Proteins for modulating symbiotic root nodule organ identity

Abstract

The present invention provides novel DNA molecules and constructs, including their nucleotide sequences, for expressing proteins in plants to promote symbiotic infections. The invention also provides plants and plant cells, transgenic plants, plant cells, plant parts, seeds, and commodity products comprising the DNA molecules operably linked to heterologous transcribable polynucleotides, as well as methods of their use.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 343,950, filed on May 19, 2022, the entire contents of which are hereby incorporated by reference.

[0003] Introduction of sequence listing

[0004] The sequence listing is contained in a file named "AGOE007WO_ST26.xml", contains 94 sequences, and is 121 KB in size (Microsoft ), and was created on May 18, 2023, which sequence listing is submitted electronically with this application and is incorporated herein by reference. Field of the Invention

[0005] The present invention relates to the fields of plant molecular biology and plant genetic engineering, DNA molecules for regulating gene expression in plants and proteins for improving agronomic performance. Background Art

[0006] Many farmers around the world are stressed by soil nitrogen or phosphate deficiencies, which can result in low yields or plant death. Symbiotic nitrogen-fixing bacteria can increase plant biomass under low nitrogen conditions. To initiate this process, legumes grow specialized nodules to house beneficial nitrogen-fixing bacteria, which provide ammonia to the plant in exchange for carbon. These symbiotic nodules, which have nodules containing cells housing nitrogen-fixing rhizobia, are morphologically and functionally distinct from lateral roots. However, the factors behind the determination of nodule organ identity are unclear. Therefore, methods are needed to promote symbiotically infected nodule formation in legumes and non-legumes in order to provide farmers with crops that exhibit improved agronomic performance under nitrogen-limited conditions.

[0007] Brief description of the invention

[0008] In one aspect, the present disclosure provides a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide fragment encoding a light-sensitive short hypocotyl protein or a fragment thereof, wherein: a. the protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; b. the protein comprises an amino acid sequence having at least 85%, or 90%, or 95%, or 98% or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 2 or 4; or c. the polynucleotide fragment hybridizes to a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7 or 8 under stringent hybridization conditions. In some embodiments, the provided recombinant DNA molecule is expressed in a plant cell to produce an increase in intercellular cortical infection, an increase in intracellular colonization of nitrogen-fixing bacteria, or an increase in nitrogen fixation of bacteria. In a further embodiment, the provided recombinant DNA molecule is operably linked to a vector, and the vector is selected from the group consisting of a plasmid, a phagemid, a bacmid, a cosmid, and a bacterial or yeast artificial chromosome. The disclosed recombinant DNA molecule can be present in a host cell, wherein the host cell is selected from the group consisting of a bacterial cell and a plant cell. For example, the bacterial host cell can be from a bacterial genus selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia. In a more specific example, the Bacillus is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus is Brevibacillus laterosperous, or the Escherichia is Escherichia coli.In other examples, the plant cell can be from a dicot or monocot plant cell, such as a plant cell selected from the group consisting of alfalfa, almond, Bambara peanut, banana, barley, bean, black currant, broccoli, blackberry, canola, cabbage, rapeseed, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cowpea, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, legume, garlic, grape, hemp, hops, indigo, leek, legume, legume tree, flatbread. Bean, lettuce, loblolly pine, lotus, lupine, millet, melon, alfalfa, nut, oat, olive, onion, ornamental plant, palm, forage, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, bean, radiata pine, radish, rapeseed, raspberry, red currant, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, zucchini, strawberry, beet, sugarcane, sunflower, liquidambar, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, walnut, watermelon, wheat and yam plant cell. In another aspect, a plant or part thereof comprising a recombinant DNA molecule as described herein is provided. In certain embodiments, the plant can be a monocot or a dicot, for example, a plant selected from the group consisting of alfalfa, almond, Bambara peanut, banana, barley, bean, black currant, broccoli, cabbage, blackberry, canola, rapeseed, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cowpea, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, legume grass, garlic, grape, hemp, hops, indigo, leek, legume, legume tree, apple, and more. Lentils, lettuce, loblolly pine, lotus, lupine, millet, melon, alfalfa, nuts, oats, olives, onions, ornamental plants, palms, forage, peas, peanuts, peppers, pigeon peas, pine, potatoes, poplars, pumpkins, beans, radiata pine, radish, rapeseed, raspberries, red currants, rice, rootstocks, rye, safflower, shrubs, sorghum, southern pine, soybeans, spinach, zucchini, strawberries, beets, sugarcane, sunflowers, liquidambar, sweet potatoes, switchgrass, tea, tobacco, tomatoes, triticale, lawn grass, walnuts, watermelons, wheat, and yams. In certain embodiments, the plants described herein or parts thereof exhibit differential expression of polynucleotide fragments encoding light-sensitive short hypocotyl proteins within 24 hours. For example, the plant or part thereof can express polynucleotide fragments encoding light-sensitive short hypocotyl proteins at increased levels during the first 12 hours of a 12 hour / 12 hour light / dark cycle. In another example, the plant or part thereof may express a polynucleotide fragment encoding a light-sensitive short hypocotyl protein at increased levels during the first 6 hours of a 12 hour / 12 hour light / dark cycle. In other embodiments, transgenic seeds comprising a recombinant DNA molecule described herein are provided.In another aspect, a method for producing offspring seeds comprising the recombinant DNA molecules provided herein is provided, the method comprising: a. planting a first seed comprising the provided recombinant DNA molecule; b. growing a plant from the seed of step a; and c. harvesting offspring seeds from the plant, wherein the harvested seeds comprise the recombinant DNA molecule. Other aspects provide plants susceptible to intercellular cortical infection or intracellular colonization of nitrogen-fixing bacteria, wherein the cells of the plant comprise the recombinant DNA molecules described herein. Also provided is a method for increasing intercellular cortical infection or intracellular colonization of nitrogen-fixing bacteria in a plant, the method comprising: a. expressing in a plant a photosensitive short hypocotyl protein or a fragment thereof having at least 70%, or 80%, or 90%, or 95%, or 99%, or about 100% sequence identity to SEQ ID NO: 2 or 4; b. contacting the plant with an effective amount of one or more rhizobia, arbuscular mycorrhizal fungi, or a combination thereof. In certain embodiments, the rhizobium is selected from the group consisting of Sinorhizobium meliloti, Mesorhizobium loti, Sinorhizobium fredii, Rhizobium sp. IRBG74 and NGR234, Bradyrhizobium sp. In further embodiments, the arbuscular mycorrhizal fungi are selected from the group consisting of R. irregularis, Glomus mosseae, and Funneliformis mosseae. In another aspect, a modified plant, plant seed, plant part, or plant cell is provided, comprising a genomic modification that modulates LSH1 or LSH2 activity compared to the activity of LSH1 or LSH2 in an otherwise identical plant, plant seed, plant part, or plant cell lacking the modification. In certain embodiments, the modification is present in at least one allele of an endogenous LSH1 or LSH2 gene. For example, the genomic modification may be in an endogenous LSH1 or LSH2 gene encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO: 2 or 4. In some instances, the modification may be in a transcribable region of an LSH1 or LSH2 gene. The plant, plant seed, plant part or plant cell may be heterozygous for the modification or may be homozygous for the modification. The modifications described herein may include deletions, insertions, substitutions, inversions, duplications or any combination thereof.For example, the modification may include at least 1, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 consecutive nucleotide deletions. The modified plants, plant seeds, plant parts, or plant cells provided herein may comprise a chromosomal sequence in a LSH1 or LSH2 gene that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 1, 3, 5, 6, 7, or 8 in regions other than deletions, insertions, substitutions, inversions, or duplications. Also provided is a method for producing a plant comprising a modified LSH1 or LSH2 gene, the method comprising: a. introducing a modification that modulates LSH1 or LSH2 activity into at least one target site in an endogenous LSH1 or LSH2 gene of a plant cell; b. identifying and selecting one or more corn plant cells of step a that comprise the modification in the LSH1 or LSH2 gene; and c. regenerating at least a first plant from the one or more cells selected in step b or their progeny comprising the modification. In another aspect, the present disclosure provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 84-93; b) a sequence comprising any one of SEQ ID NOs: 84-93; and c) a fragment of any one of SEQ ID NOs: 84-93, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA molecule. The recombinant DNA molecules described herein may comprise a sequence having at least 90% sequence identity with a DNA sequence of any one of SEQ ID NOs: 84-93, or a sequence having at least 95% sequence identity with a DNA sequence of any one of SEQ ID NOs: 84-93, or a sequence comprising a DNA sequence of any one of SEQ ID NOs: 84-93. The recombinant DNA molecules provided by the present disclosure may comprise a heterologous transcribable DNA molecule comprising an agronomic target gene. Also provided are transgenic plant cells comprising the recombinant DNA molecules disclosed herein, which may be monocotyledonous plant cells or dicotyledonous plant cells. Also provided are transgenic plants, parts thereof, progeny plants and transgenic seeds comprising the recombinant DNA molecules disclosed herein.The present disclosure also provides methods for producing a commodity, comprising obtaining a transgenic plant or part thereof according to the present disclosure, and producing a commodity therefrom, including methods for producing commodities such as: protein concentrate, protein isolate, grain, starch, seed, meal, flour, biomass or seed oil. Further provided is a method for expressing a transcribable DNA molecule, comprising obtaining a transgenic plant as described herein and cultivating the plant, wherein the transcribable DNA is expressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The patent or application file contains at least one drawing printed in color. Copies of the patent or patent application publication printed in color will be provided by the Office upon request and payment of the necessary fee.

[0010] Figure 1 : LSH1 and LSH2 are upregulated during early nodule organogenesis downstream of NIN. (A) Heatmaps showing selected genes induced during lateral root and nodule development. Fold changes compared to control are shown on a log2 scale, with a significance threshold of p value < 0.05. (B) Expression analysis of root segments treated with 100 nM 6-benzylaminopurine (BAP) for 24 h by qRT-PCR and normalized to HH3. Statistical comparison between mock (white bars) and BAP (black bars). Values ​​are mean ΔCt values ​​± SEM of 3 biological replicates for LSH1 and 2 biological replicates for LSH2 (Student t-test; asterisks indicate statistical significance *, P < 0.05; **, p < 0.01, ***, p < 0.001). (C) Expression patterns of LSH1 and LSH2 were visualized by GUS staining. Rhizobium-expressed LacZ was also stained. Ruthenium red markers distinguish cell walls. Asterisks indicate expression in vascular bundles, and arrows indicate expression in meristems. Scale bar: 500 μm.

[0011] Figure 2: LSH1 / LSH2 are required for nodule development and N-fixation. (AB) Images of dissected pedicels (A) and stipules (B) of WT, lsh1-2, lsh2-1, and lsh1-1 / lsh2-1. Scale bar: 500 μm. (C) Whole-mount images of WT, lsh1-1, lsh2-1, and lsh1-1 / lsh2-1 nodules 28 days after inoculation with S. meliloti. GUS staining (blue) indicates expression of the bacterial pNifH promoter. Scale bar: 500 μm. (D) Distribution of different nodule morphologies, expressed as the percentage of the total number of nodules per plant in WT and lsh1-1 (n=15), lsh2-1 (n=14), and lsh1-1 / lsh2-1 (n=13). (E) 28-day old nodule sections in WT, lsh1-1, and lsh1-1 / lsh2-1 with pNifH:GUS expression (blue) and ruthenium red staining of the cell wall. Scale bar: 500 μm.

[0012] Figure 3 .LSH genes are required for the development of nodule primordia that support bacterial colonization. (A) Images of WT and 1sh1 / 1sh2 nodule primordia at different developmental stages, including the first initial division (left), multilayering (center), and emergence of the primordium (right), 7 days after spray inoculation with rhizobia expressing lacZ (blue staining). Black arrows indicate that the infection thread is restricted in its progress into the inner tissue layers of the root. Squares and Figure 3 Legends to B relate. Scale bar: 500 μm. (B) Distribution of bacterial colonization phenotypes observed in WT, 1sh1, 1sh2, and 1sh1 / 1sh2 at 7 days post-inoculation, expressed as percentage of total primordium per plant, WT (n=30), 1sh1, 1sh2 (n=33), 1sh1 / 1sh2 (n=32). (C) Optical sections of WT and 1sh1 / 1sh2 roots 24 and 72 h post-inoculation with S. meliloti (n>30 per genotype and time point). At 72 hpi, Sm2011-mCherry bacteria are red, cell walls are white (fluorescent whitening agent), and EdU-labeled nuclei, indicating DNA replication, are green. White arrows indicate pericyte divisions. Scale bar: 50 μm.

[0013] Figure 4.LSH1 and LSH2 are required for upregulation of nodule organ identity genes and recruitment of shoot-expressed genes during nodule organogenesis. (A) Heat map of all differentially expressed genes (DEGs) in response to S. meliloti spot inoculation in WT and lsh1 and lsh1 / lsh2 at 24 and 72 hpi. Expression levels are expressed as log2 fold change (log2 fold change ≥ + / -1, p-value < 0.05). To compare the overall transcriptional response to S. meliloti spot inoculation between WT and mutants, all DEGs were ranked from the highest positive to the highest negative log2 fold change value. Absolute numbers represent the number of DEGs in each genotype. Percentages represent the proportion of DEGs in WT that are not expressed in mutants and are therefore dependent on LSH1 and LSH1 / 2. (B) Heatmap showing expression levels of selected functional groups of DEGs in WT, lsh1, lsh1 / lsh2, and nf-ya1 root sections at 24 and 72 hpi, as well as the response to combined ectopic expression of LSH1 / LSH2 (pUBI:LSH1 / LSH2) compared to empty vector in 3-week-old WT (M. truncatula) hairy roots. Fold changes compared to control are shown in log2 scale, with significance threshold of p value < 0.05. Green dots indicate that > ​​50% of the regulatory regions of these genes were bound by ectopically expressed LSH1-GFP in 4 biological replicates with q value < 0.05.

[0014] Figure 5 .LSH1 / LSH2 function in part via cortical activation of NF-YA1. (A) Expression patterns of NF-YA1 in WT and 1sh1 / 1sh2 were visualized by GUS staining (blue) in whole-mount images (left) and nodule sections (right). Rhizobium-expressed lacZ was stained magenta. Ruthenium red marked the cell wall in sections. Black asterisks indicate vascular expression restricted to the base of the nodule. Scale bar: 500 μm. (B) Heat map of all differentially expressed genes in response to midpoint inoculation of WT and nf-ya1 with S. meliloti at 24 and 72 hpi. Expression levels are expressed as log2 fold change (log2 fold change ≥ + / - 1, p value < 0.05). Figure 4Comparison of DEGs is depicted in A. Percentages represent the proportion of DEGs in WT that are not expressed in mutants and are therefore dependent on NF-YA1. (C). Comparison of up- and down-regulation of all DEGs according to lsh1 (light purple), lsh1 / lsh2 (dark purple), and nf-ya1 (green) at 24 and 72 hpi. Genes with a log2 fold change ≥ + / -1 and a p-value < 0.05 were included in this analysis. (D) Heatmap of selected functional groups of DEGs ectopically expressed in WT, lsh1, lsh1 / lsh2, and nf-ya1 at 24 and 72 h after S. meliloti spot inoculation compared to empty vector in 3-week-old WT (M. truncatula) hairy roots under non-symbiotic conditions. Fold changes compared to controls are expressed in log2 scale, with significance threshold at p value < 0.05. (E) Whole-mount images of nodules of hairy roots of lsh1 / lsh2 plants transformed with empty vector control, pLSH:LSH1, pLjUBI:NF-YA1, and combined pLSH1:NF-YA1 / pLSH2:NF-YA1 at 28 dpi with S. meliloti expressing pNifH:GUS. GUS staining (blue) indicates expression of the bacterial pNifH promoter. Scale bar: 500 μm.

[0015] Figure 6 .LSH1 / LSH2 promotes the expression of NOOT1 / NOOT2 and functions together with NOOT1 / NOOT2 in the same regulatory pathway. (A) Expression patterns of NOOT1 and NOOT2 in WT and lsh1 / 1sh2 nodules, visualized by GUS staining (blue) in whole-mount images (left) and nodule sections (right). LacZ expressed by rhizobia was stained magenta. Ruthenium red marks the cell wall in sections. Black asterisks indicate vascular expression at the base of nodules. Scale bar: 500 μm. (B). Heat map of all DEGs in WT, lsh1, lsh1 / lsh2, and noot1 / noot2 at 24 and 72 hpi. Expression levels are expressed as log2 fold change (log2 fold change ≥ + / - 1, p value < 0.05). Comparison as Figure 4 As shown in A. The percentages represent the proportion of DEGs in WT that are not differentially expressed in mutants and are therefore dependent on LSH1, LSH1 / 2, and NOOT1 / 2. (C) Comparison of up- and down-regulation of all DEGs according to lsh1 (light purple), lsh1 / lsh2 (dark purple), and noot1 / noot2 (orange) at 24 and 72 hpi. Genes with a log2 fold change of ≥+ / -1 and a p value <0.05 were included in the analysis.

[0016] Figure 7LSH1 / LSH2 and NOOT1 / NOOT2 act synergistically to confer nodule organ identity. (A) Whole-mount images of WT, lsh1, noot1 / noot2, and lsh1 / noot1 nodules 21 days after S. meliloti inoculation. GUS staining (blue) indicates bacterial pNifH expression. Scale bar: 500 μm. (B) Distribution of different nodule morphologies and N-fixation (pnifH:GUS staining) at 21 hpi, expressed as percentage of total nodules per plant, WT (n = 37), noot1 / noot2 (n = 44), lsh1 (n = 33), lsh1 / noot1 (n = 35), lsh1 / lsh2 (n = 40). (C) Optical sections of WT, noot1 / noot2, and lsh1 / noot1 root sections 72 h after rhizobium spot inoculation (n > 15 per genotype). Sm2011-mCherry bacteria are in red, cell wall is in white (fluorescent brightener), and EdU-labeled nuclei indicating DNA replication are in green. White arrows indicate pericyte divisions. Scale bar: 50 μm).

[0017] Figure 8 NF-YA1 and LSH1 / 2 have partially overlapping functions. (A) Distribution of nodule morphology / types 28 days after spray inoculation with S. meliloti, categorized as “white”, “blue – partial pnifH:GUS expression”, “blue – pnifH:GUS expression”, expressed as percentage per plant, for WT (n = 12) and nf-ya1-1 (n = 15). Boxplots show median (bold line), second to third quartiles (boxes), minimum and maximum range (lines), and outliers (single points). Student t-test showed that the distribution of different nodule types was dependent on genotype; asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01, ***, p < 0.001). (B) Distribution of total number of nodules per gram (g) root fresh weight of WT and nf-ya1-1 plants grown in terragreen:sand 28 days after inoculation with S. meliloti. Asterisks indicate statistical significance *, P < 0.05; **, P < 0.01, ***, P < 0.001, (Student t-test). (C) Ectopic expression of NF-YA1 partially rescues the lsh1-1 lsh2-1 nodule phenotype. The absolute number distribution and percentage of total nodules of "white", "partially blue" and "blue" nodules of transformed hairy roots grown in terragreen:sand 28 days after rhizobium spray inoculation.

[0018] Fig. 9LSH1 / 2 and NOOT1 / 2 regulate overlapping pathways to confer nodule organ identity. (A) Distribution of nodule morphology / type of WT (n = 37), noot1-1 noot2-1 (n = 44), lsh1-1 (n = 33), lsh1-1 noot1-1 (n = 35), and lsh1-1 lsh2-1 (n = 40) grown on plates 21 days after spray inoculation with S. meliloti, categorized by “white,” “blue-pnifH:GUS expressing,” “blue-pnifH:GUS expressing leafy and / or fused,” “white leafy and / or fused,” and “root-like transformation,” expressed as percentage per plant. Boxplots show median (bold line), second to third quartiles (boxes), minimum and maximum range (lines), and outliers (single points). One-way Kruskal-Wallis rank sum test showed that the distribution of nodule types depended on genotype (KW = 40.65, df = 3, p = 7.759e-09 (white), KW = 47.00, df = 3, p = 3.468e-10 (blue - pnifH:GUS expression), KW = 26.181, df = 3, p = 8.742e-06 (blue - pnifH:GUS leafy and / or fused) and KW = 41.64, df = 3, p = 4.783e-09 (white leafy and / or fused). Asterisks indicate that the means of lsh1-1, lsh2-1 and lsh1-1 lsh2-1 are significantly different compared to WT, Dunn's test (95% confidence level). (B) WT, noot1-1 grown on plates 21 days after inoculation with S. meliloti. Distribution of the total number of nodules per plant in noot2-1, lsh-1, lsh1-1 noot1-1, and lsh1-1lsh2-1 plants. One-way Kruskal-Wallis rank sum test showed that the total number of nodules per plant depended on the genotype (KW = 26.127, df = 3, p < 8.97e-06). Asterisks indicate that the means of lsh1-1 and lsh2-1 are significantly different compared to WT, Dunn's test (95% confidence level). (C) 7 dpi after inoculation with S. meliloti, the total number of nodules per plant in WT, noot1-1 noot1-2, lsh1-1, lsh1-1 The distribution of bacterial colonization phenotypes observed in seedlings grown on noot1-1 and lsh1-lsh2 plates is shown as a percentage of the total number of primordia per plant and categorized as “cortical infection in early multilayered primordia” (grey), “fully colonized emerging primordia” (black), “epidermal infection in early multilayered primordia” (white, hatched), “partially colonized emerging primordia” (grey hatched), and “uncolonized emerging primordia” (white), (WT n=23, nf-ya1-1 n=28).(D)(E) Optical sections of additional noot1-1 noot2-1 and lsh1-1 noot1-1 root sections 72 h after spot inoculation with S. meliloti. Infection with Sm2011-mCherry bacteria is labeled in red, Fluorescence Brightener (white) marks the cell wall, and EdU-labeled nuclei (green) indicate DNA replication. Scale bar: 50 μm. (F) Rhizobium-induced genes that are dependent on NOOT1 / 2 function overlap 99% with LSH1 / 2-dependent rhizobium-induced genes. Heat map of all differentially expressed genes in response to spot inoculation with S. meliloti in WT, lsh1-1 single, lsh1-1 lsh2-1 double mutant, and noot1-1 noot2-1 double mutant at 24 and 72 hpi. Expression levels are shown as log2 fold change (log2 fold change ≥ + / - 1, p value < 0.05). To compare the overall transcriptional response of WT and mutants to S. meliloti spot inoculation, all differentially expressed genes were classified based on their log2 fold change. Absolute numbers represent the number of DEGs in each genotype. Percentages represent the proportion of differentially expressed genes in WT that were not differentially expressed in mutants and therefore depended on LSH1 and LSH1 / 2 and NOOT1 / 2. (G) Comparison of up- and down-regulation of all differentially expressed genes according to lsh1-1 (light purple), lsh1-1 lsh1-2 (dark purple) and noot1-1 noot2-1 (orange) at 24 and 72 hpi. Genes with log2 fold change ≥+ / -1, p value <0.05 were included in this analysis. (H) Comparison of all differentially expressed genes dependent on lsh1-1 and / or lsh1-1 lsh1-2 and noot1-1 noot2-1 under non-symbiotic conditions with those differentially expressed in response to ectopic expression of combined LSH1 and LSH2 or combined NOOT1 and NOOT2 under the constitutive Luba promoter. Genes with log2 fold change ≥ + / - 1 and p value < 0.05 were included in this analysis.

[0019] Fig.10 . Amino acid sequences of LSH1 (SEQ ID NO: 2) and LSH2 (SEQ ID NO: 4). The ALOG domain in LSH1 (bold) extends from residue 52 to residue 179; and the ALOG domain in LSH2 (bold) extends from residue 64 to residue 191.

[0020] Fig.11 Ectopic LSH1 expression was sufficient to increase root length and diameter compared with control plants.

[0021] Fig.12 Ectopic LSH1 expression is sufficient to inhibit the progression and emergence of lateral root primordia.

[0022] Fig.13 . Overexpression of LSH1 in Medicago truncatula roots after inoculation with rhizobia.

[0023] Fig.14 Simultaneous overexpression of LSH1 and LSH2 in Medicago truncatula roots after inoculation with rhizobia.

[0024] Fig.15 .Mt LSH1 (Medtr1g069825) and Mt LSH2 (Medtr7g097030) represent the original Medicago LSH1 and LSH2 gene coding regions. HvOptMtLSH1 and HvOptMtLSH2 represent barley codon-optimized versions of LSH1 and LSH2. pOsUBI3, pPvUBI2, and pZmUBI represent the Oryza sativa (rice), Panicum virgatum (switchgrass), and Zea mays (maize) versions of the ubiquitin promoter, respectively. t35S represents the cauliflower mosaic virus (CaMV) 35S terminator; tRbcS represents the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (rbcS) terminator. nptII represents the neomycin phosphotransferase selection system.

[0025] Fig.16 .Morphological comparison of NLSs collected from roots transformed with negative GUS control, MtLSH1, and MtLSH2 in harvest 1. (A,D) NLSs harvested from negative GUS control. (B,E) NLSs harvested from roots transformed with pOsUbi::MtLSH1. (C,F) NLSs harvested from roots transformed with pOsUbi::Mt LSH2. (AC) Sections were stained with Toluidine Blue-O. (DF) Maximum projections of Z-stack confocal images of NLSs. Pink indicates the mCherry signal of the transformation visual marker. Scale bar in each image represents 200 μm. Vibratome section thickness was 100 μm.

[0026] Fig.17 .Comparison of morphology of NLSs collected from negative GUS control and pOsUbi::MtLSH1 transformed roots after auxin treatment in harvest 2. (A,C,E) NLSs harvested from negative GUS control. (B,D,F) NLSs harvested from pOsUbi::MtLSH1 transformed roots. (AB) Whole-mount images of NLSs. (CD) Sections were stained with Toluidine Blue-O. (EF) Maximum projection of Z-stack confocal images of NLSs. Pink indicates mCherry signal of the transformation visual marker. (CF) Vibratome section thickness was 100 μm. (AF) Scale bar in each image represents 200 μm.

[0027] Fig.18 .Comparison of morphology of NLSs collected from negative GUS control and pOsUbi::HvOptMtLSH1 transformed roots in harvest 1. (A,C) NLSs harvested from negative GUS control. (B,D) NLSs harvested from pOsUbi::HvOptMtLSH1 transformed roots. (AB) Sections were stained with Toluidine Blue-O. (CD) Maximum projections of Z-stack confocal images of NLSs. Pink indicates mCherry signal of the transformation visual marker. (AD) Vibratome sections were 100 μm thick. Scale bar in each image represents 200 μm.

[0028] Fig.19 .Comparison of the morphology of NLSs collected from negative GUS control and Ubi::HvOptMtLSH1 transformed roots after harvest 2 auxin treatment. (A,C) NLSs harvested from negative GUS control. (B,D) NLSs harvested from pOsUbi::HvOptMtLSH1 transformed roots. (AB) Sections were stained with Toluidine Blue-O. (CD) Maximum projections of Z-stack confocal images of NLSs. Pink indicates the mCherry signal of the transformation visual marker. (AD) Vibratome sections were 100 μm thick. Scale bar in each image represents 200 μm.

[0029] Fig. 20 Quantification of NLS in harvests before and after auxin treatment. (A) Quantification of the number of NLS per plate collected from harvest 1. (B) Quantification of the frequency of NLS in each root collected from harvest 1. NLS frequency = number of NLS / length of root (in cm).

[0030] Fig.21 . Domain tree showing protein sequences having SEQ ID NOs: 9-83 that contain conserved ALOG domain regions.

[0031] Fig. 22 .Chip-Seq data showing a high-confidence NIN binding site upstream of LSH1.

[0032] Fig.23 .ChiP-Seq data showing putative direct targets of LSH1 CRE1, IPT1, RR19, CKX3, PIN1, STYLISH, PINOID, and NOOT1.

[0033] Sequence Description

[0034] SEQ ID NO: 1 is the cDNA sequence encoding Medicago truncatula LSH1 protein.

[0035] SEQ ID NO: 2 is the polypeptide sequence of the Medicago truncatula LSH1 protein encoded by SEQ ID NO: 1. The ALOG domain extends from residue 52 to residue 179.

[0036] SEQ ID NO: 3 is the cDNA sequence encoding Medicago truncatula LSH2 protein.

[0037] SEQ ID NO: 4 is the polypeptide sequence of the Medicago truncatula LSH2 protein encoded by SEQ ID NO: 3. The ALOG domain extends from residue 64 to residue 191.

[0038] SEQ ID NO: 5 is the gDNA sequence encoding the Medicago truncatula LSH1 protein.

[0039] SEQ ID NO: 6 is the gDNA sequence encoding Medicago truncatula LSH2 protein.

[0040] SEQ ID NO: 7 is the barley (Hordeum vulgare) codon-optimized nucleotide sequence encoding the LSH1 protein.

[0041] SEQ ID NO: 8 is the barley codon-optimized nucleotide sequence encoding the LSH2 protein.

[0042] SEQ ID NOs: 9-84 are polypeptide sequences comprising a conserved ALOG domain.

[0043] SEQ ID NO:85 is the nucleotide sequence of the LSH1 promoter region including the putative NIN binding site at nucleotide 5,260.

[0044] SEQ ID NO:86 is the nucleotide sequence of LSH1 5'UTR.

[0045] SEQ ID NO:87 is the nucleotide sequence of the LSH1 intron.

[0046] SEQ ID NO:88 is the nucleotide sequence of LSH1 3'UTR.

[0047] SEQ ID NO: 89 is the nucleotide sequence of the LSH1 downstream terminator region.

[0048] SEQ ID NO:90 is the nucleotide sequence of the LSH2 promoter region including the putative NIN binding site at nucleotide 5000.

[0049] SEQ ID NO:91 is the nucleotide sequence of LSH2 5'UTR.

[0050] SEQ ID NO:92 is the nucleotide sequence of the LSH2 intron.

[0051] SEQ ID NO:93 is the nucleotide sequence of LSH2 3'UTR.

[0052] SEQ ID NO:94 is the nucleotide sequence of the LSH2 downstream terminator region. DETAILED DESCRIPTION OF THE INVENTION

[0054] Soil nitrogen or phosphorus deficiencies can lead to crop yield loss or death, which poses a major challenge worldwide. Symbiotic nitrogen-fixing bacteria can alleviate this challenge by increasing plant biomass under low nitrogen conditions. Leguminosae plants grow specialized nodules to house beneficial nitrogen-fixing bacteria that provide ammonia to the plant in exchange for carbon. These symbiotic nodules are distinct from lateral roots in morphology and function, consisting of nitrogen-fixing rhizobia housed endosymbiotically and providing favorable conditions for the biological nitrogen fixation process.

[0055] However, the gene regulatory processes that form nodules that are distinct from lateral roots have not been understood before. Therefore, the inventors investigated the gene regulatory programs that distinguish symbiotic nodules from lateral roots in order to identify key regulatory factors that establish nodule organ identity, i.e., an organ that can support infection and adaptation of nitrogen-fixing bacteria. The present disclosure provides two previously unknown members of the shoot-associated photosensitive short hypocotyl (LSH) transcription factor family and novel regulators of nodule organ identity, LSH1 and LSH2.

[0056] As described in the present disclosure, LSH1 and LSH2 are required for the development of functional nodule primordia that can support bacterial intercellular cortical infection, intracellular colonization, and nitrogen fixation. For example, the present disclosure demonstrates that LSH1 and LSH2 are required for the development of symbiotic nodules that can host bacteria intracellularly and provide an environment for nitrogen fixation. LSH1 / 2 functions include, for example, cortex-specific promotion of previously identified nodule organ identity regulators NF-YA1 and NOOT1 / 2, and thus position LSH1 / 2 as a key integrator of nodule organ identity establishment and maintenance downstream of NIN.

[0057] Thus, in some embodiments, the present invention provides a recombinant DNA molecule comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to a LSH1 polynucleotide (such as SEQ ID NO: 1, 5 or 7) or a LSH2 polynucleotide (such as SEQ ID NO: 3, 6 or 8) or a variant or fragment thereof. Also provided are plants heterologously expressing or overexpressing LSH1 or LSH2 proteins that promote symbiotic infection, such as SEQ ID NO: 2, 4 or a variant or fragment thereof. In other embodiments, further provided are plants heterologously expressing or overexpressing protein sequences that promote symbiotic infection, such as SEQ ID NO: 9-83, comprising an ALOG domain.

[0058] Symbiotic bacteria

[0059] The present invention provides DNA molecules encoding proteins that, when expressed in plants, can promote symbiotic bacterial infection, or express transcribable polynucleotide molecules that promote symbiotic bacterial infection and / or symbiotic bacterial nitrogen fixation. As used herein, "symbiotic bacteria" include nitrogen-fixing bacteria. For example, rhizobia are bacteria found in the soil that infect the roots of leguminous plants and colonize in nodules involved in nitrogen utilization. As used herein, "rhizobia" refers to any nitrogen-fixing nutrient bacteria that fix atmospheric nitrogen in plant roots.

[0060] Plants comprising the recombinant DNA molecules described herein can be inoculated with nitrogen-fixing bacteria to produce improved agronomic effects, including improved plant growth or increased yield or biomass under reduced nitrogen conditions. Symbiotic bacteria that can be used for the disclosed plants include, but are not limited to, Mesorhizobium euryale, Sinorhizobium ferretii, Rhizobium genus IRBG74 and NGR234, and Bradyrhizobium genus. Therefore, the recombinant DNA molecules provided herein can be expressed in plants, and their expression amounts can effectively produce increased intercellular cortical infection, increased intracellular colonization of symbiotic bacteria, or increased nitrogen fixation of symbiotic bacteria compared to wild-type or control plants. In addition, the recombinant DNA molecules provided herein can be expressed in plants, and their expression amounts can effectively produce rhizobium infection patterns; nodule structures, such as clustered multi-leaf nodules; upregulation of nodule organ identity genes; recruitment of bud expression genes during nodule organogenesis; detectable amounts of rhizobium-expressed LacZ; or promote cell proliferation, host cell differentiation, or endosymbiotic colonization in the primordium cell layer derived from the mesocortex of the primary root.

[0061] Compared to WT; compared to wild-type or control plants, multiple vascular bundles branch out from the base and connect to the primary root vascular system. According to further embodiments, modified plants are provided, which have 5%-20%, 5%-25%, 5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%, 75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80% or 10%-75% increased intercellular cortical infection, increased intracellular colonization of symbiotic bacteria or increased nitrogen fixation by symbiotic bacteria compared to wild-type or control plants.

[0062] DNA molecules

[0063] As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer or polynucleotide molecule of deoxyribonucleotide bases, read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The terms used herein correspond to Title 37 of the United States Code of Federal Regulations § 1.822 and are listed in the tables in WIPO Standard ST.25 (1998), Appendix 2, Table 1 and Table 3.

[0064] As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that would not naturally exist together without human intervention. For example, a recombinant DNA molecule can be a DNA molecule composed of at least two DNA molecules that are heterologous to each other, a DNA molecule containing a DNA sequence that is different from a naturally occurring DNA sequence, a DNA molecule containing a synthetic DNA sequence, or a DNA molecule that is incorporated into a host cell DNA by genetic transformation or gene editing.

[0065] As used herein, the term "separated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules, and the other molecules are usually associated with it in its natural or natural state. In one embodiment, the term "separated" refers to a DNA molecule that is at least partially separated from other molecules that are usually associated with it in a natural or natural state. Therefore, DNA molecules fused to regulatory or coding sequences that are not usually related (for example, as a result of recombinant technology) are considered to be separated in this article. When these molecules are integrated into the chromosome of the host cell or are present in a nucleic acid solution together with other DNA molecules, they are considered to be separated because they are not in a natural state.

[0066] The polynucleotides or polypeptides provided herein may further include two or more molecules that are heterologous to each other. As used herein, the term "heterologous" refers to a combination of two or more polynucleotide molecules or two or more polypeptide molecules, and this combination does not usually exist in nature. For example, the two molecules can be derived from different species and / or the two molecules can be derived from different genes, such as different genes derived from the same species or the same genes derived from different species. In some instances, the promoter is heterologous relative to the transcribable polynucleotide molecules that can be operably connected (if this combination does not usually exist in nature), that is, the transcribable polynucleotide molecules are not naturally occurring, and are operably connected to the promoter molecules.

[0067] Many methods well known to those skilled in the art can be used for separating and operating DNA molecules disclosed in the present invention or its fragment.For example, PCR (polymerase chain reaction) technology can be used for increasing specific starting DNA molecules and / or producing variants of original molecules.DNA molecules or its fragments can also be obtained by other technologies, such as directly synthesizing fragments by chemical methods, as usually implemented using automated oligonucleotide synthesizers.

[0068] As used herein, the terms "percent sequence identity," "percent identity," or "% sequence identity" refer to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (e.g., "query") sequence (or its complement) as compared to a test (e.g., "subject") sequence (or its complement) when the two sequences are optimally aligned. Optimal sequence alignments are generated by manually aligning two sequences, e.g., a reference sequence and another sequence, so that appropriate internal nucleotide insertions, deletions, or gaps are made to maximize the number of nucleotide matches in the sequence alignment. Optimal alignments for aligning sequences over a comparison window are well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search by similarity method of Pearson and Lipman, and by computerized implementations of these algorithms, such as the .NET 5000 programmable gatekeeper with default parameters. Wisconsin (Accelrys Inc., Sandiego, CA), MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715), and GAP, BESTFIT, FASTA, and TFASTA, which are part of the sequence analysis software packages of MUSCLE (version 3.6) (RC Edgar, Nucleic Acids Research (2004) 32(5): 1792-1797). The "identity score" of an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more sequences can be to the full-length sequence or a portion thereof, or to a longer sequence. As used herein, the term "sequence identity" refers to the degree of identity of two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences. As used herein, the term "reference sequence" may refer, for example, to a polynucleotide sequence provided as SEQ ID NO: 1, 3, 5, 6, 7 or 8, or a polypeptide sequence of SEQ ID NO: 2 or 4. "Reference sequence" may also refer to a polypeptide sequence of SEQ ID NO: 9-83.

[0069] Thus, one embodiment of the invention is a recombinant DNA molecule comprising a sequence having at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to a reference sequence when optimally aligned to the reference sequence, provided herein being a polynucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7, or 8. In specific embodiments, such sequences can be defined as having an activity of the reference sequence, e.g., an activity of SEQ ID NO: 1, 3, 5, 6, 7, or 8.

[0070] Similarly, another embodiment of the invention is a polypeptide molecule comprising a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to a reference sequence when optimally aligned with the reference sequence, the reference sequence provided herein being a polypeptide sequence of SEQ ID NO: 2, 4, or 9-83. In a specific embodiment, such a sequence can be defined as having an activity of the reference sequence, such as an activity of SEQ ID NO: 2, 4, or 9-83.

[0071] Also provided are fragments of the polynucleotide sequences provided herein, such as fragments of the polynucleotide sequences of SEQ ID NO: 1, 3, 5, 6, 7 or 8. In specific embodiments, fragments of polynucleotide sequences are provided that comprise at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides or longer of a DNA molecule of SEQ ID NO: 1, 3, 5, 6, 7 or 8 or a sequence encoding SEQ ID NO: 2 or 4. Methods for generating such fragments from starting molecules are well known in the art. A fragment (which may be a functional fragment) of a polynucleotide sequence provided herein may comprise the activity or function of the base sequence.

[0072] The disclosed sequences can specifically hybridize to a target DNA sequence under stringent hybridization conditions. In certain embodiments, the polynucleotides disclosed herein can hybridize to a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7 or 8 under stringent hybridization conditions. Stringent hybridization conditions are known in the art and are described, for example, in MR Green and J Sambrook, Molecular cloning: a laboratory manual, 4 th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). As used herein, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, the two nucleic acid molecules can specifically hybridize to each other. If nucleic acid molecules show complete complementarity, one of the nucleic acid molecules is the "complement" of another nucleic acid molecule. As used herein, if each nucleotide of the first molecule is complementary to each nucleotide of the second molecule when aligned, the two molecules show "complete complementarity". If two molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under at least conventional "low stringency" conditions, they are "minimally complementary". Similarly, if molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under conventional "high stringency" conditions, they are "complementary". Therefore, deviation from complete complementarity is allowed as long as this deviation does not completely exclude the ability of molecules to form double-stranded structures.

[0073] Suitable stringent conditions that promote DNA hybridization, such as 6.0x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at 2.0x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0x SSC at 50°C to a high stringency of about 0.2x SSC at 50°C. In addition, the temperature in the washing step can be increased from low stringency conditions at room temperature (about 22°C) to high stringency conditions at about 65°C. Both temperature and salt can be changed, or temperature or salt concentration can be kept constant while changing the other variable.

[0074] Also contemplated are recombinant polynucleotide sequences encoding fragments of the polypeptide sequences provided herein, including polynucleotide sequences encoding fragments of polypeptide sequences selected from SEQ ID NO: 2 or 4. In a specific embodiment, a polypeptide fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 221 or more of a polypeptide molecule of SEQ ID NO: 2 or 4 is provided. Methods for generating such fragments from starting molecules are well known in the art. Fragments of the polynucleotide sequences provided herein (which may be functional fragments) may retain the activity or function of the base sequence.

[0075] With respect to polypeptide sequences, as used herein, the term "variant" refers to a second polypeptide sequence that is similar but not identical to a first polypeptide sequence in composition, but the second polypeptide sequence still maintains the general functionality of the first polypeptide sequence, i.e., the same or similar activity. The variant can be a shorter or truncated form of the first polypeptide sequence, and / or a sequence-altered form of the first polypeptide sequence, such as a sequence with different amino acid deletions, substitutions and / or insertions. Variants having a percentage identity with a sequence disclosed herein can have the same activity as the base sequence. For example, a transcribable polynucleotide molecule can encode a protein or a variant of a protein or a fragment of a protein that is functionally defined as maintaining activity in transgenic host cells (including plant cells), plant parts, explants, and whole plants.

[0076] Similarly, with respect to polynucleotide sequences, as used herein, the term "variant" refers to a second polynucleotide sequence that is similar but not identical to a first polynucleotide sequence in composition, but the second polynucleotide sequence still retains the general functionality of the first polynucleotide sequence, i.e., the same or similar activity. The variant can be a shorter or truncated form of the first polynucleotide sequence, and / or a sequence-altered form of the first polynucleotide sequence, such as a sequence with different nucleotide deletions, substitutions, and / or insertions. A variant having a percentage identity with a sequence disclosed herein can have the same activity as the base sequence. For example, a variant polynucleotide can encode a protein sequence that is identical or similar to the base sequence, or have the same or similar gene regulatory activity as the base sequence.

[0077] As used herein, "modulation" of expression refers to the process by which overexpression or suppression of a polynucleotide or protein is achieved.

[0078] As used herein, the term "overexpression" as used herein refers to an increase in the expression level of a polynucleotide or protein in a plant, plant cell or plant tissue at any developmental or temporal stage of a gene compared to expression in a wild-type plant, cell or tissue. Overexpression can occur in plant cells that normally lack expression of a polypeptide functionally equivalent or identical to a polypeptide of the present invention. Overexpression can also occur in plant cells in which endogenous expression of a polypeptide of the present invention or a functionally equivalent molecule normally occurs, but this normal expression is at a lower level. Therefore, overexpression results in a higher than normal yield, or "overproduction" of the polypeptide in a plant, cell or tissue.

[0079] Constructs

[0080] As used herein, the term "construct" refers to any recombinant polynucleotide molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genome integration or autonomous replication, comprising a polynucleotide molecule in which one or more polynucleotide molecules are linked (i.e., operably linked) in a functionally operable manner. As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. The term includes expression cassettes isolated from any of the above molecules.

[0081] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, wherein the molecules are arranged so that the first molecule affects the function of the second molecule. Two molecules may or may not be part of a single continuous molecule and may or may not be adjacent. For example, if the promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, then the promoter is operably linked to the transcribable polynucleotide molecule.

[0082] In one embodiment, the construct of the present invention can be provided as a double Ti plasmid border DNA construct having the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmid isolated from Agrobacterium tumefaciens, comprising T-DNA, which together with the transfer molecules provided by the Agrobacterium tumefaciens cells, enables the T-DNA to be integrated into the genome of the plant cell (see, e.g., U.S. Pat. No. 6,603,061). The construct can also contain a plasmid backbone DNA fragment that provides replication function and antibiotic selection in bacterial cells, such as an E. coli replication origin (such as ori322), a broad host range replication origin (such as oriV or oriRi), and a coding region for a selectable marker (such as Spec / Strp encoding Tn7 aminoglycoside adenyltransferase (aadA) that confers spectinomycin or streptomycin resistance), or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host strain is typically A. tumefaciens ABI, C58 or LBA4404; however, other strains known to those skilled in the art of plant transformation may work in the present invention. For example, Agrobacterium rhizogenes ARqua1.

[0083] Methods for assembling and introducing constructs into cells are known in the art so that transcribable polynucleotide molecules are transcribed into functional mRNA molecules, which are translated and expressed as protein products. Conventional compositions and methods for preparing and using constructs and host cells for practicing the present invention are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3 rdEdition Volumes 1, 2, and 3 (2000) J. Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press. Methods for preparing recombinant vectors particularly suitable for plant transformation include, but are not limited to, those described in the entirety of U.S. Pat. Nos. 4,971,908; 4,940,835; 4,769,061; and 4,757,011. These types of vectors have also been described in the scientific literature (see, for example, Rodriguez, et al., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, (1988) and Glick, et al., Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, FL. (1993)). Typical vectors for nucleic acid expression in higher plants are well known in the art, including vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers, et al., Methods in Enzymology 153:253-277 (1987)). Other recombinant vectors useful for plant transformation, including the pCaMVCN transfer control vector, have also been described in the scientific literature (see, e.g., Fromm, et al., Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985)).

[0084] The constructs provided herein may further comprise other elements useful for regulating or modulating the expression of transcribable polynucleotides, including promoters, leaders, enhancers, introns and 3'UTR sequences. The constructs provided herein may also comprise one or more marker sequences for identifying the constructs in plant cells, plant tissues or plants.

[0085] Transgenic plants

[0086] Constructs, expression cassettes and vectors comprising DNA molecules disclosed herein can be constructed and introduced into plant cells according to transformation methods and techniques known in the art. For example, Agrobacterium-mediated transformation is described in U.S. Patent Application Publication Nos. 2009 / 0138985A1 (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257A1 (sugar beet), U.S. Patent Nos. 5,750,871 (rapeseed), 7,026,528 (wheat) and 6,365,807 (rice), and Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugar cane), the entire contents of which are incorporated herein by reference. Transformed cells can be regenerated into transformed plants expressing polypeptides disclosed herein and demonstrated activity by bioassays described herein and known in the art. Plants can be derived from plant cells by regeneration, seed, pollen or meristem transformation techniques. Methods for transforming plants are known in the art.

[0087] The term "plant cell" or "plant" may include, but is not limited to, dicotyledonous or monocotyledonous plants. In certain embodiments, the plants provided herein are leguminous plants, including but not limited to beans, soybeans, peas, chickpeas, peanuts, lentils, lupines, mesquite, carob, tamarind, alfalfa and clover. The plants provided herein may also be non-legumes.

[0088] The term "plant cell" or "plant" may also include, but is not limited to, alfalfa, almond, Bambara peanut, banana, barley, bean, black currant, broccoli, cabbage, blackberry, canola, rapeseed, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn (i.e., corn such as sweet corn or field corn), clover, cotton, cowpea, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, legume, garlic, grape, hemp, hops, indigo, leek, legume, legume tree, lentil, lettuce, lettuce, loblolly pine, lotus, lupine, millet, melon, alfalfa, nuts, oats, olives, onions, ornamentals, palm, forage grasses, peas, peanuts, pepper, pigeon pea, pine, potato, poplar, pumpkin, bean, radiata pine, radish, rapeseed, raspberry, red currant, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, zucchini, strawberry, sugar beet, sugar cane, sunflower, liquidambar, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, walnut, watermelon, wheat and yam plant cells or plants.

[0089] The term "plant cell" or "plant" can also include, but is not limited to, cassava (e.g., manioc, yucca, Manihot esculenta), yam (e.g., Dioscorea rotundata, Dioscorea alata, Dioscorea trifida, Dioscorea sp.), sweet potato (e.g., Ipomoea batatas), taro (e.g., Colocasia esculenta), sorrel (Oxalistuberosa), corn (e.g., Zea mays), rice (e.g., Indian rice, Japonica rice, Fragrant rice, Glutinous rice, Oryza sativa, Oryza glaberrima), wild rice (e.g., Zizania spp., Porteresia spp.), barley (e.g., Hordeum vulgare), sorghum (e.g., Sorghum bicolor), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millet, sorghum (Eleusine coracana), Panicum sumatrense, Panicum milaceum, Setaria italica, Pennisetum glaucum, Digitaria spp., Echinocloa sp.), teff (e.g., Eragrostis tef), oats (e.g., Avenasativa), triticale (e.g., Triticosecaleschlanstedtense Wittmack, Triticosecale neoblaringhemii A. Camus, Triticosecaleneoblaringhemii A.Camus, rye (e.g., Secale cereale, Secale cereanum), wheat (e.g., common wheat, spelt, durum wheat, einkorn wheat, emmer wheat, kamut, winter wheat (Triticum aestivum), spelt (Triticum spelta), durum wheat (Triticum durum), Urartu wheat (Triticum urartu), einkorn wheat (Triticum monococcum), oriental wheat (Triticum turanicum), Triticum spp.), Trema spp. (e.g., Tremacannabina, Trema cubense, Trema discolor, Trema domingensis, Trema integerrima, Trema lamarckiana, Trema micrantha, Trema orientalis, Tremaphilippinensis, Trema strigilosa), Trema tomentosa, Tremalevigata), apples (e.g. Malus domestica, Malus pumila, Pyrus malus), pears (e.g. Pyrus communis, Pyrus × bretschneideri, Pyrus pyrifolia, Pyrus sinkiangensis, Pyrus pashia), Pyrus spp.)), plums (e.g., sweet plum, green plum, black plum, European plum (Prunus domestica), oil nai (Prunus salicina), plum (Prunus mume)), apricots (e.g., wild apricot (Prunus armeniaca), Briancon plum (Prunus brigantine), Northeast apricot (Prunus mandshurica)), peaches (e.g., peach (Prunus persica)), almonds (e.g., almonds (Prunus dulcis), almonds (Prunus amygdalus)), walnuts (e.g., Persian walnut, English walnut, black walnut, English walnut (Juglans regia), black walnut (Juglans nigra), gray walnut (Juglans cinerea), California walnut (Juglans californica)), strawberries (e.g., cultivated strawberry (Fragaria×ananassa), Chilean strawberry (Fragaria chiloensis), Virginia strawberry (Fragaria virginiana), wild strawberry (Fragaria vesca), raspberry (e.g., European red raspberry, black raspberry, raspberry (Rubus idaeus L.), black raspberry (Rubus occidentalis), red raspberry (Rubus strigosus)), blackberry (e.g., evergreen blackberry, Armenian blackberry, European blackberry (Rubus fruticosus), North American blackberry (Rubus ursinus), cut leaf blackberry (Rubus laciniatus), sharp tooth blackberry (Rubus argutus), Armenian blackberry (Rubus armeniacus), European blackberry (Rubus plicatus), elm leaf blackberry (Rubus ulmifolius), Allegheny blackberry (Rubus allegheniensis), Eubatus sect.Moriferi & Ursini), redcurrants (e.g. white currant, redcurrant (Ribes rubrum), blackcurrants (e.g. black currant, blackcurrant (Ribes nigrum)), gooseberries (e.g. gooseberries (Ribes uva-crispa), gooseberries (Ribes grossulari), European gooseberries (Ribes hirtellum)), melons (e.g. watermelon, wax gourd, casaba melon, Roman melon, honeydew melon, honeydew melon, watermelon (Citrullus lanatus), winter melon (Benincasa hispida), honeydew melon (Cucumis melo), Roman melon (Cucumis melocantalupensis), winter melon (Cucumis melo inodorus), netted melon (Cucumis meloreticulatus)), cucumbers (e.g. cut cucumbers, pickling cucumbers, English cucumbers, gherkins (Cucumis sativus)), pumpkins (e.g. (Cucurbita pepo), (Cucurbita maxima), squash (e.g., Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata), grapes (e.g., Vitis vinifera, Vitis amurensis, Vitis labrusca, Vitis mustangensis, Vitis riparia, Vitis rotundifolia), beans (e.g., Phaseolus vulgaris, Phaseolus lunatus, Vigna angularis, Vigna radiate, Vigna mungo, Phaseolus coccineus, Vigna umbellate, Vigna rotundifolia), acontifolia), Phaseolus acutifolius, Vicia faba, Vicia faba equine, Phaseolus spp., Vigna spp.), soybeans (e.g. soybeans, soybeans, soybeans (Glycine max), wild soybeans (Glycine soja)), peas (e.g. (Pisum spp.), peas (Pisum sativum var. sativum), peas (Pisum sativum var. arvense)), peas (e.g. (Pisum spp.), peas (Pisum sativum var. sativum), peas (Pisum sativum var. arvense)), chickpeas (e.g. chickpeas, Indian black chickpeas, chickpeas (Cicer arietinum)), cowpeas (e.g. cowpeas (Vigna unguiculata)), pigeon peas (e.g. Indian lentils (Arhar / Toor), pigeon peas, Congo beans, peas, pigeon peas (Caganus cajan)), lentils (e.g. lentils (Lens culinaris)), Bambara peanuts (e.g. peanuts, Bambara peanuts (Vigna subterranea), lupins (e.g. Lupinus spp.), legumes (e.g. adzuki beans, Lablab purpureaus, Canavalia ensiformis, Canavalia gladiate, Psophocarpus tetragonolobus, Mucuna pruriens var. utilis, Pachyrhizus erosus), alfalfa (e.g. Medicago sativa, Medicago truncatula, Medicago arborea), lotus (e.g. Lotus japonicus), forage legumes (e.g. Leucaena spp., Albizia spp., Cyamopsis spp.), Sesbania spp. spp.), Stylosanthes spp., Trifolium spp., Vicia spp.), indigofera spp.), Indigofera tinctoria, Indigofera suffruticosa, Indigofera articulata, Indigofera oblongifolia, Indigofera aspalthoides, Indigofera suffruticosa, Indigofera arrecta, Leguminosae (e.g., Sophora japonica, Gleditsia spp., Robinia spp., Kentucky coffee tree, Gymnocladus dioicus, Acacia spp., Laburnum spp., Wisteria spp.), or Hemp (e.g., Cannabis sativa).

[0090] In certain embodiments, transgenic plants and transgenic plant parts regenerated from transgenic plant cells are provided. In certain embodiments, transgenic plants can be obtained from transgenic seeds by cutting, breaking, grinding or otherwise separating parts of plants. In certain embodiments, plant parts can be seeds, cotton bolls, leaves, flowers, stems, roots or any part thereof, or non-renewable parts of transgenic plant parts. As used herein, "non-renewable" parts of transgenic plant parts are parts that cannot be induced to form complete plants, or cannot be induced to form complete plants capable of sexual and / or asexual reproduction. In certain embodiments, the non-renewable parts of plant parts are parts of transgenic seeds, cotton bolls, leaves, flowers, stems or roots.

[0091] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to bacteria, fungi or plants, including any cell, tissue, organ or progeny of bacteria, fungi or plants. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos and pollen.

[0092] As used herein, the term "transformed" refers to a cell, tissue, organ or organism into which an exogenous DNA molecule (such as a construct) has been introduced. The introduced DNA molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ or organism, so that the introduced DNA molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism may also include progeny of the cell or organism, as well as progeny produced by a breeding process using such transgenic organisms as parents in a hybrid, and exhibiting an altered phenotype due to the presence of the exogenous DNA molecule. The introduced DNA molecule can also be transiently introduced into the recipient cell, so that the introduced DNA molecule is not inherited by subsequent progeny. The term "transgenic" refers to bacteria, fungi or plants containing one or more heterologous DNA molecules.

[0093] Many methods for introducing DNA molecules into plant cells are well known to those skilled in the art. The method generally includes selecting a suitable host cell, transforming the host cell with a vector and obtaining the transformed host cell. In the practice of the present invention, the methods and materials for transforming plant cells by introducing plant constructs into the plant genome may include any well-known and proven methods. Suitable methods may include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, drying / inhibition-mediated DNA absorption, electroporation, stirring with silicon carbide fibers, and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), etc.

[0094] The host cell may be any cell or organism, such as a plant cell, an algal cell, an algae, a fungal cell, a fungus, a bacterial cell, or an insect cell. In a specific embodiment, the host cell and transformed cells may include cells from a crop plant.

[0095] Subsequently, transgenic plants can be regenerated from the transgenic plant cells of the present invention. Seeds can be produced from the transgenic plants using conventional breeding techniques or self-pollination. Such seeds and subsequent generations of plants grown from such seeds will contain the recombinant DNA molecules of the present invention and will therefore be transgenic.

[0096] Transgenic plants of the present invention can be self-pollinated to provide seeds of homozygous transgenic plants of the present invention (homozygous for recombinant DNA molecules), or hybridized with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants of the present invention (heterozygous for recombinant DNA molecules). Such homozygous and heterozygous transgenic plants are all referred to as "offspring plants" in this article. Offspring plants are transgenic plants that are bred from original transgenic plants and contain recombinant DNA molecules of the present invention. The seeds produced using the transgenic plants of the present invention can be harvested and used to cultivate the offspring of transgenic plants, i.e., offspring plants of the present invention, which include constructs of the present invention and express agronomy target genes. Descriptions of breeding methods commonly used for different crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0097] The transformed plants can be analyzed for the presence of the gene of interest and the expression level and / or expression profile conferred by the regulatory elements of the invention. Those skilled in the art are aware of a variety of methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blot or northern blot, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Expression of transcribable DNA molecules can be performed using (Applied Biosystems, Foster City, CA) and were measured using the methods described by the manufacturer. The test matrix determines the PCR cycle time. Alternatively, other methods and reagents for measuring the expression of transcribable DNA molecules are well known in the art. For example, as described by the manufacturer Reagents and methods described by SYBR Green (Third Wave Technologies, Madison, WI) or SYBR Green (Thermo Fisher, A46012) can be used to assess transgene expression.

[0098] Transgenic plants comprising recombinant DNA molecules disclosed herein can show time-varying polynucleotide fragment expression levels, and the recombinant DNA molecules include a heterologous promoter operably connected to a polynucleotide fragment encoding a photosensitive short hypocotyl protein. For example, a plant or part thereof maintained in a 12 hour / 12 hour light / dark cycle can show an increase in the expression of a polynucleotide fragment during the 12 hour light stage of the cycle. The plant or part thereof maintained in a 12 hour / 12 hour light / dark cycle can show an increase in the expression of the polynucleotide fragment during the first 6 hours, first 5 hours, first 4 hours, first 3 hours, first 2 hours, or first 1 hour of the light period of the cycle. The plant or part thereof maintained in a 12 hour / 12 hour light / dark cycle can show an increase in the expression of the polynucleotide fragment during the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of the cycle, or any combination thereof. Recitation of discrete values ​​should be understood to include the range between each value.

[0099] Seeds of the plants of the invention can be harvested from fertile transgenic plants and used to grow progeny of transformed plants of the invention, including hybrid plant lines that comprise the construct of the invention and express the gene of agronomic interest.

[0100] The present invention also provides parts of the plants of the present invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperms, ovules and pollen. The present invention also includes and provides transformed plant cells comprising nucleic acid molecules of the present invention.

[0101] Transgenic plants can pass transgenic polynucleotide molecules to their offspring. Offspring include any regenerable plant parts or seeds that comprise the transgenic derived from ancestral plants. Transgenic plants are preferably homozygous for the polynucleotide molecules converted, and pass the sequence to all offspring through sexual reproduction. Offspring can grow from the seeds produced by transgenic plants. These other plants can be self-pollinated subsequently to produce real plant breeding lines. Among others, the gene expression situation of the offspring of these plants is also assessed. Described gene expression can be detected by several common methods, such as western blotting, northern blotting, immunoprecipitation and ELISA.

[0102] Genome modification

[0103] As an alternative to traditional transformation methods, DNA molecules (such as transgenes, expression cassettes, etc.) can be inserted or integrated into specific sites or loci in the genome of plants or plant cells by site-directed integration. Therefore, the recombinant DNA constructs and molecules of the present disclosure may include a donor template sequence, which contains at least one transgene, expression cassette, or other DNA sequence for insertion into the genome of a plant or plant cell. This donor template for site-directed integration may further include one or two homology arms (i.e., sequences to be inserted into the plant genome, transgenes, expression cassettes, etc.) located on both sides of the insertion sequence. The recombinant DNA constructs of the present disclosure may also include expression cassettes encoding site-specific nucleases and / or any related proteins for site-directed integration. These nuclease expression cassettes may be present in the same molecule or vector as the donor template (cis) or on a separate molecule or vector (trans). Several methods for site-directed integration are known in the art, involving different proteins (or complexes of proteins and / or guide RNAs) that cut genomic DNA to produce double-strand breaks (DSBs) or nicks at the desired genomic sites or loci. In short, as understood in the art, in the process of repairing the DSB or nick introduced by the nuclease, the donor template DNA can be integrated into the genome at the site of the DSB or nick. The presence of homology arms in the donor template can promote the insertion sequence to enter and be directed into the plant genome by homologous recombination during the repair process, although the insertion event can occur by non-homologous end joining (NHEJ). Examples of usable site-specific nucleases include zinc finger nucleases, engineered or natural meganucleases, TALE-endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cpf1). For methods using RNA-guided site-specific nucleases (e.g., Cas9 or Cpf1), the recombinant DNA construct will also include sequences encoding one or more guide RNAs to guide the nuclease to the desired site in the plant genome.

[0104] In certain embodiments, the present disclosure provides plants, plant parts, plant cells and seeds produced by genome modification using site-specific integration or genome editing. Genome editing can be used to perform one or more edits or mutations at a desired target site in a plant genome, such as changing the expression and / or activity of one or more genes, or integrating an insertion sequence or a transgenic at a desired position in a plant genome. Any site or locus in a plant genome can potentially be selected for genome editing (or gene editing) or site-specific integration of a transgenic, construct or transcribable DNA sequence. As used herein, a "target site" for genome editing or site-specific integration refers to the position of a polynucleotide sequence in a plant genome, which is bound and cut by a site-specific nuclease, thereby introducing a double-strand break (DSB) or a single-strand nick into the nucleic acid backbone and / or its complementary DNA chain of the polynucleotide sequence in the plant genome. "Target site" also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by any other site-specific nuclease that may not be guided by non-coding RNA molecules, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-range nucleases, etc., thereby introducing DSBs or single-stranded cuts into the polynucleotide sequence and / or its complementary DNA strand. As used herein, "target region" or "targeting region" refers to a polynucleotide sequence or region flanked by two or more target sites. In some embodiments, the target region may be mutated, deleted, inserted, substituted, inverted, or duplicated, but is not limited thereto.

[0105] As used herein, "targeted genome editing technology" refers to any method, protocol or technology that can use site-specific nucleases (such as large-range nucleases, zinc finger nucleases (ZFNs), RNA-guided endonucleases (e.g., CRISPR / Cas9 systems), TALE (transcription activator-like effector)-endonucleases (TALENs), recombinases or transposases) to perform precise and / or targeted editing (i.e., the editing is largely or completely non-random) at a specific location in a plant genome. As used herein, "editing" or "genome editing" refers to the generation of targeted mutations, deletions, insertions, substitutions, inversions or duplications of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000 or at least 25,000 nucleotides of an endogenous plant genomic nucleic acid sequence. As used herein, "editing" or "genome editing" may also encompass the targeted insertion or site-directed integration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 10,000 or at least 25,000 nucleotides into the endogenous genome of a plant. The singular form “edit” or “genome editing” refers to one such targeted mutation, deletion, insertion, substitution, inversion, or duplication, while “edit” or “genome editing” refers to two or more targeted mutations, deletions, insertions, substitutions, inversions, and / or duplications, wherein each “edit” is introduced by a targeted genome editing technique.

[0106] Genome-modified plants

[0107] As used herein, in the context of "modified" plants, plant seeds, plant parts, plant cells and / or plant genomes, refers to plants, plant seeds, plant parts, plant cells and / or plant genomes comprising expression levels of one or more target genes and / or engineered changes in endogenous sequences relative to wild-type or control plants, plant seeds, plant parts, plant cells and / or plant genomes. A modified plant refers to a plant having one or more differences compared to a native polynucleotide or amino acid sequence, the differences comprising substitutions, insertions, deletions, inversions, duplications or any desired combination of such changes. The term "modified" may further refer to plants, plant seeds, plant parts, plant cells and / or plant genomes having one or more deletions affecting endogenous LSH1 or LSH2 genes, the deletions introduced by chemical mutagenesis, transposon insertion or excision, or any other known mutagenesis techniques, or introduced by genome editing. In one aspect, the modified plant, plant seed, plant part, plant cell and / or plant genome may comprise one or more transgenes. Thus, modified plants, plant seeds, plant parts, plant cells and / or plant genomes include mutant, edited and / or transgenic plants, plant seeds, plant parts, plant cells and / or plant genomes having a modified sequence of an LSH1 or LSH2 gene relative to a wild-type or control plant, plant seed, plant part, plant cell and / or plant genome. In addition, the modification may increase, decrease, disrupt or alter the activity of a protein encoded by an LSH1 or LSH2 gene compared to the activity of the protein encoded by the LSH1 or LSH2 gene in an otherwise identical plant. As another example, a modified plant may overexpress LSH or increase LSH activity, which may result in enlarged multi-leaf and fused nodules; nodule development and N fixation; nodule primordium development that may support bacterial colonization; upregulation of nodule organ identity genes; recruitment of bud-expressed genes during nodule organogenesis; or formation of nodule-like structures (NLSs) compared to wild-type or control plants, plant seeds, plant parts, plant cells and / or plant genomes.

[0108] Modified plants, plant parts, seeds, etc. can be subjected to mutagenesis, genome editing or site-directed integration, gene transformation or a combination thereof. Such "modified" plants, plant seeds, plant parts and plant cells include descendants of modified plants, plant seeds, plant parts and plant cells that retain molecular changes (e.g., changes in expression levels and / or activity) of LSH1 or LSH2 genes or are derived from "modified" plants, plant seeds, plant parts and plant cells. The modified seeds provided herein can produce modified plants provided herein. The modified plants, plant seeds, plant parts, plant cells or plant genomes provided herein can include recombinant DNA constructs or vectors or genome editing provided herein. "Modified plant products" can be any product made from modified plants, plant parts, plant cells or plant chromosomes provided herein or any part or component thereof.

[0109] The modified plant can be further hybridized with itself or other plants to produce modified plant seeds and offspring. The modified plant can also be prepared by hybridizing a first plant comprising a DNA sequence or construct or editing (e.g., genomic deletion) with a second plant lacking a DNA sequence or construct or editing. For example, a DNA sequence or inversion can be introduced into a first plant strain suitable for transformation or editing, which can then be hybridized with a second plant strain to infiltrate the DNA sequence or editing (e.g., deletion) into the second plant strain. The offspring of these hybridizations can be further backcrossed to the desired strain for multiple times, such as by 6-8 generations or backcrossing, to produce offspring plants with genotypes substantially the same as the original parent strain, but introducing a DNA sequence or editing. The modified plant, plant cell or seed provided herein can be a hybrid plant, plant cell or seed. As used herein, a "hybrid" is produced by hybridizing two plants from different varieties, strains, inbred strains or species, so that the offspring includes genetic material from each parent. Those skilled in the art recognize that a higher-order hybrid can also be produced.

[0110] The modified plants, plant parts, plant cells or seeds provided herein can be superior varieties or superior lines."Superior varieties" or "superior lines" refer to varieties with superior agronomic performance produced by breeding and selection.

[0111] As used herein, the term "control plant" (or similar "control" plant seeds, plant parts, plant cells and / or plant genomes) refers to a plant (or plant seeds, plant parts, plant cells and / or plant genomes) used for comparison with a modified plant (or modified plant seeds, plant parts, plant cells and / or plant genomes) and having the same or similar genetic background (e.g., the same parental line, hybrid line, inbred line, experimenter, etc.) as the modified plant (or plant seeds, plant parts, plant cells and / or plant genomes), except for the genome editing (e.g., deletion) affecting the ZmDA1 gene. For example, the control plant can be the same inbred line as the inbred line used to prepare the modified plant, or the control plant can be the cross product of the same inbred parental line as the modified plant, except that there is no transgenic event or genome editing affecting the LSH1 or LSH2 gene in the control plant. Similarly, an "unmodified control plant" refers to a plant having a substantially similar or substantially identical genetic background to the modified plant, but without one or more engineered changes (e.g., mutations or edits) to the genome of the modified plant. For comparison with a modified plant, plant seed, plant part, plant cell and / or plant genome, a "wild-type plant" (or similarly a "wild-type" plant seed, plant part, plant cell and / or plant genome) refers to a non-transgenic and non-genome-edited control plant, plant seed, plant part, plant cell and / or plant genome. As used herein, a "control" plant, plant seed, plant part, plant cell and / or plant genome may also be a plant, plant seed, plant part, plant cell and / or plant genome having a similar (but not identical or completely identical) genetic background to the modified plant, plant seed, plant part, plant cell and / or plant genome if considered sufficiently similar to compare the characteristic or trait to be analyzed.

[0112] As used herein, the term "activity" refers to the biological function of a gene or protein. A gene or protein can provide one or more different functions. Therefore, the reduction, destruction or change of "activity" refers to the reduction, reduction or elimination of one or more functions of a gene or protein in a plant, plant cell or plant tissue at one or more stages of plant development compared with the activity of a gene or protein in a wild type or control plant, cell or tissue at the same stage of plant development. In addition, the increase of "activity" therefore refers to the increase of one or more functions of a gene or protein in a plant, plant cell or plant tissue at one or more stages of plant development compared with the activity of a gene or protein in a wild type or control plant, cell or tissue at the same stage of plant development. Similarly, active "regulation" refers to the process of affecting one or more functions of a gene or protein in a plant, plant cell or plant tissue at one or more stages of plant development compared with the activity of a gene or protein in a wild type or control plant, cell or tissue at the same stage of plant development.

[0113] According to some embodiments, a modified plant is provided having a genomic modification in a LSH1 or LSH2 gene, wherein the genomic modification results in an increase, decrease, disruption or alteration in the activity of a protein encoded by the LSH1 or LSH2 gene in at least one plant tissue by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or 100% compared to a control plant. According to a further embodiment, a plant having a modification of a protein encoded by a LSH1 or LSH2 gene is provided, wherein the modified plant increases, decreases, destroys or alters an activity in at least one plant tissue by 5%-20%, 5%-25%, 5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%, 75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80% or 10%-75% as compared to a control plant. Numerical ranges recited herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

[0114] According to some embodiments, modified plants are provided, wherein the level of LSH1 or LSH2 mRNA in at least one plant tissue is reduced or increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or 100% compared to a control plant. According to some embodiments, modified plants are provided, wherein the level of LSH1 or LSH2 mRNA expression in at least one plant tissue is reduced or increased by 5%-20%, 5%-25%, 5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%, 75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80% or 10%-75% compared to a control plant. According to some embodiments, a modified plant is provided, wherein the expression level of LSH1 or LSH2 protein in at least one plant tissue is reduced or increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or 100% compared to a control plant. According to some embodiments, a modified plant is provided, wherein the expression level of LSH1 or LSH2 protein in at least one plant tissue is reduced or increased by 5%-20%, 5%-25%, 5%-30%, 5%-40%, 5%-50%, 5%-60%, 5%-70%, 5%-75%, 5%-80%, 5%-90%, 5%-100%, 75%-100%, 50%-100%, 50%-90%, 50%-75%, 25%-75%, 30%-80% or 10%-75% compared to a control plant. The description of numerical ranges herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually referenced herein. The description of discrete values ​​should be understood to include the range between each value.

[0115] commodity

[0116] The present invention provides commodities comprising DNA molecules according to the present invention. As used herein, "commodity" refers to any composition or product consisting of materials derived from plants, seeds, plant cells or plant parts comprising DNA molecules of the present invention. Commodities can be sold to consumers and can be viable or non-viable. Non-viable commodities include, but are not limited to, non-viable seeds and grains; processed seeds, seed parts and plant parts; dehydrated plant tissues, frozen plant tissues and processed plant tissues; seeds and plant parts processed for animal feed, oil, meal, flour, flakes, bran, fiber, milk, cheese, paper, cream, wine and any other food for human consumption for terrestrial and / or aquatic animals; and biomass and fuel products. Viable commodities include, but are not limited to seeds and plant cells. Therefore, plants comprising DNA molecules according to the present invention can be used to make any commodity commonly obtained from plants or parts thereof.

[0117] Regulatory elements

[0118] Regulatory elements such as promoters, leader sequences (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs) play an important role in the overall expression of genes in living cells. As used herein, the term "regulatory element" refers to a DNA molecule with gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to affect the expression of an operably linked transcribable DNA molecule, for example, by affecting the transcription and / or translation of an operably linked transcribable DNA molecule. Therefore, regulatory elements (such as promoters, leader sequences, enhancers, introns, and 3'UTRs that function in plants) can be used to change plant phenotypes by genetic engineering.

[0119] The disclosure provides a regulatory element operably connected to a heterologous transcribable polynucleotide molecule, including SEQ ID NO: 84-93, or a variant or fragment thereof. Regulatory elements can be characterized by their gene expression patterns, such as positive and / or negative effects, such as constitutive expression or time, space, development, tissue, environment, physiology, pathology, cell cycle and / or chemical responsiveness expression, and any combination thereof, and quantitative or qualitative indication. As used herein, "gene expression pattern" is any pattern of operably connected DNA molecules transcribed into transcribed RNA molecules. Transcribed RNA molecules can be translated to produce protein molecules or can provide antisense or other regulatory RNA molecules, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA) etc.

[0120] As used herein, the term "protein expression" is any pattern of translation of transcribed RNA molecules into protein molecules. Protein expression can be characterized by its temporal, spatial, developmental or morphological characteristics and by quantitative or qualitative indicators.

[0121] Promoter can be used as a regulatory element for regulating the expression of operably connected transcribable DNA molecules. As used herein, the term "promoter" generally refers to a DNA molecule that participates in the recognition and combination of RNA polymerase II and other proteins (such as trans-acting transcription factors) to initiate transcription. The promoter can initially be separated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter can be a synthetically produced or manipulated DNA molecule. The promoter can also be chimeric. Chimeric promoters are produced by fusion of two or more heterologous DNA molecules. Promoters that can be used to implement the present invention include promoter elements included in SEQ ID NO:84 and 89 or its fragments or variants. In a specific embodiment of the present invention, the DNA molecules claimed for protection as described herein and any variants or derivatives thereof are further defined as comprising promoter activity, i.e., can be used as promoters in host cells, such as transgenic plant cells. In further embodiments, fragments may be defined as exhibiting the promoter activity possessed by the original promoter molecule from which they were derived, or fragments may comprise a "minimal promoter" that provides a basal level of transcription and is contained within a TATA box, other known transcription factor binding site motifs, or equivalent DNA sequences for recognition and binding to the RNA polymerase II complex to initiate transcription.

[0122] As used herein, the term "variant" refers to a second DNA molecule that is similar but not identical to a first DNA molecule in composition, such as a regulatory element, and wherein the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, by more or less equivalent transcriptional activity. Variant can be a shorter or truncated form of the first DNA molecule and / or a variation of the first DNA molecule sequence, such as a variant with different restriction enzyme sites and / or internal deletions, substitutions and / or insertions. "Variant" also encompasses regulatory elements having a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides comprising a reference sequence, wherein the derived regulatory element has more or less or equivalent transcription or translation activity than the corresponding parent regulatory molecule. Regulatory element "variants" also include variants produced by naturally occurring mutations in bacterial and plant cell transformation. In the present invention, the polynucleotide sequences of SEQ ID NO:84-93 provided can be used to produce variants that are similar but not identical to the DNA sequence of the original regulatory element in composition, while still maintaining the general functionality of the original regulatory element, i.e., the same or similar expression pattern. The generation of such variations of the present invention is well within the ordinary skill in the art in light of the present disclosure and are encompassed within the scope of the present invention.

[0123] Thus, the present disclosure provides variants of the regulatory elements disclosed herein, including SEQ ID NOs: 84-93. The variants provided have at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity with the reference sequence when optimally aligned with the reference sequence (provided herein as SEQ ID NOs: 84-93). The variants of SEQ ID NOs: 84-93 provided herein may have the activity of the reference sequence from which they are derived.

[0124] Also provided are fragments of regulatory elements disclosed herein, including SEQ ID NOs: 84-93. Fragments of regulatory elements (which may be functional fragments) may contain gene regulatory activity or function, and may be used alone or in combination with other gene regulatory elements and fragments, such as for constructing chimeric promoters. In a specific embodiment, a fragment of a regulatory element is provided, which comprises at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides, or longer DNA molecules with gene regulatory activity disclosed herein. In certain embodiments, a fragment of any one of SEQ ID NO:84-93 having full-length sequence activity is provided. Methods for generating such fragments from the starting promoter molecule are well known in the art. The expression of discrete values ​​should be understood to include the range between each value.

[0125] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from a gene, and can generally be defined as a region spliced ​​out during the processing of messenger RNA (mRNA) before translation. The present disclosure provides intron sequences including SEQ ID NO:86 and 91 and variants and fragments thereof. Alternatively, an intron can be a synthetically produced or manipulated DNA element. An intron can contain an enhancer element that affects the transcription of an operably connected gene. An intron can be used as a regulatory element for regulating the expression of an operably connected transcribable DNA molecule. A construct can include an intron, and the intron can be heterologous or not heterologous to the transcribable DNA molecule. Examples of introns in this area include rice actin introns and corn HSP70 introns.

[0126] As used herein, the term "3' transcription termination molecule", "3' untranslated region" or "3'UTR" refers to a DNA molecule used during transcription to the untranslated region of the 3' portion of an mRNA molecule. The present disclosure provides 3'UTR sequences including SEQ ID NOs: 87, 88, 92 and 93 and variants and fragments thereof. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as a polyA tail). The 3'UTR can be operably linked to and downstream of a transcribable DNA molecule, and can include a polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing or gene expression. The polyA tail is believed to play a role in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the art are the nopaline synthase 3' region; the 3' region of wheat hsp17, the 3' region of the pea ribulose bisphosphate carboxylase-oxygenase (rubisco) small subunit, the 3' region of cotton E6, and the coixin 3'UTR.

[0127] As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein typically neither the first nor the second DNA molecule exists in this structure, i.e., fused to the other. Thus, a chimeric DNA molecule is a new DNA molecule that is typically not naturally present. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulation of a DNA molecule. A chimeric promoter can combine two or more DNA fragments; for example, a fusion of a promoter with an enhancer element. Thus, the present invention encompasses designing, constructing, and using chimeric promoters for regulating expression of operably linked transcribable DNA molecules according to the methods disclosed herein.

[0128] Chimeric regulatory elements can be designed to contain various constitutive elements that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, as well as other methods known in the art. The resulting various chimeric regulatory elements can be composed of variants of the same or identical constitutive elements (but with different DNA sequences), or a DNA sequence that constitutes a connecting DNA sequence or a sequence that allows the components to be operably linked. The DNA sequences provided as SEQ ID NOs: 84-93 can provide regulatory element reference sequences, wherein the constitutive elements comprising the reference sequences can be linked by methods known in the art, and can include substitutions, deletions and / or insertions of one or more nucleotides or mutations that occur naturally in bacterial and plant cell transformation.

[0129] "Isolated DNA molecules" or equivalent terms or phrases mentioned in this application refer to that the DNA molecules exist alone or in combination with other compositions, but not in their natural environment. For example, nucleic acid elements (such as coding sequences, intron sequences, non-translated leader sequences, promoter sequences, transcription termination sequences, etc.) naturally present in the genomic DNA of an organism are not considered to be "isolated", as long as the elements are located in the genome of the organism and in the position of the genome in which they naturally occur. However, within the scope of the present disclosure, each of these elements and sub-parts of these elements are "isolated", as long as the elements are not in the genome of the organism and in the position of the genome in which they naturally occur. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a DNA nucleotide sequence inserted into the genome of a plant or bacterial cell, or present in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used to transform a cell, in the genome of a plant or bacterium, or in a detectable amount in a tissue, offspring, biological sample or commodity derived from a plant or bacterium.

[0130] The efficacy of the modifications, duplications or deletions described herein on the desired expression of a particular transgene can be tested empirically in stable and transient plant assays (such as described in the working examples herein) to verify the results, which may vary depending on the purpose of the alteration made and the starting DNA molecule alteration.

[0131] Transcriptable DNA molecules

[0132] As used herein, the term "transcribable DNA molecule" refers to any DNA molecule that can be transcribed into an RNA molecule, including, but not limited to, having a protein coding sequence and producing an RNA molecule having a sequence that can be used for gene suppression. The type of DNA molecule may include, but is not limited to, a DNA molecule from the same plant, a DNA molecule from another plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as a DNA molecule containing antisense information for a gene, or an artificial, synthetic, or other modified form of a DNA molecule encoding a transgene. Exemplary transcribable DNA molecules for introduction into constructs of the present invention include, for example, DNA molecules or genes from a species different from the species into which the DNA molecule is integrated, or genes derived from or present in the same species, but introduced into a recipient cell by genetic engineering methods rather than classical breeding techniques.

[0133] Regulatory elements, such as any one of SEQ ID NO:84-93 or variants or fragments thereof, can be operably connected to a transcribable DNA molecule heterologous to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules when such a combination does not generally exist in nature. For example, the two DNA molecules can be derived from different species and / or the two DNA molecules can be derived from different genes, such as different genes from the same species or the same gene from different species. Therefore, if such a combination does not generally exist in nature, i.e., the transcribable DNA molecule is not naturally operably connected to the regulatory element, then the regulatory element is heterologous relative to the transcribable DNA molecule operably connected.

[0134] The transcribable DNA molecule will usually be any DNA molecule that needs to express a transcript. The expression of this transcript can lead to the translation of the resulting mRNA molecule, and therefore protein expression. Or, for example, the transcribable DNA molecule can be designed to eventually cause the expression of a specific gene or protein to be reduced. In one embodiment, this can be achieved by using a transcribable DNA molecule in a directional antisense direction. Those of ordinary skill in the art are familiar with using this antisense technology. Any gene can be negatively regulated in this way, and in one embodiment, a transcribable DNA molecule can be designed to suppress a specific gene by the expression of a dsRNA, siRNA or miRNA molecule.

[0135] Thus, one embodiment of the present invention is a recombinant DNA molecule comprising a regulatory element of the present invention (such as provided SEQ ID NOs: 84-93) operably linked to a heterologous transcribable DNA molecule so that when the construct is integrated into the genome of a transgenic plant cell, the transcription of the transcribable DNA molecule is regulated at the expected level or in the desired pattern. In one embodiment, the transcribable DNA molecule comprises the protein coding region of a gene, and in another embodiment, the transcribable DNA molecule comprises the antisense region of a gene.

[0136] Agronomic target genes

[0137] Transcribable DNA molecule can be the gene with agronomy significance.As used herein, term " agronomy target gene " refers to when expressing in specific plant tissue, cell or cell type, the transcribable DNA molecule of the characteristic of giving expectation.The product of agronomy target gene can work in plant, to cause plant morphology, physiology, growth, development, yield, grain composition, nutritional characteristics, disease or pest resistance, and / or the impact of environment or chemical tolerance, or can act as insecticide in the diet of the insect that feeds on plant.In one embodiment of the invention, regulating and controlling element of the present invention is introduced in construct, makes described regulating and controlling element be operably connected on transcribable DNA molecule, described DNA molecule is agronomy target gene.In the transgenic plant containing this construct, the expression of agronomy target gene can give useful agronomy character. Beneficial agronomic traits can include, for example, but are not limited to, herbicide tolerance, insect control, improved yield, disease resistance, pathogen resistance, improved plant growth and development, improved starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit ripening, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, and desirable biofuel production.

[0138] Alternatively, agronomic genes can affect the above-mentioned plant characteristics or phenotypes by encoding RNA molecules that cause targeted regulation of gene expression of endogenous genes, such as by antisense (see, e.g., U.S. Patent No. 5,107,065); inhibitory RNA ("RNAi", including regulation of gene expression by miRNA, siRNA-, trans-acting siRNA-, and phase sRNA-mediated mechanisms, e.g., as disclosed in applications US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application No. 11 / 974,469); or co-suppression-mediated mechanisms. RNA can also be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see, e.g., US2006 / 0200878), which is engineered to cut the desired endogenous mRNA product of hunger. Methods of constructing and introducing constructs into cells are known in the art so that transcribable DNA molecules are transcribed into molecules that can cause gene suppression.

[0139] Selectable marker

[0140] Selectable marker transgenes can also be used with the regulatory elements of the present invention. As used herein, the term "selectable marker transgene" refers to any transcribable DNA molecule, the expression of which in transgenic plants, tissues or cells or its absence can be screened or scored in some way. Selectable marker genes and their associated selection and screening techniques for the practice of the present disclosure are known in the art, and include but are not limited to encoding beta-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and transcribable DNA molecules that confer herbicide tolerance proteins.

[0141] When introducing elements of the present disclosure or the embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements.

[0142] When used when listing two or more items, the term "and / or" refers to any one, any combination of the items, or all of the items associated with the term.

[0143] The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be other elements in addition to the listed elements. For example, any method that "comprising," "having," or "including" one or more steps is not limited to having only the one or more steps, and may also encompass other unlisted steps. Similarly, any composition or device that "comprising," "having," or "including" one or more features is not limited to having only the one or more features, and may encompass other unlisted features.

[0144] Having now generally described the present invention, the present invention may be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention unless otherwise stated. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, it will be appreciated by those skilled in the art based on this disclosure that many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention, and still obtaining the same or similar results, and therefore all that is described or shown in the accompanying drawings should be interpreted as illustrative rather than restrictive. DETAILED DESCRIPTION

[0145] Example 1: LSH1 and LSH2 are upregulated during early nodule organogenesis downstream of NIN

[0146] While previous comparisons between lateral roots and nodules have revealed extensive morphological and transcriptional overlap at their initiation stages, nodules become significantly distinct from lateral roots as development progresses. To better understand the gene regulatory networks that differentiate nodules from lateral roots during development, changes in gene expression were observed and correlated to time points when the first morphological differences between lateral roots and nodule primordia appear. This comparison identified a set of previously characterized regulators of nodule organ identity, in addition to LBD16, including NF-YA1 (a previously identified putative downstream target of NIN), the NF-YA1 interacting subunit NF-YB16, and the transcriptional co-activators NOOT1 and NOOT2, that were upregulated in a nodule-specific manner at these time points. Importantly, two previously unknown transcriptional regulators with as yet uncharacterized functions were identified in symbiotic nodules that displayed similar expression patterns ( Figure 1 A). Both regulatory factors contain the ALOG domain ( Fig.13 ) and showed high sequence similarity to members of the LIGHT SENSITIVE SHORTHYPOCOTYL (LSH) transcription factor family. Therefore, these new transcriptional regulators were named "MtLSH1" and "MtLSH2" and are referred to as LSH1 and LSH2 in this article. LSH1 was upregulated in roots 16 hours after rhizobium spot inoculation, while LSH2 was upregulated 36 hours after inoculation. In contrast, neither LSH1 or LSH2 was differentially expressed during lateral root development, suggesting that LSH1 and LSH2 may be part of the developmental process that distinguishes nodules from lateral roots ( Figure 1 A). Expression of LSH1 and LSH2 during rhizobium infection is dependent on CRE1 and NIN, and ectopic expression of NIN is sufficient to upregulate both genes ( Figure 1A). Furthermore, we identified DNA binding sites for NIN in LSH1 exons by chromatin immunoprecipitation using hairy roots expressing LjUBI:GFP-NIN under non-symbiotic conditions, suggesting that LSH1, but not LSH2, is a direct putative target of NIN. Furthermore, treatment of M. truncatula roots with cytokinin induced the expression of LSH1 in a CRE1- and NIN-dependent but NF-YA1-independent manner ( Figure 1 B) Taken together, this identifies LSH1 and LSH2 as putative regulators of nodule organ identity that are specifically recruited during early symbiotic nodule organogenesis in a cytokinin- and NIN-dependent manner.

[0147] To investigate the spatial expression patterns of the two LSH genes, promoter-GUS analysis of LSH1 and LSH2 was performed, and the results showed that both genes were expressed in the nodule primordium throughout development. In mature nodules, LSH1 was expressed in the apical meristem zone, and LSH2 was expressed in the infection and fixation zones. Both genes were expressed in the nodule peripheral vasculature ( Figure 1 C) Taken together, this suggests that LSH1 and LSH2 are upregulated in roots in response to symbiotic signaling and are expressed throughout nodule development in Medicago truncatula.

[0148] Example 2: The LSH gene is required for the development of nodules that support nitrogen fixation

[0149] To further evaluate the roles of LSH1 and LSH2 genes in nodule organogenesis, loss-of-function mutants of LSH1 (lsh1-1, lsh1-2) and LSH2 (lsh2-1) were identified, and the lsh1-1 lsh2-1 double mutant was generated. The lsh1 loss-of-function mutant, but not the lsh2-1 mutant, showed significantly altered shoot organ morphology, including changes in petal shape and number, and reduced stipule complexity ( Figure 8 B and E). No effects of LSH on root architecture have been reported, and we found that root morphology was not affected in lsh1 / lsh2 seedlings and that LSH1 / LSH2 were not positive regulators of lateral root development. In fact, in addition to a slight reduction in primary root length, a slight increase in lateral root number was observed in lsh1 and lsh1 / lsh2 seedlings, suggesting that in M. truncatula these genes are negative regulators of lateral root initiation.

[0150] To assess the overall effects of the loss of LSH1 and LSH2 on nodulation and nitrogen fixation, plants grown in terragreen:sand mixtures were inoculated with Sinorhizobium meliloti strain 2011 expressing GUS under the bacterial pnifH promoter, a bacterial promoter associated with the expression and activity of nitrogenase for approximating biological N-fixation in nodules. Using the pnifH:GUS reporter gene, blue, nitrogen (N)-fixing nodules were distinguished from white, non-N-fixing nodules at 28 days post inoculation (dpi), and it was found that the proportion of blue and white nodules was significantly reduced in the lsh1 mutant compared with the wild type, while the lsh2-1 mutant showed proportions comparable to the wild type (or only minor changes in the proportion of white nodules and nodules showing mature N fixation compared with the wild type) ( Figure 2 C and D, Figure 8 F and I). In the lsh1-1 lsh2-1 double mutant, almost only white nodules were observed, indicating that nitrogen fixation was severely reduced in this double mutant ( Figure 2 C and D). This was further confirmed by acetylene reduction assays, which showed that nitrogenase activity in 1sh1-1 nodules was significantly reduced compared with WT, and nitrogenase activity was completely abolished in nodules of the 1sh1-1 1sh2-1 double mutant at 21 dpi ( Figure 8 H), confirming that pnifH-GUS staining serves as a reliable approximation of nitrogen fixation in nodules. In addition, a significant increase in nodule number was observed in the 1sh1-1 1sh2-1 mutant compared to the wild type and the single mutants, a phenomenon often observed in fix- mutants ( Figure 8 I). In addition, nodule morphology was significantly altered in lsh1 and lsh1-1 lsh2-1 mutants compared with the wild type, with an increased number of enlarged, multilobed, and fused nodules observed in the lsh1 mutant and stunted, small, and fused nodules observed in the lsh1-1 lsh2-1 mutant ( Figure 2 CE and 8F and G).

[0151] Example 3: LSH genes are essential for the development of nodule primordia that support bacterial colonization

[0152] To further investigate the cause of this severe reduction in N fixation, the progression of rhizobium infection and early nodule primordium development was studied in plate-grown seedlings 7 days after inoculation with R. meliloti strain 2011lacZ. To this end, all rhizobium infection events were counted and evaluated along the entire length of the primary root of the seedling, and the analysis focused on infection events with or associated with early cell divisions in the inner root tissue layers. In the wild type, the progression of rhizobium infection through the epidermis and cortex to the dividing nodule primordium cells was observed to be temporally and spatially coordinated with the development of the nodule primordium, which begins with a few cell divisions in the inner tissue layers and develops into multilayered primordia. The coordinated progression of these two processes results in early wild-type nodule primordia that are fully colonized from the time point of primary root emergence ( Figure 3 A and B and Fig. 9 Similar to the wild type, nodule primordia were observed to form and develop after successful establishment of epidermal infection in lsh1-1 and lsh1-1 lsh2-1 mutants. However, rhizobium infection through the cortex and subsequent colonization of primordium cells was severely impaired in lsh1-1 and lsh1-1 lsh2-1 mutants ( Figure 3 AB and Fig. 9 This results in a large fraction of the primordia of lsh1-1 and lsh1-1 lsh2-1 mutants being only partially or, in the most severe cases, completely uncolonized from the time point of primary root emergence ( Figure 3 AB and 12F and G). In addition, rhizobia were observed to colonize the surface of uncolonized or partially colonized nodule primordia ( Figure 3 A and 12G ). This suggests that, unlike LBD16, LSH1 and LSH2 are not required for the initiation of nodule primordia, but they play important roles in the infectivity of the overlying cortical tissue beneath the epidermal infection site and in the habitability of the developing nodule primordium.

[0153] To investigate these early infection and colonization defects at higher temporal and spatial resolution, we used rhizobium spot inoculation combined with deep tissue imaging of the DNA synthesis marker 5-ethynyl-2-deoxyuridine (EdU) and either propidium iodide or a combination of fluorescent brighteners as cell wall markers. At 24 h after rhizobium spot inoculation (24 hpi), no obvious differences in cell cycle activity and overall primordium development were observed between the WT and 1sh1-1 1sh2-1 double mutants ( Figure 3 C, upper panel). In contrast, at 72 hpi, a severe reduction in cell cycle activity was observed in the lsh1-1 1sh2-1 double mutant primordia compared with WT ( Figure 3C and 9). This reduction in cell cycle activity was specific to primordium cells derived from the mesoderm of the primary root, while cell cycle activity in the inner tissue layer at the base of the primordium was comparable between WT and 1sh1-1 1sh2-1 double mutants. More specifically, at 72 hpi, cells in the cortical cell layer of the WT primordium primarily divided in a pericentric direction, thereby adding cortical-derived cell layers to the growing primordium. In contrast, these pericentric cell divisions were severely reduced or completely absent in 1sh1-1 1sh2-1 double mutant primordia at 72 hpi ( Figure 3 C lower panel and 9). Taken together, this suggests that LSH1 and LSH2 are required to specifically promote pericyte cell divisions in the distal cortical-derived portion of the developing nodule primordium and that these cortical divisions appear to be causally involved in the successful progression of the infection thread through the overlying cortical cell layer.

[0154] Example 4: LSH1 and LSH2 are essential for the upregulation of nodule organ identity genes and recruitment of shoot-related genes to nodule organogenesis

[0155] To better understand the regulatory functions of transcription factors LSH1 and LSH2 during rhizobium infection and nodule organogenesis, RNA-Seq was performed on root sections of 1sh1-1 single mutant, 1sh1-1 lsh2-1 double mutant and corresponding wild type (ecotype R108) spot-inoculated with rhizobia at 24 and 72 hpi. In addition, hairy roots combinatorially expressing pLjUBI:LSH1 and pLjUBI:LSH2 were generated and RNA-Seq was performed on hairy roots under non-symbiotic conditions. Hairy roots expressing pLjUBI:GFP-LSH1 and pLJUBI-NLS-GFP were prepared as controls and subjected to chromatin immunoprecipitation followed by next generation sequencing (Chromatin Immunoprecipitation Sequencing; Chip-Seq) under non-symbiotic conditions. Consistent with an early role in primordium formation, lsh1-1 and lsh1-1 lsh2-1 mutants showed severely reduced expression of nodule-related genes compared with wild type, with more than 90% of rhizobium-responsive genes in WT being dependent on LSH1 / LSH2 at 24 and 72 hpi ( Figure 4 A). Marker genes of symbiotic signaling, such as EARLY NODULIN11 (ENOD11) and NIN, are still expressed in lsh1 / lsh2, as are genes associated with early infection, such as Nodule Pectate Lyase (NPL) and Rhizobium-directed Polar Growth (RPG) ( Figure 4B). In contrast, genes related to infection progression and nitrogen fixation such as VAPYRIN (VYP) and LEGHEMOGLOBIN (LB1 / LB2) were not upregulated in the lsh1 / lsh2 mutant ( Figure 4 B). Among the genes known to be associated with nodule primordium initiation, LBD16 expression was not significantly affected by LSH mutations, whereas known nodule regulators NF-YA1 and NOOT1 / NOOT2 were partially dependent on LSH1 / LSH2 in terms of loss and gain of function. NF-YA1 has been shown to regulate the expression of the STY-1 transcription factor, which in turn promotes the expression of the YUC auxin biosynthesis genes, and both STY-1 and YUC genes were consistently downregulated in the 1sh mutant. Relatedly, several genes involved in auxin transport and binding, as well as cell cycle regulators (including A-type and B-type cyclins and the endoreduplication regulator CSS52B) were also found to be dependent on LSH1 / LSH2. Many of these genes were constitutively upregulated by overexpression of LSH1 / LSH2 and were identified as putative direct targets of LSH1 in ChiP-Seq experiments ( Figure 4 B). In summary, RNA-Seq data indicate that LSH1 / 2 promote cell proliferation in cortical-derived cell layers through upregulation of NF-YA1 and its downstream targets.

[0156] Even under non-symbiotic conditions, cytokinin signaling is required and essential for nodule formation and development, in stark contrast to its inhibitory effects on lateral root initiation and early development. Recently, it has been shown that cytokinin signaling is required for colonization of endosymbiotic host cells, promoting the transition from mitotic cell proliferation to endoreduplication through upregulation of CSS52A. However, surprisingly, we found that genes with functions in cytokinin signaling and biosynthesis were strongly affected by loss- and gain-of-function of LSH1 / LSH2 and identified as putative direct ChiP-Seq targets of LSH1, including CRE1, B-type response regulators (RESPONSE REGULATORS) RR9 and RR11, and members of the lonely guy (LOG) gene family ( Figure 4 B) Promotion of cytokinin signaling further demonstrates that LSH1 / LSH2 play a role in establishing the organ identity that distinguishes symbiotic nodules from lateral roots.

[0157] Previously, members of the LSH transcription factor family, closely related to MtLSH1 and MtLSH2, have been characterized as acting in conjunction with the transcriptional coactivator BLADE-ON-PETIOLE in shoots to control inflorescence and leaf complexity and internal asymmetry of floral organs. We consistently found that a set of developmental regulators were upregulated in an LSH-dependent manner after rhizobium spot inoculation and were also found to be expressed in shoot tissue of M. truncatula (data extracted from the MtExpressMedicago gene expression atlas (Carrere et al., 2021)). Some of these LSH-dependent and shoot-expressed genes have previously been annotated as regulators of organ growth and organ boundaries, such as KLUH and PETAL LOSS (Anastasiou et al., 2007; Brewer et al., 2004). We also discovered a set of LSH1 / LSH2-dependent genes that are repressed by rhizobia, including two members of the PLETHORA (PLT) family of root meristem regulators, PLT1 and PLT2 (Franssen et al., 2015). In summary, this study, combined with RNA-seq and ChiP-Seq analysis, demonstrates that LSH genes are master regulators of nodulation that are necessary and sufficient for upregulation of cytokinin signaling and nodulation-specific regulators such as NF-YA1, as well as for the recruitment of regulators with pleiotropic functions, including NOOT1 / NOOT2, in shoot and symbiotic nodule development.

[0158] In summary, RNA-seq analysis of gain- and loss-of-function LSH1 / LSH2 lines identified LSH genes as key regulators of rhizobial symbiosis that are necessary and sufficient for the upregulation of nodule-specific organ identity regulators such as NF-YA1 and the recruitment of shoot-associated regulators that play a role in nodule organogenesis, including NOOT1 / 2.

[0159] Example 5: LSH1 / LSH2 act in part through NF-YA1 cortical activation

[0160] To understand the spatial context of LSH1 / LSH2 function, promoter GUS analysis was performed in hairy roots expressing pNF-YA1:GUS-tNF-YA1 in wild-type and lsh1 / lsh2 backgrounds. pNF-YA1:GUS-tNF-YA1 is expressed in the inner tissue layer at the base of developing nodules and in the nodule primordium in wild-type ( Figure 5 A). In lsh1 / lsh2 mutants, NF-YA1 expression was lost in nodule primordia, but maintained in the tissue layer at the base of the nodule ( Figure 5A). This tissue-specific control of NF-YA1 is consistent with the partial LSH1 / LSH2 dependence of NF-YA1 induction observed in RNA-Seq ( Figure 4 B).

[0161] Example 6: NF-YA1 partially rescues nitrogen fixation in the lsh1 lsh2 nodule phenotype

[0162] Previously, NF-YA1 has been shown to play a key role in promoting cell proliferation, host cell differentiation, and endosymbiotic colonization in the primordium cell layer derived from the mesoderm of the primary root. Consistent with this, very similar phenotypes were observed between lsh1 / lsh2 and nf-ya1, with an increased ratio of white to blue pNifH-GUS-expressing nodules and an increase in nodule number ( Figure 2 AB and 8), and the proportion of aborted cortical infection lines increased ( Figure 3 AB). In addition, similar to 1sh1 / 1sh2, a reduction in cell divisions and cell layers was observed in nf-ya1 nodule primordia ( Figure 3 C) However, there are also differences between 1sh1 / 1sh2 and nf-ya1 nodules, particularly in terms of overall nodule morphology and maintenance of pNifH-GUS expression.

[0163] To further investigate the commonalities and differences in the functions of LSH1 / LSH2 and NF-YA1, RNA-seq was performed on rhizobium-dot-inoculated nf-ya1 and WT root sections at 24 and 72 hpi, and the gene dependency of rhizobium-induced genes between NF-YA1 and LSH1 / LSH2 was compared ( Figure 5 B, 5C, Figure 8 NF-YA1 controls a smaller subset of rhizobium-induced genes than LSH1 / LSH2: 46% and 70% of rhizobium-responsive genes are dependent on NF-YA1 at 24 and 72 hpi, respectively ( Figure 5 B, Figure 8 ): while LSH1 / LSH2, >90% ( Figure 4 A). Although at 24 hpi there was surprisingly little overlap between all NF-YA1-dependent and LSH1 / LSH2-dependent genes, <20%, the overlap of NF-YA1-dependent genes increased to 71% at 72 hpi ( Figure 5 C). In addition, NF-YA1 is constitutively expressed in hairy roots under non-symbiotic conditions (LjUBI:NF-YA1). Genes showing strong transcriptional responses in both gain and loss of LSH and NF-YA1 further emphasize the role of local auxin biosynthesis, transport and conjugation, cytokinin signaling, and cell cycle regulation during early nodulation, but also include several shoot-expressed growth regulators ( Figure 5D). In addition, this dataset confirms that LSH1 / LSH2 expression is independent of NF-YA1 ( Figure 1 B and 5D). Together, this suggests that these regulators have independent additional functions at the early nodule primordium stage and converge on similar regulatory pathways at the time points of early cortical infection and nodule differentiation. Based on this, it was hypothesized that these regulators act in similar pathways and that reduced expression of NF-YA1 in the cortex may at least partially explain the reduced bacterial colonization and nitrogen fixation phenotypes observed in lsh1 / 1sh2. To test this, NF-YA1 was ectopically expressed in LSH1 / LSH2 roots either under the constitutive LjUBI promoter or under the pLSH1 and pLSH2 promoters. Both NF-YA1 expression patterns resulted in partial rescue of lsh1 / lsh2, resulting in 25% of nodules with functional N fixation based on pNifH-GUS ( Figure 5 E), revealing a functional link between LSH1 / LSH2 and NF-YA1 during nodule organogenesis. Together, this provides evidence that LSH1 and LSH2 are required for specific promotion of NF-YA1 cortical expression during nodule organogenesis.

[0164] Example 7: LSH1 / 2 and NOOT1 / 2 function in the same pathway during nodule organogenesis

[0165] RNA-Seq results also suggested that NOOT1 / NOOT2 expression is dependent on LSH1 / LSH2. To verify this, promoter GUS analysis was performed in hairy roots expressing pNOOT1:GUS-tNOOT1 and pNOOT2:GUS-tNOOT2 in wild type and lsh1 / lsh2. Both NOOT reporter genes were expressed in the inner tissue layer and nodule primordium showing the base of developing nodules in wild type ( Figure 6 A), but in 1sh1 / 1sh2, a moderate decrease in NOOT1 expression and loss of NOOT2 expression in nodule primordia was observed ( Figure 6 A).

[0166] It has been previously shown that several orthologs of the BOP (NOOT) gene function together with members of the LSH family to regulate shoot organogenesis. To test whether LSH and NOOT function together in the same regulatory pathway during symbiotic nodule development, noot1 / noot2 were included in time-resolved expression and functional analyses. Unlike 1sh1 / 1sh2 nodule primordia that display a marked reduction in pericyte divisions in the root cortex, noot1 / noot2 primordia display cell cycle activity comparable to that of wild-type ( Figure 3C and 7B) were comparable or higher than wild-type rhizobia infection. However, at later stages of nodule development, noot1 / noot2 displayed defects in rhizobia colonization similar to lsh1 / lsh2, resulting in large numbers of partially or completely uncolonized nodules ( Figure 3 A, 3B, 9A, 9B).

[0167] Compared with the loss of LSH1 / LSH2, the loss of NOOT1 / NOOT2 had a much smaller effect on the rhizobium-inducible gene set: 25.75% and 64.45% of the rhizobium-responsive genes were not differentially expressed in the noot1 / noot2 mutant at 24 and 72 hpi, respectively ( Figure 6 B), while >90% expression in 1sh1 / 1sh2 ( Figure 4 A). There is 99% overlap in genes that are unresponsive to rhizobia inoculation in the 1sh1 / 1sh2 and noot1 / noot2 mutants, indicating that the effects on gene expression caused by loss of NOOT1 / NOOT2 are fully embedded in LSH1 / LSH2 function ( Figure 6 C). Constitutive expression of NOOT1 / NOOT2 (pLjUBI:GFP-NOOT1pLjUBI:GFP-NOOT2) showed significant (75%) overlap with genes induced by LSH1 / LSH2 overexpression ( Figure 8 B, C). Genes showing strong transcriptional responses in both gain and loss of LSH and NOOT further emphasize the role of growth regulators with pleiotropic functions in shoot development, auxin and cytokinin signaling, and the requirement for repression of root meristem regulators such as PLT1 and PLT2 during nodulation ( Figure 8 A). LSH1 / LSH2 control NOOT1 / NOOT2 gene expression during nodulation, but NOOT1 / NOOT2 have no effect on LSH1 or LSH2 expression ( Figure 4 B and Figure 8 A). These studies suggest that the lack of NOOT1 / NOOT2 function and expression downstream of LSH1 / LSH2 at least partially explains the 1sh1 / 1sh2 phenotype, particularly during the later stages of nodule development. Consistent with this observed genetic interaction between LSH and NOOT, the 1sh1 / noot1 double mutant reproduces the phenotype of the 1sh1 / 1sh2 double mutant ( Figure 7 A, 7B, 9A-C). A notable feature of the noot mutant is the emergence of lateral roots from the nodule tip. This phenotype was observed in 1sh1 / 1sh2, but at a lower frequency than that observed in noot1 / noot2 ( Figure 7 A, 7B, 9C), which is consistent with the loss of repression of root meristem genes such as PLT1 / PLT2 in both double mutants ( Figure 4 B and Figure 8 A). Phenotypic similarities between lsh1 / noot1 and lsh1 / lsh2 mutants were also observed at earlier time points, where we observed an early infection defect in lsh1 / noot1 and a marked reduction in pericyte divisions in cells derived from the root cortex, as originally observed in the lsh1 / lsh2 mutant ( Figure 3 C, 7C, 9A, 9B). This suggests that LSH1 and LSH2 control NOOT1 and NOOT2 and that this regulation partially explains the loss of function LSH1 / LSH2 phenotype.

[0168] ChiP-Seq was used to further investigate the putative direct targets of LSH1 ( Fig.23 ). Fig.23 Peaks shown are high-confidence targets found in at least 3 of 4 biological replicates and include CRE1, IPT1, RR19, CKX3, PIN1, STYLISH, PINOID, and NOOT1. These results suggest that the LSH1 transcription factor directly binds to DNA regions of cytokinin and auxin signaling and biosynthesis genes, as well as the nodule identity regulator NOOT1.

[0169] Example 8: Overexpression of LSH1 in plants

[0170] Medicago truncatula plant cells were transformed with a vector containing a sequence encoding LSH1 (SEQ ID NO: 1) under a heterologous plant promoter (pLjUBI:GFP-LSH1). Transformed plant cells were regenerated to produce LSH1 overexpressing plants. Ectopic expression of LSH1 resulted in altered transcriptional profiles of nodulation genes. In addition, root architecture changes were observed, including increased root length and diameter ( Fig.11 ). Overexpression of LSH1 also altered lateral root primordium development compared to control plants ( Fig.12 ). LSH1 overexpressing plants were further inoculated with bacteria to assess rhizobium infection and nodule formation. Inoculation resulted in altered rhizobium infection patterns and nodule architecture, including clustered, multi-leafy nodules ( Fig.13 ).

[0171] Example 9: Overexpression of LSH2 in plants

[0172] Plant cells were transformed with a vector containing a sequence encoding LSH2 (SEQ ID NO: 3) under the control of a heterologous plant promoter. The transformed plant cells were regenerated to produce LSH2 overexpressing plants, which showed altered transcriptional profiles of nodulation genes; altered root architecture (e.g., increased root length and diameter); and altered lateral root primordium development, similar to the results described in Example 8. LSH2 overexpressing plants were also inoculated with bacteria to assess rhizobium infection and nodule formation, showing altered rhizobium infection patterns, and nodulation sutures including clustered multi-leaf nodules, similar to the results described in Example 8.

[0173] Example 10: Simultaneous overexpression of LSH1 and LSH2 in plants

[0174] Medicago truncatula plant cells were transformed with a vector containing sequences encoding LSH1 and LSH2 (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) under the control of a heterologous plant promoter. The transformed plant cells were regenerated to produce plants overexpressing LSH1 and LSH2. Plants overexpressing LSH1 and LSH2 were inoculated with bacteria to assess rhizobium infection and nodule formation, showing altered rhizobium infection patterns and nodule architecture ( Fig.14 ).

[0175] Example 11: Plants with modified LSH1 activity

[0176] Non-legume plant cells are genomically modified to introduce a modification to an endogenous sequence encoding LSH1 (SEQ ID NO: 1). The modified plant cells are regenerated to produce plants having altered LSH1 activity compared to control plants not comprising the modification. Plants having altered LSH1 activity exhibit altered transcriptional profiles and altered root architecture, such as an increase in root length and diameter, and in addition to enhanced interaction with rhizobia, exhibit development of modified lateral roots resembling nodules.

[0177] Example 12: Plants with modified LSH2 activity

[0178] Non-legume plant cells are genomically modified to introduce modifications to an endogenous sequence encoding LSH2 (SEQ ID NO: 3). The modified plant cells are regenerated to produce plants having altered LSH2 activity compared to control plants not comprising the modification. Plants having altered LSH2 activity exhibit altered transcriptional profiles and altered root architecture, such as an increase in root length and diameter, and in addition to enhanced interaction with rhizobia, exhibit development of modified lateral roots resembling nodules.

[0179] Example 13: Evaluation of gene function in stably transformed roots

[0180] For preliminary functional assessment of gene function in roots, the Stable Barley Root Transformation System (STARTS) (Imani et al., 2011) was used. This method is based on callus generated from immature embryo scutellum. Through Agrobacterium tumefaciens-mediated transformation, the callus was then directly transferred to barley root induction medium containing liquid endosperm of coconut fruit, sucrose and 2 mg / l indole-3-butyric acid (IBA). Callus can be regenerated from transformed roots within 6 weeks for further rapid preliminary gene function analysis.

[0181] The 6-week-old STARTS plates were wrapped in foil and kept in a growth chamber at 25 degrees Celsius for 2 weeks to allow further growth of transformed roots. The first batch of 8-week-old STARTS transformed roots was harvested. The STARTS transformed roots were observed under a Leica MZ10F dissecting microscope equipped with a fluorescent light source and mCherry fluorescence filter. Roots with mCherry fluorescent signal were marked and the NLS was circled on the plate. Harvest from negative GUS control, pOsUbi::MtLSH1, pOsUbi::MtLSH2 and pOsUbi::HvOptMtLSH1 ( Fig.15 All visible NLSs of transformed roots of 100 μM MgCl2 were identified and immediately fixed with 50% glycerol for confocal imaging. Immediately after imaging, these NLSs were fixed with 4% PFA in PBS for 1 hour and then transferred to ClearSee solution for tissue clearing.

[0182] After the first harvest, callus with regenerated roots and shoots was immediately transferred to enriched medium plates (MS medium or MODFP medium) and regenerated STARTS plants were further grown for 7 days. For alfalfa LSH1 and LSH2 genes (MtLSH1, MtLSH2), STARTS regenerated plants were transferred to buffered nodulation medium (BNM)-N+P (nitrogen-deficient conditions). After 4 days of growth in BNM-N+P, 50 μM 2,4-dichlorophenoxyacetic acid (2,4-D) solution was sprayed on the regenerated plants for auxin treatment. For barley codon-optimized versions of LSH1 and LSH2 genes (HvOptMtLSH1 and HvOptMtLSH2), STARTS regenerated plants were directly transferred to BNM-N+P medium with 50 μM 2,4-D.

[0183] Example 14: MtLSH1 or HvOptMtLSH1 alters the development of lateral organs in barley

[0184] To evaluate the effects of the introduction of alfalfa LSH1 and LSH2 genes (MtLSH1, MtLSH2) and codon-optimized versions of LSH1 and LSH2 genes (HvOptMtLSH1 [SEQ ID NO:7] and HvOptMtLSH2 [SEQ ID NO:8]) on barley root organogenesis, constitutive overexpression constructs of these genes driven by the rice ubiquitin promoter were generated ( Fig.15 Initially, no clear differences were observed between using pOsUBI3 and pPvUBI2. Therefore, the results are presented as a combination of pUbi data. pZmUBI::mCherry( Fig.15 ) was used as a visual marker of the construct to help identify successfully transformed STARTS roots. Roots with mCherry signal were marked and collected for further analysis ( Fig.16 ).

[0185] Overexpression of MtLSH1 or MtLSH2 in barley roots alters lateral root organogenesis ( Fig.16 , 17 ). NLS harvested from negative GUS controls had central vasculature, well-defined apical meristems, and distinct root tips ( Fig.16 A, 16D). NLS harvested from roots transformed with both pOsUbi::MtLSH1 and pOsUbi::MtLSH2 had an altered overall morphology and were more spherical, but still had a central vasculature and persistent meristem ( Fig.16 BC, 16E-F).

[0186] Interestingly, the effect of introducing MtLSH1 in barley roots seemed to be more pronounced on NLS when the transformed barley roots were grown under nitrogen-deficient conditions (BNM-N+P medium) and supplemented with 50 μM 2,4-D. Based on the harvest results after auxin treatment, NLS harvested from the negative GUS control appeared to have stunted lateral roots with a broad base, central vasculature, and a distinct root tip ( Fig.17 A, 17C, 17E). On the other hand, NLS harvested from pUbi::MtLSH1 transformed roots had an enlarged spherical shape, a broad base, and enlarged meristems ( Fig.17 Surprisingly, NLSs have multiple vascular bundles branching out from the base and connecting to the primary root vasculature, similar to the morphology of alfalfa nodules ( Fig.17 D).

[0187] To evaluate whether codon optimization could improve the expression and function of MtLSH genes in barley, barley codon-optimized forms of MtLSH1 and MtLSH2 (HvOptMtLSH1 and HvOptMtLSH2) were introduced. Overexpression of HvOptMtLSH1 in barley roots also altered the organogenesis of barley lateral roots, but resulted in different morphological changes ( Fig.18 , 19 Based on the results of Harvest 1, NLS harvested from the negative GUS control had a central vasculature, a well-defined apical meristem, and a distinct root tip ( Fig.18 A, 18C). NLS collected from pOsUbi::HvOptMtLSH1 transformed roots had an altered overall morphology, lacking a persistent meristem and having a more spherical shape while still possessing a central vasculature ( Fig.17 B, 17D).

[0188] Interestingly, the effect of introducing HvOptMtLSH1 in barley roots seemed to be more pronounced on NLSs when the transformed barley roots were grown in BNM-N+P medium supplemented with 50 μM 2,4-D. Based on the results after auxin treatment, NLSs harvested from the negative GUS control had a spherical shape and a central vasculature ( Fig.19 A, 19C). On the other hand, NLS harvested from Ubi::HvOptMtLSH1 transformed roots had multi-leaf structures, dispersed meristems and dispersed vascular bundles ( Fig.19 B, 19D).

[0189] Example 15: No significant changes in the number and frequency of NLSs were observed in MtLSH1 or HvOptMtLSH1 transformed roots

[0190] To assess whether the introduction of MtLSH1 or HvOptMtLSH1 affects the frequency of lateral organ initiation in barley, the number and frequency of NLSs were also quantified. No significant changes in the number and frequency of NLSs were observed in MtLSH1 or HvOptMtLSH1 transformed roots before or after auxin treatment ( Fig. 20 ).

[0191] Example 16: Cassava plants with modified LSH1 activity

[0192] Cassava plant cells were genomic modified to introduce modifications into the endogenous sequence encoding LSH1 (SEQ ID NO: 1). The modified plant cells were regenerated to produce plants with altered LSH1 activity compared to control plants that did not contain the modification. Plants with altered LSH1 activity showed altered transcriptional profiles and altered root architecture, such as an increase in root length and diameter, and in addition to enhanced interaction with rhizobia, also showed the development of modified lateral roots similar to nodules.

[0193] Example 17: Cassava plants with modified LSH2 activity

[0194] Cassava plant cells were genomic modified to introduce modifications into the endogenous sequence encoding LSH2 (SEQ ID NO: 3). The modified plant cells were regenerated to produce plants with altered LSH2 activity compared to control plants that did not contain the modification. Plants with altered LSH2 activity showed altered transcriptional profiles and altered root architecture, such as an increase in root length and diameter, and in addition to enhanced interaction with rhizobia, also showed the development of modified lateral roots similar to nodules.

[0195] Example 18: Putative NIN binding sites in LSH1 exons

[0196] Under non-symbiotic conditions, Chip-Seq using hairy roots expressing LjUBI:GFP-NIN revealed a high-confidence DNA binding site (found in >50%, 2 out of 3 biological replicates) located 5260 bp upstream of LSH1 ( Fig. 22 ). The top panel shows the Eugene annotation of the Medicago truncatula genome version 5, the confident peak called regions, the number of reads of the LjUBI:GFP-NIN ChIP replicates mapped to the genome relative to the control, and the summarized p-value significance signal.

[0197] *******

[0198] Having illustrated and described the principles of the invention, it will be apparent to one skilled in the art that the invention can be modified in operation and detail without departing from these principles. We claim protection for all modifications within the spirit and scope of the claims. All publications and published patent documents cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

1. A recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide fragment encoding a photosensitive short hypocotyl protein or a fragment thereof, wherein: a. The protein comprises an amino acid sequence of SEQ ID NO: 2 or 4 or a fragment thereof; b. The protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4 or a fragment thereof; c. the polynucleotide fragment hybridizes with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7 or 8 or a fragment thereof under stringent hybridization conditions; or d. the polynucleotide fragment hybridizes to a polynucleotide under stringent hybridization conditions, the polynucleotide having a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or about 100% nucleotide sequence identity to SEQ ID NO: 1, 3, 5, 6, 7 or 8 or a fragment thereof.

2. The recombinant DNA molecule of claim 1, wherein: a. expressing the recombinant DNA molecule in plant cells to produce an increase in intercellular cortical infection, an increase in intracellular colonization of nitrogen-fixing bacteria or an increase in bacterial nitrogen fixation; or b. The recombinant DNA molecule is operably linked to a vector, and the vector is selected from the group consisting of a plasmid, a phagemid, a bacmid, a cosmid, and a bacterial or yeast artificial chromosome.

3. The recombinant DNA molecule of claim 1, which is present in a host cell, wherein the host cell is selected from the group consisting of a bacterial cell and a plant cell.

4. The recombinant DNA molecule of claim 3, wherein the bacterial host cell is from a bacterial genus selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia.

5. The recombinant DNA molecule of claim 4, wherein the Bacillus is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus is Brevibacillus laterosus, and the Escherichia is Escherichia coli.

6. The recombinant DNA of claim 2, wherein the plant cell is a dicotyledonous or monocotyledonous plant cell.

7. The recombinant DNA of claim 6, wherein the plant cell is selected from the group consisting of alfalfa, almond, Bambara peanut, banana, barley, bean, black currant, broccoli, cabbage, blackberry, canola, rapeseed, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cowpea, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, legume grass, garlic, grape, hemp, hops, indigo, leek, legume, legume tree, lentil, lettuce, firewood. Loblolly pine, lotus, lupine, millet, melon, alfalfa, nuts, oats, olives, onions, ornamentals, palm, forage grasses, peas, peanuts, pepper, pigeon pea, pine, potato, poplar, pumpkin, bean, radiata pine, radish, rapeseed, raspberry, red currant, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, zucchini, strawberry, sugar beet, sugarcane, sunflower, liquidambar, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, walnut, watermelon, wheat, and yam plant cells.

8. A plant or part thereof comprising the recombinant DNA molecule of claim 1.

9. The plant or part thereof of claim 8, wherein the plant is a monocot or a dicot.

10. The plant or part thereof of claim 9, wherein the plant is selected from the group consisting of alfalfa, almond, Bambara peanut, banana, barley, bean, black currant, broccoli, cabbage, blackberry, canola, rapeseed, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, cowpea, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, legume grass, garlic, grape, hemp, hops, indigo, leek, legume, legume tree, lentil, lettuce, Loblolly pine, lotus, lupine, millet, melon, alfalfa, nuts, oats, olives, onions, ornamentals, palm, forage grasses, peas, peaches, peanuts, peppers, pigeon peas, pine, potato, poplar, pumpkin, beans, radiata pine, radish, rapeseed, raspberry, red currant, rice, rootstock, rye, safflower, shrubs, sorghum, southern pine, soybeans, spinach, zucchini, strawberries, sugar beets, sugar cane, sunflower, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, walnut, watermelon, wheat, and yams.

11. The plant or part thereof of claim 8, wherein the expression of the polynucleotide fragment encoding the photosensitive short hypocotyl protein varies within 24 hours.

12. The plant or part thereof of claim 11, wherein expression of the polynucleotide fragment encoding a light-sensitive short hypocotyl protein is increased during the first 12 hours of a 12 hour / 12 hour light / dark cycle.

13. The plant or part thereof of claim 11, wherein expression of the polynucleotide fragment encoding a light-sensitive short hypocotyl protein is increased during the first 6 hours of a 12 hour / 12 hour light / dark cycle.

14. A transgenic seed comprising the recombinant DNA molecule of claim 1.

15. A method for producing a progeny seed comprising the recombinant DNA molecule of claim 1, the method comprising: a. Planting a first seed comprising the recombinant DNA molecule of claim 1; b. growing plants from the seeds of step a; and c. harvesting progeny seeds from the plant, wherein the harvested seeds contain the recombinant DNA molecule.

16. A plant susceptible to intercellular cortical infection or intracellular colonization by nitrogen-fixing bacteria, wherein cells of the plant comprise the recombinant DNA molecule of claim 1.

17. A method for increasing intercellular cortical infection or intracellular colonization of nitrogen-fixing bacteria in plants, the method comprising: a. expressing in a plant a photosensitive short hypocotyl protein or a fragment thereof having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or about 100% sequence identity with SEQ ID NO: 2 or 4; or expressing a photosensitive short hypocotyl protein or a fragment thereof encoded by a nucleic acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or about 100% sequence identity to SEQ ID NO: 1, 3, 5, 6, 7 or 8 or a fragment thereof; as well as b. contacting the plant with an effective amount of one or more rhizobia, arbuscular mycorrhizal fungi, or a combination thereof.

18. The method of claim 17, wherein: a. The rhizobium is selected from the group consisting of S. meliloti, Mesorhizobium loti, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium sp. IRBG74 and NGR234, and Bradyrhizobium sp.; or b. The arbuscular mycorrhizal fungi are selected from the group consisting of R. irregularis, Rhizophagus intraradices, Glomus mosseae and Funneliformis mosseae.

19. A modified plant, plant seed, plant part or plant cell comprising a genomic modification that modulates LSH1 or LSH2 activity compared to the activity of LSH1 or LSH2 in an otherwise identical plant, plant seed, plant part or plant cell lacking the modification.

20. The modified plant, plant seed, plant part or plant cell of claim 19, wherein the modification is in at least one allele of an endogenous LSH1 or LSH2 gene.

21. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the genomic modification is in an endogenous LSH1 or LSH2 gene encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO: 2 or 4.

22. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the modification is in the transcribable region of the LSH1 or LSH2 gene.

23. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the plant, plant seed, plant part or plant cell is heterozygous for the modification.

24. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the plant, plant seed, plant part or plant cell is homozygous for the modification.

25. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the modification comprises a deletion, an insertion, a substitution, an inversion, a duplication or any combination thereof.

26. The modified plant, plant seed, plant part or plant cell of claim 20, wherein the modification comprises the deletion of at least 1, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 consecutive nucleotides.

27. The modified plant, plant seed, plant part or plant cell of any one of claims 17-23, wherein the plant, plant seed, plant part or plant cell comprises a chromosomal sequence in a LSH1 or LSH2 gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3 in regions other than deletions, insertions, substitutions, inversions or duplications.

28. A method for producing a plant comprising a modified LSH1 or LSH2 gene, the method comprising: a) introducing a modification that modulates the activity of LSH1 or LSH2 into at least one target site in an endogenous LSH1 or LSH2 gene of a plant cell; b) identifying and selecting one or more plant cells of step (a) comprising said modification in said LSH1 or LSH2 gene; and c) regenerating at least a first plant from said one or more cells selected in step (b) comprising said modification, or their progeny.

29. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 84-93; b) a sequence comprising any one of SEQ ID NOs: 84-93; c) a fragment of any one of SEQ ID NOs: 84-93, wherein the fragment has gene regulatory activity; and d) a fragment of a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 84-93, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA molecule.

30. The recombinant DNA molecule of claim 29, wherein the sequence has at least 90% sequence identity to the DNA sequence of any one of SEQ ID NOs: 84-93 or a fragment thereof.

31. The recombinant DNA molecule of claim 30, wherein the sequence has at least 95% sequence identity to the DNA sequence of any one of SEQ ID NOs: 84-93 or a fragment thereof.

32. The recombinant DNA molecule of claim 31, wherein the sequence comprises the DNA sequence of any one of SEQ ID NOs: 84-93 or a fragment thereof.

33. The recombinant DNA molecule of claim 29, wherein the heterologous transcribable DNA molecule comprises a gene of agronomic interest.

34. A transgenic plant cell comprising the recombinant DNA molecule of claim 29.

35. The transgenic plant cell of claim 34, wherein the transgenic plant cell is a monocot cell.

36. The transgenic plant cell of claim 34, wherein the transgenic plant cell is a dicotyledonous plant cell.

37. A transgenic plant or part thereof comprising the recombinant DNA molecule of claim 29.

38. A progeny plant or part thereof of the transgenic plant of claim 37, wherein said progeny plant or part thereof comprises said recombinant DNA molecule.

39. A transgenic seed, wherein the seed comprises the recombinant DNA molecule of claim 29.

40. A method for producing a commercial product, comprising obtaining the transgenic plant or part thereof according to claim 37, and producing a commercial product therefrom.

41. The method of claim 40, wherein the commodity is a protein concentrate, a protein isolate, a grain, a starch, a seed, a meal, a flour, a biomass, or a seed oil.

42. A method for expressing a transcribable DNA molecule, comprising obtaining the transgenic plant according to claim 37 and cultivating the plant, wherein the transcribable DNA is expressed.

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