs and
Paralogs". In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
The term "equivalog" describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) World Wide Web
(www) website, " tigr.org" under the heading "Terms associated with TIGRFAMs".
In general, the term "variant" refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.
With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
Also within the scope of the invention is a variant of a transcription factor nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide. "Allelic variant" or "polynucleotide allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be "silent" or may encode polypeptides having altered amino acid sequence. "Allelic variant" and "polypeptide allelic variant" may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene. "Splice variant" or "polynucleotide splice variant" as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. "Splice variant" or "polypeptide splice variant" may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.
As used herein, "polynucleotide variants" may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. "Polypeptide variants" may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.
Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent transcription factor. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. A polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the transcription factor is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see USPN 5,840,544). "Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a transcription factor. Exemplary fragments also include fragments that comprise a conserved domain of a transcription factor. Exemplary fragments include fragments that comprise an conserved domain of a transcription factor, for example, amino acid residues 6 to 106 of G207 (SEQ ID NO: 215, or the Myb domain of SEQ ID NO: 2), 115 to 177 of G1750 (SEQ ID NO: 239, or the AP2 domain of SEQ ID NO: 50), 122 to 184 of G440 (SEQ ID NO: 240, or the AP2 domain of SEQ ID NO: 52), amino acid residues 1 10 to 166 of Gl 274 (SEQ ID NO: 256, or the WRKY domain of SEQ ID NO: 84), amino acid residues 149 to 206 of G591 (SEQ ID NO: 278, or the HLH domain of SEQ ID NO: 128), amino acid residues 13 to 115 of G233 (SEQ ID NO: 287, or the Myb domain of SEQ ID NO: 146), amino acid residues 121 to 183 of G4 (SEQ ID NO: 293, or the AP2 domain of SEQ ID NO: 160, amino acid residues 1 10 to 165 of G869 (SEQ ID NO: 309, or the AP2 domain of SEQ ID NO: 181), or amino acid residues 11 to 1 13 of G237 (SEQ ID NO: 315, or the Myb domain of SEQ ID NO: 188).
Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds Io a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length. The invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.
"Derivative" refers to the chemical modification of a nucleic acid molecule or amino acid sequence. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process that retains or enhances biological activity or lifespan of the molecule or sequence. The term "plant" includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and ceils (for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psϊlophytes, lycophytes, bryophytes, and multicellular algae (see for example, Figure 1, adapted from Daly et al. (2001) supra, Figure 2, adapted from Ku et al. (2000) supra; and see also Tudge (2000) in The Variety of Life, Oxford University Press, New York, NY pp. 547-606.
A "control plant" as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
A "transgenic plant" refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes. A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the controlled expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
"Wild type" or "wild-type", as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a transcription factor expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring resistance to pathogens or tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as extent of disease, hyperosmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
"Trait modification" refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild-type plants.
When two or more plants have "similar morphologies", "substantially similar morphologies", "a morphology that is substantially similar", or are "morphologically similar", the plants have comparable forms or appearances, including analogous features such as overall dimensions, height, width, mass, root mass, shape, glossiness, color, stem diameter, leaf size, leaf dimension, leaf density, internode distance, branching, root branching, number and form of inflorescences, and other macroscopic characteristics, and the individual plants are not readily distinguishable based on morphological characteristics alone.
"Modulates" refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.
The term "transcript profile" refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods. With regard to transcription factor gene knockouts as used herein, the term "knockout" refers to a plant or plant cell having a disruption in at least one transcription factor gene in the plant or cell, where the disruption results in a reduced expression or activity of the transcription factor encoded by that gene compared to a control cell. The knockout can be the result of, for example, genomic disruptions, including transposons, tilling, and homologous recombination, antisense constructs, sense constructs, RNA silencing constructs, or RNA interference. A T-DNA insertion within a transcription factor gene is an example of a genotypic alteration that may abolish expression of that transcription factor gene.
"Ectopic expression or altered expression" in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term "ectopic expression or altered expression" further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
The term "overexpression" as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also under the control of an inducible or tissue specific promoter. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter used. Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the transcription factor in the plant, cell or tissue.
The term "transcription regulating region" refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors of the present invention possess an conserved domain. The transcription factors of the invention also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more biotic stress resistance genes in a plant when the transcription factor binds to the regulating region.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Transcription Factors Modify Expression of Endogenous Genes
A transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. As one of ordinary skill in the art recognizes, transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding site or DNA-binding site motif (see, for example, Riechmann et al. (2000a) supra). The plant transcription factors of the present invention belong to the AP2, Myb, or HLH transcription factor families.
Generally, the transcription factors encoded by the present sequences are involved in cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to osmotic stresses. The sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.
The sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule. In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.
Expression of genes that encode transcription factors that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997) Genes Development 1 1 : 3194-3205, and Peng et al. (\999) Nature 400: 256-261. In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or a very similar phenotypic response. See, for example, Fu et al. (2001) Plant Cell 13: 1791-1802; Nandi et al. (2000) Curr. Biol. 10: 215-218; Coupland (1995) Nature 377: 482-483; and Weigel and Nilsson (1995) Λtøwre 377: 482-500. In another example, Mandel (1992) Nature 360: 273-277, and Suzuki et al. (2001) Plant J.
28: 409-418, teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al. (1992) supra; and Suzuki et al. (2001) supra). Other examples include Mϋller et al. (2001) Plant J. 28: 169-179; Kim et al. (2001) Plant J. 25: 247-259; Kyozuka and Shimamoto (2002) Plant Cell Physiol. 43: 130-135; Boss and Thomas (2002) Nature, 416: 847-850; He et al. (2000) Transgenic Res. 9: 223-227; and Robson et al . (2001 ) Plant J. 28 : 619-631.
In yet another example, Gilmour et al. (1998) Plant /. 16: 433-442 teach an Arabidopsis AP2 transcription factor, CBFl, which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al. (2001) Plant Physiol. 127: 910-917 further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues, PKK/RPAGRxKFxETRHP and DSAWR, which bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family (Jaglo et al. (2001) supra).
Transcription factors mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced transcription factor. It is well appreciated in the art that the effect of a transcription factor on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (e.g., by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor. For example, the PAP2 gene and other genes in the Myb family have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000) Plant Cell 12: 65-79; and Borevitz et al. (2000) Plant Cell 12: 2383-2393). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (e.g., cancerous vs. non-cancerous; Bhattacharjee et al. (2001) Proc. Natl. Acad. Sci. USA 98: 13790-13795; and Xu et al. (2001) Proc. Natl. Acad. Sci. USA 98: 15089-15094). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.
Polypeptides and Polynucleotides of the Invention
The present invention provides, among other things, transcription factors (TFs), and transcription factor homolog polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided in the Sequence Listing. Also provided are plants comprising novel transcription factors or variants of the transcription factors, where the plants have greater resistance to a biotrophic pathogen, a neurotrophic pathogen, or both classes of pathogens, than a control plant. The invention also includes methods for increasing a plant's resistance to disease, including disease caused by biotrophic pathogens, necrotrophic pathogens, or both. These methods are based on the ability to alter the expression of critical regulatory molecules that may be conserved between diverse plant species. Related conserved regulatory molecules may be originally discovered in a model system such as Arabidopsis and homologous, functional molecules then discovered in other plant species. The latter may then be used to confer increased resistance to pathogens in diverse plant species.
Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.
Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences, were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5' and 3' ends. The full-length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5' and 3' ends. Exemplary sequences are provided in the Sequence Listing.
Many of the sequences in the Sequence Listing, derived from diverse plant species, have been ectopically expressed in overexpressor plants. The changes in the characteristic(s) or trait(s) of the plants were then observed and found to confer increased biotic stress resistance. Therefore, the polynucleotides and polypeptides can be used to improve desirable characteristics of plants.
The polynucleotides of the invention were also ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be used to change expression levels of a genes, polynucleotides, and/or proteins of plants or plant cells.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention relates to compositions and methods for modifying a plant's traits. The compositions include plants comprising polynucleotides that encode novel plant transcription factor polypeptides first identified in Arabidopsis thaliana, a plant used experimentally as a model plant species. The methods include using the polynucleotides and their encoded polypeptides to modify a trait in a transgenic plant, such as the resistance of a plant to a biotic stress, including a plant pathogen. The data presented herein represent the results obtained in experiments with transcription factor polynucleotides and polypeptides that may be expressed in plants for the purpose of reducing reduced quality or yield losses that arise from biotic stress. A number of
Arabidopsis sequences including G207 (SEQ ID NO: 2), G 1750 (SEQ ID NO: 50), G440 (SEQ ID NO: 52), G1274 (SEQ ID NO: 84), G591 (SEQ ID NO: 128), G233 (SEQ ID NO: 146), G4 (SEQ ID NO: 160), G869 (SEQ ID NO: 181), or G237 (SEQ ID NO: 188) have been shown to confer increased disease resistance in plants, as compared to the resistance of control plants, when the sequences are overexpressed.
Polypeptide sequences that are closely-related to the reference sequences may be identified by having at least one function of each of the reference sequence to which it is compared, and having descended from a common ancestral sequence, and/or having similar conserved domains. The present invention provides for polynucleotide and polypeptide sequences that function by conferring at least one transcriptional regulatory activity of the reference sequence. The transcriptional regulatory activity will generally confer greater disease resistance to a plant overexpressing a reference or related sequence, as compared to a control plant.
Closely-related polypeptide sequences with related conserved domains may be identified by BLAST and phylogenetic analysis, as noted below. When the reference sequence conserved domain and the conserved domains of putatively related sequences are optimally aligned using a BLOSUM62 matrix, a gap existence penalty of 1 1, and a gap extension penalty of 1, similar conserved domains may be identified by virtue of having a minimum percentage identity or similarity. For example, polypeptides of the invention have conserved domains that have:
(a) a percentage identity of at least 67%, 69%, 70%, 71%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%,
83%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2;
(b) a percentage identity of at least 61%, 62%, 65%, 69%, 70%, 71%, 72%, 74%, 77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81 %, 82%, 84%,
85%, 86%, 87%, 89%, 90%, 91%, 93%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50;
(c) a percentage identity of at least 65%, 67%, 69%, 72%, 73%, 74%, 75%, 79%, 83%, 85%, or 88%, or 100%, or a percentage similarity of at least 79%, 82%, 83%, 86%,
87%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52;
(d) a percentage identity of at least 75%, 77%, 78%, 80%, 82%, or 84%, or 100%, or a percentage similarity of at least 85%, 87%, 89%, 91 %, or 92%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 256-277, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 84;
(e) a percentage identity of at least 74%, 75%, 91%, or 98%, or 100%, or a percentage similarity of at least 91%, 93%, 94%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 278-286, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 128; (f) a percentage identity of at least 82%, 84%, 58%, 86%, or 100%, or a percentage similarity of at least 93%, 94%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 287-292, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NOs: 146; (g) a percentage identity of at least 82%, 83%, 85%, 86%, 87%, 88%, or 91%, or
100%, or a percentage similarity of at least 86%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 293-308, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 160; (h) a percentage identity of at least 60%, 68%, 72%, or 100%, or a percentage similarity of at least 68%, 73%, 83%, 91%, or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 309-314, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 181 ; or
(i) a percentage identity of at least 70%, 71%, 72%, 73%, 75%, 77%, 85%, or 100%, or a percentage similarity of at least 84%, 85%, 87%, 88%, 89%, 92% or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 315-327, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 188.
G207, the G207 clade. and related sequences
G207 corresponds to AtMYBR2 and AtMYB77 (Kirik et al. (1998) Plant MoI Biol. 37:, 819-827; Stracke et al. (2001) Curr. Opin. Plant Biol. 4: 447-456). Tn earlier disease studies, a G207 knockout was found to be more susceptible to Botrytis.
Table 1 shows a number of G207 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G207 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G207 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G207, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G207 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 1. Conserved Myb domains of G207 and closely-related sequences
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Os — Oryza sativa; Zm — Zea mays.
G 1750. the G 1750 clade, and related sequences
G 1750 is a member of the ERF-B5 class of AP2 domain transcription factors. It is a putative ortholog of Tsi 1 from tobacco, which produces enhanced tolerance to multiple pathogens when overexpressed (Park et al. (2001) Plant Cell 13: 1035-1046; Shin et al. (2002) MoI. Plant Microbe Interact. 15: 983-989). It is also related to Pti6, which is implicated in Pto-dependent disease resistance in tomato (Zhou et al. (1997) EMBOJ. 16: 3207-3218). Plants overexpressing G1750 under a constitutive promoter were found to exhibit reduced size and poor fertility; therefore, dexamethasone-inducible G 1750 lines were also produced. G440 is also a member of the ERF-B5 class of AP2 domain transcription factors. Plants overexpressing G440 under a constitutive promoter were found to exhibit reduced size and poor fertility; therefore, dexamethasone-inducible G440 lines were also produced. .
Table 2 shows a number of G 1750 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier"
(Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of ύ\Q Arabidopsis G 1750 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G 1750 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of Gl 750, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G 1750 conserved domain (in Tables 2 and 3, the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 2. Conserved AP2 domains of G 1750 and closely-related sequences
Figure imgf000033_0001
Figure imgf000034_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Le — Lycopersicon esculentum; Nt — Nicotiana tabacum; Zm — Zea mays
Alternatively, the sequences in Table 2 may be compared against the conserved domain of G440 as the reference sequence rather than the similar domain of G 1750 (i.e., in Table 3, G440 is listed first, and subsequent sequences are compared to the conserved domain of G440). In this case, the clade as judged by sequences identified to data as clade member is bounded by sequences having 100% identity and similarity to the reference conserved domain sequence of G440 to 65% identity and 79% similarity. Table 3. Conserved AP2 domains of G440 and closely-related sequences
Figure imgf000035_0001
Figure imgf000036_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Le — Lycopersicon esculentum; Nt — Nicotiana tabacum; Zm — Zea mays
G 1274, the G 1274 cladc and related sequences
G 1274 (polynucleotide SEQ ID NO: 83 from Arabidopsis encodes a member of the WRKY family of transcription factors (SEQ FD NO: 84). G1274 corresponds to AtWRKYS 1 (At5g64810), a gene for which there is currently no published information .
WRKY transcription factors. WRXY genes appear to have originated in primitive eukaryotes such as Giardia lamblia, Dictyostelium discoideum, and the green alga Chlamydomonas reinhardtii, and have since greatly expanded in higher plants (Zhang and Wang (2005) BMC Evol. Biol. 5: 1). In Arabidopsis alone, there are more than 70 members of the WRKY superfamily. The defining feature of the family is the ~57 amino acid DNA binding domain that contains a conserved WRKYGQK heptapeptide motif. Additionally, all WRKY proteins have a novel zinc-finger motif contained within the DNA binding domain. There are three distinct groups within the superfamily, each principally defined by the number of WRKY domains and the structure of the zinc-finger domain (reviewed by Eulgem et al. (2000) Trends Plant Sci. 5, 199-206). Group I members have two WRKY domains, while Group II members contain only one. Members of the Group II family can be further split into five distinct subgroups (Ila-e) based on conserved structural motifs. Group III members have only one WRKY domain, but contain a zinc finger domain that is distinct from Group II members. The majority of WRKY proteins are Group II members, including G 1274 and the related genes being studied here. An additional common feature found among WRKY genes is the existence of a conserved intron found within the region encoding the C-terminal WRKY domain of group I members or the single WRKY domain of group I I/I 11 members. In G1274, this intron occurs between the sequence encoding amino acids Rl 30 and N 131.
The founding members of the WRKY family are SPFl from sweet potato (Ishiguro and Nakamura, (1994) MoI. Gen. Genet. 244: 563-571), ABF 1/2 from oat (Rushton et al. (1995) Plant MoI. Biol. 29: 691-702), PcWRKYl,2,3 from parsley (Rushton et al. (1996) EMBO J. 15: 5690- 5700) and ZAPl from Arabidopsis (de Pater et al. (1996) Nucleic Acids Res. 24: 4624-4631). These proteins were identified based on their ability to bind the so-called W-box promoter element, a motif with the sequence (T)(T)TGAC(C/T). Binding of WRKY proteins to this motif has been demonstrated both in vivo and in vitro (Rushton et al. (1995) supra; de Pater et al. (1996) supra; Eulgem et al., (1999) EMBOJ. 18: 4689-4699; Yang et al. (1999) Plant J. 18: 141 -149; Wang et al. (1998) Plant J. 16: 515-522). Additionally, the solution structure of the WRKY4 protein (ATlGl 3960) has recently been reported (Yamasaki et al. (2005) Plant Cell 17: 944-95). In this study, a DNA titration experiment strongly indicates that the conserved WRKYGQK sequence is directly involved in DNA binding. This element is remarkably conserved, and found in many genes associated with the plant defense response.
The two WRKY domains of Group I members appear functionally distinct, and it is the C- terminal sequence that appears to mediate sequence-specific DNA binding. The function of the N- terminal domain is unclear, but may contribute to the binding process, or provide an interface for protein-protein interactions. The single WRKY domain in Group II members appears more like the C-terminal domain of Group I members, and likely performs the similar function of DNA binding. Structural features of G 1274. The primary amino acid sequence for the predicted G 1274 protein is presented in the Sequence Listing as SEQ ID NO: 84, and the conserved domain of G 1274 (SEQ ID NO: 256) is shown in Table 4. Discoveries made in earlier genomics programs. G 1274 expression in wild-type plants was detected in leaf, root and flower tissue. Expression of G 1274 was also enhanced slightly by hyperosmotic and cold stress treatments, and by auxin or ABA application. Additionally, the gene appears induced by Erysiphe infection and salicylic acid treatment, consistent with the known role of WRKY family members in defense responses. The closely related gene G 1275 (SEQ ID NO: 85) is strongly repressed in wild-type plants during soil drought, and remains significantly down- regulated compared to well-watered plants even after rewatering.
In G 1274 overexpression studies, transformed lines were more tolerant to low nitrogen conditions and were less sensitive to chilling than wild-type plants. G1274 overexpress'ing seedlings were also positive in a C:N sensing screen, indicating that G1274 may alter the plant's ability to modulate carbon and/or nitrogen uptake and utilization and improve germination and/or growth in low nitrogen conditions. G 1274 overexpression also produced alterations in inflorescence and leaf morphology. Approximately 20% of overexpressors were slightly small and developed short inflorescences that had reduced internode elongation. Overall, these plants were bushier and more compact in stature than wild-type plants. In T2 populations, rosettes of some 35S::G1274 plants were distinctly broad with greater biomass than wild-type.
35S::G1274 plants also out-performed wild-type plants in a soil drought assay; the overexpressors were more tolerant to water deprivation, and also recovered better from water deprivation, than the control plants. Overexpression of G 1275 (SEQ ID NO: 85, AtWRKY50), a gene closely related to G 1274 and also being studied here, had a more severe effect on morphology than G 1274. 35S::G1275 plants were small, with reduced apical dominance and stunted inflorescences. While the plants were fertile, seed yield was low and these plants were not tested in physiological assays. In wild-type plants, this gene, similar to G 1274, appeared to be induced by various stresses, but had a different overall expression pattern. G 1275 was primarily expressed in rosettes and siliques, and had lower but detectable expression in shoots, roots, flowers and embryos.
Table 4 shows a number of G 1274 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved WRXY domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Λrabidopsis G1274 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G 1274 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G 1274, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G 1274 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 4. Conserved WRKY domains of G 1274 and closely related sequences
Figure imgf000040_0001
Figure imgf000041_0001
9124
Abbreviations: At — Arabidopsis thaliana; Sb- Sorghum bicolor; Ca - Capsicum anrtuum; Gm — Glycine max; Hv- Hordeum vulgare; Le — Lycopersicon esculentum; Ls — Latuca saliva; Os — Oiγza sativa; St - Solarium tuberosum; Zm — Zea mays
G591, the G591 clade, and related sequences G591 corresponds to AtbΗLΗ059. The only functional information that is published about this gene comes from a post-transcriptional silencing study, in which partial silencing of G591 resulted in strong stunting (Brummell et al. (2003) Plant J. 33: 793-800).
G591 has one close paralog, G793 (AtbHLH007), about which no functional information is known. Table 5 shows a number of G591 clade polypeptides of the invention and includes the SEQ
ID NO: (Column 1), the species from which the sequence was derived and the*'GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved HLH domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G591 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G591 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G591, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G591 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 5. Conserved HLH domains of G591 and closely related sequences
Figure imgf000042_0001
Figure imgf000043_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Os — Oryza sativa; Zm — Zea mays
G233. the G233 clade. and related sequences
G233 corresponds to AtMYBl 5. G233 is homologous to NtMYBl, which is involved in SA signal transduction (Yang and Klessig (1996) Proc. Natl. Acad. Sci. USA 93, 14972-14977) and NtMYB2 (LBMl), which is involved in phenylpropanoid regulation (Sugimoto el al. (2000) Plant Cell 12, 2511-2528). Published data indicate that G233 is expressed in roots (Kranz et al. (1998) Plant J. 16, 263-276).
Table 6 shows a number of G233 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G233 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G233 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G233, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G233 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 6. Conserved Myb domains of G233 and closely related sequences
Figure imgf000045_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Le — Lycopersicon esculentum; Ls — Latuca sativa; Os — Oryza sativa; Zm — Zea mays * SEQ ID NO: 146 was predicted from the complete coding sequence of SEQ ID NO: 145, whereas SEQ ID NO: 148 is predicted from the nucleotide sequence (SEQ ID NO: 147) actually used in the expression vector to create transgenic plants. Both sequences are identical except that SEQ ID NO: 148 is truncated, lacking ten residues found in SEQ ID NO: 146 at its C-terminus, and in their place SEQ ID NO: 148 has three spurious residues at its C-terminus.
G4, the G4 clade, and related sequences
G4 is a putative ortholog of CaPFl of hot pepper, which produces resistance to Pseudomonas syringae and freezing tolerance when overexpressed (Yi et al. (2004) Plant Physiol. 136: 2862-2874). G4 is a member of the ERF-B2 subfamily of AP2/ERF genes. A related ERFB-2 gene from hot pepper, CaERFLPl , also produces enhanced tolerance to P. syringae and salt stress, supporting a potential role for ERF B-2 genes in disease resistance (Lee et al. (2004) Plant MoI. Biol. 55: 61-81 ).
Table 7 shows a number of G4 clade polypeptides (or predicted polypeptides) of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G4 sequence (Column 6), the similarity in percentage terms to the conserved domain of the Arabidopsis G4 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G4, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G4 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 7. Conserved AP2 domains of G4 and closely related sequences
Figure imgf000047_0001
Figure imgf000048_0001
Abbreviations: At — Arabidopsis thaliana; Gm Glycine max; Os — Oryza saliva; Zm — Zea mays * Gene Identification number not assigned
G869. the G869 clade. and related sequences
G869 is a member of the ERF-B6 subfamily of AP2/ERF proteins. G869 overexpressing lines showed reduced growth of E. orontii in an earlier functional genomic study. Since G869 overexpressing lines were small and stunted, dexamethasone-inducible lines were also produced for analysis.
Table 8 shows a number of G869 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved AP2 domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G869 sequence (Column 6), the similarity in percentage terms to the conserved domain of the Arabidopsis G869 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G869, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G869 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 8. Conserved AP2 domains of G869 and closely related sequences
Figure imgf000049_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Os — Ory∑a sativa; Zm — Zea mays * Gene Identification number not assigned
G237. the G237 clade, and related sequences G237 corresponds to AtMYB 18 (Kranz et al. (1998) Plant J. 16, 263-276) and to LAFl, a transcriptional activator involved in phytochrome A signaling (Ballesteros et al. (2001) Genes Dev. 15: 2613-2625). G237 overexpressing lines were found to be small with developmental abnormalities. Therefore, dexamethasone-inducible G237 lines were also characterized in pathogen assays. Table 9 shows a number of G237 clade polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the "GID identifier" (Column 2), the amino acid residue coordinates for the conserved domains of each of the sequences (Column 3), the SEQ ID NO: of the conserved Myb domain (Column 4), the conserved domain sequences of the respective polypeptides (Column 5); the identity in percentage terms to the conserved domain of the Arabidopsis G237 sequence (Column 6), and the similarity in percentage terms to the conserved domain of the Arabidopsis G237 sequence (Column 7). For the latter two columns, the percentage identity or similarity for each listed GID or sequence in the first column was determined by dividing the number of identical or similar residues, respectively, between the first column sequence and the conserved domain of G237, by the total number of residue pairs in a blastp-determined alignment of the first column sequence and the G237 conserved domain (the number of identical or similar residues in an optimal alignment and the total number of residues by which the former were divided appear in the parentheses in Columns 6 and 7).
Table 9. Conserved Myb domains of G237 and closely related sequences
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Abbreviations: At — Arabidopsis thaliana; Gm — Glycine max; Os — Oryza sativa; Zm — Zea mays * Gene Identification number not assigned
Orthologs and Paralogs
Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below. As described by Eisen (1998) Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen (1998) supra). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, supra). Thus, "[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships .... After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes" (Eisen, supra).
Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. MoL Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122- 132), and a group of very similar AP2 domain transcription factors from
Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433- 442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 543)
Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenetic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994) supra; Higgins et al. (1996) supra) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence. It is often found that orthologous groups of genes identified by the methods above share particular signature sequences that are not found in other homologs, and that these signature sequences can be used to predict other functional orthologous sequences (e.g. Panstruga (2005) Plant MoI Biol. 59: 485-500). Distinct Arabidopsis transcription factors, including G482 (found in US Patent Application
US20030093837), G47 (found in US Patent Application US20040019925), and G682 (found in US Patent Application US2003021783), have been shown to confer stress tolerance or increased plant size when the sequences are overexpressed. G 1792 (found in US Patent Application US 2005- 01551 17) has been shown to confer increased disease resistance. The polypeptides sequences belong to distinct clades of transcription factor polypeptides that include members from diverse species. In each case, a significant number of clade member sequences derived from both dicots and monocots have been shown to confer increased biomass or tolerance to stress when the sequences were overexpressed. These references may serve to represent the many studies that demonstrate that conserved transcription factor genes from diverse species are likely to function similarly (i.e., regulate similar target sequences and control the same traits), and that transcription factors may be transformed into diverse species to confer or improve traits.
As shown in Tables 1-9, transcription factors that are phylogenetically related to the transcription factors of the invention have similar functions and have conserved domains that share at least 61% amino acid sequence identity or 79% amino acid sequence similarity. At the nucleotide level, the sequences of the invention will typically share at least about
30% or 40% nucleotide sequence identity, preferably at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed full-length sequences, or to a listed sequence but excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.
Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988) Gene 73: 237-244). The cluslal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST5 or ENTRJEZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1 , e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see USPN 6,262,333).
Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (see internet website at http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) J. MoI. Biol. 215: 403-410; Altschul (1993) J. MoI. Evol. 36: 290-300). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The blastn program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. Unless otherwise indicated for comparisons of predicted polynucleotides, "sequence identity" refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter "off (see, for example, internet website at http://www.ncbi.nlm.nih.gov/).
For amino acid sequences, the blastp program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Hen ikoff ( 1989) Proc. Natl. Acad. ScI USA 89:10915). When polypeptide sequences or conserved domains of polypeptides are optimally aligned using a BLOSUM62 matrix with a gap existence penalty of 1 1 and a gap extension penalty of 1, similar conserved domains may be identified by virtue of having a minimum percentage identity or similarity.
Other techniques for alignment are described by Doolittle, ed. (1996) Methods in Enzymology, vol. 266: "Computer Methods for Macromolecular Sequence Analysis" Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997) Methods MoI. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method (see, for example, Hein (1990) Methods Enzymol. 183: 626-645) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see Hillman et al., US Patent "No. 6, 168,920).
Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions. In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990) J. MoI. Biol. 215: 403-410; Altschul (1993) J. MoI. Evol. 36: 290-300), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al. (1997) Proteins 28: 405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York, NY, unit 7.7, and in Meyers (1995) Molecular Biology and Biotechnology, Wiley VCH, New York, NY, p 856-853.
A further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related transcription factors. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler and Thomashow (2002) Plant Cell 14: 1675-1690, have shown that three paralogous AP2 family genes (CBFl, CBF2 and CBF3), each of which is induced upon cold treatment, and each of which can condition improved freezing tolerance, have highly similar transcript profiles. Once a transcription factor has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function. Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and AP2, Myb or HLH domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.
Orthologs and paralogs of presently disclosed transcription factors may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present transcription factors. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present transcription factor sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Transcription factor-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed transcription factor gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
Examples of functionally similar homologs are listed in Tables 1-9 and the Sequence Listing. In addition to these sequences, the invention encompasses isolated nucleotide sequences that are phylogenetically and structurally similar to the sequences listed in the Sequence Listing, and can function in a plant by conferring a transcriptional regulatory activity of a reference sequence in the Sequence Listing and increasing resistance to pathogens when ectopically expressed in a plant. When a significant number of these sequences are phylogenetically and sequentially related to each other have been shown to increase a plant's resistance to pathogens, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of transcription factors would also perform similar functions when ectopically expressed.
Table 10 lists sequences within the UniGene database determined to be closely-related to transcription factor sequences of the present invention (reference sequences G207, G 1750, G440, G1274, G591, G233, G4, G869 or G237, SEQ ID NOs: 2, 50, 52, 84, 128, 146, 160, 181 or 188, respectively). The column headings include the transcription factors listed by: the Reference Arabidopsis sequence used to identify each clade (Column 1); the UniGene identifier of closely- related homologs found by BLAST analysis (Column 2); the species from which the homologs to the clade reference transcription factors were derived (Column 3); the SEQ ID NO: of the homolog, where provided (Column 4); the smallest sum probability relationship (probability or p-value) of the homologous sequence to the Arabidopsis reference sequence for that row, determined by tBLASTx analysis (Column 5); and the percentage identity of the homolog to the Arabidopsis reference sequence for that row, determined by tBLASTx analysis (Column 6).
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Identifying Polynucleotides or Nucleic Acids by Hybridization
Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited below (e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.; Berger and Kimmel (1987) "Guide to Molecular Cloning Techniques", in Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, CA; and Anderson and Young (1985) "Quantitative Filter Hybridisation", In: Hames and Higgins, ed., Nucleic Acid Hybridisation. A Practical Approach. Oxford, IRL Press, 73— 1 1 1).
Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-51 1). In addition to the nucleotide sequences listed in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) supra; Berger (1987) supra, pages 467-469; and Anderson and Young ( 1985) supra.
Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (Tm) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:
(I) DNA-DNA:
Tm(° C)=81.5+16.6(log [Na+])+0.41(% G+C)- 0.62(% formamide)-500/L
(II) DNA-RNA: Tm(° C)=79.8+ l 8.5(log [Na+])+0.58(% G+C)+ 0.12(%G+C)2- 0.5(% formamide) - 820/Z,
(HI) RNA-RNA:
Tm(° C)=79.8+l 8.5(log [Na+])+0.58(% G+C)+ 0.12(%G+C)2- 0.35(% formamide) - 820/1
where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C is required to reduce the melting temperature for each 1% mismatch.
Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young (1985) supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), poly vinyl-pyrrol idone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at Tm-5° C to Tm-20° C, moderate stringency at Tm-20° C to Tn,- 35° C and low stringency at Tm-35° C to Tm-50° C for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm-25° C for DNA-DNA duplex and Tm-15° C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 200C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C and about 70° C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA. Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.
The washing steps that follow hybridization may also vary in stringency; the post- hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium citrate. Thus, hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example: 6X SSC at 65° C;
50% formamide, 4X SSC at 42° C; or 0.5X SSC, 0.1% SDS at 65° C; with, for example, two wash steps of 10 - 30 minutes each. Useful variations on these conditions will be readily apparent to those skilled in the art.
A person of skill in the art would not expect substantial variation among polynucleotide species encompassed within the scope of the present invention because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides. If desired, one may employ wash steps of even greater stringency, including about 0.2x SSC, 0.1% SDS at 65° C and washing twice, each wash step being about 30 minutes, or about 0.1 x SSC, 0.1% SDS at 65° C and washing twice for 30 minutes. The temperature for the wash solutions will ordinarily be at least about 25° C, and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C to about 5° C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C to about 9° C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 500 C. An example of a low stringency wash step employs a solution and conditions of at least 25°
C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 minutes. Greater stringency may be obtained at 42° C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 minutes. Even higher stringency wash conditions are obtained at 65° C -68° C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, Hillman et al., US Patent No. 6,168,920).
Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5- 10x higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15x or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2x or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987) supra, pages 399-407, supra; and Kimmel (1987) supra). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
EXAMPLES
It is to be understood that this invention is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention.
The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. It will be recognized by one of skill in the art that a transcription factor that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the First trait.
Example I. Project Types
Director promoter fusion lines. All of the transcription factor polynucleotides were examined under the regulatory control of a direct promoter fusion; expression of a given transcription factor from a particular promoter was achieved by a direct-promoter fusion construct in which the transcription factor polynucleotide was cloned directly behind the promoter of interest.
Two-component dex-inducible lines. In order to test transcription factors under the regulatory control of a dex-inducible promoter (the two component strategy), a kanamycin resistant 35S::LexA-GAL4-TA driver line was established.
Component 1 : promoter driver lines (Promoter::LexA/GAL4). Initially, a large number of 35S::LexA-GAL4-TA independent driver lines containing construct pMEN262 (also known as P5486, SEQ ID NO: 203) were generated. Primary transformants were selected on kanamycin plates and screened for GFP (green fluorescent protein) fluorescence at the seedling stage. Any lines that showed constitutive GFP activity were discarded. At 10 days, lines that showed no GFP activity were then transferred onto MS agar plates containing 5 μM dexamethasone. Lines that showed strong GFP activation at two to three days following the dexamethasone treatments were marked for follow-up in the T2 generation. Following similar experiments in the T2 generation, a single line, line 65, was selected for future studies. Line 65 lacked any obvious background expression and all plants showed strong GFP fluorescence following dexamethasone application. A homozygous population for line 65 was then obtained and re-checked to ensure that it still exhibited induction following dexamethasone application. 35S::LexA-GAL4-TA line 65 was also crossed to an opLexA::GUS line to demonstrate that it could drive activation of targets arranged in trans.
Component 2: TF construct (opLexA::TF). Having established a driver line, the transcription factors of the invention could be expressed by super-transforming or crossing in a second construct carrying the transcription factor polynucleotide of interest cloned behind a LexA operator site
(opLexA::TF). In each case, the second construct carried a sulfonamide selectable marker and was contained within vector backbone pMEN53. For the present study, the opLexA::TF constructs prepared and used to supertransform plants were opLexA::G 1750 (construct P3963), opLexA::G440 (construct P3963), and opLexA::G869 (construct P9105).
Example II. Transformation
Transformation of Arabidopsis was performed by an Λgrobacterium-mediated protocol based on the method of Bechtold and Pelletier (1998) Methods MoI. Biol. 82: 259-266. Unless otherwise specified, all experimental work was done using the Columbia ecotype.
Plant preparation. Arabidopsis seeds were sown on mesh covered pots. The seedlings were thinned so that 6-10 evenly spaced plants remained on each pot 10 days after planting. The primary bolts were cut off a week before transformation to break apical dominance and encourage auxiliary shoots to form. Transformation was typically performed at 4-5 weeks after sowing.
Bacterial culture preparation. Agrobacterium stocks were inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics and grown until saturation. On the morning of transformation, the saturated cultures were centrifuged and bacterial pellets were re-suspended in Infiltration Media (0.5X MS, IX B5 Vitamins, 5% sucrose, I mg/ml benzylaminopurine riboside, 200 μl/L Silwet L77) until an A600 reading of 0.8 is reached.
Transformation and seed harvest. The Agrobacterium solution was poured into dipping containers. All flower buds and rosette leaves of the plants were immersed in this solution for 30 seconds. The plants were laid on their side and wrapped to keep the humidity high. The plants were kept this way overnight at 4 0C and then the pots were turned upright, unwrapped, and moved to the growth racks.
The plants were maintained on the growth rack under 24-hour light until seeds were ready to be harvested. Seeds were harvested when 80% of the siliques of the transformed plants are ripe (approximately 5 weeks after the initial transformation). This seed was deemed TO seed, since it was obtained from the TO generation, and was later plated on selection plates (either kanamycin or sulfonamide). Resistant plants that were identified on such selection plates comprised the Tl generation.
Example III. Plant Growth and Morphological Analysis Morphological analysis was performed to determine whether changes in transcription factor levels affect plant growth and development. This was primarily carried out on the Tl generation, when at least 10-20 independent lines were examined. However, in cases where a phenotype required confirmation or detailed characterization, plants from subsequent generations were also analyzed. Primary transformants were selected on MS medium with 0.3% sucrose and 50 mg/1 kanamycin. T2 and later generation plants were selected in the same manner, except that kanamycin was used at 35 mg/1. In cases where lines carry a sulfonamide marker (as in all lines generated by super-transformation), seeds were selected on MS medium with 0.3% sucrose and 1.5 mg/1 sulfonamide. KO lines were usually germinated on plates without a selection. Seeds were cold- treated (stratified) on plates for 3 days in the dark (in order to increase germination efficiency) prior to transfer to growth cabinets. Initially, plates were incubated at 22°C under a light intensity of approximately 100 microEinsteins (μE) for 7 days. At this stage, transformants were green, possessed the first two true leaves, and were easily distinguished from bleached kanamycin or sulfonamide-susceptible seedlings. Resistant seedlings were then transferred onto soil (Sunshine potting mix). Following transfer to soil, trays of seedlings were covered with plastic lids for 2-3 days to maintain humidity while they became established. Plants were grown on soil under fluorescent light at an intensity of 70-95 μE and a temperature of 18-23°C. Light conditions consisted of a 24-hour photoperiod unless otherwise stated. Under our 24-hour light growth conditions, the typical generation time (seed to seed) was approximately 14 weeks. Because many aspects of Λmbidopsis development are dependent on localized environmental conditions, in all cases plants were evaluated in comparison to controls in the same flat. Controls for transgenic lines were wild-type plants or transgenic plants harboring an empty transformation vector selected on kanamycin or sulfonamide. Careful examination was made at the following stages: seedling (1 week), rosette (2-3 weeks), flowering (4-7 weeks), and late seed set (8-12 weeks). Seedling morphology was assessed on selection plates. At all other stages, plants were macroscopically evaluated while growing on soil. All significant differences (including alterations in growth rate, size, leaf and flower morphology, coloration and flowering time) were recorded, but routine measurements were not taken if no differences were apparent.
Example IV. Plate Assay Methods
Plate-based assays were used to assess resistance to three neurotrophic pathogens. All lines were screened with a Sclerotinia sclerotiorum plate-based assay. Most lines were also subjected to Botrytis cinerea plate assays. A Fusarium oxysporum plate assay was used for selected sequences that gave an altered response to Fusarium in previous studies. Typically, eight lines were subjected to plate assays.
Unless otherwise stated, all experiments were performed with the Arabidopsis thaliana ecotype Columbia (Col-0). Assays were usually performed on non-selected segregating T2 populations (in order to avoid the extra stress of selection). Control plants for assays on lines containing direct promoter-fusion constructs were wild-type plants or CoI-O plants transformed an empty transformation vector (pMEN65). Controls for 2-component lines (generated by supertransformation) were the background promoter-driver lines (i.e. promoter: :LexA-GAL4TA lines), into which the supertransformations were initially performed.
Prior to plating, seed for all experiments were surface sterilized in the following manner: (1 ) 5 minute incubation with mixing in 70 % ethanol; (2) 20 minute incubation with mixing in 30% bleach, 0.01% Triton X-100; (3) five rinses with sterile water. Seeds were resuspended in 0.1% sterile agarose and stratified at 4 0C for 2-4 days. Sterile seeds were sown on starter plates (15 mm deep) containing the following medium:
50% MS solution, 1% sucrose, 0.05% MES, and 1% Bacto-Agar. 40 to 50 seeds were sown on each plate. Plates were incubated at 22 0C under 24-hour light (95-1 10 μE m"2 s"1) in a germination growth chamber. On day 10, seedlings were transferred to assay plates (25 mm deep plates with medium minus sucrose). For lines containing genes under the control of a dexamethasone-inducible promoter, the assay plates also contained 5 μM dexamethasone. Each assay plate had nine test seedlings and nine control seedlings on separate halves of the plate. Four plates were used per line, per pathogen. On day 14, seedlings were inoculated (specific methods below). After inoculation, plates were put in a growth chamber under a 12-hour light/12-hour dark schedule. Light intensity was lowered to 70-80 μE m"2 s"1 for the disease assay. Disease symptoms were evaluated starting four days post-inoculation (DPI) up to 10 DPI if necessary. For each plate, number of dead test plants and control plants were counted. Plants were scored as "dead" if the center of the rosette had collapsed (usually brown or water-soaked).
Sclerotinia inoculum preparation. A Sclerolinia liquid culture was started three days prior to plant inoculation by cutting a small agar plug (1/4 sq. inch) from a 14- to 21-day old Sclerotinia plate (on Potato Dextrose Agar; PDA) and placing it into 100 ml of half-strength Potato Dextrose Broth (PDB). The culture was allowed to grown in the PDB at room temperature under 24-hour light for three days. On the day of seedling inoculation, the hyphal ball was retrieved from the medium, weighed, and ground in a blender with water (50 ml/gm tissue). After grinding, the mycelial suspension was filtered through two layers of cheesecloth and the resulting suspension was diluted 1:5 in water. Plants were inoculated by spraying to run-off with the mycelial suspension using a Preval aerosol sprayer.
Botrytis inoculum preparation. Botrytis inoculum was prepared on the day of inoculation. Spores from a 14- to 21-day old plate were resuspended in a solution of 0.05% glucose, 0.03M KH2PO4 to a final concentration of 104 spores/ml. Seedlings were inoculated with a Preval aerosol sprayer, as with Sclerotinia inoculation. Fusarium inoculum preparation. Fusarium inoculum was started one day prior to plant inoculation. Spores from a 14- to 21-day old plate were resuspended in 100 ml of Czapek-Dox medium (Sigma) and grown overnight under constant light. The suspension was shaken several times throughout the day until the time of inoculation. Seedlings were inoculated with a suspension of 106 spores/ml, using a Preval aerosol sprayer. Data Interpretation
After the plates were evaluated, each line was given one of the following overall scores: (++) Substantially enhanced resistance compared to controls. The phenotype was very consistent across all plates for a given line. (+) Enhanced resistance compared to controls. The response was consistent but was only moderately above the normal levels of variability observed for that assay,
(wt) No detectable difference from wild-type controls. (-) Increased susceptibility compared to controls. The response was consistent but was only moderately above the normal levels of variability observed for that assay.
(- -) Substantially impaired performance compared to controls. The phenotype was consistent and growth was significantly above the normal levels of variability observed for that assay. (n/d) Assay not performed or data not obtained.
Example V. Soil Assays
Each project (i.e., lines from transformation with a particular construct) was tested in a soil- based assay for resistance to the biotrophic pathogen powdery mildew {Erysiphe cichoracearum). Typically, eight lines per project were subjected to the Erγsiphe assay. Unless otherwise stated, all experiments were performed with the Arabidopsis thaliana ecotype Columbia (Col-0). Erysiphe assays were usually performed on non-selected segregating T2 populations (in order to avoid the extra stress of selection). Kanamycin or sulfonamide selection was used for the Sclerotima assay to minimize variability and improve statistics on the results. Control plants for assays on lines containing direct promoter-fusion constructs were wild-type plants or CoI-O plants transformed an empty transformation vector (pMEN65). Controls for 2- component lines (generated by supertransformation) were the background promoter-driver lines (i.e. promoter::LexA-GAL4TA lines), into which the super-transformations were initially performed.
Ervsiphe soil assay. Erγsiphe inocuia were propagated on apad4 mutant line in the CoI-O background, which is highly susceptible to Erysiphe (Reuber et al. (1998) Plant J. 16: 473-485). Inocuia were maintained by using a small paintbrush to dust conidia from a 2-3 week old culture onto new plants (generally three weeks old). In addition, the inocuia were passaged through squash plants once a month to ensure the purity of the isolate; the isolate used causes disease on both squash and Arabidopsis, while most isolates are host-specific. For the assay, seedlings were grown on plates for one week under 24-hour light in a germination chamber, then transplanted to soil and grown in a walk-in growth chamber under a 12-hour light/12-hour dark regimen, 70% humidity. Each line was transplanted to two 13 cm square pots, nine plants per pot. In addition, three control plants were transplanted to each pot for direct comparison with the test line. Approximately 3.5 weeks after transplanting, plants were inoculated using settling towers, as described by Reuber et al. (1998) supra. Generally, three to four heavily infested leaves were used per pot for the disease assay. Level of fungal growth was evaluated eight to eleven days after inoculation. Data Interpretation After the pots were evaluated, each line was given one of the following overall scores: (+++) Test plants appeared to be essentially free of fungus. (++) Substantially enhanced resistance compared to controls. The phenotype was very consistent in both pots for a given line. (+) Enhanced resistance compared to controls. The response was consistent but was only moderately above the normal levels of variability observed, (wt) No detectable difference from wild-type controls. (-) Increased susceptibility compared to controls. The response was consistent but was only moderately above the normal levels of variability observed. (- -) Substantially impaired performance compared to controls. The phenotype was consistent and growth was significantly above the normal levels of variability observed, (n/d) Assay not performed or data not obtained.
Example VI. Results
G1750 {Arabidopsis SEQ ID NO: 49 and 50) and its paralog G440 {Arabidopsis SEQ ID NO: 51 and 52)
G 1750 (Arabidopsis SEO ID NO: 49 and 50\ P 1034 (SEQ ID NO: 202) contained a 35S::G 1750 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G 1750.
P5486 (SEQ ID NO: 203) and P3963 (SEQ ID NO: 204) comprised a two-component system. P5486 encoded a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker. A transgenic line carrying this construct was established and supertransformed with P3963 (carrying a sulfonamide marker) which contained an opLexA::G1750 cD"NA fragment.
Gl 750 fSEO ID NO: 49 and 50~) experimental observations. A large number of 35S::G 1750 lines were generated. Constitutive expression of G 1750 caused severe stunting, and approximately half of the plants died before reaching maturity. Typically 35S::G1750 lines were small, dark-green and vitrified early in development. Very few of the lines were fertile, and thus disease assays were performed on lines that had weak phenotypes.
Because of the severe off-types observed with constitutive expression of G 1750, we also produced two-component lines that express G 1750 under a dex-inducible promoter. A number of the two-component lines were noted to be early flowering, but the plants were otherwise wild-type. Eight 35S::G 1750 lines were tested for Erysiphe cichoracearum resistance in a soil assay. All eight lines showed moderate to strong resistance to this pathogen. It was noted that in many of the lines strong resistance was associated with some leaf chlorosis and general stunting. These 35S::G1750 lines were tested in Sclerotinia and Botrylis plate assays. All but one line showed enhanced susceptibility to Botrytis; three lines also showed enhanced susceptibility to Sclerotinia. It is important to note, however, that constitutive overexpression of Gl 750 results in plants that are tiny and developmental Iy delayed. In general, stunted plants appear more susceptible in the plate assays. The susceptibility of the 35S::G1750 lines is, therefore, likely to be an artifact of the assay.
Because of the severe morphological side effects of constitutive G 1750 expression, this transcription factor was also tested under the control of a dex-inducible promoter. Eight two- component dex-inducible Gl 750 lines were tested for Sclerotinia and Botrytis resistance. Two lines (325 and 326) showed enhanced resistance to Botrytis when grown on dex-containing plates; line 326 also showed enhanced Sclerotinia resistance. Conversely, line 322 displayed enhanced susceptibility to Botrytis. These results suggest that expression of Gl 750 does not condition susceptibility to necrotrophs, and instead contributes to resistance. These lines were not tested for resistance to Erysiphe cichoracearum.
Table 1 1. Response of G 1750 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
Figure imgf000077_0001
Potential applications: G 1750 may be useful for engineering disease resistance, particularly to biotrophic pathogens. The results from the dex-inducible lines indicate that morphological off- types can be separated from positive disease phenotypes.
G440 (Arabidopsis SEO ID NO: 51 and 52V P258 (SEQ ID NO: 205) contained a 35S::G440 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G440.
P5486 (SEQ ID NO: 203) and P5265 (SEQ ID NO: 206) comprised a two-component system. P5486 encodes a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker. A transgenic line carrying this construct was established and supertransformed with P5265 (carrying a sulfonamide marker) which contained an opLexA::G440 cDNA fragment.
Experimental observations. Twenty 35S::G440 lines were generated. These lines showed a variety of morphological alterations compared to control plants, including moderate to severe dwarfing, changes in leaf morphology, and late development.
Eight 35S::G440 lines were tested for Erysiphe resistance in a soil assay. Five of these lines showed mild to moderately enhanced resistance. These lines were also tested in Sclerotinia and Botrytis plate assays. Four lines showed mildly increased susceptibility to Botrγtis. The response to Sclerotinia was slightly variable: most lines appeared wild-type, although one line was more resistant and another line was more susceptible.
Because of the growth retardation caused by constitutive G440 expression, this transcription factor was also tested under the control of a dex-inducible promoter. Eight two-component dex- inducible G440 lines were tested for Sclerotinia and Botrytis resistance. Three of these lines showed enhanced susceptibility to Botrytis when grown on dex-containing plates, while one line was more resistant. An additional line showed mildly increased resistance to Sclerotinia.
Table 12. Response of G440 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
Figure imgf000078_0001
Figure imgf000079_0001
Potential applications: G440 has potential use for engineering resistance to biotrophic pathogens, such as powdery mildews and rusts.
G207 (Arahidopsis SEQ ID NO: 1 and 2)
P800 (SEQ ID NO: 207) contained a 35S::G207 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a genomic clone of G207.
Experimental observations. G207 overexpressing lines were found to have increased sensitivity to glucose in a germination assay, indicating a potential role for G207 in sugar signaling. G207 has previously been reported to be regulated during late embryonic development (Kirik et al. (1998) Plant MoI. Biol. 37:, 819-827), induced in secondary xylem formation (Ko et al. (2004) Plant Physiol. 135: 1069-1083), and repressed by K+ deprivation, which activates reactive oxygen species (Shin and Schachtman (2004) Proc. Natl. Acad. Sci. USA 101 : 8827-8832).
Twenty 35S::G207 lines were obtained. These lines showed no overall difference in morphology from control plants. Eight lines were tested for Botrytis, Sclerotinia, and Erysiphe resistance.
Eight 35S::G207 lines were tested for Erysiphe resistance in a soil assay. Constitutive overexpression of G207 caused enhanced resistance to Erysiphe when compared to wild-type plants, clearly evident in five of the transgenic lines. No significant difference compared to wild- type was observed in these lines in response to Botrytis and Sclerotinia. One line showed slightly enhanced susceptibility to Scleroiinia, otherwise the 35S::G207 lines appeared wild-type in response to the two pathogens.
Table 13. Response of G207 overexpressing plants in Erysiphe soil assay
Figure imgf000080_0001
Potential applications: G207 represents an excellent candidate for engineering resistance to biotrophic pathogens, such as powdery mildews and rusts. No significant negative side effects of G207 overexpression were observed, decreasing the need for extensive expression pattern optimization in engineered plants.
G233 {Arabidopsis SEQ ID NO: 145 and 146)
Pl 6 (SEQ ID NO: 208) contained a 35S::G233 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G233.
Experimental observations. Plants overexpressing G233 showed increased sensitivity to glucose in a germination assay in an earlier genomics program. In preliminary studies, knockout plants were similar to wild-type in appearance. Transcript profiling data indicated that G233 is induced by Botrytis infection, oligogalacturonides, jasmonate, and salt.
Twenty new 35S::G233 lines were generated. These lines showed no consistent differences in morphology from control plants. Eight lines were tested for Botrytis, Sclerotinia, and Erysiphe resistance. One line showed enhanced resistance to all three pathogens, one line showed enhanced resistance to Erysiphe and Botrytis, and three lines were resistant to one pathogen (one each for Botrytis, Sclerotinia, and Erysiphe).
Table 14. Response of G233 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
Figure imgf000081_0001
Potential applications: G233 shows clear potential for engineering plants for resistance to multiple pathogens, including both biotrophs and necrotrophs. Overexpression of G233 does not result in obvious negative side effects on morphology, decreasing the need for extensive expression pattern optimization in engineered plants.
G591 {Arabidopsis SEQ ID NO: 127 and 128)
P77 (SEQ ID NO: 209) contained a 35S::G591 direct promoter fusion and carries a kanamycin resistance marker. The construct contains a cDNA clone of G591.
Experimental observations. Eighteen 35S::G591 lines were generated. These lines showed no consistent difference in morphology from control plants. Eight lines were tested for resistance to Erysiphe, Sclerotinia, and Botrytis. Four of these lines showed moderate resistance to Erysiphe. The response to the two neurotrophic pathogens was more variable. Two lines were more resistant to Botrytis while two lines were more susceptible. In addition, two lines were more resistant to Sclerotinia, including one of the Botrytis resistant lines. Table 15. Response of G591 overexpressing plants in Botrγtis, Sclerotinia and Erysiphe assays
Figure imgf000082_0001
Potential applications: These results indicate that G591 has potential for engineering broad- spectrum disease resistance. The resistance to Erysiphe, a biotroph, is clear in multiple 35S::G591 lines. The response to the necrotrophs was more variable, although the fact that several lines showed resistance to Botrytis and/or Sclerotinia is promising. Overexpression of G591 does not result in obvious negative side effects on morphology, decreasing the need for extensive expression pattern optimization in engineered plants.
G4 (Arabidopsis SEQ ID NO: 159 and 160) Pl 63 (SEQ ID NO: 210) contained a 35S::G4 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G4.
Experimental observations. Lines overexpressing G4 were typically larger than controls, and they also bolted and flowered early. Eight lines were tested in Botrytis and Sclerotinia plate assays.
Three lines showed slightly enhanced resistance to Botrytis. No difference from wild-type was observed in response to Sclerotinia. AU lines also displayed a wild-type phenotype in response to
Erysiphe cichoracearum.
Table 16. Response of G4 overexpressing plants in Botrytis and Sclerotinia plate assays
Figure imgf000082_0002
Figure imgf000083_0001
Potential applications: The results obtained to date suggest that G4 might be used to increase resistance to Botrytis or related pathogens. The morphological effects suggest that G4 might also be used to regulate growth rate, biomass, and flowering time.
G237 (Arabidopsis SEQ ID NO: 187 and 188) P17 (SEQ ID NO: 21 1) contained a 35S::G237 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a genomic clone of G237.
Experimental observations. Forty 35S::G237 lines were generated. These lines showed variable degrees of dwarfing and were typically spindly, bushy, and showed reduced fertility.
Eight 35S::G237 lines were tested by Sclerotinia, Botrytis and Erysiphe disease assays. There was no significant difference compared to wild-type plants in response to Sclerotinia and
Botrytis. In the Erysiphe assay, three lines showed mildly enhanced resistance and one line showed slightly increased susceptibility. These opposing phenotypes might reflect overexpression versus silencing of G237. The lines displaying enhanced resistance showed no clear evidence of dwarfing in the T2 generation. Thus, the disease phenotypc appears separable from morphological side effects of G237 overexpression.
Because of the growth retardation seen in many of the overexpressing lines, we also tested eight dexamethasone-inducible G237 lines by Sclerotinia and Botrytis plate assays. These lines were morphologically indistinguishable from control plants and appeared similar to control plants in response to these two pathogens.
Table 17. Response of G237 overexpressing plants in Erysiphe soil assays
Figure imgf000083_0002
Potential applications: Based on the current data, it appears that modulation of G237 expression may enhance resistance to biotrophic pathogens.
G869 (Arabidopsis SEQ ID NO: 180 and 181)
P384 (SEQ ID NO: 212) contained a 35S::G869 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G869.
P5486 (SEQ ID NO: 203) and P9105 (SEQ ID NO: 213) comprised a two-component system. P5486 encodes a 35S::LexA-GAL4TA-GR fragment and carried a kanamycin resistance marker. A transgenic line carrying this construct was established and supertransformed with P9105 (carrying a sulfonamide marker) that contained an opLexA::G869 cDNA fragment. Experimental observations. Twenty 35S::G869 lines were generated. The majority of the Tl plants showed dwarfing to various degrees. Lines that were dwarfed were also relatively late developing, spindly and had decreased fertility.
Two-component lines containing a dex-inducible promoter were also isolated. Most of these plants were normal in the absence of dexamethasone, except that about half of the lines flowered early.
Eight 35S::G869 lines were tested for Erysiphe resistance in a soil assay. Three of these lines showed moderately enhanced resistance. These 35S::G869 lines were tested by Sclerotinia plate assay. Three of these lines showed enhanced susceptibility to Sclerotinia. Note that constitutive overexpression of G869 causes growth retardation in transgenic lines. The apparent enhanced susceptibility of these lines may be an artifact of the assay, since smaller plants, in general, succumb faster than larger plants in the Sclerotinia plate assay.
Because overexpression of G869 causes a reduction in plant size, this gene was also tested under the control of the dex-inducible promoter. Eight two-component dex-inducible G869 lines were tested for Sclerotinia and Botrytis resistance on dex-containing plates. Three lines showed slightly enhanced resistance to Botrytis when tested on dex plates. These results suggest that G869 expression can provide some protective effect against necrotrophic pathogens, and that the increased susceptibility to Sclerotinia observed in the 35S::G869 lines may be an artifact of the assay.
G869 Disease Assay Results Table 18. Response of G869 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
Figure imgf000085_0001
n/d not determined
Potential applications: G869 may be useful for engineering plants with enhanced disease resistance.
G 1274 (Arabidopsis SEQ ID NO: 83 and 84)
P15038 ((SEQ ID NO: 214) contained a 35S::G 1274 direct promoter fusion and carried a kanamycin resistance marker. The construct contained a cDNA clone of G 1274.
Experimental observations. G 1274 has previously been shown by us to confer tolerance to water deprivation, low nitrogen conditions, and cold. In the present study, three G 1274 lines were tested in a soil assay for resistance to Erysiphe cichor ace arum, and in plate assays for Sclerotinia sclerotiorum and Botrytis cinerea. Two lines showed moderate to strong resistance to Erysiphe. One line showed slightly enhanced susceptibility to Sclerotinia, but overall there was no significant difference from control plants in response to the two necrotrophic pathogens.
Table 19. Response of G 1274 overexpressing plants in Botrytis, Sclerotinia and Erysiphe assays
Figure imgf000085_0002
Figure imgf000086_0001
Potential applications: The results obtained to date suggest that G 1274 might be used to increase resistance to biotrophic pathogens such as powdery mildew or rusts.
Example VII. Transformation of dicots to produce increased disease resistance
Transcription factor polynucleotide sequences listed in the Sequence Listing or Tbale 10, or that encode polypeptides listed in the Sequence Listing or Table 10, recombined into, for example, one of the expression vectors of the invention, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield and/or quality. The expression vector may contain a constitutive, tissue-specific or inducible promoter operably linked to the transcription factor polynucleotide. The cloning vector may be introduced into a variety of plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. It is now routine to produce transgenic plants using most dicot plants (see Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press; Gelvin et al. (1990) Plant Molecular Biology Manual. Kluwer Academic Publishers; Herrera-Estrella et al. (1983) Nature 303: 209; Bevan (1984) Nucleic Acids Res. 12: 8711-8721 ; and Klee (1985) Bio/Technology 3: 637-642). Methods for analysis of traits are routine in the art and examples are disclosed above.
Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al. (1993), in Methods in Plant Molecular Biology and Biotechnology, p. 89-1 19, and Glick and Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, eds., CRC Press, Inc., Boca Raton, describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al. (1993) in Methods in Plant Molecular Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.
There are a substantial number of alternatives to Agrobactenwn-medlated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al. (1987) Part. ScL Technol. 5:27-37; Christou et al. (1992) Plant. J. 2: 275-281 ; Sanford (1993) Methods Enzymol. 217: 483-509; Klein et al. (1987) Nature 327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991 ; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21 , 1994). Alternatively, sonication methods (see, for example, Zhang ct al. ( 1991 ) Bio/Technology 9:
996-997); direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al. (1985) MoI. Gen. Genet. 199: 161 - 168; Draper et al. (1982) Plant Cell Physiol. 23: 451 -458); liposome or spheroplast fusion (see, for example, Deshayes et al. (1985) EMBO J., 4: 2731 -2737; Christou et al. (1987) Proc. Natl. Acad. ScL USA 84: 3962-3966); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al.( 1990) in Abstracts of VHth International Congress on Plant Cell and Tissue Culture IAPTC. A2-38: 53; D'Halluin et al. (1992) Plant Cell 4: 1495-1505; and Spencer et al. (1994) Plant MoI. Biol. 24: 51 -61) have been used to introduce foreign DNA and expression vectors into plants. After a plant or plant cell is transformed (and the latter regenerated into a plant), the transformed plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986) In Tomato Biotechnology: Alan R. Liss, Inc., 169-178, and in U.S. Patent 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μM α-naphthalene acetic acid and 4.4 μM 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the invention for 5-10 minutes, blotted dry on sterile filter paper and cqcultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to an ODβoo of 0.8. Following cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium comprising MS medium with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a kanamycin sulfate-containing medium is a positive indication of a successful transformation.
Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Patent 5,563,055 (Townsend et al., issued October 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.
Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the invention are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Patent 5,563,055).
The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.
Example VIII: Transformation of monocots to produce increased disease resistance Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may be transformed with the present polynucleotide sequences, including monocot or dicot-derived polynucleotide sequences such as those presented in the present Tables or Sequence Listing, or which encode the polypeptides found in the present Tables of Sequence Listing, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV 35S or CORl 5 promoters, or with tissue-specific or inducible promoters. The expression vectors may be found in the Sequence Listing, or any other suitable expression vector may be similarly used. For example, pMEN020 may be modified to replace the Nptll coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The Kpnl and BgIII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.
The cloning vector may be introduced into a variety of cereal plants by means well known in the art including direct DNA transfer or Agrobacterium
Figure imgf000089_0001
transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Patent No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector.
The sample tissues are immersed in a suspension of 3x10"9 cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C in the dark for several days. The calli grown on this medium are transferred to Regeneration medium. Transfers are continued every 2-3 weeks (2 or 3 times) until shoots develop. Shoots are then transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.
The transformed plants are then analyzed for the presence of the NPTlI gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, CO).
It is also routine to use other methods to produce transgenic plants of most cereal crops (Vasil (1994) Plant MoI Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al. (1993) Proc. Natl. Acad. ScL USA 90: 1 1212-1 1216), and barley (Wan and Lemeaux (1994) Plant Physiol. 104: 37-48). DNA transfer methods such as the microprojectile method can be used for corn (Fromm et al. (1990) Bio/Technol. 8: 833-839; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; Ishida (\99ϋ) Nature Biotechnol. 14:745-750), wheat (Vasil et al. (1992) Bio/Technol. 10:667-674; Vasil et al. (1993) Bio/Technol. 1 1 : 1553-1558; Weeks et al. (1993) Plant Physiol. 102:1077-1084), and rice (Christou (1991) Bio/Technol. 9:957-962; Hiei et al. (1994) Plant J. 6:271-282; Aldemita and Hodges (1996) Planta 199: 612-617; and Hici ct al. (1997) Plant MoI. Biol. 35:205-218). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al. (1997) supra; Vasil (1994) supra). For transforming corn embryogenic cells derived from immature scutellar tissue using microprojectile bombardment, the A 188XB73 genotype is the preferred genotype (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990) supra). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) supra; Gordon- Kamm et al. (1990) supra).
Example IX: Transcription factor expression and analysis of disease resistance
Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a transcription factor polypeptide or the invention and related genes that are capable of inducing disease resistance.
To verify the ability to confer biotic stress resistance, mature plants overexpressing a transcription factor of the invention, or alternatively, seedling progeny of these plants, may be challenged by a pathogen. By comparing control plants (for example, wild type) and transgenic plants similarly treated, the transgenic plants may be shown to have greater resistance to the particular pathogen.
After a dicot plant, monocot plant or plant cell has been transformed (and the latter regenerated into a plant) and shown to have resistance to disease and, possibly greater size, or produce greater yield relative to a control plant under the biotic stress conditions, the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild- type monocot plant, or another transformed monocot plant from a different transgenic line of plants.
These experiments would demonstrate that transcription factor polypeptides of the invention can be identified and shown to confer greater yield and greater disease resistance in dicots or monocots, including resistance to multiple pathogens.
Example X: Sequences that Confer Significant Improvements to non-Arabidopsis species The function of specific transcription factors of the invention (the reference sequences of the invention) have been analyzed using BLAST and phylogenetic analysis, and they or their closely related homologs may be further characterized and incorporated into crop plants. The ectopic overexpression of these sequences may be regulated using constitutive, inducible, or tissue specific regulatory elements. Genes that have been examined and have been shown to increase disease resistance encode transcription factor polypeptides found in the Sequence Listing or the present Tables. In addition to these sequences, it is expected that newly discovered polynucleotide and polypeptide sequences closely related to polynucleotide and polypeptide sequences found in the Sequence Listing can also confer alteration of traits in a similar manner to the reference sequences G207, G1750, G440, G1274, G591, G233, G4, G867, or G237, when transformed into a any of a considerable variety of plants of different species, and including dicots and monocots. Closely related sequences that have similar function include, for example, those sequences that are closely, phylogenetically related to the sequences of the invention by virtue of being within the same clade and having descended from a common ancestral sequence. The closely-related polynucleotide and polypeptide sequences derived from monocots (e.g., the rice sequences) may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy genes) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived. The sequences of the transcription factors of the invention may be overexpressed under the regulatory control of constitutive, tissue specific or inducible promoters. These transcription factors may confer disease resistance when they are overexpressed under the regulatory control of non- constitutive promoters or a transactivation domain fused to the clade member, without having a significant adverse impact on plant morphology and/or development. The lines that display useful traits may be selected for further study or commercial development.
Monocotyledonous plants, including rice, corn, wheat, rye, sorghum, barley and others, may be transformed with a plasm id containing a transcription factor polynucleotide. The transcription factor gene sequence may include dicot or monocot-derived sequences such as those presented herein. These transcription factor genes may be cloned into an expression vector containing a kanamycin-resistance marker, and then expressed constitutively or in a tissue-specific or inducible manner. The cloning vector may be introduced into monocots by, for example, means described in the previous Example, including direct DNA transfer or Agrobacterium tumefaciens-med'iated transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Patent No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector. The sample tissues are immersed in a suspension of 3x10" cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C in the dark for several days. The calli grown on this medium are transferred to Regeneration medium. Transfers are continued every 2-3 weeks (2 or 3 times) until shoots develop. Shoots are then. transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.
The transformed plants are then analyzed for the presence of the NPTIl gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Priτne Inc. (Boulder, CO). Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a transcription factor polypeptide of the invention that is capable of conferring increased disease resistance, or increased size or yield, in the transformed plants.
To verify the ability to confer disease resistance, mature plants expressing a monocot- derived equivalog gene, or alternatively, seedling progeny of these plants, may be challenged using methods described in the above Examples. By comparing wild type plants and the transgenic plants, the latter are shown be more resistant to disease as compared to wild-type or non-transformed control plants, or controls plants transformed with an empty vector, similarly treated.
It is expected that the same methods may be applied to identify other useful and valuable sequences of the present transcription factor clades, and the sequences may be derived from a diverse range of species.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The present invention is not limited by the specific embodiments described herein. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended Statements of the Invention. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the Statements of the Invention, or claims that may derive from the Statements of the Invention.

Claims

We Claim:
1. A transgenic plant comprising a recombinant polynucleotide encoding an AP2, Myb, HLH, or WRKY family polypeptide, wherein the BLOSUM62 matrix, using a gap existence penalty of 1 1 and a gap extension penalty of 1 , will generate:
(a) a percentage identity of at least 67%, 69%, 70%, 71%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%, 83%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2;
(b) a percentage identity of at least 61%, 62%, 65%, 69%, 70%, 71%, 72%, 74%, 77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81%, 82%, 84%, 85%, 86%, 87%, 89%, 90%, 91 %, 93%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50;
(c) a percentage identity of at least 65%, 67%, 69%, 72%,' 73%, 74%, 75%, 79%, 83%, 85%, or 88%, or 100%, or a percentage similarity of at least 79%, 82%, 83%, 86%, 87%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ FD NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52;
(d) a percentage identity of at least 75%, 77%, 78%, 80%, 82%, or 84%, or 100%, or a percentage similarity of at least 85%, 87%, 89%, 91%, or 92%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 256-277, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 84;
(e) a percentage identity of at least 74%, 75%, 91%, or 98%, or 100%, or a percentage similarity of at least 91 %, 93%, 94%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 278-286, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 128; (f) a percentage identity of at least 82%, 84%, 58%, 86%, or 100%, or a percentage similarity of at least 93%, 94%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 287-292, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NOs: 146; (g) a percentage identity of at least 82%, 83%, 85%, 86%, 87%, 88%, or 91%, or 100%, or a percentage similarity of at least 86%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 293-308, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 160; (h) a percentage identity of at least 60%, 68%, 72%, or 100%, or a percentage similarity of at least 68%, 73%, 83%, 91 %, or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 309-314, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 181 ; or
(i) a percentage identity of at least 70%, 71%, 72%, 73%, 75%, 77%, 85%, or 100%, or a percentage similarity of at least 84%, 85%, 87%, 88%, 89%, 92% or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 315-327, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 188; wherein the polypeptide is overexpressed in the transgenic plant relative to expression in a control plant.
2. A transgenic plant according to statement 1 , wherein said AP2, Myb, HLH5 or WRKY family polypeptide comprises a conserved domain that is at least 60% identical or at least 68% similar to a polypeptide sequence selected from the group consisting of SEQ ID NO: 215-327.
3. A transgenic plant according to statement 1, wherein the transgenic plant has greater resistance to a pathogen as compared to the control plant
4. A transgenic plant according to statement 3, wherein said pathogen is a fungal pathogen.
5. A transgenic plant according to statement 3, wherein said pathogen is selected from the group consisting of Botrytis, Sclerotinia, Etysiphe, and Fusarium.
6. A transgenic plant according to statement 1 , wherein said recombinant polynucleotide comprises a sequence derived from Arabidopsis thaliana, Capsicum annuum, Glycine max, Hordeum vulgare, Lactuca sativa, Lycopersicon esculentum, Oryza saliva, Solarium tuberosum, Sorghum bicolor, and Zea mays.
7. A transgenic plant according to statement 1 , wherein the recombinant polynucleotide comprises a constitutive, inducible, or tissue-specific promoter operably linked to a polynucleotide sequence encoding said AP2, Myb, HLH, or WRKY family polypeptide.
8. A transgenic seed produced by the transgenic plant according to statement 1.
9. A method for producing a transgenic plant, the method steps comprising:
(a) introducing into a plant an expression vector comprising a recombinant polynucleotide that encodes an AP2, Myb, HLH, or WRKY family polypeptide: wherein the BLOSUM62 matrix, using a gap existence penalty of 1 1 and a gap extension penalty of 1, will generate:
(i) a percentage identity of at least 67%, 69%, 70%, 71%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%, 83%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2; (ii) a percentage identity of at least 61 %, 62%, 65%, 69%, 70%, 71%, 72%, 74%, 77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81%, 82%, 84%, 85%, 86%, 87%, 89%, 90%, 91 %, 93%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50; (iii) a percentage identity of at least 65%, 67%, 69%, 72%, 73%, 74%, 75%, 79%, 83%, 85%, or 88%, or 100%, or a percentage similarity of at least 79%, 82%, 83%, 86%, 87%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52; (iv) a percentage identity of at least 75%, 77%, 78%, 80%, 82%, or 84%, or 100%, or a percentage similarity of at least 85%, 87%, 89%, 91%, or 92%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 256- 277, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 84;
(v) a percentage identity of at least 74%, 75%, 91%, or 98%, or 100%, or a percentage similarity of at least 91%, 93%, 94%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 278-286, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 128; (vi) a percentage identity of at least 82%, 84%, 58%, 86%, or 100%, or a percentage similarity of at least 93%, 94%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 287-292, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NOs: 146; (vii) a percentage identity of at least 82%, 83%, 85%, 86%, 87%, 88%, or 91%, or 100%, or a percentage similarity of at least 86%, 89%, 90%, 91 %, 93%, 96%, or
100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 293-308, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 160;
(viii) a percentage identity of at least 60%, 68%, 72%, or 100%, or a percentage similarity of at least 68%, 73%, 83%, 91%, or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 309-314, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 181; or (ix) a percentage identity of at least 70%, 71%, 72%, 73%, 75%, 77%, 85%, or 100%, or a percentage similarity of at least 84%, 85%, 87%, 88%, 89%, 92% or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs:
315-327, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 188; and (b) selecting a transgenic plant so produced having a transcriptional regulatory activity of SEQ
ID NO: 2, 50, 52, 84, 128, 146, 160, 181, or 188 by comparing the transgenic plant with a control plant.
10. A method according to statement 9, wherein the transgenic plant has greater resistance to a pathogen as compared to the control plant
1 1. A method according to statement 10, wherein said pathogen is a fungal pathogen.
12. A method according to statement 10, wherein said pathogen is selected from the group consisting of Botrytis, Sclerotinia, Erysiphe, and Fusarium.
13. A method according to any one of statements 9 to 12, wherein said AP2, Myb, HLH, or WRKY family polypeptide comprises a conserved domain at least 60 identical or 68% similar to a sequence selected from the group consisting of SEQ ID NO: 215-327.
14. A method according to statement 9, further comprising the steps of:
(c) crossing said transgenic plant with another plant to produce transgenic seed; and (d) growing a progeny plant from the transgenic seed thereby producing a transgenic progeny plant having increased pathogen resistance as compared to the control plant.
15. A method according to statement 14, wherein the expression levels of the polypeptide in the transgenic progeny plant are greater than in the control plant.
16. A method according to any one of statements 9 to 15, wherein said transgenic plant is selected from the group consisting of maize, rice, barley, tomato, soybean, wheat, tobacco, pepper, lettuce, and potato.
17. A transgenic seed produced by:
(a) a transgenic plant produced by any of the methods of statements 9-13 or 16; or
(b) a transgenic progeny plant produced by any of the methods of statements 14-15.
18. A method for increasing the pathogen resistance of a plant as compared to the pathogen resistance of a control plant, the method steps comprising: (a) introducing into a plant an expression vector comprising a recombinant polynucleotide that encodes an AP2, Myb, HLH, or WRKY family polypeptide comprising a conserved domain; wherein the BLOSUM62 matrix, using a gap existence penalty of 1 1 and a gap extension penalty of 1, will generate: (i) a percentage identity of at least 67%, 69%, 70%, 71%, 73%, 74%, 75%, 76%,
77%, 78%, 80%, 82%, 84%, 90%, or 100%, or a percentage similarity of 79%, 80%, 82%, 83%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 207-238, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 2; (ii) a percentage identity of at least 61 %, 62%, 65%, 69%, 70%, 71%, 72%, 74%,
77%, 80% or 88%, or 100%, or a percentage similarity of at least 80%, 81 %, 82%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 93%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 50; (iii) a percentage identity of at least 65%, 67%, 69%, 72%, 73%, 74%, 75%, 79%,
83%, 85%, or 88%, or 100%, or a percentage similarity of at least 79%, 82%, 83%, 86%, 87%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 239-255, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 52; (iv) a percentage identity of at least 75%, 77%, 78%, 80%, 82%, or 84%, or 100%, or a percentage similarity of at least 85%, 87%, 89%, 91%, or 92%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 256-277, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 84; (v) a percentage identity of at least 74%, 75%, 91%, or 98%, or 100%, or a percentage similarity of at least 91%, 93%, 94%, or 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NOs: 278-286, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 128;
(vi) a percentage identity of at least 82%, 84%, 58%, 86%, or 100%, or a percentage similarity of at least 93%, 94%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 287-292, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NOs: 146; (vii) a percentage identity of at least 82%, 83%, 85%, 86%, 87%, 88%, or 91%, or 100%, or a percentage similarity of at least 86%, 89%, 90%, 91%, 93%, 96%, or 100%, when the polypeptide is optimally aligned to a conserved domain of any of SEQ ID NO: 293-308, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 160;
(viii) a percentage identity of at least 60%, 68%, 72%, or 100%, or a percentage similarity of at least 68%, 73%, 83%, 91%, or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 309-314, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 181; or (ix) a percentage identity of at least 70%, 71%, 72%, 73%, 75%, 77%, 85%, or
100%, or a percentage similarity of at least 84%, 85%, 87%, 88%, 89%, 92% or 100%, when the polypeptide is optimally aligned to a conserved domain of SEQ ID NOs: 315-327, wherein the polypeptide has a transcriptional regulatory activity of SEQ ID NO: 188; and (b) selecting a transgenic plant so produced having greater pathogen resistance by comparing the transgenic plant with the control plant; wherein the transgenic plant has greater resistance to a pathogen or multiple pathogens as compared to the control plant.
19. A method according to statement 18, wherein said AP2, Myb, HLH, or WRKY family polypeptide comprises a conserved domain that is at least 60% identical or at least 68% similar to a polypeptide sequence selected from the group consisting of SEQ IDNO: 215-327.
20. A method according to statement 18, wherein the trangenic plant has greater resistance to a necrotrophic pathogen and/or a biotrophic pathogen.
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CN104630235A (en) * 2015-01-28 2015-05-20 南京农业大学 NAC transcription factor gene TaNACs in wheat as well as expression vector and application thereof
CN109971767A (en) * 2019-04-09 2019-07-05 贵州大学 A kind of sorghum transcription factor SbWRKY45 gene and its recombinant vector and expression
CN110229222A (en) * 2019-05-23 2019-09-13 广西壮族自治区农业科学院 Tomato anti-Meloidogyne incognita related gene and its application
CN110484544A (en) * 2019-08-31 2019-11-22 贵州大学 Tobacco gene LBM1, its screening technique and the application in regulation plant epidermal hair development
CN112813075A (en) * 2021-02-19 2021-05-18 浙江大学 Cabbage mustard BoaWRKY4 gene and application thereof
CN112831505A (en) * 2021-03-16 2021-05-25 昆明理工大学 Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications
CN113121660A (en) * 2019-12-30 2021-07-16 中国农业大学 Application of corn MYB39 protein and coding gene thereof in regulation and control of low-temperature stress tolerance of corn

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630235A (en) * 2015-01-28 2015-05-20 南京农业大学 NAC transcription factor gene TaNACs in wheat as well as expression vector and application thereof
CN109971767A (en) * 2019-04-09 2019-07-05 贵州大学 A kind of sorghum transcription factor SbWRKY45 gene and its recombinant vector and expression
CN110229222A (en) * 2019-05-23 2019-09-13 广西壮族自治区农业科学院 Tomato anti-Meloidogyne incognita related gene and its application
CN110484544A (en) * 2019-08-31 2019-11-22 贵州大学 Tobacco gene LBM1, its screening technique and the application in regulation plant epidermal hair development
CN113121660A (en) * 2019-12-30 2021-07-16 中国农业大学 Application of corn MYB39 protein and coding gene thereof in regulation and control of low-temperature stress tolerance of corn
CN112813075A (en) * 2021-02-19 2021-05-18 浙江大学 Cabbage mustard BoaWRKY4 gene and application thereof
CN112813075B (en) * 2021-02-19 2022-10-11 浙江大学 Cabbage mustard BoaWRKY4 gene and application thereof
CN112831505A (en) * 2021-03-16 2021-05-25 昆明理工大学 Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications
CN112831505B (en) * 2021-03-16 2023-04-11 昆明理工大学 Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application thereof

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