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WO2001004285A2 - Polynucleotides msi du maïs et modalites d'utilisation - Google Patents

Polynucleotides msi du maïs et modalites d'utilisation Download PDF

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Publication number
WO2001004285A2
WO2001004285A2 PCT/US2000/040371 US0040371W WO0104285A2 WO 2001004285 A2 WO2001004285 A2 WO 2001004285A2 US 0040371 W US0040371 W US 0040371W WO 0104285 A2 WO0104285 A2 WO 0104285A2
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nucleotide sequence
sequence
seq
plant
set forth
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PCT/US2000/040371
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WO2001004285A9 (fr
WO2001004285A3 (fr
WO2001004285A8 (fr
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Donald Adelphi Baldwin
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Pioneer Hi-Bred International, Inc.
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Publication of WO2001004285A3 publication Critical patent/WO2001004285A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • HC-toxin cyclic tetrapeptide which is absolutely required for pathogenicity.
  • Resistant maize genotypes produce an HC- toxin reductase encoded by the nuclear Hm locus which abolishes toxin activity by reducing the ketone group.
  • HC-toxin acts in a cytostatic manner; it is not toxic to plant cells and does not determine pathogenicity by simply killing host cells prior to colonization.
  • HC-toxin is a specific inhibitor of histone deacetylase (HD) activity.
  • Histones are proteins which bind DNA to form a complex termed the nucleosome. Nucleosomes structurally organize and compact chromosomal DNA into chromatin. In chromatin, the degree of interaction between histones and DNA varies with transcriptional activity. Chromatin regions containing active promoters often have histones which are hyperacetylated. Hyperacetylated histones are thought to adopt a chromatin structure that allows other proteins to bind promoter DNA and activate transcription. In contrast, hypoacetylated histones are associated with inactive promoters, and deacetylation of histones in normally active chromatin can repress transcription in that region.
  • Histone deacetylase is responsible for removing acetyl modifications from histones.
  • HD may be localized to promoters targeted for repression by other proteins that associate with HD and specifically bind regulatory elements in promoter DNA.
  • HD and other proteins bind retinoblastoma protein (pRb) to help form the multiprotein retinoblastoma complex.
  • pRb retinoblastoma protein
  • When the retinoblastoma complex is localized to its DNA binding site in a regulated promoter, transcription is repressed by a number of mechanisms including chromatin histone deacetylation.
  • the retinoblastoma complex is a key regulator of cell cycle progression through the Gl phase and also in cellular differentiation; loss of retinoblastoma function plays a role in many cancers.
  • histone acetylation plays a key role in fundamental cellular processes such as transcription and cell cycle progression as well as processes such as plant resistance to pathogen invasion. Mechanisms are therefore needed to modulate acetylation in order to control gene activities, cancer, and plant disease resistance.
  • the present invention provides nucleotide and amino acid sequences that find use in modulating development, developmental pathways, and the plant pathogen defense system. Particularly, the nucleotide and amino acid sequence for two maize retinoblastoma-associated-like proteins (Rb-Ap-like or MSI-like) proteins are provided.
  • the methods and compositions of the present invention can be used to modulate development in a host cell.
  • the methods and compositions of the invention can be used to modulate the plant pathogen defense system. More specifically, methods and compositions may be used for enhancing resistance to plant pathogens including fungal pathogens, plant viruses, and the like.
  • the compositions and methods of the invention can also be used to alter metabolic states of host cells.
  • methods are provided to modulate cell division, differentiation, as well as cellular processes controlling or modulating, for example, gene expression, DNA metabolism, DNA replication, DNA repair, recombination, and chromatin structure and function in host cells. Additionally, the methods can be used to promote cell death particularly in an inducible or tissue-preferred manner.
  • the MSI-like proteins of the invention additionally find use in manipulating these processes in any host cell, particularly plant cells.
  • transformed plants, plant cells, and seeds, as well as methods for making such plants, plant cells and seeds are provided.
  • promoters will be useful in the invention, the choice of which will depend in part upon the desired level of expression of the disclosed nucleotide sequences.
  • the levels of expression can be controlled to modulate the disease resistance pathway resulting in levels of immunity in the plant, which impart resistance in the plant to the pathogen or to induce cell death.
  • compositions of the invention comprise two maize MSI-like nucleotide and amino acid sequences. Particularly, the nucleotide and amino acid sequence for two maize MSI-like proteins, ZmMSIa and ZmMSIb, are provided and set forth.
  • the present invention further provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, or the nucleotide sequences encoding the DNA sequences deposited in a bacterial host as Patent Deposit No. PTA-317. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NO:l and SEQ ID NO:3, those deposited as Patent Deposit No. PTA-317, and fragments and variants thereof.
  • Plasmids containing the nucleotide sequences of the invention were deposited in a bacterial host with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, on July 9, 1999, and assigned Patent Deposit No. PTA-317. This deposit will be maintained under the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the MSI-like sequences of the invention are members of a family of molecules having sequence identity to the MSI1 (multicopy suppressor o ⁇ iral) protein of yeast.
  • Yeast MSI-1 influences the Ras-cAMP signaling pathway and therefore plays a role in controlling cellular growth.
  • Other members of the MSI- like family include, for example, Retinoblastoma-associated proteins (Rb-Ap) from a variety of organisms, including both mammals and plants.
  • Rb-Ap Retinoblastoma-associated proteins
  • family when referring to the proteins and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having sufficient amino acid or nucleotide sequence identity as defined herein.
  • the MSI-like sequences of the invention share sequence identity with other members of the MSI-like family.
  • the ZmMSIa sequence of the invention has 76% identity from nucleotide 90 to 1321 to the WD-40 repeat protein (MSI1) from Arabidopsis thaliana (GenBank Accession no. AF016846) and 76% identity from nucleotide 90 to 1327 to the WD-40 repeat protein (LeMSIl) from Lycopersicon esculentum (Accession No. AF016845).
  • ZmMISa also shares sequence identity to the retinoblastoma-binding protein (mRbAp46) from Mus musculus (Accession No. U35124) (i.e. 66% identity from nucleotides 722-1318,
  • nucleotide 304-710 60% identity from nucleotide 304-710, and 68% identity from nucleotides 86 to
  • retinoblastoma binding protein from Rattus norvegicus (Accession no. AF090306) (i.e. 65% identity from nucleotides 722-1318, 59% identity from nucleotides 304-710, and 67% from nucleotides 86-301); and the retinoblastoma- binding protein RbAp46 from Homo sapiens (Accession No. U35143) (i.e. 65% identity from nucleotides 722-1318, 59% identity from nucleotides 304-710, and 66% identity from 86-301).
  • the MSI-like sequence, ZmMSIb, of the present invention shares sequence identity, for example, to the WD-40 repeat protein MSI4 from Arabidopsis thaliana (Accession no. AF028711) (i.e. 78% identity from nucleotides 194-775, 74% identity from nucleotides 797 to 1273, and 75 % identity from nucleotides 1281 -1540).
  • ZmMSIb also shares sequence identity to the WD-40 repeat protein MSI1 from Arabidopsis thaliana (Accession no. AF016846) (i.e. 55% identity from nucleotide 485-631).
  • MSI family members play a role in the conserved retinoblastoma pathway.
  • Retinoblastoma protein is a central part of an evolutionarily conserved regulatory system. Ach et al. ((1997) Plant Cell 9:1595-1606) showed that the maize retinoblastoma protein, RRB 1 , shares significant biochemical similarity with the human retinoblastoma protein pRB, including binding of cell-cycle regulators such as D-cyclins and viral proteins, for example, simian virus SV40 (T-antigen) and adenovirus protein El A. Mutations in conserved regions of the maize protein have similar effects on function with respect to the human protein. Members of the MSI-like family are components of the highly conserved retinoblastoma (Rb) complex.
  • Rb Rb-associated protein, known as MSI in yeast
  • HD histone deacetylase
  • Components of the retinoblastoma complex including the Rb-Ap genes to which the present invention bears sequence identity, have been shown to interact with fungal and viral genes of known plant pathogens.
  • interaction has been demonstrated between histone deacetylase (HD) and the toxin of the maize fungal pathogen Cochliobolus carbonum, which inhibits HD activity.
  • HD histone deacetylase
  • compositions and methods of the present invention are useful in protecting plants and plant cells against fungal pathogens, viruses, nematodes, insects and the like; also, the compositions can be used in formulations so as to fully implement their antimicrobial activities.
  • proteins of the present invention are highly conserved with respect to a retinoblastoma-binding protein in Arabidopsis and tomato. These Arabidopsis and tomato genes have been shown to bind both the human and maize retinoblastoma homologs.
  • the proteins of the present invention are expected to bind retinoblastoma homologs and function to modulate cell cycle, DNA metabolism, and division.
  • the present invention provides utility in such exemplary applications as modification, control, or modulation of various aspects of the cell cycle, cell division, gene expression, DNA metabolism, DNA replication, DNA repair, and recombination as well as cell, organ, or whole plant differentiation and/or disease resistance.
  • Native retinoblastoma proteins in maize have been shown to localize strictly to the nucleus in maize. Sequence comparison of retinoblastoma proteins from Arabidopsis and maize show conserved regions including WD-40 repeats, A and B binding pockets, N- and C-terminal domains, conserved potential phosphorylation sites, and cysteine residues (see Ach et al. (1997) Plant Cell 9: 1595- 1606; Ach et al. (1997) Mol. Cell.
  • the present invention may provide a mechanism for modulating the functions performed by any or all of these conserved motifs, which may perform their roles in the native protein or as fragments spliced together to form a component of a designed protein.
  • conserved motifs which may perform their roles in the native protein or as fragments spliced together to form a component of a designed protein.
  • One skilled in the art will recognize that such proteins may be used alone or in conjunction with other proteins or methods to modulate cellular function.
  • the genes to which the present invention bears homology have been shown to bind to tomato golden mosaic virus AL1 protein, which induces gene transcription in differentiated cells.
  • the present invention provides compositions and methods for modulating the total levels of proteins of the present invention and/or altering their ratios in host cells, particularly plant cells.
  • the methods also comprise modulating the activity of the MSI-like proteins of the invention.
  • the functional or biological activity of the MSI-like proteins refers to an activity exerted by the MSI-like polypeptide or nucleic acid sequence on a host cell. Such activities include, for example, modulation of cell cycle, differentiation, gene expression, DNA metabolism, DNA replication, DNA repair, recombination, and chromatin structure.
  • the invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb. or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (and also referred to as "flanking DNA").
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein.
  • culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals.
  • Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence
  • fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments of a nucleotide sequence may range from at least about 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, or 1,500 nucleotides, or up to the full-length nucleotide sequence encoding the proteins of the invention.
  • a fragment of an MSI-like nucleotide sequence that encodes a biologically active portion of an MSI-like protein of the invention will encode at least 30, 50, 100, 150, 200, 250, 300, 350, or 400 contiguous amino acids, or up to the total number of amino acids present in a full-length MSI-like protein of the invention (for example, 431 total amino acids for SEQ ID NO:2 or 453 total amino acids for SEQ ID NO:4). Fragments of an MSI-like nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of an MSI-like protein.
  • a fragment of an MSI-like nucleotide sequence may encode a biologically active portion of an MSI-like protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of an MSI-like protein can be prepared by isolating a portion of one of the MSI-like nucleotide sequences of the invention, expressing the encoded portion of the MSI-like protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the MSI-like protein.
  • Nucleic acid molecules that are fragments of an MSI-like nucleotide sequence comprise at least 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450. 500, 550, 600. 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400 or 1,500 nucleotides, or up to the number of nucleotides present in a full-length MSI-like nucleotide sequence disclosed herein (for example, 1584 nucleotides for SEQ ID NO:l and 1777 nucleotides for SEQ ID NO:3).
  • MSI-like polypeptides of the invention are naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a MSI-like protein of the invention.
  • variants of a particular nucleotide sequence of the invention will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • variant protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, cell cycle control and DNA metabolism activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native MSI-like protein of the invention will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such
  • amino acid sequence variants of the MSI-like proteins can be prepared by mutations in the DNA.
  • Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA ⁇ 2:488-492; Kunkel et al. (1987) Methods in Enzymol 154:367-382; US Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof.
  • Such variants will continue to possess the desired activity (i.e., modulating cell cycle regulation, modulating gene activity, modulating disease resistance). Assays for these various cellular events are known in the art and are discussed in more detail below.
  • the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
  • deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by assays including suppression of yeast Ras signaling pathway mutations. For example, the IRAl gene product negatively regulates the Ras-cAMP pathway in yeast. Over expression of yeast MSI-1, and other MSI-like family members, suppress the iral mutant phenotype. In addition, the ras2 va 9 yeast mutant, which has a reduced intrinsic GTPase activity, is also suppressed by over expression of several of the MSI-like family
  • the activity of the sequences of the invention may be assayed by transforming iral or ras2 va yeast strains and assaying for suppression of the mutant phenotype. See, for example, Ach et al (1997) The Plant Cell 9:1595- 1606, herein inco ⁇ orated by reference. Further, in light of the role of MSI proteins in chromatin remodeling, assays to detect alteration of chromatin or histones may be used to evaluate activity. Alternatively, assays measuring the modulations in cell cycle regulation and the plant/pathogen defense system may be used to assay for the activity of the MSI-like sequences of the invention.
  • Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different MSI coding sequences can be manipulated to create a new MSI sequence or protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between the MSI gene of the invention and other known retinoblastoma-associated protein genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K m in the case of an enzyme.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751 ; Stemmer (l 994) Nature 370:389-391 ; Crameri e/ ⁇ /. (1997) Nature Biotech. 75:436-438; Moore et al. (1997) J. Mol. Biol.
  • the present invention encompasses the proteins as well as components, fragments, and variants thereof. That is, it is recognized that component polypeptides or fragments of the proteins may be produced which retain biological activity that modifies, modulates, or controls cell processes or development in a plant or plant cell as disclosed herein. These fragments include truncated sequences, as well as N-terminal. C-terminal, internal and internally deleted amino acid sequences of the proteins.
  • nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire MSI-like sequences set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are of the disclosed sequences.
  • orthologs is intended genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences have a high percentage of sequence identity and/or similarity. Functions of orthologs are often highly conserved among species.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant or organism of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press.
  • PCR Methods Manual (Academic Press, New York).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • hybridization can be made by labeling synthetic oligonucleotides based on the sequence of the invention.
  • Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • the entire sequence disclosed herein, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding sequences and messenger RNAs.
  • probes include sequences that are unique among MSI sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Such probes may be used to amplify corresponding MSI-like sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g..
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • sequences that encode an MSI-like protein and which hybridize under stringent conditions to the MSI-like nucleotide sequences disclosed herein, or to fragments thereof are encompassed by the present invention.
  • sequences will be at least about 40% to 50% homologous, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous with the disclosed sequences.
  • sequence identity of sequences may range, sharing at least about 40% to 50%, about 60%, 65%, or 70%, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • additions or deletions i.e., gaps
  • reference sequence which does not comprise additions or deletions
  • 15 comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • a gap penalty is typically introduced and is subtracted from the number of matches.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG). 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al. (1988) Gene 75:237- 244 (1988); Higgins et al.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • BLAST Gapped BLAST
  • PSI-BLAST the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison
  • the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
  • Substantial identity of amino acid sequences for these pu ⁇ oses normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1°C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) J Mol. Biol. -75:443.
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • the nucleotide sequences of the invention are provided in expression cassettes for expression in a host cell of interest, particularly a plant cell.
  • the cassette will include 5' and 3' regulatory sequences operably linked to a sequence of the invention.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the MSI-like sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a nucleotide sequence of the
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the host cell. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • heterologous is intended the transcription initiation region is not native to the coding sequence. While it may be preferable to express the sequences using heterologous promoters, the native promoter sequences may be used.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell (5-7:671-674; Sanfacon et al. (1991) Genes
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant- preferred codons for improved expression. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 77:477-498, herein inco ⁇ orated by reference. Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hai ⁇ in secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al.
  • MDMV leader Mainze Dwarf Mosaic Virus
  • Virology 154:9-20 and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al. (1991) Nature 555:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 525:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New).
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838; the
  • an inducible promoter particularly from a pathogen-inducible promoter.
  • promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta- 1,3- glucanase, chitinase, etc.
  • PR proteins pathogenesis-related proteins
  • SAR proteins beta- 1,3- glucanase
  • chitinase etc.
  • PR proteins pathogenesis-related proteins
  • promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 55:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 95:14972-14977. See also, Chen et al. (1996) Plant J. 70:955-966; Zhang et al. (1994) Proc.
  • a wound-inducible promoter may be used in the constructions of the invention.
  • wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 25:425-449; Duan et al. (1996)
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid.
  • promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc Natl. Acad Sci USA 55:10421-10425 and McNellis et al. (1998) Plant J 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol Gen Genet 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein inco ⁇ orated by reference.
  • Tissue-preferred promoters can be utilized to target enhanced MSI expression within a particular plant tissue.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol 55(7):792-803; Hansen et ⁇ /. (1997) Mol Gen Genet 254(3):337-343; Russell et al. (1997) Transgenic Res 6(2):157-168; Rinehart et al. (1996) Plant Physiol 772 ⁇ :1331-1341 ; Van Camp et al.
  • Leaf-specific promoters include, Yamamoto et al. (1997) Plant J. 720:255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7 ): 92-803; Hansen et al. (1997) Mol. Gen. Genet. 254 (3) :337 -343; Russell et al. (1997) Transgenic Res. (5f2 :157-168; Rinehart et al. (1996) Plant Physiol. 7720:1331-1341; Van Camp et al. (1996) Plant Physiol. 7720:525-535; Canevascini et al. (1996) Plant Physiol 7720:513-524; Yamamoto et al. (1994) Plant Cell Physiol.
  • Root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 200:207-218 (soybean root- specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 740:433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.
  • MAS mannopine synthase
  • seed-specific promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 70:108, herein inco ⁇ orated by reference.
  • seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); and celA (cellulose synthase).
  • Gama-zein is a preferred endosperm-specific promoter.
  • Glob-1 is a preferred embryo-specific promoter.
  • seed-specific promoters include, but are not limited to, bean ⁇ -phaseolin, napin, ⁇ -conglycinin, soybean lectin, cruciferin, and the like.
  • seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g- zein, waxy, shrunken 1, shrunken 2, globulin 1, etc.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 5:506-511 ; Christopherson et al. (1992) Proc. Natl. Acad.
  • nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the invention in a host cell, tissue, or plant.
  • nucleotide sequences, antisense constructions, complementary to at least a portion of the messenger RNA (mRNA) for the MSI- like sequences can be constructed.
  • Antisense nucleotides are constructed to hybridize with the corresponding mRNA.
  • Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, preferably 80%, more preferably 85% sequence identity to the corresponding antisensed sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene.
  • sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.
  • the nucleotide sequences of the present invention may also be used in the sense orientation to suppress the expression of endogenous genes in plants.
  • Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene.
  • a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, preferably greater than about 65% sequence identity, more preferably greater than about 85% sequence identity, most preferably greater than about 95% sequence identity. See, U.S. Patent Nos. 5,283,184 and 5,034,323; herein inco ⁇ orated by reference.
  • sequences of the invention can be used to transform any host cell, particularly plant cells. It is recognized that one skilled in the art is knowledgeable in the numerous methods available for both transient and stable expression of sequences in both prokaryotic and eukaryotic host cells. Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
  • the present invention may be used for transformation of any plant species, including, but not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa). rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica). finger millet (Eleusine), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa). rye (Secal
  • Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Locopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine (P
  • plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), more preferably corn and soybean plants, yet more preferably corn plants.
  • crop plants for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.
  • isolated nucleic acids of the present invention can also be used for recombinant expression of polypeptides, or for use as immunogens in the
  • Attachment of chemical agents, which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.
  • a primer specific to an insertion sequence e.g., transposon
  • a primer which specifically hybridizes to an isolated nucleic acid of the present invention one can use nucleic acid amplification to identity insertion sequence inactivated genes of the invention from a cDNA library prepared from insertion sequence mutagenized plants. Progeny seed from the plants comprising the desired inactivated gene can be grown to a plant to study the phenotypic changes characteristic of that inactivation.
  • non-translated 5' or 3' regions of the polynucleotides of the present invention can be used to modulate turnover of heterologous mRNAs and/or protein synthesis.
  • the codon preference characteristic of the polynucleotides of the present invention can be employed in heterologous sequences, or altered in homologous or heterologous sequences, to modulate translational level and/or rates.
  • sequences of the invention find use in modulating gene activity.
  • sequences of the invention share sequence identity to human Retinoblastoma-associated proteins, RbAp48 and RbAp46.
  • proteins contain WD-40 domains and were first reported as retinoblastoma binding proteins.
  • human RbAp48 and the yeast homolog MSI1 have been found associated with proteins involved in histone metabolism (chromatin assembly factor-I; histone acetyltransferase; histone deacetylase.)
  • chromatin assembly factor-I histone acetyltransferase
  • histone deacetylase histone deacetylase.
  • deletion of MSI 1 (renamed CAC3) reduces silencing of genes adjacent to telomeric DNA.
  • the sequences of the invention can be used to modulate gene activity in host cells, particularly plant cells. By “modulating gene activity” is intended the increase or decrease in the activity state of a gene or gene regions.
  • Molecular events that result in the modulation of gene activity include, for example, histone acetylation, heterochromatin formation, and chromatin assembly.
  • modulating gene activity also encompasses a general means of preparing a gene for transcription.
  • the nucleotide sequences of the invention can act to modulate
  • the constructs of the invention may globally modulate gene activity or alternatively, may target particular regions of the chromosome.
  • RNAase protection such as RNAase protection, Northern Blot, and mRNA profiling are known in the art and may be used to identify either global alterations in gene activity or may be used to identify specific chromosomal loci influenced by the sequences of the invention.
  • Other assays are available for determining activity. See, generally, Lusser et al. (1997) Science 277:88-91; Rundlett et al. (1996) PNAS 95:14503-14508; DeRubertis et al (1996) Nature 554:589-591; Pazin et al. (1997) Cell 59:325-328; herein inco ⁇ orated by reference. See also, Walton et al. (1993) Ann. Rev.
  • sequences of the invention can be used to modulate the cell cycle.
  • Manipulation of the cell cycle can have effects on cellular
  • compositions and methods of the present invention find use in modulating the cell cycle, DNA replication, DNA repair, and DNA recombination in a host cell or organism. While the invention is not bound by any particular mechanism of action, the gene products, probably proteins or polypeptides, function to modulate DNA metabolism. It is recognized that the present invention is not dependent upon a particular mechanism of modulation. Rather, the genes and methods of the invention work to alter the cell cycle, DNA metabolism, DNA replication, DNA repair, and recombination independently of how such modulation is achieved. The mechanisms described herein may be used alone or in combination with other proteins or agents to modulate the processes or end results affected.
  • Assays for monitoring modulations in the cell cycle are known in the art.
  • plant cell suspensions can be synchronized by chemical agents, which arrest the cell cycle by acting on, for example, CDK enzymes or regulators of the cell cycle apparatus. See, for example, Planchaisa et al. (2000) FEBS left 476:78-83 herein inco ⁇ orated by reference.
  • cells synchronized to arrest in mitosis are released from the cell cycle arrest and provided BrdU and an MSI-like amino acid sequence of the invention.
  • the level of BrdU inco ⁇ oration into the cells is reflective of progression into S phase thereby allowing the effect of the MSI-like sequences on the G-S phase transition to be assayed.
  • In vitro growth inhibition or acceleration assays may also be performed on plant cell cultures.
  • cells are incubated with 3 H-thymidine in the presence and absence of the MSI-like sequence of the invention. Cells are harvested and assayed for 3 H- thymidine inco ⁇ oration as described in Proc. Natl. Acad. Sci. USA (1986) 55:4749-4753.
  • In vivo assays to measure cell proliferation rates in plants by confocal microscopy are provided in Lauf et al. (1998) Plant Cell 70:1375-1389, herein inco ⁇ orated by reference.
  • sequences of the invention modulate cell cycle and DNA metabolism they may find use in transformation and culture protocols.
  • the sequences may work to increase transformation efficiency, particularly in plants recalcitrant to transformation and promote the establishment of plant cultures, particularly in plants which are difficult to culture.
  • regulating proteins may bind to a variety of other proteins to achieve their regulatory effect, and that the regulatory effect of a protein may depend on more than one other protein or complex or mode of action.
  • one promoter influenced by the Rb complex controls expression of E2F, a gene required for cell cycling through Gl into S phase.
  • the E2F gene is expressed when cyclin-dependent kinase phosphorylates Rb, disrupting the complex and dissociating RbAp's and histone deacetylase (HD).
  • HD has no inherent ability to bind to DNA and must be directed to a targeted promoter by co-regulatory factors. HD is known to interact directly with both Rb and RbAP.
  • E2F and Rb have potential use in therapy of human diseases resulting from inappropriate cell proliferation, such as cancer.
  • U.S. Patent No. 5,885,833, Nucleic acid constructs for the cell-cycle-regulated expression of genes and therapeutic methods utilizing such constructs"
  • U.S. Patent No. 5,851,991 "Therapeutic use of the retinoblastoma susceptibility gene product.”
  • the sequences of the invention may have potential use in such therapies by modulating the effects of E2F and Rb.
  • conserved components of this regulatory complex have also been found in plants, including homologs of plant D-type cyclins. It is understood by those skilled in the art that components of conserved regulatory complexes may differ slightly in function across organisms, and that such variations may be useful in achieving desired effects.
  • altered expression of the genes or proteins of the present invention may be used to alter flower development. Affecting RbAp48/MSIl expression by co-suppression severely disrupts normal inflorescence development, causing flowers to progressively lose their normal mo ⁇ hology.
  • different effects may be achieved with different expression levels of the present invention.
  • a maize line containing a Mutator (Mu) transposable-element-tagged allele has been identified but has no obvious developmental phenotype. It is possible, and highly likely, that the lack of obvious phenotypes in the msil-l ::Mul/msil-2::Mul homozygotes is a consequence of the functional redundancy within the MSI gene family in maize; therefore, a Mutator (Mu) transposable-element-tagged allele has been identified but has no obvious developmental phenotype. It is possible, and highly likely, that the lack of obvious phenotypes in the msil-l ::Mul/msil-2::
  • 33 phenotype may result if mutant alleles of the other MSI family members are isolated and crossed into the msil-1 or msil-2 mutant background.
  • Dominant negatives of the sequences may also be used. Deletion or point mutations could be expressed to produce altered or truncated proteins that retain the ability to interact with retinoblastoma but disrupt the normal function of the complex. For example, expression of the Rb binding domain may prevent recruitment of histone deacetylase by out-competing endogenous MSI with a peptide that does not intereact with HD. In this manner, the Rb complex is specifically targeted without affecting HD function in other complexes, such as, for example, when HC-toxin is used.
  • phenotypes of maize MSI mutants might include embryo lethality, stunted growth as the result of reduced MSI or other gene dosage, or abnormal cellular differentiation and zones of cell proliferation.
  • Assays to measure cell proliferation rates within zones of the plant using confocal sectioning are described in Laufs et al. (1998) Plant Cell 70:1375-1389.
  • the present invention improves the overall frequency and/or homologous recombination frequency of genetic transformation events.
  • the invention does not depend for its effect on a particular mechanism of increasing the overall frequency or homologous recombination frequency.
  • DNA metabolism and/or chromatin state is interrelated with both overall gene expression and frequency of recombination and/or genetic transformation. See, for example: Akhmedov et al. (1998) J. Biol. Chem. 273: 24088-24094 (showing that structural-maintenance-of-chromosomes proteins interact with DNA in chromosome condensation, DNA recombination, and gene dosage compensation); Singh et al. (1998) Mol. Cell Biol. 18: 5511-5522 (proposing that effects on gene silencing are indirect consequences of changes in
  • compositions and methods of the invention find use in controlling pathogenic agents.
  • the anti-pathogenic compositions comprise polynucleotides and proteins, particularly, the maize MSI-like nucleotide and amino acid of the present invention and fragments and variants thereof. Accordingly, the compositions and methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.
  • disease resistance is intended that the plants avoid the disease symptoms which are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened.
  • the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.
  • anti-pathogenic compositions is intended that the compositions of the invention are capable of suppressing, controlling, and/or killing the invading pathogenic organism.
  • the HC toxin of the maize fungal pathogen Cochliobolus carbonum has been found to be a potent inhibitor of histone deacetylase (HD) activity.
  • the HC- toxin of the maize pathogen C. carbonum and related cyclic tetrapeptides inhibit HDs and cause hyperacetylation of histones in susceptible, but not in resistant, maize strains.
  • the inhibition of histone deacetylation interferes with the induction of plant defense genes mediated by RNA polymerase II transcription.
  • inhibition of deacetylation by HC-toxin may lead to a rather general inhibition of host rRNA transcription, owing to inhibition of nucleolar HD2.
  • the sequences of the present invention provides methods for modulation of HD proteins and may create new possibilities for engineering fungal pathogen resistance. It is not known whether the ability of HC-toxin to overcome the maize defense response is due to prevention of defense gene expression or more general toxicity such as disruption of cell cycle control.
  • the MSI-like sequences of the present invention additionally comprises a new tool for controlling replication of agriculturally important DNA viruses. If the model for Retinoblastoma function in mammalian cells is applicable to plants, we can expect that the Retinoblastoma pathway found in maize and other plants has a central role in plant cell division and differentiation as well, and that it is the target for inactivation during replication of plant DNA viruses. See for example, Gutierrez et al. (2000) EMBO Journal 79:792-799, herein inco ⁇ orated by reference.
  • the methods involve stably transforming a plant with a DNA construct comprising an anti-pathogenic nucleotide sequence of the invention operably linked to promoter that drives expression in a plant.
  • Such methods find use in agriculture particularly in limiting the impact of plant pathogens on crop plants. While the choice of promoter will depend on the desired timing and location of expression of the anti-pathogenic nucleotide sequences, preferred promoters include constitutive and pathogen-inducible promoters.
  • the constructs of the invention can be used to selectively kill target cells or tissues. This can be accomplished through the use of inducible or tissue-preferred promoters. In this manner, the sequences of the invention may find use in enhancing pathogen resistance.
  • an antisense construct for the MSI coding sequence is operably linked to a pathogen inducible promoter. Upon contact with the pathogen, the MSI antisense construct is expressed resulting in disruption of gene expression leading to cell death and effectively preventing the invasion of the pathogen.
  • the compositions can be used in formulation use for their antimicrobial activities.
  • the proteins of the invention can be formulated with an acceptable carrier into a pesticidal composition(s) that is for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a
  • the MSI-like sequences of the invention can also be used to control resistance to pathogens by enhancing the defense mechanisms in a plant. While the exact function of the MSI-like sequences is not known, they are involved in influencing the expression of defense-related proteins. It is recognized that the present invention is not premised upon any particular mechanism of action of the MSI-like sequences. It is sufficient for pu ⁇ oses of the invention that the genes and proteins are involved in the plant defense system and can be used to increase resistance levels in the plant to pathogens. Assays to determine a modulation in the plant pathogen defense system in response to various pathogens are known in the art. For example, resistance to C. carbonum is characterized by the induction of pathogen defense genes and the appearance of only non-expanding lesions. Alternatively, sensitivity to C. carbonum is easily assayed by measuring lesion expansion.
  • plant defense mechanisms described herein may be used alone or in combination with other methods, proteins or agents to protect against plant diseases and pathogens.
  • Other plant defense proteins include those described in copending applications entitled “Methods for Enhancing Disease Resistance in Plants ', U.S. Application Serial No. 09/256,898, filed February 24, 1999, and copending application entitled “Genes for Activation of Plant Pathogen Defense Systems", U.S. Application Serial No. 09/256,158, filed February 24, 1999, all of which are herein inco ⁇ orated by reference.
  • any one of a variety of second nucleotide sequences may be utilized, some embodiments of the invention encompass those second nucleotide sequences that, when expressed in a plant, help to increase the resistance of a plant to pathogens.
  • compositions and methods for inducing resistance in a plant to plant pests are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.
  • Pathogens of the invention include, but are not limited to,
  • viruses or viroids include any plant virus, for example, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
  • Specific fungal and viral pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp.
  • syringae Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondita fsp.
  • nebraskense Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv.
  • zea Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghin
  • Nematodes include parasitic nematodes such as root-knot, cyst, lesion, and renniform nematodes, etc.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
  • Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European com borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, com earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern com borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western com rootworm; Diabrotica longicornis barberi, northern com rootworm; Diabrotica undecimpunctata howardi, southern com rootworm; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaeto
  • thief ant Tetranychus urticae, twospotted spider mite
  • Sorghum Chilo partellus, sorghum borer
  • Spodoptera frugiperda fall armyworm
  • Helicoverpa zea com
  • Mayetiola destructor Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipac wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle;
  • the present invention also provides a method of genotyping a plant using a polynucleotide of the present invention.
  • the plant is a monocot, such as maize or sorghum.
  • Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g.. Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997).
  • RFLPs restriction fragment length polymo ⁇ hisms
  • RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP.
  • the present invention further provides a means to follow segregation of an MSI-like gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.
  • Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of an MSI gene.
  • the nucleotide sequences for MSI- like can be utilized to produce the enzyme with greater purity.
  • Such enzyme preparations can be utilized for assays of enzymatic activity as well as to produce anti-MSI-like antibodies which may have pharmaceutical or therapeutic use in treatment of cell proliferation diseases such as cancer.
  • Mechanisms for antibody production are known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York) and the references cited therein.
  • Such antibodies are useful to immunoprecipitate MSI-like from cell extracts and isolate members of regulatory co-factor complexes associated with MSI and Rb in vivo.
  • Antibodies can be raised to a protein of the present invention, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill; however, one of skill will recognize that many variations upon such methods are known.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. Monoclonals antibodies are screened for binding to a protein from which the immunogen was derived. Specific monoclonal and polyclonal antibodies will usually have an antibody binding site with an affinity constant for its cognate monovalent antigen at least between 10 6 -10 7 , usually at least 10 8 ,
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing ZmMSIa or ZmMSIb sequence operably linked to a ubiquitin promoter plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows. Media recipes follow below.
  • Target Tissue The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5- cm target zone in preparation for bombardment.
  • a plasmid vector comprising the ZmMSIb or ZmMSIa nucleotide sequence operably linked to a ubiquitin promoter is made.
  • This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCl precipitation procedure as follows:
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity.
  • Plants are monitored and scored for the relevant trait affected by the present invention.
  • the plants are inoculated with C. carbonum. Modulation of the plant pathogen response can be assayed by measuring lesion expansion. When the pathogen defense genes are induced, non-expanding lesions are produced, while sensitivity to C. carbonum is characterized by lesion expansion.
  • standard gene expression assays i.e., RNase protection, Northern blot, and RNA profiling
  • Example 2 Agrobacterium-Mediated Transformation of Plants For Agrob ⁇ cterium-mediated transformation of maize with an MSI-like sequence sequence, preferably the method of Zhao is employed (U.S. Patent No. 5,981.840, and PCT patent publication WO98/32326; the contents of which are hereby inco ⁇ orated by reference).
  • immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the MSI-like sequences to at least one cell of at least one of the immature embryos (step 1 : the infection step).
  • the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation.
  • the embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step).
  • the immature embryos are cultured on solid medium following the infection step.
  • an optional "resting" step is contemplated.
  • the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step).
  • the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells.
  • inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step).
  • the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells.
  • the callus is then regenerated into plants (step 5: the regeneration step), and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.
  • Example 3 Soybean Embryo Transformation Prophetic Example Soybean embryos are bombarded with a plasmid containing the MSI-like nucleotide sequence operably linked to a ubiquitin promoter as follows. To induce somatic embryos, cotyledons. 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at
  • Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
  • Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker, 150 rp , at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium. Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 527:70- 73, U.S. Patent No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
  • a selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Vims (Odell et al. (1985) Nature 575:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T- DNA of the Ti plasmid of Agrobacterium tumefaciens.
  • the expression cassette comprising the MSI gene operably linked to the ubiquitin promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
  • Membrane mpture pressure is set at 1 100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
  • the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly.
  • Green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
  • Sunflower meristem tissues are transformed with an expression cassette containing the MSI-like sequence operably linked to ubiquitin promoter as follows (see also European Patent Number EP 0 486233, herein inco ⁇ orated by reference, and Malone-Schoneberg et al. (1994) Plant Science 103:199-207).
  • Mature sunflower seed (Helianthus annuus L.) are dehulled using a single wheat-head thresher. Seeds are surface sterilized for 30 minutes in a 20% Chlorox bleach solution with the addition of two drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice with sterile distilled water.
  • Split embryonic axis explants are prepared by a modification of procedures described by Schrammeijer et al. (Schrammeijer et ⁇ /.(1990) Plant Cell Rep. 9:55- 60). Seeds are imbibed in distilled water for 60 minutes following the surface sterilization procedure. The cotyledons of each seed are then broken off, producing a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed, cut surface up, on GBA medium consisting of
  • the explants are subjected to microprojectile bombardment prior to Agrobacterium treatment (Bidney et al. (1992) Plant Mol Biol. 75:301-313). Thirty to forty explants are placed in a circle at the center of a 60 X 20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HC1, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.
  • a binary plasmid vector comprising the expression cassette that contains the MSI-like operably linked to the ubiquitin promoter is introduced into Agrobacterium strain EHA105 via freeze-thawing as described by Holsters et al. (1978) Mol. Gen. Genet. 765:181-187.
  • This plasmid further comprises a kanamycin selectable marker gene (i.e, nptll).
  • Bacteria for plant transformation experiments are grown overnight (28°C and 100 RPM continuous agitation) in liquid YEP medium (10 gm/1 yeast extract, 10 gm/1 Bactopeptone.
  • the suspension is used when it reaches an OD 6 oo of about 0.4 to 0.8.
  • the Agrobacterium cells are pelleted and resuspended at a final OD 6 oo of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/1 NH 4 C1, and 0.3 gm/1 MgSO 4 .
  • Freshly bombarded explants are placed in an Agrobacterium suspension, mixed, and left undisturbed for 30 minutes. The explants are then transferred to GBA medium and co-cultivated, cut surface down, at 26°C and 18-hour days. After three days of co-cultivation, the explants are transferred to 374B (GBA medium lacking growth regulators and a reduced sucrose level of 1 %) supplemented with 250 mg/1 cefotaxime and 50 mg/1 kanamycin sulfate.
  • explants are cultured for two to five weeks on selection and then transferred to fresh 374B medium lacking kanamycin for one to two weeks of continued development.
  • Explants with differentiating, antibiotic-resistant areas of growth that have not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/1 cefotaxime for a second 3 -day phytohormone treatment.
  • Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of NPTII by ELISA and for the presence of transgene expression by assaying for MSI protein activity as described in Lusser et al. (1997) Science 277:88-91; Rundlett et al. (1996) PNAS 95:14503-14508; DeRubertis et al. (1996) Nature 384:589-591 ; and Pazin et al. (1997) Cell 59:325-328.
  • NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in v/ ' tro-grown sunflower seedling rootstock.
  • Surface sterilized seeds are germinated in 48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and grown under conditions described for explant culture. The upper portion of the seedling is removed, a 1 cm vertical slice is made in the hypocotyl, and the transformed shoot inserted into the cut. The entire area is wrapped with parafilm to secure the shoot.
  • Grafted plants can be transferred to soil following one week of in vitro culture. Grafts in soil are maintained under high humidity conditions followed by a slow acclimatization to the greenhouse environment.
  • Transformed sectors of T 0 plants (parental generation) maturing in the greenhouse are identified by NPTII ELISA and/or by analysis of MSI-like protein activity in leaf extracts while transgenic seeds harvested from NPTII-positive T 0 plants are identified by analysis of MSI protein activity in small portions of dry seed cotyledon.
  • An alternative sunflower transformation protocol allows the recovery of transgenic progeny without the use of chemical selection pressure. Seeds are dehulled and surface-sterilized for 20 minutes in a 20% Chlorox bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, then rinsed three times with distilled water. Sterilized seeds are imbibed in the dark at 26°C for 20 hours on filter paper moistened with water.
  • the cotyledons and root radical are removed, and the meristem explants are cultured on 374E (GBA medium consisting of MS salts, Shepard vitamins, 40 mg/1 adenine sulfate, 3% sucrose, 0.5 mg/1 6-BAP, 0.25 mg/1 IAA, 0.1 mg/1 GA, and 0.8% Phytagar at pH 5.6) for 24 hours under the dark.
  • the primary leaves are removed to expose the
  • apical meristem around 40 explants are placed with the apical dome facing upward in a 2 cm circle in the center of 374M (GBA medium with 1.2% Phytagar), and then cultured on the medium for 24 hours in the dark.
  • tungsten particles are resuspended in 150 ⁇ l absolute ethanol. After sonication, 8 ⁇ l of it is dropped on the center of the surface of macrocarrier. Each plate is bombarded twice with 650 psi rupture discs in the first shelf at 26 mm of Hg helium gun vacuum.
  • the plasmid of interest is introduced into Agrobacterium tumefaciens strain EHA105 via freeze thawing as described previously.
  • the pellet of overnight- grown bacteria at 28°C in a liquid YEP medium (10 g/1 yeast extract, 10 g/1 Bactopeptone, and 5 g/1 NaCl, pH 7.0) in the presence of 50 ⁇ g/1 kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM 2-(N-mo ⁇ holino) ethanesulfonic acid, MES, 1 g/1 NH 4 C1 and 0.3 g/1 MgSO 4 at pH 5.7) to reach a final concentration of 4.0 at OD 600.
  • Particle-bombarded explants are transferred to GBA medium (374E), and a droplet of bacteria suspension is placed directly onto the top of the meristem.
  • the explants are co-cultivated on the medium for 4 days, after which the explants are transferred to 374C medium (GBA with 1% sucrose and no BAP, IAA, GA3 and supplemented with 250 ⁇ g/ml cefotaxime).
  • the plantlets are cultured on the medium for about two weeks under 16-hour day and 26°C incubation conditions.
  • Explants (around 2 cm long) from two weeks of culture in 374C medium are screened for MSI protein activity using assays known in the art such as those described in Lusser et al. (1997) Science 277:88-91 ; Rundlett et al. (1996) PNAS 95:14503-14508; DeRubertis et al. (1996) Nature 554:589-591 ; Pazin et al. (1997) Cell 59:325-328. After positive (i.e., for MSI protein expression) explants are identified, those shoots that fail to exhibit MSI protein activity are discarded, and every positive explant is subdivided into nodal explants. One nodal explant contains at least one potential node.
  • the nodal segments are cultured on GBA medium for three to four days to promote the formation of auxiliary buds from each node. Then they are transferred to 374C medium and allowed to develop for an additional four weeks. Developing buds are separated and cultured for an additional four weeks on 374C medium. Pooled leaf samples from each newly recovered shoot are screened again by the appropriate protein activity assay.
  • the positive shoots recovered from a single node will generally have been enriched in the transgenic sector detected in the initial assay prior to nodal culture.
  • Recovered shoots positive for MSI protein expression are grafted to Pioneer hybrid 6440 in vitro-grown sunflower seedling rootstock.
  • the rootstocks are prepared in the following manner. Seeds are dehulled and surface-sterilized for 20 minutes in a 20% Chlorox bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, and are rinsed three times with distilled water. The sterilized seeds are germinated on the filter moistened with water for three days, then they are transferred into 48 medium (half-strength MS salt, 0.5% sucrose, 0.3% gelrite pH 5.0) and grown at 26°C under the dark for three days, then incubated at 16-hour-day culture conditions.
  • the upper portion of selected seedling is removed, a vertical slice is made in each hypocotyl, and a transformed shoot is inserted into a V-cut.
  • the cut area is wrapped with parafilm. After one week of culture on the medium, grafted plants are transferred to soil. In the first two weeks, they are maintained under high humidity conditions to acclimatize to a greenhouse environment.
  • Bombardment medium (560 Y) comprises 4.0 g/1 N6 basal salts (SIGMA C- 1416), 1.0 ml/1 Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/1 thiamine HC1, 120.0 g/1 sucrose, 1.0 mg/1 2,4-D, and 2.88 g/1 L-proline (brought to volume with D-I H 2 0 following adjustment to pH 5.8 with KOH); 2.0 g/1 Gelrite (added after bringing to volume with D-I H 2 0); and 8.5 mg/1 silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Selection medium (560R) comprises 4.0 g/1 N6 basal salts (SIGMA C-1416), 1.0 ml/1 Eriksson's
  • Vitamin Mix 1000X SIGMA-1511
  • 0.5 mg/1 thiamine HC1, 30.0 g/1 sucrose, and 2.0 mg/1 2,4-D brought to volume with D-I H 0 following adjustment to pH 5.8 with KOH
  • 3.0 g/1 Gelrite added after bringing to volume with D-I H 2 0
  • Plant regeneration medium (288J) comprises 4.3 g/1 MS salts (GIBCO 11117-074), 5.0 ml/1 MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/1 thiamine HCL, 0.10 g/1 pyridoxine HCL, and 0.40 g/1 glycine brought to volume
  • Hormone-free medium comprises 4.3 g/1 MS salts (GIBCO 1 11 17- 074), 5.0 ml/1 MS vitamins stock solution (0.100 g/1 nicotinic acid, 0.02 g/1 thiamine HCL, 0.10 g/1 pyridoxine HCL, and 0.40 g/1 glycine brought to volume with polished D-I H 2 0), 0.1 g/1 myo-inositol, and 40.0 g/1 sucrose (brought to volume with polished D-I H 0 after adjusting pH to 5.6); and 6 g/1 bacto-agar (added after bringing to volume with polished D-I H 0), sterilized and cooled to 60°C.

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  • Medicinal Chemistry (AREA)
  • Botany (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des procédés et des compositions permettant de moduler le cycle, le métabolisme de l'ADN et la défense contre les pathogènes de cellules végétales. L'invention concerne plus particulièrement des séquences de nucléotides codant les protéines de liaison des rétinoblastomes du maïs. Utilisées en cassettes d'expression, ces séquences permettent de moduler la réplication, la réparation et la recombinaison de l'ADN, ainsi que la défense contre les pathogènes. L'invention concerne également des plantes, des cellules et tissus végétaux ainsi que des semences transformées.
PCT/US2000/040371 1999-07-13 2000-07-13 Polynucleotides msi du maïs et modalites d'utilisation WO2001004285A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71347/00A AU7134700A (en) 1999-07-13 2000-07-13 Maize msi polynucleotides and methods of use

Applications Claiming Priority (2)

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US14343799P 1999-07-13 1999-07-13
US60/143,437 1999-07-13

Publications (4)

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WO2001004285A2 true WO2001004285A2 (fr) 2001-01-18
WO2001004285A3 WO2001004285A3 (fr) 2001-07-26
WO2001004285A9 WO2001004285A9 (fr) 2002-08-08
WO2001004285A8 WO2001004285A8 (fr) 2003-10-23

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016775A3 (fr) * 2002-08-14 2004-05-06 Cropdesign Nv Vegetaux a croissance modifiee et leur methode d'elaboration
WO2004085644A3 (fr) * 2003-03-26 2005-01-13 Basf Plant Science Gmbh Methode de production d'organismes recombinants
WO2009014462A1 (fr) * 2007-07-26 2009-01-29 Sathish Puthigae Procédés et polynucléotides pour améliorer les plantes
US7647186B2 (en) 2004-12-07 2010-01-12 Illumina, Inc. Oligonucleotide ordering system
US8669108B2 (en) 2008-04-03 2014-03-11 Vialactia Biosciences (Nz) Limited Gene expression control in plants
US8901376B2 (en) 2008-12-01 2014-12-02 Vialactia Biosciences (Nz) Limited Methods and compositions for the improvement of plant tolerance to environmental stresses
US8921538B2 (en) 2009-04-01 2014-12-30 Vialactia Biosciences (Nz) Limited Control of gene expression in plants
US9051578B2 (en) 2008-05-28 2015-06-09 Insight Genomics Limited Methods and compositions for plant improvement

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ACH ROBERT A ET AL: "A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals." PLANT CELL, vol. 9, no. 9, 1997, pages 1595-1606, XP000973437 ISSN: 1040-4651 -& DATABASE EMBL [Online] Accession Number AF016846, 20 September 1997 (1997-09-20) ACH RA ET AL.: "Arabidopsis thaliana WD-40 repeat protein (MSI1) mRNA" XP002158333 -& DATABASE EMBL [Online] Accession Number AF016845, 20 September 1997 (1997-09-20) ACH RA ET AL.: "Lycopersicon esculentum WD-40 (LeMSI1) repeat protein mRNA" XP002158334 *
ACH ROBERT A ET AL: "RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein." MOLECULAR AND CELLULAR BIOLOGY, vol. 17, no. 9, 1997, pages 5077-5086, XP000973560 ISSN: 0270-7306 *
KENZIOR ALEXANDER L ET AL: "AtMSI4 and RbAp48 WD-40 repeat proteins bind metal ions." FEBS LETTERS, vol. 440, no. 3, 4 December 1998 (1998-12-04), pages 425-429, XP000941882 ISSN: 0014-5793 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016775A3 (fr) * 2002-08-14 2004-05-06 Cropdesign Nv Vegetaux a croissance modifiee et leur methode d'elaboration
WO2004085644A3 (fr) * 2003-03-26 2005-01-13 Basf Plant Science Gmbh Methode de production d'organismes recombinants
US7647186B2 (en) 2004-12-07 2010-01-12 Illumina, Inc. Oligonucleotide ordering system
US8073666B2 (en) 2004-12-07 2011-12-06 Illumina, Inc. Systems and methods for ordering oligonucleotides
WO2009014462A1 (fr) * 2007-07-26 2009-01-29 Sathish Puthigae Procédés et polynucléotides pour améliorer les plantes
US20100242134A1 (en) * 2007-07-26 2010-09-23 Sathish Puthigae Methods and polynucleotides for improving plants
US8669108B2 (en) 2008-04-03 2014-03-11 Vialactia Biosciences (Nz) Limited Gene expression control in plants
US9051578B2 (en) 2008-05-28 2015-06-09 Insight Genomics Limited Methods and compositions for plant improvement
US8901376B2 (en) 2008-12-01 2014-12-02 Vialactia Biosciences (Nz) Limited Methods and compositions for the improvement of plant tolerance to environmental stresses
US8921538B2 (en) 2009-04-01 2014-12-30 Vialactia Biosciences (Nz) Limited Control of gene expression in plants

Also Published As

Publication number Publication date
WO2001004285A9 (fr) 2002-08-08
WO2001004285A3 (fr) 2001-07-26
WO2001004285A8 (fr) 2003-10-23
AU7134700A (en) 2001-01-30

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