Plant regulatory elements and uses thereof

Abstract

The present invention provides novel DNA molecules and constructs, including nucleotide sequences thereof, for modulating gene expression in plants and plant cells. The invention also provides transgenic plants, plant cells, plant parts, seeds, and commodity products comprising 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 / 367,703, filed on July 5, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation into the Sequence Listing

[0004] Submit electronically here a file named "AGOE010WO_ST26.xml" created on June 7, 2023 (12.5KB, Microsoft Measured in ), which is incorporated herein by reference. Technical Field

[0005] The present invention relates to plant molecular biology and plant genetic engineering and DNA molecules for regulating gene expression in plants. Background Art

[0006] Regulatory elements are genetic elements that regulate gene activity by regulating the transcription of operably linked transcribable polynucleotide molecules. Such elements include promoters, leader sequences, introns and 3' untranslated regions, and can be used in the fields of plant molecular biology and plant genetic engineering. SUMMARY OF THE INVENTION

[0008] The present invention provides novel gene regulatory elements for plants. The present invention also provides DNA constructs comprising the regulatory elements. The present invention also provides transgenic plant cells, plants and seeds comprising the regulatory elements. A sequence operably connected to a transcribable polynucleotide molecule can be provided. In one embodiment, the transcribable polynucleotide molecule can be heterologous relative to the regulatory sequence provided herein. Therefore, in a specific embodiment, the regulatory element sequence provided by the present invention can be defined as being operably connected to a heterologous transcribable polynucleotide molecule. The present invention also provides methods for preparing and using regulatory elements, DNA constructs comprising regulatory elements, and transgenic plant cells, plants and seeds comprising regulatory elements operably connected to a transcribable polynucleotide molecule.

[0009] Therefore, on the one hand, the present invention provides a DNA molecule comprising a DNA sequence selected from the following: a) a sequence having at least about 85% sequence identity with any one of SEQ ID NO: 1; b) a sequence comprising SEQ ID NO: 1; and c) a fragment of SEQ ID NO: 1 or a fragment having at least 85% sequence identity with a fragment of SEQ ID NO: 1, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule. In specific embodiments, the DNA molecule has at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the DNA sequence of SEQ ID NO: 1. In certain embodiments, if the fragment is less than 115 nucleotides, the DNA molecule has at least 87% sequence identity with the fragment of SEQ ID NO: 1. In certain embodiments of the DNA molecule, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In further embodiments, the DNA molecule has gene regulatory activity, such as promoter activity or symbiotic specific pectin methylesterase (SyPME) promoter activity. In certain embodiments, the heterologous transcribable polynucleotide molecule comprises an agronomic target gene. In some embodiments, the agronomic target gene is a gene encoding a pectin methylesterase having pectin demethylesterification activity. In a specific embodiment, the DNA sequence provides the expression of the heterologous transcribable polynucleotide molecule in response to an external stimulus. In some embodiments, the DNA sequence provides the expression of the heterologous transcribable polynucleotide molecule in root hair cells, in the cortex of the nodule primordium, in mature nodules, in the nodule infection zone of young, mature or indeterminate nodules.

[0010] The present invention also provides a transgenic plant cell comprising a heterologous DNA construct provided by the present invention, wherein the DNA construct comprises a sequence of SEQ ID NO: 1 or a fragment or variant thereof, wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0011] The present invention further provides a transgenic plant or part thereof, which comprises a DNA molecule provided herein, which comprises a DNA sequence selected from the following: a) a sequence having at least 85% sequence identity with SEQ ID NO: 1; b) a sequence comprising SEQ ID NO: 1; and c) a fragment of SEQ ID NO: 1 or a fragment having at least 85% sequence identity with a fragment of SEQ ID NO: 1, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule. In a specific embodiment, the transgenic plant can be a progeny plant of any generation comprising the DNA molecule relative to the starting transgenic plant comprising the DNA molecule. Transgenic seeds comprising the DNA molecule according to the present invention are further provided.

[0012] In another aspect, the present invention provides a method for producing a commodity, comprising obtaining a transgenic plant according to the present invention or a part thereof and producing a commodity therefrom. In one embodiment, the commodity of the present invention is a protein concentrate, a protein isolate, a grain, a starch, a seed, a meal, a flour, a biomass or a seed oil. On the other hand, the present invention provides a commodity produced using the above method. For example, in one embodiment, the present invention provides a commodity comprising a DNA molecule provided herein, the DNA molecule comprising a DNA sequence selected from the following: a) a sequence having at least 85% sequence identity with SEQ ID NO: 1; b) a sequence comprising SEQ ID NO: 1; and c) a fragment of SEQ ID NO: 1, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule.

[0013] In yet another aspect, the present invention provides a method for expressing a transcribable polynucleotide molecule, comprising obtaining a transgenic plant according to the present invention or a part thereof, such as a plant comprising a DNA molecule as described herein, and cultivating a plant in which the transcribable polynucleotide in the DNA molecule is expressed.

[0014] In the present specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to mean the inclusion of stated components, steps and / or values ​​or combinations thereof, but not the exclusion of any other components, steps and / or values ​​or combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Showing differentially modified pectins in infected cells. 14-day-old M. truncatula nodules were embedded in LR Gold and hybridized with the respective antibodies (red). DNA was counterstained with DAPI (blue). (Panel A) LM20 labels all cell wall structures. (Panel B) LM19 shows specific staining of the transcellular nodule infection line (IT; white arrows), in addition, LM19 also labels epidermal cells and exodermal cells. (Panels CD) Immunofluorescence labeling (red) with antibodies LM19 (Panel C) and LM20 (Panel D), where root (5 days after inoculation) sections were embedded in LR White. Arrows indicate infection lines. (Panel EF") CLEM analysis was performed with LM19 (Panel EE") and LM20 (Panel FF"). Scale bars represent 50 μm in AD; 2.5 μm in panel EF". TEM: transmission electron microscopy.

[0016] Figure 2 Unesterified pectin is shown to be concentrated at the IT penetration site. (Panel A) Scanning electron microscopy (SEM) and (Panel B) transmission electron microscopy (TEM) images of infected cells in nodules. Scale bar indicates 10 μm. (Panel C) Double immunogold labeling of the infection line passage site in the nodule with LM19 (12 nm) and LM20 (5 nm). Scale bar indicates 0.5 μm. (Panel D) Immunofluorescence labeling with antibody 2F4 (red) and DNA counterstained with DAPI (blue). Scale bar indicates 10 μm. Arrow indicates infection line (IT, in Panel A); arrow indicates IT penetration site (in Panels AD).

[0017] Figure 3 Showing the localization analysis of SyPME and NPL in nodules. (Panels AC) Localization of SyPME-GFP in transformed nodule sections. The SyPME-GFP signal is mainly located in the IT and strongly accumulates at the penetration site (Panels AC, indicated by arrows) and the tip of the IT (Panels DF, indicated by asterisks). SyPME-GFP is also slightly concentrated in the local area near the IT (Panels DF, indicated by arrows). The scale bar represents 5μm. (Panels GL) Localization of NPL-GFP in transformed nodule sections. The NPL-GFP signal accumulates strongly at the tip of the IT (Panels GI, indicated by asterisks) and slightly accumulates in the local area near the tip of the IT (Panels GI, indicated by arrows, the curve profile inserted in E and H indicates the GFP signal intensity marked by the red line (near the arrow) in the image). NPL-GFP is not concentrated at the IT penetration site (Panels JL, indicated by arrows). The scale bar represents 5μm.

[0018] Figure 4Show that npl mutants impair IT growth. (Panel A) Quantitative nodule number after 7 days of inoculation in open pots. (Panel B) Most infection events in npl mutants are blocked at the infection chamber stage. (Panel CJ) Phenotypic analysis of plants transformed with empty vectors (EV, panels C and D), ProUBI-NPL-RNAi constructs (panels E and F), epidermis-specific promoter (ProEXT1)-derived NPL-RNAi constructs (panels G and H) and cortex-specific promoter (ProPEP)-derived NPL-RNAi constructs (panels I and J). Cell walls were stained with Calcofluor white (white, in panels BJ). (KM") Phenotypic analysis of plants transformed with NPL promoters derived from AtPMEI12. Most infections were blocked during the IC stage (panel KK") or during the initial progression of IT formation and root hairs (panel LL") and cortex (panel MM"). Arrows indicate blocked ICs (panels B, E, G, and K''), arrowheads indicate blocked IT growth in root hairs (panel L') or cortex (panels J and M'). Scale bar represents 10 μm.

[0019] Figure 5 The proposed model of transcellular IT channel is described. CLEM analysis of cell wall modifications at IT penetration sites was performed using antibodies LM19 (unesterified pectin, panel A) and MAC265 (IT matrix, panel B). (Panel C) LM19-labeled cell wall structures were observed using transmission electron microscopy, and (Panel D) is a close-up image of the target area marked by the red box in (Panel C). (Panel E) is a proposed model of IT transcellular channel based on our observations. Scale bars represent 4μm (Panel AC) and 1μm (Panel D). ID: infection droplet.

[0020] Figure 6 Immunofluorescence labeling with different cell wall antibodies is shown. 14-day-old M. truncatula nodules were embedded in LRGold and hybridized with the respective antibodies (red). The infection thread matrix was labeled with MAC265 (panel A); LM25 recognizes the hemicellulose xyloglucan (panel B); LM5 (panel C) and LM6 (panel D) recognize different types of RG-1; LM2 (panel E), LM14 (panel F) and LM30 (panel G) all recognize arabinogalactan protein (AGP). DNA was counterstained with DAPI, scale bar represents 50 μm. Arrows indicate infection threads; CC: colonizing cells; NCC: non-colonizing cells.

[0021] Figure 7Immunofluorescence labeling with LM14 antibody is shown. 14-day-old Medicago truncatula nodules were embedded in LR Gold and hybridized with LM14 antibody (red). (Panel A) Sketch of Medicago nodule; (Panel B) indicates the area corresponding to (Panel B'); (Panel B") is a close-up of (Panel B') marked with a white box. (Panel C) indicates the area corresponding to (Panel C'); (Panel C") is a close-up of (Panel C') marked with a white box; (Panel C"') Fluorescence intensity analysis was performed using cross-sections as indicated by the lines within (Panel C"). DNA was counterstained with DAPI (blue). Arrows in (Panel B") indicate infection lines; arrows in (Panel C") indicate symbionts. Scale bars indicate 50 μm (Panel B'-C') and 5 μm (Panel C"). IZ: infection zone; FZ: fixed zone; CC: colonizing cells; NCC: non-colonizing cells.

[0022] Figure 8 Immunofluorescence labeling with LM6 antibody is shown. 14-day-old Medicago truncatula nodules were embedded in LR Gold and hybridized with LM6 antibody (red). (Panel A) Sketch of a Medicago nodule; (Panel B) indicates the area corresponding to (Panel B'); (Panel B") is a close-up of (Panel B') marked with a blue box. (Panel C) indicates the area corresponding to (Panel C'); (Panel C") is a close-up of (Panel C') marked with a magenta box; (Panel C"') Fluorescence intensity analysis was performed using cross-sections as indicated by the lines in (Panel C"). DNA was counterstained with DAPI (blue), and the scale bar represents 20 μm. IZ: infection zone; FZ: fixed zone; CC: colonizing cell; NCC: non-colonizing cell; IT: infection line.

[0023] Fig. 9 CLEM analysis showing root hair infection events. A correlative light electron microscopy (CLEM) protocol was established (Panel A); and used to visualize the cell wall of ICs (Panel BC) and ITs in root hairs (Panel D).

[0024] Fig.10Expression profile of SyPME and analysis of SyPME expression domains are shown. (Panel A) Expression patterns of NPL and SyPME are highly correlated (0.9883), as exemplified when roots were inoculated at different time points with a nodulation factor (NF)-deficient S. meliloti nodABC strain (nodABC), WT S. meliloti, and isolated NF. Data from Breakspear et al., 2014. Dpi: days post inoculation; h: hours. (Panel B) Schematic representation of the spatial expression of SyPME and different pectate lyases, including NPL, in different zones of indeterminate alfalfa nodules. Original data retrieved from Roux et al., 2014. FI: nodule meristem zone; zIId: distal infection zone; zIIp: proximal infection zone; IZ: intermediate zone; ZIII: nitrogen fixation zone. (CF) Spatial analysis of SyPME transcript accumulation by in situ hybridization on 14-day-old Medicago nodules using SyPME antisense (panels CD) and sense (control) probes (panels EF). Magenta precipitates indicate the presence of SyPME mRNA. (Panels FH) Promoter GUS (blue) analysis of ProSyPME (panels GH) and ProNPL (I) on 14-day-old transformed Medicago truncatula nodules and counterstained with toluidine blue (purple). Scale bar represents 50 μm.

[0025] Fig.11 Shown are the localization analysis of SyPME and NPL during primary infection. Images were acquired from transformed plants 7 days after inoculation. Infection chambers (AA" and CC"), growing infection lines (panels BB" and DD"), and infection line channels (EF"). Green signals represent SyPME in (panels AB" and EE"), and NPL in (panels CD" and FF"); red represents S. meliloti; scale bar represents 10 μm.

[0026] Fig.12 The genetic structure and phenotype of sypme are depicted. (Panel A) Schematic representation of the SyPME genetic structure and the Tnt1 transposon insertion site mapped. UTR, untranslated region. (Panel B) Infection chambers (IC) and infection lines (IT) were scored 10 days after inoculation, n = 10 roots per genotype. IC and IT morphology were visualized by Calcofluor-white staining (white) in R108 (Panels CD) and sypme mutants (Panels EF). Magenta indicates S. meliloti. Scale bar represents 10 μm.

[0027] Fig.13Depicted are future transcellular passage sites, as defined by cytoplasmic columns formed before the IT. TEM micrograph of a 14-day-old nodule, where the cell wall is colored in yellow, the nucleus is depicted in blue, and the cytoplasmic columns connect the infection thread (IT) to the cell wall depicted in red. IT: infection thread; R: Rhizobium. Scale bar represents 2 μm.

[0028] Sequence Description

[0029] SEQ ID NO: 1 is the promoter sequence of the Medicago truncatula SyPME gene Medtr4g087980.

[0030] SEQ ID NO: 2 is the nucleic acid sequence encoding Medicago truncatula SyPME protein Medtr4g087980.

[0031] SEQ ID NO:3 is the amino acid sequence of Medicago truncatula SyPME protein Medtr4g087980, encoded by SEQ ID NO:2.

[0032] SEQ ID NO: 4 is the nucleic acid sequence encoding the Medicago truncatula SYMREM1 protein.

[0033] SEQ ID NO:5 is the amino acid sequence of the Medicago truncatula SYMREM1 protein, encoded by SEQ ID NO:4.

[0034] SEQ ID NO: 6 is the nucleic acid genomic sequence encoding the Medicago truncatula SyPME protein. DETAILED DESCRIPTION OF THE INVENTION

[0036] The intracellular colonization of host cells by symbionts represents a mutually beneficial symbiotic relationship between host plants and soil-borne bacteria or fungi. For example, legumes are known to form a symbiotic relationship with nitrogen-fixing rhizobia, commonly known as root nodule symbiosis (RNS). When the symbiotic bacteria are initially absorbed by morphologically adapted root hairs, the rhizobia continue to progress through several root cortical tissues and later nodule cell layers within the membrane-restricted infection line. Throughout this transcellular passage, rhizobia must repeatedly pass through the host plasma membrane and cell wall (CW). In addition, spatiotemporally restricted cell wall remodeling is required to initiate and maintain infection line (IT) growth, IT transcellular passage, and bacterial release. This cell wall remodeling is partially controlled by the synergistic action of symbiotic-specific pectin methylesterase (i.e., SyPME; SEQ ID NO: 3) and pectate lyase NPL at the infection line and transcellular passage sites, which allows IT to successfully progress intracellularly through the entire root cortical tissue. Therefore, the SyPME promoter (i.e., SEQ ID NO: 1) provides beneficial gene regulatory activity for expression in plant species. For example, the present disclosure demonstrates that the SyPME promoter provides expression within the nodule primordium cortex in young and mature nodules, as well as expression within a restricted expression domain limited to infection zone II. Thus, this gene regulatory activity allows for the spatiotemporal expression of genes required for the efficient colonization of engineered nodules and / or nodule-like structures, in a symbiosis-dependent and spatially restricted manner.

[0037] Therefore, the present disclosure provides polynucleotide molecules with beneficial gene regulatory activity from plant species. The present invention provides the design, construction and use of these polynucleotide molecules. The nucleotide sequences of these polynucleotide molecules are provided herein, such as SEQ ID NO:1. These polynucleotide molecules can, for example, affect the expression of transcribable polynucleotide molecules that can be operably linked in plant tissues, and thus selectively regulate the activity of gene expression or encoded gene products in transgenic plants. The present invention also provides methods for modifying, producing and using them. The present invention also provides compositions, transformed host cells, transgenic plants and seeds containing promoters and / or other disclosed nucleotide sequences, as well as methods for preparing and using them.

[0038] The following definitions and methods are provided to better define the present invention and guide those of ordinary skill in the art to implement the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0039] Symbiotic bacteria

[0040] The present invention provides DNA molecules with gene regulatory activity, for example, a DNA molecule comprising a sequence having at least 85% sequence identity to SEQ ID NO: 1 can be expressed during symbiotic infection of rhizobia, wherein SEQ ID NO: 1 is operably linked to a transcribable polynucleotide molecule. Rhizobia are bacteria found in the soil that infect the roots of legumes and colonize nodules involved in nitrogen utilization. As used herein, "rhizobia" refers to any nitrogen-fixing bacteria that fix atmospheric nitrogen in plant roots. Symbiotic bacteria can be used with plants comprising the recombinant DNA molecules described herein. Symbiotic bacteria that can be used for the disclosed plants include, but are not limited to, Mesorhizobium loti, Sinorhizobium meliloti, Sinorhizobium fredii, and Bradyrhizobium sp.

[0041] DNA molecules

[0042] 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 of deoxyribonucleotide bases or polynucleotide molecules, 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 the terms in 37 CFR §1.822 and are shown in the tables of Tables 1 and 3 of Appendix 2 of WIPO Standard ST.25 (1998).

[0043] As used herein, the term "separated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules that are usually associated with it under its original or natural state. In one embodiment, the term "separated" refers to a DNA molecule that is at least partially separated from some nucleic acids that are located at the side of the DNA molecule under its original or natural state. Therefore, a DNA molecule that is fused to a regulatory or coding sequence that is usually not associated with it (for example, as a result of recombinant technology) is considered to be separated in this article. When integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, this molecule is considered to be separated because they are not in its original state.

[0044] Any number of 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 amplifying specific starting DNA molecules and / or producing variants of the original molecule.DNA molecules or its fragments can also be obtained by other technologies, for example, by chemical method direct synthesis of fragments, which is usually achieved by using an automatic oligonucleotide synthesizer.

[0045] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is produced by artificially aligning two sequences (e.g., a reference sequence and another sequence) to maximize the number of nucleotides that match appropriate internal nucleotide insertions, deletions, or gaps in the sequence alignment. As used herein, the term "reference sequence" refers to a sequence provided as the polynucleotide sequence of SEQ ID NO: 1.

[0046] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the identity score multiplied by 100. The "identity score" of a sequence optimally aligned with a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides in the entire length of the reference sequence. Thus, one embodiment of the present invention is a DNA molecule comprising a sequence that, when optimally aligned with the reference sequence provided herein as SEQ ID NO: 1, has 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 with the reference sequence. In specific embodiments, such a sequence can be defined as having gene regulatory activity.

[0047] Regulatory elements

[0048] Regulatory element is a DNA molecule with gene regulation activity, i.e., a DNA molecule with the ability of transcription and / or translation that can affect the transcribable polynucleotide molecule that can be operably connected.Therefore, the term "gene regulation activity" refers to the ability of affecting the expression pattern of the transcribable polynucleotide molecule that can be operably connected by affecting the transcription and / or translation of the transcribable polynucleotide molecule that can be operably connected.As used herein, the transcription regulatory sequence can include the expression element that can be operably connected, such as enhancer, promoter, leader sequence and intron.Therefore, the transcription regulatory sequence can include the 5' of the promoter that can be operably connected with the leader sequence, and the 5' of the leader sequence can be operably connected with the intron sequence again.The leader sequence and intron can actively affect the transcription of the transcribable polynucleotide molecule that can be operably connected and the translation of the resulting transcription RNA.Pretreated RNA molecule includes leader sequence and intron, and they can affect the post-transcription processing of the transcribed RNA and / or the RNA molecule that transcribes is exported to the cytoplasm from the nucleus. Following post-transcriptional processing of the transcribed RNA molecule, the leader sequence may remain as part of the final messenger RNA and may actively influence the translation of the messenger RNA molecule.

[0049] Regulatory elements such as promoters, leader sequences, introns and transcription termination regions (or 3'UTR) are DNA molecules that have gene regulatory activity and play an important role in the overall expression of genes in living cells. The term "regulatory element" refers to a DNA molecule with gene regulatory activity, i.e., a DNA molecule with the ability to affect the transcription and / or translation of an operably linked transcribable polynucleotide molecule. Therefore, isolated regulatory elements such as promoters and leader sequences that function in plants can be used to modify plant phenotypes by genetic engineering.

[0050] Regulatory elements can be characterized by their expression pattern effects (qualitative and / or quantitative), such as positive or negative effects and / or constitutive or other effects, such as their temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle and / or chemical responsive expression patterns, and any combination thereof, as well as quantitative or qualitative representations. Promoters can be used as regulatory elements that regulate the expression of operably linked transcribable polynucleotide molecules.

[0051] As used herein, "gene expression pattern" is any pattern of transcription of operably linked DNA molecules into transcribed RNA molecules. The transcribed RNA molecules can be translated to produce protein molecules or can provide antisense or other regulatory RNA molecules, such as dsRNA, tRNA, rRNA, miRNA, etc.

[0052] 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 expression.

[0053] As used herein, the term "promoter" generally refers to a DNA molecule that participates in recognizing and binding RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. The promoter can be initially isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter can be a DNA molecule produced or manipulated synthetically. The promoter can also be chimeric, i.e., a promoter produced by fusing two or more heterologous DNA molecules. Promoters that can be used to implement the present invention include SEQ ID NO: 1 or its variants or fragments. In a specific embodiment of the present invention, such molecules as described herein and any variants or derivatives thereof are further defined as comprising gene regulatory activity or promoter activity, i.e., being able to act as a promoter in a host cell (such as a transgenic plant). In a further specific embodiment, a fragment can be defined as exhibiting the gene regulatory activity or promoter activity possessed by the initiator promoter molecule derived therefrom, or a fragment can include a "minimal promoter" that provides a basal transcription level, and includes a TATA box or equivalent sequence for recognizing and binding to the RNA polymerase II complex that initiates transcription.

[0054] In one embodiment, fragments of the promoter sequences disclosed herein are provided. Promoter fragments may comprise gene regulatory activity or promoter activity as described above, and may be used alone or in combination with other promoters and promoter fragments, such as for constructing chimeric promoters. In a specific embodiment, a promoter fragment is provided that comprises at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 06, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 1 63、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、 220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345 45, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379 9, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 500, 750, 1000, 1250, 1500, 1750, or at least about 2000 consecutive nucleotides or bases, or longer. The promoter fragment can be a functional fragment having gene regulatory function or activity or promoter function or activity. The expression of discrete values ​​should be understood to include the range between each value and the discrete values ​​between these ranges. ,

[0055] Compositions derived from any promoter as shown in SEQ ID NO: 1 (e.g., internal or 5' deletions) can be generated using methods known in the art to improve or alter expression, including by removing elements that have a positive or negative effect on expression; duplicating elements that have a positive or negative effect on expression; and / or duplicating or removing elements that have a tissue or cell-specific effect on expression. Compositions derived from any promoter as shown in SEQ ID NO: 1 can be used, for example, to prepare enhancer elements, wherein the promoter comprises a 3' deletion in which the TATA box element or its equivalent and downstream sequences are removed. Further deletions can be made to remove any element that has a positive or negative; tissue-specific; cell-specific; or timing-specific (e.g., but not limited to, circadian) effect on expression. Any promoter as shown in SEQ ID NO: 1 and fragments or enhancers derived therefrom can be used to prepare chimeric transcriptional regulatory element compositions consisting of any promoter as shown in SEQ ID NO: 1 and fragments or enhancers derived therefrom that are operably linked to other enhancers and promoters. 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 those described in the working examples herein, to validate the results, which may vary depending on the alterations made and the goal of the initial molecular alteration.

[0056] As used herein, the term "leader sequence" refers to a DNA molecule separated from the non-translated 5' region (5'UTR) of the genomic copy of a gene, and is generally defined as a nucleotide segment between the transcription start site (TSS) and the start site of a protein coding sequence. Alternatively, the leader sequence can be a synthetically produced or manipulated DNA element. The leader sequence can be used as a 5' regulatory element for regulating the expression of an operably linked transcribable polynucleotide molecule. The leader molecule can be used with a heterologous promoter or its natural promoter. Therefore, the promoter molecule of the present invention can be operably linked to its natural leader sequence, or can be operably linked to a heterologous leader sequence. Leader sequences known in the art can be used to implement the present invention. The leader sequence (5'UTR) can be composed of a regulatory element or can adopt a secondary structure that has an effect on the transcription or translation of the transgene. Leader sequences known in the art can be used according to the present invention to prepare chimeric regulatory elements that affect transgenic transcription or translation. In addition, the leader sequence can be used to prepare a chimeric leader sequence that affects transgenic transcription or translation.

[0057] The introduction of foreign genes into new plant hosts does not always result in high expression of the input genes. In addition, if complex traits are treated, it is sometimes necessary to regulate several genes with spatially or temporally different expression patterns. Introns can mainly provide such regulation. However, the multiple use of the same intron in one plant has shown disadvantages. In those cases, it is necessary to have a collection of basic control elements for the construction of suitable recombinant DNA elements.

[0058] According to the present invention, promoters or promoter fragments can be analyzed for the presence of known promoter elements, i.e., DNA sequence features, such as the TATA box and other known transcription factor binding site motifs. Those skilled in the art can use the identification of such known promoter elements to design promoter variants that have similar expression patterns to the original promoter.

[0059] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting transcriptional regulatory element, also referred to as a cis-element, which imparts aspects of the overall expression pattern of an operably linked polynucleotide sequence, but is generally insufficient to drive transcription alone. Unlike a promoter, an enhancer element generally does not include a transcription start site (TSS) or a TATA box or equivalent sequence. A promoter may naturally contain one or more enhancer elements that affect the transcription of an operably linked polynucleotide sequence. An isolated enhancer element may also be fused to a promoter to produce a chimeric promoter cis-element that imparts aspects of the overall regulation of gene expression. A promoter or promoter fragment may contain one or more enhancer elements that affect the transcription of an operably linked gene. Many promoter enhancer elements are thought to bind DNA binding proteins and / or affect DNA topology, producing a local conformation that selectively allows or restricts RNA polymerase access to a DNA template or promotes selective opening of a double helix at a transcription start site. Enhancer elements may be used to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors may interact with more than one enhancer domain with different affinities. Enhancer elements can be identified by a variety of techniques, including deletion analysis, i.e., one or more nucleotides are deleted from the 5' end or inside of the promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assay, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by using known cis-element motifs or enhancer elements as target sequences or target motifs for DNA sequence similarity analysis, wherein conventional DNA sequence comparison methods such as BLAST are used. The fine structure of the enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Enhancer elements can be obtained by chemical synthesis or by separation from regulatory elements including these elements, and they can be synthesized with additional flanking nucleotides containing useful restriction enzyme sites to facilitate subsequence manipulation. Therefore, the present invention encompasses the design, construction, and use of enhancer elements according to the methods disclosed herein for regulating the expression of operably linked transcribable polynucleotide molecules.

[0060] In plants, inclusion of some introns in gene constructs results in increased mRNA and protein accumulation relative to constructs lacking introns.

[0061] This effect is called "intron-mediated enhancement" (IME) of gene expression (Mascarenhas et al., (1990) Plant Mol. Biol. 15:913-920). Introns known to stimulate expression in plants have been described in maize genes (e.g., tubA1, Adh1, Sh1, Ubi1 (Jeon et al. (2000) Plant Physiol. 123:1005-1014; Callise et al. (1987) Genes Dev. 1:1183-1200; Vasil et al. (1989) Plant Physiol. 91:1575-1579; Christiansen et al. (1992) Plant Mol. Biol. 18:675-689) and rice genes (e.g., salt, tpi: McElroy et al., Plant Cell 2:163-171 (1990); Xu et al., Plant Cell 3:164-172 (1992)). Physiol. 106:459-467 (1994)). Similarly, introns from dicot genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis (e.g., ubq3 and pat1) have been found to increase the rate of gene expression (Dean et al. (1989) Plant Cell 1:201-208; Leon et al. (1991) Plant Physiol. 95:968-972; Norris et al. (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J. 11:455-464). It has been shown that deletions or mutations within intron splice sites reduce gene expression, suggesting that splicing may be required for IME (Mascarenhas et al. (1990) Plant Mol Biol. 15:913-920; Clancy and Hannah (2002) Plant Physiol. 130:918-929). However, it has been shown by point mutations within the splice site of the pat1 gene from Arabidopsis that some IMEs in dicots do not require splicing per se (Rose and Beliakoff (2000) Plant Physiol. 122:535-542).

[0062] Enhancement of gene expression by introns is not a universal phenomenon, as some introns inserted into recombinant expression cassettes fail to enhance expression (e.g., introns from dicot genes (rbcS gene from pea, phaseolin gene from bean, and stls-1 gene from potato) and introns from maize genes (adh1 gene, ninth intron, hsp81 gene, first intron)) (Chee et al. (1986) Gene 41:47-57; Kuhlemeier et al. (1988) Mol Gen Genet 212:405-411; Mascarenhas et al. (1990) Plant Mol. Biol. 15:913-920; Sinibaldi and Mettler (1992) In WE Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academic Press, New York, pp. 11-13). 229-257; Vancanneyt et al. 1990 Mol. Gen. Genet. 220: 245-250). Therefore, not every intron can be used to manipulate the gene expression level of non-endogenous genes or endogenous genes in transgenic plants. What features or specific sequence features must be present in the intron sequence in order to increase the expression rate of a given gene is unknown in the prior art, and therefore it is impossible to predict from the prior art whether a given plant intron (when used heterologously) will result in expression enhancement at the DNA level or transcript level (IME).

[0063] 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 neither the first nor the second DNA molecule is normally found in this configuration, i.e., fused to each other. Therefore, the chimeric DNA molecule is a new DNA molecule that is not normally found in nature. 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; an example is the fusion of a promoter to an enhancer element. Therefore, the design, construction, and use of chimeric promoters for regulating the expression of operably linked transcribable polynucleotide molecules according to the methods disclosed herein are encompassed in the present disclosure.

[0064] As used herein, the term "variant" refers to a second DNA molecule that is similar but not identical to the first DNA molecule in composition, but the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern. The variant can be a shorter or truncated form of the first DNA molecule and / or an altered form of the first DNA molecule sequence, such as a form with different restriction enzyme sites and / or internal deletions, substitutions and / or insertions. "Variant" can also encompass regulatory elements having a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides of 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 encompass variants produced by naturally occurring mutations in bacterial and plant cell transformation. In the present invention, the polynucleotide sequence provided as SEQ ID NO: 1 can be used to produce variants that are similar but not identical to the polynucleotide 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. According to the present disclosure, the generation of these variants of the present invention is completely within the ordinary skill of the art, and is encompassed within the scope of the present invention. Chimeric regulatory element "variants" contain the same constituent elements as the reference sequence, but the constituent elements comprising the chimeric regulatory element 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 chimeric regulatory element "variant" may be composed of the same constituent elements as the reference sequence or variants thereof, but differs in one or more sequences containing one or more linker sequences that allow the constituent parts to be operably linked. In the present invention, the polynucleotide sequence provided by SEQ ID NO: 1 provides a reference sequence, wherein the constituent elements comprising the reference sequence can be linked by methods known in the art, and may contain substitutions, deletions and / or insertions of one or more nucleotides or mutations that naturally occur in bacterial and plant cell transformation.

[0065] Constructs

[0066] As used herein, the term "construct" refers to any recombinant polynucleotide molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or a linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule derived from any source, which is capable of genomic integration or autonomous replication, comprising a polynucleotide molecule, wherein one or more polynucleotide molecules are linked in a functionally operable manner, i.e., operably linked. As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes, i.e., introducing heterologous DNA into a host cell. The term includes expression cassettes isolated from any of the aforementioned molecules.

[0067] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, wherein the molecules are arranged in such a way that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single continuous molecule and may or may not be adjacent. For example, if a promoter regulates transcription of a target transcribable polynucleotide molecule in a cell, the promoter is operably linked to a transcribable polynucleotide molecule. For example, a leader sequence is operably linked to a coding sequence when it is capable of serving as a leader sequence for a polypeptide encoded by the coding sequence.

[0068] 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 a Ti plasmid isolated from Agrobacterium tumefaciens containing T-DNA, which, together with the transfer molecules provided by the Agrobacterium tumefaciens cells, allows the T-DNA to be integrated into the genome of the plant cell (e.g., see U.S. Patent No. 6,603,061). The construct may also include a plasmid backbone DNA segment that provides replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli replication origin such as ori322, a broad host replication origin such as oriV or oriRi, and a selective marker (e.g., Spec / Strp) encoding a Tn7 aminoglycoside adenylyltransferase (aadA) that confers spectinomycin or streptomycin resistance, or a coding region of a gentamicin (Gm, Gent) selective marker gene. For plant transformation, the host strain is typically Agrobacterium tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation may also function in the present invention.

[0069] Methods for assembling and introducing constructs into cells in a manner whereby a transcribable polynucleotide molecule is transcribed into a functional mRNA molecule which is translated and expressed as a protein product are known in the art. Conventional compositions and methods for preparing and using constructs and host cells for the practice of the present invention are well known to those skilled in the art, for example, see Molecular Cloning: A Laboratory Manual, 3 rded. 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, methods described in U.S. Pat. Nos. 4,971,908; 4,940,835; 4,769,061; and 4,757,011 in their entireties. These types of vectors are also reviewed in the scientific literature (e.g., see 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 expressing nucleic acids in higher plants are well known in the art and include 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 (e.g., see Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985)).

[0070] Various regulatory elements may be included in the construct, including any regulatory element provided herein. Any such regulatory element may be provided in combination with other regulatory elements. This combination may be designed or modified to produce desired regulatory characteristics. In one embodiment, construct of the present invention comprises at least one regulatory element operably connected to a transcribable polynucleotide molecule, which is operably connected to a 3'UTR.

[0071] Constructs of the present invention may include any promoter or leader sequence provided herein or known in the art. For example, a promoter of the present invention may be operably linked to a heterologous non-translated 5' leader sequence, such as a leader sequence from a heat shock protein gene (see, e.g., U.S. Patent Nos. 5,659,122 and 5,362,865). Alternatively, a leader sequence of the present invention may be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcriptional promoter (see, U.S. Patent No. 5,352,605).

[0072] As used herein, the term "intron" refers to a DNA molecule that can be separated or identified from the genomic copy of a gene, and can generally be defined as a region spliced ​​out during the processing of pre-translation mRNA. Alternatively, an intron can be a DNA element that is synthetically produced or manipulated. 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 polynucleotide molecule. A DNA construct can include an intron, and the intron may or may not be heterologous to a transcribable polynucleotide molecule sequence. Examples of introns in this area include rice actin introns (U.S. Patent number 5,641,876) and corn HSP70 introns (U.S. Patent number 5,859,347). Furthermore, when modifying intron / exon boundary sequences, it is preferred to avoid the use of the nucleotide sequence AT or the nucleotide A just before the 5' end of the splice site (GT), and the nucleotide G or the nucleotide sequence TG just after the 3' end of the splice site (AG) to eliminate the possibility of forming an unwanted start codon during the processing of the messenger RNA into the final transcript. Thus, the sequence surrounding the splice site at the 5' or 3' end of the intron can be modified in this way.

[0073] As used herein, the term "3' transcription termination molecule" or "3'UTR" refers to a DNA molecule that is used to produce the 3' untranslated region (3'UTR) of an mRNA molecule during transcription. The 3' untranslated region of an mRNA molecule can be produced by specific cleavage and 3' polyadenylation (also known as a polyA tail). The 3'UTR can be operably linked to a transcribable polynucleotide molecule and located downstream thereof, and can include a polynucleotide that provides a polyadenylation signal and other regulatory signals that can affect 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 (see Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803-4807 (1983)); the 3' region of wheat hsp17; the pea ribulose bisphosphate carboxylase-oxygenase small subunit 3' region; the cotton E6 3' region (U.S. Pat. No. 6,096,950); the 3' region disclosed in WO 0011200A2; and the coixin 3'UTR (U.S. Pat. No. 6,635,806).

[0074] Transcriptable polynucleotide molecule

[0075] As used herein, the term "transcribable polynucleotide molecule" refers to any DNA molecule that can be transcribed into an RNA molecule, including but not limited to molecules having a protein coding sequence and molecules that produce RNA molecules having a sequence for gene suppression. "Transgenic" refers to a transcribable polynucleotide molecule that is heterologous to a host cell (at least its location in the genome) and / or a transcribable polynucleotide molecule that is artificially introduced into the host cell genome in the current or any previous generation of cells.

[0076] Promoter of the present invention can be operably connected to the transcribable polynucleotide molecule heterologous to the promoter molecule. As used herein, the term "heterologous" refers to a combination of two or more polynucleotide molecules when this combination does not usually exist in nature. For example, the two molecules can come from different species and / or the two molecules can come from different genes, such as different genes from the same species or the same gene from different species. In addition, the two molecules can be derived from a separation position in the same gene, wherein this combination of molecules does not usually exist in nature. If this combination does not usually exist in nature, the transcribable polynucleotide molecule is not a naturally occurring combination operably connected to the promoter molecule, so the promoter is heterologous relative to the transcribable polynucleotide molecule that is operably connected.

[0077] As used herein, the term "overexpression" refers to an increase in the expression level of a transcribable polynucleotide molecule or protein in a plant, plant cell or plant tissue compared to the expression at any developmental or temporal stage of a gene in a wild-type plant, cell or tissue. Overexpression can occur in plant cells that typically lack expression of a target transcribable polynucleotide molecule. Overexpression can also occur in plant cells, where transcribable polynucleotide molecules or functionally equivalent molecules typically undergo endogenous expression, but such endogenous expression is at a lower level compared to overexpression. Therefore, overexpression results in a greater output of a polypeptide in a plant, cell or tissue than endogenous output or "overproduction".

[0078] In certain embodiments, the expression or overexpression of the transcribable polynucleotide molecules disclosed herein can directly or indirectly affect enhanced traits or altered phenotypes. In some cases, this can be achieved, for example, by promoting the effective colonization of symbiotic bacteria. In certain exemplary embodiments, the protein produced by the transcribable polynucleotide molecules can regulate the cell wall structure in cells colonized by bacteria.

[0079] The transcribable polynucleotide molecule can generally be any DNA molecule that needs to express RNA transcripts. This expression of RNA transcripts can lead to the translation of the resulting mRNA molecules and therefore lead to protein expression. Or, for example, the transcribable polynucleotide molecule can be designed to ultimately lead to a reduction in the expression of a specific gene or protein. In one embodiment, this can be achieved by using transcribable polynucleotide molecules directed to the antisense direction. Those of ordinary skill in the art are familiar with the use of this antisense technology. In short, when the antisense transcribable polynucleotide molecule is transcribed, the RNA product hybridizes with the complementary RNA molecule in the cell and isolates it. This double-stranded RNA molecule cannot be translated into protein by the translation mechanism of the cell and is degraded in the cell. Any gene can be negatively regulated in this way.

[0080] Thus, one embodiment of the present invention is a regulatory element of the present invention, such as a regulatory element provided by SEQ ID NO: 1, which is operably linked to a transcribable polynucleotide molecule so as to regulate the transcription of the transcribable polynucleotide molecule at a desired level or in a desired pattern when the construct is integrated into the genome of a plant cell. In one embodiment, the transcribable polynucleotide molecule comprises a protein coding region of a gene, and the promoter affects the transcription of an RNA molecule that is translated and expressed as a protein product. In another embodiment of the present invention, the transcribable polynucleotide molecule comprises a sequence encoding a protein, wherein 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 to any one of SEQ ID NOs: 3 and 5. In a specific embodiment, such a sequence can be defined as having the activity of a reference sequence, such as the activity of any one of SEQ ID NOs: 3 and 5.

[0081] In another embodiment, the transcribable polynucleotide molecule comprises an antisense region of a gene, and the promoter effects the transcription of an antisense RNA molecule, double-stranded RNA, or other similar inhibitory RNA molecule to inhibit the expression of a specific RNA molecule of interest in a target host cell.

[0082] Agronomic target genes

[0083] Transcribable polynucleotide molecules may be agronomy target genes. As used herein, term "agronomy target gene" refers to a transcribable polynucleotide molecule that gives desired characteristics when expressed in specific plant tissues, cells or cell types, such as the characteristics related to plant morphology, physiology, growth, development, yield, product, nutritional profile, disease or insect pest resistance and / or environment or chemical tolerance. Agronomy target genes include but are not limited to encoding yield proteins, stress-resistant proteins, developmental control proteins, tissue differentiation proteins, meristem proteins, environmental response proteins, aging proteins, hormone response proteins, shedding proteins, source proteins, library proteins, flower suppression proteins, seed proteins, herbicide-resistant proteins, disease-resistant proteins, fatty acid biosynthetic enzymes, tocopherol biosynthetic enzymes, amino acid biosynthetic enzymes, insecticidal proteins or any other reagent (such as the gene of antisense or RNAi molecules for suppressing targeted specific genes). In some embodiments, the agronomic target genes include, but are not limited to, those encoding pectate lyases (e.g., NPL) and pectin methylesterases (e.g., SyPME) to regulate cell wall structures in cells colonized by bacteria; genes encoding proteins with scaffolding functions (e.g., SYMREM1); genes encoding high-affinity cytochrome oxidases (e.g., cbb3-type oxidases) to increase cellular respiration under low free oxygen conditions; and genes encoding legumin to generate a low oxygen environment within an engineered nodule-like structure. In specific embodiments, the agronomic target gene may comprise any one of SEQ ID NOs: 2, 4, and 6; or may comprise a polynucleotide fragment encoding a protein, wherein the protein comprises an amino acid sequence of any one of SEQ ID NOs: 3 and 5. The product of the agronomic target gene may act within the plant to affect plant physiology or metabolism, or may act to promote the formation and development of nodule-like structures on the plant.

[0084] Alternatively, the agronomic target gene can affect the above-mentioned plant characteristics or phenotypes by encoding a targeted regulatory RNA molecule that causes gene expression of an endogenous gene, for example, by antisense (see, for example, U.S. Patent No. 5,107,065); Inhibitory RNA ("RNAi", including regulating gene expression via miRNA-, siRNA-, trans-acting siRNA- and phase sRNA-mediated mechanisms, such as disclosed in US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application No. 11 / 974,469); or a co-suppression-mediated mechanism. RNA can also be a catalytic RNA molecule (e.g., a ribozyme or riboswitch; see, for example, US2006 / 0200878) that is engineered to cut a desired endogenous mRNA product. Therefore, any transcribable polynucleotide molecule that encodes a transcription RNA molecule that affects an important agronomic phenotype or morphological change can be used to implement the present invention. It is known in the art to construct a construct and introduce it into a cell in a manner that a transcribable polynucleotide molecule is transcribed into a molecule that can cause gene inhibition. For example, U.S. Patent Nos. 5,107,065 and 5,759,829 disclose post-transcriptional gene inhibition of gene expression in plant cells using constructs of transcribable polynucleotide molecules with antisense orientation, and U.S. Patent Nos. 5,283,184 and 5,231,020 disclose post-transcriptional gene inhibition of gene expression in plants using constructs of transcribable polynucleotide molecules with sense orientation. The expression of transcribable polynucleotides in plant cells can also be used to inhibit plant pests that feed on plant cells, such as compositions (U.S. Patent Publication No. US20070124836) separated from coleopteran pests and compositions (U.S. Patent Publication No. US20070250947) separated from nematode pests. Plant pests include, but are not limited to, arthropod pests, nematode pests, and fungi or microbial pests. Exemplary transcribable polynucleotide molecules for incorporation into constructs of the invention include, for example, DNA molecules or genes from species other than the target species, or genes derived from or present in the same species, but incorporated into recipient cells by genetic engineering methods rather than classical propagation or breeding techniques. The types of polynucleotide molecules can include, but are not limited to, polynucleotide molecules already present in plant cells, polynucleotide molecules from another plant, polynucleotide molecules from a different organism, or exogenously produced polynucleotide molecules, such as polynucleotide molecules containing antisense information for a gene, or polynucleotide molecules encoding artificial, synthetic or other modified forms of transgenes.

[0085] Selective markers

[0086] As used herein, the term "marker" refers to any transcribable polynucleotide molecule whose expression or lack of expression can be screened or scored in some way. Marker genes used to implement the present invention include, but are not limited to, encoding β-glucuronidase (GUS described in U.S. Pat. No. 5,599,670), green fluorescent protein and its variants (GFP described in U.S. Pat. Nos. 5,491,084 and 6,146,826), proteins that confer antibiotic resistance, or proteins that confer herbicide tolerance.

[0087] The term "selective marker" also includes genes encoding secretable markers, whose secretion can be detected as a means of identifying or selecting transformed cells. Examples include markers encoding secretable antigens that can be identified by antibody interactions, or even secretable enzymes that can be catalytically detected. Selectable secretory marker proteins can be divided into multiple categories, including small, diffusible proteins that can be detected (e.g., by ELISA), small active enzymes that can be detected in extracellular solution (e.g., α-amylase, β-lactamase, phosphinothricin transferase), or proteins that are inserted or restricted in the cell wall (e.g., proteins containing leader sequences, such as those found in extended expression units or related to tobacco pathogenesis, also referred to as tobacco PR-S). Other possible selectable marker genes are obvious to those skilled in the art and are included in the present invention.

[0088] Cell transformation

[0089] The present invention also relates to methods of producing transformed cells and plants comprising a promoter operably linked to a transcribable polynucleotide molecule.

[0090] The term "transformation" refers to the introduction of a nucleic acid 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.

[0091] As used herein, the term "transformed" refers to a cell, tissue, organ or organism into which an exogenous polynucleotide molecule (e.g., a construct) has been introduced. The introduced polynucleotide molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ or organism so that the introduced polynucleotide molecule is inherited to subsequent progeny. "Transgenic" or "transformed" cells or organisms may also include progeny of the cell or organism, as well as progeny produced by a breeding program that uses such a transgenic organism as a hybrid parent and exhibits phenotypic changes caused by the presence of an exogenous polynucleotide molecule. The term "transgenic" refers to bacteria, fungi or plants that contain one or more heterologous polynucleotide molecules.

[0092] There are many methods for introducing polynucleic acid molecules into plant cells. The method generally includes the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector, and obtaining the transformed host cell. Suitable methods include bacterial infection (e.g., Agrobacterium), binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, drying / inhibition-mediated DNA uptake, electroporation, stirring and accelerating DNA-coated particles with silicon carbide fibers, etc. (reviewed in Potrykus, et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205 (1991)).

[0093] The technology of introducing DNA molecules into cells is well known to those skilled in the art. In the practice of the present invention, the methods and materials for transforming plant cells by introducing plant constructs into plant genomes may include any well-known and proven methods. Any transformation method can be used to transform host cells with one or more promoters and / or constructs of the present invention. The host cell can be any cell or organism, such as a plant cell, an algae cell, an algae, a fungal cell, a fungus, a bacterial cell or an insect cell. Preferred hosts and transformed cells include cells from: plants, Aspergillus, yeast, insects, bacteria and algae.

[0094] The regenerated transgenic plants can be self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated transgenic plants can be crossed with non-transgenic plants, preferably with inbred lines of agronomically important species. Descriptions of breeding methods generally used for different traits and 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). Conversely, pollen from non-transgenic plants can be used to pollinate regenerated transgenic plants.

[0095] The transformed plants can be analyzed for the presence of the gene of interest and the expression level and / or 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 or northern blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Expression of transcribable polynucleotide molecules can be performed using the methods described by the manufacturer. (Applied Biosystems, Foster City, CA) and were measured using PCR cycle times were determined using the TestingMatrix. Alternatively, the manufacturer's (Third Wave Technologies, Madison, WI) reagents and methods can be used for transgene expression.

[0096] 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.

[0097] The present invention also provides plant parts 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.

[0098] Transgenic plants can pass transgenic polynucleotide molecules to their progeny. Progeny includes any regenerable plant part or seed, which comprises the transgene derived from ancestral plants. Transgenic plants are preferably homozygous for the polynucleotide molecules converted, and the sequence is passed to all progeny by sexual reproduction. Progeny can be grown from the seeds produced by transgenic plants. Then, these extra plants can be self-pollinated to produce real plant propagation lines. The progeny of these plants are evaluated by gene expression, etc. Gene expression can be detected by several common methods, such as western blotting, northern blotting, immunoprecipitation and ELISA.

[0099] The transgenic plants, plant cells, plant parts or progeny thereof described herein can be selected from 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, garlic, forage legumes, grapes, hops, indigo, leek, legume trees, lentils, 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, dry beans, radiata pine, radish, rapeseed, raspberries, rice, rootstock, rye, red currant, safflower, shrubs, sorghum, southern pine, soybeans, spinach, zucchini, strawberries, sugar beets, sugar cane, sunflower, corn, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, walnuts, watermelon, wheat, or yams.

[0100] commodity

[0101] 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 according to the present invention. Commodities can be sold to consumers, and can also be active or inactive. Inactive commodities include, but are not limited to, inactive 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 for terrestrial and / or aquatic animals, oils, noodles, flours, flakes, bran, fibers, milk, cheese, paper, cream, wine and any other food for human consumption; and biomass and fuel products. Active 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.

[0102] Now that the present invention has been generally described, it will be more readily understood by reference to the following examples, which are provided as illustrations and are not intended to limit the present invention unless otherwise specified. 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 specific embodiments disclosed without departing from the spirit and scope of the present invention, and similar or similar results may still be obtained, and therefore, all that is described or shown in the accompanying drawings should be interpreted as illustrative rather than restrictive. Example

[0103] Example 1: Analysis of the cell wall structure around the infection line.

[0104] To investigate cell wall structure and composition during rhizobium infection and transcellular IT passage, a set of immunolabeling experiments was performed on indeterminate M. truncatula nodules using a panel of selected antibodies against different cell wall components (Table 1). Briefly, seedlings were grown directly after seed germination in open pots (1:1 quartz sand:vermiculite mixture, 2 plants / pot). Liquid The plants were watered once a week with medium (nitrate-free, 30 ml / pot) and tap water (30 ml / pot). Seven days after transfer, the plants were then inoculated with Sinorhizobium meliloti (OD600=0.003) (20 ml / pot). After another 10 days, the plants were harvested for quantification of infection structure.

[0105] Table 1. List of cell wall antibodies used to study cell wall structure and composition during rhizobium infection and transcellular IT passage.

[0106]

[0107]

[0108] To identify IT, the IT matrix glycoprotein was first labeled with the MAC265 antibody (VandenBosch et al., 1989). As expected, nodule IT was specifically labeled by this method, while the peripheral cell wall of nodule cortical cells did not show any fluorescent signal ( Figure 6 , panel A). The situation was different when targeting xyloglucan as the most abundant hemicellulose by LM25. Here, ubiquitous labeling of the cell periphery of all nodule cells, including IT, was found ( Figure 6 , Panel B). In contrast, different arabinogalactan proteins (marked by LM2, LM14, LM30) reported to play a role in plant-microbe interactions were found to accumulate specifically within the infection zone and around the symbionts ( Figure 6 , small picture EG and Figure 7 Next, the presence of different pectins was investigated, including rhamnogalacturonan I (RG-I), homogalacturonan (HG), rhamnogalacturonan II (RG-II), and xylogalacturonan. Linear (1-4)-β-D-galactan (recognized by LM5), an epitope of RG-I, was barely detectable in sections of alfalfa nodules ( Figure 6 , panel C). This is consistent with previously published data in which this epitope was almost absent in nodule sections from Medicago spp. In contrast, (1-5)-α-L-arabinosyl (the epitope of RG-I recognized by LM6) was present in most cell walls of cells within the infection zone of the nodule ( Figure 6 , panel D), and mainly accumulated around the colonized cells in the fixed zone ( Figure 6 , small figure D and Figure 8 ), while the uninfected cells in this area did not accumulate (1-5)-α-L-arabinosyl ( Figure 8 , panels C'-C"'). To distinguish HG isoforms, two antibodies, LM20 and LM19, were used to recognize methyl-esterified and unesterified HG, respectively. Although esterified pectin is present in most cell walls ( Figure 1 , panel A), but unesterified pectin mainly accumulated in epidermal cells, exodermal cells, and around infection lines, while the cell walls of central uninfected and infected cells lacked this processed form of pectin ( Figure 1 , Panel B). In addition, as shown in the nodule tissue, unesterified pectin ( Figure 1 , panel C) instead of methylated pectin ( Figure 1, panel D) accumulates around the IT of root cortical cells. Labeling of unesterified pectin was also noted, often extending slightly from the IT to adjacent cells. These results provide insight into the cell wall remodeling that occurs during rhizobium infection and transcellular IT passage, and indicate the expression and activity of pectin demethylesterase during this process.

[0109] Example 2: Dissecting cell wall patterns at the site of transcellular IT channels.

[0110] In order to further analyze the cell wall pattern at the site of transcellular IT channels, a photoelectric correlation microscopy (CLEM) protocol was established. In brief, CLEM was applied to 70nm LowicrylHM20 ultrathin sections obtained using a Reichert-Jung ultramicrotome and collected in a viewfinder grid. The grid with the sections was washed with PBS buffer for 5 minutes and then incubated with 0.12M glycine in PBS for 10 minutes. After washing in PBS for 5 minutes, the grid was incubated in a blocking solution (4% BSA in PBS) for 10 minutes, followed by incubation with the first antibody in the blocking solution for 30 minutes. After washing 6 times in PBS for 3 minutes each time, the grid was incubated in a blocking solution containing a fluorescently labeled second antibody for 30 minutes. The grid was washed 6 times in PBS for 3 minutes each time and incubated in a 1% DAPI solution for 5 minutes, then mounted on a microscope slide for observation under a fluorescence microscope (ZEISS ApoTome.2). Additionally, immunogold staining for CLEM was performed as described below, but with protein A-gold conjugated instead of the secondary antibody, and sections were contrasted after washing with 2% uranyl acetate in water.

[0111] The use of this CLEM protocol allows for the visualization of the Figure 1 , Panels E, F) to search for events and subsequently use transmission electron microscopy (TEM) ( Figure 1 , panels E', F') perfectly retrieve these sites in ultrathin sections. Superimposing these images shows the presence of unesterified pectin along the IT and small fragments of the host cell wall in close proximity to the IT ( Figure 1 , panels E”, F”). These sites can be transcellular passage sites, as shown using scanning electron microscopy (SEM) ( Figure 2 , panel A) and TEM ( Figure 2 , panel B). These observations were further confirmed by double immunogold labeling, which showed that unesterified pectin (LM19, 12 nm) was concentrated at the IT penetration site, while only a few gold particles were detected using LM20 (methyl-esterified pectin, 5 nm). Figure 2 , panel C). These images also show fusion and thickening of CW structures at the sites of transcellular IT channels ( Figure 2, Panel B). To assess whether this was the result of cell wall loosening and subsequent expansion or represented a hardened structure, these samples were probed with the 2F4 antibody that recognizes the 'egg crate' pectin dimer. 2F4 immunofluorescence indeed confirmed that Ca 2+ -Accumulation of complexed pectin ( Figure 2 , panel D). This is consistent with the observed enrichment of unesterified pectin around IT, as Ca 2+ - Complexation requires demethylation of HG ( Figure 1 , panels B and C).

[0112] To visualize the cell walls of IC and IT in root hairs, a modified CLEM setup ( Fig. 9 , panel A). In stark contrast to nodule IT, primary infections are rather rare and cannot be routinely retrieved by classical ultrathin sectioning and TEM. Therefore, infected roots were initially embedded in low-melting-point agarose and searched for curled root hairs using bacterial fluorescence, after which the sample blocks were trimmed and transferred to EM resin. Semi-thin sectioning was performed until curled root hairs were obtained. Subsequent ultrathin serial sections were performed for TEM analysis. As expected, the IC was surrounded by a thick and electron-dense cell wall that was much thinner on the possible IT initiation side ( Fig. 9 , panel BC). Further sample sectioning also revealed this cell wall structure with multiple vesicles flanking the root hair IT ( Fig. 9 , Panel D). Although this method allows unprecedented observation of IC and IT morphology in root hairs, its throughput is limited. Therefore, nodule samples were used in the following experiments.

[0113] Example 3: Identification of Symbiotic Pectin Methylesterase (SyPME).

[0114] As described in Examples 1 and 2, unesterified pectin, but not methyl-esterified pectin, is present along the IT and small fragments of the host cell wall are in close proximity to the IT. Pectin demethylesterification is enzymatically mediated by pectin methylesterase. The Medicago PME family, which consists of more than one hundred members, was searched for suitable candidates. Using publicly available transcriptome data, a gene (Medtr4g087980 or MtrunA17_Chr4g0069841) was identified that was consistently induced after application of nodulation factor and inoculation of roots with Sinorhizobium meliloti ( Fig.10 , panel A). The gene also maintains the highest expression in the nodule meristem and cells in the distal infection zone zIId ( Fig.10 , panel B). Therefore, it was named 'Symbiotic PME' (SyPME). The transcriptome data were first verified by in situ hybridization and expression of transcriptional reporters using 2 kb upstream of the transcription start site as a putative promoter region. When nodule sections were hybridized with antisense in situ probes, the cortical cells of the nodule primordium ( Fig.10 , panel C) and the zIId region of mature nodules ( Fig.10 , panel D). No signal was observed when the sensor probe was used as a control in the same tissue ( Fig.10 , Panels EF). Thus, SyPME promoter activity was found throughout the cortex of nodule primordia as depicted by β-glucuronidase (GUS) staining ( Fig.10 , panel F), whereas a restricted expression domain confined to infection zone II was observed in young and mature nodules ( Fig.10 , Panel G). These results demonstrate the ability of the SyPME promoter to spatially and temporally regulate expression in developing and mature nodules, as well as within the nodule infection zone of indeterminate nodules during symbiotic infection.

[0115] To evaluate the localization pattern of SyPME protein, SyPME-GFP translational fusion under the control of the ubiquitin 10 promoter was generated and used for hairy root transformation.

[0116] Briefly, after sterilization with pure sulfuric acid (H2SO4) for 20 minutes, the seeds of Medicago truncatula were washed 6 times with sterile tap water. The seeds were then treated with a bleach solution (12% NaCl, 0.1% SDS) for 60 seconds and washed 6 times with sterile tap water. The sterilized seeds were covered with sterile tap water for 2 hours and then transferred to 1% agar plates and stratified in the dark at 4°C for 3 days. After stratification, the seeds were kept in the dark at 24°C for 24 hours for germination. The seed coat was removed and the seeds were placed in a controlled environment chamber at 24°C with a 16h / 8h light / dark photoperiod, and the seedlings were then used for hairy root transformation as described previously (Boisson-Dernier et al., 2001). The complex was first placed on a solid Medium (containing 0.5 mM NH4NO3) and incubated in the dark (22°C) for 3 days, then incubated under white light at 22°C for 4 days, with the roots kept in the dark. After one week, the seedlings were transferred to fresh The cells were incubated on medium (0.5 mM NH4NO3) for another 10 days. Then, transformed roots were selected and positive plants were transferred to open pots for phenotypic analysis.

[0117] For phenotypic analysis, seedlings were grown directly after germination in open pots (1:1 quartz sand:vermiculite mixture, 2 plants / pot). The plants were watered once a week with medium (nitrate-free, 30 ml / pot) and tap water (30 ml / pot). Seven days after transfer, the plants were then inoculated with Sinorhizobium meliloti (OD600=0.003) (20 ml / pot). After another 10 days, the plants were harvested for quantification of infection structure.

[0118] In the root hairs surrounding the infection chamber ( Fig.11 , panel A) and along the growth of the primary IT ( Fig.11 , Panel B) observed clear and confined fluorescence. Consistent with this, nodule IT was also decorated with SyPME proteins ( Figure 3 , panel A), while the strongest accumulation was observed at the transcellular channel site ( Figure 3 , panels B, C). Here, SyPME localization is strictly delineated in the peripheral cell wall at cell junctions with intersecting ITs ( Figure 3 , panel B), and is therefore rich in Ca 2+ - Sites of complexed unesterified pectin ( Figure 2 , Panel D). In addition, SyPME accumulates at the tip region of growing ITs and at spatially restricted sites at the cell periphery marking the site of subsequent transcellular IT passage ( Figure 3 , thumbnail DF, Fig.11 , Panel E and Fig.12 , panel A-A').

[0119] Example 4: SyPME and NPL synergistically regulate IT growth.

[0120] Since unesterified pectin serves as a substrate for pectate lyase (PL) or polygalacturonase (PG), the interaction between alfalfa nodule pectate lyase (NPL; Medtr3g086320) and SyPME was evaluated. SyPME and NPL were significantly co-expressed in infected root hairs (correlation coefficient = 0.9883, Fig.10 , panel A). Compared with other members of the pectate lyase family present in the nodule transcriptome data, NPL was found to be the most highly expressed, mainly restricted to the nodule meristem and zIId ( Fig.10 , Panel B). This spatially controlled expression in nodules was also confirmed when the transcriptional GUS reporter was generated using 2 kb (2038 bp) upstream of the NPL transcription start site ( Fig.10 , panel I). At the protein level and consistent with the pattern observed for SyPME, the NPL-GFP fusion protein driven by the endogenous NPL promoter also localized to the IC, the primary IT in the root hair, and the aforementioned spatially restricted sites to be penetrated by the IT ( Fig.11 , panels CD, F). This suggests that SyPME precedes NPL-mediated pectin degradation in the IC, the apical region of growing IT, and the local cell wall site that initially prepares IT channels. Interestingly, older parts of these ITs show reduced NPL accumulation ( Fig.11 , panel D), while SyPME protein levels remained high in these regions ( Fig.11, panel B), suggesting that residual IT in root hairs may become rigid rather than loose. Although NPLs were also localized at the tip of nodule IT and in localized cell wall regions near nodule IT, the protein was not present at the transcellular passage site itself ( Figure 3 , panels C and D). Therefore, unesterified pectin is likely not degraded by NPLs at these transcellular passage sites, confirming that these regions are provided by Ca2+, a function of SyPME. 2+ - Complexed pectin is stabilized and probably occluded. This would also limit the extracellular diffusion of rhizobia and other microorganisms that interfere with the intercellular colonization of plants grown in their natural habitat. These experiments confirm the co-localization of SyPME and NPL during symbiotic infection.

[0121] Example 5: Genetic framework regulates IT growth and transcellular passage.

[0122] To analyze the crosstalk between SyPME and NPL in more detail, we used loss- and gain-of-function approaches to assess their effects on infection. Compared with R108 WT plants, nodules formed on the alfalfa npl mutant were significantly reduced ( Figure 4 , Panel A). In addition, most infection events terminate at the IC stage ( Figure 4 , panel B), observations consistent with previously published data. In order to be able to distinguish the requirement for NPL in the epidermis and root cortex, an RNA interference (RNAi) approach was also performed, which expressed silencing constructs under the control of different promoters in transgenic roots. Briefly, roots transformed with empty vectors and NPL-RNAi constructs were harvested 10 days after inoculation and then fixed in a PBS solution containing 4% PFA under vacuum for 15 minutes (twice) and kept at room temperature for 2 hours before being transferred to Clearsee solution. The roots were kept in Clearsee for 2-3 days, and then the solution was renewed and provided with 0.1% Calcofluor white before imaging.

[0123] To demonstrate the validity of the constructs used in general, an empty control vector was first expressed, in which no changes in IT formation and progression were observed (EV; Figure 4 , panels C and D). However, constitutive overexpression of the NPL-RNAi construct often resulted in the entrapment of rhizobia within the IC ( Figure 4 , Panel E), while only some IT successfully extended ( Figure 4 , Panel F). The same pattern was observed when the Solanum lycopersicum (tomato) expansin 1 (ProEXT1) promoter, previously shown to mediate epidermis-specific expression in Medicago truncatula, drove the silencing construct ( Figure 4, panels G, H). In contrast, cortex-specific silencing of NPL using the Arabidopsis endopeptidase PEP-promoter resulted primarily in the infection of normally developing ITs in root hairs, which subsequently terminated in the root cortex ( Figure 4 , panels I, J). These results further demonstrate that NPLs are required during IT initiation and transcellular progression.

[0124] To genetically test whether SyPME and / or other members of this protein family are required for successful infection, the TNT1 transposon insertion collection was searched and a single sypme allele carrying an insertion in the first intron was identified (NF2281_high_35; Fig.12 , Panel A). However, homozygous individuals did not show any symbiotic phenotype ( Fig.12 , panels BF), which is probably due to the large size of this gene family. To target functionally redundant PMEs with spatiotemporal precision, Arabidopsis PME inhibitor 12 (PMEI12), which has previously been shown to effectively inhibit PME activity, was expressed under the control of the NPL promoter and these transgenic roots were inoculated with S. meliloti. In fact, most infections occurred at the IC stage ( Figure 4 , Inset LL") and root cortex ( Figure 4 , Panel MM") is blocked during IT progression and thus phenocopies the npl mutant. This supports the proposed dependence of NPL on the activity of preceding PMEs and, therefore, the coordinated expression of SyPME throughout the infection process.

[0125] In another set of experiments, to unambiguously reveal cell wall changes during transcellular IT passage, a CLEM approach was used to monitor pectin and changes within the cell-cell interface at different stages of transcellular IT progression. To this end, labeled unesterified pectin (LM19, Figure 5 , panel A) and IT matrix (MAC265, Figure 5 , panel B), and search for ultrathin sections ( Figure 5 , panels CD) to find the moment of IT and IT passage close to the basal cell membrane / cell wall. Unesterified pectin accumulates around the future transcellular passage site within the cell-cell interface, as defined by the cytoplasmic column formed before the IT ( Figure 5 , Phase I; Fig.13 The channel site further increases in size, expanding laterally and then in the central region ( Figure 5 , stage II). After this, the rhizobia enter the closed apoptotic compartment (apopolastic compartment) before entering the neighboring cells that have formed a pre-infection line ( Figure 5 , stage III). Finally, the rhizobia enter the adjacent cell through the apoptotic space, where there is no membrane restriction ( Figure 5 , Stage IV).

[0126] Given that the estimated diameter of the channel site is in the range of 1 nm to 2 nm, the IT membrane is disrupted when the IT fuses with the basal membrane, and reinvaginates at the adjacent cell, transcellular passage is a continuous series of events ( Figure 5 E), which includes 1) targeted secretion of PME and initial secretion of NPL, 2) localized pectin demethylesterification and partial pectin degradation at the IT tip and at the local host cell wall in preparation for penetration, 3) maintenance of SyPME but reduction of NPL protein levels at the passage site, 4) restriction of cell wall expansion at the passage site, 5) spatial release of rhizobia into enclosed apoptotic compartments, and 6) uptake into neighboring cells.

[0127] Having illustrated and described the principles of the invention, it will be apparent to those skilled in the art that the arrangements and details of the invention may be modified without departing from the 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, as if each individual publication or patent application was expressly and individually indicated to be incorporated herein by reference.

Claims

1. A DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to SEQ ID NO: 1; b) a sequence comprising SEQ ID NO: 1; and c) a fragment of SEQ ID NO: 1 or a fragment having at least 85% sequence identity to a fragment of SEQ ID NO: 1, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule. 2 . The DNA molecule according to claim 1 , wherein the DNA molecule has at least 90% sequence identity with the DNA sequence of any one of SEQ ID NO: 1 . 3 . The DNA molecule according to claim 1 , wherein the DNA molecule has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NO:

1. 4 .

4. The DNA molecule of claim 1, wherein if the fragment is less than 115 nucleotides, the fragment has at least 87% sequence identity with a fragment of SEQ ID NO:

1.

5. The DNA molecule of claim 1, wherein the DNA sequence comprises gene regulatory activity.

6. The DNA molecule of claim 5, wherein the gene regulatory activity is promoter activity.

7. The DNA molecule of claim 5, wherein the gene regulatory activity is symbiosis-specific pectin methylesterase (SyPME) promoter activity.

8. The DNA molecule of claim 1, wherein the heterologous transcribable polynucleotide molecule comprises a gene of agronomic interest.

9. The DNA molecule according to claim 8, wherein the agronomic target gene is a gene encoding pectin methylesterase having pectin demethylesterification activity.

10. The DNA molecule of claim 8, wherein the DNA sequence provides for expression of the heterologous transcribable polynucleotide molecule in response to an external stimulus.

11. The DNA molecule of claim 10, wherein the DNA sequence provides for expression of the heterologous transcribable polynucleotide molecule in root hair cells, in the cortex of nodule primordium, in mature nodules, in the nodule infection zone of young, mature or indeterminate nodules.

12. A transgenic plant cell comprising a heterologous DNA molecule comprising a sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to any one of SEQ ID NO: 1; b) a sequence comprising any one of SEQ ID NO: 1; and c) a fragment of SEQ ID NO: 1 or a fragment having at least 85% sequence identity to a fragment of SEQ ID NO: 1, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule.

13. The transgenic plant cell of claim 12, wherein the transgenic plant cell is a monocotyledonous plant cell.

14. The transgenic plant cell of claim 12, wherein the transgenic plant cell is a dicotyledonous plant cell.

15. A transgenic plant or part thereof, comprising the DNA molecule of claim 1.

16. A progeny plant or part thereof of the transgenic plant of claim 15, wherein the progeny plant or part thereof comprises the DNA molecule.

17. A transgenic seed, wherein the transgenic seed comprises the DNA molecule of claim 1.

18. A method for producing a commodity, comprising obtaining the transgenic plant or part thereof according to claim 15 and producing a commodity therefrom.

19. The method of claim 18, 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.

20. A commercial product comprising the DNA molecule of claim 1.

21. The commodity product of claim 20, wherein the commodity product is a protein concentrate, a protein isolate, a grain, a starch, a seed, a meal, a flour, a biomass or a seed oil.

22. A method of expressing a transcribable polynucleotide molecule, comprising obtaining the transgenic plant or part thereof according to claim 15, and growing the plant in which the transcribable polynucleotide molecule is expressed.

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