Application of FLOE2 protein and FLOE3 protein in regulation and control of plant disease resistance
By regulating the expression and activity of FLOE2 and FLOE3 proteins, the problem of plant resistance to Pseudomonas syringae was solved, and the plant disease resistance was significantly improved or reduced, providing new breeding resources and methods.
Patent Information
- Application Number
- CN202511237000.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2026-03-03
AI Technical Summary
Existing technologies are insufficient to effectively regulate plant resistance to Pseudomonas syringae, leading to frequent plant diseases.
By regulating the expression and activity of FLOE2 and FLOE3 proteins, and utilizing FLOE2 and FLOE3 protein-related biomaterials and substances, plant disease resistance can be improved or reduced. This includes methods such as overexpressing or inhibiting these proteins, as well as gene editing technologies to alter plant disease resistance.
Significantly increasing or decreasing plant resistance to Pseudomonas syringae provides new breeding resources and methods, promoting the cultivation of disease-resistant plant varieties.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering, specifically to the application of FLOE2 and FLOE3 proteins in regulating plant disease resistance. Background Technology
[0002] Pseudomonas syringae ( Pseudomonas syringae *Pseudomonas syringae* is a common member of the microbial community on plant leaves and stems. It can colonize the surfaces of leaves and stems of various plants, including crops such as wheat, corn, and rice; vegetables such as tomatoes, cucumbers, and lettuce; and many other plants such as flowers and trees. It is also widely present in soil, especially in the rhizosphere. In soil, it can interact with other microorganisms and participate in processes such as soil nutrient cycling. Besides plants and soil, *Pseudomonas syringae* may also exist in water bodies, air, and other environmental environments.
[0003] Pseudomonas syringae ( Pseudomonas syringae Bacillus subtilis (BSD) is a pathogen causing many plant diseases, leading to symptoms such as necrosis and rot in various plants. For example, it can cause bacterial leaf spot in tomatoes, resulting in brown necrotic spots on the leaves, affecting photosynthesis and growth; in wheat, it can cause wheat leaf blight, causing the leaves to turn yellow and reducing wheat yield and quality. Its pathogenic mechanism mainly involves its surface attachment factors, secreted extracellular enzymes, and some toxic factors acting on plant cells, damaging the plant's cellular structure and physiological functions.
[0004] As research into the interaction mechanisms between plants and *Pseudomonas syringae* deepens, breeding disease-resistant varieties using resistance genes has become one of the most effective, safest, and most economical ways to control *Pseudomonas syringae*. In-depth exploration of genes related to *Pseudomonas syringae* resistance using the model plant *Arabidopsis thaliana* as the research subject will provide genetic resources for crop disease resistance breeding and contribute to disease resistance breeding. Summary of the Invention
[0005] The technical problem to be solved by this invention is how to regulate plant disease resistance.
[0006] To address the aforementioned technical problems, this invention first provides new uses for FLOE2 and FLOE3 proteins.
[0007] This invention provides the use of FLOE2 and FLOE3 proteins in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Prepare products that regulate plant disease resistance; A3) Cultivate plants with altered disease resistance; A4) Prepare products from plants with altered disease resistance; A5) Plant breeding; A6) Preparation of plant breeding products; The FLOE2 protein is any one of the following M1)-M4): The amino acid sequence of M1 is the protein shown in sequence 2; M2) is a fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; M3) is a protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of M1. M4) is a protein that shares more than 75% identity with M1 and is associated with plant disease resistance; The FLOE3 protein is any one of the following (N1)-N4): The amino acid sequence of N1 is the protein shown in sequence 5; N2) A fusion protein associated with plant disease resistance is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 5; N3) is a protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of N1. N4) is a protein that shares more than 75% identity with N1 and is associated with plant disease resistance.
[0008] In the proteins described in M2) or N2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.
[0009] In the protein described in M3 or N3 above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.
[0010] In the proteins described above (M4) or (N4), the identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences having 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequences shown in Sequence 2 or Sequence 4 of the present invention.
[0011] The proteins described in M1)-M4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.
[0012] To address the aforementioned technical problems, the present invention provides new uses for biomaterials related to the aforementioned FLOE2 protein and biomaterials related to the aforementioned FLOE3 protein.
[0013] This invention provides the application of biomaterials related to the aforementioned FLOE2 protein and biomaterials related to the aforementioned FLOE3 protein in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Prepare products that regulate plant disease resistance; A3) Cultivate plants with altered disease resistance; A4) Prepare products from plants with altered disease resistance; A5) Plant breeding; A6) Prepare products for plant breeding.
[0014] In the above applications, the biomaterials related to the FLOE2 protein are nucleic acid molecules encoding the FLOE2 protein or expression cassettes, recombinant vectors, or recombinant microorganisms containing the nucleic acid molecules.
[0015] The biological material associated with the FLOE3 protein is a nucleic acid molecule encoding the FLOE3 protein or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.
[0016] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA.
[0017] In this invention, the nucleic acid molecule is a DNA molecule represented by sequence 1, sequence 3, sequence 4, or sequence 6, or a DNA molecule that has 75% or more of the same identity as sequence 1, sequence 3, sequence 4, or sequence 6, and encodes the aforementioned FLOE2 protein or FLOE3 protein.
[0018] Wherein, sequence 1 and sequence 3 are respectively FLOE2 The coding sequence (CDS) of a gene and the genome sequence.
[0019] Among them, sequence 4 and sequence 6 are respectively FLOE3 The coding sequence (CDS) of a gene and the genome sequence.
[0020] Those skilled in the art can readily mutate the nucleotide sequences encoding the FLOE2 or FLOE3 proteins of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity with the nucleotide sequences of the FLOE2 or FLOE3 proteins isolated according to this invention, provided they encode the FLOE2 or FLOE3 protein and have the same function, are derived from and are equivalent to the nucleotide sequences of this invention. The identity refers to sequence similarity to natural nucleic acid sequences, including nucleotide sequences that have 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the protein whose coding sequence is shown in Sequence 2 or Sequence 5 of this invention. Identity can be evaluated visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
[0021] The expression cassette may include a promoter, the aforementioned nucleic acid molecule, and a terminator. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters, and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.
[0022] The vector refers to a vector capable of delivering the aforementioned nucleic acid molecules into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecules with a plant expression vector. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). Nos The untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing recombinant vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics).nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer resistance to glyphosate) or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.
[0023] The microorganisms may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The recombinant microorganisms refer to those whose genes have been manipulated and modified to obtain recombinant microorganisms with altered functions. For example, recombinant microorganisms obtained by introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.
[0024] In the above applications, regulating plant disease resistance can improve plant disease resistance.
[0025] The regulation mechanism is positive regulation. Specifically, when the content and / or activity of FLOE2 and FLOE3 proteins in plants increase, plant disease resistance increases; when the content and / or activity of FLOE2 and FLOE3 proteins in plants decrease or are absent, plant disease resistance decreases.
[0026] In the above applications, the indicator for plant breeding is disease resistance (such as resistance to Pseudomonas syringae).
[0027] The purpose of the plant breeding is to cultivate disease-resistant plant varieties (such as plant varieties resistant to Pseudomonas syringae).
[0028] To address the aforementioned technical problems, the present invention also provides novel uses for substances that inhibit the FLOE2 protein and substances that inhibit the FLOE3 protein.
[0029] This invention provides the use of substances that inhibit the above-mentioned FLOE2 protein and substances that inhibit the above-mentioned FLOE3 protein in any of the following B1)-B6): B1) Reduces plant disease resistance; B2) Prepare products that reduce plant disease resistance; B3) Cultivate plants with reduced disease resistance; B4) Prepare products from plants that have reduced disease resistance; B5) Plant breeding; B6) Preparation of plant breeding products; The substance that inhibits the above-mentioned FLOE2 protein is any one of the following P1)-P3): P1) Substances that reduce the activity and / or content of the aforementioned FLOE2 protein; P2) Substances that inhibit the expression of the FLOE2 protein-coding gene; P3) The substance that knocks out the FLOE2 protein-coding gene mentioned above; The substance that inhibits the above-mentioned FLOE3 protein is any one of the following Q1)-Q3): Q1) Substances that reduce the activity and / or content of the above-mentioned FLOE3 protein; Q2) Substances that inhibit the expression of the FLOE3 protein-coding gene; Q3) The substance that knocks out the FLOE3 protein-coding gene mentioned above.
[0030] The substances that reduce the activity and / or content of the FLOE2 protein include substances that inhibit the expression of the FLOE2 protein-coding gene and substances that knock out the FLOE2 protein-coding gene. The substance that inhibits the FLOE2 protein-coding gene can be any nucleic acid molecule capable of inhibiting or interfering with the expression of the FLOE2 protein-coding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc. The substance that knocks out the FLOE2 protein-coding gene can be any substance that prevents the host cell from producing... FLOE2 Substances that are functional protein products of genes, such as zinc finger protein (ZFN) gene editing systems, TALENs gene editing systems, CRISPR / Cas9 gene editing systems, T-DNA insertion, etc.
[0031] The substances that reduce the activity and / or content of the aforementioned FLOE3 protein include substances that inhibit the expression of the FLOE3 protein-coding gene and substances that knock out the FLOE3 protein-coding gene. The substance that inhibits the FLOE3 protein-coding gene can be any nucleic acid molecule capable of inhibiting or interfering with the expression of the FLOE3 protein-coding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc. The substance that knocks out the FLOE3 protein-coding gene can be any substance that prevents the host cell from producing... FLOE3 Substances that are functional protein products of genes, such as zinc finger protein (ZFN) gene editing systems, TALENs gene editing systems, CRISPR / Cas9 gene editing systems, T-DNA insertion, etc.
[0032] To address the aforementioned technical problems, this invention also provides a method for improving plant disease resistance or cultivating transgenic plants with enhanced disease resistance.
[0033] The method for improving plant disease resistance or cultivating transgenic plants with improved disease resistance provided by the present invention includes the following steps: increasing the activity and / or content of the above-mentioned FLOE2 protein and the above-mentioned FLOE3 protein in the target plant to obtain a transgenic plant; the transgenic plant has higher disease resistance than the target plant.
[0034] Furthermore, the method for increasing the activity and / or content of the FLOE2 and FLOE3 proteins in the target plant is to overexpress the FLOE2 and FLOE3 proteins in the target plant.
[0035] Furthermore, the overexpression method involves introducing the coding genes for the FLOE2 protein and the FLOE3 protein into the target plant.
[0036] To address the aforementioned technical problems, the present invention also provides a method for reducing plant disease resistance or cultivating transgenic plants with reduced disease resistance.
[0037] The method for reducing plant disease resistance or cultivating transgenic plants with reduced disease resistance provided by this invention is either Method 1 or Method 2 as follows: Method 1 includes the following steps: reducing the activity and / or content of the above-mentioned FLOE2 protein and the above-mentioned FLOE3 protein in the target plant to obtain a transgenic plant; the disease resistance of the transgenic plant is lower than that of the target plant.
[0038] The second method includes the following steps: extracting the genome of the target plant... FLOE2 Gene mutation, resulting in FLOE2 Mutated plants; plants with genetically modified genomes; FLOE3 Gene mutation, resulting in FLOE3 Mutant plants; the aforementioned FLOE2 Mutant plants and the above FLOE3 By hybridizing genetically mutated plants, the following results were obtained: FLOE2 Genes and FLOE3 Double mutant plants; the aforementioned FLOE2 Genes and FLOE3 The disease resistance of double-mutated plants is lower than that of the target plants.
[0039] In Method 1 above, the method for reducing the activity and / or content of the FLOE2 protein and the FLOE3 protein in the target plant is to introduce substances that inhibit the FLOE2 protein and substances that inhibit the FLOE3 protein into the target plant.
[0040] In method two above, the step of removing the target plant genome FLOE2 Gene mutation to change the genome of the target plant FLOE2 Gene mutation and loss of activity, even if not produced in the target plant FLOE2 Functional protein products of genes.
[0041] The target plant genome FLOE3 Gene mutation to change the genome of the target plant FLOE3 Gene mutation and loss of activity, even if not produced in the target plant FLOE3 Functional protein products of genes.
[0042] The gene mutations include base substitutions and / or base insertions and / or base deletions. The bases include single bases and / or multiple bases.
[0043] The gene mutation method can be any method known in the art, such as zinc finger protein ZFN gene editing system, TALENs gene editing system, CRISPR / Cas9 gene editing system, T-DNA insertion, etc.
[0044] The FLOE2 Mutant plants are FLOE2 Homozygous mutant plants, i.e., those with homologous chromosomes FLOE2 The same mutation occurs in the gene.
[0045] The FLOE3 Mutant plants are FLOE3 Homozygous mutant plants, i.e., those with homologous chromosomes FLOE3 The same mutation occurs in the gene.
[0046] The FLOE2 Genes and FLOE3 Double mutant plants are FLOE2 Genes and FLOE3 Homozygous double mutant plants, i.e., those with homologous chromosomes FLOE2 The same mutation occurs in the gene and FLOE3 The same mutation occurs in the gene.
[0047] In some implementations, the FLOE2 The homozygous mutant plant is shown in the examples below. floe2 Homozygous mutant.
[0048] In some implementations, the FLOE3 The homozygous mutant plant is shown in the examples below. floe3 Homozygous mutant.
[0049] In some implementations, the FLOE2 Genes andFLOE3 The homozygous double mutant plant is shown in the examples below. floe23 Homozygous double mutant.
[0050] It should be noted that although this invention yields susceptible material, it represents the first confirmation of the involvement of FLOE2 and FLOE3 proteins in the infection cycle of *Pseudomonas syringae*. This susceptible material can be utilized as a potential breeding resource. The discovery and enrichment of breeding resources will greatly accelerate the breeding process of disease-resistant plant varieties and strengthen disease control. Furthermore, those skilled in the art can also achieve resistance to *Pseudomonas syringae* by overexpressing FLOE2 and FLOE3 proteins in cruciferous plants through transgenic or other means. In addition, this susceptible material can also be used as germplasm material with loss of function of the FLOE2 and FLOE3 protein genes. The plant's resistance to *Pseudomonas syringae* is a cascade reaction involving many pathways. The susceptible material obtained in this invention can be used to study which genes involved in the *P. syringae* infection cycle still make plants more susceptible to *P. syringae* infection after the loss of function of the FLOE2 and FLOE3 protein genes. Therefore, the confirmation that FLOE2 and FLOE3 proteins are involved in the resistance to *P. syringae* infection cycle also provides new ideas and methods for revealing and applying the research on genes related to FLOE2 and FLOE3 proteins involved in resistance to *P. syringae*.
[0051] The disease resistance mentioned above can be resistance to pathogenic bacteria.
[0052] Furthermore, the pathogenic bacteria is *Pseudomonas syringae*.
[0053] Furthermore, the *Pseudomonas syringae* is a *Pseudomonas syringae* carrying the hopz1a effector protein.
[0054] Furthermore, the *Pseudomonas syringae* carrying the hopz1a effector protein is *Pseudomonas syringae* (…). Pseudomonas syringae ) Psthopz1a strains.
[0055] The amino acid sequence of any of the above-mentioned hopz1a effector proteins is shown in Sequence 7.
[0056] Any of the plants mentioned above are monocotyledonous or dicotyledonous plants.
[0057] Furthermore, the dicotyledonous plant is a plant of the Brassicaceae family.
[0058] Furthermore, the cruciferous plant in question is a plant of the Arabidopsis genus.
[0059] Furthermore, the Arabidopsis species mentioned are Arabidopsis thaliana.
[0060] In some implementations, the Arabidopsis thaliana is the Columbia ecotype col-0.
[0061] This invention demonstrates through experiments that, compared to wild-type Arabidopsis thaliana, FLOE2 Genes and FLOE3 All genes have mutated floe23 The double mutant material showed significantly reduced resistance to *Pseudomonas syringae* carrying the hopz1a effector protein when infected. This indicates that FLOE2 and FLOE3 proteins, or substances regulating their content, can be used to regulate plant resistance to *P. syringae* carrying the hopz1a effector protein. The plants with significantly reduced resistance to *P. syringae* carrying the hopz1a effector protein produced by this invention can not only serve as breeding resources to accelerate the cultivation of disease-resistant plants, but also be used to study which genes participate in the disease resistance response after the loss of function of the FLOE2 and FLOE3 protein genes. This invention provides new germplasm resources for genetic breeding work, new materials for the selection of disease-resistant plant varieties, and plays a positive role in accelerating the improvement of disease resistance in plant varieties. Attached Figure Description
[0062] Figure 1 For wild-type Arabidopsis thaliana and floe23 Statistical results of bacterial counts in double mutant materials after inoculation with Pseudomonas syringae. Detailed Implementation
[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0065] The KB liquid culture medium (1L) formulation in the following examples is as follows: 29g peptone, 8mL glycerol, 1.96g dipotassium hydrogen phosphate, 1.52g magnesium sulfate heptahydrate, and the remainder is water.
[0066] The KB solid medium (1L) in the following examples is obtained by mixing 15g of agar with KB liquid medium (1L).
[0067] The following examples of Pseudomonas syringae (Pseudomonas syringae ) Pst The strain (which does not carry the hopz1a effector protein) is described in the literature “Dong X*., Feng F., Li Y., Li L., Chen S., Zhou J*. 14-3-3 proteins facilitate the activation of MAP kinase cascades by upstream immunity-related kinases. Plant Cell 2023, 35(6):2413-2428”.
[0068] The following examples of Pseudomonas syringae ( Pseudomonas syringae ) Psthopz1a The strain (carrying the hopz1a effector protein) is described in the literature "Hu M., Qi J., Bi G*., Zhou J*. Bacterial effectors induce oligomerization of immune receptor ZAR1 in vivo. MolPlant 2020, 13, 793-801".
[0069] Example 1 floe23 Obtaining double mutant Arabidopsis thaliana and analyzing its resistance to Pseudomonas syringae. one, floe23 Obtaining double mutant Arabidopsis thaliana 1. floe2 Single mutant and floe3 Obtaining and Sequence Analysis of Single Mutants 1) floe2 Obtaining and Sequence Analysis of Single Mutants floe2 The single mutant seed was purchased from ABRC (https: / / abrc.osu.edu / ). floe2 The seed number for the single mutant is SALK_145362C.
[0070] Wild-type Arabidopsis thaliana (Col-0) and floe2 Genomic DNA of the single mutant was amplified by PCR using primer pair FLOE2-LP / FLOE2-LP and primer pair LBb1.3 / FLOE2-RP, respectively. The PCR products were then subjected to agarose gel electrophoresis for identification. floe2 Homozygous mutants. Mutant plants that amplified a 750bp band with primer pair LBb1.3 / FLOE2-RP and showed no amplification band with primer pair FLOE2-LP / FLOE2-RP are considered homozygous mutants. floe2Homozygous mutant. Primer sequences are as follows: FLOE2-LP: 5'-CTCACCACGCTTTAGATCTCG-3'; FLOE2-RP: 5'-CTGCGCCTCAACTATTTCTTG-3'; LBb1.3: 5'-ATTTTGCCGATTTCGGAAC-3'; FLOE2-RP: 5'-CTGCGCCTCAACTATTTCTTG-3'.
[0071] 2) floe3 Obtaining and Sequence Analysis of Single Mutants floe3 All single mutant seeds were purchased from ABRC (https: / / abrc.osu.edu / ). floe3 The seed number for the single mutant is SALK_105072C.
[0072] Wild-type Arabidopsis thaliana (Col-0) and floe3 Genomic DNA of the single mutant was amplified by PCR using primer pair FLOE3-LP / FLOE3-RP and primer pair LBb1.3 / FLOE3-RP, respectively. The PCR products were then subjected to agarose gel electrophoresis for identification. floe3 Homozygous mutants. Mutant plants that amplified a 750bp band with primer pair LBb1.3 / FLOE3-RP but showed no amplification band with primer pair FLOE3-LP / FLOE3-RP are considered homozygous mutants. floe3 Homozygous mutant. Primer sequences are as follows: FLOE3-LP: 5'-TTGAGATCATCCACCAGGTTC-3'; FLOE3-RP: 5'-TCATTTCTTTACAAAGGGGCC-3'; LBb1.3: 5'-ATTTTGCCGATTTCGGAAC-3'; FLOE3-RP: 5'-TCATTTCTTTACAAAGGGGCC-3'; 2. floe23 Obtaining and Sequence Analysis of Double Mutant Materials 1) floe2 homozygous mutants and floe3 Hybridization of homozygous mutants is performed using the following steps: Select bolting mutants... floe2 The homozygous mutant Arabidopsis flower buds were emasculated, and floe3 The pollen of the homozygous mutant was stained with pollen from the emasculated pollen. floe2F1 generation seeds were obtained from the pistils of homozygous mutants; 2) Plant the obtained F1 generation seeds in a plant culture room, harvest individual plants, and obtain F2 generation seeds; 3) The obtained F2 generation seeds were planted in a plant culture room to obtain F2 generation plants. Genomic DNA was extracted from individual F2 generation plants, and PCR amplification was performed using primer pair LBb1.3 / FLOE2-RP and primer pair LBb1.3 / FLOE3-RP, respectively. The PCR products were then subjected to agarose gel electrophoresis for identification. floe23 Homozygous double mutant. Plants that show no amplification bands with both primer pair LBb1.3 / FLOE2-RP and primer pair LBb1.3 / FLOE3-RP are considered homozygous double mutants. floe23 Homozygous double mutant Arabidopsis thaliana. Primer sequences are as follows: LBb1.3: 5'-ATTTTGCCGATTTCGGAAC-3'; FLOE2-RP: 5'-CTGCGCCTCAACTATTTCTTG-3'; FLOE3-RP: 5'-TCATTTCTTTACAAAGGGGCC-3'.
[0073] two, floe23 Resistance analysis of homozygous double mutant Arabidopsis thaliana to Pseudomonas syringae Wild-type Arabidopsis thaliana (Col-0) and wild-type Arabidopsis thaliana (Col-0) were planted under normal conditions (photoperiod of 12 hours of light / 12 hours of darkness, temperature of 22℃). floe23 Homozygous double mutant Arabidopsis thaliana, taken from wild-type Arabidopsis thaliana (Col-0) at approximately 5 weeks of age and floe23 A homozygous double mutant of Arabidopsis thaliana was used for inoculation experiments with *Pseudomonas syringae*. The specific inoculation steps are as follows: 1. Select appropriate amounts of frozen *Pseudomonas syringae* carrying the hopz1a effector protein. Pseudomonas syringae ) Psthopz1a strain (denoted as) Pst hopz1a ) and *Pseudomonas syringae* that does not carry the hopz1a effector protein ( Pseudomonas syringae ) Pst strain (denoted as) Pst EVs were inoculated onto KB culture plates containing kanamycin and rifampicin antibiotics and placed in an incubator at 28°C to activate the strain.
[0074] 2. Pick an appropriate amount of bacterial cells from the KB plate and inoculate them into 2 mL of KB liquid medium containing kanamycin and rifampicin antibiotics. Incubate overnight at 28°C for 12 h.
[0075] 3. Centrifuge at 4000 rpm for 5 min at room temperature, discard the supernatant, collect the bacterial cells, wash three times with sterile ddH2O, centrifuging at 4000 rpm for 5 min each time, resuspend the bacterial cells with sterile ddH2O, and adjust the OD. 600nm The value was 0.001, and the bacterial solution to be injected was obtained.
[0076] 4. Take wild-type Arabidopsis thaliana (Col-0) at approximately 5 weeks of age and floe23 For homozygous double mutant Arabidopsis plants, select well-maintained, flat leaves and mark them. Inject the bacterial solution prepared in step 3) into the underside of the leaves using a 1 mL syringe (without the needle). Select 8 plants of each material, and select 2 leaves from each plant.
[0077] 5. Three days after inoculation, two leaves from each plant were stacked together, and leaf samples of the same area were taken using a punch and placed in a 1.5 mL centrifuge tube. 100 μL of sterile ddH2O was added.
[0078] 6. Grind the leaves thoroughly with an electric drill, then add 900 μL of sterile ddH2O. After dilution to an appropriate ratio, take 20 μL of the bacterial solution and spread it on a TSA medium plate containing the corresponding antibiotic. Incubate at 28℃ for 2 days and count the number of colonies.
[0079] The results are as follows Figure 1 As shown, the results indicate that inoculation with *Pseudomonas syringae*... Pst The mean bacterial count of wild-type Arabidopsis thaliana Col-0 EV was 5.31, after inoculation with Pseudomonas syringae. Pst EV floe23 The mean number of homozygous double mutant bacteria was 5.23, after inoculation with *Pseudomonas syringae*. EV wild-type Arabidopsis thaliana Col-0 and There was no significant difference in bacterial numbers between the homozygous double mutants. However, inoculation with *Pseudomonas syringae*... The average bacterial count in wild-type Arabidopsis thaliana Col-0 was 3.27, after inoculation with *Pseudomonas syringae*. of The mean number of homozygous double mutant bacteria was 3.81, after inoculation with *Pseudomonas syringae*. of The number of bacteria in the homozygous double mutant was significantly higher than that inoculated with *Pseudomonas syringae*. Wild-type Arabidopsis thaliana Col-0. (Note: This likely refers to a specific variety of Arabidopsis thaliana.) Homozygous double mutants against Pseudomonas syringae carrying the hopz1a effector protein The resistance to phytotoxicity is weakened, and the FLOE2 and FLOE3 proteins can regulate the plant's resistance to Pseudomonas syringae carrying the hopz1a effector protein. Resistance.
[0080] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. Applications of FLOE2 and FLOE3 proteins in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Prepare products that regulate plant disease resistance; A3) Cultivate plants with altered disease resistance; A4) Prepare products from plants that have been modified to have disease resistance; A5) Plant breeding; A6) Preparation of plant breeding products; The FLOE2 protein is any one of the following M1)-M4): The amino acid sequence of M1 is the protein shown in sequence 2; M2) is a fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; M3) is a protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of M1. M4) is a protein that shares more than 75% identity with M1 and is associated with plant disease resistance; The FLOE3 protein is any one of the following (N1)-N4): The amino acid sequence of N1 is the protein shown in sequence 5; N2) A fusion protein associated with plant disease resistance is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 5; N3) is a protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of N1. N4) is a protein that shares more than 75% identity with N1 and is associated with plant disease resistance.
2. Use of biomaterials related to the FLOE2 protein of claim 1 and biomaterials related to the FLOE3 protein of claim 1 in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Prepare products that regulate plant disease resistance; A3) Cultivate plants with altered disease resistance; A4) Prepare products from plants that have been modified to have disease resistance; A5) Plant breeding; A6) Prepare products for plant breeding.
3. The application according to claim 2, characterized in that: The biological material related to the FLOE2 protein of claim 1 is a nucleic acid molecule encoding the FLOE2 protein of claim 1 or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule; Alternatively, the biological material associated with the FLOE3 protein of claim 1 may be a nucleic acid molecule encoding the FLOE3 protein of claim 1 or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.
4. The use of the substance that inhibits the FLOE2 protein of claim 1 and the substance that inhibits the FLOE3 protein of claim 1 in any one of the following B1)-B6): B1) Reduces plant disease resistance; B2) Prepare products that reduce plant disease resistance; B3) Cultivate plants with reduced disease resistance; B4) Prepare products from plants that have reduced disease resistance; B5) Plant breeding; B6) Preparation of plant breeding products; The substance that inhibits the FLOE2 protein of claim 1 is any one of the following P1)-P3): P1) Substances that reduce the activity and / or content of the FLOE2 protein as described in claim 1; P2) A substance that inhibits the expression of the FLOE2 protein-coding gene as described in claim 1; P3) The substance that knocks out the FLOE2 protein-coding gene as described in claim 1; The substance that inhibits the FLOE3 protein of claim 1 is any one of the following Q1)-Q3): Q1) Substances that reduce the activity and / or content of the FLOE3 protein as described in claim 1; Q2) A substance that inhibits the expression of the FLOE3 protein-coding gene as described in claim 1; Q3) The substance that knocks out the FLOE3 protein-coding gene as described in claim 1.
5. The application according to any one of claims 1-4, characterized in that: The disease resistance refers to resistance to pathogenic bacteria; Alternatively, the pathogenic bacteria may be *Pseudomonas syringae*. Alternatively, the *Pseudomonas syringae* may be a *Pseudomonas syringae* carrying the hopz1a effector protein.
6. The application according to claims 1-5, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.
7. A method for improving plant disease resistance or cultivating plants with improved disease resistance, comprising the following steps: increasing the activity and / or content of the FLOE2 protein and the FLOE3 protein of claim 1 in the target plant to obtain a transgenic plant; wherein the transgenic plant has higher disease resistance than the target plant.
8. A method for reducing plant disease resistance or cultivating plants with reduced disease resistance, said method being either method one or method two: Method 1 includes the following steps: reducing the activity and / or content of the FLOE2 protein and the FLOE3 protein of claim 1 in the target plant to obtain a transgenic plant; the disease resistance of the transgenic plant is lower than that of the target plant; The second method includes the following steps: extracting the genome of the target plant... FLOE2 Gene mutation, resulting in FLOE2 Mutated plants; plants with genetically modified genomes; FLOE3 Gene mutation, resulting in FLOE3 Mutant plants; the aforementioned FLOE2 Mutant plants and the above FLOE3 By hybridizing genetically mutated plants, the following results can be obtained: FLOE2 Genes and FLOE3 Double mutant plants; the aforementioned FLOE2 Genes and FLOE3 The disease resistance of double-mutated plants is lower than that of the target plants.
9. The method according to claim 7 or 8, characterized in that: The disease resistance refers to resistance to pathogenic bacteria; Alternatively, the pathogenic bacteria may be *Pseudomonas syringae*. Alternatively, the *Pseudomonas syringae* may be a *Pseudomonas syringae* carrying the hopz1a effector protein.
10. The method according to any one of claims 7-9, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.