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WO2015071749A1 - Gènes régulateurs de végétaux favorisant l'association à une bactérie de fixation de l'azote - Google Patents

Gènes régulateurs de végétaux favorisant l'association à une bactérie de fixation de l'azote Download PDF

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WO2015071749A1
WO2015071749A1 PCT/IB2014/002488 IB2014002488W WO2015071749A1 WO 2015071749 A1 WO2015071749 A1 WO 2015071749A1 IB 2014002488 W IB2014002488 W IB 2014002488W WO 2015071749 A1 WO2015071749 A1 WO 2015071749A1
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Prior art keywords
plant
amino acid
polynucleotide
nucleic acid
seq
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Tatiana KRAISER
Bernardo GONZALES
Rodrigo GUTIÉRREZ
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Pontificia Universidad Catolica de Chile
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Pontificia Universidad Catolica de Chile
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Priority claimed from US14/083,193 external-priority patent/US20150143578A1/en
Priority claimed from CL2013003314A external-priority patent/CL2013003314A1/es
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Publication of WO2015071749A1 publication Critical patent/WO2015071749A1/fr
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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

Definitions

  • the present disclosure relates to plant biochemistry.
  • Embodiments relate to genetic factors regulating the association of plants with nitrogen-fixing bacteria.
  • Nitrogen is an essential macronutrient for plant growth and development, and it is a major limiting factor with regard to plant growth and crop productivity.
  • Traditional agriculture is based on nitrogen fertilizers to support world nutritional needs. However, such fertilizers are damaging to the environment and to human health when used in excess.
  • nitrogen-fixing bacteria NFB
  • NFB nitrogen-fixing bacteria
  • the main process transforming atmospheric N 2 into biologically useful nitrogen forms such as ammonium in natural ecosystems ⁇ See, e.g., Olivares et al. (2013) Mol. Plant Microbe Interact. 26:486).
  • nitrogen is often limiting in soils, some plant species have evolved molecular mechanisms to associate with nitrogen fixing bacteria (NFB). Zehr et al. (2003) Env. Microbiol. 5:539; Sprent and James (2007) Plant Physiol. 144:575; Kraiser et al. (201 1) J.
  • Bacteria and archaea are the only types of organisms capable of biological nitrogen fixation. Reduction of atmospheric N 2 to ammonium is catalyzed by the nitrogenase complex, composed of dinitrogenase and dinitrogenase reductase subunits. Joerger et al. (1991) J. Bacterid. 173:4440.
  • Plant-bacteria interactions associated with nitrogen nutrition have been primarily studied in legumes. Legumes are able to symbiotically associate with a phylogenetically diverse group of bacteria, collectively called rhizobia. Kistner and Parniske (2002) Trends Plant Sci. 7:511. Association of these plant species with their bacterial partners induces formation of a specialized organ called a "nodule.” Nodules harbor bacteria and provide appropriate conditions for nitrogen fixation to occur. Markmann and Parniske (2009) Trends Plant Sci. 14:77. In these nodules, the plant provides carbon sources in exchange for nitrogen fixed by the bacteria. Kistner and Parniske (2002), supra; Masson-Boivin et al. (2009) Trends Mirobiol.
  • AtNSPl-like, AtNLP4, and AtNLP9 Classes of genes that regulate a functional interaction for N-nutrition between non-nodulating plant species and NFB (e.g., Sinorhizobium meliloti) are disclosed.
  • non-natural nucleic acid molecules comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide is a polynucleotide that is at least 80% identical to SEQ ID NO:4; a polynucleotide that hybridizes under stringent (e.g., highly stringent) conditions to a nucleic acid consisting of SEQ ID NO:4; a polynucleotide that is at least 80% identical to SEQ ID NO:5; a polynucleotide that hybridizes under stringent (e.g., highly stringent) conditions to a nucleic acid consisting of SEQ ID NO:5; a polynucleotide that is at least 80% identical to SEQ ID NO: 6; or a polynucleotide that hybridizes under stringent (e.g., highly stringent) conditions to a nucleic acid consisting of SEQ ID NO:6.
  • the method may comprise introducing at least one heterologous polypeptide into the plant to produce a transgenic plant, wherein the heterologous polypeptide is a Nodulation Signaling Pathway-like (NSP) or NIN-like Protein (NLP).
  • the heterologous polypeptide is NSP1, NLP4, or NLP9.
  • Such a heterologous polypeptide may be, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs:l , 3, and 4.
  • the method for increasing nitrogen efficiency in a plant may comprise transforming the plant with a polynucleotide encoding the heterologous polypeptide.
  • the polynucleotide may be substantially identical to one of SEQ ID NOs:4-6, and/or be a homolog or ortholog thereof.
  • the polynucleotide encoding the heterologous polypeptide hybridizes under stringent (e.g., highly stringent) conditions to at least one of SEQ ID NOs:4-6.
  • Also described herein are methods for increasing nitrogen efficiency in a plant comprising introducing at least one at least one means for promoting association with NFB into the plant, for example, to produce a transgenic plant.
  • means for promoting association with NFB include a polypeptide consisting of SEQ ID NO:l ; a polypeptide consisting of SEQ ID NO:2; a polypeptide consisting of SEQ ID NO:3; a polynucleotide consisting of SEQ ID NO:4; a polynucleotide consisting of SEQ ID NO:5; and a polynucleotide consisting of SEQ ID NO:6.
  • transgenic plant in particular embodiments exhibits increased growth under limited nitrogen growing conditions, as compared to a wild-type plant of the same species.
  • FIG. 1A and IB includes a representation of the effect of NFB on plant growth under limiting N conditions.
  • N 40 mM NO3 + 20 mM NH 4
  • inoculated or not with different bacteria NFB: Burkholderia vietnamensis G4, B. xenovorans LB400, Cupriavidus taiwanensis LMG19424, Rhizobium etli CFN42, Sinorhizobium meliloti RMPl lO; and non-NFB: B. phytofirmans PSJN, C.
  • FIG. 1A Plant dry weight was measured for plants grown in N-containing medium for seven days and that were transplanted to limiting N conditions and inoculated with live or dead S. meliloti, or non-inoculated.
  • FIG. IB The number of lateral roots per plant were measured for plants grown and treated in the same conditions that are detailed above for FIG. 1A (mean of three independent biological replicates ⁇ SE; asterisk indicates means that differ significantly as compared to non-inoculated plants grown without N (P ⁇ 0.05)).
  • FIG. 2A and 2B includes a representation of the enhancement of plant growth by N fixation under N-limiting conditions.
  • Biomass was measured as dry weight of plants grown under sufficient (2.5 mM NH4NO 3 ) or limiting N conditions, inoculated or not with S. meliloti RMP1 10 wild-type or with the mutant type unable to fix N.
  • FIG. 2A Biological nitrogen fixation was measured by the 15 N dilution technique. Plants were grown in a medium with sufficient N (5% 15 N). After seven days, plants were transferred to plates with different treatments. Using mass spectrometry, the amounts of 14 N and 15 N were determined in harvested and dried plants 7 days after treatment, ⁇ ,5 N represents the 14 N: I5 N isotope ratio, relative to the non-inoculated condition (mean of three independent biological replicates ⁇ SE (P ⁇ 0.05)).
  • FIG. 2A Biological nitrogen fixation was measured by the 15 N dilution technique. Plants were grown in a medium with sufficient N (5% 15 N). After seven days, plants were transferred to plates with different treatments. Using mass spectrometry, the
  • FIG. 3A and 3B includes a representation of the induction of certain Arabidopsis genes upon bacterial inoculation.
  • NSP1 FIG. 3 A
  • NTN-like transcription factors FIG. 3A
  • Plants were grown under sufficient (2.5 mM NH4NO3) or limiting N conditions, and inoculated or not with NFB. Values plotted correspond to the mean of three independent biological replicates ⁇ SE (asterisk indicates means that differ significantly as compared to non-inoculated plants (P ⁇ 0.05)).
  • FIG. 4 includes a representation of the effect of mutations in Arabidopsis thaliana of genes essential for a functional association with S. meliloti RMP1 10; AtNSPl-like, AtNLP4, AtNLP8, and AtNLP9 transcription factors. Biomass is expressed as dry weight measured in wild-type and mutant plants grown under limiting nitrogen conditions and inoculated or not with S. meliloti (mean of three independent biological replicates ⁇ SE; asterisk indicates means that differ significantly as compared to non-inoculated plants (P ⁇ 0.05)).
  • FIG. 5 includes an image showing the effect of NFB on root hair length. Plants grown in complete MS salt medium for seven days were transplanted to MS medium without N, and inoculated or not with NFB. Pictures were taken seven days after treatment.
  • FIG. 6A and 6B includes a representation of the effect of S. meliloti on
  • FIG. 6A Lateral root density (FIG. 6A) and primary root length (FIG. 6B) were measured in plants grown under sufficient/limiting N conditions, inoculated or not with S. meliloti RMP1 10 wild-type or with the mutant type unable to fix N. Values plotted correspond to the mean of three independent biological replicates ⁇ SE.
  • FIG. 7A and 7B includes a representation of the response to bacterium inoculation of AtNSP2-like, AtNLPl, AtNLP2, and AtNLP5.
  • Gene expression was measured by real-time quantitative reverse transcription PCR at the third or seventh day post treatment (mean of three independent biological replicates ⁇ SE; asterisk indicates means that differ significantly as compared to non-inoculated plants (P ⁇ 0.05)). Plants were grown under sufficient or limiting N conditions, and inoculated or not with S. meliloti.
  • nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. ⁇ 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
  • SEQ ID NO: l shows an NSPl-like (SCL29; At3gl3840) polypeptide:
  • SEQ ID NO:3 shows an NLP9 (At3g59580) polypeptide:
  • MENPSASRDNKGFCFPDIPVEE DGWVKNLISEED FSSSSTSEL MNFESFASWCNSPSAADILFTQYGLSTSQSIIPFGGLEGSYACEKRPLD CTSVPRSLSHSLDEKMLKALSLFMEFSGEGILAQFWTPIKTGDQYMLST CDQAYLLDSRLSGYREASRRFTFSAEANQCSYPGLPGRVFISGVPEWTS NVMYYKTAEYLRMKHALDNEVRGSIAIPVLEASGSSCCAVLELVTCREK PN FD VEMN S VCRALQAVNLQT ST I PRRQYLS SNQKEALAE I RDVLRAVC YAHRLPLALAWIPCSYSKGANDELVKVYGKNSKECSLLCIEETSCYVND MEMEGFVNACLEHYLREGQGIVGKALISNKPSFSSDVKTFDICEYPLVQ HARKFGLNAAVATKLRSTFTGDNDYILEFFLPVSMKGSSEQQLLLDSLS GTMQRL
  • EEEWVMLVTDSDLHECFEILNGMRKHTVKFLVRDIPNTAMGSSAG SNGYLGTGT SEQ ID NO:4 shows an NSPl-like (SCL29; At3gl3840) polynucleotide coding sequence (CDS):
  • SEQ ID NO-.5 shows an NLP4 (Atlg20640) polynucleotide coding sequence (CDS):
  • NFB Newcastle disease virus
  • a symbiotic signal transduction pathway is activated to induce nodule development in the plant.
  • calcium oscillation generates the induction of the primary transcription factors, NSP1 and NSP2, which induce N7N gene expression.
  • NSP1 and NSP2 Some components of the symbiotic transduction pathway are shared between rhizobium and arbuscular mycorrhizal fungi associations. Oldroyd (2013), supra. However and unlike NSP2, NSP1 and NIN genes are specifically implicated in the association with NFB.
  • Described herein is a mechanism that promotes association of non-nodulating plant species with NFB for improved N-nutrition.
  • Arabidopsis associates with Sinorhizobium meliloti, a NFB capable of providing reduced nitrogen for plant nutrition, contributing to plant growth under N-limiting conditions.
  • Embodiments herein employ plant transcription factors identified for the first time as essential for functional association between non-nodulating plants and S. meliloti. We have discovered that homologous genes of transcription factors that are specific for the Iegume:NFB association are necessary to mediate plant growth promotion induced by S. meliloti in non-nodulating plants.
  • the conservation of the association mechanism across plant species allows for the association between Arabidopsis and S. meliloti to be recapitulated in other plants through genetic modification, to govern beneficial interactions with NFB for N-nutrition, for example, to enhance nitrogen use efficiency in a cropping system.
  • S. meliloti a bacterium that associates with the legume, Medicago sativa. Marsh et al. (2007) Plant Physiol. 144:324; Peiter et al. (2007) Plant Physiol. 145:192. Under free-living conditions and in association with legumes, S. meliloti performs N-fixation only under N-limiting conditions. Szeto et al. (1987) J. Bacteriol. 169: 1423. As further described herein, S. meliloti promotes plant growth under N-limiting conditions (e.g., in a medium without an additional N-source), and is thus suitable for N-fixation and the promotion of plant growth due to BNF.
  • N-limiting conditions e.g., in a medium without an additional N-source
  • Backcrossing methods may be used to introduce a nucleic acid sequence into plants.
  • the backcrossing technique has been widely used for decades to introduce new traits into plants. Jensen, N., Ed. Plant Breeding Methodology, John Wiley & Sons, Inc., 1988.
  • the original variety of interest recurrent parent
  • a second variety non-recurrent parent
  • the resulting progeny from this cross are then crossed again to the recurrent parent, and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent plant are recovered in the converted plant, in addition to the transferred gene from the non-recurrent parent.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins), while effecting a chemical or functional change in the component (e.g., a nucleic acid may be isolated from a chromosome by breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome).
  • Nucleic acid molecules and proteins that have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods, wherein there has been a chemical or functional change in the nucleic acid or protein. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically-synthesized nucleic acid molecules, proteins, and peptides.
  • nucleic acid molecule may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide.
  • a "nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.”
  • a nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA.
  • a nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.).
  • the term "nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially du
  • Oligonucleotide An oligonucleotide is a short nucleic acid molecule. Oligonucleotides may be formed by cleavage of longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred base pairs in length. Because oligonucleotides may bind to a complementary nucleotide sequence, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a technique for the amplification of small DNA sequences. In PCR, the oligonucleotide is typically referred to as a "primer," which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.
  • a nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, inter-nucleotide modifications (e.g.
  • nucleic acid molecule also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
  • coding sequence refers to a nucleotide sequence that is transcribed into RNA when placed under the control of appropriate regulatory sequences.
  • a "protein coding sequence” is a nucleotide sequence (DNA or RNA) that is ultimately translated into a polypeptide, via transcription and mRNA.
  • coding sequence refers to a nucleotide sequence that is translated into a peptide, polypeptide, or protein. The boundaries of a coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding sequences include, but are not limited to: genomic DNA; cDNA; EST; and recombinant nucleotide sequences.
  • Genome refers to chromosomal DNA found within the nucleus of a cell, and also refers organelle DNA found within subcellular components of the cell.
  • a DNA molecule may be introduced into a plant cell such that the DNA molecule is integrated into the genome of the plant cell.
  • the DNA molecule may be either integrated into the nuclear DNA of the plant cell, or integrated into the DNA of the chloroplast or mitochondrion of the plant cell.
  • Endogenous refers to one or more nucleic acid(s) that are normally (e.g., in a wild-type cell of the same type and species) present within their specific environment or context.
  • an endogenous gene is one that is normally found in the particular cell in question and in the same context (e.g., with regard to regulatory sequences).
  • Endogenous nucleic acids can be distinguished from exogenous and/or heterologous, for example and without limitation, by detection in the latter of sequences that are consequent with recombination from bacterial plasmid; identification of atypical codon preferences; and amplification of atypical sequences in a PCR reaction from primers characterized in a wild-type cell.
  • exogenous refers to one or more nucleic acid(s) that are not normally present within their specific environment or context. For example, if a host cell is transformed with a nucleic acid that does not occur in the untransformed host cell in nature, then that nucleic acid is exogenous to the host cell.
  • exogenous also refers to one or more nucleic acid(s) that are identical in sequence to a nucleic acid already present in a host cell, but that are located in a different cellular or genomic context than the nucleic acid with the same sequence already present in the host cell.
  • a nucleic acid that is integrated in the genome of the host cell in a different location than a nucleic acid with the same sequence is normally integrated in the genome of the host cell is exogenous to the host cell.
  • a nucleic acid e.g., a DNA molecule
  • a nucleic acid that is present in a plasmid or vector in the host cell is exogenous to the host cell when a nucleic acid with the same sequence is only normally present in the genome of the host cell.
  • Heterologous as applied to nucleic acids (e.g., polynucleotides, DNA, RNA, and genes) herein, means of different origin. For example, if a host cell is transformed with a nucleic acid that does not occur in the untransformed host cell in nature, then that nucleic acid is heterologous (and exogenous) to the host cell. Furthermore, different elements (e.g., promoter, enhancer, coding sequence, terminator, etc.) of a transforming nucleic acid may be heterologous to one another and/or to the transformed host.
  • nucleic acids e.g., polynucleotides, DNA, RNA, and genes
  • heterologous may also be applied to one or more nucleic acid(s) that are identical in sequence to a nucleic acid already present in a host cell, but that are now linked to different additional sequences and/or are present at a different copy number, etc.
  • sequence identity refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • the term "percentage of sequence identity” may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences, and amino acid sequences) over a comparison window, wherein the portion of the 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 nucleotide 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 comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
  • NCBI National Center for Biotechnology Information
  • BLASTTM Altschul et al. (1990)
  • BLASTTM Altschul et al. (1990)
  • Bethesda, MD National Center for Biotechnology Information
  • Blastn the "Blast 2 sequences" function of the BLASTTM (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
  • hybridizable/specifically complementary are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and a target nucleic acid molecule.
  • Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleic acid sequences of the two nucleic acid molecules. The two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that, if it is sufficiently stable, is detectable using methods well known in the art.
  • a nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. However, the amount of sequence complementarity that must exist for hybridization to be specific is a function of the hybridization conditions used.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na + and/or Mg ++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are known to those of ordinary skill in the art, and are discussed, for example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the hybridization molecule and a homologous sequence within the target nucleic acid molecule.
  • Stringent conditions include further particular levels of stringency.
  • “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize;
  • conditions of "high stringency” are those under which sequences with more than 10% mismatch will not hybridize;
  • conditions of "very high stringency” are those under which sequences with more than 5% mismatch will not hybridize.
  • High Stringency condition detects sequences that share at least 90% sequence identity: Hybridization in 5x SSC buffer at 65°C for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer at 65°C for 20 minutes each.
  • Moderate Stringency condition detects sequences that share at least 80% sequence identity: Hybridization in 5x-6x SSC buffer at 65-70°C for 16-20 hours; wash twice in 2x SSC buffer at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer at 55-70°C for 30 minutes each.
  • Non-stringent control condition sequences that share at least 50% sequence identity will hybridize: Hybridization in 6x SSC buffer at room temperature to 55°C for 16-20 hours; wash at least twice in 2x-3x SSC buffer at room temperature to 55°C for 20-30 minutes each.
  • substantially homologous or “substantial homology,” with regard to a polynucleotide, refers to polynucleotides that hybridize under stringent conditions to the reference nucleic acid sequence.
  • polynucleotides that are substantially homologous to a reference DNA coding sequence are those polynucleotides that hybridize under stringent conditions (e.g., the Moderate Stringency conditions set forth, supra) to the reference DNA coding sequence.
  • Substantially homologous sequences may have at least 80% sequence identity.
  • substantially homologous sequences may have from about 80% to 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%.
  • the property of substantial homology is closely related to specific hybridization.
  • a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions.
  • ortholog refers to a gene in two or more species that has evolved from a common ancestral nucleotide sequence, and may retain the same function in the two or more species.
  • nucleic acid sequence molecules are said to exhibit "complete complementarity" when every nucleotide of a sequence read in the 5' to 3' direction is complementary to every nucleotide of the other sequence when read in the 3' to 5' direction.
  • a nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence identical to the reverse complement sequence of the reference nucleotide sequence.
  • substantially identical may refer to nucleotide sequences that are more than 85% identical.
  • a substantially identical nucleotide sequence may be at least 85.5%; at least 86%; at least 87%; at least 88%; at least 89%; at least 90%; at least 91 %; at least 92%; at least 93%; at least 94%; at least 95%; at least 96%; at least 97%; at least 98%; at least 99%; or at least 99.5% identical to the reference sequence.
  • expression of a coding sequence refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell (e.g., a protein).
  • Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases expression of a gene comprised therein. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein.
  • Gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, and/or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations of any of the foregoing.
  • Gene expression can be measured at the RNA level or the protein level by methods known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, and in vitro, in situ, or in vivo protein activity assay(s).
  • Increase expression refers to initiation of expression, as well as to a quantitative increase in the amount of an expression product produced from a template construct.
  • at least one heterologous gene may be provided to a cell or organism that otherwise comprises an endogenous copy of the same gene, so as to increase the expression of the polypeptide encoded by the gene.
  • the increase in expression may be determined by comparison of the amount of the polypeptide produced in the cell comprising the heterologous and endogenous genes, with the amount produced in the cell comprising only the endogenous gene.
  • a first polypeptide that affects transcription may be provided to a cell or organism, so as to increase the expression of a second polypeptide encoded by a gene under the control of the first polypeptide.
  • the increase in expression may be determined by comparison of the amount of the polypeptide produced from the gene in the presence of the first polypeptide, with the amount produced from the gene in the absence of the first polypeptide.
  • a regulatory sequence may be operably linked to a gene, so as to increase the expression of the gene.
  • the increase in expression may be determined by comparison of the amount of the polypeptide produced from the gene after operable linkage of the regulatory sequence thereto, with the amount produced from the gene before operable linkage or introduction of the regulatory sequence.
  • a first nucleotide sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence.
  • operably linked nucleic acid sequences are generally contiguous, and, where necessary to join two protein-coding regions, in the same reading frame (e.g., in a polycistronic ORF). However, nucleic acids need not be contiguous to be operably linked.
  • operably linked when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence.
  • regulatory sequences or “control elements,” refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
  • promoter refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
  • a plant promoter is a promoter that is capable of initiating transcription in a plant cell.
  • Some embodiments herein include a "tissue-preferred promoter.”
  • a tissue-preferred promoter is a promoter that is capable of initiating transcription under developmental control, and include, for example and without limitation: promoters that preferentially initiate transcription in leaves, pollen, tassels, roots, seeds, fibers, xylem vessels, tracheids, and sclerenchyma. Promoters that initiate transcription essentially only in certain tissues are referred to as "tissue-specific.”
  • 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” promoter may be a promoter which may be under environmental control. Examples of environmental conditions that may initiate transcription by inducible promoters include anaerobic conditions and the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters.
  • any inducible promoter may be used in some embodiments herein. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With an inducible promoter, the rate of transcription increases in response to an inducing agent.
  • exemplary inducible promoters include, but are not limited to: Promoters from the ACEI system that responds to copper; In2 gene from maize that responds to benzenesulfonamide herbicide safeners; Tet repressor from TnlO; and the inducible promoter from a steroid hormone gene, the transcriptional activity of which may be induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-5).
  • a "constitutive" promoter is a promoter that is active under most environmental conditions.
  • Exemplary constitutive promoters include, but are not limited to: promoters from plant viruses, such as the 35S promoter from CaMV; promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter; and the ALS promoter, Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xbal Ncol fragment) (PCT International Patent Publication No. WO 96/30530).
  • tissue-specific or tissue-preferred promoter may be utilized in some embodiments of the invention. Plants transformed with a nucleic acid molecule comprising a coding sequence operably linked to a tissue-specific promoter may produce the product of the coding sequence exclusively, or preferentially, in a specific tissue.
  • tissue-specific or tissue-preferred promoters include, but are not limited to: a root-preferred promoter, such as that from the phaseolin gene; a leaf-specific and light-induced promoter such as that from cab or rubisco; an anther-specific promoter such as that from LAT52; a poiien-specific promoter such as that from Zml3; and a microspore-preferred promoter such as that from apg.
  • conservative substitution refers to a substitution where an amino acid residue is substituted for another amino acid in the same class.
  • a non-conservative amino acid substitution is one where the residues do not fall into the same class, for example, substitution of a basic amino acid for a neutral or non-polar amino acid.
  • Classes of amino acids that may be defined for the purpose of performing a conservative substitution are known in the art.
  • a conservative substitution includes the substitution of a first aliphatic amino acid for a second, different aliphatic amino acid. For example, if a first amino acid is one of Gly; Ala; Pro; He; Leu; Val; and Met, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ala; Pro; He; Leu; Val; and Met. In particular examples, if a first amino acid is one of Gly; Ala; Pro; He; Leu; and Val, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ala; Pro; He; Leu; and Val.
  • a first amino acid is one of Ala; Pro; He; Leu; and Val
  • the first amino acid may be replaced by a second, different amino acid selected from Ala; Pro; He; Leu; and Val.
  • a conservative substitution includes the substitution of a first aromatic amino acid for a second, different aromatic amino acid. For example, if a first amino acid is one of His; Phe; Trp; and Tyr, the first amino acid may be replaced by a second, different amino acid selected from His; Phe; Trp; and Tyr. In particular examples involving the substitution of uncharged aromatic amino acids, if a first amino acid is one of Phe; Trp; and Tyr, the first amino acid may be replaced by a second, different amino acid selected from Phe; Trp; and Tyr.
  • a conservative substitution includes the substitution of a first hydrophobic amino acid for a second, different hydrophobic amino acid. For example, if a first amino acid is one of Ala; Val; He; Leu; Met; Phe; Tyr; and Tip, the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; He; Leu; Met; Phe; Tyr; and Tip.
  • the substitution of non-aromatic, hydrophobic amino acids if a first amino acid is one of Ala; Val; He; Leu; and Met, the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; He; Leu; and Met.
  • a conservative substitution includes the substitution of a first polar amino acid for a second, different polar amino acid. For example, if a first amino acid is one of Ser; Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid may be replaced by a second, different amino acid selected from Ser; Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu.
  • the first amino acid may be replaced by a second, different amino acid selected from Ser; Thr; Asn; Gin; Cys; Gly; and Pro.
  • the substitution of charged, polar amino acids if a first amino acid is one of His; Arg; Lys; Asp; and Glu, the first amino acid may be replaced by a second, different amino acid selected from His; Arg; Lys; Asp; and Glu.
  • the first amino acid may be replaced by a second, different amino acid selected from Arg; Lys; Asp; and Glu.
  • the substitution of positively charged (basic), polar amino acids if a first amino acid is one of His; Arg; and Lys, the first amino acid may be replaced by a second, different amino acid selected from His; Arg; and Lys.
  • the substitution of positively charged, polar amino acids if a first amino acid is Arg or Lys, the first amino acid may be replaced by the other amino acid of Arg and Lys.
  • the substitution of negatively charged (acidic) polar amino acids if a first amino acid is Asp or Glu, the first amino acid may be replaced by the other amino acid of Asp and Glu.
  • a conservative substitution includes the substitution of a first electrically neutral amino acid for a second, different electrically neutral amino acid. For example, if a first amino acid is one of Gly; Ser; Thr; Cys; Asn; Gin; and Tyr, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ser; Thr; Cys; Asn; Gin; and Tyr. In some embodiments, a conservative substitution includes the substitution of a first non-polar amino acid for a second, different non-polar amino acid.
  • a first amino acid is one of Ala; Val; Leu; He; Phe; Tip; Pro; and Met
  • the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; Leu; He; Phe; Tip; Pro; and Met.
  • the selection of a particular second amino acid to be used in a conservative substitution to replace a first amino acid may be made in order to maximize the number of the foregoing classes to which the first and second amino acids both belong.
  • the second amino acid may be another polar amino acid (i.e., Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; or Glu); another non-aromatic amino acid (i.e., Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; He; Leu; Val; or Met); or another electrically-neutral amino acid (i.e., Gly; Thr; Cys; Asn; Gin; or Tyr).
  • the second amino acid in this case be one of Thr; Asn; Gin; Cys; and Gly, because these amino acids share all the classifications according to polarity, non-aromaticity, and electrical neutrality. Additional criteria that may optionally be used to select a particular second amino acid to be used in a conservative substitution are known in the art. For example, when Thr; Asn; Gin; Cys; and Gly are available to be used in a conservative substitution for Ser, Cys may be eliminated from selection in order to avoid the formation of undesirable cross- linkages and/or disulfide bonds. Likewise, Gly may be eliminated from selection, because it lacks an alkyl side chain. In this case, Thr may be selected, e.g., in order to retain the functionality of a side chain hydroxyl group. The selection of the particular second amino acid to be used in a conservative substitution is ultimately, however, within the discretion of the skilled practitioner.
  • Nitrogen-limiting conditions refers to conditions wherein there is a limited amount of nitrogen sources (e.g., nitrate and ammonium) in the soil or culture medium.
  • the amount that is "limiting” is in some examples a range of nitrogen concentration from 0.0 to 0.2 mM; e.g., from 0 to 0.1 mM, from 0 to 0.03 mM, and from 0 to 0.05 mM.
  • traits of particular interest include agronomically important traits, as may be expressed, for example, in a crop plant.
  • transformation refers to the transfer of one or more nucleic acid molecule(s) into a cell.
  • a cell is "transformed” by a nucleic acid molecule introduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication.
  • transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al.
  • transgene is an exogenous nucleic acid sequence.
  • a transgene may be a sequence that encodes one or both strand(s) of a dsRNA molecule that comprises a nucleotide sequence that is complementary to a target nucleic acid.
  • a transgene may be an antisense nucleic acid sequence, the expression of which inhibits expression of a target nucleic acid.
  • a transgene may be a gene sequence ⁇ e.g. , a herbicide-resistance gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait.
  • a transgene may contain regulatory sequences operably linked to the coding sequence of the transgene ⁇ e.g. , a promoter).
  • a vector refers to a nucleic acid molecule as introduced into a cell, for example, to produce a transformed cell.
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. Examples of vectors include, but are not limited to: a plasmid; cosmid; bacteriophage; and a virus that carries exogenous DNA into a cell.
  • a vector may also include one or more genes, antisense molecules, and/or selectable marker genes and other genetic elements known in the art.
  • a vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and/or proteins encoded by the vector.
  • a vector optionally includes materials to aid in achieving entry of the nucleic acid molecule into the cell (e.g., a liposome, protein coating, etc.).
  • NSPl-like and NSP2-like, and NIN-like genes e.g., NLPsl-9.
  • NSPl-like, NLP4, and NLP9 influence the functional interaction between plants (e.g., non-nodulating plants, such as Arabidopsis) and NFB (e.g., S. meliloti, such as RMP110).
  • NSPl-like, NLP4, and/or NLP9 may be used to regulate the association of a NFB with a crop plant.
  • NSPl-like, NLP4, and NLP9 described herein may be used, for example, to provide transgenic plants with an altered BNF phenotype.
  • NSPl-like (SCL29), NLP4, and/or NLP9 may be expressed or over-expressed in a plant to initiate and/or increase the ability of the plant to associate with NFB under N-limiting conditions, and/or to increase the efficiency of the plant's utilization of environmental nitrogen.
  • NSPl -like (SCL29), NLP4, and/or NLP9 may be introduced into a plant or over-expressed in a plant that normally comprises the polypeptide (e.g., by introducing additional copies of a gene encoding the polypeptide, and/or changing the regulatory control of the normally present gene).
  • NSPl-like polypeptides comprise an amino acid sequence showing increasing percentage identities when aligned with SEQ ID NO:l (Arabidopsis thaliana NSPl-like).
  • Specific amino acid sequences within these and other embodiments may comprise sequences having, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1.
  • NIN-like protein polypeptide examples include an NIN-like protein polypeptide.
  • Particular embodiments include an NLP4 polypeptide, and/or an NLP9 polypeptide.
  • NLP4 polypeptides comprise an amino acid sequence showing increasing percentage identities when aligned with SEQ ID NO:2 ⁇ A. thaliana NLP4).
  • Specific amino acid sequences within these and other embodiments may comprise sequences having, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%), or 100%) identity with SEQ ID NO:2.
  • some embodiments include an AtNLP4 ortholog.
  • NLP9 polypeptides comprise an amino acid sequence showing increasing percentage identities when aligned with SEQ ID NO:3A. thaliana NLP9).
  • Specific amino acid sequences within these and other embodiments may comprise sequences having, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO:9.
  • some embodiments include an AtNLP9 ortholog.
  • a polypeptide comprising an amino acid sequence having the aforementioned sequence identity when aligned with SEQ ID NO:l (NSPl-like polypeptides) or SEQ ID NO:2 (NLP4 polypeptides) and/or SEQ ID NO:3 (NLP9 polypeptides) is comprised within a protein that is required for the association of a plant comprising the protein and NFB.
  • NSPl-like polypeptides may be identified, for example, by searching a sequence database for polypeptide sequences having a threshold sequence identity with SEQ ID NO:l .
  • NLP4 polypeptides may be identified, for example, by searching a sequence database for polypeptide sequences having a certain sequence identity with SEQ ID NO:2.
  • NLP9 polypeptides may be identified, for example, by searching a sequence database for polypeptide sequences having a certain sequence identity with SEQ ID NO:3.
  • Useful sequence databases may be searched by any of many methods known to those of skill in the art ⁇ e.g., utilizing NCBI's BLAST ® tool). Other databases are available for many plants and other organisms through a variety of public and private commercial sources.
  • NLP4 and NLP9 are homologous proteins, and thus, a particular polypeptide identified as comprising an amino acid sequence sharing sequence identity with SEQ ID NO:2 or SEQ ID NO:3 may also share sequence identity with the other of SEQ ID NOs:2 and 4.
  • Structural equivalents include, but are riot limited to, conservative substitutions of amino acid residues within the amino acid sequence of the NSPl-like, NLP4, and NLP9 polypeptides herein.
  • a "conservative substitution” is a substitution where an amino acid residue is substituted for another amino acid in the same class.
  • a non-conservative amino acid substitution is one where the residues do not fall into the same class, for example, substitution of a basic amino acid for a neutral or non-polar amino acid.
  • Classes of amino acids that may be defined for the purpose of performing a conservative substitution are known in the art.
  • a conservative substitution includes the substitution of a first aliphatic amino acid for a second, different aliphatic amino acid. For example, if a first amino acid is one of Gly; Ala; Pro; He; Leu; Val; and Met, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ala; Pro; He; Leu; Val; and Met. In particular examples, if a first amino acid is one of Gly; Ala; Pro; He; Leu; and Val, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ala; Pro; He; Leu; and Val.
  • a conservative substitution includes the substitution of a first aromatic amino acid for a second, different aromatic amino acid.
  • a first amino acid is one of His; Phe; Trp; and Tyr
  • the first amino acid may be replaced by a second, different amino acid selected from His; Phe; Trp; and Tyr.
  • the substitution of uncharged aromatic amino acids if a first amino acid is one of Phe; Trp; and Tyr, the first amino acid may be replaced by a second, different amino acid selected from Phe; Trp; and Tyr.
  • a conservative substitution includes the substitution of a first hydrophobic amino acid for a second, different hydrophobic amino acid. For example, if a first amino acid is one of Ala; Val; He; Leu; Met; Phe; Tyr; and Trp, the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; He; Leu; Met; Phe; Tyr; and Trp. In particular examples involving the substitution of non-aromatic, hydrophobic amino acids, if a first amino acid is one of Ala; Val; He; Leu; and Met, the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; lie; Leu; and Met.
  • a conservative substitution includes the substitution of a first polar amino acid for a second, different polar amino acid. For example, if a first amino acid is one of Ser; Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid may be replaced by a second, different amino acid selected from Ser; Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu.
  • the first amino acid may be replaced by a second, different amino acid selected from Ser; Thr; Asn; Gin; Cys; Gly; and Pro.
  • the substitution of charged, polar amino acids if a first amino acid is one of His; Arg; Lys; Asp; and Glu, the first amino acid may be replaced by a second, different amino acid selected from His; Arg; Lys; Asp; and Glu.
  • the first amino acid may be replaced by a second, different amino acid selected from Arg; Lys; Asp; and Glu.
  • the substitution of positively charged (basic), polar amino acids if a first amino acid is one of His; Arg; and Lys, the first amino acid may be replaced by a second, different amino acid selected from His; Arg; and Lys.
  • the substitution of positively charged, polar amino acids if a first amino acid is Arg or Lys, the first amino acid may be replaced by the other amino acid of Arg and Lys.
  • the substitution of negatively charged (acidic) polar amino acids if a first amino acid is Asp or Glu, the first amino acid may be replaced by the other amino acid of Asp and Glu.
  • a conservative substitution includes the substitution of a first electrically neutral amino acid for a second, different electrically neutral amino acid. For example, if a first amino acid is one of Gly; Ser; Thr; Cys; Asn; Gin; and Tyr, the first amino acid may be replaced by a second, different amino acid selected from Gly; Ser; Thr; Cys; Asn; Gin; and Tyr.
  • a conservative substitution includes the substitution of a first non-polar amino acid for a second, different non-polar amino acid. For example, if a first amino acid is one of Ala; Val; Leu; He; Phe; Trp; Pro; and Met, the first amino acid may be replaced by a second, different amino acid selected from Ala; Val; Leu; He; Phe; Trp; Pro; and Met.
  • the selection of a particular second amino acid to be used in a conservative substitution to replace a first amino acid may be made in order to maximize the number of the foregoing classes to which the first and second amino acids both belong.
  • the second amino acid may be another polar amino acid (i.e., Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; or Glu); another non-aromatic amino acid (i.e., Thr; Asn; Gin; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; He; Leu; Val; or Met); or another electrically-neutral amino acid (i.e., Gly; Thr; Cys; Asn; Gin; or Tyr).
  • the second amino acid in this case be one of Thr; Asn; Gin; Cys; and Gly, because these amino acids share all the classifications according to polarity, non-aromaticity, and electrical neutrality. Additional criteria that may optionally be used to select a particular second amino acid to be used in a conservative substitution are known in the art. For example, when Thr; Asn; Gin; Cys; and Gly are available to be used in a conservative substitution for Ser, Cys may be eliminated from selection in order to avoid the formation of undesirable cross-linkages and/or disulfide bonds. Likewise, Gly may be eliminated from selection, because it lacks an alkyl side chain. In this case, Thr may be selected, e.g., in order to retain the functionality of a side chain hydroxy! group. The selection of the particular second amino acid to be used in a conservative substitution is ultimately, however, within the discretion of the skilled practitioner.
  • NSPl-like, NLP4, and NLP9 coding polynucleotides can be made to generate expression products that are better suited for, for example, expression and scale up, in a host cell comprising the polynucleotide(s).
  • cysteine residues can be deleted or substituted with another amino acid in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • Hydrophilic amino acids generally include and generally have the respective relative degree of hydrophobicity (at pH 7.0; kcal/mol) as follows: aspartic acid (D),-7.4; glutamic acid (E)-9.9; asparagine (N),-0.2; glutamine (Q),-0.3; lysine (K),-4.2; arginine (R),-1 1.2; serine (S),-0.3; and cysteine (C),-2.8.
  • Hydrophobic amino acids generally include and generally have the respective relative degree of hydrophobicity as follows: histidine (H), 0.5; threonine (T), 0.4; tyrosine (Y), 2.3; tryptophan (W), 3.4; phenylalanine (F), 2.5; leucine (L), 1.8; isoleucine (I), 2.5; methionine (M), 1.3; valine (V), 1.5; and alanine (A), 0.5.
  • Glycine has a relative degree of hydrophobicity of 0 and may be considered to be hydrophilic or hydrophobic.
  • amino acid homology of peptides can be readily determined by contrasting the amino acid sequences thereof as is known in the art.
  • amphiphilic homology of peptides can be determined by contrasting the hydrophilicity and hydrophobicity of the amino acid sequences.
  • nucleic acid comprising a nucleotide sequence encoding a NSPl-like (a "NSPl -like polynucleotide"), NLP4 (a “NLP4 polynucleotide”), and/or NLP9 polypeptide (a "NLP9 polynucleotide”), such as are described above.
  • NSPl -like polynucleotide a nucleotide sequence encoding a NSPl-like polynucleotide
  • NLP4 a “NLP4 polynucleotide”
  • NLP9 polypeptide a “NLP9 polynucleotide”
  • nucleic acid sequences within these and other embodiments may comprise sequences having, for example and without limitation, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identity SEQ ID NO:4 or SEQ ID NO:5 and/or SEQ ID NO:6.
  • nucleic acids comprising a nucleotide sequence encoding a NSPl-like, NLP4, and/or NLP9 polypeptide can be readily identified by those of skill in the art.
  • nucleic acid molecules may be modified without substantially changing the amino acid sequence of the encoded polypeptide, for example, by introducing permissible nucleotide substitutions according to codon degeneracy.
  • any NSPl-like, NLP4, and/or NLP9 polypeptide with a given amino acid sequence may be immediately reverse-engineered to any of many redundant nucleotide sequences.
  • genes encoding a NSPl-like, NLP4, and/or NLP9 polypeptide may be selected from any of the many available plant genomic libraries, cDNA libraries, EST libraries, and the like ⁇ e.g., by homology to one of SEQ ID NOs:4-6), or by sequence similarity of an encoded polypeptide with SEQ ID NO: l or SEQ ID NOs:2 and/or 3, or such genes may be cloned from an organism according to reliable and well-known techniques in molecular biology.
  • NSPl-like, NLP4, and NLP9 polypeptides may be utilized in certain embodiments of the invention.
  • a nucleic acid comprising a nucleotide sequence encoding a NSPl-like, NLP4, and/or NLP9 polypeptide comprises a gene regulatory element ⁇ e.g., a promoter).
  • Promoters may be selected on the basis of the cell type into which the vector construct will be inserted. Promoters which function in bacteria, yeast, and plants are well-known in the art. The promoters may also be selected on the basis of their regulatory features. Examples of such features include enhancement of transcriptional activity, inducibility, tissue-specificity, and developmental stage-specificity. In plants, promoters that are inducible, of viral or synthetic origin, constitutive ly active, temporally regulated, and spatially regulated have been described. See, e.g., Poszkowski et al. (1989) EMBO J. 3:2719; Odell et al. (1985) Nature 313:810; and Chau et al. (1989) Science 244:174-81).
  • a heterologous gene(s) it may be preferred to reengineer the gene(s) so that it is more efficiently expressed in the expression host cell ⁇ e.g., a plant cell, for example, canola, rice, tobacco, maize, cotton, and soybean). Therefore, an optional additional step in the design of a gene encoding a NSPl-like or NLP4 and/or NLP9 polypeptide for plant expression ⁇ i.e., in addition to the provision of one or more gene regulatory elements) is reengineering of a heterologous gene protein coding region for optimal expression.
  • Particular examples include a redesigned Arabidopsis gene that has been optimized to increase the expression level ⁇ i.e., produce more protein) in a transgenic plant cell from a second plant species than in a plant cell from the second plant species transformed with the original ⁇ i.e., unmodified) Arabidopsis gene sequence.
  • NSPl -like or NLP4 and/or NLP9 polypeptide for expression in a plant cell ⁇ e.g., rice, tobacco, maize, cotton, and soybean
  • a plant cell e.g., rice, tobacco, maize, cotton, and soybean
  • codon bias of the prospective host plant(s) it is helpful if the codon bias of the prospective host plant(s) has been determined.
  • the codon bias is the statistical distribution of codons that the expression host uses for coding the amino acids of its proteins.
  • the codon bias can be calculated as the frequency at which a single codon is used relative to the codons for all amino acids.
  • the codon bias may be calculated as the frequency at which a single codon is used to encode a particular amino acid, relative to all the other codons for that amino acid (synonymous codons).
  • the primary (“first choice”) codons preferred by the plant should be determined, as well as the second, third, fourth, etc., choices of preferred codons when multiple choices exist.
  • a new DNA sequence can then be designed which encodes the amino sequence of the NSPl-like or NLP4 and/or NLP9 polypeptide, wherein the new DNA sequence differs from the native DNA sequence (encoding the polypeptide) by the substitution of expression host-preferred (first preferred, second preferred, third preferred, or fourth preferred, etc.) codons to specify the amino acid at each position within the amino acid sequence.
  • the new sequence is then analyzed for restriction enzyme sites that might have been created by the modifications.
  • the identified putative restriction sites are further modified by replacing these codons with a next-preferred codon to remove the restriction site.
  • Other sites in the sequence which may affect transcription or translation of heterologous sequence are exon:intron junctions (5' or 3'), poly-A addition signals, and/or RNA polymerase termination signals.
  • the sequence may be further analyzed and modified to reduce the frequency of TA or CG doublets. In addition to these doublets, sequence blocks that have more than about six G or C nucleotides that are the same may also adversely affect transcription or translation of the sequence. Therefore, these blocks are advantageously modified by replacing the codons of first or second choice, etc., with the next-preferred codon of choice.
  • a method such as that described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants.
  • the method is further illustrated in PCT International Patent Publication No. WO 97/13402 Al .
  • optimized synthetic genes that are functionally equivalent to NSPl-like, NLP4, and/or NLP9 polynucleotides of some embodiments may be used to transform hosts, including plants and plant cells.
  • NSPl-like, NLP4, and NLP9 polynucleotides may also be generated, in silico, from an initial amino acid sequence. Additional guidance regarding the production of synthetic genes can be found in, for example, U.S. Patent 5,380,831.
  • nucleic acid molecules comprising the polynucleotide sequence can be synthesized in the laboratory to correspond in sequence precisely to the designed sequence.
  • synthetic DNA molecules may be cloned and otherwise manipulated exactly as if they were derived from natural or native sources.
  • NSPl-like, NLP4, and NLP9 are necessary for plants to maintain and/or increase growth through an association with NFB in N-limiting growth conditions.
  • NSPl-like, NLP4, and/or NLP9 polypeptides may be introduced or heterologously expressed in a plant cell, for example and without limitation, to increase the association of a plant comprising the plant cell with NFB; to increase the growth of the plant under N-limited conditions; and to increase the efficiency with which the plant utilizes environmental nitrogen.
  • a NSPl-like, NLP4, and/or NLP9 polypeptide may be expressed or over-expressed in a cell or organism, for example and without limitation, by introducing a NSPl-like, NLP4, and/or NLP9 polynucleotide into the cell or organism; by introducing the NSPl-like, NLP4, and/or NLP9 polypeptide into the cell or organism; and/or by providing positive or negative signals sufficient to promote expression of the NSPl-like, NLP4, and/or NLP9 polypeptide through an interaction of the signal(s) with regulatory elements operably linked to a NSPl-like, NLP4, and/or NLP9 polynucleotide in the cell or organism.
  • a NSPl-like, NLP4, and/or NLP9 polypeptide may be knocked-out or under-expressed in a cell or organism, for example and without limitation, by disrupting, mutating, or inactivating a NSPl-like, NLP4, and/or NLP9 polynucleotide; introducing an antisense nucleic acid into the cell or organism that targets a NSPl-like, NLP4, and/or NLP9 polynucleotide; by physically removing the NSPl-like, NLP4, and/or NLP9 polypeptide from the cellular machinery of the cell or organism by binding the NSPl -like, NLP4, and/or NLP9 polypeptide with antibodies or other specific binding proteins; and/or by providing positive or negative signals sufficient to reduce or eliminate expression of the NSPl-like, NLP4, and/or NLP9 polypeptide through an interaction of the signal(s) with regulatory elements operably linked to a NSPl-like, NLP4, and
  • an NSPl-like polypeptide may be expressed or over-expressed in a plant cell or organism, so as to promote the expression of at least one NIN gene(s); for example and without limitation, NLP4 and NLP9.
  • an NSPl-like polypeptide may be removed or under-expressed in a plant cell or organism, so as to decrease or eliminate the expression of at least one NIN gene(s), for example, to study the mechanism of NFB association in non-nodulating plants.
  • an NLP4 and/or NLP9 polypeptide may be expressed or over-expressed in a plant cell or organism, so as to directly increase the ability of the plant cell to associate with NFB.
  • an NLP4 and/or NLP9 polypeptide may be removed or under-expressed in a plant cell or organism, so as to decrease or eliminate the ability of the plant cell to associate with NFB.
  • an NSPl-like, NLP4, and/or NLP9 polypeptide is expressed from a polynucleotide that is operably linked to regulatory elements that direct the expression of the polypeptide(s) in conditions other than those where nitrogen is growth limiting, thereby increasing the efficiency with which the plant utilizes environmental nitrogen under those other conditions.
  • the conservation of the NFB association mechanism across and between different plant species is leveraged to introduce a new NFB association phenotype into a plant via heterologous expression of a NSPl -like, NLP4, and/or NLP9 polypeptide.
  • a NSPl-like, NLP4, and/or NLP9 polypeptide may be expressed in a non-nodulating plant not normally expressing the NSPl-like, NLP4, and/or NLP9 polypeptide, so as to confer increased ability to associate with NFB upon the plant.
  • a plant cell, plant part, and/or plant may be genetically modified to comprise at least one NSPl-like, NLP4, and/or NLP9 polynucleotide by any of several methods of introducing a heterologous molecule known in the art, thereby producing a non-natural transgenic plant cell, plant part, or plant.
  • a heterologous molecule is introduced into a plant cell, plant part, and/or plant by a method selected from, for example and without limitation: transformation and selective breeding (e.g., backcross breeding).
  • the NSPl-like, NLP4, and/or NLP9 polynucleotide is introduced such that it is operably linked to a constitutive promoter, so as to direct the expression of the gene products under conditions where they are not normally expressed ⁇ e.g., when nitrogen is not limited).
  • the NSPl-like, NLP4, and/or NLP9 polynucleotide is introduced such that it is operably linked to a non-constitutive promoter, so as to direct the expression of the gene products in a tissue-preferred ⁇ e.g., in root tissue) or tissue-specific manner.
  • the NSPl-like, NLP4, and/or NLP9 polynucleotide is introduced such that it is operably linked to an inducible promoter, so as to direct the expression of the gene products in a controlled manner.
  • any plant species or plant cell may be genetically modified to comprise a heterologous nucleic acid herein.
  • the plant cell that is so genetically modified is capable of regeneration to produce a plant.
  • plant cells that are genetically modified ⁇ e.g., host plant cells) include cells from, for example and without limitation, a higher plant, a dicotyledonous plant, a monocotyledonous plants, a consumable plant, a crop plant, a plant utilized for its oils ⁇ e.g., an oilseed plant), and a non-nodulating plant.
  • Such plants include, for example and without limitation: alfalfa; soybean; cotton; rapeseed (canola); linseed; corn; rice; brachiaria; wheat; safflower; sorghum; sugarbeet; sunflower; tobacco; and grasses ⁇ e.g., turf grass).
  • a genetically modified plant cell or plant herein includes, for example and without limitation: Brassica napus; indian mustard ⁇ Brassica juncea); Ethiopian mustard ⁇ Brassica carinatd); turnip ⁇ Brassica rapd); cabbage ⁇ Brassica oleracea); Glycine max; Linum usitatissimum; Zea mays,' Carthamus tinctorius; Helianthus annuus; Nicotiana tabacum; Arabidopsis thaliana, Brazil nut ⁇ Betholettia excelsa); castor bean ⁇ Ricinus communis); coconut ⁇ Cocus nucifera); coriander ⁇ Coriandrum sativum); Gossypium spp.; groundnut ⁇ Arachis hypogaea); jojoba ⁇ Simmondsia chinensis); oil palm ⁇ Elaeis guineeis); olive (Olea eurpaea); Oryza
  • the plant may have a particular genetic background, as for elite cultivars, wild-type cultivars, and commercially distinguishable varieties.
  • nucleic acids can be introduced into essentially any plant.
  • Embodiments herein may employ any of the many methods for the transformation of plants (and production of genetically modified plants) that are known in the art. Such methods include, for example and without limitation, biological and physical transformation protocols for dicotyledenous plants, as well as monocotyledenous plants. See, e.g., Goto-Fumiyuki et al. (1999) Nat. Biotechnol. 17:282; Miki et al. (1993) Methods in Plant Molecular Biology and Biotechnology (Glick, B. R. and Thompson, J. E., Eds.), CRC Press, Inc., Boca Raton, FL, pp. 67-88.
  • vectors and in vitro culture methods for plant cell and tissue transformation and regeneration of plants are described, for example, in Gruber and Crosby (1993) Methods in Plant Molecular Biology and Biotechnology, supra, at pp. 89-1 19.
  • Plant transformation techniques available for introducing a nucleic acid into a plant host cell include, for example and without limitation: transformation with disarmed T-DNA using Agrobacterium tumefaciens or A. rhizogenes as the transformation agent; calcium phosphate transfection; polybrene transformation; protoplast fusion; electroporation (D'Halluin et al. (1992) Plant Cell 4: 1495); ultrasonic methods ⁇ e.g., sonoporation); liposome transformation; microinjection; contact with naked DNA; contact with plasmid vectors; contact with viral vectors; biolistics ⁇ e.g., DNA particle bombardment ⁇ see, e.g., Klein et al.
  • a heterologous nucleic acid may be introduced directly into the genomic DNA of a plant cell.
  • a widely utilized method for introducing an expression vector into a plant is based on the natural transformation system of Agrobacterium. Horsch et al. (1985) Science 227: 1229.
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria known to be useful to genetically transform plant cells.
  • Agrobacterium vector systems and methods for Agrobacterium-medm ' ted gene transfer are also available in, for example, Gruber et al., supra, Miki et al., supra, Moloney et al. (1989) Plant Cell Reports 8:238, and U.S. Patent Nos. 4,940,838 and 5,464,763.
  • the DNA to be inserted typically is cloned into special plasmids; either into an intermediate vector or a binary vector. Intermediate vectors cannot replicate themselves in Agrobacterium.
  • the intermediate vector may be transferred into A. tumefaciens by means of a helper plasmid (conjugation).
  • the Japan Tobacco Superbinary system is an example of such a system (reviewed by Komari et al. (2006) Methods in Molecular Biology (K. Wang, ed.) No. 343; Agrobacterium Protocols, 2 nd Edition, Vol. 1, Humana Press Inc., Totowa, NJ, pp.15-41 ; and Komori et al. (2007) Plant Physiol.
  • Binary vectors can replicate themselves both in E. coli and in Agrobacterium.
  • Binary vectors comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacterium (Holsters, 1978).
  • the Agrobacterium comprises a plasmid carrying a vir region.
  • the Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.
  • the vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of a T-strand containing the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:871 1) or the co-cultivation procedure (Horsch et al. (1985) Science 227: 1229).
  • a binary T DNA vector Bevan (1984) Nuc. Acid Res. 12:871 1
  • the co-cultivation procedure Horsch et al. (1985) Science 227: 1229.
  • the Agrobacterium transformation system is used to engineer dicotyledonous plants. Bevan et al. (1982) Ann. Rev. Genet 16:357; Rogers et al. (1986) Methods Enzymol. 1 18:627.
  • the Agrobacterium transformation system may also be used to transform, as well as transfer, nucleic acids to monocotyledonous plants and plant cells. See U.S. Patent No. 5,591 ,616; Hernalsteen et al. (1984) EMBO J 3:3039; Hooykass-Van Slogteren et al. (1984) Nature 311 :763; Grimsley et al. (1987) Nature 325: 1677; Boulton et al. (1989) Plant Mol. Biol. 12:31 ; and Gould et al. (1991) Plant Physiol. 95:426.
  • a recombinant host herein may be performed using standard genetic techniques and screening, and may be carried out in any host cell that is suitable to genetic manipulation.
  • a recombinant host cell may be any organism or microorganism host suitable for genetic modification and/or recombinant gene expression.
  • a recombinant host may be a plant.
  • Standard recombinant DNA and molecular cloning techniques used here are well-known in the art and are described in, for example and without limitation: Sambrook et al. (1989), supra; Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York, NY.
  • the plant cell may be grown, and upon emergence of differentiating tissue such as shoots and roots, mature plants can be generated. In some embodiments, a plurality of plants can be generated. Methodologies for regenerating plants are known to those of ordinary skill in the art and can be found, for example, in: Plant Cell and Tissue Culture (Vasil and Thorpe, Eds.), Kluwer Academic Publishers, 1994. Genetically modified plants described herein may be cultured in a fermentation medium or grown in a suitable medium such as soil.
  • a suitable growth medium for higher plants may be any growth medium for plants, including, for example and without limitation; soil, sand, any other particulate media that support root growth (e.g., vermiculite, perlite, etc.) or hydroponic culture, as well as suitable light, water and nutritional supplements that facilitate the growth of the higher plant.
  • soil e.g., soil, sand, any other particulate media that support root growth (e.g., vermiculite, perlite, etc.) or hydroponic culture, as well as suitable light, water and nutritional supplements that facilitate the growth of the higher plant.
  • Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype, and thus the desired phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans et al. (1983) "Protoplasts Isolation and Culture," in Handbook of Plant Cell Culture, Macmillian Publishing Company, New York, pp. 124-176; and Binding (1985) Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73. Regeneration can also be performed from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. Plant Phys. 38:467.
  • the plant cells which are transformed are not capable of regeneration to produce a plant.
  • Such cells may be employed, for example, in developing a plant cell line having a relevant phenotype, for example, NFB association.
  • a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection can be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells can also be identified by screening for the activities of any visible marker genes (e.g., the ⁇ -glucuronidase, luciferase, or gfp genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
  • any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, or gfp genes
  • a transgenic plant containing a heterologous molecule herein can be produced through selective breeding, for example, by sexually crossing a first parental plant comprising the molecule, and a second parental plant, thereby producing a plurality of first progeny plants.
  • a first progeny plant may then be selected that is resistant to a selectable marker (e.g., glyphosate, resistance to which may be conferred upon the progeny plant by the heterologous molecule herein).
  • the first progeny plant may then by selfed, thereby producing a plurality of second progeny plants.
  • a second progeny plant may be selected that is resistant to the selectable marker.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating, added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes.
  • Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Other breeding methods commonly used for different traits and crops are known in the art. Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line, which is the recurrent parent.
  • the resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent.
  • the resulting parent is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • a nucleic acid may also be introduced into a predetermined area of the plant genome through homologous recombination.
  • Methods to stably integrate a polynucleotide sequence within a specific chromosomal site of a plant cell via homologous recombination have been described within the art.
  • site specific integration as described in US Patent Publication No. 2009/01 1 1188 Al involves the use of recombinases or integrases to mediate the introduction of a donor polynucleotide sequence into a chromosomal target.
  • WO 2008/021207 describes zinc finger mediated-homologous recombination to stably integrate one or more donor polynucleotide sequences within specific locations of the genome.
  • recombinases such as FLP/FRT as described in US Patent 6,720,475, or CRE/LOX as described in US Patent 5,658,772, can be utilized to stably integrate a polynucleotide sequence into a specific chromosomal site.
  • meganucleases for targeting donor polynucleotides into a specific chromosomal location is described in Puchta et al. (1996) Proc. Natl. Acad. Sci. USA 93:5055.
  • site-specific recombination systems that have been identified in several prokaryotic and lower eukaryotic organisms may be applied for use in plants. Examples of such systems include, but are not limited too; the R/RS recombinase system from the pSRl plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191), and the Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170).
  • nucleic acid molecule of certain embodiments herein can be employed in connection with the nucleic acid molecule of certain embodiments herein.
  • the following techniques are useful in detecting the presence of a nucleic acid molecule in a plant cell.
  • the presence of the molecule can be determined by using a primer or probe of the sequence, an ELISA assay to detect an encoded protein, a Western blot to detect the protein, or a Northern or Southern blot to detect RNA or DNA.
  • Additional techniques such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of a recombinant construct in specific plant organs and tissues.
  • Southern analysis is a commonly used detection method, wherein DNA is cut with restriction endonucleases and fractionated on an agarose gel to separate the DNA by molecular weight and then transferring to nylon membranes. It is then hybridized with the probe fragment which was radioactively labeled with 32 P (or other probe labels) and washed in an SDS solution.
  • RNA is cut with restriction endonucleases and fractionated on an agarose gel to separate the RNA by molecular weight and then transferring to nylon membranes. It is then hybridized with the probe fragment which was radioactively labeled with 32 P (or other probe labels) and washed in an SDS solution.
  • Analysis of the RNA ⁇ e.g., mRNA) isolated from the tissues of interest can indicate relative expression levels. Typically, if the mRNA is present or the amount of mRNA has increased, it can be assumed that the corresponding transgene is being expressed.
  • Northern analysis, or other mRNA analytical protocols can be used to determine expression levels of an introduced transgene or native gene.
  • Nucleic acids herein, or segments thereof may be used to design primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Mutations, insertions, and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.
  • Hydrolysis probe assay is another method of detecting and quantifying the presence of a DNA sequence.
  • a FRET oligonucleotide probe is designed with one oligo within the transgene and one in the flanking genomic sequence for event-specific detection.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe.
  • a fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • Some embodiments herein provide plants comprising at least one heterologous NSPl-like, NLP4, and/or NLP9 polynucleotide, such as may be regenerated from stably transformed plant cells or tissues, or may be produced by introgression of such a nucleic acid from a donor line. Such plants may be used or cultivated in any manner, wherein presence of the transforming polynucleotide(s) of interest is desirable. Accordingly, transgenic plants may be engineered to, inter alia, have one or more desired traits ⁇ e.g., NFB association), by transformation, and then may be cropped and cultivated by any method known to those of skill in the art. Particular embodiments herein provide parts, cells, and/or tissues of such transgenic plants. Plant parts, without limitation, include seed, endosperm, ovule and pollen. In some embodiments, the plant part is a seed.
  • plants include non-nodulating plants; Arabidopsis; field crops ⁇ e.g. alfalfa, barley, bean, clover, corn, cotton, flax, lentils, maize, pea, rape/canola, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops ⁇ e.g., asparagus, beet, Brassica, broccoli, Brussels sprouts, cabbage, carrot, cauliflower, celery, cucumber (cucurbits), eggplant, lettuce, mustard, onion, pepper, potato, pumpkin, radish, spinach, squash, taro, tomato, and zucchini); fruit and nut crops ⁇ e.g., almond, apple, apricot, banana, blackberry, blueberry, cacao, cassava, cherry, citrus, coconut, cranberry, date, hazelnut, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit
  • assays include, for example and without limitation: biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISA and/or Western blots) or by enzymatic function; plant part assays (e.g., leaf or root assays); and analysis of the phenotype of the plant.
  • biochemical assays such as detecting the presence of a protein product, e.g., by immunological means (ELISA and/or Western blots) or by enzymatic function
  • plant part assays e.g., leaf or root assays
  • analysis of the phenotype of the plant e.g., phenotype of the plant.
  • a transgenic plant comprising at least one NSPl-like, NLP4, and/or NLP9 polynucleotide may be used in a plant breeding and/or germplasm development program.
  • Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety an improved combination of desirable traits from the parental germplasm. These important traits may include higher seed yield, resistance to diseases and insects, better stems and roots, tolerance to drought and heat, and better agronomic quality.
  • breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F ⁇ hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants.
  • Popular selection methods include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.
  • breeding method The complexity of inheritance influences choice of the breeding method.
  • Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cuitivars.
  • Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross.
  • Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cuitivars produced per unit of input (e.g. , per year, per dollar expended, etc.).
  • Pedigree breeding and recurrent selection breeding methods are used to develop cuitivars from breeding populations. Breeding programs combine desirable traits from two or more cuitivars or various broad-based sources into breeding pools from which cuitivars are developed by selfing and selection of desired phenotypes. The new cuitivars are evaluated to determine which have commercial potential.
  • Pedigree breeding is used commonly for the improvement of self-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F ⁇ . An F 2 population is produced by selfing one or several ⁇ ⁇ S. Selection of the best individuals may begin in the F 2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families can begin in the F 4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., Fe and F 7 ), the best lines or mixtures of phenotypically similar lines are tested for potential release as new cultivars.
  • Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops.
  • a genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.
  • Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent.
  • the source of the trait to be transferred is called the donor parent.
  • the resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent.
  • the resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • the single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation.
  • the plants from which lines are derived will each trace to different F 2 individuals.
  • the number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F 2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.
  • a NSPl-like, NLP4, and/or NLP9 polynucleotide may be introduced into a plant germplasm, for example, to develop novel inbred lines that are characterized by increased association with NFB, under the control of regulatory elements that are operably linked to the polynucleotide(s).
  • a particular advantage of such a development program may be that the expression of an NFB phenotype, for example, in a non-nodulating plant, results in increased nitrogen utilization and/or growth.
  • Arabidopsis thaliana Columbia 0 ecotype was used for all experiments unless otherwise indicated.
  • A. thaliana seedlings were grown on 0.8% agar plates with Murashige and Skoog (MS) salt media or MS salt media without nitrogen (N) supplemented with 5 mM KN0 3 as indicated, with 16:8 ligh dark photoperiod (hrs.) and 22°C constant temperature. After seven days, plants were transferred to MS salt media complete, or without N, supplemented or not with 2.5 mM NH4NO3.
  • plants where inoculated or not with NFB or non-NFB. All bacteria were previously grown in diluted 869 medium, and 20 mL overnight culture that reached an optical density at 600 nm of 0.4 was used for inoculation. Prior to inoculation, bacteria were washed with sterile water and resuspended in 5 mL. 20 mL of plant's agar medium were inoculated with 5 mL bacteria, or alternatively non-inoculated with sterile water.
  • A. thaliana establishes beneficial interactions for N-nutrition with NFB, we assessed the effect of different NFB species on plant growth under N-limiting conditions. We selected five different NFB species shown to fix N in association with plants:
  • Burkholderia phytofirmans PsJN known to enhance Arabidopsis growth (Zuniga et al. (2013) Mol. Plant Microbe Interact. 26:546), and
  • Cupriavidus pinatubonensis JMP134 a soil bacterium capable of associating with plants but without a positive impact on plant growth under our experimental conditions (Ledger et al. (2012) Antonie Van Leeuwenhoek 101 :713).
  • Plants were grown on vertical plates with MS medium for seven days, and were transferred to plates with the same MS medium, but without N (MS-N), or MS-N inoculated with 104 colony forming units (cfu)/mL of the different bacteria. We evaluated dry weight of the plants seven days after transferring to MS-N.
  • Plant dry weight was significantly higher in the presence of S. meliloti RMPl 10 as compared to non-inoculated medium under N-limiting conditions.
  • FIG. 1. Moreover, plant growth was comparable to that achieved in full MS medium, under our experimental conditions.
  • FIG. 1. These results indicate S. meliloti RMPl lO can promote plant growth in the absence of an N source. None of the other bacterial species utilized were able to increase Arabidopsis dry weight, indicating mere presence of neutral or beneficial bacteria cannot explain this observation. Effective interactions between Arabidopsis and different bacteria were confirmed by a non-specific increase in root hair length observed in response to bacterial inoculation in all cases.
  • NSPl-like and NSP2-like genes At3gl3840 (AtNSPl-like) and At4g08250 (AtNSP2-like), respectively. These genes have no known function in Arabidopsis thaliana.
  • AtNSPl-like and AtNSP2-like genes were evaluated in plants grown under N-sufficient conditions (5 mM KNO3) and then transferred to 2.5 mM NH 4 NO 3 or to MS-N medium in the presence or absence of S. meliloti RMPl l O. Plants were harvested 3 and 7 days after the transfer. Total RNA was prepared, and transcript levels for the genes of interest were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).
  • FIG. 3 A This result suggests that AtNSPl-like function is required under N-limiting conditions when S. meliloti is present. In contrast, AtNSP2-like was not regulated under the experimental conditions tested.
  • FIG. 7A This result suggests that AtNSPl-like function is required under N-limiting conditions when S. meliloti is present. In contrast, AtNSP2-like was not regulated under the experimental conditions tested.
  • NSP1 and NSP2 regulate the expression of N genes.
  • the Arabidopsis genome encodes nine NZN-like genes (NLPs).
  • NLP1 At2gl 7150
  • NLP2 At4g35270
  • NLP3 At4g38340
  • NLP4 Atlg20640
  • NLP5 ⁇ Atlg76350
  • NLP8 At2g43500
  • NLP9 At3g59580
  • AtNLP4, AtNLP8, and AtNLP9 is induced under ⁇ -limiting conditions and in the presence of S. meliloti RMPl 10 (FIG. 3B), similar to AtNSPl-like.
  • AtNLP3 showed a different expression pattern, being induced under N-sufficient conditions but only in the presence of bacteria.
  • FIG. 3B Gene expression of AtNLPl, AtNLP2, and AtNLPS did not change significantly under the experimental conditions evaluated.
  • AtNSPl-like, AtNLP4, AtNLP8, and AtNLP9 genes in the context of Arabidopsis and S. meliloti RMPl 10 interactions, two independent homozygous mutant lines of A. ihaliana for AtNSPl-like (salk_036071 C - salk_023595C), AtNLP4 (salk_100786C - salk_063595C), AtNLP8 (salk_140298 - salk_031064C) and AtNLP9 (salk_025839C - salk_042082C) were obtained from the Arabidopsis Biological Resource Center (ABRC).
  • ABRC Arabidopsis Biological Resource Center
  • AtNSPl-like, AtNLP4, and AtNLP9 are required for a functional interaction between Arabidopsis and S. meliloti RMP110 for enhanced growth under N-limiting conditions. These results also indicate that a conserved mechanism exists in plants to mediate plan bacteria beneficial interactions for N nutrition that can be employed to provide BNF to non-nodulating plant species.

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Abstract

L'invention concerne des réponses à l'azote des végétaux. Selon certains modes de réalisation, l'invention concerne les facteurs régulateurs qui contribuent à l'association fonctionnelle des végétaux (par ex., des plantes non-nodularisantes) à la bactérie de fixation de l'azote.
PCT/IB2014/002488 2013-11-18 2014-11-18 Gènes régulateurs de végétaux favorisant l'association à une bactérie de fixation de l'azote Ceased WO2015071749A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US14/083,193 2013-11-18
US14/083,193 US20150143578A1 (en) 2013-11-18 2013-11-18 Plant regulatory genes promoting association with nitrogen fixing bacteria
CL2013-3314 2013-11-18
CL2013003314A CL2013003314A1 (es) 2013-11-18 2013-11-18 Ácidos nucleicos aislados codificantes para proteínas de las familias nsp y nin, método para incrementar la eficiencia del nitrógeno en una planta sin nódulos basado en la expresión heteróloga de los polinucleótidos antes descritos.

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WO2015071749A1 true WO2015071749A1 (fr) 2015-05-21

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PCT/IB2014/002488 Ceased WO2015071749A1 (fr) 2013-11-18 2014-11-18 Gènes régulateurs de végétaux favorisant l'association à une bactérie de fixation de l'azote

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TW (1) TW201522641A (fr)
UY (1) UY35843A (fr)
WO (1) WO2015071749A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272904A (zh) * 2018-03-15 2019-09-24 中国科学技术大学 水稻氮高效利用基因OsNLP4及其编码蛋白的应用
WO2021170794A1 (fr) * 2020-02-28 2021-09-02 Cambridge Enterprise Limited Méthodes, plantes et compositions pour surmonter la suppression des nutriments de la symbiose mycorhizienne

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008118394A1 (fr) * 2007-03-23 2008-10-02 New York University Procédés pour influencer l'assimilation de l'azote dans des plantes

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2008118394A1 (fr) * 2007-03-23 2008-10-02 New York University Procédés pour influencer l'assimilation de l'azote dans des plantes

Non-Patent Citations (6)

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Title
BARBULOVA, ANI ET AL.: "Differential effects of combined N sources on early steps of the nod factor-dependent transduction pathway in Lotus japonicus", MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 20, no. 8, August 2007 (2007-08-01), pages 994 - 1003 *
DATABASE GENBANK 18 September 2002 (2002-09-18), accession no. AM51312.1 *
DATABASE GENBANK 18 September 2002 (2002-09-18), accession no. Y117237.1 *
KRAISER, TATIANA: "NSP-like and NIN-like genes are involved in the association between Arabidopsis thaliana and nitrogen fixing bacteria", THESIS, 2013 *
SMIT, PATRICK ET AL.: "NSP1 of the GRAS protein family is essential for rhizobial nod factor-induced transcription", SCIENCE, vol. 308, no. 5729, 17 June 2005 (2005-06-17), pages 1789 - 1791 *
ZHU, HONGYAN ET AL.: "Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses", GENETICS, vol. 172, no. 4, April 2006 (2006-04-01), pages 2491 - 2499 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272904A (zh) * 2018-03-15 2019-09-24 中国科学技术大学 水稻氮高效利用基因OsNLP4及其编码蛋白的应用
WO2021170794A1 (fr) * 2020-02-28 2021-09-02 Cambridge Enterprise Limited Méthodes, plantes et compositions pour surmonter la suppression des nutriments de la symbiose mycorhizienne
US12503707B2 (en) 2020-02-28 2025-12-23 Cambridge Enterprise Limited Methods, plants and compositions for overcoming nutrient suppression of mycorrhizal symbiosis

Also Published As

Publication number Publication date
UY35843A (es) 2015-06-30
TW201522641A (zh) 2015-06-16

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