Method for increasing plant disease resistance

Abstract

Provided is a method for increasing plant disease resistance. Specifically, provided is a method for increasing plant disease resistance by means of reducing or inhibiting the expression level and / or activity of the SWEET11a gene or a protein encoded thereby. The method can increase plant disease resistance, especially increase the resistance to gray leaf spot, leaf blight, bacterial wilt and stem rot, and has important application value in biological breeding.

Description

A method for improving disease resistance of plants

[0001] The present application claims priority to the Chinese patent application with the application date of June 3, 2024, the application number of CN 2024107040234, and the invention title of "A method for improving disease resistance of plants". The present application incorporates the full text of the above-mentioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology and crop genetic breeding, and relates to a method for improving disease resistance of plants, in particular a method for improving disease resistance of plants by reducing or inhibiting the expression amount and / or activity of SWEET11a gene or its encoded protein in the plants. BACKGROUND

[0003] Maize is one of the main food crops in China, and its planting area is very wide in China. Various factors affect the yield and quality of maize, such as climate, soil conditions, planting varieties, diseases and pests, etc. Among them, maize diseases have a greater impact on maize yield and quality. Common diseases of maize include large spot disease, small spot disease, gray spot disease, smut disease, sheath blight disease, rust disease, stem rot disease, and bacterial wilt disease, etc.

[0004] Gray spot disease, also known as Cercospora leaf spot disease or Mycosphaerella leaf spot disease, can infect not only maize but also various gramineous plants such as sorghum, lemongrass, and awn grass. Maize gray spot disease is one of the diseases that have risen rapidly and caused serious damage in recent years. Gray spot disease is commonly found in southern regions and has a tendency to spread northward.

[0005] Large spot disease is one of the important diseases of maize and is widely distributed in all maize cultivation areas around the world. In years of high incidence, it generally reduces yield by 15-20%, and in severe cases, it reduces yield by more than 50%.

[0006] Bacterial wilt disease is caused by several Fusarium or Pythium fungi alone or in combination, and occurs in maize. The symptoms are sudden wilting and wilting, with the whole leaf appearing water scalded and dry; the ear droops, and the bract leaf dies; the stem base is initially waterlogged, then gradually turns light brown, and feels hollow when pinched, often leading to lodging.

[0007] Improving the resistance of maize to different diseases has a broad market prospect. In order to improve the disease resistance of maize, we studied the SWEET gene of maize in order to obtain maize plants with improved disease resistance. SUMMARY

[0008] The present application aims to provide a method for improving disease resistance of plants.

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

[0010] In another preferred embodiment, the SWEET11a gene encodes an amino acid sequence selected from the group consisting of:

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

[0012] (ii) a polypeptide derived from (i) by substitution, deletion, or addition of one or several (e.g. 1-10) amino acid residues to the amino acid sequence shown in SEQ ID No. 1, having the same or similar function;

[0013] (iii) a polypeptide having an amino acid sequence with 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%) homology to the amino acid sequence shown in SEQ ID No. 1, having the same or similar function.

[0014] In another preferred embodiment, the SWEET11a gene has a nucleotide sequence selected from the group consisting of:

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

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

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

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

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

[0020] In another preferred embodiment, the SWEET11a gene encodes an amino acid sequence having 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 comprises an increased resistance of the plant to gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot), large patch, bacterial wilt, or stalk rot.

[0022] In another preferred embodiment, the improved disease resistance of the plant comprises an increased resistance of the plant to gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot) and / or to bacterial wilt and / or to stalk rot.

[0023] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot), large patch, bacterial wilt, or stalk rot related pathogen.

[0024] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot) related pathogen.

[0025] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to Cercospora zeina.

[0026] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to large patch related pathogen.

[0027] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to Exserohilum turcicum (Pass.) Leonay et Suggs.

[0028] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to bacterial wilt related pathogen.

[0029] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to stalk rot related pathogen.

[0030] In another preferred embodiment, the improved disease resistance of the plant refers to an increased resistance of the plant to Fusarium and / or Pythium.

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

[0032] In another preferred embodiment, the improved disease resistance of the plant includes the increased resistance of the plant to any one or several of Cercospora zeina, Exserohilum turcicum (Pass.) Leonay et Suggs, Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, or 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 an inhibitor of SWEET11a gene or 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 amount and / or activity of SWEET11a gene or its encoded protein.

[0037] In another preferred embodiment, the "reducing or inhibiting" refers to the reduction of the expression or activity of SWEET11a gene or its encoded protein to satisfy the following conditions: the SWEET11a gene or its encoded protein completely loses activity or partially loses activity in the plant, or the ratio of A1 / A0 is ≤ 80%, preferably ≤ 60%, more preferably ≤ 40%, and most preferably 0-30%; wherein A1 is the expression or activity of SWEET11a gene or its encoded protein in the plant, and A0 is the expression or activity of the same SWEET11a gene or its encoded protein in a wild-type plant of the same type.

[0038] In another preferred embodiment, the reduction or inhibition refers to that the expression level El of the SWEET11a gene or its encoded protein in the plant is 0-80%, preferably 0-60%, more preferably 0-40%, more preferably 0-30% of 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 group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of an inhibitor of the gene or protein, 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 UV mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrous acid, hydroxylamine, EMS, nitroso guanidine, 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 group consisting of CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

[0042] 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 mutation of the SWEET11a gene.

[0043] In another preferred embodiment, the mutation comprises an insertion mutation, a deletion mutation, a frameshift mutation, a substitution mutation.

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

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

[0046] In another preferred embodiment, the mutation is a deletion of one or more bases in the nucleotide sequence of the SWEET11a gene 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 in 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 an insertion of one base g 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 a deletion of bases from 241st to 249th base and an insertion of one base a after the 278th base of the nucleotide sequence of the SWEET11a gene relative to the sequence shown in SEQ ID No. 2.

[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 compared with the nucleotide sequence of the parent SWEET11a gene corresponding to the nucleotide sequence shown in SEQ ID No. 2.

[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, Linaceae, Asteraceae, Malvaceae, Rosaceae, Linaceae, Convolvulaceae, Dioscoreaceae, Umbelliferae, Liliaceae, Zingiberaceae, Palmae.

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

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

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

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

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

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

[0059] In another preferred embodiment, the nucleotide sequence of the mutated SWEET11a gene is as shown in any one of 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 as shown in any one of SEQ ID No. 3 or SEQ ID No. 5.

[0061] In the present application, SWEET11a genes from different plant sources, for example, SWEET11a genes naturally existing in different plants or SWEET11a genes from different plant sources artificially modified, can all be used as parent SWEET11a genes; those skilled in the art can obtain the nucleotide sequences and amino acid sequences of SWEET11a genes from different sources through routine technical knowledge, and mutate the SWEET11a genes based on the methods described in the present application or the prior art to achieve the purpose of reducing or inhibiting the expression amount and / or activity of the SWEET11a gene or the protein encoded thereby in the plant.

[0062] In another preferred embodiment, the plant includes 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, Linaceae, Asteraceae, Malvaceae, Rosaceae, Linaceae, Convolvulaceae, Dioscoreaceae, Umbelliferae, Liliaceae, Zingiberaceae, Palmae.

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

[0065] In another preferred embodiment, the plant is selected from soybean, Arabidopsis thaliana, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber, 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 application provides a composition for improving the disease resistance of a plant, the composition comprising:

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

[0071] (b) an agriculturally acceptable carrier.

[0072] In preferred embodiments, the inhibitor is capable of reducing or inhibiting the expression and / or activity of SWEET11a gene or its encoded protein.

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

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

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

[0076] In another preferred embodiment, the inhibitor is selected from the group consisting of a gene editing reagent, an antisense nucleic acid, an antibody, a small molecule compound, a Crispr reagent, a small molecule ligand, or a combination thereof.

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

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

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

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

[0081] In another aspect, the present application provides use of the above-mentioned composition for enhancing plant disease resistance.

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

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

[0084] reducing or inhibiting the expression and / or activity of SWEET11a gene or its encoded protein in the plant cell, or the plant seed, or the plant tissue, or the plant part, or the plant.

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

[0086] (1) introducing a gene editing tool into a plant cell, a plant seed, a plant tissue, a plant part;

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

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

[0089] Further, the above method further comprises the steps of screening the mutated plant cell, plant tissue, plant part, and optionally, isolating the gene editing tool.

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

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

[0092] In another preferred embodiment, the gene editing tool is a CRISPR reagent. Preferably, the CRISPR reagent comprises a gene editing enzyme. Preferably, the gene editing enzyme is a Cas9 protein, and the CRISPR reagent further comprises a Scaffold sequence that can specifically bind to the Cas9 protein. After the Scaffold sequence is operably linked to the guide sequence, a gRNA is formed.

[0093] In another aspect, the present application provides a method for improving the disease resistance of a plant, comprising the steps of:

[0094] Regenerating the disease resistance improved plant cell, or plant seed, or plant tissue, or plant part prepared by the above method into a plant body, thereby obtaining a disease resistance improved plant.

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

[0096] In another aspect, the present application provides a disease resistance improved plant cell, or plant seed, or plant tissue, or plant part, or plant, 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 application provides a method for improving the plant traits, comprising the steps of:

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

[0099] (b) regenerating a plant from the plant cell, plant tissue, plant part in step (a).

[0100] In another preferred embodiment, in step (a), the plant cell, plant tissue, plant part is engineered by gene editing technology to reduce the expression or activity of SWEET11a gene or its encoded protein in the plant cell, plant tissue, plant part.

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

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

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

[0104] In another preferred embodiment, the method comprises reducing or inhibiting the expression and / or activity of SWEET11a gene or its encoded protein to obtain a genetically engineered plant tissue or plant cell.

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

[0106] regenerating a plant from the genetically engineered plant tissue or plant cell prepared by the above method to obtain a genetically engineered plant.

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

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

[0109] In another preferred embodiment, the detection site of the plant comprises callus, fruit, seed, flower, stem, leaf, ear, root of the plant.

[0110] In another aspect, the present application provides a method for producing a plant seed with enhanced disease resistance, the method comprising the step of producing a plant seed with enhanced disease resistance using the plant produced by the above method.

[0111] In another aspect, the present application provides a plant seed with enhanced disease resistance, the plant seed with enhanced disease resistance being produced by the above method for producing a plant seed with enhanced disease resistance.

[0112] In another aspect, the present application provides a method for producing a hybrid plant, the method comprising the step of crossing the plant produced by the above method with another plant.

[0113] In another aspect, the present application provides a method for inhibiting or killing a pathogenic fungus, the method comprising the steps of:

[0114] (a) using the plant seed, plant tissue, plant part, or plant with enhanced disease resistance produced by the above method;

[0115] (b) contacting the plant seed, plant tissue, plant part, or plant produced in step (a) with the pathogenic fungus.

[0116] In another preferred embodiment, the pathogenic fungus is a pathogenic fungus that causes large patch disease, gray leaf spot disease (also known as Cercospora leaf spot disease, or Mycosphaerella leaf spot disease), and / or bacterial wilt disease.

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

[0118] In another preferred embodiment, the pathogenic fungus that causes gray leaf spot disease (also known as Cercospora leaf spot disease, or Mycosphaerella leaf spot disease) comprises Cercospora zeina.

[0119] In another preferred embodiment, the pathogenic fungus that causes large patch disease comprises Exserohilum turcicum (Pass.) Leonay et Suggs.

[0120] In another preferred embodiment, the pathogenic fungus that causes bacterial wilt disease comprises Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme.

[0121] In another preferred embodiment, the pathogenic fungi are any one or several of Cercospora zeina, Exserohilum turcicum (Pass.) Leonay et Suggs, Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, Fusarium moniliforme.

[0122] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be repeated one by one here. DETAILED DESCRIPTION

[0123] Unless otherwise defined, 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 terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which can be double-stranded or single-stranded.

[0125] The term "homology" or "identity" is used in reference to the matching of sequences between two polypeptides or two nucleic acids. When a position in each of two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules occupied by adenine, or a position in each of two polypeptides occupied by lysine), then the molecules are homologous at that position. A degree of homology between two sequences is expressed as a percentage of matching or identical positions. Typically, two sequences are compared when aligned to produce maximum homology. Alignment methods are known in the art, such as the BLAST algorithm.

[0126] The term "genetic engineering" refers to the technology of modifying and utilizing nucleotides that control the genetic information of organisms through artificial intervention to obtain new genetic characteristics, or new varieties, or new products, including all genetic modification technologies disclosed in the art, such as gene mutagenesis, transgenic or gene editing methods. Methods of gene mutagenesis include but are not limited to physical mutagenesis (such as UV mutagenesis), chemical mutagenesis (such as acridine dyes), biological mutagenesis (such as viral, phage mutagenesis) and the like.

[0127] The specific amino acid positions (numbering) within the protein according to the present application are determined by aligning the amino acid sequence of the protein of interest with the sequence of SEQ ID No. 1 using standard sequence alignment tools, such as aligning the two sequences using the Smith-Waterman algorithm or using the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is highest. The alignment score can be calculated according to the method described in Wilbur, W. J. and Lipman, D. J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. In the CLUSTALW2 (1.82) algorithm the use of the default parameters is preferred: Protein gap open penalty = 10.0; Protein gap extension penalty = 0.2; Protein matrix = Gonnet; Protein / DNA end gap = -1; Protein / DNA GAP DIST = 4. Preferably the AlignX program (part of the vector NTI suite) is used to determine the position of a specific amino acid within the protein according to the present application by aligning the amino acid sequence of the protein with the sequence of SEQ ID No. 1 using the default parameters for multiple alignment (gap open penalty: 10; gap extension penalty 0.05).

[0128] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having the specific sequences of nucleotides, i.e. rRNA, tRNA, and mRNA, or the specific sequences of amino acids and the biological processes by which they are generated. Thus, if a mRNA corresponding to a gene is translated into a protein, the gene is said to encode the protein.

[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 of the amino acid residues are chemical analogs of naturally occurring amino acid residues. Proteins and polypeptides of the present application can be produced recombinantly or by chemical synthesis.

[0131] In the present application, the amino acid residues can be represented by single letter or three letter, 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 "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, Calif. (1990). In certain instances, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neuronal, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In certain instances, regulatory elements can also direct expression in a temporal-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which can or can not be tissue- or cell type-specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as the WPRE; the CMV enhancer; the R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., vol. 78(3), pp. 1527-31, 1981).

[0133] The term "promoter" has its art-understood meaning and refers to a non-coding nucleotide sequence located upstream from a gene that initiates transcription of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or defining a gene product, results in 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 with a polynucleotide encoding or defining a gene product, results in production of the gene product in a 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 with a polynucleotide encoding or defining a gene product, results in production of the gene product in a cell essentially only when the cell is of the tissue type to which the promoter corresponds.

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

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

[0136] The term "vector" is a vehicle that comprises elements that allow the vector to integrate into the genome of a host cell or to replicate autonomously within the cell independent of the genome. The vector can comprise any element that ensures self-replication. It is typically in the form of double-stranded DNA and carries genes that are not part of the cell's central metabolism. The choice of vector is typically dependent 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 is dependent on the method used to transform the host cell, which is well known to those skilled in the art. For example, a plasmid vector can be used.

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

[0138] The nucleic acid sequence, nucleic acid construct or expression vector of the present application can be introduced into a host cell by a variety of techniques, including transformation, transfection, transduction, viral infection, biolistics or Ti-plasmid mediated gene delivery, and 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 calli, plant clumps, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, shoots, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

[0140] The term "plant cell" is understood to mean any cell from or found in a plant that is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, a component part of a plant, a plant or a seed.

[0141] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants, in particular monocotyledonous or dicotyledonous plants, at any stage of development or maturation, vegetable crops, including artichokes, Brussels sprouts, cress, leeks, asparagus, lettuce (e.g. head lettuce, leaf lettuce, long-leaf lettuce), bok choy, yellow fleshed yam, melons (e.g. muskmelons, watermelons, crenshaw melons, cantaloupes, honeydews), oilseed crops (e.g. Brussels sprouts, cabbage, cauliflower, broccoli, collards, kale, Chinese cabbage, bok choy), artichokes, carrots, napa, okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g. zucchini, cucumbers, crookneck squash, calabaza, pumpkin), radishes, dry bulb onions, rutabaga, eggplant (also known as aubergine), burdock, endive, green onions, endive, garlic, spinach, green onions, squash, greens, sugar beets (sugar beet and fodder beet), sweet potatoes, Swiss chard, wasabi, tomatoes, turnips, and spices; fruits and / or vine crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, aronia, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, goji berries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomes, melons, mangoes, papayas, and lychees; field crops, such as clover, alfalfa, evening primrose, meadow grass, maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, cotton, legumes (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa, groundnuts), Arabidopsis, fiber plants (cotton, flax, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broad-leaved trees and evergreens, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other young plants.

[0142] The term "gene editing" technology includes CRISPR technology, TALEN technology, ZFN technology. CRISPR technology refers to Clustered regularly interspaced short palindromic repeats, which comes from the immune system of microorganisms. Among them, the gene editing tools include guide RNA and Cas protein (such as Cas9, Cpf1, Cas12b, Cas12i, Cas12j, etc.). The gene editing tool referred to in TALEN technology is a restriction enzyme that can cut specific DNA sequences, which includes a TAL effector DNA binding domain and a DNA cutting domain. The gene editing tool referred to in ZFN technology is also a restriction enzyme that can cut specific DNA sequences, which includes a zinc finger DNA binding domain and a DNA cutting domain. Those skilled in the art are familiar with that nucleotides encoding gene editing tools and other regulatory elements are constructed in suitable vectors, and then transformed into cells, which can achieve editing of the genome in cells, and the types of editing include gene knockout, insertion, base editing.

[0143] As used herein, the term "gene editing enzyme" refers to a nuclease suitable for use in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), ZFN (Zinc finger nuclease) and the like editing tools. Preferably, the gene editing enzyme is a CRISPR enzyme, also known as Cas protein, which includes but is not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein. The Cas protein refers to a protein family, which can have different structures depending on its source, such as SpCas9 derived from Streptococcus pyogenes, SaCas9 derived from Staphylococcus aureus; it can 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 can have double-stranded or single-stranded or no cleavage activity. The Cas protein of the present application can be wild type or its mutant, the mutation type of the mutant includes substitution, substitution or deletion of amino acid, the mutant can change or not change the cleavage activity of the Cas protein. As known by those skilled in the art, a variety of Cas proteins with nucleic acid cleavage activity have been reported in the prior art, and the known proteins or their modified variants can achieve the function of the present application, which are incorporated by reference herein.

[0144] As used herein, the terms "guide RNA" (gRNA), "mature crRNA", "guide sequence", "sgRNA", "gRNA" are used interchangeably and have the meaning generally understood by one of skill in the art. Generally, sgRNA includes a first segment and a second segment; the first segment is also known as "scaffold region" or "Scaffold sequence"; the second segment is also known as "targeting sequence of targeting nucleic acid" or "targeting segment of targeting nucleic acid", or "guide sequence", or "spacer sequence". The first segment of the gRNA, "scaffold region", or "Scaffold sequence" can interact with the Cas enzyme of the present application, so that the Cas enzyme and the gRNA form a complex. The gRNA of the present application is guided by the targeting sequence of the targeting nucleic acid to the specific nucleotide sequence in the target nucleic acid.

[0145] In certain instances, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of a CRISPR / Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned, 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 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 in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0146] SWEET gene is a sugar transporter gene, there are multiple SWEET genes in corn, and some SWEET gene family members have been proved to play an important role in the process of pathogenic bacteria infecting hosts. The accession number of SWEET11a gene in corn is GRMZM2G368827, the amino acid sequence is shown as SEQ ID No. 1, and the CDS sequence is shown as SEQ ID No. 2.

[0147] Gray leaf spot (GLS), also known as Cercospora leaf spot, corn leaf spot, in addition to infecting corn, it can also infect sorghum, sweet grass, and many other gramineous plants. One of the main pathogens causing corn gray leaf spot is Cercospora zeina. The main harm of corn gray leaf spot pathogen is to the leaf. The initial lesion is a small yellow spot with a needle-shaped faded green under transmitted light, and after 1 week, long rectangular lesions are formed on the susceptible varieties, most of which spread along the corn leaf veins. The lesion junction is clear. The central part of the lesion is gray, and the edge has a brown necrotic line. Gray mold layer can be produced on both sides of the leaf, mostly on the back. When the humidity is high, gray mold appears on the back of the lesion, which is the conidiophore and conidium of the pathogen. In severe years, the leaves of the plant die early.

[0148] Large spot disease is one of the important diseases of corn, and the pathogen is Exserohilum turcicum (Pass.) Leonay et Suggs. Large spot disease mainly harms the leaves, and also harms the leaf sheaths and bracts in severe cases. The lower leaves of the plant are infected first, and then spread upwards.

[0149] Bacterial wilt is also a disease that occurs in corn. The pathogenic bacteria of corn bacterial wilt are various, in China, the main ones are Fusarium and Pythium, including Pythium aphanidermatum, Pythium infawm, Pythium graminicola, Fusarium graminearum, and Fusarium moniliforme. The symptoms are sudden wilting, the whole leaf is water scalding and dry, the ear is drooping, the bract leaf is dead, the stem base is water immersion at first, then gradually turns into light brown, and the hand feels hollow, often leading to lodging.

[0150] It should be understood that although the genes provided in the examples of the present application are derived from corn, gene sequences derived from other similar plants, which have certain homology (such as 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 application (preferably, the sequences are as shown in any one of SEQ ID Nos. 1-2) are also included within the scope of the present application, as long as the skilled person can easily isolate the sequences from other plants according to the information provided in the present application after reading the present 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 loss of protein activity.

[0152] Agricultural inhibitors

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

[0154] These formulations can be produced in known manner, for example, by mixing the active substances with extenders, that is to say liquid or liquefied gaseous or solid diluents or carriers, and if desired surfactants, that is to say emulsifiers and / or dispersants and / or foam formers. It is possible for organic solvents to be used as auxiliaries, for example in the case of water as extender.

[0155] Suitable as diluents or carriers with liquid solvents are, for example, aromatic hydrocarbons, such as xylene, toluene or alkyl naphthalenes; chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, chloroethene or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffins, for example mineral oil fractions; alcohols, such as ethanol or glycol and their ethers and esters; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; or less commonly used polar solvents, such as dimethylformamide and dimethyl sulphoxide, and also water.

[0156] By diluents or carriers for liquid gases are meant liquids which at normal temperatures and pressures will become gaseous, for example aerosol propellants, such as halogenated hydrocarbons, and also butane, propane, nitrogen and carbon dioxide.

[0157] Solid carriers can be used ground natural minerals, such as kaolins, clays, talc, quartz, attapulgite, or silica and ground synthetic minerals, such as highly dispersed silicic acid, aluminium oxide and silicates. Solid carriers for granules are crushed and fractionated natural rocks such as pumice, dolomite, calcite, argillite, sepiolite and meerschaum, and also organic and inorganic, finely divided synthetic granules, and also organic materials such as sawdust, coconut shells, corn cobs and tobacco stalks.

[0158] Non-ionic and anionic emulsifiers and / or foam formers can be used as emulsifiers and / or foam formers. Examples are polyoxyethylene-fatty acid esters, polyoxyethylene-fatty alcohol ethers, such as alkylaryl polyglycol ethers, alkylsulphates, alkyl sulphates, arylsulphates and albumin hydrolysates. Dispersants include, for example, lignosulphite waste liquors and methylcellulose.

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

[0160] It is possible to use colourants such as inorganic pigments, such as iron oxide, titanium oxide and Prussian Blue, organic dyes, such as alizarin dyes or metal phthalocyanine dyes, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, aluminium and zinc.

[0161] In the present application, 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 (e.g., increasing plant disease resistance); and an agriculturally acceptable carrier.

[0162] As used herein, the "agriculturally acceptable carrier" is a pesticidally acceptable solvent, suspending agent or excipient for delivering the active material of the present application to a plant. The carrier can be liquid or solid. The agriculturally acceptable carrier suitable for use in the present application is selected from the group consisting of water, buffer, DMSO, a surfactant such as Tween-20, or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present application.

[0163] The agricultural inhibitor of the present application can include an agricultural composition.

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

[0165] The dosage form of the agricultural formulation of the present application can be various, as long as the active ingredient can be effectively delivered to the plant, and from the standpoints of ease of preparation and application, a preferred agricultural formulation is a spray or solution formulation.

[0166] The agricultural formulation of the present application generally contains 0.0001 to 99 wt%, preferably 0.1 to 90 wt% of the active ingredient of the present application based on the total weight of the agricultural formulation. The concentration of the active ingredient of the present application in the commercial formulation or use form can vary within a wide range. The concentration of the active ingredient of the present application in the commercial formulation or use form can be from 0.0000001 to 100% (g / v), preferably between 0.0001 and 50% (g / v).

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

[0168] The present application has found that the plant disease resistance is improved by inhibiting the expression or activity of SWEET11a gene or its encoded protein. BRIEF DESCRIPTION OF DRAWINGS

[0169] FIG. 1. Bacterial wilt resistance of different corn plants.

[0170] FIG. 2. Bacterial wilt incidence of different corn plants after inoculation with pathogenic bacteria.

[0171] FIG. 3. Incidence of stalk rot of different corn plants.

[0172] SEQUENCE INFORMATION

[0173] DETAILED DESCRIPTION

[0174] The present application is further described below in connection with the embodiments. The following description is only a preferred embodiment of the present application and is not intended in any way to limit the present application. Any person skilled in the art can make equivalent changes to the above-mentioned technical content to obtain equivalent embodiments. Any simple modification or equivalent change to the following embodiments without departing from the technical essence of the present application falls within the scope of the present application.

[0175] Example 1, obtaining of gene edited corn

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

[0177] In this embodiment, the vector construction method can refer to the reference (“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] The amino acid sequence encoded by ZmSWEET11a gene:

[0179] The nucleotide sequence of ZmSWEET11a gene:

[0180] In this embodiment, sgRNA targeting SWEET11a gene is designed by using target Design (http: / / skl.scau.edu.cn / targetdesign / ). The guide sequence of sgRNA is shown in Table 1.

[0181] Table 1 sgRNA sequence information

[0182] The constructed vector is transformed into Agrobacterium, and the corn embryo is infected. After culture, screening, differentiation, and rooting, the complete plant is grown. Then, the primer is used to confirm the editing form of the gene edited corn (the corn variety used in this embodiment is wild type Chang 7-2).

[0183] The SWEET11a gene mutant inactivated corn plants 23SFLN002 and 23SFLN004 were obtained by the above method, and the editing types were respectively:

[0184] The editing result of the SWEET11a gene of the edited plant 23SFLN002 is that one base g is inserted after the 245th base of the CDS sequence (the sequence shown in SEQ ID No. 2) of the SWEET11a gene. The predicted amino acid sequence of the SWEET11a gene of the edited plant 23SFLN002 is shown in SEQ ID No. 3, and the CDS sequence is shown in SEQ ID No. 4.

[0185] The editing result of the edited plant 23SFLN004 is that 9 bases from 241st to 249th of the CDS sequence (the sequence shown in SEQ ID No. 2) of the SWEET11a gene are deleted, and one base a is inserted after the 278th base, 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.

[0186] The amino acid sequence encoded by the ZmSWEET11a gene of the edited plant 23SFLN002:

[0187] The nucleotide sequence of the ZmSWEET11a gene of the edited plant 23SFLN002:

[0188] The amino acid sequence encoded by the ZmSWEET11a gene of the edited plant 23SFLN004:

[0189] The nucleotide sequence of the ZmSWEET11a gene of the edited plant 23SFLN004:

[0190] Example 2, detection of disease resistance of gene edited corn

[0191] The edited plants 23SFLN002, 23SFLN004 obtained in Example 1 and Chang 7-2 wild type plants were sent to Liaoning Academy of Agricultural Sciences for gray spot and large spot resistance determination, and the results are shown in the following table: Chang 7-2 wt wild type plants have certain resistance to gray spot, and the edited plants 23SFLN002 and 23SFLN004 have high resistance to gray spot; Chang 7-2 wt wild type plants are susceptible to large spot, and the edited plant 23SFLN002 is susceptible to large spot, and the edited plant 23SFLN004 has moderate resistance to large spot.

[0192] Note: The fungus for gray spot inoculation in the above table is Cercospora zeina; I and II refer to the first and second repeats; Arabic numerals refer to disease resistance grades, and the smaller the number, the higher the resistance.

[0193] In addition, the SWEET11a gene mutant plants (23SFLN002, 23SFLN004) obtained in Example 1 were subjected to bacterial wilt resistance detection, and the disease resistance results are shown in Figure 1. The incidence of bacterial wilt of Chang 7-2 wild type corn (WT in Figure 1) is about 30%; the SWEET11a gene mutant plants (sweet11a in Figure 1) are almost not diseased. That is, the incidence of bacterial wilt of the SWEET11a gene mutant inactivated plant is significantly reduced.

[0194] Chang 7-2 wild type corn and mutant plants (23SFLN002, 23SFLN004) were planted in the field, and the materials 10 days after pollination were subjected to F. graminearum puncture inoculation. The inoculation steps are as follows:

[0195] (1) Culture of pathogen spore solution:

[0196] F. graminearum grown on PDA medium was inoculated into a spore production culture solution, and the spore solution was enriched to a concentration of 10 7 / mL after 3 days of culture at 28°C in a 200 rpm shaker in the dark.

[0197] (2) Stem inoculation identification:

[0198] Before and after flowering of the corn plants, a 3 cm long and 0.5 cm deep wound was made in the middle of the 4th stem node of the plant using a scalpel, and 2 mL of spore solution was injected into the wound at a 45-degree angle. After inoculation, the field was watered once, and then normal irrigation management was carried out.

[0199] The stem interior of different strains was photographed to identify the disease degree 12 days after inoculation with the pathogen Fusarium graminearum. The occurrence of bacterial wilt was counted, and the lesion area of 23SFLN002 and 23SFLN004 mutant plants was significantly lower than that of the wild type control (as shown in Figure 2).

[0200] In addition, in summer 2024, Chang 7-2 wild type corn and mutant plants (23SFLN002, 23SFLN004) were planted in the field; three plot repeats were planted for each strain, and each repeat was 15 square meters. The natural occurrence of stalk rot was counted, and the results are shown in Figure 3. Compared with the wild type plants, the occurrence rate of stalk rot of 23SFLN002 and 23SFLN004 mutant plants was significantly reduced.

[0201] In summary, the SWEET11a gene mutation inactivation can significantly improve the resistance of corn to gray spot, large spot, bacterial wilt and stalk rot.

[0202] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually. In addition, it should be understood that, after reading the above teaching of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A method for increasing disease resistance in a plant, comprising, The method comprises the steps of: reducing or inhibiting the expression amount and / or activity of the SWEET11a gene or the encoded protein thereof in the plant; Preferably, the SWEET11a gene encodes an amino acid sequence having at least 70% sequence identity compared with SEQ ID No.

1.

2. The method of claim 1, wherein, The improved plant disease resistance includes increased resistance of the plant to gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot), large patch, bacterial wilt, and / or stem rot.

3. The method of claim 2, wherein, The improved plant disease resistance includes increased resistance of the plant to the pathogen associated with gray leaf spot (or called as Cercospora leaf spot, mycosphaerella leaf spot), the pathogen associated with large patch, the pathogen associated with bacterial wilt, and / or the pathogen associated with stem rot.

4. The method according to any of claims 1 to 3, characterized in that, The plant is a monocotyledon or a dicotyledon; Preferably, the plant is corn.

5. A composition for improving disease resistance of a plant, characterized by comprising the plant growth regulator according to claim 1 or 2. The composition comprises: (a) an inhibitor of the SWEET11a gene or the encoded protein thereof; and, (b) an agronomically acceptable carrier; Preferably, the SWEET11a gene encodes an amino acid sequence having at least 70% sequence identity compared with SEQ ID No.

1. Preferably, the inhibitor is selected from the group consisting of a gene editing reagent, an antisense nucleic acid, an antibody, a small molecule compound, a Crispr reagent, a small molecule ligand, or a combination thereof.

6. Use of a 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 of making a plant cell, or a plant seed, or a plant tissue, or a plant part, or a plant with increased disease resistance, characterized in that, The method comprises the steps of: reducing or inhibiting the expression amount and / or activity of the SWEET11a gene or the encoded protein thereof in the plant; Preferably, the SWEET11a gene encodes an amino acid sequence having at least 70% sequence identity compared with SEQ ID No.

1. Preferably, the method further comprises the step of regenerating the plant cell, or the plant seed, or the plant tissue, or the plant part into a plant body, thereby obtaining the plant with improved disease resistance.

8. A method of modifying a trait of a plant, comprising: The method comprises the steps of: (a) providing a plant cell, a plant tissue, a plant part, introducing an inhibitor of the SWEET11a gene or the encoded protein thereof into the plant cell, the plant tissue, or the plant part; or, reducing or inhibiting the expression and / or activity of the SWEET11a gene or the encoded protein thereof in the plant cell, the plant tissue, or the plant part; (b) regenerating the plant cell, the plant tissue, or the plant part in step (a) into a plant; Preferably, the SWEET11a gene encodes an amino acid sequence having at least 70% sequence identity compared with SEQ ID No.

1. Preferably, the improved plant trait is improved plant disease resistance.

9. A method of making a genetically engineered plant tissue or plant cell, characterized by, The method comprises the steps of: (i) providing a plant or a plant cell; and (ii) introducing the plant or the plant cell with an sgRNA targeting the SWEET11a gene and a corresponding Cas protein; Preferably, the method comprises reducing or inhibiting the expression amount and / or activity of the SWEET11a gene or the encoded protein thereof, thereby obtaining a genetically engineered plant tissue or plant cell.

10. A method of making a hybrid plant, the method comprising the step of crossing a plant made using the method of any one of claims 7-9 with another plant.

11. A method of inhibiting or killing pathogenic bacteria, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-10. 5 The method comprises the steps of: (a) making a plant seed, plant tissue, plant part, or plant with increased disease resistance using the method of any one of claims 7-10; (b) contacting the plant seed, plant tissue, plant part, or plant of step (a) with a pathogenic fungus; Preferably, the pathogenic fungus is a pathogenic fungus of gray leaf spot (or Cercospora leaf spot, mycosphaerella leaf spot), large patch, goss' wilt, and / or stem decay.

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