US20120278929A1 - miRNA396 AND GROWTH REGULATING FACTORS FOR CYST NEMATODE TOLERANCE IN PLANTS - Google Patents

miRNA396 AND GROWTH REGULATING FACTORS FOR CYST NEMATODE TOLERANCE IN PLANTS Download PDF

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US20120278929A1
US20120278929A1 US13/457,775 US201213457775A US2012278929A1 US 20120278929 A1 US20120278929 A1 US 20120278929A1 US 201213457775 A US201213457775 A US 201213457775A US 2012278929 A1 US2012278929 A1 US 2012278929A1
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plant
expression
grf
mirna396
genes
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Thomas J. Baum
Tarek Abdel Fattah Hewezi
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Iowa State University Research Foundation ISURF
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    • 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
    • C12N15/8285—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 nematode 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates generally to the field of plant molecular biology.
  • Nematodes are a very large group of invertebrate animals generally referred to as roundworms, threadworms, eelworms, or nemas. Some nematodes are plant parasites and can feed on stems, buds, leaves, and in particular on roots. Cyst nematodes (principally Heterodera and Globodera spp.) are key pests of major crops. Cyst nematodes are known to infect tobacco, cereals, sugar beets, potato, rice, corn, soybeans and many other crops. Heterodera schachtii principally attacks sugar beets, and Heterodera avenae has cereals as hosts.
  • the soybean cyst nematode Heterodera glycines ) infests every soybean-producing state in the U.S., with total soybean yield loss estimates approaching $1 billion per year.
  • Plant-parasitic nematodes change shape as they go through their life cycle. In its juvenile form, the animals penetrate plant roots. The number of juveniles entering the plant root soon after plant emergence can have a dramatic effect on plant growth and development. Plant damage occurs from juvenile feeding which removes cell materials and disrupts the vascular tissue by inducing the formation of novel plant cell types that are associated in a unique feeding organ, the syncytium. Due to the sedentary nature of their parasitism, cyst nematodes need to obtain all their nourishment from one location, in fact, through the contact with the initial feeding cell.
  • Cyst nematodes infect as second-stage juveniles (J2), which initiate the induction/formation of the syncytium. During this phase, J2s begin feeding on the growing syncytium and then develop into third-stage (J3) and fourth-stage juveniles (J4) followed by the adult stage.
  • J2s begin feeding on the growing syncytium and then develop into third-stage (J3) and fourth-stage juveniles (J4) followed by the adult stage.
  • Syncytium formation encompasses reprogramming of differentiated plant root cells, and these redifferentiations are accompanied and mediated by massive gene expression changes, which have been documented in diverse research approaches using soybean and the soybean cyst nematode Heterodera glycines (Alkharouf et al., 2006; Ithal et al., 2007; Klink et al., 2009) and probably most extensively in Arabidopsis infected by the sugar beet cyst nematode H. schachtii (Szakasits et al., 2009). Regulatory networks governing gene expression patterns in nematode-infected roots and particularly in the developing syncytium are very poorly understood.
  • Crop rotation has also been used to control nematode disease. Rotating non-host plants can be effective in controlling nematode disease. Unfortunately, these non-host crops are often less valuable. Cover crops grown between the main crops is another alternative management strategy. Ryegrain, barley, oats, sudangrass, tall fescue, and annual ryegrass have been shown to be non- or poor hosts for some nematodes. Using cover crops, however, can be costly because the cover crops occupy space that could be used to grow more valuable crops.
  • Biological control organisms have also been used to try to control nematode disease in crops. Commercially available preparations of biological control organisms are limited in their use to regions that can support the growth of the control organism. Moreover, the outcome of using one organism to control another is unpredictable and subject to a variety of factors such as weather and climate.
  • the present invention includes methods to alter the genetic composition of crop plants, particularly those that are susceptible to nematode infection, thereby improving tolerance to nematode infection and reducing the effects thereof in plants.
  • This invention provides methods and compositions for modulating key pathways involved in the syncytial event of nematode infection and for preventing the cascade of differential gene expression caused by the same.
  • Applicants have found that the microRNA miR396 acts as a master switch of syncytial gene expression changes in plants after infection, and further that miR396 and growth regulating transcription factors (GRF) with miRNA396 binding sites are connected through a negative feedback loop to establish an irreversible plant gene regulatory switch from syncytium initiation and maintenance.
  • GRF growth regulating transcription factors
  • This invention in one embodiment relates to modulation of expression of miRNA396 and GRFs with miRNA396 binding sites to engineer improved tolerance to cyst nematode infection in plants as well as the hinder the development and maintenance of the syncytium, essential for plant pathogen survival.
  • miR396 and GRF1/GRF3 are connected through a negative feedback loop from a low miR396 high GRF1/3 state during syncytium initiation, to high miR396 low GFR1/3 during maintenance. Modulated expression of this interaction alters the outcome of the plant pathogen interaction and alters plant susceptibility. In particular, overexpression of miRNA396 reduces plant susceptibility to nematode infection by more than half. Other methods of interfering with this miRNA396 and GRF interaction would also be included within the scope of this invention, whether by increasing activity of the same, through such mechanisms as overexpression, inhibition of activity, such as through inhibition of translation or transcription, or introduction of heterologous interfering or competing proteins.
  • the invention contemplates the regulation of miRNA396 and the pathway of regulatory transcription factors associated with the same to engineer tolerance to nematode infection in plants, preferably by modulation of miRNA sequences or activity in plants.
  • miRNA396 or “miR396” shall be interpreted to include genes such as miR396a ( Arabidopsis ATG10606, Glycine max MI0001785, MIMAT0001687); miR396b ( Arabidopsis AT5G35407, Glycine max MI0001786, MIMAT0001688); miR396c ( Glycine max MI0010572, MIMAT0010079); and miR396e ( Glycine max MI0016586, MIMAT0018345) which regulate expression of growth regulating transcription factor genes that have an miR396-binding site such as GRF 1 through 4 and 7 through 9 in Arabidopsis , See Jones-Rhoades and Bartel, 2004, “Computational identification of plant microRNAs and their targets, including a stress-induced miRNA” Mol.
  • miR396a Arabidopsis ATG10606, Glycine max MI0001785, MIMAT0001687
  • miR396b Arabidopsis AT5G3540
  • Soybean GRFs include GRF8, 9, 12, 13, 15, 16, and 19, Mi396 is a highly conserved micro RNA as many are, and has been found in many other nematode susceptible plants including Citrus unshiu, Glycine max (soybean), Lactuca sativa (lettuce), Lotus japonicus, Medicago truncatula, Nicotiana benthaminiana (tobacco), Oryza sativa (rice), and Populus euphratica . See, Zhang et al., “Conservation and Divergence of Plant MicroRNA Genes” The Plant Journal (2006) 46 243-259. Additionally, other miRNA396 homologs may be identified thought databases such as Genbank, and the mircoRNA database, at world wide web mirbase.org.
  • At 2g22840 AtGRF1 transcription activator (GRF1) At 2g22840 AtGRF1 transcription activator (GRF1), At2g36400 AtGRF3 transcription activator (GRF3), At3g52910 AtGRF4 expressed protein, growth-regulating factor, At3g13960 AtGRF5 transcription activator (GRF5), At2g06200 AtGRF6 expressed protein, At5g53660 AtGRF7 hypothetical protein At4g24150 AtGRF8 hypothetical protein.
  • GmGRF8 (Glyma10g07790); GRF9 (XM — 003537618); GmGRF12 (Glyma13g16920); GmGRF13 (Glyma13g21630); GmGRF15 (XM — 003547454); GmGRF16 (Glyma16g00970) and GmGRF19 (XM — 003553541). All GFR transcription factors useful for the invention, will have an miRNA396 sequence (CAAGUUCUUUCGNACACCUU) (SEQ ID NO:27) binding site AAGGUGUNCGAAAGAACUUGC (SEQ ID NO:28) in common.
  • miRNA396 sequence CAAGUUCUUUCGNACACCUU
  • the invention is exemplified herein with specific Arabidopsis and soybean genes, the invention is not so limited and has applicability to any plant susceptible to nematode or other plant pathogen infection by interaction with miRNA396 and corresponding GRF transcription factors.
  • the invention provides methods for improving plant tolerance to cyst nematode infection by modulating miRNA 396 interacting pathway, such as, for example, increasing/modulating the activity of at least one miRNA396.
  • miRNA 396 interacting pathway such as, for example, increasing/modulating the activity of at least one miRNA396.
  • other steps along the signaling pathway could be modulated, such as the miRNA396 binding sites including GRF1, GRF 3 and other GRFs.
  • the methods for modulation include modification of a plant cell by introducing at least one polynucleotide sequence comprising a plant miRNA396 or plant GRF nucleic acid sequence, or subsequence thereof, into said plant cell, such that the polynucleotide sequence is operably linked to a promoter functional in said plant cell.
  • the method of modulating the production of miRNA396 or a GRF protein by increasing/modulating includes a miRNA396 or GRF gene which comprises, e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 99.5% or more sequence identity to miR396a ( Arabidopsis ATG10606 (SEQ ID NO:1), Glycine max MI0001785 (SEQ ID NO:12), or MIMAT0001687 (SEQ ID NO:13); miR396b ( Arabidopsis AT5G35407 (SEQ ID NO:2), Glycine max MI0001786 (SEQ ID NO:14), MIMAT0001688) (SEQ ID NO:15); or miR396c ( Glycine max MI0010572 (SEQ ID NO:116), MIMAT0010079 (SEQ ID NO:17)); or miR396e ( Glycine max MI00
  • the invention relates to methods for improving plant tolerance to cyst nematode infection by providing an isolated or recombinant modified plant cell comprising at least one modification that increases, decreases or otherwise modulates miRNA396 or GRF activity.
  • a plant cell resulting from the methods of the invention is from a dicot or monocot.
  • the plant cell is in a plant comprising a sterility phenotype, e.g., a male sterility phenotype.
  • the methods of the invention are practiced with an isolated or recombinant polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide, or a complement thereof, comprising, e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 99.5% or more sequence identity to an miRNA396 or GRF transcription factor or a subsequence thereof, or a conservative variation thereof; (b) a polynucleotide, or a complement thereof, encoding a polypeptide sequence of a (c) a polynucleotide, or a complement thereof, that hybridizes under stringent conditions over substantially the entire length of a polynucleotide subsequence comprising at least 100 contiguous nucleotides of SEQ a, or that hybridizes to a polynucleotide sequence
  • Such polynucleotides for practice of the methods of the invention can comprise or be contained within an expression cassette or a vector (e.g., a viral vector).
  • the vector or expression cassette can comprise a promoter (e.g., a constitutive, tissue-specific, or inducible promoter) operably linked to the polynucleotide.
  • the promoter is a root specific promoter.
  • Detection of expression products is performed either qualitatively (by detecting presence or absence of one or more product of interest) or quantitatively (by monitoring the level of expression of one or more product of interest). Aspects of the invention optionally include monitoring an expression level of a nucleic acid, polypeptide or chemical as noted herein for detection of the same in a plant or in a population of plants.
  • the present invention is directed to a transgenic plant or plant cells with improved performance under nematode infecting conditions, containing the nucleic acids described herein.
  • Preferred plants containing the polynucleotides of the present invention include but are not limited to soybean, sunflower, maize, sorghum, canola, wheat, alfalfa, cotton, oat, rice, barley, tomato, cacao and millet.
  • the transgenic plant is a soybean plant or plant cells.
  • Plants produced according to the invention can have at least one of the following phenotypes in nematode infecting conditions as compared to a non-modified control plant, including but not limited to: increased root mass, increased plant survival, increased root length, increased leaf size, increased ear size, increased seed size, absence of syncytia, smaller or decreased syncytia, or increased plant size when compared to a non-modified plant under conditions of nematode infection.
  • levels of miRNA396 or GRF proteins or mutant polynucleotide or polypeptide (where appropriate) sequences may be used as markers or selection traits to identify and select nematode tolerant plants even in the absence of transformation for breeding of tolerant lines, plants seeds, varieties and the like. Marker assisted selection protocols are thus included herein.
  • FIG. 1 Characterization of transgenic plants overexpressing miR396 or the target genes GRF1 and GRF3.
  • A Overexpression of miR396 reduces GRF gene expression.
  • the mRNA expression level of GRF1-9 was measured by quantitative real-time RT-PCR in the root tissues of 10 d-old wild-type (Col-0) and transgenic plants overexpressing miR396b (line 16-4). The expression levels were normalized using Actin8 as an internal control. The relative fold-change values represent changes of mRNA levels in the transgenic plants relative to the wild-type control. Data are averages of three biologically independent experiments ⁇ SE.
  • G Overexpression of GRF1 or GRF3 negatively regulates GRF gene expression.
  • the mRNA expression levels of GRF1 through 9 were quantified in the root tissues of the transgenic plants overexpressing the wild-type forms of GRF1 and GRF3 ( 35 S:wtGRF1 and 35 S:wtGRF3) or the miR396-resistant forms ( 35 S:rGRF1 and 35 S:rGRF3) using qPCR. The expression levels were normalized using Actin8 as an internal control.
  • the relative fold-change values represent changes of GRF expression levels in the transgenic plants relative to the wild-type control. Data are averages of three biologically independent experiments ⁇ SE. Note that the expression levels of GRF1 and GRF3 in the 35 S:rGRF1 and 35 S:rGRF3 plants include the endogenous transcripts.
  • FIG. 2 Promoter activity of miR396a, miR396b and the target genes GRF1 and GRF3 during Heterodera schachtii infection.
  • Time course experiments comparing the expression of miR396a:GUS (A-D), miR396b:GUS (E-H), GRF1:GUS (1-L), and GRF3: GUS (M-P) transgenic plants at the second-stage (J2), early and late third-stage (J3), and fourth-stage juvenile (J4) time points.
  • N indicates nematode and S indicates syncytium. See also Figure S2.
  • FIG. 3 Post-transcriptional regulation of GRF1 and GRF3 by miR396 in response to H. schachtii infection.
  • the expression level of pre-miR396a, pre-miR396b, mature miR396, GRF1 and GRF3 was measured by qPCR in wild-type (Col-0) root tissues. Infected and noninfected tissues were collected at 1, 3, 8, and 14 days after inoculation (dpi). Down regulation of miR396 at 1 and 3 dpi was associated with up regulation of both GRF1 and GRF3.
  • FIG. 4 Nematode susceptibility assays of miR396 overexpression lines and GRF mutants (A) and (B) Nematode susceptibility assays of miR396 overexpression lines.
  • Homozygous T3 lines overexpressing miR396a (lines 22-5, 13-10, and 10-12) or miR396b (lines 16-4, 15-1 and 8-16) were planted on modified Knop's medium, and 10-d-old seedlings were inoculated with ⁇ 200 surface-sterilized J2 H. schachtii nematodes.
  • FIG. 5 Overexpression of miR396, GRF1 or GRF3 negatively impacts syncytium size and nematode development.
  • A Transgenic plants overexpressing miR396, rGRF1 or rGRF3 developed smaller syncytia than the wild type. Homozygous T3 lines overexpressing miR396b (line 16-4), rGRF1 (lines 12-3) or rGRF3 (line 12-5) as well as wild-type (Col-0) were planted on modified Knop's medium, and 10-d-old seedlings were inoculated with ⁇ 200 surface-sterilized J2 H. schachtii nematodes.
  • FIG. 6 Functional classification of the differentially expressed genes identified in 35S:rGRF1, 35S:rGRF1 and grf1/grf1/grf3 mutants.
  • A Venn diagram showing overlaps between differentially expressed genes in 35S:rGRF1, 35S:rGRF3 and grf1/grf2/grf3 mutants. The total number of differentially expressed genes in each set is shown in parentheses. Genes are listed in Table S1A-C.
  • B and
  • C Venn diagram comparing the overlapping differentially expressed genes between 35S:rGRF1 and grf1/grf2/grf3 (B) or 35S:rGRF1 and grf1/grf2/grf3 (C).
  • FIG. 7 Expression profiles of GRF gene family members in Arabidopsis roots.
  • FIG. 8 (A-L): Spatial expression patterns of miR396a and miR396b and the target genes GRF1 and GRF3.
  • FIG. 9 Quantification of transgene expression levels in the transgenic Arabidopsis lines described in this study using qPCR.
  • FIG. 10 Characterization of Arabidopsis grf1 and grf3 mutants.
  • FIG. 12 Soybean miR396/target GRFs Expression Analyses with qRT-PCR after SCN Infection.
  • nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole. In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
  • amplified is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template.
  • Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, Persing, et al., eds., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
  • conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation.
  • Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine; one exception is Micrococcus rubens , for which GTG is the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” when the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered.
  • 1, 2, 3, 4, 5, 7 or 10 alterations can be made.
  • Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived.
  • substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably 60-90% of the native protein for its native substrate.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • “consisting essentially of” means the inclusion of additional sequences to an object polynucleotide where the additional sequences do not selectively hybridize, under stringent hybridization conditions, to the same cDNA as the polynucleotide and where the hybridization conditions include a wash step in 0.1 ⁇ SSC and 0.1% sodium dodecyl sulfate at 65° C.
  • nucleic acid encoding a protein comprising the information for translation into the specified protein.
  • a nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • the information by which a protein is encoded is specified by the use of codons.
  • amino acid sequence is encoded by the nucleic acid using the “universal” genetic code.
  • variants of the universal code such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.
  • nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference).
  • the maize preferred codon for a particular amino acid might be derived from known gene sequences from maize.
  • Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.
  • control plant is a plant without recombinant DNA disclosed herein.
  • a control plant is used to measure and compare trait improvement in a transgenic plant with such recombinant DNA.
  • a suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein.
  • a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement.
  • a control plant may also be a negative segregant progeny of hemizygous transgenic plant.
  • gene refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression.
  • heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • host cell is meant a cell, which comprises a heterologous nucleic acid sequence of the invention, which contains a vector and supports the replication and/or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli , or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells.
  • host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, lawn grass, barley, millet, and tomato.
  • a particularly preferred monocotyledonous host cell is a soybean host cell.
  • hybridization complex includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
  • “improved trait” refers to a trait with a detectable improvement in a transgenic plant relative to a control plant or a reference.
  • the trait improvement can be measured quantitatively.
  • the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference.
  • the trait improvement is only measured qualitatively. It is known that there can be a natural variation in a trait.
  • the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein.
  • Trait improvement includes, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.
  • the term “introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • isolated or “isolated nucleic acid” or “isolated protein” refer to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment.
  • isolated material optionally comprises material not found with the material in its natural environment.
  • Nucleic acids which are “isolated”, as defined herein, are also referred to as “heterologous” nucleic acids.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • nucleic acid library is meant a collection of isolated DNA or RNA molecules, which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, (1987) Guide To Molecular Cloning Techniques, from the series Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vols. 1-3; and Current Protocols in Molecular Biology, Ausubel, et al., eds, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).
  • operably linked includes reference to a functional linkage between a first sequence, such as a promoter, and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA 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.
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
  • Plant cell as used herein includes, without limitation, cells in or from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Bro
  • yield may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and/or the volume of biomass generated (for forage crops such as alfalfa, and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated.
  • polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a “plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium . Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma.
  • a “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • An “inducible” or “regulatable” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light.
  • Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development.
  • Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of “non-constitutive” promoters.
  • a “constitutive” promoter is a promoter, which is active under most environmental conditions.
  • recombinant includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention; or may have reduced or eliminated expression of a native gene.
  • the term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
  • a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
  • amino acid residue or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”).
  • the amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
  • Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
  • stringent conditions or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.
  • 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., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5 ⁇ to lx 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 0.1 ⁇ SSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • T m can be approximated from the equation of Meinkoth and Wahl, (1984) Anal.
  • 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.
  • T m hybridization and/or wash conditions
  • T m hybridizes to sequences of the desired identity.
  • the T m can be decreased 10° C.
  • 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.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C.
  • T m thermal melting point
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (T m ).
  • T m thermal melting point
  • high stringency is defined as hybridization in 4 ⁇ SSC, 5 ⁇ Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and a wash in 0.1 ⁇ SSC, 0.1% SDS at 65° C.
  • “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
  • transgenic plant includes reference to a plant, which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • transgenic does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • transgenic seed refers to a plant seed whose genome has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein.
  • transgenic plant is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains the recombinant DNA in its genome.
  • vector includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
  • sequence relationships between two or more nucleic acids or polynucleotides or polypeptides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial 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 means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of 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.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • GAP uses the algorithm of Needleman and Wunsch, supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or greater.
  • GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity.
  • the Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Clayerie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity When 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. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences which 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., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein 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.
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more 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 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • the degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. 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 that the polypeptide, which the first nucleic acid encodes, 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 between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, 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 and Wunsch, supra.
  • 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.
  • a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical.
  • Peptides which are “substantially similar” share sequences as noted above, except that residue positions which are not identical may differ by conservative amino acid changes.
  • agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits.
  • Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
  • transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
  • “increased yield” of a transgenic plant of the present invention may be evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, kilo per hectare.
  • maize yield may be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture.
  • Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens.
  • Trait-improving recombinant DNA may also be used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
  • the present invention provides, inter alia, for the use of isolated nucleic acids of RNA, DNA, homologs, paralogs and orthologs and/or chimeras thereof, comprising a plant miRNA396 and plant GRF encoding polynucleotide. This includes naturally occurring as well as synthetic variants and homologs of the sequences.
  • homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn (maize), potato, cotton, rice, rape, oilseed rape (including canola), sunflower, alfalfa, clover, sugarcane, and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, tomatillo, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum
  • Other crops including fruits and vegetables, whose phenotype can be changed and which comprise homologous sequences include barley; rye; millet; sorghum; currant; avocado; citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries; nuts such as the walnut and peanut; endive; leek; roots such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato; and beans.
  • the homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates.
  • homologous sequences may be derived from plants that are evolutionarily-related to crop plants, but which may not have yet been used as crop plants. Examples include deadly nightshade ( Atropa belladona ), related to tomato; jimson weed ( Datura strommium ), related to peyote; and teosinte ( Zea species), related to corn (maize).
  • deadly nightshade Atropa belladona
  • jimson weed Datura strommium
  • peyote Datura strommium
  • teosinte Zea species
  • Homologous sequences as described above can comprise orthologous or paralogous sequences.
  • Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. Three general methods for defining orthologs and paralogs are described; an ortholog, paralog or homolog may be identified by one or more of the methods described below.
  • Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
  • gene duplication may result in two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs.
  • a paralog is therefore a similar gene formed by duplication within the same species.
  • Paralogs typically cluster together or in the same Glade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360).
  • orthologs genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species.
  • CLUSTAL Thimpson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) supra
  • potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.
  • Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J. Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence).
  • the plant miRNA396 or GRF1/3 nucleotide sequences maybe used to generate variant nucleotide sequences having the nucleotide sequence of the 5′-untranslated region, 3′-untranslated region, or promoter region that is approximately 70%, 75%, 80%, 85%, 90% and 95% identical to the original nucleotide sequence. These variants are then associated with natural variation in the germplasm for component traits related to nematode infection. The associated variants are used as marker haplotypes to select for the desirable traits.
  • Variant amino acid sequences of the plant GRF polypeptides are generated. For one example, one amino acid is altered. Specifically, the open reading frames are reviewed to determine the appropriate amino acid alteration. The selection of the amino acid to change is made by consulting the protein alignment (with the other orthologs and other gene family members from various species). An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using a protein alignment, an appropriate amino acid can be changed. Once the targeted amino acid is identified, the procedure outlined herein is followed. Variants having about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequence identity are generated using this method. These variants are then associated with natural variation in the germplasm for component traits related to plant pathogen infection. The associated variants are used as marker haplotypes to select for the desirable traits.
  • the present invention also includes polynucleotides optimized for expression in different organisms.
  • the sequence can be altered to account for specific codon preferences and to alter GC content as according to Murray, et al, supra.
  • Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.
  • the plant miRNA398 or GRF1/GRF3 nucleic acids which may be used for the present invention comprise isolated plant polynucleotides which are inclusive of:
  • the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof.
  • the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a fungus or bacteria.
  • the nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
  • nucleic acids include such vectors as: M13, lambda ZAP Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/ ⁇ , pSG5, pBK, pCR-Script, pET, pSPUTK, p3′SS, pGEM, pSK+/ ⁇ , pGEX, pSPORTI and II, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45, p
  • Optional vectors for the present invention include but are not limited to, lambda ZAP II, and pGEX.
  • pGEX a description of various nucleic acids see, e.g., Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
  • the isolated nucleic acids used in the methods of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., (1979) Meth. Enzymol. 68:90-9; the phosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-51; the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra. Letts.
  • RNA Ribonucleic Acids Res. 13:7375.
  • Positive sequence motifs include translational initiation consensus sequences (Kozak, (1987) Nucleic Acids Res. 15:8125) and the 5 ⁇ G> 7 methyl GpppG RNA cap structure (Drummond, et al., (1985) Nucleic Acids Res. 13:7375).
  • Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell. Biol. 8:284). Accordingly, the present invention provides 5′ and/or 3′ UTR regions for modulation of translation of heterologous coding sequences.
  • polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage.
  • Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in maize.
  • Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group. See, Devereaux, et al., (1984) Nucleic Acids Res. 12:387-395); or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
  • the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention.
  • the number of polynucleotides (3 nucleotides per amino acid) that can be used to determine a codon usage frequency can be any integer from 3 to the number of polynucleotides of the present invention as provided herein.
  • the polynucleotides will be full-length sequences.
  • An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
  • the present invention also includes the use of sequence shuffling using polynucleotides disclosed for the methods of the present invention, and compositions resulting therefrom.
  • Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-9; and Zhao, et al., (1998) Nature Biotech 16:258-61.
  • sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic, which can be selected or screened for.
  • Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides, which comprise sequence regions, which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • the population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method.
  • the characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
  • the selected characteristic will be an altered K m and/or K cat over the wild-type protein as provided herein.
  • a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide.
  • a protein or polynucleotide generated from sequence shuffling will have an altered pH optimum as compared to the non-shuffled wild-type polynucleotide.
  • the increase in such properties can be at least 110%, 120%, 130%, 140% or greater than 150% of the wild-type value.
  • the present invention provides the use of recombinant expression/transcription cassettes comprising a polynucleotide for a plant microRNA396, or a GRF useful for the methods of the present invention.
  • a nucleic acid sequence coding for the desired polynucleotide for example a cDNA or a genomic sequence encoding a polypeptide long enough to code for an active GRF protein, or for a desired mircor RNA can be used to construct a recombinant expression cassette which can be introduced into the desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
  • plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker.
  • plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant.
  • Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
  • Examples of constitutive promoters include the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens , the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.
  • ubiquitin is the preferred promoter for expression in monocot plants.
  • the plant promoter can direct expression in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as “inducible” promoters.
  • Environmental conditions that may affect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adh1 promoter, which is inducible by hypoxia or cold stress, the Hsp70 promoter, which is inducible by heat stress, and the PPDK promoter, which is inducible by light.
  • promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
  • the operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region.
  • the polyadenylation region can be derived from a variety of plant genes, or from T-DNA.
  • the 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • regulatory elements include, but are not limited to, 3′ termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res. 14:5641-50; and An, et al., (1989) Plant Cell 1:115-22); and the CaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).
  • PINII potato proteinase inhibitor II
  • An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
  • Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg, (1988) Mol. Cell. Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev. 1:1183-200).
  • Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit.
  • Use of maize introns Adh1-S intron 1, 2 and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, eds., Springer, N.Y. (1994).
  • Plant signal sequences including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., (1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana plumbaginifolia extension gene (DeLoose, et al., (1991) Gene 99:95-100); signal peptides which target proteins to the vacuole, such as the sweet potato sporamin gene (Matsuka, et al., (1991) Proc. Natl. Acad. Sci.
  • the vector comprising the sequences from a plant nicroRNA396, GRF1 or GRF3 will typically comprise a marker gene, which confers a selectable phenotype on plant cells.
  • the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4
  • Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al. (1987), Meth. Enzymol. 153:253-77. These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant.
  • Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al., (1987) Gene 61:1-11, and Berger, et al., (1989) Proc. Natl. Acad. Sci. USA, 86:8402-6.
  • Another useful vector herein is plasmid pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).
  • an miRNA396 or GRF protein in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells.
  • the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
  • the expression of isolated nucleic acids encoding a GRF 1 or GRF3 protein or microRNA will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
  • a strong promoter such as ubiquitin
  • Constitutive promoters are classified as providing for a range of constitutive expression. Thus, some are weak constitutive promoters, and others are strong constitutive promoters.
  • weak promoter is intended a promoter that drives expression of a coding sequence at a low level.
  • low level is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts.
  • strong promoter drives expression of a coding sequence at a “high level,” or about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts.
  • modifications could be made to a GRF protein or MicroRNA without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
  • Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli ; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.
  • selection markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA.
  • Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-35; Mosbach, et al., (1983) Nature 302:543-5).
  • the pGEX-4T-1 plasmid vector from Pharmacia is the preferred E. coli expression vector for the present invention.
  • eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
  • yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris .
  • Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a plant protein, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates or the pellets.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
  • sequences encoding plant GRF proteins or miRNA396 can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
  • Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7 th ed., 1992).
  • Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus.
  • suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth, and Drosophila cell lines such as a Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-65).
  • polyadenylation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-81 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors (Saveria-Campo, “Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNA Cloning: A Practical Approach, vol. II, Glover, ed., IRL Press, Arlington, Va., pp. 213-38 (1985)).
  • the plant GRF or miRNA396 gene placed in the appropriate plant expression vector can be used to transform plant cells.
  • the polypeptide can then be isolated from plant callus or the transformed cells can be used to regenerate transgenic plants.
  • Such transgenic plants can be harvested, and the appropriate tissues (seed or leaves, for example) can be subjected to large scale protein extraction and purification techniques.
  • the methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch et al., Science 227:1229-31 (1985)), electroporation, micro-injection, and biolistic bombardment.
  • the isolated plant polynucleotides or polypeptides may be introduced into the plant by one or more techniques typically used for direct delivery into cells. Such protocols may vary depending on the type of organism, cell, plant or plant cell, i.e. monocot or dicot, targeted for gene modification. Suitable methods of transforming plant cells include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, direct gene transfer (Paszkowski et al., (1984) EMBO J.
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which genetically transform plant cells.
  • the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes carry genes responsible for genetic transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant Sci. 10:1.
  • the gene can be inserted into the T-DNA region of a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes , respectively.
  • expression cassettes can be constructed as above, using these plasmids.
  • Many control sequences are known which when coupled to a heterologous coding sequence and transformed into a host organism show fidelity in gene expression with respect to tissue/organ specificity of the original coding sequence. See, e.g., Benfey and Chua, (1989) Science 244:174-81.
  • Particularly suitable control sequences for use in these plasmids are promoters for constitutive leaf-specific expression of the gene in the various target plants.
  • NOS nopaline synthase gene
  • the NOS promoter and terminator are present in the plasmid pARC2, available from the American Type Culture Collection and designated ATCC 67238. If such a system is used, the virulence (vir) gene from either the Ti or Ri plasmid must also be present, either along with the T-DNA portion, or via a binary system where the vir gene is present on a separate vector.
  • vir nopaline synthase gene
  • Such systems, vectors for use therein, and methods of transforming plant cells are described in U.S. Pat. No. 4,658,082; U.S. Pat. No. 913,914, filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993; and Simpson, et al., (1986) Plant Mol. Biol. 6:403-15 (also referenced in the '306 patent); all incorporated by reference in their entirety.
  • these plasmids can be placed into A. rhizogenes or A. tumefaciens and these vectors used to transform cells of plant species, which are ordinarily susceptible to Fusarium or Alternaria infection.
  • transgenic plants include but not limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper.
  • the selection of either A. tumefaciens or A. rhizogenes will depend on the plant being transformed thereby. In general A. tumefaciens is the preferred organism for transformation.
  • European Patent Application No. 604 662 A1 discloses a method for transforming monocots using Agrobacterium .
  • European Application No. 604 662 A1 discloses a method for transforming monocots using Agrobacterium .
  • 672 752 A1 discloses a method for transforming monocots with Agrobacterium using the scutellum of immature embryos. Ishida, et al., discuss a method for transforming maize by exposing immature embryos to A. tumefaciens (Nature Biotechnology 14:745-50 (1996)).
  • these cells can be used to regenerate transgenic plants.
  • whole plants can be infected with these vectors by wounding the plant and then introducing the vector into the wound site. Any part of the plant can be wounded, including leaves, stems and roots.
  • plant tissue in the form of an explant, such as cotyledonary tissue or leaf disks, can be inoculated with these vectors, and cultured under conditions, which promote plant regeneration. Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A.
  • tumefaciens containing the gene coding for the fumonisin degradation enzyme, can be used as a source of plant tissue to regenerate fumonisin-resistant transgenic plants, either via somatic embryogenesis or organogenesis. Examples of such methods for regenerating plant tissue are disclosed in Shahin, (1985) Theor. Appl. Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et al., supra; and U.S. Pat. Nos. 913,913 and 913,914, both filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993, the entire disclosures therein incorporated herein by reference.
  • a generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 ⁇ m.
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate the plant cell walls and membranes (Sanford, et al., (1987) Part. Sci. Technol. 5:27; Sanford, (1988) Trends Biotech 6:299; Sanford, (1990) Physiol. Plant 79:206; and Klein, et al., (1992) Biotechnology 10:268).
  • Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang, et al., (1991) BioTechnology 9:996.
  • liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, e.g., Deshayes, et al., (1985) EMBO J. 4:2731; and Christou, et al., (1987) Proc. Natl. Acad. Sci. USA 84:3962.
  • Direct uptake of DNA into protoplasts using CaCl 2 precipitation, polyvinyl alcohol, or poly-L-ornithine has also been reported. See, e.g., Hain, et al., (1985) Mol.
  • Some embodiments may involve the improvement in nematode tolerance by modulating the expression of a plant miRNA396, GRF1/GRF3 in a way that decreases the activity/expression of the protein or mircroRNA.
  • Methods are also provided to reduce or eliminate the activity of a plant GRF Polypeptide or MicroRNA by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the plant polypeptide or microRNA.
  • the polynucleotide may inhibit the expression of the plant a plant GRF Polypeptide or MicroRNA directly, by preventing transcription or translation of the plant messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of an plant a plant GRF Polypeptide or MicroRNA gene encoding an plant a plant GRF Polypeptide or MicroRNA.
  • Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of the plant a plant GRF Polypeptide or MicroRNA. Many methods may be used to reduce or eliminate the activity of GRF polypeptides. In addition, more than one method may be used to reduce the activity of a plant GRF Polypeptide or MicroRNA.
  • a plant is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of a plant GRF Polypeptide or MicroRNA of the invention.
  • an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one a plant GRF Polypeptide or MicroRNA is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one plant a plant GRF Polypeptide or MicroRNA of the invention.
  • Examples of polynucleotides that inhibit the expression of a plant GRF Polypeptide or MicroRNA include sense suppression/cosuppresion.
  • an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a plant GRF Polypeptide or MicroRNA in the “sense” orientation. Over expression of the RNA molecule can result in reduced expression of the native gene.
  • the polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the a plant GRF Polypeptide or MicroRNA, all or part of the 5′ and/or 3′ untranslated region of a plant GRF Polypeptide or MicroRNA transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding a plant GRF Polypeptide or MicroRNA.
  • the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.
  • inhibition of the expression of a plant GRF Polypeptide or MicroRNA may be obtained by antisense suppression.
  • the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the a plant GRF Polypeptide or MicroRNA. Over expression of the antisense RNA molecule can result in reduced expression of the native gene.
  • the polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the a plant GRF Polypeptide or MicroRNA, all or part of the complement of the 5′ and/or 3′ untranslated region of the plant a plant GRF Polypeptide or MicroRNA transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the plant a plant GRF Polypeptide or MicroRNA.
  • the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence.
  • inhibition of the expression of a plant GRF Polypeptide or MicroRNA may be obtained by double-stranded RNA (dsRNA) interference.
  • dsRNA interference a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
  • Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of plant a plant GRF Polypeptide or MicroRNA. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which is herein incorporated by reference.
  • inhibition of the expression of a plant GRF Polypeptide or MicroRNA may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.
  • hpRNA hairpin RNA
  • ihpRNA intron-containing hairpin RNA
  • the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem.
  • the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence.
  • the base-paired stem region may correspond to a portion of a promoter sequence controlling expression of the gene to be inhibited.
  • the base-paired stem region of the molecule generally determines the specificity of the RNA interference.
  • hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci.
  • the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed.
  • the use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al., (2000) Nature 407:319-320. In fact, Smith, et al., show 100% suppression of endogenous gene expression using ihpRNA-mediated interference.
  • the expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA.
  • the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene.
  • it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00904; Mette, et al., (2000) EMBO J. 19:5194-5201; Matzke, et al., (2001) Curr. Opin. Genet. Devel. 11:221-227; Scheid, et al., (2002) Proc. Natl. Acad.
  • Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus.
  • the viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication.
  • the transcripts produced by the amplicon may be either sense or antisense relative to the target sequence.
  • the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of the plant miRNA396 or GRF polypeptide.
  • the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the plant GRF polypeptide or miRNA396. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.
  • inhibition of the expression of a plant GRF Polypeptide or MicroRNA activity may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA).
  • miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier, et al., (2003) Nature 425:257-263, herein incorporated by reference.
  • the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene.
  • the miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence).
  • miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
  • the polynucleotide encodes a zinc finger protein that binds to a gene encoding a plant GRF Polypeptide or MicroRNA, resulting in reduced expression of the gene.
  • the zinc finger protein binds to a regulatory region a plant GRF Polypeptide or MicroRNA gene.
  • the zinc finger protein binds to a messenger RNA encoding a plant GRF Polypeptide or MicroRNA and prevents its translation.
  • the polynucleotide encodes an antibody that binds to at least one a plant GRF Polypeptide or MicroRNA, and reduces the activity of the a plant GRF Polypeptide or MicroRNA.
  • the binding of the antibody results in increased turnover of the antibody-GRF Polypeptide or MicroRNA complex by cellular quality control mechanisms.
  • the activity of a plant GRF Polypeptide or MicroRNA is reduced or eliminated by disrupting the gene encoding a plant GRF Polypeptide or MicroRNA.
  • the gene encoding the plant a plant GRF Polypeptide or MicroRNA may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have increased nematode tolerance.
  • transposon tagging is used to reduce or eliminate a plant GRF Polypeptide or MicroRNA activity of one or more plant GRF Polypeptides or MicroRNA polypeptides.
  • Transposon tagging comprises inserting a transposon within an endogenous plant a plant GRF Polypeptide or MicroRNA gene to reduce or eliminate expression of the plant a plant GRF Polypeptide or MicroRNA.
  • the expression of one or more a plant GRF Polypeptide or MicroRNA is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding a plant GRF Polypeptide or MicroRNA.
  • a transposon that is within an exon, intron, 5′ or 3′ untranslated sequence, a promoter, or any other regulatory sequence of a plant GRF Polypeptide or MicroRNA gene may be used to reduce or eliminate the expression and/or activity of the encoded a plant GRF Polypeptide or MicroRNA.
  • mutagenesis such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted.
  • Mutations that impact gene expression or that interfere with the function of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the activity of the encoded protein. conserveed residues of plant GRF polypeptides and/or miRNA396 suitable for mutagenesis with the goal to eliminate activity have been described. Such mutants can be isolated according to well-known procedures, and mutations in different loci can be stacked by genetic crossing. See, for example, Gruis, et al., (2002) Plant Cell 14:2863-2882.
  • the methods of the invention provides for improved plant tolerance to nematode infection. This performance may be demonstrated in a number of ways including the following.
  • Root development is intended any alteration in the development of the plant root under nematode infection when compared to a control plant.
  • Such alterations in root development include, but are not limited to, alterations in the growth rate of the primary root, the fresh root weight, the extent of lateral and adventitious root formation, the vasculature system, meristem development, or radial expansion.
  • the methods comprise modulating the level and/or activity of a miRNA396, GRF1 or GRF3 and their interaction in the plant.
  • a plant miRNA396 sequence expression construct is provided to the plant.
  • root development is modulated by increasing the level or activity of the GRF proteins that interact with miRNA396 in the plant.
  • a change in plant GRF activity can result in at least one or more of the following alterations to root development, including, but not limited to, alterations in root biomass and length when the plant is grown under nematode infection.
  • root growth encompasses all aspects of growth of the different parts that make up the root system at different stages of its development in both monocotyledonous and dicotyledonous plants. It is to be understood that enhanced root growth can result from enhanced growth of one or more of its parts including the primary root, lateral roots, adventitious roots, etc.
  • exemplary promoters for this embodiment include constitutive promoters and root-preferred promoters. Exemplary root-preferred promoters have been disclosed elsewhere herein.
  • Stimulating root growth and increasing root mass in the presence of nematode infection by increasing the activity and/or level of miRNA396 or its targets such as the GRF proteins also finds use in improving the standability of a plant.
  • the term “resistance to lodging” or “standability” refers to the ability of a plant to fix itself to the soil. For plants with an erect or semi-erect growth habit, this term also refers to the ability to maintain an upright position under adverse (environmental) conditions. This trait relates to the size, depth and morphology of the root system. Furthermore, higher root biomass production has a direct effect on the yield and an indirect effect of production of compounds produced by root cells or transgenic root cells or cell cultures of said transgenic root cells.
  • Methods are also provided for modulating shoot and leaf development in a plant, particularly under nematode infection.
  • modulating shoot and/or leaf development is intended any alteration in the development of the plant shoot and/or leaf in nematode infection.
  • Such alterations in shoot and/or leaf development include, but are not limited to, alterations in shoot meristem development, in leaf number, leaf size, leaf and stem vasculature, internode length, and leaf senescence.
  • leaf development andshoot development encompasses all aspects of growth of the different parts that make up the leaf system and the shoot system, respectively, at different stages of their development, both in monocotyledonous and dicotyledonous plants.
  • the method for modulating shoot and/or leaf development in a plant in nematode infected conditions comprises increasing the activity and/or level of plant mrRNA396 or its target GRF proteins.
  • the plant nucleotide sequences can be provided by introducing into the plant a polynucleotide comprising an plant expression construct, expressing the same, and thereby modifying shoot and/or leaf development in nematode infected plants.
  • the plant expression nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
  • An increase in plant tolerance to nematode infection can result in at least one or more of the following alterations in shoot and/or leaf development under nematode infection when compared to a nonmodified plant, including, but not limited to, changes in leaf number, altered leaf surface, altered vasculature, internodes and plant growth, and alterations in leaf senescence, when compared to a control plant in the same conditions.
  • promoters for this embodiment include constitutive promoters, shoot-preferred promoters, shoot meristem-preferred promoters, and leaf-preferred promoters. Exemplary promoters have been disclosed elsewhere herein.
  • nucleotides, expression cassettes and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant.
  • Various other changes in phenotype are of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's stress tolerance, and the like.
  • These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants.
  • the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant.
  • genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly.
  • General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.
  • the plant miRNA/GRF nucleic acid sequences of can be used in combination (“stacked”) with other polynucleotide sequences of interest in order to create plants with a desired phenotype.
  • the combinations generated can include multiple copies of any one or more of the polynucleotides of interest.
  • the polynucleotides of the present invention may be stacked with any gene or combination of genes to produce plants with a variety of desired trait combinations, including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat. Nos.
  • polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene 48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S.
  • modified oils e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)
  • modified starches e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)
  • polymers or bioplastics e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference.
  • PHAs polyhydroxyalkanoates
  • polynucleotides of the present invention could also combine with polynucleotides affecting agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
  • agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
  • sequences of interest improve plant growth and/or crop yields.
  • sequences of interest include agronomically important genes that result in improved primary or lateral root systems. Such genes include, but are not limited to, nutrient/water transporters and growth induces.
  • genes include but are not limited to, maize plasma membrane H + -ATPase (MHA2) (Frias, et al., (1996) Plant Cell 8:1533-44); AKT1, a component of the potassium uptake apparatus in Arabidopsis , (Spalding, et al., (1999) J Gen Physiol 113:909-18); RML genes which activate cell division cycle in the root apical cells (Cheng, et al., (1995) Plant Physiol 108:881); maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J.
  • MHA2 maize plasma membrane H + -ATPase
  • AKT1 a component of the potassium uptake apparatus in Arabidopsis , (Spalding, et al., (1999) J Gen Physiol 113:909-18
  • sequence of interest may also be useful in expressing antisense nucleotide sequences of genes that that negatively affects root development.
  • Additional, agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson, et al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are herein incorporated by reference.
  • Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide.
  • the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference.
  • Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley, et al., (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed.
  • Applewhite American Oil Chemists Society, Champaign, Ill.), pp. 497-502; herein incorporated by reference
  • corn Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359; both of which are herein incorporated by reference
  • rice agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.
  • Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art.
  • the bar gene encodes resistance to the herbicide basta
  • the nptII gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
  • Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like.
  • the level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
  • the next steps generally concern identifying the transformed cells for further culturing and plant regeneration.
  • identifying the transformed cells for further culturing and plant regeneration.
  • one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the invention.
  • DNA is introduced into only a small percentage of target cells in any one study.
  • a means for selecting those cells that are stably transformed is to introduce into the host cell, a marker gene which confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide.
  • antibiotics which may be used include the aminoglycoside antibiotics neomycin, kanamycin and paromomycin, or the antibiotic hygromycin.
  • aminoglycoside antibiotics Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.
  • aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I
  • hygromycin phosphotransferase Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I
  • NPT II neomycin phosphotransferase II
  • hygromycin phosphotransferase Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes such as
  • surviving cells are those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA.
  • Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus and is composed of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. Upon removal of the L-alanine residues by intracellular peptidases, the PPT is released and is a potent inhibitor of glutamine synthetase (GS), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al., 1973). Synthetic PPT, the active ingredient in the herbicide LibertyTM also is effective as a selection agent Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.
  • PPT phosphinothricin
  • GS glutamine synthetase
  • Synthetic PPT the active ingredient in the herbicide LibertyTM also is effective as a selection agent Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of
  • the organism producing bialaphos and other species of the genus Streptomyces also synthesizes an enzyme phosphinothricin acetyl transferase (PAT) which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes .
  • PAT phosphinothricin acetyl transferase
  • the use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642 829 A, wherein the gene is isolated from Streptomyces viridochromogenes.
  • Glyphosate inhibits the action of the enzyme EPSPS which is active in the aromatic amino acid biosynthetic pathway Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived thereof.
  • U.S. Pat. No. 4,535,060 describes the isolation of EPSPS mutations which confer glyphosate resistance on polypeptides encoded by the Salmonella typhimurium gene for EPSPS, aroA.
  • the EPSPS gene was cloned from Zea mays and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate resistant EPSPS enzymes are described in, for example, International Patent WO 97/4103. The best characterized mutant EPSPS gene conferring glyphosate resistance comprises amino acid changes at residues 102 and 106, although it is anticipated that other mutations will also be useful (PCT/WO97/4103).
  • transformed tissue can be cultured for 0-28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg/l bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or 1-3 mM glyphosate may be preferred, it is proposed that ranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will find utility.
  • Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in media that supports regeneration of plants.
  • MS and N6 media may be modified by including further substances such as growth regulators.
  • growth regulators is dicamba or 2,4-D.
  • other growth regulators may be employed, including NAA, NAA+2,4-D or picloram.
  • Media improvement in these and like ways has been found to facilitate the growth of cells at specific developmental stages. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least 2 wk, then transferred to media conducive to maturation of embryoids. Cultures are transferred every 2 wk on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators.
  • the transformed cells identified by selection or screening and cultured in an appropriate medium that supports regeneration, will then be allowed to mature into plants.
  • Developing plantlets are transferred to soiless plant growth mix, and hardened, e.g., in an environmentally controlled chamber, for example, at about 85% relative humidity, 600 ppm CO 2 , and 25-250 microeinsteins m ⁇ 2 s ⁇ 1 of light.
  • Plants may be matured in a growth chamber or greenhouse. Plants can be regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue.
  • cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Cons.
  • Regenerating plants can be grown at about 19 to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
  • Seeds on transformed plants may occasionally require embryo rescue due to cessation of seed development and premature senescence of plants.
  • To rescue developing embryos they are excised from surface-disinfected seeds 10-20 days post-pollination and cultured.
  • An embodiment of media used for culture at this stage comprises MS salts, 2% sucrose, and 5.5 ⁇ l agarose.
  • embryo rescue large embryos (defined as greater than 3 mm in length) are germinated directly on an appropriate media. Embryos smaller than that may be cultured for 1 wk on media containing the above ingredients along with 10 ⁇ 5M abscisic acid and then transferred to growth regulator-free medium for germination.
  • assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCRTM; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • Genomic DNA may be isolated from cell lines or any plant parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note, that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
  • the presence of DNA elements introduced through the methods of this invention may be determined, for example, by polymerase chain reaction (PCRTM). Using this technique, discrete fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not prove integration of the introduced gene into the host cell genome. It is typically the case, however, that DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCRTM analysis.
  • PCRTM polymerase chain reaction
  • PCRTM techniques it is not typically possible using PCRTM techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCRTM techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced gene.
  • Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome.
  • the technique of Southern hybridization provides information that is obtained using PCRTM, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.
  • RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
  • PCRTM techniques also may be used for detection and quantitation of RNA produced from introduced genes. In this application of PCRTM it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCRTM techniques amplify the DNA. In most instances PCRTM techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
  • Southern blotting and PCRTM may be used to detect the gene(s) in question, they do not provide information as to whether the corresponding protein is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
  • Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
  • bioassays Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes which change starch quantity which may be analyzed by near infrared reflectance spectrometry. Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
  • transgenic plants may be made by crossing a plant having a selected DNA of the invention to a second plant lacking the construct.
  • a selected polypeptide coding sequence can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the current invention not only encompasses a plant directly transformed or regenerated from cells which have been transformed in accordance with the current invention, but also the progeny of such plants.
  • progeny denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a selected DNA construct prepared in accordance with the invention.
  • Crossing a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the invention being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene of the invention. To achieve this one could, for example, perform the following steps:
  • step (d) repeating steps (b) and (c) for the purpose of transferring a desired DNA sequence from a plant of a first genotype to a plant of a second genotype.
  • Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion.
  • a plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid.
  • a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.
  • cyst nematodes are obligate parasitic roundworms that induce the formation of novel plant cell types that are associated in a unique feeding organ, the syncytium.
  • Cyst nematodes infect as second-stage juveniles (J2), which initiate the induction/formation of the syncytium.
  • J2s begin feeding on the growing syncytium and then develop into third-stage (J3) and fourth-stage juveniles (J4) followed by the adult stage.
  • Syncytium development can be separated into an induction/formation phase followed by a maintenance phase.
  • Induction/formation involves effector-mediated communication between the nematode and plant cells leading to cytoplasmic and nuclear changes followed by successive cell-to-cell fusions of the cells surrounding an initial feeding cell (IFC). Through continuous cell fusions, syncytium formation and enlargement continues.
  • IFC initial feeding cell
  • cyst nematodes Due to their sedentary nature of parasitism, cyst nematodes need to obtain all their nourishment from one location, in fact, through the contact with the IFC.
  • the severity of this constraint becomes obvious when considering that the worm-shaped infective J2 nematode has a body length of approximately 500 um and then grows to a large lemon-shaped sphere that produces several hundred eggs, each containing a fully infective nematode.
  • the sheer logistics of nutrient availability and flux appear unrivalled for an individual plant pathogen. This association is also impressive with regard to the complete dependence of nematode survival on the well-being and survival of the IFC and the syncytium.
  • a single hypersensitive response or an interruption of the newly induced developmental programs of syncytium formation would eliminate nematode parasitism.
  • co-evolution of nematode and plant has resulted in an uncannily robust and successful pathosystem in which nematode contact with the IFC does not trigger effective defenses.
  • syncytial cells are dedicated to nematode nourishment, and their plant defenses have been suppressed by the nematode.
  • Syncytium formation encompasses reprogramming of differentiated root cells, and these redifferentiations are accompanied and mediated by massive gene expression changes, which have been documented in diverse research approaches using soybean and the soybean cyst nematode Heterodera glycines (Alkharouf et al., 2006; Ithal et al., 2007; Klink et al., 2009) and probably most extensively in Arabidopsis infected by the sugar beet cyst nematode H. schachtii (Szakasits et al., 2009).
  • miRNAs initially have been shown to be involved in the regulation of a variety of plant developmental processes including phase transition, hormone synthesis and signaling, pattern formation, and morphogenesis (Chen, 2009). Recent studies indicate that miRNAs and small endogenous RNAs also are involved in biotic stress responses in plants (Navarro et al. 2006; Li et al., 2010; He et al., 2008; Lu et al., 2007; Fahlgren et al. 2007; Hewezi et al., 2008a; Pandey et al., 2008; Katiyar-Agarwal et al., 2006 and 2007).
  • the miR396 family governs the expression of seven growth regulating transcription factor genes (GRFs) (Jones-Rhoades and Bartel, 2004).
  • GRFs growth regulating transcription factor genes
  • the GRF gene family in Arabidopsis is known to act in a functionally redundant fashion to positively control cell proliferation and size in leaves (Kim et al., 2003; Kim and Kende, 2004; Horiguchi et al., 2005; Kim and Lee, 2006).
  • miR396 acts as a negative regulator of GRF gene expression
  • overexpression of miR396 negatively impacted cell proliferation in leaves and meristem size
  • miR396/GRF regulatory module roles of the miR396/GRF regulatory module in controlling developmental events during plant-pathogen interactions or in root developmental processes are completely unknown.
  • miR396 is differentially expressed in the syncytium, that the miR396-GRF regulatory unit is subject to extensive feedback regulation, and that this microRNA functions as a true master switch in syncytium formation.
  • miR396 is encoded by two genes, miR396a (AT2G10606) (SEQ ID NO:1) and miR396b (AT5G35407) (SEQ ID NO:2) and regulates the expression of seven of the nine Arabidopsis growth regulating transcription factor genes (GRF1 through 4 and 7 through 9), which share the miR396-binding site (Jones-Rhoades and Bartel, 2004).
  • GRF1 Arabidopsis growth regulating transcription factor genes
  • qPCR quantitative real-time RT-PCR
  • miR396a and miR396b have Similar Spatial Expression Patterns and Overlap With GRF1 and GRF3 Expression in Roots
  • miRNA396 and GRF Transcription Factors Represent a Complex Regulatory Unit Governed by Multiple Mechanisms Including Feedback Regulation.
  • the miRNA396-GRF gene family system constitutes a non-trivial and complex, multi-dimensional regulatory network.
  • miR396a/b and GRF1 and GRF3 are Expressed in Syncytia of Heterodera schachtii.
  • GRF1 and GRF3 mRNA abundance should markedly decrease in syncytia during the maintenance phase as a function of miRNA396-mediated post-transcriptional transcript degradation.
  • GRF1 and GRF3 are Post-Transcriptionally Regulated by miR396 During Nematode Infection
  • schachtii and root tissues were collected from inoculated and non-inoculated control plants at 1, 3, 8, and 14 days post inoculation (dpi) for RNA isolation and cDNA synthesis.
  • miR396 and GRF1 and GRF3 are differentially expressed in syncytia strongly suggests that miR396-mediated regulation of GRFs is of importance in the plant-nematode interaction, and the timing of these expression changes implies a possible function in the early events of syncytium induction/formation and even a delineation of the transition from a period of syncytium initiation/formation to the period of syncytium maintenance.
  • the average reduction in syncytium size was up to 33% in miR396-overexpression plants and 19% and 14% in the transgenic plants expressing rGRF1 and rGRF3, respectively.
  • GRF1 and GRF3 function as transcription factors, identifying their direct or indirect target genes will elucidate the pathways in which these transcription factors function.
  • Arabidopsis Affymetrix ATH1 GeneChips to compare the mRNA profiles of root tissues of the grf1/grf2/grf3 triple mutant and transgenic plants expressing rGRF1 or rGRF3 with those of the corresponding wild-type (Col-0 or Ws).
  • GRFs in Arabidopsis function redundantly in controlling various aspects of plant development (Kim et al., 2003; Kim and Kende, 2004; Horiguchi et al., 2005; Kim and lee, 2006).
  • To address the potential redundant function of GRF1 and GRF3 in regulating gene expression we compared the 1,098 candidate target genes of GRF1 with the 600 candidate target genes of GRF3 to identify genes that are common to both.
  • the 264 overlapping target genes all showed the same trend of expression in the rGRF1 and rGRF3 overexpression lines, in which 124 genes were up regulated and 140 genes were down regulated in both lines, indicating that GRF1 and GRF3 activate and inhibit gene expression in a similar manner.
  • the candidate GRF1 and GRF3 target genes are regulated by these transcription factors and have a role in mediating syncytium induction/formation, these genes should exhibit differential regulation in the syncytium when compared with other root tissues because we have documented differential regulation of GRF1 and GRF3 in the syncytium. Therefore, we next compared the candidate targets of GRF1 and GRF3 with the 7,225 genes differentially expressed in Arabidopsis syncytia reported by Szakasits et al. (2009).
  • miR396 and its target genes GRF1 and GRF3 showed opposite expression patterns in the early developing syncytium at the parasitic J2 and early J3 stages when miR396 was down regulated and GRF1 and GRF3 showed up regulation.
  • up regulation of miR396 at 8 and 14 dpi is accompanied by a posttranscriptional down regulation of GRF1 and GRF3 ( FIG. 3 ).
  • miRNA expression being positively or negatively regulated by the transcription factors they target through negative or positive feedback loops (Gutierrez et al., 2009; Wu et al., 2009; Wang et al, 2009; Yant et al., 2010; Marin et al., 2010).
  • the miR396/GRF1 and GRF3 regulatory module is under a tightly fine-tuned regulation to ensure adequate expression of GRF1 and GRF3 and their negative regulator miR396.
  • Our data suggest that maintenance of the homeostasis of miR396 and the target genes at specific threshold levels is critical for syncytium development. This suggestion is supported by our finding that down regulation of GRFs through overexpression of miR396a/b, or overexpression of wild-type or miR396-resistant versions of GRF1/GRF3 resulted in reduced nematode susceptibility.
  • GRF1 and GRF3 are part of a highly interconnected network of GRF transcription factors that fine tune downstream signaling pathways in the syncytium, and that disturbance of this interconnected network impacts normal differentiation and developmental processes in the syncytium.
  • miR396 The opposite expression patterns of miR396 during syncytium initiation/formation and maintenance stages are similar to those of Arabidopsis miR156 and miR172 during the juvenile-to-adult phase transition where miR156 is expressed at high levels during shoot development and then decreases with time, while miR172 has an inverse expression pattern (Aukerman and Sakai, 2003; Jung et al., 2007; Wu and Poethig, 2006).
  • the enrichment of transcription factors belonging to zinc finger, Myb, WRKY, bHLH, AP2 domain-containing, CCAAT-binding, or NAC domain transcription factor families among the GRF1 or GRF3 target genes represents a powerful mechanism to trigger a massive signaling response to GRF1 or GRF3 expression.
  • syncytium formation has to be associated with a modulation of host defense responses (Davis et al., 2004; Gheysen and Fenoll 2002; Williamson and Kumar 2006) and we found a number of genes involved in different aspects of plant defenses among the putative targets of GRF1 or GRF3.
  • auxin have been implicated in syncytium development (Grunewald et al., 2009), and GRF1 or GRF3 appear to regulate a set of genes involved in hormone biosynthesis or signaling pathways of auxin, brassinosteroids, cytokinins, ethylene, gibberellins, and jasmonates.
  • cell wall modifications are obvious mechanisms of syncytium formation and a high proportion of genes with cell wall related functions also are enriched among the putative GRF target genes.
  • GRF1 and GRF3 likely are impacting a very wide spectrum of physiological processes associated with syncytium formation.
  • Arabidopsis thaliana Wild type Columbia-0 (Col-0) was used in all experiments except for the grf1/grf2/grf3 triple knockout mutant, which is in the Wassilewskija (Ws) background (Kim et al., 2003). Plants were grown in long days (16 h light/8 h dark) at 23° C.
  • Plants were grown on modified Knop's medium in 12-well culture plates. At 10 days, each plant was inoculated with 200 surface-sterilized J2 of H. schachtii , and plants were assessed at 5 and 21 days post infection for parasitic-stage juveniles and females, respectively. Average numbers of developing nematodes were calculated for each time point, and statistically significant differences were determined in a modified test using the statistical software package SAS.
  • the synthesized cDNAs then were diluted to a concentration equivalent to 10 ng total RNA/ ⁇ L and used as a template in real-time RT-PCR reactions to quantify both miRNA and GRF expression levels using the two-step RT-PCR kit (Bio-Rad) according to the manufacturer's protocol. PCR conditions and primer sequences are provided in the Supplemental Experimental Procedures.
  • Arabidopsis plants were grown vertically on modified Knop's medium for 2 weeks and then root tissues were collected for RNA extraction.
  • Affymetrix Arabidopsis gene chips (ATH1) were used to compare the gene expression in the wild type to gene expression in the triple mutant and the rGRF1 or rGRF3 plants.
  • Probe preparation was performed as described in the GeneChip® 3′ IVT Express Kit (Affymetrix, part number 901229) technical manual. Hybridization and washes were performed as described by Affymetrix in the GeneChip facility at Iowa State University.
  • Statistical analyses of gene expression levels are detailed in the Supplemental Experimental Procedures. Testing for the significance of gene list overlaps was determined using Chi-square tests. See Supplemental Experimental Procedures for details.
  • miR396a (AT2G10606) (SEQ ID NO:1), miR396b (AT5G35407)) (SEQ ID NO:2), GRF1 (At2g22840) (SEQ ID NO:3), GRF2 (At4g37740)) (SEQ ID NO:4), GRF3 (At2g36400)) (SEQ ID NO:5), GRF4 (At3g52910)) (SEQ ID NO:6), GRF5 (At3g13960)) (SEQ ID NO:7), GRF6 (At2g06200)) (SEQ ID NO:8), GRF7 (At5g53660) (SEQ ID NO:9), GRF8 (At4g24150) (SEQ ID NO:10), GRF9 (At2g45480)) (SEQ ID NO:11),
  • miR396 plays during soybean infection by the soybean cyst nematode ( Heterodera glycines ; SCN)
  • expression analyses were performed on primary and mature sequences for all miR396 paralogs (miR396a, miR396b, miR396c and miR396e) and seven of its predicted target Growth Regulating Transcription Factors (GRF8, GRF9, GRF12, GRF13, GRF15, GRF16 and GRF19) using quantitative real-time PCR (qRT-PCR).
  • GRF8 predicted target Growth Regulating Transcription Factors
  • GRF9 GRF9, GRF12, GRF13, GRF15, GRF16 and GRF19
  • qRT-PCR quantitative real-time PCR
  • RNA levels for miR396 and its target genes in soybean during SCN infection very closely resembled the observations made in Arabidopsis : an early downregulation of mature miR396 with a simultaneous increase in GRF mRNA at the time of syncytium formation. At later time points, likely coinciding with the end of syncytium formation, abundance of mature miR396 increases and GRF target gene expression is turned off. Consequently, there is a high probability that the manipulations we performed in Arabidopsis and that resulted in decreased plant susceptibility will have similar effects on susceptibility of soybean to SCN. Results are shown in FIG. 12 .
  • Sequences for soybean miRNA396 may be found at miRBase dot org at world wide web including accession numbers MIMAT0020922 (gma-miR3961-3p), MIMAT0001688 (gma-miR396B-5p), MIMAT0020923 (gma-miR396b-3p). MIMAT0010079 (gma-miR396c), MIMAT0018262 (gma-miR396d). Other miR396 sequences available from different plant species include but are not limited to:
  • “miR396a” Accession ID MI0001013 ath-MIR396a MI0001046 osa-MIR396a MI0001539 sbi-MIR396a MI0001785 gma-MIR396a MI0001801 zma-MIR396a MI0002325 ptc-MIR396a MI0005621 mtr-MIR396a MI0005650 ghr-MIR396a MI0005773 bna-MIR396a MI0006569 vvi-MIR396a MI0012094 aqc-MIR396a MI0014581 aly-MIR396a MI0016122 pab-MIR396a MI0016706 csi-MIR396a MI0016983 bgy-MIR396a MI0016987 bcy-MIR396a MI0017511 tcc-MIR396a MI0018111 bdi-MIR
  • “miR396b” Accession ID MI0001014 ath-MIR396b MI0001047 osa-MIR396b MI0001538 sbi-MIR396b MI0001786 gma-MIR396b MI0001800 zma-MIR396b MI0002326 ptc-MIR396b MI0005622 mtr-MIR396b MI0005651 ghr-MIR396b MI0006570 vvi-MIR396b MI0012095 aqc-MIR396b MI10014582 aly-MIR396b MI0016123 pab-MIR396b MI0016707 csi-MIR396b MI0016984 bgy-MIR396b MI0016988 bcy-MIR396b MI0017512 tcc-MIR396b M10018125 bdi-MIR396b MIMAT0001688 gma-M
  • “miR396c” Accession ID MI0001048 osa-MIR396c MI0001540 sbi-MIR396c MI0002327 ptc-MIR396c MI0007955 vvi-MIR396c MI0010569 zma-MIR396c MI0010572 gma-MIR396c MI0016124 pab-MIR396c MI0016735 csi-MIR396c MI0017513 tcc-MIR396c MI0018101 bdi-MIR396c
  • “miR396d” Accession ID MI0001702 osa-MIR396d MI0002328 ptc-MIR396d MI0006571 vvi-MIR396d MI0010570 zma-MIR396d MI0010897 sbi-MIR396d MI0016503 gma-MIR396d MI0017514 tcc-MIR396c MI0018096 bdi-MIR396d MI0001013 ath-MIR396a MI0001014 ath-MIR396b MI0001046 Osa-MIR396a MI0001047 osa-MIR396b MI0001048 osa-MIR396c

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160108411A1 (en) * 2014-10-14 2016-04-21 Clemson University Methods and compositions for transgenic plants with enhanced cold tolerance, ability to flower without vernalization requirement and impacted fertility
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US20220033841A1 (en) * 2018-09-27 2022-02-03 University Of Tennessee Research Foundation Discovery of soybean cyst nematode resistance genes based on epigenetic analysis
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060174380A1 (en) * 2005-01-07 2006-08-03 State Of Or. Acting By & Through The State Board Of Higher Education On Behalf Of Or. State Univ. Method to trigger RNA interference

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006298844B2 (en) * 2005-09-20 2012-01-12 Basf Plant Science Gmbh Methods for controlling gene expression using ta-siRAN
EP2330202A3 (en) * 2006-01-06 2011-09-14 University Of Georgia Research Foundation, Inc. Cyst nematode resistant transgenic plants
WO2010002984A1 (en) 2008-07-01 2010-01-07 Monsanto Technology, Llc Recombinant dna constructs and methods for modulating expression of a target gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060174380A1 (en) * 2005-01-07 2006-08-03 State Of Or. Acting By & Through The State Board Of Higher Education On Behalf Of Or. State Univ. Method to trigger RNA interference

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Hewezi et al, 2008, MPMI, 21:1622-1634 *
Hewezi et al, 2012, Plant Phys., 159:321-335 *
Liu et al, 2009, Physiologica Plantarum, 136:223-236 *
Mazarei et al, 2004, Mol. Plant Path., 5:409-423 *

Cited By (6)

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US20160108411A1 (en) * 2014-10-14 2016-04-21 Clemson University Methods and compositions for transgenic plants with enhanced cold tolerance, ability to flower without vernalization requirement and impacted fertility
US10023873B2 (en) * 2014-10-14 2018-07-17 Clemson University Methods and compositions for transgenic plants with enhanced cold tolerance, ability to flower without vernalization requirement and impacted fertility
CN111630174A (zh) * 2018-01-03 2020-09-04 科沃施萨特有限及两合公司 遗传修饰植物的再生
CN110066823A (zh) * 2018-01-22 2019-07-30 中国科学院上海生命科学研究院 Mim396在增大水稻穗形及提高水稻株高中的应用
US11873499B2 (en) * 2018-02-14 2024-01-16 Institute Of Genetics And Developmental Biology Chinese Academy Of Sciences Methods of increasing nutrient use efficiency
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