Enhancement of nitrogen fixation by FUN

Abstract

The present disclosure relates to enhancing nitrogen fixation in legumes grown under high nitrate or nitrate stress conditions. In particular, the present disclosure relates to genetically modified plants with altered levels or expression of FUN or downstream targets of FUN, as well as methods for producing and growing the same. The present disclosure also relates to nodule senescence regulated by FUN and its downstream targets, and the regulation of FUN activity or expression by cellular zinc.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 483,248, filed February 3, 2023, and U.S. Provisional Application No. 63 / 580,171, filed September 1, 2023, both of which are incorporated by reference in their entireties.

[0002] [Reference to Electronic Sequence Listing] The contents of the electronic sequence listing (794,542,002,240SEQLIST.xml; size: 175,320 bytes; and creation date: February 1, 2024) are incorporated herein by reference in their entirety.

[0003] [Technical Field] The present disclosure relates to enhanced nitrogen fixation in legumes grown under high nitrate or nitrate stress conditions. In particular, the present disclosure relates to genetically modified plants with altered levels or expression of FUN or downstream targets of FUN, and methods for producing and cultivating the same. The present disclosure also relates to root nodule senescence regulated by FUN and its downstream targets, and the regulation of FUN activity by zinc in cells.

[0004] [Background technology] Plant growth and development depend on aboveground carbon dioxide and sunlight, as well as water and mineral nutrients in the soil. Nutrient accessibility in the soil depends on many factors, and nutrient availability varies spatially and temporally. Local nutrient sensing and awareness of the global nutrient situation shape plant responses to the nutrient environment and act to coordinate plant development, with the participation of microorganisms, to optimize nutrient acquisition and regulate plant growth. One of the major nutrients limiting plant productivity is nitrogen (N).

[0005] Nitrogen fixation is important for the sustainable and profitable production of legumes. The symbiosis between legumes and nitrogen-fixing microorganisms is regulated by the plant in several ways, including the number of nodules allowed to form (Nishimura, R. et al. HAR1 mediates systemic regulation of symbiotic organ development. Nature 420, 426-429 (2002); Krusell, L., Madsen, LH, Sato, S. & Aubert, G. Shoot control of root development and nodulation is mediated by a receptor-like kinase. Nature 420, 422-426 (2002); Searle, IR et al. Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science 299, 109-112 (2003); Tsikou, D. et al. Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA. Science 362, 233-236). (2018)), and the function of the resulting organs. Nitrogen fixation in legumes can meet all of the plant's nitrogen requirements, balanced with nitrogen acquisition from available soil resources. However, nitrogen supplies in the soil fluctuate, and so does the plant's demand for nitrogen.

[0006] In intensive agriculture, nitrogen is often applied in high concentrations in the form of inorganic fertilizers to increase crop productivity. These concentrations are generally higher than plants need or can store in the soil. As a result, these nutrients are released into the environment, affecting ecosystems and biodiversity and contributing to climate change (CJ Stevens, Nitrogen in the environment. Science 363, 578-580 (2019); J. A Foley et al., Solutions for a cultivated planet. Nature 478, 337-342 (2011); J. Rockstrom et al., A safe operating space for humanity. Nature 461, 472-475 (2009)). However, high levels of nitrate in soil have been shown to inhibit or suppress the nitrogen fixation ability of legumes.

[0007] There is a general need to enhance the nitrogen fixation capacity of legumes and other plants that have a symbiotic relationship with nitrogen-fixing bacteria to accomplish nitrogen fixation under conditions of high nitrate or nitrate stress, which would inhibit, suppress, or reduce nitrogen fixation. For example, there is a need for the ability to engineer legume or other plant responses to nitrate and the corresponding regulation of nitrogen fixation. In particular, there is a need for the identification of transcription factors that can regulate nitrogen fixation under high nitrate conditions to provide a means for engineering legumes to fix nitrogen even when grown under high nitrate conditions. Further, there is a need for the ability to engineer root nodule senescence in legumes or other plants in response to nitrate concentration and / or nitrogen stress. In particular, there is a need for the identification of transcription factors that can regulate root nodule senescence under high nitrate or nitrogen stress conditions to provide a further means for engineering legumes to fix nitrogen even when grown under high nitrate or nitrogen stress conditions.

[0008] Summary of the Invention To address these needs, the present disclosure provides a transcription factor, FUN, that regulates nitrogen fixation in legumes under high nitrate conditions. The disclosure also provides downstream targets of FUN that act to regulate nitrogen fixation under high nitrate conditions. Mutation or silence of either FUN or its downstream targets can produce plants with nitrate-tolerant nitrogen fixation capabilities. This provides an opportunity to increase biologically fixed nitrogen in fields or cropping conditions where soil nitrate concentrations are high.

[0009] One aspect of the present disclosure includes a genetically modified plant or portion thereof that includes one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: The polypeptides include those having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or a conserved domain thereof, or a combination thereof.In additional embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof.

[0010] Further aspects of the present disclosure include genetically modified plants or portions thereof that include one or more genetic modifications that decrease the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same cultivation conditions. In further embodiments of this aspect, the protein is the NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof; and the protein is the bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75, or a conserved domain thereof;wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is the HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; wherein the protein is the NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; or wherein the protein is the AS1 protein, and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a conserved domain thereof.In additional embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74, or a conserved domain thereof; wherein the bZIP28 protein comprises SEQ ID NO:75, or a conserved domain thereof; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73, or a conserved domain thereof; wherein the HO1 protein comprises SEQ ID NO:77, or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78, or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a conserved domain thereof, or a combination thereof.

[0011] Another aspect of the present disclosure includes a transgenic plant comprising one or more genetic modifications that decrease the activity or expression of one or more of a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein compared to the activity or expression of a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions, wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:21,SEQ ID NO:22,SEQ ID NO:23,SEQ ID NO:24,SEQ ID NO:25,SEQ ID NO:26,SEQ ID NO:27,SEQ ID NO:28,SEQ ID NO:29,SEQ ID NO:30,SEQ ID NO:31,SEQ ID NO:32,SEQ ID NO:33,SEQ ID NO:34,SEQ ID NO:35,SEQ ID NO:36,SEQ ID NO:37,SEQ ID NO:38,SEQ ID NO:39,SEQ ID NO:40,SEQ ID NO:41,SEQ ID NO:42,SEQ ID NO:43,SEQ ID NO:44,SEQ ID NO:45,SEQ ID NO:46,SEQ ID NO:47,SEQ ID NO:48,SEQ ID NO:49,SEQ ID NO:50,SEQ ID NO:51,SEQ ID NO:52,SEQ ID NO:53,SEQ ID NO:54,SEQ ID NO:55,SEQ ID NO:56,SEQ ID NO:57,SEQ ID NO:58,SEQ ID NO:59,SEQ ID NO:60,SEQ ID NO:61,SEQ ID NO:62,SEQ ID NO:63,SEQ ID NO:64,SEQ ID NO:65,SEQ ID NO:66,SEQ ID NO:67,SEQ ID NO:68,SEQ ID NO:69,SEQ ID NO:70,SEQ ID NO:71,SEQ ID NO:72,SEQ ID NO:73,SEQ ID NO:74,SEQ ID NO:75,SEQ ID NO:76,SEQ ID NO:77,SEQ ID and wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein has enhanced expression in nodules of roots lacking the one or more genetic modifications.

[0012] In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In still other embodiments of this aspect that can be combined with any of the preceding embodiments, the reduction is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene, preferably the binding site is a transcriptional activator protein binding site or a TATA box. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the cultivation conditions include a moderate nitrate concentration, a high nitrate concentration, or a nitrate concentration around the plant that reduces or inhibits nitrogen fixation. In still other embodiments of this aspect, the nitrate concentration is from about 10 mM to about 250 mM nitrate, or alternatively, at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate.In still other embodiments of this aspect, the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same conditions. In further embodiments of this aspect, the nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In still other embodiments of this aspect that may be combined with any of the preceding embodiments, the plant forms nodules. In additional embodiments of this aspect, the plant exhibits an increased number of nodules, increased hemoglobin content, or increased acetylene reduction assay (ARA) activity compared to a control plant grown under the same cultivation conditions.

[0013]

[0010] Additional aspects of the present disclosure include methods for cultivating genetically modified plants that result in increased nitrogen fixation under conditions comprising nitrate concentrations around the roots of the plants that repress nitrogen fixation, the method comprising: (a) providing the genetically modified plant, wherein the plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions, and wherein the one or more genetic modifications decrease nitrate concentration repression of nitrogen fixation; and (b) cultivating the genetically modified plant under the nitrate concentrations around the roots of the plant, wherein the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same cultivation conditions. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:The method includes the step of obtaining a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or a conserved domain thereof, or a combination thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof. In yet other embodiments of this aspect, the protein is the NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof; and wherein the protein is the bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof;and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein said protein is said HO1 protein, and said HO1 protein is selected from the group consisting of SEQ ID NO:wherein the protein is the NRT2.1 protein and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; or wherein the protein is the AS1 protein and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a conserved domain thereof. In further embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74, or a conserved domain thereof; wherein the bZIP28 protein comprises SEQ ID NO:75, or a conserved domain thereof; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDwherein the HO1 protein comprises SEQ ID NO:77, or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78, or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79, or a conserved domain thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:NO:42, or a conserved domain thereof, or a combination thereof. Additional embodiments of this aspect that can be combined with any of the preceding embodiments include the nitrate concentration in step (c) being about 10 mM to about 250 mM nitrate, alternatively at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. Further embodiments of this aspect that can be combined with any of the preceding embodiments include the nitrogen fixation being increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules or hemoglobin content is increased compared to control plants grown under the same cultivation conditions. In additional embodiments of this aspect, the increased amount of nitrogen fixation is measured using a method selected from the group consisting of measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants.

[0014] A further aspect of the present disclosure includes a method of cultivating a genetically modified plant capable of fixing nitrogen when cultivated under nitrogen fertilized conditions, said method comprising: (a) providing said genetically modified plant, wherein said plant or portion thereof has a higher activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same cultivation conditions; (b) cultivating the plant under conditions in which the area around the roots of the plant contains a standard nitrate concentration; and (c) using nitrogen fertilizer to create conditions in which the area around the roots of the plant contains a nitrate concentration that inhibits nitrogen fixation, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same conditions. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:The polypeptides include those having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or a conserved domain thereof, or a combination thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof. In yet other embodiments of this aspect, the protein is the NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof; and wherein the protein is the bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof;and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein said protein is said HO1 protein, and said HO1 protein is selected from the group consisting of SEQ ID NO:wherein the protein is the NRT2.1 protein and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; or wherein the protein is the AS1 protein and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a conserved domain thereof. In further embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74, or a conserved domain thereof; wherein the bZIP28 protein comprises SEQ ID NO:75, or a conserved domain thereof; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDwherein the HO1 protein comprises SEQ ID NO:77, or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78, or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79, or a conserved domain thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:NO:42, or a conserved domain thereof, or a combination thereof. Additional embodiments of this aspect that can be combined with any of the preceding embodiments include the nitrate concentration in step (c) being about 10 mM to about 250 mM nitrate, alternatively at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. Further embodiments of this aspect that can be combined with any of the preceding embodiments include the nitrogen fixation being increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules or hemoglobin content is increased compared to control plants grown under the same cultivation conditions. In additional embodiments of this aspect, the increased amount of nitrogen fixation is measured using a method selected from the group consisting of measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants. In further embodiments of this aspect that may be combined with any of the previous embodiments, the genetically modified plants are grown in an intercropping system with non-nitrogen fixing plants or in a rotational cropping system after non-nitrogen fixing plants.

[0015] Additional aspects of the present disclosure include methods for delaying root nodule senescence, the methods comprising: (a) providing a genetically modified plant, wherein the plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions, and wherein the one or more genetic modifications delay root nodule senescence; and (b) cultivating the genetically modified plant under stress conditions, wherein the genetically modified plant exhibits delayed root nodule senescence compared to the control plant grown under the same conditions. Further embodiments of this aspect include the stress conditions selected from the group of: moderate nitrate concentration, high nitrate concentration, peripheral nitrate concentration that promotes nodule senescence, moderate heat level, high heat level, peripheral heat level that promotes nodule senescence, moderate water deficit level, high water deficit level, peripheral water deficit level that promotes nodule senescence, moderate waterlogging level, high waterlogging level, peripheral waterlogging level that promotes nodule senescence.In further embodiments of this aspect which may be combined with any of the preceding embodiments, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO: The present invention also includes polypeptides having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:81, or SEQ ID NO:82, or a conserved domain thereof, or a combination thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: NO:82, or a conserved domain thereof, or a combination thereof.In still other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof. In still other embodiments of this aspect, which may be combined with any of the preceding embodiments with a NAC domain-containing protein. wherein the protein is the NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof; wherein the protein is the bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75, or a conserved domain thereof;wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is the HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof;wherein the protein is the NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; or wherein the protein is the AS1 protein, and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a conserved domain thereof. In further embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74, or a conserved domain thereof; and wherein the bZIP28 protein comprises SEQ ID NO:75, or a conserved domain thereof;wherein the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO:77, or a conserved domain thereof;wherein the NRT2.1 protein comprises SEQ ID NO:78, or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79, or a conserved domain thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a conserved domain thereof, or a combination thereof. In further embodiments of this aspect, nodule senescence is delayed by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0016]

[0010] A further aspect of the present disclosure includes a method of inducing FUN protein filament formation, the method comprising: (a) providing a FUN protein; and (b) increasing the amount of zinc or manganese around the FUN protein, wherein the increased amount of zinc or manganese induces filament formation compared to a control FUN protein in an environment without the increased amount of zinc or manganese. In additional embodiments of this aspect, the filament formation is induced under high nitrate conditions. In still other embodiments of this aspect that can be combined with any of the preceding embodiments, the method is performed in vitro.

[0017] Further aspects of the present disclosure include methods of inducing filamentation, the methods comprising: (a) providing a plant containing a FUN protein; and (b) cultivating the plant under conditions of elevated zinc or manganese, wherein filamentation of the FUN protein in the plant is induced compared to FUN protein in a control plant in an environment without elevated amounts of zinc or manganese. In other embodiments of this aspect, the plant comprises a genetic modification. In additional embodiments of this aspect, the filamentation is induced under high nitrate conditions. In yet other embodiments of this aspect, which can be combined with any of the above embodiments in which the plant comprises a genetic modification, the genetic modification reduces the activity of the FUN protein without eliminating the activity of the FUN protein. In a further embodiment of this aspect, the induction of filamentation increases nitrogen fixation, reduces the activity, or inactivates the FUN protein in the genetically modified plant compared to a control plant grown under the same conditions. In still other embodiments of this aspect that may be combined with any preceding embodiment or aspect with a method of inducing filament formation, the number of nodules is increased, the hemoglobin content is increased, or the acetylene reduction assay (ARA) activity is increased compared to a control plant grown under the same cultivation conditions.

[0018] A further aspect of the present disclosure includes a method for modulating nodule function in response to available nitrogen in soil, the method comprising: a) providing a genetically modified plant containing a FUN protein with altered nitrate-induced activation; and b) cultivating the genetically modified plant under nitrate conditions, wherein the genetically modified plant exhibits reduced FUN activity or expression and / or a reduced active form of FUN compared to a wild-type plant grown under the same nitrate conditions. In additional embodiments of this aspect, altering nitrate-induced FUN protein activation comprises silencing FUN, reducing FUN activity, knocking out FUN by mutation, knocking down FUN expression, knocking out a FUN promoter element, or a combination thereof. In a further embodiment of this aspect, altering nitrate-induced FUN protein activation comprises manipulating environmental or cellular zinc concentrations, wherein the manipulation maintains the FUN protein in an inactive filamentous form. In yet other embodiments of this aspect, altering nitrate-induced FUN protein activation comprises genetically modifying the FUN protein sequence to alter its sensitivity to zinc.

[0019] In a further embodiment of this aspect which may be combined with any of the preceding embodiments or aspects, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO: The present invention also includes polypeptides having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:81, SEQ ID NO:82, or a conserved domain thereof, or a combination thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: NO:82, or a conserved domain thereof, or a combination thereof.In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof.

[0020] Further aspects of the present disclosure include methods for producing genetically modified plants that result in increased nitrogen fixation under conditions that include nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the methods comprising introducing into the plant or part thereof one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions. In additional embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: and a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, Sor the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof.

[0021] Additional aspects of the present disclosure include methods for producing genetically modified plants that result in increased nitrogen fixation under conditions involving nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the methods comprising introducing into the plant or portion thereof one or more genetic modifications that reduce the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In still other embodiments of this aspect, wherein the protein is the NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74, or a conserved domain thereof; and wherein the protein is the bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75, or a conserved domain thereof;wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is the HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; wherein the protein is the NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; or wherein the protein is the AS1 protein, and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a conserved domain thereof; and wherein the NRT3.wherein the bZIP28 protein comprises SEQ ID NO:75, or a conserved domain thereof; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO:77, or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78, or a conserved domain thereof; or wherein the AS1 protein comprises SEQ ID NO: or the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a conserved domain thereof, or a combination thereof.

[0022] Yet another aspect of the present disclosure includes a method for producing a genetically modified plant that results in increased nitrogen fixation under conditions that include nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or portion thereof one or more genetic modifications that reduce the activity or expression of one or more of the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In some embodiments of this aspect, the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein has enhanced expression in nodules of roots lacking the one or more genetic modifications.

[0023]

[0010] Further aspects of the present disclosure include methods for producing a transgenic plant or part thereof according to any preceding embodiment, the method comprising introducing into a plant cell a genetic modification that reduces or knocks out the activity or expression of a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein. In further embodiments of this aspect, the genetic modification comprises a first nucleic acid sequence capable of reducing or knocking out a second nucleic acid sequence encoding a FUN protein, a FUN-like protein, an NAC domain-containing protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein operably linked to a promoter. In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the genetically modified plant is selected from one or more of the group consisting of alfalfa, bambara bean, pulses (e.g., kidney bean, black bean, etc.), black currant, chickpea, white clover, cowpea, forage legume, legume tree, lentil, lotus, lupine, Medicago, pea, peanut, pigeon pea, soybean, Parasponia, alder, and elm. In other embodiments of this aspect, the nucleic acid comprises a short RNA, antisense RNA, siRNA, miRNA, dsRNA, tasiRNA, or secondary siRNA associated with RNA silencing. In yet other embodiments of this aspect, the promoter is a nodule-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof. In a further embodiment of this aspect, the promoter is a constitutive promoter, and wherein the promoter is selected from the group comprising the CaMV35S promoter, a derivative of the CaMV35S promoter, a maize ubiquitin promoter, a polyubiquitin promoter, a vein mosaic cassava virus promoter, or an Arabidopsis UBQ10 promoter.In yet other embodiments of this aspect, the nucleic acid sequence is inserted into the genome of the plant such that it is operably linked to an endogenous promoter. In further embodiments of this aspect, the endogenous promoter is a nodule-specific promoter or a root-specific promoter.

[0024]

[0010] Further aspects of the present disclosure include a method for producing a transgenic plant or part thereof according to any of the preceding embodiments, the method comprising: genetically modifying a plant cell by transforming the plant cell with one or more gene editing components targeting an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, a NAC domain-containing protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, wherein the endogenous nuclear genomic sequence or part thereof is knocked out. In other embodiments of this aspect, the one or more gene editing components include a ribonucleoprotein complex targeting the nuclear genomic sequence; a vector comprising a sequence encoding a TALEN protein targeting the nuclear genomic sequence; a vector comprising a sequence encoding a ZFN protein targeting the nuclear genomic sequence; an oligonucleotide donor (OND) targeting the nuclear genomic sequence; or a vector comprising a sequence encoding a CRISPR / Cas enzyme and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

[0025] Yet other aspects of the present disclosure include an expression vector or isolated DNA molecule, the expression vector or isolated DNA molecule comprising: (i) one or more nucleotide sequences encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; (ii) a nucleotide sequence encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof; or (iii) one or more nucleotide sequences that can reduce or knock out a nucleic acid sequence, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; or (iv) one or more nucleotide sequences that have a mutation in a gene for a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the mutation reduces or knocks out the activity or expression of the protein, and the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site. In a further embodiment of this aspect, the protein is a FUN protein, and the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, Sor the FUN protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82; or wherein said FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or a conserved domain thereof, or a combination thereof; wherein said protein is a FUN-like protein and said FUN-like protein is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:83, and SEQ ID NO:94. or a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:4, SEQ ID NO:84, or a conserved domain thereof, or a combination thereof;and / or wherein said protein is an NRT3.1 protein and said NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74 or a conserved domain thereof; wherein said protein is a bZIP28 protein and said bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75 or a conserved domain thereof; wherein said protein is a NAC domain-containing protein and said NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDor a combination thereof; wherein said protein is an HO1 protein, and said HO1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77 or a conserved domain thereof; wherein said protein is an NRT2.1 protein, and said NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78 or a conserved domain thereof; or wherein said protein is an AS1 protein, and said AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79 or a conserved domain thereof. wherein said NRT3.1 protein comprises SEQ ID NO:74 or a conserved domain thereof; wherein said bZIP28 protein comprises SEQ ID NO:75 or a conserved domain thereof; and wherein said NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQwherein the HO1 protein comprises SEQ ID NO:77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79 or a conserved domain thereof; or wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a conserved domain thereof, or a combination thereof.

[0026] Some aspects of the present disclosure relate to a bacterial or Agrobacterium cell comprising an expression vector or an isolated DNA molecule according to any of the previous embodiments.

[0027] An additional aspect of the present disclosure relates to a transgenic plant, plant part, plant cell, or seed comprising an expression vector or isolated DNA molecule according to any of the previous embodiments.

[0028] A further aspect of the present disclosure relates to a kit comprising an expression vector or an isolated DNA molecule according to any of the previous embodiments, or a bacterial cell or an Agrobacterium cell according to the previous embodiments.

[0029] Further aspects of the present disclosure relate to methods for increasing nitrogen fixation, delaying nodule senescence, or inducing FUN filament formation in a plant, the methods comprising: (a) introducing a genetic modification via an expression vector or isolated DNA molecule described in any of the previous embodiments; and, optionally, (b) treating the plant with zinc or manganese or growing the plant under high zinc, high manganese, or high nitrate conditions.

[0030] Additional aspects of the present disclosure relate to the genome of a genetically modified plant, wherein the genome of said genetically modified plant comprises (i) one or more genetic modifications in a genetically modified plant or part thereof according to any one of the preceding embodiments, or (ii) one or more genetic modifications in a genetically modified plant or part thereof produced by a method according to any one of the preceding embodiments.

[0031] A further aspect of the present disclosure pertains to a non-regenerable part or cell of a transgenic plant or part thereof according to any one of the preceding embodiments.

[0032] This patent or this patent application contains one or more drawings executed in color. Copies of this patent or this patent application including color drawing(s) will be provided by the Patent Office upon request and payment of the appropriate fee.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] Figures 1A-1V show the nodule phenotype and nitrogen fixation activity of fun mutant Lotus plants under nitrate-limited conditions, demonstrating that Fun is specifically expressed in nodules. Figure 1A shows representative photographs of the nodule phenotype of wild-type Lotus plants (WT; Gifu) under KCl conditions (control; upper left), wild-type Lotus plants under 10 mM KNO3 conditions (top center), and fun mutant Lotus plants under 10 mM KNO3 conditions (top right and bottom). The fun mutations include fun (top right), fun-2 (bottom left), fun-3 (bottom center), and fun-4 (bottom right). Scale bar = 1 cm.

[0034] [Figure 1B] Figure 1B shows a diagram of the FUN gene and LORE1 insertion in each genetic background phenotyped in Figure 1A. In fun and fun-4, LORE1 is inserted in the promoter region (light gray line). In fun-2, LORE1 is inserted at the end of the fourth intron (the intron is represented by a black line). In fun-3 (30099638), LORE1 is inserted at the end of the seventh intron. Arrows indicate the insertion point of LORE1 along the gene.

[0035] [Figure 1C] Figure 1C shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (colored box, labeled "Pink") formed in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0036] [Figure 1D] Figure 1D shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0037] [Figure 1E] Figure 1E shows the ARA measurements (vertical axis) from Figure 1D over a 14-day course (top) and followed up to 20 mM KNO3 (bottom). The light gray line with medium gray circles represents the ARA results for wild-type plants. At the top, the medium gray line with medium gray circles represents the ARA results for fun mutant plants, and the black line with medium gray circles represents the ARA results for fun-3 mutant plants. At the bottom, the black line with medium gray circles represents the ARA results for fun mutant plants.

[0038] [Figure 1F] Figure 1F shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, fun mutant Lotus plants, fun-2 mutant Lotus plants, fun-3 mutant Lotus plants, and fun-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0039] [Figure 1G] Figure 1G shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, fun mutant Lotus plants, fun-2 mutant Lotus plants, fun-3 mutant Lotus plants, and fun-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0040] [Figure 1H] Figure 1H shows the number of pink nodules per plant for wild-type (WT), fun, fun-3, and fun-4 mutants sprayed with 5 mM nitrate before inoculation (gray box, labeled "5 mM") or without nitrate before inoculation (white box, labeled "0 mM"). Plants were grown on plates supplemented with 0 mM or 5 mM KNO3 and then inoculated with rhizobia.

[0041] [Figure 1I] Figure 1I shows the total number of nodules per plant for wild-type (WT), fun, fun-3, and fun-4 mutants in the presence of 5 mM nitrate (gray box, labeled "5 mM") and absence of nitrate (white box, labeled "0 mM") before inoculation. Plants were grown on plates containing 0 or 5 mM KNO3 and then inoculated with rhizobia; nodules were counted 3 weeks after inoculation.

[0042] [Figure 1J] Figure 1J shows nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT), fun, fun-3, and fun-4 mutant plants treated with 5 mM nitrate before inoculation (gray box, labeled "5 mM") and without nitrate before inoculation (white box, labeled "0 mM"). Plants were grown on plates supplemented with 0 or 5 mM KNO3 and then inoculated with rhizobia; ARA was performed 3 weeks after inoculation. In Figures 1F–1J, letters indicate significant differences (p<0.05) between the compared plant groups.

[0043] [FIG. 1K] FIG. 1K shows the leghemoglobin content (μg leghemoglobin per plant) of wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0044] [Figure 1L] Figure 1L shows complementation of fun mutants grown under 10 mM KNO3 exposure for 2 weeks. The gray boxes indicate empty vector (labeled "EV") transformed into the WT background (left) or the fun mutant background (center), while the white boxes indicate proUbi:fun-GFP transformed into the fun mutant background (right). Letters indicate significant differences between the compared plant groups.

[0045] [Figure 1M] Figure 1M shows a representative photograph of a plant root with nodules in a plant expressing the proFun:GUS reporter construct, used to visualize Fun gene expression in situ. The root was stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). Blue staining indicates GUS reporter expression. Scale bar = 2 cm.

[0046] [Figure 1N] Figure 1N shows a mid-longitudinal section of a nodule photographed using a light microscope. Blue staining indicates expression of the GUS reporter; scale bar = 200 μm.

[0047] [Figure 1O] Figure 1O is an enlarged view of Figure 1N, where "ic" indicates infected cells, "uc" indicates uninfected cells, and "nc" indicates the nodule cortex; scale bar = 200 μm. In Figures 1M-1O, proFun:GUS is a construct in which the endogenous Fun promoter sequence (proFUN) is fused to the coding sequence for β-glucuronidase (GUS), followed by the endogenous Fun termination sequence (tFUN). [FIG. 1P] FIG. 1P is a schematic diagram of the FUN protein showing the bZIP DNA-binding domain (gray oval) and the sensor domain (dark gray star).

[0048] [Figure 1Q] Figure 1Q shows a bar graph comparing normalized RNA measurements of FUN transcripts among different plant tissues in Lotus japonicus Gifu, based on data calculated by Kamal et al. (2020). ("Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence." DNA Res. 27(3)) Plant tissues of L. japonicus Gifu are listed along the vertical axis. "Nodule 21d" and "Nodule 10d" refer to measurements obtained from nodules 21 and 10 days after inoculation with Mesorhizobium loti R7A, respectively.

[0049] [Figure 1R] Figure 1R shows a bar graph of genes that were upregulated (top) or downregulated (bottom) and significantly downregulated (DE) in WT (black box) or fun mutant (gray box) plants. The number of DE genes is plotted on the horizontal axis.

[0050] [Figure 1S] Figure 1S presents a bar graph showing the elevated levels (log2 fold change relative to WT plant expression levels, horizontal axis) of differential expression of selected genes (vertical axis) in fun mutant plants (black box) and fun-3 mutant plants (gray box).

[0051] [Figure 1T] Figure 1T depicts a bar graph showing the reduced levels (log2 fold change relative to WT plant expression, horizontal axis) of differential expression of selected genes (vertical axis) in fun mutant plants (black box) and fun-3 mutant plants (gray box).

[0052] [Figure 1U] Figure 1U shows that the ontologies identified by GO-MWU are enriched among upregulated genes (the first three major branches) and downregulated genes (the fourth to seventh major branches). The size and thickness of the text indicate the p-value.

[0053] [Figure 1V] Figure 1V shows the relative expression levels of downstream targets of FUN, identified with TGA motifs in their promoters, that are differentially expressed in fun compared to wild-type. Relative expression levels are shown in the RNA-seq time series by Wang et al. (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi:10.1111 / nph.18896). In Figures 1C-1D and 1F-1L, circles represent individual plants. In Figures 1C-1E and 1K-1L, asterisks indicate significant differences between the compared groups. "**" indicates a p-value <0.01, and "*" indicates a p-value <0.05.

[0054] [Figure 2A] Figures 2A–2S show that FUN controls the expression of downstream genes Nrt2.1, Ho1, NAC094, Nrt3.1, and AS1 to regulate nitrate signaling and nitrogen fixation in root nodules. Figure 2A shows the expression levels of Nrt2.1 (left), Ho1 (center), and NAC094 (right) genes in nodules of wild-type Lotus plants (Gifu; white), fun mutant Lotus plants (gray), and fun-3 mutant Lotus plants (dark gray) at 0, 3, and 24 hours after treatment with 10 mM KNO3 nitrate.

[0055] [Figure 2B] Figure 2B shows the expression levels of Nrt3.1 (left) and AS1 (right) genes in nodules of wild-type Lotus plants (Gifu; white), fun mutant Lotus plants (gray), and fun-3 mutant Lotus plants (dark gray) at 0, 3, and 24 hours after treatment with 10 mM KNO3 nitrate.

[0056] Figure 2C shows a schematic diagram of the promoters of Nrt2.1 (proNRT2.1; top), Ho1 (proHO1; second from the top), NAC094 (proNAC094; middle), Nrt3.1 (proNRT3.1; second from the bottom), and AS1 (proAS1; bottom). The Nrt2.1 promoter has four putative FUN-binding sites (FBSs), designated p1, p2, p3, and p4; the Ho1 promoter has two putative FBSs, designated p1 and p2; the NAC094 promoter has one putative FBS, designated p1; the Nrt3.1 promoter has three putative FBSs, designated p1, p2, and p3; and the AS1 promoter has one putative FBS, designated p1.

[0057] [Figure 2D] Figure 2D shows gel images of EMSA assays demonstrating the binding of FUN protein to DNA probes containing FBS p1, p2, p3, and p4 from the Nrt2.1 promoter (left), FBS p1 and p2 from the Ho1 promoter (center), and FBS p1 from the NAC094 promoter (right).

[0058] [Figure 2E] Figure 2E shows a gel image of an EMSA assay demonstrating binding of FUN protein to DNA probes containing FBS p1 from the Nrt2.1 promoter, FBS p1, p2, and p3 from the Nrt3.1 promoter, and FBS p1 from the AS1 promoter.

[0059] [Figure 2F] Figure 2F shows a gel image of MSA targeting p1 (top) and p4 (bottom) FBS of the Nrt2.1 promoter in a competition assay. The competitor DNA was 50-, 150-, and 500-fold more concentrated than WT DNA without a tag attached to the probe. The label "m" corresponds to a DNA probe with a mutation in the core binding site, TGACG.

[0060] [Figure 2G] Figure 2G shows the results of a transcriptional activation assay of the Nrt2.1 (left), Ho1 (center), and NAC094 (right) promoters by FUN in N. benthamiana leaves. White bars represent GFP, and gray bars represent pro35S:FUN-GFP. pro35S:FUN-GFP was expressed as an effector, and GUS was driven by either the Nrt2.1 promoter, Ho1 promoter, or NAC094 promoter as a reporter.

[0061] [Figure 2H] Figure 2H shows the results of a transcriptional activation assay of the Nrt3.1 (left) and AS1 (right) promoters by FUN in N. benthamiana leaves. White bars represent GFP, and gray bars represent pro35S:FUN-GFP. pro35S:FUN-GFP was expressed as an effector, and a GUS reporter was driven by either the Nrt3.1 or AS1 promoter.

[0062] [Figure 2I] Figure 2I shows representative photographs of root nodules from wild-type (Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, ho1-4 mutant Lotus plants, or nac094-3 mutant Lotus plants exposed to 10 mM KNO for 2 weeks. The scale bar is 1 cm in all four photographs.

[0063] [Figure 2J] Figure 2J shows the total number of nodules (gray box, labeled "Total") and the number of functional pink nodules (white box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0064] [Figure 2K] Figure 2K shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0065] [Figure 2L] Figure 2L shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0066] [Figure 2M] Figure 2M shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, ho1-4 mutant Lotus plants, and ho1-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0067] [Figure 2N] Figure 2N shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0068] [Figure 2O] Figure 2O shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0069] [Figure 2P] Figure 2P shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, hol-4 mutant Lotus plants, and hol-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0070] [Figure 2Q] Figure 2Q shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, hol-4 mutant Lotus plants, and hol-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0071] [Figure 2R] Figure 2R shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks.

[0072] [Figure 2S] Figure 2S shows normalized read counts of FUN downstream targets with identified TGA motifs in their promoters in mock-treated wild-type plants (labeled "WT mock") and wild-type, fun, and fun-3 mutant plants 24 days after nitrate exposure (labeled "WT 24h," "fun 24h," and "fun-3 24h," respectively). The graph shows, from left to right, the normalized read counts of NRT2.1, HO1, NAC094, NRT3.1, and AS1. In Figures 2A-2B, 2G-2H, and 2J-2R, circles represent individual plants. In Figures 2A-2B, 2G-2H, and 2J-2R, asterisks indicate significant differences between the compared groups. "**" indicates a p-value <0.01, and "*" indicates a p-value <0.05.

[0073] Figures 3A-3L show that the FUN sensor domain forms filamentous structures in the presence of physiological concentrations of zinc (Zn). Figure 3A shows dynamic light scattering (DLS) analysis of the FUN sensor domain exposed to 4 mM MgCl, CaCl, MnCl, ZnCl, NH, Cl, KNO, KNO, KCl, or a blank sample ("FUN sensor" alone, control).

[0074] [Figure 3B] Figure 3B shows the results of DLS analysis of FUN(bZIP) with added MnCl2 at concentrations of 8 mM, 4 mM, 2 mM, 1 mM, 500 μM, 250 μM, 62.5 μM, 15.6 μM, 3.9 μM, or 0 μM.

[0075] Figure 3C shows the DLS analysis of FUN (bZIP) with ZnCl at concentrations of 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, 7.8 μM, 3.9 μM, 2.0 μM, 0 μM, or 8 mM in the absence of FUN (8 mM b no ZIP; control).

[0076] [Figure 3D] Figure 3D shows the results of DLS analysis of FUN bZIP alone ("FUN sensor"), FUN in the presence of 100 μM ZnCl2 ("FUN sensor + Zn"), or FUN in the presence of 100 μM ZnCl2 and 5 mM ethylenediaminetetraacetic acid (EDTA) ("FUN sensor + Zn + EDTA").

[0077] [Figure 3E] In Figure 3E, the vertical axis shows the scattering intensity as "I(q)" in cm -1 The horizontal axis is expressed in units of q (Å -1) shows plots of SAXS analysis. Scattering is plotted for FUN sensor alone (bZIP only) (“FUN sensor”, gray), FUN bZIP bound to Zn (“FUN sensor + Zn”, light gray), or FUN bZIP with Zn removed using EDTA (“FUN sensor + Zn + EDTA”, black).

[0078] [Figure 3F] Figure 3F shows a plotted histogram of interpair distances between points within particles based on the analysis in Figure 3E. The pair distance distribution model p(r) is plotted on the vertical axis, and the distance r (Å) is plotted on the horizontal axis. The arrow "394 Å" represents the maximum diameter (Dmax) of 394 Å for Zn-bound FUN bZIP ("FUN sensor + Zn"). The arrow "118 Å" represents the Dmax of 118 Å for FUN bZIP alone ("FUN sensor") or FUN bZIP with Zn removed ("FUN sensor + Zn + EDTA").

[0079] Figure 3G shows the Guinier plot calculated from Figures 3E and 3F, where the radius of gyration is calculated through the scattering intensity as a function of the scattering vector q (vertical axis vs. horizontal axis). Closed circles represent the data used in the fit, and open circles represent omitted data points. The p(r) function shows that the radius of gyration for the pure FUN sensor sample and the EDTA + zinc-containing sample is 39 ± 1 Å, while it is 125 ± 1 Å for the zinc-bound sample. Guinier analysis showed slightly lower values ​​for all samples.

[0080] [Figure 3H] Figure 3H shows representative electron micrographs of the FUN sensor domain alone (bZIP1 sensor; top row), the FUN sensor domain with 300 μM ZnCl2 added (bZIP1 sensor + 300 μM Zn; middle row), and FUN with 300 μM ZnCl2 and 5 mM EDTA added (bZIP1 sensor + 300 μM Zn + 5 mM EDTA; bottom row).

[0081] [Figure 3I] Figure 3I shows the relative expression level of Fun (vertical axis) over time (horizontal axis) in 3-week-old root nodules exposed to 10 mM KNO3 for 0, 0.5, and 3 hours (left) and for 0, 1, 3, and 7 days (right).

[0082] [Figure 3J] Figure 3J shows the purification and thermal stability of the FUN sensor domain. The left side shows a chromatogram of the FUN sensor domain obtained by size-exclusion chromatography (Superdex 200 increase 10 / 300), plotting absorbance (vertical axis) against elution volume (horizontal axis). The right side shows sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the SEC fractions. Fractions 14–17 were pooled and stored, as indicated by the dashed line on the chromatogram and the horizontal line above the SDS-PAGE.

[0083] [Figure 3K] Figure 3K shows the inflection point temperature (T i The thermal stability of the purified FUN sensor domain, measured in °C, is shown either alone ("FUN sensor only") or in the presence of different ions (vertical axis) at a concentration of 4 mM: MgCl2, CaCl2, MnCl2, ZnCl2, NH4Cl, KNO3, KNO2, or KCl.

[0084] [Figure 3L] Figure 3L shows DLS analysis of the zipper domain and sensor domain-containing FUN protein in the absence (gray line) and presence (medium gray line) of 100 μM ZnCl2. The change in hydrodynamic radius induced by zinc is reversed by 5 mM EDTA (black line). In Figures 3C–3G, the gray “FUN Sensor” represents measurements of the FUN sensor alone, the medium gray “FUN Sensor + Zn” represents measurements of the FUN sensor treated with 100 μM ZnCl2, and the black “FUN Sensor + Zn + EDTA” represents the FUN sensor treated with 100 μM ZnCl2 and then 5 mM EDTA to remove Zn. In Figures 3C–3G, the four-pointed star labeled “apo” represents the apo-conformation (“apo”) FUN sensor. The chains with overlapping four-pointed stars labeled “Zn-bound” represent combinations of Zn-bound FUN sensors in larger oligomers.

[0085] [Figure 4A] Figures 4A-4H show that zinc regulates the subcellular localization and function of FUN. Figure 4A shows representative images of the subcellular localization of pro35S:FUN-GFP in N. benthamiana leaves.

[0086] [FIG. 4B] FIG. 4B shows the fluorescence distribution of the subcellular localization of FUN-GFP in N. benthamiana leaves.

[0087] [FIG. 4C] FIG. 4C shows the ratio of punctate nuclei (dark gray) to total nuclei (homogeneous; light gray) for subcellular localization in N. benthamiana leaves. In Figures 4A-4C, results were obtained under conditions of 500 μM MgCl2 (mock), MnCl2 (Mn), and ZnCl2 (Zn).

[0088] Figure 4D shows the results of a FUN-mediated transcriptional activation assay of the Nrt2.1 promoter in N. benthamiana leaves treated with 500 μM MgCl (mock) and ZnCl (Zn). White bars represent GFP, and gray bars represent pro35S:FUN-GFP. FUN-GFP was expressed as an effector, and GUS was driven by the Nrt2.1 promoter (proNrt2.1:GUS) as a reporter.

[0089] [Figure 4E] Figure 4E shows nitrogen fixation activity, quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant), in wild-type (WT; Gifu accession) Lotus plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) in combination with KCl (left) or 10 mM KNO3 (middle and right).

[0090] Figure 4F shows nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) with 10 mM KNO3.

[0091] Figure 4G shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 500 µM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) with 10 mM KNO3.

[0092] [Figure 4H] Figure 4H shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants exposed to 10 mM KCl (left), 10 mM KNO (middle), or ZnCl treatment plus 10 mM KNO (right) for 2 weeks. In Figures 4D-4H, circles represent individual plants.

[0093] Figures 5A-5H show that nitrate promotes zinc transport from nodule cells using the zinc-sensitive dye Zinpyr-1. Figure 5A shows that expression of two putative zinc transporter genes, Zip2 and Zip4, is induced in nodules after 24 hours of treatment with 10 mM KNO3 (nitrate).

[0094] [Figure 5B] Figure 5B depicts the mechanism of nodule function regulated by FUN. On the left is the mechanism that operates when soil nitrate concentrations are low, where zinc accumulates in the nodules, keeping FUN in inactive filaments and allowing nitrogen fixation to continue. On the right, the mechanism operates under high soil nitrate concentrations, where a decrease in cellular zinc concentration releases active FUN from the filaments, increasing target gene expression. Arrows indicate the direction of action of these conditions. "NAC094," "HO1," and "NRT2.1" indicate the activity of these three target genes that induce nodule senescence. Dark gray indicates the presence of inactive FUN filaments, and pink nodules represent the corresponding phenotype. Light gray indicates the presence of active FUN.

[0095] Figure 5C shows cellular zinc concentration levels in root nodules, as indicated by Zinpyr-1 fluorescent dye, 24 hours after mock treatment (left, KCl) and nitrate treatment (right, KNO). Scale bar = 200 μm.

[0096] FIG. 5D shows the average intensity of the fixation zone indicated by the dashed circle in FIG. 5C.

[0097] Figure 5E shows X-ray fluorescence (XRF) microscopy images of nodule cut surfaces taken 24 hours after mock treatment (left, KCl) and nitrate treatment (right, KNO). Scale bar = 20 μm.

[0098] [FIG. 5F] FIG. 5F shows the image of the white boxed area analyzed for zinc quantification in FIG. 5E. Scale bar = 20 μm.

[0099] [Figure 5G] Figure 5G shows fluorescence images of the FUN-GFP construct in control (left, labeled "MgCl") and zinc (right, labeled "ZnCl")-treated Lotus roots. Scale bar = 20 μm. Chi-squared test values ​​are shown below, *** = p-value < 0.01.

[0100] [Figure 5H] Figure 5H shows confocal images of N. benthamiana leaves expressing the FUN-GFP construct and co-infiltrated with either MgCl2 (left) or ZnCl2 (right) 2 days before confocal observation. Scale bar = 5 μm. Chi-squared test values ​​are shown below; * = p-value < 0.05.

[0101] Figure 6A-6E show the phylogenetic tree of FUN proteins and the relative expression patterns of soybean FUN orthologs. Figure 6A shows the phylogenetic tree of FUN and LjFUN-like genes as well as FUN orthologs identified using shoot.bio. Tree support values ​​are plotted at branch points.

[0102] Figure 6B shows the relative expression (vertical axis) patterns across various tissues (horizontal axis) of the closest FUN soybean orthologs, Glyma.02G097900 and Glyma.01G084200. For each tissue, the expression level of Glyma.02G097900 is shown on the left, and the expression level of Glyma.01G084200 is shown on the right.

[0103] [Figure 6C] Figure 6C shows a schematic diagram of the LjFUN protein. The DNA-binding bZIP domain is shown in the left box, and the zinc sensor domain is shown in the right box.

[0104] [FIG. 6D] FIG. 6D shows the first half of a protein alignment of selected orthologs of FUN and FUN-like proteins.

[0105] [FIG. 6E] FIG. 6E shows the second half of a protein alignment of selected orthologs of FUN and FUN-like proteins. In Figures 6A, 6D, and 6E, plant species names correspond to the following abbreviations: Prunus persica: Prupe; Lotus japonicus: Lj; Glycine max: Glyma; Manihot esculenta: Manes; Gossypium raimondii: Gorai; Eucalyptus grandis: Eucgr; Brassica oleracea: Bol; Arabidopsis thaliana: AT; Solanum lycopersicum: Solyc; Aquilegia coerulea: Aqcoe; Amborella trichopoda: AmTr; Spirodela polyrhiza: Spipo; Musa acuminata: GSMUA; Zea mays: GRMZM; ​​Setaria italica: Seita; Triticum Aestivum: Traes; Hordeum vulgare: HORVU; and Oryza sativa: Os. In Figures 6D-6E, the protein sequences arranged from top to bottom are the consensus sequence (SEQ ID NO: 150), Lotus japonicus LjFUN (SEQ ID NO: 1), Glycine max Glyma.02G097900.1 (SEQ ID NO: 8), Glycine max Glyma.01G084200.1 (SEQ ID NO: 9), Glycine max Glyma.10G276100 (SEQ ID NO: 6), Glycine max Glyma.20G113600.1 (SEQ ID NO: 7), Lotus japonicus LjFUNL (SEQ ID NO: 83), and Arabidopsis thaliana AT1G68640.1 (SEQ ID NO: 4).

[0106] [Figure 7] Figure 7 shows a phylogenetic tree of the Lotus japonicus NAC domain-containing protein Nac094 (labeled "LjNAC094") and orthologous NAC domain-containing proteins from other species. Species names and protein identifiers are indicated at the ends of the branches. Tree support values ​​are plotted at the branch points.

[0107] [Figure 8A] Figures 8A-8C show evidence of a role for FUN in drought and heat tolerance. Figure 8A shows RNAseq counts of NAC094 (left graph) and HO1 (right graph) in Medicago truncatula root nodules under the following conditions (from left to right): after irrigation, day 2 of drought, and day 4 of drought.

[0108] [Figure 8B] Figure 8B shows RNAseq counts of NAC094 (left graph) and HO1 (right graph) in Lotus japonicus root nodules under the following conditions (from left to right): control, 2 days of drought, and 4 days of drought.

[0109] [Figure 8C] Figure 8C shows nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type plants (left) and fun-3 mutant plants (right) before heat stress (gray box) and 7 days after heat stress (white box). 'ns' indicates no significant difference.

[0110] [Mode for Carrying Out the Invention] Exemplary methods, parameters, etc. are described below, but it should be recognized that these descriptions are not intended to limit the scope of the present disclosure, but rather are provided as descriptions of exemplary embodiments.

[0111] [FUN protein] One embodiment of the present disclosure includes the FUN (nitrate fixation) protein, where FUN is a transcriptional regulator expressed in root nodules, with related uses disclosed throughout. Example 1 describes the identification of the basic leucine zipper transcription factor FUN as a novel master regulator of nitrogen fixation in legumes. Examples of FUN proteins include, but are not limited to, the originally identified Lotus japonicus FUN protein (LotjaGi2g1v0279100; SEQ ID NO: 1), as well as orthologs of the Lotus japonicus FUN protein (both of which are also expressed in the root nodules of their respective plants), soybean FUNa and FUNb proteins (Glyma.02G097900; SEQ ID NO: 8, and Glyma.01G084200; SEQ ID NO: 9), Vicia faba FUN protein (Vfaba.Hedin2.R1.1g203360.1; SEQ ID NO: 81), and V. unguiculata (cowpea) FUN (Vigun02g036100.1.p; SEQ ID NO: 82). FUN proteins that exhibit activity, expression, or enhanced activity or expression in root nodules are readily distinguishable from FUN-like proteins, or paralogs of FUN proteins, which are distinguished by a lack of enhanced expression or activity in root nodules. FUN-like proteins also form independent paralogous branches on the phylogenetic tree. Exemplary FUN-like proteins include Lotus genus FUN-like protein (LotjaGi5g1v0341400; SEQ ID NO:83); cowpea FUN-like protein (Vigun07g272100.1.p; SEQ ID NO:84); soybean FUN-like A (G. max Wm82.a2.v1|Glyma.10G276100.1.p; SEQ ID NO:6); soybean FUN-like B (G. max Wm82.a2.v1|Glyma.20G113600.1.p; SEQ ID NO:7), and Arabidopsis thaliana PAN (A. thaliana Araport11|AT1G68640.1; SEQ ID NO:4). The FUN protein may be overexpressed in root nodules.FUN or FUN-like proteins can be inactive, filamentous (e.g., large filaments), or active, oligomeric, non-filamentous. The FUN or FUN-like genes encode proteins of the TGA family of transcription factors. TGA transcription factors belong to the bZIP family of transcription factors, which are sometimes characterized by the presence of an N-terminal leucine zipper (bZIP) DNA-binding domain and a C-terminal DOG1 domain, referred to as the sensor domain in Figure 1P (Tomaz, S., Gruden, K. & Coll, A. TGA transcription factors—Structural characteristics as a basis for functional variability. Front. Plant Sci. 13, 935819 (2022)). The protein domains and precise locations of the FUN and FUN-like protein sequences described herein were analyzed and are listed in Table 1.

[0112] [Table 1] TIFF2026504437000003.tif228169TIFF2026504437000004.tif227169TIFF202 6504437000005.tif230169TIFF2026504437000006.tif230169TIFF20265044370 00007.tif229169TIFF2026504437000008.tif232169TIFF2026504437000009.t if225169TIFF2026504437000010.tif231169TIFF2026504437000011.tif216169

[0113] Protein domains and motifs are both conserved sequence patterns. A domain is an independent folding unit of a protein. A motif is a small region or set of small regions of three-dimensional structure, nucleotide sequence, or amino acid sequence that is shared among proteins. A conserved domain or motif is a recurring unit of molecular evolution, the extent of which can be determined by sequence and structural analysis. A conserved domain or motif may contain a conserved sequence pattern or sequence motif, which allows the detection of the domain or motif in a polypeptide sequence.

[0114] [Downstream targets of FUN protein] FUN is a transcriptional regulator that controls the expression of downstream genes, including high-affinity nitrate transporter 2.1 (NRT2.1), heme oxygenase (HO1), NAC domain-containing protein 94 (NAC transcription factor 94, or NAC094), high-affinity nitrate transporter 3.1 (NRT3.1), basic leucine zipper transcription factor 28 (bZIP28), and asparagine synthase 1 (AS1), to regulate nitrate signaling and nitrogen fixation in root nodules. An exemplary NRT2.1 protein includes SEQ ID NO:78. An exemplary HO1 protein includes SEQ ID NO:77. An exemplary NRT3.1 protein includes SEQ ID NO:74. An exemplary bZIP28 protein includes SEQ ID NO:75. An exemplary AS1 protein includes SEQ ID NO:79. NAC094 (SEQ ID NO:31; also called FEZ protein) of L. japonicus, which is a downstream target of FUN (Fig. 7).Exemplary NAC094 homologs (NAC domain-containing proteins) are SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:76.

[0115] [Genetically modified plants and related methods] One aspect of the present disclosure includes a genetically modified plant or portion thereof that includes one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: The present invention also includes polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or domains thereof, e.g., the bZIP1, bZIP2, or DOG1 domains listed in Table 1, or combinations thereof.In additional embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: NO:82, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or each domain listed in Table 1, or a combination thereof.

[0116] Further aspects of the present disclosure include genetically modified plants or portions thereof that include one or more genetic modifications that reduce the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In further embodiments of this aspect, wherein the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; and wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75. wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: and wherein the HO1 protein is a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is an HO1 protein, and the HO1 protein is selected from the group consisting of SEQ ID NO: and a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to NRT2. NO:77; wherein said protein is an NRT2.1 protein, and said NRT2.or wherein the protein is an AS1 protein and the AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78; or wherein the protein is an AS1 protein and the AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79, or a domain thereof, e.g., a domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to each domain set forth in Table 1, or a combination thereof. In an additional embodiment of this aspect, the NRT3.wherein the bZIP28 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or the AS1 protein comprises SEQ ID NO:79, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or each of the domains set forth in Table 1, or a combination thereof. The protein domains and detailed locations of the NAC domain-containing NRT3.1, NRT2.1, AS1, and HO1 protein sequences described herein have been analyzed and are shown in Table 1.

[0117] Additional aspects of the present disclosure relate to the genome of a genetically modified plant, including (i) one or more genetic modifications in a genetically modified plant or portion thereof according to any one of the preceding embodiments, or (ii) one or more genetic modifications in a genetically modified plant or portion thereof produced by a method according to any one of the preceding embodiments. In still other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: In another embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82, or each of the domains listed in Table 1, or a combination thereof. In further embodiments of this aspect, the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; and wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDand wherein said protein is an HO1 protein, and said HO1 protein is selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; or wherein said protein is an NRT2.1 protein and said NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77; wherein said protein is an NRT2.1 protein and said NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78; or wherein said protein is an AS1 protein and said AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79.The polypeptides include those having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to NO:79, or to domains thereof, e.g., each domain listed in Table 1, or combinations thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity. In additional embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ IDwherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or the AS1 protein comprises SEQ ID NO:79, or each domain set forth in Table 1, or a combination thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or each domain set forth in Table 1, or a combination thereof.

[0118] In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In still other embodiments of this aspect that can be combined with any of the preceding embodiments, the reduction is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in a promoter region of the gene, preferably the binding site is a transcriptional activator protein binding site or a TATA box. In further embodiments of this aspect that can be combined with any of the preceding embodiments, the cultivation conditions include a moderate nitrate concentration, a high nitrate concentration, or a nitrate concentration around the plant that reduces or inhibits nitrogen fixation. In still other embodiments of this aspect, the nitrate concentration is from about 10 mM to about 250 mM nitrate, or alternatively, at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate.In still other embodiments of this aspect, the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same conditions. In further embodiments of this aspect, the nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In still other embodiments of this aspect that may be combined with any of the preceding embodiments, the plant forms nodules. In additional embodiments of this aspect, the plant exhibits an increased number of nodules, increased hemoglobin content, or increased acetylene reduction assay (ARA) activity compared to a control plant grown under the same cultivation conditions.

[0119] In certain embodiments of any of the foregoing aspects, and various embodiments thereof, the plant part may be a seed, pod, fruit, leaf, flower, stem, root, part or cell of any of the foregoing, or a non-regenerable part or cell of a transgenic plant part. As used in this context, a non-regenerable part or cell of a transgenic plant or part thereof is a part or cell that cannot be induced to form a whole plant or cannot be induced to form a whole plant capable of sexual and / or asexual reproduction. In certain embodiments, the non-regenerable part or cell of the plant part is a part or cell of a transgenic seed, pod, fruit, leaf, flower, stem, or root. In other embodiments, the non-regenerable part or cell of the plant part is part of a processed plant product.

[0120] Also provided are processed plant products containing detectable amounts of nucleotide fragments, expressed RNA, and / or proteins comprising the genetic modifications described herein. Such processed products include, but are not limited to, plant biomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, and processed seeds. The processed products may be non-renewable. The plant products may include trade items and other commercial products derived from transgenic plants or transgenic plant parts, which trade items and other products are traceable through commerce by detecting the nucleotide fragments, expressed RNA, and / or proteins comprising identifiable portions of the genetic modifications described herein.

[0121] Additional aspects of the present disclosure include methods for cultivating genetically modified plants that result in increased nitrogen fixation under conditions comprising nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the method comprising: (a) providing the genetically modified plant, wherein the plant or portion thereof exhibits an increased activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also referred to as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein, compared to the activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also referred to as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same cultivation conditions; (b) cultivating the genetically modified plant under conditions where the nitrate concentration is at the root of the plant, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same cultivation conditions. In yet other embodiments of this aspect, the decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene, preferablythe binding site is a transcriptional activator protein binding site or a TATA box. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: The present invention also includes polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or domains thereof, e.g., the bZIP1, bZIP2, or DOG1 domains listed in Table 1, or combinations thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,In still other embodiments of this aspect, the FUN protein comprises SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or each domain listed in Table 1, or a combination thereof. In still other embodiments of this aspect, the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; and wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID or wherein said protein is an HO1 protein and said HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:73; wherein said protein is an HO1 protein, and said HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77; wherein said protein is an NRT2.1 protein, and said NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78; or wherein said protein is an AS1 protein, and said AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78. or a domain thereof, e.g., a domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to NO:79, or a domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a domain thereof, e.g., a domain listed in Table 1, or a combination thereof.It includes polypeptides having at least 95%, or at least 99% identity. In further embodiments of this aspect, wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78; or wherein the AS1 protein comprises SEQ ID NO:79, or each domain set forth in Table 1, or a combination thereof. In still other embodiments of this aspect, the NAC domain-containing protein is SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or each domain set forth in Table 1,Additional embodiments of this aspect that can be combined with any of the preceding embodiments include those in which the nitrate concentration in step (c) is from about 10 mM to about 250 mM nitrate, alternatively at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, or at least about 60 mM nitrate. The nitrogen fixation concentration may be at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. Further embodiments of this aspect that may be combined with any of the preceding embodiments include the nitrogen fixation being increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules or hemoglobin content is increased compared to a control plant grown under the same cultivation conditions. In additional embodiments of this aspect, the increased nitrogen fixation is measured using a method selected from the group consisting of measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour (acetylene reduction assay (ARA)) compared to control plants, or measuring micrograms of hemoglobin per plant compared to control plants (e.g., as described in Example 1).

[0122] A further aspect of the present disclosure includes a method of cultivating a genetically modified plant capable of fixing nitrogen when grown under nitrogen fertilized conditions, said method comprising: (a) providing said genetically modified plant, wherein said plant or portion thereof has a higher activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also referred to as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also referred to as FEZ protein) in a control plant grown under the same cultivation conditions; (b) cultivating the plant under conditions in which the root zone of the plant contains a standard nitrate concentration; and (c) using nitrogen fertilizer to create conditions in which the root zone of the plant contains a nitrate concentration that inhibits nitrogen fixation, wherein the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same conditions. In yet other embodiments of this aspect, the decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or the genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene.Alternatively, it comprises knocking out a binding site in the promoter region of said gene, preferably said binding site being a transcription activator protein binding site or a TATA box. In a further embodiment of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: The present invention also includes polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of NO:82, or domains thereof, e.g., each domain set forth in Table X, or combinations thereof. In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,In still other embodiments of this aspect, the FUN protein comprises SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or each domain listed in Table 1, or a combination thereof. In still other embodiments of this aspect, the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; and wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID or wherein said protein is an HO1 protein and said HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:73; wherein said protein is an HO1 protein, and said HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77; wherein said protein is an NRT2.1 protein, and said NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78; or wherein said protein is an AS1 protein, and said AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78. In further embodiments of this aspect, the NRT3.1 protein comprises SEQ ID NO:74; the bZIP28 protein comprises SEQ ID NO:75; and the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 ...60, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79, or each domain set forth in Table 1, or a combination thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or each domain set forth in Table 1, or a combination thereof. Additional embodiments of this aspect that can be combined with any of the previous embodiments include those in which the nitrate concentration in step (c) is from about 10 mM to about 250 mM nitrate, alternatively at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate,or at least about 250 mM nitrate. Further embodiments of this aspect that can be combined with any of the preceding embodiments include at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1, The nitrogen fixation is increased by 50%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules or hemoglobin content is increased compared to a control plant grown under the same cultivation conditions. In additional embodiments of this aspect, the increased nitrogen fixation is measured using a method selected from the group consisting of measuring the number of pink nodules per plant compared to a control plant, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour (acetylene reduction assay (ARA)) compared to a control plant, or measuring micrograms of hemoglobin per plant compared to a control plant (e.g., as described in Example 1). In further embodiments of this aspect that may be combined with any of the previous embodiments, the genetically modified plant is grown in an intercropping system with a plant that does not fix nitrogen, or in a crop rotation system after a plant that does not fix nitrogen.

[0123] Additional aspects of the present disclosure include a method for delaying root nodule senescence, the method comprising: (a) providing a genetically modified plant, wherein the plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions, and wherein the one or more genetic modifications delay root nodule senescence; and (b) cultivating the genetically modified plant under stress conditions, wherein the genetically modified plant exhibits delayed root nodule senescence compared to the control plant grown under the same cultivation conditions. Yet other embodiments of this aspect include the step of: said decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene, preferably wherein said binding site is a transcriptional activator protein binding site or a TATA box. Further embodiments of this aspect include: moderate nitrate concentrations; high nitrate concentrations; nitrate concentrations around the plant that promote nodule senescence;The stress conditions include those selected from the group consisting of moderate heat, high heat, heat around the plant that promotes nodule senescence, moderate water deficit (i.e., drought), high water deficit, water deficit around the plant that promotes nodule senescence, moderate waterlogging, high waterlogging, and waterlogging around the plant that promotes nodule senescence. For each of these conditions, the stress level is believed to be sufficient to inhibit nitrogen fixation in the particular plant species. In further embodiments of this aspect which may be combined with any of the preceding embodiments, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO: In another embodiment of this aspect, the FUN protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:81, or SEQ ID NO:82, or to a domain thereof, e.g., each domain set forth in Table 1, or a combination thereof.SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or each domain listed in Table 1, or a combination thereof. In still other embodiments of this aspect that may be combined with any of the preceding embodiments having a NAC domain-containing protein, wherein the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; and wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID and wherein the protein is an HO1 protein and the HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is an HO1 protein and the HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77; wherein the protein is an NRT2.1 protein and the NRT2.1 protein is selected from the group consisting of SEQ ID NO: or wherein the protein is an AS1 protein and the AS1 protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79.or at least 99% identical to, or comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a domain thereof, e.g., each domain set forth in Table 1, or a combination thereof. In further embodiments of this aspect, wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; and wherein the NRT2.1 protein comprises SEQ ID NO:78,Alternatively, wherein the AS1 protein comprises SEQ ID NO:79, or each domain set forth in Table 1, or a combination thereof. In yet other embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID N, O:41, or SEQ ID NO:42, or each domain set forth in Table 1, or a combination thereof. In further embodiments of this aspect, nodule senescence is delayed by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0124]

[0010] A further aspect of the present disclosure includes a method of inducing FUN protein filament formation, comprising: (a) providing a FUN protein; and (b) increasing the amount of zinc or manganese around the FUN protein, wherein the increased amount of zinc or manganese induces filament formation compared to a control FUN protein in an environment without the increased amount of zinc or manganese. In additional embodiments of this aspect, the filament formation is induced under high nitrate conditions. In still other embodiments of this aspect that can be combined with any of the preceding embodiments, the method is performed in vitro.

[0125] A further aspect of the present disclosure includes a method of inducing filamentation, comprising: (a) providing a plant comprising a FUN protein; and (b) cultivating the plant under conditions of elevated zinc or manganese, wherein filamentation of the FUN protein in the plant is induced compared to FUN protein in a control plant in an environment without elevated amounts of zinc or manganese. In other embodiments of this aspect, the plant comprises a genetic modification. In additional embodiments of this aspect, the filamentation is induced under high nitrate conditions. In yet other embodiments of this aspect, which can be combined with any of the above embodiments in which the plant comprises a genetic modification, the genetic modification reduces the activity of the FUN protein without eliminating its activity. In a further embodiment of this aspect, the induction of filamentation increases nitrogen fixation, reduces the activity, or inactivates the FUN protein in the genetically modified plant compared to a control plant grown under the same conditions. In still other embodiments of this aspect that may be combined with any preceding embodiment or aspect with a method of inducing filament formation, the number of nodules is increased, the hemoglobin content is increased, or the acetylene reduction assay (ARA) activity is increased compared to a control plant grown under the same cultivation conditions. A further aspect of the present disclosure includes a method for modulating nodule function in response to available nitrogen in soil, comprising: a) providing a genetically modified plant containing a FUN protein with altered nitrate activation; and b) cultivating the genetically modified plant under nitrate conditions, wherein the genetically modified plant exhibits reduced FUN activity or expression and / or a reduced active form of FUN compared to a wild-type plant grown under the same nitrate conditions. In additional embodiments of this aspect, altering nitrate-induced FUN protein activation comprises silencing FUN, reducing FUN activity, knocking out FUN by mutation, knocking down FUN expression, knocking out a FUN promoter element, or a combination thereof. In a further embodiment of this aspect, altering nitrate-induced FUN protein activation comprises manipulating environmental or cellular zinc concentrations, wherein the manipulation maintains the FUN protein in an inactive filamentous form. In yet another embodiment of this aspect, altering nitrate-induced FUN protein activation comprises genetically modifying the FUN protein sequence to alter its sensitivity to zinc.

[0126] In further embodiments of this aspect which may be combined with any of the preceding embodiments, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO: or a domain thereof, e.g., each domain set forth in Table 1, or a combination thereof.In other embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO: NO:82, or each domain listed in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:9, or each domain listed in Table 1, or a combination thereof.

[0127] Further aspects of the present disclosure include methods for producing genetically modified plants that result in increased nitrogen fixation under conditions that include nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, comprising introducing into the plant or part thereof one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions. In yet other embodiments of this aspect, the decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene, preferably wherein said binding site is a transcriptional activator protein binding site or a TATA box. In additional embodiments of this aspect, the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDand a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82; or the FUN protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:82; or the FUN protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:83. NO:9 or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a domain thereof, e.g., each domain listed in Table 1, or a combination thereof.

[0128] Additional aspects of the present disclosure include methods for producing genetically modified plants that result in increased nitrogen fixation under conditions, including nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, comprising introducing into the plant or portion thereof one or more genetic modifications that reduce the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein, compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In yet other embodiments of this aspect, the decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene, preferably wherein said binding site is a transcriptional activator protein binding site or a TATA box. In yet other embodiments of this aspect, wherein the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74;wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID and a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:73; wherein said protein is an HO1 protein, and said HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:77;wherein the protein is an NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:78; or wherein the protein is an AS1 protein, and the AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79; wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77;wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein is SEQ ID NO:79; or wherein the NAC domain-containing protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a domain thereof, e.g., a domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to each domain listed in Table 1, or a combination thereof;

[0129] Yet another aspect of the present disclosure includes a method for producing a genetically modified plant that results in increased nitrogen fixation under conditions comprising nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, comprising introducing into the plant or portion thereof one or more genetic modifications that reduce the activity or expression of one or more of FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In yet other embodiments of this aspect, the decrease in activity or expression is due to knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene; and / or said genetic modification comprises knocking out a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knocking out a protein domain, introduction of a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of said gene, preferably wherein said binding site is a transcriptional activator protein binding site or a TATA box. In some embodiments of this aspect, the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein,Alternatively, the AS1 protein may be selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO: NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,or a combination thereof, wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein has enhanced expression in nodules of roots lacking the one or more genetic modifications.

[0130] A further aspect of the present disclosure includes a method for producing a genetically modified plant or part thereof according to any of the preceding embodiments, comprising introducing into a plant cell a genetic modification that reduces or knocks out the activity or expression of a FUN protein, a FUN-like protein, a NAC domain-containing protein (also referred to as a FEZ protein), an NRT3.1 protein, a bZIP28 protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein. In a further embodiment of this aspect, the genetic modification comprises a first nucleic acid sequence capable of reducing or knocking out a second nucleic acid sequence encoding a FUN protein, a FUN-like protein, a NAC domain-containing protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein operably linked to a promoter. In additional embodiments of this aspect that may be combined with any of the preceding embodiments, the genetically modified plant is selected from one or more of the group consisting of alfalfa, bambara bean, pulses (e.g., kidney bean, black bean, etc.), black currant, chickpea, white clover, cowpea, forage legume, legume tree, lentil, lotus, lupine, Medicago, pea, peanut, pigeon pea, soybean, Parasponia, alder, and elm. In other embodiments of this aspect, the nucleic acid comprises a short RNA, antisense RNA, siRNA, miRNA, dsRNA, tasiRNA, or secondary siRNA associated with RNA silencing. In yet other embodiments of this aspect, the promoter is a nodule-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof.In additional embodiments of this aspect, the promoter is a root-specific promoter, and wherein the promoter is selected from the group consisting of NFR1 promoter, NFR5 promoter, LYK3 promoter, CERK6 promoter, NFP promoter, Lotus japonicus NFR5 promoter (SEQ ID NO:85), Lotus japonicus NFR1 promoter (SEQ ID NO:89), Lotus japonicus CERK6 promoter (SEQ ID NO:87), Medicago truncatula NFP promoter (SEQ ID NO:86), Medicago truncatula LYK3 promoter (SEQ ID NO:88), maize metallothionein promoter, chitinase promoter, maize ZRP2 promoter, tomato LeExtl promoter, glutamine synthetase soybean root promoter, RCC3 promoter, rice antiquitin promoter, LRR receptor kinase promoter, and Arabidopsis pCO2 promoter. In yet other embodiments of this aspect, the nucleic acid sequence is inserted into the genome of the plant such that it is operably linked to an endogenous promoter. In further embodiments of this aspect, the endogenous promoter is a nodule-specific promoter or a root-specific promoter.

[0131] Further aspects of the present disclosure include a method for producing a transgenic plant or portion thereof according to any of the preceding embodiments, comprising genetically modifying the plant cell by transforming the plant cell with one or more gene editing components targeting an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, a NAC domain-containing protein (also referred to as a FEZ protein), an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, wherein the endogenous nuclear genomic sequence or portion thereof is knocked out. In other embodiments of this aspect, the one or more gene editing components include a ribonucleoprotein complex targeting the nuclear genomic sequence; a vector comprising a sequence encoding a TALEN protein targeting the nuclear genomic sequence; a vector comprising a sequence encoding a ZFN protein targeting the nuclear genomic sequence; an oligonucleotide donor (OND) targeting the nuclear genomic sequence; or a vector comprising a sequence encoding a CRISPR / Cas enzyme and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

[0132] As used herein, a control refers to a control or reference sample obtained from a wild-type, azygous, or null-segregant plant, species, or sample, or a population thereof. A control plant, as used herein, can also refer to a plant identical to a genetically modified or transgenic plant but without modification, or a wild-type plant grown under the same cultivation conditions, soil, and / or growth medium. A reference value can be used in place of a control or reference sample previously obtained from a wild-type, azygous, or null-segregant plant, species, or sample, or a population thereof, or a group of wild-type, azygous, or null-segregant plants, species, or samples. A control or reference sample can also be a sample containing a known amount of a detectable composition, or a spiked sample.

[0133] The recitation of each discrete value set forth herein is understood to include ranges between each value. The recitation of a range of values ​​set forth herein is understood to include each discrete value within the range.

[0134] [Expression vectors or isolated DNA molecules, cells or kits containing same, and related methods] Yet other aspects of the disclosure include an expression vector or isolated DNA molecule, (i) one or more nucleotide sequences encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein (also referred to as a FEZ protein), a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; and (ii) a nucleic acid sequence encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, is reduced or knocked out. or (iii) one or more nucleotide sequences comprising a mutation in a gene for a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the activity or expression of the protein is reduced or knocked out, and wherein the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site. In another embodiment of this aspect, the expression control sequence comprises a nodule-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof. In yet other embodiments of this aspect, wherein the promoter is a root-specific promoter, and wherein the promoter is selected from the group consisting of NFR1 promoter, NFR5 promoter, LYK3 promoter, CERK6 promoter, NFP promoter, Lotus japonicus NFR5 promoter (SEQ ID NO:85), Lotus japonicus NFR1 promoter (SEQ ID NO:89), Lotus japonicus CERK6 promoter (SEQ ID NO:87), Medicago truncatula NFP promoter (SEQ ID NO:No. 86), Medicago truncatula LYK3 promoter (SEQ ID No. 88), maize metallothionein promoter, chitinase promoter, maize ZRP2 promoter, tomato LeExtl promoter, glutamine synthetase soybean root promoter, RCC3 promoter, rice antiquitin promoter, LRR receptor kinase promoter, and Arabidopsis thaliana pCO2 promoter, or wherein the promoter is a constitutive promoter, and wherein the promoter is selected from the group consisting of CaMV35S promoter, derivatives of CaMV35S promoter, maize ubiquitin promoter, polyubiquitin promoter, vein mosaic cassava virus promoter, and Arabidopsis thaliana UBQ10 promoter. In a further embodiment of this aspect, the protein is a FUN protein, and the FUN protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO: and SEQ ID NO:82; wherein the FUN protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 ...or wherein said FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82; or wherein said FUN protein comprises SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO: NO:9; wherein the protein is a FUN-like protein, and the FUN-like protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:83, and SEQ ID NO:84; wherein the FUN-like protein comprises SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:83, or SEQ ID NO:84; and / or wherein the protein is an NRT3.1 protein, and the NRT3.1 protein is selected from the group consisting of SEQ ID NO: and a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; wherein the protein is a bZIP28 protein, and the bZIP28 protein has SEQ ID NO:74.and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID and wherein the protein is an HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:73; wherein the protein is an HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:77; wherein the protein is an NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group of SEQ ID NO:73.or wherein the protein is an AS1 protein and the AS1 protein comprises a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:79; wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; and wherein the NAC domain-containing protein is SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:79, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO: NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID wherein said HO1 protein comprises SEQ ID NO:77; wherein said NRT2.1 protein comprises SEQ ID NO:78, or wherein said AS1 protein comprises SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73; wherein said HO1 protein comprises SEQ ID NO:77; wherein said NRT2.1 protein comprises SEQ ID NO:78, oror wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a domain thereof, e.g., a respective domain set forth in Table 1, or a combination thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity.

[0135] Some aspects of the present disclosure relate to a bacterial or Agrobacterium cell comprising an expression vector or an isolated DNA molecule according to any of the previous embodiments.

[0136] An additional aspect of the present disclosure relates to a transgenic plant, plant part, plant cell, or seed comprising an expression vector or isolated DNA molecule according to any of the previous embodiments.

[0137] A further aspect of the present disclosure relates to a kit comprising an expression vector or an isolated DNA molecule according to any of the previous embodiments, or a bacterial cell or an Agrobacterium cell according to the previous embodiments.

[0138] Further aspects of the present disclosure relate to methods for increasing nitrogen fixation, delaying root nodule senescence, or inducing FUN filament formation in plants, comprising: (a) introducing a genetic modification via an expression vector or isolated DNA molecule described in any of the preceding embodiments; and, optionally, (b) treating the plant with zinc or manganese or cultivating the plant under high-zinc, high-manganese, or high-nitrate conditions. The high-zinc, high-manganese, or high-nitrate conditions may be higher than ambient conditions in the soil in which the plant is growing, higher than optimal conditions for plant growth, not natural or naturally occurring, or at an amount sufficient to induce or maintain FUN protein filament formation or aggregation. In some embodiments, the elevated levels may be 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 250%, or 500% higher than ambient conditions, optimal conditions, or naturally occurring amounts.

[0139] [Plant breeding method] Plant breeding begins with an analysis of current germplasm, a definition of problems and weaknesses in the current germplasm, setting program goals, and defining specific breeding objectives. The next step is the selection of germplasm with traits that will achieve the program goals. The selected germplasm is crossed to recombine the desired traits, and through further selection, varieties or parent lines are developed. The goal is to integrate an improved combination of desirable traits from the parent germplasm into a single variety or hybrid. These important traits may include increased yield, field performance, improved fruit or agronomic quality, resistance to biological stresses such as disease and pests, and tolerance to environmental stresses such as drought and heat.

[0140] Every breeding program should include periodic, objective evaluation of the efficiency of breeding procedures. Evaluation criteria vary depending on goals and objectives, but should include gains per year from selection compared to appropriate standards, the overall value of advanced breeding lines, and the number of successful cultivars produced per unit of input (e.g., per year, per cost, etc.). Promising advanced breeding lines are thoroughly tested for at least three years in an environment representative of the commercial target area and compared to appropriate standards. The best lines become candidates for new commercial cultivars, and lines still lacking some traits are used as parents to produce new populations for further selection. These processes lead to the final stages of marketing and distribution, which typically take five to ten years after the initial crossing or selection.

[0141] The choice of breeding or selection method depends on the plant's breeding system, the heritability of the trait being improved, and the type of variety used commercially (e.g., F1 hybrid variety, inbred variety, etc.). For highly heritable traits, selection of superior individual plants evaluated at a single location is effective, while for traits with low heritability, selection should be based on averages obtained from repeated evaluations of related plant lines. Genetic complexity also influences the choice of breeding method. Backcross breeding is used to transfer one or a few genes for highly heritable traits into a desirable variety (e.g., breeding for disease resistance), while recurrent selection techniques are used for quantitative traits controlled by multiple genes. A variety of recurrent selection techniques are used. Commonly used selection methods include pedigree selection, recombinant pedigree selection, mass selection, and recurrent selection.

[0142] Pedigree selection is commonly used to improve self-pollinated crops and inbred lines of cross-pollinated crops. Two parents with advantageous, complementary traits are crossed to produce the F1. The F2 population is created by selfing one or more F1s or by crossing two F1s (sib mating). Selection of the best individuals usually begins with the F2 population; the best individuals within the best lines are then selected from the F3 population. In the F4 generation, replicate testing of lines and hybrid combinations using individuals from these lines is often performed to improve the effectiveness of selection for traits with low heritability. At advanced stages of inbreeding (i.e., F6 and F7), the best phenotypically similar lines or blends thereof are tested for potential sale as new cultivars.

[0143] Mass and recurrent selection may be used to improve populations of self- or cross-pollinated crops. A genetically diverse population of heterozygous individuals is created or identified by crossing several different parents. The best plants are those that exhibit individual superiority, especially Selection is based on superior offspring or superior combining ability. The selected plants are then crossed to create new populations, and the cycle of selection continues.

[0144] Backcross breeding (i.e., recurrent selection) may be used to transfer genes for simply inherited, highly heritable traits into a desirable homozygous cultivar or line, the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plants are expected to possess the characteristics of the recurrent parent (e.g., cultivar) and the desired traits transferred from the donor parent. After the initial cross, individuals with the donor parent phenotype are selected and then repeatedly crossed (backcrossed) to the recurrent parent. The resulting plants are expected to possess the characteristics of the recurrent parent (e.g., cultivar) and the desired traits transferred from the donor parent.

[0145] In the strict sense, single seed inheritance refers to planting a segregating population, harvesting one seed sample from each plant, and using this one seed sample to plant the next generation.When the population advances to a desired inbreeding level from the F2 generation, the plants from which the lineage is derived are each derived from different F2 individuals.Because some seeds do not germinate or some plants do not produce at least one seed, the number of plants in the population decreases with each generation.As a result, as generations progress, not all of the F2 plants originally sampled in the parent population become offspring.

[0146] In addition to observing phenotypes, plant genotypes can also be examined. Many laboratory-based techniques are available for analyzing, comparing, and characterizing plant genotypes; these include isozyme electrophoresis, restriction fragment length polymorphisms (RFLPs), randomly amplified polymorphic DNA (RAPDs), arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence-characterized amplified regions (SCARs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs, also known as microsatellites), fluorescently tagged intersimple sequence repeats (ISSRs), single nucleotide polymorphisms (SNPs), genotyping by sequencing (GbS), and next-generation sequencing (NGS).

[0147] Molecular markers, or "markers," can also be used to select for qualitative traits during the breeding process. Markers that are closely linked to or contain sequences within the allele of interest can be used to select plants containing the allele of interest. The use of markers in the selection process is often referred to as genetic marker-enhanced selection or marker-assisted selection. Methods for performing marker analysis are generally known to those skilled in the art. Mutation breeding can also be used to introduce new traits into plant varieties. Spontaneous or artificially induced mutations can be a valuable resource for plant breeders. The goal of artificial mutagenesis is to increase the mutation rate for a desired trait. Mutation rates can be increased by many different means, including temperature, long-term seed storage, tissue culture conditions, radiation (e.g., X-rays, gamma rays, neutrons, beta rays, ultraviolet light), chemical mutagens (e.g., the base analog 5-bromouracil), antibiotics, alkylating agents (e.g., sulfur mustard, nitrogen mustard, epoxides, ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azides, hydroxylamines, nitrites, or acridines. Once a desired trait is observed through mutagenesis, it can be incorporated into existing genetic resources using conventional breeding techniques. Details of mutation breeding are described in Principles of Cultivar Development: Theory and Technique, Walter Fehr (1991), Agronomy Books, 1 (https: / / lib.dr.iastate.edu / agron_books / 1).

[0148] Diploid production is also used to develop homozygous lines in breeding programs. Diploids are generated by doubling the chromosome set from heterozygous plants to produce fully homozygous individuals. See, e.g., Wan, et al., Theor. Appl. Genet., 77:889-892, 1989.

[0149] Additional non-limiting examples of breeding methods that may be used include, but are not limited to, those described in Principles of Plant Breeding, John Wiley and Son, pp. 115-161 (1960); Principles of Cultivar Development: Theory and Technique, Walter Fehr (1991), Agronomy Books, 1 (https: / / lib.dr.iastate.edu / agron_books / 1), which are incorporated herein by reference.

[0150] [Molecular biological methods for producing genetically modified plant cells, plant parts and plants] One embodiment of the present disclosure provides a genetically modified or transgenic plant, or portion thereof, that contains one or more genetic modifications that result in reduced activity or expression of a FUN protein. Another embodiment of the present disclosure includes a genetically modified plant, or portion thereof, that contains one or more genetic modifications that reduce the activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein (also known as a FEZ protein), an HO1 protein, an NRT2.1 protein, or an AS1 protein.

[0151] FUN is a member of the TGA transcription factor family. TGA transcription factors are characterized by an N-terminal DNA-binding bZIP domain and a C-terminal DOG1 domain (Tomaz, S., Gruden, K. & Coll, A. TGA transcription factors—Structural characteristics as a basis for functional variability. Front. Plant Sci. 13, 935819 (2022)). This disclosure redefines the DOG1 domain as a sensor domain in L. japonicus FUN (SEQ ID NO:1), as this domain senses zinc (Figure 1P). FUN is highly conserved in legumes, as evidenced by the presence of both FUN and FUN-like paralogous proteins in the PAN ortholog group in all analyzed legumes (Figure 6A). Exemplary FUN homologs include SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. Exemplary FUN-like homologs include SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84. Additional TGA transcription factors related to the FUN protein are the FUN-like proteins.FUN-like proteins can be distinguished from FUN proteins by their lack of enhanced expression in root nodules (although there may be expression in roots in general, possibly including nodules), and by the fact that they form independent paralogous branches on the phylogenetic tree.

[0152] L. japonicus NAC094 (SEQ ID NO:31; also called FEZ protein) is a downstream target of FUN (Figure 7). Exemplary NAC094 homologs are SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73.

[0153] Transformation and production of genetically modified monocotyledonous and dicotyledonous plant cells are well known in the art. See, for example, Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); US Patent 5,679,558; Agrobacterium Protocols, ed.: Gartland, Humana Press Inc. (1995); Wang, et al. Acta Hort. 461:401-408 (1998), and Broothaerts, et al. Nature 433:629-633 (2005). The choice of method will depend on the type of plant to be transformed, the particular application, and / or the desired results. One skilled in the art can easily select an appropriate transformation technique.

[0154] Methods known in the art for deleting, inserting, or otherwise modifying cellular DNA (e.g., genomic and organelle DNA) can be used in practicing the compositions, methods, and processes described herein. For example, CRISPR / Cas-9 systems and related systems (e.g., TALENs, ZFNs, ODNs, etc.) can be used to insert heterologous genes into target sites in genomic DNA, or to subsequently edit endogenous genes to express heterologous genes, or to modify promoters to increase or alter expression of endogenous genes, for example, by removing repressor binding sites or introducing enhancer binding sites. For example, in Agrobacterium tumefaciens, a disarmed Ti plasmid containing a genetic construct for deleting or inserting a target gene can be used to transform plant cells, and then transformed plants can be regenerated from the transformed plant cells using procedures described in the art. For example, as described in EP 0116718, EP 0270822, WO 84 / 02913 and European Patent Application EP 0242246. Ti plasmid vectors each contain the gene between the border sequences or at least to the left of the right border sequence of the T-DNA of the Ti plasmid.Of course, other types of vectors can be used in procedures such as direct gene transfer (e.g., as described in EP 0233247), pollen-mediated transformation (e.g., as described in EP 0270356, WO 85 / 01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (e.g., as described in EP 0067553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Pat. No. 4,536,475), and in the transformation of maize (e.g., U.S. Pat. No. 6,140,553; Fromm et al., Bio / Technology (1990) 8, 833-839; Gordon-Kamm et al., The Plant Cell, (1990) 2, 603-618), rice (Shimamoto et al., Nature, (1989) 338, 274-276; Datta et al., Other methods can be used to transform plant cells, such as the method for transforming specific lines of (Bio / Technology, (1990) 8, 736-740) and general methods for transforming monocotyledonous plants (WO 92 / 09696). For cotton transformation, the method described in WO 00 / 71733 can be used. For soybean transformation, see methods known in the art, such as those described by Hinchee et al. (Bio / Technology, (1988) 6, 915) and Christou et al. (Trends Biotech, (1990) 8, 145), or WO 00 / 42207.

[0155] The genetically modified plants of the present disclosure can be used in conventional plant breeding systems to further produce genetically modified plants with the same characteristics or to introduce the genetic modification into other varieties of the same or related plant species. Seeds obtained from the modified plants preferably contain the genetic modification as a stable insertion in chromosomal DNA or as a recombinant relative to an endogenous gene or promoter. Plants containing the genetic modification according to the present disclosure include plants that contain or are derived from a plant rootstock containing the genetic modification of the present disclosure, such as fruit trees and ornamental plants. Thus, non-transgenic grafted plant parts inserted into a transformed plant or plant part are also included in the present disclosure.

[0156] The genetic modifications of the present disclosure contained within expression vectors or expression cassettes that result in expression of introduced genes or altered expression of endogenous genes generally utilize plant-expressible promoters. As used herein, the term 'plant-expressible promoter' refers to a promoter that ensures expression of the genetic modifications of the present disclosure in plant cells. Examples of constitutive promoters frequently used in plant cells include the cauliflower mosaic virus (CaMV) 35S promoter (Kay et al. Science, 236, 4805, 1987), the minimal CaMV 35S promoter (Benfey & Chua, Science, (1990) 250, 959-966), various other derivatives of the CaMV 35S promoter, the fig mosaic virus (FMV) promoter (Richins, et al., Nucleic Acids Res. (1987) 15:8451-8466), the maize ubiquitin promoter (Christensen & Quail, Transgenic Res. 5, 213-8, 1996), and the polyubiquitin promoter (Ljubql, Maekawa et al. Mol Plant Microbe Interact. 21, 375-82, 2008), the vein mosaic cassava virus promoter (WO 97 / 48819), and the Arabidopsis UBQ10 promoter (Norris et al. Plant Mol. Biol. 21, 895-906, 1993).

[0157] Additional examples of promoters that confer constitutive expression in plants are known in the art, and include: The strong constitutive 35S promoter of cauliflower mosaic virus (CaMV) ("35S promoter", e.g., the CM1841 isolate (Gardner et al., Nucleic Acids Res, (1981) 9, 2871-2887), CabbB S (Franck et al., Cell (1980) 21, 285-294), and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter (Christensen et al., Plant Mol Biol, (1992) 18, 675-689), the gos2 promoter (de Pater et al., The Plant J (1992) 2, 834-844), the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581-588), actin promoters such as those described by An et al. (The Plant J, (1996) 10, 107), rice actin promoters such as those described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); fig mosaic virus (FMV) promoter (Richins, et al., Nucleic Acids Res. (1987) 15:8451-8466), cassava vein mosaic virus promoter (WO 97 / 48819, Verdaguer et al. (Plant Mol Biol, (1998) 37, 1055-1067), subterranean clover ( clovers) stunt virus-derived promoters, such as the pPLEX series (WO 96 / 06932, especially the S4 promoter or S7 promoter), alcohol dehydrogenase promoters, such as pAdh1S (GenBank accession numbers: X04049, X00581), and T-DNA (Velten et al., EMBO J, (1984) 3, 2723 2730) and the TR1' promoter and TR2' promoter ("TR1' promoter" and "TR2' promoter", respectively) that drive the expression of the 1' and 2' genes of the ribosomal RNA. Includes:

[0158] Alternatively, the plant-expressible promoter may be a tissue-specific promoter, i.e., a promoter that confers high expression levels in certain cells or tissues of the plant, such as root epidermal cells or root cortex cells. In a preferred embodiment, the LysM receptor promoter is used. Non-limiting examples include the NFR1 promoter, NFR5 promoter, LYK3 promoter, NFP promoter, Lotus japonicus NFR5 promoter (SEQ ID NO: 27), Lotus japonicus NFR1 promoter (SEQ ID NO: 27), Medicago truncatula NFP promoter (SEQ ID NO: 29), Lotus japonicus CERK6 promoter (SEQ ID NO: 46), and Medicago truncatula LYK3 promoter (SEQ ID NO: 28). In an additional preferred embodiment, a root-specific promoter is used. Non-limiting examples include the maize metallothionein promoter (De Framond et al, FEBS 290, 103-106, 1991 Application EP 452269), chitinase promoter (Samac et al. Plant Physiol 93, 907-914, 1990), glutamine synthetase soybean root promoter (Hirel et al. Plant Mol. Biol. 20, 207-218, 1992), RCC3 promoter (PCT Application WO 2009 / 016104), rice antiquitin promoter (PCT Application WO 2007 / 076115), LRR receptor kinase promoter (PCT application WO 02 / 46439), maize ZRP2 promoter (US Pat. No. 5,633,363), tomato LeExtl promoter (Bucher et al. Plant Physiol. 128, 911-923, 2002), and the Arabidopsis pCO2 promoter (Heidstra et al., Genes Dev. 18, 1964-1969, 2004).These plant promoters may be combined with enhancer elements, may be combined with minimal promoter elements, or may contain repetitive elements to ensure expression in the desired expression profile.

[0159] Examples of constitutive promoters often used in plant cells are the cauliflower mosaic (CaMV) 35S promoter (Kay et al. Science, 236, 4805, 1987) and various other derivatives of the promoter, the vein mosaic cassava virus promoter (WO 97 / 48819; Christensen & Quail, Transgenic Res, 5, 213-8, 1996), polyubiquitin (Ljubql, Maekawa et al. Mol Plant Microbe Interact. 21, 375-82, 2008) and Arabidopsis thaliana UBQ10 (Norris et al. Plant Mol. Biol. 21, 895-906, 1993).

[0160] In some embodiments, genetic modifications can be used to increase expression in plant cells, such as introns at the 5' or 3' end of the transgene or within the coding sequence of the transgene (e.g., the hsp70 intron). Other such genetic elements can include, but are not limited to, promoter-enhancer elements, duplicated or tripled promoter regions, 5' leader sequences that differ from the leader sequences of other transgenes or endogenous (plant host) genes, and 3' trailer sequences that differ from the trailer sequences of other transgenes or endogenous (plant host) genes in the same plant.

[0161] A transgene of the present disclosure can be introduced such that the inserted gene portion is upstream (i.e., 5') of an appropriate 3'-terminal transcriptional regulatory signal (i.e., transcript formation and polyadenylation signal). This is preferably accomplished by inserting the gene into the genome (nucleus or chloroplast) of the plant cell. Preferred polyadenylation and transcript formation signals include those from the nopaline synthase gene (Depicker et al., J. Molec Appl Gen, (1982) 1, 561-573), octopine synthase gene (Gielen et al., EMBO J, (1984) 3:835-845), SCSV or malic enzyme terminator (Schunmann et al., Plant Funct Biol, (2003) 30:453-460), and T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981-6998), which act as 3' non-translated DNA sequences in transformed plant cells. In some embodiments, one or more transgenes are stably integrated into the nuclear genome. Stable integration occurs when the nucleic acid sequence remains integrated into the nuclear genome and continues to be expressed (i.e., detectable mRNA transcripts or proteins are produced) through subsequent plant generations. Stable integration into the nuclear genome can be accomplished by any method known in the art (e.g., particle bombardment, Agrobacterium-mediated transformation, CRISPR / Cas9, electroporation of protoplasts, microinjection, etc.).

[0162] The term "recombinant nucleic acid" or "recombinant nucleic acid" refers to a polynucleotide created by combining two segments of sequence. This combination is accomplished by the artificial manipulation of isolated segments of polynucleotides, either by genetic engineering techniques or chemical synthesis. In doing so, desired functional polynucleotide segments can be joined to potentially create a desired combination of functions.

[0163] As used herein, the term "overexpression" refers to increased expression (e.g., expression of mRNA, polypeptides, etc.) relative to expression in a wild-type organism (e.g., a plant) as a result of genetic modification, and can refer to expression of a heterologous gene at a level sufficient to achieve a desired result, such as increased yield. In some embodiments, the increase in expression is a small increase of about 10% over wild-type expression. In some embodiments, the increase in expression is 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) over wild-type expression. In some embodiments, an endogenous gene is enhanced. In some embodiments, an exogenous gene is enhanced by its expression. Enhanced gene expression in plants can be achieved by any method known in the art, including, but not limited to, the use of constitutive promoters with added inducible response elements, inducible promoters, high expression promoters with added inducible response elements (e.g., the PsaD promoter), enhancers, transcriptional and / or translational regulatory sequences, codon optimization, recombinant transcription factors, and / or mutant or recombinant genes that control expression of the enhanced gene in response to stimuli such as cytokine signaling.

[0164] When a recombinant nucleic acid is designed for the expression, cloning, or replication of a specific sequence, the DNA construct prepared for introduction into a host cell typically contains a replication system (e.g., a vector) recognized by the host and carries a DNA fragment of interest encoding a desired polypeptide. The DNA construct may also contain transcriptional and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Furthermore, the construct may contain a cellular localization signal (e.g., a plasma membrane localization signal). In a preferred embodiment, these DNA constructs are introduced into the genomic DNA, chloroplast DNA, or mitochondrial DNA of the host cell.

[0165] In some embodiments, non-integrative expression systems can be used to induce expression of one or more transgenes. Expression systems (expression vectors) can include, for example, an origin of replication or an autonomously replicating sequence (ARS) and expression control sequences, promoters, enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilization sequences. Where appropriate, a signal peptide from a secreted polypeptide of the same or related species can also be included, allowing the protein to cross and / or remain in the cell membrane or cell wall, or be secreted extracellularly.

[0166] The selectable marker useful in carrying out the methods described herein can be a positive selectable marker. Typically, positive selection refers to when genetically modified cells can survive in the presence of a toxic substance as long as the recombinant polynucleotide of interest is present in the cells. Negative selectable markers and screening markers are also known in the art and are contemplated in the present disclosure. In carrying out the compositions, methods, and processes described herein, those skilled in the art will understand that any available relevant marker can be used.

[0167] Screening and molecular analysis of the recombinant strains of the present disclosure can be performed using nucleic acid hybridization techniques. Hybridization procedures aid in the identification of polynucleotides recombined using the techniques described herein that have sufficient homology to the regulatory sequences of interest to be useful as demonstrated herein. A specific hybridization technique is not essential to the present disclosure. Improvements in hybridization techniques are continually being made and can be readily utilized by those skilled in the art. Any suitable label known to those skilled in the art can be used to label the hybridization probe. Hybridization and wash conditions, such as temperature and salt concentration, can alter the stringency of detection. For more information regarding hybridization conditions, see, for example, Sambrook et al. (1989) vide infra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, NY.

[0168] In addition to screening and molecular analysis of genetically modified strains, the production of desired isolated nucleic acids can also be performed using polymerase chain reaction (PCR). PCR is a repetitive, enzymatic, primer-assisted synthesis of nucleic acid sequences. This procedure is known to those skilled in the art and is commonly used (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230:1350-1354). PCR is based on the enzymatic amplification of a target DNA fragment flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with their 3' ends facing each other. Repeated cycles of heat denaturation of the template, annealing of the primers to complementary sequences, and extension of the annealed primers by DNA polymerase result in the amplification of fragments defined by the 5' ends of the PCR primers. Because the extension product of each primer can serve as a template for other primers, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of a specific target fragment, reaching millions of times over the course of a few hours. The amplification process can be fully automated by using a thermostable DNA polymerase, such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Other enzymes that can be used are known to those skilled in the art.

[0169] The nucleic acids and proteins of the present disclosure may also encompass homologs of the specifically disclosed sequences. Homology (e.g., sequence identity) may be 50% to 100%. In some instances, such homology is greater than 80%, greater than 85%, greater than 90%, or greater than 95%. The degree of homology or identity required for any intended use of the sequences is readily identified by those of skill in the art. As used herein, the percent sequence identity of two nucleic acids is determined using algorithms known in the art, such as those disclosed in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 and recombined in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN, BLASTP, and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. To obtain nucleotide sequences with a desired percentage of sequence identity, BLAST nucleotide searches are performed using the BLASTN program, score = 100, and word length = 12. To obtain gapped alignments for comparison purposes, gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (BLASTN and BLASTX) are used. See www.ncbi.nih.gov. Those skilled in the art will appreciate that default settings (e.g., for BLASTP, gap opening penalty: 11, gap extension penalty: 1, expectation: 10, word size: 3, maximum score: 25, maximum alignment: 15, and matrix: blosum62) are used.By aligning a sequence of interest with a reference sequence using an appropriate BLAST program (for BLASTN, Gap Opening Penalty: 5, Gap Extension Penalty: 2, Nucleic Match: 1, Nucleic Mismatch: -3, Expectation: 10, Word Size: 11, Max Score: 25, and Max Alignment: 15), positions in the reference sequence that correspond to amino acids or nucleic acids can be easily identified.

[0170] Preferred host cells are plant cells. As used herein, a recombinant host cell is one that has been genetically modified to contain an isolated nucleic acid molecule, one that has one or more genes that are normally present and functional in the host cell deleted or rendered non-functional, or one that contains one or more genes and produces at least one recombinant protein. The nucleic acid encoding the protein of the present disclosure can be introduced by any means known to those skilled in the art that is appropriate for the particular cell type, including, but not limited to, transformation, lipofection, electroporation, or other methods known to those skilled in the art.

[0171] The terms "isolated," "isolated DNA molecule," or equivalent terms or phrases refer to a DNA molecule or other moiety that is present alone or in combination with other compositions but that has been altered from or is not present in its natural state. For example, nucleic acid elements such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, and the like that are present in the genomic DNA of an organism as found in nature are not considered "isolated" as long as they are present in the genome of the organism as found in nature and in the location within the genome. However, these elements, and portions of these elements, are considered "isolated" from their natural context within the scope of this disclosure unless they are present in the genome of the organism as found in nature, or they are altered from their natural form, or they are not present in the location within the genome as found in nature. Similarly, a nucleotide sequence that encodes a protein or a naturally occurring variant of that protein is an "isolated nucleotide sequence" if the nucleotide sequence was not present in the DNA of the organism as the sequence encoding the protein is found in that location in nature, or if the nucleotide sequence is altered from its natural form. A synthetic nucleotide sequence that encodes the amino acid sequence of a naturally occurring protein is considered "isolated" for purposes of this disclosure. For purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant, algae, fungus, or bacterial cell, or a nucleotide sequence present in an extrachromosomal vector, is considered an "isolated nucleotide sequence," regardless of whether it is present in a plasmid or similar structure used to transform the cell, in the genome of the plant or bacteria, or in detectable amounts in tissues, progeny, biological samples, or commercial products obtained from the plant or bacteria.

[0172] [plant] The compositions and methods described herein can be used with plants, plant cells, plant parts, or their progeny, such as plants capable of nodulation and plants with endogenous FUN (e.g., legumes). Legumes are plants belonging to the legume (Fabaceae) family and can be characterized by their ability to fix nitrogen in the soil through a symbiotic relationship with nitrogen-fixing bacteria in their root nodules. As such, the plants, plant cells, plant parts, or their progeny described herein can be selected from the group consisting of alfalfa, bambara beans, pulses (e.g., kidney beans, black beans, etc.), black currants, chickpeas, white clover, cowpeas, forage legumes, legume trees, lentils, lotus, lupine, Medicago, peas, peas, peanuts, pigeon peas, soybeans, Parasponia, alder, and elm.

[0173] Plants with identified FUN orthologs include Prunus persica (peach), Lotus japonicus (e.g., lotus grass or king laurel), Glycine max (soybean), Manihot esculenta (cassava), Gossypium raimondii (wild cotton), Eucalyptus grandis (e.g., flooded eucalyptus or rose eucalyptus), Brassica oleracea (wild cabbage; this species includes various cultivated forms such as broccoli, cauliflower, and cabbage), Arabidopsis thaliana (Arabidopsis), Solanum lycopersicum (tomato), Aquilegia coerulea (columbine), Amborella trichopoda, Spirodela polyrhiza (duckweed), Musa acuminata (banana), Zea mays (corn), and Setaria It may be Triticum italica (foxtail millet), Triticum Aestivum (wheat), Hordeum vulgare (barley), or Oryza sativa (rice).

[0174] Cover crops and cover crop combinations can be used to add nitrogen to soil and further benefit from the increased nitrogen content, thereby reducing the expression of FUN or its downstream targets. For example, the cover crop can be a legume. In some embodiments, the legume can be soybean, cowpea, clover (e.g., red clover, white clover, red clover, balansa clover, berseem clover, Egyptian clover, arrowleaf clover, ball clover, subterranean clovers), vetch (e.g., veronica japonica, alfalfa), or pea (e.g., Austrian winter pea, snow pea). As another example, the cover crop can be grass. In some embodiments, the grass can be rye (e.g., winter rye, cereal ryegrass, Italian ryegrass), triticale, fescue grass (e.g., orchard grass, meadow fescue grass), or sudan grass (e.g., sudan grass, sorghum-sudangrass hybrids), or alfalfa. As yet another example, the cover crop can be a Brassicaceae plant. In some embodiments, the Brassicaceae plant can be mustard (e.g., white mustard), radish (e.g., radish, oilseed radish), turnip (e.g., purple-necked turnip, fodder turnip), rapeseed (e.g., rapeseed), phacelia, sunflower, sunn hemp, kale, or a grain (e.g., oats, buckwheat, or millet (e.g., pearl millet)).

[0175] Having provided a general description of the compositions, methods, and processes in this disclosure, the disclosure will be better understood by reference to specific examples, which are included herein to further illustrate the disclosure and are not intended to limit the scope of the invention, which is defined by the claims.

[0176] [Example] The present disclosure will be described in more detail in the following examples. However, these examples are not intended to limit the scope of the present disclosure as described in the claims in any way. The accompanying drawings are created to be considered as an essential part of the specification and description of the present disclosure. The following examples are provided for illustrative purposes and do not limit the scope of the present disclosure as described in the claims.

[0177] Example 1: FUN regulates nitrate-mediated repression of nitrogen fixation. This example describes the identification of Fun genes as regulators of nitrogen fixation in Lotus japonicus and further describes experiments to characterize the expression patterns of Fun genes.

[0178] [material and method] (Mutation Screening) To identify mutants that maintain nitrogen fixation under nitrate-limiting conditions, the Lotus japonicus Gifu ecotype background was used in a forward genetic screen. Functional nodules were able to form before the application of limiting nitrate conditions, indicating that the screen clearly identified mutants with impaired regulation of nodule function. The characteristic color of nitrogen-fixing nodules (functional nodules are pink) was used to screen for mutants that maintain nodule function when watered with KNO3 for 2 weeks. Under these cultivation conditions, most nodules in wild-type plants turned green and senesced, whereas fixation under nitrate (fun) mutant plants continued to form pink nodules even under these high nitrate concentrations.

[0179] (Plant strains and cultivation conditions) The Lotus japonicus Gifu ecotype was used as the wild type (WT). LORE1 insertion mutants were ordered through LotusBase (lotus[dot]au[dot]dk), and homozygotes were isolated for phenotypic analysis and generation of higher mutants as described in (Emms, D.M. & Kelly, S. SHOOT: phylogenetic gene search and ortholog inference. Genome Biol. 23, 85 (2022)). The mutant lines Fun, Fun-2, Fun-3, and Fun-4 were tested, and the specific insertions generating the mutant genotypes are illustrated in Figure 1B. The line numbers and genotyping primers used are listed in Table 2 below.

[0180] [Table 2]

[0181] All plants were grown at 21°C under a 16-h light / 8-h dark regime. For germination, Lotus seeds were scratched with sandpaper and surface-sterilized with 1% sodium hypochlorite for 10 min. Seedlings were washed five times with sterile water and then germinated upright in sterile rectangular Petri dishes on moist filter paper (AGF 651; Frisenette ApS) at 21°C for 2 days. Seedlings were then transferred to a substrate mixture (Reca:vermiculite = 3:1).

[0182] (Mutant screening and sequence analysis) A pool of LORE1 mutants, each with a random LORE1 insertion in the genome, was germinated in a substrate mixture (Reca:vermiculite = 3:1) and inoculated with M. loti NZP2235. Four weeks after inoculation, the plants were treated with KNO3 for three weeks. Most nodules were green or black, and plants with pink nodules were isolated for rescreening in the next generation. DNA from mutant plants was isolated and flanking sequences of LORE1 were determined to identify the LORE1 insertion site, as previously described (Urbanski, D.F., Malolepszy, A., Stougaard, J. & Andersen, S.U. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J. 69, 731-741 (2012)).

[0183] (Strains and culture conditions) Chemically competent E. coli TOP10 (ThermoFisherScientific) was used for molecular cloning and grown in LB medium at 37°C.

[0184] For all hairy root transformation experiments, Agrobacterium rhizogenes strain AR1193 (Stougaard, J. Methods Mol Biol 1995 49:49-61) was used and cultured in LB medium at 28°C.

[0185] (Construction of plant expression vectors) To verify the function of the Fun gene, we constructed expression constructs expressing FUN fused to green fluorescent protein (GFP) under the control of the ubiquitin promoter (pUbi). These constructs were designated pUbi:FUN, proUbi:FUN-GFP, or FUN-GFP. For tobacco assays, we used the 35S promoter (pro35S), and the constructs were designated pro35S:FUN, pro35S:FUN-GFP, or FUN-GFP.

[0186] To study the expression pattern of the Fun gene in situ, the coding sequence of β-glucuronidase (GUS) was constructed in combination with the native Fun promoter sequence (proFUN) and the native Fun terminator sequence (tFUN), and the construct was designated proFUN:GUS.

[0187] (hairy root transformation) The pIV10 expression vector (Hansen, J. et al. Plant Cell Rep 1989 8: 12-15) was used for transformation of L. japonicus hairy roots. This expression vector contains a sequence encoding triple YFP fused to a nuclear localization signal (pIV10_tYFP-NLS), which served as a control for transformation. The Lotus ubiquitin promoter and 35S terminator were also cloned into the pIV10 expression vector.

[0188] L. japonicus seeds were wounded with sulfuric acid for 15 minutes, washed five times with ddH20, and spread on moist filter paper for germination. Three-day-old seedlings were transferred to square plates containing 1 / 2 solid B5 medium. A. rhizogenes strain AR1193 (Stougaard, 1987 #432) carrying the desired construct was cultured on LB agar medium containing ampicillin, rifampicin, and spectinomycin for two days. For each construct, cells grown on one plate were resuspended in 4 ml YMB medium. The bacterial suspension was then used to transform the hypocotyls of 6-day-old seedlings using a 1 ml syringe with a Sterican® needle (0.40 x 20 mm). A hole was pierced in the hypocotyl and a drop of the solution was placed on the wound. The square plates containing transformed seedlings were sealed and placed in the dark for 2 days before being transferred to a 16-h / 8-h light / dark regime at 21°C. After 3 weeks, untransformed roots were removed and the seedlings were transferred to the substrate mixture described above or 1 / 4x B&D plates. After transformation, plants were inoculated with rhizobia and fed nitrate as described above. All plants were grown under a 16-h / 8-h light / dark regime at 21°C.

[0189] (Nodulation assay) After 2 weeks of exposure to 10 mM KNO, the number of pink functional nodules per plant and total nodule counts were taken. Photographs were taken using a Leica M165FC fluorescence stereo microscope equipped with a Leica DFC310 FX digital color camera. Mean values ​​between treatment groups were compared using ANOVA and Tukey's post-hoc test.

[0190] (Nitrogen fixation assay) As previously described in (Reid, DE, Heckmann, AB, Novak, O., Kelly, S. & Stougaard, J. Cytokinin oxide / dehydrogenase3 maintains cytokinin homeostasis during root and nodule development in Lotus japonicus. Plant Physiol. 170, 1060-1074 (2016)), nitrogen fixation activity was quantified using the acetylene reduction assay (ARA), which measures the amount of acetylene (C2H2) reduced to ethylene (C2H4) per plant per hour in Lotus spp. after 2 weeks of exposure to 10 mM KNO3. Nodulated roots from a single plant were placed in a 5 ml glass GC vial. 500 μl of air in the vial was replaced with 2% acetylene using a syringe. Samples were incubated for 30 min at room temperature, after which ethylene was quantified using a SensorSense (Nijmegen, NL) ETD-300 ethylene detector at a flow rate of 2.5 L / h in sample mode and a detection time of 6 min. Mean values ​​between treatment groups were compared using ANOVA and Tukey's post-hoc test.

[0191] (Leghemoglobin content assay) Leghemoglobin content was measured after 2 weeks of exposure to 10 mM KNO3, as previously described (Du, M., Gao, Z., Li, X., and Liao, H. (2020). Excess nitrate induces nodule greening and reduces transcript and protein expression levels of soybean leghemoglobin. Ann. Bot. 126: 61-72). Fresh root nodules from each plant were first crushed and homogenized in 16 volumes of pre-chilled 0.1 M PBS (NaHPO4-NaHPO4 buffer, 5°C, pH 6.8). The resulting slurry was then centrifuged at 12,000 g for 15 minutes, and the supernatant was measured spectrophotometrically at wavelengths of 540, 520, and 560 nm. Leghemoglobin content was calculated from a standard curve using bovine hemoglobin as a protein standard. Mean values ​​between treatment groups were compared using ANOVA and Tukey's post-hoc test.

[0192] (GUS staining) Three weeks after inoculation, hairy roots were placed in GUS staining buffer containing 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 100 mM potassium phosphate buffer (pH 7.0), 10 mM EDTA (pH 8.0), 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, and 0.1% Triton X-100. The roots were incubated overnight at 37°C. Roots were washed twice with 70% ethanol before imaging.

[0193] (statistical analysis) Mean values ​​between treatment groups were compared using ANOVA and Tukey's post-hoc test.

[0194] [result] (FUN controls the inhibition of nitrogen fixation by nitrate) To identify environmental regulators of nodule formation, we envisioned that applying restrictive conditions after functional nodules had formed would allow us to identify mutants with specific defects in regulating nodule function. Taking advantage of the pink color characteristic of nitrogen-fixing nodules, which differ from green senescent nodules, we screened a population of LORE1 (Fukai, E. et al. Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1. Plant J. 69, 720-730 (2012); Urbanski, D.F., Malolepszy, A., Stougaard, J. & Andersen, S.U. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J. 69, 731-741 (2012); Malolepszy, A. et al. The LORE1 insertion mutant resource. Plant J. 88, 306-317 (2016)) insertion mutants in the model legume Lotus japonicus (Lotus) to identify genotypes that maintained nodule function even under repressive nitrate conditions. We identified a mutant that retained a higher number of pink nodules compared to the wild-type (WT) and named it fixation under nitrate (fun) (Figures 1A and 1C). Additional LORE1 insertion mutants were also identified (Figure 1B). We examined the amount of functional pink nodules and total nodule number per plant in Lotus plants carrying the fun mutation compared to WT (Gifu) Lotus plants. We found that fun, fun-2, fun-3, and fun-4 plants retained significantly more pink nodules than WT plants (Figures 1C and 1F).

[0195] The functionality of these pink nodules was confirmed by increased nitrogen fixation rates as measured by the acetylene reduction assay (ARA) (Figures 1D, 1E, and 1G). Furthermore, Lotus plants with the fun mutation exhibited increased leghemoglobin content compared to wild-type (WT) Lotus plants (Figure 1K). Analysis of FUN transcript abundance from an available expression atlas for Lotus japonicus Gifu (Kamal et al., DNA Res. 7 (2020)) showed significantly increased expression of FUN activity in the nodules where nitrogen fixation occurs (Figure 1Q).

[0196] (Structure of FUN protein) A LORE1 retrotransposon insertion was identified in the promoter region of a bZIP transcription factor, designated FUN, as described above. Subsequently, additional LORE1 insertions were identified within the promoter and FUN gene regions, as shown in Figure 1B. The FUN gene encodes a protein belonging to the TGA family of transcription factors, and this protein was found to share the highest similarity with the Arabidopsis PERIANTHIA (PAN) transcription factor (Running, MP & Meyerowitz, E.M. Mutations in the PERIANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern. Development 122, 1261-1269 (1996); Maier, A.T., Stehling-Sun, S., Offenburger, S.-L. & Lohmann, J.U. The bZIP Transcription Factor PERIANTHIA: A Multifunctional Hub for Meristem Control. Front. Plant Sci. 2, 79 (2011)). The TGA family belongs to group D bZIP transcription factors (Droge-Laser, W., Snoek, BL, Snel, B. & Weiste, C. The Arabidopsis bZIP transcription factor family—an update. Curr. Opin. Plant Biol. 45, 36-49 (2018)) and is characterized by the presence of an N-terminal basic-leucine zipper (bZIP) DNA-binding domain and a C-terminal DOG1 domain of unknown function (Tomaz, S., Gruden, K. & Coll, A. TGA transcription factors—Structural characteristics as a basis for functional variability. Front. Plant Sci. 13, 935819 (2022)).As explained in detail below, the DOG1 domain is referred to as the sensor domain of FUN. The protein structure of FUN is shown in Figure 1P, depicting the bZIP domain and the sensor domain.

[0197] (Fun is specifically expressed in root nodules) In Lotus spp., Fun transcripts were detected at high levels in root nodules (Figure 1Q), and promoter activity was evident in the nodules (Figures 1M, 1N, and 1O). To assess Fun expression in situ, a GUS transcriptional reporter construct driven by the native Fun promoter was transformed into wild-type (Gifu) Lotus plants (Figures 1M-1O). After GUS staining of nodule-bearing Lotus roots, Fun was found to be expressed exclusively in the nodules (Figure 1M). Stained root nodules were sectioned and photographed under a light microscope to confirm the cellular localization of Fun expression. The results indicated that Fun was predominantly expressed in uninfected cells of the nodules (Figures 1N-1O).

[0198] (Complementation of Lotus plants carrying fun mutations) FUN was validated as a causative gene by complementing the fun mutation with constitutively expressed FUN (Figure 1L) and by confirming that the nodulation phenotype remained unchanged in three independent LORE1 mutant alleles that reduced gene expression by promoter insertions (fun and fun-4) or disrupted function by exon insertions (fun-3) (Figures 1A and 1E-1G). The intron-inserted allele (fun-2) showed no impairment compared to the wild type (Figures 1F-1G). Regulation of FUN was restricted to mature, functional nodules; application of nitrate prior to inoculation suppressed nodule formation in fun mutant plants to the same extent as in wild-type plants (Figures 1H-1J). Furthermore, cloning the Fun promoter together with the Fun genomic sequence demonstrated that it was sufficient to complement the fun mutation (data not shown).

[0199] These results suggest that the Fun gene is specifically expressed in root nodules and regulates the repression of nitrogen fixation by nitrate.

[0200] Example 2: FUN initiates nodule senescence via multiple pathways. This example describes the identification of FUN binding sites in FUN target genes and in their promoters, and describes the phenotypes of loss-of-function mutants of FUN targets in the genus Lotus.

[0201] [material and method] (Gene Expression) For RNA-seq analysis, 3 weeks after inoculation, plants were acclimated by overnight immersion in 1 / 4 Long Ashton liquid medium before treatment and then treated with 0 or 10 mM potassium nitrate (KNO3) for 24 hours. Mature nodules were harvested. mRNA was extracted using the NucleoSpin RNA Plant kit (Macherey-Nagel), and RNA sequencing (PE-150 bp Illumina sequencing) was performed by Novogene. RNA-seq analysis was performed by mapping reads to reference transcripts using Salmon 39 and quantification using DESeq2 (Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)). Published time series data for nitrate-treated root nodules (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896) were obtained from GEO (Gene Expression Omnibus) using accession number GSE197362. GO enrichment analysis was performed using GO_MWU with GO terms obtained from lotus[dot]au[dot]dk.

[0202] For target gene expression, RevertAid Reverse Transcriptase (Thermo) was used to synthesize first-strand cDNA. For qRT-PCR, a LightCycler 480 instrument and LightCycler 480 SYBR Green I master (Roche Diagnostics) were used. Ubiquitin-conjugating enzyme was used as a reference. Target gene cDNA concentrations were calculated using LinRegPCR amplicon PCR efficiency calculations (Ramakers, C., Ruijter, JM, Deprez, RHL & Moorman, AFM. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62-66 (2003)). Target genes were compared with the reference for five biological replicates (each consisting of only 8–10 nodules). At least two technical replicates were performed for each analysis. The primers used are listed in Table 3 below.

[0203] [Table 3]

[0204] (Plant strains and cultivation conditions) The plant strains and cultivation conditions were as described in Example 1.

[0205] (Strains and culture conditions) The strain and culture conditions were as described in Example 1.

[0206] (hairy root transformation) Hairy root transformation was as described in Example 1.

[0207] (Nodulation assay) The nodulation assay was as described in Example 1.

[0208] (Nitrogen fixation assay) Nitrogen fixation assays were performed as described in Example 1.

[0209] (Leghemoglobin content assay) Leghemoglobin content assay was as described in Example 1.

[0210] (Electrophoretic mobility shift assay (EMSA)) The 5'-terminal 6-FAM-labeled DNA probes were synthesized by Eurofins and are shown in Table 4. The purified FUN DNA-binding domain (residues 178–237) was incubated with the probe in EMSA buffer (25 mM Tris-HCl pH 8.0, 80 mM NaCl, 35 mM KCl, 5 mM MgCl) at 37°C for 60 min. After incubation, the reaction mixture was electrophoresed on a 6% native polyacrylamide gel, and the labeled DNA was then detected using a Typhoon scanner (Fujifilm). The probe without the 6-FAM label served as the competitor, and the probe with a mutation in the core binding site (TGACG) served as the mutant.

[0211] [Table 4]

[0212] (Transient activation assay) The promoters of candidate FUN target genes (NRT2.1, HO1, NAC094, NRT3.1, and AS1), the glucuronidase (GUS) CDS, and the 35S terminator were cloned into compatible Golden Gate vectors as reporters; whereas, the 35S promoter, FUN CDS, eGFP, and the 35S terminator were cloned as effectors. The reporter and effector constructs were then cloned into the p50507 Golden Gate binary vector. These constructs were then transformed into A. tumefaciens strain AGL1. These A. tumefaciens strains were grown at OD .600 The solution was diluted to 0.2 and infiltrated into N. benthamiana leaves. Three days after infiltration, approximately 20 mg of sample was collected for protein extraction. GUS activity was measured using a Thermo Scientific Varioskan flash with 4-methylumbelliferyl-β-D-glucuronide (Sigma-Aldrich) as the substrate. For Zn treatment, two days after A. tumefaciens infiltration, N. benthamiana leaves were infiltrated with 500 μM MgCl2 (mock), 500 μM ZnCl2, or 2.5 mM EDTA. GUS activity was measured one day after treatment.

[0213] [result] FUN is the master regulator of nodule senescence. Because FUN is a transcriptional regulator, we performed RNA-seq analysis to explore gene targets that may directly regulate nitrate signaling or nodule function. RNA-seq analysis identified 587 genes with a ≥2-fold change in expression in WT nodules exposed to nitrate. Comparison with the fun mutant revealed that 106 of these genes were differentially regulated in fun nodules (Figures 1R–1T). RNA-seq analysis identified multiple downstream targets of FUN, some of which were upregulated and others downregulated. GO-MWU analysis detected multiple gene ontology clusters within both upregulated and downregulated gene clusters (Nielsen, R. et al. A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol. 3, e170 (2005)) (Figure 1U). Among these, 29 genes with increased expression levels and 22 genes with decreased expression levels predicted as FUN targets are shown in Tables 5A and 5B below.

[0214] [Table 5A]

[0215] [Table 5B]

[0216] From this long list, six genes with elevated expression were selected for further investigation. These six genes are shown in Table 6 below. Particularly noteworthy among these genes were the heme oxygenase HO1, which degrades leghemoglobin during nodule senescence; the nitrate transporter NRT3.1; and asparagine synthase 1 (AS1), which is important for nitrogen absorption (Figure 1V). In addition, numerous putative TGA-type binding motifs (TGACG; Bartlett, A. et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat. Protoc. 12, 1659-1672 (2017)) were identified in the promoter regions of two genes that, when mutated, exhibit a phenotype similar to fun: the nitrate transporter NRT2.1 (Misawa, F. et al. Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus. Plant Cell 34, 1844-1862 (2022)), and the NAC transcription factor NAC094, which causes nodule senescence (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896).

[0217] [Table 6]

[0218] To verify whether the expression of these genes is regulated by FUN, we examined their relative expression levels in fun mutant nodules after nitrate treatment. qRT-PCR (Figures 2A-2B) and RNA-seq analysis (Figure 2S) revealed that nitrate induction of all these genes was attenuated in fun mutants. While FUN was coexpressed with NAC094 and HO1 in uninfected cells, nitrate regulation of NAC094, which also occurred in infected cells (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896), may require additional regulatory factors.

[0219] The promoter region of Nrt2.1 with four putative FBSs (P1–P4), the promoter region of Ho1 with two FBSs (P1 and P2), the promoter region of NAC094 with one FBS (P1), the promoter region of Nrt3.1 with three FBSs (P1, P2, and P3), and the promoter region of AS1 with one FBS (P1) are all shown in Figure 2C. EMSA was performed to examine whether the FUN DNA-binding domain binds to probes representing FBSs. As shown in Figure 2D, the FUN DNA-binding domain bound to P1 and P4 of the Nrt2.1 promoter, P1 and P2 of the Ho1 promoter, and P1 of the NAC094 promoter. As shown in Figure 2E, the FUN DNA-binding domain also bound to P1, P2, and P3 of the Nrt3.1 promoter, and P1 of the AS1 promoter. A competition assay using excess unlabeled probe demonstrated the specificity of this interaction (Figure 2F).

[0220] To verify the in vivo validity of the EMSA binding results, transient activation experiments were performed on the NRT2.1, HO1, NAC094, NRT3.1, and AS1 promoters in N. benthamiana. In this system, all of these promoters linked to the GUS reporter were significantly induced by FUN. A FUN-GFP construct was expressed as the effector, and promoter-driven GUS was expressed as the reporter. As shown in Figure 2G, in this system, the NRT2.1, HO1, and NAC094 promoters linked to the GUS reporter were significantly induced by FUN. Similar effects were observed with Nrt3.1 and AS1 (Figure 2H).

[0221] Further supporting the notion that FUN is a master regulator controlling these pathways, the resulting mutants in nrt2.1, ho1, and nac094 exhibited nodule phenotypes similar to the original FUN mutants, including enhanced nitrogen fixation and leghemoglobin content. Figure 2I shows the nodule phenotypes of nrt2.1-3, ho1-4, and nac094-3 mutants compared to WT. Figures 2J–2L show the results of nodule number, ARA activity, and leghemoglobin content assays for nrt2.1-3 and nrt2.1-4 mutants compared to WT. Figures 2M–2Q show the results of nodule number, ARA activity, and leghemoglobin content assays for ho1-4 and ho1-5 mutants, and nac094-3 and nac094-4 mutants compared to WT. Figure 2R shows the results of ARA activity assays for nac094-3 and nac094-4 mutants compared to WT. Taken together, these data indicate that FUN targets nodule senescence and the nitrate signaling pathway to regulate nodule function in relation to the environment. Thus, FUN regulation of the nitrate signaling pathway may play a role in altering the sensitivity of nodules to nitrate compared with other root tissues.

[0222] Example 3: The oligomeric state of FUN is regulated by zinc. This example describes the structural characterization of the FUN sensor domain and experiments evaluating the contribution of manganese and zinc as ligands for the sensor domain.

[0223] [material and method] (Protein production and purification) The FUN sensor domain (residues 244-480) with a 3C-cleavable N-terminal tag consisting of 10 histidines, 7 arginines, and a SUMO tag was ordered from GenScript, along with a FUN sensor construct with a zipper domain (residues 178-480) tagged at the N-terminus with 7 histidines and a GB1 tag. This plasmid was transformed into E. coli LOBSTR cells (Andersen, KR, Leksa, NC & Schwartz, TU. Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81, 1857-1861 (2013)). Expression cultures were grown in LB medium supplemented with 0.1 mg / mL ampicillin and 0.034 mg / mL chloramphenicol at OD . 600Cells were grown at 37°C and 110 rpm until a pH of 0.6 was reached. Cells were cold-shocked on ice for 30 min before induction with 0.4 mM IPTG overnight at 18°C. The cells were pelleted (4400 g, 4°C, 10 min), resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine), and disrupted by sonication. The lysate was clarified by centrifugation (30600 g, 4°C, 30 min), and protein was purified from the clarified lysate using a Protino Ni-NTA 5 mL column (Machery-Nagel). The protein was eluted with a high-imidazole buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5% glycerol, 500 mM imidazole, 5 mM β-mercaptoethanol). The zippered FUN sensor was not further purified. The FUN sensor was dialyzed overnight against 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5% glycerol, and 5 mM β-mercaptoethanol with 3C protease at a 1:50 molar ratio. The cleaved tag and protease were then removed by a second Ni-IMAC step. The FUN sensor was further purified by SEC using a Superdex 200 Increase 10 / 300 GL (GE Healthcare) in minimal buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol).

[0224] For SAXS analysis, the FUN sensor was further purified using a ResourceQ 1 mL column (GE Healthcare) and eluted with a linear gradient of 10–500 mM NaCl and 10 mM Tris-HCl pH 8.0 with 5 mM β-mercaptoethanol. The eluted fractions were pooled and dialyzed against minimal buffer.

[0225] (Dynamic Light Scattering (DLS) and Nano Differential Scanning Fluorometry (nanoDSF) Analysis) FUN protein was analyzed for changes in thermal unfolding (nanoDSF) and size (DLS) upon ligand addition using a Prometheus Panta instrument (NanoTemper Technologies). 0.8 mg / mL purified protein was incubated with 4 mM of different potential ligands or a 0-4 mM ZnCl2 series for 20 minutes, followed by the addition of 5 mM EDTA to analyze reversible filament formation. Before addition, ZnCl2 was filtered through a VivaSpin 5 kDa MWCO and immediately added to the protein sample. For each sample, 10 consecutive DLS measurements were performed at 25°C with 100% laser power, followed by a nanoDSF experiment using a temperature ramp from 25°C to 90°C at 1°C / min with 100% excitation power. All measurements were performed in triplicate.

[0226] (Small angle X-ray scattering (SAXS) analysis) SAXS measurements were performed on a NanoSTAR instrument (Pedersen, J.S. A. flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J. Appl. Crystallogr. 37, 369-380 (2004); Lyngso, J. & Pedersen, J.S. A. high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering. J. Appl. Crystallogr. 54, 295-305 (2021)). The instrument uses a Cu rotating anode, a scattering-free pinhole in front of the sample, and a two-dimensional position-sensitive gas detector (Vantec 500, Bruker AXS). Sample and buffer were measured with a homemade flow-through capillary. The scattering intensity I(q) is expressed as a function of the magnitude of the scattering vector, Q (and Q = 4π sin(2θ) / λ), where 2θ is the scattering angle and λ is the X-ray wavelength. The scattering of the buffer was subtracted from the scattering of the sample, and the intensities were converted to an absolute scale. Also, variations in detector efficiency were corrected by normalizing based on the scattering of pure water (Pedersen, J.S. A. flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J. Appl. Crystallogr. 37, 369-380 (2004)). The data were analyzed by Guinier analysis, ln(I(q)) = q 2 Plot against the radius of gyration R gwas determined, and an indirect Fourier transform (IFT) was performed (Glatter, O. A new method for the evaluation of small-angle scattering data. J. Appl. Crystallogr. 10, 415-421 (1977); Pedersen, J. S., Hansen, S. & Bauer, R. The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering. Eur. Biophys. J. 22, 379-389 (1994)) to obtain the pair distance distribution function p(r). This p(r) is a histogram of interpair distances between points within a particle, weighted by the excess scattering length density. Note that the resolution of the SAXS data is approximately 400 Å, so the total length of the zinc-induced fibrils cannot be resolved. In this case, the p(r) function is related to the cross-sectional structure of the filaments.

[0227] (negative staining electron microscopy) For electron microscopy, 0.1 mg / mL of purified FUN sensor domain was incubated with or without 100 μM ZnCl2 and with or without 5 mM EDTA at room temperature for 20 minutes. Negatively stained samples were prepared on 400-mesh copper grids with a carbon-coated collodion support membrane using a Leica EM SCD 500 High Vacuum Sputter Coater. Prior to staining, the grids were glow-discharged for 45 seconds at 25 mA negative polarity using a PELCO easiGlow glow discharge system. 3 μL of FUN sensor was placed on the grid, incubated for 30 seconds, and then removed with Whatman paper. After blotting, the grids were floated three times in 2% uranyl formate solution for 15 seconds each and then dried. Negative staining photomicrographs were recorded at a cooled electron microscopy facility using a Tecnai G2 Spirit microscope equipped with a TemCam-F416 (4kx4k) TVIPS CMOS camera and a Veleta (2kx2k) CCD camera operating at 120 kV. Photomicrographs were recorded at magnifications of 42,000x and 52,000x.

[0228] (Microscopy and Confocal Imaging) To determine the FUN expression pattern, roots were observed after GUS staining using a Leica M165FC fluorescence stereomicroscope. Root nodules were embedded in 3% agarose and cut into 100 μm slices using a vibratome. The nodule slices were then observed using a Zeiss Axioplan 2 light microscope. To determine the subcellular localization of FUN, hairy roots of Lotus sp. expressing FUN-GFP and leaves of N. benthamiana were treated with 500 μM ZnCl2 (Zn) or MgCl2 (mock) for 3 days and observed for fluorescence using a Zeiss LSM 710 confocal microscope with a 491-535 nm filter. For zinc biosensor (eCALWY and eCALWYnls) assays, root nodules were embedded in 3% agarose and cut into 75 μm slices using a vibratome. Using a Zeiss LSM 710 confocal microscope, cerulean was excited at 458 nm, citrine fluorochrome was detected with a 514–550 nm filter, and DsRed from M. loti was detected with a 587–665 nm filter.

[0229] (Imaging and quantification of Zinpyr-1) Plants bearing pink nodules (3 wpi) were acclimatized by overnight immersion in 1 / 4 Long Ashton liquid medium prior to treatment and then treated with 0 or 10 mM MKNO3 for 24 hours. Mature nodules were embedded in 3% agarose and cut into 80 μm slices using a vibratome. Slides were stained with 5 μM Zinpyr-1 for 3 hours and then rinsed three times with water. Fluorescence was observed using a Zeiss LSM 710 confocal microscope using excitation at 488 nm and emission at 505–550 nm. Fluorescence intensity was quantified using ImageJ.

[0230] (micro X-ray fluorescence microscopy) Micro-X-ray (mXRF) images were acquired using a scanning X-ray microscope equipped with a liquid nitrogen passively cooled freezing stage (Cotte, M. et al. The ID21 X-ray and infrared microscopy beamline at the ESRF: status and recent applications to artistic materials. J. Anal. At. Spectrom. 32, 477-493 (2017)). Samples were prepared as described by Escudero et al. (Escudero, V. et al. Medicago truncatula Ferroportin2 mediates iron import into nodule symbiosomes. New Phytol. 228, 194-209 (2020)). Briefly, root nodules were embedded in OCT medium and cryofixed by immersion in liquid nitrogen-cooled isopentane. 20 μm sections of frozen samples were obtained using a Leica LN22 cryomicrotome, placed between two pieces of Ultralene (Spex SamplePrep, Rickmansworth, UK) film in a liquid nitrogen-cooled sample holder. The beam was focused to a 0.9 × 0.6 μm area using Kirkpatrick-Baez mirror optics. 2 The emitted fluorescent signal was collected using a large-area energy-dispersive microscope (80 mm) equipped with a beryllium window. 2 The images were captured with an SDD detector (XFlash SGX, RaySpec, High Wycombe, UK) at a fixed energy of 9.8 keV. The sample was imaged in a 2x2 μm 2The image was acquired by raster scanning with a step of 100 ms and a dwell time of 220 ms. Elemental distributions were calculated using the PyMca software package (Sole, VA et al. A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra. Spectrochim. Acta Part B At. Spectrosc. 62, 63-68 (2007)).

[0231] [result] The sensor domain of FUN forms filamentous structures in the presence of physiological concentrations of zinc. The FUN sensor domain shares distant homology with metal-binding proteins (Trepreau, J. et al. Structural basis for metal sensing by CnrX. J. Mol. Biol. 408, 766-779 (2011)). Since no transcriptional regulation of FUN was observed in root nodules (Figure 3I), we hypothesized that its activity might be regulated at the protein level. To understand this mechanism, we expressed and purified the FUN sensor domain (Figure 3J) and screened it with common cellular metal ions and nitrogen compounds to see whether they affected the FUN sensor. We found that the thermal stability of FUN (nanoDSF; Figure 3K) and its molecular size as measured by dynamic light scattering (DLS; Figures 3A–3C) increased in the presence of zinc and manganese, whereas the other compounds tested did not alter the response. Concentration-dependence experiments revealed that zinc increased the molecular size of the FUN sensor at low, physiologically relevant concentrations (3.9–7.8 μM), whereas manganese increased the molecular size only at very high, non-physiological concentrations (2–4 mM), indicating that zinc was the relevant ligand (Figures 3A and 3B).

[0232] The zinc-induced changes were reversible when zinc was chelated using EDTA (Figure 3D). Similar zinc sensitivity and reversibility were confirmed for proteins containing both DNA-binding and sensor domains (Figure 3L). Further investigations using small-angle X-ray scattering (SAXS) experiments, combining scattering data with pairwise distance distribution functions (histograms of distances between pairs of points within the structure), confirmed that the presence of zinc shifts the FUN sensor from a small molecular size to a large oligomeric form, and that this effect is reversible when zinc is removed with EDTA (Figures 3E-3G).

[0233] The oligomeric structure of the FUN sensor was examined by electron microscopy. Negatively stained samples revealed that large filamentous structures were formed when the FUN sensor bound zinc, and these filaments disassembled upon zinc removal using EDTA (Fig. 3H).

[0234] Taken together, these data indicate that FUN binds to physiologically low concentrations of zinc, transforming its oligomeric structure into large filaments, and that this process is dynamic and reversible, potentially representing a mechanism for regulating activity.

[0235] Example 4: Zinc regulation of FUN is important in vivo This example describes an in vivo study of zinc regulation of FUN.

[0236] [material and method] (Subcellular localization assay) The subcellular localization of the FUN-GFP construct described in Example 1 was examined in N. benthamiana (tobacco) leaves. Tobacco leaves expressing the pro35S:FUN-GFP protein were infiltrated with 500 μM mock, Mn, and Zn. Fluorescent images were taken with a confocal microscope (Zeiss SP5), and cell nuclei were counted based on their punctate or homogenous distribution.

[0237] (Plant strains and cultivation conditions) The plant strains and cultivation conditions were as described in Example 1.

[0238] (Strains and culture conditions) The strain and culture conditions were as described in Example 3.

[0239] (hairy root transformation) Hairy root transformation was as described in Example 1.

[0240] (Nodulation assay) The nodulation assay was as described in Example 1.

[0241] (Nitrogen fixation assay) Nitrogen fixation assays were performed as described in Example 1.

[0242] (Leghemoglobin content assay) Leghemoglobin content assay was as described in Example 1.

[0243] (Transient activation assay) The transient activation assay was as described in Example 2.

[0244] (Zinc biosensor) The response of root nodule cells to Zn treatment was assayed using the FRET-based zinc biosensor eCALWY (Lanquar, V., Grossmann, G., Vinkenborg, JL, Merkx, M., Thomine, S., and Frommer, WB (2014). Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology. New Phytol. 202: 198-208) and eCALWYnls, which contained a nuclear localization signal (nls).

[0245] (Expression analysis) We used RNA sequencing to compare the expression of putative zinc transporter genes Zip1 and Zip2 under normal and nitrate-stress cultivation conditions. The original data were from the RNA sequencing analysis described in Example 3.

[0246] [result] (Zinc is a second messenger that regulates FUN activity) The identification of zinc-induced FUN filaments raised the possibility that zinc may play a role in regulating the protein's activity. Notably, zinc infiltration caused a change in the nuclear fluorescence of FUN-GFP in N. benthamiana leaves. The results of a subcellular localization assay demonstrating the altered fluorescence are shown in Figures 4A-4C. In these figures, the addition of zinc promoted FUN aggregation.

[0247] Using the NRT2.1 promoter as a readout for FUN activity, zinc co-infiltration significantly reduced FUN activity in N. benthamiana leaves compared to mock (MgCl2) (Figure 4D). This suggests that the zinc-bound filamentous state of FUN is an inactive form of the protein. Considering the phenotype of the fun mutant, zinc may function as a messenger linking nitrate to FUN activity and nodule regulation.

[0248] To examine whether nitrate affects cellular zinc concentrations, we performed experiments using the zinc-sensitive Zinpyr-1 dye (reference 1) to evaluate nodule segments of Lotus spp. grown under nitrate-free conditions and those exposed to 10 mM KNO3 for 24 h (Figures 5C-5D). Results revealed a significant decrease in zinc concentration, particularly in the nitrogen-fixing zone of nitrate-treated nodules. Independent confirmation of this decrease was obtained by micro-X-ray fluorescence microscopy of nodule segments treated with 10 mM KNO3 for 24 h, which showed a ring-like distribution of zinc within infected cells associated with the radial distribution and dense packaging of symbiosomes (Figures 5E-5F). Density measurements of 10 cells from each condition confirmed a halving of zinc concentration relative to untreated nodules (0.54 ± 0.06; Figure 5F). To confirm the in vivo relevance of FUN's zinc-dependent filament formation, we expressed a FUN-GFP construct in Lotus spp. roots. FUN-GFP showed dispersed nuclear localization under control conditions (500 μM MgCl2), but addition of zinc (500 μM ZnCl2) induced relocalization into distinct nuclear condensates (Figure 5G). Consistent with the effect of zinc on protein activity in N. benthamiana, a zinc-dependent increase in condensate frequency was also detected in leaves infiltrated with zinc along with the FUN-GFP construct (Figure 5H). To further confirm the relationship between nitrate, zinc, and FUN activity, addition of 500 μM zinc significantly increased nodule function in nitrate-exposed WT plants, recapitulating the phenotype of the fun knockout mutant as determined by acetylene reduction (Figure 4E) and leghemoglobin content (Figure 4H). This increase was dependent on the presence of FUN, and no further increase in nodule function was observed in the fun mutant (Figures 4F and 4G). Finally, Figure 5A shows the expression analysis of two putative zinc transporters, Zip2 and Zip4. Both Zip2 and Zip4 were induced in nodules after nitrate treatment.

[0249] Taken together, these results indicated that changes in zinc concentration in response to soil nitrate were sufficient to alter FUN activity and, consequently, the nitrogen fixation phenotype of root nodules.

[0250] [discussion] The genetic screen described in Example 1 identified the basic leucine zipper transcription factor FUN as a novel master regulator of nitrogen fixation in legumes. A sensor domain within FUN was identified as critical for its activity, and intracellular zinc levels were shown to determine protein activity through ligand-dependent protein filament formation. Examples 2-4 demonstrate that FUN forms inactive filaments under high zinc concentrations and acts as a molecular reservoir that releases the active protein when zinc levels decrease (Figure 5J). Cellular zinc levels are inversely related to nitrate, indicating that zinc signals nitrate availability and acts as a second messenger that controls the transition of the FUN protein between its filamentous (inactive) and active states.

[0251] In plants, changes in zinc concentration have been shown to affect FUN protein activity and nodule function, linking soil nitrate supply to the transcriptional regulation of nodule metabolism. This post-translational regulation of FUN activity is thought to enable plants to respond to nitrate gradients by releasing more activated FUN through gradually decreasing zinc concentrations, thereby adjusting nodule function in response to the environment. The exact mechanisms by which intracellular zinc concentrations are affected by nitrate, such as modulation of transporters, sequestration in organelles, or export, remain unclear. FUN is a transcription factor of the TGA family, whose members are involved in nitrate uptake (Alvarez, JM et al. Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots. Plant J. 80, 1-13 (2014), Ruffel, S. et al. Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters. Plant Physiol. 186, 696-714 (2021)), pathogen response (Kumar, S. et al. Structural basis of NPR1 in activating plant immunity. Nature 1-6 (2022)), and flower development (Maier, AT, Stehling-Sun, S., Offenburger, S.-L. & Lohmann, JU The bZIP Transcription Factor PERIANTHIA: A Multifunctional Hub for Meristem Control. Front. Plant Sci. 2, 79 (2011)).Given the presence of sensor domains identified within TGA family homologs, it is plausible that zinc or other metal ions and metabolites could confer similar graded responses to environmental stimuli, linking the environment and plant development through metal ion signaling. Manipulating metal ion accumulation or the responsiveness of protein filament formation to these metal ions may provide new avenues for optimizing these important plant traits.

[0252] Nitrogen fixation is an energy-demanding process that requires the supply of fixed carbon to symbiotic rhizobia. The regulated senescence program limits carbon supply to the nodule, allowing it to resupply nutrients to support plant growth and reproduction (Puppo, A. et al. Legume nodule senescence: roles for redox and hormone signaling in the orchestration of the natural aging process. New Phytol. 165, 683-701 (2005)). Recently, several NAC transcription factors have been shown to regulate pathways required for root nodule senescence (Yu, H. et al. GmNAC039 and GmNAC018 activate the expression of cysteine ​​protease genes to promote soybean nodule senescence. Plant Cell (2023) doi: 10.1093 / plcell / koad129; Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896). The identification of FUN as a novel regulator of senescence-related processes through multiple pathways, including NAC094, opens new avenues for fine-tuning these pathways, providing opportunities for enhancing soil nitrate tolerance in legumes and increasing the supply of fixed nitrogen to agriculturally important crops.Importantly, the specificity of this identified pathway for regulating nodule function allows mutants to regulate nodule number (Krusell, L., Madsen, LH, Sato, S. & Aubert, G. Shoot control of root development and nodulation is mediated by a receptor-like kinase. Nature 420, 422-426 (2002), Nishimura, R. et al. HAR1 mediates systemic regulation of symbiotic organ development. Nature 420, 426-429 (2002), Huault, E. et al. Local and systemic regulation of plant root system architecture and symbiotic nodulation by a receptor-like kinase. PLoS Genet. 10, e1004891 (2014)) or nitrate uptake and signaling (Lin, J.-S. et al. NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula. Nat Plants 4, 942-952 (2018), Misawa, F. et al. Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus. Plant Cell 34, 1844-1862 (2022), Jiang, S. et al. NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules. Science 374, 625-628 (2021)).

[0253] Example 5: Identification of FUN and NAC094 orthologs and construction of a phylogenetic tree This example describes the identification of orthologues of FUN and NAC094 in other plant species and the construction of phylogenetic trees using these sequences.

[0254] [material and method] (Identification of FUN and NAC094 orthologues and construction of phylogenetic trees) The FUN protein sequence was used as a BLAST query against the target species. Similarly, the NAC094 protein sequence was used as a BLAST query against the target species. BLAST hit candidates were aligned to a phylogenetic tree using Shoot.bio (Emms, D.M., Kelly, S. SHOOT: phylogenetic gene search and ortholog inference. Genome Biol 23, 85 (2022)).

[0255] The FUN orthologous protein sequences were SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0256] The NAC094 orthologous protein sequences include SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, The sequences were SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73.

[0257] Protein sequences were aligned using MAFFT 7.490, and trees were constructed using FastTree 2.1.11. The trees were visualized using iTOL 6.7.3 (Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293-W296 (2021)).

[0258] [result] Phylogenetic analysis revealed that FUN is highly conserved in legumes, which have both FUN and FUN-like paralogs in the PAN ortholog cluster (Figure 6A). Duplication of FUN in legumes resulted in two copies of the FUN gene in L. japonicus: FUN, which is expressed in nodules and is described in Example 1, and FUN-like, which is not expressed in nodules and has orthologs in non-legumes. A second duplication occurred in Glycine max, resulting in two FUN orthologs and two FUN-like orthologs.

[0259] The FUN orthologs were found to be in a phylogenetic clade with Lotus FUN (LotjaGi2g1v0279100; SEQ ID NO:1) and Glycine max (soybean) FUNa (Glyma.02G097900; SEQ ID NO:8) and FUNb (Glyma.01G084200; SEQ ID NO:9). The closest non-legume orthogroup member was Arabidopsis PAN (AT1G68640.1; SEQ ID NO:4). The orthology was further supported by measuring gene expression in root nodules by RNA-seq analysis (data not shown).

[0260] To assess whether Glycine max FUN orthologs also exhibit nodule-specific expression, we measured the expression levels of two orthologs, FUNa and FUNb, in various soybean tissues (Fig. 6B). Indeed, both orthologs were predominantly expressed in symbiotic nodules. These observations suggested that the function of FUN may be conserved in soybean and other legumes.

[0261] FUN paralogs that do not function in nodule regulation are found in a phylogenetic clade with Lotus FUN-like (LotjaGi5g1v0341400; SEQ ID NO: 83) and soybean FUN-like (Glyma.20G113600 (SEQ ID NO: 7) and Glyma.10G276100 (SEQ ID NO: 6)).

[0262] We also constructed a phylogenetic tree of the NAC domain-containing protein Nac094 and identified its orthologs and paralogs in legumes and non-legumes (Figure 7). Nac094 (LotjaGi2g1v0259200; SEQ ID NO: 31) was orthologous to soybean Glyma.19G021900.1 (SEQ ID NO: 42) and Glyma.13G063300.1 (SEQ ID NO: 41).

[0263] Example 6: Fun mutants in soybean and cowpea exhibit enhanced nitrogen fixation. This example describes the generation and characterization of Fun mutants in soybean and cowpea, specifically experiments evaluating nitrogen fixation activity and yield performance under various stress conditions in soybean and cowpea.

[0264] [material and method] (Plant materials and cultivation conditions) Glycine max (soybean) and Vigna unguiculata (cowpea) lines were used for Agrobacterium transformation and regeneration of CRISPR Fun knockout mutants.

[0265] (nitrogen fixation activity) Nitrogen fixation was assessed using the method of Example 1.

[0266] (Construction of plant expression vectors) Expression constructs were generated to express CRISPR / Cas and multiple guide RNAs targeting the coding sequence of the Fun gene in G. max (soybean) and V. unguiculata (cowpea).

[0267] (Plant transformation and regeneration) Plant transformation and regeneration was carried out using standard methods for soybean and cowpea.

[0268] (Yield performance) Yield performance was evaluated using standard methods for soybean and cowpea under a range of nitrogen applications, from low to high, and also under scenarios with companion crops.

[0269] [result] (Improved nitrogen fixation increases yields in soybean and cowpea) To investigate whether the function of Lotus Fun genes is conserved across legumes, we generated CRISPR knockouts of FUN orthologs in soybean (Figure 6A) and cowpea. Multiple knockout lines were selected and crossed to generate homozygous FUN mutants. Once homozygotes were generated, the nitrogen fixation activity of fun mutants in soybean and cowpea was evaluated under nitrate-limited conditions. The nodule phenotype and nitrogen fixation activity were assayed in the same manner as for Lotus fun mutants. If the function of Lotus Fun genes is conserved in soybean and cowpea, fun mutants in both species will exhibit a pink (active) nodule phenotype and improved nitrogen fixation activity under nitrate-limited conditions. Furthermore, to investigate whether the absence of functional Fun genes enhances nitrogen fixation activity in other stress environments, the nitrogen fixation rates of Lotus, soybean, and cowpea Fun mutants will be evaluated under drought, heat stress, and flooding conditions.

[0270] Soybean and cowpea varieties with enhanced nitrogen fixation activity under stress conditions have the potential to sustainably increase the yield of both crops. Promising fun mutant lines in soybean and cowpea identified from the stress assays described above were evaluated for field performance. The yield characteristics of fun mutants in soybean and cowpea were evaluated in the field to determine whether enhanced nitrogen fixation leads to increased yield.

[0271] Example 7: Engineered FUN mutants improve nitrogen fixation in legumes. This example describes the structure-function characterization of FUN for the design and characterization of FUN mutants in Lotus and target crops.

[0272] [material and method] (Plant materials and cultivation conditions) The fun mutants of Lotus genus described in Example 1 and those identified in soybean and cowpea in Example 6 were used for Agrobacterium transformation and regeneration of modified FUN mutants. Cultivation conditions were as described in Examples 1 and 6.

[0273] (Experimental Technology) The experimental techniques are as described in any of Examples 1-6.

[0274] [result] (Structure-based engineering of FUN mutants) Structural characterization of the FUN mutant protein and its alleles identified regions important for protein function. This structural analysis, combined with computational modeling, pinpointed key residues for designing novel mutants in the FUN protein that further improved its nitrogen fixation properties. The engineered FUN protein mutants were introduced into Lotus spp., and their nitrogen fixation properties were evaluated.

[0275] (The function of the engineered FUN mutant is conserved in soybean and cowpea) To assess whether structure-based engineering of FUN could improve crop yield, we introduced mutations corresponding to modified Lotus FUN mutants into soybean and cowpea. The performance of soybean and cowpea lines with modified FUN was evaluated under various stress conditions and in the field.

[0276] Example 8: The FUN sensor domain mechanism is conserved in TGA transcription factors. This example describes the structural characterization of a TGA-type transcription factor that shares a conserved domain structure with FUN, experiments assessing the conformational modulation of the TGA sensor domain by metal ion ligands, and the identification of a nitrate-responsive metal ion transporter.

[0277] [material and method] The experimental techniques are as described in any of Examples 1-7.

[0278] [result] Sensor domain filament formation is a conserved feature of TGA transcription factors. Lotus FUN is a member of the TGA family of transcriptional regulators, which play broad roles in plant development, immunity, and nitrate signaling. All TGA family members share a conserved domain structure, with a DNA-binding domain and a sensor domain. To determine whether sensor domain filament formation is conserved among the TGA family of transcription factors, we determined the sensor domain structure of representative TGA family members. We also investigated the ability of metal ion ligands to induce sensor domain multimerization. If metal ion ligands can regulate sensor domain complex formation and activity, as with FUN, family-wide regulation may be achieved by metal ion treatments targeting various pathways relevant to crop improvement.

[0279] (Identification of nitrate-responsive metal ion transporters that regulate nitrogen fixation) To identify metal ion transporter genes regulated by nitrate, differential gene expression in the genus Lotus was analyzed under three conditions: nitrate treatment versus mock, zinc treatment versus mock, and simultaneous nitrate and zinc treatment versus mock.

[0280] Differentially expressed candidate genes were further evaluated for expression, activity, and zinc concentration within nodules in response to nitrate conditions. Knockout lines of Lotus species for identified metal ion transporter genes were obtained or generated and evaluated for improved nitrogen fixation characteristics. Identification of these genes provided mechanistic insight into zinc regulation by nitrate and provided additional tools for modulating nitrogen fixation.

[0281] Example 9: Effect of FUN on heat and drought tolerance This example describes the generation and characterization of fun mutants in the genera Medicago and Lotus, as well as experiments to evaluate the nitrogen fixation activity and yield performance of fun mutants in Medicago and Lotus under heat and drought conditions.

[0282] [material and method] The experimental techniques are as described in any of Examples 1-8.

[0283] [result] (FUN manipulation improves drought tolerance) RNA sequencing revealed that NAC094 and HO1 were strongly upregulated after 4 days of drought in both Medicago and Lotus (Figure 8A). To determine whether FUN regulation of NAC094 and / or HO1 plays a role in drought tolerance, fun mutants were generated in Medicago and Lotus and tested under drought conditions. Plant drought tolerance was assessed.

[0284] (Heat resistance improves with FUN operation) As quantified using an acetylene reduction assay (ARA), fun mutant plants showed increased nitrogen fixation activity after 7 days of heat stress (Figure 8C). To determine whether manipulation of FUN improves heat tolerance, fun mutants were tested under heat conditions. The heat tolerance of the plants was assessed. [Brief explanation of the drawings]

[0285] [Figure 1A]Figures 1A-1V show the nodule phenotype and nitrogen fixation activity of fun mutant Lotus plants under nitrate-limited conditions, demonstrating that Fun is specifically expressed in nodules. Figure 1A shows representative photographs of the nodule phenotype of wild-type Lotus plants (WT; Gifu) under KCl conditions (control; upper left), wild-type Lotus plants under 10 mM KNO3 conditions (top center), and fun mutant Lotus plants under 10 mM KNO3 conditions (top right and bottom). The fun mutations include fun (top right), fun-2 (bottom left), fun-3 (bottom center), and fun-4 (bottom right). Scale bar = 1 cm. [Figure 1B] Figure 1B shows a diagram of the FUN gene and LORE1 insertion in each genetic background phenotyped in Figure 1A. In fun and fun-4, LORE1 is inserted in the promoter region (light gray line). In fun-2, LORE1 is inserted at the end of the fourth intron (the intron is represented by a black line). In fun-3 (30099638), LORE1 is inserted at the end of the seventh intron. Arrows indicate the insertion point of LORE1 along the gene. [Figure 1C] Figure 1C shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (colored box, labeled "Pink") formed in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 1D] Figure 1D shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 1E]Figure 1E shows the ARA measurements (vertical axis) from Figure 1D over a 14-day course (top) and followed up to 20 mM KNO3 (bottom). The light gray line with medium gray circles represents the ARA results for wild-type plants. At the top, the medium gray line with medium gray circles represents the ARA results for fun mutant plants, and the black line with medium gray circles represents the ARA results for fun-3 mutant plants. At the bottom, the black line with medium gray circles represents the ARA results for fun mutant plants. [Figure 1F] Figure 1F shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, fun mutant Lotus plants, fun-2 mutant Lotus plants, fun-3 mutant Lotus plants, and fun-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 1G] Figure 1G shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, fun mutant Lotus plants, fun-2 mutant Lotus plants, fun-3 mutant Lotus plants, and fun-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 1H] Figure 1H shows the number of pink nodules per plant for wild-type (WT), fun, fun-3, and fun-4 mutants treated with 5 mM nitrate (gray box, labeled "5 mM") or no nitrate (white box, labeled "0 mM") before inoculation. Plants were grown on plates supplemented with 0 or 5 mM KNO and then inoculated with rhizobia. [Figure 1I] Figure 1I shows the total number of nodules per plant for wild-type (WT), fun, fun-3, and fun-4 mutants treated with 5 mM nitrate before inoculation (gray box, labeled "5 mM") and without nitrate before inoculation (white box, labeled "0 mM"). Plants were grown on plates containing 0 or 5 mM KNO3 and then inoculated with rhizobia; nodules were measured 3 weeks after inoculation. [Figure 1J] Figure 1J shows nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT), fun, fun-3, and fun-4 mutant plants treated with 5 mM nitrate before inoculation (gray box, labeled "5 mM") and without nitrate before inoculation (white box, labeled "0 mM"). Plants were grown on plates supplemented with 0 or 5 mM KNO3 and then inoculated with rhizobia; ARA was performed 3 weeks after inoculation. In Figures 1F–1J, letters indicate significant differences (p<0.05) between the compared plant groups. [Figure 1K] FIG. 1K shows the leghemoglobin content (μg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants and fun mutant Lotus plants exposed to 10 mM KNO 3 for 2 weeks. [Figure 1L] Figure 1L shows the complementation of fun mutants grown under conditions exposed to 10 mM KNO for 2 weeks. The gray boxes indicate empty vector (labeled "EV") transformed into the WT background (left) or the fun mutant background (center), while the white boxes (right) indicate when proUbi:fun-GFP was transformed into the fun mutant background. Letters indicate significant differences between the compared plant groups. [Figure 1M] Figure 1M shows a representative photograph of a plant root with nodules in a plant expressing the proFun:GUS reporter construct, used to visualize Fun gene expression in situ. The root was stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). Blue staining indicates GUS reporter expression. Scale bar = 2 cm. [Figure 1N] Figure 1N shows a mid-longitudinal section of a root nodule photographed using a light microscope. Blue staining indicates GUS reporter expression; scale bar = 200 μm. [Figure 1O]Figure 1O is an enlarged view of Figure 1N, where "ic" indicates infected cells, "uc" indicates uninfected cells, and "nc" indicates the nodule cortex; scale bar = 200 μm. In Figures 1M-1O, proFun:GUS is a construct in which the endogenous Fun promoter sequence (proFUN) is fused to the coding sequence for β-glucuronidase (GUS), followed by the endogenous Fun termination sequence (tFUN). [Figure 1P] FIG. 1P is a schematic diagram of the FUN protein showing the bZIP DNA-binding domain (gray oval) and the sensor domain (dark gray star). [Figure 1Q] Figure 1Q shows a bar graph comparing normalized RNA measurements of FUN transcripts among different plant tissues in Lotus japonicus Gifu, based on data calculated by Kamal et al. (2020). (Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence. DNA Res. 27(3)) Plant tissues of L. japonicus Gifu are listed along the vertical axis. "Nodule 21d" and "Nodule 10d" refer to measurements obtained from nodules 21 and 10 days after inoculation with Mesorhizobium loti R7A, respectively. [Figure 1R] Figure 1R shows a bar graph of genes that were upregulated (top) or downregulated (bottom) and significantly altered in expression (DE) levels in WT (black box) or fun mutant (gray box) plants. The number of DE genes is plotted on the horizontal axis. [Figure 1S] Figure 1S presents a bar graph showing the elevated levels (log2 fold change relative to WT plant expression levels, horizontal axis) of differential expression of selected genes (vertical axis) in fun mutant plants (black box) and fun-3 mutant plants (gray box). [Figure 1T]Figure 1T presents a bar graph showing the reduced levels (log2 fold change relative to WT plant expression, horizontal axis) of differential expression of selected genes (vertical axis) in fun mutant plants (black box) and fun-3 mutant plants (gray box). [Figure 1U] Figure 1U shows that the ontologies identified by GO-MWU are enriched among upregulated genes (the first three major branches) and downregulated genes (the fourth to seventh major branches). Text size and thickness indicate p-values. [Figure 1V] Figure 1V shows the relative expression levels of downstream targets of FUN, identified with TGA motifs in their promoters, that are differentially expressed in fun compared to wild type. The relative expression levels are shown in the RNA-seq time series from Wang et al. (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi:10.1111 / nph.18896). In Figures 1C-1D and 1F-1L, circles represent individual plants. In Figures 1C-1E and 1K-1L, asterisks indicate significant differences between the compared groups. "**" indicates a p-value <0.01, and "*" indicates a p-value <0.05. [Figure 2A] Figures 2A–2S show that FUN regulates the expression of downstream genes Nrt2.1, Ho1, NAC094, Nrt3.1, and AS1 to regulate nitrate signaling and nitrogen fixation in root nodules. Figure 2A shows the expression levels of Nrt2.1 (left), Ho1 (center), and NAC094 (right) in nodules of wild-type Lotus plants (Gifu; white), fun mutant Lotus plants (gray), and fun-3 mutant Lotus plants (dark gray) after 10 mM KNO3 nitrate treatment at 0, 3, and 24 hours. [Figure 2B]Figure 2B shows the expression levels of Nrt3.1 (left) and AS1 (right) genes in nodules of wild-type Lotus plants (Gifu; white), fun mutant Lotus plants (gray), and fun-3 mutant Lotus plants (dark gray) at 0, 3, and 24 hours after treatment with 10 mM KNO3 nitrate. [Figure 2C] Figure 2C shows a schematic diagram of the promoters of Nrt2.1 (proNRT2.1; top), Ho1 (proHO1; second from the top), NAC094 (proNAC094; middle), Nrt3.1 (proNRT3.1; second from the bottom), and AS1 (proAS1; bottom). The Nrt2.1 promoter has four putative FUN-binding sites (FBSs), designated p1, p2, p3, and p4; the Ho1 promoter has two putative FBSs, designated p1 and p2; the NAC094 promoter has one putative FBS, designated p1; the Nrt3.1 promoter has three putative FBSs, designated p1, p2, and p3; and the AS1 promoter has one putative FBS, designated p1. [Figure 2D] Figure 2D shows gel images of EMSA assays demonstrating the binding of FUN protein to a DNA probe containing FBS p1, p2, p3, and p4 from the promoter of Nrt2.1 (left), a DNA probe containing FBS p1 and p2 from the promoter of Ho1 (center), and a DNA probe containing FBS p1 from the promoter of NAC094 (right). [Figure 2E] Figure 2E shows a gel image of an EMSA assay demonstrating binding of FUN protein to DNA probes containing FBS p1 from the promoter of Nrt2.1, FBS p1, p2, and p3 from the promoter of Nrt3.1, and FBS p1 from the promoter of AS1. [Figure 2F]Figure 2F shows gel images of MSA targeting p1 (top) and p4 (bottom) FBS of the Nrt2.1 promoter in a competition assay. Competitor DNA was at 50-, 150-, and 500-fold concentrations compared with WT DNA without a tag attached to the probe. The label "m" corresponds to a DNA probe with a mutation in the core binding site, TGACG. [Figure 2G] Figure 2G shows the results of a FUN-induced transcriptional activation assay of the Nrt2.1 (left), Ho1 (center), and NAC094 (right) promoters in N. benthamiana leaves. The white bars represent GFP, and the gray bars represent pro35S:FUN-GFP. pro35S:FUN-GFP was expressed as an effector, and GUS was driven by either the Nrt2.1, Ho1, or NAC094 promoter as a reporter. [Figure 2H] Figure 2H shows the results of a transcriptional activation assay of the Nrt3.1 (left) and AS1 (right) promoters by FUN in N. benthamiana leaves. The white bars represent GFP, and the gray bars represent pro35S:FUN-GFP. pro35S:FUN-GFP was expressed as an effector, and a GUS reporter was driven by either the Nrt3.1 or AS1 promoter. [Figure 2I] Figure 2I shows representative photographs of root nodules from wild-type (Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, ho1-4 mutant Lotus plants, or nac094-3 mutant Lotus plants exposed to 10 mM KNO for 2 weeks. The scale bar is 1 cm in all four photographs. [Figure 2J] Figure 2J shows the total number of nodules (gray box, labeled "Total") and the number of functional pink nodules (white box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2K]Figure 2K shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2L] Figure 2L shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, nrt2.1-3 mutant Lotus plants, and nrt2.1-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2M] Figure 2M shows the total number of nodules (white box, labeled "Total") and the number of functional pink nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, ho1-4 mutant Lotus plants, and ho1-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2N] Figure 2N shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2O] Figure 2O shows the total number of nodules (white box, labeled "Total") and the number of pink functional nodules (gray box, labeled "Pink") formed in wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2P] Figure 2P shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, hol-4 mutant Lotus plants, and hol-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2Q]Figure 2Q shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants, hol-4 mutant Lotus plants, and hol-5 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2R] Figure 2R shows the nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type (WT; Gifu) Lotus plants, nac094-3 mutant Lotus plants, and nac094-4 mutant Lotus plants exposed to 10 mM KNO3 for 2 weeks. [Figure 2S] Figure 2S shows normalized read counts of FUN downstream targets with identified TGA motifs in their promoters in mock-treated wild-type plants (labeled "WT Mock") and wild-type, fun, and fun-3 mutant plants 24 days after nitrate exposure (labeled "WT 24h," "fun 24h," and "fun-3 24h," respectively). The graphs show normalized read counts for NRT2.1, HO1, NAC094, NRT3.1, and AS1 from left to right. In Figures 2A-2B, 2G-2H, and 2J-2R, circles represent individual plants. In Figures 2A-2B, 2G-2H, and 2J-2R, asterisks indicate significant differences between the compared groups. "**" indicates a p-value <0.01, and "*" indicates a p-value <0.05. [Figure 3A] Figures 3A-3L show that the FUN sensor domain forms filamentous structures in the presence of physiological concentrations of zinc (Zn). Figure 3A shows dynamic light scattering (DLS) analysis of the FUN sensor domain exposed to 4 mM MgCl, CaCl, MnCl, ZnCl, NH, Cl, KNO, KNO, KCl, or a blank sample ("FUN sensor" alone, control). [Figure 3B] Figure 3B shows the DLS analysis results of FUN(bZIP) with added MnCl2 at concentrations of 8 mM, 4 mM, 2 mM, 1 mM, 500 μM, 250 μM, 62.5 μM, 15.6 μM, 3.9 μM, or 0 μM. [Figure 3C]Figure 3C shows the DLS analysis results of FUN (bZIP) with ZnCl2 at concentrations of 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, 7.8 μM, 3.9 μM, 2.0 μM, 0 μM, or 8 mM in the absence of FUN (8 mM b no ZIP; control). [Figure 3D] Figure 3D shows the results of DLS analysis of FUN bZIP alone (“FUN sensor”), FUN in the presence of 100 μM ZnCl2 (“FUN sensor + Zn”), or FUN in the presence of 100 μM ZnCl2 and 5 mM ethylenediaminetetraacetic acid (EDTA) (“FUN sensor + Zn + EDTA”). [Figure 3E] Figure 3E shows a plot of the SAXS analysis, with scattering intensity on the vertical axis expressed as "I(q)" in cm-1 and q (Å-1) on the horizontal axis. Scattering is plotted for FUN sensor alone (bZIP only) ("FUN Sensor", gray), FUN bZIP bound to Zn ("FUN Sensor + Zn", light gray), or FUN bZIP with Zn removed using EDTA ("FUN Sensor + Zn + EDTA", black). [Figure 3F] Figure 3F shows a plotted histogram of interpair distances between points within particles based on the analysis in Figure 3E. The pair distance distribution model p(r) is plotted on the vertical axis, and the distance r (Å) is plotted on the horizontal axis. The arrow "394 Å" represents the maximum diameter (Dmax) of 394 Å for Zn-bound FUN bZIP ("FUN sensor + Zn"). The arrow "118 Å" represents the Dmax of 118 Å for FUN bZIP alone ("FUN sensor") or FUN bZIP with Zn removed ("FUN sensor + Zn + EDTA"). [Figure 3G]Figure 3G shows the Guinier plot calculated from Figures 3E and 3F, where the radius of gyration is calculated through the scattering intensity as a function of the scattering vector q (vertical axis vs. horizontal axis). Closed circles represent the data used in the fit, and open circles represent omitted data points. The p(r) function shows that the radius of gyration for the pure FUN sensor sample and the EDTA + zinc-containing sample is 39 ± 1 Å, while it is 125 ± 1 Å for the zinc-bound sample. Guinier analysis showed slightly lower values ​​for all samples. [Figure 3H] Figure 3H shows representative electron micrographs of the FUN sensor domain alone (bZIP1 sensor; top row), the FUN sensor domain with 300 μM ZnCl2 added (bZIP1 sensor + 300 μM Zn; middle row), and FUN with 300 μM ZnCl2 and 5 mM EDTA added (bZIP1 sensor + 300 μM Zn + 5 mM EDTA; bottom row). [Figure 3I] Figure 3I shows the relative expression level of Fun (vertical axis) over time (horizontal axis) in 3-week-old root nodules exposed to 10 mM KNO3 for 0, 0.5, and 3 h (left) and for 0, 1, 3, and 7 days (right). [Figure 3J] Figure 3J shows the purification and thermal stability of the FUN sensor domain. The left side shows a chromatogram of the FUN sensor domain obtained by size-exclusion chromatography (Superdex 200 increase 10 / 300), plotting absorbance (vertical axis) against elution volume (horizontal axis). The right side shows sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the SEC fractions. Fractions 14–17 were pooled and stored, as indicated by the dashed line on the chromatogram and the horizontal line above the SDS-PAGE. [Figure 3K] Figure 3K shows the thermal stability of the purified FUN sensor domain as measured by inflection point temperature (Ti) (°C) either alone ("FUN sensor only") or with different ions (vertical axis) at a concentration of 4 mM: MgCl2, CaCl2, MnCl2, ZnCl2, NH4Cl, KNO3, KNO2, or KCl. [Figure 3L]Figure 3L shows DLS analysis of the zipper domain and sensor domain-containing FUN protein in the absence (gray line) and presence (medium gray line) of 100 μM ZnCl2. The change in hydrodynamic radius induced by zinc is reversed by 5 mM EDTA (black line). In Figures 3C-3G, the gray "FUN Sensor" represents measurements of the FUN sensor alone, the medium gray "FUN Sensor + Zn" represents measurements of the FUN sensor treated with 100 μM ZnCl2, and the black "FUN Sensor + Zn + EDTA" represents the FUN sensor treated with 100 μM ZnCl2 and then 5 mM EDTA to remove Zn. In Figures 3C-3G, the four-pointed star labeled "apo" represents the apo-conformation ("apo") of the FUN sensor. The chains with overlapping four-pointed stars labeled “Zn-bound” represent combinations of Zn-bound FUN sensors in larger oligomers. [Figure 4A] Figures 4A–4H show that zinc regulates the subcellular localization and function of FUN. Figure 4A shows a representative image of the subcellular localization of pro35S:FUN-GFP in N. benthamiana leaves. [Figure 4B] Figure 4B shows the fluorescence distribution of the subcellular localization of FUN-GFP in N. benthamiana leaves. [Figure 4C] Figure 4C shows the ratio of punctate nuclei (dark gray) to total nuclei (homogeneous; light gray) for subcellular localization in N. benthamiana leaves. In Figures 4A-4C, results were obtained under conditions of 500 μM MgCl2 (mock), MnCl2 (Mn), and ZnCl2 (Zn). [Figure 4D] Figure 4D shows the results of a FUN-mediated transcriptional activation assay of the Nrt2.1 promoter in N. benthamiana leaves after treatment with 500 μM MgCl (mock) and ZnCl (Zn). The white bars represent GFP, and the gray bars represent pro35S:FUN-GFP. FUN-GFP was expressed as an effector, and GUS was driven by the Nrt2.1 promoter (proNrt2.1:GUS) as a reporter. [Figure 4E]Figure 4E shows nitrogen fixation activity, quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant), in wild-type (WT; Gifu accession) Lotus plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) in combination with KCl (left) or 10 mM KNO3 (middle and right). [Figure 4F] Figure 4F shows nitrogen fixation activity, quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant), in wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) with 10 mM KNO3. [Figure 4G] Figure 4G shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) and fun mutant Lotus plants exposed to 500 µM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) with 10 mM KNO3. [Figure 4H] Figure 4H shows the leghemoglobin content (µg leghemoglobin per plant) of wild-type (WT; Gifu) Lotus plants exposed to 10 mM KCl (left), 10 mM KNO (middle), or ZnCl plus 10 mM KNO (right) for 2 weeks. In Figures 4D–4H, circles represent individual plants. [Figure 5A] Figures 5A-5H show that nitrate promotes zinc transport from nodule cells using the zinc-sensitive dye Zinpyr-1. Figure 5A shows that after 24 hours of treatment with 10 mM KNO (nitrate), the expression of two putative zinc transporter genes, Zip2 and Zip4, is induced in nodules. [Figure 5B]Figure 5B depicts the mechanism of nodule function regulated by FUN. On the left, the mechanism operates when soil nitrate concentrations are low, where zinc accumulates in the nodule, retaining FUN in inactive filaments and allowing nitrogen fixation to continue. On the right, the mechanism operates when soil nitrate concentrations are high, where a decrease in cellular zinc releases active FUN from the filaments and increases target gene expression. Arrows indicate the direction of action of these conditions. "NAC094," "HO1," and "NRT2.1" indicate the activity of these three target genes that induce nodule senescence. Dark gray indicates the presence of inactive FUN filaments, and pink nodules represent the corresponding phenotype. Light gray indicates the presence of active FUN. [Figure 5C] Figure 5C shows cellular zinc concentration levels in root nodules, as indicated by Zinpyr-1 fluorescent dye, 24 hours after mock treatment (left, KCl) and nitrate treatment (right, KNO). Scale bar = 200 μm. [Figure 5D] FIG. 5D shows the average intensity of the fixation zone indicated by the dashed circle in FIG. 5C. [Figure 5E] Figure 5E shows X-ray fluorescence (XRF) microscopy images of nodule cut surfaces taken 24 h after mock treatment (left, KCl) and nitrate treatment (right, KNO). Scale bar = 20 μm. [Figure 5F] Figure 5F shows the image of Figure 5E with the area analyzed for zinc quantification boxed in white. Scale bar = 20 μm. [Figure 5G] Figure 5G shows images of fluorescence produced by the FUN-GFP construct in control (left, labeled "MgCl") and zinc (right, labeled "ZnCl")-treated Lotus roots. Scale bar = 20 μm. Chi-squared test values ​​are shown below, *** = p-value < 0.01. [Figure 5H] Figure 5H shows confocal images of N. benthamiana leaves expressing the FUN-GFP construct and co-infiltrated with either MgCl2 (left) or ZnCl2 (right) 2 days before confocal observation. Scale bar = 5 μm. Chi-squared test values ​​are shown below; * = p-value < 0.05. [Figure 6A] Figures 6A-6E show the phylogenetic tree of FUN proteins and the relative expression patterns of soybean FUN orthologs. Figure 6A shows the phylogenetic tree of FUN and LjFUN-like genes identified using shoot.bio. Tree support values ​​are plotted at branch points. [Figure 6B] Figure 6B shows the relative expression (vertical axis) patterns across various tissues (horizontal axis) of the closest FUN soybean orthologs, Glyma.02G097900 and Glyma.01G084200. For each tissue, the expression level of Glyma.02G097900 is shown on the left, and the expression level of Glyma.01G084200 is shown on the right. [Figure 6C] Figure 6C shows a schematic diagram of the LjFUN protein, with the DNA-binding bZIP domain in the left box and the zinc-sensor domain in the right box. [Figure 6D] Figure 6D shows the first half of a protein alignment of selected orthologs of FUN and FUN-like proteins. [Figure 6E]Figure 6E shows the second half of the protein alignment of selected orthologs of FUN and FUN-like proteins. In Figures 6A, 6D, and 6E, plant species names correspond to the following abbreviations: Prunus persica: Prupe; Lotus japonicus: Lj; Glycine max: Glyma; Manihot esculenta: Manes; Gossypium raimondii: Gorai; Eucalyptus grandis: Eucgr; Brassica oleracea: Bol; Arabidopsis thaliana: AT; Solanum lycopersicum: Solyc; Aquilegia coerulea: Aqcoe; Amborella trichopoda: AmTr; Spirodela polyrhiza: Spipo; Musa acuminata: GSMUA; Zea mays: GRMZM; ​​Setaria italica: Seita; Triticum Aestivum: Traes; Hordeum vulgare: HORVU; and Oryza sativa: Os. In Figures 6D-6E, the protein sequences arranged from top to bottom are the consensus sequence (SEQ ID NO: 150), Lotus japonicus LjFUN (SEQ ID NO: 1), Glycine max Glyma.02G097900.1 (SEQ ID NO: 8), Glycine max Glyma.01G084200.1 (SEQ ID NO: 9), Glycine max Glyma.10G276100 (SEQ ID NO: 6), Glycine max Glyma.20G113600.1 (SEQ ID NO: 7), Lotus japonicus LjFUNL (SEQ ID NO: 83), and Arabidopsis thaliana AT1G68640.1 (SEQ ID NO: 4). [Figure 7]Figure 7 shows a phylogenetic tree of the Lotus japonicus NAC domain-containing protein Nac094 (labeled "LjNAC094") and orthologous NAC domain-containing proteins from other species. Species names and protein identifiers are indicated at the branch ends. Tree support values ​​are plotted at the branch points. [Figure 8A] Figures 8A-8C show evidence for a role of FUN in drought and heat tolerance. Figure 8A shows RNAseq counts of NAC094 (left graph) and HO1 (right graph) in root nodules of Medicago truncatula under the following conditions (from left to right): post-irrigation, day 2 of drought, and day 4 of drought. [Figure 8B] Figure 8B shows RNAseq counts of NAC094 (left graph) and HO1 (right graph) in Lotus japonicus root nodules, with the conditions (from left to right) being control, drought day 2, and drought day 4. [Figure 8C] Figure 8C shows nitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / hour / plant) in wild-type plants (left) and fun-3 mutant plants (right) before heat stress (gray box) and 7 days after heat stress (white box). 'ns' indicates no significant difference.

Claims

1. A transgenic plant or part thereof comprising one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions, wherein the FUN protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, S 1. A genetically modified plant or part thereof comprising a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:

82.

2. 2. The transgenic plant or part thereof according to claim 1, wherein the FUN protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID A genetically modified plant or part thereof comprising SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:

82.

3. A genetically modified plant or portion thereof, comprising one or more genetic modifications that decrease the activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein compared to the activity of the NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions, wherein the protein is an NRT3.1 protein and comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 74; wherein the protein is a bZIP28 protein and comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 75;wherein the protein is a NAC domain-containing protein, and is selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID or a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 73; wherein the protein is an HO1 protein and comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 77; wherein the protein is an NRT2.1 protein and comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 78; orwherein the protein is an AS1 protein, and the transgenic plant or part thereof comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 79;

4. 4. The transgenic plant or part thereof of claim 3, wherein the NRT3.1 protein comprises SEQ ID NO: 74; wherein the bZIP28 protein comprises SEQ ID NO: 75; and wherein the NAC domain-containing protein comprises SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:

76. NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID wherein the HO1 protein comprises SEQ ID NO: 77; wherein the NRT2.1 protein comprises SEQ ID NO: 78; or wherein the AS1 protein comprises SEQ ID NO:

79.

5. 1. A genetically modified plant or portion thereof, comprising one or more genetic modifications that decrease the activity or expression of one or more of a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein compared to the activity or expression of the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein in a control plant grown under the same conditions, wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 3 NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID A genetically modified plant or part thereof, wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein is selected from the group of polypeptides having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to NO:84, and wherein expression of the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein is enhanced in nodules of roots that do not have the one or more genetic modifications.

6. 6. The transgenic plant or part thereof according to any one of claims 1 to 5, wherein the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, and wherein optionally the reduction is due to knockout of a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knockout of a protein domain, introduction of a transcriptional repressor protein binding site, or knockout of a binding site in the promoter region of said gene; and / or the genetic modification comprises knockout of a protein gene, introduction of a premature stop codon in the coding region of a protein gene, silencing by RNAi, knockout of a protein domain, introduction of a transcriptional repressor protein binding site, or knockout of a binding site in the promoter region of said gene, preferably wherein said binding site is a transcriptional activator protein binding site or a TATA box.

7. 7. The genetically modified plant or part thereof of any one of claims 1 to 6, wherein the cultivation conditions comprise a moderate nitrate concentration, a high nitrate concentration, or a nitrate concentration around the plant that reduces or inhibits nitrogen fixation, and optionally wherein the nitrate concentration comprises from about 10 mM to about 250 mM nitrate, or at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate.

8. 8. The genetically modified plant or part thereof of claim 7, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same cultivation conditions, optionally wherein nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

9. 9. The genetically modified plant or part thereof of any one of claims 1 to 8, wherein the plant forms nodules and has an increased number of nodules, an increased hemoglobin content, or an increased acetylene reduction assay (ARA) activity compared to a control plant grown under the same cultivation conditions.

10. 1. A method for cultivating a genetically modified plant with increased nitrogen fixation under conditions of nitrate concentrations that inhibit nitrogen fixation around the roots of the plant, comprising: a) providing the genetically modified plant, wherein the genetically modified plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions; and b) cultivating the genetically modified plant at said nitrate concentration around the roots of the plant, wherein the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same cultivation conditions; A method for cultivating genetically modified plants, comprising:

11. 11. A genetically modified plant or part thereof produced by the method of claim 10, wherein the number of nodules or hemoglobin content is increased compared to a control plant grown under the same cultivation conditions, and wherein the number of pink nodules per plant compared to the control plant, the amount of acetylene (C) released per hour compared to the control plant, or the amount of hemoglobin released per hour compared to the control plant are increased. ２ H ２ ) is ethylene (C ２ H ４ ), measuring the amount of hemoglobin reduced to nitrogen (acetylene reduction assay (ARA)), and measuring micrograms of hemoglobin per plant compared to control plants.

12. 1. A method of cultivating a genetically modified plant capable of fixing nitrogen when grown under nitrogen fertilization conditions, comprising: a) providing the genetically modified plant, wherein the plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions; b) cultivating the plant under conditions in which the root zone of the plant contains a standard nitrate concentration; and c) creating conditions around the roots of the plant by using nitrogen fertilizer, which conditions include nitrate concentrations that inhibit nitrogen fixation, wherein the genetically modified plant exhibits increased nitrogen fixation compared to a control plant grown under the same conditions; A method for cultivating genetically modified plants, comprising:

13. 13. The method of claim 12, wherein the genetically modified plants are grown in an intercropping system with non-nitrogen fixing plants or in a rotational cropping system after non-nitrogen fixing plants.

14. A method for delaying root nodule senescence, comprising: a) providing the genetically modified plant, wherein the genetically modified plant or portion thereof comprises one or more genetic modifications that decrease the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, compared to the activity or expression of a FUN protein, an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same cultivation conditions, and wherein the one or more genetic modifications delay nodule senescence; and b) cultivating the genetically modified plants under stress conditions, wherein the genetically modified plants exhibit delayed nodule senescence compared to control plants cultivated under the same conditions; A method for delaying root nodule senescence, comprising:

15. 15. The method of claim 14, wherein the stress conditions are selected from the group of: moderate nitrate concentration, high nitrate concentration, peripheral nitrate concentration that promotes nodule senescence, moderate heat level, high heat level, peripheral heat level that promotes nodule senescence, moderate water deficit level, high water deficit level, peripheral water deficit level that promotes nodule senescence, moderate waterlogging level, high waterlogging level, peripheral waterlogging level that promotes nodule senescence.

16. 1. A method for inducing filament formation of a FUN protein, comprising: a) providing the FUN protein; and b) increasing the amount of zinc or manganese around said FUN protein, wherein the increased amount of zinc or manganese induces filament formation compared to a control FUN protein in an environment without increased amounts of zinc or manganese, and optionally, said filament formation is induced under high nitrate conditions and / or said method is performed in vitro; A method for inducing filament formation of a FUN protein, comprising:

17. 1. A method for inducing filament formation, comprising: a) providing a plant containing a FUN protein; and b) cultivating said plant under conditions of elevated zinc or manganese, wherein filamentation of said FUN protein in said plant is induced compared to FUN protein in a control plant in an environment without elevated amounts of zinc or manganese, and wherein optionally said plant comprises a genetic modification and / or said filamentation is induced under high nitrate conditions; A method for inducing filament formation, comprising:

18. 18. The method of claim 16 or 17, wherein the genetic modification reduces the activity of the FUN protein without eliminating the activity of the FUN protein, and / or the induction of filament formation increases nitrogen fixation in the genetically modified plant compared to a control plant grown under the same conditions, or reduces the activity, or inactivates the FUN protein.

19. 1. A method for adjusting nodule function in response to the amount of nitrogen available in the soil, comprising: a) providing a genetically modified plant containing a FUN protein with altered activation by nitrate; and b) cultivating said genetically modified plant under nitrate conditions, wherein said genetically modified plant has reduced activity or expression of FUN and / or reduced active forms of FUN compared to a WT plant grown under the same nitrate conditions, optionally comprising: (i) Altering the activation of FUN protein by nitrate includes silencing FUN, reducing FUN activity, knocking out FUN by mutation, knocking down the expression of FUN, knocking out or disrupting the promoter element of FUN, or a combination thereof; (ii) altering the activation of FUN proteins by nitrate involves manipulating environmental or cellular zinc or manganese concentrations, where the manipulation maintains the FUN proteins in the form of inactive filaments; or (iii) altering the activation of a FUN protein by nitrate includes genetically modifying the sequence of said FUN protein to alter its sensitivity to zinc or manganese; Methods for modulating root nodule function.

20. A method for producing a genetically modified plant that results in increased nitrogen fixation under conditions including nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic modifications that reduce the activity or expression of a FUN protein compared to the activity or expression of a FUN protein in a control plant grown under the same conditions.

21. A method for producing a genetically modified plant that results in increased nitrogen fixation under conditions including nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic modifications that reduce the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions.

22. A method for producing a genetically modified plant that results in increased nitrogen fixation under conditions including nitrate concentrations around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic modifications that reduce the activity or expression of one or more of the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to the activity or expression of the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions.

23. 10. A method for producing a genetically modified plant or part thereof according to any one of claims 1 to 9, comprising introducing into a plant cell a genetic modification that reduces or knocks out the activity or expression of a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, wherein the genetic modification is a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, or an AS1 protein operably linked to a promoter. , HO1 protein, NRT2.1 protein, or AS1 protein, and a second nucleic acid sequence encoding the same, wherein the first nucleic acid sequence is capable of reducing or knocking out a second nucleic acid sequence encoding the same, wherein the genetically modified plant is selected from one or more of the group consisting of alfalfa, bambara bean, pulses (e.g., kidney bean, black bean, etc.), black currant, chickpea, white clover, cowpea, forage legume, legume tree, lentil, lotus, lupine, Medicago, pea, pea, peanut, pigeon pea, soybean, Parasponia, alder, and elm.

24. 20. A method for producing a genetically modified plant or part thereof according to any one of claims 1 to 17, comprising genetically modifying the plant cell by transformation of the plant cell with one or more gene editing components targeting an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, an HO1 protein, an NRT2.1 protein, or an AS1 protein, wherein the endogenous nuclear genomic sequence or part thereof is knocked out, wherein the one or more gene editing components comprise a ribonucleoprotein complex targeting the nuclear genomic sequence; a vector comprising a sequence encoding a TALEN protein targeting the nuclear genomic sequence; a vector comprising a sequence encoding a ZFN protein targeting the nuclear genomic sequence; an oligonucleotide donor (OND) targeting the nuclear genomic sequence; or a vector comprising a sequence encoding a CRISPR / Cas enzyme and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

25. An expression vector or isolated DNA molecule, comprising: (i) a sequence encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; (ii) one or more nucleotides capable of reducing or knocking out the nucleic acid sequence encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof. an expression vector or isolated DNA molecule comprising: (i) a sequence, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; or (ii) one or more nucleotide sequences comprising a mutation in a gene encoding a FUN protein, a FUN-like protein, an HO1 protein, a NAC domain-containing protein, a bZIP28 protein, an NRT2.1 protein, an NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the mutation reduces or knocks out the activity or expression of the protein, and further wherein the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site.

26. 26. The expression vector or isolated DNA molecule of claim 25, wherein the protein is a FUN protein, and the FUN protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ or a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO: Contains NO:82;Alternatively, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9; wherein the protein is a FUN-like protein, and the FUN-like protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83, and SEQ ID NO: 84; wherein the FUN-like protein comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83, or SEQ ID NO: 84; and / or wherein the protein is an NRT3.1 protein, and the NRT3.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:74; wherein the protein is a bZIP28 protein, and the bZIP28 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:75; and wherein the protein is a NAC domain-containing protein, and the NAC domain-containing protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:59, SEQ ID NO:59, SEQ ID NO:59, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73;wherein the protein is an HO1 protein, and the HO1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 77; wherein the protein is an NRT2.1 protein, and the NRT2.1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 78; or wherein the protein is an AS1 protein, and the AS1 protein comprises a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 79; wherein the NRT3.1 protein comprises SEQ ID NO: 74; and wherein the bZIP28 protein comprises SEQ ID NO: 75;Here, the NAC domain-containing protein is selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID an expression vector or an isolated DNA molecule comprising SEQ ID NO: 73; wherein the HO1 protein comprises SEQ ID NO: 77; wherein the NRT2.1 protein comprises SEQ ID NO: 78, or wherein the AS1 protein comprises SEQ ID NO: 79; or wherein the NAC domain-containing protein comprises SEQ ID NO: 31, SEQ ID NO: 41, or SEQ ID NO: 42;

27. 27. A bacterial or Agrobacterium cell comprising the expression vector or isolated DNA molecule of claim 25 or 26.

28. 27. A transgenic plant, plant part, plant cell, or seed comprising the expression vector or isolated DNA molecule of claim 25 or 26.

29. 28. A kit comprising an expression vector or isolated DNA molecule according to claim 25 or 26, or a bacterial cell or Agrobacterium cell according to claim 27.

30. A method for increasing nitrogen fixation, delaying nodule senescence, or inducing FUN filament formation in a plant, comprising: (a) introducing a genetic modification via an expression vector or isolated DNA molecule described in claim 25 or 26; and, optionally, (b) treating the plant with zinc or manganese or cultivating the plant under high zinc, high manganese, or high nitrate conditions.

31. 25. The genome of a genetically modified plant comprising one or more genetic modifications in a genetically modified plant or part thereof according to any one of claims 1 to 9, or (ii) a genetically modified plant or part thereof produced by the method according to any one of claims 20 to 24.

32. A non-regenerable part or cell of a genetically modified plant or part thereof according to any one of claims 1 to 9.

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