Protein capable of reducing disease resistance of plant powdery mildew and application of protein

Abstract

The invention discloses a protein capable of reducing disease resistance of plant powdery mildew and application of the protein, and belongs to the field of biological agriculture. The invention aims to solve the technical problem of how to regulate and control the disease resistance of plants. The protein disclosed by the invention is CSEP03399, and the CSEP03399 is the protein from the 31st site to the 192nd site of which the amino acid sequence is SEQ ID No.3. Experiments prove that after inoculation of the hevea brasiliensis powdery mildew, compared with an empty vector transgenic plant, the hevea brasiliensis powdery mildew can form a dense hypha network and a small amount of conidia on the CSEP03399 transgenic plant, and stimulated cell death, active oxygen generation and PR1 gene expression are also obviously weakened, which indicates that the CSEP03399 plays a toxic function in arabidopsis thaliana, so that the hevea brasiliensis powdery mildew can be used as a transgenic plant of the arabidopsis thaliana. Infection of powdery mildew is promoted. The CSEP03399 disclosed by the invention can be used for preparing plants sensitive to powdery mildew so as to screen anti-powdery mildew drugs.

Description

Technical Field

[0001] The invention belongs to the field of biological agriculture, and in particular relates to a protein for reducing the disease resistance of plant powdery mildew and an application thereof. Background Art

[0002] In order to resist the infection of pathogens, plants have evolved a multi-layered and interrelated innate immune system. First, the pattern recognition receptors (PRRs) located on the plant cell membrane recognize pathogen-associated molecular patterns (PAMPs) and activate PTI (pattern-triggered immunity), which resists the infection of most pathogens and prevents the occurrence of diseases. [1-2] The generation of PTI triggers a series of basic defense responses, including the burst of cellular reactive oxygen species, callose deposition on the cell wall of the infected site, activation of mitogen-activated protein kinases, and expression of genes related to the pathogenesis, such as PR1 (Pathogenesis related 1), FRK1 (FLG22-induced receptor-like kinase 1) and WRKY Transcription factors, etc. [3-4] In order to suppress the plant PTI immune response, some pathogens secrete a large number of effector proteins into host cells to interfere with the immune process and make the plant susceptible to disease. Accordingly, plants have evolved a second level of immune system, using their own resistance genes to recognize pathogen effector proteins, thereby stimulating ETI (effector-triggered immunity), which is usually accompanied by a large number of cell death, limiting the infection of pathogens to a certain part. [5-6] .

[0003] Powdery mildew is one of the common diseases that affect plant growth and causes harm to many crops, such as wheat, grapes, soybeans, melons, barley, etc. [7] The pathogen that causes powdery mildew is Powdery mildew, which belongs to the Ascomycetes, Powdery mildew order. Powdery mildew is an obligate parasitic fungus that requires living host cells to survive and cannot be cultured in vitro. All steps of its life cycle, including spore production, spore accumulation and germination, occur on living tissues. [8-9]. During the interaction with the host plant, the invasive spikes are produced to penetrate the leaf epidermal cells and form haustoria to absorb water and nutrients from the host plant. At the same time, the powdery mildew secretes a large number of effector proteins into the plant cells through the haustoria, inhibiting the plant's innate immune response and thus achieving successful infection of the host plant. The genome, transcriptome and proteome sequencing analysis of some similar grass powdery mildew identified approximately 350-800 candidate secreted effector proteins (CSEPs) [10-15] At present, only a few effector proteins have been explored. Using host-induced gene silencing (HIGS) and transgenic overexpression methods, it was found that nearly 20 barley powdery mildew effector proteins can play a toxic function and promote the infection of powdery mildew.

[16] , 14 effector proteins can be recognized by host resistance genes to stimulate disease resistance

[17] . Rubber tree ( Heveabrasiliensis ) Powdery mildew is one of the important diseases that affect the yield of natural rubber. Oidiumheveae ) mainly infects immature tissues such as new leaves and flowers of rubber trees. When the disease is severe, the entire leaf surface of the rubber tree is covered with hyphae, resulting in large-scale leaf fall and canopy dryness. Through genome and transcriptome sequencing analysis of rubber tree powdery mildew species HO-73, 133 candidate effector proteins were identified

[18] Among them, the effector protein CSEP01276 can interfere with the cellular localization of epoxycarotenoid dioxygenase in plants, inhibit the synthesis of abscisic acid, and promote the infection of powdery mildew.

[19] The effector protein CSEP04187 is recognized by plants and triggers disease resistance in Arabidopsis, tobacco, and rubber tree protoplasts

[20] However, the mechanism of action of most of the rubber tree powdery mildew effector proteins remains unknown.

[0004] Rubber trees are native to Brazil and have limited genetic resources. So far, no genetic subspecies has been found that is resistant to rubber tree powdery mildew. In addition, rubber trees are large trees of the Euphorbiaceae family, and are difficult to genetically manipulate. The operation cycle is long and difficult, which seriously hinders the study of the interaction mechanism between the two. Recent studies have found that rubber tree powdery mildew stimulates disease resistance in Arabidopsis thaliana.

[21] , while Arabidopsis TIR-NB-LRR (Toll-Interleukin1 Receptor-nucleotidebinding-leucine-rich repeat) resistance gene WRR4B(White rust resistance 4B) Partial resistance to powdery mildew in rubber trees

[22] , which provides clues and tools for in-depth research on the pathogenic mechanism of rubber tree powdery mildew. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to regulate the disease resistance of plants.

[0006] In order to solve the above technical problems, the present invention first provides any of the following applications of a protein or a substance for regulating the content or activity of the protein: D1) Regulate plant disease resistance; D2) Preparation of products for regulating plant disease resistance; D3) Breeding plants with altered disease resistance; D4) Preparation of products for breeding plants with altered disease resistance; The protein is derived from rubber tree powdery mildew Oidium heveae , its name is CSEP03399, CSEP03399 is as follows: A1), A2) or A3): A1) a protein whose amino acid sequence is positions 31 to 192 of SEQ ID No. 3; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 3 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added; A3) A protein obtained by attaching a tag to the N-terminus or / and C-terminus of A1) or A2).

[0007] The CSEP03399 protein in A2) above is a protein having 75% or more identity with the amino acid sequence of the protein shown in positions 31-192 of SEQ ID No.3 and having the same function. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated, and then the identity value (%) can be obtained. The 75% or greater identity refers to 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0008] The CSEP03399 protein in A2) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0009] The gene encoding the CSEP03399 protein in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown at positions 91-576 of SEQ ID No.2, and / or performing missense mutations of one or several base pairs, and / or attaching a coding sequence of a tag at its 5′ end and / or 3′ end. The DNA molecule shown at positions 91-576 of SEQ ID No.2 encodes the protein shown at positions 31-192 of SEQ ID No.3.

[0010] The tag in A3) can be a polypeptide or protein fused with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a signal peptide, Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag and / or SUMO tag, etc.

[0011] The protein described in A3) above may be a protein having an amino acid sequence of SEQ ID No. 3.

[0012] In the above application, the substance may be any one of the following B1) to B4): B1) Nucleic acid molecule encoding CSEP03399; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0013] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0014] Those skilled in the art can easily mutate the nucleotide sequence encoding the CSEP03399 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the CSEP03399 protein isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the CSEP03399 protein and have the function of the CSEP03399 protein.

[0015] In the above application, the nucleic acid molecule in B1) may be as follows: b11) or b12) or b13): b11) The coding sequence is the DNA molecule at positions 91 to 576 of SEQ ID No. 2 in the sequence list; b12) The coding sequence is the DNA molecule of SEQ ID No. 2 in the sequence list; b13) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) or b12) and encodes CSEP03399.

[0016] b11) The DNA molecule is the DNA molecule shown at positions 91 to 576 of SEQ ID No. 2. b12) The DNA molecule may be the DNA molecule shown in SEQ ID No. 1 or SEQ ID No. 2.

[0017] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein consisting of the amino acid sequence shown in positions 31-192 of the coding SEQ ID No.3 of the present invention. Identity can be evaluated by the naked eye 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.

[0018] The above 75% or more identity may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0019] In the above application, the expression cassette containing the nucleic acid molecule encoding the CSEP03399 protein (CSEP03399 gene expression cassette) described in B2) refers to a DNA capable of expressing the CSEP03399 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the CSEP03399 gene, but also a terminator for terminating transcription of the CSEP03399 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or a LAP promoter (both of which can be induced by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); a seed-specific promoter, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent No. 200710099169.7)), a promoter specific for seed storage proteins (e.g., phaseolin, napin, oleosin and soybean beta conglycin promoters (Beachy et al. (1985) EMBO J. 4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated in their entirety.Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0020] Existing expression vectors can be used to construct CSEP03399 Recombinant vector of gene expression cassette. The plant expression vector includes binary Agrobacterium vector and vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., contain polyadenylic acid signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as Agrobacterium crown gall induction (Ti) plasmid gene (such as nopaline synthase gene Nos), the non-translated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes ( GUS genes, luciferase genes, etc.), antibiotic marker genes (such as those that confer resistance to kanamycin and related antibiotics nptII Gene that confers resistance to the herbicide phosphinothricin bar Gene that confers resistance to the antibiotic hygromycin hph genes, and those conferring resistance to methotrexate dhfr Genes, such as EPSPS genes that confer resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0021] In the above application, the vector may be a plasmid, a cosmid, a phage or a virus vector. Specifically, the plasmid may be a pER8 vector.

[0022] B3) The recombinant vector may specifically be pER8-CSEP03399. The pER8-CSEP03399 is a pER8 vector XOt and B i The recombinant vector is obtained by replacing the DNA fragment between the recognition sequences with the DNA fragment shown at positions 91-579 of SEQ ID No.2.

[0023] In the above application, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Agrobacterium, such as Agrobacterium GV3101.

[0024] In the above application, the disease resistance may be resistance to powdery mildew.

[0025] In one embodiment of the present invention, the powdery mildew is rubber tree powdery mildew.

[0026] In the above application, the substance for regulating the content or activity of CSEP03399 may be a substance for increasing the content or activity of CSEP03399, the regulating plant disease resistance may be a substance for reducing the disease resistance of the plant, and the cultivating plants with altered disease resistance may be a substance for cultivating plants with reduced disease resistance.

[0027] The present invention also provides any of the following methods: X1) A method for reducing plant disease resistance, comprising: increasing the content or activity of CSEP03399 protein in a recipient plant to obtain a target plant with reduced disease resistance compared with the recipient plant, thereby reducing plant disease resistance; X2) A method for breeding plants with reduced disease resistance, comprising: increasing the content or activity of CSEP03399 protein in a recipient plant to obtain a target plant with reduced disease resistance compared with the recipient plant.

[0028] Specifically, the methods X1) and X2) can be achieved by introducing the gene encoding CSEP03399 into the recipient plant.

[0029] In the above method, the encoding gene may be the nucleic acid molecule described in B1).

[0030] The coding gene of CSEP03399 can be introduced into a recipient plant using a recombinant expression vector containing the coding gene of CSEP03399. The recombinant expression vector can specifically be the pER8-CSEP03399.

[0031] The recombinant expression vector can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).).

[0032] The target plant is understood to be not only the first generation plant containing the CSEP03399 gene, but also its progeny. For the target plant, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus, complete plants and cells.

[0033] In the above, the plant may be any one of M1)-M6): M1) dicots or monocots; M2) Magnolia; M3) Solanales or Cruciferales; M4) Solanaceae or Cruciferae; M5) Nicotiana or Arabidopsis; M6) tobacco or Arabidopsis thaliana.

[0034] CSEP03399 or the substance that regulates the content or activity of CSEP03399 also falls within the scope of protection of the present invention.

[0035] The present invention clones the candidate effector protein gene of rubber tree powdery mildew CSEP03399 , constructed transgenic plants in the wild-type Col-0 background of Arabidopsis thaliana, and found that overexpression of CSEP03399 did not induce visible cell necrosis in Arabidopsis thaliana and Nicotiana benthamiana. CSEP03399 The transgenic plants formed dense mycelial networks and a small number of conidia, and stimulated cell death, reactive oxygen production and PR1 The gene expression was also significantly weakened, indicating that the effector protein CSEP03399 played a toxic function in Arabidopsis and promoted the infection of powdery mildew. hC - CSEP03399 did not promote the bacterial growth of Pseudomonas sp., nor did it inhibit the expression of DC3000 avrB and DC3000 avrRpt2 as well as Arabidopsis thaliana. TIR- NB-LRR (Toll-Interleukin1 Receptor-nucleotide binding-leucine-rich repeat) disease resistance gene WRR4B (White rust resistance 4B) induced hypersensitivity reaction. CSEP03399 After transgenic plants, Q-PCR revealed that CSEP03399 could inhibit chitin stimulation FRK1 (FLG22-induced receptor-like kinase 1) and WRKY22 Gene expression indicates that CSEP03399 may exert toxicity by inhibiting the PTI (PAMP triggered immunity) signaling pathway. The CSEP03399 of the present invention can be used to prepare powdery mildew-sensitive plants to screen anti-powdery mildew drugs.

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A is the detection of transgenic plants. A is the result of protein immunoblotting, and B is the plant phenotype. EmptyVector is the empty vector transgenic control plant.

[0038] Figure 2 .Candidate effector protein CSEP03399 enhances disease susceptibility in Arabidopsis thaliana.

[0039] (A) Symptoms on leaves 10 days post inoculation (dpi). (B) Fungal structures stained with Coomassie blue and imaged using light microscopy at 10 dpi. Bars, 50 μm. (C) Leaves stained with Coomassie blue and imaged using light microscopy at 48 hours post inoculation (hpi). Bars, 50 μm. (D) Leaves stained with Coomassie blue and imaged using light microscopy at 48 hours post inoculation (hpi). Oidium heveae Quantitative analysis of HN1106 growth. Data represent the mean ± standard deviation (SD) of three experiments, each based on at least 20 fungal colonies per genotype. The hyphal length of each colony was measured from the photographs using MIE3.1 software. * indicates significant difference from the empty vector transgenic control plants (Student's t-test, P < 0.05). (E) The number of conidia per colony was calculated. Data represent the mean ± standard deviation of at least 20 fungal colonies per genotype. ** indicates significant difference from the empty vector transgenic control plants (Student's t-test, P < 0.01). The experiment was repeated twice with similar results. Wild-type Col-0 and Empty Vector transgenic plants were used as control plants.

[0040] Figure 3 .CSEP03399 inhibition O. heveae Induced ROS production, cell death and PR1 Gene expression.

[0041] (A) Leaves were stained with DAB and imaged using light microscopy. Each is based on 8 leaves per genotype. Bars, 100 μm. (B) Leaves were stained with trypan blue and imaged using light microscopy. Each is based on 8 leaves per genotype. Bars, 100 μm. (C) Quantitative PCR assay O. heveae Specified time point after inoculation PR1Abundance of mRNA. Data represent mean ± standard deviation of 3 replicates. ** indicates significant difference from empty vector transgenic control plants (Student's t-test, P<0.01). The experiment was repeated twice with similar results. Empty Vector is the empty vector transgenic control plant.

[0042] Figure 4 .CSEP03399 did not enhance the virulence of P. syringae in Arabidopsis thaliana.

[0043] After 24 hours of estrogen treatment, Pseudomonas syringae DC3000, DC3000 hC - , DC3000 avrB and DC3000 avrRpt2 were inoculated into 5-week-old plants. (A) Pseudomonas syringae Growth results of DC3000. (B) Pseudomonas syringae DC3000 hC - (C) HR symptoms triggered by DC3000 avrB and DC3000 avrRpt2 24 hours after inoculation. Empty Vector is the empty vector transgenic control plant.

[0044] Figure 5 .CSEP03399 inhibits chitin-induced FRK1 and WRKY 22 Gene expression.

[0045] (A) CSEP03399 did not inhibit WRR4B-triggered hypersensitivity in N. benthamiana. (B) Protein abundance was measured by immunoblotting using anti-FLAG antibody 24 h after estradiol treatment. The experiment was repeated twice with similar results. FRK1 Gene (C) and WRKY22 Expression level of gene (D). Data represent the mean ± standard deviation of 3 replicates. ** indicates significant difference from empty vector (Student's t-test, P<0.01). The experiment was repeated twice with similar results. Empty Vector is an empty vector. DETAILED DESCRIPTION

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are all repeated at least three times, and the results are averaged.

[0047] Rubber tree powdery mildew in the following embodiments Oidium heveae HN1106 (MEI S, HOU S, CUI H, et al. Characterization of the interaction between Oidium heveae and Arabidopsisthaliana[J]. Molecular plant pathology, 2016, 17(9):1331-1343.), the public can obtain this biological material from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0048] The Pseudomonas DC3000 in the following example (ZHANG J, LI W, XIANG T, et al. Receptor-like Cytoplasmic Kinases Integrate Signaling from Multiple Plant Immune Receptors and Are Targeted by a Pseudomonas syringae Effector [J]. Cell Host Microbe, 2010, 22; 7(4): 290-301.) is a biological material that the public can obtain from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0049] Pseudomonas DC3000 in the following examples hC - (ZHANG J, LI W, XIANG T, et al. Receptor-like Cytoplasmic Kinases Integrate Signaling from Multiple Plant Immune Receptors and Are Targeted by a Pseudomonas syringae Effector [J]. Cell Host Microbe, 2010, 22; 7 (4): 290-301.), the public can obtain the biological material from the applicant, and the biological material can only be used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0050] The Pseudomonas DC3000 avrB in the following example (SHANG Y, LI X, CUI H, et al. RAR1, a central player in plant immunity, is targeted by Pseudomonas syringaeeffectorAvrB [J]. PNAS (track II), 2006, 103: 19200-19205.) is available to the public from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0051] The Pseudomonas DC3000 avrRpt2 in the following examples (CHEN Z, KLOEK A, CUZICK A, et al. The Pseudomonas syringaetype III effector AvrRpt2 functions downstream or independently of SA to promote virulence on Arabidopsis thaliana [J]. Plant journal. 2004, 40(5): 790-8.) is a biological material that the public can obtain from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0052] Example 1: CSEP03399 can exert toxicity in Arabidopsis thaliana and promote the infection of powdery mildew 1.1 Test materials and culture conditions Rubber tree powdery mildew Oidium heveae HN1106, stored in the leaves of rubber tree 73397. Culture conditions: temperature 28℃, relative humidity 85%, light 16 h, dark 8 h. Wild-type Arabidopsis thaliana Col-0, stored in the laboratory. Culture conditions: temperature 23℃, relative humidity 85%, light-dark cycle of 10 h, dark 14 h.

[0053] 1.2 Candidate effector proteins CSEP03399 Gene cloning Rubber tree leaves were inoculated with powdery mildew HN1106 at the bronze stage, and leaves were cut 8 days later to extract total RNA and obtain cDNA by reverse transcription. O. heveae HN1106 genomic DNA and reverse transcribed cDNA were used as templates for amplification using CSEP03399 F and CSEP03399 R. The primers were connected to a blunt-end vector for sequencing. The primer sequences are as follows: CSEP03399 F: 5′-atgctaacctgtagatctca-3′; CSEP03399 R: 5′-ttatcgggtcatctcgacct-3′.

[0054] The sequencing results showed that CSEP03399 The genomic DNA contains one intron, which is 668 bp in total and its sequence is SEQ ID No.1. CSEP03399 The gene coding sequence is 579 bp, and its sequence is SEQ ID No. 2, encoding the CSEP03399 protein shown in SEQ ID No. 3. Further, the signal peptide analysis software (SignalIP-5.0) predicted that the 1-30 amino acids of the CSEP03399 protein are its secretion signal peptide sequence, suggesting that CSEP03399 may be secreted into the plant body as an effector protein to function.

[0055] 1.3 CSEP03399 Construction of transgenic plants Remove the signal peptide, design primers (CSEP03399 Clone F, CSEP03399 Clone R), use the cDNA obtained in step 1.2 as a template for PCR amplification, and use XOt and B i The PCR product was digested by NEB and the digested product was combined with pER8 vector (estrogen-induced transgenic vector) XOt and B i The vector backbone obtained by enzyme digestion was connected, and the obtained recombinant vector with correct sequence was recorded as pER8-CSEP03399. The recombinant vector contained the DNA fragment shown at positions 91-576 of SEQ ID No.2, and could express the fusion protein of the protein shown at positions 31-192 of SEQ ID No.3 and FLAG tag.

[0056] pER8-CSEP03399 was introduced into Agrobacterium GV3101 to obtain the recombinant bacteria GV3101 / pER8-CSEP03399; then the wild-type Col-0 inflorescence of Arabidopsis thaliana was infected by the floral dip method during the flowering period of Arabidopsis thaliana. After the seeds matured, the Arabidopsis thaliana seeds were harvested. The seeds were dried and sterilized, and spread on MS medium containing hygromycin and carbenicillin. After about two weeks, the Arabidopsis thaliana plants with roots were selected and transplanted to nutrient soil for further growth. The primer sequences are as follows: CSEP03399 Clone F: 5′-AAA CTCGAG atgaattcaacattttatacctc-3′; CSEP03399 Clone R: 5′-AAA TTCGAA tcgggtcatctcgacct-3′.

[0057] According to the above method, the empty vector transgenic control plants were obtained using the pER8 vector.

[0058] 1.4 Protein immunoblotting of transgenic plants After 5 weeks of growth, the leaves of Arabidopsis were sprayed with estrogen (i.e. estradiol). After 36 hours of induction, one leaf was cut, protein was extracted, and SDA-PAGE electrophoresis was performed. Then, FLAG antibody (Biyuntian Company, product number: AF519) was used as the primary antibody, and HRP-labeled goat anti-mouse was used as the secondary antibody (Biyuntian Company, product number: A0216) for western blot, and ECL color development and photography were performed using a chemiluminescence instrument. Rubisco was used as an internal reference.

[0059] After estrogen induction, three independent CSEP03399-expressing transgenic lines in the Col-0 background were identified by immunoblotting, namely CSEP03399-1, CSEP03399-4, and CSEP03399-7 (Figure 1A). After estrogen induction, the three transgenic lines did not show obvious cell necrosis symptoms compared with the empty vector transgenic control plants ( Figure 1 Middle B), indicating that the candidate effector protein CSEP03399 does not induce disease resistance in Arabidopsis plants.

[0060] 1.5 Mycelial growth observation To explore the mechanism of action of the candidate effector protein CSEP03399 in plants, the rubber tree powdery mildew HN1106 was inoculated on wild-type Col-0, empty vector transgenic control plants and CSEP03399-expressing transgenic lines. After 5 weeks of growth, the leaves of Arabidopsis were sprayed with estrogen. After 24 hours of estrogen treatment, fresh rubber tree powdery mildew was inoculated into Arabidopsis leaves using a nylon membrane inoculation box with a pore size of 50 μM and a brush. After 2 days of inoculation, Arabidopsis leaves were cut and placed in a decolorizing solution (ethanol: phenol: water: lactic acid = 2:1:1:1) for decolorization. When the leaves were decolorized to colorless and transparent, the residual decolorizing solution was rinsed with clean water. The leaves were placed in Coomassie Brilliant Blue staining solution (G250, concentration 6 g·L −1 The spores were stained in ethanol solution for 30 s, and then the residual staining solution was washed off with water. The growth distribution of hyphae was observed using an optical microscope, and the hyphae length was measured using MIE3.1 software. The hyphae length of at least 20 spores of each plant was counted and measured in three independent experiments.

[0061] The results show that O. heveaeTwo days after HN1106 inoculation, two hyphae were formed on the wild-type Col-0 and empty vector transgenic control plants ( Figure 2 C), length 100 μM ( Figure 2 D) around, and in 3 CSEP03399 Four hyphae can be formed on the transgenic plants ( Figure 2 C), length 400 μM ( Figure 2 (D), indicating that CSEP03399 inhibited the growth of Arabidopsis thaliana O. heveae Resistance to early mycelial growth.

[0062] 1.6 Conidia count observation Ten days after the rubber tree powdery mildew inoculation in step 1.5, cut the Arabidopsis leaves, put them in the prepared decolorizing solution for decolorization, stain them in Coomassie brilliant blue staining solution, and observe them under a microscope. The number of conidia formed was counted, and the number of conidia produced by at least 20 spores of each plant was counted and determined in three independent experiments.

[0063] The results showed that 10 days after inoculation, CSEP03399 The transgenic plants showed mild typical powdery mildew symptoms ( Figure 2 In the middle (A), wild-type Col-0 and empty vector transgenic control plants did not show powdery mildew symptoms, but showed yellowing leaves ( Figure 2 Middle A). Coomassie Brilliant Blue staining and microscopic observation showed that wild-type Col-0 and empty vector transgenic control plants could not produce conidia, and only collapsed hyphae could be observed ( Figure 2 B, E), while CSEP03399 Transgenic plants can form hyphal networks and produce a small amount of conidia ( Figure 2 (B, E).

[0064] Explanation: CSEP03399 can enhance O. heveae Susceptibility in Arabidopsis thaliana.

[0065] 1.7 Observation of leaf cell death Cell death was determined by trypan blue staining. 8 days after the rubber tree powdery mildew inoculation in step 1.5, the infected leaves were cut and completely immersed in trypan blue staining solution, and then heated and boiled at 100 °C for 5 min. After natural cooling, the staining solution was discarded, the leaves were rinsed with water, decolorized with chloral hydrate until the blue color faded, and immersed in 50% glycerol. The cell death status of the leaves was observed under a microscope. At least 8 inoculated leaves of each plant were observed and measured in three independent experiments.

[0066] 1.8 Observation of reactive oxygen species generation and PR1 Gene expression analysis 8 days after the rubber tree powdery mildew inoculation in step 1.5, Arabidopsis leaves were cut for DAB staining and fluorescence quantitative PCR analysis. The leaves were placed in the prepared diaminobenzidine dye solution (1 mg mL -1 ) in a flask until the staining solution covers the leaves, and stain for 12 h in the dark. Discard the staining solution and add 95% ethanol aqueous solution to decolorize. After decolorization until the chlorophyll fades, use a microscope to observe the production of reactive oxygen. At least 8 inoculated leaves of each plant were observed and measured in three independent experiments. The primers used for fluorescent quantitative PCR are as follows: ACTIN : 5′-TGGTGGAAGCACAGAAGTTG-3′; 5′-GATCCATGTTTGGCTCCTTC-3′; PR1 : 5′-TACGCAGAACAACTAAGAGG-3′; 5′-TCGTTCACATAATTCCCACG-3′.

[0067] The results show that O. heveae HN1106 produced extensive reactive oxygen species and cell death in the empty vector transgenic control plants ( Figure 3 Compared with the 0th day after inoculation, the empty vector transgenic plants inoculated 8th day PR1 Gene expression is about 250 times ( Figure 3 However, in CSEP03399 Only sporadic cell death and reactive oxygen species ( Figure 3 (A, B) Compared with 0 day, at 8 days CSEP03399 Transgenic plants PR1 Gene expression is only about 50 times ( Figure 3 (C), the difference is extremely significant, indicating overexpression CSEP03399 Genes can significantly reduce O. heveae Disease resistance responses elicited in Arabidopsis thaliana.

[0068] 1.9 CSEP03399 does not promote the virulence function of Pseudomonas In order to verify whether CSEP03399 also exerts toxicity against other pathogens, the inventors further tested the empty vector transgenic control plants and CSEP03399 Transgenic plants were inoculated with virulent Pseudomonas DC3000 and PAMPs pool DC3000 hC - The steps are as follows: After 5 weeks of growth, the leaves of Arabidopsis were sprayed with estrogen. 24 hours after estrogen treatment, the leaves were inoculated with toxin-producing Pseudomonas DC3000 and DC3000. hC -Samples were taken, ground, and plated on 0 and 3 days after inoculation, and the bacteria per unit area were counted.

[0069] The results showed that Pseudomonas sp. DC3000 and DC3000 hC - In the empty vector transgenic control plants and CSEP03399 There was no significant difference in bacterial growth on the transgenic plants ( Figure 4 (A, B), indicating that CSEP03399 does not promote the virulence function of Pseudomonas.

[0070] The inventors further found that in the empty vector transgenic control plants and CSEP03399 Transgenic plants were inoculated with DC3000 avrB and DC3000 avrRpt2 strains containing hypersensitive response effector proteins: after 5 weeks of growth, the leaves were sprayed with estrogen; 24 hours after estrogen treatment, the leaves were inoculated with DC3000 avrB and DC3000 avrRpt2 strains, respectively. 24 hours after inoculation, both strains could be expressed in empty vector transgenic control plants and CSEP03399 Transgenic plants induced a clear and visible hypersensitivity reaction ( Figure 4 (C) indicates that CSEP03399 does not inhibit the hypersensitivity reaction induced by Pseudomonas.

[0071] 1.10 CSEP03399 inhibits chitin-activated FRK1 and WRKY22 Gene expression Previous literature reported that rubber tree powdery mildew stimulated disease resistance in Arabidopsis thaliana. TIR-NLR (TNL) Disease resistance gene WRR4 Involved in resistance to powdery mildew in rubber trees

[22] . WRR4B Gene in Nicotiana benthamiana triggers hypersensitivity reaction

[22] To further explore whether CSEP03399 inhibits WRR4B Genetically triggered hypersensitivity reactions.

[0072] The pER8 vector XOt and B i The DNA fragment between the recognition sequences is replaced with the one shown in SEQ ID No.4 WRR4B pER8-WRR4B was introduced into Agrobacterium GV3101 to obtain the recombinant bacterium GV3101 / pER8-WRR4B; pER8 vector was introduced into Agrobacterium GV3101 to obtain the recombinant bacterium GV3101 / pER8.

[0073] GV3101 / pER8-CSEP03399, GV3101 / pER8-WRR4B, GV3101 / pER8-CSEP03399+GV3101 / pER8-WRR4B, and GV3101 / pER8 were used as the strains to be injected, and the experiments were carried out: the strains were cultured in 5 mL of LB liquid medium (spectinomycin); after 48 h of culture, 2 ml of Agrobacterium culture solution was taken out and 4000 g , centrifuge for 10 min, remove the supernatant, and use buffer (10 mmol·L -1 MgCl2) was rinsed three times, adjusted to an OD value of 0.3 to 0.6, and injected into 5-week-old Nicotiana benthamiana leaves; 24 hours after injection, estrogen treatment was performed; 36 hours after treatment, the injection site was cut and protein was extracted for immunoblotting analysis. FLAG antibody (Biyuntian Company, product number: AF519) was used as the primary antibody, and HRP-labeled goat anti-mouse was used as the secondary antibody (Biyuntian Company, product number: A0216).

[0074] The results showed that after 72 h of estrogen treatment, the empty vector alone and the estrogen-containing CSEP03399 Agrobacterium strains expressing the gene cannot induce a significant hypersensitive response ( Figure 5 A). Contains WRR4B Gene Agrobacterium injection site and WRR4B Gene, CSEP03399 The site of co-injection of Agrobacterium tumefaciens showed a typical hypersensitive reaction ( Figure 5 There is no significant difference between the two, indicating that the effector protein CSEP03399 does not inhibit WRR4B Genetically triggered hypersensitivity reactions.

[0075] To further investigate whether the effector protein CSEP03399 inhibits the immune response stimulated by PAMPs, we inoculated empty vector transgenic control plants and CSEP03399 The leaves of transgenic plants were injected with fungal PAMP chitin. At 0 h and 3 h after injection, the leaves were cut, total RNA was extracted, reverse transcribed, and fluorescent quantitative PCR analysis was performed. The primers used are as follows: ACTIN : 5′-TGGTGGAAGCACAGAAGTTG-3′; 5′-GATCCATGTTTGGCTCCTTC-3′; FRK1 :5'-TCTGAAGAATCAGCTCAAGGC-3';5'-TGTTGGCTTCACATCTCTGTG-3'; WRKY22: 5'-AAGCTCATCAGCTACTACC-3'; 5'-TCTAGAGGTATTGGAGCCAC-3'.

[0076] The results showed that 3 h after injection, chitin CSEP03399 PTI signaling pathway marker genes activated in transgenic plants FRK1 and WRKY22 Compared with the empty vector transgenic plants, the expression was greatly reduced, and the difference was extremely significant ( Figure 5 (C and D) indicate that the effector protein CSEP03399 can inhibit the PTI immune response stimulated by chitin.

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[25] RAMONELL K, BERROCAL-LOBO M, KOH S, et al . Loss-of-functionmutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphecichoracearum [J]. Plant physiology, 2005, 138(2):1027-1036. The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Any of the following uses of a protein or a substance for regulating the content or activity of the protein: D1) Regulate plant disease resistance; D2) Preparation of products for regulating plant disease resistance; D3) Breeding plants with altered disease resistance; D4) Preparation of products for breeding plants with altered disease resistance; The protein is as follows: A1), A2) or A3): A1) a protein whose amino acid sequence is positions 31 to 192 of SEQ ID No. 3; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 3 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added; A3) A protein obtained by attaching a tag to the N-terminus or / and C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that: A3) The protein is a protein whose amino acid sequence is SEQ ID No.

3.

3. The use according to claim 1 or 2, characterized in that: The substance is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

4. The use according to claim 3, characterized in that: B1) The nucleic acid molecule is as follows: b11) or b12) or b13): b11) The coding sequence is the DNA molecule at positions 91 to 576 of SEQ ID No. 2 in the sequence list; b12) The coding sequence is the DNA molecule of SEQ ID No. 2 in the sequence list; b13) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) or b12) and encodes the protein.

5. The use according to any one of claims 1 to 4, characterized in that: The disease resistance is resistance to powdery mildew.

6. The use according to any one of claims 1 to 5, characterized in that: The substance for regulating the content or activity of the protein is to increase the content or activity of the protein, the substance for regulating the disease resistance of the plant is to reduce the disease resistance of the plant, and the substance for cultivating plants with altered disease resistance is to cultivate plants with reduced disease resistance.

7. Any of the following methods: X1) Methods of reducing plant disease resistance, including: Increasing the content or activity of the protein of claim 1 or 2 in a recipient plant to obtain a target plant with reduced disease resistance compared with the recipient plant, thereby achieving a reduction in plant disease resistance; X2) A method for breeding plants with reduced disease resistance, comprising: increasing the content or activity of the protein of claim 1 or 2 in a recipient plant to obtain a target plant with reduced disease resistance compared to the recipient plant.

8. The method according to claim 7, characterized in that: The methods X1) and X2) are achieved by introducing the gene encoding the protein into the recipient plant.

9. The use according to any one of claims 1 to 6, or the method according to claim 7 or 8, characterized in that: The plant is any one of M1)-M6): M1) dicots or monocots; M2) Magnolia; M3) Solanales or Cruciferales; M4) Solanaceae or Cruciferae; M5) Nicotiana or Arabidopsis; M6) tobacco or Arabidopsis thaliana.

10. The protein according to claim 1 or 2, or the substance for regulating the content or activity of the protein according to any one of claims 1 to 4.

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