Method for improving disease resistance of plants

Abstract

The invention provides a method for improving the disease resistance of plants, and particularly provides a method for improving the disease resistance of the plants by reducing or inhibiting the expression quantity and / or activity of SWEET11a genes or encoded proteins thereof. According to the method, the disease resistance of the plants can be improved, particularly the resistance to gray speck disease, northern leaf blight, bacterial wilt and stem rot is improved, and the method has important application value in biological breeding.

Description

[0001] This application claims priority to Chinese patent application number CN 2024107040234, filed June 3, 2024, entitled “A Method for Improving Disease Resistance in Plants.” This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the fields of biotechnology and crop genetic breeding, and relates to a method for improving plant disease resistance, in particular to improving plant disease resistance by reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein in the plant. Background Art

[0003] Corn is one of my country's major grain crops, cultivated on a vast acreage. A variety of factors influence corn yield and quality, including climate, soil conditions, crop varieties, pests and diseases. Among these, corn diseases have a significant impact on both yield and quality. Common corn diseases include large leaf spot, small leaf spot, gray leaf spot, powdery mildew, sheath blight, rust, stem rot, and bacterial wilt.

[0004] Gray leaf spot, also known as Cercospora leaf spot or mildew, affects not only corn but also a variety of grasses, including sorghum, lemongrass, and Andropogon citratus. Gray leaf spot in corn has been a rapidly increasing and devastating disease in recent years. It is common in southern China and is showing signs of spreading northward.

[0005] Large leaf spot is a major maize disease, widespread in maize-growing regions around the world. In severe cases, it can cause yield reductions of 15-20%, and in severe cases, over 50%.

[0006] Bacterial wilt is a corn disease caused by several Fusarium or Pythium fungi, either alone or in combination. Symptoms include sudden wilting, with the entire plant's leaves becoming scalded, dry, and discolored; drooping ears and dead husks; and a water-soaked appearance at the base of the stem, which gradually turns light brown and feels hollow when pinched, often leading to lodging.

[0007] Improving corn's resistance to different diseases has broad market prospects. In order to improve corn's disease resistance, we studied the corn SWEET gene in order to obtain corn plants with improved disease resistance. Summary of the Invention

[0008] The present invention aims to provide a method for improving plant disease resistance.

[0009] In one aspect, the present invention provides a method for improving plant disease resistance, comprising the steps of reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein in the plant.

[0010] In another preferred embodiment, the amino acid sequence encoded by the SWEET11a gene is selected from the following group:

[0011] (i) a polypeptide having the amino acid sequence shown in SEQ ID No. 1;

[0012] (ii) a polypeptide derived from (i) having the same or similar function as described above, formed by substituting, deleting or adding one or several (e.g., 1-10) amino acid residues of the amino acid sequence shown in SEQ ID No. 1;

[0013] or (iii) a polypeptide having an amino acid sequence that is 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99% or 100%) identical to the amino acid sequence of SEQ ID No. 1 and having the same or similar functions.

[0014] In another preferred embodiment, the nucleotide sequence of the SWEET11a gene is selected from the following group:

[0015] (a) a polynucleotide encoding the polypeptide shown in SEQ ID No. 1;

[0016] (b) a polynucleotide whose sequence is shown in SEQ ID No. 2;

[0017] (c) a polynucleotide having a nucleotide sequence homology of ≥95% (preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID No. 2;

[0018] (d) a polynucleotide having 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID No. 2;

[0019] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0020] In another preferred example, the amino acid sequence encoded by the SWEET11a gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No. 1.

[0021] In another preferred embodiment, the improved disease resistance of the plant includes increasing the plant's resistance to gray leaf spot (also known as cercospora leaf spot, mildew), large leaf spot, bacterial wilt or stem rot.

[0022] In another preferred embodiment, the improved disease resistance of the plant includes increasing the plant's resistance to gray leaf spot (also known as cercospora leaf spot, mildew) and / or to bacterial wilt and / or stem rot.

[0023] In another preferred embodiment, the improved plant disease resistance refers to an increase in the plant's resistance to pathogens related to gray leaf spot (also known as cercospora leaf spot, mildew), large leaf spot, bacterial wilt or stem rot.

[0024] In another preferred embodiment, the improved plant disease resistance refers to an increase in the plant's resistance to pathogens related to gray leaf spot (also known as Cercospora leaf spot or mildew).

[0025] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to Cercosporazeina.

[0026] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to pathogens related to northern leaf spot disease.

[0027] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to Exserohilum turcicum (Pass.) Leonay et Suggs.

[0028] In another preferred embodiment, the improved plant disease resistance refers to an increase in the plant's resistance to pathogens related to bacterial wilt.

[0029] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to stem rot-related pathogens.

[0030] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to Fusarium and / or Pythium.

[0031] In another preferred embodiment, the improved plant disease resistance refers to an increased resistance of the plant to any one or more of Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, or Fusarium moniliforme.

[0032] In another preferred example, the improved plant disease resistance includes increasing the plant's resistance to any one or more of Cercosporazeina, Exserohilum turcicum (Pass.) Leonay et Suggs, Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme.

[0033] In another preferred embodiment, the method comprises the steps of:

[0034] (i) providing a plant or plant cell; and

[0035] (ii) introducing the SWEET11a gene or an inhibitor of its encoded protein into the plant or plant cell, thereby obtaining a modified plant or plant cell.

[0036] In a preferred embodiment, the method comprises reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein.

[0037] In another preferred example, the "reduction or inhibition" means that the expression or activity of the SWEET11a gene or its encoded protein is reduced to meet the following conditions: the SWEET11a gene or its encoded protein in the plant is completely inactive or partially inactive, or the ratio of A1 / A0 is ≤80%, preferably ≤60%, more preferably ≤40%, and optimally 0-30%; wherein A1 is the expression or activity of the SWEET11a gene or its encoded protein in the plant; A0 is the expression or activity of the same SWEET11a gene or its encoded protein in the wild-type plant of the same type.

[0038] In another preferred example, the reduction or inhibition refers to that the expression level E1 of the SWEET11a gene or its encoded protein in the plant is 0-80% of the wild type, preferably 0-60%, more preferably 0-40%, and more preferably 0-30% compared with the expression level E0 of the SWEET11a gene or its encoded protein in the wild type plant.

[0039] In another preferred embodiment, the reduction or inhibition of the expression and / or activity of the SWEET11a gene or its encoded protein is achieved by a method selected from the following group: gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of gene or protein inhibitors, or a combination thereof.

[0040] In another preferred embodiment, the gene mutation is obtained by one or more of the following methods: natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), gene editing or biosynthesis.

[0041] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

[0042] In another preferred embodiment, the reduction or inhibition of the expression level and / or activity of the SWEET11a gene or its encoded protein is achieved by mutation of the SWEET11a gene.

[0043] In another preferred embodiment, the mutation includes insertion mutation, deletion mutation, frameshift mutation, and substitution mutation.

[0044] In another preferred embodiment, the mutation causes complete loss or partial loss of function and / or activity of the SWEET11a gene or its encoded protein; preferably, the mutation causes complete loss of function and / or activity of the SWEET11a gene or its encoded protein.

[0045] In another preferred embodiment, the mutation is that the nucleotide sequence of the SWEET11a gene has base substitution, base deletion and / or base insertion relative to the sequence shown in SEQ ID No. 2.

[0046] In another preferred embodiment, the mutation is that the nucleotide sequence of the SWEET11a gene is missing one or more bases relative to the sequence shown in SEQ ID No. 2.

[0047] In another preferred embodiment, the mutation is an insertion of one or more bases into the nucleotide sequence of the SWEET11a gene relative to the sequence shown in SEQ ID No. 2.

[0048] In another preferred embodiment, the mutation is that a base g is inserted after the 245th base of the nucleotide sequence of the SWEET11a gene relative to the sequence shown in SEQ ID No. 2.

[0049] In another preferred embodiment, the mutation is that the nucleotide sequence of the SWEET11a gene is deleted from bases 241 to 249 relative to the sequence shown in SEQ ID No. 2, and a base a is inserted after base 278.

[0050] In another preferred embodiment, the mutation refers to that the nucleotide sequence of the mutated SWEET11a gene has base substitution, base deletion and / or base insertion in the nucleotide sequence corresponding to SEQ ID No. 2 compared with the nucleotide sequence of the parent SWEET11a gene.

[0051] In another preferred embodiment, the parent SWEET11a gene is derived from a monocotyledonous plant or a dicotyledonous plant.

[0052] In another preferred embodiment, the plant is selected from one or more plants of the following group: Leguminosae, Cruciferae, Poaceae, Solanaceae, Cucurbitaceae, Chenopodiaceae, Polygonaceae, Pedulaceae, Asteraceae, Malvaceae, Rosaceae, Pedulaceae, Convolvulaceae, Dioscoreaaceae, Apiaceae, Liliaceae, Zingiberaceae, and Palmaceae.

[0053] In another preferred embodiment, the plant is derived from one or more plants selected from the group consisting of soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, alfalfa, sorghum, barley, wheat, millet, sweet potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, peanut, watermelon, cabbage, strawberry, cucumber, coconut, or a combination thereof.

[0054] In another preferred embodiment, the plant is selected from soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber, and coconut.

[0055] In another preferred embodiment, the parent SWEET11a gene is derived from corn.

[0056] In another preferred example, the accession number of the parent SWEET11a gene is GRMZM2G368827.

[0057] In another preferred example, the amino acid sequence encoded by the parent SWEET11a gene is shown as SEQ ID No.1.

[0058] In another preferred example, the nucleotide sequence of the parent SWEET11a gene is shown as SEQ ID No.2.

[0059] In another preferred embodiment, the nucleotide sequence of the mutated SWEET11a gene is shown as either SEQ ID No. 4 or SEQ ID No. 6.

[0060] In another preferred embodiment, the amino acid sequence encoded by the mutated SWEET11a gene is shown in either SEQ ID No. 3 or SEQ ID No. 5.

[0061] In the present invention, SWEET11a genes from different plant sources, for example, SWEET11a genes naturally present in different plants or SWEET11a genes from different plant sources that have been artificially modified can all be used as parent SWEET11a genes; those skilled in the art can obtain the nucleotide sequence and amino acid sequence of SWEET11a genes from different sources through conventional technical knowledge, and mutate the SWEET11a gene based on the method described in the present invention or the existing technology to achieve the purpose of reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein in the plant.

[0062] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0063] In another preferred embodiment, the plant is selected from one or more plants of the following group: Leguminosae, Cruciferae, Poaceae, Solanaceae, Cucurbitaceae, Chenopodiaceae, Polygonaceae, Pedulaceae, Asteraceae, Malvaceae, Rosaceae, Pedulaceae, Convolvulaceae, Dioscoreaaceae, Apiaceae, Liliaceae, Zingiberaceae, and Palmaceae.

[0064] In another preferred embodiment, the plant is derived from one or more plants selected from the group consisting of soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, alfalfa, sorghum, barley, wheat, millet, sweet potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, peanut, watermelon, cabbage, strawberry, cucumber, coconut, or a combination thereof.

[0065] In another preferred embodiment, the plant is selected from soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber, and coconut.

[0066] In another preferred embodiment, the plant is corn.

[0067] In another preferred embodiment, the corn is Chang 7-2 or silage corn.

[0068] In another preferred embodiment, the corn is Chang 7-2.

[0069] In another aspect, the present invention provides a composition for improving plant disease resistance, comprising:

[0070] (a) an inhibitor of the SWEET11a gene or its encoded protein; and

[0071] (b) an agriculturally acceptable carrier.

[0072] In a preferred embodiment, the inhibitor can reduce or inhibit the expression level and / or activity of the SWEET11a gene or its encoded protein.

[0073] In another preferred embodiment, the composition comprises an agricultural composition.

[0074] In another preferred embodiment, the inhibitor includes an agricultural inhibitor.

[0075] In another preferred embodiment, the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.

[0076] In another preferred embodiment, the inhibitor is selected from the group consisting of gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, Crispr reagents, small molecule ligands, or a combination thereof.

[0077] In one embodiment, the gene editing reagent includes a Cas enzyme and a gRNA capable of targeting the SWEET11a gene.

[0078] In another preferred embodiment, the antisense nucleic acid is selected from the group consisting of antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.

[0079] In another preferred embodiment, the interfering RNA is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.

[0080] In another preferred embodiment, the composition further comprises other substances that improve plant disease resistance.

[0081] In another aspect, the present invention provides use of the above composition for improving plant disease resistance.

[0082] In another preferred embodiment, the composition is used for preparing a reagent or a kit for improving plant disease resistance.

[0083] In another aspect, the present invention provides a method for preparing a plant cell, or plant seed, or plant tissue, or plant part, or plant with enhanced disease resistance, comprising the steps of:

[0084] Reducing or inhibiting the expression and / or activity of the SWEET11a gene or its encoded protein in plant cells, plant seeds, plant tissues, plant parts, or plants.

[0085] In another preferred embodiment, the method comprises the following steps:

[0086] (1) Introducing gene editing tools into plant cells, plant seeds, plant tissues, or plant parts;

[0087] (2) The step of allowing the gene editing tool to act on its endogenous SWEET11a gene and causing it to mutate in the nucleotide sequence corresponding to SEQ ID No. 2.

[0088] Furthermore, the mutation results in a decrease in the expression level and / or activity of the SWEET11a gene or its encoded protein in the plant.

[0089] Furthermore, the above method also includes the steps of screening mutated plant cells, plant tissues, plant parts, and optionally, isolating the gene editing tool.

[0090] In another preferred embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0091] In another preferred embodiment, the gene editing tool can produce the mutated SWEET11a gene in plants.

[0092] In another preferred embodiment, the gene editing tool is a CRISPR reagent. Preferably, the CRISPR reagent includes a gene editing enzyme. Preferably, the gene editing enzyme is a Cas9 protein, and the CRISPR reagent also includes a scaffold sequence that can specifically bind to the Cas9 protein. The scaffold sequence is operably linked to the guide sequence to form the gRNA.

[0093] In another aspect, the present invention provides a method for producing plants with improved disease resistance, comprising the steps of:

[0094] The plant cells, plant seeds, plant tissues, or plant parts with improved disease resistance prepared by the above method are regenerated into plants, thereby obtaining plants with improved disease resistance.

[0095] In another preferred embodiment, the method further comprises the step of harvesting plant seeds from the plant having improved disease resistance.

[0096] On the other hand, the present invention provides a plant cell, or plant seed, or plant tissue, or plant part, or plant with improved disease resistance, characterized in that the plant cell, or plant seed, or plant tissue, or plant part, or plant is prepared by the above method.

[0097] In another aspect, the present invention provides a method for improving plant traits, the method comprising the steps of:

[0098] (a) providing a plant cell, plant tissue, or plant part, and introducing an inhibitor of the SWEET11a gene or its encoded protein into the plant cell, plant tissue, or plant part; or reducing or inhibiting the expression and / or activity of the SWEET11a gene or its encoded protein in the plant cell, plant tissue, or plant part;

[0099] (b) regenerating the plant cells, plant tissues, or plant parts of step (a) into plants.

[0100] In another preferred embodiment, in step (a), the plant cells, plant tissues, and plant parts are modified using gene editing technology, thereby reducing the expression or activity of the SWEET11a gene or its encoded protein in the plant cells, plant tissues, and plant parts.

[0101] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.

[0102] In another preferred embodiment, the improved trait is enhanced disease resistance.

[0103] On the other hand, the present invention provides a method for preparing genetically engineered plant tissue or plant cells, comprising the steps of: (i) providing a plant or plant cell; and (ii) introducing an sgRNA targeting the SWEET11a gene and a corresponding Cas protein into the plant or plant cell.

[0104] In another preferred embodiment, the method includes reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein, thereby obtaining genetically engineered plant tissues or plant cells.

[0105] In another aspect, the present invention provides a method for preparing a genetically engineered plant, comprising the steps of:

[0106] The genetically engineered plant tissues or plant cells prepared by the above method are regenerated into plant bodies, thereby obtaining genetically engineered plants.

[0107] In another aspect, the present invention provides a genetically engineered plant, which is prepared by the above method.

[0108] In another aspect, the present invention provides a method for screening or identifying plant disease resistance, comprising the step of detecting the expression level of the SWEET11a gene.

[0109] In another preferred embodiment, the detection parts of the detection plant include plant callus, fruit, seeds, flowers, stems, leaves, ears, and roots.

[0110] In another aspect, the present invention provides a method for preparing plant seeds with improved disease resistance, the method comprising the step of preparing plant seeds with improved disease resistance using the plants prepared by the above method.

[0111] On the other hand, the present invention also provides a plant seed with improved disease resistance, wherein the plant seed with improved disease resistance is prepared by the above method for preparing the plant seed with improved disease resistance.

[0112] On the other hand, the present invention also provides a method for preparing a hybrid plant, which comprises the step of hybridizing the plant prepared by the above method with other plants.

[0113] On the other hand, the present invention also provides a method for inhibiting or killing pathogens, the method comprising the steps of:

[0114] (a) preparing plant seeds, plant tissues, plant parts, or plants with improved disease resistance using the above method;

[0115] (b) contacting the plant seeds, plant tissues, plant parts, or plants obtained in step (a) with pathogenic bacteria.

[0116] In another preferred embodiment, the pathogen is a pathogen that causes large leaf spot disease, gray leaf spot disease (also known as cercospora leaf spot disease, mildew disease) and / or bacterial wilt disease.

[0117] In another preferred embodiment, the pathogen is a pathogen that causes gray leaf spot (also known as Cercospora leaf spot, mildew) and / or bacterial wilt.

[0118] In another preferred embodiment, the pathogen causing gray leaf spot (also known as Cercospora leaf spot or mildew spot) includes Cercospora zeina.

[0119] In another preferred embodiment, the pathogen causing the large spot disease includes Exserohilumturcicum (Pass.) Leonay et Suggs.

[0120] In another preferred embodiment, the pathogens causing bacterial wilt include Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme.

[0121] In another preferred embodiment, the pathogen is any one or more of Cercospora zeina, Exserohilum turcicum (Pass.) Leonay et Suggs, Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme.

[0122] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. DETAILED DESCRIPTION

[0123] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0124] As used herein, the "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.

[0125] The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. Between two sequences. Typically, comparison is made when the two sequences are aligned for maximum identity. Alignment methods are conventional methods known to those skilled in the art, such as the BLAST algorithm.

[0126] The term "genetic engineering" refers to the technology of artificially modifying and utilizing the nucleotides that control the genetic information of organisms to obtain new genetic traits, new varieties, or new products. This includes all genetic modification technologies disclosed in the art, such as gene mutagenesis, transgenic technology, and gene editing. Genetic mutagenesis methods include, but are not limited to, physical mutagenesis (such as ultraviolet mutagenesis), chemical mutagenesis (such as acridine dyes), and biological mutagenesis (such as viral and bacteriophage mutagenesis).

[0127] The specific amino acid position (number) within the protein of the present invention is determined by aligning the amino acid sequence of the target protein with the sequence of SEQ ID No. 1 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. The default parameters are preferably used in the ClustalW2 (1.82) algorithm: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used to determine the position of specific amino acids in the protein of the present invention by aligning the amino acid sequence of the protein with the sequence of SEQ ID No. 1 using default parameters suitable for multiple alignment (gap opening penalty: 10; gap extension penalty 0.05).

[0128] The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0129] The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

[0130] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues is a chemical analog of a naturally occurring amino acid residue. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0131] In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0132] The term "regulatory element," also known as a "regulatory element," as used herein, is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a temporally dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0133] The term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0134] The term "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" proteins for import into the cell nucleus via nuclear transport. That is, proteins with an NLS are transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the protein surface. Exemplary NLSs include, but are not limited to, NLSs from the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

[0135] The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0136] The term "vector" refers to a vector that contains elements that allow the vector to be integrated into the host cell genome or to replicate autonomously within the cell independently of the genome. The vector may contain any elements that ensure self-replication. It usually carries genes that are not part of the central metabolism of the cell and is usually in the form of double-stranded DNA. The choice of vector usually depends on the compatibility of the vector with the host cell into which the vector is to be introduced. If a vector is used, the choice of vector depends on methods well known to those skilled in the art for transforming host cells. For example, a plasmid vector can be used.

[0137] Vectors suitable for use in the present invention include commercially available plasmids such as, but not limited to, pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA), pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript IIKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0138] The nucleic acid sequence, nucleic acid construct or expression vector of the present invention can be introduced into the host cell by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid-mediated gene delivery, as well as calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation.

[0139] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

[0140] The term "plant cell" is to be understood as any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0141] The term "plant" is to be understood as meaning any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., cantaloupe, watermelon, Crenshaw melon, honeydew melon, cantaloupe), oilseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, bok choy), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, cabbage, Onions, rutabagas, eggplant (also known as eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, erry), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pome fruits, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, meadowsweet, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas beans, soybeans), oil plants (rapeseed, mustard, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, as well as trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0142] The term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. CRISPR technology refers to clustered, regularly interspaced short palindromic repeats, which come from the immune system of microorganisms. Among them, gene editing tools include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, Cas12i, Cas12j, etc.). The gene editing tool referred to in TALEN technology is a restriction enzyme that can cut a specific DNA sequence, which includes a TAL effector DNA binding domain and a DNA cleavage domain. The gene editing tool referred to in ZFN technology is also a restriction enzyme that can cut a specific DNA sequence, which includes a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that by constructing the nucleotides encoding the gene editing tool and other regulatory elements into a suitable vector and then transforming the cell, the editing of the genome in the cell can be achieved. The types of editing include gene knockout, insertion, and base editing.

[0143] As used herein, the term "gene editing enzyme" refers to nucleases suitable for editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nuclease technology), and ZFN (Zinc finger nuclease technology). Preferably, the gene editing enzyme is a CRISPR enzyme, also known as a Cas protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein. The Cas protein refers to a family of proteins, which may have different structures depending on their sources, such as SpCas9 derived from Streptococcus pyogenes, SaCas9 derived from Staphylococcus aureus; it may also be classified according to structural features (such as domains), such as the Cas12 family including Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12i, etc. The Cas protein may have double-stranded or single-stranded or no cutting activity. The Cas protein of the present invention may be a wild type or a mutant thereof, and the mutant type of the mutant includes amino acid replacement, substitution or deletion, and the mutant may or may not change the enzymatic activity of the Cas protein. As known to those skilled in the art, a variety of Cas proteins with nucleic acid cleavage activity reported in the prior art, the known protein or its modified variants can achieve the function of the present invention, and are herein incorporated by reference into the scope of protection.

[0144] As used herein, the terms "guide RNA", "mature crRNA", "guide sequence", "sgRNA", and "gRNA" are used interchangeably and have meanings generally understood by those skilled in the art. Generally speaking, sgRNA includes a first segment and a second segment; the first segment is also called a "skeleton region" or a "Scaffold sequence"; the second segment is also called a "targeting sequence for targeting nucleic acid" or a "targeting segment for targeting nucleic acid", or a "guide sequence", or a "spacer sequence". The first segment, "skeleton region", or "Scaffold sequence" of the gRNA can interact with the Cas enzyme of the present invention, so that the Cas enzyme and the gRNA form a complex. The gRNA of the present invention guides its interacting Cas enzyme to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

[0145] In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity to the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of those of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0146] The SWEET gene is a sugar transporter gene. Multiple SWEET genes are found in maize, and certain members of the SWEET gene family have been shown to play important roles in pathogen infection of hosts. The accession number for the SWEET11a gene in maize is GRMZM2G368827, its amino acid sequence is shown in SEQ ID No. 1, and its CDS sequence is shown in SEQ ID No. 2.

[0147] Gray leaf spot (GLS), also known as Cercospora leaf spot and corn mildew, infects not only corn but also a variety of grasses such as sorghum, lemongrass, and bluestem. One of the main pathogens causing corn gray leaf spot is Cercospora zeina. The pathogen of corn gray leaf spot mainly harms leaves. The initial lesions appear as small, needle-shaped, chlorotic yellow spots under transmitted light. After one week, long rectangular lesions form on susceptible varieties, most of which extend along the veins of corn leaves. The junction of the lesions is clear. The center of the lesion is gray, with brown necrotic lines on the edge. A gray mold layer can form on both sides of the leaf, mostly on the back of the leaf. When the humidity is high, gray mold-like substances, namely conidiophores and conidia of the pathogen, grow on the back of the lesion. In years with severe disease, the leaves of the plants die and age prematurely.

[0148] Large leaf spot is a major disease of corn. The pathogen is Exserohilum turcicum (Pass.) Leonay et Suggs. Large leaf spot primarily affects leaves, but in severe cases, it can also affect sheaths and bracts. The disease first affects the lower leaves and then spreads upward.

[0149] Bacterial wilt is also a disease of corn. There are many pathogens responsible for corn wilt, primarily Fusarium and Pythium species in China, including Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme. Symptoms include sudden bacterial wilt and wilting, with leaves appearing scalded, dry, and discolored. The cobs droop, and the bracts die. The base of the stem initially appears waterlogged, then gradually turns light brown and feels hollow when pinched, often leading to lodging.

[0150] It should be understood that although the genes provided in the examples of the present invention are derived from corn, gene sequences derived from other similar plants and having a certain homology (e.g., having more than 70%, such as 70%, 75%, 80%, 85%, 90%, 95% or even 98%, 99%, or 100% sequence identity) with the sequences of the present invention (preferably, the sequences are shown in any of SEQ ID No. 1-2) are also included within the scope of the present invention, as long as those skilled in the art can easily isolate the sequence from other plants based on the information provided in this application after reading this application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0151] The “same or similar function” mainly refers to the activity of improving plant disease resistance by losing protein activity.

[0152] Agricultural inhibitors

[0153] The active substances of the present invention (such as inhibitors of the SWEET11a gene) can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, and coatings for seeds.

[0154] These preparations can be produced by known methods, for example, by mixing the active substance with an extender, i.e., a liquid, liquefied gas, or solid diluent or carrier, and optionally a surfactant, i.e., an emulsifier and / or dispersant and / or foam former. When, for example, water is used as the extender, an organic solvent can also be used as an auxiliary agent.

[0155] When a liquid solvent is used as a diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons, for example xylene, toluene or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, for example chlorobenzene, vinyl chloride or dichloromethane; aliphatic hydrocarbons, for example cyclohexane or paraffin, for example mineral oil fractions; alcohols, for example ethanol or ethylene glycol and their ethers and lipids; ketones, for example acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; or less commonly used polar solvents, for example dimethylformamide and dimethyl sulfoxide, and water.

[0156] As for the diluent or carrier of liquefied gas, it refers to the liquid that will become gas at normal temperature and pressure, such as aerosol propellants, such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.

[0157] Solid carriers can be ground natural minerals such as kaolin, clay, talc, quartz, atavistic clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals such as highly dispersed silicic acid, alumina, and silicates. Solid carriers for particles are crushed and graded natural zircons such as calcite, marble, pumice, sepiolite, and dolomite, as well as particles synthesized from inorganic and organic coarse powders, and particles of organic materials such as sawdust, coconut shells, corn cobs, and tobacco stems.

[0158] Nonionic and anionic emulsifiers can be used as emulsifiers and / or foam formers. Examples include polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, such as alkylaryl polyethylene glycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates, and albumin hydrolysates. Dispersants include, for example, lignin sulfite waste liquor and methylcellulose.

[0159] Binders such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or emulsions, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, may be used in the formulations.

[0160] Colorants such as inorganic dyes, such as iron oxide, cobalt oxide and Prussian blue; organic dyes, such as azo dyes or metal phthalocyanine dyes; and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, aluminum and zinc can be used.

[0161] In the present invention, the "agricultural formulation" is generally an agricultural plant growth regulator, which contains an inhibitor of the SWEET11a gene or its encoded protein as an active ingredient for improving plant traits (such as improving plant disease resistance); and an agriculturally acceptable carrier.

[0162] As used herein, the term "agriculturally acceptable carrier" is an agrochemically acceptable solvent, suspending agent, or excipient for delivering the active substance of the present invention to plants. The carrier can be liquid or solid. Suitable agriculturally acceptable carriers for use in the present invention are selected from the group consisting of water, buffer, DMSO, surfactants such as Tween-20, or combinations thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.

[0163] The agriculturally acceptable inhibitors of the present invention may comprise agricultural compositions.

[0164] The agricultural formulation of the present invention can be used in combination with other substances that improve plant disease resistance. The other substances that improve plant disease resistance can be plant growth regulators known to those skilled in the art.

[0165] The agricultural formulation of the present invention can be in a variety of forms, as long as the active ingredients can effectively reach the plant body. From the standpoint of ease of preparation and application, the preferred agricultural formulation is a spray or solution formulation.

[0166] The agricultural formulations of the present invention typically contain 0.0001-99 wt%, preferably 0.1-90 wt%, of the active ingredient of the present invention, based on the total weight of the agricultural formulation. The concentration of the active ingredient of the present invention in commercial formulations or dosage forms can vary over a wide range. The concentration of the active ingredient of the present invention in commercial formulations or dosage forms can range from 0.0000001-100% (g / v), preferably between 0.0001 and 50% (g / v).

[0167] The main advantages of the present invention are:

[0168] The present invention has found through research that inhibiting the expression or activity of the SWEET11a gene or its encoded protein can improve plant disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0169] Figure 1 . Resistance to bacterial wilt in different maize plants.

[0170] Figure 2 .The incidence of bacterial wilt in different corn plants after inoculation with pathogens.

[0171] Figure 3 .The incidence of stalk rot in different corn plants.

[0172] Sequence information

[0173] Serial number content SEQ ID No.1 Amino acid sequence of the wild-type SWEET11a gene SEQ ID No. 2 Nucleotide sequence of the wild-type SWEET11a gene SEQ ID No. 3 Editing the amino acid sequence of the SWEET11a gene in plant 23SFLN002 SEQ ID No.4 Editing the nucleotide sequence of the SWEET11a gene in plant 23SFLN002 SEQ ID No.5 Editing the amino acid sequence of the SWEET11a gene in plant 23SFLN004 SEQ ID No.6 Editing the nucleotide sequence of the SWEET11a gene in plant 23SFLN004 DETAILED DESCRIPTION

[0174] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0175] Example 1: Obtaining Gene-Edited Corn

[0176] In this example, Cas9 and sgRNA targeting the ZmSWEET11a gene were used to edit the SWEET11a gene in corn. The specific operation method can be carried out according to conventional methods in the art. The accession number of the ZmSWEET11a gene is GRMZM2G368827, the amino acid sequence is shown in SEQ ID No. 1, and the nucleotide (CDS) sequence is shown in SEQ ID No. 2.

[0177] In this embodiment, the vector construction method can refer to the reference literature ("High-efficiency CRISPR / Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize", Weiwei Qi et al., "BMC Biotechnology", 2016).

[0178] Amino acid sequence encoded by the ZmSWEET11a gene:

[0179] magglfsmahpavtlsgiagniisflvflapvatflqvyrkkstggfssvpyvvalfssvlwifyalvktnsrplltinafgcgveaayivlyl

[0180] ayaprrarlrtlayfflldvaafalvvavtlfavrephrvkflgsvclafsmavfvaplsiivkvvktksveflpislsfcltlsavawfcyglftk

[0181] dpfvmypnvggfffscvqmglyfwyrkprpaaknnavlptttdggnavqvqgqvielapntvailsvspipivgvhkievveqqhkeaavaaetrrmaaanpdgampevieivpaaaav(SEQ ID No.1);

[0182] Nucleotide sequence of ZmSWEET11a gene:

[0183] atggcaggaggcctcttctccatggctcacccggccgtcaccctctccggcatcgcaggaaacatcatctccttcctggtgttccttgcaccag

[0184] tggcgacgttcctgcaggtgtaccggaagaagtcgacgggcgggttcagctcggtgccgtacgtggtggcgctcttcagctcggtgctgtgg

[0185] atcttctacgcgctggtgaagaccaactcgaggccgctgctgaccatcaacgccttcggctgcggcgtggaggcggcctacatagtcctcta

[0186] cctggcgtacgcgccgcggcgggcgcgcctgcggactctggcctacttcttcctgctggacgtggcggccttcgcgctcgtcgtcgccgtc

[0187] acgctcttcgccgtccgcgagccccaccgcgtcaagttcctcggcagcgtctgcctcgccttctccatggccgtcttcgtcgcgccgctcagc

[0188] atcatcgtcaaggtggtcaagaccaagagcgtcgagttcctgcccatcagcctctccttctgcctcacgctcagcgccgtcgcctggttctgct

[0189] acggcctcttcaccaaggacccctttgtcatgtaccccaacgtcggcggcttcttcttcagctgcgtccagatgggcctctacttctggtaccgc

[0190] aagccccgcccggcggccaagaacaacgccgtgctgccgacgaccacggacggcggcaacgcggtgcaggtgcaggggcaggtcatc

[0191] gagctggcgcccaacacggtggccatcctgtcggtgagccccatccccatcgtgggcgtgcacaagatcgaggtggtggagcagcagca

[0192] caaggaggccgccgtggccgccgagacccgccggatggccgccgcaaacccggacggcgccatgccggaggtcatcgagatcgtccccgccgccgccgcggtgtga (SEQ ID No. 2).

[0193] In this embodiment, sgRNA targeting the SWEET11a gene was designed using target Design (http: / / skl.scau.edu.cn / targetdesign / ), and the guide sequence of the sgRNA is shown in Table 1.

[0194] Table 1 sgRNA sequence information

[0195]

[0196] The constructed vector was transferred into Agrobacterium and used to infect maize embryos. The embryos were cultured, screened, differentiated, and rooted to grow into complete plants. Primers were then used to confirm the editing form of the gene-edited maize (the maize variety used in this example was the wild type of Chang 7-2).

[0197] The maize plants 23SFLN002 and 23SFLN004 with inactivated SWEET11a gene mutation were obtained by the above method, and their editing types were:

[0198] The editing result of the SWEET11a gene in the edited plant 23SFLN002 was the insertion of a base g after base 245 in the CDS sequence of the SWEET11a gene (sequence shown in SEQ ID No. 2). The predicted amino acid sequence of the SWEET11a gene in the edited plant 23SFLN002 is shown in SEQ ID No. 3, and the CDS sequence is shown in SEQ ID No. 4.

[0199] The editing result of the edited plant 23SFLN004 was a deletion of nine bases from positions 241 to 249 of the CDS sequence of the SWEET11a gene (sequence shown in SEQ ID No. 2), and an insertion of a base a after base 278, resulting in premature termination of the sequence. The predicted amino acid sequence of the SWEET11a gene of the edited plant 23SFLN004 is shown in SEQ ID No. 5, and the CDS sequence is shown in SEQ ID No. 6.

[0200] Edit the amino acid sequence encoded by the ZmSWEET11a gene of plant 23SFLN002:

[0201] magglfsmahpavtlsgiagniisflvflapvatflqvyrkkstggfssvpyvvalfssvlwifyalvktnsrplltinafglrrggglhsplp

[0202] gvraaagapadsglllpagrgglrarrrrhalrrprapprqvprqrlprllhgrlrraaqhhrqggqdqerrvpahqplllphaqrrrlvllrpl

[0203] hqgplchvpqrrrlllqlrpdgplllvpqappggqeqrraadhgrrqrgagagaghragaqhgghpvgephphrgraqdrgggaaaqggrrgrrdppdgrrkpgrrhagghrdrprrrrgvtqrqspctvhtllasy(SEQ IDNo.3);

[0204] Edit the nucleotide sequence of the ZmSWEET11a gene of plant 23SFLN002:

[0205] atggcaggaggcctcttctccatggctcacccggccgtcaccctctccggcatcgcaggaaacatcatctccttcctggtgttccttgcaccag

[0206] tggcgacgttcctgcaggtgtaccggaagaagtcgacgggcgggttcagctcggtgccgtacgtggtggcgctcttcagctcggtgctgtgg

[0207] atcttctacgcgctggtgaagaccaactcgaggccgctgctgaccatcaacgccttcgggctgcggcgtggaggcggcctacatagtcctct

[0208] acctggcgtacgcgccgcggcgggcgcgcctgcggactctggcctacttcttcctgctggacgtggcggccttcgcgctcgtcgtcgccgt

[0209] cacgctcttcgccgtccgcgagccccaccgcgtcaagttcctcggcagcgtctgcctcgccttctccatggccgtcttcgtcgcgccgctcag

[0210] catcatcgtcaaggtggtcaagaccaagagcgtcgagttcctgcccatcagcctctccttctgcctcacgctcagcgccgtcgcctggttctgc

[0211] tacggcctcttcaccaaggacccctttgtcatgtaccccaacgtcggcggcttcttcttcagctgcgtccagatgggcctctacttctggtaccg

[0212] caagccccgcccggcggccaagaacaacgccgtgctgccgacgaccacggacggcggcaacgcggtgcaggtgcaggggcaggtcat

[0213] cgagctggcgcccaacacggtggccatcctgtcggtgagccccatccccatcgtgggcgtgcacaagatcgaggtggtggagcagcagc

[0214] acaaggaggccgccgtggccgccgagacccgccggatggccgccgcaaacccggacggcgccatgccggaggtcatcgagatcgtccccgccgccgccgcggtgtgacccaacgccaatcaccatgcaccgtacacaccctgctagcttcttatta (SEQ ID No. 4);

[0215] Edit the amino acid sequence encoded by the ZmSWEET11a gene of plant 23SFLN004:

[0216] magglfsmahpavtlsgiagniisflvflapvatflqvyrkkstggfssvpyvvalfssvlwifyalvktnsrplltinagveaayivl(SEQ ID No. 5);

[0217] Edit the nucleotide sequence of the ZmSWEET11a gene of plant 23SFLN004:

[0218] atggcaggaggcctcttctccatggctcacccggccgtcaccctctccggcatcgcaggaaacatcatctccttcctggtgttccttgcaccag

[0219] tggcgacgttcctgcaggtgtaccggaagaagtcgacgggcgggttcagctcggtgccgtacgtggtggcgctcttcagctcggtgctgtgg atcttctacgcgctggtgaagaccaactcgaggccgctgctgaccatcaacgccggcgtggaggcggcctacatagtcctctaa (SEQ ID No. 6).

[0220] Example 2: Disease resistance testing of gene-edited corn

[0221] The edited plants 23SFLN002, 23SFLN004 and Chang 7-2 wild-type plants obtained in Example 1 were sent to the Institute of Plant Protection, Liaoning Academy of Agricultural Sciences for resistance to gray spot and large spot disease. The results are shown in the following table: Chang 7-2wt wild-type plants have a certain resistance to gray spot disease, and the edited plants 23SFLN002 and 23SFLN004 have improved resistance to gray spot disease to high resistance; Chang 7-2wt wild-type plants are susceptible to large spot disease, the edited plant 23SFLN002 is susceptible to large spot disease, and the edited plant 23SFLN004 has moderate resistance to large spot disease.

[0222]

[0223] Note: The strain used for gray leaf spot inoculation in the above table is Cercospora zeina; I and II refer to the first and second repetitions; Arabic numerals refer to the disease resistance level; the smaller the number, the higher the resistance.

[0224] In addition, the SWEET11a gene mutant plants (23SFLN002, 23SFLN004) obtained in Example 1 were tested for bacterial wilt resistance. The disease resistance results are as follows: Figure 1 As shown, Chang 7-2 wild-type maize ( Figure 1 The bacterial wilt incidence of SWEET11a mutant plants (WT) was about 30%; Figure 1 In other words, the incidence of bacterial wilt in plants with the SWEET11a gene mutation was significantly reduced.

[0225] Chang 7-2 wild-type corn and mutant plants (23SFLN002 and 23SFLN004) were planted in the field and inoculated with Fusarium graminearum 10 days after pollination. The inoculation steps were as follows:

[0226] (1) Culture of pathogenic bacteria spores:

[0227] The spore-forming culture medium was inoculated with Fusarium graminearum grown on PDA medium and cultured in a shaker at 200 rpm in the dark at 28°C for 3 days to enrich the spore solution to a concentration of 10 7 / mL.

[0228] (2) Stem inoculation identification:

[0229] Before and after flowering, corn plants were inoculated. A 3cm long and 0.5cm deep wound was made in the middle of the fourth stem node near the ground using a scalpel. 2mL of spore fluid was drawn up with a syringe and inserted downward at a 45-degree angle into the wound. After inoculation, the field was watered once, and normal fertilization and water management was continued thereafter.

[0230] Twelve days after inoculation with Fusarium graminearum, the interior of the stems of different strains were photographed to identify the degree of disease susceptibility. The incidence of bacterial wilt was counted. The lesion area of ​​23SFLN002 and 23SFLN004 mutant plants was significantly lower than that of the wild-type control (e.g. Figure 2 shown).

[0231] In addition, in the summer of 2024, wild-type corn strains of Chang 7-2 and mutant plants (23SFLN002 and 23SFLN004) were planted in the field. Three replicates of each strain were planted, each replicate was 15 square meters, and the natural incidence of stalk rot was recorded. The results are as follows: Figure 3 As shown, the incidence of stem rot in 23SFLN002 and 23SFLN004 mutant plants was significantly reduced compared with wild-type plants.

[0232] In summary, the inactivation of the SWEET11a gene mutation can significantly improve the resistance of corn to gray leaf spot, large leaf spot, bacterial wilt and stalk rot.

[0233] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for improving plant disease resistance, characterized in that: The method comprises the steps of: Reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein in the plant; Preferably, the amino acid sequence encoded by the SWEET11a gene has at least 70% sequence identity with SEQ ID No.

1.

2. The method according to claim 1, characterized in that The improved disease resistance of plants includes increasing the resistance of plants to gray leaf spot (also known as cercospora leaf spot, mildew), large leaf spot, bacterial wilt and / or stem rot.

3. The method according to claim 2, characterized in that The improved plant disease resistance includes increasing the plant's resistance to pathogens related to gray leaf spot (also known as cercospora leaf spot, mildew), pathogens related to large leaf spot, pathogens related to bacterial wilt and / or pathogens related to stem rot.

4. The method according to any one of claims 1 to 3, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant; Preferably, the plant is corn.

5. A composition for improving plant disease resistance, characterized in that: The composition comprises: (a) an inhibitor of the SWEET11a gene or its encoded protein; and (b) an agriculturally acceptable carrier; Preferably, the amino acid sequence encoded by the SWEET11a gene has at least 70% sequence identity with SEQ ID No. 1; Preferably, the inhibitor is selected from the group consisting of gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, Crispr reagents, small molecule ligands, or a combination thereof.

6. The use of the composition according to claim 5, characterized in that The composition is used for improving plant disease resistance; or for preparing a reagent or a kit for improving plant disease resistance.

7. A method for preparing a plant cell, plant seed, plant tissue, plant part, or plant with improved disease resistance, characterized in that: Including steps: Reducing or inhibiting the expression and / or activity of the SWEET11a gene or its encoded protein in plant cells, plant seeds, plant tissues, plant parts, or plants; Preferably, the amino acid sequence encoded by the SWEET11a gene has at least 70% sequence identity with SEQ ID No. 1; Preferably, the method further comprises the step of regenerating the plant cells, or plant seeds, or plant tissues, or plant parts prepared by the method into a plant body, thereby obtaining the plant with improved disease resistance.

8. A method for improving plant traits, characterized in that: The method comprises the steps of: (a) providing a plant cell, plant tissue, or plant part, and introducing an inhibitor of the SWEET11a gene or its encoded protein into the plant cell, plant tissue, or plant part; or reducing or inhibiting the expression and / or activity of the SWEET11a gene or its encoded protein in the plant cell, plant tissue, or plant part; (b) regenerating the plant cell, plant tissue, or plant part from step (a) into a plant; Preferably, the amino acid sequence encoded by the SWEET11a gene has at least 70% sequence identity with SEQ ID No. 1; Preferably, the improved plant trait is to improve plant disease resistance.

9. A method for preparing genetically engineered plant tissues or plant cells, characterized in that: Including steps: (i) providing a plant or plant cell; and (ii) introducing the sgRNA targeting the SWEET11a gene and the corresponding Cas protein into the plant or plant cell; Preferably, the method comprises reducing or inhibiting the expression level and / or activity of the SWEET11a gene or its encoded protein, thereby obtaining genetically engineered plant tissues or plant cells.

10. A method for preparing a hybrid plant, comprising the step of hybridizing a plant prepared by the method according to any one of claims 7 to 9 with another plant.

11. A method for inhibiting or killing pathogenic bacteria, characterized in that: The method comprises the steps of: (a) preparing a plant seed, plant tissue, plant part, or plant with improved disease resistance using the method according to any one of claims 7 to 10; (b) contacting the plant seeds, plant tissues, plant parts, or plants obtained in step (a) with pathogenic bacteria; Preferably, the pathogen is a pathogen of gray leaf spot (also known as cercospora leaf spot, mildew spot), large leaf spot, bacterial wilt and / or stem rot.

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