TW201522641A - Plant regulatory genes promoting association with nitrogen fixing bacteria - Google Patents

Abstract

This disclosure concerns plant nitrogen responses. Embodiments concern regulatory factors that contribute to the functional association of plants (e.g., non-nodulating plants) with nitrogen-fixing bacteria.

Description

Plant regulatory genes that promote the association with nitrogen-fixing bacteria Priority claim

The application claims are based on the filing date of U.S. Application No. 14/083,193, filed on November 18, 2013, and the filing date of the patent application No. 2013-3314, "PLANT REGULATORY GENES PROMOTING ASSOCIATION WITH NITROGEN FIXING BACTERIA".

Technical field

This disclosure relates to plant biochemistry. Specific examples are genetic factors that regulate the association between plants and nitrogen-fixing bacteria.

background

Nitrogen is a fundamentally large nutrient for plant growth and development, and nitrogen is a major limiting factor for plant growth and crop productivity. Marschner (1995) Mineral Nutrition of Higher Plants , Academic Press, Harcourt, San Diego, CA, p. 889; Epstein (2005) Mineral Nutrition of Plants: Principles and Perspectives , 2 ^nd Ed., Sinauer Associates, Inc., Sunderland, MA; Galloway and Cowling (2002) AMBIO 31:64. Traditional agriculture is based on nitrogen fertilizer to support the world's nutrient needs. However, such fertilizers can damage the environment and destroy human health when used excessively.

Despite the abundance of nitrogen in the atmosphere, plants cannot directly access the nitrogen reserves of the biological world. Plants obtain nitrogen directly from the soil, in some cases by nitrogen-fixing bacteria (NFB) via biological nitrogen fixation processes; the main process of converting atmospheric N ₂ into biologically available nitrogen forms - such as ammonium in natural ecosystems (see, for example, Olivares et Al. (2013) Mol . Plant Microbe Interact. 26: 486). Due to the often limited nitrogen in the soil, some plant species evolved molecular mechanisms associated with nitrogen-fixing bacteria (NFB). Zehr et al. (2003) Env . Microbiol. 5: 539; Sprent and James (2007) Plant Physiol. 144: 575; Kraiser et al. (2011) J. Exp. Bot. 62: 1455. Bacteria and archaea are the only species that can biologically fix nitrogen. Reduction of atmospheric N ₂ to ammonium is catalyzed by a nitrogenase complex - consisting of a diazinase and a diazotase reductase subunit. Joerger et al. (1991) J. Bacteriol . 173: 4440.

The plant-bacteria interactions associated with nitrogen nutrients are primarily in legume research. Leguminos can be associated with phylogenetically diverse bacterial groups - collectively referred to as Rhizobium - in a symbiotic manner. Kistner and Parniske (2002) Trends Plant Sci. 7: 511. The association of such plant species and their bacterial partners involves the formation of specialized organs known as "nodulation". Nodulation encompasses bacteria and provides appropriate conditions for nitrogen fixation. Markmann and Parniske (2009) Trends Plant Sci. 14:77. In these nodules, plants provide a source of carbon that exchanges nitrogen fixed by the bacteria. Kistner and Parniske (2002), supra; Masson-Boivin et al. (2009) Trends Mirobiol. 17:458. The symbiotic relationship between bacteria and legumes is highly regulated and occurs only when plants are grown under nitrogen-limited conditions. Low levels of nitrate and ammonium stimulate the formation of nodules, while high levels of these nutrients also inhibit the number of infection sites in the roots and the N-fixation of existing nodules. Eaglesham (1989) Crop Sci. 29: 115; Zahran (1999) Microbiol. Mol. Biol. Rev. 63: 968; Bisseling et al. (1978) Bio . Biophys. Acta 539: 1.

The plant recognizes the Nod factor secreted by bacteria, which activates the signal transduction pathway of NODULATION SIGNALING PATHWAY 1 (NSP1) and NSP2. Smit et al. (2005) Science 308: 1789; Kalo et al. (2005) Science 308: 1786; Heckmann et al. (2006) Plant Physiol. 142: 1739. In summary, these transcription factors regulate the expression of the nodulation initiation ( NIN ) gene, which is required for bacterial infection and nodulation organogenesis. Smit et al. (2005), supra; Hirsch et al. (2009) Plant Cell 21: 545; Schauser et al. (1999) Nature 402: 191. Activation of these transcription factors in response to bacterial Nod factors occurs when the plant is in a N-limited condition. Barbulova et al. (2007) Mol . Plant Microbe Interact. 20:994.

In addition to legumes, a limited number of other plant species from different taxonomies have been reported to be associated with NFB. Although no tumors can be established, some plants (such as wheat and sugar cane) can absorb their significant nitrogen requirements through biological nitrogen fixation under N-limited conditions. Iniguez et al. (2004) Mol . Plant Microbe Interact. 17: 1078; Boddey et al. (1991) Plant Soil 137:111.

Reveal content

Gene classes that regulate N-nutrient interactions between non-nodulated plant species and NFB (such as Sinorhizobium meliloti ) (eg, AtNSP1-like , AtNLP4 , and AtNLP9 ) are revealed .

Disclosed in a particular embodiment is a non-native nucleic acid molecule comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide is at least 80% identical to SEQ ID NO: 4 An acid; a polynucleotide that hybridizes under stringent (eg, 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 to a nucleic acid consisting of SEQ ID NO: 5, for example, under highly stringent conditions; a polynucleotide that is at least 80% identical to SEQ ID NO: 6; or in harsh (eg, highly stringent) A polynucleotide that hybridizes to the nucleic acid consisting of SEQ ID NO: 6 under the conditions.

This case also illustrates methods for increasing the nitrogen efficiency of plants such as non-nodulated plants. In some embodiments, the method can comprise introducing at least one heterologous polypeptide into a plant to produce a gene transfer plant, wherein the heterologous polypeptide is a nodulation signal transduction pathway-like (NSP) or NIN-like protein (NLP). In a particular embodiment, the heterologous polypeptide is NSP1, NLP4, or NLP9. Such a heterologous polypeptide can be, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, with one or more of SEQ ID NOs: 1, 3, and 4. 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% consistent.

In some embodiments, a method of increasing plant nitrogen efficiency can comprise transforming a plant with a polynucleotide encoding the heterologous polypeptide. In a particular embodiment, the polynucleotide can be substantially identical to one of SEQ ID NOs: 4-6 And/or homologs or heterologs of them. In a particular embodiment, the polynucleotide encoding the heterologous polypeptide is hybridized to at least one of SEQ ID NOs: 4-6 under stringent (e.g., highly stringent) conditions.

The present invention also illustrates a method of increasing the nitrogen efficiency of a plant, such as a non-nodulated plant, comprising introducing the plant into at least one of at least one mode that promotes its association with NFB, for example, to produce a genetically transformed plant. Examples of means for promoting the association with NFB include a polypeptide consisting of SEQ ID NO: 1; a polypeptide consisting of SEQ ID NO: 2; a polypeptide consisting of SEQ ID NO: 3; a polynucleus consisting of SEQ ID NO: a nucleotide; a polynucleotide consisting of SEQ ID NO: 5; and a polynucleotide consisting of SEQ ID NO: 6.

Further, the present invention describes plant cells, plant parts, plant material, plant tissues, plant seeds, and whole plants which comprise or stably transform any of the aforementioned polypeptides and/or nucleic acid constructs. Such genetically transgenic plants exhibit increased growth under nitrogen-limited growth conditions in specific embodiments compared to wild-type plants of the same species.

The above and other features will be apparent from the following detailed description of the embodiments.

Figures 1A and 1B are schematic diagrams showing the effect of NFB on plant growth under N-limited conditions. Plant biomass (dry weight; n=15) was transferred to complete MS medium to MS medium with N (40 mM NO ₃ + 20 mM NH ₄ ) or no N and with different bacteria (NFB: Burk Bunge G4, phagocytosis) Heterologous Burkholderia LB400, Taiwan Copperophilus LMG19424, Rhizopus arrhizus CFN42, Rhizobium meliloti RMP110; and Non-NFB: Burkholderia PSJN, Pseudomonas aeruginosa JMP134) (sequence by left To the right) 7 days after inoculation or no measurement. Figure 1A. Plant dry weight was measured on plants grown in N-containing medium for 7 days and transplanted to N-limited conditions and inoculated with live or dead S. meliloti, or uninoculated. Figure 1B. The number of lateral roots per plant is measured by plants grown and treated under the same conditions as detailed above in Figure 1A. Figure 1A (mean ± SE of three independent biological replicates; asterisk indicates a significantly different average (P < 0.05) compared to uninoculated plants without N growth).

Figures 2A and 2B include schematic diagrams of N-immobilization enhancing plant growth under conditions of limited N. Bio-mass system to grow in a well (2.5mM NH ₄ NO ₃₎ or N limiting conditions, to a wild-type S. meliloti RMP110 inoculated plant or whether or not the mature N fixed inoculated on a dry weight measurement. Figure 2A. Biological nitrogen fixation was measured by a ¹⁵ N dilution technique. Plant lines are grown in a sufficient N (5% ¹⁵ N) medium. After seven days, the plants were moved to different treatment dishes. Using mass spectrometry, ¹⁴ N and ¹⁵ N portions were measured on harvested and dried plants after 7 days of treatment. δ ¹⁵ N represents the ¹⁴ N: ¹⁵ N isotope ratio compared to uninoculated conditions (mean ± SE of three independent biological replicates (P < 0.05)). Figure 2A.

Figures 3A and 3B include schematic representations of the introduction of certain Arabidopsis genes via bacterial vaccination. The NSP1 (Fig. 3A) and NIN-like transcription factor (Fig. 3A) gene expression lines were measured by real-time quantitative reverse transcription PCR after the third or seventh day of treatment. Plant lines were grown in sufficient (2.5 mM NH ₄ NO ₃ ) or N-limited conditions and inoculated with NFB or no. The values are equivalent to the mean ± SE of three independent biological replicates (the asterisk indicates a significantly different average (P < 0.05) compared to the uninoculated plants).

Figure 4 includes Arabidopsis thaliana - a gene critical for the association with S. meliloti RMP110; a schematic representation of the mutational effects of AtNSP1-like , AtNLP4 , AtNLP8 , and AtNLP9 transcription factors. Biomass was expressed as the dry weight measured by wild-type and mutant plants grown under nitrogen-limited conditions and inoculated with N. faecalis (average ±SE of three independent biological replicates; asterisk indication compared to unvaccinated Plants were significantly different (P < 0.05)).

Figure 5 includes an image showing the effect of NFB on root hair length. Plant lines grown in complete MS salt medium for several days were transplanted to N-free MS medium and inoculated with NFB or not. Take a photo after a few days of processing.

Figures 6A and 6B are schematic diagrams showing the effect of Sinorhizobium meliloti on the Arabidopsis root architecture. The lateral root density (Fig. 6A) and the major root length (Fig. 6B) were measured on plants grown in a sufficient/limited N condition, inoculated with R. sphaeroides RMP110 wild type or no or inoculated with a mature type in which N could not be fixed. The plotted values are equivalent to the mean ± SE of three independent biological replicates.

Figures 7A and 7B include schematic representations of bacterial responses to AtNSP2-like , AtNLP1 , AtNLP2 , and AtNLP5 . Gene expression was measured by real-time quantitative reverse transcription PCR after the third or seventh day of treatment (mean ± SE of three independent biological replicates; asterisk indicates a significantly different average (P < 0.05) compared to uninoculated plants). Plant lines are grown in sufficient or limited N conditions and inoculated with S. meliloti or no.

Sequence table

The nucleic acid sequences listed in the accompanying sequence listing are shown using the nucleotide base standard letter abbreviations as defined in 37 CFR § 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strands are included with any reference to the display strand. In the accompanying sequence listing: SEQ ID NO: 1 shows the NSP1-like (SCL29; At3g13840) polypeptide: SEQ ID NO: 2 shows the NLP4 (At1g20640) polypeptide: SEQ ID NO: 3 shows the NLP9 (At3g59580) polypeptide: SEQ ID NO: 4 shows the NSP1-like (SCL29; At3g13840) polynucleotide coding sequence (CDS): SEQ ID NO: 5 shows the NLP4 (At1g20640) polynucleotide coding sequence (CDS): SEQ ID NO: 6 shows the NLP9 (At3g59580) polynucleotide coding sequence (CDS):

Mode for carrying out the invention I. Overview of several specific examples

In legumes, the functional association between plants and NFB is highly regulated. Molecular regulatory systems prevent the formation of nodules in plants grown in the presence of nitrogen. The symbiotic signal transduction pathway is activated to induce nodulation in plants only in the presence of N-limited conditions and in the presence of NFB. After recognition of the Nod factor, calcium oscillations induce the induction of the primary transcription factors NSP1 and NSP2, which induce NIN gene expression. Some components of the symbiotic signal transduction pathway are shared between Rhizobium and arbuscular mycorrhizal fungi. Oldroyd (2013), see above. However, unlike the NSP2 , NSP1 and NIN gene lines, it is explicitly related to NFB.

This case illustrates the promotion of non-nodulated plant species and NFB Linkage to promote N-nutrients. Under the condition of N, Arabidopsis is associated with Sinorhizobium meliloti, and NFB can provide reducing nitrogen as plant nutrients, which is beneficial to plant growth under N-limited conditions. The specific case of this case uses the first identification of plant transcription factors that are critical for the functional relationship between non-nodulated plants and S. meliloti. We have found that homologous genes specific for leguminous: NFB-related transcription factors are essential for plant growth promoting effects induced by Sinorhizobium meliloti in non-nodulated plants. In a specific example, the retention of the cross-linkage mechanism between plant species allows the association between Arabidopsis and S. meliloti to be replicated in other plants via genetic modification to manage beneficial N-nutrient interactions with NFB For example, to enhance the efficiency of nitrogen use in farming systems.

As described in this case, the Arabidopsis genus has a functional association with S. meliloti - a bacterium associated with the leguminous alfalfa. Marsh et al. (2007) Plant Physiol. 144: 324; Peiter et al. (2007) Plant Physiol . 145: 192. In free-living conditions and related to legumes, Sinorhizobium meliloti is only N-fixed under N-limited conditions. Szeto et al. (1987) J. Bacteriol. 169: 1423. As described in the present case, Sinorhizobium meliloti is resistant to plant growth under conditions of limited N (for example, in the absence of an N-source medium), and is therefore suitable for N-immobilization of BNF and plant growth promotion.

II. Abbreviation

III. Terminology

In the following descriptions and tables, many terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the categories given by such terms, the following definitions are provided: Backcrossing: Backcrossing methods can be used to introduce nucleic acid sequences into plants. Backcrossing techniques have been widely used for decades to introduce novel traits into plants. Jensen, N., Ed. Plant Breeding Methodology , John Wiley & Sons, Inc., 1988. In a typical backcrossing process, the original cultivar of interest (recurrent parent) is mated with a second cultivar (non-recurrent parent) carrying the gene of interest to be transferred. The progeny thus mated are then re-matured with the recurrent parent, and the process is repeated until the plant is obtained, wherein - in addition to the transfer gene from the non-recurrent parent - substantially all of the reincarnation of the plant is recovered in the transformed plant Morphological and physiological characteristics.

Isolating: "Isolated" biological components (such as nucleic acids or proteins) have been derived from other biological components (ie, other chromosomes and extrachromosomal DNA and RNA, and proteins) within the organism's cells in which the component naturally exists. Isolation, manufacture, or purification, while causing chemical and functional changes in the components (eg, nucleic acids can be isolated from the chromosome by breaking the chemical bonds of the nucleic acid to the rest of the DNA in the chromosome). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods in which chemical and functional changes have been made in the nucleic acid or protein. The term also encompasses nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins, and Peptide.

Nucleic Acid Molecule: As used herein, the term "nucleic acid molecule" may refer to a polymeric form of a nucleotide, which may include the sense and antisense strands of RNA, cDNA, genomic DNA, and the above synthetic forms and mixed polymers. A nucleotide can refer to a modified form of a ribonucleotide, a deoxyribonucleotide, or any type of nucleotide. The "nucleic acid molecule" used in this context is synonymous with "nucleic acid" and "polynucleotide". Nucleic acid molecules are typically at least 10 bases in length unless otherwise indicated. The term includes both mono- and bi-strand forms of DNA. A nucleic acid molecule can include any one or both of the naturally occurring and modified nucleotides joined together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified by chemical or biochemical means, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides into an analog cross-nucleotide modification (eg, uncharged linkage: for example, methylphosphonate) , a phosphate triester, an amino phosphate, a urethane, etc.; a charged linkage: for example, a phosphorothioate, a phosphorodithioate, etc.; a pendant moiety: for example, a peptide; embedded Agents: for example, acridine, psoralen, etc.; chelating agents; alkylating agents; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological configuration, including single-strand, double-strand, partially double helix, triple helix, hairpin, ring, and lock configurations.

Oligonucleotide: An oligonucleotide is a short nucleic acid molecule. Oligonucleotides can be formed by cleavage of longer nucleic acid fragments, or by polymerization of individual nucleotide precursors. Automated synthesizers allow synthesis of up to hundreds of base pairs in length Oligonucleotides. Because oligonucleotides can bind to complementary nucleotide sequences, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) composed of DNA can be used for PCR, a technique for amplifying small DNA sequences. In PCR, oligonucleotides are often referred to as "primers" which allow the DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

A nucleic acid molecule can include any one or both of the naturally occurring and modified nucleotides joined together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified by chemical or biochemical means, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides into an analog cross-nucleotide modification (eg, uncharged linkage: for example, methylphosphonic acid) Ester, phosphotriester, amino phosphate, urethane, etc.; charged linkage: for example, phosphorothioate, phosphorodithioate, etc.; pendant moiety: for example, peptide; Inserts: for example, acridine, psoralen, etc.; chelating agents; alkylating agents; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological configuration, including single-strand, double-strand, partially double helix, triple helix, hairpin, ring, and lock configurations.

The term "coding sequence" when used in reference to DNA in this context refers to a nucleotide sequence that is transcribed into RNA upon administration of the 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. In the case of RNA, the term "coding sequence" refers to a nucleotide sequence that is translated into a peptide, polypeptide, or protein. The boundaries of the coding sequence are determined by the 5'-end translation start codon and the 3'-end translation stop codon. Coding sequences include, but are not limited to, genomic DNA; cDNA; EST; and recombinant nucleotide sequences.

Genome: As used herein, the term "genome" refers to chromosomal DNA found in the nucleus, and also refers to organelle DNA found within the subcellular components of a cell. In some embodiments of the invention, a DNA molecule can be introduced into a plant cell such that the DNA molecule is embedded in the genome of the plant cell. In these and other specific examples, the DNA molecule can be embedded in the nuclear DNA of the plant cell, or embedded in the chloroplast or mitochondrial DNA of the plant cell.

Endogenous: The term "endogenous" applied to nucleic acids (such as polynucleotides, DNA, RNA, and genes) in this case refers to the habitual (such as wild-type cells in the same species and species) appearing in their specificity. One or more (plural) nucleic acids within the environment or background. For example, an endogenous gene is a gene that is routinely found in a particular cell in question and in the same context, such as for regulatory sequences. Endogenous nucleic acids can be distinguished from exogenous and/or heterologous - by way of example and without limitation - by detecting the latter sequence obtained by bacterial plastid recombination; identifying atypical codon bias; and in PCR reactions Amplifying the atypical sequence of primers characterization from wild-type cells.

Exogenous: The term "exogenous" as applied to a nucleic acid of the present invention refers to one or more (plural) nucleic acids that are not normally found in their specific environment or context. For example, if the host cell is transformed by a nucleic acid that is not naturally found in the untransformed host cell, the nucleic acid is exogenous to the host cell. The term exogenous - as used herein - also refers to one or more of the cell or genomic backgrounds that are identical to the nucleic acid sequence already present in the host cell but that are different from the nucleic acid already present in the host cell with the same sequence ( Multiple) Nucleic acid. For example, nucleic acids embedded in and having the same sequence of nucleic acids that are conventionally embedded in the host cell genome at various locations in the host cell genome are exogenous to the host cell. Furthermore, nucleic acids (such as DNA molecules) present in the host cell plastid or vector are exogenous to the host cell when the nucleic acid having the same sequence is customarily present only in the host cell genome.

Heterologous: The term "heterologous" as applied to a nucleic acid (such as a polynucleotide, DNA, RNA, and gene) of the present invention means having a different source. For example, a host cell is heterologous (and exogenous) to the host cell if the host cell is transformed by a nucleic acid that is not naturally found in the untransformed host cell. Furthermore, different elements of the transmorphic nucleic acid (such as promoters, enhancers, coding sequences, terminators, etc.) may be heterologous to the other and/or to the transpogic host. The term heterologous - as used herein - also applies to one or more (plural) of nucleic acid sequences already present in a host cell, but now joined to different additional sequences and/or present in different copies, and the like. Nucleic acid.

Sequence Consistency: The term "sequence identity" or "consistency" as used in this context for two nucleic acid or polypeptide sequences can be referred to - when aligned at a particular alignment window with maximum correspondence - the same residues within both sequences.

As used herein, the term "percent sequence identity" can refer to a value determined by aligning two aligned alignment sequences (eg, a nucleic acid sequence, with an amino acid sequence) in an alignment window, wherein in comparison to a reference sequence ( When it does not include additions or deletions for optimal alignment of the two sequences, additions or deletions (ie, gaps) may be included in the sequence portion of the alignment window. The percentage is calculated by determining the number of positions in which the consensus nucleotide or amino acid residue is present in both sequences to generate the number of paired positions, dividing the number of paired positions by the alignment window The number of all positions, and the result is multiplied by 100 to generate a sequence consistency percentage.

Methods for aligning sequences for alignment are well known in the art. Various programs and alignment algorithms are described, for example: Smith and Waterman (1981) Adv. Appl. Math. 2: 482; Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; Higgins and Sharp (1988) Gene 73: 237-44; Higgins and Sharp (1989) CABIOS 5: 151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8: 155-65; Pearson et al. (1994) Methods Mol . Biol. 24: 307 31; Tatiana et al. ) FEMS Microbiol. Lett. 174: 247-50. Detailed considerations for sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

National Center for Biotechnology Information (NCBI) nucleotide local alignment search tool ^{(BLAST TM;. Altschul et al} (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the web to Used in conjunction with several sequence analysis programs. Use this program to illustrate how to determine sequence identity can be achieved in the BLAST ^TM chapter "Help (help)" on the web. To a nucleic acid sequence alignments, the default parameters can be used using BLAST ^TM (Blastn) of the program "Sequence alignment of bis (Blast 2 sequences)" function. Nucleic acid sequences with even greater similarity to the reference sequence will show an increased percent identity when evaluated by this method.

Specific hybridization/specific complementation: as used in this case, surgery The terms "specifically hybridize" and "specifically complement" are terms that indicate a sufficient degree of complementarity to result in a stable specific binding between a 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 capable of forming hydrogen bonds with corresponding bases on the opposite strand to form a double helix molecule detectable using methods well known in the art, provided that the molecule is sufficiently stable. The nucleic acid molecule does not have to be 100% complementary to the target sequence to be specifically hybridized. However, there must be a function to make the sequence complementary portion of the hybrid specificity a function of the hybridization conditions used.

Hybridization conditions that produce a particular stringency will vary depending on the nature of the hybridization method selected and the composition and length of the hybrid nucleic acid sequence. In general, the hybridization temperature and the ionic strength of the hybridization buffer (especially the Na ⁺ and/or Mg ⁺⁺ concentration) will determine the stringency of the hybridization, although the wash time will also affect the severity. The calculation of the hybridization conditions necessary to achieve a particular severity is well known to those skilled in the art and is exemplified by Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual , 2 ^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11; discussed in Hames and Higgins (eds.) Nucleic Acid Hybridization , IRL Press, Oxford, 1985. Further details and guidance regarding nucleic acid hybridization can be, for example, Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes , Part I, Chapter 2, Elsevier, NY, 1993; found in Ausubel et al. , Eds., Current Protocols in Molecular Biology , Chapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.

As used herein, "stringent conditions" encompasses hybridization only - in the case of a hypothesis that there is less than 20% unpaired-occurrence between the hybrid molecule and the homologous sequence within the target nucleic acid molecule. “Strict conditions” include additional levels of particular severity. Thus, as used in this context, "moderately harsh" conditions are those conditions in which more than 20% of the unpaired molecules will not hybridize; "highly stringent" conditions are such that they have more than 10% unpaired Conditions under which the sequence will not hybridize; and "very high severity" conditions are those conditions in which more than 5% of the unpaired sequences will not hybridize.

The following are representative, non-limit hybridization conditions.

Highly stringent conditions (detecting sequences sharing 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 at 0.5 x SSC buffer was washed twice at 65 ° C for 20 minutes each.

Moderately harsh conditions (detecting sequences sharing at least 80% sequence identity): hybridization in 5x-6x SSC buffer at 65-70 °C for 16-20 hours; washing twice in 2x SSC buffer at room temperature, each For 5-20 minutes; and wash twice in 1x SSC buffer at 55-70 ° C for 30 minutes each.

Non-rigid control conditions (sequences that share at least 50% sequence identity will hybridize): 16-20 hours in 6x SSC buffer at room temperature to 55 °C; in room temperature to 55 °C in 2x-3x SSC buffer Wash at least twice, each for 20-30 minutes.

As used herein, the term "substantially homologous" or "substantially homologous" with respect to a polynucleotide refers to a polynucleotide that hybridizes under stringent conditions to a reference nucleic acid sequence. For example, a polynucleotide that is substantially homologous to a reference DNA coding sequence is a polynucleoside that hybridizes to a reference DNA coding sequence under stringent conditions (such as the list of moderately harsh conditions, see above). acid. Substantially homologous sequences can have at least 80% sequence identity. For example, a substantially homologous sequence can 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%; 99%; about 99.5%; and about 100%. The properties of substantial homology are closely related to specific hybridization. For example, a nucleic acid molecule specifically hybridizes when there is sufficient complementarity to avoid non-specific binding of the nucleic acid to non-specific under stringent hybridization conditions under conditions where the specific binding system is desired. Target sequence.

As used herein, the term "heterologous" refers to a gene that has evolved from a common prior nucleotide sequence within two or more species and that retains the same function within the two or more species.

As used herein, two nucleic acid sequence molecules are read when the nucleotide is read in the 5' to 3' direction and each nucleotide in the other sequence is read in the 3' to 5' direction. It is called to show "complete complementarity." A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence that is identical to the reverse complement of the reference nucleotide sequence. These terms and descriptions are expressly defined in the art and are readily understood by those skilled in the art.

As used in this case, the term "substantially consistent" may refer to more than 85%. Consistent nucleotide sequence. For example, a substantially identical nucleotide sequence can 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% consistent.

Performance: As used in this context, the "expression" of a coding sequence (for example, a gene or a transgene) refers to the manipulation, non-operation, or structure of the coding information (including, for example, genomic DNA or cDNA) of a nucleic acid transcription unit into a cell. Part of the process (such as protein). Gene expression can be affected by extrinsic signals; for example, exposing cells, tissues, or organisms to agents that increase or decrease the gene expression contained in this case. Gene expression can also be regulated anywhere in the DNA to RNA to protein pathway. Regulation of gene expression can occur, for example, via activation, inactivation by transcription, translation, RNA delivery and processing, control of degradation of intervening molecules such as mRNA, and/or via specific protein molecules. , compartment, or degradation, or by any combination of the foregoing. Gene expression can be measured at RNA level or protein level by methods known in the art, including, but not limited to, Northern blotting, RT-PCR, Western blotting, and in vitro, in situ, Or in vivo protein activity test.

Increased performance: As used in this case, the term "increase performance" refers to the initial performance and also refers to an increase in the number of performance product parts produced by the template structure. In some embodiments, at least one heterologous gene can be provided to a cell or organism that otherwise comprises an endogenous replica of the same gene, thereby increasing the performance of the polypeptide encoded by the gene. In such specific cases, the table The current increase can be determined by aligning the amount of polypeptide produced by cells containing the heterologous and endogenous genes and the amount of the part produced by only the endogenous gene cells. In some embodiments, a first polypeptide (eg, NSP1-like and/or NSP2-like) that affects transcription can be provided to a cell or organism, thereby increasing a second polypeptide encoded by a gene controlled by the first polypeptide. Performance. In such embodiments, the increase in performance can be determined by aligning the portion of the polypeptide made from the gene in the presence of the first polypeptide and the portion produced from the gene in the absence of the first polypeptide. In some embodiments, the regulatory sequences are operably linked to a gene, thereby increasing the performance of the gene. In such a specific example, the increase in expression can be determined by aligning the amount of the polypeptide produced by the gene after the regulatory sequence is operatively linked to the gene and the amount of the moiety produced by the gene prior to the operative linkage or introduction of the regulatory sequence. .

Operably linked: when the first nucleic acid sequence is functionally associated with the second nucleic acid sequence, the first nucleotide sequence and the second nucleic acid sequence are operatively linked. When recombinantly produced, and where it is necessary to join two protein-coding regions in the same reading frame (e.g., in a polycistronic ORF), the operably linked nucleic acid sequences are generally contiguous. However, nucleic acids do not need to be contiguous to be operatively linked.

The term "operatively linked" as used in reference to a control sequence and a coding sequence means that the regulatory sequence affects the performance of the linked coding sequence. A "regulatory sequence" or "control element" refers to a nucleotide sequence that affects the timing and level of transcription, the processing or stability of RNA, or the translation of a contiguous coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; backbone-loop structures; suppressor binding sequences; termination sequences; polyadenylation Don't sequence; wait. The particular regulatory sequences may be located upstream and/or downstream of the coding sequence operably linked to them. Further, a particular regulatory sequence operably linked to a coding sequence can be located on a contiguous complementary strand of a double stranded nucleic acid molecule.

Promoter: As used herein, the term "promoter" refers to a region of DNA that is upstream of the start of transcription and that may be involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. An promoter is operably linked to a coding sequence for expression in a cell, or a promoter is operably linked to a nucleotide sequence encoding a signal sequence operably linked to a coding sequence for expression in a cell .

Some specific examples of this case include "plant promoters". Plant promoters are promoters that initiate transcription in plant cells.

Some specific examples of this case include the "organization-preference facilitator". Tissue-preference promoters are promoters that initiate transcription under developmental control, including, by way of example and not limitation: preferred initiation leaves, pollen, ears, roots, seeds, fibers, xylem vessels, tracheids, and Promoter of transcription of thick-walled tissues. Promoters that initiate transcription only in certain tissues are referred to as "tissue-specific". The "cell type-specific" promoter primarily drives the expression of certain cell types in one or more organs, for example, vascular cells in the root or leaf. An "inducible" promoter can be an environmentally motivated promoter. Examples of environmental conditions by which promoters can be induced to initiate transcription include anaerobic conditions and illumination. Tissue-specificity, tissue-preference, cell type-specificity, and inducible promoters constitute a "non-composition facilitator subclass.

Any inducible promoter can be used in some specific cases in this case. See Ward et al. (1993) Plant Mol. Biol. 22: 361-366. By inducing the promoter, the rate of transcription of the response elicitor is increased. Exemplary inducible promoters include, but are not limited to, promoters from the ACEI system that respond to copper; In2 genes from maize that respond to sulfonamide herbicide safeners ; Tet inhibitors from Tn10; and genes from steroid hormones Promoters can be induced whose transcriptional activity can be induced by glucocorticosteroids (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-5).

In contrast to non-composed promoters, the "composition" promoter is a promoter that is active under most environmental conditions. Exemplary compositional promoters include, but are not limited to, promoters derived from plant viruses, such as the 35S promoter from CaMV; promoters from rice actin; ubiquitin promoter; pEMU; MAS; maize H3 histone promoter; With the ALS promoter, Xba1/NcoI is segmented 5' to the canola ALS3 structural gene (or a nucleotide sequence similar to the Xba1/NcoI segment) (PCT International Patent Publication No. WO 96/30530).

Furthermore, any tissue-specific or tissue-preferential promoter can be utilized in some specific examples of the invention. A plant transformed with a nucleic acid molecule comprising a coding sequence operably linked to a tissue-specific promoter can specifically or preferentially produce a product of the encoded sequence in a particular tissue. Exemplary tissue-specific or tissue-preferential promoters include, but are not limited to, root-preference promoters, such as those from the bean gene; leaf-specific and light-inducible promoters, such as those from cab or rubisco ; of facilitator, for example those derived from LAT52; promote pollen-specific promoters, such as those derived from Zm13; and a microspore-preferred facilitator, for example, those derived from apg.

Conservative substitution: As used herein, the term "conservative substitution" refers to a substitution of an amino acid residue by another amino acid of the same class. use. The non-conservative amino acid substitution is a substitution in which the residue does not fall within the same class, for example, the basic amino acid is substituted with a neutral or non-polar amino acid. The class of amino acids that can be defined for the purpose of conservative substitutions are well known in the art.

In some embodiments, conservative substitutions include the substitution of a first aliphatic amino acid with a second, different aliphatic amino acid. For example, if the first amino acid is Gly; Ala; Pro; Ile; Leu; Val; and Met, the first amino acid can be substituted to be selected from Gly; Ala; Pro; Ile; Leu; Val; a second different amino acid from Met. In a specific example, the first amino acid is Gly; Ala; Pro; Ile; Leu; The second different amino acid of Val. In a specific example involving the substitution of a hydrophobic aliphatic amino acid, if the first amino acid is Ala; Pro; Ile; Leu; and one of Val, the first amino acid can be substituted to be selected from Ala; ; Ile; Leu; a second different amino acid from Val.

In other embodiments, conservative substitutions include the substitution of the first aromatic amino acid with a second, different aromatic amino acid. For example, if the first amino acid is His; Phe; Trp; and Tyr, the first amino acid can be substituted with a second different amino acid selected from the group consisting of His; Phe; Trp; and Tyr. In a specific example involving the substitution of an uncharged aromatic amino acid, if the first amino acid is Phe; Trp; and Tyr, the first amino acid can be substituted with a moiety selected from Phe; Trp; and Tyr. The second different amino acid.

In an alternative embodiment, the conservative substitution comprises the substitution of the first hydrophobic amino acid with a second, different hydrophobic amino acid. For example, if The first amino acid is Ala; Val; Ile; Leu; Met; Phe; Tyr; and Trp, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; Met; Phe; Tyr a second different amino acid from Trp. In a specific example involving the substitution of a non-aromatic hydrophobic amino acid, if the first amino acid is Ala; Val; Ile; Leu; and Met, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; a second different amino acid with Met.

In some embodiments, conservative substitutions include the substitution of a first polar amino acid by a second, different polar amino acid. For example, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from the group consisting of Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; second different amino acid with Glu. In a specific example involving the substitution of an unsubstituted amino acid, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; and Pro, the first amino acid can be replaced From Ser; Thr; Asn; Gln; Cys; Gly; a second different amino acid from Pro. In a specific example involving substitution of a charged amino acid, if the first amino acid is His; Arg; Lys; Asp; and Glu, the first amino acid can be substituted to select from His; Arg; Lys; Asp; a second different amino acid from Glu. In another example involving substitution of a charged amino acid, if the first amino acid is Arg; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from Arg; Lys; Asp; A second amino acid different from Glu. In a specific example involving substitution of a positively charged (basic) polar amino acid, the first amino acid is His; Arg; and one of Lys, the first amino acid may be substituted to be selected from His; Arg; A second amino acid different from Lys. Involving a positive electrode with a positive electrode In another example of a base acid, if the first amino acid is Arg or Lys, the first amino acid can be substituted with another amino acid among Arg and Lys. In a specific example involving the substitution of a negatively charged (acidic) polar amino acid, if the first amino acid is Asp or Glu, the first amino acid can be substituted with another amino acid among Asp and Glu.

In additional embodiments, conservative substitutions include the substitution of a first electrically neutral amino acid by a second, different electrically neutral amino acid. For example, if the first amino acid is Gly; Ser; Thr; Cys; Asn; Gln; and Tyr, the first amino acid can be substituted to be selected from Gly; Ser; Thr; Cys; Asn; Gln; a second amino acid different from Tyr.

In some embodiments, conservative substitutions include the substitution of a first non-polar amino acid by a second, different, non-polar amino acid. For example, if the first amino acid is Ala; Val; Leu; Ile; Phe; Trp; Pro; and Met, the first amino acid can be substituted to be selected from Ala; Val; Leu; Phe; Trp; Pro; a second different amino acid with Met.

In many instances, a particular second amino acid to be used for conservative substitution to replace the first amino acid can be selected in order to maximize the number of the aforementioned classes to which both the first and second amino acids are attached. Thus, if the first amino acid is Ser (polar, non-aromatic, and electrically neutral amino acids), the second amino acid can be another polar amino acid (ie, Thr; Asn; Gln; Cys; Gly). ;Pro;Arg;His;Lys;Asp; or Glu); another non-aromatic amino acid (ie, Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; Ile ; Leu; Val; or Met); or another electrically neutral amino acid (ie, Gly; Thr; Cys; Asn; Gln; or Tyr). However, it is preferred that the second amine group The acid may in this case be one of Thr; Asn; Gln; Cys; and Gly, since the amino acids share all classes according to polarity, non-aromaticity and electrical neutrality. Additional points that may optionally be used in selecting a particular second amino acid to be used for conservative substitutions are well known in the art. For example, when Thr; Asn; Gln; Cys; and Gly are both useful for conservative substitution of Ser, Cys can be excluded from selection to avoid formation of undesired cross-linking and/or disulfide bonds. Likewise, Gly can be excluded from the selection because it lacks an alkyl side chain. In this case, Thr can be selected, for example, to retain the functionality of the side chain hydroxyl groups. However, the selection of a particular second amino acid to be used for conservative substitution is ultimately at the discretion of the skilled artisan.

Nitrogen-limited conditions: As used herein, the term "nitrogen-limited conditions" refers to conditions in which the nitrogen source (such as nitrates and ammonium salts) in the soil or medium is limited in part. The "limited" portion is in the range of 0.0 to 0.2 mM; such as 0 to 0.1 mM, 0 to 0.03 mM, and 0 to 0.05 mM nitrogen concentration in some examples.

Traits or phenotypes: The terms "trait" and "phenotype" are used interchangeably in this case. For purposes of this disclosure, traits of particular interest include, for example, agronomically important traits that crop plants can exhibit.

Transformation: As used herein, the term "transformation" refers to the transfer of one or more (plural) nucleic acid molecules into a cell. When a nucleic acid molecule becomes stable to be replicated by a cell, whether it is incorporated into the genome of the cell by the nucleic acid molecule, or by replication of the free gene, the cell is "transformed" by the nucleic acid molecule introduced into the cell. As used in this context, the term "transformation" encompasses all techniques by which nucleic acid molecules can be introduced into such cells. Examples include, but are not limited to, transfection with viral vectors; transformation with plastid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipid transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); Microinjection (Mueller et al. (1978) Cell 15:579 85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl .Acad.Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

Transgenic gene: The transgenic gene is an exogenous nucleic acid sequence. In some examples, the transgenic gene can be one of a dsRNA molecule or a double (multiple) strand comprising a nucleotide sequence that is complementary to the target nucleic acid. In some examples, the transgenic gene can be an antisense nucleic acid sequence that exhibits inhibition of the performance of the target nucleic acid. In still other examples, the transgenic gene can be a genetic sequence (such as a herbicide resistant gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding an desired agricultural trait. In these and other examples, the transgene may contain regulatory sequences (e.g., promoters) operably linked to the coding sequence of the transgene.

Vector: A vector refers to a nucleic acid molecule that introduces a cell, for example, to produce a transforming cell. A vector can include a nucleic acid sequence that allows it to replicate within a host cell, such as an origin of replication. Examples of vectors include, but are not limited to, plastids that carry exogenous DNA into the cell; plastids; phage; and viruses. The vector may also include one or more genes, antisense molecules, and/or selectable marker genes and other genetic elements as are known in the art. The vector can transduce, transform, or transfect the cell, thereby causing the cell to express the nucleic acid molecule and/or protein encoded by the vector. The vector optionally includes materials that help achieve entry of the nucleic acid molecule into a cell, such as a liposome, a protein coat, and the like.

Unless explicitly indicated or implied, the terms "a", "an", And "the" is used in this case to symbolize "at least one."

Unless otherwise expressly stated, all technical and scientific terms used in this context have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of commonly used terms in molecular biology can be found, for example, by Lewin B., Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers RA (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference , VCH Publishers, Inc., 1995 (ISBN 1-56081 -569-8) Find it. All percentages refer to weight and all solvent mixture ratios refer to volume unless otherwise noted. All temperatures are in degrees Celsius.

IV. BNF gene of non- noduce plants

The present disclosure provides compositions and methods for the development of novel and unintended uses of transcription factor-NSP1-like and NSP2-like, and NIN-like genes (such as NLPs1-9). As disclosed herein, NSP1-like, NLP4, and NLP9 affect functional interactions between plants (such as non-noduce plants, such as Arabidopsis) and NFB (such as S. meliloti, such as RMP110). Thus, for example, NSP1-like, NLP4, and/or NLP9 can be used to modulate the association of NFB with crop plants. The characteristics of NSP1-like, NLP4, and NLP9 described herein can be used, for example, to provide genetically transgenic plants having an adjusted BNF phenotype. For example, NSP1-like (SCL29), NLP4, and/or NLP9 may behave or overexpress in plants to initiate and/or increase the ability of plants to associate with NFB under conditions of limited N, and/or to increase The efficiency of the use of nitrogen in the environment. In certain instances, NSP1-like (SCL29), NLP4, and/or NLP9 can be introduced into a plant or overexpressed in a plant that routinely comprises the polypeptide (eg, by introducing an additional copy of the gene encoding the polypeptide, and/or Change the regulatory control of genes that are routinely present).

Some specific examples include NSP1-like transcription factor polypeptides. The NSP1-like polypeptide according to a particular embodiment comprises an amino acid sequence which shows an increased percent identity when aligned with SEQ ID NO: 1 (Arabid mustard NSP1-like). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 1, 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%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity.

Other specific examples include NIN-like protein polypeptides. Particularly specific examples include NLP4 polypeptides, and/or NLP9 polypeptides.

An NLP4 polypeptide according to some examples comprises an amino acid sequence that exhibits increased percent identity when aligned with SEQ ID NO: 2 (Arabid mustard NLP4). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 2, 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%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity. For example, some specific examples include AtNLP4 xenogenic homologs.

An NLP9 polypeptide according to some examples comprises an amino acid sequence that exhibits increased percent identity when aligned with SEQ ID NO: 3 (Arabid mustard NLP9). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 9, 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%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity. For example, some specific examples include AtNLP9 xenogenes.

In some embodiments, comprising the foregoing sequence identity when aligned with SEQ ID NO: 1 (NSP1-like polypeptide) or SEQ ID NO: 2 (NLP4 polypeptide) and/or SEQ ID NO: 3 (NLP9 polypeptide) The polypeptide of the amino acid sequence is contained within a protein essential for the association of the plant containing the protein with the NFB. An NSP1-like polypeptide can be identified by, for example, searching in a sequence library for a polypeptide sequence having a critical sequence identity to SEQ ID NO: 1. The NLP4 polypeptide can be identified by, for example, searching in a sequence library and a polypeptide sequence having certain sequence identity of SEQ ID NO: 2. A NLP9 polypeptide polypeptide can be identified by, for example, searching in a sequence library and a polypeptide sequence having certain sequence identity in SEQ ID NO:3. Useful sequence databases can be by any number of methods apparent to those skilled in the art having conventional (such as the use of the NCBI BLAST ^® tool) to search. Access to numerous databases of plants and other organisms is available through a variety of public and private commercial sources. It will be understood by those skilled in the art that NLP4 and NLP9 are homologous proteins, and thus, a specific polypeptide which is recognized to contain an amino acid sequence which shares sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3 is also recognized. Sequence identity can be shared with the other of SEQ ID NOs: 2 and 4.

Other specific examples include structural equivalents of NSP1-like polypeptides and/or structural equivalents of NLP4 polypeptides and/or NLP9 polypeptides. Structural equivalents include, but are not limited to, conservative substitutions of amino acid residues within the amino acid sequence of the NSP1-like, NLP4, and NLP9 polypeptides of the present invention. As used herein, "conservative substitution" is the substitution of an amino acid residue by another amino acid of the same class. The non-conservative amino acid substitution is a substitution in which the residue does not fall within the same class, for example, the basic amino acid is substituted with a neutral or non-polar amino acid. The class of amino acids that can be defined for the purpose of conservative substitutions are well known in the art.

In an alternate embodiment, conservative substitutions include the substitution of the first aliphatic amino acid with a second, different aliphatic amino acid. For example, if the first amino acid is Gly; Ala; Pro; Ile; Leu; Val; and Met, the first amino acid can be substituted to be selected from Gly; Ala; Pro; Ile; Leu; Val; a second different amino acid from Met. In a specific example, the first amino acid is Gly; Ala; Pro; Ile; Leu; The second different amino acid of Val. In a specific example involving the substitution of a hydrophobic aliphatic amino acid, if the first amino acid is Ala; Pro; Ile; Leu; and one of Val, the first amino acid can be substituted to be selected from Ala; ; Ile; Leu; a second different amino acid from Val.

In other embodiments, conservative substitutions include the substitution of the first aromatic amino acid with a second, different aromatic amino acid. For example, if you The monoamino acid is His; Phe; Trp; and one of Tyr, the first amino acid can be substituted with a second different amino acid selected from the group consisting of His; Phe; Trp; and Tyr. In a specific example involving the substitution of an uncharged aromatic amino acid, if the first amino acid is Phe; Trp; and Tyr, the first amino acid can be substituted with a moiety selected from Phe; Trp; and Tyr. The second different amino acid.

In some embodiments, conservative substitutions include the first hydrophobic amino acid being substituted with a second, different hydrophobic amino acid. For example, if the first amino acid is Ala; Val; Ile; Leu; Met; Phe; Tyr; and Trp, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; Met; Phe; Tyr; a second different amino acid from Trp. In a specific example involving the substitution of a non-aromatic hydrophobic amino acid, if the first amino acid is Ala; Val; Ile; Leu; and Met, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; a second different amino acid with Met.

In additional embodiments, conservative substitutions include the substitution of a first polar amino acid for a second, differently polar amino acid. For example, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from the group consisting of Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; second different amino acid with Glu. In a specific example involving the substitution of an unsubstituted amino acid, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; and Pro, the first amino acid can be replaced From Ser; Thr; Asn; Gln; Cys; Gly; a second different amino acid from Pro. In a specific example involving substitution of a charged amino acid, if the first amino acid is His; Arg; Lys; Asp; and one of Glu, the first amino acid can be substituted It is selected from the group consisting of His; Arg; Lys; Asp; and a second different amino acid with Glu. In another example involving substitution of a charged amino acid, if the first amino acid is Arg; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from Arg; Lys; Asp; A second amino acid different from Glu. In a specific example involving the substitution of a positively charged (basic) polar amino acid, if the first amino acid is His; Arg; and one of Lys, the first amino acid can be substituted to be selected from His; a second different amino acid from Lys. In another example involving the substitution of a positively charged amino acid, the first amino acid can be substituted with another amino acid among Arg and Lys, provided that the first amino acid is Arg or Lys. In a specific example involving the substitution of a negatively charged (acidic) polar amino acid, if the first amino acid is Asp or Glu, the first amino acid can be substituted with another amino acid among Asp and Glu.

In some embodiments, conservative substitutions include the substitution of a first electrically neutral amino acid by a second, different electrically neutral amino acid. For example, if the first amino acid is Gly; Ser; Thr; Cys; Asn; Gln; and Tyr, the first amino acid can be substituted to be selected from Gly; Ser; Thr; Cys; Asn; Gln; a second amino acid different from Tyr.

In another embodiment, the conservative substitution comprises the first non-polar amino acid being the second different non-polar amino acid. For example, if the first amino acid is Ala; Val; Leu; Ile; Phe; Trp; Pro; and Met, the first amino acid can be substituted to be selected from Ala; Val; Leu; Phe; Trp; Pro; a second different amino acid with Met.

In many instances, a particular second amino acid to be used for conservative substitution to replace the first amino acid may be for the first and second amino acids to be The number of the aforementioned categories is maximized to make a selection. Thus, if the first amino acid is Ser (polar, non-aromatic, and electrically neutral amino acids), the second amino acid can be another polar amino acid (ie, Thr; Asn; Gln; Cys; Gly). ;Pro;Arg;His;Lys;Asp; or Glu); other non-aromatic amino acids (ie, Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; Ile; Leu; Val; or Met); or another electrically neutral amino acid (ie, Gly; Thr; Cys; Asn; Gln; or Tyr). However, it is preferred that the second amino acid may in this case be Thr; Asn; Gln; Cys; and Gly, since the amino acids are shared according to polarity, non-aromaticity and electrical neutrality. All categories. Additional elements that may optionally be used in selecting a particular second amino acid to be used for conservative substitution are well known in the art. For example, when Thr; Asn; Gln; Cys; and Gly are both useful for conservative substitution of Ser, Cys can be excluded from selection to avoid formation of undesired cross-linking and/or disulfide bonds. Likewise, Gly can be excluded from the selection because it lacks an alkyl side chain. In this case, Thr can be selected, for example, to retain the functionality of the side chain hydroxyl groups. However, the selection of a particular second amino acid to be used for conservative substitution is ultimately at the discretion of the skilled artisan.

Other mutations can be performed on plant NSP1-like, NLP4, and NLP9 encoding polynucleotides to generate more suitable, for example, expression and scale-up in host cells comprising the polynucleotide(s). Performance product. For example, a cysteine residue can be deleted or replaced with another amino acid to exclude a disulfide bridge; the N-linked glycosylation site can be modified or eliminated to achieve, for example, performance Yeast hosts that are known to over-glycosylate N-linkage sites are more likely to recover and purify homogenous products. For this purpose, in the extracellular region (ECD) a wide variety of amino acid substitutions (NXS or NXT) occurring in one or both of the first or third amino acid positions of any one or more glycosylation recognition sequences, and/or at ECD Amino acid deletion at the second position of any one or more of such recognition sequences will prevent glycosylation of the mutant polypeptide of the modified tripeptide sequence.

Hydrophilic amino acids generally include and generally have the following individual relative hydrophobicities (at pH 7.0; kcal/mol): aspartic acid (D), -7.4; glutamic acid (E)-9.9; aspartame ( N), -0.2; branamine (Q), -0.3; lysine (K), -4.2; arginine (R), -11.2; serine (S), -0.3; and cysteamine Acid (C), -2.8. Hydrophobic amino acids generally include and generally have the following individual relative degrees of hydrophobicity: 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; with propylamine Acid (A), 0.5. Glycine has a degree of hydrophobicity of 0 and can be considered hydrophilic or hydrophobic.

As is known in the art, the amino acid homology of a peptide can be readily determined by comparison to its amino acid sequence. Likewise, the amphoteric homology of the peptide can be determined by comparing the hydrophilicity and hydrophobicity of the amino acid sequence.

Some specific examples include nucleosides comprising NSP1-like ("NSP1-like polynucleotides"), NLP4 ("NLP4 polynucleotides"), and/or NLP9 polypeptides ("NLP9 polynucleotides"). A nucleic acid of an acid sequence, such as those described above. For example, when the nucleic acid sequence in some specific examples is aligned with SEQ ID NO: 4 ( Arabid mustard NSP1-like ) or SEQ ID NO: 5 ( Arabid mustard NLP4 ) and/or SEQ ID NO: 6 ( Arabid mustard NLP9 ) Shows increased percentage consistency. The particular nucleic acid sequences in these and other specific examples can include, by way of example and not 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% identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5 and/or SEQ ID NO: 6.

A number of nucleic acids comprising nucleotide sequences encoding NSP1-like, NLP4, and/or NLP9 polypeptides can be readily identified by those skilled in the art. For example, a nucleic acid molecule can be modified without substantially altering the amino acid sequence encoding the polypeptide, for example, by introducing a permissible nucleotide substitution based on codon degeneracy. Thus, it will be appreciated that any NSP1-like, NLP4, and/or NLP9 polypeptide of a given amino acid sequence can be immediately reverse engineered into any of a number of redundant nucleotide sequences. By way of further example, the gene encoding the NSP1-like, NLP4, and/or NLP9 polypeptide can be selected from any of a number of available plant genomic libraries, cDNA libraries, EST libraries, and the like (eg, by SEQ ID NOs: 4-6 Homology, or by sequence similarity of the encoded polypeptide to SEQ ID NO: 1 or SEQ ID NOs: 2 and/or 3, or such genes can be selected according to reliable and well-known techniques of molecular biology Since the organism.

Any and all of the NSP1-like, NLP4, and NLP9 polypeptides, as well as nucleic acid molecules encoding the same, can be utilized in certain embodiments of the invention.

In some embodiments of the present invention, a nucleic acid comprising a nucleotide sequence encoding an NSP1-like, NLP4, and/or NLP9 polypeptide comprises a gene regulatory element (such as a promoter). Promoters can be selected based on the type of cell into which the vector construct is to be inserted. Promoters acting on bacteria, yeast, and plants are well known in the art. Promoters can also be selected based on their regulatory characteristics. Examples of such features include increased transcriptional activity, inducing ability, tissue-specificity, and developmental phase-specificity. In plants, promoters that can be induced, have a viral or synthetic origin, are constitutively active, are regulated in a temporal manner, and are spatially regulated are described. See, for example, Poszkowski et al. (1989) EMBO J. 3:2719; Odell et al. (1985) Nature 313:810; and Chau et al. (1989) Science 244: 174-81).

In order to obtain a higher degree of expression of the heterologous gene(s), it may be preferred to reprogram the gene(s) to render the host cell more efficiently (eg, plant cells, for example, rapeseed, rice) , tobacco, corn, cotton, and soybeans). Thus, an additional step in designing a gene encoding a NSP1-like or NLP4 and/or NLP9 polypeptide for expression in a plant (ie, in addition to providing one or more gene regulatory elements) is a reprogramming of the heterologous gene protein coding region To optimize performance. Specific examples include redesigned Arabidopsis genes that have been optimized to increase performance levels in gene transfer plant cells from a second plant species (ie, to make more protein) compared to the second plant species Plant cells transformed with the original (ie unmodified) Arabidopsis gene sequence.

Genomic evolution in different organisms or organism classes has resulted in differences in the use of synonymous codons due to the plasticity conferred by gene redundancy/degenerate (ie, some amino acids are specified by more than one codon). This "codon bias" reflects the average base composition of the protein coding region. For example, a biological system with a relatively low G+C content of the genome utilizes more codons with A or T at the third position of the synonymous codon, and such Relatively high G+C content utilizes more codons with G or C at the third position. Furthermore, it is believed that the presence of "trace" codons in the mRNA reduces the absolute translational rate of the mRNA, especially when the relative redundancy of charged tRNAs corresponding to the microcodons is extremely low. An extension of this discussion is that the reduced translation rate due to individual microcodons will add at least for multiple microcodons. Thus, mRNAs with particularly high levels of microcodons in a particular expression host have a correspondingly low translation rate. This ratio can be reflected by the corresponding low level of the encoded protein.

In engineering an optimized gene encoding an NSP1-like or NLP4 and/or NLP9 polypeptide for expression in plant cells (eg, rice, tobacco, corn, cotton, and soybean), if the desired host plant(s) have been determined Codon bias is helpful. There are a number of publicly available databases of DNA sequences in which we can find information about the distribution of plant genome codons or protein coding regions of various plant genes.

Codon bias is a codon statistical distribution that represents the amino acid that the host uses to encode its protein. Codon bias can be calculated as the frequency with which a single codon is used relative to the codons of all amino acids. Alternatively, codon bias can be calculated as the frequency of use of a single codon encoding a particular amino acid relative to all remaining codons (synonymous codons) of the amino acid.

When designing an optimized coding region for a plant to express NSP1-like or NLP4 and/or NLP9 polypeptides, when multiple options exist, the plant's preferred major codon ("first option") should be determined, and a second Preferred codons for options such as third, fourth, etc. A novel DNA sequence encoding an amino sequence of an NSP1-like or NLP4 and/or NLP9 polypeptide can then be designed, wherein A novel DNA sequence differs from a native DNA sequence (encoding the polypeptide) by the substitution of a host-preferred (first preference, second preference, third preference, or fourth preference, etc.) codon to specify the amine Amino acids at various positions within the base acid sequence. The restriction enzyme sites that may be created by modification in the novel sequences are then analyzed. The identified putative restriction sites are then modified by replacing the codons with a preferred codon to remove the restriction sites. Other sites in the sequence that may affect transcription or translation of the heterologous sequence are exons: intron junctions (5' or 3'), poly-A addition signals, and/or RNA polymerase termination signals. The sequence can be reanalyzed and modified to reduce the frequency of TA or CG doublets. In addition to the duplexes, sequence blocks having more than about six identical G or C nucleotides can also adversely affect sequence transcription or translation. Thus, the blocks are advantageously modified by replacing the first or second codons of the codons with the next preferred codon option.

A method such as that described above enables a person skilled in the art to modify a gene(s) that is foreign to a particular plant, such that the gene is optimally expressed in the plant. This method is further exemplified in PCT International Patent Publication No. WO 97/13402 A1. Thus, some specific examples of optimized synthetic genes that are functionally equivalent to NSP1-like , NLP4 , and/or NLP9 polynucleotides can be used in transformed hosts, including plant and plant cells. Furthermore, also generated in the computer (in silico) from the initial amino acid sequence of NSP1- like, nLP4, and NLP9 polynucleotide. Additional guidance for the manufacture of synthetic genes can be found, for example, in U.S. Patent 5,380,831.

Once the NSP1-like , NLP4 , or NLP9 polynucleotide sequence has been designed on paper or on a computer, the actual nucleic acid molecule comprising the polynucleotide sequence can be synthesized in the laboratory such that the sequence corresponds exactly to the designed sequence. Such synthetic DNA molecules are selected or otherwise manipulated, as are derived from natural or native sources.

V. Adjusting plant growth with NSP1-like , NLP4 , and/or NLP9 under N-limited conditions

Some specific examples use NSP1-like, NLP4, and NLP9 to be necessary for plants to maintain and/or increase growth via association with NFB under N-limited growth conditions. For example, NSP1-like, NLP4, and/or NLP9 polypeptides can introduce or heterologously express plant cells, by way of example and without limitation: increasing the association of plants comprising plant cells with NFB; increasing plant growth under conditions of limited N And increase the efficiency of plants using environmental nitrogen.

In a particular embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptides can be expressed or overexpressed in a cell or organism, by way of example and without limitation: by NSP1-like , NLP4 , and/or NLP9 polynucleotides Introducing a cell or organism; introducing a NSP1-like, NLP4, and/or NLP9 polypeptide into a cell or organism; and/or by providing a signal or operatively linked to NSP1 via the signal(s) in the cell or organism The regulatory elemental interaction of the -like , NLP4 , and/or NLP9 polynucleotides is sufficient to promote positive or negative signaling of NSP1-like, NLP4, and/or NLP9 polypeptide expression. In another embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptides can be eliminated or rendered low in a cell or organism, by way of example and without limitation: by disrupting, mutating, or inactivating NSP1-like , NLP4 And/or NLP9 polynucleotide; introducing an antisense nucleic acid into a cell or organism that targets an NSP1-like , NLP4 , and/or NLP9 polynucleotide; binding NSP1- by an antibody or other specific binding protein , NLP4, and/or NLP9 polypeptides physically remove NSP1-like, NLP4, and/or NLP9 polypeptides from cellular machinery or cells of a living organism; and/or via (s) provided in a cell or organism Signaling and operably linked to a regulatory elemental interaction of NSP1-like , NLP4 , and/or NLP9 polynucleotides sufficient to reduce or eliminate positive or negative signaling of NSP1-like, NLP4, and/or NLP9 polypeptide expression .

In some embodiments, the NSP1-like polypeptide can be expressed or overexpressed in a cell or organism, thereby promoting the expression of at least one NIN (s) gene; by way of example and without limitation, NLP4 and NLP9 . In another embodiment, the NSP1-like polypeptide can be removed or expressed in a low degree in a cell or organism, thereby reducing or eliminating the performance of at least one NIN (s) gene, for example, to study in a non-junction The mechanism of NFB association in plants. In some embodiments, the NLP4 and/or NLP9 polypeptide can be expressed or overexpressed in a cell or organism, thereby directly increasing the ability of the plant cell to be associated with NFB. In additional embodiments, the NLP4 and/or NLP9 polypeptides can be removed or understated in cells or organisms, thereby reducing or eliminating the ability of plant cells to be associated with NFB. In a particular embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptide is a nucleoside operatively linked to a regulatory element that directs expression of the polypeptide(s) in conditions other than growth restriction of the nitrogen. The acid is expressed thereby increasing the efficiency of the plant in utilizing environmental nitrogen under these other conditions.

In a particular embodiment, the conservation of the NFB-related mechanism between different plant species and different plant species is amplified to correlate novel NFB via heterologous expression of NSP1-like, NLP4, and/or NLP9 polypeptides. Sexual phenotype introduces plants. For example, NSP1-like, NLP4, and/or NLP9 polypeptides may be non-expressing non-NSP1-like, NLP4, and/or NLP9 polypeptides. Expressed in nodulation plants, thereby conferring increased ability to bind the plant to NFB.

In an alternative embodiment of the present invention, plant cells, plant parts, and/or plants can be genetically engineered to include at least one NSP1-like , NLP4 , and/ by any number of methods known in the art to introduce heterologous molecules. Or a NLP9 polynucleotide whereby a non-native gene transfer plant cell, plant part, or plant is made. In a particular embodiment of the present invention, the heterologous molecule is introduced into a plant cell, a plant part, and/or a plant by a method selected from the group consisting of, but not limited to, -transformation and selective breeding, such as backcross breeding.

In some embodiments, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to a compositional promoter, thereby directing the gene product to generally not exhibit (such as nitrogen non-limited) Performance under the conditions. In a specific embodiment, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to a non-constituting promoter to direct gene product to tissue-preference (eg, in root tissue) ) or tissue-specific performance. In a particular embodiment, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to an elicitible promoter to direct the expression of the gene product in a regulated manner.

Any plant species or plant cell can be genetically engineered to comprise a heterologous nucleic acid of the present invention. In some embodiments, the genetically engineered plant cells are capable of being regenerated to produce plants. In some embodiments, genetically engineered plant cells (eg, host plant cells) include cells from, for example, without limitation: higher plants, dicots, monocots, consumable plants, crop plants, oil plants ( Such as oilseed plants, and non-nodulated plants. Such plants include, by way of example and not limitation:: soy; soybean; cotton; rapeseed (rapeseed); flaxseed; corn; rice; arm-shaped grass; wheat; safflower; sorghum; beet; sunflower; tobacco; Lawn grass). In a specific example, the genetically modified plant cell or plant of the present invention includes, by way of example and not limitation: Brassica napus ; Brassica juncea ; Brassica carinata ; Brassica rapa ; Cauliflower ( Brssica oleracea ); soybean ( Glycine max ); flax ( Linum usitatissimum ); corn ( Zea mays ); safflower ( Carthamus tinctorius ); sunflower ( Helianthus annuus ); tobacco ( Nicotiana tabacum ); arabian mustard, brazil nut ( Betholettia excelsa ); Ricinus communis ; Cocus nucifera ; Coriandrum sativum ; Gossypium spp .; Arachis hypogaea ; Simmondsia chinensis ; Elaeis guineeis Olea eurpaea ; Oryza sativa ; Cucurbita maxima ; Hordeum vulgare ; Saccharum officinarum ; Triticum durum and Triticum aestivum ; With the genus Lemnaceae ( Lemnaceae sp. ). In some embodiments, plants may have in particular a genetic background as an excellent cultivar, a wild-type cultivar, and a commercially distinguishable species.

Nucleic acids can be introduced into essentially any plant according to methods known in the art. Specific examples of the present invention can employ any of a wide variety of methods known in the art for plant transformation (and for the manufacture of genetically modified plants). Such methods include, by way of example and not limitation, biological and physical transformation procedures for dicotyledonous plants, as well as monocotyledons. See, for example, Goto-Fumiyuki et al. (1999) Nat. Biotechnol . 17:282; Miki et al. (1993) Methods in Plant Molecular Biology and Biotechnology (Glick, BRand Thompson, JE, Eds.), CRC Press, Inc. , Boca Raton, FL, pp. 67-88. In addition, vectors and in vitro culture methods for plant cell and tissue transformation and regenerative plants are described, for example, in Gruber and Crosby (1993) Methods in Plant Molecular Biology and Biotechnology , supra, at pp. 89- 119.

Plant transformation techniques for introducing nucleic acids into plant host cells include, by way of example and not limitation, the use of Agrobacterium tumefaciens or A. rhizogenes as a transforming agent to de-trace T-DNA transformation; calcium phosphate Transfection; polyamine transduction; protoplast fusion; electroporation (D'Halluin et al. (1992) Plant Cell 4: 1495); ultrasonic methods (such as ultrasonic perforation); liposome transformation; microinjection Contact with naked DNA; contact with plastid vectors; contact with viral vectors; biological ballistics (such as DNA particle bombardment (see, for example, Klein et al. (1987) Nature 327:70) and microprojectile bombardment (Sanford et al. (1987) Part. Sci. Technol. 5:27; Sanford (1988) Trends Biotech. 6:299, Sanford (1990) Physiol. Plant 79:206; and Klein et al. (1992) Biotechnology 10:268); 碳WHISKERS-mediated 碳Transformation (Kaeppler et al. (1990) Plant Cell Rep. 9: 415); nanoparticle transformation (see, for example, US Patent Publication No. US 2009/0104700 A1); aerosol emission; and polyethylene glycol (PEG)- Mediated uptake. In a particular example, a heterologous nucleic acid can be introduced directly into the base of a plant cell. Group DNA.

The widely used method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. Horsch et al. (1985) Science 227:1229. Agrobacterium tumefaciens and Agrobacterium rhizogenes are phytopathogenic soil bacteria which are conventionally used for genetically transforming plant cells. The Agrobacterium tumefaciens and the Ti and Ri plastids of Agrobacterium tumefaciens carry genes responsible for plant gene transformation, respectively. Kado (1991) Crit. Rev. Plant. Sci. 10:1. Details of the Agrobacterium vector system and methods for Agrobacterium-mediated gene transfer can also be obtained, for example, from Gruber et al. , supra; Miki et al. , supra; Moloney et al. 1989) Plant Cell Reports 8: 238, and US Patent Nos. 4,940,838 and 5,464,763.

In case the Agrobacterium is used as a transformation, the DNA to be inserted is usually selected into a special plastid; into an intermediate vector or a binary vector. The intermediate vector cannot replicate itself in Agrobacterium. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of an auxiliary plastid (conjugate). The Japan Tobacco Superbinary System is an example of such a system (reviewed in Komari et al. (2006) Methods in Molecular Biology (K. Wang, ed.) No. 343; Agrobacterium Protocols , ^2nd Edition , Vol. 1, Humana Press Inc., Totowa, NJ, pp. 15-41; and Komori et al. (2007) Plant Physiol . 145: 1155). The binary vector can replicate itself in both E. coli and Agrobacterium. The binary vector comprises a selectable marker gene and a linker or polylinker that is sandwiched by the right and left T-DNA border regions. They can be directly transformed into Agrobacterium (Holsters, 1978). Agrobacterium contains a plastid carrying the vir region. The Ti or Ri plastid also contains the vir region necessary for T-DNA transfer. The vir region is required for the transfer of T-DNA to plant cells. May contain additional T-DNA.

The viral function of the Agrobacterium tumefaciens host will direct the inclusion of the construct and the adjacent marker T-strand into the plant cell DNA when the cell is infected with a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12: 8711) or co-culture program (Horsch et al. (1985) Science 227: 1229). In general, Agrobacterium transformation systems are used to engineer dicots. Bevan et al. (1982) Ann. Rev. Genet 16:357; Rogers et al. (1986) Methods Enzymol. 118:627. Agrobacterium transformation systems can also be used for transformation, as well as transfer of nucleic acids to monocots and plant cells. See US 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.

Genetic manipulation of recombinant hosts in this case can be performed using standard genetic techniques. It can be carried out in any host cell suitable for gene manipulation. In some embodiments, the recombinant host cell can be any organism or microbial host suitable for use in gene manipulation and/or recombinant gene expression. In some embodiments, the recombinant host can be a plant. The standard recombinant DNA and molecular selection techniques used in this case are well known and described in the art, by way of example and not 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.

After introduction of the nucleic acid into the plant cell, the plant cell can grow and, after emergence of meristems such as buds and roots, can produce mature plants. In some embodiments, a plurality of plants can be produced. The methodology for regenerating plants is well known to those skilled in the art and can be found, for example, in Plant Cell and Tissue Culture (Vasil and Thorpe, Eds.), Kluwer Academic Publishers, 1994. The genetically modified plants described in this case can be cultured in a fermentation medium or grown in a suitable medium, such as soil. In some embodiments, a suitable growth medium for higher plants may be any growth medium for the plant, including, by way of example and not limitation: soil, sand, supporting root growth (such as vermiculite, perlite, etc.) or hydroponic cultivation Any other specific medium, as well as suitable light, water and nutrient supplements that help the growth of higher plants.

The transformed plant cells produced by any of the above-described transformation techniques can be cultured to regenerate whole plants having a transformed phenotype and thus a desired phenotype. Such regeneration techniques rely on manipulation of certain plant hormones in tissue growth media, typically relying on biocides and/or herbicide markers that have been introduced with the desired nucleotide sequence. Regeneration of plant lines 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 carried out by plant mites, explants, organs, pollen, embryos or a part thereof. Such regeneration techniques are generally described in Klee et al. (1987) Ann. Rev. Plant Phys. 38:467.

In other specific examples, the transformed plant cells cannot be regenerated to make Planting. Such cells can be used, for example, in the development of plant cell lines with associated phenotypes - for example, NFB-related properties.

The transformed plant cell, sputum, tissue or plant can be identified and isolated by selecting or sieving the plant material by the trait encoded by the marker gene present on the transmorphic DNA. For example, selection can be carried out by growing the engineered plant material on a medium containing an antibiotic or herbicide inhibiting amount that confers resistance to the transforming gene construct. Furthermore, transformed plants and plant cells can also be screened for the activity of any visible marker gene (such as beta-glucuronidase , luciferase , or gfp gene) that can be present on the recombinant nucleic acid construct. Identification. Such selection and screening methods are well known to those skilled in the art.

A genetically transgenic plant comprising a heterologous molecule of the present invention can be produced by selective breeding, for example, by sexually crossing a first parental plant comprising the molecule with a second parental plant, thereby producing a plurality of first sub-species Generation of plants. A first progeny plant can then be selected that is resistant to a selection marker (such as glyphosate, which is conferred to the progeny plant by the heterologous molecule in this case). The first progeny plant can then be selfed, thereby producing a plurality of second progeny plants. Subsequently, a second progeny plant resistant to the selection marker can be selected. The steps may further comprise backcrossing the first progeny plant or the second progeny plant to the second parental plant or the third parental plant.

It is also understood that two different genetically transformed plants can also be mated to produce offspring containing two independently segregating, plus exogenous genes. Selfing of appropriate offspring can produce two plants that are homozygous for the exogenous gene. Backcrossing of parental plants and outcrossing with non-genetically transformed plants are also envisaged. Asexual reproduction is also true. Other breeding methods commonly used for different traits and crops are well known in the art. Backcross breeding has been used to transfer genes that are simply inherited highly heritable traits into the desired homozygous cultivar or inbred line, which is a recurrent parent. The resulting plant line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent. After the initial mating, the individuals who have the representative type are given and the mating (backcrossing) is repeated to the recurrent parent. The resulting parental line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent.

Nucleic acids can also be introduced into predetermined blocks of the plant genome via homologous recombination. Methods for the stable integration of polynucleotide sequences within specific chromosomal sites of plant cells via homologous recombination have been described in the art. For example, the site-specific integration described in U.S. Patent Publication No. 2009/0111188 A1 relates to the use of a recombinase or integrase to mediate the introduction of a chromosomal target into a polynucleotide sequence. In addition, PCT International Patent Publication No. WO 2008/021207 describes zinc finger structure-mediated homologous recombination to stably integrate one or more donor polynucleotide sequences within a genome-specific location. The use of a recombinase, such as the FLP/FRT described in U.S. Patent No. 6,720,475, or the CRE/LOX described in U.S. Patent No. 5,658,772, can be used to stably integrate a polynucleotide sequence into a specific chromosomal site. Finally, the use of meganucleases to target a polynucleotide to a specific chromosomal location is described in Puchta et al. (1996) Proc. Natl. Acad. Sci. USA 93:5055.

Other various methods for site-specific integration within plant cells are generally known and applicable. Kumar et al. (2001) Trends Plant Sci. 6(4): 155. Furthermore, site-specific recombination systems that have been identified in several prokaryotic and lower eukaryotes can be applied to plants. Examples of such systems include, but are not limited to, the R/RS recombinase system derived from the pSR1 plastid of Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol . Biol . 182: 191), and Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170).

Various assays can be used in conjunction with nucleic acid molecules of certain embodiments of the present invention. In addition to phenotypic observation, the following techniques can be used to detect the presence of nucleic acid molecules in plant cells. For example, the presence of a molecule can be determined by using a primer or probe of the sequence, an ELISA assay to detect the encoded protein, a Western blotting method for detecting the protein, or a northern or southern dot method for detecting RNA or DNA. . Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, can also be used to detect the presence or expression of recombinant constructs within specific plant organs and tissues.

Southern analysis is a commonly used detection method in which DNA is cleaved by restriction endonucleases and fractionated on an agar gel to separate DNA by molecular weight and then transferred to a nylon membrane. The radiolabeled probe fragment is then hybridized with ³² P (or other probe label) and washed in the SDS solution.

Similarly, Northern analysis employed a similar procedure in which RNA was cleaved by restriction endonucleases and fractionated on an agar gel to isolate RNA by molecular weight and subsequently transferred to a nylon membrane. The radiolabeled probe fragment is then hybridized with ³² P (or other probe label) and washed in the SDS solution. Analysis of RNA isolated from tissue of interest (eg, mRNA) can indicate relevant performance levels. In general, if mRNA is present or the amount of mRNA is increased, it can be inferred that the corresponding transgene is being expressed. Northern analysis, or other mRNA analysis procedures, can be used to determine the level of expression of the introduced transgene or native gene.

The nucleic acids of this case, or fragments thereof, can be used to design primers for PCR amplification. When performing PCR amplification, some degree of unpairing can be tolerated between the primer and the template. Mutations, insertions, and deletions can be made in a given primer by a method known to those skilled in the art.

Hydrolysis probe assay - or conventionally known as TAQMAN ^® (Life Technologies, Foster City, Calif.) - is another method for detecting and quantifying the presence of DNA sequences. Briefly, FRET oligonucleotide probes are designed such that an oligo is located within the transgene and one is located in the flanking genomic sequence for event specific detection. The FRET probe and the PCR primer (one primer located in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe causes the fluorescent moiety on the FRET probe to cleave and release from the quenching moiety. Fluorescent signals indicate flanking/transgenic gene insertion sequences that are present due to successful amplification and hybridization.

VII. Plants, plant parts , and plant materials containing NSP1-like , NLP4 , and NLP9

Some specific examples of the present invention include plants comprising at least one heterologous NSP1-like , NLP4 , and/or NLP9 polynucleotide, such as may be produced from stably transformed plant cells or tissues, or may be derived from a donor line Genetic introgression of nucleic acids. Such plants can be used or cultured in any manner in which the desired polynucleotide(s) of interest are present. Accordingly, the genetically transformed plant can be engineered and cultured, inter alia, by transformation into one or more desired traits (e.g., NFB affinity) and then by any method known to those skilled in the art. Particular embodiments of the present invention provide portions, cells, and/or tissues of such genetically transformed plants. Plant parts include seeds, endosperms, ovules and pollen without limitation. In some embodiments, the plant parts are seeds.

Representative, non-limited plants include non-nodulated plants; Arabidopsis; field crops (such as alfalfa, barley, beans, clover, maize, cotton, flax, lentils, corn, peas, rapeseed/rapeseed, rice, rye) , safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (such as asparagus, beets, canola, broccoli, Brussels sprouts, cabbage, carrots, broccoli, celery, cucumber (melon crops), eggplant, lettuce) , mustard, onions, sweet peppers, potatoes, pumpkins, turnips, spinach, squash, taro, tomatoes, and zucchini; fruit and nut crops (such as almonds, apples, apricots, bananas, blackberries, blueberries, cocoa, cassava, Cherry, citrus, coconut, cranberry, date, hazelnut, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, Pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut, and watermelon); trees and ornamental plants (such as alder, ash, white poplar, rhododendron, birch Tree, boxwood, camellia, carnation, chrysanthemum, eucalyptus, fir, ivy, jasmine, juniper, oak, palm, willow, pine, rosewood, azalea, rose, and rubber).

To confirm the presence of the polynucleotide of interest(s) used to regenerate the plant, a variety of assays can be performed. Such tests include, by way of example and not limitation, biochemical tests, such as detecting the presence of a protein product, such as By immunological means (ELISA and / or Western blotting method) or by the function of enzymes; plant part tests (such as leaf or root test); and analysis of plant phenotype.

In the development of any novel source of desired plant, there are numerous steps that can be initiated to produce a genetically transformed crop plant. In some embodiments, a genetically transgenic plant comprising at least one NSP1-like , NLP4 , and/or NLP9 polynucleotide can be used in a plant breeding and/or provenance development program.

Plant breeding begins with the analysis and definition of current sources of problems and weaknesses, the establishment of planning goals, and the definition of specific breeding purposes. The next step is to select the source that has the traits that meet the planned goals. The goal is to combine improved combinations of desirable traits from the parental source in a single variant. Such traits may include higher seed yield, resistance to disease and insects, preferred stem and root, drought and heat tolerance, and better agronomic qualities.

The selection of breeding or selection methods depends on the reproductive mode of the plant, the heritability of the trait to be improved, and the type of commercial cultivar (such as F ₁ hybrid cultivar, pure cultivar, etc.). In the case of highly heritable traits, superior individual plant options evaluated at a single location will be effective, but for low heritability traits, the selection should be based on an average obtained from repeated assessments of the relevant plant family. Popular selection methods include pedigree selection, modified lineage selection, quality selection, and recurrent selection.

Genetic complexity affects the choice of breeding methods. Backcross breeding lines are used to transfer one or several desirable genes of highly hereditary traits into cultivars. This method has been widely used for breeding of disease resistant cultivars. Various recurrent selection techniques are used to improve the quantitative genetic traits regulated by numerous genes. In self The use of recurrent selection in flower pollination crops depends on the ease of pollination, the frequency of successful hybridizations for each pollination, and the number of hybrid progeny that have been successfully mated.

Each breeding program should include a regular, objective assessment of the efficiency of the breeding program. Assessment points are subject to change by purpose and purpose, but should include selection gains based on the appropriate standard value of the comparison of advanced breeding lines, and the number of successful cultivars per input unit (eg, annual, per dollar, etc.) .

Promising advanced breeding lines are thoroughly tested and compared to appropriate standards for three or more years in an environment representing the business target area(s). The best strains are candidates for novel commercial cultivars; those who still lack a number of traits can be used as parents to make new populations for further selection.

From the time of the first mating, these processes - the final steps leading to sales and distribution - usually take eight to twelve years. Therefore, the development of novel cultivars is a time-consuming process that requires precise forward-looking planning, efficient use of resources, and little change of direction.

Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. The breeding program integrates the desired traits or broad-based resources from two or more cultivars into the breeding pool of the cultivar by self-intersection and selection of desired phenotypes. Evaluate novel cultivars to determine which has commercial potential.

Lineage breeding systems are commonly used to improve self-pollination crops. So has the desirable traits of two complementary parental mating to produce F _1. The F ₂ population is made by selfing one or several F ₁ s. The best choice may be subject to a F ₂ population started; then, begins at F _3, select the best individual optimal family. These tests can be repeated beginning family generations F _4, to improve with low heritability of the trait selection effectiveness. In the later stages of breeding (ie F ₆ and F ₇ ), testing the mixture of the best line or phenotypically similar lines is released as a potential for novel cultivars.

Quality and recurrent selection can be used to improve the population of self-pollinated crops or cross-pollinated crops. The genetically variable population of a heterozygous individual is either recognized or created by cross-mating of several different parents. The best plant lines are selected based on individual strengths, outstanding offspring, or outstanding binding abilities. The selected plants are cross-matured to create a novel population in which a further selection cycle continues.

Backcross breeding has been used to transfer genes that are simply inherited highly heritable traits into the desired homozygous cultivar or inbred line, which is a recurrent parent. The source of the trait to be transferred is called the parental parent. The resulting plant line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent. After the initial mating, the individuals who have the representative type are given and the mating (backcrossing) is repeated to the recurrent parent. The resulting parental line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent.

A single seed descendant program refers in the strict sense to planting isolated populations, harvesting one seed sample per plant, and using the one seed sample to plant the next generation. When the evolution of the population from the F ₂ to the desired time position inbred derived plants produced based traced back to each different F ₂ individuals. The number of plants in the population is attenuated with generations because several seeds are unable to germinate or several plants are unable to produce at least one seed. As a result, when the completion of the evolution of generations, not all of the initial sample population of F ₂ progeny plants will be represented.

In the specific case of the present invention, NSP1-like , NLP4 , and/or NLP9 polynucleotides can be introduced into a plant provenance, for example, by development characterized by - operatively linked to the polynucleotide(s) A novel inbred line that regulates meta-regulation and is associated with increased NFB. A particular advantage of such development programs may be, for example, the expression of the NFB phenotype in non-nodulated plants to produce increased nitrogen utilization and/or growth.

The following examples are provided to illustrate certain specific features and/or specific examples. The examples should not be construed as limited to the particular features or specific examples illustrated.

Example Example 1: Materials and Methods

Arabidopsis Columbia 0 Ecotype was used for all experiments unless otherwise indicated.

Growth and treatment conditions. Arabidopsis seedlings were grown at a constant temperature of 16:8 light: dark light cycle (hrs.) and 22 °C in a medium with MS or salt of Murashige and Skoog (MS), no The 5 mM KNO ₃ nitrogen (N) was added to a 0.8% agar dish. After seven days, the plants were moved to the full, or no N, supplemented with or without 2.5mM NH MS medium salts of ₄ NO _3.

Depending on the treatment, the plant is inoculated with NFB or non-NFB or not. All bacteria were first grown in diluted 869 medium and 20 mL overnight cultures at an optical density of 0.4 at 600 nm were used for inoculation. Prior to inoculation, the bacteria were washed with sterile water and resuspended in 5 mL. 20mL plant agar medium inoculation 5 mL of bacteria, or uninoculated as sterile water.

Example 2: Functional association between non-nodulated plants and NFB

To determine whether Arabidopsis has established a beneficial N-nutrient interaction with NFB, we evaluated the effects of different NFB species on plant growth under N-limited conditions. We have chosen to display five different NFB species that are fixed N when associated with plants: Rhizobium meliloti RMP110 (Pichon et al. (1992) Plant Cell 4: 1199), Rhizopus genus CFN42 (Poupot et al. (1995) J .Biol. Chem. 270: 6050), Taiwan's copper bacterium LMG 19424 (Marchetti et al. (2011) Appl . Env . Microbiol. 77: 2161), B. plasmophilus LB400 (Perin et al. (2006) ) Appl. Env. Microbiol. 72: 3103), and B. vietii G4 (Perin et al. (2006), supra).

We also tested two additional bacteria that could not be used as BFN as a control group: Burkholderia psJN, which is known to enhance Arabidopsis growth (Zuniga et al. (2013) Mol . Plant Microbe Interact . 26: 546), and Neisseria pallidum JMP134, a soil bacterium that is associated with plants, but has no positive effects on plant growth under our experimental conditions (Ledger et al. (2012) Antonie Van Leeuwenhoek 101: 713).

Plant the plants on vertical dishes with MS media for seven days. And moved to an MS-N dish with the same MS medium but no N (MS-N), or inoculated with 104 colony forming units (cfu) / mL of different bacteria. We evaluated the dry weight of the plants seven days after moving to MS-N.

Under the condition of limited N, the dry weight of the plant was significantly higher in the presence of R. sphaeroides RMP110 than the uninoculated medium. figure 1. Moreover, under our experimental conditions, the growth of plants is similar to that achieved with intact MS media. figure 1. These results indicate that R. sphaeroides RMP110 promotes plant growth in the absence of N source. None of the other bacterial species utilized could increase the dry weight of Arabidopsis, indicating that the presence of only neutral or beneficial bacteria could not explain this observation. The effective interaction between Arabidopsis and different bacteria was confirmed by a non-specific increase in the length of the root hair observed in response to bacterial inoculation in all cases. Figure 5.

BNF was confirmed by the ¹⁵ N dilution technique under plants in the N-limited condition with plants inoculated with wild-type and Δ nifH mutants. Our results indicate that S. meliloti can fix atmospheric nitrogen and make it useful as plant nutrients.

In order to determine whether N fixation is necessary for plant growth under N-limited conditions, we used a Rhizobium meliloti RMP110 mutant (ΔnifH) that could not fix N. Bobik et al. (2006) J. Bacteriol. 188: 4890. As shown in Fig. 2A, under the condition of N, the Rhizobium meliloti RMP110ΔnifH had a significant effect on plant growth compared to the wild type bacteria.

In order to confirm that the growth promoting effect of R. sphaeroides RMP110 is partly due to the fact that the immobilized N line contributes to Arabidopsis nutrients, we have grown plants on MS-N supplemented with 5 mM KNO ₃ for seven days. The -N is labeled with a 5% ¹⁵ N isotope. The plants are then exposed to wild-type S. meliloti RMP110 or ΔnifH mutants in MS-N medium. Compared to △ nifH inoculated plants were inoculated plant or, in the presence of wild-type S. meliloti to RMP110 supplemented with ¹⁵ N isotope ratio KNO medium processing plant MS-N labeled ₃ exhibit reduced. Figure 2B. This result indicates that Arabidopsis requires atmospheric N, with the ¹⁴ N isotope being the majority. Both wild-type and ΔnifH mutant strains had similar effects on lateral root density and root hair elongation, indicating that both plants could interact with plants. Figure 6A.

These results indicate that Arabidopsis can be functionally associated with S. meliloti RMP110 to enhance plant growth under N-limited conditions. This plant: bacterial interaction represents an outstanding model system for the non-legume mechanisms discussed to promote the association with bacterial species to cause nutrient-related biological N fixation.

Example 3: Genes regulating the interaction between Arabidopsis and Sinorhizobium meliloti

In Arabidopsis, homologs of the NSP1 and NIN genes are regulated by the presence of bacteria only when the plants are grown under N-limited conditions. This suggests that the availability of N is a major factor in regulating plant susceptibility to bacterial susceptibility. By utilizing a functional genomic approach in which plants carry insertional mutations in NSP1-like and selected NLPs genes, we have found that these transcription factors are critical for functional connectivity between Arabidopsis and S. meliloti.

Nitrogen fixation in legume species depends on the precise molecular mechanisms governing when and how to establish symbiotic association with Rhizobium. Oldroyd (2013) Nat. Rev. Microbiol. 11: 252). To determine whether the mechanism of interaction in the model system that regulates our recent discovery - the interaction between Arabidopsis and R. sphaeroides RMP110 - can exist in Arabidopsis, we separately analyzed NSP1-like and NSP2-like genes, At3g13840 ( AtNSP1-like ) and At4g08250 ( AtNSP2-like ). These genes do not have a known function in Arabidopsis.

The expression of AtNSP1-like and AtNSP2-like genes was assessed by plants grown in N-sufficient conditions (5 mM KNO ₃ ) and subsequently transferred to 2.5 mM NH ₄ NO ₃ or moved to MS-N medium with or without sputum Chinese root nodule The presence of the bacterium RMP110. Plants were harvested 3 and 7 days after transfer. Total RNA was prepared and the transcriptional level of the gene of interest was measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Our results show that the AtNSP1-like gene expression is induced when the plants are moved to the MS-N medium with bacteria, but not under other experimental conditions. Figure 3A. This result suggests that AtNSP1-like function is required under the condition of N and in the presence of S. meliloti. Conversely, AtNSP2-like samples were not regulated under the experimental conditions tested. Figure 7A.

In Leguminosae, NSP1 and NSP2 regulate the performance of the NIN gene. The Arabidopsis genome encodes nine NIN -like genes ( NLPs ). As a first step in assessing the role of NLPs in the interaction between Arabidopsis and S. meliloti, we analyzed these 9 NLPs ( NLP1 ( At2g17150 ); NLP2 ( At4g35270 ); NLP3 ( NLP3 ( At4g38340 ); NLP4 ( At1g20640 ); NLP5 ( At1g76350 ); NLP8 ( At2g43500 ); and NLP9 ( At3g59580 )).

Our results showed that the gene expression of AtNLP4 , AtNLP8 , and AtNLP9 was induced in the presence of N-limited conditions and Rhizobium meliloti RMP110 (Fig. 3B), similar to AtNSP1-like . Conversely, AtNLP3 showed a different appearance, which was induced under N-sufficient conditions but only in the presence of bacteria. Figure 3B. The gene expression of AtNLP1 , AtNLP2 , and AtNLP5 did not change significantly under the experimental conditions evaluated. Figure 7B.

In order to discuss the interaction of AtNSP1-like , AtNLP4 , AtNLP8 , and AtNLP9 genes against Arabidopsis and R. sphaeroides RMP110, AtNSP1-like (salk_036071C-salk_023595C), AtNLP4 (salk_100786C-salk_063595C), AtNLP8 (salk_140298-salk_031064C Two independent homozygous mutant lines of AtNLP9 (salk_025839C-salk_042082C) are obtained from the Arabidopsis Bioresource Center (ABRC).

Mutations that were grown under N-limited conditions were inoculated with wild-type S. meliloti RMP110 (or mock imitation as a control group). Our results showed that under the condition of N, the plant growth promoting effect of R. sphaeroides RMP110 was lost in the atnsp1 mutant. Figure 4A. Similarly, mutant plants of AtNLP4 and AtNLP9 genes did not exhibit increased growth in the presence of R. sphaeroides RMP110. Figure 4B. Conversely, the atnlp8 mutant plant did not show a plant growth promoting effect induced by R. sphaeroides RMP110 under N-limited conditions. These results indicate that AtNSP1-like , AtNLP4 , and AtNLP9 are required for functional interaction between Arabidopsis and S. meliloti RMP110 to enhance growth under N-limited conditions. These results also indicate that conserved mechanisms are present in plants to mediate plant-to-bacterial beneficial interactions with respect to N-nutrients, which can be applied to provide BNF to non-nodulated plant species.

<110> Pontificia Universidad Catolica de Chile Kraiser, Tatiana Gonzalez, Bernardo Guitierrez, Rodrigo

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Claims (22)

A method for increasing nitrogen efficiency of a non-nodulated plant, the method comprising introducing at least one heterologous polypeptide into a plant to produce a genetically transformed plant, wherein the heterologous polypeptide is selected from a nodulation signal transduction pathway Groups of (NSPs) and NIN-like proteins (NLPs). The method of claim 1, wherein the heterologous polypeptide is selected from the group consisting of NSP1-like, NLP4, and NLP9. The method of claim 2, wherein the heterologous polypeptide is at least 90% identical to one or more of SEQ ID NOs: 1-4. The method of claim 2, wherein the heterologous polypeptide is introduced into a plant comprising a polynucleotide-transformed plant encoding a heterologous polypeptide. The method of claim 4, wherein the polynucleotide is at least 80% identical to one or more of SEQ ID NOs: 4-6. The method of claim 4, wherein the polynucleotide hybridizes under stringent conditions to one or more of SEQ ID NOs: 4-6. The method of claim 4, wherein the polynucleotide is codon-optimized to display a heterologous polypeptide in the plant. The method of claim 4, wherein the polynucleotide is operatively linked to a plant promoter selected from the group consisting of: a composition promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducer Promoter. The method of claim 1, wherein the increased nitrogen efficiency comprises increased growth of the genetically transformed plant under nitrogen-limited conditions. The method of claim 1, wherein the heterologous polypeptide is introduced into the root of the plant Department organization. The method of claim 1, wherein the method comprises growing the genetically transformed plant under nitrogen-limited conditions. A genetically modified plant produced by the method of claim 1. A cell, tissue, seed, or material obtained by the genetically transgenic plant of claim 12. A nucleic acid molecule comprising an agronomically functional polynucleotide, wherein the polynucleotide is selected from the group consisting of: a polynucleotide that is at least 80% identical to SEQ ID NO: 4; in harsh conditions a polynucleotide that hybridizes to a nucleic acid consisting of SEQ ID NO: 4; a polynucleotide that is at least 80% identical to SEQ ID NO: 5; hybridizes under stringent conditions to the nucleic acid consisting of SEQ ID NO: Polynucleotide; a polynucleotide that is at least 80% identical to SEQ ID NO: 6; and a polynucleotide that hybridizes under stringent conditions to the nucleic acid consisting of SEQ ID NO: 6; wherein the nucleic acid sequence is Operatively linked to a heterologous promoter. A gene transfer plant cell which is stably transformed with the nucleic acid molecule of claim 14. A method for producing a genetically transgenic plant, the method comprising introducing a nucleic acid into a plant cell, wherein the nucleic acid comprises a heterologous nodulation signal transduction pathway 1-like (NSP1-like), NIN-like protein-4 ( The nucleotide sequence of NLP4), or NIN-like protein-9 (NLP9) polypeptide, thereby producing a genetically transformed plant. The method of claim 16, wherein the plant is a non-nodulated plant. A genetically modified plant produced by the method of claim 16. A cell, tissue, seed, or material obtained by the genetically transgenic plant of claim 17. The gene transfer plant of claim 17, wherein the gene transfer plant comprises increased growth of the genetically transformed plant under nitrogen-limited conditions compared to wild-type plants of the same species. A method for increasing the nitrogen efficiency of a non-nodulated plant, the method comprising introducing into the plant at least one mode that promotes the association with a nitrogen-fixing bacteria (NFB), wherein the nitrogen efficiency of the plant is increased via biological nitrogen fixation (BNF). The method of claim 21, wherein the means for promoting the association with the nitrogen-fixing bacteria NFB is selected from the group consisting of: a polypeptide consisting of one of SEQ ID NOs: 1, 3, and 4, and SEQ ID NOs: A polynucleotide consisting of one of 4-6.